
Proceedings of the AACR

Volume 60 | April 2019

Part A: Abstracts 2749-5314

TABLE OF CONTENTS

BIOINFORMATICS AND SYSTEMS BIOLOGY

  * Applications of Cancer Computational Biology 1
  * Applications of Cancer Computational Biology 2
  * Convergence
  * Methods and Tools for Cancer Analysis

CANCER CHEMISTRY

  * Cancer Chemical Biology and Therapeutics Development
  * Drug Delivery and Nanoparticles
  * Next-Generation Small Molecules: From Hits to Leads to Candidates
  * Cancer Proteomics

CLINICAL RESEARCH

  * Immune Infiltration and Immune Therapy
  * New Discoveries in Childhood Cancer
  * Novel Strategies for Biomarker Identification and Use in Cancer 1
  * Tumor Markers to Assess the Biology and Clinical Course of Cancer 1
  * Immunomodulation by Chemotherapy and Targeted Agents
  * Novel Strategies for Biomarker Identification and Use in Cancer 2
  * Special Populations / Biostatistics in Clinical Trials
  * Tumor Markers to Assess the Biology and Clinical Course of Cancer 2
  * Molecular and Cell-Based Circulating Biomarkers to Guide Optimal Anticancer Treatment
  * Advances in Radiation Therapy / Surgical Oncology
  * Novel Strategies for Biomarker Identification and Use in Cancer 3
  * Supportive Care and Survivorship Research / Outcome Research
  * Tumor Markers to Assess the Biology and Clinical Course of Cancer 3

EPIDEMIOLOGY

  * Factors Influencing Cancer Outcomes
  * Cancer Predisposition
  * Descriptive Epidemiology: Risk, Outcomes, and Disparities
  * Factors Influencing Cancer Outcomes
  * Influences on Cancer Risk and Outcomes

EXPERIMENTAL AND MOLECULAR THERAPEUTICS

  * Cellular and Molecular Pharmacology, Pharmacogenomics, Pharmacogenetics, and Therapeutic Response
  * Cellular Responses to Anticancer Agents 3: Novel Targets
  * Diagnostics, Biomarkers, and the Tumor Microenvironment
  * Drug Resistance 4
  * New Target Identification
  * Novel Antitumor Agents 1
  * Drug Resistance 5
  * Epigenetic Targets
  * Novel Antitumor Agents 2
  * Pharmacokinetics and Pharmacodynamics / Preclinical Toxicology
  * PI3K/AKT Inhibitors
  * Preclinical Radiotherapeutics
  * Genomic Correlates of Therapy Response
  * Novel Therapeutics
  * DNA Repair and Reactive Agents / HDAC / Demethylating Agents
  * Drug Resistance 6
  * Gene- and Vector-Based Therapy
  * Novel Antitumor Agents 3
  * Targeted Therapies
  * Targeted Therapies and Immunological/Tumor Microenvironment Effects

IMMUNOLOGY

  * Adoptive Cell Therapy 3
  * Combination Immunotherapies 2
  * Immune Checkpoints 1
  * Novel Immunomodulatory Agents 1
  * Biomarkers and Immune Monitoring
  * Combination Immunotherapies 3
  * Immune Checkpoints 2
  * Novel Immunomodulatory Agents 2
  * Tumor Immune Microenvironment
  * Adaptive Immune Cells in the Tumor Microenvironment
  * Immunomodulators and Response to Therapy
  * Tumor Immune Microenvironment

MOLECULAR AND CELLULAR BIOLOGY / GENETICS

  * Cancer Genomics 4
  * Cell Signaling 1
  * Deregulated Tumor Suppressors and Carcinogenesis
  * DNA Damage and Repair 3
  * High-Throughput Sequencing
  * miRNAs as Tumor Suppressors/Oncogenes
  * Noncoding RNAs 2
  * Targeting Metabolism for Cancer Therapy
  * Autophagy
  * Cell Signaling 2
  * Epigenetic Changes as Molecular Markers
  * Functional Genomics
  * Metabolic Reprogramming in Cancer
  * Post-transcriptional and Translational Control of Gene Regulation
  * Targeting the Cell Cycle: Development of Preclinical Models and Therapeutic Targets
  * Ubiquitin Ligases
  * Deregulated Transcription and RNA Processing in Cancer
  * Noncoding RNAs in Cancer Biology
  * Oncogenes, Tumor Suppressors, and Carcinogenesis
  * Advanced Omics
  * Cellular and Stromal Interactions of Tumor Cells
  * Changes in Chromatin Structure
  * Deregulation of Histone Modifications
  * Mechanisms and Consequences of Transcriptional Deregulation
  * Regulation of Gene Expression in Cancer
  * Tracking Metabolic Profiles and Subtype-specific Metabolic Interventions
  * Tumor Suppressors, Carcinogenesis, and Tumor Cell Resistance

PREVENTION RESEARCH

  * Clinical Prevention, Early Detection, and Interception 1
  * Science and Health Policy 1
  * Science and Health Policy 2 / Regulatory Science and Policy
  * Clinical Prevention, Early Detection, and Interception 2
  * Cancer Chemoprevention and Interception: From Mechanism to Translation

TUMOR BIOLOGY

  * Expression Profiling and Biomarkers of Tumor Progression and Metastasis
  * Immune Cells in the Tumor Microenvironment 2
  * Inflammation and Microbiome
  * Invasion and Migration 1
  * Targets and Therapies in Pediatric Cancer
  * Tumor Evolution and Heterogeneity 1
  * Tumor Radiosensitivity or Resistance
  * Biology and Signaling in Pediatric Cancer
  * Epigenetic Regulation, Plasticity, and DNA Repair of Cancer Stem Cells
  * Novel Model Organisms and Approaches
  * Radiation Tissue Tolerance, Immunity, and in Vivo Effects of Radiation
  * Tumor Dormancy, Metastasis, and the Metastatic Niche
  * Tumor Evolution and Heterogeneity 2
  * Viral and Microbiome-associated Carcinogenesis
  * Invasion and Metastasis 2: Tumor Microenvironment
  * Nonclinical Models of Cancer
  * Immune Cells in the Tumor Microenvironment 3
  * Immunity in the Microenvironment
  * Invasion and Migration 2
  * Mouse Models
  * Mutagenesis, Chemical Carcinogenesis, and Tumor Initiation/Promotion
  * Signaling Cascades in Cancer Stem Cells
  * Tumor Evolution and Heterogeneity 3

# Tuesday, April 2, 2019

## CANCER CHEMISTRY

### Cancer Chemical Biology and Therapeutics Development

#2749

Defining structure activity relationships for GPCR engagement and anti-cancer efficacy of imipridone small molecules.

Varun V. Prabhu,1 Abed Rahman Kawakibi,1 Neel S. Madhukar,2 Lakshmi Anantharaman,3 Sean Deacon,3 Neil S. Charter,3 Mathew J. Garnett,4 Ultan McDermott,4 Cyril H. Benes,5 Wolfgang Oster,1 Olivier Elemento,2 Martin Stogniew,1 Joshua E. Allen1. 1 _Oncoceutics, Inc, Philadelphia, PA;_ 2 _Weill Cornell Medicine, New York, NY;_ 3 _Eurofins DiscoverX, CA;_ 4 _Wellcome Trust Sanger Institute, United Kingdom;_ 5 _Massachusetts General Hospital, Boston, MA_.

G protein-coupled receptors (GPCRs) represent the most widely exploited superfamily of drug targets for FDA-approved therapies for many diseases, however, these receptors are underexploited for oncology. ONC201 is a selective antagonist of GPCRs dopamine receptor D2 (DRD2) and DRD3 that has been shown to induce tumor regressions with a benign safety profile in high grade glioma patients. ONC201 (benzyl-2-methylbenzyl-imipridone) is the founding member of the imipridone class of small molecules that share a unique tri-heterocyclic core chemical structure. Imipridones share several chemical and biological properties that are desirable drug-like characteristics: oral administration, wide therapeutic window, chemical stability and blood brain barrier penetrance. In this study, we profiled a series of imipridones for GPCR engagement and anti-cancer efficacy. Several imipridones were screened against a large panel of human GPCRs using a β-arrestin recruitment assay. The imipridones tested resulted in GPCR agonist/antagonist activity (threshold set at >20% activity) that was heterogenous, but exclusive among Class A GPCRs that represent the largest class. Minor chemical modifications to the ONC201 chemical structure caused large shifts in agonist versus antagonist activity and selectivity for GPCRs. Specifically, switching the ONC201 imipridone core from an angular to a linear isomer resulted in loss of DRD2 antagonist activity and impaired inhibition of cancer cell viability, indicating the imipridone core structure is critical for GPCR engagement and anti-cancer effects. The addition of electron withdrawing groups (e.g. di- or tri-halogen substitution) to the methyl benzyl ring improved potency for GPCR engagement and anti-cancer effects, but not for the benzyl ring. Loss of the benzyl ring impaired anti-cancer effects. Among all of the GPCR hits identified, maximal variance in imipridone GPCR engagement was identified for DRD2/DRD3 antagonism and GPR132 agonism that were prioritized considering their known biological relevance in oncology. ONC206 (benzyl-2,4-difluoromethylbenzyl-imipridone) emerged as the most selective and potent antagonist for D2-like dopamine receptors that are overexpressed and critical for survival in several cancers. ONC212 (benzyl-4-trifluoromethylbenzyl-imipridone) was the most selective and potent agonist for tumor suppressor GPR132. Both compounds were tested in the GDSC panel of >1000 cancer cell lines and demonstrated broad spectrum nanomolar inhibition of cancer cell viability and a wide therapeutic window. GPCR target expression correlated with anti-cancer efficacy in the GDSC panel for both compounds, providing potential biomarkers of response. Thus, chemical derivatization of ONC201 has generated a class of novel GPCR-targeting agents with promising preclinical efficacy and safety profiles in oncology.

#2750

Physical and functional landscape of deubiquitinating enzymes (DUBs) in KRAS mutant lung cancer.

Emma Adhikari, Shikha Mahajan, Harshani Lawrence, Yan Yang, Bin Fang, Eric Haura. _Moffitt cancer center, Tampa, FL_.

DUBs are involved in tumorigenesis and are of high interest as potential targets for cancer therapy. However, target discovery is problematic due to the absence of clear DUB activity within subsets of human cancers. Our goal was to profile DUBs and their activity in KRAS-mutated lung cancer cell lines using chemical biology strategies. Approximately 25% of patients with Lung adenocarcinoma have tumor associated KRAS mutations in non-small lung cancers (NSCLC), yet specific RAS inhibitors against KRAS-mutated lung cancer have not yet been successfully developed.

We used activity-based protein profiling (ABPP) to profile DUBs in 25 distinct human lung cancer cell lines harboring KRAS mutations. ABPP uses chemical probes that are active-site directed and covalently bind to a class of enzymes in complex proteome. Whole cell lysates were incubated with HA-UB-VME and HA-UB-PA probes and ABPP pull-down was performed. Enriched proteins were trypsin digested and peptides were analyzed using liquid chromatography and tandem mass spectrometry (LC-MS/MS). MAXQUANT software was used to quantify DUBs. We related activity of observed DUBs to effects on cell viability by examining publicly available RNAi (Project DRIVE) and CRISPR (PICKLES) databases.

A total of 50 DUBs were identified in our ABPP screen. Each cell line expressed at least 32 different DUBs and 22 DUBs were represented in all cell lines. The identified DUBs include 48% USP, 36% OTU, 8% JAMM and 8% UCH & MJD family. USP5, USP7, USP14, USP15, UCHL5, UCHL3, OTUB1, PRP8, PSMD7 and PSMD14 are the highly expressed DUBs. Functional enrichment approach and protein-protein interaction network reveal that 70% of the active DUBs are involved in cell cycle regulation. DDR, RNA splicing, TGF-beta receptor, NF-kB and Wnt signaling are some other enriched pathways. Of the 35 cell cycle DUBs, CRISPR screens identify 14 DUBs and DRIVE data identified 11 DUBS as having effects on cell viability. Combining the ABPP data, shRNA and CRISPR results, OTUD5, BRCC3, UCHL5, UFD1L, USP5, YY1, USP3, USP39, USP37, OTUB1, USP9X and COPS5 have activity in KRAS mutant lung cancer cell lines and demonstrate reduced cell viability with loss of function through CRISPR or RNAi.

We have identified and prioritized DUB targets in KRAS mutant lung cancer. Future studies will further validate DUB as targets and identify mechanisms of activity in KRAS mutant lung cancers, as well as examine DUB activity using ABPP in human lung cancer tumor tissues, including adenocarcinoma (KRAS mutant and wild-type) and squamous cell lung cancer.

#2751

Development of a specific Wee-1 inhibitor.

Mandy Watson, Tom Pesnot, Andrew Scott, Anthony Huxley, Gary Nelson, Montserrat Shelbourne, Jen Morton, Tilly Bingham. _Concept Life Sciences, Manchester, United Kingdom_.

Somatic mutations in the TP53 gene are one of the most frequent alterations in human cancers. The resulting TP53-deficient cancer cells rely on WEE1 to trigger a G2/M arrest, which allows for DNA repair and survival. Inhibition of WEE1 has therefore emerged as an attractive therapeutic strategy to selectively sensitise TP53-deficient tumours to DNA damaging agents. The WEE1 kinase inhibitor adavosertib is the only agent undergoing evaluation in a range of clinical trials to validate this hypothesis. It has also been the tool compound of choice to interrogate WEE1 biology for over a decade.

However, recent reports show adavosertib has antiproliferative single agent activity, which is counter intuitive considering its postulated mode of action. Other studies suggest adavosertib exerts poor kinase selectivity, and inhibits PLK1 with similar potency as WEE1. Since PLK1 is a well-established mitotic regulator with biological functions throughout the cell cycle, its inhibition may contribute to the clinical efficacy and toxicity observed for adavosertib. Other reported WEE1 inhibitors also suffer from low selectivity and single agent toxicity, making then unsuitable tool compounds. There is a clear need to identify a selective Wee1 tool compound.

Using a structure-based drug design approach, we exploited subtle active site differences to identify novel, potent WEE1 inhibitors that display high selectivity over PLK1. These compounds allowed us to demonstrate the distinct effects of WEE1 and PLK1 inhibition on cellular toxicity, and confirm that adavosertib's single agent efficacy is unlikely to be mediated solely through the inhibition of WEE1. Our ongoing studies aim to provide a WEE1 selective tool compound that more rigorously probes WEE1 biology, and eventually leads to the development of less toxic therapeutic agents.

#2752

Recognition of the hybrid-1 human telomeric G-quadruplex by a platinum(II)-based compound.

Wenting Liu,1 Clement Lin,1 Zong-wan Mao,2 Danzhou Yang1. 1 _Purdue University, West Lafayette, IN;_ 2 _Sun Yat-Sen University, Guangzhou, China_.

The human telomeric DNA, which consists of tandem repeat sequences of d(TTAGGG)n, plays an important role in cancers and cell aging. Previous studies in human cancer cells have demonstrated DNA G-quadruplex (G4) structure formation in telomeres. G-quadruplexes are four-stranded structures formed in guanine-rich sequences that are held together by guanine-guanine Hoogsteen hydrogen bonding, and G-quadruplex stabilization by small molecules induces tumor cell senescence and apoptosis by repressing telomerase activity and the DNA damage response pathway. Additionally, telomeric-overhang DNA can form biologically relevant higher-order DNA structures containing consecutive G-quadruplexes which provides additional binding sites for small molecules. Thus, telomeric DNA G-quadruplexes have attracted increasing interest as potential drug targets for cancer therapy. Here, we found that a novel platinum (II)-based tripod (Pt-tripod) specifically recognizes the hybrid-1 human telomeric G4, which is formed in the physiologically relevant K+ solution. Pt-tripod strongly represses telomerase activity and exhibits DNA-targeted photodynamic therapy anticancer activity. Using NMR, we show that Pt-tripod binds the hybrid-1 human telomeric G4 over other G-quadruplex structures and dsDNA. We determined solution structures of the 1:1 and the dimeric 4:2 Pt-tripod-hybrid-1 telomeric G4 complexes by NMR. Our 1:1 complex structure shows preferential binding of the Pt-tripod to the 5ʹ-end of hybrid-1 telomeric G4. At higher ligand ratio, Pt-tripod binds to the 3ʹ-end of the hybid-1 G4 and induces an unprecedented dimeric 4:2 structure interlocked by a non-canonical A:A pair at the 3ʹ-end. The specific binding of the Pt-tripod with the hybrid-1 G4 is achieved by unique interaction modes including the π-π stacking, hydrogen bonding, and electrostatic interactions with the loop and flanking segments. The non-planar tertiary amine conformation and three properly sized cationic platinum arms are critical for the specific and strong binding. Our structures provide significant insights into understanding the dynamic binding of small molecules with G-quadruplexes, and a structural basis for future rational anticancer drug design of non-planar small molecules targeting the human telomeric G4. Our studies also provide a potential handle to study the specific protein interactions and biological functions of hybrid-1 human telomeric G4.

#2753

The molecular basis of blocking the TIM-3 checkpoint with the LY3321367 mAb in cancer immunotherapy.

Jaafar N. Haidar,1 Stephen Antonysamy,2 Sneha Mathew,1 Lan Wu,1 Yi Zhang,1 Margaret C. Kearins,2 Leyi Shen,1 J. Michael Sauder,2 David Schaer,1 Kyla E. Driscoll,1 Michael Kalos1. 1 _Lilly Research Laboratories, Eli Lilly & Company, New York, NY; _2 _Lilly Biotechnology Center, Eli Lilly & Company, San Diego, CA_.

The anti-T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) antibody LY3321367 is a promising immune-checkpoint inhibitor that is being clinically tested for the treatment of multiple solid tumors (NCT03099109). LY3321367 was phenotypically selected from a large set of phage library Fabs that are TIM-3 binders. Tim3 has multiple protein and non-protein binding partners; hence, we attempt in this study to deconvolute the molecular mechanism of action of LY3321367. Surprisingly, LY3321367 in ELISA assays partially blocks the TIM-3/GAL-9 complex but does not block the TIM-3/CEACAM-1 complex. We also utilized X-ray crystallography to solve the structure the LY3321367-Fab complex with the IgV domain of TIM-3 at 2.0Å resolution. After solving the unbound structure of the LY3321367-Fab, we demonstrated that its CDRs do not undergo any conformational changes upon binding human TIM-3. The structural alignment of the human TIM-3/LY3321367-Fab complex with the available murine TIM-3 structure unexpectedly suggests that the 6.0Å epitope of LY3321367 overlaps the phosphatidylserine (PS) binding site on TIM-3. Consequently, we developed a cell-based assay to demonstrate that LY3321367 completely blocks the binding of soluble human TIM-3 to PS displayed on the surface of Campothecin-treated DO11.10 cells.

Our structural results suggest an important role for PS in TIM-3 biology which is blocked by LY3321367. When combined with the partial blocking of GAL-9, the structural data of LY3321367 suggests that the binding sites of PS and GAL-9 may be proximal to each other on TIM-3.

#2754

Affinity-based probe reveal Bim negatively regulates the chaperone functions of Hsp70, a non-Bcl-2 BH3 receptor.

Ziqian Wang, Zhichao Zhang, Ting Song, Zongwei Guo. _Dalian University of Technology, Dalian, China_.

The identification of novel non-Bcl-2 BH3 receptors is still challenge owing to frequently weak, transient and reversible binding character of the PPIs mediated by them. Herein, we designed and synthesized an affinity-based probe (AfBP), S1b-probe, by introducing a photo-crosslinker to an artificial BH3 mimetic. Hsp70 proteins were identified as novel BH3 receptors in situ. The PPIs between Bcl-2 proteins and Hsp70 proteins were further validated by fluorescent polarization (FP), isothermal titration calorimetry (ITC) and Co-immunoprecipitation (Co-IP) experiments. Structural and functional analysis by photo-crosslinking/LC-MS/MS sequencing, 1H-15N transverse relaxation optimized spectroscopy (TROSY-HSQC), trypsin proteolysis, single-turnover ATPase rates and denatured rhodanese aggregation measurement demonstrated that BimBH3 bind in the NBD domain of Hsp70 proteins to act as a co-chaperone that negatively regulates the ATPase activity and chaperone functions. S1g, derived from BH3 mimetics, was screened as a specific Hsp70 inhibitor in native physiological context by using S1b-probe.

#2755

Limited proteolysis coupled to mass spectrometry (LiP-MS), a novel drug target deconvolution strategy.

Nigel Beaton,1 Roland Bruderer,1 Kristina Beeler,1 Nicholas Dupuis,1 Ilaria Piazza,2 Paola Picotti,2 Lukas Reiter1. 1 _Biognosys AG, Schlieren, Switzerland;_ 2 _ETH Zurich, Zurich, Switzerland_.

Background High attrition rates in target-centric drug development approaches, as well as a limited number of targets, have shifted the focus of drug development back towards phenotypic screening. In parallel, novel proteomics-based target deconvolution approaches to drug target identification have gained popularity. Limited proteolysis coupled with mass spectrometry (LiP-MS) is a new target deconvolution technique that exploits protein structural alterations driven by drug binding. A major advantage of LiP-MS is its unique focus on detection of peptides that report on ligand binding induced structural changes that are generated by a limited digestion and identified by proteomic analysis. Here we demonstrate the performance of LiP-MS using the protein phosphatase inhibitor Calyculin A, as well as two well known kinase inhibitors, selumetinib (SE) and staurosporine (ST), in HeLa cell lysate.

Materials and Methods Mechanically sheared HeLa cell lysate was incubated with compound at multiple concentrations. Next, a limited digest was performed using proteinase K. Finally, the limited digests were processed to peptides with trypsin for mass spectrometry analysis. A project-specific spectral library was generated using data-dependent acquisition (DDA) mass spectrometry and for quantitative analysis data-independent acquisition (DIA) data were recorded and analyzed using Spectronaut Pulsar X.

Results LiP-MS identifies several peptides for the phosphatase inhibitor Calyculin A, with IC50 values of 52 nM and 17 nM for PP1A and PP2A respectively. Additionally, a previously unknown target PP1B was also identified among the same family, although with a higher IC50 (74 nM). Through structural inspection of ligand-sensitive peptides we were able to map the drug's binding site within the phosphatases and predict distal conformational changes, demonstrating that LiP-MS can be used to provide structural insights to ligand-protein binding. Similar dose response relationships were observed for both specific (SE) and broad (ST) kinase inhibitors. Amongst the top 200 identified target candidate peptides ranked by LiP score, GO enrichment analysis confirmed a highly significant 3-fold enrichment for kinase targets (p < 0.00002) in ST-treated lysate, while no such enrichment was observed for SE. However, in SE-treated lysate robust identification of multiple MEK1 peptides, one of the compound's main targets, was observed. In the case of both kinase inhibitors LiP peptides could be successfully mapped to ATP binding sites, confirming the ability of LiP-MS to model drug-bound protein structure.

Conclusions This data demonstrates that LiP-MS can be used to effectively identify protein drug targets and characterize the binding properties, regardless of the specificity of the compound. These capabilities make LiP-MS a powerful target deconvolution and identification strategy.

#2756

**A high-resolution G-quadruplex structure involved in** cmyc **oncogene regulation.**

Jonathan Dickerhoff, Luying Chen, Guanhui Wu, Buket Onel, Clement Lin, Danzhou Yang. _Purdue University, West Lafayette, IN_.

Gene expression needs strict regulation in human cells to prevent tumorigenesis. Among other control mechanisms, guanine-rich sequences which are clustered in promoter regions of oncogenes can form four stranded nucleic acid structures, called G-quadruplexes (G4), and regulate their transcription. The cmyc protein is the most important cellular regulator for proliferation and is often overexpressed in cancer cells. The DNA G-quadruplex formed in the cmyc promoter is a transcriptional silencer element and stabilization of this G4 by small molecules suppresses cmyc transcription, making it a promising target for cancer therapy. High-resolution structures of the highly diverse G4s provide the opportunity to understand their molecular interactions with transcription factors and to design small molecules that specifically bind a particular G4 conformer. Characteristically, multiple G4s can be formed in gene promoter regions because their G-rich sequence generally contains more than the minimum of four G-stretches. While all reported cmyc G4s share the same core with parallel oriented strands, their overall shape is individualized by distinct combinations of varying flanking and loop motifs. In this study, we present a new high-resolution NMR structure of a G4 found to form in the cmyc promoter region. The determined cmyc G4 has a 1:6:1 loop-size arrangement and its conformation is largely defined by its long central loop of 6 nt. We show that this G4 structure is the preferred target of nucleolin protein in comparison to other G4s formed in the cmyc promoter, demonstrating how the polymorphism of a G-rich sequence can modulate the interaction with transcription factors and other regulators. Our NMR structure shows that, despite the length of the 6-nt loop motif and the associated increased flexibility, the folding converges well due to a base pairing between the central loop and the 5'-flanking. Thus, the long loop is pulled towards the 5'-end while analogous loop-flanking interactions are prevented at the opposite 3'-site. In conclusion, the presented new addition to the ensemble of possible cmyc promoter G4 conformations extends the folding landscape for this promoter region, in particular due to the long central loop. This may shed light on how proteins such as nucleolin can target specific G4 and shift the equilibrium of folding to fine-tune gene expression. Finally, this new structure is another promising target for cancer drug development.

#2757

Discovery and characterization of covalent Pin1 inhibitors targeted to an active site cysteine.

Benika Pinch,1 Zainab Doctor,2 Christopher M. Browne,2 Hyuk-Soo Seo,3 Behnam Nabet,3 Shingo Kozono,4 Xiaolan Lian,4 Daniel Zaidman,5 Dina Daitchman,5 Nir London,5 Lu Gong,4 Theresa Manz,3 Yujin Chun,6 Li Tan,7 Jarrod Marto,3 Stephen Buratowski,6 Sirano Dhe-Paganon,3 Xiao Zhou,4 Kun Ping Lu,4 Nathanael S. Gray2. 1 _Harvard University, Cambridge, MA;_ 2 _Dana-Farber Cancer Institute; Harvard Medical School, Boston, MA;_ 3 _Dana-Farber Cancer Institute, Boston, MA;_ 4 _Beth Israel Deaconess Medical Center; Harvard Medical School, Boston, MA;_ 5 _Weizmann Institute of Science, Rehovot, Israel;_ 6 _Harvard Medical School, Boston, MA;_ 7 _Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, China_.

Proline-directed phosphorylation at serine or threonine residues (pSer/Thr-Pro) regulates numerous cellular processes, including the cell cycle, transcription, and differentiation. Deregulation of such signaling networks is a hallmark of transformation and oncogenesis. Pin1, a peptidyl-prolyl isomerase, regulates the function and stability of phosphoproteins by catalyzing the cis/trans isomerization of pSer/Thr-Pro motifs. Pin1 is frequently overexpressed in human cancers, including pancreatic ductal adenocarcinoma (PDAC), and Pin1 is required for activated Ras to induce tumorigenesis. While mutations in KRAS are observed in 90-95% of human PDAC cases, it has historically proven very challenging to develop small molecules that inhibit mutant Ras function. Consequently, drug discovery efforts have turned to targets required for Ras-mediated transformation, such as Pin1. However, existing Pin1 inhibitors lack the potency, selectivity, and/or cell permeability to serve as informative cellular probes. We report a highly potent, cell-permeable Pin1 inhibitor that covalently targets Cys113, a conserved cysteine residue in the Pin1 active site. Through iterative rounds of synthesis and characterization, we developed inhibitor 1b. With a Ki of 15 nM as measured in biochemical binding and isomerase inhibition assays, 1b is currently the most potent Pin1 inhibitor available. Furthermore, in a chemoproteomic study using Covalent Inhibitor Target Site Identification (CITe-Id) to quantify the dose-dependent covalent labeling of 1b to individual cysteines across the proteome, Pin1 Cys113 was the only identified target, highlighting the pronounced selectivity of 1b for Pin1. We show that treatment with 1b diminishes viability of human PDAC cell lines, which can be fully rescued in corresponding Pin1 knockout cells generated using CRISPR/Cas9, showing that this phenotype is on-target. In parallel to inhibitor development, we used CRISPR/Cas9 GFP-dropout screens to further validate the dependence of these cell lines on Pin1. Genetic disruption of Pin1 led to antiproliferative effects, confirming the results of 1b treatment. We also employed the degradation tag (dTAG) approach to assess the effects of rapid and selective targeted Pin1 degradation through generation of FKBP12F36V-Pin1, Pin1-/- human PDAC cell lines. Treatment with a small molecule FKBP12F36V-degrader led to rapid ubiquitination and degradation of FKBP12F36V-Pin1, enabling comparisons of targeted inhibition and Pin1 degradation. Through the development of a selective Pin1 inhibitor coupled with genetic approaches and the chemical-genetic dTAG strategy, we demonstrate that Pin1 inhibition represents a tractable strategy in PDAC.

#2758

Domain swapping and SMYD1 interactions with the PWWP domain of human hepatoma-derived growth factor.

Chun-Jung Chen,1 Li-Jin Hsu2. 1 _National Synchrotron Radiation Research Center, Hsinchu, Taiwan;_ 2 _National Cheng Kung University, Tainan, Taiwan_.

The human hepatoma-derived growth factor (HDGF), containing the chromatin-associated N-terminal PWWP domain capable of binding the SMYD1 promoter, participates in various cellular processes and is involved in human cancers, including hepatocellular carcinoma, pancreatic cancer, oral cancer, breast cancer and non-small cell lung cancer. However, the functional mechanisms of human HDGF had been puzzling because of a lack of essential knowledge of its intact structure in complex with DNA. We present the first crystal structures of the human HDGF PWWP domain (residues 1 - 100) in a complex with SMYD1 of 10 bp at 2.84 Å resolution and its apo form at 3.3 Å, respectively. The structure of the apo PWWP domain comprises mainly four β-strands and two α-helices. The PWWP domain undergoes domain swapping to dramatically transform its secondary structures, altering the overall conformation from monomeric globular folding into an extended dimeric structure upon DNA binding. The flexible loop2, as a hinge loop with the partially built structure in the apo PWWP domain, notably refolds into a visible and stable α-helix in the DNA complex. The swapped PWWP domain interacts with the minor grooves of the DNA through residues Lys19, Gly22, Arg79 and Lys80 in varied ways on loops 1 and 4 of the two chains, and the structure becomes more rigid than the apo form. These novel structural findings, together with physiological and activity assays of HDGF and the PWWP domain, provide new insights into the DNA-binding mechanism of HDGF during nucleosomal functions.

#2759

Chemoproteomic profiling of the oncometabolite fumarate.

Sarah E. Bergholtz,1 Chloe A. Briney,1 Rhushikesh A. Kulkarni,1 David Wei,2 Daniel W. Bak,3 Jonathan H. Shrimp,1 Eranthie Weerapana,3 W. Marston Linehan,2 Jordan L. Meier1. 1 _National Cancer Institute, Frederick, MD;_ 2 _National Cancer Institute, Bethesda, MD;_ 3 _Boston College, Chestnut Hill, MA_.

Dysregulated metabolism is a feature of many diseases, including cancer. In addition to providing energy and building blocks to growing cells, altered metabolism can assist in tumor growth by producing "oncometabolites". For example, in hereditary leiomyomatosis and renal cell carcinoma (HLRCC) inactivating mutations in fumarate hydratase (FH) leads to accumulation of millimolar levels of an oncometabolite, fumarate. At such high concentrations, fumarate can spontaneously react with protein cysteine residues leading to a post-translational modification (PTM) called cysteine succination. This PTM can directly stimulate tumorigenic signaling, however, current methods to study this PTM are limited. Our lab has developed chemical proteomic methods to investigate proteome-wide targets of cysteine succination. Using these methods, we have identified hundreds of targets of fumarate in normal kidney cells as well as HLRCC cells. One interesting target identified that has implications in cancer progression is SMARCC1, a core subunit of a nucleosome remodeling complex known as the SWI/SNF complex. The SWI/SNF complex is a well-known tumor suppressor and mutation of this complex leads to cancer development. We have demonstrated that excess fumarate levels result in hyper-modification of SMARCC1 and decrease in SWI/SNF complex stability. This instability also results in an increase in EZH2 expression as well as activity, and an increase in H3K27Me3. Our current efforts are aimed at understanding how fumarate affects the function of this complex in vivo and exploring the potential of using EZH2 inhibitors in the treatment of HLRCC.

#2760

Photoinducible detection of the oncometabolite fumarate.

Chloe Briney,1 Sarah Bergholtz,1 Rhushikesh Kulkarni,1 Daniel Crooks,2 Chandrasekhar Mushti,3 Stephen Lockett,1 Rolf Swenson,3 W. Marston Linehan,2 Jordan Meier1. 1 _National Institutes of Health, Frederick, MD;_ 2 _National Institutes of Health, Bethesda, MD;_ 3 _National Institutes of Health, Rockville, MD_.

Dysregulated metabolism is an important marker of many disease states, including cancer. For example, in hereditary leiomyomatosis and renal cell cancer (HLRCC), inactivating mutations in fumarate hydratase (FH) lead to accumulation of high levels of fumarate, a so-called "oncometabolite." Substantial evidence indicates that fumarate stimulates various oncogenic signaling pathways, necessitating sensitive methods to detect the oncometabolite in order to more rapidly diagnose HLRCC as well as to identify new disease settings in which fumarate may play a signaling role. Here, we report development of novel photoactivatable, fluorogenic chemical probes for detection and profiling of fumarate in biological systems. These chemical probes, diaryl tetrazoles, are by themselves inert towards fumarate. However, upon irradiation with UV light, they release nitrileimines that can form fluorescent cycloadducts with fumarate. We have demonstrated that diaryl tetrazoles can sensitively detect FH activity as well as low micromolar levels of fumarate in complex biological samples. We have also shown that diaryl tetrazoles can be used to monitor changes in intracellular fumarate levels in biological samples by live-cell imaging and flow cytometry. Moreover, these compounds are capable of visualizing differences between patient-derived primary HLRCC tumors lacking FH activity and the adjacent normal kidney, highlighting their potential utility in clinical diagnostics. By offering new insights into fumarate reactivity, our studies provide the chemical basis for novel approaches to therapy and diagnosis in cancers driven by oncometabolite accumulation.

#2761

Development of synthetic guide RNA libraries for CRISPR-mediated transcriptional activation screening for gain-of-function studies.

Annaleen Vermeulen, Zaklina Strezoska, Sarah M. Dickerson, Maren M. Gross, Eldon T. Chou, Elena Maksimova, Matthew R. Perkett, Shawn McClelland, Anja V. Smith. _Dharmacon, a Horizon Discovery Company, Lafayette, CO_.

Functional gene analysis studies have been empowered by development of CRISPR-Cas9 gene knockout tools, however the CRISPR-Cas9 system has also been adapted for inhibition or activation of gene transcription. A nuclease-deactivated Cas9 (dCas9) can be fused to various effector domains to produce an RNA-guided transcription factor for either inhibition (CRISPRi) or activation (CRISPRa) of target genes. For overexpression studies, CRISPRa holds significant advantages over traditional vector-based gene expression, because genes are upregulated from their native promoter and endogenous genomic context. The majority of CRISPRa research performed to date has utilized single guide RNA (sgRNA) expressed from a DNA vector, primarily in the context of pooled lentiviral screening approaches. Here we describe the development of CRISPRa synthetic guide RNAs for the use in arrayed screening, so that we combine this next-generation transcriptional activation method with the ability to support more complex assays in a one-gene-per well format. Considerations for arrayed activation screens will be shown. The combination of gain-of-function from CRISPRa with loss-of-function using RNAi or canonical CRISPR-Cas9 for gene knockout allows for robust characterization of gene mechanisms and pathways.

#2762

Development of a novel peptide-formed nanoparticle for pancreatic cancer treatment.

Qiongling Wang, Kevin F. Staveley-O'Carroll, Guangfu Li. _University of Missouri, Columbia, MO_.

Background and objective: Pancreatic ductal adenocarcinoma (PDAC) is the most common malignant tumor of pancreas with an overall 5-year survival rate of less than 5%. Surgical operation is an curable treatment, however, only 10-20% of patients with PDAC are respectable at diagnosis. Taking advantage of the progression in nanoparticle study, we developed a novel peptide-formed nanoparticle (PF-Nano) and investigate its therapeutic effect in PDAC treatment.

Methods: Phage display peptide library was used to screen a high-affinity tumor-homing peptide (THP) for pancreatic cancer (PC) cells. Ligation of THP with cell penetrating peptide was used to prepare a tandem peptide, which automatically formed a nanoparticle. We used this PF-Nano as a vehicle to load and deliver siRNA. Dynamic light scattering (DLS) was employed to define nanoparticle size; and fluorescence imaging was applied to assess PF-Nano siRNA delivery efficiency. In vitro, we used MTT assay and colony formation to evaluate cell growth; wound healing assay to detect cell migration, and flow cytometry to evaluate cell cycle and apoptosis. Therapeutic effect was evaluated by measuring final tumor weight in the mice receiving different treatments. qPCR and Western Blot (WB) were used to detect gene expression in mRNA and protein level. Immunoprecipitation in combination with mass spectrometry and immunofluorescence were used to identify THP's ligands.

Results: The screened peptide has a high-affinity with PC cells. DLS identified evenly distributed PF-Nano with a diameter of approximately 230nm. Detection of fluorescence signal from labeled siRNAs in PC cells and tumor tissue suggested that PF-Nano owned the capacity to successfully deliver siRNA in vitro and in vivo. In vitro treatment with PF-Nano significantly suppressed PC cell proliferation, colony formation, and cell migration. Also it caused cycle arrest in S phase (DNA synthesis) and increase in cell apoptosis. Treatment of tumor-bearing mice with PF-Nano by intraperitoneal injection markedly suppressed tumor growth, evidenced by the reduction of tumor weight to 25% relevant to that in control mice without treatment. Immunoprecipitation and mass spectrometry revealed that protein phosphatase 1 catalytic subunit gamma (PP1C) was a potential binding target of the specific THP. Mechanistically, PF-Nano treatment strongly suppressed RAC-alpha serine/threonine-protein kinase (AKT) phosphorylation,

suggesting PP1-AKT signal pathway may be responsible for peptide effect in pancreatic tumor.

Conclusion: The established PF-Nano is able to specifically deliver siRNAs to PC tumors and therapeutically suppresses PC tumor growth.

#2763

Raf-1 cysteine-rich domain (CRD) promotes active KRas4B membrane orientation facilitating dimerization through the allosteric lobe interface.

Hyunbum Jang, Ruth Nussinov. _National Cancer Institute at Frederick, Frederick, MD_.

Ras proteins are classical members of small GTPases, controlling signal transduction pathways and promoting cell proliferation. Ras consists of highly homologous catalytic domains and flexible C-terminal hypervariable regions (HVRs) that differ significantly across Ras isoforms. Ras activation is regulated by guanine nucleotide exchange factors that catalyze the exchange of GDP by GTP, and activation is terminated by GTPase-activating proteins that induce the GTP hydrolysis. Ras has multiple partners, signals through several key pathways and fulfills critical functions in the cell life. Mutations in Ras are common in a variety of cancers; yet it is still undruggable. KRas4B is frequently mutated in cancer. Elucidation of Ras conformational ensembles at the signaling platforms in the plasma membrane including the ligand-bound conformations, membrane orientations, the activated (or inactivated) allosteric modulated states, post-translationally modified states, mutational states, and transition states are essential for deciphering Ras functions from its conformational landscapes. In the MAPK pathway, KRas4B preferentially recruits Raf-1 and activates it. Raf kinases contain Ras binding domain (RBD) and cysteine-rich domain (CRD) at conserved region 1 (CR1), and kinase domain at CR3. The high-affinity interaction of Raf RBD with Ras was solved in the absence of CRD. It is known that Raf CRD play a key role in the membrane anchoring mechanism. Recently, pivotal roles of CRD in the membrane interactions of KRas4B-Raf-1 RBD-CRD complex were reported. Here, we employ all-atom molecular dynamics (MD) simulations to investigate dimeric KRas4B-Raf-1 complex at the anionic lipid bilayer composed of DOPC and DOPS (4:1). Active KRas4B dimer modulates Raf-1 activation, promoting dimerization of Raf-1's kinase domain. In the dimeric complex of KRas4B-Raf-1, Raf-1 CRD stably binds anionic lipid bilayers inserting its positively-charged loop into the amphipathic interface. The key basic residues at the loop are responsible for the membrane association, suggesting that CRD is an intrinsic membrane binding domain of Raf kinase. Our simulations suggest that Raf-1 CRD not only offers an additional membrane anchor for the KRas4B-Raf-1 complex, but also reduces the fluctuations of Ras-RBD, enhancing the high affinity interaction between Ras and Raf. Raf-1 CRD supports the active-state Ras orientation, promoting Ras dimerization through the allosteric lobe helical interface at the membrane. The enhanced stability of the complex at the membrane rendered by CRD promotes Raf dimerization in the MAPK signaling, which is the key Ras proliferative pathway. Here we suggest these significant roles of CRD at the membrane in the Raf activation. Funded by Frederick National Laboratory for Cancer Research, National Institutes of Health, under contract HHSN261200800001E.

#2764

Small activating RNA induced expression of VHL gene in renal cell carcinoma.

Jong Soon Kang,1 Moo RIm Kang,2 Ki Hwan Park1. 1 _Korea Research Inst. of Biosci. Biotech., Cheongju, Republic of Korea;_ 2 _Ractigen Therapeutics, Nantong, China_.

Recent studies have reported that chemically synthesized double-stranded RNAs (dsRNAs), also known as small activating RNA (saRNAs), can specifically induce gene expression by targeting promoter sequences in a mechanism termed RNA activation (RNAa). In the present study, we designed four candidate dsRNAs targeting the Von Hippel-Lindau (VHL) gene promoter. Among these dsRNAs, dsVHL-821 significantly inhibited cell growth by up-regulating VHL at both the mRNA and protein levels in renal cell carcinoma 769-P cells. Functional analysis showed that dsVHL-821 induced apoptosis by increasing p53, decreasing Bcl-xL, activating caspase 3/7 and poly-ADP-ribose polymerase in a dose-dependent manner. Chromatin immunoprecipitation analysis revealed that dsVHL-821 increased the enrichment of Ago2 and RNA polymerase II at the dsVHL-821 target site. In addition, Ago2 depletion significantly suppressed dsVHL-821-induced up-regulation of VHL gene expression and related effects. Consistent with previous studies on RNAa of other genes, single transfection of dsVHL-821 caused long-lasting (14 days) VHL up-regulation. Furthermore, the activation of VHL by dsVHL-821 was accompanied by an increase in dimethylation of histone 3 at lysine 4 (H3K4me2) and acetylation of histone 4 (H4ac) and a decrease in dimethylation of histone 3 at lysine 9 (H3K9me2) and lysine 27 (H3K27me2) in the dsVHL-821 target region. Taken together, these results demonstrate that dsVHL-821, a novel saRNA for VHL, induces the expression of the VHL gene by epigenetic changes, resulting in inhibition of cell growth and induction of apoptosis. Targeted activation of VHL by dsVHL-821 may be explored as a novel treatment of renal cell carcinoma.

## TUMOR BIOLOGY

### Expression Profiling and Biomarkers of Tumor Progression and Metastasis

#2765

Exosomal miRNAs as circulating biomarkers for prediction of metastasis in stage II/III gastric cancer.

Xin Liu,1 Kent-Man Chu2. 1 _Univ. of Hong Kong Faculty of Medicine, Hong Kong, Hong Kong;_ 2 _Univ. of Hong Kong Faculty of Medicine, Queen Mary Hospital, Hong Kong, Hong Kong_.

Background: Metastasis is the fatal character of cancer, leading to mortality of gastric cancer. In recent years, exosomes have been reported as extracellular vesicles secreted by cells. They contain various biological molecules including miRNAs from the original cells. Exosomal miRNAs are significant for pre-metastatic niche formation, as they can change the microenvironment and make it favorable for future metastasis. Therefore, the aim of this study was to identify dysregulated exosomal miRNAs as circulating biomarkers for prediction of metastasis of gastric cancer.

Materials and Methods: Pre-treatment serum samples of 36 metastatic patients and 55 non-metastatic patients with stage II/III gastric cancer were collected. Exosomes were extracted and validated by western blot, transmission electron microscope and nanoparticle tracking analysis. MiRCURY LNATM miRNA miRNome PCR Array (containing 752 miRNAs) was performed in three pairs of serum exosomes. Each pair samples were with the same stage, race, gender and similar age. These three pairs of samples represented stage II, IIIA or IIIB gastric cancer, respectively. A panel of commonly dysregulated exosomal miRNAs was released from the PCR Array. Expressions of exosomal miRNAs were validated in the remaining 33 metastatic and 52 non-metastatic patients by RT-qPCR, as well as in gastric cancer cell culture medium and cell lines.

Results: By comparing the expressions of exosomal miRNAs between the metastatic patients and matched non-metastatic ones, 13 upregulated and 6 downregulated miRNAs were released after normalization with miR-16-5p and miR-93-5p. Among these miRNAs, 7 of them were selected for further validation according to their fold changes, p-values and association with cancer development. MiR-379-5p and miR-410-3p were validated to be significantly upregulated in metastatic patients (P<0.01). Sensitivity, specificity and AUC were 67.31%, 69.70% and 0.6894 for miR-379-5p, and 65.38%, 66.67% and 0.6719 for miR-410-3p. Higher expression of serum exosomal miR-379-5p or miR-410-3p showed shorter overall survival of the patients (P<0.05). It was also found that miR-379-5p and miR-410-3p expressed significantly higher in gastric cancer cell culture medium comparing with gastric cancer cells.

Conclusions: Exosomal miRNAs are dysregulated in the serum of metastatic gastric cancer patients before they are clinically diagnosed. Upregulation of serum exosomal miR-379-5p and/or miR-410-3p are promising circulating biomarkers for prediction of metastasis in stage II/III gastric cancer.

#2766

Amplification-associated upregulation of genes involved in oxidative phosphorylation for disseminated prostate cancer cells.

Chun-Lin Lin, Tan Xi, Chia-Nung Hung, Pawel A. Osmulski, Chih-Wei Chou, Meizhen Chen, Chiou-Miin Wang, Kohzoh Mitsuya, Nameer Kirma, Maria E. Gaczynska, Chun-Liang Chen, Tim H.-M. Huang. _University of Texas Health San Antonio, San Antonio, TX_.

Purpose: Little is known about adaptive selection of tumor cells transiting from in situ proliferation to distant colonization through blood circulation. This study used genomic, transcriptomic, and biophysical analyses at single-cell levels to explore biological and physical properties of circulating tumor cells (CTCs) undergoing hemodynamic stress. The aims are trying to understand how CTCs strive to survive in the bloodstream and to develop strategies for therapeutic intervention.

Experimental Design: We compared genomic profiles of CTCs in blood and primary tumor cells shed in urine of prostate cancer patients to identify amplified regions preferentially retained in CTCs. Single-cell RNA-seq was used to confirm amplification-associated overexpression of genes in CTCs.

Results: Among 261 recurrently amplified genomic regions in the analysis, 70 were found predominantly shown in CTCs relative to primary tumor cells. In line with the results at the genomic level, the transcriptomic data of CTCs demonstrate a great amount of cells showing high expression levels in the oxidative phosphorylation (OXPHOS) pathway compared to other hallmark gene sets. The finding suggests that tumor cells with these pre-existing genomic alterations were adaptively selected for transcription reprogramming during blood circulation. Specifically, amplified genes associated with OXPHOS were exploited by CTCs for alternative fuels. As to the upstream of this transcription event, we found the expression of MEN1, encoding menin known to form a transcription factor complex with the mixed-lineage leukemia protein (MLL) in prostate cancer cells, was positively correlated with 11 of the 14 OXPHOS loci in the Cancer Genomic Atlas prostate cancer cohort. In vitro assay showed that MEN1 knockdown by shRNA resulted in attenuation of both mRNA and protein expression of OXPHOS loci in PC-3 cells. Taken together pre-existing amplification of OXPHOS loci can be used as a transcription apparatus by menin for metabolic reprogramming of tumor cells in response to harsh microenvironments in the bloodstream.

Conclusions: Single-cell profiling identified signaling pathways that are crucial for CTCs to survive in the bloodstream. The finding suggests that a metabolic shift from Warburg to OXPHOS metabolism can be associated with a hybrid mesenchymal-epithelial phenotype of CTCs. Moreover, the study demonstrates the feasibility of routinely conducting single-cell analysis of exfoliated tumor cells for minimally invasive monitoring of disease progression and treatment response in prostate cancer patients

#2767

Expression of defensin-associated genes may be correlated with lymph node metastasis of early stage tongue cancer.

Doh Young Lee. _Seoul National University, Seoul, Republic of Korea_.

OBJECTIVES: Appropriate patient selection for elective neck dissection in N0 tongue cancer has been controversial, while there has been no genetic consideration so far. We aimed to analyze genetic difference between early and late cervical lymph node metastasis in oral tongue cancer patients using the Seoul National University Hospital (SNUH) cancer cohort and cancer genome atlas (TCGA) data, and to suggest genetic background for choosing eligible patient for elective neck dissection. METHODS: A total of 35 cases with RNAseq in SNUH cancer cohort with tongue cancer were also enrolled in this study. To investigate gene expression in early cervical lymph node metastasis, genomic data of following 2 groups was compared; 1) N0 group: T1/2 and N0, 2) N+ group: T1/2 and N+. Differentially expressed genes (DEGs) were extracted using R and limma package in bioconductor program. Gene ontology and pathway enrichment analysis were performed using DAVID online tool. Immune cell infiltration was analyzed using CIBERSORT online program. Additionally, 70 cases with matched RNAseq data of primary tumor and clinical TCGA data were analyzed to validate the role of DEGs. RESULTS: N0 and N+ groups showed no difference in age, gender, size and thickness of tumor, and nearest resection margin. In addition, there was no significant difference in 22 types of immune cell infiltration. A total of 51 DEGs were identified, and 14 genes were significantly upregulated and 37 genes were significantly downregulated (p<0.01, fold-change >2). Significant genes were PLA2G4D, NELL2, PCOLCE2, PI3, and PLA2G3. On KEGG pathway analysis, calcium, muscle contraction, and arachidonic acid metabolism-related pathway was significantly correlated. The most significant difference was found in 6 genes as follows; DEFB4A, SPRR2B, DEFB103B, SPRR2G, DEFB4B, FAM25A.DEFB which was the most significantly difference in expression was associated with 'antibiotic', 'antimicrobial', 'beta defensing type', defense reponse to bacterium', and 'defensin'. In TCGA data, DEFB4A and DEFB103B were more highly expressed in N0 group than N+ group, although it did not attain significant significance. CONCLUSIONS: Defensin (DEFB4A, DEFB103B, DEFB4B) may be a novel biomarker for early regional metastasis in T1/2 tongue cancer. Considering that defensin can be detected in saliva, simple saliva test for prediction of metastasis can be developed in the future.

#2769

A squalene epoxidase-cholesterolaxis drives colorectal cancer progression and metastatic dissemination.

Soo Young Jun,1 Andrew J. Brown,2 Ji-Yong Yoon,1 Jeong-Ju Lee,1 Ngee Kiat Chua,3 Jin OK Yang,1 Ju-Sik Min,1 Insu Jang,1 Su-Jin Jeon,1 Min-Hyuk Choi,1 Tae-Ik Choi,4 Cheol-Hee Kim,4 Nam-Soon Kim1. 1 _Korea Research Inst. of Biosci. and Biotech., Daejeon, Republic of Korea;_ 2 _School of Biotechnology and Biomolecular Science, University of New South Wales, Sydney, Australia;_ 3 _School of Biotechnology and Biomolecular Sciences, University of NEw South Wales, Sydney, Australia;_ 4 _Department of Biology, Chungnam National University, Daejeon, Republic of Korea_.

Links between cholesterol and cancer are well-documented, but the mechanisms remain unclear. Squalene epoxidase (SQLE), a key enzyme in cholesterol biosynthesis degraded by excess cholesterol, is suggested as a proto-oncogene. Paradoxically, we found reduced SQLE in aggressive colorectal cancer (CRC); low SQLE being associated with a shortened survival of CRC patients. This was confirmed in a spontaneous CRC metastasis mouse model where SL-15 reduction, by either a high-cholesterol regimen or genetic knockdown, strikingly promotes CRC aggressiveness through the production of migratory cancer stem cells. Experiments in CRC cell-lines demonstrated that SQLE reduction helps overcome constraints for malignant transformation. Specifically, we uncovered a surprising interaction of SQLE with GSK3β and p53. Depletion of SQLE disrupted the GSK3β/p53 complex, resulting in a metastatic phenotype. Our findings provide mechanistic insights into the link between cholesterol and CRC, identifying SQLE as a key regulator in CRC progression and potentially a biomarker for risk assessment.

#2770

Spatio-temporal genetic heterogeneity and clonal evolution in advanced papillary thyroid carcinomas and matched distant metastases.

Sara Gil-Bernabe, Noa Feas Rodríguez, Miriam Vega Herrero, Jose Javier Estébanez García, Ginesa M. Garcia-Rostan. _Institute of Molecular Biology and Genetics (IBGM), Valladolid, Spain_.

Papillary Thyroid Carcinoma (PTC) represents 65-80% of all thyroid cancers. Though the vast majority of PTCs are indolent tumors, around 5-15% behave aggressively, developing blood-borne metastases, which cause patient´s death. The molecular mechanisms underlying metastatic spread are poorly understood. Little it is known about the contribution of intratumor molecular heterogeneity to distant metastases (DM). Dynamic changes in mutation distribution through space and time have not been in deep characterized. In this study, by genotyping 13 cases of matched primary tumors (PrT) and DMs, we sought to determine the prevalence of mutations in genes that have been associated with tumor progression and aggressiveness in follicular thyroid carcinogenesis (TERTp, MED12, BRAF, NRAS, KRAS, HRAS, PIK3CA). To asses the contribution of intratumor heterogeneity and clonal evolution to DMs, 54 tumor areas, including different areas across space and time within the PrTs and the DMs were characterized. Mutational analysis was approached by means of PCR and SSCP or direct sequencing. Twelve cases (92%) were mutated in at least one of the genes screened [TERTp=9 cases (69%), BRAF=7 cases (54%), KRAS=3 cases (23%), NRAS=3 cases (23%), HRAS=2 cases (15.4%) and PIK3CA=2 cases (15.4%)]. No mutations were seen at MED12. Among the mutated cases 67% exhibited more than 1 gene activated. Three mutated genes co-existed in 62.5% of the cases with concomitant mutations [3 cases (60%) TERTp+RAS+BRAF and 2 cases (40%) TERTp+BRAF+PIK3CA]. Concurrent activation of TERTp+RAS or TERTp+BRAF was seen in 5 cases each event (62.5%). Simultaneous activation of BRAF and RAS was found in 4 cases (50%). In all the cases in which more than 1 area of DM, across space and time, was analyzed, the mutations at TERTp, KRAS and HRAS resulted clonal. De novo mutations at DM, not present in the PrT, were seen in 3 cases mutated at TERTp, 2 cases mutated at NRAS, 1 case mutated at KRAS. Among the mutated cases, in which more than one area of PrT was analyzed, clonality was seen in 80% of the cases mutated at TERTp and subclonality was seen in 80% of the cases mutated at BRAF. The activation of RAS within the PrT tend to be a clonal event. Conclusions: The number of mutational events in PTC with DM is strikingly higher than in in PTC without DM. While TERTp and RAS mutations tend to be clonal within the PrT and the DMs, BRAF mutations tend to be subconal. TERTp and RAS mutations may appear the novo at DM. PTC with DM display a much higher rate of genetic heterogeneity (67%). The coexistence of mutations in different genes is in agreement with the hypothesis that tumor progression relies on progressive accumulation of genetic alterations. MED12 does not play a role in aggressive papillary thyroid carcinomas. Concurrent mutations at TERTp, and different PI3K/AKt and MAPK pathway genes are common in poorly differentiated and anaplastic carcinomas.

#2771

Identification of novel genes associated with metastasis from TCGA transcriptomic data.

Takahiko Koyama, Laxmi Parida. _IBM T.J. Watson Research Center, Yorktown Heights, NY_.

Introduction: Many cancer patients suffer from metastasis in bone, brain, liver and lung. However, understanding of metastasis in molecular level is quite limited. To improve survival rate and quality of life, preventing metastasis is crucial. In this study, we analyzed transcriptomic data with tumor node metastasis (TNM) classification from The Cancer Genome Atlas (TCGA) to identify genes associated with metastasis.

Method: TCGA samples in 20 solid tumor types with both transcriptome and TNM annotation data are used. To obtain the best cut point separating cohorts of high and low gene expression, Hothorn and Lausen's method is used. A contingency table is created with expression separated by the the cut point and presence of staging T, N, M for each gene. Association between the staging category and the gene was calculated with Fisher's exact test.

Results: In total, 13,220 distinct genes including miRNAs are associated with either lymph node or distant metastasis for some cancer types with a loose criteria of p-value < 1.0e-3. Among them, we identified 1,357 genes with p-value < 1.0e-8. ADAMTS12, AURKB, BUB1, C17orf53, CCNA2, CEP55, EPR1, KIF4A, KIF11, KRT80, MELK, MKI67, NCAPG, NEIL3, PBK, PLK1, RRM2, TPX2, miR139, miR30a, miR375, and miR379 appear most commonly in lymph node metastasis for many cancer types. Likewise, NOP56, PKD1L2, miR150, miR365, miR9, miR149, miR210, miR425, miR675, and miR937 are most commonly associated genes in distant metastasis. Many of these genes have been already reported in association with metastasis.

Conclusions: This study created comprehensive list of metastasis associated genes for the 20 solid tumors in TCGA. They would be useful as diagnostic and prognostic biomarkers; however, we found therapeutically relevant genes such as PLK1, AURKB, MET, MELK, CDK1, BUB1, and PBK. Some adhesion molecules associated with a particular cancer type might explain its organ destination preference. Among distant metastatic miRNAs discovered, metastasis promoting miRNAs in high abundance might be engaged in homing as exosomal miRNAs by reprogramming destination niche in favor of migrating tumor cells. Further work to reveal metastasis mechanism and to discover some pharmaceutical agents to prevent metastasis will be conducted.

#2772

Recurrent alterations of the TenascinC in highly aggressive neuroendocrine sub-type of prostate cancer.

Prachi Mishra,1 Michael Kebish,2 Jennifer Cullen,1 Amina Ali,1 Alagasamy Srinivasan,1 Inger Rosner,1 David McLeod,1 Leonardo Rodrigues,2 Viatcheslav Akmaev,2 Rangaprasad Sarangarajan,2 Shiv Srivastava,1 Niven Narain,2 Albert Dobi1. 1 _Center for Prostate Disease Research, Uniformed Service University of Health Sciences, Rockville, MD;_ 2 _BERG Health, Farmingham, MA_.

Introduction: Early detection and prognosis of prostate cancer (CaP) is challenging due to its wide spectrum of biological features. Multi-analytic prognostic marker panels have increased sensitivity than single analytes among the biopsy tests and require advanced bioinformatics platform. We aimed to develop a panel of serum biomarkers using multi-omics approach and further characterized their involvement in prostate cancer progression. In a collaborative study between CPDR, Department of Surgery, USU and Berg Health, serum samples (N=385) were examined by multi-omics (proteomics, lipidomics and metabolomics) and Tenascin C (TNC) was identified as one of the promising markers for disease progression in combination with three other analytes, Apolipoprotein AIV, 1-Methyladenosine and a phosphatidic acid (PA 18:0-22:0). The combination of two clinical features, pathological measurement of T-Stage and Gleason score, along with the four molecular analytes further increased the AUC to 0.89 with a NPV of 0.96 and an odds ratio of 12.4. TNC, an extracellular matrix protein, is poorly studied in prostate cancer, though it is expressed in several cancer tissues such as the breast, lung, colon, and the gastrointestinal tract.

Methods: Publically available prostate cancer databases (cBioportal Version 1.17.1, http://www.cbioportal.org/index.do) were queried for TNC, and known driver oncogenes in prostate cancer, ERG, AR and MYC.

Results: Publically available prostate cancer databases (cBioPortal) demonstrated alterations (either amplifications or mutations) in TNC in 10 out of 16 datasets. Query of TNC along with the driver oncogenes of prostate tumorigenesis, including ERG, MYC and AR, in the aggressive neuroendocrine prostate cancer (NEPC) tumor whole genome sequencing datasets (N=77) demonstrating significant alterations (predominantly amplifications) in 74% of patient samples. CaP genomic levels TNC (30%) was significantly co-amplified (P < 0.001) with ERG (27%), AR (56%) and MYC (53%). TNC protein expression was detected in all examined prostate cancer cell lines VCaP, LNCaP, PC3 and DU145.

Conclusion: Genomic alterations of TNC was thus associated with major oncogenic drivers of CaP, such as ERG, AR and MYC in NEPC genomic datasets. Multi-analyte serum biomarkers offers new opportunities with potential impact on primary treatment and surveillance strategies. Functional involvement of the analytes in disease progression to address their mechanistic link with major CaP oncogenic pathways will be further investigated.

#2773

**Prognostic value of** KRASP53PIK3CA **in non-small cell lung cancer patients.**

Yiping Han. _Shanghai Changhai Hospital, Shanghai, China_.

We explored the relationship of KRAS, PIK3CA, and TP53 mutations with the clinical features and survival prognosis in 50 non-small cell lung cancer (NSCLC) patients. The most common concurrent single gene mutation was TP53, followed by KRAS and PIK3CA. Coexisting mutations were found in 17 patients. KRAS, PIK3CA, and TP53 mutations were related to Ca19-9 expression, invasive growth, vacuolar signs, and margin lobulation in chest CT imaging. The incidence of distant metastasis (bone and adrenal) with KRAS and TP53 mutations was greater than that of local metastasis (pleura). Patients the wild-type gene had longer progression-free survival than those with KRAS, TP53, KRAS/TP53, or PIK3CA/TP53 mutations. Patients with KRAS/TP53 or PIK3CA/TP53 mutations had shorter progression free survival than those with a single KRAS or TP53 mutation. KRAS, PIK3CA, and TP53 mutations were associated with distant metastases and poor prognosis. NSCLC patients should receive routine KRAS, PIK3CA, and TP53 gene sequencing to determine mutations for the analysis of clinical characteristics and prognosis.

#2774

Pain signals oral cavity cancer metastasis.

Aditi Bhattacharya,1 Malvin N. Janal,1 Hyesung Kim,1 Susanna Wang,1 Angie K. Wu,1 Mari Hagiwara,2 Alexander R. Kerr,1 Brian L. Schmidt,1 Donna G. Albertson1. 1 _NYU College of Dentistry, New York, NY;_ 2 _NYU School of Medicine, New York, NY_.

Metastasis to the cervical (neck) lymph nodes is the primary determinant of survival for patients diagnosed with oral cavity cancer. Imaging (MRI, PET, CT) and clinical examination lack sensitivity to accurately detect cervical metastasis. Therefore most clinically node negative patients receive surgery to remove the cervical lymph nodes (elective neck dissection) at the time of surgical tumor removal. Half of these patients do not benefit from this additional surgery, highlighting the need for improved preoperative assessment in predicting metastatic risk. Oral cancer patients suffer severe chronic and mechanically-induced pain at the site of the cancer. We asked whether patient reported pain measured prior to surgery by a validated instrument, the University of California San Francisco Oral Cancer Pain Questionnaire (UCSFOCPQ), was an indicator of risk for lymph node metastasis. Seventy-two oral cancer patients were consented and enrolled, patients rated their pain using the UCSFOCPQ. Clinical and pathological characteristics of the patient cohorts were collected from pathology reports and medical records. Inclusion criteria included diagnosis of an oral squamous cell carcinoma, planned curative resection of the cancer and completion of the UCSFOCPQ within six weeks of surgery. Exclusion criteria included prior chemo- or radiation therapy for cancer, failure to complete or understand the UCSFOCPQ and less than one year post surgery follow-up. Sixty-six patients met the inclusion/exclusion criteria (35 node negative and 31 node positive). Patients with metastasis reported higher pain scores prior to surgery. Low pain score and depth of invasion each predicted low metastatic risk with high sensitivity and negative predictive values. Amongst patients who were clinically staged N0 by current standard of care imaging and physical exam prior to surgery, low pain scores correctly identified patients determined by pathology to be N0. Conclusions: Low pain scores assessed prior to surgery could identify patients at low risk for metastases who would not benefit from neck dissection. Pain score might be added to the standard of care preoperative assessments and decision making processes for determining whether to recommend an elective neck dissection.

#2775

Investigating myeloid-derived suppressor cells (MDSC) gene expression in predicting outcome in stage matched colorectal cancer patients.

Saleh G. Heneidi, Chance Bloomer, Pankaj Ahluwalia, Ashis Mondal, Ravindra Kolhe. _Augusta University, Augusta, GA_.

Colorectal cancer (CRC) has emerged as the third most commonly diagnosed cancer in males and second most common in females. Although surgery, chemotherapy, and emerging immunotherapies have reduced mortality rates, tools that can assist in tailoring individualized treatments are needed. MDSC are immune-suppressive cells that interfere with the functioning of T cells and immune-inflammatory pathways. An extensive literature search identified 8 MDSC-centric genes which were tested for their prognostic significance in CRC. These genes; CEBPB, IL10, NOS2, RORC, S100A8, SOCS1, SOCS3, and TGFB1, along with their expression in tumor microenvironments could give new insights when correlated with clinical-pathologic features of colon cancer patients. The aim of the study is to quantify MDSC gene signatures in colorectal cancer patients. Under an IRB approved protocol, a total of 750 colon cancer patients at the Medical College of Georgia with 5 years of follow-up were initially selected. A total of 88 patients fit in our inclusion criteria, on the basis of survival duration after diagnosis. The FFPE blocks were acquired and patients were stratified on the basis of overall survival in two groups, with higher (>5 years) and lower survival (<1 year), as well as AJCC staging (I to IV), grade, gender and age. Total RNA was isolated and quantified through Nanodrop method. The statistical analysis of data was performed using student t-test and Pearson correlation. SOCS1 (p = 0.00*) was expressed in higher levels in patients with high survival (>5 years). Patients with low expression of TGFB1 survived longer compared to high expression group (p = 0.02*). On race comparison, CEBPA (p = 0.01*) showed significant expression in Caucasian population while NOS2 (p = 0.01*) expressed at higher levels in African Americans. These findings points to the clinical significance of MDSC based genes and tested the utility of a MDSC-related gene expression based prognostic biomarker panel for CRC patients.

#2776

Mitochondrial calcium uniporter in pancreatic cancer metastasis and metabolic stress resistance.

Xiuchao Wang,1 Shengchen Lin,1 Jianwei Sun,1 Jiaxin Kang,1 Jihui Hao,2 Shengyu Yang1. 1 _Penn State College of Medicine, Hershey, PA;_ 2 _Tianjin Cancer Hospital, Tianjin, China_.

Despite recent advances in cancer diagnosis and therapeutic modalities, the prognosis for pancreatic ductal carcinoma (PDAC) remains very poor. Most PDAC patients succumb to local and distant metastasis soon after the initial diagnosis. Therefore understanding molecular mechanisms underlying PDAC metastasis is crucial to the development of more effective PDAC therapies. Here we examined the role of mitochondrial calcium uniporter (MCU), a mitochondrial inner membrane calcium channel responsible for mitochondrial Ca2+ uptake, in pancreatic cancer metastasis and progression. We discovered that MCU is overexpressed in PDAC when compared to adjacent pancreatic tissues. MCU overexpression in PDAC patients is significantly associated with PDAC lymph node metastasis, histologic grade and pTNM stage. We also found that higher levels of MCU in PDAC patients was associated with worse overall survival (OS, P<0.01) and relapse-free survival (RFS, P<0.05) after surgical resection of tumors. MCU knockdown inhibits the PDAC cell motility, invasiveness, metabolic stress resistance and chemoresistance in cell culture models and PDAC metastasis in an orthotopic xenograft mouse model. Mechanistically, MCU controls the activation of the Keap1-Nrf2 signaling pathway through promoting mitochondrial ROS production and the oxidation of mitochondrion-recruited Keap1. Our data support that MCU regulates cancer cell metabolic stress resistance and oxidative stress response to drive PDAC metastasis and progression. Inhibiting MCU expression or blocking mitochondrial calcium influx may be an attractive approach for treating metastatic PDAC.

#2777

LOXL4-expressing neutrophils are found in colorectal cancer liver metastases resistant to anti-angiogenic therapy.

Vincent Palmieri,1 Anthoula Lazaris,2 Stephanie Petrillo,2 Hussam Alamri,1 Abdellatif Amri,2 Woong-Yang Park,3 Zu-hua Gao,1 Peter Metrakos1. 1 _McGill University Health Centre, Montreal, Quebec, Canada;_ 2 _Research Institute of the McGill University Health Centre, Montreal, Quebec, Canada;_ 3 _Samsung Genome Institute, Seoul, Republic of Korea_.

Three different histopathological growth patterns (HGP) have been identified in colorectal cancer liver metastases (CRCLM) resected from patients: the desmoplastic HGP, the pushing HGP, and the replacement HGP. Evidence suggests that the predominant growth pattern with which a CRCLM presents has clinically relevant prognostic implications. We have shown that patients with replacement HGP lesions who received bevacizumab plus chemotherapy had a worse pathological response and five-year overall survival than those with desmoplastic HGP lesions receiving the same treatment. We have also demonstrated that CRCLM with the replacement HGP promote vessel co-option for vascularization, rather than sprouting angiogenesis as seen in desmoplastic HGP metastases. These findings point to the growth patterns in CRCLM possibly serving as predictive biomarkers of response to anti-angiogenic therapy, when no such biomarker has been validated to date. However, HGP scoring is performed on resected liver metastatic tissue, implying that preoperative treatment precedes growth pattern assessment. Therefore, surrogate markers for the HGPs that can be appraised prior to surgery would be helpful to inform clinical decision-making about whether a patient with CRCLM may benefit from anti-angiogenic treatment.

This project aims to identify genes that are differentially expressed between CRCLM presenting with either the replacement or desmoplastic HGP. RNA sequencing (RNA-Seq) was used to compare the transcriptional profiles of these distinct CRCLM. A gene expression signature was generated from the RNA-Seq data to include genes that were upregulated in the replacement HGP liver metastases. Immunostaining of select genes from this signature was performed to validate these findings on human tumor tissue.

Initial analysis of our RNA-Seq data identified 525 genes that are differentially expressed between the replacement and desmoplastic HGP CRCLM, of which 53 genes met the criteria for the gene expression signature. Pathway analysis of these genes identified pathways involved in the immune system and extracellular matrix (ECM) to be upregulated in the replacement HGP lesions. Subsequent immunostaining revealed the expression pattern of lysyl oxidase like-4 (LOXL4) protein to be distinct between the HGPs - significantly greater quantities of LOXL4-expressing neutrophils were detected in the replacement HGP tumor microenvironment.

Characterizing differences in gene expression between replacement and desmoplastic HGP CRCLM is expected to provide some insight into the mechanisms responsible for generating these distinct growth patterns. Our findings suggest that further investigations into the role of tumor-associated leukocytes and ECM remodelling may ultimately lead to the development of biomarkers and new therapeutic targets for replacement HGP CRCLM.

#2778

ADD3-loss in glioblastoma is associated with increased angiogenesis and malignancy.

Mei-Yee Kiang, Gilberto Ka-Kit Leung. _The University of Hong Kong, Hong Kong, Hong Kong_.

Introduction: Adducin 3 (ADD3) is one of the major functional component in the cytoskeleton. Dysregulation of ADD3 has been implicated in various diseases including cancer and participate in different signaling transductions. However, little is known about the functional properties of ADD3 in cancer, its role in cancer biology remains unclear and controversial. We aim to delineate the role of ADD3 dysregulation in GBM pathogenesis and to unravel the underlying molecular mechanisms.

Methods: Microarray datasets were used to analyze the gene expression of ADD3 in normal tissue and grade II, III, IV gliomas. Gene expression levels were verified by western blot, qPCR and immuno-histological analyses by using human clinical specimens. Luciferase-expressing ADD3 stable knockdown U87 malignant glioma cells were injected to immunocompromised mice subcutaneously and orthotopically for xenograft models. ADD3 stable knockdown U87 cells were co-cultured with human endothelial cells (HUVEC) to assess the functional changes.

Findings: Our study demonstrated significant downregulation of ADD3 in human GBM when compared with lower-grade gliomas. Downregulation of ADD3 was only shown in GBM (i.e., WHO grade IV lesion) but not in grade II and grade III tumor. Importantly, we found that ADD3 downregulation is associated with poor clinical outcome in GBM patients. And that ADD3 knock-down enhanced tumor growth in vivo, possibly mediated by promoting angiogenesis.

Conclusions: These findings suggest that ADD3 is associated with glioma malignancy, implying that cancer progression may occur along with the loss of ADD3. It may contribute towards tumor growth by enhancing angiogenesis.

#2779

miR-142-3p as a regulator of RAC1 and a prognostic factor in melanoma.

Adriana T. Cruz,1 Júlia A. Morata,1 Ana C. Monteiro,1 Ana Luísa P. Ayub,1 Roseli S. Soares,1 André Fujita,2 Fernando Andrade,2 Victor Tron,3 Miriam G. Jasiulionis1. 1 _Universidade Federal de São Paulo, São Paulo, Brazil;_ 2 _Universidade de São Paulo, São Paulo, Brazil;_ 3 _Queen's University, Kingston, Ontario, Canada_.

In order to identify miRNAs able to distinguish melanoma patients with good or poor prognosis, the expression profile of a panel of miRNAs evaluated in primary melanoma samples from 65 patients was compared to clinicopathological data from these patients (Breslow's depth, mitotic counts, metastasis progression and survival). The miR-142-3p was identified as presenting at least 2-fold reduced expression in melanoma samples from patients with poor prognosis for 3 of 4 clinical parameters analyzed compared to melanoma samples from patients with good prognosis. The miR-142-3p expression was validated by RT-qPCR in primary human melanoma samples, confirming a significant lower expression in melanoma patients with poor prognosis compared to those with good prognosis. Searching for miR-142-3p mRNA targets in melanoma, the gene expression profile was analyzed by gene microarrays in a wild type and miR-142-3p overexpressing melanoma cell line. RAC1, ITGB8 e GNB2 were found as potential targets of this miRNA. Protein expression analyses in melanoma cell lines overexpressing the miR-142-3p reinforced the regulation of RAC1 by this miRNA. Functional assays showed that the overexpression of miR-142-3p leads to a reduced migratory ability compared to control cells, indicating its role in cell migration process. Finally, multivariate COX analyses demonstrated that high levels of RAC1 and reduced levels of miR-142-3p predict poor progression-free survival of melanoma patients, revealing these molecules as potential prognostic biomarkers for melanoma patients.

Supported by FAPESP, CNPQ and CAPES

Short title: miR-142-3p as a prognostic factor in melanoma

#2780

IL13RA2 is differentially regulated in papillary thyroid carcinoma versus follicular thyroid carcinoma.

Siao Ting Chong,1 Catherine Y. Kok,1 Khee Ming Tan,1 Shou Ping Guan,1 Siang Hui Lai,2 Cindy Lim,1 Jiancheng Hu,1 Charles Sturgis,3 Charis Eng,4 Paula Y. Lam,1 Joanne Ngeow1. 1 _National Cancer Centre, Singapore;_ 2 _Singapore General Hospital, Singapore;_ 3 _Cleveland Clinic, Cleveland, OH;_ 4 _Case Western Reserve University School of Medicine, Cleveland, OH_.

The interleukin-13 receptor alpha2 (IL13RA2), which is known to overexpressed in glioblastoma multiforme, plays a role in various cellular processes such as cell migration that may contribute to tumor progression. Studies have attributed IL13RA2 to invasion and metastasis in cancers of the ovary, breast, and pancreas but the pathological role of IL13RA2 in thyroid cancer is still unclear. This study aims to evaluate the expression of IL13RA2 in thyroid carcinomas and examine the role of IL13RA2 in progression of papillary thyroid cancer (PTC). We performed IL13RA2 immunochemical staining on tissue microarrays of 137 thyroid carcinomas and observed that IL13RA2 expression was significantly correlated with advanced tumor stage (pT3 / pT4; p=0.001) and regional lymph node metastasis (pN1; p<0.001). Moreover, the staining scores of IL13RA2 were significantly higher in PTC compared to follicular and anaplastic subtypes (p<0.02) and correlated with advanced tumor stage amongst PTC samples (pT3 / pT4; p=0.028). This differential profile of IL13RA2 in PTC was further validated in thyroid cancer cell lines overexpressing IL13RA2 to assay the effects on cell proliferation, cell migration and epithelial-mesenchymal transition (EMT) using CCK-8, transwell migration assay, qRT-PCR and western blot analyses. Knockdown of IL13RA2 in the PTC subtype B-CPAP cell line showed significantly reduced cell viability, cell migration and EMT markers including N-cadherin, Vimentin and Snail. Exogenous overexpression of IL13RA2 in another PTC cell line, K1 increased cell migration and EMT although cell proliferation was not affected. In summary, we demonstrated that IL13RA2 is differentially regulated in PTC and is involved in cell migration by enhancing EMT. The underlying molecular mechanisms on how IL13RA2 drives progression of thyroid cancer remains to be further investigated.

#2781

Gene expression in colorectal liver metastases: Distinct immune signatures and opportunities for immune modulating therapy.

Vigdis Nygaard,1 Vegar Johansen Dagenborg,1 Sheraz Yaqub,1 Åsmund Avdem Fretland,1 Olga Østrup,2 Laxmi Siwal-Pandit,1 Krzysztof Grzyb,1 Anne-Lise Børresen-Dale,1 Gunhild Maelandsmo,1 Anne Ree,3 Bjørn Edwin,1 Kjersti Flatmark1. 1 _Oslo University Hospital, Oslo, Norway;_ 2 _Rigshospitalet, Copenhagen, Copenhagen, Denmark;_ 3 _Akershus University Hospital, Oslo, Norway_.

Colorectal cancer (CRC) is one of the most common cancers and a leading cause of cancer death in the Western world. Up to 50% of CRC patients develop metastatic disease and the liver is the most common site. The recently identified consensus molecular subtypes (CMS1-4) based on analyses of primary CRC have prognostic and therapeutic implications, but it is unclear whether these molecular subtypes are valid for metastatic disease. The tumor microenvironment plays a major role in the dynamic shaping of the tumor phenotype and the clinical impact of the immune contexture within given CMS subgroups is emerging.Surgically resected CLMs (n=44) were analyzed by genome-wide transcription profiling. Clustering approaches were applied to define subgroups. Differential gene expression analyses were performed to reveal altered expression patterns in subgroups selected by clinicopathological parameters. Functional annotation of gene signatures was conducted. Validation in public data sets and by proteomic analyses (RPPA) is ongoing. Quantification of T cell subsets by immunohistochemistry was scored. The CMS classifier tool revealed the cohort to be highly enriched for CMS2 and thus of limited stratification utility. In contrast, intrinsic subgroups were revealed by alternative computational approaches. Distinct immune profiles were associated with the subgroups identified. Unsupervised clustering based on high-variance genes identified a 55-gene cluster that split the cohort near evenly. Functional annotation of the 55 genes identified one subgroup to display dysregulated cholesterol/lipid homeostasis with reciprocal links to immune regulatory genes. Supervised clustering using EMT/mesenchymal signatures identified a mesenchymal subgroup (20% of the samples). Functional annotation suggested co-existence of immunosuppression with the mesenchymal phenotype. Elevated expression of immune checkpoint molecules including TIM-3, and M2 macrophage markers correlated with the EMT/mesenchymal signature. The subgroup showed relative high density of T cell subset infiltration (CD3, CD4, CD8, FOXP3). In differential gene expression analysis, CLMs exposed to neoadjuvant chemotherapy showed activation of interferon gamma (IFNG) signaling and genes linked to immunogenic cell death. Co-expression of immune regulatory genes suggested counter-balance of the immune responses triggered by IFNG. Immune-related gene expression signatures were associated with the CLM subgroups identified. These observations underline the integration and importance of the immune interactome in CLM. The coordinated expression of immunosuppressive factors suggest not only a rational for immune therapy, but also that development of immune-modulating strategies may be required to increase efficacy of current therapy for selected CLM patients.

#2782

MicroRNAs response to concurrent chemoradiotherapy predicts oligo- and polymetastatic progression in patients with Stage IV colorectal adenocarcinoma.

Sung Hak Lee,1 Nima Pouladi,2 Colleen Kenost,2 Francesca Vitali,2 Yves A. Lussier2. 1 _The Catholic University of Korea, Seoul, Republic of Korea;_ 2 _University of Arizona, Tucson, AZ_.

Colorectal cancer (CRC) is the third most common cancer and an important contributor to cancer mortality and morbidity worldwide. The survival rate (11%) in patients with stage IV CRC remain suboptimal. Among these, an intermediate state of distal metastasis termed oligometastasis(es) is characterized by a less aggressive biology with limited tumor progression. Oligometastases are amenable to metastasis-directed local treatments; however, a significant portion of these progresses to polymetastases. The purpose of this study is to identify predictors of oligometastatic progression that could improve patient selection and survival for metastasis-directed localized therapy. microRNAs (miRs) classifiers have previously been shown to predict oligometastatic progression in smaller studies with a heterogeneous primary tumor. Here, we focus on a larger and first study comprising exclusively stage IV CRC to determine CRC-specific classifier of oligometastic progression. We analyzed microRNAs microarray expression patterns from initial oligometastatic patients (≤5 initial metastases) with metastatic CRCs resected with curative intent. 96 stage IV CRC Korean subjects (70 males, 26 females; mean age 60 years) were stratified into subgroups based on their rate of recurrent metastatic progression over a follow-up period. The criteria for these subgroups were previously validated (PloS one, 7(12), p.e50141): (i) patients with no and/or low rate of progression (LRP; <0.6 new metastases/year) and (ii) patients with a high rate of progression (HRP; >3.6 new metastases/year). microRNA expression (Affymetrix GeneChip microRNA 4.0) was analyzed with quantile normalization, COMBAT, and LIMMA, and then validated using PAM. 47 microRNAs were prioritized (FDR<5%) between HRP (n=22) and LRP (n=60) and were associated with the rate of metastatic progression and survival in the current dataset. Six miroRNAs of this study were previously shown to discriminate between HRP vs LRP among lung metastases of heterogeneous primary tumor types (miR-127-5p, miR-154-5p, miR-199b-5p, miR-485-5p, miR-491-5p, and miR-502-5p). In addition, we observe an overrepresentation of microRNAs from the 14q32.31 locus and members of the miR-154 family from this loci that had previously been implicated in oligo- vs poly- metastasis progression (miR-154-5p, miR-337-5p, miR-381-3p, miR-382-5p, miR-409-5p, and miR-487a-3p). These results further corroborate the hypothesis that HRP and LRP are distinct entities at both clinical and molecular levels.

#2783

OMICS **analysis of breast cancer PDX tumors to determine CTC-cluster-specific signature in predicting breast cancer metastasis.**

Hariprasad Thangavel,1 Carmine De Angelis,2 Suhas Vasaikar,3 Raksha Bhat,1 Mohit Kumar Jolly,4 Lacey Elizabeth Dobrolecki,3 Jason T. George,4 Mario Giuliano,2 Michael Lewis,3 Herbert Levine,4 Bing Zhang,3 Rachel Schiff,3 Meghana V. Trivedi1. 1 _University of Houston, Houston, TX;_ 2 _University of Naples Federico II, Naples, Italy;_ 3 _Baylor College of Medicine, Houston, TX;_ 4 _Rice University, Houston, TX_.

Circulating tumor cell clusters (CTCcl) have been shown to have a significantly higher metastatic potential compared to single CTCs and to predict long-term outcomes in breast cancer patients. A characterization of primary tumors that give rise to CTCcl hold significant promises for better diagnosis and may uncover targets for prevention and treatment of metastatic breast cancer. In our study, we utilized the available transcriptomic and proteomic data (RNA-Seq and RPPA) of 10 triple-negative breast cancer patient-derived xenograft (TNBC-PDX) transplantable models. The sample set consisted of 6 CTCcl-negative (-) and 4 CTCcl-positive (+) models. Genome-wide transcriptomic analysis revealed 549 differentially expressed genes in CTCcl+ TNBC-PDX models vs. CTCcl- models (316 up-regulated and 233 down-regulated, p<0.05). Gene set enrichment analysis (GSEA) terms significantly enriched (FDR<0.05) in CTCcl+ TNBC-PDXs included Cytoplasmic Ribosomal Proteins (NES=2.53), Angiogenesis (NES=1.95), Type II Interferon signaling (NES=-2.52), Antigen processing and presentation (NES=-2.38), TNF signaling pathway (NES=-2.04), Jak-STAT signaling pathway (NES=-1.91), and apoptosis (NES=-1.69). The proteomic analysis revealed elevated levels of Bcl2 expression in PDX tumors associated with CTCcl positivity, which substantiated the inhibited apoptosis pathway as seen in GSEA analysis. The increase in Bcl2 was also validated independently by IHC staining in primary PDX tumors that were CTCcl+. The distribution of EMT score based on the inferential EMT metric using canonical epithelial and mesenchymal markers was not significantly different between CTCcl+ and CTCcl- TNBC-PDX tumors suggesting no differential regulation of EMT in tumors that give rise to clusters versus only single cells. In summary, our results suggest that primary tumors with active anti-apoptotic and/or survival pathways may promote CTC clusters formation and increase the risk of distant metastasis.

#2784

Driver genes network promotes mesenchymal to epithelial transition and organ-specific metastasis.

Ming Chang,1 Xin Huang,1 Songjian Lu,2 Wei Zhang,1 Weiping Liang,1 Evan Keller,3 Xinghua Lu,2 Jian Zhang1. 1 _Southern University of Science and Technology, Shenzhen, China;_ 2 _University of Pittsburgh, Pittsburgh, PA;_ 3 _University of Michigan, Ann Arbor, MI_.

Metastasis, a major characteristic of malignancy, causes high mortality. Accumulating evidence suggests that cancer intends to metastasize to specific organ, however the molecular mechanisms remain unknown. Others and us have demonstrated that epithelial to mesenchymal transition (EMT) plays key roles in initiation of primary tumor adherent, migration, and invasion, whereas mesenchymal to epithelial transition (MET) revokes "sleeping" disseminated tumor cell (DTC) to colonization at distant metastatic organ sites and facilitates the tumor cell proliferation. We have successfully generated the lung-specific metastatic subclones of human prostate cancer PC3 and murine RM1 cells. Through omic bench work and data analysis and comparison with TCGA database, we, for the first time, identified a preliminary network of "driver genes" that potentially promotes the reversible EMT/MET process We utilized cutting-edge tools of data analysis to identify the selected driver genes (LAMA, SPP1, ITGB3, S100A8, SERPINE, DKK2) and further characterized the gene functions via in vitro bioassay of cell proliferation, migration, invasion, cell cycle, and apoptotic testing etc, and in vivo with our unique cell and animal metastatic models. We observed that the rate of organ-specific metastasis was dramatically reduced when these gene expression levels were changed. Finally, we confirmed our findings using TMA slides, Our results may provide novel mechanisms of metastasis and clinical targets of cancer therapy. Supported by NSFC projects 81773146; Guangdong Provincial Key Laboratory of Cell Microenvironment and Disease Research (2017B030301018); JCYJ20170412152943794, JCYJ20170412154619484, JCYJ20170307105128101, JCYJ2017030711041760

### Immune Cells in the Tumor Microenvironment 2

#2785

Metabolic autofluorescence microscopy of 3D microscale macrophage-tumor co-cultures.

Tiffany M. Heaster,1 Jiaquan Yu,1 Margaret Edman,1 David J. Beebe,1 Melissa C. Skala2. 1 _University of Wisconsin-Madison, Madison, WI;_ 2 _Morgridge Insitute for Research, Madison, WI_.

Macrophages are ideal treatment targets due to their tumor-regulatory functions and high infiltration in the tumor microenvironment (TME). However, macrophage heterogeneity prevents effective therapeutic reprogramming. Macrophages have high plasticity and adopt phenotypes with diverse behavior and metabolism. The complex TME can drive this plasticity but is not well understood. Standard functional assays (e.g. flow cytometry, ELISA) lack sensitivity to heterogeneous cell populations. Also, their destructive sample processing limits spatial and temporal assessment. Thus, nondestructive, single cell imaging and analysis tools are needed to study macrophage heterogeneity within the TME.

Optical metabolic imaging (OMI) measures two-photon excited fluorescence from the coenzymes NAD(P)H and FAD to resolve cellular metabolism within intact, 3D samples. The optical redox ratio (NAD(P)H intensity/FAD intensity) and fluorescence lifetimes reflect cell redox state and intracellular protein binding, respectively. Previous studies have shown that OMI detects spatial and temporal changes in stromal cells across in vivo and 3D in vitro models. Microscale models closely mimic the TME, are high throughput, and enable precise environmental control. Thus, microenvironmental stimulation of macrophage polarization and migration was mimicked in 3D microscale cultures of mouse macrophages (RAW264.7) and mammary carcinoma cells (PyVMT) in a collagen matrix. OMI captured redox ratio, NAD(P)H and FAD mean lifetime (τm) changes in mono-cultured and co-cultured macrophages 24-72 hours post-seeding. Intensity and lifetime volumes of macrophage layers further assessed metabolic changes in macrophages during migration.

Co-cultured macrophages exhibited significantly increased (p<0.05) redox ratio and NAD(P)H τm compared to mono-cultured macrophages by 24 hours. Decreasing redox ratio in co-cultures over time suggested an oxidative metabolic shift, characteristic of M2-like phenotype. Distribution curves of redox ratio revealed macrophage subpopulations after 24 hours co-culture that disappear by 72 hours. Immunofluorescence markers of M1- or M2-like macrophages indicated heterogeneous polarization at 24 hours then transition to a homogeneous M2-like phenotype by 72 hours, consistent with metabolic imaging. Migration increased in co-cultured macrophages over time. Decreased redox ratio and increased NAD(P)H and FAD τm in migratory macrophages suggested proximity to the tumor affects macrophage metabolism. These results establish OMI for noninvasive monitoring of cell-level macrophage dynamics in 3D, in vitro TME models. Microscale co-culture and OMI were also demonstrated with primary mouse macrophage/Panc02 co-culture and human THP-1/MDA-MB-231 co-culture. Overall, these tools could better characterize heterogeneous macrophage behavior to improve regulation of anti-tumor activity.

#2786

Effects of tumor microenvironment on the efferocytic and phagocytic genes of human macrophages.

Hirendra N. Banerjee,1 Kayla Johnston,1 Christopher Krauss,2 Santanu Dasgupta,3 Somiranjan Ghosh,4 Santosh Mandal5. 1 _Elizabeth City State University, Elizabeth City, NC;_ 2 _Morgan State University, Baltimore, MD;_ 3 _University of Texas Health Science Center at Tyler, Tyler, TX;_ 4 _Howard University Medical School, Washington DC, DC;_ 5 _Morgan State University, Baltimore, MD_.

Macrophages are the first line of defense in the cellular environment in response to any antigenic or foreign invasion. Since cancer cells express antigenic molecules and create a tumor microenvironment quite different from the normal cellular environment, macrophages will attack this cancer cells as foreign invaders. However, the cancer cells suppress the macrophage activity by secreting compounds unknown to the cancer, biologists even today and recruit these macrophages to their benefit as tumor associated macrophages or TAM. In this study, we were interested to find out the genes and proteins that are involved in this conversion of cancer-fighting macrophages to cancer friendly macrophages. We co-cultured the prostate cancer cells along with the human macrophages, then collected these tumor micro environment (TME) exposed human macrophages, and isolated RNA from these cells. We further analyzed these tumor associated macrophages (TAM's) along with control macrophages RNA by Microarray analysis using Affymetrix platform for gene expression studies. Our Phagocytosis results showed decreased phagocytic index in the tumor medium exposed macrophage cells as evidenced by the much lower relative fluorescence unit (RFU) recorded by the fluorometer in comparison to macrophages grown in absence of tumor medium. Further analysis of the gene expression studies by PARTEK coupled with Ingenuity Pathway Analysis (IPA®) showed down-regulation of several genes helping in the phagocytosis process and differential expression of several noncoding RNAs that control the expression of such phagocytic genes. The following oncomiR's, miR 148,615,515,130,139 were -upregulated and tumor suppressor miR's3130, let7c,101,103,383 were downregulated. The TARGET SCAN Software results showed these differential expression of noncoding RNA's causing down regulation of phagocytosis promoting genes elf5A, Meg3, Tubb5, Sparcl-1, Uch-1, Bsg (CD147), Ube2v, and Pamr1. There is an up-regulation of RAP1GAP gene that causes downregulation of the tubulin genes that promote cytoskeletal assembly in forming phagosomes. Ingenuity Software analysis of the gene expression data also showed upregulation of anti-phagocytic genes IL10, CD16.IL18 and MMP9. IPA core canonical pathways analysis revealed the pathophysiology of complex cellular signaling, by which, the rogue cancer cells tame their enemies, the macrophages and actually make them their helper cells to survive and propagate in the tumor microenvironment and thus prepare for epithelial mesenchymal transition for future metastasis and cancer stem cell formation and progression. Further work is underway in our laboratory to decipher the complete biology of TAM formation. Acknowledgement: Supported by ECSU-NIH-MARC2T34GM100831-06 , ECSU-NSF-LSAMP, and 5G12MD007597-25(NIMHD)

#2787

Artificial intelligence approach identifies IL2RB as a common prognostic and potential predictive biomarker associated with immune checkpoints in colorectal cancer.

Matthew Alderdice, Stephanie Craig, Matt Humphries, Alan Gilmore, Victoria Bingham, Nicole Johnston, Stephen McQuaid, Manuel Salto-Tellez, Mark Lawler, Darragh G. McArt. _Queen's University Belfast, Belfast, United Kingdom_.

Identifying robust predictive biomarkers to enable stratification of colorectal cancer (CRC) patients, based on their response to immune checkpoint therapy, is an area of unmet clinical need. Genetic algorithms represent an exciting branch of artificial intelligence which can be used to extract meaningful associations from 'big data' now emerging more frequently in oncological research. We have employed Atlas Correlation Explorer (ACE), a user-friendly workbench that utilises a genetic algorithm to mine data deposited in The Cancer Genome Atlas (TCGA). Our aim was to establish common intersections between gene expression analyses in ACE using nine well established immune checkpoint markers (CD274, PDCD1, CTLA4, LAG3, TIM3, TIGIT, ICOS, IDO1 and BTLA). We observed IL2RB to be the common gene associated with immune checkpoints in both microarray and RNA sequencing data from the TCGA (7/9 gene lists). Assessment of IL2RB indicates that it is highly expressed on CD56+ natural killer cells and is associated with an increased infiltration of cytotoxic lymphocytes and a decreased infiltration of fibroblasts. It is also significantly enriched in the immune consensus molecular subtype group CMS1. We next demonstrated that patients with high IL2RB gene expression have better relapse free survival in the TCGA CRC cohort (n = 322, log-rank p = 0.011) and an all stage CRC validation cohort GSE39582 (n = 519, log-rank p = 0.006). It is also an independent prognostic factor by multivariate analysis (p = 0.01). We next observed strong correlations between IL2RB gene expression in CRC and previously published predictive gene signatures for anti-PD1 therapies in other solid tumours (Pearson correlation, R = 0.88). Finally, we optimised assessment of IL2RB immunohistochemistry in a large CRC cohort (n=661) using a digital pathology approach with the open-source QuPath software. To conclude, we have validated IL2RB as prognostic biomarker and have provided evidence to demonstrate that IL2RB expression could be used for CRC patient stratification in future immunotherapy based clinical trials.

#2788

The interplay between tumor-cell derived FoxO1 and macrophage in the tumor microenvironment of esophageal squamous cell carcinoma.

Ying Wang, Xinyuan Guan, Woon Ngar Kam. _Department of Clinical Oncology, Hong Kong, Hong Kong_.

Introduction: Esophageal squamous cell cancer (ESCC) is the predominant histologic type in China with high incidence and mortality. FOXO1 is considered as a tumor suppressor in several cancers, but its role in tumor immunology still remain elusive. A better understanding of how FOXO1 mediates the secretion of downstream chemokines, such as CCL20, and thus its ability in regulating macrophage function would be of crucial for further investigation.

Materials and Methods: Quantitative PCR and immunohistochemistry were used to study the expression of FoxO1 in clinical ESCC samples. The survival analysis was performed based on the result of tissue microarray and corresponding clinicopathological information. Stable foxo1-transfected cell lines were constructed in human ESCC cell lines, and ELISA, western blotting and Quantitative PCR were conducted to evaluate the expression of foxo1 and its downstream molecule CCL20. The subsets of macrophages were identified through Flow cytometry and qPCR.

Results: The expression of FOXO1 was significantly upregulated in tumorous tissue when comparing to adjacent normal tissue and predicted a poor prognosis in ESCC patients. Besides, the analysis of TCGA database indicated that the markers of M2 macrophage were upregulated in foxo1-high expressing tumor tissues when compared with foxo1-low expressing tumor tissues. In vitro, we have successfully differentiated monocytic cell line THP-1 into macrophage M0, M1 and M2. The M1 and M2 markers (CD80, CD163 and CD68) were measured by flow cytometry, while their major cytokines (IL6, TNF-α, CCL17, CCL18, IL1β and IL10) were detected by qPCR. The ELISA, western blot and qPCR results showed that the expression of CCL20 was upregulated in foxo1-high expressing tumor cells when compared to negative control. The migration assay showed that ESCC cells transfected with foxo1 could recruit more M2 macrophages when compared with foxo1-low expression ESCC cells and the effect was diminished upon blockade with anti-CCL20 neutralizing antibody in tumor cells. Besides, we found out that M2 macrophage could promote the migration and EMT of tumor cells by using M2 conditioned medium.

Conclusions: FoxO1 displays an important role in M2 macrophage migration through the modulation of CCL20. This increased CCL20 reported in ESCC may become specific molecular/cellular processes operative in the TME of ESCC patients.

#2789

Highly multiplexed imaging mass cytometry reveals immune cell composition and spatial heterogeneity in diffuse large B cell lymphoma associated with treatment outcome.

Monirath Hav, Erik Gerdtsson, Mohan Singh, Anthony Colombo, James Hicks, Peter Kuhn, Imran Siddiqi, Akil Merchant. _University of Southern California, Los Angeles, CA_.

Diffuse large B cell lymphoma (DLBCL) is an aggressive and heterogenous entity characterized by its variable clinical and biological behaviour, and approximately 30% of patients experience relapsed or refractory disease after first-line therapy. We hypothesize that a better characterization of the tumor microenvironment (TME) might help identify patients who may benefit from individualized immunotherapies. Similar studies in this area have been limited by technical challenges - conventional highly multiplexed techniques require tissue disruptions that lose spatial information, while those that retain tissue architecture can only examine 6-8 markers simultaneously. We and others have previously reported that PD-L1 expression is correlated with decreased survival in a cohort of 85 DLBCL patients. In the present study, we characterized TME components, including their types, frequency and spatial interaction, in DLBCL using imaging mass cytometry (IMC), which allows high-dimensional, single-cell and spatial analysis of FFPE tissues at sub-cellular resolution. Using a panel of 32 antibodies, IMC was performed on a subset of our previously studied cohort. We examined 41 cores from 33 DLBCL cases, 17 GCB and 16 non-GCB, by Hans criteria. Clinical outcome data were available for 29 patients, 22 of whom had complete response (CR) to R-CHOP therapy while 7 had primary refractory disease. Using both supervised gating and unsupervised clustering algorithm, IMC data were analyzed for relevant immunophenotypes and compared across clinical outcome groups. The TME was mainly composed of 13.1% ± 1.9% (mean ± SE) CD4+ T-helper cells, 10.8% ± 1.1% CD8+ cytotoxic T cells, 6.3% ± 0.9% CD68+ macrophages, 2.7% ± 0.5% FoxP3+ regulatory T cells, and 58.1% ± 3.4% tumor cells. In non-GCB group, higher ratio of regulatory T cells was associated with refractory disease. In contrast, activated granzyme-B+/CD8+ cytotoxic T cells were more frequent in CR group, while markers of exhaustion (Tim3, Lag3) were found in patients with refractory disease. To gain functional insight into the various immune subsets, we performed spatial analysis of the immune cells and their relation to blood vessels and tumor cells. Nearest distance analysis showed that CD4+ cells were most tightly clustered around blood vessels in patients with CR, while in those with refractory diseases, CD4+ cells were further away from the vessels (p=0.03). On the contrary, distances between cytotoxic T cells and regulatory T cells showed no difference between CR and refractory patients. Together, these results show variable composition of the different immune cells and their spatial heterogeneity to be associated with the clinical outcome of DLBCL patients and that spatial analysis of immune cells should be explored as a potential biomarker for patients treated with immunotherapies.

#2790

Characterization of the immune microenvironment in brain metastasis from non-small cell lung cancer.

Yujin Kudo,1 Cara Haymaker,1 Jiexin Zhang,1 Alexandre Reuben,1 Dzifa Yawa Duose,1 Junya Fujimoto,1 Sinchita Roy-Chowdhuri,1 Luisa Maren Solis,1 Hitoshi Dejima,1 Edwin Roger Cuentas,1 Barbara Mino,1 Ronald Abraham,1 Norihiko Ikeda,2 Rajyalakshmi Luthra,1 Jack J. Lee,1 Humam Kadara,3 Jason T. Huse,1 Ignacio I. Wistuba1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _Tokyo Medical University, Tokyo, Japan;_ 3 _American University of Beirut, Beirut, Lebanon_.

Purpose Brain metastases can induce life-threatening events and their immune microenvironment is still unrevealed. To elucidate the probability of immune-oncology therapy for brain metastases, we utilized immune profiling of resected brain metastases and paired non-small cell lung cancer (NSCLC).

Methods To compare pure tumor-infiltrating lymphocytes (TILs) or tumor tissues between NSCLC and brain metastases, macro-dissection was performed before extracting DNA and RNA from 39 patients who received surgical resection of local NSCLC tumor lesions and brain metastases from matched patients. We applied gene expression analysis of mRNA by using a 770 gene panel related with cancer immune cells. Targeted sequencing of 50 cancer hotspot genes on extracted DNA was performed for genotyping. TCRß sequencing of extracted DNA was utilized for profiling of TCR repertoire.

Results We identified 161 differentially expressed genes, which reflected inhibition of dendritic cell maturation pathway, Th1 pathway, and leukocyte extravasation signaling pathway in brain metastases (p < 0.01). VCAM1 was one of highly inter-connected network genes with low expression level in brain metastases. Immune cell profiling analysis illustrated low T-cell infiltration and higher ratio of macrophages against TILs in the brain metastases (p < 0.001). The detected mutations in genotyping analysis were completely shared in 28 patients (71%). TCR sequencing analysis revealed that T-cell clones were expanded in 64% of patients with brain metastases. The majority of TCR repertoire in brain metastases were principally shared with the paired NSCLC, while the density of T cells was sparse in the brain metastases.

Conclusion The microenvironment in brain metastases from NSCLC was revealed to be immunosuppressive, with the majority of T-cell clones in brain metastases being shared with the primary lesion.

#2791

RelB upregulates PD-L1 in advanced prostate cancer: An insight into tumor immunoescape.

Yanyan Zhang,1 Shuyi Zhu,1 Peipei Qian,1 Xiumei Wang,1 Zhi Xu,2 Wenbo Sun,1 Yong Xu1. 1 _The Affiliated Cancer Hospital of Nanjing Medical University, Nanjing, China;_ 2 _The First Affiliated Hospital of Nanjing Medical University, Nanjing, China_.

Interaction between programmed death (PD)-1 receptor and its ligand (PD-L1) is essential for suppression of activated T-lymphocytes. Thus, the high level of PD-L1 in tumors is critical for triggering the inhibitory effect on T cell responses leading to immunoescape. However, the mechanism underlying PD-L1 overexpression in cancers remains fully elucidated. Hence, we demonstrate that PD-L1 is uniquely expressed at a high level in advanced prostate cancer with high constitutive RelB, a member to drive the NF-κB alternative pathway. Clinical investigation indicated that the high levels of RelB/PD-L1 are correlated to the patient's Gleason scores, which was confirmed by quantification of their levels in several prostate cancer cell lines vs. normal prostate epithelial cell. Knocked out RelB in androgen-independent PC-3 cells resulted in reducing PD-L1 expression. Consistently, ectopic expression of RelB in androgen-dependent 22Rv1 cells led to increasing PD-L1 level, implicating that RelB participates in PD-L1 regulation in prostate cancer. Cytokine IFN-γ is able to induce PD-L1 expression through promoting RelB nuclear translocation, revealing that RelB may directly regulate PD-L1 transcription. Accordingly, an NF-κB enhancer element was identified in the promoter region, which is responsible for RelB-mediated PD-L1 transcriptional activation. These results suggest that overexpression of PD-L1 in advanced prostate cancer is, at least in part, through RelB activation. The finding from this study provides a potential approach for enhancing immunotherapeutic efficiency by targeting RelB-activated NF-κB alternative pathway.

#2792

**PD-1 modulates CD8** + **T cell function in prostate tumor microenvironment.**

James Stokes,1 Eric Berry,1 Rajesh Singh,2 Upender Manne,3 Manoj K. Mishra1. 1 _Alabama State University, Montgomery, AL;_ 2 _Morehouse School of Medicine, Atlanta, AL;_ 3 _University of Alabama Birmingham, Birmingham, AL_.

In general, the Regulatory T (Treg) suppresses the immune response. However, their role and immunosuppressive mechanism(s) in context to programmed death 1 (PD-1) are not completely understood. This study aims to determine the role of PD-1 on Treg cells and their impact on CD8+ T cell function in prostatic tumor microenvironment using transgenic adenocarcinoma of the mouse prostate (TRAMP) cells (TRAMP C1, C2, and C3) as a model system. To execute the above aim, tumor induction studies were performed. Briefly, the C57BL/6 mice were administered with serial log concentrations of TRAMP (C1-3) cells. Interestingly, the TRAMP-C3 cells do not form a tumor, however, TRAMP-C1 and TRAMP-C2 cells do form a tumor. After tumor development, mice were sacrificed by cervical dislocation; tumor, lung, spleen, and draining lymph nodes were harvested when the tumor size reached approximately 20mm. Single cell suspensions were prepared from different organs, cells were then stained with specific antibodies, and flow analyzed for the expression of different immune markers. Our preliminary findings demonstrated that PD-1 expression on Foxp3+ Treg cells displayed greater suppressive capacity against CD8+ T cell function in tumor, lung, spleen, and draining lymph node when compared to the control. More importantly, Foxp3+ Treg(high) PD-1(high) interaction with PDL-1 induced immunosuppression by blocking CD8+ T cells function in the prostatic tumor microenvironment. Our data suggest that the expression of Foxp3 and PD-1 may enhance tumor progression; thus, targeting the PD-1 on Treg cells may be a possible therapy to treat prostate cancer.

#2793

Mismatch repair proficient colorectal cancer and adaptive immunosuppression of endogenous anti-tumor immune response: Implications for immunotherapy.

Nicolas Llosa, Anas H. Awan, Ada J. Tam, Hongni Fan, Kellie N. Smith, Cynthia L. Sears, Hao Wang, Drew M. Pardoll, Robert A. Anders, Franck Housseau. _Johns Hopkins University School of Medicine, Baltimore, MD_.

The tumor immune microenvironment (TiME) of mismatch-repair deficient (MMRd) colorectal cancer (CRC) is characterized by a cytotoxic T cell immune signature and counter-expression of IFNγ -inducible T cell checkpoints, features underlining intratumoral adaptive immunosuppression and sensitivity to immune checkpoint blockade. Since single-agent checkpoint inhibitors have not demonstrated, so far, similar meaningful clinical activity for MMR proficient (MMRp) CRC, we compared the tumor immune microenvironment (TiME) in CRC patients that responded to anti-PD1 therapy with the TiME of patients that did not (NCT01876511) to identify immune signatures and biomarkers that could help deciding on combinatorial immunotherapies to treat patients with MMRp CRC. With this approach, we detected immunoreactive MMRp (irMMRp) tumors characterized by a dense infiltration with cytotoxic T cells (CTL) as well as high expression of IFNγ and CD274 (PD-L1), despite their low tumor mutation burden. We observed that these irMMRp colon tumors are characterized by a negative association between Indolamine 2,3 dioxygenase 1 (IDO1) expression and Th17 infiltration. Despite the high expression of CD8 and PD-L1 expression, only irMMRp CRC with high expression of IDO1 on tumor cells and low infiltration with Th17 cells have a TiME resembling the TiME of CRC responding to anti-PD1. Since IDO1-derived tryptophan metabolites, kynurenines, are known to repress Th17 differentiation, these results suggested that the use of IDO1 small molecule inhibitors may derepress Th17 infiltration in CRC and blunt the effect of anti-PD1. Combining therapy with IDO1/PD1 inhibitors with drugs inhibiting IL-17 signaling pathway may help to treat irMMRp CRC patients.

#2794

Expression of LAP, latency-associated peptide of TGFb, on immune cell subsets.

Stavros Kopsiaftis, Patricia E. Rao, Randall Burton, Jessie M. English, Barbara S. Fox, Kenneth J. Simon. _Tilos Therapeutics, Lexington, MA_.

LAP, latency-associated peptide of TGFβ1, is a known marker of regulatory T cells in the tumor microenvironment. TGFβ is a major tolerogenic cytokine that is co-opted by tumors to evade the immune system and is implicated in resistance to checkpoint inhibitors. LAP cages TGFβ in an inactive, latent state until it is activated and released by integrins, MMPs or other activators. Anti-LAP antibodies inhibit the release of TGFβ, inhibit tumor growth in mouse models (Gabriely et al., 2017), and have promise as novel cancer therapeutics. Anti-LAP antibodies are thought to mediate anti-tumor activity both by inhibiting the release of active TGFβ and by reducing the number of LAP+ immunosuppressive cells in the tumor microenvironment (TME). To provide insight into the cellular sources of TGFβ in the TME and further delineate the mechanism of action of anti-LAP antibodies in tumor growth inhibition we determined the expression profile of LAP on the surface of both Treg and other immune cell subsets derived from tumor, spleen, and human PBMCs.

LAP expression on the surface of various mouse and human immune cell subsets was determined using flow cytometry. Tumor infiltrating leukocytes were profiled from CT26 and 4T1 syngeneic mouse tumor models. Expression of LAP was detected on a subset of regulatory T cells, M2 macrophages, dendritic cells, G-MDSC and M-MDSC populations in both CT26 and 4T1 tumors suggesting that each of these cell types are a potential source for TGFβ activation in the tumor microenvironment. Striking differences were detected in LAP expression on immune cell subsets in the TME versus spleens of CT26 tumor bearing mice, consistent with TGFβ expression being increased upon immune cell activation. Additionally, CD4+ cells were isolated from human PBMCs from normal healthy donors and activated using CD3/CD28 +IL2 stimulation. Consistent with previous reports, activated Foxp3+ T cells were positive for cell surface LAP. In addition, LAP expression was also assessed on various human macrophage subsets (M1, M2a, M2b and M2c) that were skewed from CD14+ cells isolated from PBMCs. LAP expression was detected in varying amounts on the surface of the different macrophage subsets with M2a macrophages demonstrating the highest expression.

Taken together these results demonstrate that LAP is expressed on a variety of immune cells with known immunosuppressive function. The location of the LAP-TGFβ1 complex is of critical biological and clinical importance because, once the mature TGFβ1 cytokine, which has a short half-life in solution, is released, it acts locally, either in an autocrine or near paracrine fashion. These results warrant further research to determine the effectiveness of anti-LAP in inhibiting the release of TGFβ and its effects on immunosuppression in the tumor microenvironment.

#2795

Dissemination score of CD8+ TILs by automated image analysis is a potential marker of immune activity in human cancers.

Stefan Bentink,1 Andreas Spitzmueller,1 Tze Heng Tan,1 Hadassah Sade,1 Song Wu,2 Brandon W. Higgs,2 Keith E. Steele2. 1 _Definiens AG, Munich, Germany;_ 2 _MedImmune, LLC, Gaithersburg, MD_.

The overall density of CD8+ tumor-infiltrating lymphocytes (TILs) is important for characterizing the level of immune activity in the tumor microenvironment (TME). Beyond the densities of CD8+ TILs, both their location and distributional patterns may also have relevance to immune activity. We evaluated 645 resected tumors encompassing seven cancer types, and correlate location and spatial patterns of CD8+ TILs to immune pathway activity.

We integrated image analysis results from digitized immunohistochemistry (IHC) slides with gene expression data from a targeted Ion Torrent Panel. Overall density of CD8+ TILs and the exact position of individual CD8+ lymphocytes were determined from IHC slides. A dissemination score was defined as ratio of global density and average local density of CD8+ TILs. This score is the inverse of the Ripley's K statistic and becomes high for disseminated spatial patterns. We used this quotient as a continuous metric to identify tumors with a disseminated TIL pattern and to distinguish them from tumors with focal distribution of CD8+ TILs. Within a subset of tumors, the continuous dissemination metric was correlated with biological pathways using targeted mRNA sequencing and gene set enrichment analysis. In addition, association of the dissemination score with overall survival was tested on a subset of cases.

CD8+ TIL distributional patterns differed significantly between tumor types. Breast and pancreatic cancers more frequently showed a focal distribution of CD8+ TILs, while lung tumors comparatively exhibited a disseminated pattern. Transcriptional profiling data revealed differences between both image analysis phenotypes. On average, cases with more disseminated patterns of CD8+ T cells were associated with mRNA expression of genes that fall in pathways related to motility, migration and activation status of tumor infiltrating T cells. We also found a trend to better overall survival in patients whose tumors had a disseminated TIL score compared to those with a focal pattern. This trend was significant in non-small cell adenocarcinoma of the lung (log rank p = 0.018).

We demonstrate the value of spatial image analysis to automatically score CD8+ TIL dissemination as a marker of immune activity in the TME. Jointly analyzing transcriptional profiles appears to identify a biologically meaningful activation phenotype in tumors with high dissemination scores. Our data further suggests that this phenotype is associated with improved overall survival in some cancer patients.

#2796

Validation of Brightplex, a multiplexed IHC solution for immune cell phenotyping of the tumor microenvironment.

Aurélie Collignon, Assil Benchaaben, Alboukadel Kassambara, Felipe Guimaraes, Emmanuel Prestat, Christophe Haond, Jacques Fieschi. _HalioDx, Marseille, France_.

Brightplex is a multiplexing solution developed by HalioDx to analyze the tumor microenvironment on FFPE tissue. It leverages chromogenic immunohistochemistry performed by iterative automated immunostaining on the same slide followed by whole slide digital pathology analysis based on proprietary software combined to Halo from Indica Lab. Stained cells can be quantified in various regions of interest (tumor, stroma, invasion margin). The Brightplex T cell exhaustion (TCE) panel includes 5 biomarkers (CD3, CD8, PD1, TIM3 and LAG3) to assess the expression of three immune checkpoints on T cells. This panel is intended to be used in clinical research to identify patients susceptible to respond to new immunotherapies especially in the context of the resistance to anti PD(L)1 treatment. Therefore, it is critical to thoroughly demonstrate the robustness and the accuracy of such complex assay. For the validation study of the TCE panel, tissue sections were sequentially immunostained for TIM3, PD1, LAG3, CD8 and CD3. Following each immunostaining, the slides were digitized, de-stained and the antibodies stripped before the next immunostaining. Images of the whole slide were then coregistered and analyzed by digital pathology to determine the densities of various T cell populations: CD8\+ T lymphocytes expressing of 1, 2 or 3 immune checkpoints. The precision of the assay was evaluated in terms of intra- and inter-run repeatability and the accuracy was evaluated by comparison of the automated multiplex assay with singleplex staining. Results demonstrate the reliability of Brightplex to analyze tumor sections. The precision study showed that Brightplex was reproductible on several tumor types such as NSCLC, colon and breast and the accuracy was demonstrated by comparison of cell density for each biomarker following either multiplex or singleplex staining. The high degree of robustness could be explained notably by the iterative chromogenic immunohistochemistry technology which limits the risks of steric hindrance when antibodies and amplification systems are used simultaneously and by the intrinsic robustness of this routine workflow including IVD grade instrument and reagents.

#2797

Accumulation of neutrophils in the tumor microenvironment promotes resistance to immunotherapy in pancreatic cancer.

Despina Siolas, Emily Vucic, Jane Cullis, Mark Choi, Dafna Bar-Sagi. _New York University Medical Ctr., New York, NY_.

The prognosis for pancreatic cancer patients is unequivocally poor. This is due in part to the fibrotic and immunosuppressive microenvironment of pancreatic tumors, which serves as a physical barrier to chemotherapy and immunotherapy. Intratumoral genetic alterations can cause remodeling of the immune environment through distinct cellular mechanisms. Oncogenic Kras gene mutations are the most frequently occurring genetic event in pancreatic cancer, followed by transforming alterations in TP53. We show that CRISPR/Cas engineered KrasG12D;Trp53R172H pancreatic ductal tumors have a distinct immune profile characterized by an increase in neutrophils and a concomitant decrease in cytotoxic and Th1+ T cells. In addition, KrasG12D;Trp53R172H pancreatic ductal tumors have an increase in CXCL5, a chemokine involved in neutrophil accumulation. Analysis of publicly available datasets shows that increased neutrophil numbers significantly correlate with decreased survival in pancreatic cancer patients. Of clinical relevance, p53-Crispr/Cas engineered pancreatic tumors in mice are resistant to immunotherapy/chemotherapy combination treatment. This data supports the possibility of personalizing immunotherapy treatments based on immune profiles.

#2798

**High density of CD68** + **HLA-DR** - **macrophages in the stroma of primary melanoma correlates with an unfavorable immune microenvironment as assessed by Digital Spatial Profiling.**

Emanuelle M. Rizk,1 Andrew Chen,1 Andrew M. Silverman,2 Douglas K. Marks,3 Raul Rabadan,1 Kit Fuhrman,4 Alison VanSchoiack,4 Yan Liang,4 Joseph Beechem,5 Yvonne M. Saenger,1 Robyn D. Gartrell2. 1 _Columbia University, New York, NY;_ 2 _Columbia University/New York Presbyterian, New York, NY;_ 3 _New York University Winthrop Hospital, New York, NY;_ 4 _NanoString Technologies, Seattle, WA;_ 5 _NanoString, Seattle, WA_.

Introduction: The role of macrophages (Mφ) in melanoma progression is controversial, as they have been shown to both favor and inhibit anti-tumor immunity. Density of Mφ at the leading tumor edge has been shown to be a marker of poor prognosis. In previous work, we used quantitative multiplexed immunofluorescence (qmIF) to discover the ratio of CD8+ T lymphocytes (CTLs) to CD68+ Mφ in the peritumoral stroma predicts a favorable prognosis. Further, we found that increased proximity of HLA-DR- Mφ to CTLs in the stroma predicts poor prognosis. These findings highlight the importance of the location of Mφ within the tumor microenvironment (TME). Here, we further analyze and compare the TME of patients with high and low stromal densities of HLA-DR- Mφ.

Methods: From a cohort of 104 patients with primary stage II-III melanoma and known survival information, we selected 8 patients for Digital Spatial Profiling (DSP) analyses. Of this subcohort, 4 patients had a high density of HLA-DR- Mφ and close proximity of HLA-DR- Mφ to CTLs in the stroma while the other 4 had a high CTL to Mφ ratio with low HLA-DR- Mφ density, as determined by qmIF (methods published). FFPE slides were stained with antibodies conjugated to UV-photocleavable DNA barcodes and specific to 34 proteins, including CD45, CD4, CD8, CD68, PD-1, and PD-L1. Twelve regions of interest (ROIs) per patient were selected based on high Mφ density as determined by qmIF. ROIs were then analyzed using UV excitation, which releases DNA barcodes for downstream quantitation on the nanoString nCounter® platform. Protein expression was compared between patients and statistical analysis performed using Mann-Whitney test.

Results: We found that ROIs from patients with higher density of HLA-DR- Mφ had a lower immune infiltration overall as assessed by quantitation of CD45 per ROI (p<0.0001). As expected, the ratio of CD68 to CD45 was higher in these patients than in patients with lower density of HLA-DR- Mφ (p<0.0001). Interestingly, patients with higher density of HLA-DR- Mφ also had a significantly higher ratio of CD4 to CD45 (p<0.0001), but a similar CD8A to CD45 ratio. Patients with a higher HLA-DR- Mφ density had a higher CD4 to CD8A ratio (p<0.0001). Further, PD-L1 and PD1 levels per CD45 were significantly higher in patients with higher HLA-DR- Mφ density (p<0.0001 and p=0.0002, respectively).

Conclusion: Patients with higher densities of HLA-DR- Mφ in the stroma and increased proximity of HLA-DR- Mφ to CTLs in the tumor stroma have lower levels of CD45, a higher ratio of CD4 to CD8, and a higher ratio of PDL1 and PD1 to CD45 by assessment of Mφ-rich areas using DSP. These findings highlight the close relation between Mφ and the local immune microenvironment in primary stage II-III melanoma.

#2799

Targeting CXCR2-mediated neutrophil recruitment to lung cancer.

Roni F. Rayes,1 Jack G. Mouhanna,1 Claire Wang,1 Simon Milette,1 Carson Wong,1 Mariana Usatii,1 Betty Giannias,1 France Bourdeau,1 Rachel Mot,2 Arvind Chandrasekaran,2 Christopher Moraes,2 Sidong Huang,2 Daniela Quail,2 Logan Walsh,2 Veena Sangwan,1 Nicholas Bertos,1 Pierre-Olivier Fiset,1 Jonathan Cools-Lartigue,1 Lorenzo E. Ferri,1 Jonathan D. Spicer1. 1 _McGill University Health Center, Montreal, Quebec, Canada;_ 2 _McGill University, Montreal, Quebec, Canada_.

With recent advances in immunotherapy, it is evident that targeting the tumor microenvironment (TME) is an effective strategy to treat lung cancer (LC), however, more than half LC patients are still resistant to therapy. Limited attention was given to the relevance of the innate immune system despite its critical role in triggering adaptive responses. Neutrophils (PMNs) are the predominant circulating leukocyte in humans. PMNs are associated with developing lesions and are the main immune component of primary non-small cell LC (NSCLC). Multiple studies support the notion that PMNs promote tumor progression, however, the exact mechanisms in which these PMNs are recruited to the primary and metastatic lung TMEs remain unclear. To this end, we examined available genomic databases of > 1,000 NSCLC primary adenocarcinoma (ADC) patients and observed that high expression of all CXCR2 ligands (CXCL1-8 and MIF) correlate with poor survival in lung ADC. Lung ADC patients display one of the highest fold increases of these ligands as compared to all other cancers. We then performed shRNA knock down (KD) of CXCL1 and MIF in A549 and tested the migration of PMNs towards treated and control cell lines using the novel microfluidic device. We observe 3-fold increase of PMN migration towards A549 compared to control. This increase was significantly inhibited in MIF and CXCL1 KDs as well as using MIF and CXCL1 neutralizing antibodies (NA) as compared to controls. PMN migration was higher to A549 then to PC9EN, and treatment of PMNs with a CXCR2 NA led to a decrease in their migration to A549 while unaffecting their migration to PC9EN. Due to the lack of similar genomic databases on LC metastasis, we profiled liver homogenates of mice intrasplenically injected with liver-metastatic Lewis lung carcinoma (LLC) and observed that Cxcl1 was the most overexpressed gene as compared to non-tumor bearing mice (non-TBM). We then KD CXCL1 from the liver metastatic LLC cell line and compared its capacity to recruit PMNs in live mice using intravital microscopy. We observe a decrease in the number of PMNs around developing CXCL1 KD LLC tumors compared to control LLC. We also observe a decrease in PMN migration toward the CXCL1 KD LLC tumors as compared the control LLC. This resulted in a significant decrease in liver metastasis of the CXCL1 KD LLC as compared to control LLC injected mice. Altogether, our data highlight the importance of CXCR2-mediated PMN migration in primary LC and the establishment of liver metastasis from LC. Thus, inhibiting CXCR2 represents a promising strategy to impede primary tumor growth and metastatic dissemination of LC.

#2800

Oral cancer cell-derived extracellular vesicles can modulate an immunosuppressive microenvironment through M2 phenotype polarization.

Ana Karina Oliveira,1 Thiago Andrade Patente,2 Rodrigo Nalio Ramos,3 Ariane Busso Lopes,1 Romênia Ramos Domingues,1 Mariane Tami Amano,4 Eliana Blini Marengo,5 Jose Alexandre Barbuto,2 Adriana Franco Squina1. 1 _Brazilian Center for Research in Energy and Materials - CNPEM, Campinas, Brazil;_ 2 _University of Sao Paulo, Sao Paulo, Brazil;_ 3 _Institut Curie Research Center, Paris, France;_ 4 _Instituto Sírio-Libanês de Ensino e Pesquisa, Sao Paulo, Brazil;_ 5 _Butantan Institute, Campinas, Brazil_.

Oral squamous cell carcinoma (OSCC) is the most prevalent malignant tumor among head and neck cancers, with 300,000 new cases and 145,000 deaths around the world per year, with less than 50% survival rate of advanced cases after five years of diagnosis. The molecular mechanisms involved in immunosurveillance and oral cancer development are poorly understood and, neither molecular markers nor immunotherapy response predictors are available. However, recent findings in molecular biology and immunology of cancer cell have shown that tumor-stroma communication promotes intense suppressive signaling in the microenvironment, which may contribute to failure of therapy. Tumor-stroma communication is not an unidirectional process, driven only by cancer cells. Other resident cells, like cancer-associated fibroblasts (CAFs), immune cells as macrophages and lymphocytes, endothelial cells and tumor stem cells, which are in an intense cross-talk by cell-cell contact, secreted factors and other components of the extracellular matrix (ECM), can alter and be altered by the microenvironment. In this context, the cancer-derived extracellular vesicles (EVs) acquire a significant pathological role in cell-cell communication by changing the phenotype of the recipient cells, promoting tumor growth and metastasis. Here, we explored the role of three different source of EVs, originated from normal gingival keratinocyte (HMK) and from less (SCC9) and high (HSC3) aggressive OSCC cell lines in the modulation of monocyte-derived macrophage M1 or M2 phenotypes, and their ability to contribute to the suppressive phenotype in the tumor microenvironment. CD163 expression, a marker of M2 macrophages, was significantly increased in CD14+ monocytes stimulated by HSC3-EVs, in comparison with non-stimulated monocytes, HMK- and SCC9-EVs-stimulated cells. In addition, CD64+/CD86+/CD80+ M1 macrophage subset, differentiated by GM-CSF/IFN-gamma, showed a decreased expression of the T-cell co-stimulatory molecule, CD86 after stimulus with HSC3-EVs. Furthermore, the mass spectrometry analysis of HSC3-EV protein contents identified a set of up- and down-regulated candidate proteins that may be involved in immunoregulation and suppressive pathways. Since the M2 macrophage phenotype is associated with increased angiogenesis, immunological tolerance and development of neoplastic cells, the M1 subset, the classically activated macrophages, may recognize and eliminate cancer cells. Thus, our results indicate that EVs from aggressive cells HSC3 may contribute to immunosuppressive tumor microenvironment in oral cancer by deviating macrophages into a tumor-promoting instead of a tumor-controlling phenotype.

#2801

Comparison of immunostaining quality for 3D volumes of tissue processed by various clearing methods.

Yi Chen, Qi Shen, Sharla L. White, Laurie J. Goodman. _ClearLight Biotechnologies, LLC, Sunnyvale, CA_.

Background: There are several tissue clearing techniques available and each technique has its own advantages and disadvantages with respect to downstream tissue processing. Adapting these cleared tissues to subsequent 3D immunostaining/imaging followed by re-embedding into traditional pathology is challenging and has never been evaluated across the methods. This study was undertaken to compare the quality of immunostaining and its possible adaptation to the formalin-fixed paraffin-embedded (FFPE)-based immunohistochemistry (IHC) staining with tissues cleared by four different methods: CLARITY, Visikol, iDISCO, and CUBIC.

Methods: Normal mouse kidneys, mouse cell line xenograft tumors (MCF7) and human tonsil tissues (5mm*5mm*1mm) were processed using for each types of clearing method according to their standard protocol. Clearing was preceded or followed by immunostaining of multiple biomarkers (mouse kidney: α-SMA, CD31; MCF7: Pan-CK, Ki67; human tonsil: Pan-CK, CD3, CD20, CD8, PD-L1) depending on the clearing protocol. All samples were counterstained with DAPI. The samples were imaged on a Leica SP8 confocal microscope and Imaris 9.2 software was used for image visualization and analysis in 3D. The cleared samples were subsequently re-embedded into traditional FFPE blocks for H&E and IHC staining as a comparison.

Results: All the tissues reached visual optical transparency for all the clearing methods. Visikol and iDISCO techniques resulted in ~30%-50% tissue-dependent shrinkage, while CLARITY processed tissue demonstrated ~30% tissue size expansion which disappeared after refractive index matching. Among these clearing methods, CLARITY, whose process is clearing the tissue first followed by immunostaining, demonstrated the most even staining and the best signal/noise ratio in a 2D field of view and also achieved the highest imaging depth. The remaining methods displayed varying levels of fluorescent signal damage, especially the nucleic acid stain, DAPI as well as nuclear protein markers. CLARITY was superior in fluorescence preservation for over 6 months without significant signal loss compared to all other techniques. Of all the cleared tissue re-embedded into the FFPE blocks for IHC staining, CLARITY showed the best epitope protection and comparable staining pattern with traditional FFPE processed tissue.

Conclusions: Based on this study, CLARITY has proven itself to be one of the best clearing techniques for 3D immunostaining and adaption to traditional pathology workflow.

#2802

In situ **detection of PD-1** + **CXCL13** + **CD8** + **T cells in non-small cell lung cancer.**

Na Li, Bingqing Zhang, Niyati Jhaveri, Zhifu Zhang, Xin Wang, Hongzhe Sun, Ying Zhou, Courtney Anderson, Xiao-Jun Ma. _Advanced Cell Diagnostics, Inc., Newark, CA_.

Recent reports identified one subset of intratumoral CD8+ cytotoxic T lymphocytes (CTLs) in non-small cell lung cancer (NSCLC) that are PD-1high with distinct molecular and functional properties. Strikingly, these cells produce very high levels of CXCL13 mRNA and protein, which may mediate immune recruitment. Furthermore, the presence of PD-1high CD8+ T lymphocytes are strongly predictive for both response and survival in NSCLC patients treated with PD-1 blockade. Thus, it is of great value to develop a practical biomarker assay to specifically detect these cells in formalin-fixed paraffin-embedded (FFPE) tumor biopsies. In this study, we combined the highly sensitive and specific RNAscope multiplex fluorescent RNA in situ hybridization (ISH) assay detecting CXCL13 and PDCD1 mRNAs with immunohistochemistry (IHC) detecting CD8 protein in a single tissue section to directly visualize PD-1+CXCL13+CD8+ T lymphocytes in NSCLC tissues. Two NSCLC tissue microarrays (TMAs) consisting of 63 independent patient FFPE samples were stained with full automation using the Leica BOND RX instrument. The resulting slides were scanned, and the images were analyzed using the Perkin Elmer Phenochart software. 57 of the 63 TMA cores were available for image analysis. Each tissue core was first examined under 4X magnification, then snapshot images of three independent 40X fields with enriched CD8+ cells (if present) were taken. CD8+ cells, CXCL13+ cells, and PD-1+ cells in each snapshot were counted. Every snapshot contained both stromal and tumor regions. 43 samples contained high (>20) CD8+ CTLs whereas 14 samples contained low (≤20) CD8+ CTLs across the three snapshots. Interestingly, PD-1+CXCL13+CD8+ cells were detected in both high and low CTL tumors. Five of 57 tumors carried high percentages of PD-1+CXCL13+CD8\+ cells (>10% of CD8+ CTLs), with three from high CTL tumors and two from low CTL tumors. These results demonstrate that this fully automated multiplexed RNAscope dual ISH/IHC assay allows for co-localization of RNA and protein biomarkers in single cells with morphological context. The ability to detect RNA and protein in a single slide-based assay enables immune profiling applications to include biomarkers such as secreted proteins and non-coding RNAs that are difficult or impossible to detect by IHC.

#2803

CSF-1 receptor-mediated macrophage depletion can induce immunomodulatory resistance mechanisms in murine tumor models.

Sarah A. O'Brien, Katarzyna Skrzypczynska, Jessica Orf, Brian Belmontes, Hong Tan, Daniel Lu, Ian Driver, Jackson Egen. _Amgen, San Mateo, CA_.

Tumor associated macrophages (TAMs) are present within the stroma of most solid tumors where they can act to inhibit immune responses and promote disease progression through expression of immunosuppressive factors and modulation of tumor stroma. Tumor immunotherapies aimed at depletion of TAMs, for instance through the use of blocking antibodies against the CSF1 receptor (CSF1R), have been widely explored, with multiple studies demonstrating that blocking TAM function can inhibit tumor growth/metastasis and stimulate anti-tumor immunity. In order to further dissect the functions of TAM populations and define conditions under which antiCSF1R immunotherapy may have benefit, we evaluated the effect of antiCSF1R treatment on multiple subcutaneous murine tumor models with varying dosing parameters. Despite robust macrophage depletion, some tumor models, such as Renca renal cell adenocarcinoma, showed only a modest change in tumor volume and minimal effector T cell recruitment or activation in response to anti-CSF1R therapy. Comprehensive flow cytometry-based immunophenotyping and single-cell RNA sequencing on immune cells isolated from antiCSF1R and isotype control-treated Renca tumors revealed shifts in gene expression patterns related to vascular remodeling, hypoxic responses, and enhanced regulatory T cell activity. These studies indicate macrophage depletion may, under some conditions, result in compensatory anti-tumor immune responses that could limit the clinical benefit of this therapeutic approach.

#2804

Metabolic-immunoregulatory role of CCRK-mTOR signaling in NAFLD-associated hepatocellular carcinoma.

Wenshu TANG, Jingying ZHOU, Weiqin YANG, Yu FENG, Myth MOK, Alfred Sze CHENG. _The Chinese University of HongKong, HongKong, Hong Kong_.

Obesity increases the risks of steatosis, non-alcoholic steatohepatitis (NASH) and HCC development. Non-alcoholic fatty liver disease (NAFLD) associated-hepatocellular carcinoma (HCC) is a male-predominant cancer mainly caused by metabolic disorder. Our previous findings demonstrated that cell cycle-related kinase (CCRK), through upregulates mTORC1 pathway, resulting in deregulated lipid/glucose metabolism and tumorigenesis (Nat Communs accepted). In this study, we further elucidate that CCRK activates mTORC1 through SLC3A2 and SLC7A5 dependent amino acid transportation. We performed comprehensive immune cell profiling in a high-fat high-carbohydrate diet (HFHC)-induced obesity and NASH murine model and uncovered activation of CCRK-mTORC1 signaling cascade and specific induction of myeloid-derived suppressor cell (MDSC), reduction in CD8+ T cells, which subsequently enhanced tumor growth of orthotopically-implanted HCC tumor cells. Notably, treatment of the mTORC1/C2 dual kinase inhibitor vistusertib (AZD2014), currently undergone phase I/II clinical trials, abrogated mTORC1/SREBP2 signaling and metabolic dysregulation especially cholesterol levels. Further immune profiling in liver and blood of AZD2014 treated mice will be performed. Furthermore, mTORC1/SREBP2 positively associated with cholesterol level in the streptozotocin (STZ)-HFHC induced spontaneous NAFLD-HCC mouse model, which will be utilized to further study the therapeutic effect of vistusertib in vivo. Collectively, this study will elucidate the metabolic-immunosuppressive role of CCRK-mTOR signaling in NAFLD-associated HCC and further provide insights into development of therapeutic strategies against the CCRK-mTOR reinforced NAFLD-HCC. This project is supported by Collaborative Research Fund C4017-14G and the Focused Innovations Scheme 1907309.

#2805

DFMO and 5-azacytidine increase M1 macrophages in the tumor microenvironment of an ovarian cancer mouse model.

Meghan Travers, Stephen Brown, Matthew Dunworth, Cassandra Holbert, Robert Casero, Cynthia Zahnow. _Johns Hopkins School of Medicine, Baltimore, MD_.

Although ovarian cancer has a low incidence rate encompassing roughly 1% of all new cancers, it remains the most deadly gynecologic malignancy. Our lab has designed a novel combination therapy which combines a DNA methyl transferase inhibitor (DNMTi) and ornithine decarboxylase inhibitor that together, alter the tumor microenvironment to inhibit ovarian tumor growth. Previous work has demonstrated that the DNMTi 5-Azacytidine (AZA) activates type I interferon signaling (Li, 2014, Chiappinelli, 2015) to increase IFNγ+ T cells and NK cells and reduce the percentage of macrophages and myeloid derived suppressor cells (MDSCs) in the tumor microenvironment (Stone, 2017). To improve the efficacy of epigenetic therapy, we hypothesized that the addition of α-difluoromethylornithine (DFMO), an ornithine decarboxylase inhibitor may decrease activity of MDSCs through inhibition of arginase. Immunocompetent C57Bl/6 mice were injected with syngeneic mouse ovarian surface epithelial (MOSE) tumor cells (ID8-VEGF-Defensin) and subsequently treated with AZA, DFMO, or combination AZA+DFMO. After several weeks, mice developed hemorrhagic ascites that was drained, processed and stained for cell-surface markers and analyzed by flow cytometry to identify T cells, NK cells, and macrophages. We found that in vivo AZA, DFMO, and AZA+DFMO significantly increased survival in mice, decreased tumor burden, and caused recruitment of activated (IFNγ+) CD4+ T cells, CD8+ T cells, and NK Cells compared to vehicle. The combination therapy had a striking increase in survival when compared to single agent treatment; however, the difference in recruited lymphocytes was less striking. Instead, combination therapy led to the most dramatic decrease in immunosuppressive cells such as M2 polarized macrophages and increase in tumor-killing M1 macrophages. Using a CSF1R blocking antibody, we found that depleting macrophages in this model reduced the efficacy of AZA+DFMO treatment, and resulted in fewer M1 macrophages in the tumor microenvironment. We thus conclude that our novel combination therapy modifies macrophage polarization in the tumor microenvironment, recruiting M1 macrophages and prolonging survival. We hypothesize that this treatment regimen could have a widespread impact on macrophage-rich tumors in general, and plan to test this hypothesis in additional tumor models.

#2806

Progression of ductal carcinoma in situ (DCIS), is it in the immune microenvironment.

Mathilde M. Almekinders,1 Lindy Visser,1 Bram Thijssen,1 Rianne van der Linden,1 Charlotte van Rooijen,1 Petra Kristel,1 Annegien Broeks,1 Tycho Bismeijer,1 Lodewyk Wessels,2 Erik Hooijberg,1 Karin de Visser,1 Esther Lips,1 Jelle Wesseling,1 on behalf of the PRECISION team (PREvent ductal Carcinoma In Situ Invasive Overtreatment Now). 1 _Netherlands Cancer Institute, Amsterdam, Netherlands;_ 2 _Netherlands Cancer Institute, Oncode Institute, Netherlands Cancer Institute, Amsterdam, Netherlands_.

Introduction: DCIS is a non-obligate precursor to invasive breast cancer (IBC). DCIS patients are treated similarly to breast cancer with surgery, often followed by radiotherapy and/or endocrine treatment. However, most DCIS lesions will never progress to IBC, indicating that overdiagnosis and overtreatment exists. DCIS lesions show variable amounts of immune cells, particularly in the periductal stroma. Immune escape might be a critical step for transition from DCIS to IBC. We aim to identify factors within the immune microenvironment of DCIS lesions that distinguish harmless from potentially hazardous DCIS.

Methods: A case-control study is being conducted consisting of women with pure DCIS diagnosed between 1989-2005 with median follow-up of 12 years, treated with breast conserving surgery only. Cases are defined as women with DCIS developing subsequent ipsilateral breast cancer (iIBC), controls as women with DCIS without subsequent iIBC. Multispectral immunohistochemical imaging was performed on primary DCIS lesions, aiming at detection of CD20+ B-cells, CD8+ T-cells, CD3+ T-cells, CD3+Foxp3+ regulatory T-cells, and CD68+ macrophages. Density of immune cell subsets in cells/mm2, immune cell ratios and spatial relationships were calculated for 27 cases and 28 controls. These immune cell related factors were correlated to outcome and integrated with RNAseq data of pure microdissected DCIS. We performed gene set enrichment analysis on the correlation between DCIS gene expression and density of immune cell types with sample permutation (flexgsea R package).

Results: Stromal lymphocyte, B-cell, CD8+ T-cell, regulatory T-cell and macrophage density did not significantly differ between cases and controls. Immune cell composition (CD8+ T-cell/lymphocyte, CD8+ T-cell/CD3+Foxp3+ regulatory T-cell and CD20+/lymphocyte ratio) and fraction of regulatory T-cells in close proximity of a CD8+ T-cell did not differ between cases and controls. We find a negative association between stromal B-cell density and DCIS gene expression of estrogen receptor (ESR1) targets. Higher stromal T-cell density was associated with proliferation and expression of genes characteristic for luminal B and basal-like subtypes. Furthermore, higher density of specific immune cell subsets within the DCIS compartment was associated with several immune and cancer pathways.

Conclusion: A first set of analyzed DCIS cases (n=27) and controls (n=28) show no significant differences regarding immune cell density, composition and spatial relationships. Considering the entire group of DCIS patients (n=55), a negative association between stromal B-cell density and gene expression of ESR1 targets was found. Higher density of lymphocytes was associated with proliferation and expression of genes characteristic for luminal B and basal-like subtypes. The full set of 175 DCIS lesions will be presented at AACR Annual Meeting 2019.

#2807

Accessing the transcriptome of low-abundance tumor-infiltrating lymphocytes with SPRI beads.

Aiqing He, Manling Ma-Edmonds, Chan Gao, Vishal Patel, David Galbraith, Yan Zhang, Qi Guo, Heshani DeSilva, Gennaro Dito, Amy Truong, Sunil Kuppasani, Kandasamy Ravi, Omar Jabado. _Bristol-Myers Squibb, Princeton, NJ_.

Background: Multicolor flow cytometry has provided deep characterization of tumor-infiltrating lymphocytes (TILs). Routine fluorescence-activated cell sorting (FACS) methods discard cells after counting, precluding transcriptome analysis, which is key to understanding the activation states of immune cells. FACS-sorted cells pose 3 problems for transcriptomics: low cell count, cell rupture from high-pressure sort process, and inefficient centrifugation and recovery of cells from large collection volumes. SMART-Seq (TaKaRa/Clontech) is a highly sensitive, reverse transcriptase-based method to amplify low-input RNA that is commonly used to generate RNA-Seq libraries. After FACS, cell RNA can be purified by Qiagen RNeasy or nucleic acid binding solid-phase reversible immobilization (SPRI) magnetic beads. Alternatively, cells can be spun down after FACS then directly lysed as input to the SMART-Seq method. We sought to compare different methods and design a high-throughput protocol to capture the transcriptome from FACS-sorted, rare immune cell types.

Methods: A dilution series of 50-5000 human T cells was generated to test efficiency and sensitivity of RNA extraction. We compared RNA extraction yields by column (Qiagen RNeasy Low Input Micro Kit) or SPRI bead (Agencourt RNA-Advanced Cell v2, Beckman Coulter). Direct lysis of cells in SMART-Seq buffer was tested as an alternative to extraction. RNA yields were quantified by High Sensitivity Agilent Bioanalyzer or digital droplet PCR (BioRad). Extracted RNA was reverse transcribed and amplified using SMART-Seq v2 followed by library preparation (Nextera XT DNA kit) and sequencing (NovaSeq Illumina). To obtain TILs, mice were implanted subcutaneously with MC38 tumor cells, then tumors were harvested, disassociated, and FACS sorted to isolate T cells (CD4+, CD8+) and macrophages (yields ranged from 1000-8000 cells).

Results: Direct lysis followed by SMART-Seq was found to work well with diluted T cells; however, the method was sensitive to large buffer volumes and was unreliable for small numbers of pelleted, FACS-sorted cells (<5000). SPRI beads provided higher yields than Qiagen columns and were amenable to plate-based automation. RNA-Seq analysis of a T cell dilution series showed high concordance between SPRI and Qiagen columns, although more genes expressed at very low levels were detected in SPRI-purified samples. We used the SPRI bead method to characterize the transcriptome of TILs from implanted mouse tumors. Expected patterns of immune marker expression by immune cell type were observed.

Conclusions: Use of SPRI beads to extract RNA from FACS-sorted immune cells was sensitive and reproducible, enabling transcriptomic characterization of <5000 cells. Successful profiling of TILs derived from mouse syngeneic tumors suggests that the method can be used to probe activation states of rare immune cell types in the tumor microenvironment.

#2808

Investigating the role of macrophage-mediated suppression in the efficacy of NK cell immunotherapy for metastatic outgrowth of breast cancer cells.

Demi Brownlie, Dahlia Doughty-Shenton, Jeffrey W. Pollard, Takanori Kitamura. _University of Edinburgh, Edinburgh, United Kingdom_.

Breast cancer is the leading cause of cancer-related death in females worldwide. Although 5-year survival of breast cancer patients in early stages is 89-100%, that of patients with metastatic tumours is reduced to just 21%, suggesting the requirement of more effective therapies for metastatic breast cancer (MBC). As MBC is refractory to current therapies, NK cell infusion has been suggested as an alternative therapy. However, its efficacy is modest in MBC possibly due to the suppressive nature of the tumour microenvironment, especially through the tumour-associated macrophages (TAMs) within it. Here we investigated the effects of TAMs in metastatic tumours on NK cell cytotoxicity, as well as the mechanism behind TAM-mediated NK cell suppression.

To evaluate NK cell cytotoxicity, we established a method whereby mouse breast cancer cells were cultured with splenic NK cells, and the resultant tumour cell apoptosis was determined by quantitative fluorescence microscopy. To investigate the effects of TAMs on this NK cytotoxicity, we purified them from lungs with metastatic tumours and added them to this co-culture. We also investigated the suppressive effects of bone marrow-derived macrophages cultured with M-CSF (M-BMMs). To further investigate the mechanism, we depleted TAMs in mice with metastatic tumours and detected inhibitory receptor expression on NK cells as well as NK cell inhibitory ligands on TAMs in the metastatic lung and on M-BMMs by flow cytometry.

TAMs from metastatic tumours significantly reduced NK cell cytotoxicity toward tumour cells. M-BMMs (resembling TAMs) also reduced NK cell cytotoxicity. We also found that TAMs and M-BMMs expressed high levels of NK cell inhibitory ligands. There was no difference in the expression of inhibitory receptors on NK cells between tumour bearing lungs with or without TAMs.

To conclude, we have shown that TAMs in metastatic tumours, as well as their mimetic M-BMMs, can suppress NK cell cytotoxicity towards breast cancer cells in vitro. Our data suggest that TAMs express higher levels of inhibitory ligands and thereby transmit suppressive signals possibly through direct contact with NK cells. Further investigation of mechanisms behind TAM-mediated NK suppression might lead to the improvement of NK cell-based immunotherapy for MBC.

#2809

**The induction of CD8** + **T lymphocytes and PD-L1** + **immune cells by neoadjuvant chemoradiotherapy for rectal cancer.**

Eiji Fukuoka, Kimihiro Yamashita, Yutaka Sugita, Akira Arimoto, Tetsu Nakamura, Yoshihiro Kakeji. _Kobe University Graduate School of Medicine, Kobe, Japan_.

Background; The relationship between tumor immunological microenvironment and irradiation has been studied so far. Based on these findings, we examined a tumor microenvironment of rectal cancer performed by neoadjuvant chemoradiotherapy (NACRT) with immunohistochemical analysis of surgical specimens. But, for introduction to clinical settings, the analysis is not yet sufficient. So, in more detail, we investigate tumor infiltrating CD8\+ T cells by irradiation in mouse CT26 colon tumor model.

Methods; Locally advanced rectal cancer were enrolled and 68 were eligible for this study. Immunohistochemical staining of programmed cell death- lignad1(PD-L1), CD8, and CD163 was performed on specimens of all cases. In a mouse model, CT26 tumor cells were inoculated in mouse, tumors are irradiated with a single dose of 10Gy after 14 days. We analyzed CD8+ T cells infiltration of both irradiated mouse and no treated mouse by flow cytometry.

Results; There were 44 cases in which NACRT was performed (NA group) and 24 cases in which surgery alone was performed (SA group). In the SA group, there was no significant change in the infiltration of PD-L1+immune cells (PD-L1+IC) nor CD8 +cells before and after treatment, but in the NA group, the infiltrations of them were significantly increased. In multivariate analysis, low infiltration of CD8 +cells were identified as poor prognostic factors in disease-free survival. Moreover, in our mouse model, elevated numbers of tumor-infiltrating CD8+ T cells between day11 and day18 after irradiation were observed in tumors of irradiated mice compared to no treated mouse. We observed that among CD8\+ T cells, cells that coexpress PD-1 and Tim-3 comprise the major population in irradiated mouse. PD-1+Tim-3\+ CD8\+ T cells produced more INF-γ compared to that by PD-1+Tim-3-CD8\+ T cells and PD-1-Tim-3\- CD8\+ T cells in irradiated mouse and no treated mouse. Among the three populations PD-1+Tim-3+ population contained the largest fraction of central memory (CD44hiCD62Llow) cells, but the lowest fraction of central memory (CD44hiCD62Lhi) cells.

Conclusion; The infiltration of PD-L1+IC and CD8 +cells was induced by NACRT. In the mouse model, we confirmed the kinetical and phenotypical changes of tumor-infiltrating CD8+ T cells.

#2810

Exploring T cell- repertoire in the tumor microenvironment during check-point inhibition in patients with metastatic solid tumors.

Vinicius Araujo Barbosa de Lima,1 Morten Hansen,2 Kristoffer Staal Rohrberg,1 Iben Spangaard,1 Inge Marie Svane,2 Ulrik Lassen1. 1 _Rigshospitalet, Copenhagen, Denmark;_ 2 _Herlev Hospital, Copenhagen, Denmark_.

Background: Immunomodulation has improved anti-tumor activity. However, this treatment modality is only effective in a proportion of patients. Hence, analyzing changes in the tumor microenvironment is essential for understanding the variety of response patterns seen among patients undergoing the treatment. It also provides clinicians with valuable knowledge of possible new therapeutic targets.

Aim: Assessment of the repertoire of T-cells (tumor-infiltrating lymphocytes -TIL) in the tumor microenvironment during check-point inhibition in patients with metastatic solid tumors.

Material and methods: 35 patients with different metastatic solid tumors were included in the study at our Unit for Experimental Treatment at Rigshospitalet, Denmark. Patients were given treatment with check-point inhibitors targeting PD-1/PD-L1/CTLA-4 either in monotherapy or combined with other agents. Fresh tumor biopsies were taken at two time-points, namely, at baseline and on-treatment (50 days interval). T-cells were expanded in vitro with high doses of IL2 (6,000IU/mL IL2), harvested and cryopreserved for later use. Samples were analyzed using a multicolor flow cytometry panel. Response to the treatment was assessed radiologically using to RECIST criteria 1.1 and patients were divided in responder and non-responders.

Results: Expansion was successful in seventeen patients and five other follow-up samples are still in culture at the moment. The current data analysis was run based on the seventeen cryopreserved samples.

Among responders, we observed that baseline samples showed a statistically significant high percentage of effector CD8+(CD45RO+CCR7-) and CD8+naïve T cells(CD62L+), with p value 0.034 and 0.009, respectively. The presence of a high proportion of naïve CD8+Tcells in tumor microenvironment of responders could be explained by the fact that there is a high proportion of T cells surviving and becoming memory T cells and eventually effector T cells.

No statistical difference was seen at the baseline expression of PD-1, either in the CD8 (p 0.66) or in the CD4 compartment (p 0.88).

For follow-up samples, CD4+ effector memory T cells seemed to be more present among responders (p 0.009). Interestingly, NK-cells expressing CD56 (p 0.046) and co-expressing CD137 (p 0.005), known as an activation marker, appeared to be more present in samples from the responder group. CD4+T cells deriving form follow-up samples were also co-stained for LAG-3 revealing that responders had a low percentage of TILs expressing both markers (p 0.016).

Paired-sample test will be run as the expansion phase for the remaining 5 samples still in culture is over.

Conclusion: The preliminary findings of our study suggest that response achieved during check-point inhibition is reliant on the subset of T cells present in the tumor microenvironment and on how its composition changes over time.

#2811

**A translational immuno-oncology platform to model the tumor microenvironment** in vitro **.**

Louise S. Brackenbury,1 S. Rhiannon Jenkinson,1 Shilina Roman,2 Robert D. Nunan,1 Sylvie D. Hunt,1 Anna Willox,1 Neil A. Williams,1 Omar Aziz,2 Ian Waddell2. 1 _Charles River (Portishead), Bristol, United Kingdom;_ 2 _Charles River, Harlow, United Kingdom_.

The tumor microenvironment (TME) is a complex network, consisting of the tumor, blood vessels, stromal and immune cells, and soluble factors. The immune system plays an important role in combating tumor growth, and multiple studies associate raised immune infiltrate with beneficial outcome. Various tractable immuno-oncology targets have been identified, both in the TME and immune cells. It is critical to test novel immuno-modulators in assays involving multiple cell types, to understand the MOA and identify biomarkers before moving into the clinic. Charles River have developed a range of primary human assays to model the TME in vitro. This platform models multiple anti-tumor immune effector pathways and has been validated with standard of care therapeutics. The assays include: T cell or NK cell-mediated tumor killing, myeloid/macrophage assays, Th1/Th17/iTreg differentiation and regulatory T cell suppression assays. Tumor killing assays were performed using an IncuCyte ZOOM and PBMC were cultured with tumor monolayers with TCR ligation. Keytruda and Yervoy raised levels of tumor cell death, indicating that they enhanced T cell killing. To further model the TME, 3D 'spheroids' were used to screen for either T cell mediated or ADCC-mediated NK cell killing. For ADCC, PBMC were co-cultured with tumor spheroids and killing measured by monitoring spheroid diameter in the presence of Herceptin, which potentiated NK-driven tumour killing. Myeloid/macrophage cells were differentiated from monocytes in tumor-conditioned media (TCM). TCM drove the generation of immature cells which were CD25lo, CD127lo, CD184hi, CD80lo, CD163hi, CD68lo and MHCIIlo. These cells produced IL-10 and VEGF and were suppressive in a T cell assay. Phagocytosis assays were also performed using anti-CD47 as a control. The TME is associated with increased Treg numbers and low levels of Th1 or Th17 cells. Many therapeutics aim to shift the balance away from Treg, towards Th1 or Th17 cells. Assays were therefore performed by differentiating naïve CD4+ T cells into iTreg, in the presence or absence of a USP7 inhibitor. Reduced iTreg generation, without significant alteration in Th1/17 frequency was observed. The resulting iTreg were less able to suppress T cell proliferation when compared to non-treated iTreg. Suppression assays were also performed using nTreg. As before, the USP7 inhibitor partially reversed nTreg-mediated suppression. Charles River is pleased to present an immuno-oncology platform to model the TME in human cells in vitro, enabling partners to rapidly assess the immunomodulatory capacity of their therapeutics. The 3D assays represent an important complex cell model to support translational drug discovery, sitting alongside T cell-mediated tumor killing, myeloid/macrophage assays, Th1/Th17/ iTreg differentiation and nTreg assays and helps to define the diverse aspects of micro-environmental control of immune response.

#2812

Triple-negative breast cancer cell-intrinsic glucocorticoid receptor activity and modulation of the immune microenvironment.

Deniz N. Dolcen,1 Diana West Szymanski,1 Ananth Panchamukhi,1 Xiaomeng Wang,2 Randy F. Sweis,1 Rita Nanda,1 Suzanne Conzen1. 1 _University of Chicago, Chicago, IL;_ 2 _Bates College, ME_.

Background: Variation in the infiltration of TNBC immune microenvironment is not well understood. Our lab previously demonstrated that high glucocorticoid receptor (GR; nuclear hormone receptor) activity in TNBC models is associated with tumor cell-intrinsic anti-inflammatory gene expression pathways. In addition, high GR expression in early-stage TNBC is associated with a significantly lower rate of recurrence-free survival suggesting a role for GR-associated mechanisms in TNBC progression. GR activation in lymphocytes is well known to result in their apoptosis; however, a current gap in knowledge regarding GR biology is whether tumor-cell intrinsic GR activity modulates immune cells in the microenvironment. We hypothesized that TNBC cell-intrinsic GR activity may inhibit antitumor T cell activity in the tumor microenvironment.

Methods and Results: We divided TCGA primary TNBC RNAseq expression data based on GR expression and determined enrichment for immune cells using two transcriptome parsing algorithms. We found that TNBCs with high GR expression are significantly enriched for the T cell-inflamed gene expression signature, suggesting the presence of T cells in the microenvironment. The xCell algorithm found that TNBCs with high GR expression are significantly enriched for the CD4+ naïve T cell transcriptome, which is absent in TNBCs with low GR-expression. To begin to determine whether GR activation in TNBC cells induces the secretion of cytokines and/or growth factors that result in the chemotaxis of CD4+ naïve T cells, we collected conditioned cell culture media from control versus GR-active TNBC cells and performed multiplex cytokine ELISA. We found that G-CSF secretion by TNBC cells was significantly increased following GR activation; G-CSF has been shown to promote immunosupressive Treg cell differentiation.

Conclusions: Prior data suggest that breast tumor-infiltrating immunosuppressive Tregs arise from chemotaxis of circulating naive CD4+ T cells that differentiate into Tregs in situ. Thus targeting GR in TNBC may be an important therapeutic approach to potentiate antitumor T cell responses and suppress the Treg population in the tumor microenvironment. Ongoing work is aimed at further delineating a mechanism by which GR can modulate the immune tumor microenvironment.

#2813

Clinical impact of immune microenvironment according to intratumoral locations of esophageal squamous cell carcinoma.

Ken Hatogai,1 Satoshi Fujii,1 Takashi Kojima,1 Shigehisa Kitano,2 Hiroyuki Daiko,1 Takayuki Yoshino,1 Atsushi Ohtsu,1 Toshihiko Doi,1 Atsushi Ochicai1. 1 _National Cancer Center Hospital East, Kashiwa, Japan;_ 2 _National Cancer Center Hospital, Tokyo, Japan_.

Background: The immune microenvironment is thought to differ according to the intratumoral location of esophageal squamous cell carcinomas (ESCCs), depending on cancer genomics and environmental factors. The aim of this study was to clarify the clinical impact of the immune microenvironment in different intratumoral locations in patients with ESCC.

Methods: The expressions of CD8 and PD-1 on tumor-infiltrating immune cells (TIICs) and the expression of PD-L1 on tumor cells (PD-L1TC) were immunohistochemically examined in the surface area (Surf), the center area (Cent), and the invasive front area (Inv) of tumors that had been surgically resected at our institution from 192 patients with ESCC. The expressions of each of the molecules were compared, and the relationships between the expressions of each of the molecules and overall survival (OS) were investigated for each of the three intratumoral locations.

Results: The median numbers of CD8+ TIICs were 132/mm2 in Surf, 118/mm2 in Cent, and 122/mm2 in Inv, while those of PD-1+ TIICs were 98/mm2 in Surf, 96/mm2 in Cent, and 92/mm2 in Inv, with no significant differences between each of the intratumoral locations for either type of TIICs. In addition, the number of TIICs was moderately to highly correlated between each intratumoral location for both CD8 (correlation coefficients [ρ] were 0.682 [Surf/Cent], 0.758 [Cent/Inv], and 0.669 [Inv/Surf]) and PD-1 (ρ were 0.617 [Surf/Cent], 0.753 [Cent/Inv], and 0.611 [Inv/Surf]). In contrast, the PD-L1 positive rate was 14.6% in Surf, 18.2% in Cent, and 12.0% in Inv, showing a significantly low positive rate in Inv compared with that in Cent (P = 0.012). Among patients with higher CD8+ TIICs, the PD-L1 positive rate was lower in Inv (22.1%) than that in Surf (26.3%) and Cent (29.2%), and it was also lower in Inv (21.1%) than that in Surf (27.4%) and Cent (25.5%) among patients with higher PD-1+ TIICs, with no significant differences. A high number of CD8+ TIICs and PD-1+ TIICs and PD-L1TC positivity were significantly related to a better OS in Surf and Cent, but not in Inv (CD8: stage-adjusted hazard ratio [HR] = 0.602 [P = 0.012, logrank] in Surf, HR = 0.649 [P = 0.018] in Cent, HR = 0.806 [P = 0.165] in Inv; PD-1: HR = 0.637 [P = 0.028] in Surf, HR = 0.645 [P = 0.021] in Cent, HR = 0.815 [P = 0.208] in Inv; PD-L1: HR = 0.442 [P = 0.044] in Surf, HR = 0.509 [P = 0.026] in Cent, HR = 0.778 [P = 0.718] in Inv). Among the 43 patients with PD-L1TC positivity in at least one intratumoral location, 20 patients with positive PD-L1TC in Surf and/or Cent but not in Inv demonstrated a strong tendency toward a better OS than the 23 patients with PD-L1TC positivity in Inv (HR = 0.339 [P = 0.053]).

Conclusions: Despite the homogeneous distribution of TIICs, PD-L1 expression and the relationship between the immune microenvironment and OS differed according to the intratumoral location of ESCC. The immune microenvironments in Surf and Cent have a clinical impact.

#2814

Systemic and local bone metastasis models for immuno-oncology drug development.

Tiina E. Kähkönen,1 Mari I. Suominen,1 Jenni H. Mäki-Jouppila,1 Jussi M. Halleen,1 Arne Scholz,2 Jenni Bernoulli1. 1 _Pharmatest Services, Turku, Finland;_ 2 _Bayer AG, Berlin, Germany_.

Bone metastases are common and count 30-70% of metastases in breast, lung, and bladder cancer, and 80% of multiple myeloma patients have bone disease. Despite recent progress in cancer treatment, bone metastases remain incurable. Novel immunotherapies have the potential to cure also bone metastatic disease. Here we have established and validated syngeneic models with a focus on bone metastasis that could be used in preclinical efficacy studies. Syngeneic models were established for breast (4T1-GFP), multiple myeloma (5TGM1), bladder (MBT-2) and lung cancer (KLN-205). The cells were inoculated into systemic circulation (4T1 intracardially and 5TGM1 into tail vein) or bone marrow (MBT-2 and KLN-205). In the 4T1 model, tumor burden was assessed by ex vivo GFP imaging and in the 5TGM1 model by measuring serum IgG2b paraprotein levels during the study. Tumor-induced bone changes were followed by X-ray imaging in all models. Hind limbs were analyzed by histology and immunohistochemistry for tumor-infiltrating lymphocytes (TILs). The effects of standard-of-care compounds were assessed in the 4T1 (cyclophosphamide, 100 mg/kg or zoledronic acid, 0.1 mg/kg) and 5TGM1 (bortezomib, 1 mg/kg) models. The effect of anti-PD-1 treatment (200 µg/dose) was evaluated in the MBT-2 model. In the 4T1 model, osteolytic bone lesions formed within 13 days. In addition to bone metastases, about 50% of the mice had metastases in lungs, ovaries, kidneys and adrenal glands based on GFP imaging. Cyclophosphamide decreased the tumor burden and the area of osteolytic bone lesions. Zoledronic acid decreased the osteolytic lesion area but had no effect on tumor burden. Low number or no TILs were observed. In the 5TGM1 model, osteolytic lesions were observed and the study was ended at day 35. Metastases in ovaries, kidneys and adrenal glands were observed in about 30% of the mice. Bortezomib decreased serum paraprotein compared to vehicle treated mice. In both models, cachexia and paraplegia were occasionally observed. Moderate number of CD3+ TILs were observed in the tumors. In the intratibial MBT-2 and KLN-205 models, large osteolytic lesions were observed within 25 days, and in the KLN-205 model also lung metastases were observed. Anti-PD-1 treatment decreased osteolytic tumor area in the MBT-2 model. Moderate number of CD3+ and low number of CD4+ and CD8+ TILs were observed in the tumors growing in bone and also in the lung metastases. A high incidence of bone metastases was observed in all models. The use of systemic models allows studying the effects of test compounds in prevention or treatment of metastases. Intratibial models can be used when the primary interest is in tumor growth in bone microenvironment. Mimicking the clinical situation, none of the SOC compounds could prevent tumor growth completely, and therefore combination therapies are warranted for better overall efficacy. 

### Inflammation and Microbiome

#2815

Sulindac reverses an immunosuppressive tumor microenvironment associated with obesity-driven metastatic mammary tumors in mice.

Shannon B. McDonell,1 Alyssa J. Cozzo,1 Lydia K. Eisenbeis,1 Andrew J. Dannenberg,2 Stephen D. Hursting1. 1 _The University of North Carolina at Chapel Hill, Chapel Hill, NC;_ 2 _Weill Cornell Medical College, New York, NY_.

Obesity is an established risk factor for several breast cancer (BC) subtypes. Obese BC patients also show poorer response to therapy, increased metastases, and increased mortality. While high levels of inflammation are thought to be a driver for BC metastasis, it is unknown how chronic exposure to low levels of obesity-associated inflammation contributes to BC progression and metastasis. We previously showed that treatment with the non-steroidal anti-inflammatory drug (NSAID) Sulindac (140 ppm mixed into control [10% kcal/fat] or diet induced obesity [DIO, 60% kcal/fat] diets) offset both the pro-growth and pro-metastatic effects of obesity on BC using three different mouse models of metastatic BC - basal-like E0771, and claudin-low metM-Wnt-lung and metM-Wnt-liver. To characterize the effect of Sulindac treatment on the tumor microenvironment, we conducted microarray analysis on E0771 tumors from mice in all four diet groups (Control, Control+Sulindac, DIO, and DIO+Sulindac). Ingenuity Pathway Analysis revealed significant Sulindac-induced differences (q<0.01) in several pathways relating to immune function. Specifically, Th1/Th2 pathways, CD28 signaling, and markers of dendritic cell maturation were significantly downregulated in DIO tumors compared to all other diet groups. Similarly, immune burden calculation using CIBERSORT showed lower levels of CD4+ T memory cells (p<0.05) and higher levels of resting dendritic cells (p<0.001) in DIO tumors compared to all other diet groups. To further explore Sulindac-associated effects on immune and cancer cell gene expression, we performed single-cell RNA-seq on E0771 tumors. Preliminary analyses showed marked differences in gene expression between DIO and DIO+Sulindac tumor cells. Of the top 25 significant (q<0.01) genes upregulated in DIO vs DIO+Sulindac tumor cells, 15 have been previously shown to be drivers of breast cancer progression or metastasis, with only 2 showing tumor suppressive functions. Similarly, 13 of the top 25 genes upregulated in DIO+Sulindac compared to DIO tumor cells have previously shown to have tumor suppressive functions, while only 2 are pro-tumorigenic. Immune analyses showed fewer immune cells in DIO tumors as well as increased pro-tumorigenic or immunosuppressive gene expression in DIO immune cells, while DIO+Sulindac immune cells showed increased markers of immune activation. Finally, T cell receptor (TCR) analysis showed greater TCR diversity in DIO+Sulindac T cells compared to DIO. Taken together, these preclinical findings indicate that obesity-driven mammary tumor progression and metastatic burden are associated with an immunosuppressive tumor microenvironment that is largely reversed by Sulindac treatment. This suggests treatment with NSAIDs like Sulindac should be further assessed as a possible strategy for improving outcomes in obese breast cancer patients.

#2816

SB1703 has therapeutic effect on syngeneic mouse model as an immune modulator in TME.

Hyo Young Kim,1 Hee-Jung Kim,1 Jae Hoon Jung,1 So Young Jo,1 Ju Hee Kim,1 Wansang Cho,2 Mingi Kim,2 Junhyeong Yim,2 Seung Bum Park2. 1 _Sparkbiopharma, Seoul, Republic of Korea;_ 2 _Seoul National University, Seoul, Republic of Korea_.

Although stage I melanoma is highly curable, the prognosis of late stage melanoma is poor and treatment options are limited to immune checkpoint modulators (ICMs). In this study, we synthesized a novel small molecule, SB1703, and identified high-mobility group box (HMGB) proteins as the direct binding target proteins. SB1703 inhibited melanoma growth in B16F10 syngeneic mouse model. To elucidate the tumor growth inhibitory mechanism, we determined the changes in myeloid-derived suppressor cell (MDSC) number during drug treatment. MDSCs expand in disease states such as cancer and have immune suppressive functions. Serum HMGB1 and interleukin (IL)-6 levels were increased in vehicle administered mice and decreased in SB1703 administered mice. We examined the possibility of combinatorial treatment with SB1703 and ICMs. SB1703 showed synergistic effect with ICMs. In conclusion, SB1703 exhibited therapeutic effects against melanoma in both single and combination treatments with ICMs, which might be due to modulation of myeloid cells such as MDSCs

#2817

Epithelial memory of resolved inflammation cooperates with activated oncogene to limit tissue damage and promote pancreatic cancer.

Andrea Viale,1 Edoardo Del Poggetto,1 I-Lin Ho,1 Chiara Balestrieri,2 Denise Corti,1 Wantong Yao,1 Giuseppe Diaferia,3 Chieh-Yuan Li,1 Sara Loponte,1 Federica Carbone,1 Shan Jiang,1 Hong Jiang,1 Angela Deem,1 Haoqiang Ying,1 Genovese Giannicola,1 Giulio Draetta,1 Alessandro Carugo,1 Gioacchino Natoli4. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _Humanitas University, Milano, Italy;_ 3 _IEO_European Institute of Oncology, Milano, Italy;_ 4 _Humanitas Clinical and Research Institute, Milano, Italy_.

The association between tumors and inflammation is a long-established clinical observation. Although many studies demonstrated that the inflammatory microenvironment can promote tumor growth through the activation of survival and proliferation programs in cancer cells, the reason why inflammation, an evolutionarily conserved response to damage aimed at reestablishing tissue integrity upon injury, might be integral to tumorigenesis still remains unknown.

Pancreatic ductal adenocarcinoma (PDAC), a tumor characterized by poor prognosis, represents a distinctive example of cooperation between inflammation and activated oncogenes. Frequently developed in a context of chronic pancreatitis, PDAC is always associated with an inflammatory microenvironment. As supported by a substantial body of evidence across a multitude of experimental models, when occurring in the context of pancreatitis, mutations of KRAS, the most frequent driver oncogene of pancreatic cancer, lead to accelerated tumor development inducing the appearance of neoplastic precursor lesions, such as acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN), which can evolve into invasive tumors. Interestingly, preneoplastic pancreatic alterations, specifically ADM, have been previously identified in acute and chronic pancreatitis in the absence of oncogene activation.

To better understand the relationship between inflammatory processes and pancreatic tumorigenesis, we investigated the long-term effects of transient inflammatory events in response to acute pancreatic damage, and how resolved inflammation cooperates with activated oncogenes to drive tumor progression in normal epithelial cells. Using a well-characterized mouse model of PDAC in which oncogenic KRAS-G12D expression can be induced in the pancreas, we discovered that even long after their complete resolution, transient inflammatory events prime epithelial cells to cooperate with oncogenic KRAS. Indeed, after recovery from a single inflammation, epithelial cells activate an enduring adaptive response associated with sustained transcriptional reprogramming. Such epithelial memory facilitates the prompt and massive activation of ADM upon subsequent inflammatory events, likely representing a physiological mechanism for limiting tissue damage via rapid decrease of zymogen production.

We propose that since activating mutations of KRAS maintain an irreversible ADM and thus limit cellular and tissue damage, they may be beneficial and under strong positive selection in the context of recurrent pancreatitis.

#2818

Neutrophil extracellular traps regulate mitochondrial quality control in cancer cells to promote tumor growth.

Samer Tohme, Hamza Yazdani, Richard L. Simmons, Allan Tsung, David Bartlett. _University of Pittsburgh, Pittsburgh, PA_.

Introduction: Evidence is accumulating that neutrophil extracellular traps (NETs) are present within tumors and confer a worse outcome. The exact mechanisms of their role in tumor progression have been far from answered.

Methods: Preoperative serum and tumor tissue were obtained from colorectal cancer liver metastases (CCLM) patients. In vivo, subcutaneous and liver metastasis tumor models were done in WT and nu/nu mice. Hepatocellular and colon murine and human cancer cell lines were used. DNase injection or PAD4KO mice were used to suppress NETs. In vitro, human and mice neutrophils were isolated and stimulated with PMA to form NETs to treat multiple cancer cell lines and examine NETs effect on their bioenergetics.

Results:Tumors from CCLM patients showed increased NETs. Preoperative serum MPO-DNA, a NET marker, was found to be significantly elevated in CCLM patients compared to healthy controls. Patients with high MPO-DNA had significantly shorter overall and disease-free survival than patients with low MPO-DNA. The median disease-free survival was 5.8 times shorter (5.5vs.31 months, p<0.001). In vivo, we compared the growth rates of tumors in the subcutaneous and liver metastases models in C57BL/6 WT and PAD4KO mice. Tumors grew slower and were significantly smaller in PAD4 KO mice. Similarly, human cancer cell lines HCT116 and Huh7, grew slower in DNAse-treated nu/nu mice. PAD4KO or DNAse treated tumors showed decreased proliferation and increased in apoptosis and oxidative stress. These tumors had decreased mitochondrial density and mitochondrial DNA and lesser degree of ATP production. There was also a significant decrease in mitochondrial biogenesis proteins PGC-1α, TFAM and NRF-1 in PAD4KO tumors. In vitro, cancer cells treated with NETs showed a significant upregulation in the mitochondrial biogenesis associated genes, increased mitochondrial density and increased ATP production compared to control. NETs significantly enhanced the percentage of cancer cells with reduced mitochondrial membrane potential and increased the oxygen consumption rate. Furthermore, NETs increased cancer cell's expression of fission and fusion associated proteins DRP-1 and MFN-2 and expression of mitophagy-linked proteins, PINK1 and Parkin. All of which were decreased in PAD4KO tumors. Mechanistically, we demonstrated that neutrophil elastase (NE) released from NETs activates TLR-4 on cancer cells that in turn phosphorylates p38 and activates PGC-1α to upregulate mitochondrial biogenesis and tumor growth.

Conclusion:NETs actively recruited to the tumor microenvironment can alter the metabolic programming of cancer cells by upregulating biogenesis and maintain mitochondrial fission, fusion and mitophagy which result in increased tumor growth. NETs represent a promising therapeutic target to halt cancer progression.

#2819

Upstream activation of anti-tumor immunity via dendritic cell subsets.

Eynav Klechevsky. _Washington University School of Medicine, Saint Louis, MO_.

Dendritic cells (DCs) are key antigen-presenting cells that control both immunity and tolerance due to their ability to orchestrate either effector or regulatory T cell responses. DCs are localized anatomically in most tissues and at surface barriers, where they function as sentinels for pathogen recognition. In the skin, they are the main target of vaccines and therefore understanding the features of skin DCs is critical for developing novel vaccines. Three main subsets of DCs exist in human skin: Langerhans Cells (LCs) in the epidermis and CD1a+DCs or CD14+DCs, which reside in the dermis. Our group has recently demonstrated that LCs and dermal CD1a+DCs can be subdivided into two distinct subtypes based on their CD5 expression. The CD5+DCs are highly efficient at priming type I helper T cells and multi-functional cytotoxic CD8+T cells compared to their CD5-counterparts. Interestingly, CD5\+ DCs were found to be significantly elevated in inflamed psoriatic skin plaques, while their numbers were dramatically reduced in malignant tissues. Consistent with this notion, we demonstrate that CD5 functions to trigger an inflammatory pathway of DC maturation and inflammatory T cell activation. CD5-deficient DCs produce less inflammatory cytokines upon activation, and activates T cells less efficiently than WT DCs in vitro and in vivo. Overall our studies highlight a role for CD5 on DCs in immune activation and the potential exploration of this pathway in cancer therapy.

#2820

Macrophages recruited to and function to promote activation of cancer stem cells in response to chemotherapy.

Xi C. He, Linheng Li. _Stowers Inst. for Medical Research, Kansas City, MO_.

Failure of chemoradiotherapy for treating colorectal cancer (CRC) is often caused by the relapse of residual tumor cells that enrich cancer stem cells (CSCs). However, how CSCs are regulated by tumor microenvironment (TME), particularly myeloid derived cells (MDCs), remains unclear. Here, we used the APCMin/+ adenoma model to study the dynamic interaction between MDCs and CSCs in vivo following chemoradiotherapy. Using 3D scanning electronic microscopy, we observed that while proliferating tumor cells were dying following chemoradiotherapy, MDCs or its derivative tumor associated macrophages (TAMs) were recruited to the site where CSCs paired with necroptotic cells. TAMs functioned to promote activation of slow-cycling CSCs. Dissected with single cell RNA-seq, we observed that while proliferating tumor cells were declining following chemotherapy, an increase in MDCs was accompanied with a reduction of immune cells as well as an activation and short-term expansion of initially quiescent CSCs. Depletion of TAMs using a drugs or genetic means resulting in reduced CSCs and attenuated tumorigenesis. Mechanistically, we uncovered that TAM with secreted PGE2 activated Akt and Wnt signaling to promote activation of CSCs. Hence, targeting TAMs will benefit clinical treatment of CRC by reducing drug-resistant CSCs.

#2821

Gut commensal bacteria modulate functions of tumor-associated neutrophils in human colorectal cancer.

Valeria Governa,1 Eleonora Cremonesi,1 Diego Calabrese,1 Valentina Mele,1 Emanuele Trella,1 Raoul A. Droeser,2 Martin Bolli,3 Serenella Eppenberger-Castori,1 Salvatore Piscuoglio,4 Charlotte K. y. Ng,4 Jesus Francisco Glaus Garzon,5 Lubor Borsig,5 Klaus-Peter Janssen,6 Luigi M. Terracciano,1 Giulio Cesare Spagnoli,7 Dimitri Christoforidis,8 Pietro Edoardo Majno-Hurst,8 Giandomenica Iezzi9. 1 _University of Basel, Switzerland;_ 2 _University Hospital Basel, University of Basel, Switzerland;_ 3 _St. Claraspital Basel, Switzerland;_ 4 _Basel University Hospital and University of Basel, Switzerland;_ 5 _University of Zürich, Switzerland;_ 6 _Technical University of Münich, Germany;_ 7 _National Research Council, Italy;_ 8 _Ente Ospedaliero Cantonale Lugano and Università Svizzera Italiana, Switzerland;_ 9 _University of Basel and Ente Ospedaliero Cantonale Lugano, Switzerland_.

Tumor infiltration by immune cells critically impacts on clinical outcome of human colorectal cancer (CRC). While high densities of CD8+ T cells and T-helper type 1 cells are associated with prolonged patient survival, the role of tumor-associated neutrophils (TANs) is debated.

CRC arise in an environment heavily populated by microrganisms. Upon CRC oncogenesis, gut commensal bacteria translocate into the submucosa where they directly interact with residing immune cells. Neutrophils represent a front-line arm of the immune system in the response to bacteria. However, little is known about their interaction with commensal bacteria within CRC tissues. We investigated the interplay between neutrophils and commensal bacterial species present within the CRC microenvironment.

Using an orthotopic CRC xenograft model, we found that commensal bacteria stimulate CRC cells to produce neutrophil recruiting chemokines. We then compared in vitro chemokine induction capacity of Fusobacterium nucleatum (Fn) and Bacteroides fragilis (Bf), two most abundant bacterial species of CRC microenvironment. Fn induced significantly higher levels of IL-8 by CRC cells than Bf, thus more effectively promoting neutrophil recruitment. Accordingly, in human CRC samples, abundance of Fn, but not Bf, significantly correlated with high infiltration by CD66b+ cells. Functional studies indicate that neutrophils cultured in the presence of Fn, but not Bf, lose their ability to enhance proliferation and cytokine release by CD8+ T cells undergoing antigenic stimulation. Moreover, neutrophils exposed to Fn, but not Bf, stimulate release of IL-6 by tumor-associated stromal cells, leading to enhanced tumor cell proliferation.

Our data cumulatively suggest that distinct bacterial components of the human gut flora might differentially modulate functions of CRC infiltrating neutrophils, thus ultimately influencing their prognostic significance.

#2822

Low intestinal microbial diversity is associated with severe immune-related adverse events and lack of response to neoadjuvant combination antiPD1, anti-CTLA4 immunotherapy.

Marcel Batten,1 Erin R. Shanahan,2 Ines P. Silva,1 Chandra Adhikari,3 Jordan Conway,1 Annie Tasker,1 Alexander M. Menzies,1 James S. Wilmott,1 Robyn P. Saw,1 Andrew J. Spillane,1 Kerwin F. Shannon,1 Christian U. Blank,4 Andrew J. Holmes,2 Richard A. Scolyer,1 Georgina V. Long1. 1 _Melanoma Institute Australia, Sydney, Australia;_ 2 _The University of Sydney, Sydney, Australia;_ 3 _Royal Prince Alfred Hospital, Sydney, Australia;_ 4 _Netherlands Cancer Institute, Amsterdam, Netherlands_.

Background: Immunotherapies targeting PD-1/PD-L1 and CTLA4 have revolutionized the treatment of advanced melanoma. Combining the two therapeutics increases the response rates compared to either treatment alone. However, this increased efficacy is accompanied by a higher incidence of severe immune-related adverse events (irAEs). Metrics of the intestinal microbiome are associated with cancer patients' responses to immunotherapy but the value of microbiome metrics as predictors for irAEs, are unknown. In an effort to reduce irAEs during combination neoadjuvant therapy, the OpACIN-neo trial (Rozeman et al. ESMO 2018) was initiated wherein Stage III melanoma patients were treated with 2 doses each of ipilimumab and nivolumab in the neoadjuvant setting, according to three dosing regimen. Involved lymph nodes were resected after 6 weeks.

Aims: (1) To determine whether intestinal microbial components are associated with response to antiPD1/anti-CTLA4 immunotherapy in the neoadjuvant setting or with the development of severe irAEs. (2) To determine the effects of antiPD1/anti-CTLA4 immunotherapy on the microbiome over the 6 week course of treatment.

Methods: Of Melanoma Institute Australia patients enrolled in the OpACIN-neo trial (n=38), 68% of patients experienced complete, or near complete pathological responses and 61% experienced at least one irAE of grade 3 or more. Faecal microbiomes at baseline, and at resection (6 weeks immunotherapy), were analyzed using 16S ribosomal gene and metagenomic sequencing and the results compared with patient response and development of G3-G5 irAEs. Results: In baseline samples, Inverse Simpson's diversity index indicated significantly lower overall microbial diversity in non-responders (p=.014), as well as those who went on to experience severe irAEs (p=.002). Importantly, the group of patients who were both non-responders and experienced severe irAEs had the lowest microbial diversity of all patients (p=.0033). Specific taxa associated with irAEs are distinct from those described for response. The 6 week course of immunotherapy led to a slight increase in microbial diversity but few specific taxa were observed to be significantly altered between the timepoints.

Conclusions: The findings suggest that not only are patients with extremely low microbial diversity predisposed to develop severe irAEs but they are also unlikely to respond to combination immunotherapy. Thus, microbial diversity may delineate patients who are likely to have poor outcomes and would benefit from microbial modulation.

#2823

Sample storage conditions alter microbiome profiles - its potential relevance for cancer patients undergoing fecal microbial transplants.

Samir V. Jenkins,1 Kieng B. Vang,2 Allen Gies,1 Robert J. Griffin,1 Se-Ran Jun,1 Intawat Nookaew,1 Ruud Petrus Dings1. 1 _UMAS Winthrop P. Rockefeller Cancer Inst., Little Rock, AR;_ 2 _University of Arkansas at Little Rock, Little Rock, AR_.

Background and Objective: Fecal microbiota transplantation helps restore beneficial bacteria in cancer patients after intense antibiotics administration. Here we investigated the influence of different stabilization and storage strategies on the quality and composition of the fecal microbial community.

Methods: Same-day isolated murine DNA was compared to samples stored for one month in air at ambient temperature, with or without preservative buffers (i.e. EDTA and lysis buffer), different temperatures (i.e. 4°C, -20°C, and -80°C), and hypoxic conditions.

Results: Only storage in lysis buffer significantly reduced DNA content, yet without integrity loss. Storage in EDTA affected alpha diversity the most, which was also reflected in cluster separation. Distinct changes were also seen in the phyla and bacterial species abundance per storage strategy. Metabolic function analysis showed 22 pathways not significantly affected by storage conditions, whereas the tyrosine metabolism pathway was significantly changed in all strategies except by EDTA.

Conclusions: Overall, each long-term storage strategy introduced a unique post-collection bias, which is important to take into account when interpreting data. Funded by COBRE P20GM103625.

#2824

Modulation of oral cancer cells survival and invasiveness by fusobacteria.

Amani Harrandah, Sasanka S. Chukkapalli, William JR Dunn, Ann Progulske-Fox, Edward K. Chan. _University of Florida, Gainesville, FL_.

Oral cancer represents a significant health burden worldwide while periodontitis is a common chronic infection of the periodontium. Many studies have suggested the association between periodontal disease and oral cancer. Studies have shown that biofilms on oral cancer lesions are rich in anaerobic bacteria, known to be associated with periodontal disease. This establishes that periodontal bacteria are present in the oral cancer microenvironment and likely directly interact with cancer cells. Therefore, it is vital to understand the interactions between the oral bacteria and cancer cells in order to improve treatment outcomes. The aim of this study was to investigate the effect of periodontal bacteria on oral cancer cells. Three oral cancer cell lines (OQ01, BHY and HN) were polyinfected with 4 bacteria associated with chronic periodontitis (Tanneralla forsynthia, Treponema denticola, Porphyromonas gingivalis and Fusobacterium nucleatum). Likely because BHY and HN already have a high baseline level of IL-8, OQ01 alone showed significantly enhanced IL-8 secretion (P< 0.05) but enhanced TFG-β secretion was detected in all cell lines tested. Polybacterial infection of oral cancer cells also upregulated mRNA for MMP1 (40 fold) and MMP9 (more than 60 fold), which are known to enhance cancer cell invasiveness. In addition, the expression of ZEB1 (4 fold higher), an oncogene known to induce epithelial mesenchymal transition in cancer cells, and MYC, JAK1 and STAT3, oncogenes involved in cell survival, were all significantly enhanced in polybacterial infected cancer cells. Further analysis using OQ01 cells in a single bacterial infection showed that F. nucleatum alone had the same or greater effect (4 fold higher IL-8 secretion) as the polybacterial infection with all 4 bacteria. All three strains of F. nucleatum tested enhanced the invasiveness of the oral cancer cells in vitro. Interestingly, Fusobacterial supernatant alone was enough to induce the same invasive phenotype as live bacteria in OQ01. These results suggest that F. nucleatum is the main periodontal pathogen responsible for inducing an invasive phenotype in these oral cancer cells. For in vivo studies, we used a mouse model of infection-associated oral tumorigenesis, which included the induction of chronic periodontitis by P. gingivalis and F. nucleatum and the administration of an oral carcinogen 4-nitroquinoline-1-oxide (4NQO). The results demonstrated that infection with Fusobacteria and P. gingivalis promoted oral cancer progression and enhanced invasion compared to controls. Our data also showed that Fusobacteria enhanced both survival and invasiveness of oral cancer cells. Collectively, this is the first study revealing the interaction between oral cancer cells and multiple periodontal bacteria. The modulation of the tumor microenvironment by oral bacteria has the potential to identify target genes for cancer treatment and improve patients' prognosis.

#2825

**Two classic TCM formulas suppressed polyp counts in concurrence with improved gut microenvironment, and remodeled gut microbial composition of the Apc** Min+ **mice.**

Wenrui Xia, Imran Khan, Xiaoang Li, Guoxin Huang, Wai Kit Leong, Wei lin Liao, W.L. Wendy Hsiao. _State Key Laboratory of Quality Research in Chinese Medicines, Macao, Macao_.

Background: In this study we evaluated the anti-cancer effects and gut modulating properties of the two Chinese medicinal formulas, known as HuaiHuaJinYinJiu (SL) and Floris Sophorae Powder (FSP). SL is comprised of Sophorae Flos (HuaiHua) and Lonicerae Japonicae Flos (JinYinHua) and has been traditionally prescribed for carbuncle treatment. The FSP is constituted of Sophorae Flos and Gardenia Jasminoides Ellis (ZhiZi) and has been used for the cure of hemafecia and abdominal pain.

Methodology: A total of 27 ApcMin/+ mice were equally divided into three groups and each group were continuously gavage for 28 days with daily dosage of SL (1g/kg), FSP (1g/kg), and water respectively. At the end of the treatments, fecal samples were collected; mice were terminated and sampled for tissues for later investigations.

Results: We observed that SL and FSP substantially reduced polyps by 32% and 37% in ApcMin/+ mice, respectively. Additionally, both formulas greatly strengthened the intestinal barrier of the ApcMin/+ mice by enhancing the population of Paneth and goblet cells, up-regulating E-cadherin, and down-regulating N-cadherin expressions. Meanwhile, SL and FSP remodeled the gut microbial composition by facilitating the growth of bacteria belonged to phyla Bacteroidetes and Verrucomicrobia, whereas suppressed the abundance of phyla Fusobacteria and Deferribacteres which are generally known for their pathogenesis. The growth of sulfate-reducing bacteria and bacteria in genus Helicobacter were inhibited in SL and FSP treated mice. We also detected the downregulation of the CagA - which is a cytotoxin-associated gene expressed highly in Helicobacter pylori and has been implicated with CRC development. SL and FSP, on the other hand, facilitated the growth of short-chain fatty (SCFA) producing bacteria that was positively correlated with upregulated expressions of the SCFAs-sensing receptors GPR41, GPR43, and GPR109a. We further found the downregulation of p-AKT, p-ERK, p-STAT3, and beta-catenin in the formula-treated mice. Inflammatory milieu of the SL and FSP treated mice were greatly improved by the polarization of M1 to M2 macrophages and modified with upregulation of anti-inflammatory cytokines (IL-4, IL-10, IL-12) and suppression of pro-inflammatory cytokines (iNOS, IL-1β, IL-17, IL-18, IL-23).

Conclusion: Collectively, this study presents strong evidences that the modulation of gut microbiota and the associated metabolites as well as the reversal of the inflammatory gut epithelial barrier might account for the anti-cancer effects of SL and FSP TCM formulas in the ApcMin/+ mice. [This study is supported by Macau FDCT 103/2016/A3 to WLW Hsiao]

#2826

Understanding associations among local microbiome, immune response, and efficacy of immunotherapy in esophageal cancer.

Chao Zhang,1 Prashant V. Thakkar,1 Prateek Sharma,2 Sreekar Vennelaganti,2 Doron Betel,1 Manish A. Shah1. 1 _Weill Cornell Medical College, New York, NY;_ 2 _Veterans Affairs Medical Center and University of Kansas School of Medicine, Kansas city, KS_.

Although Barrett's esophagus is the primary precursor of esophageal adenocarcinoma, the annual incidence of adenocarcinoma is 0.12% in a Barrett's esophagus population. We hypothesized that the esophageal microbiome may be associated with mucosal immunity changes that may determine subsequent carcinogenesis. To better understand the association among the local microbiota composition, immune response and progression of esophageal cancer, we performed whole genome sequencing (WGS) and bulk RNASeq in parallel, from endoscopic biopsies (n=23) collected from patients with normal squamous cell (n=9), non-dysplastic Barrett's esophagus (n=8) and esophageal adenocarcinoma (n = 6). Using a novel customized computational pipeline to identify and characterize bacteria from low microbial content endoscopic samples (Zhang et al, Genome Biology, 2015). WGS analysis revealed a higher microbial diversity in normal squamous cell carcinomas compared to the non-dysplastic Barrett's esophagus with high abundance of Veilonella Parvula, Prevotella melaninogenica and Streptococcus parasanguinis. Unsupervised clustering analysis of the entire data set revealed high abundance of Fusobacterium nucleatum in adenocarcinoma samples, which has been associated with a shorter survival in esophageal cancer. To characterize the immune infiltration in the mucosal biopsy samples, we defined a 784-gene immune panel. Unsupervised clustering analysis of gene expression revealed a much higher immune infiltration in most adenocarcinoma and some non-dysplastic Barrett's compared with normal esophageal squamous tissue samples. Especially CD4+ Th1 and Th2 helper cell as well as CD8+ T cell associated immune response are highly enriched in some adenocarcinoma and non-dysplastic Barrett's esophageal samples. To further investigate the effect of immune tumor microenvironment on efficacy of immunotherapy, we performed single cell sequencing on matching normal and tumor tissues collected at baseline and post-treatment from 6 patients subjected to anti-PD1 therapy. Using this approach, we can identify both innate and adaptive immune cells in both pre and post treatment biopsies. We specifically observe upregulation of MHCII associated cells in post-treatment biopsies. Together, our data suggest that esophageal cancers display distinct microbial patterns associated with chronic inflammation and a tumor-promoting pro-inflammatory microenvironment.

#2827

Identification of chemotherapeutic resistant microbiota profile in colorectal cancer.

Shuyun Rao, Charles Hadley King, Raja Mazumder, Shoujun Gu, Richard Amdur, Bibhuti Mishra, Lopa Mishra. _George Washington Univ., Washington, DC_.

Background: Emerging data shows a rise in CRC incidence in men and women under 55 years of age, with a potential new risk factors including alterations in the microbiome. While recent advances have significantly reduced CRC morbidity and mortality, such as screening and new therapeutic strategies, how the microbiome modulates CRC chemotherapy response remains understudied. This alarming trend presents an urgent need for further understanding of the molecular mechanisms and "signatures" of the gut microbiome in CRC patients. Key driving pathways in CRC include WNT, RAS, RAF, APC and TGF-β signaling. Modulating the gut immune response includes multiple mechanisms, including sensors through carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) that serve as receptors to microbes, and crosstalk with signaling pathways such as TGF-β signaling pathway. Interestingly, disruption of TGF-β leads to increased sensitivity to cisplatin and other DNA cross-linking agents.

Hypothesis and Aims: Our hypothesis is that the gut microbiome plays a key role in modulating the TGF-β pathway by CEACAMs binding to specific microbial species, thereby impeding the TGF-β pathway and altering chemotherapy response. Our aims are to explore TCGA genomics, and functional interactions between gut microbiota, CEACAMs, and TGF-β signaling in processing chemotherapy response.

Methods and Results: We first analyzed microbiome signatures in normal and CRC patients, and identified a signature pattern. TCGA analyses in 9,125 human cancers reveal alterations of TGF-β with CEACAM members in over 40% of CRC and correlate with a cancer stem cell signature. Mechanistic studies reveal that CEACAMs inhibit TGF-β tumor suppressor signaling by blocking TGFBR1, and promoting invasive CRC. Likewise, our TGF-β signaling-deficient mouse models spontaneously develop CRC, as well as metastases, likely modulated in part by the microbiome. Furthermore, key defects in the TGF-β pathway alter sensitivity to 5-FU and DNA cross-linking agents, such as cisplatin, through disruption of DNA repair pathways.

Conclusion: The TGF-β pathway is important in maintaining colon epithelial cell homeostasis potentially through interactions with CEACAMs and gut microbiota. When the pathway is disrupted, epithelial cells are more susceptible to transformation and invasion, potentially identifying specific populations that are more sensitive to chemotherapy such as cisplatin and 5FU.

#2828

Bacterial diversity correlates with tumor stage and survival in stomach and lung TCGA cancer cohorts.

Rebecca M. Rodriguez,1 Vedbar S. Khadka,1 Mark Menor,1 Youping Deng,1 Brenda Y. Hernandez2. 1 _John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, HI;_ 2 _University of Hawaii at Manoa, Honolulu, HI_.

Microbiological infections account for up to 20% of the total global cancer burden. Infection-associated cancers are commonly attributed to viral, and to a lesser extent, parasitic and bacterial etiologies. There is growing evidence that microbial variation can influence cancer development, progression, response to therapy, and outcome. We wanted to examine the microbial composition of paired tumor and adjacent normal tissue across various cancer types in order to provide an improved understanding of microbial diversity and abundance patterns of the tumor microenvironment and their influence on clinical presentation and survival. We hypothesized that adjacent normal will be associated with greater species diversity. Methodology: 2234 TCGA tumor & adjacent normal sequencing files (phs000178.v.9.p8; Project ID 14778) representing 1532 cases from stomach adenocarcinoma (TCGA -STAD) and bronchus & lung adenocarcinoma and squamous cell carcinoma (TCGA-LUAD, LUSC ) primary tumor sites were processed through a bioinformatics pipeline designed to generate microbial profiles from raw DNA sequences using PathoScope 2.0 (http://sourceforge.net/projects/pathoscope/) and visualized with KRONA™ plots (Ondov BMC Bioinformatics 2011,12:385). Diversity metrics were calculated to compare taxa between tumor & its paired adjacent normal using vegan R-package (version 2.5.2). Differential abundance was examined using Wilcoxon Signed-Rank Sum test. Bacterial taxa with false discovery rate (FDR) adjusted P-value< 0.05 were considered significantly different at both genus and species level. Results: 509 paired tumor and adjacent normal samples (1:1) were selected for analyses. In stomach we identified 706 distinct bacteria taxa in STAD tumor and adjacent normal while 1004 distinct bacteria taxa were identified in lung cancers tumor and adjacent normal. Average reads ratio per patient was 25% higher in tumor compared to normal adjacent for STAD and 30% lower in tumor compared to normal in lung cancers. Distinct bacterial patterns were observed in tumor versus normal adjacent. In STAD, Helicobacter pylori was significantly less abundant in tumor compared to adjacent normal (log2fc = -4.8, p=<0.0001 FDR= 0.01). None of the taxa detected in lung cancers showed significant differences in abundance in tumor compared to adjacent normal. In lung cancers, relative abundance of certain taxa was associated with tumor stage and years survived (p=<0.001). Alpha diversity varied widely by tumor stage and histology after controlling for age at diagnosis and smoke (p=0.0001). Conclusion: Diversity was correlated to tumor stage and survival. Significant differential abundance was observed for certain taxa for tumor when compared to adjacent normal. Differences in clinical presentation and survival among cancer patients may be explained by microbial abundance patterns.

#2829

Pancreatic tumor microbiome and associated immune responses determine clinical outcomes.

Erick M. Riquelme. _UT MD Anderson Cancer Ctr., Houston, TX_.

Most patients diagnosed with pancreatic adenocarcinoma (PDAC) survive less than 5 years, but a very small subset of patients survive longer. The factors that determine the long-term survivorship remain elusive. Recently, studies have shown that bacteria can be found in PDAC which may influence therapy responses. In this study, we aimed to determine if the tumor microbiome, and its associated immune responses, can guide long-term survivorship in resected PDAC patients. Using 16S rRNA gene sequencing, we analyzed the tumor microbiome composition and immunoprofile in PDAC patients who survived less than 5 years (short term survivors, STS) versus those who survived more than 5 years (long term survivors, LTS) in two independent cohorts of patients from two institutions (MD Anderson Cancer Center and Johns Hopkins University). We found higher alpha-diversity in the tumor microbiome from LTS compared to STS PDAC patients. Additionally, we found greater densities of immune cells in the LTS compared to STS, with significant correlation with alpha-diversity. Taken together, our study demonstrates that the PDAC microbiome composition may influence the host immune response and the natural history of the disease.

#2830

Breast cancer and the human oral and gut microbiomes.

Michael J. Campbell,1 Emma McCune,1 Breanna Johnson,1 Tess O'Meara,1 Diane Heditsian,2 Susie Brain,1 Laura Esserman1. 1 _UCSF, San Francisco, CA;_ 2 _deClarity, CA_.

The human body harbors ten times more bacterial cells than human cells - a stunning figure that suggests a likely dynamic between our bodies and the bacteria we carry, both in health and disease. In this study, we characterized and compared the gut and oral microbiota from women with invasive breast cancer, women with ductal carcinoma in situ (DCIS), and healthy women. Samples were collected prior to any systemic therapy to avoid therapy-associated effects on the microbiomes studied. Kits for collecting oral and stool swab samples were distributed to patients for self-collection. DNA was isolated from these samples and bacterial 16S rRNA was PCR amplified and sequenced. Based on the sequencing results, bacterial taxa present in the samples were enumerated. The gut microbiota of women with breast cancer demonstrated significantly lower alpha-diversity compared to the gut microbiomes of healthy women. In contrast, there was no difference in gut microbiota alpha-diversity of women with DCIS compared to healthy women. There were no differences in alpha-diversity of the oral microbiota between any of the cohorts. Discriminant analysis of principal components (DAPC) at the genus level of both the gut and oral microbiota demonstrated distinct clustering of the DCIS, breast cancer, and healthy cohorts. LEfSe analyses were performed to detect differences in relative abundance of bacterial taxa across the gut and oral samples. The genus Bacteroides was significantly enriched in breast cancer compared to healthy samples, as were the related taxa Bacteroidetes (phylum), Bacteroidia (class), Bacteroidales (order), and Bacteroidaceae (family). The genera Fusicatenibacter and Butyrivibrio, both from the phylum Firmicutes, class Clostridia, order Clostridiales, family Lachnospiraceae, were more abundant in the healthy cohort. Only 2 genera, Fusicatenibacter and Clostridium were differentially abundant between the DCIS and healthy gut samples, both being enriched in the healthy samples. Numerous taxa in the oral microbiota were found to be differentially abundant between cohorts. In particular, 7 genera (Bacteroides, Blautia, Faecalibacterium, Roseburia, Pseudobutyrivibrio, Anaerostipes, and Subdoligranulum) were all significantly enriched in the healthy oral samples compared to the DCIS and breast cancer oral samples. To determine whether the taxonomic differences we observed between the cohorts' gut and oral microbiota corresponded to functional differences, we performed a predictive functional analysis using Piphillin which identified 35 KEGG pathways differentially present in the gut microbiota and 8 pathways differentially present in the oral samples. Understanding how gut and oral microbiomes relate to breast cancer may open up new opportunities for the development of novel markers for early detection (or markers of susceptibility) as well as new strategies for prevention and/or treatment.

#2831

Detection of methane-producing archaea in the cervix of Hispanics using shotgun metagenomics.

Filipa Godoy-Vitorino, Frances Vazquez-Sanchez, Anelisse Dominicci-Maura, Josefina Romaguera. _University of Puerto Rico, School of Medicine, San Juan, PR_.

The human cervix is colonized by microbial communities that have traditionally been regarded as a protective barrier against infections. Dysbioses disrupting the balance of the normal microbiota, leads to the appearance of anaerobic taxa that have been related to sexual transmitted infections and cancer. The complexity of the diversity and composition of cervicovaginal communities has been recently highlighted by 16S rDNA surveys, although these have limitations to either select mostly bacteria and the lack of strong lower taxonomic resolution such as being species-specific. Metagenomic shotgun sequencing overcomes these limitations, and gives us access to the extent of diversity beyond bacterial communities. This is the first project to characterize the bacterial and archaeal communities of the cervix of Puerto Ricans using shotgun metagenomics. Women coming for gynecology and colposcopy evaluation at the UPR and San Juan City clinics (San Juan Metropolitan area), and who did not meet several excluding criteria including having taken antibiotics for the last month, were recruited for the study. Samples were acquired by inserting a speculum for access and visualization of the cervix and a sterile swab was rotated along the lumen with a circular motion and frozen immediately for posterior genomic DNA extractions and sequencing. Library construction and shotgun sequencing of 10 cervical patient samples was done with the Illumina HiSeq platform An average of 1,690,000 good quality reads per sample were analyzed. Approximately 90% of the contigs had more than 1,000bp and 10% had sizes ranging from 1,000-5,000bp. Microbial communities were dominated by Lactobacillus, including L. iners, Gardnerella vaginallis, Nocardia and Atapobium vaginae. Archaeal diversity was dominated by Methanosarcina (namely, M. barkeri) with other less dominant taxa such as Methanolobus, Methanococcus, Natrococcus and Methanobrevibacter. The dominance of Lactobacillus was expected, and as Archaea are environmental organisms that have been associated with the mucosa in mammals, including protecting the human gut, they must have an important role in the cervix. As methanogenic archaea are known to remove fermentation end products, such as methanol and ethanol, as end-products of other fermentative bacteria, methanogenesis maybe important in preventing the accumulation of gases and other reaction end products that could damage the cervical mucosa.

#2832

Comparison of bacteriome in plaque dental, saliva and tumor tissue in oral squamus carcinoma.

Dabeiba A. García Robayo, Herlinto Alveiro Tupaz Erira, Fredy Omar Gamboa Jaimes. _Pontificia Universidad Javeriana, Bogotá, Colombia_.

Introduction: Bacterial microbiota found in the oral cavity, facilitates or promotes its carcinogenesis to Oral Squamous Cell Carcinoma (OSCC), by persistent inflammatory and production of acetaldehyde.

Objective: To determine and compare the microbiome and functional potential of dental plaque, saliva and tumor tissue of patients with OSCC with that found in dental plaque and saliva of healthy people.

Methods: After signed the informed consent 10 patients diagnosed with OSCC were selected. Samples were saliva, dental plaque and tumor tissue; sociodemographic data were collected from each patient, which were used to search for their corresponding control as age, gender, alcohol and tobacco consumptions. Subsequently, DNA was extracted from all the samples and 16S amplification and sequencing by ion-torrent. Once the data assembled paired-end reads were clustered into Operational Taxonomic Units (OTUs) algorithm with the open reference OTU picking pipeline in QIIME 1.9.1. The taxonomic assignment was Greengenes Database version 13.8. The α diversity and β diversity were calculated. To determine the metabolic potential of the oral microbiota, we used the PICRUSt significance was less 0.05.

Results: It was observed that the greatest bacterial diversity in dental plaque samples compared to the samples of saliva and tumor tissue. Multiple comparisons were made between the microbiome present between the different types of samples and between the two groups (cancer vs control). A total of 25 bacterial genera were found in exclusively in patients with OSCC and in more than 30% of patients who were Geobacillus, Anaerotruncus, Comamonas, Sphingomonas, Sphaerochaeta, Dorea, Delftia, Pseudoxanthomonas, Luteibacter, Pseudomonas, Peptoniphilus, Novosphingobium, Bradyrhizobium, Faecalibacterium, Clostridium, Dechloromonas, Methylobacterium, Proteus, Finegoldia, Enterococcus, Turicibacter, Desulfovibrio, Pyramidobacter, Morganella and Staphylococcus, and 2 those found exclusively in healthy people in more than 30% of controls that were Odoribacter and Helicobacter. Regarding the functional potential, statistically significant differences were observed between patients with OSCC and healthy people, increased ion-coupled transporter signaling pathways in patients with OSCC and the signaling pathways related to increased replication, recombination and repair in the group of healthy people.

Conclusions: Bacterial genera associated with OSCC and associated with health were described and propose them as non-invasive biomarkers. Additionally, signaling pathways increased by the microbiota associated with oral cancer are related to transporters associated with ions, while signaling pathways increased by the microbiota associated with health are related to replication, recombination and repair.

#2833

Alterations in intestinal microbiota of colorectal cancer patients receiving radical surgery combined with adjuvant CapeOx therapy.

Cheng Kong, Renyuan Gao, Huanlong Qin. _Shanghai Tenth People's Hospital Affiliated to Tongji University, Shanghai, China_.

Background: An intricate relationship exists and interactions occur between gut microbiota and colorectal cancer (CRC). Radical surgery combined with adjuvant chemotherapy (AC) serves as the mainstream therapeutic scheme for most CRC patients. However, the impact of surgery or chemotherapy on gut microbiota remains elusive.

Methods: Forty-three CRC patients who received radical surgery and AC were enrolled in the present study. Fecal samples of these patients were collected preoperatively, postoperatively, and after the first to fifth cycles of postoperative chemotherapy. The microbial community of each sample was analyzed using high throughput 16S rRNA amplicon sequencing.

Results: Compared with preoperative samples, fecal samples collected postoperatively exhibited a significant decrease of obligate anaerobes (Bacteroides, Parabacteroides, Faecalibacterium, Prevotella_9 and Bifidobacterium), tumor-related bacteria (Enterococcus and Fusobacterium), and butyric acid-producing bacteria (Bacillus, Bilophila, Barnesiella and Enterococcus). However, a significant increase of some conditional pathogens was observed (Escherichia-Shigella, Enterobacteriaceae_unclassified, Veillonella, Morganella, Streptococcus, and Proteus). In addition, the AC regimen (CapeOx) was found to alter intestinal microbiota dramatically. In particular, several changes were observed after chemotherapy including an increase of pathogenic bacteria (Collinsella, Anaerostipes, Bilophila, Comamonas, Weissella, and Eggerthella), the "rebound effect" of chemotherapy-adapted bacteria (Dorea, Ruminococcaceae_UCG-010, Streptococcus, Prevotella_9, Mogibacterium and Roseburia), the shift of lactate-utilizing microbiota from Veillonella to Butyricimonas and Butyricicoccus, as well as the decrease of probiotics (such as Lactobacillus).

Conclusions: Both radical surgery and CapeOx chemotherapy exert a non-negligible effect on the gut microbiota of CRC patients. Microbiota-based intervention may be beneficial for these patients during postoperative clinical management.

#2834

Bacteroides fragilis: **A potential pathogen orchestrating EMT and stemness in breast epithelial cells via concomitant activation of Notch and βcatenin axes.**

Sheetal Parida, Shaoguang Wu, Nethaji Muniraj, Sumit Siddharth, Arumugam Nagaligam, Christina Hum, Panagiotis Mistriotis, Konstantinos Konstantopoulos, Cynthia L. Sears, Dipali Sharma. _Johns Hopkins University School of Medicine, Baltimore, MD_.

Background: Last decade established significant contributions of microbiome to organ specific cancers. A few recent studies suggested the existence of distinct breast microbiota and a shift in microbial community composition in diseased breast compared to normal breast. However, their functional impact and underlying mechanisms are unknown. Present study was designed to examine the contribution of pro-carcinogenic bacteria in breast cancer initiation, growth and progression.

Results: Utilizing extensive data mining and metagenomic analyses, we discovered presence of toxin producing Bacteriodes fragilis in malignant breast. B. fragilis is known for its potential to initiate and/or promote colon cancer. Its pathogenicity has been attributed to its unique toxin BFT. Mice infected with B. fragilis exhibited significant circulating BFT and distinct morphological alterations in mammary gland. While no changes were observed in cell growth and clonogenicity upon BFT treatment, significant increase in migration and invasion potential and decreased adhesion of MCF10A and MCF7 cells were observed. BFT-treated cells displayed acquisition of fibroblast-like appearance and increased formation of pseudopodia/microtentacles emanating from the cell membrane along with molecular markers of epithelial-to-mesenchymal transition. Decreased expression of epithelial marker, E-cadherin along with elevated levels of mesenchymal markers, N-cadherin and vimentin were observed. BFT also increased expression of EMT-related transcription factors, Snail, Slug and Twist. BFT-treated cells attained stem cell-like phenotype exhibiting an increased ability to form secondary and tertiary mammospheres and elevated expression of pluripotency-factors (Oct4, Nanog and Sox2). Mechanistic studies showed BFT induced expression and nuclear translocation of cleaved NOTCH1 and βcatenin resulting in activation of downstream targets. Inhibition of Notch1 and βcatenin using γ-secretase inhibitor and ICG001 successfully inhibited functional effects of BFT. Further, BFT-pretreated MCF7 cells exhibit increased tumor growth and form multifocal tumors in mice. MCF10A-KRas cells, pretreated with BFT, also showed increased tumor progression and multifocal tumors in mice. In vivo limiting dilution assay using breast tumors from BFT-pretreated MCF7 cells exhibited a striking increase in tumor-initiating cells. Follow-up analyses of these tumors demonstrated increased migratory, invasive, and mammospheres-forming behavior confirming that brief BFT exposure elicits long-term molecular changes.

Conclusion: Collectively, these findings present the first in vitro and in vivo evidence to show that Bacteriodes fragilis Toxin induces EMT, invasion/migration and stem cell-like phenotype and leads to concomitant activation of Notch and βcatenin axes.

#2835

Breast tumor microbiota populations are modulated by chemotherapy and may be indicative of outcome.

Akiko Chiba, Alaa Bawaneh, Christine Velazquez, Edward Levine, Katherine L. Cook. _Wake Forest University, Winston Salem, NC_.

Breast tumors were identified to have their own microbiota population distinct from mammary gland tissue. Breast cancer patients that present in the clinic with larger tumors often undergo neo-adjuvant chemotherapy to reduce tumor burden before surgery. The purpose of our study was to evaluate whether chemotherapy can modulate the tumor microbiome and the potential impact of microbes in the development of distant metastases. Using snap-frozen aseptically collected breast tumor tissue from women that underwent neo-adjuvant chemotherapy or women with no prior therapy at time of surgery, we performed 16S rRNA sequencing to identify tumoral bacterial populations. We also stained breast tumor microarrays (normal breast tissue, primary breast tumor, and lymph node metastases) to confirm presence of identified microbiota. Our data indicates that chemotherapy administration significantly increased breast tumor Pseudomonas and reduced the proportional abundance of Streptococcus. Primary breast tumors from patients that developed distant metastases later on in life (regardless of therapy) displayed increased tumoral abundance of Acinetobacter, Brevundimonas, and Staphylococcus. Stratifying patients based upon BMI indicates that obesity modulates breast tumor microbiota populations and needs to be considered in analyses. Furthermore, we confirm presence of Pseudomonas in breast tumor tissue by immunohistochemical staining. Our results indicate breast tumor microbiota populations can be modified by chemotherapy and specific microbes correlate with tumor recurrence. Further studies with a larger patient cohort may provide greater insights into the role of microbiota in therapeutic outcome and for the development of novel bacterial biomarkers that could predict distant metastases.

#2836

The commensal urinary microbiome as a predictor of response to Bacillus Calmette-Guerin (BCG) immunotherapy in non-muscle invasive bladder cancer.

Randy F. Sweis,1 Shay Golan,2 Nimrod Barashi,1 Elle Hill,1 Ciro Andolfi,1 Ryan Werntz,1 Jeffrey Bloodworth,1 Thomas Gajewski,1 Gary D. Steinberg1. 1 _University of Chicago, Chicago, IL;_ 2 _Rabin Medical Center, Israel_.

Bacillus Calmette-Guerin (BCG) is an intravesical immunotherapy that is standard of care to prevent recurrence of non-muscle invasive bladder cancer. Even with adequate BCG treatment, recurrences occur in up to 50% of patients within 5 years. The identification of a commensal urinary microbiome has been recently reported, but it has not been studied in the context of bladder cancer treatment. We hypothesized that variation in the urine microbiome is associated with response to BCG immunotherapy. A clinical study was initiated that enrolled patients with a newly diagnosed bladder tumor to evaluate urinary biomarkers. Urine samples were collected via catheterization to minimize contamination at baseline and up to 8 additional time points. Samples were centrifuged and DNA was extracted. The microbial composition for each sample determined by the 16S rRNA gene sequencing-based method and analyzed by Operational Taxonomic Units (OTUs). Interferon gamma and interleukin-2 levels were measured using the Invitrogen ProcartaPlex immunoassay. Thirty-one patients were enrolled with a median age of 69 years, including 9 females and 22 males. All patients were treated with BCG and 10 patients were diagnosed with recurrent disease at a median follow up of 6 months. In baseline samples, a significant difference in microbial composition was observed by distance matrix computation between patients with and without recurrence (Bonferroni-corrected P=0.017). Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, and Tenericutes accounted for >99% of the phyla detected across all samples. Proteobacteria had a significantly higher abundance in patients who developed recurrence (P=0.035). In particular, the Enterobacteriacae family was significantly more abundant among patients with recurrence (P=0.001). Lactobacillales was lower in abundance in patients with recurrence versus those without recurrence (P=0.049). A subset of 13 patients, including 6 with recurrence, were analyzed for changes in urine gamma interferon and interleukin-2 levels after BCG induction (week 6), and no difference was observed between patients with and without recurrence (P=0.79 and P=0.96, respectively). Characterizing the commensal urine microbiome in bladder cancer patients is feasible, and variation in composition may predict response to BCG therapy. Further studies are ongoing to validate these findings and determine longitudinal patterns over time.

#2837

Fusobacterium nucleatum and T cells in colorectal cancer liver metastasis.

Yuki Sakamoto, Kosuke Mima, Nobuya Daitoku, Yukiharu Hiyoshi, Katsunori Imai, Masaaki Iwatsuki, Takatsugu Ishimoto, Yoshifumi Baba, Shiro Iwagami, Yuji Miyamoto, Yoichi Yamashita, Naoya Yoshida, Hideo Baba. _Kumamoto University, Kumamoto, Japan_.

Background: Accumulating evidence links the intestinal microbiota and colorectal carcinogenesis. Fusobacterium nucleatum has been shown to promote colorectal tumor growth and inhibit antitumor immune responses. Emerging evidence demonstrates an enrichment of Fusobacterium species in colorectal cancer live metastasis (CRLM).

Aim: To test the hypothesis that higher amount of Fusobacterium nucleatum is associated with lower density of CD8+ T-cells in CRLM.

Method: Genomic DNA was extracted from CRLM and adjacent normal liver tissues. We measured the amount of Fusobacterium nucleatum DNA in 164 CRLM tissues using a quantitative polymerase chain reaction assay. The density of CD8+ T-cells in CRLM was determined by immunohistochemical staining.

Result: Fusobacterium nucleatum was detected in 8 (4.9%) of 164 CRLM cases. A higher density of CD8+ T-cells in CRLM was significantly associated with better relapse-free survival (P =0.036). Fusobacterium nucleatum-negative cases, Fusobacterium nucleatum-positive cases were inversely associated with the density of CD8+ T-cells (P =0.020).

Conclusions: The presence of Fusobacterium nucleatum is inversely associated with CD8+ T-cells density in CRLM. Upon validation, our data may have implications for preventing the formation of liver metastases in colorectal cancer through targeting intestinal microflora.

#2838

The gut microbiome of melanoma patients is distinct from healthy controls, and associations with treatment outcomes are influenced by host lifestyle factors.

Christine N. Spencer*,1 Vancheswaran Gopalakrishnan*,2 Jennifer McQuade*,2 Miles C. Andrews,2 Beth Helmink,2 M.A. Wadud Khan,2 Elizabeth Sirmans,2 Lauren Haydu,2 Alexandria Cogdill,2 Elizabeth Burton,2 Rodabe Amaria,2 Sapna Patel,2 Isabella Glitza,2 MIchael Davies,2 Eliza Posada,2 Wen-Jen Hwu,2 Adi Diab,2 Kelly Nelson,2 Hussein Tawbi,2 Michael Wong,2 Robert R. Jenq,2 Lorenzo Cohen*,2 Carrie Daniel-MacDougall*,2 Jennifer A. Wargo*2. 1 _Parker Institute for Cancer Immunotherapy, San Francisco, CA;_ 2 _MD Anderson Cancer Center, Houston, TX_.

Background: There is a growing appreciation for the GM in response to melanoma therapy. Recent work identified a favorable (T1) GM signature associated with checkpoint benefit. However, little is known about how lifestyle factors may influence features of the GM or about the influence of the GM on therapeutic outcomes to other common melanoma therapies.

Methods: We prospectively collected fecal samples melanoma patients at MD Anderson Cancer Center and characterized the GM via 16S rRNA sequencing (n=146). In a subset initiating systemic therapy (n= 113), we also collected baseline diet information via the NCI dietary screener questionnaire, and probiotic and antibiotic use and followed for response (RECISTv1.1). We compared GM beta diversity (BD)(Bray-Curtis) with ANOSIM and descriptive statistics and Wilcoxon tests were applied to alpha diversity (AD)(Chao1) among treatment response groups. Spearman correlation was used to test relationships of dietary groups and GM composition. Univariate and multivariate logistic regression models were used to determine the individual and joint effects of lifestyle factors on response. Receiver operating characteristic (ROC) curves were constructed for area under the curve (AUC) quantification.

Results: Considering all treatment groups, baseline AD was highest in patients with complete/partial response (mean=355), compared to patients with stable (mean=330) and progressive disease (mean=318). The GM did not significantly differ by age, sex or BMI. Report of probiotic (42%) and antibiotic (29%) use at baseline was associated with lower AD (p=0.02). Whole grains and overall diet quality correlated positively with pro-response bacteria, added sugars and processed meat negatively correlated. GM community structure (BD) differed by high and low fiber intake (p<0.05). Patients with high vs. low fiber diet had higher odds of response to aPD1 (OR=5.3, 95% CI: 1.02-26.3) and AUC for fiber in predicting response was 0.65. Joint effects analysis showed greatest odds of response among patients with a T1 signature and high fiber diet (OR=3.6, 95% CI: 0.7-17.8) vs. low fiber diet (OR=1.9, 95%CI 0.6-6.4), compared to patients without a T1 signature. Similar results were seen for overall diet score; and for both fiber and diet score, results reached statistical significance excluding patients on antibiotics.

Conclusion: AD of the GM varies by response regardless of treatment type. While preliminary, this data points to the idea that the GM in melanoma patients may be negatively influenced by probiotics and could be targeted by dietary manipulation. Larger prospective and interventional studies are needed to assess the relationships between host lifestyle factors, the GM and response to melanoma therapies.

#2839

Leveraging gut microbiota networks to impact tumor immunotherapy.

Lata Jayaraman, Jaclyn Sceneay. _Seres Therapeutics, Cambridge, MA_.

The human gut microbiota forms a diverse, dynamic and complex ecosystem that modulates numerous host processes including metabolism, inflammation and cellular and humoral immune responses. Emerging data suggest that the gut microbiota of cancer patients may predict tumor response to immune checkpoint inhibitors (ICI). To better understand how the microbiome may impact response to ICI, we have developed and validated robust tumor models using either conventional mice treated with antibiotics or germ-free (GF) mice. We have confirmed in both models, that mice lacking a diverse microbiome fail to mount an efficient anti-tumor immune response after treatment with anti-PD-1, primarily due to T cell dysfunction. This response to anti-PD-1 can be restored by introduction of a microbiome using fecal microbial transplant (FMT) prepared from stool from healthy donors in GF mice, and is driven by increased tumor-infiltrating lymphocytes (TILs), specifically CD8+ T cells. Importantly, for the first time, we show that the bacterial spore fraction from healthy donor stool can also restore response to anti-PD-1 and increase CD8+ TILs in both conventional mice treated with antibiotics and GF mice. We have also tested cancer patient-derived microbiome samples that are efficacious or non-efficacious in GF mouse models. We have sequenced the input material as well as the engrafted gut microbiota in these mice via 16S rDNA sequencing. Comparison of these sequences has enabled us to develop a "microbiome signature" of response that is being used in the development of a microbiome-based therapy for combination with ICI. Based on these encouraging animal model data we plan to initiate a randomized, placebo-controlled clinical study in patients with advanced metastatic melanoma. The clinical trial will evaluate the impact of an anti-PD-1 checkpoint inhibitor with adjunctive microbiome therapy on patient outcomes. Seres is developing SER-401, a preclinical stage oral microbiome therapy to improve the efficacy and safety of immunotherapy. Our drug discovery strategy combines computational analyses and empirical in vitro, in vivo and ex-vivo screening of strains and consortia to inform selection and drive microbiome drug design. Data from such a comprehensive approach are invaluable for designing compositions of bacteria that form "functional ecological networks" that can impact response to ICI therapy. We believe these data will provide insight into how microbiome drugs can be developed in the setting of immunotherapy to augment the efficacy of ICIs by altering the cancer-immune set point.

#2840

Fusobacterium nucleatum **stimulates colorectal cancer progression by activating Annexin A1 in cancerous cells.**

Mara R. Rubinstein,1 Jung Eun Baik,1 Stephen M. Lagana,1 William J. Raab,1 Dabashis Sahoo,2 Piero Dalerba,1 Timothy C. Wang,1 Yiping W. Han1. 1 _Columbia University, New York, NY;_ 2 _University of California San Diego, San Diego, CA_.

Colorectal cancer (CRC) is the second leading cause of cancer death in the U.S., affecting one in 20 individuals. Numerous studies have implicated Fusobacterium nucleatum (Fn), a Gram-negative oral commensal, in CRC; however, mechanistic insight on the role of Fn in this debilitating disease is scarce.

Previously, we have reported that Fn stimulates CRC growth through its unique adhesin FadA, which binds to E-cadherin and modulates β-catenin signaling. In the present study, we tested the specificity of Fn-induced cell growth. Fn did not promote the growth of lung, prostate, breast and bladder cancer cells, indicating specific component(s) on CRC cells are required for stimulation. To identify such component(s), we utilized a CRC progression model consisting of a

series of cell lines sequentially derived from a human colonic adenoma. Fn specifically stimulates growth of the cancerous cells without affecting the non-cancerous cells. The growth stimulation is mediated by Annexin A1 (ANXA1), a member of the Annexin family of Ca2+-dependent phospholipid-binding proteins. ANXA1 is specifically expressed in the proliferating cancerous cells, but not in non-cancerous or non-proliferating cells. Analysis of a database of 466 colon cancer patients reveals that increased level of ANXA1 is associated with cancer reoccurrence independent of cancer stage, grade, sex and age. Knocking down of ANXA1 in CRC cells inhibits cell proliferation due to down-regulation of Cyclin D1. An E-cadherin mediated positive feedback loop between FadA and ANXA1 is identified in the cancerous cells. Fn preferentially binds to ANXA1-expressing cancerous cells, which in turns stimulates ANXA1 expression. FadA, E-cadherin and Annexin A1 form a complex in CRC cells leading to activation of β-catenin signaling.

The correlation between FadA and ANXA1 was investigated in vivo using the APCmin/+ mice. Mice gavaged with wild-type Fn produced significantly increased number of tumors in the colon, compared to those gavaged with sterile PBS, E. coli DH5α, or the fadA-deletion mutant US1. In both mice and humans, increased ANXA1 mRNA levels were detected in the tumors compared to the matching normal colonic tissues, and a positive correlation between the fadA gene levels and ANXA1 mRNA levels was identified in the colorectal tumors. Immunofluorescent staining of paired normal and carcinoma tissues from CRC patients confirmed this finding and revealed co-localization of FadA and Annexin A1 in the carcinomas.

We have thus elucidated a novel mechanism by which Fn promotes CRC, i.e. through stimulating Annexin A1, a novel Wnt/β-catenin modulator, in the cancerous cells. Given the non-cancerous cells are not affected by Fn, we propose a "two-hit" model in which the first hit would be the somatic mutation(s) causing normal cells to become cancerous, and Fn serves as the second hit to exacerbate cancer progression.

#2841

Differential immune responses to HPV-positive and HPV-negative head and neck cancer.

Hannan A. Qureshi, Xiaodong Zhu, Sylvia M. Lee, Eduardo Mendez, A. McGarry Houghton. _Fred Hutchinson Cancer Research Ctr., Seattle, WA_.

Background: Immune checkpoint inhibitors (ICIs) have been effective in treating several solid tumor malignancies, including head and neck squamous cell carcinoma (HNSCC); however, less than 20% of patients benefit from even the most promising ICI drugs. There is a lack of accurate predictors for response to ICI treatment in HNSCC and little is known about which types of immune cells have a more dominant impact on T-cell infiltration in HPV-positive and HPV-negative HNSCC. The main objective of our study was to identify the unique immune signatures of HPV-positive and HPV-negative HNSCC and to determine the major immune suppressive entities within their respective tumor microenvironments.

Methods: We retrospectively analyzed a cohort of 102 HNSCC patients (44 HPV-positive, 58 HPV-negative), a subset of whom went on to receive ICI therapy later in their treatment course. We determined an immune signature by HPV-status using multiplex gene expression analysis (NanoString Immune Profiler). We also used multiplex immunohistochemistry (MIHC) to simultaneously evaluate markers for macrophages (CD68/CD163), neutrophils (CD66b), cytotoxic T-cells (CD8), B-cells (CD19/CD20), tumor cells (cytokeratin), PD-L1 expression, and DAPI. Immune cell distributions and their spatial relationships were quantified with HALO image analysis software (Indica Labs).

Results: Nanostring analysis revealed increased differential expression of genes related to B-cell functions in HPV-positive HNSCC, such as MS4A1, CD79B, and CD27, which were significant at a Benjamini-Yekutieli adjusted p-value of <0.001. T-cell function genes (e.g. CTLA4 and LAG3) also had significantly increased expression in HPV-positive HNSCC compared to HPV-negative HNSCC. MIHC displayed increased CD8+ T-cell and CD19/CD20+ B-cell densities in the tumor region of HPV-positive HNSCC as opposed to HPV-negative HNSCC (p<0.01). There was a negative correlation in the densities of neutrophils and macrophages in the stroma region compared to density of T-cells in the tumor region in HPV-negative tumors (p<0.05), but not for HPV-positive tumors.

Conclusions: HPV-positive HNSCC appears to have a B-cell and T-cell dominant gene expression profile with increased infiltration of these cells in the tumor region compared to HPV-negative HNSCC. The infiltration of CD8+ T-cells appears to be affected by myeloid cell burden in the stroma in HPV-negative HNSCC. The B-cell signature in HPV-positive HNSCC is particularly of interest, given that B-cells typically play a major role in resolving viral infections. Further analysis will involve correlating the differential patterns of gene expression with immune cell infiltration data to identify which immune cells have a more dominant effect on T-cell infiltration, and subsequently determine the effect on prognosis and response to ICI treatment.

#2842

Vesal Yaghoobi.

Vesal Yaghoobi, Vasiliki Pelekanou, Tess O'Meara, Andrea Silber, Lajos Pusztai, David Rimm. _Yale University, New Haven, CT_.

Background: It has been shown that even after adjusting for clinical stage at presentation and socio-economic variables, Triple Negative Breast Cancer (TNBC) still has a worse overall outcome in African American (AA) compared to Non-African American (NAA) patients. The abundance and composition of immune cells in the tumor microenvironment is a powerful prognostic factor in TNBC. Our goal was to assess if differences exist in CD8, CD68 and PD-L1 protein expression between AA and NAA TNBC. We hypothesize that the microenvironment of African AA TNBC patients may be different than that of NAAs.

Method: We selected N=43 AA and N=43 NAA TNBC samples from the Yale Pathology archives that were matched by diagnosis date. We measured CD8, CD68 and PD-L1 protein expression in both the tumor and stromal compartments in whole slides from formalin fixed paraffin embedded (FFPE) tissues using multiplexed quantitative immunofluorescence (QIF). The average of each marker expression was calculated in all Fields of View (FOV), and the top 10% brightest FOV of each slide (i.e. hotspot).

Results: The frequency of macrophages, as assessed by the expression of CD68 was significantly higher in AA compared to NAA. This was seen by overall assessment for all FOV (mean NAA=2273au versus mean AA=3627au; p = 0.0052), and in hotspot FOVs (mean NAA=4858au versus mean AA=6371au; p = 0.0411), and also for assessments in tumor and stromal subcompartments. Expression of CD8 positive cytotoxic T cells was significantly lower in AA compared to NAA, but only when measuring hotspots in the tumor compartment (p=0.017).

Conclusion: The significantly higher CD68 and lower CD8 expression in AA compared to NAA TNBC might contribute to a more immune attenuated microenvironment. We are currently further characterizing the macrophage polarity and the cytokine milieu of these samples.

#2843

Advanced age and obesity separately and interactively potentiate triple negative breast cancer progression.

Laura A. Smith, Magdalena A. Rainey, Nishita T. Sheth, Stephanie A. Montgomery, Stephen D. Hursting. _University of North Carolina at Chapel Hill, Chapel Hill, NC_.

Advanced age and obesity are major risk factors for breast cancer (BC) mortality that are independently associated with aggressive disease and therapeutic resistance. Despite this, preclinical models of BC mainly utilize young lean mice. The mechanisms underlying age- and obesity-related BC progression are currently unknown, highlighting a critical gap in our understanding of BC biology. To address this gap, we generated aged (16 mos) and young (6 mos) cohorts of lean and obese mice by placement on either low-fat control (10% kcal from fat) or diet-induced obesity (DIO; 60% kcal from fat) diet, respectively, beginning at 8-weeks of age. This resulted in four experimental groups: aged lean, aged DIO, young lean, young DIO. Mice were orthotopically injected with one of two triple negative BC cell lines (E0771 or Met-MWNTLung), which represent aggressive BC subtypes associated with poor prognosis in humans. Outcomes included primary tumor weight and lung metastasis quantification. Biochemical and molecular analyses included serum cytokines and gene expression within tumor and adjacent normal mammary tissue. Our TNBC models indicate tumor growth is similarly increased in young DIO and aged lean mice, with both significantly higher than young lean mice. Moreover, the combination of advanced age and obesity in aged DIO mice significantly increased tumor growth relative to all other experimental groups. Preliminary pathology analyses in Met-MWNTLung-bearing mice reveal that advanced age enhances lung metastasis beyond that of young mice regardless of diet. Increased tumor burden in both the aged lean and young DIO mice is accompanied by increased serum proinflammatory cytokines (IL-1b, CXCL13, CCL11, among others). Consistent with these findings, Gene Set Enrichment Analysis (GSEA) of Met-MWNTLungtumor and adjacent normal mammary microarray data demonstrated enrichment of inflammatory pathways, including IFNγ response, IFNα response, IL-2/STAT5 signaling, and IL-6/JAK/STAT3 signaling in aged lean and young DIO mice relative to young lean mice. Interestingly, tumor-adjacent mammary from aged lean and young DIO mice was also enriched for markers of EMT, consistent with age- and DIO-associated phenotypic switching of cells within the tumor-adjacent microenvironment. Taken together, our findings reveal that obesity and advanced age augment BC progression and promote inflammation within the local (mammary & tumor) and systemic (serum) microenvironments. Ongoing analyses are investigating the role of cellular senescence, a stress-induced cell cycle arrest in which cells acquire a proinflammatory secretome. Knowledge gained from this study will help to elucidate the mechanisms underlying advanced age- and obesity-related BC progression, which is urgently needed to develop new strategies for reducing adverse TNBC outcomes associated with advanced age and obesity in human populations.

#2844

In vitro **recapitulation of** in vivo **obesity-promoted colorectal cancer growth using a patient-derived xenograft engineered tumor model.**

Iman Hassani, Benjamin Anbiah, Bulbul Ahmed, Nicole L. Habbit, Michael W. Greene, Elizabeth A. Lipke. _Auburn University, Auburn, AL_.

Strong epidemiological evidence links obesity to an increased risk of colorectal cancer (CRC). However, the precise molecular mechanisms underlying such an association have not been fully elucidated, partly due to the lack of physiologically relevant models. Here, we established a novel PEG-fibrinogen-based engineered tumor model using patient-derived xenograft (PDX) CRC co-cultured with 3T3-L1 adipocytes and an orthotopically implanted PDX CRC model of obesity.

The PDX CRC cells were isolated from PDXs propagated subcutaneously in NOD-SCID mice and encapsulated within a biomimetic polymer, PEG-fibrinogen, to create 3D engineered PDX CRC tumors (3DePCCTs) or cultured in a standard 2D manner. We compared cell viability, colony area, and cell subpopulations within the 3DePCCTs and stiffness of the 3DePCCTs to the in vivo propagated tumors. A stage IV CRC tumor was employed in vivo and in vitro to study obesity-promoted tumor growth. Responsiveness of the 3DePCCTs to the growth promoting effects of obesity was investigated through continuous co-culture with insulin sensitive (IS) and resistant (IR) (treated with TNFα and 1% hypoxia) 3T3-L1 adipocytes (adipocytes replaced every 72 hrs). The responsiveness of the stage IV CRC PDX line was validated using an orthotopically implanted CRC model of obesity in which Rag1tm1Mom mice were fed a high fat Western diet + 4% sugar water (HFWD+S) to induce obesity or a chow diet to maintain a lean phenotype.

The cells within the 3DePCCTs remained viable and the PDX-line dependent differential growth of tumor colony area within the 3DePPCTs recapitulated line-dependent difference in in vivo tumor growth. Based on flow cytometry, human (70±3%) and Cytokeratin 20+ (31±1) cell subpopulations within the 3DePCCTs were similar to the original in vivo tumor tissue and maintained over time, whereas supporting mouse stromal cells took over 2D cultured cells (n=3 batches). Stiffness of the 3DePCCTs was within the range of the in vivo tumor tissue stiffness (0.3 to3.6 KPa, n=3 tissues). In vitro adipocytes maintained IR for at least 72 hrs based on significantly higher MCP1 (at least 10 folds) and lower GLUT4 (maximally 0.1 fold) (n=3 batches). The in vitro coculture model revealed a significantly (p < 0.05) greater number of cancer cell colonies within 3DePCCTs after 8 days of co-culture with IR adipocytes compared to IS adipocytes and this difference increased through day 29. In the in vivo model, a significant (p < 0.05) greater than 2-fold increase in weight of PDX CRC tumors grown in mice fed the HFWD+S diet was observed.

We have established the ability to maintain PDX CRC cells in 3D culture long-term (29 days) and generated a novel in vitro obesity-mimetic engineered tumor model that recapitulated the growth promoting effects of the in vivo orthotopic, IR tumor microenvironment and could be used to examine mechanistic questions and therapeutic targets.

### Invasion and Migration 1

#2845

The role of PSAT1 in triple negative breast cancer metastasis.

Stephanie Metcalf, Traci Kruer, Susan Dougherty, Rumeysa Biyik-Sit, Carolyn Klinge, Brian Clem. _University of Louisville, Louisville, KY_.

Breast cancer is the number one diagnosed cancer type among women in the United States (NCI). This disease can be stratified into different molecular subtypes, including triple negative breast cancer (TNBC), each of which can have different therapeutic options, patient prognosis, and survival outcomes. TNBC disease readily metastasizes and different processes have been implicated in the development of metastatic disease. The serine synthesis pathway (SSP) is responsible for de novo production of serine and consists of three enzymes, including phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase 1 (PSAT1). PSAT1 catalyzes the second step in this pathway, is elevated in more aggressive tumor types such as basal breast cancer (Pollari, 2011) and has clinically been associated with poorer distant metastatic free survival and overall survival (unpublished from KMPlotter) in breast cancer patients. These prior studies combined with our own preliminary data underscores the hypothesis that PSAT1 promotes the metastatic potential of triple negative breast cancer. This hypothesis was first evaluated through immunohistochemistry examination of patient samples which demonstrated that expression of PSAT1 increases with TNBC tumor grade. In vitro assays of proliferation showed no difference upon loss of PSAT1, however, metastasis (migration and invasion) was significantly inhibited upon PSAT1 suppression. In a tail-vein experimental metastasis model, suppression of PSAT1 significantly inhibited the formation of tumor nodules. Mechanistically, the relevance for de novo serine synthesis pathway was examined in vitro via suppression of PHGDH. Decreased PHGDH did not affect proliferation or the metastatic capability of either cell line. Further, addition of exogenous serine could not rescue metastatic deficiencies upon loss of PSAT1. Our results suggest that PSAT1 does contribute to metastasis in TNBC and that this affect appears to be independent of its metabolic role in serine synthesis. While studies are ongoing, this points to the potential of a non-canonical function of PSAT1 being a therapeutic target for patients with TNBC that are currently lacking treatments options.

#2846

Chemerin, as a serum biomarker, promotes tumorigenesis and metastasis in oral squamous cell carcinoma.

Anxun Wang. _Sun Yat-sen Univ., Guangzhou, China_.

Chemerin encoded by retinoic acid receptor responder 2 (RARRES2) has been found to be related with malignant tumors, but its causal role in the development of oral squamous cell carcinoma (OSCC) is largely unexplored. In the present study, clinical data showed that higher serum levels of chemerin were observed in OSCC patients compared to the healthy and significantly decreased after tumor resection, and were positively associated with advanced tumor stage and lymph node metastasis. The expression level of chemerin was positive correlated with the migration and invasion of OSCC cell lines. Recombinant chemerin dose-dependently enhanced the migration, invasion and proliferation of OSCC cells in vitro, and shRNA targeting RARRES2 inhibited chemerin expression, OSCC cells metastasis and proliferation in vitro and vivo. Additionally, Chemerin activated the Manganese superoxide dismutase (SOD2) activity and increased intracellular H2O2, led to a significant decrease of E-cadherin expression and a dramatically increasing expression of p-ERK1/2, Slug, Vimentin and N-cadherin. This study suggested that chemerin was a novel biomarker for OSCC patients. Chemerin promoted the tumorigenesis and metastasis of OSCC and would be a new therapeutic target for OSCC.

Keywords: Chemerin; Oral squamous cell carcinoma; Tumorgenesis; Metastasis; SOD2

#2847

Tenascin C expression in neural microenvironment enhances perineural invasion and is associated with locoregional recurrence-related poor prognosis in pancreatic ductal adenocarcinoma.

Satoru Furuhashi, Takanori Sakaguchi, Tomohiro Murakami, Mayu Fukushima, Yoshifumi Morita, Koji Ikegami, Hirotoshi Kikuchi, Megumi Baba, Mitsutoshi Setou, Hiyoya Takeuchi. _Hamamatsu University School of Medicine, Shizuoka, Japan, Hamamatsu, Japan_.

Background: Perineural invasion (PNI) is commonly seen in pancreatic ductal adenocarcinoma (PDAC) and worsens the postoperative prognosis. However, the associated mechanisms in cancer progression remain unclear. Tenascin C (TNC), an extracellular matrix glycoprotein, is abundant in cancer stroma and modulates tumor progression. The aim of this study was to evaluate the functional roles of TNC in the neural microenvironment of PDAC.

Methods: We immunohistochemically examined TNC expression in 78 resected PDAC specimens. TNC staining in perineural sites at the invasive front was classified as low or high intensity, by comparison with adjacent non-cancerous tissues in the same section. The relationships between TNC expression and clinicopathological features were retrospectively analyzed. Furthermore, interactions between cancer cells and nerves after supplementation with TNC were investigated when co-culturing a PDAC cell line with a mouse dorsal root ganglion (DRG) in vitro.

Results: High perineural TNC expression at the invasive front, seen in 30 (38%) patients, was associated with PNI (p = 0.006), pathological T stage ≥ 3 (p = 0.01), and postoperative locoregional recurrence (p = 0.002). It was independently associated with postoperative, poor recurrence-free survival in multivariate analysis (p = 0.045). In the in vitro co-culture model, supplementation with TNC significantly enhanced both neurotropism of PDAC cells and tumor tropism of a DRG. On the other hand, when PDAC cells and a DRG were cultured separately, TNC did not affect cancer cell proliferation or neural outgrowth.

Conclusions: Strong perineural TNC expression was a prognostic indicator of locoregional recurrence-related poor prognosis. The neurotropism of PDAC induced by TNC suggests that TNC could serve as a therapeutic target for PDAC.

#2848

TSPAN8-EPS8L3 signaling play a role in EGF-dependent pancreatic cancer cell invasion and migration.

Junjian Li,1 Rongxuan Zhu,1 Gaoda Ju,1 Weiyu Ge,1 Mengyi Jiang,2 Tianhao Zhou,1 Hongxia Wang1. 1 _Shanghai General Hospital, Shanghai, China;_ 2 _Nanjing Medical University, Nanjing, China_.

The mechanism and function of TSPAN8 on the invasion and metastasis of cancer cells is poorly understood. In present study, we demonstrated that TSPAN8 over-expressed in pancreatic cancer (Paca) tissues (37.98%) comparing with normal tissue (0%). TSPAN8 expression levels were reversely correlated with the overall survival time of Paca patients (p = 0.021). In vitro, over-expressed or knockdown TSPAN8 expression in Paca cells SW1990 or Capan-2 and AsPC-1 modulated invasion, immigration and clone formation ability. Mechanistically, we revealed that SOX9, a downstream transcription factor of EGFR signaling, activated TSPAN8 transcription under EGF condition. Meanwhile, we uncovered a previously uncharacterized mechanism underlying EGF-dependent Paca cells invasion and migration. In brief, TSPAN8 was phosphorylated at Ser129 by AKT, resulting in the binding of TSPAN8 with BCAFL1, TSPAN8 entering nucleus and regulating the EPS8L3 transcription, remodeling of cytoskeleton, and enhancing cell invasion and migration. By single cell seq analysis of Paca cells derived from patients and tissue array assay, we validated that the EGFR and EPS8L3 expression in mRNA and protein level were highly correlated with TSPAN8 expression. In vivo and in vitro study demonstrated that EPS8L3 depletion or overexpression rescued the invasion ability induced by TSPAN8. These findings reveal a molecular basis of EGF-TSPAN8-EPS8L3 signaling and highlight the critical role of TSPAN8 in tumor metastasis.

#2849

Axl: A promising therapeutic target that leads to multimodal inhibition of the metastatic pathway.

Mai Tanaka, Dietmar W. Siemann. _University of Florida, Gainesville, FL_.

Cancer is the second leading cause of death in the United States and approximately 90% of cancer-related deaths result from metastasis, the spread of cancer cells to secondary sites. In tumors, the upregulation of certain signaling pathways promotes the metastatic phenotypes of cancer cells characterized by enhanced invasion, migration, survival, proliferation and induction of angiogenesis. A key pathway involved in metastasis is the receptor tyrosine kinase Axl. Axl is expressed in a variety of tumor types and is associated with poor prognosis, metastasis, and outcome. The purpose of the current study was to evaluate the efficacy of genetic and pharmacologic inhibitions of Axl on the metastatic phenotypes, specifically cell migration, invasion and angiogenesis.

Human breast and prostate cancer cell lines (MDA-MB-231 and PC3-ML, respectively), and human lung microvascular endothelial cell (HMVEC-L) were used in this study. Stable Axl short hairpin RNA (shRNA) knockdown MDA-MB-231 and PC3-ML cell lines were generated by Mission Lentiviral transduction particles (Sigma). The efficacy of genetic and pharmacologic inhibition of Axl on tumor cell migration and invasion was evaluated by transwell migration and invasion assays. The effect of Axl knockdown on prostate cancer cell metastasis in vivo was evaluated by an intracardiac bone metastasis model. Conditioned media of Axl knockdown or control MDA-MB-231 cells were analyzed for angiogenic factors using the human angiogenesis array. The effect of Axl knockdown conditioned medium on angiogenic properties in vitro were assessed by endothelial cell tube formation and sprouting. Induction of angiogenesis in vivo was measured by performing intradermal assay.

Genetic and pharmacologic inhibition of Axl decreases tumor cell migration and invasion. Axl knockdown inhibits metastatic disease burden of prostate cancer. Axl knockdown conditioned medium secretes less pro-angiogenic factors compared to its scramble control cells, suggesting that tumor cells utilize Axl pathway to promote the induction of angiogenesis. Axl knockdown conditioned medium impairs angiogenic properties, including endothelial cell tube formation and sprouting. Axl knockdown of tumor cells suppress tumor cell-induced angiogenesis in vivo. Collectively, these data indicate that Axl is involved in multiple steps of the metastatic pathway and that Axl is a promising anti-metastatic agent to inhibit tumor cell dissemination and initiation of angiogenesis. Further studies are ongoing to understand the downstream molecular signaling of Axl.

#2850

Characterization of cadherin 6 as a novel therapeutic target in metastatic ovarian carcinoma.

Miranda Burdiel, Ruben Bartolomé, Marta Jaén, Ignacio Casal. _Centro de Investigaciones Biológicas (CIB), Madrid, Spain_.

Cadherin 6 (CDH6) is a type II cadherin containing a RGD motif in a similar way to LI-cadherin (CDH17) and VE-cadherin (CDH5), which are able to activate α2β1 integrin signalling to promote lung and liver metastasis. CDH6 gene is frequently overexpressed in late stages of some types of ovarian carcinoma, clear cell renal carcinoma, renal papillary carcinoma and thyroid cancers. Therefore, as reported for CDH17 and CDH5, we hypothesized that CDH6 might be a potential therapeutic target for metastatic ovarian cancer. Indeed, an "in silico" analysis revealed a significant association between CDH6 expression, tumour stage and poor survival in renal and ovarian cancer patients.

We confirmed a high CDH6 expression in ovarian cancer cell lines, although with significant differences in cell surface availability, as determined by flow cytometry. In addition, we noticed a variable α2β1 integrin expression in the same cell lines. Indeed, CDH6 RGD peptide caused β1 integrin activation in OVCAR3 cells but not in SKOV-3, suggesting the existence of alternative mechanisms. In this regard, as CDH6 has been reported to be a αIIbβ3 integrin ligand in platelets, we investigated and confirmed the expression of αIIbβ3 integrin in SKOV3 but not in OVCAR3 cells. Remarkably, CDH6 was able to activate αIIbβ3 integrin in SKOV3 cells. Moreover, silencing of CDH6 or β3 integrin triggered a significant loss of prometastatic traits in ovarian cells, such as cell adhesion, migration, invasion and proliferation. Signalling pathway analysis demonstrated that β3 integrin activation was triggering the phosphorylation of AKT and SRC, but not FAK, promoting cell invasion. Finally, treatment with monoclonal antibodies against the cadherin RGD motif inhibited the metastatic behaviour in ovarian cancer cell lines "in vitro". Experiments with metastatic mouse models to confirm the inhibitory capacity of cadherin RGD-specific monoclonal antibodies are in progress.

In summary, these findings suggest that CDH6 RGD motif could activate α2β1 or αIIbβ3 integrins, according to the cell context, and be targeted to avoid metastatic dissemination and cancer progression in those ovarian tumours overexpressing CDH6.

#2851

The effects of nidogen-1 on proliferation and migration in claudin-low mammary tumor cells.

Rebecca Jagroop, Roger Moorehead. _University of Guelph, Guelph, Ontario, Canada_.

Breast cancer is the most common type of cancer among women, with one subset of the triple-negative subtype, claudin-low, known to be very aggressive and metastatic. For invasion and metastasis to occur, cancer cells must cross basement membranes, which contain structural proteins such as laminin and collagen IV and linking proteins such as perlecan and nidogen, to colonize distant tissues. Nidogen is a glycoprotein that makes up 2-3% of basement membranes and has two types: nidogen-1 (NID1) and nidogen-2 (NID2). There have been limited studies on NID1 in cancer, with results demonstrating decreased invasiveness and metastatic capabilities in Nid1 silenced cells of various cancer types. In previous work, a murine cell line representative of the claudin-low subtype, known as RJ423, was developed; it demonstrated a 5000-fold increase in Nid1 expression compared to the luminal subtypes. To test whether high Nid1 expression contributes to the aggressive, metastatic nature of claudin-low tumors, Nid1 levels were knocked down in RJ423 cells and proliferation and migratory capabilities were assessed. Phospho-histone H3 immunofluorescence demonstrated that suppressing NID1 reduced RJ423 cell proliferation significantly. Also, proliferation was explored using flow cytometry to obtain cell cycle profiles, and it was observed that there was a reduction in the G2 phase of NID1-suppressed cells. Additionally, apoptosis was assessed through flow cytometry to determine annexin V levels, but suppression of NID1 levels did not alter apoptosis. Furthermore, invasion assays demonstrated a reduction in migration through collagen IV-coated wells using NID1-suppressed cells. Knockdown of Nid1 expression in RJ423 cells reduced the level of NID1 in conditioned media, and preliminary studies suggest that the levels of NID1 in conditioned media can influence tumor cell growth. These findings suggest that NID1 could be targeted to lessen the metastatic nature of claudin-low breast cancer.

#2852

PKM2 phosphorylates and stabilises Matrin 3 which promotes FOXC2-mediated tumorigenesis.

Richa Kumari, Sanjeev Das. _National Institute of Immunology, Delhi, India_.

PKM2 is tumor specific isoform of Pyruvate kinase essential for Warburg effect. Apart from its well-known function in aerobic glycolysis, it has non-metabolic functions as well. Under oncogenic/mitogenic stimulation conditions it can translocate into the nucleus and acts as co-transactivator or kinase and promotes tumorigenesis. In order to explore its interactome we carried out a proteomic screen and found several interacting proteins. However, in vitro and in vivo studies confirmed Matrin 3 as a novel interacting partner of PKM2. Kinase assays revealed Matrin 3 as novel substrate of the PKM2-mediated phosphorylation. Further, we mapped the site of phosphorylation to the highly conserved T239 residue in Matrin 3. Intriguingly; this phosphorylation is inhibitory in nature as it prevents Matrin 3 degradation by abrogating K48-linked ubiquitylation. Moreover, the expression of a phospho-dead mutant of Matrin 3 or kinase-dead mutant of PKM2 increases ubiquitylation and leads to reduced level of Matrin 3.We also identified K132 as site of ubiquitylation which is conserved across species. Further, we screened a shRNA library targeting nuclear E3 ubiquitin ligases and identified FBXO11 as the cognate E3 ligase involved in this degradation. Matrin 3 is a RNA binding protein which mediates post-transcriptional regulation of various mRNAs. Further studies using qPCR array for EMT related genes and RNA immunoprecipitation assays established that Matrin 3 binds to FOXC2 and promotes post-transcriptional stability of FOXC2. FOXC2 is a transcription factor which regulates EMT, angiogenesis, invasion/migration by regulating Integrin β-3, ZEB1 and c-MET. Decreased Matrin 3 level leads to reduced proliferation capacity, migration and invasiveness of tumor cells. Furthermore, mouse tumor models confirmed that Matrin 3 promotes tumorigenesis, metastasis and invasion in FOXC2 dependent manner. Taken together, our findings add to the substrate repertoire of nuclear PKM2 kinase and provide key insights into FOXC2-mediated tumorigenesis.

#2853

Cell motility and invasiveness are promoted by guanylate binding protein 1, GBP-1 in lung adenocarcinoma.

Takahiro Mimae,1 Ichiko Yamakita,1 Yasuhiro Tsutani,1 Yoshihiro Miyata,1 Akihiko Ito,2 Morihito Okada1. 1 _Hiroshima University, Hiroshima, Japan;_ 2 _Kinki University, Japan_.

Previously, we identified candidate molecules involved in human lung adenocarcinoma progression from non-invasive to invasive tumor by comparing gene expression profiles of each component in the same tumor using laser microdissection and DNA microarray analysis. Among them, guanylate-binding protein 1 (GBP-1) was selected for further analysis because RT-PCR for normal lung and invasive human lung adenocarcinoma tissue samples showed that the relative GBP-1 gene expression levels normalized to GAPDH for invasive lung adenocarcinoma were approximately three-fold higher than those for normal lung samples. In addition, GBP-1 gene and protein expression levels were also higher in mesenchymal-like than in epithelial-like lung cancer cell lines. To determine whether GBP-1 is involved in lung adenocarcinoma cell motility and invasion, we performed migration and wound healing assays using RERF-LC-OK, human lung adenocarcinoma cells transfected with siRNAs. The relative migration of transfected GBP1-siRNA cells was significantly lower than that of control-siRNA cells. The relative wound healing capacities were assessed after scratching. Cells transfected with GBP1-siRNAs were also significantly lower than those of the control-siRNAcells. Moreover, Immunohistochemistry of 80 patients with stage I lung adenocarcinoma demonstrated that non-invasive component cells showed clearly negative GBP-1 expression in all cases. On the other hand, invasive component cells were GBP-1 positive in 10 cases (12.5%) and GBP-1 negative in 70 cases (87.5%). Lymphatic-vascular invasion was positive in 20 patients (25%) and positively correlated with GBP-1 expression (P< 0.05). In conclusion, GBP-1 might enhance lung adenocarcinoma cell motility and invasiveness and controlling of GBP-1 expression has the potential to contribute to the development of novel therapeutic strategies for lung adenocarcinoma.

#2854

Clinical significance of Cofilin-1 expression in pancreatic cancer.

Rumi Itoyama, Shigeki Nakagawa, Toshihiko Yusa, Yosuke Nakao, Takanobu Yamao, Naoki Umezaki, Tatsunori Miyata, Hirohisa Okabe, Hiromitsu Hayashi, Katsunori Imai, Yo-ichi Yamashita, Akira Chikamoto, Hideo Baba. _Department of Gastroenterological Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan_.

Background: Pancreatic cancer is one of the most malignant tumors. The prognosis of patients with pancreatic cancer remains poor, with a 5-year survival rate of less than 10%. Therefore, identification of new biomarkers and development of therapeutic drugs are urgent issues. As a result of genetic research using public database, we focused on Cofilin-1. In recent years, it has been revealed that cancer cells form invadopodia to destroy extracellular matrix and cause hematogenous metastasis, and it is suggested that Cofilin-1 has a key role in stabilizing the structure of the invadopodia. However the significance of Cofilin-1 expression in cancer invasion is not clear. The purpose of this study is to verify the relationship between the expression of Cofilin-1 and patient's prognosis in pancreatic cancer.

Methods: At first, we examined the relationship between Cofilin-1 expression level and clinicopathological factors or prognosis using GSE21501 and TCGA, which are public databases of patients with pancreatic cancer. We set the cut-off value, and divided into the two groups, the high Cofilin-1 and low groups. Then, we evaluated expression of Cofilin-1 with immunohistochemical (IHC) staining in 200 pancreatic cancer patients who underwent curative surgery during April 2004 to March 2018 at the department of gastroenterological surgery, Kumamoto University. After that, we examined the relationship between Cofilin-1 expression and prognosis.

Results: In silico analyses using GSE21501 and TCGA dataset, high expression of Cofilin-1 is associated with early recurrence and poor prognosis. Then, we evaluated expression of Cofilin-1 with IHC staining using original scoring. The score is calculated by multiplying each scores classified into 4 stages from 0 to 3 in intensity of Cofilin-1 expression and area of that. 0 to 3 of the score was the low expression group (n=93), 4 to 9 is the high expression group (n=107). The result showed that high Cofilin-1 expression is associated with high occurrence of lymph node metastasis. In addition, both the relapse free and overall survivals were significantly worse in the high Cofilin-1 group. Cox proportional hazard analysis revealed that the high Cofilin-1 expression was an independent poor prognostic predictor.

Conclusion: In both silico analysis and IHC staining, high Cofilin-1 expression is associated with poor prognosis, especially high recurrence rate in patients with pancreatic cancer. Cofilin-1 may be useful as a biomarker for early recurrence in pancreatic cancer.

#2855

Oleic acid-induced ANGPTL4 facilitates metastasis of human colorectal cancer via up-regulation of NOX4 expression.

Chih-Jie Shen,1 Yu-Han Liao,1 Jhih-Peng Tsai,1 Liang-Yi Hung,1 Wen-Chang Chang,2 Ben-Kuen Chen1. 1 _National Cheng Kung Univ., Tainan, Taiwan;_ 2 _Taipei Medical University, Tainan, Taiwan_.

Colorectal cancer (CRC) is highly associated with metabolic diseases, such as obesity and diabetes. Elevated free fatty acids (FFAs) in cancer patients with metabolic disorders may be associated with cancer progression. In addition, increased levels of reactive oxygen species (ROS) are also observed in dyslipidemia which induces expression of NADPH oxidase 4 (NOX4) by FFAs in aorta, kidney, and white adipocyte in physiology condition. Although the expression of NOX4 has been also up-regulated in various cancer cell types, the correlation between up-regulation of NOX4 and dyslipidemia-regulated CRC metastasis remains unclear. Here, we found that oleic acid (OA) induced-angiopoietin-like 4 (ANGPTL4) expression was through the activation of PPARs pathways, resulting in promoting the metastasis of CRC. The recombinant protein of human ANGPTL4 rescued the OA-induced invasive ability in ANGPTL4 knockdown cells. It is worthy to note that the knockdown of ANGPTL4 not only significantly inhibited OA-induced NOX4 expression, but also reduced ROS production. In addition, the depletion of ROS by using NAC or knockdown of NOX4 inhibited OA-induced extravasation of CRC cells in vivo. OA-induced MMP1 and MMP9 expressions were also dependent on the expression of ANGPTL4 and NOX4 in CRC cells. The transcriptional activation of NOX4 gene by OA-induced ANGPTL4 was regulated by activation of c-Jun and dependent on AP-1 site of NOX4 promoter. These results reveal that OA-promoted CRC metastasis was through the activation of ANGPTL4/NOX4 axis, suggesting that ANGPTL4 and NOX4 may be potential therapeutic targets and diagnostic markers to improving outcomes for patients with CRC.

#2856

Secretion modification region (SMR) peptide blocks migration and invasion in human breast cancer cells.

Ming Bo Huang,1 Jennifer Y. Wu,2 William W. Roth,1 James Lillard,1 Vincent C. Bond1. 1 _Morehouse School of Medicine, Atlanta, GA;_ 2 _Columbia College, Columbia University, New York, NY_.

Background: Our group has discovered and developed a novel antagonist peptide, SMRwt. This peptide has dual roles as a tumor and HIV antagonist, through interaction with mortalin. It effectively suppresses breast cancer cell activity, growth, and release of EV [1], and inhibits release of HIV virus and EV during infection [2].

Aims: This study continued to explore the function and mechanism of interaction of the SMRwt peptide with mortalin during migration and invasion of breast cancer cells. It is known that changes in mortalin affect regulation of migration and invasive properties of human breast cancer epithelial cells, resulting in promoting metastatic potential.

Methods: Breast cancer cells, either treated with SMRwt or SMRmut peptides, were assayed via the MTT assay, a cell wound-healing assay, and for migration, and invasion to determine the inhibitory role of these peptides on cell proliferation and migration/invasion. Flow cytometry and Western blot analysis were used to track expression of the peptides and mortalin and to measure expression of EV proteins. NanoSight analysis and acetylcholinesterase (AchE) assay were used for measuring EV size and concentration.

Results: Our results demonstrate that the modified SMRwt peptides not only affected breast cancer cell proliferation and arrested breast cancer cell growth, but they also inhibited migration of breast cancer cells. In the wound-healing assay, an evident delay was observed in the wound closure of breast cancer cells treated for 24 h with SMRwt peptide while no delay was observed using the negative control peptide. Both SMRwt-CPP and PEG-SMRwt-CLU peptides decreased breast cancer cells migration ability in the tumor transendothelial migration assay. Proliferation of breast cancer cells was inhibited after a 24 h exposure to the SMRwt peptide as measured by MTT assay. Increased apoptosis rates were observed in tumor cells exposed to the SMRwt peptide and either paclitaxel or a mortalin inhibitor via flow cytometry. The above data indicate that SMRwt peptide/mortalin antagonism resulted in decreased capability for migration, invasion, and proliferation of tumor cells as well as increasing their capability to be killed via apoptosis.

Conclusions: Breast cancer cells treated with SMRwt peptide displayed decreased ability of mortalin to do its function, which significantly affects tumor cell proliferation and reduces tumor cell invasion and migration. These results further support the role of mortalin in progression of breast cancer and support the role of the SMRwt peptide as an antagonist of mortalin function ultimately negatively modulating tumor metastasis. The peptide also antagonizes tumor EV release and reduces the expression of mortalin all of which could play a role in its effect on breast cancer cell invasion and metastasis suggesting the potential clinical applications of the peptide in late stage breast cancer chemotherapy.

#2857

Vimentin is a novel PKC-ι target facilitating migration and invasion in melanoma.

Wishrawana Sarathi Ratnayake, Sloan Breedy, Christopher Apostolatos, Robert Hill, Clare Dennison, Mildred Acevedo-Duncan. _Univ. of South Florida, Tampa, FL_.

Vimentin plays an important role in gaining rear-to-front polarity for mesenchymal cells, making it a hallmark of cancer metastasis. Epithelial-mesenchymal transition (EMT) is a vital process in tumor progression that converts stationary epithelial cells into motile mesenchymal phenotype by downregulating E-cadherin and upregulating Vimentin expression. Molecular dynamics of Vimentin intermediate filaments (VIFs) play several key roles in cancer metastasis. Vimentin is activated via phosphorylation through various kinases. Recently, we reported that Protein Kinase C-iota (PKC-ι), activates Vimentin in melanoma while inducing EMT. We also reported the effects of PKC-ι inhibition on mitigating EMT in melanoma based on PKC- ι specific inhibitors {Ratnayake, W.S., et al. Int. J. Oncol. 51(5), (2017), 1370-1382 https://doi.org/10.3892/ijo.2017.4131 and Ratnayake, W.S., et al. Cell Adhes. Migr. (2018) https://doi.org/10.1080/19336918.2018.1471323}. This novel finding is very important with respect to cancer progression since PKC-ι is a well-established oncogene in various tumor types. Here, we analyzed the VIF dynamic changes upon PKC-ι inhibition using specific inhibitors targeting multiple activation sites on Vimentin. In addition to siRNA knockdown of expression of PKC-ι, we also report the effects of siRNA knockdown of two transcription factors, Paired related homeobox1 (PRRX1) and Zinc finger protein SNAI1 (SNAIL1) on the expressions of Vimentin, PKC-ι and E-cadherin. Herein we show that inhibition of PKC-ι downregulate the transcriptional activities of PRRX1 and SNAIL1, which was demonstrated with diminished levels of Vimentin and elevated levels of E-cadherin. Western blot, confocal immunofluorescence and immune-gold electron microscopic results suggested that PKC-ι targets multiple activation sites (S33, S39 and S56) on Vimentin without having an effect on S6 and S71 phosphorylation sites. Results also suggest that PKC-ι inhibition leads to diminution of activities of PRRX1 and SNAIL1 thereby further downregulation of Vimentin while upregulating E-cadherin in order to continue retardation of EMT in-vitro. Overall results indicate that PKC-ι is essential for VIF dynamics regulation during the metastasis in-vitro and therefore can be used as a novel biomarker to assess and mitigate melanoma metastasis.

#2858

Thymidine kinase 1 variability in primary and metastatic human breast cell lines and its correlation to metastatic potential.

Eliza E. Bitter, Michelle H. Townsend, Kelsey A. Bennion, Juan Mejia, Gajendra Shrestha, Kelsey Hirschi, Juan Arroyo, Kim O'Neill. _Brigham Young University, Provo, UT_.

The purpose of this study is to determine the role of Thymidine Kinase 1 (TK1) in the cellular invasion of breast cancer cells in vitro. TK1 is a cytosolic DNA salvage pathway enzyme responsible for the conversion of thymidine to thymidine monophosphate; it is known to increase during G1/S phase of the cell cycle and is significantly elevated in the serum of multiple cancer patients including breast, lung, and colon. As such, TK1 has been implicated as a useful biomarker for cancer prognosis and patient monitoring. In addition to TK1 upregulation in cancer serum, recent findings have shown that TK1 also localizes to the surface of ALL, AML, and colon cancer cells in patients. However, minimal studies have determined how the surface localization of TK1 is an indicator of metastatic potential. In addition, the function and timing of TK1 expression on the cellular surface has not been determined. When comparing the TK1 expression in metastatic cancer cell lines we found that there was a trend of overall elevation in metastatic cell lines (n = 39) when compared to primary cell lines (n = 31). This expression was extremely irregular between cell lines and TK1 showed significant variability between samples. We tested several breast cancer cell lines (primary: HCC 1806, HCC 1937, and JIMT-1; metastatic: T47D, BT 549, and MDA-MB-231) for their TK1 surface localization, more specifically the difference between metastatic cells and primary cells. We found significant TK1 expression in all cell lines tested with the highest expression in HCC 1806 primary breast cancer cells (90.36%; p = .0001) and the lowest expression in MDA-MB-231 metastatic breast cancer cells (47.2%; p = .01). We hypothesized that the expression of TK1 on the surface of cancer cells was dependent on the proliferative state of the cells and would be highest in the S phase of the cell cycle. To evaluate this hypothesis, cell cycle analysis was performed utilizing a PI stain to determine DNA content within the cells. Preliminary analysis of this data indicates that surface localized TK1 may not be dependent on the cell cycle but may be dependent on the proliferative capacity of individual patient's tumors. Currently under investigation is the direct effect of surface TK1 on invasion potential. To accomplish this, TK1-/- cells are actively being produced and their invasion potential against TK1WT cells will be compared to determine whether TK1 surface localization is influential in the metastatic ability of cancer cells. We plan to test the correlation between TK1 expression for a potential indicator of tumor aggressiveness and invasion potential.

#2859

Targeting the calcium sensing receptor in breast cancer cells diminishes colonization and migratory response in a trabecular bone explant model, and reduces osteolytic lesions in vivo.

Souvik Das,1 Antoine Gabrion,2 Isabelle Six,1 Cedric Boudot,1 Said Kamel,1 Philippe Clezardin,3 Michel Brazier,1 Romuald Mentaverri1. 1 _University of Picardie Jules Verne, Amiens, France;_ 2 _Centre Hospitalier Universitaire d'Amiens-Picardie, Amiens, France;_ 3 _Université de Lyon, Lyon, France_.

Breast cancer is the most common form of cancer affecting women worldwide with bones being the prevalent site of metastasis. The calcium sensing receptor (CaSR), whose primary physiological role is calcium homeostasis, has emerged as a new target in the "vicious cycle" that amplifies the metastatic cascade. Our team had already shown that overexpression of the CaSR dramatically increased the osteolytic potential of MDA-MB-231 cells in vivo. Our objective here was to study the involvement of CaSR in the metastasis of breast cancer cells to the bone using human femur tissue explants and to abate it with calcilytics (CaSR antagonists). Methods: To investigate the role of CaSR in migration of breast cancer cells, MDA-MB-231 cells were made to overexpress either a full-length wild-type CaSR (CaSR-WT) or a dominant negative mutant (CaSR-DN). For control, cells were transfected with empty pcDNA™3.1/Zeo(+) plasmid (CaSR-EV). NPS 2143 was used as a calcilytic in relevant assays. ERK phosphorylation was checked across all cell types in response to calcium. Trabecular bone fragments, extracted from human femoral heads, were used as explants to study the osteoinvasion in vitro. Transwell inserts seeded with the transfected cells were exposed to the bone fragment supernatant before quantifying migration. Treated/untreated cells were seeded directly onto the bone fragments, and zeocin-selective colony formation assay was performed with the extracted marrow population. Mice were inoculated intra-tibially with MDA-B02 cells and received NPS-2143 (IP) for 7 days. Results: The cultured supernatant elicited a strong migratory response across all transfected cell types. In these culture conditions, the CaSR-WT and CaSR-EV cells had a 50% increase in migration levels over the CaSR-DN (p<0.01). The difference between CaSR-WT and CaSR-EV was not statistically significant. Interestingly, similar results were obtained when cells were directly added to the bone fragment and allowed to colonize. The CaSR-WT and CaSR-EV had a 3-fold increase in colony formation over CaSR-DN. This led us to select the CaSR-EV in further experiments, where treating the cells with NPS 2143 decreased their colony formation by around 42%, diminished migration toward bone supernatants (p< 0.01), and blunted proliferation sensitized by the conditioned media (p<0.01). In vivo, administration of calcilytics reduced incidence (4/5 vs. 1/6) and extent (by 86%, p<0.05) of osteolytic lesions. Perspectives: In a co-culture model benefitting from an architecturally intact tissue micro-environment, we demonstrate that the CaSR in breast cancer cells is involved in engraftment and chemoattraction to the bone. Further experiments are underway in vivo to see if calcilytics can be repurposed as a therapeutic intervention for bone metastases from breast cancer.

#2860

RCP augments oral cancer cell invasion through Zeb1 and Slug expression.

Jin Young Kim, Kyung Hwa Cho, Bo Young Jeong, Zi Hae Song, Chang Gyo Park, Hoi Young Lee. _College of Medicine Konyang University, Daejeon, Republic of Korea_.

Background: Rab coupling protein (RCP), also called family interacting protein 1 (Rab11fip1) is a protein encoded by the RAB11FIP1 gene and has been known to augment cancer tumorigenesis, invasion, and metastasis. Recently, RCP was reported to aggravate cancer cell invasion and metastasis by stabilizing beta1 integrin and inducing epithelial-mesenchymal transition (EMT) in breast and ovarian cancer. Further, a high RCP expression was significantly correlated with shorter overall survival and disease-free survival of HNSCC patients. However, the molecule mechanisms by which RCP increases HNSCC invasion and metastasis are unclear. In the present study, we demonstrate the role of RCP in oral cancer cell invasiveness and its underlying mechanism.

Methods: Oral cancer YD-10B and YD-38 cells were transiently transfected with RCP constructs. siRNAs were used to analyze the role of specific proteins for Slug and Zeb1 expression. Immunoblotting and immunofluorescence were used to analyze protein expression. Matrigel-coated in vitro insert and wound healing assay were used to analyze cancer cell invasion and migration, respectively.

Results: We observed that ectopic expression of RCP induces invasion of oral cancer cells. Conversely, silencing of RCP expression increased E-cadherin expression and reduced oral cancer cell invasion. In addition, RCP upregulated Zeb1 and Slug expression that is important for RCP-induced oral cancer cell EMT and invasion. Furthermore, we identified that silencing of beta1 integrin efficiently attenuates RCP-induced Zeb1 and Slug expression. In addition, we observed that a selective inhibitor of EGFR kinase activity, Gefitinib, significantly reduces RCP-induced Zeb1 and Slug expression and that RasN17 recovers RCP-induced Zeb1 expression. Finally, RCP increased the viability of oral cancer cells against a chemotherapeutic agent cisplatin.

Conclusion: Collectively, we demonstrate that RCP augments oral cancer cell EMT and invasiveness through a beta1 integrin/EGFR/Ras/Slug and Zeb1 signaling axis, providing potential biomarkers for oral cancer patients therapy.

#2861

ACKR3 promotes cell metastasis via activating TGF-beta 1/Smad signaling in head and neck squamous cell carcinoma.

Nayoung Kim, Solbi Kim, Mina Joo, Heung Jin Jeon, Myung-Won Lee, Hyewon Ryu, Hyo Jin Lee. _Chungnam National University, Daejeon, Republic of Korea_.

Background: The atypical chemokine receptor, ACKR3, has been shown to play as a tumor promoter, which is upregulated in various types of cancer. However, the biological role of ACKR3 and its underlying mechanism in head and neck squamous cell carcinoma (HNSCC) is still unknown. In this study, we investigated the functional role of ACKR3 and the underlying molecular mechanism of tumor progression in HNSCC.

Methods: We examined the ACKR3 expression and its association with clinicopathological characteristics in 103 cases of HNSCC by immunohistochemical staining. The biological roles of ACKR3- and ACKR3-induced downstream signaling were investigated in HNSCC cell through ACKR3 overexpression and knockdown of ACKR3 in vitro and in vivo.

Results: We detected differential expression of ACKR3 in HNSCC cells and tissues. High ACKR3 expression was significantly associated with invasion depth of tumor (T status; P = 0.007), lymph node involvement (N status; P = 0.004), and higher stage III/IV (P = 0.02). Overexpression of ACKR3 dramatically enhanced cell migration and invasion in HNSCC cells in vitro, and promoted lymph node metastasis in vivo. ACKR3 overexpression also induced the epithelial-mesenchymal transition. Vimentin, Slug, and Twist were increased but E-cadherin and Ep-CAM were decreased by ACKR3 expression. Upregulation of TGF-β1 was observed in ACKR3 overexpression, and TGF-β1/Smad signaling activation and Akt phosphorylation were induced in HNSCC cells with ACKR3 overexpression. Treatment with a PI3K inhibitor reduced Slug and Twist levels while suppression of Smad2/3 signaling by siRNA reduced Akt phosphorylation, as well as Slug and Twist. Furthermore, inhibition of Smad2/3 decreased tumor cell migration and invasion in HNSCC. ACKR3 knockdown using siRNA in HNSCC cells reduced the secretion of TGF-β1 and also recovered the cell migratory and invasive behavior of HNSCC cells.

Conclusions: ACKR3 expression was associated with an aggressive tumor behavior in HNSCC. ACKR3 contributed to migration and invasion of HNSCC cell through the TGF-β1/Smad signaling axis and epithelial-mesenchymal transition in vitro, and was involved in lymph node metastasis in vivo, suggesting that ACKR3 plays an important role during HNSCC progression and metastasis.

#2862

Long isoform of VEGF stimulates cell migration of breast cancer by filopodia formation via NRP1/ARHGAP17/Cdc42 regulatory network.

Marina Kiso, National Hospital Organization Himeji Medical Center, Sunao Tanaka, Masakazu Toi, Fumiaki Sato. _Kyoto Univ. Graduate School of Medicine, Kyoto, Japan_.

Background and objective: Bevacizumab is an antibody of VEGF and suppresses tumor angiogenesis. It exhibited therapeutic efficacy for metastatic breast cancer, but impact on overall survival has not been proved. VEGF is a multi-function molecule. We revealed VEGF-related molecular mechanisms on breast cancer (BC).

Methods and Results: A VEGF-expressing BC cell line, MDA-MB-231 cells (WT231) were used. VEGF of WT231 cells was knocked out using Crisper-Cas9 system (231VEGFKOex3). 231VEGFKOex3 cells showed small and rounded morphology. Migration assay demonstrated the impaired cell migration of 231VEGFKOex3 cells. Bevacizumab treatment did not induce this morphologic change and impaired migration. Next, we generated soluble neuropilin-1 (sNRP1) overexpressed 231 cells (231sNRP1). sNRP1 traps VEGF to function as an antagonist. 231sNRP1 cells exhibited small and rounded morphology and impaired cell migration similar to that of 231VEGFKOex3 cells. VEGF165 (contains a NRP-binding domain) addition to 231VEGFKOex3 cells recovered WT231 cell-like morphology and induced cell migration. On the other hand, VEGF121 (doesn't contain NRP-binding domain) addition could not recover morphology and migration. As WT231 cells express almost no VEGFR, these results indicate that the interaction between NRP1 and long isoform of VEGF containing a NRP-binding domain regulates the morphology and migration ability of 231 cells. Genome-wide gene expression profiling identified ARHGAP17 as one of the target genes in the downstream of the VEGF/NRP1 signal. The expression of ARHGAP17 was upregulated in 231VEGFKOex3 cells. ARHGAP17 is a RhoGAP which inactivates Cdc42. Cdc42 is important for the formation of filopodia which required for cell functions such as migration. Scanning electron microscope revealed diminished filopodia formation in 231VEGFKOex3 cells compared to WT231 cells. As a result, 231VEGFKOex3 cells showed reduced migration. Our results indicate that VEGF/NRP1/ARHGAP17/Cdc42 signaling network controls filopodia formation and migration of the BC cells. Among 450 hormone-receptor-negative BC (HR(-)BC) cases from a public database, the ratio of VEGF and SEMA3A in primary tumor is associated with ARHGAP17 expression inversely, and with disease free survival.

Discussion: Bevacizumab and NRP1 bind VEGF at amino acid motifs from exon 3-4 and exon 7 of VEGF, respectively. Thus, our findings suggested that long isoform of VEGF could play a role in filopodia formation via VEGF/NRP1/ARHGAP17/Cdc42 singaling network for WT231 cells, and that bevacizumab could not block the signal. In conclusion, long isoform of VEGF can generate a signal through NRP1 without VEGFR into BC cells. In addition, we identified a target molecule of this signal, ARHGAP17, that regulates Cdc42 activity. VEGF/NRP1/ARHGAP17/Cdc42 regulatory network may contribute to malignant behaviors of HR(-)BC.

#2863

**Long non-coding RNA** LINC00152 **plays an oncogenic role via targeting MET, STAT3 signaling in esophagus adenocarcinoma.**

Matthew Xiao,1 Zhuwen Wang,1 Jules Lin,1 David G. Beer,1 Andrew C. Chang,1 Guoan Chen2. 1 _University of Michigan, Ann Arbor, MI;_ 2 _Southern University of Science and Technology, Shenzhen, China_.

Esophageal cancer remains a threat to public health with an increasing incidence and low survival rate world wide. Long non-coding RNA, LINC00152 has been linked to several human cancers and promotes cell proliferation in lung, gastric, hepatocellular, colorectal, and clear cell renal carcinoma. The expression of LINC00152 and its roles in esophagus adenocarcinomas (EAC), however, are unknown. The objective of this study is to investigate the role of LINC00152 in EAC. We used the esophageal cancer cell lines Flo-1, OE19, and OE33 for in vitro study. siRNA of specific to LINC00152 was used to knockdown LINC00152 followed by cell proliferation, colony formation, invasion and migration assays. Real-time PCR assay was used for detecting mRNA expression and Western blot for examining altered proteins expression affected by LINC00152 knockdown. Data analysis were performed using excel and Prism. The difference of variables was analyzed using the Student's T-test. We found that LINC00152 was increased in EAC as compared to Barrett, low-grad displasia or high-grad dysplasia. The LINC00152 expression was decreased more than 90% after LINC00152 knockdown with siRNA on Flo, OE19 and OE33 cells measured by RT-PCR. Cell proliferation, colony and invasion were decreased significantly after LINC00152 siRNA transfection on these EAC cell lines. Apoptosis was also induced. Mechanistic study indicated that MET protein was significantly decreased in OE33 cells after LINC00152 knockdown, while STAT3 decreased in Flo cells. P21, p27 and Myc was decreased in all 3 cell lines. In this study we found that LINC00152 knockdown could inhibit cell proliferation, colony formation, cell migration and invasion, as well as trigger apoptosis via targeting multiple oncogenic signaling. We also found LINC00152 was increased in EAC tumors. Our study indicates that LINC00152 plays an important role in EAC and is potentially a diagnostic marker. Further characterization of LINC00152 as a novel therapeutic target of EAC is warranted.

#2864

MAEA (macrophage erythroblast attacher) suppresses migration, invasion and enhances chemosensitivity in colorectal cancer cell lines.

Jae Ho Lo, Shivani Soni, Ryuma Tokunaga, Francesca Battaglin, Madiha Naseem, Alberto Puccini, Hiroyuki Arai, Tricia Ning, Martin D. Berger, Shu Cao, Wu Zhang, Josh Millstein, Heinz-Josef Lenz. _USC Norris Comprehensive Cancer Center, Keck School of Medicine, Los Angeles, CA_.

Purpose: MAEA, also known as Emp (Erythroblast Macrophage protein), plays an important role in actin cytoskeleton rearrangement in macrophages and erythroid cells. A recent study indicated that down-regulation of MAEA in mouse monocyte macrophage results in abnormal cell motility and higher expression of mitogen-activated protein kinase 1 (MAPK 1) and thymoma viral proto-oncogene 1 (Akt 1). However, the precise mechanism of MAEA in cancer remains unclear. In this study, we examined the role of MAEA in cell motility, invasion and chemosensitivity in colorectal cancer (CRC) cell lines.

Experimental Procedure: CRC cell lines were selected based on MSI/MSS status. MAEA was overexpressed in HCT116, SW480, LoVo, SW626 cell lines using vector pRP[Exp]-EGFP/Puro-CMV>hMAEA[ORF013172]. MAEA expression was quantified by qRT-PCR and immunoblotting. To determine the role of MAEA in migration and invasion, wound closure and transwell invasion assays were performed. Immunofluorescent staining was carried out to study actin cytoskeletal remodeling. The effect of MAEA on chemosensitivity was evaluated by using MTS assay after treatment with 5-FU, oxaliplatin and irinotecan. Both cell motility and DNA repair related genes expression profile were analyzed by human RT2 Profiler PCR array.

Result: Our results showed that up-regulation of MAEA significantly suppressed migration and invasion in HCT116 cell line (MSI, RAS mutant, BRAF wild type, APC wild type). Furthermore, immunofluorescence staining indicated actin cytoskeleton projections were disrupted with up-regulation of MAEA. In MTS assay, MAEA overexpressed HCT116 demonstrated enhanced chemo sensitivity to 5-FU, oxaliplatin, and irrinotecan (35%, 40%, and 40% respectively). PCR array analysis showed significant downregulation of cell motility related genes AKT1, BCAR1, HGF, ITGA4, ITGB3, MAPK1, RHOB, SRC (2.4, 2.4, 50, 9.1, 2.0, 1.56, 2.38, and 2.38 folds respectively). Similarly, DNA repair related genes DMC1, ERCC1, ERCC2, ERCC3, ERCC4, RAD51D, RAD52 were downregulated (1.63, 1.64, 1.56, 1.66, 1.66, 2.0, 1.75 folds respectively).

Conclusion: Our findings show MAEA suppresses migration, invasion and enhances chemo sensitivity to first-line of treatment in colorectal cancer cell lines. Downregulation of both MAPK1 and AKT1 suggests that MAEA may be involved in PI3K-ATK and/or RAS-ERK pathways regulating cell motility and invasion. Downregulation of DNA repair related genes can be a plausible reason for enhanced chemosensitivity in MSI-high CRC cell lines. 

### Targets and Therapies in Pediatric Cancer

#2865

**AsiDNA** TM **, a novel DNA repair inhibitor to sensitize aggressive medulloblastoma subtypes.**

Sofia Ferreira, Chloe Foray, Sophie Heinrich, Celio Pouponnot, Marie Dutreix. _Institut Curie, Orsay, France_.

Purpose: Medulloblastoma is paediatric tumor of the cerebellum. It represents the most frequent malignant brain tumor in childhood. It can be classified into four disparate molecular subgroups but current therapies are not yet tailored to their specificities. This is particularly important for subgroups with worse prognosis as Group3. Patients who survive often present severe treatment-related morbidity, which can be largely attributed to brain radiation in ages of development. It is therefore important to improve treatment efficacy in more aggressive subgroups as well as reduce treatment-related morbidity across all subgroups. We investigated whether AsiDNATM, a radiosensitizer in clinical trials which works through DNA repair inhibition, could be used to improve radiotherapy efficacy with no additional toxicity allowing radiation dose de-escalation in groups with better prognosis and improved radiotherapy efficacy in those with high-risk disease.

Experimental design: The cytotoxic effect of AsiDNATM was determined in a panel of 4 human medulloblastoma cell lines from different subgroups and different P53 status. We analyzed the ability of AsiDNATM to reach brain tissue and toxicity of treatments with or without irradiation in pups (10 days old mice). Distribution of AsiDNA was estimated by quantification of Cy-5-AsiDNA in brain tissues. Radiation toxicity was estimated by survival to high doses, monitoring of circulating growth hormone (GH), weight loss, bones size at adult age and neuro histology analysis. We assessed the AsiDNATM radio-enhancing efficacy in Group3 xenografted preclinical models using X-RAD 320 (PXI Inc) X-ray irradiator. Local brain irradiation was performed with an image-guided X-ray system (SARPP, Xstrahl Ldt), in which dosimetry and animal setting were controlled at each treatment session.

Results: AsiDNATM treatments exhibit an additive effect to radiation in all cell lines, leading to an approximate 2-fold reduction of the radiation dose to reach 50% survival. Cy5-AsiDNA distributed to pup brain but not to adult brain devoid of tumors. However, in adults AsiDNATM distributed in tumors in brain through permeability of tumor vessels. Irradiation toxicity in pups was confirmed by a significant decrease in GH secretion and associated reduction of bone size. The lethal dose after irradiation was 20 Gy. No increase of irradiation toxicity was observed with AsiDNATM. In vivo, AsiDNATM alone significantly enhances survival rates (p=0.005) and increases radiotherapy efficacy. When combined with radiotherapy, AsiDNATM led to delay in tumor growth and survival improvement as compared to radiotherapy alone.

Conclusion: Overall, our current results show AsiDNATM as an attractive candidate to improve radiation therapy in medulloblastoma in both standard- and high-risk patients with no indication of additional toxicity in healthy developing brain tissues.

#2866

Surfaceome profiling in osteosarcoma: Identification of the candidate immunotherapeutic target.

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

Osteosarcoma (OS) is the most common primary malignant bone tumor in pediatric patients. Current developments in targeted therapeutic strategies show potential clinical implications in cancer treatments. However, immunotherapies such as antibody-drug conjugates (ADCs) or CAR-T cell therapy have not been sufficiently studied in OS because of the lack of tumor-specific target. In this study, we developed a multi-step RNA-seq based pipeline. First, we did RNA sequencing for our OS PDX models and 17 patient-derived OS cell lines. The results are then pooled with the RNQ-seq data from 111 OS patient via the Therapeutically Applicable Research to Generate Effective Treatments project (TARGET). All these tumor data are subsequently compared with normal tissue RNA-sequencing data from NIH Genotype-Tissue Expression (GTEx) database. The significantly differentially expressed genes (log fold change tumor versus normal >1 for each tissue, p < 0.01) are selected. We further filtered this gene list by cell surface protein prediction based on Gene Ontology, the TransMembrane prediction using hidden Markov models (TMHMM), and glycosylphosphatidylinositol (GPI)-anchored protein annotations. Based on the transcriptomic result, ranks of differentially expressed genes were generated. CD276, LCLAT1, and OR8B12 are the surface markers with high ranks. To validate the transcriptomic results, we prepared surface protein extraction of the PDX models and patient-derived OS cell lines. We are profiling these surface proteins by proteomic mass spectrometry. Further validation for the expression level and location of these markers are underway. Our current finding is not previously reported in OS. The results warrant further development of ADC and car-T cell therapy.

#2867

MDM2 and MDM4 are therapeutic vulnerabilities in malignant rhabdoid tumors.

Thomas P. Howard,1 Taylor E. Arnoff,1 Melinda R. Song,1 Andrew O. Giacomelli,1 Xiaofeng Wang,2 Andrew L. Hong,1 Neekesh V. Dharia,1 Su Wang,3 Francisca Vazquez,4 Minh-Tam Pham,1 Ann M. Morgan,1 Franziska Wachter,1 Gregory H. Bird,1 Guillaume Kugener,4 Elaine M. Oberlick,1 Matthew G. Rees,4 Hong Tiv,1 Justin H. Hwang,1 Katherine H. Walsh,4 April Cook,1 John M. Krill-Burger,4 Aviad Tsherniak,4 Prafulla C. Gokhale,1 Peter J. Park,3 Kimberly Stegmaier,1 Loren D. Walensky,1 William C. Hahn,1 Charles W. Roberts5. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Dartmouth Geisel School of Medicine, Lebanon, NH;_ 3 _Harvard Medical School, Boston, MA;_ 4 _Broad Institute, Cambridge, MA;_ 5 _St. Jude Children's Research Hosptial, Memphis, TN_.

Malignant rhabdoid tumors (MRT) are aggressive cancers of early childhood that are largely resistant to traditional therapies. MRT exhibit a remarkably low mutation rate, with no recurrent mutations beyond the defining biallelic inactivating mutation in SMARCB1, a core subunit of the SWI/SNF (BAF) chromatin-remodeling complex. Thus, MRT do not display traditional oncogenic mutations that are amenable to targeted therapies, limiting their use for this disease. In order to nominate new drug targets for MRT, we screened MRT cell lines with large-scale RNAi, CRISPR-Cas9, and small-molecule libraries. The most significant vulnerabilities consistent across all three screens were MDM2 and MDM4, the major negative regulators of p53. We found that MRT cell lines are more sensitive than other p53 wild-type cancer cell lines to both idasanutlin (MDM2-specific) and ATSP-7041 (MDM2/4-dual) in vitro. Both inhibitors induced substantial activation of the p53 pathway in MRT cell lines, which responded with permanent apoptotic or senescent cell fate decisions. CRISPR-Cas9-mediated inactivation of TP53 caused a significant resistance to these compounds, confirming that on-target mechanisms were responsible for MRT sensitivity. We found that loss of SMARCB1 sensitizes MRT cells to idasanutlin by shifting the p53 response towards apoptosis. In MRT xenograft studies, both idasanutlin and ATSP-7041 slowed tumor growth. Most strikingly, an idasanutlin pulse of only five days was sufficient to induce sizable regression of all tumors, which remained complete and durable in 50% of mice. Finally, gene expression analysis of primary MRT predicts that, like cell lines and xenografts, MRT in patients are likely to be sensitive to MDM2 inhibition. Collectively, these studies describe a genetic link between SWI/SNF complex mutations and p53, while providing evidence to support the use of MDM2 and MDM2/4 inhibitors that have already entered clinical trials for the treatment of this devastating pediatric cancer.

#2868

CD99 inhibition by clofarabine induces Ewing sarcoma cytotoxicity through activation of FGFR1 and downstream kinase pathways.

Haydar Celik, Levent Dusunceli, Anna Molotkova, David V. Allegakoen, Erin J. Conn, Jeff R. Petro, Jeffrey A. Torestky, Aykut Uren. _Georgetown University Medical Center, Washington, DC_.

Ewing sarcoma (ES) cells express high levels of the cell surface protein CD99, which is used for diagnosis of ES. More importantly, the inhibition of CD99 activity results in reduced growth of ES cells in vitro and in vivo. In an earlier publication, we have identified clofarabine and cladribine as selective inhibitors of CD99, which inhibited ES growth both in vitro and in vivo. Clofarabine and cladribine directly bound to CD99, inhibited its molecular interactions, and regulated CD99 specific intracellular signaling pathways. In order to gain further insight into how clofarabine may regulate cellular functions through inhibiting CD99, we utilized a phospho-kinase array to identify changes in phosphorylation levels of 43 proteins in response to clofarabine or CD99 antibody treatment. We identified ERK1/2-MSK1/2-CREB signaling axis as one of the signaling pathways downstream of CD99. These findings were validated in four different ES cell lines and in xenograft lysates that were harvested from clofarabine-treated mice compared to control mice. The phosphorylation events induced by clofarabine treatment was significantly diminished when CD99 was knocked down either by siRNA-mediated knockdown or CRISPR/Cas9-mediated genomic disruption. Time-course experiments on ES cells showed that clofarabine triggers a very rapid signaling cascade through CD99. Cytarabine, a structurally similar pyrimidine analog and an inhibitor of the EWS-FLI1 transcriptional activity that has been failed in clinical trials on ES patients, did not activate ERK1/2, MSK1/2 or CREB phosphorylation in ES cells. In order to test whether the observed changes in phosphorylation levels are required for cell death, we screened a kinase inhibitor small molecule library for their ability to rescue clofarabine-induced death of ES cells. We identified TG100-115 (PI3K inhibitor) and rebastinib (c-abl inhibitor) as the most potent kinase inhibitors that showed significant reversal of clofarabine-induced ES cell death, suggesting that clofarabine-induced death of ES cells requires specific phosphorylation cascades. We then discovered that CD99 can be co-immunoprecipitated with FGFR1 and clofarabine treatment of ES cells results in phosphorylation of FGFR1. In conclusion, we discovered a novel mechanism of action for clofarabine and cladribine in that their selective cytotoxic effect on ES is through inhibiting CD99 on the cell surface, and it is not dependent on their ability to inhibit DNA synthesis. CD99 inhibition resulted in increased phosphorylation of FGFR and two downstream signaling pathways (PI3K/Akt and MEK1/ERK1). Inhibition of PI3K pathway rescues clofarabine-induced ES cell cytotoxicity. These findings provide additional mechanistic support for repurposing clofarabine and cladribine for ES indication.

#2869

Zika virus is a potent oncolytic agent against aggressive human ependymoma.

Carolini Kaid,1 Patrícia Semedo-Kuriki,1 Ernesto Goulart,1 Luiz Caires-Júnior,1 Renato Astray,2 Oswaldo Keith Okamoto,1 Mayana Zatz1. 1 _Human Genome and Stem Cell Research Center, Institute of Biosciences - University of São Paulo, São Paulo, Brazil;_ 2 _Butantan Institute, São Paulo, Brazil_.

Ependymoma is a metastatic CNS tumor responsible for 10% of all pediatric brain tumors. Displaying a poor survival rate of only 32.5%, the conventional ependymoma treatment is surgery and radiation therapy associated to significant morbidity. Previous studies found significantly increased expression of NESTIN, a neural progenitor cell (NPC) marker, in aggressive ependymoma. Zika virus (ZIKV) has recently emerged as an oncolytic therapy against adult and pediatric CNS tumors due to its ability to infect NPCs and neural stem-like tumor cells. We showed for the first time that Brazilian Zika virus strain efficiently destroyed pediatric CNS tumor cells in vitro and in vivo, resulting in significantly mice longer survival and fewer metastasis. Once tumor cells overexpressing NPCs markers are more susceptible to ZIKV infection, here we evaluated the oncolytic properties of Brazilian ZIKV against human ependymoma. A patient-derived ependymoma cell line (USP21-EPE) has been established and found to abnormally express the pluripotency markers CD133, SOX2 and NESTIN. USP21-EPE monolayer culture showed massive cell death three days after ZIKV infection in four tested MOIs: 0.01, 0.1, 1 and 2. At MOI 0,1 50% of cell population decreased viability while at higher MOIs was observed 100% of cell death 72 hours post ZIKV infection. Moreover, ZIKV significantly disrupted CNS ependymoma tumorspheres reinforcing its oncolytic effect against the ependymoma tumor cells. Considering the poor effectiveness and severe side effects of available treatments for ependymoma and that most ZIKV infections are associated to very mild or no symptoms, our findings open new avenues for novel therapies against this aggressive pediatric tumor.

#2870

Genetic mechanisms of primary chemotherapy resistance in pediatric acute myeloid leukemia.

Nicole McNeer,1 John Philip,1 Heather Geiger,2 Rhonda E. Ries,3 Vincent-Philippe Lavallee,1 Michael Walsh,1 Minita Shah,2 Kanika Arora,2 Anne-Katrin Emde,2 Nicolas Robine,2 Todd A. Alonzo,4 E. Anders Kolb,5 Alan S. Gamis,6 Malcom Smith,7 Daniela Se Gerhard,7 Jaime Guidry-Auvil,7 Soheil Meshinchi,3 Alex Kentsis1. 1 _Memorial Sloan Kettering Cancer Center, Manhattan, NY;_ 2 _New York Genome Center, Manhattan, NY;_ 3 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 4 _University of Southern California, Los Angeles, CA;_ 5 _Nemours/Alfred Dupont Hospital for Children, Wilmington, DE;_ 6 _University of Missouri-Kansas City School of Medicine, Kansas City, MO;_ 7 _National Cancer Institute, Rockville, MD_.

Survival for children with acute myeloid leukemia (AML) remains low, with a primary induction remission failure rate of 10-15%. Although some mutations have been identified that are associated with increased relapse risk, the genomic landscape of pediatric AML resistant to primary chemotherapy is not well characterized. As part of the TARGET initiative (Therapeutically Applicable Research To Generate Effective Treatments), we assembled a cohort of 28 pediatric AML patients with primary chemotherapy resistance and failure of induction chemotherapy, uniformly treated as part of the COG AAML0531 study. We analyzed whole-genome DNA, mRNA, and miRNA sequence data obtained at diagnosis and after induction chemotherapy. At least three genetically distinct groups were apparent in this cohort, including those with NUP98 rearrangements, somatic mutations of WT1 in the absence of NUP98 variants, and additional recurrent variants including those in KMT2C and MLLT10. Comparisons of mutations before and after chemotherapy revealed both common and distinct gene expression programs. On chemotherapy exposure, these leukemias exhibited diverse forms of clonal evolution. We observed selection for mutant alleles of FRMD8, DHX32, PIK3R1, SHANK3, MKLN1, as well as persistence of WT1 and TP53 mutant clones, and elimination or contraction of FLT3, PTPN11, and NRAS mutant clones. These findings suggest diverse genetic mechanisms for primary chemotherapy resistance in pediatric AML, which should guide new approaches for its diagnosis and therapy.

#2871

Global reduction of H3K4me3 improves chemotherapeutic efficacy for pediatric ependymomas.

Guifa Xi,1 Nitin Wadhwani,1 Rintaro Hashizume,2 Barbara Mania-Farnell,3 Marcelo Bento Soares,4 Charles D. James,2 Tadanori Tomita1. 1 _Ann & Robert H. Lurie Children's Hosp. of Chicago, Chicago, IL; _2 _Northwestern University Feinberg School of Medicine, Chicago, IL;_ 3 _Purdue University Northwest, Hammond, IN;_ 4 _University of Illinois College of Medicine at Peoria, Peoria, IL_.

Background Ependymomas (EPNs) are the third most common brain tumor in children. These tumors are resistant to available chemotherapeutic treatments, therefore new effective targeted therapeutics must be identified. Increasing evidence shows epigenetic alterations including histone posttranslational modifications (PTMs), are associated with malignancy, chemotherapeutic resistance and prognosis for pediatric EPNs. In this study we examined histone PTMs in EPNs and identified potential targets to improve chemotherapeutic efficacy.

Methods Global histone H3 lysine 4 trimethylation (H3K4me3) levels were detected in pediatric EPN tumor samples with immunohistochemistry and immunoblots. Candidate genes conferring therapeutic resistance were profiled in pediatric EPN tumor samples with micro-array. Promoter H3K4me3 occupancy was examined for two candidate genes, CCND1 and ERBB2, with chromatin-immunoprecipitation coupled with real-time PCR (ChIP-PCR). These methods and MTS assay were used to verify a relationship between H3K4me3 levels and CCND1 and ERBB2, and to investigate cell viability in response to chemotherapeutic drugs in primary cultured pediatric EPN cells.

Results H3K4me3 levels positively correlate with WHO grade malignancy in pediatric EPNs and are associated with progression free survival in patients with posterior fossa group A EPNs (PF-EPN-A). Reduction of H3K4me3 by silencing its methyltransferase SETD1A, in primary cultured EPN cells, increased cell response to chemotherapy.

Conclusions Our results support the development of a novel treatment that targets H3K4me3 to increase chemotherapeutic efficacy in pediatric PF-EPN-A tumors.

Key words: promoter, H3K4me3, human SETD1A, chemotherapy, pediatric ependymoma

#2872

Acquisition of drug resistance mutations during chemotherapy treatment in pediatric acute lymphoblastic leukemia.

Benshang Li,1 Samuel W. Brady,2 Xiaotu Ma,2 Shuhong Shen,1 Yingchi Zhang,3 Yongjin Li,2 Yu Liu,2 Ningling Wang,4 Diane Flasch,2 Matthew Myers,5 Heather Mulder,2 Lixia Ding,1 Yanling Lu,2 Liqing Tian,2 Kohei Hagiwara,2 Ke Xu,2 Edgar Sioson,2 Tianyi Wang,1 Liu Yang,1 Jie Zhao,1 Hui Zhang,2 Ying Shao,2 Hongye Sun,6 Lele Sun,6 Jiaoyang Cai,1 Ting-Nien Lin,2 Lijuan Du,1 Fan Yang,1 Michael Rusch,2 Michael Edmonson,2 John Easton,2 Xiaofan Zhu,3 Jingliao Zhang,3 Cheng Cheng,2 Benjamin Raphael,5 Jingyan Tang,1 James Downing,2 Bin-Bing Zhou,1 Ching-Hon Pui,2 Jun Yang,2 Jinghui Zhang2. 1 _Shanghai Children's Medical Center, Shanghai, China;_ 2 _St. Jude Children's Research Hospital, Memphis, TN;_ 3 _Chinese Academy of Medical Sciences, Tianjin, China;_ 4 _Second Hospital of Anhui Medical University, Hefei, China;_ 5 _Princeton University, Princeton, NJ;_ 6 _WuXi NextCODE Co., Shanghai, China_.

Acute lymphoblastic leukemia (ALL) is a leading cause of cancer-associated death in children. To study the mechanisms of drug resistance in ALL, we performed whole-genome sequencing of diagnosis-relapse-germline trios from 103 Chinese patients and ultra-deep sequencing of 208 serial bone marrow samples from 17 of them. Relapse-specific somatic alterations were enriched in 12 genes (NR3C1, NR3C2, TP53, NT5C2, FPGS, CREBBP, MSH2, MSH6, PMS2, WHSC1, PRPS1, and PRPS2), which were predominantly involved in response to thiopurines, glucocorticoids, methotrexate, and other drugs. Four lines of evidence indicate that these resistance mutations frequently developed during treatment, rather than pre-existing at diagnosis. First, two novel, relapse-specific mutational signatures (novel signatures 1 and 2), most likely caused by chemotherapeutic regimens, were detected in 15% and 14% of relapsed cases, respectively. Drug resistance mutations frequently appeared at novel signature-associated trinucleotide contexts, indicating that chemotherapy may directly cause drug resistance mutations in ALL. The signatures were validated in NCI TARGET relapsed ALL samples, 2% and 23% of which harbored novel signatures 1 and 2, respectively. The varying signature prevalence between cohorts may reflect treatment differences. The novel signatures were not detected in >2,000 adult cancers from the PCAWG study. Novel signature 1 induced C>G transversions, particularly at GCC and TCT trinucleotides, and showed transcription-strand bias indicating guanine adducts. Novel signature 2 favored C>T and C>G mutations at CCG, and correlated with relapse-specific dinucleotide variants and structural variants, indicating an agent causing multiple mutation types. The drugs inducing these novel signatures are being explored in vitro. Second, mathematical modeling using growth curves of drug-resistant ALL indicated that drug resistance mutations occur, in some cases, long after diagnosis, during active treatment. Third, some patients acquired multiple drug resistance mutations sequentially through successive relapses, a finding inconsistent with their pre-existence at diagnosis. Indeed, 20% of relapses had multiple drug resistance mutations targeting different drug classes. Fourth, most relapsed ALLs derived from a subclone detected at diagnosis, which then evolved additional mutations, including drug resistance mutations, not detectable at diagnosis using 2000X targeted sequencing. Drug resistance mutations were often subclonal at relapse, suggesting later appearance. Together these data indicate that fully drug-resistant clones may not necessarily pre-exist at diagnosis in ALL, but may be acquired later during treatment. Thus, early intensive or targeted treatment strategies in slow responders may forestall the subsequent development of drug resistance mutations.

#2873

Elimination of MYCN-amplified neuroblastoma cells by telomerase-targeted oncolytic virotherapy as novel precision medicine.

Morimichi Tani,1 Hiroshi Tazawa,1 Terutaka Tanimoto,1 Hiroshi Nouso,1 Yasuo Urata,2 Shunsuke Kagawa,1 Takuo Noda,1 Toshiyoshi Fujiwara1. 1 _Okayama University, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan;_ 2 _Oncolys BioPharma, Inc., Japan_.

Background: Neuroblastoma (NB) is a primary malignant tumor of the peripheral sympathetic nervous system. Recent whole-genome sequencing studies have demonstrated that high-risk NB with unfavorable clinical outcome is characterized by amplification of MYCN oncogene and rearrangement of human telomerase reverse transcriptase (hTERT) gene. Since hTERT expression is transcriptionally and commonly activated in high-risk NB, hTERT activation would be an attractive therapeutic target for the treatment of high-risk NB. To eliminate hTERT-activated malignant tumor cells, we have developed two types of hTERT-driven oncolytic adenoviruses, OBP-301 and tumor suppressor p53-armed OBP-702. In this study, we investigated the therapeutic potential of hTERT-driven oncolytic adenoviruses OBP-301 and OBP-702 in MYCN-amplified NB cells.

Methods: We used 4 human NB cell lines with MYCN amplification (IMR-32, CHP-134, NB-1, LA-N-5). The expression of cell surface coxsackie and adenovirus receptor (CAR) and hTERT was analyzed using flow cytometric analysis and real-time RT-PCR. We assessed the in vitro antitumor effect of OBP-301 and OBP-702 using a XTT assay. The molecular mechanism of virus-mediated antitumor effect was investigated in aspect of apoptosis, autophagy, and MYCN expression. To address the underlying mechanism of virus-mediated MYCN modulation, we analyzed the role of E2F1 and p53 in the virus-infected NB cells. The in vivo antitumor effect of OBP-301 and OBP-702 was assessed using a subcutaneous CHP-134 xenograft tumor model.

Results: All MYCN-amplified NB cells showed CAR and hTERT expression. OBP-301 and OBP-702 showed profound antitumor effect in association with autophagy-related cell death in all MYCN-amplified NB cells. In the virus-infected NB cells, E2F1 expression was inversely correlated with MYCN expression. Virus-induced E2F1 and p53 expression suppressed the MYCN expression in the MYCN-amplified NB cells. OBP-301 and OBP-702 significantly suppressed tumor growth in a subcutaneous CHP-134 xenograft tumor model.

Conclusion: Our results suggest that hTERT-driven oncolytic adenoviruses OBP-301 and OBP-702 are promising antitumor agents for eliminating the MYCN-amplified NB cells through E2F1- and p53-mediated suppression of oncogenic MYCN protein. Further clinical study is warranted to evaluate the safety and feasibility of hTERT-driven oncolytic virotherapy as novel precision medicine for high-risk NB patients.

#2874

De novo **acquisition of RD3 loss with intensive multi-modal therapy harmonize neuroblastoma relapse.**

Dinesh Babu Somasundaram,1 Karthikeyan Subramanian,1 Sheeja Aravindan,2 Zhongxin Yu,1 Mohan Natarajan,3 Terence S. Herman,1 Natarajan Aravindan1. 1 _University of Oklahoma Health Sciences, Oklahoma City, OK;_ 2 _Stephenson Cancer Institute, Oklahoma City, OK;_ 3 _University of Texas Health Sciences Center, San Antonio, TX_.

The majority of high-risk neuroblastoma that initially responds to therapy will ultimately relapse. Acquired genetic/molecular rearrangement contributes to tumor relapse and currently, no salvage treatment regimens are known to be curative. Our studies identified the loss of retinal-degeneration-protein-3 (RD3) in progressive disease (PD) and its association with poor clinical outcomes. Herein we investigated the acquisition of RD3-loss after current standard of care (SOC, intensive multimodal therapy) in neuroblastoma. Acquired transcriptional (RNA in situ hybridization, QPCR) and translational (IHC on a custom archived cell micro array, immunoblotting) loss of RD3 with SOC was investigated with bed-to-bench approach utilizing a panel of 15 stage-4 patient derived cell-lines with known therapy status, diagnosis [DX] vs. PD after SOC). Further, RD3-loss in progressive disease, its direct role in the pathogenesis disease progression (cell migration, invasion, tumorosphere formation and metastatic potential) was investigated using mouse model of progressive neuroblastoma and in experimental in vitro and ex vivo settings coupled with gene manipulation strategies. In addition, RD3 status in response to therapy were also affirmed by in silico analysis of independent studies on bed-to-bench and experimental models. Results demonstrated RD3-loss (transcriptional/translational) acquisition with SOC in human PD. Experimental in vitro, in vivo, ex vivo studies affirmed RD3 loss in PD and, further forced RD3-rearrangements in these models defined its functional role in PD pathogenesis. Data-mining experimental studies with salvage therapeutic agents affirmed the acquisition of RD3-loss in resistant cells and in residual tumors. For the first time, these results demonstrate the de novo acquisition of RD3 loss in PD after intensive multi-modal therapy. Defining the criticality of RD3-loss in PD and the defined role of RD3 loss in PD pathogenesis, benefit of targeted reinforcement could lead to improved salvage therapy for high-risk neuroblastoma.

#2875

Targeted therapy of Ewing's sarcoma by human anti CD99 targeted hybrid polymerized liposomal nanoparticles (HPLNs) encapsulating anticancer agents.

HyungGyoo Kang,1 Jon Nagy,2 Sheetal Mitra,1 Triche Triche1. 1 _USC/Children's Hospital Los Angeles, Los Angeles, CA;_ 2 _NanoValent Pharmaceuticals Inc., Bozeman, MT_.

Most anticancer therapies are unspecific and therefore inevitably lead to off-target toxicity. To overcome these shortcomings, targetable hybrid polymerization liposomal nanoparticles (HPLNs) have been invented. Multiple innovative and proprietary technologies have been developed to enable creation of this unique form of nanoparticle therapy with covalent attachment of targeting molecules such as tumor specific antibodies or peptides. Human monoclonal anti-CD99 antibody-targeted HPLNs encapsulating irinotecan (CD99-HPLN/Ir) can efficiently reach implanted Ewing sarcoma tumors in xenograft mice and dramatically reduce and/or eliminate the tumor burden. Complete tumor ablation has been observed at doses as low as 1 mg irinotecan/kg, treated twice per week. In other cases, animals who failed untargeted (HPLN/Ir) treatment showed complete tumor ablation after 'salvage' treatment with CD99-HPLN/Ir. Our CD99 targeted HPLN/irinotecan formulation, termed NV103 showed much better efficacy than the commercial untargeted liposomal irinotecan (Onivyde™) and doxorubicin (Doxil™). Toxic side effects, normally associated with systemic administration of free irinotecan, were minimized or undetectable. Our nanoparticles showed excellent bioavailability. Even the untargeted HPLN/Ir improve drug bioavailability six-fold in a comparison experiment with the drug in liposome form (Onivyde™), with no discernable systemic toxicity. Experiments using human antiCD99 antibody targeted HPLNs demonstrated they can be used for delivery of therapeutic nucleic acids including siRNA, ASO or functional CRISPR-Cas9 systems against EWS-FLI1. Targeted HPLN/ siRNA and ASO against EWS-Fli1 reduced the protein expression of EWS-Fli1 by 35% and 65%, respectively. Our results of HPLN/CRISPR-Cas9 systems showed the high efficient delivery of CRISPR-Cas9 components and a 70% knockdown of EWS-FLI1 expression in vitro. In the initial animal study our work showed the systemic administration of human monoclonal anti-CD99 antibody-targeted HPLNs encapsulating CRISPR-Cas9 against EWS-Fli1 could reduce the tumor growth of Ewing tumor successfully. In conclusion, our targeted HPLNs can be used as a clinically viable method for the targeted delivery of varied therapeutics including chemotherapeutic agents (Doxorubicin and Irinotecan), therapeutic nucleic acids (siRNA and ASO) and CRISPR-Cas9 systems. Ultimately, after development of other tumor-specific targeting agents and varied encapsulated therapeutics, this technology can be usefully adapted for the treatment of various cancers as well.

#2876

p21-activated kinase 1 is a novel therapeutic target for metastatic osteosarcoma harboring p27 mislocalization.

Xiang Chen,1 Justin Cates,2 Yuchen Du,1 Yun Jung Sung,1 Xiao-nan Lo,3 John Hicks,1 Tsz-Kwong Man1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _Vanderbilt University Medical Center, TN;_ 3 _Northwestern University, IL_.

Lung metastasis is the leading cause of death in osteosarcoma (OS), the most common bone cancer in children and young adults. Due to a lack of biomarkers and therapeutic targets, significant progress has not been made in the treatment of metastatic OS over the past three decades. The survival of metastatic patients remains dismal. We have previously reported that the tumor suppressor protein p27 (KIP1, CDKN1B) is frequently mislocalized in the cytoplasm of OS, which promotes the motility and invasiveness of the tumor cells and the development of pulmonary metastases in mice. In this study, we showed that a higher proportion score of cytoplasmic p27 significantly correlated with metastases developed within 3- or 5-year of follow-up, and poor disease-specific and event-free survival in a large OS cohort (n=136). Using immunoprecipitation followed by mass spectrometry, we demonstrated that cytoplasmic p27 interacted with p21-activated kinase 1 (PAK1), where the two proteins co-localized in the cytoplasm of OS cells. PAK1 is a serine/threonine kinase that has been implicated in various cancers; however, its role in OS is still elusive. We showed that the phosphorylations at serine-144 and threonine-423 residues of PAK1 were higher in the p27-mislocalized cells relative to the control cells. The PAK1 phosphorylations increased actin stress fiber formation in the tumor cells. Gene silencing or therapeutic inhibition of PAK1 reduced the motility of p27-mislocalized cells in vitro. Similar results were observed in other cancer cell lines known to have the p27 mislocalization, suggesting PAK1 is a common and potential downstream effector of p27-mediated metastasis. An experimental metastasis mouse model further showed that a PAK1-silenced mutant had a significant impairment in the development of pulmonary metastasis. In summary, our study indicates that cytoplasmic p27 is a novel prognostic biomarker in OS. In the tumor cell, the mislocalization activates PAK1-mediated cell motility and metastasis. Targeting PAK1 in metastatic OS harboring the p27 mislocalization may be exploited as a potential therapeutic intervention to improve the poor outcome of the affected children.

#2877

Genomic cfDNA analysis of aqueous humor in retinoblastoma (RB) predicts eye salvage.

Liya Xu,1 Jesse L. Berry,2 Irsan Kooi,3 A Linn Murphree,2 Rishvanth K. Prabakar,1 Mark W. Reid,4 Kevin Stachelek,4 Bao Han A. Le,2 Lisa Welter,1 Rima Jubran,4 Thomas C. Lee,2 Jonathan W. Kim,2 Peter Kuhn,1 David Cobrinik,2 James B. Hicks1. 1 _University of Southern California, Los Angeles, CA;_ 2 _Children's Hospital Los Angeles and the USC Roski Eye Institute, Los Angeles, CA;_ 3 _Leiden, the Netherlands, Leiden, Netherlands;_ 4 _Children's Hospital Los Angeles, Los Angeles, CA_.

Background: Retinoblastoma (RB) was one of the first tumors to demonstrate a genetic basis to the development of cancer. However, unlike other cancers, RB cannot be directly biopsied due to the high risk of extraocular cancer spread. Therefore, unless the eye is enucleated, tumor tissue is not evaluated for genetic and genomic changes and these alterations are not used to inform diagnosis or prognosis for this disease. However, in a 2017 publication in JAMA Ophthalmology, we demonstrated that tumor-derived cell-free DNA can be extracted from the aqueous humor (AH) of RB eyes, which is safe to remove even with active intraocular disease. The purpose of this current study (Berry JL, Xu L et al. Molec Canc Res 2018) was to identify somatic chromosomal copy number alterations (SCNA) in tumor-derived cell-free DNA in the AH of RB eyes and to correlate with clinical outcomes particularly tumor relapse requiring enucleation.

Methods: AH was extracted via paracentesis from RB eyes during intravitreal injection of chemotherapy or post enucleation. Shallow whole genome sequencing was performed to assess for cell-free tumor DNA fractions and highly-recurrent SCNAs in RB which include gain of 1q, 2p, 6p and loss of 13q and 16q. Globe salvage was recorded.

Results: 26 patients were included; 3 patients had both eyes included for 29 eyes. From these, 63 samples of AH were analyzed; 5 post-enucleations and 58 during intravitreal chemotherapy injection. Ultimately 13 eyes required enucleation and 16 eyes were salvaged. Follow-up ranged from 8-43 months (median 17 months).

The presence of any detectable SCNA was 92% in enucleated eyes versus 38% in salvaged eyes (p=0.006). 6p gain was the most common SCNA found in 77% of enucleated eyes versus 25% of salvaged eyes (p=0.0092). 6p gain was associated with a ten-fold increased odds of enucleation (OR=10, 95% CI:1.8-55.6). The mean amplitude of 6p gain was 1.47 in enucleated eyes versus 1.07 in salvaged eyes (p=0.001). The probability of ocular survival was higher in eyes without detectable SCNAs in the AH (p=0.0028).

Serum testing was positive for a germline mutation in 17 eyes of 14 patients with the following incidence of RB SCNAs 1q (35%); 2p (18%); 6p (47%); 13q (18%) and 16q (35%) versus 12 eyes of 12 patients without germline disease with 1q (33%); 2p (8%); 6p (50%); 13q (17%) and 16q (33%) (p=0.71).

Conclusions: This is the first study to show that clinical outcomes correlate with highly-recurrent SCNAs in the AH from RB eyes. This study suggests that the AH can reliably serve as a surrogate to tumor biopsy and improves upon current clinical staging to predict tumor response to therapy and the ability to salvage the eye. Unlike previous studies that suggest a greater incidence of RB SCNAs in patients with non-germline disease, this was not seen in this evaluation which may be because all previous work was done on tumor from enucleated eyes instead of salvaged eyes.

#2878

**CRISPR-Cas9 screens identify the nuclear export factor NXT1 as a novel therapeutic target in** MYCN- **amplified neuroblastoma.**

Clare F. Malone,1 Neekesh V. Dharia,1 Guillaume Kugener,2 Brenton Paolella,2 Michael Rothberg,2 Mai Abdusamad,2 Alfredo Gonzalez,2 Nancy Dumont,2 Scott Younger,2 David Root,2 Francisca Vazquez,2 Kimberly Stegmaier1. 1 _Dana Farber Cancer Institute, Boston, MA;_ 2 _The Broad Institute, Cambridge, MA_.

Pediatric cancers, such as neuroblastoma, have a relative lack of oncogenic mutations and the known drivers of these cancers are largely transcription factors, a target class notoriously difficult to "drug." In order to identify novel therapeutic targets for high-risk, MYCN-amplified neuroblastoma, we employed functional genomic screening using a CRISPR-Cas9 approach. We generated genome-scale CRISPR dependency data to identify a candidate gene list of 197 putative genetic dependencies in neuroblastoma. We next created a focused sgRNA library targeting these genes and performed time-course dropout screens using CRISPR and CRISPRi and Annexin-V-based positive-selection cell death screens in four MYCN-amplified neuroblastoma cell lines. We also screened this library in vivo in two xenograft models of MYCN-amplified neuroblastoma. At the intersection of these screens, we identified the nuclear export factor NXT1 as a top candidate for therapeutic development in neuroblastoma. NXT1 scores as a strong dependency using both CRISPR and CRISPRi, is strongly enriched in our Annexin-V based positive selection cell death screen and scores in our in vivo screen. We have validated that NXT1 is indeed a genetic dependency in neuroblastoma in low-throughput and that NXT1 loss induces apoptosis in these cell lines. We were next interested in identifying biomarkers of dependency on NXT1, and by integrating RNAseq data with CRISPR dependency data, we identified low expression of NXT2, a paralog of NXT1, as the top predictive feature of NXT1 dependency. Importantly, while NXT1 itself is not currently druggable, inhibitors against another nuclear export protein, CRM1, are currently in Phase II clinical trials, demonstrating this class of proteins has the potential to be effectively drugged. Together, we show that CRISPR-Cas9 functional screens can be used to identify new therapeutic targets, particularly in diseases that lack "druggable" oncogenic drivers, such as many pediatric cancers.

#2879

TYRO3 is a potential therapeutic target in pediatric acute myeloid leukemia.

Sherri K. Smart,1 Manali Rupji,2 Bhakti Dwivedi,2 Jeanne Kowalski,3 Deborah DeRyckere,1 Douglas K. Graham1. 1 _Emory University/Aflac Cancer and Blood Disorders Center, Children's Health Care of Atlanta, Atlanta, GA;_ 2 _Winship Cancer Institute, Atlanta, GA;_ 3 _Winship Cancer Institute/Emory University, Atlanta, GA_.

BACKGROUND: While outcomes for children with acute myeloid leukemia have improved dramatically in recent years, a subset of patients continue to have poor prognosis with <60% 5-year overall survival and the high intensity chemotherapy used to treat these patients is associated with significant short and long-term side effects. New less toxic and more effective treatments are needed. TYRO3, a member of the TAM (TYRO3, AXL, MERTK) family of receptor tyrosine kinases, is overexpressed in many types of cancer and functions to promote tumor cell survival and/or proliferation, metastasis, and resistance to chemotherapy. In addition, higher levels of TYRO3 expression are associated with decreased overall survival in patients with colorectal, hepatocellular or breast cancer. We hypothesize that TYRO3 plays a critical role in survival and proliferation of AML cells and may be a therapeutic target in pediatric AML.

METHODS: The TARGET database was utilized to determine expression of mRNAs encoding the TAM family kinases and their ligands, GAS6 and PROS1, in pediatric AML patient samples. Samples (n=145) were categorized into two groups with "high" expression greater than the median or "low" expression less than or equal to the median and overall survival was compared between the two groups using the log-rank test. Expression of TAM family kinases and ligands was determined in AML cell lines by immunoblot. Derivatives of the NB4 AML cell line with shRNA-mediated inhibition of TYRO3 were generated and the impact of TYRO3 inhibition on MERTK levels was determined by immunoblot.

RESULTS: High levels of TYRO3 or PROS1 mRNAs in patient samples were independently associated with decreased overall survival in children with AML (p<0.001 and p= 0.042, respectively). Patients with leukemias that expressed higher levels of TYRO3 had a median survival of 870 days with only 40% of patients surviving for 10 years. In contrast, 70% of patients with leukemias that expressed lower levels of TYRO3 were long-term survivors. A hazard ratio of 2.60 (1.56-4.33 95% confidence interval, p<0.001) was associated with high levels of TYRO3. Similarly, patients whose leukemias had higher levels of PROS1 expression had a median survival of 1229 days with 46% 10-year overall survival compared to 62% 10-year survival in patients whose tumors express low levels of PROS1. High levels of PROS1 were associated with a hazard ratio of 1.65 (1.01-2.68, p=0.044). TYRO3 protein was detected in all 9 AML cells lines tested; however, MERTK, GAS6, and PROS1 were differentially expressed. Inhibition of TYRO3 in NB4 cells using shRNA resulted in upregulation of MERTK.

CONCLUSIONS: TYRO3 was ubiquitously expressed in all AML cell lines tested and higher levels of TYRO3 and its ligand PROS1 were associated with significantly poorer prognosis in children with AML. These data suggest a role for TYRO3 in pediatric AML and implicate TYRO3 as a therapeutic target in this context.

#2880

Targeted drug therapies for osteosarcoma.

Leanne C. Sayles, Amanda Koehne, Kieren Marini, Alex G. Lee, Stanley G. Leung, Avanthi T. Shah, Marcus R. Breese, E. Alejandro Sweet-Cordero. _University of California San Francisco, San Francisco, CA_.

Osteosarcoma (OS) is a highly aggressive cancer that has had no new treatments options in over 30 years. OS is a heterogeneous disease which is characterized by widespread and recurrent somatic copy-number alterations (SCNAs) with relatively few recurrent point mutations. We have previously reported that SCNAs contain key oncogenic drivers that can be used to identify patient-specific candidates for targeted therapies (Sayles and Breese, et al. Cancer Discovery 2018). Using patient-derived tumor xenografts (PDX), we demonstrated that targeting of patient-specific oncogenes within SCNAs leads to significant decrease in tumor burden. However, no single agent therapy was able to regress tumors completely suggesting that combination therapies would be required for disease management. In order to assess the applicability of OS PDX models to the patient tumor and the stability of the oncogenes within SCNAs, we preformed whole genome sequencing (WGS). We observed that SCNAs are highly stable across samples from the same patient in addition to multiple PDX passages and PDX derived cells lines, highlighting the equivalence between the PDX models and their derived cell lines to the human disease. This allows us to use the PDX cell lines as a surrogate for the identification of combination drug therapies that may be of benefit in OS. Currently, we have generated six PDX cell lines that encompass the most common SCNAs observed in patients including MYC, CCNE1 and CDK4 gain and alterations in the PI3K/PTEN pathway. We have performed single agent drug screens in these cell lines and have identified heterogeneous responses as would be anticipated by the genomic diversity observed in OS. We are currently testing combination drug therapies and will validate using the PDX xenografts in vivo. We have found that MYC driven OS PDX cell lines and PDX xenografts show a striking re-sensitization to cisplatin after AURKB inhibitor (barasertib) pretreatment. We have two PDX and derived cell lines that were generated from highly aggressive metastatic disease. Both of these PDX have high MYC amplification and were resistant to cisplatin, which is part of the standard of care. We pretreated the PDX cell lines for 24hrs with barasertib then added cisplatin and saw a synergistic effect in vitro. Further studies in the PDX model demonstrated a decrease in tumor volume for the combination therapy compared to vehicle or either single agent alone. Additionally, we observed retention of the platinum adduct 24 hrs after cisplatin dosing only when tumors were pretreated with barasertib. Further work is needed to assess the mechanism of this synergy and whether it can be of use in other SCNA driven OS and to identify other possible combination therapies that could be of importance in this disease.

#2881

Antiproliferative effect of copper complex of clotam in neuroblastoma cell lines.

Yazmin Hernandez,1 Sarah Sarah Grebennikov,1 Emily Zakhary,1 Ashni Dudhia,1 Rajasekhar Maram,1 Diego Casanova,1 Umesh T. Sankpal,1 W. Paul Bowman,1 Deondra T. Brown,2 Jaya Chhabra,2 Alvin A. Holder,2 Riyaz M. Basha1. 1 _Univ. of North Texas Health Science Ctr., Fort Worth, TX;_ 2 _Old Dominion University, Norfolk, VA_.

Neuroblastoma (NB) is the most common extracranial solid tumor in infants and young children. NB often metastasizes to lymph nodes and bone marrow. When compared to low or intermediate risk tumors, high risk neuroblastoma (HRNB) is more aggressive with low (40-50%) 5 year survival rates. Furthermore, current intensive treatment methods induce higher toxicity and side effects. In search of identifying less-toxic agents to induce anti-cancer activity in NB cells, our group reported the anti-cancer activity of a non-steroidal anti-inflammatory drug, Clotam (TA) using HRNB cell lines potentially inhibiting the transcription factor, Specificity protein 1 (Sp1) and an inhibitor of apoptosis protein, survivin. Recently, our laboratories tested a copper complex of TA (Cu-TA) using 12 cell lines and found that Cu-TA's IC50 values were 30-80% lower when compared to TA. In this study, we investigated the cytotoxicity of SH-SY 5Y, LA1-55n and SMS-KCNR cells in the presence of vehicle (Dimethyl sulfoxide) or TA or Cu-TA using CellTiter-Glo kit and determined the IC50 values using SigmaPlot software. The effect of TA or Cu-TA on apoptosis and cell cycle phase distribution was evaluated by flow cytometry using Annexin-V and Propidium Iodide kits respectively. The characterization and stability of the Cu-TA was also determined. SH SY5Y and SMS-KCNR cells were treated with IC50 values of TA or Cu-TA and the expression of Sp1 and survivin was measured by qPCR and Western blot analysis. The results showed that while both TA and Cu-TA inhibited NB cell growth, Cu-TA's IC50 values were 60-70% less than TA. The inhibition of NB cells with Cu-TA was accompanied by induced apoptotic cell population and cell cycle arrest at G2M phase. Western blot and qPCR results confirmed that Cu-TA inhibited the expression of both Sp1 and survivin with predominant effect on SMS-KCNR cells. These results demonstrate that Cu-TA is more effective than TA for inhibiting Sp1 and survivin in NB cells, which was consistent with the anti-proliferative activity against NB cells. Since TA and Cu-TA are less toxic than standard chemotherapeutic agents, employing these agents for the treatment of pediatric malignancies could be useful. Eventually, combination of Cu-TA and chemotherapeutic agents will be tested. If Cu-TA sensitizes NB cells to chemotherapy, it will help to address the issues associated with the side effects of chemotherapy.

#2882

Mithramycin impedes EWS-FLI1 driven transcription by preventing target promoter clearance.

Guillermo Flores, Susan Kitchen-Goosen, Brandon Oswald, Elissa Boguslawski, Marie Adams, Ian Beddows, Zachary Madaj, Patrick Grohar. _Van Andel Institute, Grand Rapids, MI_.

Background: Ewing sarcoma (ES) is a primary bone tumor of adolescence with poor prognosis. ES is characterized by the presence of EWS-FLI1 which acts as an oncogenic transcription factor to promote and maintain a tumorigenic phenotype. All ES cells express EWS-FLI1, however, other actionable mutations are exceedingly rare. We previously identified mithramycin (MMA) as a potent and specific EWS-FLI1 inhibitor. MMA effectively decreases ES tumor cell viability and tumor volume in vivo by reversing the EWS-FLI1 transcriptional signature. Structural and biochemical data suggest that sub-micromolar MMA increases the stability of the DNA binding domain of EWS-FLI1 at GGAA repeats. These data suggest that MMA may prevent EWS-FLI1 from successfully promoting Pol II transcriptional elongation.

Methods: We used ChIP-qPCR to detect EWS-FLI1 binding at the promoter of well characterized targets. We used a Pol II processivity assay to measure the efficiency of transcriptional elongation at EWS-FLI1 targets. We use RT-qPCR, western blotting, and RNAseq to confirm that MMA sensitizes ES cells to elongation inhibitors. Finally, we use GROseq to identify the changes to Pol II dynamics at EWS-FLI1 targets following MMA treatment.

Results: Our ChIP-qPCR data demonstrate an increased fraction of EWS-FLI1 DNA binding at the promoters of target genes with GGAA microsatellite regions. MMA significantly inhibits the processivity of Pol II at EWS-FLI1 targets when added both into a run-on reaction or following ES cell pretreatment. Combining MMA with elongation inhibitors induces strong synergy as measured by Bliss-Independence, renders multiple ES cell lines unviable by inhibiting the effects of EWS-FLI1 at the mRNA and protein level, and is effective in vivo. MMA biases GROseq reads toward the 5' end of genes, a result consistent with a promoter trapping mechanism. We are currently correlating GROseq data to global EWS-FLI1 DNA binding using ChIPseq.

Conclusions: We characterized a mechanism of MMA-induced EWS-FLI1 inhibition that involves increased stabilization of the fusion protein at target promoter regions. This mechanism is supported by changes to EWS-FLI1 DNA-binding, EWS-FLI1 target transcriptional processivity, and a reversal of the EWS-FLI1 transcriptional signature by combining MMA with downstream elongation inhibition. We hope that this work will inform future endeavors to develop a targeted therapy for ES that inhibits the main driver of disease, EWS-FLI1.

#2883

Mithramycin inhibits KDM6A (UTX) and SWI/SNF to induce apoptosis in malignant rhabdoid tumor.

Maggie H. Chasse,1 Elissa Boguslawski,1 Katie Sorensen,1 Ian Beddows,1 Kristin Rybski,2 Zachary Madaj,1 Susan M. Kitchen-Goosen,1 Patrick J. Grohar1. 1 _Van Andel Institute, Grand Rapids, MI;_ 2 _Oakland University William Beaumont School of Medicine, Rochester, MI_.

BACKGROUND: Rhabdoid tumor (RT) is an aggressive pediatric malignancy defined by the genetic deletion of SMARCB1 (BAF47/SNF5/INI1), a subunit of the SWI/SNF chromatin remodeling complex. SMARCB1 loss redistributes the SWI/SNF complex to favor a proliferative non-differentiated cellular state. Nevertheless, while EZH2, the catalytic subunit of PRC2, is an established, synthetically lethal therapeutic target, other vulnerabilities that result from this imbalance between the SWI/SNF and PRC2 complexes are relatively unexplored.

METHODS: In a screen of multiple pediatric cancer cell lines, we identified a heightened sensitivity of RT cells to mithramycin (MMA), a small molecule transcription inhibitor. We hypothesized the hypersensitivity of rhabdoid tumor to mithramycin was due to the disruption of SWI/SNF and PRC2 dynamics. Here, we describe an epigenetic mechanism of action for mithramycin in rhabdoid tumor. We characterize KDM6A, a H3K27me3 histone demethylase, as a novel therapeutic vulnerability in rhabdoid tumor with live cell imaging, western blot, small molecule inhibition, and chromatin immunoprecipitation (ChIP) sequencing. We identify SWI/SNF eviction from chromatin as a novel mechanism of action for mithramycin with biochemical fractionation, ChIP-seq, and ATAC-seq. Finally, we demonstrate these findings in vivo using RT xenograft models.

RESULTS: Mithramycin induced apoptosis and a striking increase in H3K27me3, the catalytic mark of PRC2, in rhabdoid tumor cell lines in a dose and time-dependent manner. This amplification of H3K27me3 preceded suppression of cellular proliferation and induction of apoptosis suggesting H3K27me3 amplification may drive the apoptotic phenotype. In addition, knockdown of KDM6A, a H3K27me3 histone demethylase, potentiates the effects of MMA providing further evidence that KDM6A loss and H3K27me3 amplification lead to apoptosis. We have identified SP1 as the transcription factor driving KDM6A and mithramycin interferes with SP1-dependent activation of KDM6A by evicting SWI/SNF from chromatin. We are currently working on the mechanism of action for mithramycin in rhabdoid tumor cells, including H3K27me3 ChIP-seq and ATAC-seq to elucidate global transcription and chromatin changes. We have recapitulated these results in vivo with xenograft mouse models.

CONCLUSIONS: Mithramycin inhibits KDM6A, a histone demethylase, and SWI/SNF leading to apoptosis through the amplification of H3K27me3. Eviction of SWI/SNF from chromatin is a novel mechanism of action for mithramycin. Overall, this study indicates KDM6A and SWI/SNF loss are novel therapeutic vulnerabilities in rhabdoid tumor and mithramycin may be a promising clinical candidate for the treatment of rhabdoid tumor.

#2884

Establishment of patient-derived xenograft models for high-risk neuroblastoma.

Alvin Kamili,1 Andrew J. Gifford,1 Nancy Li,1 Chelsea Mayoh,1 Georgina L. Eden,1 Jinhan Xie,1 Robyn E. Lukeis,2 Murray D. Norris,1 Michelle Haber,1 Geoffrey McCowage,3 Toby N. Trahair,4 Jamie I. Fletcher1. 1 _Children's Cancer Institute Australia, Randwick, NSW, Australia;_ 2 _Cytogenetics Laboratory, SydPath, St Vincent's Hospital, Darlinghurst, NSW, Australia;_ 3 _Cancer Centre for Children, Children's Hospital at Westmead, Westmead, NSW, Australia;_ 4 _Kids Cancer Centre, Sydney Children's Hospital, Randwick, NSW, Australia_.

Background: High-risk neuroblastoma (HR-NB) accounts for 15% of all pediatric oncology deaths, with long-term survival approximately 50%. Predictive preclinical models play an important role in the assessment of new treatment strategies and as avatar models for personalized medicine. We investigated the feasibility of establishing patient-derived xenograft (PDX) models of HR-NB from a range of tumor-bearing patient materials at diagnosis and relapse, and assessed techniques that may improve engraftment times and success rates.

Methods and Results: Tumor materials were obtained from 12 HR-NB patients, including primary and metastatic solid tumor biopsies, bone marrow mononuclear cells, pleural fluid and residual cells from cytogenetic analysis. Samples were processed and implanted subcutaneously into immunocompromised mice. The engraftment success rates were 33% (4/12) for diagnosis samples and 100% (6/6) for relapse samples. Median engraftment time to 1000mm3 tumor was 93.5 days (range 33–210 days). Engraftment at diagnosis was associated with poor outcome. PDX models were validated against primary material by histology, short-tandem repeat analysis, and single nucleotide polymorphism (SNP) array. PDXs matched the donor patient specimen in all but one case, where attempted engraftment from pleural fluid resulted in EBV-associated lymphoid proliferation. Attempted engraftment of tumor derived from a lymph node metastasis was associated with human T-lymphocyte infiltration of the mouse liver and spleen. Matched PDX models established directly from tumor biopsy and via short-term cytogenetics culture were equally similar to the donor tumor by SNP array and histological analysis. We directly compared engraftment rates for three different patient tumors at subcutaneous, intramuscular and orthotopic implantation sites. For two patient tumors, orthotopic engraftment was significantly faster than other sites, while the third model is ongoing.

Conclusion: HR-NB PDX models can be established from diverse sample types, allowing us to develop a preclinical testing platform for new therapies and the capacity to model personalized therapy. Engraftment of relapse samples can be readily achieved, while engraftment at diagnosis may portend poor prognosis. The engraftment time of neuroblastoma patient samples might be accelerated by orthotopic implantation.

#2885

Angiotensin-(1-7) prevents doxorubicin-induced cardiotoxicity by reducing oxidative stress and fibrosis.

Patricia E. Gallagher, Omeed Rahimi, Brian Westwood, Jay Kirby, Jasmina Varagic, Elisabeth Ann Tallant. _Wake Forest School of Medicine, Winston-Salem, NC_.

Doxorubicin (Dox) is an effective chemotherapeutic agent for various malignancies including childhood leukemias and lymphomas. Unfortunately, Dox administration often results in a cumulative dose-dependent cardiotoxicity that manifests as cardiomyopathy with marked oxidative stress and fibrosis leading to a long-term reduction in cardiovascular function and heart failure. Adjunct therapies to mitigate Dox-induced cardiotoxicity and enhance long-term quality-of-life in cancer patients are needed, particularly for pediatric patients. Angiotensin-(1-7) [Ang-(1-7)] is an endogenous heptapeptide hormone of the renin-angiotensin system with cardioprotective properties. We investigated whether adjunct Ang-(1-7) attenuates cardiotoxicity resulting from acute (6 week) exposure to Dox (21-24 mg/kg) in male and female juvenile rats (n = 7-10). Dox treatment reduced body/heart mass in male and female rats and co-administration of Ang-(1-7) had no effect on these morphological changes. However, co-administration of the heptapeptide hormone prevented Dox-mediated diastolic dysfunction, including decreased passive filling (E; 16%, p<0.05) and mitral annulus tissue movement (e'; 45% in males and 31% in females, p<0.001) and the subsequent increased filling pressure (E/e'; 47% in males and 43% in females, p<0.001). In both male and female rat hearts, Dox administration increased NADPH oxidase 4 (Nox4; 2-fold in males and females, p<0.05), the major Nox isozyme that generates ROS in the heart, as well as 4-hydroxynonenal (4-HNE; 7.5-fold in males and 3.5-fold in females, p<0.001) and malondialdehyde (MDA; 20-fold in males and 15-fold in females, p<0.001), markers of lipid peroxidation. Co-administration of Ang-(1-7) prevented these effects of Dox while Ang-(1-7) alone had no effect. Treatment of rats with Dox and Ang-(1-7) also increased cardiac superoxide dismutase 1 (SOD1; 4-fold in males and 5-fold in females, p<0.05 and p<0.01 respectively) and catalase (10-fold in males and 4-fold in females, p<0.05), further contributing to reduced oxidative stress. Oxidative stress is associated with an increase in cardiac fibrosis, a maladaptive change that exacerbates cardiac dysfunction. Treatment of male and female rats with Dox increased collagen deposition in the heart which was associated with an increase in the fibrogenic cytokine TGF-β1 and TGF-β1-activated phospho-SMAD2; co-administration of Ang-(1-7) mitigated the Dox-induced increase in fibrosis while the heptapeptide hormone alone had no effect. The anti-oxidant and anti-fibrotic effects of Ang-(1-7) may account for the improvement in diastolic function by the heptapeptide hormone, suggesting that treatment with Ang-(1-7) may serve as an effective adjuvant to improve Dox-induced cardiac dysfunction.

#2886

The Hippo pathway protein YAP mediates resistance to MEK1/2 inhibition in neuroblastoma.

Grace E. Coggins,1 Alvin Farrel,1 Komal Rathi,1 Colin M. Hayes,1 Tracy Tang,2 Leonard Post,2 John M. Maris1. 1 _Children's Hospital of Philadelphia, Philadelphia, PA;_ 2 _Vivace Therapeutics, San Mateo, CA_.

Background: Relapsed NBs harbor an enrichment in mutations activating RAS-MAPK signaling pathway. The MEK1/2 inhibitor trametinib has shown tumor growth delay but not regressions in NB preclinical models. Recent studies have implicated the Hippo pathway transcriptional coactivator protein YAP in relapsed NBs as well as trametinib resistance in other cancers. We hypothesized that increased YAP transcriptional activity is a mechanism of MEK1/2 inhibition resistance in RAS-driven NBs.

Methods: We screened a panel of NB cell lines for mutations predicted to hyperactive the RAS pathway and for high YAP expression. NLF and SK-N-AS cells were treated with the MEK1/2 inhibitor trametinib (20 nM) over 72hrs, and changes in YAP protein expression and cellular localization were observed via immunoblot. YAP protein expression was modulated in NLF, SK-N-AS, and SK-N-FI cells by siRNA-mediated knockdown, CRISPR-Cas9 genetic knockout, and constitutive overexpression. Effects on survival, proliferation, and signaling were observed with and without trametinib treatment by CellTiter-Glo cell cycle analysis, RT-qPCR, and immunoblot. Synergy studies were performed to assess the combination of trametinib and a YAP/TEAD transcriptional activity inhibitor (Vivace Therapeutics) using Incucyte and CellTiter-Glo.

Results: In nuclear extracts of NLF (biallelic NF1 inactivation) and SK-N-AS (NRAS Q61K), trametinib caused a near-complete translocation of YAP protein into the nucleus after 72 hrs. Transient knockdown and genetic knockouts of YAP in untreated NLF cells had a modest effect by decreasing proliferation by 20% (p<0.01), while combinatorial inhibition of MEK and YAP signaling caused a significant reduction in cellular proliferation by 70% (p<0.001). Overexpression of the constitutively-active form of the YAP protein, YAP-5SA, rescued this effect. Finally, synergy assays between trametinib and a YAP-TEAD transcriptional activity inhibitor produced combination index values< 1, indicating strong synergy.

Conclusion: Prolonged trametinib exposure causes a marked nuclear enrichment of active YAP, and co-inhibition of MEK and YAP in NB models effectively reduces proliferation and survival. Genetic depletion of YAP in NB cell lines caused a significant increase in sensitivity to trametinib, which was rescued with overexpression of a constitutively-active YAP-5SA. We have identified a potential drug combination to target a YAP-driven mechanism of trametinib resistance in RAS-driven NBs to support future development of a novel combination therapy for NB patients.

#2887

**Patient-derived 3-dimensional models of pediatric tumors for** in situ **study of cancer biology, immunotherapy, and drug treatment.**

Padraic P. Levings, Juan M. Urueña, Eric O. McGhee, Alex J. McGhee, Derek L. Hood, Kylie E. Van Meter, Steve H. Ghivizzani, Colin J. Anderson, Wallace G. Sawyer. _University of Florida, Gainesville, FL_.

The 2D culture systems are currently the mainstay of pre-clinical testing of potential therapeutics, however, these models fail to recapitulate the dynamic and heterogenous conditions found within the in vivo tumor microenvironment and are poor predictors of clinical activity. Patient derived xenograft models of cancer excel recapitulating these complexities but are not the most tractable approaches for high throughput efficacy testing, particularly of immuno-oncologic therapies. In addition, small animal imaging techniques to noninvasively and longitudinally monitor PDX tumor growth require costly equipment and a high level of technical expertise. Thus, there is a tremendous need for in vitro model systems with improved diagnostic accuracy to better predict therapeutic responses earlier in drug development. This need is even more acute for pediatric cancers such as osteosarcoma (OS) and glioblastoma (GB), for which there are few or no viable non-clinical models to investigate basic science or for pre-clinical drug efficacy studies.We have developed a 3D culture system integrating 3D printing in Liquid Like Solids (LLS) materials to enable the precise arrangement of delicate and highly detailed assemblies of cells, that include, fibroblasts, vascular endothelial cells, and/or activated T-cells and extra-cellular matrix components-with a modular perfusion system that uses a negative pressure chamber to draw fluid through the culture chamber and assay workspace without disturbing the positioning of the experiments. Real-time imaging in these 3D assays is performed in situ using confocal microscopy under controlled perfusion. In this study we present preliminary results from in situ studies of drug cytotoxicity, angiogenesis, and fibroblast activation, as well as, T cell migration and infiltration in bio-fabricated patient derived 3D tumor microtissues of OS and GB. We assessed the impact of oxygen tension, DNA damage, ER stress, and stromal composition (matrix and cell types) on response to frontline therapies, tumor angiogenesis, and T cell activation. Secretion of cytokines, pro-inflammatory factors and the release of damage-associated molecular patterns by microtumor constructs cultured under each condition was measured by ELISA. These data highlight the unique capabilities of our integrated confocal microscope and 3D passive perfusion culture system for studies of cancer biology, immunotherapy, and drug treatment regimens to provide in situ measurements of tumor-stroma interactions and immune cell invasion dynamics in precision fabricated OS and GB patient derived 3D microtissues.

#2888

Targeting the fatal pediatric brain tumors AT/RT and DIPG with the DNA binding agent quinacrine.

Harpreet Kaur, Huizi Guo, Peter Green, Sepehr Akhtarkhavari, Smit Shah, Charles G. Eberhart, Eric H. Raabe. _Johns Hopkins Univ. School of Medicine, Baltimore, MD_.

Atypical teratoid/rhabdoid tumors (AT/RT) and diffuse intrinsic pontine glioma (DIPG) are incurable pediatric brain tumors. Developing new targets and novel therapeutics are urgently needed. We have previously shown that multiple primary brain tumors and cell lines express increased amounts of the epigenetic modifier high mobility group AT-hook 2 (HMGA2). HMGA2 is a DNA-binding oncoprotein that regulates transcription during normal embryogenesis and in cancer stem cells. Targeting HMGA2 using short hairpins significantly decreased AT/RT and glioma growth and increased survival of xenografted mice. We hypothesized that pharmacological inhibition of HMGA proteins using DNA minor-groove binding drugs like quinacrine will decrease AT/RT and DIPG growth due to displacement of HMGA proteins from the DNA. We used quinacrine in ten patient-derived cell lines: five AT/RT (BT37, CHLA-05, CHLA-06, BT-12, CHLA-266) and five DIPG (JHHDIPG1, SUDIPGXIII, JHHDIPG16A, SF7761, HSJD-007). Quinacrine has been used in millions of humans to treat malaria and parasitic infections, has a well-known safety profile and can penetrate the brain. Using quinacrine fluorescence as a surrogate, we can achieve therapeutically efficacious micromolar concentration of quinacrine in the mouse and zebrafish brain after oral administration without overt toxicity. In both tumor cell lines, quinacrine causes a dose-dependent reduction in growth (MTS) and proliferation (BrdU) compared to vehicle-treated cells (P<0.01). Treatment of both tumor lines with quinacrine significantly increased apoptosis (cleaved caspase-3 and cleaved PARP) compared to control cells (P<0.01). Quinacrine had no effect on growth of normal hindbrain cells. Our results suggest that minor groove binding drugs like quinacrine are a viable potential treatment strategy for these lethal tumors. Ongoing studies include validating the in vivo efficacy of quinacrine using zebrafish and mouse models of AT/RT and DIPG. Future studies are aimed at investigating the mechanism of quinacrine in these devastating tumors.

#2889

The Yes-Associated Protein plays a role in tumor formation, stemness, and therapy response in high risk neuroblastoma.

Jenny Shim, Yoo Joo Lee, Kaitlyn Janssen, Alexa Fritz, Kelly C. Goldsmith. _Emory University/CHOA, Atlanta, GA_.

Neuroblastoma (NB) is the most common extra-cranial solid tumor of childhood. Despite intensive multimodal therapy, >50% of high risk (HR) NB patients relapse with chemotherapy-resistant disease. RAS/RAF/MAPK pathway mutations and increased Yes-Associated Protein (YAP) transcriptional activity are more prevalent in relapsed NB compared to diagnostic tumors. YAP is a transcriptional co-activator, regulated by the Hippo pathway that binds with TEAD in the nucleus to activate genes promoting cell growth, self-renewal, and survival. YAP aberrant transcription has been implicated in many tumor types, yet the role for YAP in NB is unknown. YAP protein is heterogeneously expressed in HR NB cell lines. We used human derived NB cell lines stably transduced to express (NGPYAP) or inhibit (SK-N-ASshYAP, NLFshYAP) YAP to further characterize YAP's role in NB. While TEAD expression increases with YAP genetic inhibition, expression of YAP paralog TAZ did not change. SK-N-ASshYAP xenografts in NSG mice showed significantly delayed time to tumor formation and slower growth compared to empty vector transduced xenografts. Unmodified NB cells grown as neurospheres in neural basal media showed increased YAP expression and stemness markers, OCT4 and SOX2, by RT-qPCR. OCT4 and SOX2 were dependent on YAP expression as they did not increase in NLFshYAP neurospheres. Furthermore, NLFshYAP had decreased number and size of neurospheres compared to control. Given YAP's role in tumor stemness, we treated unmodified NB cells with 13-cis-retinoic acid (RA) to see if YAP-induced stemness would overcome RA-induced differentiation. Interestingly, YAP expression and downstream targets, OCT4 and SOX2, increased in response to RA treatment. This suggests that RA paradoxically induces YAP transcriptional activity and may be selecting for a more chemoresistant tumor initiating phenotype. Lastly, YAP knockdown sensitized NRAS- and NF-1-mutated NBs to the MEK inhibitor, Trametinib, and to chemotherapy in vitro. In adult cancers, YAP's role in therapy resistance is due to its regulation of anti-apoptotic Bcl-2 family proteins. However, YAP knockdown or induced expression in NBs did not change Bcl-2 family protein levels, nor did it affect Bim binding patterns to Mcl-1 or Bcl-2. Thus, investigations are ongoing to understand how YAP negatively regulates Trametinib response. We treated NB cells with Verteporfin (VP), an FDA-approved drug used to treat macular degeneration that inhibits YAP-TEAD interactions in other cancers. While VP inhibited YAP expression in NBs, it also had significant off target effects, potently killing YAP null NBs via light-activated generation of reactive oxygen species. Given the relevance of YAP in relapsed NBs and its effects on NB tumor formation, stemness, and therapy resistance, the need for targeted and specific YAP inhibitors is critical to reinstating therapy sensitivity at relapse.

#2890

Paternal pre-conception stress as a potential risk factor for neuroblastoma.

Sung-Hyeok Hong, Susana Galli, Raquel Santana da Cruz, Lu Jin, Larissa Wetlisbach, Shiya Zhu, Abir Malik, Jason Tilan, Sonia de Assis, Joanna B. Kitlinska. _Georgetown University, Washington, DC_.

Neuroblastomas (NBs) are pediatric tumors that lack adequate treatment for their aggressive form, despite many efforts focusing on novel therapies. Although the disease is known to have a genetic background, its etiology cannot be explained solely by genetics. Yet, the role of environment in NB development is understudied. Thus, there are no established risk factors for NB aside from rare genetic predispositions and therefore no preventative measures. NBs arise due to defects in sympathetic neuron (SN) differentiation, which occurs in the fetus. Thus, we hypothesize that prenatal or pre-conception exposures may interfere with this process and promote NB. Maternal stress during pregnancy and paternal pre-conception stress induce neurodevelopmental defects in offspring by direct effects on fetal development or epigenetic changes, respectively. Indeed, we have previously shown that in a mouse model of NB prenatal stress interferes with SN differentiation in offspring, accelerating NB development and leading to its increased frequency and malignancy. The goal of the current study was to determine if a similar increase in risk of NB development can result from paternal pre-conception stress. To test the effect of paternal stress on NB, we subjected TH-MYCN males to 6 weeks of chronic unpredictable stress (CUS) prior to mating with wild type females and assessed the impact of stress on 1) miRNA content in the germline (RNAseq); 2) fertility and fetal survival; 3) NB frequency, latency, growth and metastasis in their hemizygous TH-MYCN offspring (periodic MRI, histological analyses). CUS resulted in significant changes in non-coding RNA content of the germline, including miRNAs and tsRNAs. Of particular interest was a stress-induced down-regulation of sperm miRNAs, including miR-200b and miR-128, which are involved in neuroblast differentiation, inhibit NB invasiveness and are inactivated in NB tumors. Paternal stress decreased the rate of successful pregnancies from 75% in controls to 37.5% and increased frequency of death in utero from 5.5% to 29.2% (p=0.02). Moreover, offspring of stressed fathers exhibited accelerated NB development (60% vs 0% of mice with tumors at 6 weeks of age for CUS and control groups, respectively; p=0.03) and growth (p=0.3), higher tumor frequency (60% vs 25% at 14 weeks of age, p=0.03), as well as a trend toward a more metastatic phenotype. However, these effects on NB formation and phenotype were observed solely in male offspring. In summary, our data provide the first evidence for the role of paternal preconception stress in NB development. If confirmed, our findings may open new avenues for NB prevention and identifying biomarkers of increased NB risk.

#2891

Landscape of infiltrating immune repertoire in pediatric solid tumors.

Arash Nabbi,1 Natalie Jäger,2 Sumedha Sudhaman,3 Pengbo Sun,2 S. Y. Cindy Yang,1 Kelsey Zhu,1 Marcel Kool,2 Komal Rathi,4 Karthik Kalletla,4 Pichai Raman,4 Yuankun Zhu,4 Joseph N. Paulson,5 David T. Jones,2 Uri Tabori,3 Adam C. Resnick,4 Stefan M. Pfister,2 Trevor J. Pugh1. 1 _Princess Margaret Cancer Centre, Toronto, Ontario, Canada;_ 2 _Hopp-Children's Cancer Center at the NCT Heidelberg (KiTZ), Heidelberg, Germany;_ 3 _The Hospital for Sick Children, Toronto, Ontario, Canada;_ 4 _Children's Hospital of Philadelphia, Philadelphia, PA;_ 5 _Genentech Inc, San Francisco, CA_.

Introduction: Immune repertoire is a highly diverse pool of B and T cell receptors (B/TCR) that determine boundaries of immune surveillance. Pre-existing immune clones within Tumor MicroEnvironment (TME) suggest existence of a functional antigen presenting machinery and recognizing T cells, which fail to eliminate tumor, likely due to immunosuppressive environment. Shifts in composition of immune repertoire upon immune checkpoint blockade have been reported in adult cancers and shown to be associated with clinical response. As immunotherapy is being introduced to pediatric oncology, there is a need to better understand immunogenomic aspects of TME in childhood cancers.

Objective: We hypothesize that characteristics of TCRs in conjunction with gene expression profile of immune cells are key determinants of immune response in pediatric patients.

Methods: We compiled a pan-pediatric cohort and associated RNAseq datasets through multiple research initiatives. Participating programs include NCI TARGET (n ~ 270), International Cancer Genome Consortium (ICGC, n ~ 250) and Children's Brain Tumor Tissue Consortium (CBTTC, n ~ 790). As comparator, we analyzed 7 adult cancer types as well as data from constitutive mismatch repair deficiency (CMMRD) consortium. We devised a simple and reliable index to estimate immune diversity. We used CIBERSORT tool to infer immune fraction of TME and investigated immune-related gene expression. In partnership with Gabriela Miller's Kids First Data Resource Centre, analyses were performed on CAVATICA computational platform.

Results: While median number of RNAseq reads were comparable across TARGET, CBTTC and TCGA datasets (~ 67-90 million reads), ICGC dataset contained a median of ~ 208 million reads. However, number of reads mapped to immune loci were comparable across all datasets and appeared to be confounded by infiltration extent rather than technical discordance. Our preliminary results indicate Neuroblastoma harbored the highest median of inferred diversity across pediatric cancers (TRβ=59.38). Inferred immune content indicated a range of infiltration from 0.59 in Teratoma/Germinoma to 0.15 in Medulloblastoma. Immune checkpoint gene expression across pediatric cancers show overall downward trend compared to adult counterparts.

Conclusions: Our comprehensive study will serve as a common ground for researchers to delineate immune component of pediatric tumor microenvironment. Covered in this study are common solid tumors as well as rare malignancies, characterization of which will aid rational design of immunotherapy-related clinical trials of pediatric cancers.

#2892

Genetic inhibition of STAT3 increases medulloblastoma chemosensitivity.

Sutapa Ray, Donald Coulter, Phillip Wilder, Jason Sughroue, Nagendra Chaturvedi, Timothy Mcguire, Santharam Joshi, Graham Sharp, Kishor Bhakat, Angie Rizzino. _Univ. of Nebraska Medical Ctr., Omaha, NE_.

Medulloblastoma (MB) is the most common malignant brain tumor in children that arises from cerebellar neuronal progenitor cells. Post-surgical radiotherapy in combination with adjuvant chemotherapy are considered as standard treatment of care for MB. However, this long-term treatment often leads to harmful neurologic effects to the developing brain in children. Therefore, identification of novel therapeutic target to increase efficacy and reduce toxicity in MB represent an unmet clinical need. Signal transducers and activators of transcription-3 (STAT3) is constitutively activated in MB, where it functions as an oncoprotein, mediating cancer cell proliferation. Here, to evaluate the biological consequences of specific targeting of STAT3 in detail, we engineered MYC expressing Group 3 MB cells to stably express STAT3 shRNA under a doxycycline (Dox) inducible promoter and assessed the its effect on STAT3 function. Treatment of MB cells with Dox leads to growth inhibition and cell cycle arrest. Dox treatment downregulated expression of STAT3 and its target genes/proteins including MYC and delayed migration of MB cells. Expression of STAT3 shRNA decreased association of STAT3, BRD4, phospho-RNA Pol II to the proximal promoter of MYC and CCND1. It further downregulated occupancy of acetylated histone H3 K27 to the MYC and CCND1 promoter indicating gene repression. Moreover, we showed that combination Dox and cisplatin significantly decreased highly aggressive MYC-amplified MB cell growth and induced apoptosis by downregulating STAT3 regulated proliferation and anti-apoptotic gene expression. Together, our results revealed an important role of STAT3 in regulating MB pathogenesis. Disruption of this pathway with STAT3 inhibitor, therefore, may serves as a promising candidate for targeted MB therapy by enhancing chemo-sensitivity of MB cells, potentially improving outcomes in high-risk patients.

#2893

ALK amplification and rearrangements are recurrent targetable events in glioblastoma.

Anne-Florence Blandin. _Dana-Farber Cancer Institute, Boston, MA_.

Anaplastic Lymphoma Kinase (ALK) expression, rearrangements, and single nucleotide variants have been reported in several brain tumor types, but the significance and function of each aberration in adults and children have not been clearly established. To determine the degree to which ALK represents a relevant therapeutic target in gliomas, we first examined ALK expression using immunohistochemistry (IHC) in a panel of 148 adult GBM and 49 pediatric GBM/high grade gliomas. We identified high ALK expression was most frequent in pediatric gliomas (32%, 16/49, IHC score 2+ or 3+). Copy arrays/sequencing and FISH for the ALK locus revealed high levelALK amplification in 31% of ALK-expressing cases (5/16) but was a rare event in gliomas overall. Whole-genome sequencing and RNA sequencing identified novel and recurrent PPP1CB-ALKfusions in 43% of ALK-expressing pediatric GBM (7/16), suggesting IHC may be an efficient means of screening for ALK aberrations as in the identification of EML4-ALK lung cancer. All ALK- amplified cases harbored PPP1CB-ALK fusion but 2 PPP1CB-ALK cases were copy neutral. The phosphatase PPP1CB was fused in-frame to ALK at exon 20 with preservation of the ALK kinase domain and predicted to activate via the same mechanism as other ALK rearranged cancers. ALKfusion proteins promoted cell proliferation and constitutive kinase activity and upregulated STAT and AKT signaling pathways. However, expression of novel ALK missense mutations in NSCs did not promote proliferation. Administration of ALK inhibitor Crizotinib reduced tumor growth in a PDX GBM from a patient with a novel ALK fusion. This work validates amplification and rearrangement of ALK as a highly recurrent driver event and therapeutic target in GBM, particularly in young children where it may be the sole driver event.

### Tumor Evolution and Heterogeneity 1

#2894

**Overcoming intrinsic resistance to** BRAF **-inhibitors by modulating redox balance in** BRAF **-mutated melanoma cells.**

B. Bishal Paudel, Leonard A. Harris, Keisha N. Hardeman, Corey E. Hayford, Christian T. Meyer, Arwa A. Abugable, Darren R. Tyson, Joshua P. Fessel, Vito Quaranta. _Vanderbilt University, Nashville, TN_.

Post-resistant tumor analyses or static measurements provide most of our current knowledge of tumor recurrence. Unfortunately, these studies neither elucidate critical events that precede resistance nor explain the dynamics of drug response in cancer cells. Filling this knowledge gap may engender key advances in cancer therapy. Therefore, we coupled single-cell time-lapse microscopy, bioinformatics, and mathematical modeling to study the dynamics of drug response in BRAF-mutated melanoma cells. We recently reported a novel non-quiescent idling state that melanoma cells exhibit under continued drug exposure. Although Idling cells exhibit a constant population size, they continue to divide and die at the level of the single cell. We speculate that idling state, primed to acquire resistance, could be a new type of drug tolerance, and may constitute the bulk of minimum residual disease (MRD)often seen in the clinic in remission. Melanoma cells we considered, exhibited heterogeneous responses prior to convergence to idling state. These differential responses in melanoma can be defined within a mathematical framework of epigenetic landscape: initial heterogeneity reflects an early re-equilibration among distinct phenotypic basins while idling is a final equilibrated state. Thus, describing the molecular signatures that define basins in BRAF-mutated melanoma landscape, both drug-naïve and drug-induced, would provide rational strategies to alter the landscape and enhance the clinical benefits. To this end, we utilized single-cell-derived clones to find gene-signatures that correlate to drug-sensitivity and discovered that drug-naïve landscape could be defined with respect to distinct redox metabolism. We further confirmed and validated these signatures in CCLE dataset across a range of BRAF-mutated melanoma cells. Specifically, the antioxidants such as NADPH and GSH provide an early survival advantage to melanoma cells under BRAF-inhibition. We show that modulating expression of these metabolic genes changes the initial landscape, and synergizes with BRAF-inhibitors. The enhanced effects of combination can be mitigated by antioxidants specific to glutathione redox balance. These observations suggest that disruption of redox balance, in combination with BRAF-inhibition, may improve the outcomes of BRAF-targeted therapy. Additionally, this strategy will maximize initial cell killing, and hence reduce the number of cells that constitute MRD or idling state. Taken together, our work provides a unifying view of how melanomas respond to BRAF-inhibition and suggest that targeted landscaping is a rational way to suppress both an initial tumor and idling cells.

#2895

MBD4 inactivation results in an alternative evolutionary route in uveal melanoma.

Manuel Rodrigues, Lenha Mobuchon, Alexandre Houy, Samar Alsafadi, Sophie Gardrat, Sophie Piperno-Neumann, Nathalie Cassoux, Gaelle Pierron, Sergio Roman-Roman, Pascale Mariani, Marc-Henri Stern. _Institut Curie, Paris, France_.

Introduction

Uveal melanomas (UM) are chemoresistant tumors that carry few copy number variations (CNV) and a low mutation burden. Genomics of UM metastases have been scarcely described. We assessed genetic heterogeneity in UM at primary and metastatic stages, in order to evaluate if tumor heterogeneity may explain this chemoresistance.

Methods

Whole-exome sequencing of primary and metastatic UM tumors.

Results

We obtained 97 tumor samples from 26 patients (16 samples from 15 primary tumors and 81 samples from 50 metastases). MBD4 was inactivated in two cases (MBD4-deficient; MBD4def). All samples presented high variant allelic frequencies of UM driver mutations (Gαq pathway mutations, BAP1, SF3B1 and EIF1AX). Indeed, mutational statuses of these genes were identical between primary tumors and metastases, except for one MBD4def case that carried both SF3B1 and BAP1 mutations in the primary tumor, while the metastasis only carried a different BAP1 mutation. MBD4-proficient (MBD4pro) primary tumors presented a median of 11.5 single nucleotide variants (SNV; range 5-22); while MBD4pro metastases carried a median of 14 SNV (range 6-23) with a very high genetic homogeneity between samples. No mutation was recurrently acquired during the metastatic process. Metastases presented a higher number of copy number variations (CNV; median = 11; range 2-25) than matched primary (median = 5; range 2-18). Metastases-associated CNV were similar to that usually associated with UM primary tumors, including 8q gain, 1p loss, 1q gain, 6q loss and isodisomy 3. MBD4def samples were hypermutated with at least 266 mutations/sample (>20-fold increase compared to MBD4pro) with >70% of CpG>TpG. MBD4def cases presented a higher heterogeneity with less than 60% of common SNV concordance between tumors in a given patient. No major difference in CNV profiles was observed between MBD4def and MBD4pro samples.

Conclusion

MBD4pro are homogeneous diseases that evolve as metastases with few non-recurrent SNV and recurrent UM-typical CNV. These characteristics lead to simple evolutionary trees with a limited number of short clades and branches, supporting a punctuated evolutionary process. Genetic heterogeneity thus cannot explain the resistance to chemotherapy. In contrast, MBD4 inactivation in UM results in genetic heterogeneity, subclonality and risk of secondary resistance to anticancer drugs.

#2896

Impact of intratumoral heterogeneity on gene expression profiles and known biomarkers in FFPE tumor biopsies from early stage breast cancer patients.

Lucas Delmonico,1 Joan Chen,2 Trushna Desai,1 Lisa Barnett,1 Aladdin Shadyab,2 John Obenauer,2 Gilda Alves Brown,3 Mauricio Magalhaes Costa,4 Marcia Fournier1. 1 _Bioarray Genetics Inc, Farmington, CT;_ 2 _Rancho BioSciences, San Diego, CA;_ 3 _State University of Rio de Janeiro, Rio de Janeiro, Brazil;_ 4 _Americas Centro de Oncologia Integrada, and Academia Nacional de Medicina, Rio de Janeiro, Brazil_.

Background: Cancer is a dynamic disease that becomes more heterogeneous during its course, with cellular subpopulations with distinct molecular profiles across different regions. Because this intratumoral heterogeneity may result in varying levels of gene expression patterns and sensitivity to treatment across different regions of the tumor, it is important to understand patterns of heterogeneity to develop effective biomarkers. We conducted multiregion sampling within single breast tumor biopsies to evaluate the effect of intratumoral heterogeneity on expression of known and novel biomarkers.

Methods: We sampled three sequential regions of breast tumor biopsies to compare gene expression patterns determined by a novel biomarker panel (BA355), 5 known biomarkers (ERS1, ERBB2, PGR, AR, and MKi67) and a novel predictive classifier (BA100). RNA expression levels were measured using a proprietary custom designed nanoString nCounter assay. We investigated formalin-fixed paraffin-embedded (FFPE) breast tumor biopsies from a cohort of 40 early stage breast cancer patients (T1 N0 M0) collected prospectively. RNA was extracted from upper, middle, and lower regions of each biopsy, resulting in a total of 120 samples quantified and QC using standard procedures. Each layer consisted of 40 micrometers, with an interval of 50 micrometers of tissue. BA100 linear regression models were used to generate prediction scores. Analyses were done in R.

Results: Eighteen of the 120 samples had poor data quality (sample median = 0 or inter quantile range > mean + 2*sd). Further analyses only included 102 good quality samples corresponding to 34 FFPE biopsies. Among 33 of 34 samples, gene expression patterns between the three regions of the biopsies were highly correlated, with correlation coefficients ranging from 0.90-0.99 (p-values ranged from 2.07e-19 to 8.45e-319). Among 31 of 34 samples, the three regions were clustered together in cluster analysis. The coefficient of variation (CV) for ESR1 ranged from 0.37%-15.5%, with a majority (32 of 34) of the samples having CV <10%. While the CVs of ERBB2, PGR, and AR were within the same range as ESR1, MKI67 had much larger variation (CV ranged from 0.99%-173%). BA100 scores yielded consistent predictions for the three regions in 33 of 34 samples.

Conclusion: Examination of gene expression patterns across multiple regions of breast tumor biopsies did not reveal a significant impact of intratumoral heterogeneity on expression patterns using a novel gene signature panel. However, at the individual gene level, while a number of genes including ESR1, ERBB2, PGR, and AR displayed consistent gene expression, genes such as MKI67 showed significant variation, indicating that tumor heterogeneity may impact individual gene biomarkers. Further studies are ongoing to evaluate intratumoral heterogeneity.

#2897

Dynamic transdifferentiation programs in small cell lung carcinoma.

Priyanka Gopal, Kevin Rogacki, Craig D. Peacock, Mohamed E. Abazeed. _Cleveland Clinic, Clevleand, OH_.

Tumors must also have the ability to rapidly adjust to a mutable environment during the lifespan of the cell. This form of phenotypic plasticity is typified by biological programs regulated by a host of transcription factors in a well-coordinated "switch" that is initiated by both cell autonomous and microenvironmental cues. We sought to study these dynamics in small cell lung carcinoma (SCLC). First, we used k-means clustering to identify discrete transcriptional programs that are represented across SCLC using gene expression values from 53 and 71 SCLC cell lines and primary tumors, respectively. To investigate these programs further, we used our inventory of 67 patient-derived xenografts (PDX) to establish an ex vivo cell culture system that reconstitutes with high fidelity the gene expression values observed in the PDXs and the primary tumors from which these cells were derived. Our data demonstrates that gene expression groupings or clusters across samples mark distinct SCLC transcriptional states, representing intertumoral variation. However, in an intriguing demonstration of intratumoral heterogeneity, the very same groupings define morphologically and phenotypically distinct subpopulations within individual samples. Subpopulations were represented by suspending aggregates of very small cells (neuroendocrine or NEn), pre-suspension aggregates that are nested above mesenchymal-like cells and appear to give rise to the suspending aggregates (proneural or PN), pleomorphic cells growing as a monolayer composed of larger cells with visible cytoplasm and spindle-like membrane extensions (mesenchymal or MS), and semi-adherent cells that are physically positioned over the latter (neuroepithelial or NEp). WES demonstrated high genetic fidelity across all subpopulations, indicating that the significant morphologic and phenotypic differences were non-genetic. RNAseq and gene ontology was applied to differentially expressed genes to inform classifications. Using network analyses, we identified transcription factors (TFs) that function as central nodes in the gene expression networks of each population. Namely, we show that ASCL1 marks a rapidly proliferating neuroendocrine population (NEn), NEUROD1 marks a primitive neural population (PN) and YAP1 marks a slow-growing mesenchymal population (MS). We also developed a promoter-reporter fluorescent protein system that positions distinctly colored fluorescent proteins under the regulatory control of the promoters of the three TFs. We show that a Markov process models the transition probability of states. Taken together, our results show that human SCLC is comprised of distinct tumor subpopulations characterized by significant, non-genetic plasticity. We posit that understanding of the SCLC intratumoral ecosystem can create new opportunities to evolutionarily steer this tumor toward improved therapeutic results.

#2898

Stochastic variation in gene expression is selected during clonal evolution of spontaneous mouse mammary tumors.

Sara J. Felts, Xiaojia Tang, Virginia P. Van Keulen, Krishna R. Kalari, Larry R. Pease. _Mayo Clinic College of Medicine and Science, Rochester, MN_.

Molecular and cellular diversity in cancer cells enables evolutionary processes that govern tumor progression and contributes to treatment failures, but the mechanisms governing this variability remain incompletely understood. We previously described tumor-specific epigenetic alterations in DNA devoid of canonical CpG methylation sites throughout the genomes of spontaneous breast tumors which arise in genetically uniform, yet heterozygous, [FVB X BALB/c MMTV-NeuT+] F1 mice. Those changes were detected in purified tumor DNA using a template-assisted quantitative ligation assay employing locus-specific and SNP-specific oligonucleotide genotyping probes, and the assay was subsequently shown to be sensitive to epigenetic modifications of the templates. We now describe losses of heterozygosity similarly distributed throughout the transcriptomes of tumors in this same model. We quantified expressed alleles across a population of clonal tumors and tested each expressed variant for the occurrence of allelic ratio outliers. The results revealed variegated patterns of expression in hundreds of genes, highly significantly enriched in pathways typically co-opted by tumors (Molecular Mechanisms of Cancer, P < 10-10, IPA analysis). An astounding 87% (2634 of 3044) of expressed genes that could be measured using this approach were represented in at least one tumor. Furthermore, the frequency of these outliers in any one individual tumor was found to correlate strongly with the transcriptional repression of an additional large set of non-polymorphic genes. These findings reveal underappreciated, latent mechanisms driving sporadic errors in epigenetic programming that promote repression of a multiple cis-linked transcripts. This genome-wide molecular chaos presents as dysregulated cellular homeostasis and is the foundation for heritable diversity as tumors evolve.

#2899

Single-cell analyses reveal increasing intratumoral heterogeneity as an essential component of treatment resistance in small cell lung cancer.

C. Allison Stewart,1 Carl M. Gay,1 Yuanxin Xi,1 Junya Fujimoto,1 Neda Kalhor,1 Patrice M. Hartsfield,1 Hai Tran,1 Luisa Fernandez,2 David Lu,2 Yipeng Wang,2 Ryan Dittamore,2 Jianjun Zhang,1 Stephen G. Swisher,1 Jack A. Roth,1 Trudy G. Oliver,3 John V. Heymach,1 Ignacio I. Wistuba,1 Bonnie S. Glisson,1 Paul Robson,4 Jing Wang,1 Lauren A. Byers1. 1 _University of Texas M.D. Anderson Cancer Center, Houston, TX;_ 2 _Epic Sciences, San Diego, CA;_ 3 _University of Utah, Huntsman Cancer Institute, Salt Lake City, UT;_ 4 _The Jackson Laboratory, Farmington, CT_.

Small cell lung cancer (SCLC) is an aggressive malignancy characterized by rapid onset of platinum-resistance. However, mechanisms underlying platinum-resistance remain obscure due to scarcity of tissue samples from relapsed patients. We generated circulating tumor cell (CTC)-derived xenograft (CDX) models from SCLC patients that recapitulate patient tumor genomics and response to platinum chemotherapy. Little is known about whether intratumoral heterogeneity (ITH) exists in SCLC and how it may contribute to clinical outcomes and development of treatment resistance. To investigate this, we performed baseline single-cell RNAseq analyses of platinum-sensitive and -resistant CDX models, as well as longitudinal single-cell RNAseq analyses of CDX models and patient CTCs over the course of therapy. Within each CDX model, we observe not only increased ITH with resistance (variance-based metric, P=0.018), but distinct cellular populations with unique gene signatures associated with resistance (e.g. EMT, DNA damage repair, MYC activation, etc.). To confirm this relationship between ITH and resistance, platinum-sensitive CDX models were subjected to extended treatment with DNA damage response targeted therapies until relapse occurred. Single-cell RNAseq confirmed that, as predicted, untreated tumors were molecularly homogeneous, while relapse was associated with increased ITH and multiple, concurrent mechanisms of resistance, including TGF beta signaling and G2/M checkpoints. Unexpectedly, we found variations in the mechanisms of resistance within replicate treatment-relapsed mice, suggesting that resistance even to molecularly targeted therapies does not follow a predictable, reproducible pathway. For example, onset of resistance to a PARP inhibitor resulted in upregulation of NOTCH signaling in one tumor, but not others. Similarly, longitudinal single-cell profiling of CTCs directly from patient blood before, during, and after platinum-relapse confirmed increased ITH post-relapse accompanying unique mechanisms of resistance within specific cell populations (e.g., MYC activation, EMT, and TNFα signaling). We independently found ITH of protein expression (e.g., SLFN11, EZH2, EMT) in CTCs isolated from patient blood, signifying a method for measuring ITH clinically. These data suggest that treatment resistance in SCLC entails a fluid process of shifting expression profiles to generate an increasingly heterogeneous tumor with multiple, disparate mechanisms of resistance. Clinically, these findings imply that drug development efforts in this disease should focus on combination or maintenance therapies for treatment-naïve SCLC tumors to maximize depth of initial responses and delay the onset of resistance defined by ITH.

#2900

Dissection of clonal heterogeneity unmasks pre-existing chemoresistance and new metabolic vulnerabilities in pancreatic cancer.

Sahil Seth, Chieh-Yuan Li, Sara Loponte, I-Lin Ho, Denise Corti, Luigi Sapio, Edoardo Del Poggetto, Michael Peoples, Tatiana Karpinets, Frederick S. Robinson, Shan Jiang, Prasanta Dutta, Joseph Marszalek, Maria E. Di Francesco, Timothy P. Heffernan, Virginia Giuliani, Pratip K. Bhattacharya, Giannicola Genovese, Andrew Futreal, Giulio Draetta, Andrea Viale, Alessandro Carugo. _UT MD Anderson Cancer Ctr., Houston, TX_.

Adaptive drug-resistance mechanisms allow human tumors to evade treatment through selection and expansion of treatment-resistant clones. Modeling the functional heterogeneity of tumors can unmask critical contributions of distinct tumor cell sub-populations toward identifying rational drug combinations. Here, studying clonal evolution of tumor cells derived from human pancreatic tumors, we demonstrate that in vitro adherent cultures and in vivo tumors are maintained by a common set of long term self-renewing tumorigenic cells that can be used to establish clonal replica tumors (CRTs), large cohorts of animals bearing human tumors with identical clonal composition. Using CRTs to conduct quantitative assessments of clonal dynamics and adaptive responses to therapeutic challenge over time, we uncovered that the tumorigenic compartment of pancreatic tumors maintains a multitude of functionally heterogeneous subpopulations of cells with differential degrees of sensitivity to therapeutics. High-throughput isolation and deep characterization of unique clonal lineages showed genetic and transcriptomic diversity underlying the functionally diverse subpopulations, positioning the origins of tumor heterogeneity within the long-term self-renewing compartment. Molecular annotation of gemcitabine-naïve clonal lineages with distinct responses to treatment in the context of CRTs generated signatures that can predict the response to chemotherapy and exposed pre-existing functional mechanisms of clonal resistance, primarily associated to DNA damage tolerance and mitochondrial respiration (OXPHOS). Further transcriptomic and metabolic characterization of residual tumor cells in patient derived xenograft models as well as in patients after chemoradiation showed that resistant cells that contribute to tumor relapse are metabolically rewired to upregulate OXPHOS. Combining a novel inhibitor of oxidative phosphorylation (IACS-10759) developed at the MD Anderson Institute for Applied Cancer Science, and currently in phase I clinical trial in acute myeloid leukemia and solid tumors, with standard of care drugs drastically reduces tumor clonal complexity, underscoring the promise of inhibiting mitochondrial respiration as a new therapeutic strategy to prolong patient survival by eradicating resistant clones that survive chemoradiation. Our study, correlating genomic and transcriptomic traits with specific functional phenotypes, uncovered new mechanisms that underlie intra-tumor sub-clonal heterogeneity, influence treatment response to drugs and sustain tumor relapse.

#2901

Clonal dynamics and phenotypic evolution during chemoresistance and metastasis revealed by patient-derived xenograft models of triple negative breast cancer.

Gloria V. Echeverria,1 Zhongqi Ge,1 Sahil Seth,1 Emily Powell,2 Xiaomei Zhang,1 Sabrina Jeter-Jones,1 Xinhui Zhou,1 Yan Jiang,1 Aaron McCoy,3 Shirong Cai,1 Yizheng Tu,1 Michael Peoples,1 Yuting Sun,1 Huan Qiu,4 Christopher Bristow,1 Alessandro Carugo,1 Jiansu Shao,1 Stacy L. Moulder,1 William F. Symmans,1 Timothy P. Heffernan,1 Jeffrey T. Chang,4 Helen M. Piwnica-Worms1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _Parkview Hospital, Fort Wayne, IN;_ 3 _Washington University, St. Louis, MO;_ 4 _UT Health Science Center, Houston, TX_.

Half of all triple negative breast cancer (TNBC) patients harbor significant residual cancer burden following standard neoadjuvant chemotherapy treatment, resulting in distant metastasis and death for most of these patients. Intra-tumor heterogeneity (ITH) is pervasive in TNBC as is a barrier to development of effective therapeutic strategies, but the relative contributions of heterogeneous tumor cell populations to chemoresistance and metastasis are not well understood. To investigate the clonal dynamics that accompany chemotherapy treatment and metastasis, we employed orthotopic patient-derived xenograft (PDX) models of treatment-naïve TNBC, thus enabling experimentation with heterogeneous populations of human tumor cells that have undergone minimal manipulation.

To monitor the fates of PDX tumor cell lineages as they metastasized, we introduced a pooled lentiviral barcode library (Cellecta) into freshly dissociated PDX tumor cells which were orthotopically engrafted into recipient mice. Genomic analyses, including barcode enumeration, whole-exome sequencing, custom targeted DNA sequencing, and transcriptome sequencing, were conducted to characterize the clonal dynamics that accompanied metastasis. Of the thousands of diverse primary tumor clones, only ~2% harbored metastatic capacity. Of those, a rare population of the exact same clones predominated metastases in lung, liver, and brain, the three most common sites of human TNBC metastasis. These studies provide a quantitative map of the clonal architecture of multi-organ metastasis in TNBC and reveal that identical subclones can thrive in diverse secondary organ microenvironments.

NACT resistance leads to metastasis and death for most patients, yet the origins of chemoresistance in TNBC are unclear. We modeled NACT resistance in an array of PDX models derived from treatment-naïve TNBC biopsies in alignment with an ongoing neoadjuvant clinical trial (NCT02276443). Upon partial response to NACT, tumors entered a transient drug-tolerant state characterized by distinct histologic, proteomic, and transcriptomic features that were reverted as tumors regrew after cessation of treatment. Barcode-mediated lineage tracking and whole-exome sequencing revealed that the drug-tolerant state was not mediated by clonal selection. Based on transcriptomic and metabolic features of the drug-tolerant state, we conducted preclinical trials with an inhibitor of mitochondrial oxidative phosphorylation (IACS-010759), which significantly delayed the regrowth of residual tumors in PDX models. Together, these studies revealed that TNBCs can resist NACT through non-selective adaptation of a reversible phenotypic state, and that inhibition of oxidative phosphorylation may be a promising therapy in the neoadjuvant setting for TNBC.

#2902

Non-genetic TPX2/AURKA signaling facilitates tumor evolution in EGFR-TKI resistance in NSCLC.

Khyati N. Shah, Roma Bhatt, Julia Rotow, Julia Rohrberg, Victor Olivas, Victoria E. Wang, Jonathan Kuhn, Sophie Dumont, Frank Mccormick, Andrei Goga, Collin M. Blakely, Trever G. Bivona, Sourav Bandyopadhyay. _UCSF, San Francisco, CA_.

Background: EGFR tyrosine kinase inhibitors are effective for treatment of non-small cell lung cancer. Benefit from these therapies is often transient due to residual disease leading to acquired resistance. Osimertinib now used in first-line setting, but mechanisms of resistance are ill-defined and no effective second-line therapies exist. Disease progression occurs through tumor evolution that involves the emergence of distinct genetic alterations and non-genetic changes in cell state. In this study, we determine the molecular drivers of drug sensitivity and tumor evolution which forms the basis of clinical genomic heterogeneity. We show that the increase in genomic instability correlates with increased chromosomal alterations or rate of mutation which creates evolutionary dependencies.

Method: Using our previously established EGFR-TKI acquired resistance models, we observed heightened TPX2/AURKA activity. We measured the temporal activation of TPX2/AURKA in sensitive phase, a drug-tolerant phase (residual phase) and acquired resistant phase. Chromosomal segregation errors, mitotic deformation and aneuploidy were used as a functional read-out to the measure genomic instability upon EGFR-TKI treatment. Using clinical specimens, the contribution of non-genetic TPX2/AURKA axis in disease progression was evaluated.

Results: In-vitro models of residual disease exhibited increased AURKA activation via upregulation of its co-activator TPX2. Pharmacological inhibition of AURKA activation by MLN8237 together with osimertinib enhanced the magnitude of response and forestalled the emergence of resistance. Therefore, tumor cells reply on AURKA to transition from a sensitive to drug-tolerant phase and is maintained in acquired resistance.

Since high levels of TPX2 and AURKA are known to cause mitotic errors and polyploidy, we surveyed for mitotic defects induced by EGFR-TKI treatment. EGFR-TKI resulted in a marked accumulation of errors in centrosome biogenesis, spindle assembly, and chromosome segregation resulting in polyploid cells with abnormal DNA content indicating persistent mitotic stress is a feature of EGFR inhibition. We conclude that the abnormal activation of TPX2/AURKA leaves a signature of defects associated with mitotic stress, a prominent feature of genomic instability. Examination of clinical samples with T790M mutation or MET amplification upon disease progression had high levels of TPX2. This nominates TPX2 as a novel biomarker therapeutic resistance in a significant fraction of EGFR-mutant lung cancers.

Conclusion: Our results indicate that heightened TPX2/AURKA activation is required for tumor cells to transition out of sensitive cell state and persist upon drug pressure. This deregulation creates a highly genomically unstable environment and give rise to genomic heterogeneity providing a fertile ground for the subsequent survival of fittest subclones.

#2903

Genomic evolution of uveal melanoma arising in ocular melanocytosis.

Michael A. Durante,1 Matthew G. Field,1 Margaret I. Sanchez,1 Kyle R. Covington,2 Christina L. Decatur,1 Sander R. Dubovy,1 J. William Harbour1. 1 _Bascom Palmer Eye Institute, Sylvester Comprehensive Cancer Center and Interdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, Miami, FL;_ 2 _Castle Biosciences Inc., Friendswood, TX_.

Introduction: Ocular melanocytosis is an congenital condition characterized by the presence of excessive hyperpigmented melanocytes in the uveal tract. Epidemiological studies have shown that this condition is the strongest predisposing risk factor for development of uveal melanoma (UM). In UM patients, the presence of ocular melanocytosis doubles the risk of metastasis. UM has well-characterized driver mutations consisting of two groups. The first group consists of mutually exclusive gain-of-function mutations in members of the Gq signaling pathway (GNAQ, GNA11, CYSLTR2 and PLCB4), and are thought to represent initiating oncogenic events that are insufficient alone to cause full malignant transformation. The second group consists of near-mutually exclusive mutations in BAP1, SF3B1, and EIF1AX, which are thought to occur later in tumor progression. The genomics of ocular melanocytosis and matched UM have not been characterized previously. The purpose of this study was to investigate the genomic evolution of matched ocular melanocytosis and UM tumors.

Methods: Exome and deep-targeted sequencing data from two matched ocular melanocytosis and primary uveal melanomas were evaluated using a previously developed bioinformatic pipeline optimized to call mutations and CNVs in UM. Data from this analysis were used in downstream mutation subclone detection algorithms to determine genomic evolutionary patterns within these matched samples.

Results: For both cases, the initiating driver mutations that are characteristic of UM occur at the same genomic site in matched ocular melanocytosis and UM samples. This finding suggests that they arose within the melanocytosis and subsequently expanded in the tumor. Monosomy of chromosome 3 was absent from the melanocytosis but present in ~100% of tumor cells, indicating that it arose early during clonal tumor expansion. In Patient 1, the BAP1 mutation was present in a tumor subclone and chromosome 8q gain was present in a smaller subclone, indicating that they arose later during tumor evolution. In Patient 2, the BAP1 mutation and other copy number variations were present in ~100% of tumor cells indicating that tumor evolution was completed in this tumor. Additionally, no Gq signaling pathway driver mutations were found in the germline DNA of these patients indicating that that the mutations in ocular melanocytosis occurred somatically during development.

Conclusions: This study provides the first genomic evidence relating ocular melanocytosis to UM. This data shows that Gq signaling pathway driver mutations are early genomic events that are necessary but not sufficient for UM tumor development.These insights help further refine our genomic model of UM tumor evolution and may help to address persistent challenges in the field such as the failure of targeted therapies aimed at inhibiting the Gq pathway.

#2904

Intratumor δ-catenin heterogeneity driven by genomic rearrangement dictates growth factor dependent prostate cancer cell fate and behavior.

Mingchuan Li,1 Jongdee Nopparat,2 Jiao Zhang,1 Byron J. Aguilar,3 Yan-Hua Chen,3 Jie Du,1 Xin Ai,4 Yong Luo,1 Yongguang Jiang,1 Christi Boykin,3 Qun Lu5. 1 _Beijing An Zhen Hospital at Capital Medical University, China;_ 2 _Prince of Songkla University, Thailand;_ 3 _East Carolina University Brody School of Medicine, Greenville, NC;_ 4 _PLA Army General Hospital, China;_ 5 _East Carolina Univ. Brody School of Medicine, Greenville, NC_.

Only a small number of genes are bona fide oncogenes and tumor suppressors such as Ras, Myc, p53 and PTEN. However, targeting these cancer drivers frequently fail to demonstrate sustained cancer remission. Tumor heterogeneity and evolution contribute to cancer resistance and pose challenges for cancer therapy due to differential gene expression profiles driving distinct tumor responses to treatments. Here we report that intratumor heterogeneity of Wnt/β-catenin/armadillo modulator δ-catenin controls individual cell behavior to promote cancer. The differential intratumor subcellular localization of δ-catenin mirrors its compartmentalization in prostate cancer xenograft cultures as a result of mutation-rendered δ-catenin truncations. Wildtype and δ-catenin mutants displayed non-overlapping protein interactomes that highlight rewiring of signal networks. Localization specific δ-catenin mutants influenced p120ctn-dependent Rho GTPase phosphorylation and shifted cells towards bFGF-responsive growth, a known signal to bypass androgen receptor dependence. Mutant δ-catenin promoted Myc-induced prostate tumorigenesis by increasing proliferation over apoptosis as well as β-catenin stabilization and HIF-1α expression. Therefore, intratumor δ-catenin heterogeneity by genetic remodeling promotes prostate cancer expansion towards androgen independence, supporting a neomorphism model paradigm for targeting tumor progression.

#2905

Clinical features in relation to age at diagnosis in luminal breast cancer patients: A hospital-based case series study in Beijing, China.

Hela Koka,1 Changyuan Guo,2 Mustapha Abubakar,1 Hyuna Sung,3 Hyuna Sung,1 Bin Zhuo,2 Eric Tang,1 Joseph Deng,1 Nan Hu,1 Ning Lu,2 Xiaohong R. Yang1. 1 _NIH/NCI, Rockville, MD;_ 2 _Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China;_ 3 _American Cancer Society, Atlanta, GA_.

Background

Breast cancer (BC) is a heterogenous disease that can be classified into various molecular subtypes. Patients with younger age at diagnosis are less likely to develop luminal A cancers. However, a large proportion of early-onset luminal BCs are seen among Asian BC patients. While this may be explained by the increasing exposure to Westernized lifestyle, germline genetics, breast density, or immune factors may also give rise to a distinct age-related biology in Asian BC. We investigated several tumor characteristics in luminal BC patients with respect to age, among women in Beijing, China.

Methods

Subjects in this study were diagnosed with invasive breast cancer and treated at the Cancer Hospital, Chinese Academy of Medical Sciences, Beijing, between 2008-2016. 7,543 luminal cases (ER+ and/or PR+), were further defined as Luminal A: ER+, PR+, HER2-, low grade (1/2); Luminal B/high grade: ER+ and/or PR+, HER2-, high grade (3); Luminal B/HER2+: ER+ and/or PR+, HER2+. Patients were divided into four age groups: <40 (12.1%), 40-50 (34.2%), 50-60 (32.2%), and 60+ (21.5%) years old. We investigated the associations between age and tumor markers (ER, PR, HER2, KI67, P53, CK5/6, and EGFR) and features (tumor size, lymph node involvement, and histologic grade) using unconditional logistic regression.

Results

The mean age at diagnosis among these cases was 51.5 years (SD=10.8). Compared to older women (60+), patients diagnosed <40 years old were more likely to have lower ER percentage/intensity, to be positive for HER2, P53, and EGFR, to have higher KI67, and luminal B/HER2+ tumors (p-values=<.0001). PR showed a different and interesting pattern among patients in age group 40-50, who had higher values of PR percentage/intensity compared to women 60+ years old. After mutually adjusting for the significant tumor features, the associations for ER [OR<40 vs 60+=0.34 (0.21, 0.55), OR40-50 vs 60+=0.22 (0.15, 0.32), strong vs. weak staining] and PR [OR<40 vs 60+=2.95 (2.00, 4.35), OR40-50 vs 60+=4.22 (3.13, 5.68), strong vs. weak staining], HER2 positivity [OR<40 vs 60+=1.60 (1.15, 2.23)], KI67 [OR<40 vs 60+=1.52 (1.04, 2.21), quartile 4 vs. 1], and lymph nodal involvement [OR<40 vs 60+=1.87 (1.38, 2.51)] remained significant. While younger patients in general were more likely to have luminal B/HER2+ tumors, women in age group 40-50 were less likely to develop luminal B/high grade BC [OR40-50 vs 60+=0.78 (0.63, 0.97)] compared to women 60+ years old.

Conclusion

We observed the known associations between younger age at diagnosis and more aggressive tumor features. We also found that patients in the age 40-50 group showed interesting associations with PR and the luminal B/high grade subtype. We intend to replicate these results in other Asian datasets and to explore the age associations with etiologic factors.

#2906

Intratumor heterogeneity of non-small-cell lung cancer with EGFR mutations.

Sitan Qiao,1 Xin Zhao,1 Xinlan Zhou,1 Yaxiong Zhang,2 Li Zhang,2 Kui Wu1. 1 _BGI-Research, Shenzhen, China;_ 2 _Sun Yat-sen University Cancer Center, Guangzhou, China_.

The mutation frequency of epidermal growth factor receptor (EGFR) is approximately 40% among Asian patients with NSCLC. Guidelines for the diagnosis and treatment of primary lung cancer in China recommended epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) as a first-line therapy for advanced NSCLC patients carrying EGFR mutation including exon 19 deletion (19Del) and L858R mutation on exon 21 (L858R). Although survival periods in clinical practice of EGFR-TKIs treatment have been getting longer, the patients with different subtype of EGFR mutations exhibited diverse drug response to EGFR-TKIs. The reasons why are still unclear.

In this study, we performed whole genome and panel sequencing of 239 spatially distinct regions from 42 lung adenocarcinoma of Chinese patients, of which 14 patients carried EGFR 19del mutation (EGFR-19Del), 15 patients carried L858R mutation (EGFR-L858R) and 13 patients did not carry any of these two mutation types (Control). We found the intra-tumor heterogeneity (ITH) levels were significantly higher in EGFR-mutant patients compared with control (P<0.05). Average fraction of nonubiquitous mutation was 93.63%, 93.87% and 71.34% in EGFR-19Del, EGFR-L858R and Control, respectively. Remarkably, mutational process preferentially associated with different genes on different pathways were observed between two EGFR mutant subtypes, in which cell cycle related pathways were more likely to mutate in EGFR-L858R patients, while cell adhesion associated pathways were more susceptible to mutations in EGFR-19Del patients. Furthermore, two distinct evolutionary patterns were identified in EGFR-mutant patients based on whether TP53 mutations occurred simultaneously. In EGFR-mutant patients with stage I, the prognosis of patients with TP53 mutations was significantly worse than those without TP53 mutations. Meanwhile, we found that in the absence of TP53 mutations, more than 70% of EGFR-L858R patients showed multiple recurrent clonal driver mutations regardless of the stage of the patients. In contrast, EGFR-19Del patients only carried recurrent clonal driver mutation of EGFR in stage I, and began to accumulate other clonal driver mutations in later stage, which may indicate different evolutionary strategies underlying these two subtypes.

These preliminary results suggested that intratumor genetic diversity provided a substrate for tumor adaptation and evolution in the subtype of EGFR mutation. It could provide genetic basis for diagnosis and prognosis in EGFR-TKIs therapeutic response and to guide stratification of lung cancer treatment and clinical research in future.

#2907

Exosome secretion is an inheritable property of cancer cells: Single-cell profiling of exosome secretion.

Mohsen Fathi,1 Robiya Joseph,2 Melisa Martinez-Paniagua,1 Jay R Adolacion,1 Xingyue An,1 Ankit Mahendra,1 Konrad Gabrusiewicz,2 Sujash Chatterjee,1 Sendurai A. Mani,2 Navin Varadarajan1. 1 _University of Houston, Houston, TX;_ 2 _University of Texas MD Anderson Cancer Center, Houston, TX_.

Decades of research on exosomes, nano-sized vesicles secreted by cells, have revealed novel roles of these vesicles in the formation of pre-metastasis niches that enhances the migration of tumor cells to those sites. Paradoxically, more recent work has suggested that these tumor-derived exosomes can also have an immunostimulatory role, depending on the model studied. The rate of secretion of exosomes by single cells is likely heterogeneous, and a deep profiling of the secretion capacity of individual tumor cells has been largely unexplored.

We have developed a high-throughput single-cell methodology to quantify the dynamic secretion of exosomes from single cells using a modified immunoassay and sought to define: (a) heterogeneity among individual cells within the tumor cell population, and (b) the nature of the cells secreting exosomes within a tumor cell population. In order to investigate these, we chose to study models of triple negative breast cancer: the metastatic tumor lines, 4T1 (mouse) and MDA-MB-231(human); and the non-metastatic lines 67NR (mouse) and MCF7 (human). Our method revealed that MDA-MB-231 single cells secreted exosomes at a rate, ~2 fold higher than MCF7 cells. Surprisingly, the non-metastatic 67NR cells showed a higher secretion rate (~1.5 fold) than metastatic 4T1 cells. Next, we performed single-cell RNA-seq on 67NR and 4T1 single cells. Consistent with our single-cell exosomal profiling results, 67NR cells were significantly (p-value < 0.01) enriched for genes correlated to exosome formation in comparison to the 4T1 cells. Next, in order to study exosomes of single cells, we isolated single cells using an automated micromanipulator which allowed us to establish monoclonal cell lines. Measurement of these monoclonal cells showed that secretor cells of 67NR secrete exosomes with ~2 fold higher rate than non-secretor cells.

Since our in vitro results clearly demonstrated that the secretor clonal cell lines had a higher frequency of exosome secreting single cells, we sought to define the in vivo relevance of these results. Consistent with the hypothesis that the exosomes from 67NR are immune-stimulatory, injection of the secretor clones into Balb/c mice led to lack of primary tumor formation (2/15 mice had tumors) whereas the injection of the non-secretor clones led to tumor formation in 7/15 mice.

In aggregate, our results show that the higher rate of secretion of exosomes from non-metastatic cells can facilitate tumor rejection in vivo. We are currently performing in vitro studies with the 67NR and MDA-MB-231 exosomes to study their impact on immune cells.

#2908

Mesothelioma phylogeny reveal MTAP as a solitary clonal deletion, exposing vulnerability to the PRMT5 perturbagen, quinacrine.

Sara Busacca,1 Lee Brannan,1 Apostolos Nakas,2 Annabel Sharkey,3 Chiara Riganti,4 David Waller,5 Cathy Richards,2 Iris Salaroglio,4 Vladan Milosevic,4 Peter Wells-Jordan,2 Alan Dawson,1 Michael Sheaff,5 John LeQuesne,1 Aarti Gaba,1 Robert Hastings,1 Luke Martinson,1 Jin-Li Lo,1 Amrita Bajaj,2 Paul Boutros,6 Tom John,7 Bibhusal Thapa,7 Gareth Wilson,8 Jacqui Shaw,1 Charles Swanton,8 Frank Dudbridge,1 Edward Hollox,1 Dean A. Fennell1. 1 _University of Leicester, Leicester, United Kingdom;_ 2 _University Hospitals of Leicester NHS Trust, Leicester, United Kingdom;_ 3 _Sheffield Teaching Hospitals NHS Foundation Trust, United Kingdom;_ 4 _University of Torino, Torino, Italy;_ 5 _Barts and the London NHS Trust, London, United Kingdom;_ 6 _Ontario Institute for Cancer Research, Toronto, Ontario, Canada;_ 7 _Olivia Newton-John Cancer Research Institute, Melbourne, Australia;_ 8 _Francis Crick Institute, London, United Kingdom_.

Background: Malignant Pleural Mesothelioma (MPM) remains an incurable cancer that is caused by asbestos, and for which there is a paucity of effective therapy. Stratified medicine for MPM is in its infancy. We hypothesized that deciphering the phylogenetic architecture of mesothelioma would yield a census of recurrent clonal homozygous copy number losses as potential therapeutic vulnerabilities.

Methods and Results: We prospectively enrolled 125 patients with MPM undergoing radical pleurectomy decortication, into the MEDUSA (Mesothelioma Evolution: DrUgging Somatic Alterations) study. Multi-region whole exome sequencing was conducted on 106 tumours from 20 patients (Medusa20 cohort). Up to 5 consistent regions were sampled: apex , pericardium, anterior/ posterior costophrenic angles, and the oblique fissure. For each patient, matching whole blood DNA was also whole exome sequenced to allow identification of tumour- specific somatic variations. Somatic copy number alterations (SCNAs) in each tumour region were called using SEQUENZA. We inferred phylogeny for each patient's tumour using the SCNA calls by maximum parsimony (TUMULT), which revealed branched evolution in all MPMs. The total number of SCNAs ranged from 78 to 380 across the cohort with biphasic MPMs exhibiting a significantly larger total and clonal SCNA burden compared to epithelioid MPMs (p=0.024) . Only 9p21 which harbours CDKNA and methylthioadenosine phosphorylase (MTAP), exhibited clonal homozygous loss in 3 patients (15%). Clonal heterozygous loss was seen in 2 patients (10%). A further 5 patients showed with evidence of parallel evolution involving MTAP loss in distant MPM regions (25%), with one patient's MPM having late homozygous deletion in a single branch (5%). MTAP loss was validated by array based SCNA analysis and was found to be negatively prognostic in an independent cohort. Protein arginine methyltransferase 5 (PRMT5) has been recently identified as a vulnerability in MTAP deleted cancer. We found that siRNA silencing of PRMT5, caused MTAP selective loss of clonogenicity with proliferative arrest. Utilizing the connectivity map, quinacrine was validated as a PRMT5 perturbagen, which suppressed c-jun-dependent PRMT5 expression without inhibiting its methyltransferase activity. Quinacrine phenocopied PRMT5 siRNA, reducing global symmetrical arginine dimethylation of histone H4 (H4R3me2S). Finally, exogenous wild-type PRMT5 rescued quinacrine-mediated cell arrest in MTAP-negative cells, an effect not seen using the PMRT5 E444Q methyltransferase dead mutant.

Conclusion: MTAP deletion is a clonal homozygous event in mesothelioma, with potential as a therapeutically tractable Achilles heel, via PRMT5 silencing using a repurposed small molecule, quinacrine.

#2909

Rapid autopsy of metastatic cholangiocarcinoma demonstrates diverse patterns of clonal heterogeneity.

Melanie A. Krook,1 Russell Bonneville,1 Hui-Zi Chen,1 Julie W. Reeser,1 Michele R. Wing,1 Jharna Miya,1 Amy M. Smith,1 Anoosha Paruchuri,1 Thuy Dao,1 Dorrelyn M. Martin,1 Eric Samorodnitsky,1 Kaitlin R. Baker,1 Cynthia Timmers,2 Kristin Dittmar,1 Sharon Cole,3 Lianbo Yu,1 Aharon G. Freud,1 Patricia Allenby,1 Sameek Roychowdhury1. 1 _The Ohio State University, Columbus, OH;_ 2 _Medical University of South Carolina, Charleston, SC;_ 3 _Orion Cancer Care, Inc., Findlay, OH_.

Background: Cholangiocarcinoma is an aggressive and deadly bile duct cancer. Due to its poor prognosis and limited treatments beyond first-line therapy, in-depth characterization of cholangiocarcinoma is needed to facilitate the development of novel therapies. Tumor heterogeneity has emerged as a contributor to treatment resistance, but it is largely unknown how it contributes to cholangiocarcinoma. Characterization of tumor heterogeneity in cholangiocarcinoma has largely been limited to biopsies and surgical resections and may not accurately reflect the complex and heterogeneous nature of the disease. A partnership with patients and their families for body donation is a powerful strategy to characterize the landscape of tumor heterogeneity in cholangiocarcinoma.

Study Design: We performed whole exome sequencing (WES) of a pre-treatment tumor biopsy and multiple tumor samples collected through rapid research autopsy from 9 patients with metastatic cholangiocarcinoma. This will allow us to determine spatial heterogeneity, delineate cancer phylogeny, and identify spatial origin of treatment-resistant clones as well as drivers of metastasis and recurrence.

Results and Conclusions: We completed research autopsies of 9 patients with metastatic cholangiocarcinoma including 5 with FGFR alterations, 1 with an IDH1 mutation, 1 with a BRCA mutation, 1 with a BAP1 splice site mutation, and 1 with hypermutation by an unknown mechanism. We observed varying patterns of clonal evolution in this cohort including persistent, sensitive, and emergent clones. One patient with metastatic cholangiocarcinoma with a novel FGFR2-CLIP1fusion received the FGFR inhibitor, INCB054828 and had a 5.5-month response but unfortunately progressed and succumbed to his disease. Multi-site WES and analysis of tumor heterogeneity through subclonal inference identified four genetically distinct clonal cancer cell populations, two of which were only observed in post-treatment samples (emergent clones). Additionally, WES revealed an FGFR2N549H mutation in a single tumor sample hypothesized to confer resistance to INCB054828. This hypothesis was corroborated in vitro, in which cells expressing the FGFR2-CLIP1 fusion protein were sensitive to INCB054828 (IC50of 10.16 nM), whereas cells expressing FGFR2-CLIP1 N549H were resistant (IC50of 1257.57 nM). Furthermore,the FGFR2 N549H mutation displayed cross-resistance to other selective FGFR inhibitors, but remained sensitive to the non-selective inhibitor, ponatinib.

Cancer Relevance: These studies will augment our understanding of tumor heterogeneity in cholangiocarcinoma. The broader impact of this research includes opportunities to develop new therapies for cholangiocarcinoma, centralized storage of cholangiocarcinoma tumor samples, and demonstration of how research autopsy can be applied to study tumor heterogeneity in other cancer types.

#2910

Multi-sites rapid-autopsy data reveals aggressive metastatic colonization and metastasis phenotypes.

Xiaomeng Huang,1 Yi Qiao,1 Samuel W. Brady,1 Adam Cohen,1 Andrea H. Bild,2 Gabor T. Marth1. 1 _University of Utah, Salt Lake City, UT;_ 2 _City of Hope, Duarte, CA_.

Metastatic breast cancer is a deadly disease with low 5-year survival rate (22%). Because it is extremely difficult to track metastatic spread in a living patient, the patterns of new organ colonization are currently poorly understood. In this study, we have collected tumor biopsies at initial diagnosis and at mastectomy necessitated by the patient's relapse, and autopsies from twenty-six metastatic tumors across seven organs and two skin tissues as normal controls via a rapid autopsy procedure within hours after the patient's death. All samples were subjected to 60X Illumina whole genome sequencing and bulk RNA sequencing. We used copy number variation (CNV) and short variant (SNP, INDEL) data to reconstruct the phylogenetic relationships among the tumor samples, and established how the tumor evolved at the subclone level across different metastatic sites, using our SubcloneSeeker algorithm. We selected tumor samples with the lowest normal tissue contamination for transcriptomic analysis. Differentially expressed gene and pathways were identified from RNA-seq data. Our analysis revealed that the cancer first escaped to the lung, an event that occurred before the time of initial diagnosis. This was followed by four distinct 'waves' (G1, G2, G3 and G4) of massive metastatic expansion, beginning with the abdominal organs (involving samples in G1), then the lymph nodes (G2 and G4), brain and bones (G3). We see clear evidence for metastatic "recolonization" where further evolved subclones invaded already established, earlier metastatic sites. Subclone analysis also revealed a bifurcating pattern of evolution in the primary tumor, in which two lineages gave rise to the G1 and G2-G4 metastases respectively. Consistent with the genomic findings, unsupervised clustering based on the gene expression data showed that samples in G1, and samples in G2-G4 formed distinct clusters. Comparing gene expression profiles between G1 (n=4) and G3 (n=9) samples showed that samples in G1 exhibited enrichment in Integrin activation pathways and samples in G3 presented a signature for chromosome 17 amplification. These distinct metastatic phenotypes indicate different survival mechanisms for G1 and G3 metastatic tumors. The high number of sampled sites in this study, 30 in all, allowed us to reconstruct the evolution of the disease in this patient with unprecedented resolution, identify novel patterns of metastatic colonization, and identify distinct metastatic phenotypes. This study suggests the need for more comprehensive collection and evaluation of metastatic sites to ensure that therapies chosen to target specific genomic aberrations are able to combat the tumor at all metastatic sites.

#2911

Genome-wide copy number profiling of single circulating multiple myeloma cells (CMMCs) reveals intra-patient convergent copy-number alterations (CNAs).

Claudio Forcato,1 Andrea Raspadori,1 Alberto Ferrarini,1 Mario Terracciano,1 Valentina del Monaco,1 Marianna Garonzi,1 Carrie Morano,2 Steven Gross,2 Genny Buson,1 Chiara Bolognesi,1 Francesca Fontana,1 Gianni Medoro,1 Mark Connelly,2 Nicolò Manaresi1. 1 _Menarini Silicon Biosystems S.p.A, Bologna, Italy;_ 2 _Menarini Silicon Biosystems Inc., PA_.

Introduction: Multiple Myeloma (MM) evolution and heterogeneity is interesting for its potential translational relevance. Recent studies using bulk sequencing of Smoldering MM cells obtained from Bone Marrow (BM) report recurring CNA patterns. By analyzing single-CMMCs isolated from enriched peripheral blood, we show here, for the first time in MM, evidence of frequent convergent lesions, i.e. alterations developed independently across different evolutionary branches, including CNAs often found in MM and previously reported as common truncal alterations.

Methods: Peripheral blood samples (4.0 ml) were obtained from n=3 patients with MM. CMMCs were enriched with CellSearch® AutoPrep® using a custom kit with anti-CD138 or anti-CD138/CD38 antibody-conjugated ferrofluids for positive enrichment and CD38-PE, CD19/CD45-APC immunofluorescent staining for detection. Cell enumeration was based on the co-localization of nuclear DAPI staining and CD38-PE on CellSearch CTAII®. Single CMMCs (CD38+/CD19- and CD45-/DAPI+) and White Blood Cells (WBCs: CD38-/CD19+ or CD45+/DAPI+) were then isolated with DEPArray NxT system. Single-cell genomic DNA was amplified using Ampli1™ Whole Genome Amplification (WGA) kit, Illumina®-compatible libraries were obtained using Ampli1™ LowPass kit and the process was automated on a Hamilton STARLet Liquid handler. Multiplexed, low-pass whole-genome sequencing was performed on HiSeq 2500 Illumina® platform. Genome-wide single-cell Low-Pass Copy Number Alteration (LPCNA) analysis was performed using the cloud-based bioinformatic suite MSBio Suite (Menarini Silicon Biosystems).

Results: 186/215 (86%) single CMMC in total passed QC criteria and were included in the analysis. Single WBCs were also included as normal controls. Cumulatively, CNA profiles of single CMMCs showed patterns typical of MM, including 1q gain, 13 monosomy, sub-chromosomal gain or trisomy 3, 5, 7, 9, 11, 15, 21. Of these, intra-patient single-cell profiling surprisingly revealed -in all three patients- convergent lesions, i.e. alterations developed independently across different evolutionary branches, along with conserved (common truncal), and divergent alterations (found only in specific sub-clusters). In addition, we found evidence that 1q gain, 13q deletion and 6p gain were actually subclonal, in contrast with recent publications reporting them as truncal early-onset lesions.

Conclusion: Single CMMCs CNA profiling reveals patterns of frequent convergent alterations developed independently through branched evolution, undetectable through bulk sequencing.

#2912

The impact of primary tumor sidedness on the effect of regorafenib in refractory metastatic colorectal cancer.

Sang Eun Yoon, Joon Young Hur, Su Jin Lee, Jeeyun Lee, Se Hoon Park, Joon Oh Park, Ho Yeong Lim, Won Ki Kang, Young Suk Park, Seung Tae Kim. _Samsung Medican Center, Seoul, Republic of Korea_.

Recently, the sidedness of the primary tumor (right versus left) has been investigated for its ability to prognosticate and predict outcomes. We evaluated the effect of regorafenib based on KRAS mutation status and the sidedness of the primary tumor in patients with metastatic colorectal cancer (mCRC). We analyzed 135 patients with refractory metastatic colorectal cancer (mCRC) being treated with regorafenib at Samsung Medical Center, between January 2014 and January 2018. Primary tumors originating in the splenic flexure, descending colon, sigmoid colon, rectum, or proximal third of the transverse colon were defined as left-sided CRC (LC). Primary tumors originating in the appendix, cecum, ascending colon, hepatic flexure, or distal two-thirds of the transverse colon were defined as right-sided CRC (RC). Among all 135 patients, 100 (74.1%) had left sided colon cancer and 35 (25.9%) had right-sided colon cancer. No patients achieved a complete response, but four achieved a partial response, revealing a response rate (RR) of 3.0%. Thirty-seven patients had stable disease, yielding a disease control rate (DCR) of 30.4%. There was no difference in RR or DCR according to the location of the primary tumor (LC vs. RC). A significant difference in progression free survival (PFS) with regorafenib was observed between the LC and RC groups (2.6 months; 95% CI, 2.0 to 3.1 vs. 1.9 months; 95% CI, 1.6 to 2.3; P = 0.04, respectively). In a subpopulation with wild type KRAS, PFS with regorafenib was also significantly different between the LC and RC groups (2.9 months; 95% CI, 1.5 to 4.3 vs. 2.1 months; 95% CI, 0.6 to 3.6; P = 0.04). On multivariate analysis, the sidedness of the primary tumor (LC vs. RC) and the number of metastatic sites (≤1 vs. 2>) had a prognostic effect on PFS (P = 0.01 and P = 0.01, respectively). Regorafenib is a current standard treatment for CRC, but treatment outcomes may be improved if regorafenib is administered based on the appropriate biomarker. 

### Tumor Radiosensitivity or Resistance

#2913

Serpinb3a-/- mice are radioresistant.

Stephanie Thermozier,1 Michael Epperly,1 Darcy Franicola,1 Xichen Zhang,1 Renee Fisher,1 Donna Shields,1 Hong Wang,1 John Willis,1 Cliff Luke,2 Gary Silverman,2 Joel Greenberger1. 1 _University of Pittsburgh, Pittsburgh, PA;_ 2 _Washington University School of Medicine, St. Louis, MO_.

Serpins are a superfamily of serine protease inhibitors that regulate proteolytic pathways by utilizing a conformational change to bind to and inhibit their target peptidases. Currently 36 human serpins have been identified, but the biological roles of most are not fully understood (Silverman et al., J Biol Chem. 285(32):24299-305, 2010). Serpinb3a (the mouse orthologue of human SERPINB3 and -B4) is an intracellular serpin and inhibits both serine and cysteine peptidases invitro. We now report radioresistance of Serpinb3a-/- mice and their hematopoietic progenitor cells. Serpinb3a-/- and control Balb/c mice were irradiated to a dose of 8.0 Gy total body irradiation and followed for the development of the hematopoietic syndrome. Long Term Bone Marrow Cultures (LTBMCs) were established from the bone marrow flushed from the femurs and tibias of Serpinb3a-/- and Balb/c mice. Irradiation survival curves were performed on bone marrow stromal cell lines established from the LTBMCs and on fresh bone marrow isolated from the femurs of sacrificed Serpinb3a-/- and Balb/c mice. Colony forming unit granulocyte-macrophage (CFU-GM) colonies of 50 cells or greater were counted. The data from 3 experiments were analyzed using linear quadratic and single-hit, multi-target models. Serpinb3a-/- mice showed increased survival following 8.0 Gy TBI compared to Balb/c mice (p < 0.0001). CFU-GM from Serpinb3a-/- were radioresistant compared to Balb/c marrow (ň = 4.9 ± 1.4 and 1.4 ± 0.1, p = 0.0451, respectively). Serpinb3a-/- bone marrow stromal cells were radioresistant with an increased Do (1.97 + 0.11 Gy) compared to bone marrow stromal cells from Balb/c mice (Do = 1.45 + 0.03 Gy, p = 0.0113). Loss of Serpinb3a provides a radiation survival advantage, increases radioresistance of both hematopoietic progenitor cells and marrow stromal cells, and may lead to identification of new targets for design of radiation mitigator drugs.

This research was supported by grant NIAID/NIH U19-A168021.

#2914

Hexokinase 2-mediated metabolic reprogramming and apoptosis inhibition supports hepatocellular carcinoma radiation resistance.

Yuan Fang, Yizhi Zhan, Yiyi Li, Wei Wang, Dehua Wu, Yi Ding. _Southern Medical Univ., Guangzhou, China_.

Purpose: Hepatocellular carcinoma (HCC) cells are metabolically distinct from normal hepatocytes by selectively upregulating hexokinase 2 (HK2), a key enzyme that catalyzes the first committed step of aerobic glycolysis, which provides genetic proof of concept that HK2 can be inhibited to treat HCC without adverse physiological consequences. The present study was designed to investigate the role of HK2-mediated metabolic reprogramming and apoptosis inhibition in radiation resistance of HCC cells.

Experimental design: Acquired radiation-resistant cells were generated by sequential irradiation and recovery, and proteomics approach was used to illustrate the biological changes and molecular mechanisms involved. qRT-PCR was performed to evaluate the expression of glycolysis genes including HK2. TCGA and GEO analysis were used to investigate the prognostic value of HK2. Intrinsic radiation responsive and resistant HCC cells were also used to further identify the resistant role of HK2. Either lentivirus-mediated HK2 knockdown or pharmacological blockade by 3-bromopyruvate (BrPA) was used to inhibit HK2. Radiation resistance was evaluated by DNA damage response, in vitro cytotoxicity and colony formation assay. Apoptosis and ROS levels were measured with Annexin V and DC-FDA. Cytochrome c release and HK2 mitochondrial translocation were detected by immunofluorescence and western blots. 2-NBDG uptake, HK2 activity, lactate production, and 13C isotopomer tracing experiment were conducted to illustrate the glycolytic activities. HK2 translation efficiency was measured by polyribosome analysis. The upstream AKT-mTOR signaling were detected by western blots and rescued by pharmacological inhibitors. Xenografts were treated with radiation, BrPA, or a combination of radiation and BrPA.

Results: Aerobic glycolysis was significantly activated and was indispensable for cell survival in both acquired and intrinsic radiation-resistant HCC cells. Mechanically, both up-regulation of mRNA expression of hexokinase (HK2) after sequential irradiation and increased translation of HK2 mRNA after one-fractional irradiation were mediated by the activation of AKT-mTOR axis. In addition, high HK2 expression was significantly associated with poor overall survival of HCC patients. Inhibition of HK2 expression or its mitochondrial translocation re-sensitized HCC cells to radiation therapy by promoting mitochondrial cytochrome c release and inhibiting of glycolysis both in vitro and in vivo.

Conclusion: AKT-mTOR-HK2 axis plays crucial roles in both acquired and intrinsic radiation resistance of HCC cells by facilitating aerobic glycolysis and inhibiting cytochrome c release. Targeting HK2 could be an attractive and beneficial approach to improve the therapeutic effects of radiotherapy in HCC patients.

#2915

A circulating stromal cell subpopulation that accurately predicts resistance and progression in treatment naïve lung cancer patients receiving definitive radiotherapy.

Daniel L. Adams,1 Jianzhong He,2 Yawei Qiao,2 Hui Gao,2 James Reuben,2 Ignacio I. Wistuba,2 Cha-Mei Tang,3 Steven H. Lin2. 1 _Creatv MicroTech, Inc., Monmouth Junction, NJ;_ 2 _MD Anderson Cancer Center, Houston, TX;_ 3 _Creatv MicroTech, Inc., Potomac, MD_.

Background: Cancer Associated Macrophage-Like cells (CAMLs) are recently described circulating stromal cells common in the peripheral blood of cancer patients hypothesized to be a mechanism in cancer pathogenesis. We previously described that treatment naive patients with circulating CAMLs ≥50µm is a significant independent prognostic indicator of shorter progression free survival (PFS) in a variety of cancers. However, the clinical value of CAMLs in specific diseased cohorts for predicting response to treatment has not been evaluated. We present the results of a prospective study on treatment naive lung cancer patients before induction and directly after completion of definitive radiotherapy to determine if CAMLs are predictive of treatment response and cancer progression.

Methods: A 2 year single blind prospective study was undertaken, testing the relationship of enlarged CAMLs (≥50µm) to PFS of lung cancer patients before & after induction of definitive radiation therapy. To achieve a 2-tailed 95% power (α=0.05) we recruited a training set of 55 patients, all with pathologically confirmed lung cancer: Stage I (n=13), Stage II (n=7), Stage IIIa (n=10), Stage IIIb (n=18) & Stage IV (n=7). Baseline (BL) blood samples were taken prior to start of therapy. If possible, a second blood sample (T1) was taken after completion of radiotherapy (~60 days), n=46 patients. Blood was filtered by CellSieveTM filtration and CAMLs quantified. Analysis by CAML size of <50 µm or ≥50 µm was used to evaluate PFS hazard ratios (HRs) by censored univariate & multivariate analyses.

Results: CAMLs were found in 93% of BL samples averaging 3.2 CAMLs/7.5mL. Patients with at least one CAML ≥50 µm had reduced PFS (HR=2.9, 95%CI 1.4-6.0, p=0.010). 46 patients agreed to a follow up blood draw. 13 of the 46 patients had an increase of CAML size to ≥50 µm, but 3 patients had a decrease to <50 µm, resulting in an increase of PFS (HR=7.7, 95%CI 2.6-12.5, p<0.001). Combined, 90% of patients with a ≥50 µm CAML at BL progressed within 2 years vs 46% of patients with <50 µm CAMLs. Enlarged CAMLs at T1 was more predictive of progression, with 92% of patients with a ≥50 µm CAML progressing vs 21% of patients with <50 µm CAMLs. Notably, 100% of patients that had a ≥50 µm CAML at both BL and T1 progressed. By comparison, only 11% of patients with <50 µm CAMLs at both BL and T1 progressed. In a multivariate analysis, CAML size was the most significant indicator of PFS and OS, independent of all other clinical variables, including stage.

Conclusions: Our data suggests that for lung cancer patients undergoing definitive radiation therapy, the presence of enlarged CAMLs appears to predict patients that are resistant to treatment. Furthermore, these circulating stromal cells appear useful for sequential monitoring of patients that may prognosticate who are likely to progress after treatment.

#2916

Delineating the radiogenomic landscape of cancer through systematic annotation of genetic variants.

Brian Yard, Aaron Petty, Mohamed Abazeed. _Cleveland Clinic, Cleveland, OH_.

Radiation is a fundamental pillar of treatment for many cancer patients. However, there are still no established means by which to identify patients who are more or less likely to respond to treatments. In addition, there are few synergistic drug-radiotherapy combinations currently in clinical use. To address these needs, we sought to discover genetic alterations that can predict the vulnerability of cancer to radiation. Here, we report on a large-scale study to identify and classify tumor associated genetic variants that determine survival to radiation.

To gain insight into the genetic landscape of radioresistance, we previously established a high-throughput platform that measures cellular survival to radiation. We then used this technology to analyze the radiosensitivity of more than 500 cancer cell lines and identified mutant alleles associated with radiation resistance. To functionally annotate a multitude of these variants, mutant constructs were generated using a high-throughput, site-directed mutagenesis approach and shuffled into lentiviral vectors for stable expression in immortalized BEAS2B lung cells. The effect of individual variants on radiation survival was assessed using our high-throughput platform and benchmarked against the radio-protective NRF2 E79K gain-of-function mutation and validated by colony forming assay. Radioresistant variants were further profiled in TP53 deficient BEAS2B cells and in cells across multiple lineages. For a subset of variants predicted to confer resistance by a loss of gene function, expression of the endogenous gene was knocked out by CRISPR directed to an exon-intron junction, thus allowing for the expression of the intron-less variant ORF.

We successfully generated and profiled over 550 tumor associated genetic variants. Top radioresistant conferring alleles were comprised of both rare and recurrent mutations. In many instances, rare variants were more impactful than common mutations in the same domain or even the same amino acid position. Therapeutically targetable radioresistant variants were discovered within ARAF, BRAF, CTNNB1, EGFR, MAP2K1 and PIK3CA, among others. Loss of gene function variants were profiled in KEAP1, STK11, and TP53. A subset of KEAP1 loss of gene function variants were found to confer a dominant-negative phenotype by stabilizing NRF2 levels.

Determining the impact of gene variants remains a major obstacle in the implementation of personalized radiotherapy. Here, we report on a large-scale profiling effort to identify and classify mutant alleles that govern the sensitivity of cells to radiation. We demonstrate that new genetic features responsible for mediating cellular survival to DNA damage can be discovered. We also show that a substantial proportion of variants that confer resistance to radiation are druggable, advancing drug-radiation combinations in "baskets" of patients with these alterations.

#2917

Inhibition of Annexin A6 sensitizes triple negative breast cancer cells to radiation.

Gladys N. Nangami, Amos Sakwe, Josiah Ochieng. _Meharry Medical College, Nashville, TN_.

Introduction: Despite vast evidence that tumor genetics play a dominant role in determining radiosensitivity and ultimate benefit from radiation therapy (RT), current RT delivery algorithms have not successfully incorporated tumor biology. Instead, clinical decisions regarding the use of RT are still based on features of tumor aggressiveness such as stage, grade, degree of metastasis and type of surgery. We provide evidence showing that most aggressive basal-like TNBC cells characteristically expresses low levels of the Ca2+-dependent membrane-binding tumor suppressor Annexin A6 (AnxA6) as compared to mesenchymal-like TNBC cell lines. We have also shown that depletion of AnxA6 in TNBC cells induced severe morphological changes from the slow growing mesenchymal-like to rapidly growing basal-like phenotypes and that this was not only associated with enhanced anchorage independent cell proliferation, but significantly inhibited cell adhesion, motility and invasiveness. Given that rapidly proliferating cells are more radiosensitive, we hypothesized that the expression status of AnxA6 in triple negative breast cancer cells underlies their response to RT.

Materials and Methods: For clonogenic assay, cells were grown to 70-80% confluency then were allowed to settle and attach for 8 hours before being exposed to irradiation at various doses. Dishes were then incubated at 37oC in 5% CO2 in a humidified incubator to form colonies for 14 days. Colonies (50 cells or more) were stained with crystal violet and enumerated under a Fisher Stereomaster II 10X stereoscopic microscope. Radiation induced cell cycle arrest was assessed by flow cytometry.

Results: Our studies show that depletion of AnxA6, significantly increased the sensitivity of triple negative breast cancer cells via up-regulation of cell cycle pathways as demonstrated by lack of G2/M cell cycle arrest in cells with low level of AnxA6. Additionally, we demonstrate that knockdown of AnxA6 enhanced radiation induced DNA damage.

Conclusions: Our findings demonstrate that reduced expression of AnxA6 enhances radiation sensitivity of triple negative breast cancer cells.

#2918

**Molecular analysis of the mechanism of action of AsiDNA** TM **brings new clues on DNA damage response regulation.**

Nathalie Berthault, Kozlac Maria, Sergey Alekseev, Pierre-Marie Girard, Marie Dutreix. _Institut Curie, Orsay, France_.

Purpose: Our laboratory succeeded at developing an original DNA repair inhibitor, called AsiDNATM, developed and tested in clinic by Onxeo. AsiDNATM is a small modified double-stranded DNA molecule vectorised by a covalently bound cholesterol. It acts by inhibiting DNA repair and sensitizes tumors to genotoxic treatments. Here we investigate the different steps involved in its activity.

Experimental design: SK28, MRC-5 and MDA-MB-231 cells were treated by AsiDNATM. Cellular uptake of AsiDNATM was monitored by FACS and electron microscopy. AsiDNATM interacting proteins were identified by mass spectrometry in cell extracts pooled down with AsiDNA. Kinetics of activation of the two main targeted enzyme, DNA-PK and PARP, were monitored by measuring phosphorylation of H2AX and formation of poly-adenyl-ribose polymers (PAR) and NAD consumption. Inhibition of DNA repair enzymes recruitment at damage site (53BP1, XRCC4, BRCA1, Rad51 and NBS1) was analyzed by confocal microscopy. DNA damage were induced at different times after AsiDNATM addition. The role of PARP, DNA-PK and ATM enzymes in the recruitment inhibition was tested using specific inhibitors or deficient cells. DNA repair activity was analyzed by alcalin comet assay and survival curves were established. Transcriptomes were monitored 24h after AsiDNATM treatment.

Results: AsiDNATM enters the cells via cholesterol route using LDL receptors. It binds DNA-PK and PARP and triggers their activation for more than 24h. The levels of DNA-PK and PARP activation as well as NAD consumption were always higher than those induced by a 10 Gy irradiation. Survival rate to AsiDNATM alone was between 90% and 70%. No significant effect was observed on cell cycle or proliferation. Pretreatment of tumor cells with AsiDNATM inhibits DNA repair and increase cell death induced by irradiation. The earliest event observed after irradiation was a transitory phosphorylation of ATM(ser1981) disappearing after 2h. It was followed by the inhibition of 53BP1and XRCC4 recruitment in a PARP dependent manner. Then, concomitantly with γ-H2AX and PAR increase, recruitment of BRCA1 and RAD51 was inhibited in cells with high level of DNA-PK activation. This inhibition was dependent of DNA-PK and not of PARP or ATM. Transcriptome analysis showed large change in the expression of genes involved in metabolism.

Conclusion: AsiDNATM inhibits NHEJ and HR double-strand break DNA repair by preventing the recruitment of key enzymes at break sites. The inhibition of NHEJ proteins recruitment is the earliest event and requires PARP activity. The inhibition of HR proteins appears lately and is dependent upon DNA-PK activation. PARP activation induces metabolism change that might participate to the antitumoral activity of AsiDNATM. These results highlight the unique mechanism of action of AsiDNATM through the activation of two complementary key enzymes involved in DNA damage response.

#2919

p16 modulates a novel ubiquitin signaling cascade which regulates radioresponse and offers clinically relevant therapeutic targets in squamous cell carcinoma.

David Molkentine,1 Kathleen Bridges,2 Aakash Sheth,3 David Valdecanas,2 Curtis Pickering,2 Heath D. Skinner1. 1 _The University of Pittsburgh, UPMC Hillman Cancer Center, Pittsburgh, PA;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 3 _Rutgers University, Robert Wood Johnson Medical School, New Brunswick, NJ_.

Human papilloma virus (HPV) is associated with multiple types of squamous cell carcinoma, including that of the head and neck (HNSCC). The presence of HPV leads to increased expression of p16, to the extent that p16 is used as a surrogate marker of HPV positivity. HPV/p16 expression is associated with improved response to genotoxic therapy and improved outcome; however, the mechanism of this phenomenon is unclear. Previously, we showed that HPV/p16 expression repressed the DNA damage repair protein TRIP12 and led to radiosensitivity. In the current project we identify USP7 and HUWE1 as key links in this signaling pathway. Ubiquitin-specific-processing protease 7 (USP7) is a deubiquitinating enzyme that has previously been shown to bind to TRIP12, which we confirmed in HPV (-) HNSCC cell lines. We found that forced p16 overexpression in a panel of SCC cell lines led to decreased levels of USP7 and TRIP12 protein, while inhibition of p16 in HPV (+) HNSCC cell lines led to their increase. Forced p16 expression did not affect USP7 mRNA expression, however did lead to increased K48-linked ubiquitinylation and degradation of USP7. Chemical or shRNA mediated inhibition of USP7 in p16/HPV (-) cells led to decreased 53BP1 and BRCA1 foci and increased cell death following radiation. Expression of USP7 shRNA in a HPV (-), radioresistant cell derived xenograft (HN5) lead to a tumor growth delay (TGD) of 10 days following radiation (4 Gy x 5d), compared to a TGD of 1.4 days in control tumors (p<1x10-15). To determine how p16 regulates USP7 ubquitinylation and degradation, we performed immunoprecipitation/mass spectrography (IP/MS) analysis. In 3 HPV (-) and 3 HPV (+) cell lines, pulldown of USP7 identified several E3 ubquitin ligases, however only HUWE1 and TRIM21 were identified in all cell lines tested. We then examined HUWE1 expression following forced expression of p16 and found that both HUWE1 mRNA and protein were increased. Moreover, inhibition of p16 expression in HPV (+) HNSCC cell lines led to decreased HUWE1 expression. Moreover, inhibition of HUWE1 via shRNA could partially rescue the effects of p16 overexpression on expression of DNA damage repair proteins. As HUWE1 is known to be mutated in ~10% of HNSCC, we then examined the effects of HUWE1 on outcome in the TCGA HNSCC patient cohort. In HPV (+) patients, neither HUWE1 mutation nor its gene expression were associated with survival. However, in HPV (-) patients, low HUWE1 expression was associated with worse disease-free survival (DFS)(p=0.048). Additionally, truncating mutations in HUWE1 led to a median survival of 9.4 mos, compared to 67.7 mos in the remaining patients (p=0.008). In conclusion, p16-HUWE1-USP7 signaling can modulate response to radiation. These data suggest that agents under investigation to target USP7, or other members of this signaling cascade, have the potential to improve outcomes in HNSCC.

#2920

Lymphoblast cells secrete putative protein factors in response to high-dose radiation causing anti-tumor effect in lung cancer cells.

Kamila Rawojc,1 Anthony Yeung,2 Mansoor M. Ahmed,3 Seema Gupta4. 1 _The University Hospital, Kraków, Poland;_ 2 _Fox Chase Cancer Center, Philadelphia, PA;_ 3 _Division of Cancer Treatment and Diagnosis, Rockville, MD;_ 4 _, Sylvester Comprehensive Cancer Center, University of Miami Leonard Miller School of Medicine, Miami, FL_.

High-dose ionizing radiation is known to induce non-targeted effects through bystander, abscopal, or immunological factors. As a complex niche, tumor microenvironment (TME) consists of several types of cells including cancer cells, immune cells, cancer-associated fibroblasts, endothelial cells etc. During the focal irradiation of the tumors, these cells may get exposed and release specific/unique factors, resulting in enhanced anti-tumor effects. The aim of this study was to identify the protein factors secreted by irradiated lymphoblasts and investigate their bystander/abscopal effects on lung cancer cells.

Lymphoblasts (GM3798) were irradiated (10 Gy) at the density of 1x106/mL. After 24 h, conditioned medium (CM) from radiation exposed (L-RCM) and un-exposed (L-CCM) flasks was collected and centrifuged to remove debris. H460 cells pre-plated on the microelectronic sensors of the 16-well plate were either incubated with L-CCM or L-RCM and their growth was continuously monitored by real time-cell electronic sensing system. Furthermore, lymphoblasts were grown in serum-free media for 24 h and following irradiation, 200 mL of the L-RCM and L-CCM was concentrated and fractionated by ultrafiltration (10, 30, 50 and 100KDa fractions) to identify the protein factors secreted by the cells. The effects of different fractions on A549 cell survival were assessed by colony forming assay. To obtain a proteomic comparison of >100 KDa fractions of L-RCM vs. L-CCM, following 1D PAGE, each gel slice was subjected to tryptic digestion and protein identification by mass spectrometry.

We found that L-RCM exerted significant growth inhibition of H460 lung tumor cells compared to L-CCM. Based on these results, we analyzed the secretory proteome profile of high-dose irradiated lymphoblasts. The protein factors responsible for 10 Gy-specific cell killing of A549 cells were found to reside in the 100 KDa fraction in a manner proportional to protein concentration. 277 proteins were identified with confidence in the protein fraction retained by the >100 KDa fractions, representing both large proteins and protein complexes. A number of statistically reliable proteins were found only in the 10 Gy RCM fraction. The highest eMPAI score was assigned to HLA-DQB2 protein involved in immune response of CD4+ T lymphocytes.

Our findings strongly suggest that high-dose radiation to TME can facilitate the release of secretory factors that can render a robust anti-tumor effect through bystander/abscopal signaling. Furthermore, identification of secretory proteins in response to high-dose radiation by the use of systematic and high through-put proteomics will help in identifying biomarkers in cancer patients. These proteins will serve as a marker for tailoring the use of adjuvants such as conventional subsequent radiation or chemotherapy or biological modifying drugs.

#2921

A dual role of genomic instability on radiosensitivity in cancer cells.

Yuji Seo, Keisuke Tamari, Yutaka Takahashi, Kazumasa Minami, Keisuke Otani, Fumiaki Isohashi, Kazuhiko Ogawa. _Osaka University Graduate School of Medicine, Suita, Japan_.

Background: Genomic instability is a hallmark of cancer, which may lead to heterogeneity and provide an advantage on tumor survival and proliferation. However, we previously found that a greater number of genomic mutations were associated with reduced tolerance to radiation. The aim of this study is to elucidate a role of genomic instability on cellular radiosensitivity.

Materials and Methods: We used 59 cancer cell lines derived from various types of solid tumors. Radiation-induced cell death was measured by the standard colony-forming assay at the dose range of 0 to 10 Gy. Based on the resulting survival curves, the mean inactivation dose (MID) was calculated as a single value representing cellular radiosensitivity. Somatic gene aberration data for the 59 cell lines were obtained from the Catalogue Of Somatic Mutations In Cancer cell line project v.80. The genomic analysis focused on cancer-related pathways defined by Kyoto Encyclopedia Genes and Genomes. The genomic mutations included nucleotide variations (substitutions, insertions, or deletions) and copy number variations (CNV). Only pathogenic mutations were selected and neutral mutations were disregarded according to the functional analysis through hidden Markov model. Both gain and loss in CNVs were included.

Results: There were either nucleotide variations or copy number variations in 598 genes in the 59 cancer cell lines. The mutations were found in 16 caretakers, 51 gatekeepers, 105 oncogenes, and 449 passengers. There were significant positive correlations between the MIDs and the proportions of mutations in the gatekeepers or the oncogenes to the whole mutations. Namely, radioresistant cell lines harbored gatekeeper mutations or oncogene mutations at higher proportions. Conversely, radiosensitive cell lines showed significantly higher proportions of passenger mutations. No correlation was found in between the MIDs and the proportions of caretaker mutations.

Conclusions: Genomic instability caused by caretaker mutations may result in not only increased tolerance to radiation through mutations in gatekeeper genes and oncogenes but also reduced tolerance to radiation through accumulating passenger mutations. A balance between the two opposing phenomena is a significant determinant of cancer cell radiosensitivity.

#2922

The radioprotector GC4419 enhances the response of squamous cell carcinoma of the head and neck tumors to ionizing radiation and enhances radioimmune therapy.

Brock J. Sishc, Elizabeth M. Polsdofer, Yuanyuan Zhang, Debabrata Saha, Michael D. Story. _UT Southwestern Medical Center, Dallas, TX_.

Locoregional recurrence (LRR) is the major cause of morbidity and mortality in patients with squamous cell carcinoma of the head and neck (HNSCC). For those patients that experience surgically unresectable LRR, median overall survival (OS) is less than one year. Approaches utilizing stereotactic ablative radiotherapy (SAbR) and immune oncology agents such as Nivolumab (α-PD-1 agonist) in the salvage setting have become alternatives to conventional chemoradiation therapy with one randomized, Phase II clinical trial combining these two modalities currently recruiting (clinicaltrials.gov identifier: NCT02684253). Despite improvements in OS, rates of acute oral mucositis (OM) and late toxicities (skin fibrosis) remain a concern with SAbR, particularly in the context of re-irradiation. Here we present pre-clinical evidence that GC4419 (Galera Therapeutics), a highly selective superoxide dismutase mimetic that recently received FDA Breakthrough therapy status following a Phase II, randomized, placebo controlled trial for reducing the duration and incidence of severe OM in patients undergoing chemoradiation therapy for locally advanced HNSCC (NCT02508389) also enhances the anti-tumor radiation response of HNSCC tumors to irradiation. GC4419 slows the growth of a panel of HNSCC cell lines in vitro, and utilizing clonogenic survival assays, demonstrates single agent anti-cancer activity at physiologically achievable concentrations, while also enhancing the response of HNSCC lines to irradiation. In tumor growth delay (TGD) experiments utilizing the syngeneic HNSCC tumor model, AT-84, when GC4419 is delivered starting 30-60 minutes prior to irradiation with biologically equivalent fractionation schedules of 17 Gy x 1 fxn, 10.24 Gy x 2 fxn, or 5 Gy x 5 fxn, a significant enhancement of the radiation response is achieved. Furthermore, when GC4419 is combined with radiation and α-PD-1 inhibitor, a further enhancement of the radiation response is observed, indicating that GC4419 compliments radiotherapy combined with immune oncology therapies. This potentiation of the anti-tumor response with GC4419 is more pronounced at doses that exceed the threshold to be considered for intensity modulated radiation therapy (IMRT). Results from clinical trials and the accompanying pre-clinical data strongly suggest that GC4419 should be combined with radio-immune therapy to not only enhance local tumor control, but that the potential for normal tissue protection with SAbR also creates the opportunity for dose escalation and may further improvement in treatment outcome.

#2923

DNA-PK inhibitor, M3814, is a potent inducer of inflammatory micronucleation in irradiated p53-deficient cancer cells: Implications for combination radio-immunotherapy.

Michael Carr,1 Astrid Zimmermann,2 Yige Guo,1 Xiaohong Liu,1 Patrick Steiner,2 Susanne Hahn,2 Frank Zenke,2 Andree Blaukat,2 Lyubomir T. Vassilev1. 1 _EMD Serono, Billerica, MA;_ 2 _Merck KGaA, Darmstadt, Germany_.

A novel, clinical-stage DNA-PK inhibitor, M3814, potently and selectively blocks the non-homologous end-joining pathway for repair of DNA double strand breaks (DSB) and synergizes with ionizing radiation (IR) and DSB-inducing chemotherapy. We have previously shown that M3814 boosts ATM/p53 signaling and reinforces cell cycle checkpoint controls in irradiated p53 wild-type cancer cells, inducing irreversible premature senescence. However, in the absence of functional p53 this protective mechanism is disrupted, cancer cells progress through the cell cycle with unrepaired DSBs, leading to aberrant mitotic division and ultimately cell death. Here, we investigated the mechanistic consequences of DNA-PK inhibition in p53-dysfunctional cancer cells exposed to IR. We found that cell cycle progression with persistent DSB damage, promoted by M3814, causes severe chromosome aberrations, misalignment, and mis-segregation. This leads to the formation of micronuclei after each abnormal mitotic division. Live cell imaging and immunofluorescence analysis revealed high numbers of ɣH2AX foci, marking unrepaired DSBs inside the micronuclei, and lamin-B1-deficient micronuclei lamina. Cyclic GMP-AMP synthase (cGAS) was found to colocalize with micronuclei, suggesting that compromised laminar integrity exposes damaged DNA to the cytosol and activates the STING pathway. Gene expression analyses revealed activation of over 60 immune-related genes in cells exposed to IR+M3814, consistent with a STING-driven inflammatory response. Indeed, many cyto-/chemokines with immunomodulatory properties were detected in culture media from IR+M3814 treated cells. In addition, elevated expression of PD-L1 protein was found in irradiated p53-deficient cancer cells exposed to M3814. Together, these results provided a clear rationale for combination with PD-L1 inhibitory therapy. Murine p53-mutant cancer cells, MC38, were grown subcutaneously bilaterally in syngeneic mice. One of the tumors was exposed to 5 x 3.6 Gy daily IR fractions given locally and daily oral M3814 at previously established efficacious doses, followed by three applications (every 3-4 days) of the PD-L1 inhibitory antibody, avelumab. The other tumors were left unirradiated to monitor for abscopal effect. Study results demonstrated a superior benefit of adding M3814 to the IR/avelumab combination, including enhancement of the abscopal effect of IR/avelumab combination. Altogether, our results revealed that selective DNA-PK inhibition by M3814 provides a powerful new mechanism for induction of micronucleation and STING inflammatory signaling in p53-disfunctional cancer cells and offers a new approach to combination radio-immunotherapy of cancer.

#2924

Evaluation of the pan-FGFR inhibitor AZD4547 with radiation in non-small cell lung cancer.

Margot Miller, Michael Fisher, Gopika Senthilkumar, Saakshi Kaushik, Lindsey Able, Sean Brennan, Gopal Iyer, Randall Kimple, Andrew M. Baschnagel. _University of Wisconsin, Madison, WI_.

Objective: The fibroblast growth factor receptors (FGFR) are commonly altered in non-small cell lung cancer (NSCLC), including high level of amplification of FGFR1 in lung squamous cell carcinoma. FGFR signaling may play a role in the response to radiation. We investigated the radiosensitizing effects of the selective tyrosine kinase inhibitor AZD4547 in a NSCLC model using cell lines and tumor xenografts.

Methods: A panel of six NSCLC cell lines were screened for FGFR1/2 DNA amplification, RNA expression, and protein expression. All cells were assessed for in vitro response to AZD4547. Immunoblots were used to examine the effect of AZD4547 on downstream signaling proteins. Apoptosis was measured by Annexin V staining and autophagy with acridine orange assay. Radiation clonogenic survival assays and xenograft growth delays experiments were performed to investigate radiosensitization. In vivo mechanistic studies were conducted using immunohistochemistry.

Results: Cell lines demonstrated varying degree of FGFR1/2 RNA and protein expression. In sensitive cell lines, AZD4547 inhibited p-MAPK in a time dependent manner. In vitro clonogenic survival assays showed robust radiosensitivity with AZD4547 in three out of six NSCLC cell lines. All three cell lines overexpressed FGFR1 and two of the cells had high FGFR1 copy number. There was no radiosensitization seen in an immortalized normal human bronchial epithelial cell line. A significant increase in autophagy and apoptosis was observed with combined radiation and AZD4547. Significant tumor growth delay was observed with the administration of radiation and AZD4547 compared to radiation or drug alone in two NSCLC xenograft tumor models. IHC analysis revealed modulation of FGFR-downstream signaling (p-Erk and p-S6) and proliferative (Ki67) and apoptotic markers (Cleaved Caspace 3).

Conclusion: These findings suggest that AZD4547 can augment the response of radiation in NSCLC model systems. FGFR1 and FGFR2 expression may be a potential targets for radiosensitization in NSCLC. Additional studies are underway to understand the mechanism of radiosensitization.

#2925

Targeting histone acetyltransferase (HAT) function for synthetic cytotoxicity in CREBBP/EP300 mutant tumors.

Curtis Pickering,1 Manish Kumar,1 Kathleen Bridges,1 Tongxin Xie,1 David Molkentine,2 Aakash Sheth,3 Liang Yang,1 Mitchell Frederick,4 Tim Heffernan,1 Sahil Seth,1 Jeffrey Myers,1 Heath D. Skinner2. 1 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _The University of Pittsburgh, UPMC Hillman Cancer Center, Pittsburgh, PA;_ 3 _Rutgers University, Robert Wood Johnson Medical School, New Brunswick, NJ;_ 4 _Baylor College of Medicine, Houston, TX_.

CREB-binding protein (CREBBP) and E1A binding protein p300 (EP300) share significant homology and are collectively mutated in ~14% of head and neck cancers (HNSCC). Recently we have identified CREBBP/EP300 mutations being associated with poor outcome. Thus, tumors which harbor these mutations may be an ideal subset to study for agents that lead to synthetic cytotoxicity. We have performed in vivo shRNA screening combined with radiation in tumors derived from human HNSCC cell lines (CREBBP/EP300 mutant: Cal27, UMSCC22A, UMSCC47; CREBBP/EP300 wild type: UPCISCC152, HN31) to identify targets that are synthetically cytotoxic when combined with radiation in the CREBBP/EP300 mutant background. This identified CREBBP and EP300 proteins as the most significant targets. Additionally, inhibition of either CREBBP or EP300 with shRNA, combined with radiation, led to increased γ-H2AX, decreased BRCA1, increased apoptosis and increased cell death in CREBBP/EP300 mutant, but not wild type cells. In vivo studies of CREBBP mutant tumors using both HPV (+) and HPV (-) xenograft models showed dramatically increased tumor growth delay (TGD) following radiation in CREBBP knockdown tumors, but not controls, with 40-80% of tumors cured after low doses of XRT. This was accompanied by increased TUNEL staining in shCREBBP tumors following radiation (p<0.001), as well as an increase in pro-apoptotic proteins on RPPA. We then evaluated chemical inhibitors of CREBBP and EP300. A CREBBP specific inhibitor led to significant in vitro radiosensitization, irrespective of HPV status, but only in CREBBP mutant HNSCC cell lines. A similar finding was seen using shRNA-mediated inhibition, namely that specific inhibition of either CREBBP or EP300 led to synthetic cytotoxicity only in HNSCC lines harboring the individual mutant. We next turned our attention to agents that affect specific functions of EP300 and CREBBP. We initially used a bromodomain inhibitor in vitro in combination with radiation and observed minimal effect at therapeutic doses. We next examined a HAT inhibitor specific for CREBBP and EP300. This agent, but not its inactive sister compound, led to a profound radiosensitization on clonogenic assay in a panel of EP300 and/or CREBBP mutant HNSCC cell lines (p<0.001 for each of 6 HNSCC lines), but not in 3 double wild type lines. This effect was accompanied by increased apoptosis and decreased BRCA1 foci following radiation. We also observed decreased H3K18 and H3K27 acetylation following HAT inhibitor treatment, but only in CREBBP/EP300 mutant cell lines. In conclusion, the observed synthetic cytotoxicity of radiation and CREBBP/EP300 inhibition in mutated background is seen both in vitro and in vivo, largely due to increased apoptosis, and may be primarily due to the HAT function of the protein. As HAT inhibitors are being developed for clinical use, these observations could have a direct positive clinical impact.

#2926

Nomination and characterization of TTK for radiosensitization in basal-like breast cancers.

Benjamin C. Chandler, Leah Moubadder, Cassie Ritter, Kari Wilder-Romans, Meleah Cameron, Meilan Liu, Shyam Nyati, Andrea Pesch, Anna Michmerhuizen, Eric Olsen, Yashar Niknafs, Arul Chinnaiyan, Corey Speers. _University of Michigan, Ann Arbor, MI_.

Background: Increased rates of locoregional recurrence have been observed in basal-like breast cancer despite the use of radiation therapy (RT), therefore approaches that result in radiosensitization of basal-like breast cancer are critically needed. Studies detailing the poor response of basal-like tumors to adjuvant RT underscore the biologic differences and as of yet undefined oncogenic drivers of these particular types of breast cancer.

Methods: 4 independent datasets were used to correlate gene expression with local recurrence (LR) after 3 years. Kaplan-Meier analysis was used to validate the impact of TTK expression on LR. The TCGA and institutional breast cancer dataset were used to determine TTK expression in BC subtypes. TTK RNA and protein levels were measured using qPCR and western blot at baseline and correlated to intrinsic radiosensitivity. Clonogenic survival assays were used to determine the radiosensitization of cell lines after TTK inhibition (TTKi). Mice models were used to assess TTKi in combination with RT in vivo. DNA damage was quantified using γH2AX staining. HR and NHEJ efficiency assays were performed using HR/NHEJ specific reporter systems. HR competency was also assessed using RAD51 foci formation assays.

Results: Ten genes were found to significantly correlate with early LR (≤3 years) across 4 distinct datasets (N=896 pts) (OR of recurrence > 2, p-value <0.000001), with TTK, a cell cycle kinase, ranked the highest. Kaplan-Meier survival analysis in 3 cohorts demonstrated that higher than median TTK expression correlates with decrease LR free survival after RT (HR 1.70-2.42, p<0.01 for all 3 cohorts). Subtype association analysis demonstrated that TTK expression was most elevated in basal-like breast cancer (p<0.0001). Clonogenic survival assays, using inducible shRNA models, show the combination of TTK knockdown and RT increases radiosensitivity in MDA-MB-231 and BT-549 basal-like breast cancer cell lines (rER 1.21-1.63). Additionally, TTKi using, Bayer 1161909 (B909), enhanced radiosensitivity in both cell lines (rER 1.10-1.39). In vivo, TTKi, using shRNA and B909 in combination with RT led to a significant increased time to tumor tripling. Using γH2AX foci formation assays, increased DNA damage was found after combination treatment of TTKi and RT compared to RT alone, indicating that DNA damage repair mechanisms may be compromised by TTKi. We found that the efficiency of the double strand DNA damage repair mechanism, homologous recombination (HR), but not non-homologous end joining (NHEJ), was reduced upon TTKi in HR/NHEJ specific reporter systems. Additionally, RAD51 foci formation was reduced by TTKi after RT compared to RT alone.

Conclusion: These data support TTK inhibition as a rationale radiosensitizing strategy for clinical development in basal-like breast cancer patients and that the mechanism of radiosensitization is, at least in part, through impaired HR repair.

#2927

Papaverine and its novel derivatives radiosensitize solid tumors by inhibiting mitochondrial metabolism.

Martin Benej,1 Xiangqian Hong,2 Sandip Vibhute,3 Sabina Scott,1 Jinghai Wu,1 Edward Graves,4 Quynh-Thu Le,4 Albert C. Koong,5 Amato J. Giaccia,4 Ching-Shih Chen,6 Bing Yu,2 Ioanna Papandreou,1 Nicholas C. Denko1. 1 _The Ohio State Univ.y Wexner Medical Ctr., Columbus, OH;_ 2 _Marquette University and Medical College of Wisconsin, Milwaukee, WI;_ 3 _The Ohio State Univ.y, Columbus, OH;_ 4 _Stanford University School of Medicine, Stanford, CA;_ 5 _MD Anderson Cancer Center, Houston, TX;_ 6 _China Medical University, Taichung, Taiwan_.

Radiation therapy is a standard type of treatment modality used to achieve local control in more than 50% of all cancer patients. However, tumor hypoxia reduces the effectiveness of radiation therapy by limiting the biologically effective dose. Limited distribution of oxygen is a direct consequence of abnormalities in vascular structure and angiogenesis that fails to provide sufficient oxygen to meet the demands of metabolically hyperactive cancer cells. An acute increase in tumor oxygenation prior to radiation treatment should therefore significantly improve the tumor cell kill after radiation. Nonetheless, therapeutic efforts to increase oxygen delivery to the tumor have not shown positive clinical results to this day. We have taken an alternative route by targeting the demand for oxygen rather than its supply. In the cell, mitochondrial respiration is the major oxygen-consuming process. We show that pharmacological inhibition of mitochondrial oxygen consumption (OCR) temporarily reduces the tumor cells' demand for oxygen leading to increased tumor oxygenation and enhanced radiation response. We identified a previously unrecognized activity of the FDA-approved drug papaverine as an inhibitor of mitochondrial complex I. In vivo, a single clinically achievable dose of papaverine increased tumor, but not normal tissue oxygenation within 45 minutes and strikingly enhanced tumor response to radiation therapy in both ortho- and heterotopic rodent tumor models. Moreover, our GI tract studies show that this can be achieved without exacerbating normal tissue toxicity, as papaverine does not radiosensitize hypoxic normal tissues. Papaverine is an ergot alkaloid originally isolated from Papaver somniferum in 1848. It was used for decades as smooth muscle relaxant to treat vasospasms and erectile dysfunction. Its vascular effects were believed to be mediated by its ability to inhibit phosphodiesterase 10A (PDE10A). We provide genetic evidence that papaverine's complex I inhibition, not its activity as a PDE10A inhibitor is directly responsible for increased oxygenation and enhanced radiation response. Furthermore, we describe novel derivatives of papaverine that have the potential to become a new generation of clinical radiosensitizers with potentially fewer side effects. Papaverine has a short half-life of 90-120 minutes, is cell-permeable, reversible and quickly cleared from the patient. In vitro, all 28 cancer and normal cell lines tested were sensitive to papaverine regardless of their oncogenic landscape, suggesting possible application for a broad spectrum of cancers that depends primarily on their level of hypoxia. In conclusion, PPV or one of its novel derivatives appear to be ideal candidates for clinical radiosensitization, applicable primarily in cancers where local control increases the overall survival.

#2928

New insight into signaling contexts for the administration of the imipridone ONC201 to prostate cancer cells.

Francesca Amoroso,1 Adam Pickard,2 Kimberly Glass,1 Rohinton Tarapore,3 Josh Allen,3 Ian G. Mills4. 1 _Queen's Univ. Belfast, Belfast, United Kingdom;_ 2 _University of Manchester, Manchester, United Kingdom;_ 3 _Oncoceutics Inc., Philadelphia, PA;_ 4 _University of Oxford, Oxford, United Kingdom_.

Prostate cancer is the most common non-cutaneous cancer in men and a notable cause of cancer mortality. In the majority of the cases localised disease radical treatment consists of surgery or radiotherapy. Upon progression androgen deprivation therapy can extend survival however metastatic disease is incurable. The prostate is a specialized accessory gland with a high secretory capacity. As prostate cancer develops and treatment resistance emerges, the unfolded protein response (UPR) is an important adaptive biology which co-amplifies with key cancer drivers1. The UPR can be cytoprotective but when acutely activated can lead to cell death. In this study we sought to enhance the acute activation of the UPR using radiation and ONC201, previously reported to be a UPR activator2. We found that treating prostate cancer cells with ONC201 induced increases in the expression of components in all arms of the UPR - ATF4, ATF6 and IRE1-XBP1 - at protein and transcript levels at a 24-hour time point and this was followed at 72 hours by cell death. This delay in the induction of cell death provided a time window for the short-term administration of ONC201 to prime an enhanced cytotoxic response to radiation. Pre-treatment with ONC201 for 24 hours followed by withdrawal and subsequent irradiation led to enhanced cytotoxicity assessed by cell viability and clonogenic assays. In order to decipher the impact of ONC201 and radiation we undertook RNA-seq under a range of treatment conditions. We noted a durable suppression of transcripts encoding cell cycle regulatory genes when ONC201 pre-treatment was used to enhance radiation response and this translated into cell cycle arrest and induction of both necrotic and apoptotic cell death as assessed by flow cytometry. Pathway analysis of the RNA-seq suggests alternative targets that could replicate this priming effect to enhance radiation response.

1.Storm, M. et al., Oncotarget 7(33): 54051-54066 (2016).

2.Allen, J. et al., Oncotarget (2016). doi:10.18632/oncotarget.11814.

#2929

Radiosensitization of head and neck cancer by FGFR inhibition.

Gopika SenthilKumar, Margot Miller, Michael Fisher, Sean Brennan, Saakshi Kaushik, Lindsey Able, Paul M. Harari, Gopal Iyer, Randall J. Kimple, Andrew M. Baschnagel. _University of Wisconsin, Madison, Madison, WI_.

Objective:

Fibroblast growth factor receptors are frequently amplified or overexpressed in head and neck squamous cell carcinomas (HNSCC). We assessed the potential of a selective FGFR-kinase inhibitor, AZD4547, to act as a radiosensitizer in head and neck cancer cell lines and xenografts.

Methods:

A panel of head and neck cancer (n=10) and normal oral mucosa (HTE) cell lines were screened for FGFR1, 2, and 3 gene copy number, RNA expression, and protein. Sensitivity to the FGFR inhibitor AZD4547, alone or in combination with radiation, was assessed using proliferation and clonogenic survival assays. Putative mechanisms of radiosensitization were investigated by immunoblot and by assessing for DNA repair capacity, cell cycle effects, apoptosis, senescence, and autophagy. Tumor response of cell line xenografts and patient derived xenografts was assessed in vivo.

Results:

Cells demonstrated varying responses to FGFR inhibition but this did not correlate with FGFR gene copy number, mRNA expression, or protein expression. Three cell lines which responded to AZD4547 alone (CCL30, Tu-138, and SCC6) were selected for further investigation. TU-138 and CCL30 featured high FGFR expression and were radiosensitized by AZD4547 in vitro. SCC6 was not. Cell lines which were insensitive to FGFR inhibition (including HTE) did not demonstrate radiosensitization by AZD4547. In the sensitive cell lines, enhanced p-MAPK inhibition was seen in a time-dependent manner following drug treatment. When added to radiation, AZD4547 resulted in increased apoptosis, autophagy, and senescence but did not alter the kinetics of DNA repair as assessed by resolution of gH2AX foci. No difference in the cell cycle distribution for irradiated cells treated with or without the addition of AZD4547 was seen. Treatment of two in vivo tumor xenografts with AZD4547 and radiation showed significant delay in tumor growth compared to treatment with radiation or drug alone.

Conclusion:

Our findings indicate that AZD4547 can augment the response of FGFR expressing HNSCC to radiation both in vitro and in vivo. Improved approaches to identify tumors most likely to benefit from FGFR inhibition could enable the selection of patients for combination therapy and has the potential to improve outcomes in these difficult to treat cancers.

#2930

Baicalein attenuates radiation-induced enteritis by improving endothelial dysfunction.

Hyosun Jang, Sunhoo Park, Sehwan Shim, Jae Kyung Myung. _Korea Institute of Radiological and Medical Sciences, Seoul, Republic of Korea_.

Background: Radiation-induced intestinal injury is frequently observed following clinical application of radiation for abdominal and pelvic cancers or occurs secondary to radiation exposure following a nuclear accident. Radiation-induced enteritis may be considered an ideal model of gastrointestinal inflammation. The endothelium is a crucial component of inflammation, and the endothelial dysfunction following radiation exposure induces the intestinal proinflammatory response and progression of radiation enteritis. Baicalein (5,6,7-trihydroxyflavonoid) is a flavonoid from Scutellaria baicalensis used in oriental herbal medicine. Baicalein has been found to have multiple beneficial properties including antioxidant, anti-inflammatory, anti-allergic, and anti-cancer activities. Here, we investigated the therapeutic effects of baicalein on endothelial dysfunction in radiation-induced intestinal inflammation. Methods: We performed histological analysis, bacterial translocation assays, and intestinal permeability assays, and also assessed infiltration of leukocytes and inflammatory cytokine expression using a radiation-induced enteritis model. In addition, to investigate the effect of baicalein in endothelial dysfunction, we analyzed endothelial-derived adherent molecules in human umbilical vein endothelial cells and irradiated intestinal tissue. Results: Histological damage such as shortening of villi length and impaired intestinal crypt function was observed in the radiation-induced enteritis model. However, intestinal damage was attenuated in baicalein-treated groups with improvement of intestinal barrier function. Baicalein inhibited the expression of radiation-induced adherent molecules in the endothelial cells and increased leukocyte infiltration in the irradiated intestine. We also determined that baicalein could accelerate crypt regeneration via recovery of endothelial damage. Conclusions: Baicalein has a therapeutic effect on radiation-induced intestinal inflammation by attenuating endothelial damage. <!--EndFragment-->

#2931

**The compounding effects of coenzyme q10 and radiation treatment on glial fibrillary acidic protein network of glioma** in vitro **.**

Chong-Chun Liao, Megha Jhunjhunwala, Li-Han Chung, Chi-Shuo Chen. _National Tsing Hua University, R.O.C, Hsinchu, Taiwan_.

Introduction: Astrocytoma is an aggressive primary malignant brain tumor in adults. Radiation therapy is an effective medical treatment, but one of its undesired side-effects is the induced genomic instability of surrounding astrocytes, which can lead abnormal distribution of GFAP and associates with severe brain injury (BI).

Purpose: In order to cope with this serious difficulty, various antioxidants have been applied with radiation therapy. Among antioxidants, Coenzyme Q10 (Q10) is one of the few candidates without double-edged sword effect. However, the understand about Q10 therapeutic effect on radiological protection is limited. In this study, we aimed to investigate the potential therapeutic effects of Q10 on brain tumor radiation treatment.

Materials and Methods: Astrocyte and glioma cell lines were cultivated with Q10 (10μM~ 50 μM) and exposed to 10 Gy single dose from Cobalt-60 (60Co). After the radiation exposure, cell viability was analyzed using MTT assay, GFAP responses was examined by immunofluorescence staining and qPCR, and other cell phenotype indexes, such as the morphology, migration, and invasion in vitro, were observed by microscopy.

Results: Our results indicated that Q10 treatment was capable of maintaining GFAP network of astrocyte after radiation exposure. Besides, Q10 synergistically eliminated glioma proliferation after radiation treatment. By extending horizontally along the base of GFAP remodeling, we further investigated the ability of 3D tumor invasion in co-culture model which simulated the microenvironment composed of reactive astrocyte and glioma in clinical research. Results indicated that radiation therapy with Q10 can further suppress the invasive ability in vitro.

Conclusions: In sum, our results demonstrated the potential radiological protection of Q10 on astrocyte and amplified the therapeutic effectiveness for brain tumor therapy.

#2932

Withanolide D enhances radiosensitivity of human cancer cells to X-rays radiation.

Jerome Lacombe,1 Titouan Cretignier,2 Laetitia Meli,2 Jean-Luc Veuthey,2 Leslie Gunatilaka,1 Muriel Cuendet,2 Frederic Zenhausern1. 1 _University of Arizona, Phoenix, AZ;_ 2 _University of Geneva, Switzerland_.

Along with surgery and chemotherapy, radiation therapy (RT) is an important modality in cancer treatment, with about half of cancer patients receiving RT during their treatment. However, there are still a lot of challenges to improve its efficiency by minimizing normal tissue toxicity while maximizing tumor control. In this perspective, radiosensitizers are a promising approach in order to create a better tumor response to ionizing radiation (IR) while allowing for better dose modulation. Withanolides are natural steroidal lactones found in the plant Withania somnifera and already known for their numerous biological effects, in particular an antitumor activity due to induction of ROS production, cell cycle arrest or cytoskeleton destabilization. Hence, we tested in this study our hypothesis that withanolide D (WD), a compound with an important antitumor effect, could also act as a radiosensitizer. Clonogenic assays showed that 1-hour WD pretreatment (0.7µM) before IR decreased surviving fraction of several cancer cell lines. To determine the mechanisms by which WD achieves its radiosensitizing effect, we then assessed whether WD could promote radiation-induced DNA damages and inhibit double-strand breaks (DSBs) repair in SKOV3 cells. Comet and γH2AX/53BP1 foci formation assays confirmed that DSBs are higher from 1 hour up to 24 hours after 2Gy-irradiation in WD-treated cells compared to vehicle-treated cells, suggesting that WD induces the persistence of radiation-induced DNA damages. We then performed immunoblotting to investigate protein expression involved in DNA repair pathways. Interestingly, DNA-PK, ATM and their phosphorylated forms appeared to be inhibited at 24 hours post-irradiation in WD-treated samples. XRCC4 expression is also decreased while RAD51 expression does not change compared to vehicle-treated cells suggesting that only non-homologous end joining pathways is altered by WD. In order to assess the consequence of such inhibition, we then investigated cell death, and especially delayed death, referred to as mitotic catastrophe (MC), which was described as the main form of epithelial cell death induced by IR. MC is induced after IR and but is predominant in WD-treated samples as showed by the few numbers of cells pursing into anaphase. Interestingly, our results showed that cells preferentially undergo necrosis-like death after MC instead of apoptosis. This can be explained by the p53 mutated profile of SKOV3 in addition to the WD-induced ATM inhibition. Together, these data demonstrate that WD is a promising radiosensitizer candidate for RT and additional studies are required to investigate its effect in more relevant clinical models.

## EXPERIMENTAL AND MOLECULAR THERAPEUTICS

### Cellular and Molecular Pharmacology, Pharmacogenomics, Pharmacogenetics, and Therapeutic Response

#2933

Application of a mouse model humanized for the major pathways of drug disposition in anticancer drug development and use.

Colin J. Henderson,1 Nico Scheer,2 Yury Kapelyukh,1 Aileen McLaren,1 Kenneth MacLeod,1 Anja Rode,2 De Lin,1 C Roland Wolf1. 1 _University of Dundee, Dundee, United Kingdom;_ 2 _Taconic Biosciences, Cologne, Germany_.

A vast number of targeted anticancer drugs are being developed in the pharmaceutical industry whose efficacy will only become fully realized through their combination with other established or novel anti-tumor agents. This is particularly the case because combination chemotherapy is a major approach being taken to overcome the rapid onset of drug resistance which current dosing regimens can cause. This has raised the conundrum of which of the large number of possible drug combinations, which may involve combinations of more than two drugs, have the greatest chance of success. It is not possible to test all the possible combinations by clinical trial alone so more informative preclinical models are badly needed. Nearly all targeted anti-tumor agents are substrates for the cytochrome P450-dependent monooxygenase system. The testing of novel drug regimens in mice is severely compromised by the major species differences in this enzyme system both in catalytic function, in the pattern of metabolite formation and the regulation by transcription factors such as CAR and PXR. In order to circumvent this problem we have created a mouse model where thirty four murine P450's have been deleted from the mouse genome and substituted for the major enzymes involved in drug disposition in man ie CYP1A1, CYP1A2, CYP2C9, CYP2D6, CYP3A4 and CYP3A7. The mice have also been humanized for the transcription factors CAR and PXR. CYP3A4 and CYP2D6 are expressed off the human promoters. We report the validation the utility of this model by studying the in vivo metabolism and disposition of model drugs and the metabolism and disposition of the EGFR inhibitor, osimertinib and the BRAF inhibitor, dabrafenib. We show that the use of this model allows accurate prediction of clinically observed drug exposures, in the generation of human metabolites and drug/drug interactions. This model has therefore great potential for the development of combination therapies involving complex drug regimens and in the design of clinical trials targeted at overcoming drug resistance.

#2934

AKT mutant allele-specific activation dictates pharmacologic sensitivities.

Tripti Shrestha Bhattarai,1 Tambudzai Shamu,1 Swati Patel,1 Alexander Gorelick,1 Matthew T. Chang,2 Elena I. Gavrila,1 JianJong Gao,1 Mark T. Donoghue,1 Paul S. Gao,1 Tara Soumerai,1 Wassim Abida,1 Lilian M. Smyth,1 David M. Hyman,1 David B. Solit,1 Barry S. Taylor1. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _Genentech Inc., South San Francisco, CA_.

The objective of this study is to explore the biochemical characteristics and therapeutic sensitivity profiles of novel, non-E17K, less frequent AKT1 mutations, and thereby expand the biomarker of sensitivity to AKT inhibition in molecularly defined cancer patients. AKT is a critical signaling node that translates phosphoinositide 3-kinase (PI3K) pathway stimulation into diverse cellular effects. Gain of function AKT1 mutations arise in diverse human cancers, of which E17K is the most common. Presence of AKT1 E17K mutation renders these tumors susceptible to AKT inhibition. Nevertheless, the long tail of potentially activating mutations in AKT is largely uncharacterized, thereby limiting our ability to act clinically in prospectively sequenced advanced cancer patients. We performed a population-scale candidate driver mutation discovery analysis in AKT1, AKT2 and AKT3 in a cohort of 41,075 retrospectively and prospectively sequenced primary and metastatic cancers, and explored both their functional and biological impact as well as their therapeutic sensitivity. Our results demonstrated that some, but not all of the identified AKT missense mutations activated PI3K signaling in a growth factor-independent manner, and sensitized tumor cells to diverse AKT inhibitors. By contrast, we discovered a different class of small in-frame paralogous AKT duplication mutants that induced distinctive structural changes, leading to a far greater degree of membrane affinity, AKT activation, pathway dependence, and hyper-sensitivity to ATP-competitive AKT inhibitors, while conferring resistance to allosteric AKT inhibitors. Leveraging a co-clinical trial framework, we are now enrolling patients on the basis of these mutations in a basket study involving AKT alterations. One such case was that of a castration-resistant metastatic prostate cancer patient who harbored AKT2 duplication mutant, and subsequently responded to AKT inhibition. Collectively, our data indicate that the degree and mechanism of activation of oncogenic AKT mutants vary, thereby dictating allele-specific pharmacological sensitivities to AKT inhibition.

#2935

Novel combination of repurposed drugs induces complete cell invasion arrest of glioblastoma in vitro.

Liliana Oancea-Castillo,1 Kerstin Bahr,2 Anne Régnier-Vigouroux,1 Maximilian Ackermann,2 Isaac R. Gabriel,3 Samuel T. Pereles,3 Madeleine M. Li,3 Dana W. Lowitt,3 William W. Li,3 Vincent W. Li3. 1 _Institute of Developmental Biology & Neurobiology Johannes Gutenberg-Universität, Mainz, Germany; _2 _Institute of Functional and Clinical Anatomy, University Medical Center, Johannes Gutenberg University, Mainz, Germany;_ 3 _The Angiogenesis Foundation, Cambridge, MA_.

Introduction: Glioblastoma multiforme (GBM) is an aggressive and lethal cancer with a poor prognosis even after conventional treatment (surgery, radiation, chemotherapy). Temozolomide (TMZ) is a standard cyotoxic agent used, despite resistance leading to recurrence. Therefore, additional strategies for targeting the tumor environment are needed. We demonstrate a combination of approved drugs (CAD) repurposed to target GBM leads to complete arrest of GBM cell invasion. Each drug in the combination individually targets diverse tumor pathways: 1) invasion via MMP2 (doxycycline); 2) angiogenesis, inflammation, and proliferation via p53-dependent G1 cell-cycle arrest (celecoxib); 3) autophagy (tamoxifen); 4) tumor metabolism and angiogenesis (metformin).

Material and Methods: Multicellular Tumor Cell Spheroids (MTCS) were generated from two GBM cell lines (LN18, U87MG) and embedded in collagen (three-dimensional in vitro assay) and mixed with the drug combination at 2 doses + TMZ. GBM cell invasion was monitored and quantified over 96h. Flow cytometry analysis using Propidium-Iodine was performed (24h, 48h, 7h2 and 96h) to quantify the effect of the drug cocktail. Scanning Electron Microscopy (SEM) of MTCS was used to evaluate cell death at the ultrastructural level (96h after treatment).

Results: The CAD induced complete cell invasion arrest in both U87MG and LN18 MTCS regardless of drug dose or presence of TMZ. GBM cell death was observed after 96h. TMZ in combination with CAD showed a significant increase in cell death, suggesting synergism. SEM images demonstrated large-scale changes in the tumor spheroid morphology showing GBM cell death.

Conclusions: The combination of approved drugs (CAD) with TMZ can effectively halt GBM cell invasion and induce cell death in vitro. These results suggest a sensitizing effect of CAD to potentiate TMZ's effects. Because CAD components have well established safety profiles, further investigation to define the anti-GBM mechanisms and effects on the tumor microenvironment are warranted. This CAD has potential for future therapeutic approaches. The generic nature of the drugs makes CAD an intriguing cost-effective GBM adjunctive therapy.

#2936

Increased PARP1-DNA binding due to autoPARylation inhibition of PARP1 on DNA rather than PARP1-DNA trapping is correlated with PARP1 inhibitor's cytotoxicity.

Hua-Dong Chen, Chuan-Huizi Chen, Yu-Ting Wang, Ne Guo, Yu-Nan Tian, Xia-Juan Huan, Shan-Shan Song, Jin-Xue He, Ze-Hong Miao. _Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China_.

PARP1 inhibitors (PARPis) have been successfully used in the clinic for cancer therapy. The mechanisms by which they elicit cytotoxicity in cancer cells remain to be clarified. Initially, inhibition of cellular polymerase activity of PARP1 (reflected as inhibition of cellular poly (ADP-ribose) (PAR) formation or PARylation) was proposed as their primary mechanism. However, either cell-free histone-based or cellular PARylation inhibition displays very low correlation with PARPis' cytotoxicity or clinical anticancer effects. Subsequently, the Pommier's group proposed the mechanism of PARP1-DNA trapping and demonstrated that this mechanism showed much higher correlation with PARPis' cytotoxicity. Nevertheless, contradictory results have been reported since then. Here, we showed no significant correlation between PARP1-DNA trapping and cytotoxicity induced by PARPis. We complemented PARP1-knockout sublines with wild-type PARP1 and 11 mutants with different point mutations that affect the polymerase activity. When examining the PARPi talazoparib, the induced cytotoxicity was highly significantly correlated with cellular PARP1 polymerase activity, but not with its PARP1-DNA trapping or polymerase inhibition. Similarly, talazoparib's PARP1-DNA trapping revealed significant correlation with the polymerase activity rather than its inhibition. Differently, however, when evaluating purified wild-type and mutated PARP1, we identified an almost linear relationship between PARPis' inhibiting PARP1 dissociation from DNA and their cytotoxicity in 17 cancer cell lines. In contrast, no significant correlation existed between PARP1 polymerase inhibition in the histone-based systems and the cytotoxicity. After careful comparisons on different methods and detection targets, we conclude that the PARPi-mediated increase in PARP1-DNA binding by inhibiting autoPARylation of PARP1 on DNA rather than in PARP1-DNA trapping is correlated with PARPi's cytotoxicity. Based on this conclusion, we established a new PARPi screening model that more closely predicts cytotoxicity (r=0.9984; p<0.0001).

#2937

**Preclinical evaluation of new therapeutic strategies on** SDHB **invalidated clones from human pheochromocytoma cells.**

Constance Lamy, Julien Hadoux, Sylvere Durand, Abir Alghuzlan, Julie Riviere, Deborah Lefevre, Amaury Bongers, Jean-Yves Scoazec, Eric Baudin, Angelo Paci, Sophie Broutin. _Inst. Gustave Roussy, Villejuif, France_.

Introduction: Malignant paraganglioma/pheochromocytoma (MPP) are very rare neuroendocrine tumors with heterogeneous prognostic and no gold-standard treatment. MPP can be associated with germline mutations at SDHB gene which encode for a TCA enzyme, the succinate dehydrogenase that catalyzes the oxidation of succinate to fumarate. When mutated, SDHB losses its function, leading to succinate accumulation by inhibiting 2‐oxoglutarate dependent dioxygenases involved in methylation and angiogenesis. Thus alkylating agents, antiagniogenic and demethylating drugs are potential strategies for MPP patients.

Aim: This project aimed to establish preclinical models of SDHB invalidated human cell line and to characterize new therapeutic strategies.

Material & Methods: Invalidation of SDHB gene has been performed using the CRISPR-Cas9 technology on a human pheochromocytoma cell line (hPHEO1). Isogenic clones have been characterized at the genetic, cellular, proteic, and enzymatic levels through sequencing, immunohistochemistry, western-blotting and enzymatic assays, respectively. The antiproliferative effects of three drugs (temozolomide, sunitinib, azacytidine) have been explored and half maximal inhibitory concentrations (IC50) have been determined on the parental cell line and the 4 clones using the WST1® assay.

Results: Four heterozygote isogenic clones have been obtained with 3 different genetic alterations (deletion-insertion). Expression of Ki67 was increased in the SDHB+/- clones. SDHB expression was partially reduced in clones 1 and 2 and almost totally suppressed in clones 3 and 4. For temozolomide and sunitinib, IC50 were significantly lower in SDHB+/- clones (~140 µM for temozolomide and ~1.4 µM for sunitinib) compared to the parental cell line (~1300 µM for temozolomide and ~6 µM for sunitinib) whereas IC50 were similar for azacytidine (~3.5 µM). No significant difference was observed between clones.

Conclusion: In this study we obtained 4 SDHB invalidated isogenic clones from the human hPHEO1 cell line, pretty useful for preclinical evaluations in this rare disease. In agreement with literature clones were more sensitive to temozolomide but also to sunitinib. Metabolomic characterization of hPHEO1 and SDHB+/- clones is currently under investigation at baseline and after IC50 treatment of the 3 therapeutic strategies. Drugs' combinations will be studied in a further step.

#2938

Transmembrane chloride intracellular channel 1 (tmCLIC1) correlates with metastatic potential of colorectal cancer cells.

Valentina Carlini, Francesca Cianci, Ivan Verduci, Michele Mazzanti. _University of Milan, Milan, Italy_.

Colorectal cancer (CRC) is the third most common and lethal tumor worldwide. Although CRC can be efficiently eradicated when diagnosed in the early stage, no effective therapies are currently available for advanced metastatic disease. Therefore, targeting metastasis represents a critical challenge for the successful treatment of CRC. To achieve this purpose, our laboratory has found a promising candidate inthe transmembrane localized Chloride Intracellular Channel 1 (tmCLIC1) protein. Growing scientific evidence have suggested an involvement of tmCLIC1 in different human malignancies, including CRC, with an unknown mechanism. CLIC1 is a metamorphic protein able to shuttle between a soluble cytoplasmic and a membrane integrated form, the latter characterized by ionic channel function. In normal cells, it is mostly cytoplasmic and cooperates to the maintenance of several physiological processes. Otherwise, in cancer cells CLIC1 undergoes to chronic membrane insertion, where contributes to the abnormal cellular hyperproliferative rate.In the present investigation, using CRC human cell lines at different stage of tumor development, we demonstrate that tmCLIC1 functional expression correlates with CRC cells increasing aggressiveness, while it is almost absent in normal colon cells. We also show that CLIC1 enrichment in plasma membrane is involved in promotion of CRC metastasis. CLIC1 inhibition using different pharmacological treatments led to drastic reduction of cell proliferation, migration and invasiveness.tmCLIC1 function permits to discriminate between normal and malignant cells and its downregulation is able to arrest metastatic cells. This is not the case of other upregulated proteins not only involved in tumorigenesis but also in physiological cell functions. Since metastasis are considered the leading cause of treatment failure and tumor relapse, CLIC1 inhibition coupled to conventional therapies can represent a novel strategy to successfully treat CRC with reduced side effects. (This work is supported by a GRANT from AIRC to MM)

#2939

Developing new therapeutic strategy to bladder cancer by targeting CK1 delta.

Yu-Chen Lin, Mei-chuan Chen, Jing-Ping Liou, Chun-Han Chen. _Taipei Medical University, Taiwan_.

Bladder cancer is a common malignancy with high recurrent rate, but targeted therapy other than immune checkpoint inhibitors is currently unavailable. Hence, there is an unmet medical need for the discovery of new therapeutic approach to bladder cancer. By investigating Oncomine database, the mRNA level of CK1 delta is higher in bladder cancer compared to normal tissue, which correlates with survival rate. The aim of this study is to develop new CK1 delta inhibitors and examine their therapeutic potential to bladder cancer as well as the pharmacological mechanisms. Cell viability, cell cycle progression, apoptosis, migration and downstream signaling pathway were analyzed. The results showed that treatment of CK1 delta inhibitors decreased cell viability and inhibited wnt/β-catenin pathway in bladder cancer cell lines. CK1 delta inhibitors increased the accumulation of sub-G1 phase by flow cytometry analysis, as well as the activation of caspase-3, 8, 9 and PARP cleavage by western blot, indicating the promotion of cell apoptosis. Meanwhile, necroptosis was also involved as evident by the rescuing effects by knocking down MLKL in bladder cancer cells. In conclusion, this study demonstrates that CK1 delta is a potential target for bladder cancer therapy in the future.

#2940

**4-Hydroxytamoxifen enhances sensitivity of estrogen receptor α-positive breast cancer to docetaxel in an estrogen and** ZNF423 **SNP-dependent fashion.**

Gen Wang,1 Sisi QIN,1 Jacqueline Zayas,1 James N. Ingle,1 Mohan Liu,1 Richard Weinshilboum,1 Kunwei Shen,2 Liewei Wang1. 1 _Mayo Clinic, Rochester, MN;_ 2 _Shanghai Jiao Tong University School of Medicine, Shanghai, China_.

Background: In early stage, ERα-positive breast cancer, the concurrent use of endocrine therapy and chemotherapy has not been shown to be superior to sequential use. We hypothesized that genetic biomarkers can aid in selecting patients who would benefit from chemo-endocrine therapy. Our previous genome-wide association study (GWAS) and functional studies revealed that a single nucleotide polymorphism (SNP) in ZNF423, rs9940645, determines tamoxifen response. Specifically, 4-hydroxytamoxifen (4-OH-TAM) increases the gene expression of ZNF423, a transcription factor for BRCA1, in cells that are carry the rs9940645 variant, but not the wildtype, genotype. In this study, we set out to identify additional genes regulated by ZNF423, which, upon identifying a relationship between ZNF423 and cell cycle genes, led us to investigate taxane response in a SNP- and tamoxifen-dependent fashion. Method: Gene correlation analysis using The Cancer Genome Atlas (TCGA) breast cancer dataset was used to identify genes with expression correlated with that of ZNF423. Quantitative reverse transcription PCR, chromatin immunoprecipitation, and luciferase reporter assays were used to validate the gene associations. To test ZNF423 rs9940645-specific phenotypes, we used ZR75-1 cells, which are homozygous for the variant SNP, and ZR75-1 cells that were generated to be homozygous wild type (WT) using CRISPR-Cas9 technology, in addition to Hs578T breast cells with ERα overexpression (WT) and HCC1500 cells (variant). Cell cycle was assessed by propidium iodide staining. Cytotoxicity was assessed using a colorimetric assay. Results: Mitosis-related genes VRK1 and PBK, which encode histone H3 kinases, were experimentally validated to be regulated by ZNF423. Specifically, ZNF423 knockdown resulted in decreased VRK1 and PBK expression and activity. Additionally, ZNF423 knockdown resulted in enhanced docetaxel-induced G2/M arrest and cytotoxicity which could be rescued by VRK1 or PBK overexpression. Lastly, breast cancer cells carrying the rs9940645 variant SNP genotype had increased G2-M arrest and decreased cell viability when treated with docetaxel in combination with estradiol and 4-OH-TAM. Conclusion: We identified ZNF423 regulated genes involved in the G2/M phase of the cell cycle. We found that 4-OH-TAM sensitized ERα-positive breast cancer cells to docetaxel treatment in a ZNF423 SNP-dependent manner. Our findings suggest that patients with rs9940645 variant genotype may benefit from concurrent tamoxifen endocrine therapy and docetaxel chemotherapy. This would impact a substantial proportion of patients given the minor allele frequency of 0.47 for this SNP.

#2941

**Combination of hemiasterlin derivative** (R)(S)(S) **-BF65 and PI3K/mTOR dual inhibitor NVP-BEZ235 as a new strategy to inhibit ovarian cancer growth and metastasis.**

Po-Chia Su, Tzu-En Huang, Lih-Ching Hsu. _National Taiwan University, Taipei, Taiwan_.

Metastasis is a big issue to cancer therapy and is the major cause of therapeutic failure and cancer death. Therefore, inhibiting cancer metastasis is a key challenge in cancer treatment. Ovarian cancer is the most deadly cancer among women and combination treatment strategy is usually used to fight against this disease not only to reduce the toxicity and side effects, but also to enhance the anticancer effect.

(R)(S)(S)-BF65 is a hemiasterlin derivative and potent anti-tubulin agent with IC50 values in the low nanomolar range against various cancer cells. Previous studies indicated that (R)(S)(S)-BF65 in combination with an allosteric Akt inhibitor MK-2206 synergistically inhibited the growth of SKOV3 human ovarian cancer cells. Using the MTT assay, we demonstrated that (R)(S)(S)-BF65 could also enhanced the growth inhibitory effect of NVP-BEZ235, a PI3K/mTOR dual inhibitor in SKOV3 cells. The IC50 was 11.6 μM for NVP-BEZ235 and 20.6 nM for (R)(S)(S)-BF65 after 48 h-treatment. With a molar ratio of 1:1000 or 1:500, the combination of (R)(S)(S)-BF65 and NVP-BEZ235 displayed an additive to mild synergistic cytotoxic effect with CI50 values of 0.96 and 0.91, respectively.

Interestingly, the combination of (R)(S)(S)-BF65 and NVP-BEZ235, at concentrations much lower than those required for growth inhibition, significantly inhibited cell migration and invasion as demonstrated by wound healing migration, transwell migration and invasion assays. The combinatorial effect was much more effective than single treatments.

Epithelial-mesenchymal transition (EMT) plays an important role in cancer metastasis. It has been reported that activation of the PI3K/Akt pathway leads to phosphorylation and inactivation of GSK3β, which may in turn induce EMT. Western blot analysis of EMT markers revealed that NVP-BEZ235 downregulated mesenchymal markers β-catenin and Slug, and upregulated the epithelial marker, E-cadherin. (R)(S)(S)-BF65 also downregulated mesenchymal markers β-catenin and Snail, but had no effect on E-cadherin. The combination of both led to downregulation of more mesenchymal markers including β-catenin, Slug, Snail and ZEB1, and upregulation of E-cadherin, which may at least in part explain why the combination was more effective than single treatments to suppress cell migration and invasion.

Taken together, the combination of (R)(S)(S)-BF65 and NVP-BEZ235 may be a promising strategy to inhibit ovarian cancer growth and metastasis.

#2942

Cathepsin S inhibition alleviates oxaliplatin-induced peripheral neuropathy through regulation of Ca2+ homeostasis.

Szu-Jung Chen,1 Meng-Ru Shen,2 Li-Hsien Chen,2 Hsiao-Han Lin,2 Shih-Han Hsu,2 Jang-Yang Chang1. 1 _National Institute of Cancer Research, National Health Research Institutes, Tainan City, Taiwan;_ 2 _Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan City, Taiwan_.

Chemotherapeutic drug oxaliplatin is frequently causing chemotherapy-induced peripheral neuropathy. Currently, there is no effective drug to prevent neuropathy in the clinic. Therefore, identification of the potential target to treat to alleviate oxaliplatin-induced neuropathy is urgent. Previously, the release of cathepsin S (CTSS) from microglial cells, which causes neuropathic pain, has been reported, but the detailed mechanism remains unclear. In this study, we discovered that oxaliplatin treatment triggers CTSS expression accompanies with prompted calcium influx from SOCE in vitro. And, the oxaliplatin treated mice displayed increased tail-withdrawal temperature hyperalgesia and impaired locomotor activity along with the loss of neuromuscular function. Furthermore, oxaliplatin-induced peripheral neuropathy was inhibited by the highly selective CTSS inhibitor, 58, which significantly improved the thermal sensitivity and alleviated mechanical allodynia on the heat tail-withdrawal and von Frey tests in oxaliplatin-treated mice, respectively. The grip strength test showed that 58 significantly improved the motor strength and neuromuscular function of oxaliplatin-treated mice. The ultrastructure of sciatic nerve myelin integrity through G ratio quantification supported the observations in our oxaliplatin-induced peripheral neuropathy mouse behavioral tests. Also, inhibition of CTSS enzymatic activity by 58 resulted in the suppression of STIM1 aggregation and decreasing the oxaliplatin-induced calcium influx from SOCE. These results revel a novel molecular mechanism of CTSS in the involvement of oxaliplatin-induced peripheral neuropathy. (This study was supported by the following grants: the National Health Research Institutes (CA-107-PP-22), the Ministry of Science and Technology (MOST 106-2314-B-006-076-MY3)

#2943

Severe hepatotoxicity of mithramycin therapy caused by altering expression of hepatocellular bile transporters.

Tristan M. Sissung,1 Phoebe A. Huang,1 Ralph Hauke,1 Edel McCrea,1 Cody J. Peer,1 Roberto H. Barbier,1 Jonathan D. Strope,1 Ariel M. Ley,1 Mary Zhang,1 Julie A. Hong,1 David Venzon,1 Jonathan P. Jackson,2 Kenneth R. Brouwer,2 Patrick Grohar,1 John Glod,1 Brigitte C. Widemann,1 Theo Heller,3 David S. Schrump,1 William D. Figg1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _BioIVT, Bethesda, NC;_ 3 _National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD_.

Mithramycin has shown significant preclinical anticancer activity, but its therapeutic dose is limited by the development of hepatotoxicity that remains poorly characterized. A pharmacogenomics characterization of mithramycin-induced transaminitis revealed that hepatotoxicity is associated with inter-individual variation in genes involved in bile disposition: ABCB4(MDR3) rs2302387 and ABCB11(BSEP) rs4668115 variants reduce transporter expression (P<0.05) and were associated with ≥Grade 3 liver function test (LFT) elevations developing 24 hours after the third infusion of mithramycin (25mcg/kg, 6hr/infusion, qdx7, every 28 days;P<0.0040). A similar relationship was observed in a pediatric cohort genotyped for ABCB11. We therefore undertook to characterize the mechanism of mithramycin-induced acute transaminitis. As mithramycin affects cellular response to bile acid treatment by altering the expression of multiple bile transporters (e.g., ABCB4, ABCB11, NTCP, OSTα/β) in several cell lines (Huh7, HepaRG, HepaRG BSEP (-/-)) and primary human hepatocytes, we hypothesized that mithramycin inhibited bile-mediated activation of the farnesoid X receptor (FXR). FXR was downregulated in all hepatocyte cell lines and primary human hepatocytes (P<0.0001), and mithramycin inhibited CDCA- and GW4046-induced FXR-GAL4 luciferase reporter activity (P<0.001). Mithramycin promoted GCDC-induced cytotoxicity in cells lacking the BSEP transporter and increased the overall intracellular concentration of bile acids in primary human hepatocytes grown in sandwich culture (P<0.01). Mithramycin is an FXR expression and FXR transactivation inhibitor that inhibits bile flow and potentiates bile-induced cellular toxicity, particularly in cells with low BSEP function. These results suggest that mithramycin causes hepatotoxicity through derangement of bile acid disposition; results also suggest that pharmacogenomic markers may be useful to identify patients who may tolerate higher mithramycin doses.

#2944

Identification of key transcriptomic and epigenomic factors influencing conventional and metronomic dosing in aggressive and non-aggressive prostate cancer.

Taraswi Mitra Ghosh,1 Joshua Davis,1 Grafton S. Barnett,1 Elena Skarupa,1 Jeff D. Warner,1 Brian S. Cummings,2 Robert D. Arnold1. 1 _Auburn University, Auburn, AL;_ 2 _University of Georgia, Athens, GA_.

Repetitive, low-dose drug administration (metronomic; METRO) shows ability to overcome drug resistance and increased drug efficacy in many cancers, but the mechanisms are not understood fully. Previously we showed topotecan (TOPO) METRO dosing was more effective than conventional (CONV) dosing in aggressive prostate cancer cell lines and xenograft mouse models. To explore possible mechanisms by which METRO dosing alters tumor growth and metastases, we performed targeted mRNA and miRNA expression studies and identified potential candidate cancer pathway genes and miRNA-mRNA pairs as unique treatment-related biomarkers for alternative TOPO-METRO therapy. To determine drug response, Androgen-independent human prostate cancer cells (PC3), androgen-dependent (LNCaP) cell lines were treated with TOPO following CONV and METRO dosing schedules. Cancer pathway gene and micro-RNA expression profiles were assessed at the calculated IC50 of TOPO after each treatment. Expression signatures were identified using differential expression (DE) analysis software and a Spearman's rank-based correlation was used to assess the association of drug response, mRNA and miRNA expression. Gene signatures associated with TOPO-METRO therapy were validated against patient profiles in The Cancer Genome Atlas (TCGA) and Genomic Data Commons (GDC) and protein expression of most significant genes by immunoblotting. We identified disparate treatment-related mRNA and miRNA expression signatures for TOPO-METRO vs CONV treatment. We also identified a gene signature (top five gene: SERPINB5, CDKN1A, TNF, FOS, & ANGPT1) for both PC-3 and LNCaP following TOPO-METRO dosing. Ingenuity Pathway Analysis identified that upregulation of tumor suppressor, anti-proliferative genes (eg, SERPINB5), genes involved in activation of the immune system (RPL13A) and down regulation of genes involved in apoptosis, invasion, metastasis, and inflammation (TNF, FOS, and MMP1) are likely vital to the observed treatment response. Epigenetic analysis showed distinct miRNA expression changes may influence differential gene expression (miRNA-mRNA pairs) for TOPO-METRO dosing. 20 miRNAs genes (including miR-30c, miR-19a, mir-20a, mir-17, let7i & let7b) were associated with TOPO cytotoxicity (p<0.05); seven of these bind to 28 mRNAs (p<0.05) as mRNA-miRNA pairs. Furthermore, we confirmed the top 5 significant genes (e.g. FOS, SERPINEB5, MMP9) for TOPO-Metro therapy by in-silico validation (TCGA). Overall, these studies address a fundamental gap in knowledge related to the effect of METRO dosing on genomic and epigenetic influences and overall treatment efficacy. Using genomics, transcriptomics and epigenomics approaches we determined gene signatures that may be used in identifying patients and personalizing their treatment with the goal of improving overall survival.

#2945

Clinical implementation of precision systems oncology in the treatment of ovarian cancer based on ex-vivo drug testing and molecular profiling.

Astrid Murumägi,1 Daniela Ungureanu,2 Suleiman Khan,1 Akira Hirasawa,3 Mariliina Arjama,1 Katja Välimäki,1 Piia Mikkonen,1 Wilhelmiina Niininen,2 Ashwini Kumar,1 Samuli Eldfors,1 Teijo Pellinen,1 Vilja Pietiäinen,1 Andrus Mägi,4 Riitta Koivisto-Korander,5 Johanna Tapper,5 Mikko Loukovaara,5 Tero Aittokallio,1 Ralf Bützow,6 Olli Kallioniemi1. 1 _Institute for Molecular Medicine Finland (FIMM), University of Helsinki, Helsinki, Finland;_ 2 _Institute of Biosciences and Medical Technology, Tampere University, Tampere, Finland;_ 3 _Department of Obstetrics & Gynecology, Keio University School of Medicine, Keio, Japan; _4 _The Hematology–Oncology Clinic, Tartu University Hospital, Tartu, Estonia;_ 5 _Department of Obstetrics and Gynecology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland;_ 6 _Department of Pathology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland_.

Ovarian cancer (OC) is difficult to treat and has a high mortality rate. There is a strong need for new therapies, but OC is often not the main indication for drug development activities. Hence, here we explored systematic drug repurposing based on clues from ex-vivo testing of human OC patient cells. We established patient-derived cancer cell (PDCs) cultures using ascites or tumor tissue from serous OC patients and applied them to ex vivo Drug Sensitivity and Resistance Testing (DSRT) with a panel of 528 approved and investigational oncology drugs. The representativity of the PDCs was confirmed by genomic and phenotypic profiling in comparison to the original tumor sample. Both 2D and 3D culture conditions were applied and compared for their drug responses. 9 PDCs and 11 high-grade serous OC cell lines were examined. High grade serous OC (HGSOC, 6 cases) with TP53 mutation and CCNE1, CCNE1/KRAS or MYC/KRAS amplifications showed highly resistant drug response profiles, which reflects the clinical challenges in treating this OC subtype. Some HGSOC PDCs showed vulnerability to SMAC mimetics, inhibitors of Apoptosis Protein Antagonists, (e.g. birinapant) and to prexasertib, a checkpoint kinase 1 and 2 inhibitor. In contrast, low-grade serous OC (LGSOC, 3 cases) showed subtype-specific sensitivity to several kinase inhibitors, including EGFR/Her2, MEK, mTOR and PI3K inhibitors. In a metastatic LGSOC patient, RNA-sequencing revealed a CLU-NRG1 fusion gene, which creates an autocrine activation loop of the ErbB family of receptor kinases. DSRT assay confirmed a strong sensitivity of the PDCs ex-vivo to dual-kinase inhibitors, including afatinib. Based on these data, the patient has received approved kinase inhibitors, including afatinib monotherapy followed by a combination of herceptin and pertuzumab. This strategy has managed to keep the disease in control for over 3 years as measured by clinical criteria, including imaging and serum CA125 antigen levels. In conclusion, systems precision cancer medicine with PDCs could provide a valuable approach to reveal patient-specific drug efficacies that can be translated to the clinic in real-time.

#2946

AI-driven stratification of novel synergistic drug combinations for glioblastoma.

Vasileios Stathias, Anna M. Jermakowicz, Marie Maloof, Winston M. Walters, Regina M. Graham, Nagi Ayad, Stephan Schürer. _Univ. of Miami Miller School of Medicine, Miami, FL_.

Glioblastoma is the most common malignant primary adult brain tumor. Despite the medical advances in cancer treatment, drug resistance is inevitable, thus necessitating novel and patient-specific therapeutic approaches. Combination therapies offer the potential to combat glioblastoma recurrence through different mechanisms, such as the simultaneous inhibition of multiple oncogenic pathways and the increase of efficacy through synergism. However, the number of possible combinations prohibits their comprehensive in vitro and in vivo testing. To overcome this limitation, we computationally scored all possible combinations according to their predicted efficacy. To accomplish this we developed an artificial intelligence (AI) model that combines several large datasets: (i) consensus transcriptional perturbation response signatures for 20,000 small molecule and 7,000 genetic perturbations across many cell lines processed from from the Library of Network-based Cellular Signatures (LINCS), (ii) glioblastoma-specific disease signatures obtained by integrating bulk TCGA RNA-seq data with single-cell RNA-seq data obtained from glioblastoma samples, (iii) chemical structure information, and (iv) cell viability datasets. This model provided the necessary resolution and scope to prioritizing novel drug combinations considering different glioblastoma sub-populations and related transcriptional, structural and molecular features. The improved efficacy of such combination treatments over individual drugs was confirmed via synergy screening experiments using GBM PDX cell lines. In conclusion, our study highlights the therapeutic relevance of computationally prioritizing compound combinations based on the integration of both transcriptional- response signatures and patient specific signatures on the single-cell level.

#2947

Discovering novel secondary drug combinations in sex hormone-related cancers using large-scale pharmacogenomics databases through computational modeling and molecular genetics.

Suman Mazumder,1 Li Chen,1 Ujjal K. Mukherjee,2 Amit K. Mitra1. 1 _Auburn University Harrison School of Pharmacy, Auburn, AL;_ 2 _University of Illinois at Urbana-Champaign, Champaign, IL_.

Response to drugs is heterogeneous - not all patients respond equally well to treatment and those who do often acquire resistance over the course of treatment. Therefore, predicting the efficacy of anti-cancer drugs and identifying secondary regimens to circumvent drug resistance is essential to prevent delay in the selection of more effective alternative strategies.

Epidemiologic studies have demonstrated a tendency for common cancers to aggregate in families. For example, breast, prostate and ovarian cancers are sometimes grouped together owing to shared etiology, common genetic susceptibility regions and similar effects of sex hormones (estrogen or testosterone) exposure on their progression. Our objective is to use the vast array of human cancer cell lines in Genomics of Drug Sensitivity in Cancer (GDSC1000), the largest public database of drug sensitivity in human cancer cell lines - 265 anti-cancer drugs in 1074 cell lines (224,510 IC50 values), to apply a computational prediction algorithm that will identify successful secondary drug combinations in sex-hormone related cancers resistant to standard-of-care drugs.

We used a greedy algorithm-based set-covering computational optimization method followed by a regularization technique to seek all secondary drugs that could kill maximum number of cancer cell lines resistant to the standard-of-care drug in a sequential manner ordered by the number of cell lines killed. A greedy algorithm constructs a solution to an optimization problem piece by piece through a sequence of choices to find the overall, or globally, optimal solution. For the purpose of this study, we used 136 potentially sex-hormone related cancer subtypes: breast (n=52), cervix (n=14), endometrium (n=11), ovary (n=45), prostate (n=8), testis (n=3), vulva (n=3). To validate our in silico prediction results, we treated the drug-resistant cancer cell lines with the predicted best secondary drugs, alone or as combination, and performed in vitro chemo-sensitivity assays to evaluate the effect of combination treatments using Chou-Talalay's combination index (CI) method and the isobologram algorithm. Furthermore, we performed gene expression profiling (GEP) analysis using llumina's next-generation sequencing system to identify differentially expressed (DE) genes and pathways underlining novel secondary therapies in chemotherapy-resistant cancers.

Our preliminary data suggests promising early breakthrough and supports our hypothesis that the predicted drug combinations can circumvent drug resistance in human cancers - which we will be presenting at the annual meeting. This study is part of our unique research endeavor that aims to create a universally applicable software application for predicting novel secondary therapies in drug-resistant cancers for any cancer type and any test drug.

#2948

Novel cell line barcoding method reveals tepoxalin as a selective drug against MDR1-high tumor cells.

Steven M. Corsello,1 Ryan D. Spangler,2 Rohith T. Nagari,2 Mustafa Kocak,2 Jordan Rossen,2 Patrick O'Hearn,2 Jennifer Roth,2 Alfredo Gonzalez,2 Nancy Dumont,2 John Doench,2 Jesse S. Boehm,2 Francisca Vazquez,2 Aviad Tsherniak,2 Todd R. Golub2. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Broad Institute, Cambridge, MA_.

Drug repurposing for cancer therapy has seen notable success, such as the reintroduction of thalidomide. To discover new indications, all existing drugs known to be safe should be systematically evaluated for anti-cancer properties. However, given cost and throughput considerations, phenotypic screens are typically limited along the cell line (e.g., NCI-60 cell lines) or compound dimension (e.g., testing only several hundred cancer drugs). To identify new repurposing opportunities at scale, we tested more than 4,600 existing drugs for cytotoxicity against 578 diverse cancer cell lines using PRISM, a recently developed multiplexed cell viability assay. High gene expression of the ABCB1 transporter (MDR1/p-glycoprotein) was strongly correlated with resistance to numerous approved cancer therapeutics including taxanes, anthracyclines, vinca alkaloids, and proteasome inhibitors. Surprisingly, we identified a single drug, tepoxalin, with the opposite profile: selective activity against MDR1-high cancer cell lines.

Tepoxalin, an oral cyclooxygenase inhibitor, is FDA-approved for treatment of osteoarthritis in dogs and was previously found to be safe in human trials. To evaluate for alternative mechanism of action and genomic mediators of tepoxalin resistance, we conducted pooled genome-wide CRISPR-Cas9 modifier screens in the LS1034 colorectal cancer cell line. The CRISPR knockout screen revealed ABCB1 as the top enriched gene mediating tepoxalin resistance, while the CRISPR activation screen revealed ABCB1 as the top depleted gene. Overexpression of ABCB1 in ovarian cancer cell lines also sensitized to tepoxalin killing. Small molecule combination testing demonstrated rescue of tepoxalin killing by known ABCB1 inhibitors. Future studies will include biophysical characterization of the interaction between tepoxalin and the ABCB1 protein as well as in vivo efficacy testing. Our results demonstrate that tepoxalin or related derivatives may be useful in the treatment of chemotherapy-resistant colorectal cancer, ovarian cancer, lymphoma, and other malignancies.

#2949

The impact of drug perturbations on neuroblastoma disease pathways: A pharmaco-transcriptomics approach.

Ramy Elgendy, Elin Almstedt, Michael Vanlandewijck, Sven Nelander. _Uppsala University, Uppsala, Sweden_.

Neuroblastoma (NB) cells exhibit a complex spectrum of pathway changes associated with oncogene activation, chromosome events, tumor micro-environment and super-enhancer states. So far, elucidating which pharmaceutical compounds could modulate the activation level of each known pathway in NB cells has not been feasible. To solve this problem, we have combined transcriptome profiling of drug-perturbed NB cells with mathematical modeling of public data to create a first map of drug- and NB-specific transcriptional signatures for more than 7000 pharmaceutical compounds. We treated 2 patient-derived xenograft (PDX) NB cell lines with different chemical compounds, at 3 different doses (IC50, IC20, and IC10) and 2 time-points (6 and 24h). The whole-transcriptome of the treated cells was analyzed by a cost-effective hybrid 'PLATE-Seq/SMART-Seq2' method. Using factor analysis and supervised machine learning, the data was used to build a model that predicts the NB-specific outcome of 7035 drugs studied in other (non-NB) cell lines in the L1000/LINCS project. Analysis of 768 NB-specific transcriptome profiles, showed that our method accurately detects dose-dependent drug-induced pathway changes in the NB cells, including alteration of characteristic pathways and risk signatures. From the 768 profiles, our best-fitted machine learning model was then extended to 7035 compounds by in silico extrapolation, providing a first panel of estimated drug effects in NB cells. Our constructed NB pharmacological map provides a detailed view of both predicted and in vitro-validated drug-NB interactions. Effective treatments for high-risk NB are currently lacking. Using a new and cost-effective strategy, we could build an accurate map of drug-induced pathway alterations, for network pharmacology and drug repositioning for NB patients.

#2950

Association of p53 gene polymorphisms with the cervical cancer risk in the Bangladeshi women.

Mohammad Safiqul Islam, Md. Saddam Hussain, Md. Salah Uddin Millat, Mohammad Sarowar Uddin, Md. Giash Uddin. _Noakhali Science and Technology University, Noakhali, Bangladesh_.

Cervical cancer (CC) is the fourth most frequently diagnosed cancer and one of the leading causes of cancer related death in women. The number of cervical cancer cases is increasing day by day in Bangladesh. The rs1042522 (pro/arg) polymorphism of p53 gene at codon 72 plays an important role in the cervical cancer development due to the inactivation of its arginine containing protein by the E6 oncoprotein of human papillomavirus. Though several pharmacogenomics studies were conducted in the different population, no such type of study has been yet conducted in the Bangladeshi cervical cancer women. This case control study has been designed to investigate the presence or absence of association of p53rs1042522 polymorphism with the Bangladeshi population. Total 104 CC cases were recruited from the different hospitals of Bangladesh and 104 healthy women were recruited as controls matching age with the cases. After taking the written consent from the patients and controls 3 ml blood sample was collected from each of them. DNA extraction was performed by a validated method routinely used in our laboratory. After PCR amplification, PCR products were digested with BstUI restriction enzyme and finally, different genotypes were detected by visualizing the digested products on 2% agarose gel electrophoresis after staining with ethidium bromide. The frequencies of pro/pro, pro/arg and arg/arg genotypes were 19.23%, 24.04%, 56.73% and 38.46%, 14.42%, 47.12% among the cases and controls, respectively. Women carrying pro/arg shown 3.33 times more risk (OR=3.33, 95% Cl= 1.45-7.69, p=0.005) and women carrying arg/arg genotype shown 2.41 times more risk (OR=2.41, 95% Cl= 1.25-4.65, p=0.009) whereas women carrying one variant allele (dominant model, pro/arg + arg/arg ) shown 2.63 times more risk (OR=2.63, 95% Cl= 1.40-4.91, p=0.003) for the cervical cancer development in compared to the controls carrying pro/pro genotype. The frequency of 'arg' allele was 68.75% and 54.33% in case of CC women and controls respectively. Women carrying 'arg' allele shown 1.85 times more risk (OR=1.85, 95% Cl= 1.24-2.76, p=0.003) for the development of CC in compared to the women carrying 'pro' allele. Our result indicates that arg/pro, arg/arg, arg/pro+arg/arg genotypes and 'arg' allele significantly (p<0.05) increased the CC risk in the Bangladeshi women. We can conclude that rs1042522 SNP of the p53 gene is associated with CC risk in the Bangladeshi population.

#2951

A genome-wide association study identifies five novel genetic markers for trastuzumab-induced cardiotoxicity.

Chihiro Udagawa,1 Mari Hara,2 Arata Shimo,3 Yasuyuki Kojima,3 Reiko Yoshie,3 Hisamitsu Zaha,4 Norie Abe,4 Tokiwa Motonari,4 Mikiko Unesoko,4 Kenji Tamura,5 Tatsunori Shimoi,5 Masayuki Yoshida,5 Teruhiko Yoshida,6 Hiroshi Okamura,1 Taisei Mushiroda,7 Koichiro Tsugawa,3 Hitoshi Zembutsu8. 1 _Toyo Kohan, Tokyo, Japan;_ 2 _Japanese Foundation for Cancer Research, Tokyo, Japan;_ 3 _St. Marianna University School of Medicine, Kawasaki, Japan;_ 4 _Nakagami Hospital, Okinawa, Japan;_ 5 _National Cancer Center Hospital, Tokyo, Japan;_ 6 _National Cancer Center Research Institute, Tokyo, Japan;_ 7 _RIKEN Center for Integrative Medical Sciences, Yokohama, Japan;_ 8 _Cancer Institute, Japanese Foundation for Cancer Research, Tokyo, Japan_.

Purpose: Trastuzumab has been administered to HER2-positive breast or gastric cancer patients as an effective treatment, however, the cardiotoxicity is identified as one of the life-threatening side effects. To identify a novel genetic marker(s) determining the risk of trastuzumab-induced cardiotoxicity, we performed a genome-wide association study (GWAS).

Experimental Design: We carried out a GWAS using 11 cases (with trastuzumab-induced cardiotoxicity) and 257 controls (showing no sign of trastuzumab-induced cardiotoxicity). Top 100 single nucleotide polymorphisms (SNPs) which revealed smallest P value in GWAS (P = 1.61 x 10-7 -1.97 x 10-4) were further examined using replication samples consisted of 14 cases and 199 controls. Results: The combined analysis of the GWAS and replication study indicated possible association of five loci with trastuzumab-induced cardiotoxicity (chromosome 13q14.3, 4q25, 15q26.3, 17q25.3 and another 15q26.3, Pcombined = 6.00 x 10-6, 8.60 x 10-5, 1.01 x 10-4, 1.07 x 10-4, 1.60 x 10-4, respectively). Furthermore, we developed a risk prediction model for trastuzumab-induced cardiotoxicity, using five SNPs which revealed smallest P value at each locus. The incidence of trastuzumab-induced cardiotoxicity in patients with risk score ≥5 was 42.5% (P =8.69 x 10-15) . This scoring system using five candidate loci could be used to predict the risk of the adverse event before administration of trastuzumab.

Conclusion: We identified five novel loci, which could be associated with trastuzumab-induced cardiotoxicity. These findings provide new insights into personalized trastuzumab therapy for patients with HER2-positive cancer.

#2952

Predicting concentration of PARP inhibitors in human tumor tissue using PBPK modeling.

Rachel H. Rose,1 Kaiming Sun,2 Linzhong Li,1 Keyur Gada,2 Jing Yu Wang,2 Yongchang Qiu2. 1 _Certara UK Limited, Simcyp Division, Sheffield, United Kingdom;_ 2 _TESARO, Inc., Waltham, MA_.

Poly (ADP-ribose) polymerase (PARP) inhibitors exert their effect intracellularly within tumor, thus sufficient tumor penetration is essential for a pharmacological response. Preclinical mouse xenograft data show a 3.3-fold higher tumor versus plasma exposure of niraparib, while for olaparib tumor exposure was less than plasma. This study aimed to build a physiologically-based pharmacokinetic (PBPK) model extended with a tissue composition-based permeability-limited tumor model to: (a) gain a mechanistic understanding of the differences in tumor exposure of niraparib and olaparib; and (b) to predict clinical tumor exposure in ovarian cancer patients at clinically relevant dosing regimens. A permeability-limited tumor model was developed that integrates data on tumor composition and drug physicochemical properties analogous to the established permeability-limited organ model available for the liver in the Simcyp Simulator [1,2]. The model assumes that unbound unionized drug is in equilibrium between the vascular and interstitial compartments and movement of the drug between the interstitial and intracellular space is via passive permeability. Total tumor concentration is dependent on passive permeability of the drug, drug binding to PARP and nonspecific binding to neutral lipids, neutral phospholipids, and acidic phospholipids in the intracellular space, albumin in the interstitial and pH of the tumor interstitial and intracellular spaces. Clinical and preclinical tumor physiological parameters such as volume, blood flow, and tissue composition are defined using published data. The model was developed using the Simcyp Simulator V17.1 and R [3]. Consistent with preclinical data, the model predicts a 5-7-fold higher tumor exposure relative to plasma, as measured by the AUC tumor to plasma ratio of niraparib compared with olaparib. Significant binding to acidic phospholipids contributes to the increased tumor exposure to niraparib, a basic drug, a mechanism that is not relevant to the neutral drug olaparib. Ongoing work aims to extrapolate the model to predict the clinical tumor concentration of olaparib and niraparib in ovarian cancer patients and to investigate the sensitivity of the model to key tumor attributes, including blood flow and interstitial pH that may contribute to variability in tumor drug exposure. A similar modeling approach may be used to predict the tumor exposure of other small molecule anticancer drugs from their plasma concentration and physicochemical properties in different types of solid tumor.

References

  1. Jamei M et al, Clin Pharmacokinet. 2014;53:73-87
  2. Poulin P et al, J Pharm Sci. 2015;104:1508-21
  3. R Core Team (2018). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R-project.org/.

### Cellular Responses to Anticancer Agents 3: Novel Targets

#2953

Selective activity of XPO1 inhibitors in TET2-mutant myeloid malignancies.

Nicole Prutsch, Chang-Bin Jing, Julia Etchin, Alla Berezovskaya, Hong Tiv, Michael J. Poitras, Prafulla Gokhale, Yosef Landesman, A. Thomas Look. _Dana Farber Cancer Institute, Boston, MA_.

TET2 is among the most frequently mutated genes in hematopoietic malignancies. Inactivating mutations in TET2 are found in ∼30% of patients with myelodysplastic syndrome (MDS), ∼19% with de novo acute myeloid leukemia (AML), and ~15% with myeloproliferative neoplasm (MPN). TET2 mutations are also identified in a subset of individuals over 50 years of age with clonal hematopoiesis of indeterminate potential (CHIP), a condition that predisposes affected individuals to progression to myeloid malignancy and atherosclerotic heart disease with heart attack or stroke. TET2 mutations represent an early genetic lesion in hematopoietic stem and progenitor cells (HSPCs), inducing a premalignant state of clonal dominance that predisposes to the acquisition of additional mutations. Thus, effective therapy for patients with TET2 mutations in HSPCs will require identifying drugs that are selectively lethal to TET2 mutant HSPCs but spare normal HSPCs, a property analogous to the widely studied genetic relationship called "synthetic lethality". Using a tet2-mutant zebrafish model created in our laboratory, we screened for drugs from libraries of FDA-approved compounds and drugs in Phase I/II testing. We found that nuclear exporter XPO1 inhibitors, selinexor (KPT-330) and eltanexor (KPT-8602), were among the most promising, in that they are selectively lethal to HSPCs in tet2-mutant compared to wild-type fish. Moreover, we treated HSPCs of wild-type and Tet2-deficient mice with both selinexor and eltanexor in a methylcellulose colony formation assay. WT HSPCs lose their replating capacity at passage 3 (P3), while Tet2-mutant clones demonstrate aberrant sustained self-renewal capacity over multiple replatings. We found that both drugs selectively kill primary Tet2-mutant murine HSPCs and also block the aberrant self-renewal of these cells. By transplanting WT and Tet2-mutant CD45.2 donor cells into CD45.1 recipient mice, we monitored peripheral blood cells before and after treatment with selinexor or eltanexor to explore synthetic lethality of these drugs in vivo. Moreover, the human AML cell line K562 with TET2 homozygous mutations generated with CRISPR-Cas9, was more sensitive to selinexor and eltanexor than the parental cell line. To elucidate the mechanism behind the selective targeting of Tet2-mutant blood stem cells, we will use PRO-Seq and START-Seq methods to identify transcriptional elongation and initiation sites at single-nucleotide resolution. These preclinical studies must be done to show a therapeutic efficacy and mechanism of action of these new drugs before they can be translated to improve therapy of patients who have TET2 inactivating mutations.

#2954

Mithramycin analogues disrupt ETS transcription factor DNA binding.

Reiya C. Hayden, Caixia Hou, Prithiba Mitra, Abhisek Mandal, Jurgen Rohr, Jon Thorson, Oleg Tsodikov, Markos Leggas. _University of Kentucky, Lexington, KY_.

Introduction: Ewing sarcoma, prostate cancer, and leukemia are a few examples where ETS transcription factors drive tumorigenesis. The transcription factors EWS-FLI1 and EWS-ERG are common translocations in Ewing sarcoma and bind DNA at GGAA repeats leading to expression of genes that drive tumor growth. Pharmacologic inhibition of EWS-FLI1 with mithramycin (MTM) was shown to inhibit expression of downstream genes and tumor growth in mice. But despite this specific inhibitory activity, MTM has a narrow therapeutic window with hematologic and hepatic toxicity attributed to displacement of the ubiquitously acting Sp1 transcription factor. Thus, a synthetic effort was initiated to develop MTM analogues with reduced toxicity and increased specificity for ETS binding sites. Structural studies informed the design of MTM analogues that may stabilize transcriptional complexes leading to the disruption of transcriptional activity and DNA damage. In vitro cytotoxicity assays demonstrated that MTM analogues have significantly higher cytotoxicity in EWS-ETS expressing cell lines. Here we present mechanistic evidence for the differences in biochemical activity among MTM and its novel analogues.

Methods: Qualitative interactions between drug-DNA-protein were assessed and optimized by electrophoretic mobility shift assays (EMSA). Time-resolved fluorescence energy transfer (TR-FRET) assays were used to quantitatively determine ERG displacement from DNA in the presence of MTM and analogues. Expression of proteins indicating DNA damage (c-PARP, γ-H2AX) and phosphorylation at the C-terminal domain (CTD) of RNAPII was determined by western blot following drug treatments in ETS and non ETS expressing cell lines.

Results: Using TR-FRET, we observed that MTM displaced DNA bound ERG more potently and in a concentration dependent manner as compared to MTM analogues. As compared to MTM, treatment with MTM analogues resulted in higher expression of DNA damage markers, γ-H2AX and c-PARP, specifically in cell lines containing EWS-ETS translocations in a concentration dependent manner.

Conclusion: These studies provide insights regarding differences among MTM and analogues in DNA binding and interactions with DNA associated proteins in the presence and absence of EWS-ETS expression. Our results suggest that MTM analogues may bind and stabilize transcriptional complexes. These differences will provide the basis for structure activity relationships and for the development of analogues with decreased in vivo toxicity. Future work will incorporate co-immunoprecipitation studies to determine if physical protein interactions are being disrupted by MTM and analogues and cellular thermal shift assays to directly probe drug interactions with EWS-ETS proteins and with RNAPII.

#2955

Development of non-apoptotic, caspase-independent cell death inducers against cancer cells.

Mariah A. Pasternak,1 Noor Hussein,1 Karthikeyan Chandrabose,2 Paul W. Erhardt,1 Amit K. Tiwari1. 1 _University of Toledo, Toledo, OH;_ 2 _Indira Gandhi National Tribal University, Amarkantak (MP), India_.

Since most cancer therapeutics currently exert their effects using apoptotic pathways to which cancer cells can become resistant, it is important to develop new drug molecules that work through non-apoptotic pathways. A novel, non-apoptotic cell death mechanism called "methuophagy," which combines features of both autophagy and methuosis, is being examined using structure-activity relationship (SAR) studies of synthesized compounds. Methuosis is a non-apoptotic cell death that includes macropinocytosis and the fusing of smaller vesicles into larger vesicles that is seen along with cell death. BAPT compounds that induce methuophagy have been synthesized, and minor changes on a primary scaffold are being used to determine the SAR for the series. The series retains the 4-pyridinyl moiety that induced methuosis with the compound MOMIPP. Screening experiments with HCT-116 colorectal cancer cells (CRC), BT-20 triple-negative breast cancer cells (TNBC), and U-251 glioblastoma cancer cells (GBM) have shown that compounds having a rigid, completely aromatic structure with three aromatic centers induce methuophagy more potently than compounds with a "hydrazone-like" moiety or a non-aromatic linker. The cell death caused by methuophagy can be seen as early as 6 hours, with small vacuoles that coalesce together to form larger vacuoles. From these screening experiments, it has been concluded that the completely aromatic compounds are the most promising candidates for methuophagy drug development and will be used in experiments to determine protein targets and in vivo to further determine efficacy.

#2956

Inhibition of CHK1 sensitizes Ewing sarcoma cells to CDK1-dependent cell death in S-phase.

David Gordon, Kelli Goss, Stacia Koppenhafer. _University of Iowa, Iowa City, IA_.

Ewing sarcoma is a highly aggressive bone and soft tissue cancer that is caused by the EWS-FLI1 fusion protein. The treatment of Ewing sarcoma has changed very little in the past two decades and novel treatment approaches are needed. We recently identified that Ewing sarcoma cells are uniquely vulnerable to inhibitors of ribonucleotide reductase (RNR), the rate limiting enzyme in the synthesis of deoxyribonucleotides. We subsequently found that the inhibition of checkpoint kinase 1 (CHK1) increases the sensitivity of Ewing sarcoma cells to inhibitors of RNR, such as gemcitabine. The ATR-CHK1 pathway, from a mechanistic standpoint, regulates multiple stages of the cell cycle, including S-phase, the G2/M transition, and M phase. In the current work, we used cell synchronization to identify that the inhibition of CHK1 in S-phase cells results in premature entry into mitosis and cell death. Similarly, the inhibition of ATR serine/threonine kinase (ATR), the upstream activator of CHK1, also causes Ewing sarcoma cells to inappropriately enter mitosis. Moreover, we have found in Ewing sarcoma cells that this aberrant entry into mitosis is mediated, in part, by cyclin dependent kinase 1 (CDK1). In addition, activation of CDK1 by inhibiting the WEE1 kinase with AZD1775 also results in entry into mitosis and cell death in Ewing sarcoma cells. Currently, ongoing work is focused on the in vivo testing of gemcitabine in combination with CHK1 and WEE1 inhibitors as a novel therapeutic approach for the treatment of Ewing sarcoma.

#2957

Nasopharyngeal cancer.

Ngar Woon Kam, See Wing Chan, George Sai-Wah Tsao, Xin-Yuan Guan, Dora Lai Wan Kwong. _The University of Hong Kong, Hong Kong, Hong Kong_.

Background:

Nasopharyngeal cancer (NPC) is associated with a poor survival rate. The ability of cancer cells to evade apoptosis and exhibit limitless replication potential allows for the progression of cancer. The aim of this study was to investigate in vitro the effect of the isoflavone phenoxodiol on human NPC cell line and T cells, which has previously been shown to inhibit cell proliferation and promote apoptosis in a range of cancer cell lines and in anti-CD3/anti-CD28-activated murine splenocytes.

Methods:

Four NPC cancer cell lines-C666, C17, and paired NPC43 EBV-positive and its respective EBV-negative cell lines were cultured in vitro, and then treated with phenoxodiol for 72h or 5 days. The migration and proliferation was investigated by transwell and XTT, respectively. Conditioned medium of treated NPC cell lines were collected and incubated with human PBMC for 5 days. CD4 and CD8 surface staining were analyzed by flow cytometry.

Result:

We demonstrated that phenoxodiol inhibited NPC migration and cell proliferation (IC50 4µM). Among all the NPC cell lines, the proliferation effect on EBV-negative cell line (NPC43-ve) is least responsive to phenoxodiol. A dose-dependent of apoptosis in NPC cell lines by phenoxodiol [early apoptotic cells (Annexin V+/PI- at 1µM); late apoptotic cells (Annexin V+/PI+ at 2µM), and necrotic cells (Annexin V-/PI+, at 4µM) were distinguished. Interestingly, phenoxodiol reduced frequency of CD4 T cells while promoting the number of CD8 T cells.

Conclusions:

Phenoxodiol demonstrates an ability in NPC cancer cells to induce its apoptosis while inhibit its migration and proliferation. Moreover, the ability of phenoxodiol to modulate T cells population indicates that phenoxodiol would be effective as a potential future treatment for NPC.

#2958

Evaluating the therapeutic effects of methylene blue against prostate cancer.

Priyadarshini Thiruvalluvan Shanthi,1 Abigail Foes,2 Gnanasekar Munirathinam1. 1 _UIC college of Medicine- Rockford, Rockford, IL;_ 2 _Boylan Central Catholic High School, Rockford, IL_.

Prostate cancer (PCa) is the second most commonly occurring cancer among men and the fourth most commonly occurring cancer overall. According to American Cancer Society, in the year 2018, it was estimated that there will be 164,690 new cases and 29,430 deaths from PCa. The treatment options currently available for PCa are found to be ineffective with varied side effects and complications associated with the development of resistance among patients. Therefore, there is an unmet need to find a safe and potent agent to treat PCa. In our study, we focused on studying the anticancer potential of Methylene Blue (MB) which belongs to the class of phenothiazinium salt. MB has been widely used to treat the condition of methemoglobinemia, emerging studies have shown that it has been effectively used as a photosensitizer in the treatment of cancer by means of photodynamic therapy (PDT). Our initial analysis showed that MB effectively reduced the viability of androgen-dependent (LNCaP) and androgen-independent (PC3 and DU145) PCa cells. Further experimental evaluations showed that MB inhibited the colony forming ability of PCa cells in-vitro suggesting its tumor suppressive potential. In addition, our studies showed that MB treatment disrupted the migration potential of PCa cells in a wound healing assay indicating the anti-metastatic function of MB. Moreover, confocal and FACS analysis using Annexin V FITC and propidium iodide staining revealed that MB effectively targeted the PCa cell lines by inducing apoptotic cell death. To delineate the underlying anticancer mechanism of MB, apoptosis protein array was performed employing LNCaP cells, and the results of which showed that key apoptotic molecules such as Bax, TRAIL

R2/D5, and phospho p53 (Serine 15, Serine 46, Serine 392) were robustly upregulated in LNCaP cells following MB treatment. In conclusion, our findings suggest that MB induces apoptosis in PCa cells and thus could serve as a potential anticancer agent for treating both hormone-dependent and -independent PCa.

#2959

Deoxysappanchalcone inhibits cell growth by regulation of TOPK signaling pathway in colon cancer.

Ran Zhao,1 Hai Huang,1 Bu Young Choi,2 Mengqiu Song,1 Fanxiang Yin,3 Xiaorong Fu,3 Hanyong Chen,4 Jung Hyun Shim,5 Mee-Hyun Lee,1 Zigang Dong4. 1 _China-US(Henan) Hormel Cancer Institute, Zhengzhou, China;_ 2 _Seowon University, Chungbuk, Republic of Korea;_ 3 _Zhengzhou University, Zhengzhou, China;_ 4 _The Hormel Institute, Austin, MN;_ 5 _Makpo National University, Jeonnam, Republic of Korea_.

Background: Colorectal cancer is one of the most common cancer death causes in the worldwide. Still systemic chemotherapies are limited to many complications and relapse. T-lymphokine-activated killer cell-originated protein kinase (TOPK) is highly expressed and activated in colon cancer, and plays important roles in inflammation, proliferation and survival of cancer cells. Therefore, suppression of the TOPK activity and its downstream signaling cascades is considered to be a rational therapeutic/preventive strategy of colon cancers.

Purpose: Deoxysappanchalcone (DSC), a component of Caesalpinia sappan L., is a natural oriental medicine. In the present study, we investigated the effects of DSC on colon cancer cell growth and elucidated its underlying molecular mechanism by targeting TOPK.

Study Design and Methods: To evaluate the effects of DSC on colon cancers, we performed cell proliferation assay, propidium iodide- or annexin V-staining analysis and western blotting. Targeting TOPK by DSC was identified by kinase/binding assay and computational docking models. Results: DSC inhibited the kinase activity of TOPK but not mitogen-activated protein kinase (MEK). The direct binding of DSC with TOPK was explored using a computational docking model and binding assay in vitro & ex vivo. DSC inhibited colon cancer cell proliferation, anchorage-independent cell growth, and induced G2/M cell cycle arrest and apoptosis. Treatment of colon cancer cells with DSC induced protein expressions which are involved in cell cycle (cyclin B1) and apoptosis (cleaved-PARP, cleaved-caspase-3 and cleaved-caspase-7), and suppressed protein expressions of extracellular signal-regulated kinase (ERK)-1/2, ribosomal S6 kinase (RSK) or cellular jun (c-Jun) which are regulated by the upstream kinases TOPK.

Conclusion: DSC suppresses colon cancer cell growth by direct targeting TOPK-mediated signaling pathway.

#2960

NCX4040, a nitric oxide (NO) donating form of aspirin as a potential treatment for prostate cancer.

SOMAIAH CHINNAPAKA, Gnanasekar Munirathinam. _University of Illinois at Chicago, ROCKFORD, IL_.

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been shown to be beneficial in preventing various types of cancers including prostate cancer (PCa). Of all the NSAIDs in use, Aspirin appears to be one of the promising anticancer agents for PCa management. However, experimental and epidemiological studies indicated that larger doses of Aspirin and long term supplementation is required to achieve anticancer effect with accompanied side effects. Therefore, our study focused on evaluating Aspirin derivative, namely NCX4040 which is a NO releasing form of Aspirin as a potent alternative for PCa treatment while minimizing the side effects. In the present study, we assessed the anticancer effects of NCX4040 on PC-3, a bone metastatic PCa cell line employing various in-vitro cellular assays. In our preliminary studies, we compared the effects of NCX4040 with that of Aspirin and NO releasing compound DETA/NO on PC-3 cells using MTT as read out. These results showed that NCX4040 is highly potent in inhibiting the viability of PC-3 cells when compared to Aspirin or DETA/NO treatments. Hence, further studies were focused on characterizing the anticancer properties as well as the underlying anticancer mechanism of NCX4040. Of note, our results showed that NCX4040 robustly inhibited the colony forming ability of PC-3 cells in-vitro implicating its antitumorigenic potential. In addition to inhibiting colony formation of PC-3 cells, NCX4040 also suppressed the Transwell matrigel invasion of PC-3 cells suggesting the antimetastatic effects of NCX4040. The observed antimetastatic effect of NCX4040 in PC-3 PCa cells appears to be via targeting actin cytoskeleton as confirmed by confocal microscopic analysis using phalloidin stain. Following these findings, we performed cell cycle analysis to determine whether NCX4040 enforces cell cycle arrest in PC-3 cells. These results showed that NCX4040 induced robust G0 cell cycle arrest in a dose dependent manner. Moreover, flow cytometry analysis using Annexin V and PI staining revealed that NCX4040 induced potent apoptotic cell death in PC-3 cells. To delineate the underlying anticancer mechanism of NCX4040, PC-3 cells treated with NCX4040 was subjected to apoptotic protein array analysis. Results of this study inferred that NCX4040 down regulated the expression of key anti-apoptotic proteins such as Survivin, XIAP and IAPs. In conclusion, our study suggests that NCX4040 is a promising alternative agent for PCa treatment.

#2961

Ganoderma lucidum extracts inhibit mTORC1/2 by activating AMPK and inhibiting IGFR/PI3K/Rheb in tumor cells.

Shile Huang, Didem Sohretoglu, Chao Zhang, Jun Luo. _Louisiana State Univ. Health Sciences Ctr., Shreveport, LA_.

Ganoderma lucidum (G. lucidum) extracts, as dietary supplements, have been found to possess potent anticancer activity, which is attributed to the presence of polysaccharides and triterpenes. However, the molecular mechanism underlying the anticancer action of G. lucidum extracts remains to be elucidated. Here we show that ReishiMax GLp, containing both polysaccharides and triterpenes of G. lucidum (GLPT), inhibited cell proliferation and induced cell death in human lung cancer cells (A549 and A427), and concurrently suppressed the signaling pathways mediated by the mammalian target of rapamycin (mTOR), a central regulator of cell proliferation and survival. Interestingly, GLPT not only inhibited mTORC1-mediated phosphorylation of p70 S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1), but also repressed mTORC2-mediated phosphorylation of Akt. Mechanistically, GLPT downregulated the phosphorylation and protein levels of insulin-like growth factor 1 receptor (IGFR) and phosphoinositide 3-kinase (PI3K), as well as the protein level of RAS homolog enriched in brain (Rheb). Besides, GLPT also activated AMP-activated protein kinase (AMPK) network. This is supported by the findings that GLPT increased the phosphorylation of AMPKα (T172), as well as its substrates tuberous sclerosis complex 2 (TSC2, S1387) and regulatory-associated protein of mTOR (raptor, S792). Ectopic expression of dominant negative AMPKα partially attenuated the inhibitory effect of GLPT on mTORC1, indicating that GLPT inhibits mTORC1 partly via activating AMPK. The results suggest that G. lucidum extracts execute the anticancer action at least partly by inhibiting the mTORC1/2 signaling through activation of AMPK and inhibition of IGFR/PI3K/Rheb in tumor cells. Supported by the Feist-Weiller Cancer Center, LSU Health Sciences Center, Shreveport, LA, USA.

#2962

Targeting BCL2 as a therapeutic strategy in neuroendocrine prostate cancer.

Alexandra N. Corella,1 Jared M. Lucas,1 Arja Kaipainen,1 Ilsa M. Coleman,1 Colm Morrissey,2 Eva Corey,2 Paul Nghiem,2 David MacPherson,1 Peter S. Nelson1. 1 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 2 _University of Washington, Seattle, WA_.

Small-cell neuroendocrine prostate carcinoma (SC/NEPC) is an aggressive subtype of prostate cancer (PC) with poor response to conventional therapies and no approved targeted therapies. The molecular mechanisms that drive the growth and survival of these tumors are poorly understood due to disease rarity and a lack of model systems for the subtype. SC/NEPC shares defining characteristics with other aggressive small-cell neuroendocrine tumors, such as "small-blue round cell" morphology and expression of chromogranin A. In this study, we compare whole transcriptome RNA-sequencing data from metastatic PCs and two additional SC/NE tumor types, small cell lung cancer (SCLC) and Merkel Cell carcinoma (MCC). By differential expression analysis of each neuroendocrine tumor type to Androgen Receptor-responsive Prostate Cancer (AR-PC), we identify 4,300 genes exhibiting shared expression patterns in multiple neuroendocrine tumor types. Among these "pan-neuroendocrine" genes are known and proposed drivers of the neuroendocrine transcriptional program including MYCN (p=8.7E-07, upregulated in SC/NEPC over AR-PC) and potential novel drivers of the neuroendocrine phenotype in PC such as MYCL (p=3.5E-05, upregulated in SC/NEPC over AR-PC). We also identify conserved expression patterns of druggable targets including BCL2 which has previously been reported as highly expressed in subsets of SCLC and MCC, but not SC/NEPC (p=1.24E-05, upregulated in SC/NEPC over AR-PC). We then analyzed a panel of cell lines and novel PDX models, which further confirm that the BCL2 protein is highly expressed specifically in the SC/NEPC phenotype. We determined that SC/NEPC cell lines and PDX models are sensitive to BCL2 family member inhibitors targeting BCL2, BCLXL, BCLW, including, ABT263, ABT199 and ABT737, at sub-micromolar concentrations, whereas models of ARPC were resistant. These inhibitors lead to apoptotic cell death in SC/NEPC. Collectively, our data suggests that BCL2 inhibition may present a novel targeted approach for SC/NEPC that warrants further evaluation.

#2963

**CD44** + **/CD133** + **colorectal cancer stem cells are sensitive to trifluridine.**

Kenta Tsunekuni,1 Masamitsu Konno,2 Jun Koseki,2 Ayumu Asai,2 Kazuaki Matsuoka,1 Teiji Takechi,1 Yuichiro Doki,2 Masaki Mori,2 Hideshi Ishii2. 1 _Taiho Parmaceutical, Tokushima, Japan;_ 2 _Osaka University, Osaka, Japan_.

Background: Cancer stem cells (CSCs) are involved in disease recurrence, metastases, and therapeutic resistance. However, anticancer agents that target CSCs are not currently available. Trifluridine (FTD)/tipiracil (TPI), a novel oral antitumor drug, was approved for the treatment of patients with metastatic colorectal cancer who had been previously treated with anti-vascular endothelial growth factors; anti-epidermal growth factor receptors (in patients with ras wild-type genes); or fluoropyrimidine-, oxaliplatin-, irinotecan-based chemotherapies. FTD/TPI improves overall survival, and FTD is the key component of FTD/TPI. Here, we demonstrated the efficacy of FTD against CD44 and CD133 high-expressing (CD44+CD133+) colorectal cancer cells that possess CSC-like properties.

Method: CD44+CD133+ and other populations of DLD-1, a colorectal cell line, were separated by fluorescence-activated cell sorting (FACS). The sphere-forming activity of each population, and anti- sphere-forming effects of FTD and fluorouracil (5-FU) on CD44+CD133+ cells, were measured. In addition, DLD-1 and HCT-116, two colorectal cell lines, were treated with FTD for 5 months. The CSC markers CD44 and CD133 were measured by FACS, and the cells' in vitro and in vivo tumor-initiating properties were evaluated.

Results: CD44+CD133+ DLD-1 cells formed significantly more spheres than did CD44-CD133- cells (sphere ratio CD44+CD133+/CD44-CD133- = 4.0) and CD44+CD133- cells (sphere ratio CD44+CD133+/CD44+CD133- = 1.7). In the in vitro proliferation assay, CD44+CD133+ cells had greater 5-FU resistance, but not higher FTD resistance, than did unsorted cells. In addition, treatment of CD44+CD133+ DLD-1 cells with subtoxic concentrations of FTD (1 µM) disrupted sphere formation, which was superior to the effect of treatment with 1 µM 5-FU. The inhibition rates for FTD and 5-FU were 59.5% and 14.3%, respectively. DLD-1 and HCT-116 cell lines treated with FTD for 5 months had smaller CD44+CD133+ populations and lower sphere-forming activity than did untreated cell lines; population decline rates were 93.8% and 74.7%, respectively. Furthermore, 5 months of in vitro exposure to FTD reduced in vivo tumor formation potential in both cell lines. These results suggest that FTD is effective against CSC-like cells, and that treatment with FTD might reduce CSC-like populations.

Conclusion: FTD had a direct effect on the sphere-forming activity of CSC-like CD44+CD133+ cells that was superior to that of treatment with 5-FU. Its effectiveness against CSC-like CD44+CD133+ cells suggests that FTD might be useful for CSC-targeted chemotherapy in tumors that highly express CD44 and CD133.

#2964

Pharmacological inhibition of WIP1 by GSK2830371 sensitizes AML cells to MDM2 inhibitor Nutlin-3a.

Maria Chiara Fontana,1 Jacopo Nanni,1 Giovanni Marconi,1 Martina Pazzaglia,2 Matteo Bocconcelli,1 Antonella Padella,1 Simona Soverini,1 Ilaria Iacobucci,3 Cristina Papayannidis,1 Anna Ferrari,4 Maria Teresa Bochicchio,4 Enrica Imbrogno,4 Michele Cavo,2 Andrea Ghelli Luserna di Rora,2 Giorgia Simonetti,4 Giovanni Martinelli4. 1 _Institute of Hematology "L. and A. Seragnoli", University of Bologna, Bologna, Italy;_ 2 _Institute of Hematology, Bologna, Italy;_ 3 _St. Jude Children's Research Hospital, Memphis, TN;_ 4 _Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy_.

Introduction: PPM1D (wild-type p53-inducible protein phosphatase WIP1) is a member of the PP2C family serine/threonine phosphatase involved in negative regulation of cell stress response pathways, leading to suppression of p53 after stress. To restore p53 function through MDM2 inhibition (Nutlin-3a) is a promising approach in AML. Promising results of the combination of MDM2 inhibitors and WIP1 inhibitor (WIP1i), obtained in many preclinical studies of solid tumors, paved the way for their application in AML. We investigated whether the inhibition of WIP1 (GSK2830371) could sensitize AML cell lines and primary cells to Nutlin-3a (Nut-3a) in order to obtain a novel therapeutic strategy for AML patients, based on restoration of p53 activity.

Methods: In vitro viability (by WST-1 reagent) and Annexin-V/PI apoptosis assay were performed on a panel of TP53-wt AML cells (MOLM-13, MV-4-11 and OCI-AML3), on TP53-mutated NOMO-1 AML cells and on primary AML samples. Gene expression profile (GEP) and Western Blot (WB) analyses were performed on MV-4-11 and NOMO-1 after 16h.

Results: Combined inhibition of increasing dosage of Nut-3a (0.5 to 5 uM) and WIP1i (5 to 20 uM) synergistically reduces TP53-wt AML cells viability, while NOMO-1 resulted to be insensitive to the combination. The combination index analyses showed a synergistically effect of the combination of both compounds on TP53-wt AML cells. Annexin V/PI staining showed that WIP1i sensitizes TP53-wt AML cell lines and primary samples to Nut-3a-induced apoptosis, when compared with single treatment. No effect was seen in NOMO-1. GEP demonstrated that MV-4-11 cells exhibits a major response to drug combination with an upregulation of cell cycle control genes, a downregulation of DNA repair machinery genes, upregulation of MDM2 and of TP53-downstream genes (eg.CDKN1A), confirming the activation of p53 pathway. NOMO-1 cells showed upregulation of resiliency-mechanism and confirmed insensitivity to both treatment. WB analysis confirmed GEP data showing an increased expression of p53 and p21 in wt-TP53 cell line after 16h of combined-treatment.

Conclusions: We identified a novel synergistic drug combination between Nut-3a and WIP1i that induces apoptosis in AML cell lines and primary samples. GEP and WB of TP53-wt MV-4-11 and TP53-mutated NOMO-1 cells showed mechanisms underlying drug sensitivity and resistance giving novel insights on potential markers of response and novel drugable targets. In vivo studies are needed to confirm these preclinical data. Supported by: AIRC, FP7-NGS-PTL, Fondazione del Monte, HARMONY.

#2965

Induction of DNA damage responses by T-oligo and 6-thio-dG via modulating telomere associated proteins and telomerase.

Zachary Schrank, Nabiha Khan, Joseph Kellen, Sanjana Singh, Chike Osude, Neelu Puri. _University of Illinois College of Medicine, Rockford, IL_.

T-oligo, a guanine-rich oligonucleotide (GRO) homologous to the 3ʹ telomeric overhang, has been shown to elicit potent DNA-damage responses (DDRs) in numerous cancer cell lines. However, the detailed molecular mechanism by which T-oligo induces these responses in cancer cells remains elusive. Additionally, it has recently been reported that the novel drug 6-Thio-2'-Deoxyguanosine (6-thio-dG), a nucleoside analogue of the approved drug 6-thioguanine, inhibits growth of cancer cells by its incorporation into the telomere via telomerase followed by subsequent shelterin disruption. This study aims to investigate the mechanism of action of T-oligo and 6-thio-dG at the telomere, as well as to assess the therapeutic potential of T-oligo in combination with 6-thio-dG. Finally, we also investigated the therapeutic efficacy of T-oligo in combination with Vemurafenib, an FDA-approved BRAF inhibitor, in BRAF-mutant melanoma cells as a potential combinatorial therapy for aggressive BRAF-mutant melanoma. Previous studies have shown that T-oligo is able to reduce expression of hTERT, the catalytic subunit of telomerase, by 50% in MM-AN melanoma cells. Results from this study showed that 2.5µM of 6-thio-dG increased expression of hTERT by 50% in this cell line, as seen by qPCR. This suggests that the mechanism of action of 6-thio-dG and T-oligo may involve modulation of telomerase in different ways. Treatment of MM-AN melanoma cells with 2.5μM 6-thio-dG induced a 1.54- and 1.75-fold upregulation of TRF2 at 48 and 72 hours, respectively, and an upregulation of POT1 by 1.63- and 2.15-fold at 48 and 72 hours, respectively, as seen by immunoblotting. These results suggest that 6-thio-dG may induce shelterin dissociation events similar to T-oligo, which has been shown to upregulate TRF2 and POT1 in this cell line. Our studies showed that 1.25μM and 2.5μM 6-thio-dG significantly inhibited growth of melanoma cells by 1.6- and 3.8-fold, respectively (p<0.01), by MTT assay. However, treatment of melanoma cells with 10μM T-oligo in combination with 6-thio-dG (1.25μM or 2.5μM) did not significantly inhibit cell growth in comparison to T-oligo alone, suggesting that these drugs may induce DDRs by similar mechanisms. Preliminary results indicate that, in MU melanoma cells, which are positive for the V600E BRAF mutation, treatment with T-oligo and Vemurafenib induced an additive effect in comparison to Vemurafenib or T-oligo alone. These results suggest that T-oligo and Vemurafenib may be potential candidates for combinatorial therapy for aggressive BRAF-mutant melanomas.

#2966

Isatuximab-induced multiple myeloma cell killing through effector functions is dependent on CD38 expression and complement inhibitors.

Zhili Song,1 Guang Yang,1 Anlai Wang,1 Rita Greco,1 Joachim Theilhaber,1 Elvis Shehu,1 Daniel Ajona,2 Bruno Paiva,2 Chen Zhu,1 Dmitri Wiederschain,1 Marielle F. Chiron Blondel,3 Francisco Adrian1. 1 _Sanofi, Cambridge, MA;_ 2 _Universidad de Navarra, Pamplona, Spain;_ 3 _Sanofi, Vitry-Sur-Seine, France_.

Isatuximab (Isa) is an IgG1 monoclonal antibody (Ab) that specifically recognizes human CD38. Once Isa engages multiple myeloma (MM) cells expressing a high level of CD38, it can induce tumor cell killing via Fc-dependent mechanisms including Ab-dependent cell-mediated cytotoxicity (ADCC), Ab-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). To better understand the mechanism of Isa-mediated cytotoxicity, we studied CD38 levels in 16 established MM lines and measured Isa-mediated ADCC, ADCP, and CDC in tumor cell killing. The cytotoxic functions of Isa were dependent on CD38 receptor density (RD) in most cell lines. Isa-mediated ADCC was observed in a subset (7/16) of MM cell lines that displayed CD38 RD >100,000 molecules/cell. Similarly, the same subset of MM cells, with 1 exception, were sensitive to Isa-mediated ADCP, indicating a similar CD38 RD is also required for this killing effect. A much higher CD38 RD (>250,000 molecules/cell) alone was insufficient for Isa-mediated CDC. Cell lines MOLP-8 and MOLP-2 have CD38 RD >250,000 molecules/cell, but are resistant to Isa-mediated CDC. Overexpression of CD38 in cell lines with low endogenous CD38 expression did not always sensitize the cells to Isa-mediated CDC. These results suggest that additional mechanisms are involved in the regulation of such cytotoxic effects. We further investigated the expression of complement-cascade inhibitors CD46, CD55, and CD59. High-level expression of at least one of these molecules was associated with resistance to Isa-mediated CDC, even when cells were CD38 high (hi). By comparing 4 selected MM cell lines, we found that a minimal RD level of 50,000 molecules/cell for CD46, CD55 or CD59 appears to be an important threshold to suppress Isa-mediated CDC. When CD59 function was neutralized by an anti-CD59 antibody, we were able to re-sensitize the cells to Isa-mediated CDC in CD38hi MOLP-8 cells. Neutralizing CD59 function alone did not rescue Isa-mediated CDC in CD38-low expressing NCI-H929 cells. However, overexpressing CD38 and inhibiting CD59 rendered NCI-H929 cells sensitive to CDC. Taken together, the high-level of CD38 expression and low-level of CD59 (and perhaps other inhibitors) expression are important for Isa-mediated CDC in killing of target tumor cells. In conclusion, the main immune effector mechanisms involved in Isa-mediated killing of MM cells include ADCC and ADCP. These effects are dependent on high levels of CD38 RD in MM cell lines in vitro. Further confirmation is under way using samples from MM patients.

#2967

Activity of G-quadruplex stabilizing small molecule that downregulates MYC and synergizes with Navitoclax in acute myeloid leukemia cells.

Justin J. Montoya,1 Megan A. Turnidge,2 Daniel H. Wai,1 David W. Lee,1 Apruvi Patel,1 Vijay Gokhale,3 Laurence J. Hurley,3 David O. Azorsa1. 1 _Phoenix Children's Hospital, Phoenix, AZ;_ 2 _University of Arizona College of Medicine - Phoenix, Phoenix, AZ;_ 3 _University of Arizona, Tucson, AZ_.

Acute Myeloid Leukemia (AML) is a malignancy of myeloid precursor cells that can occur when genomic changes alter expression of key genes, causing cells to resume an undifferentiated state and proliferate. Ongoing efforts have focused on developing therapies that specifically target the protein products of aberrantly expressed genes. However, many of the identified proteins are difficult to target because of structural challenges, protein overexpression, or mutations that confer resistance to therapy. A type of therapy that circumvents these issues is the use of small molecules that stabilize DNA secondary structures called G-quadruplexes, which are present in the promoters of many potential oncogenes, and have regulatory roles in their transcription. This study analyzes the activity of G-quadruplex stabilizing small molecule GQC-05 that has been shown to down-regulate MYC, which is commonly misregulated in AML. Treatment of MYC-expressing AML cell lines KG-1a, CMK and TF-1 with GQC-05 resulted in decreased expression of MYC mRNA and protein, with the effect more pronounced in KG-1a cells. GQC-05 treatment of the AML cells decreased cell viability, while increasing apoptosis and DNA damage. Combinational drug screening was performed to identify compounds that potentiated the anti-proliferative effects of GQC-05. Results from the screen identified the BCL-2 inhibitor Navitoclax as a compound that potentiated GQC-05 activity. Co-treatment with GQC-05 and Navitoclax showed synergistic effect on cell viability of AML cells as determined by Chou-Talalay analysis. Furthermore, co-treatment with Navitoclax did not affect the downregulation of MYC by GQC-05, but did increase apoptosis and DNA damage leading to rapid cytotoxicity. These results indicate that the G-quadruplex stabilizing small molecule GQC-05 may function more as a DNA damaging agent in AML cells and that combining GQC-05 with a Bcl-2/Bcl-XL inhibitor such as Navitoclax can result in increased cytotoxic activity.

#2968

Trim25 and KLF5 expression predict AFPep-mediated inhibition of estrogen-dependent growth.

Kanthi J. Bommareddy,1 Anusri Kadakuntla,1 Michael Kuna,1 Priya Shivraj,1 Roman Ginnan,1 Ann Hohenhaus,2 James Bennett,1 Thomas Andersen1. 1 _Albany Medical College, Albany, NY;_ 2 _Animal Medical Center, New York City, NY_.

Introduction: Alpha-fetoprotein (AFP) functions in utero to inhibit estrogen-mediated growth in fetal tissue. AFPep is a 9-amino acid cyclized form of the active site in AFP that impedes phosphorylation and activation of ERα. Recent studies demonstrate that AFPep inhibits estrogen-dependent growth in MCF-7 and T47D xenografts and decreases mammary tumor burden in estrogen-exposed ACI rats. It is important to identify biomarkers that predict the efficacy of AFPep. Dimerized, phosphorylated ERα (pERα) transcribes TRIM25, which is associated with breast cancer metastasis and decreased survival. KLF5 is a transcription factor downstream of pERα and is associated with breast and cervical cancer proliferation, invasiveness, and migration. We hypothesize that treatment with AFPep inhibits expression of TRIM25 and KLF5 and that these are predictive biomarkers for AFPep-mediated inhibition of estrogen-dependent growth.

Methods: Expression of ERα, pERα, TRIM25, and KLF5 were assessed in three estrogen-sensitive models: immature mouse uterus, MCF7 xenografts, and canine breast tumors. Immature mice were treated with saline only, AFPep (100 μg) only, estrogen, or estrogen plus AFPep (100 μg). Twenty-four hours post treatment, uteri were removed, weighed, and blotted for biomarkers. Mice bearing MCF7 xenografts were treated with estrogen or estrogen with AFPep and biopsied after 14 days. Finally, two dogs, one with a simple tubular mammary adenoma and the other with a grade II mixed mammary carcinoma, were treated with AFPep for seven days.

Results: Immature mice treated with estrogen had greater uterine-to-body weight ratio, increased ratio of pERα to total ERα, and increased KLF5 and TRIM25 expression compared to mice treated with saline alone or AFPep alone. Uteri from mice treated with estrogen and AFPep showed decreased uterine-to-body weight ratio, decreased ratio of pERα to total ERα, and decreased KLF5 and TRIM25 expression compared to uteri treated with estrogen alone. MCF7 xenografts from mice treated with estrogen and AFPep showed decreased ratio of pERα to total ERα compared to mice treated with estrogen alone. AFPep treatment of xenograft-bearing mice decreased expression of TRIM25 and KLF5 in the tumor. In the clinical canine mammary tumor study, a mixed carcinoma expressed pERα and treatment of that dog with AFPep decreased the expression of both KLF5 and TRIM25 in that tumor. A dog with a simple tubular adenoma did not express pERα, and treatment of that dog with AFPep had little impact on the expression of TRIM25 and KLF5 in the tumor.

Conclusion: We conclude that AFPep inhibits estrogen-mediated growth and the ratio of pERα to total ERα. KLF5 and TRIM25 also serve as predictive markers for the efficacy of AFPep in uterus and breast tissue respectively.

#2969

The antiviral agent Cidofovir induces DNA damage and mitotic catastrophe in HPV-positive and -negative head and neck squamous cell carcinomas in vitro.

Ernst-Jan M. Speel, Femke Verhees, Dion Legemaate, Robin Jacobs, Wisse E. Haakma, Mat Rousch, Bernd Kremer. _Maastricht UMC, Maastricht, Netherlands_.

Objectives: Cidofovir (CDV) is an antiviral agent with anti-proliferative properties and is suggested as treatment option in head and neck squamous cell carcinoma (HNSCC) patients to improve outcome and quality of life. The mechanisms underlying the effectivity of CDV are not completely understood. The aim of our study was to investigate the efficacy of CDV in HPV-positive and -negative HNSCC cell lines in vitro and whether it is caused by a difference in response to DNA damage.

Materials and methods: Upon CDV treatment of HPV-positive and -negative HNSCC-, uterine cervical carcinoma (UCC)- and immortalized NOK- cell lines, cell viability was assessed with a MTT assay. Accumulation of DNA double strand breaks (DSBs) and activation of the DNA repair pathway were analyzed using immunofluorescence (IF) and Western blotting (WB), respectively. Apoptosis was detected by cleaved-PARP (WB) and mitotic catastrophe by phospho-Aurora Kinase and Cyclin B1 (IF).

Results: All cell lines responded to CDV. Treatment resulted in γ-H2AX accumulation and upregulation of DNA repair proteins, especially in the HPV-positive cells. CDV did not induce PARP cleavage but induced Cyclin B1 expression. Phospho-Aurora Kinase immunostaining showed a decrease in number of mitoses but an increase in aberrant mitoses suggesting mitotic catastrophe upon CDV treatment.

Conclusion: CDV inhibits cell growth in HPV-positive and -negative HNSCC and UCC cell lines which was more profound in HPV-positive cell lines. CDV treated cells showed accumulation of DNA DSBs. Although the DNA repair was activated, apoptosis did not occur. Rather our data indicate the occurrence of mitotic catastrophe.

#2970

Comparison of anti-CD38 antibodies in vitro mechanisms of action in multiple myeloma.

Michelle Kinder,1 Mi Ta,1 Amy Axel,1 Amy Wong,1 Stephen Rudnick,1 Bart de Goeij,2 Jeroen Lammerts van Bueren,2 Alex Babich,1 Mark Mendonca,1 Jocelyn Sendecki,1 Christopher Chiu,1 Kevin Bellew,1 Colleen Kane1. 1 _Janssen Research & Development, Spring House, PA; _2 _Genmab B.V., Utrecht, Netherlands_.

Background: The outcome of multiple myeloma (MM) patients has been dramatically improved by the monoclonal antibody (mAb) daratumumab (DARA). DARA is a human IgG1κ mAb specific for CD38 and approved for the treatment of newly diagnosed and relapsed/refractory MM as a monotherapy and in combination with standard of care. Isatuximab and TAK-079 are additional CD38 targeting mAbs in clinical development by Sanofi and Takeda, respectively. These mAbs are reported to bind different epitopes and induce lysis of MM tumor cells via multiple mechanisms. It is unclear how the pleiotropic mechanisms collectively impact tumor cytolysis and exhibit anti-tumor effects in a comprehensive ex vivo immune milieu.

Methods: For research purposes, DARA was compared with representative mAbs based on the published patent sequences for isatuximab and TAK-079, these surrogate analogs will hereafter be referred to as Sanofi anti-CD38 and Takeda anti-CD38. Mechanism of action (MOA) studies were done to compare antibody dependent cell-mediated cytotoxicity (ADCC), antibody dependent cell phagocytosis (ADCP), complement mediated cytotoxicity (CDC), and early and late detection of apoptosis. In addition, fresh whole blood from healthy donors was used to assess the cumulative effect of the MOAs on MM cell lines.

Results: We tested the CDC activity of the anti-CD38s on multiple MM cell lines with a range of CD38 surface expression and CDC sensitivity levels. In LP-1 and MOLP-8 MM cell lines, DARA resulted in higher levels CDC activity as compared to the other anti-CD38 mAbs. In ADCC and ADCP assays, all 3 anti-CD38 mAbs induced similar levels of MM cell death and phagocytosis. Apoptosis was also assessed in the presence and absence of FcR crosslinking. The Annexin V assay replicated published results that only the Sanofi anti-CD38 was able to induce phosphatidylserine translocation to the cell surface without FcR crosslinking. However, in a robust 5-day cytotoxicity assay detecting metabolically active cells, all 3 antibodies elicited comparable high levels of cell death in the presence of the FcR crosslinker and low levels in its absence. We also performed assays with fresh whole blood from healthy volunteers (n=6) and determined the cumulative effect of the anti-CD38 mAbs on LP-1 and MOLP-8 MM cell lines. DARA demonstrated a higher maximal cytotoxicity than the other mAbs. Moreover, DARA had a significantly lower EC50 than Takeda anti-CD38 in both cell lines and lower than Sanofi-anti-CD38 in MOLP-8.

Conclusion: DARA and surrogate analogs of Sanofi anti-CD38 and Takeda anti-CD38 have similar MOAs with the exception of higher CDC demonstrated by DARA which may contribute to the observed improved efficacy of DARA in a comprehensive immune milieu in vitro. As DARA is the only approved CD38 mAb, it remains to be determined in clinical trials if these in vitro differences lead to differences in clinical benefit.

#2971

Ellagic acid and its metabolite Urolithin A induce prostate cancer cell death in p53 dependent and independent manner.

Yasir I. Mohammed Saleem, Mustafa Selim. _East Carolina University, Greenville, NC_.

Carcinoma of the prostate (CaP) is the most common cancer in men and the second leading cause of cancer-related death in men worldwide. Treatment of early stages of CaP often involves the surgical removal of all or part of the prostate gland; whereas malignant cases of advanced CaP generally requires a combination of therapies including removal of the entire prostate, radiotherapy and androgen deprivation. However, significant number of CaP patients relapse the CaP as the disease becomes hormonal independent. Therefore, it is important to target cell death pathways that function independently of androgen signaling. p53 is a common tumor suppressor that mediates apoptosis, cell cycle arrest and DNA repair. Inactivation of p53 is common in CaP cells and this repression of p53 function diminishes androgen receptor signaling. The most common negative regulator of p53 is MDM2, which is itself a target gene of p53 to form an autoregulatory negative feedback loop. MDM2 inhibits p53 transcriptional activity through the induction of p53 polyubiquitination and degradation in the proteasome. This loss of p53 may permit CaP cells to undergo uncontrolled cell growth and cancer progression. Previous studies have shown that certain polyphenols, derived from natural products, possess anticancer activity that may be attributed to their repression of MDM2 ligase activity and activation of p53. Pomegranates, berries, and walnuts contain several bioactive compounds, including the Ellagitannins (ETs). ETs are polyphenolic compounds that are hydrolyzed in the stomach to form Ellagic acid (EA) which is itself metabolized in the gut microbiota to Urolithin A (UA). The purpose of this study was to investigate the influence of EA and UA on the p53-MDM2 signaling pathway in CaP cells. Three models of CaP cell lines were used because each harbor different p53 genotypes: LNCaP (p53+/+), 22RV1(p53-/+) and PC3 (p53-/-). Here we found that, when 22RV1 and LNCaP were treated with EA and UA, the interaction between p53 and MDM2 was disrupted. As a result, both EA and UA caused an increase in p53 protein levels and increased the steady-state concentration of p21, a main downstream target gene of p53 that mediates cell cycle arrest. In addition, EA and UA increased the levels of PUMA and NOXA proteins, both target genes of p53 which confer p53's pro-apoptotic function. Moreover, we confirmed UA inhibits MDM2-mediated polyubiquitination and degradation of p53. Finally, the data show that EA and UA induce apoptosis in PC3 cells (p53-/-), indicating p53 independent role of these compounds. These results suggest that EA and UA may have potential anti-neoplastic activity in CaP cells that may be at least partially attributed to the stabilization and activation of p53.

#2972

Effect of novel copper-tolfenamic acid complex on medulloblastoma cells.

Julie Hong,1 Melissa Schullek,1 John Kolton,1 Joseph Reitman,1 Amal Dudhia,1 Nihant Tallapaka,1 Rassil Basha,1 Deondra T. Brown,2 Jaya Chhabra,2 Alvin Holder,2 Umesh T. Sankpal1. 1 _Univ. of North Texas Health Science Ctr., Fort Worth, TX;_ 2 _Old Dominion University, Norfolk, VA_.

Introduction. Medulloblastoma is the most common malignant brain tumor in children. New treatment strategies are urgently needed as the current options often result in significant long term neurocognitive and growth defects among survivors. We have previously demonstrated the anti-cancer activity of small-molecule non-steroidal anti-inflammatory drug tolfenamic acid (TA) in medulloblastoma cells and tumor model. The anti-cancer activity of TA can be correlated to its ability to downregulate the anti-apoptotic protein Survivin, cause cell cycle arrest, and induce apoptosis. A recent publication has shown that the activity of TA can be enhanced by forming a complex with copper (II). In this study we tested the function of the copper complex as an anti-cancer agent against pediatric medulloblastoma.

Methods. Pediatric medulloblastoma cell lines DAOY and D283 were used in this study. We have previously demonstrated the synthesis, stability, and anti-cancer activity of Copper-Tolfenamic acid (Cu-TA) complex using various cancer cell lines. The efficacy of Cu-TA against medulloblastoma cells was determined by studying the effect of increasing dose on cell viability, using the CellTiter-Glo Luminescent Cell Viability Assay. Further experiments, using optimized doses of Cu-TA, were carried out to determine its effect on cell cycle and its ability to induce apoptosis. Based on our previous results with TA, we also investigated the effect of Cu-TA on survivin protein levels.

Results. Inhibition of cell viability was observed with increasing doses of Cu-TA as well as with increasing time. The IC50 values were 2-fold lower compared to that of TA and were used in further experiments. Cell cycle analysis using propidium iodide staining and flow cytometry revealed that treatment with Cu-TA results in accumulation of cells in G0/G1 phase, as early as 12h after treatment. Cu-TA induced apoptosis was studied by flow cytometric analysis of AnnexinV stained cells and by western blot analysis of cleaved-PARP. At 48h post-treatment, significant increase in apoptosis was detected by flow cytometry, that also correlated with increased levels of c-PARP. Cu-TA treatment also resulted in a significant decrease in survivin levels compared to TA at 24 and 48h post-treatment.

Conclusion. Our earlier studies with TA have demonstrated its potential as an anti-cancer agent with relatively safe toxicity profile, both in vitro and in pre-clinical mouse model studies. TA also has the capability to enhance the response of standard treatments when used in combination. Here, we demonstrate the ability to increase the efficacy of TA when complexed with copper (II). Cu-TA, with its increased anti-cancer activity and potential to sensitize cells to chemotherapy and radiation, provides a novel therapeutic option for the treatment of medulloblastoma.

#2973

Growth response of human liver cancer cell lines to treatment with various agents targeting different members of the HER family and CDKs.

Ozlem H. Ozyamaci,1 Alan M. Seddon,1 Satvider Mudan,2 Helmout Modjtahedi3. 1 _Kingston University London, Kingston-upon-Thames, United Kingdom;_ 2 _St George's University of London,, London, United Kingdom;_ 3 _Kingston University/School of Life Science, Pharmacy and Chemistry, Kingston-upon-Thames, United Kingdom_.

Liver cancer is one of the most lethal types of cancer in the world. The heterogeneous nature of liver cancer, together with primary and secondary resistance to existing drugs are some of the important contributing factors. In the past three decades, abnormal expression and activation of human epidermal growth factor receptor (HER) family members have been reported in a wide range of epithelial cancers and several monoclonal antibody (mAb)-based drugs and small molecule tyrosine kinase inhibitors (TKIs) targeting the HER family members have been approved for the treatment of patients with a wide range of cancers. However, none of the HER inhibitors have yet been approved for the treatment of patients with liver cancer. In some studies, the expression of other members of the HER family or other growth factor receptors has been associated with the resistance to therapy with the HER inhibitors. In this study, we investigated the relative expression of all HER family members, other growth factor receptors (e.g. IGF-IR, C-MET) and the putative cancer stem cell biomarker CD44 in a panel of human liver cancer cell lines (LCCLs). We also investigated the sensitivity of these LCCLs to treatment with various agents including different types of TKIs with specificity to one or more members of the HER family, C-MET or IGFI-IR, as well as inhibitors of CDK4/6 (palbocicilib) and CDK1/2/5/9 (dinacicilib) and compared our data to those obtained with the FDA approved agents sorafenib and regorafenib for the treatment of liver cancer. While the great majority of the LCCLs were positive for the HER family members, overexpression of the EGFR, HER-3, and HER-4 were only present in SNU475, PLC/PRF5 and PLC/PRF5 LCCLs with mean fluorescence intensity (MFI) values of 620, 130 and 84 respectively. In contrast, the expression levels of C-MET and IGF-IR were the highest in SNU449 and HEPG-2 with MFI values of 95 and 99 respectively. Four of the seven LCCLs had overexpression of CD44 with the MFI values ranging from 329 to 704. With the exception of PLC/PRF5 which was found to be more sensitive to treatment with the pan-HER TKI afatinib (IC50 = 86nM) than treatment with sorafenib (IC50= 370nM) and regorafenib (372 nM), most of the other LCCLs were insensitive to treatment with the other types of HER inhibitors. Moreover, out of all the targeted therapies, the CDK inhibitors Dinacicilib and Palbocicilib were the most effective treatment for inhibiting the growth in vitro of these LCCLs. However, using linear regression analysis, we did not find any significant association between the expression level of different growth factor receptors and the response to therapy with various agents. Taken together our results show the heterogeneous nature of human LCCLs and the need for further investigation on the therapeutic potential of afatinib in combination with other agents in liver cancer.

#2974

Preclinical evaluation of Ozanimod for prostate cancer treatment.

Reshmii Venkatesan,1 Somaiah Chinnapaka,1 John Schappert,2 Gnanasekar Munirathinam1. 1 _University Of Illinois, College Of Medicine, Rockford, IL;_ 2 _University Of Notre Dame, Notre Dame, IN_.

Prostate cancer (PCa) is the second leading cause of cancer-related death in men of United States, after lung cancer. There are various treatment options such as surgery, radiation therapy, chemotherapy, and androgen deprivation therapy are availabale for PCa. However, invariably patients develop resistance to these therapies, resulting in the manifestation of aggressive and recurrent PCa. Hence, there is an urgent need for an effective treatment strategy for managing PCa. Previous studies have identified that the sphingosine 1-phosphate receptor (S1PR) as a potential target to treat prostate cancer. Ozanimod (OZM) is a selective sphingosine 1- phosphate receptor 1 (S1PR1) and (S1PR5) modulator, which is in phase 3 clinical trial for patients with relapsing multiple sclerosis and ulcerative colitis. The objective of this study is to determine the anticancer effects of OZM, an S1PR modulator using LNCaP, PC-3, and DU-145 as cellular models of PCa. These PCa cell lines were subjected to cell viability, colony formation and wound healing assays as endpoints to assess the anticancer effects of OZM. The cell viability assay results revealed that OZM had a differential effect on PCa cell lines with LNCaP cells being the most sensitive and DU-145, PC-3 showing similar sensitivities to OZM treatment. Furthermore, OZM treatment inhibited the colony formation characteristics of PCa cell lines in-vitro when compared with control in all the three cell lines. In wound healing assay, the migratory characteristics of PCa cell lines were inhibited by OZM in a dose dependent manner. These findings suggested that OZM has both tumor growth suppression and metastasis inhibition potential. Furthermore, Annexin V and Propidium Iodide staining assay revealed that OZM

induces apoptosis in PC-3 and DU-145 cells. Following these findings, human apoptotic array study using LNCaP cells showed that anti-apoptotic protein molecules such as c-IAP1, clusterin, and livin were downregulated by OZM treatment in addition to inhibition of HIF-1α expression. The mechanistic study conducted using LNCaP cells by Western blot analysis revealed that OZM treatment upregulated BiP expression while downregulated PERK, IRE-1α which are protein markers associated with endoplasmic reticulum (ER) stress pathway. Furthermore, our results showed that c-myc was down-regulated and MAD, MAX transcription factors were upregulated when treated with OZM. Based on these findings, we suggest that OZM treatment modulates ER stress pathway and anti-apoptotic proteins leading to prostate tumor suppression. In conclusion, OZM might be a potential chemotherapeutic agent for treating both indolent and aggressive PCa.

#2975

CXCR4 blockade of T cell acute lymphoblastic leukemia causes systemic disease in an NSG model allowing ruxolitinib and venetoclax to synergistically treat cancer burden.

Kirsti L. Walker,1 Sabrina A. Kabakov,1 Fen Zhu,1 Sydney L. Olson,2 Lixin Rui,1 Christian M. Capitini1. 1 _University of Wisconsin-Madison, Madison, WI;_ 2 _UW Madison, Madison, WI_.

T-cell acute lymphoblastic leukemia (T-ALL) is a hematologic malignancy that accounts for 25% of adult and 15% of pediatric acute lymphoblastic leukemia (ALL) cases. Relapsed or refractory T-ALL is difficult to salvage with chemotherapy, which causes long-term toxicity, and is often fatal. The JAK/STAT and BCL-2 pathways are upregulated in T-ALL and promote increased T-ALL proliferation and survival. Currently, targeted therapies of the JAK/STAT and BCL-2 pathways have not been investigated in combination. I propose that dual inhibition of the JAK/STAT and BCL-2 pathways, with ruxolitinib and venetoclax respectively, will lead to maximal T-ALL cell death. Jurkat cells were treated with single doses of ruxolitinib (0.156µM - 5µM) or venetoclax (1.56nM - 50nM) in vitro, and analyzed by trypan blue exclusion, MTT and flow cytometry at 24, 48 and 72h post-treatment. Results demonstrate decreased proliferation by MTT, decreased viability by trypan blue exclusion, and increased apoptosis by flow cytometry for the three highest doses of ruxolitinib (1.25µM, 2.5µM and 5µM) and venetoclax (12.5nM, 25nM and 50nM). A synergistic effect was achieved for all three assays at 48 and 72h when cells were treated with a combination dose of ruxolitinib (1.25µM) and venetoclax (25nM; CI<1). This optimal in vitro combined dose significantly decreased proliferation (p<0.0001) and viability (p<0.0001) of Jurkat cells compared to vehicle and single drug treatment groups. Jurkat-GFP cells were injected intravenously into NSG mice to mimic a systemic in vivo xenograft model of T-ALL, and followed for clinical scores and survival. End organs were analyzed for GFP+CD45+ cells by flow cytometry and GFP+ cells by immunohistochemistry (IHC). Compared to single treatments of ruxolitinib and venetoclax, all mice treated with ruxolitinib and/or venetoclax combination therapy developed hind-limb paralysis and died of CNS disease in the spinal cord and brain as shown by IHC. This suggests for the first time that ruxolitinib or venetoclax cannot penetrate the blood brain barrier (BBB). LC-MS-MS studies were performed to confirm that ruxolitinib and venetoclax cannot penetrate the BBB. Previously published data suggests that T-ALL exploits the CXCR4-CXCL12 chemokine pathway to relapse into the CNS. Jurkat cells were analyzed by flow cytometry and showed high expression of the CXCR4 surface receptor while NSG brain tissue showed presence of CXCL12 mRNA and protein by in situ hybridization (ISH) and western blot analysis. Antibody blockade of CXCR4 in vivo prevented the migration of Jurkat cells into the CNS and suggests that disruption of the CXCR4-CXCL12 pathway will result in a systemic model of T-ALL that could allow for ruxolitinib and venetoclax to eliminate T-ALL at primary sites of disease.

#2976

**A comparative study between the therapeutic efficacy of vitamin D** 3 **and its analogue, paricalcitol, in the treatment of colon cancer in rat.**

Mohammed Abbas Baghdadi,1 Mohammed Aslam,2 Jawwad Ahmad,2 Shakir Idris,2 Bassem Refaat2. 1 _King Faisal Specialist Hospital & Research Center, Jeddah, Saudi Arabia; _2 _Umm Al-Qura University, Makkah, Saudi Arabia_.

Background: Vitamin D3 (VD) and its analogue, Paricalcitol (Pcal), have previously shown anti-tumorigenic activities against colorectal cancer (CRC). Hence, this study compared the therapeutic efficacy of both VD types with or without chemotherapy (5-FU) in azoxymethane (AOM)-induced CRC in rat.

Methods: seventy male Wistar rats were equally allocated into 7 groups: Control, AOM, VD, Pcal, 5-FU, 5-FU/VD and 5-FU/Pcal. AOM was subcutaneously injected for 2 weeks (15mg/kg/week). The study duration was 25 weeks. VD (500 IU/rat/day; 3 days/week) and Pcal (2.5 μg/kg/day; 3 days/week) were injected subcutaneously from week-15 till the end of the study. 5-FU intraperitoneal injections started in week-21 (12 mg/kg/day; 4 successive days) and continued in week-22 (6 mg/kg/4 doses every other day). The colons were collected for gross and histopathological examination. The expression of VD signalling system, Wnt, β-catenin, NF-κB, COX-2, iNOS, VEGF, smad4, p21 and p27 proteins was measured by immune-histochemistry/fluorescence. The mRNA expression of Wnt, β-catenin, DKK-1 and COX-2 genes was measured by Quantitative RT-PCR. ELISA was used to measure the concentrations of TGF-β1, VEGF and COX-2 proteins in tissues.

Results: The AOM and 5-FU groups showed marked inhibitions in the VD receptor and binding protein compared with controls (P < 0.05). VD and Pcal monotherapies significantly increased the expression of the VD molecules and the VD group showed the utmost elevations. Moreover, the tumour numbers and sizes were equally and significantly reduced in the 5-FU, VD and Pcal groups compared with the AOM group (P < 0.05). Interestingly, the 5-FU/VD and 5-FU/Pcal resulted in significantly lower numbers of tumours compared with the remaining study groups and the highest significant reduction was observed in the former group. At the molecular level, the 5-FU/VD markedly inhibited the pro-oncogenic molecules (Wnt, β-catenin, NF-κB, COX-2, iNOS & VEGF) as well as significantly upregulated the anti-tumorigenic molecules (DKK-1, TGF-β1, smad4, p21 and p27) compared with the other study groups (P < 0.05).

Conclusions: Vitamin D3 showed a better synergism than Pcal with 5-FU. The enhanced anti-tumorigenic actions of 5-FU/VD involved the modulation of the Wnt/β-catenin and TGF-β1/smad4 pathways, angiogenesis and cell cycle arrest. Further studies are required to elucidate the therapeutic significance of VD in clinical settings

#2977

Targeting multiple cancer metabolic pathways simultaneously with a novel natural compound trans-gnetin H in prostate cancer cells.

Yixuan Gong,1 Uma Chippada-Venkata,1 Li Wang,1 Haocheng Yu,1 Sudeh Izadmeh,1 Husnu Kaniskan,1 He Chen,1 Jun Zhu,1 Matthew Galsky,1 Jian Jin,1 Geoffrey Girnun,2 William Oh1. 1 _Icahn School of Medicine at Mount Sinai, New York, NY;_ 2 _Stony Brook School of Medicine, Stony Brook, NY_.

Metabolic reprogramming is now recognized as a hallmark of cancer. Aerobic glycolysis (aka the Warburg Effect) represents one of the most well-studied dysregulated metabolic pathways in cancer, whereby cells rapidly convert glucose to ATP and lactate, even in the presence of oxygen. It is now appreciated that in addition, aerobic glycolysis enables cells to generate intermediates for biosynthetic pathways. We sought to identify agents that alter cell metabolism in cancer without significant toxicity to normal cells, focusing on purified compounds from natural plant extracts. Trans-gnetin H (TGH) has been shown to inhibit the growth of several cancer cell lines, although its effects in PCa have not been reported. We found that TGH was more effective in inhibiting cell growth of androgen-independent cell lines PC3 and DU145 than the androgen sensitive line, LNCaP. TGH induced G1 cell cycle arrest and apoptosis in 2D and 3D cultures, respectively. Mechanistically, we observed a significant decrease in ATP, as well as activation of AMPK, and decreased activation of pathways downstream of mTOR. TGH potently inhibited glycolysis as determined by ECAR at sub-micromolar concentrations in a Seahorse Glycolysis Stress Assay. Stable isotope tracer analysis showed that TGH decreased glucose incorporation into glycolytic as well as TCA cycle intermediates. Furthermore, TGH dramatically reduced biosynthetic pathways as determined by intermediates for nucleotide synthesis and de novo fatty acid synthesis. RNA-seq of TGH-treated PC3 cells demonstrated that TGH induced the unfolded protein response and disrupted protein hemostasis. In summary, TGH profoundly disrupted carbohydrate, lipid and protein hemostasis in prostate cancer cells in vitro. TGH administered to mice with PC3 tumor xenografts significantly reduced tumor growth without adverse effects. Immunohistochemistry showed disrupted tumor morphology, reduced Ki67 staining and increased apoptosis in TGH treated tumors. RNA-seq data from xenograft tumors showed that TGH reduced the expression of gene sets related to cancer progression including hypoxia, mTORC1 signaling, epithelia mesenchymal transition (EMT), and MYC-targets, consistent with anti-tumor nature of the compound. Altogether, our study identified a potent metabolic inhibitor in prostate cancer that should be further evaluated as a potential therapeutic agent. (*We acknowledge Dr. Ying Gao and Dr. Elliot Altman from Middle Tennessee State University for provision of trans-gnetin H for some of the study.)

#2978

Clinical evaluation of lapatinib induced BCL-2 adaptive responses.

Jason J. Zoeller,1 Sara A. Hurvitz,2 Michael F. Press,3 Laura M. Selfors,1 Judy Dering,2 Dennis J. Slamon,2 Joan S. Brugge1. 1 _Harvard Medical School, Boston, MA;_ 2 _University of California Los Angeles, Los Angeles, CA;_ 3 _University of Southern California, Los Angeles, CA_.

Our pre-clinical data identified BCL-2 upregulation as a critical component and biomarker of the adaptive response to blockade of PI3K/mTOR in vitro and inhibition of HER2 via lapatinib in vivo. We therefore evaluated whether a similar BCL-2 upregulation could be identified in patient clinical samples. Gene expression profiles of patient biopsies collected before and after lapatinib treatment from the TRIO-TORI-B-07 clinical trial were evaluated. Eighteen cases with matched samples were available for analysis. BCL-2 transcriptional upregulation was detected in both HER2+ER- (7 out of 11) as well as HER2+ER+ (5 out of 7) patient tumor samples. To address whether RNA expression correlated with BCL-2 protein expression, pre- and post-treatment FFPE tumors were evaluated for BCL-2 via IHC. We evaluated BCL-2 by intensity and proportion scores to calculate an H-score on a case-by-case basis. Twenty-three cases with matched samples were evaluated by blinded pathological assessment. Despite BCL-2 mRNA upregulation within lapatinib-treated HER2+ER- tumors, only 2 out of 12 cases displayed a minor increase in BCL-2 protein levels following treatment. BCL-2 protein levels were undetectable in most of the lapatinib-treated HER2+ER- tumors. 11 matched cases were HER2+ER+ (baseline ER H-score > 0). All of these cases were BCL-2-postive before treatment. Post-treatment, BCL-2 upregulation was evident in 9 out of 11 HER2+ER+ cases (p = 0.0107). Evaluation of ER status by SP1 IHC in these specimens indicated parallel upregulation of ER in a subset of the HER2+ER+ tumors (p = NS). ER was upregulated in 6 out of the 9 cases where BCL-2 was upregulated in response to lapatinib. To determine whether treatment altered other prosurvival proteins, we performed BCL-XL IHC on the HER2+ER+ cases. BCL-XL was upregulated in 4 out of the 9 cases where BCL-2 was upregulated in response to lapatinib (p = NS). To determine whether BCL-2 upregulation correlated with HER2 inhibition, we performed Ki67 IHC on the HER2+ER+ samples. We observed an overall reduction in Ki67+ tumor cells post-lapatinib (p = 0.0273). Ki67 was reduced in 7 out of the 9 cases where BCL-2 was upregulated in response to lapatinib. Together, these results indicate that BCL-2 mRNA is upregulated following HER2 inhibition via lapatinib; however, protein levels post-treatment are undetectable in most HER2+ER- tumors. Within HER2+ER+ tumors, BCL-2 upregulation correlated with ER upregulation, consistent with evidence that BCL-2 is a direct target of ER transcriptional regulation; however, BCL-2 upregulation was also observed in tumors without ER upregulation, suggesting ER-independent regulation of BCL-2. These findings support evaluation of BCL-2 inhibitors to enhance the therapeutic efficacy of HER2 receptor tyrosine kinase inhibitors.

### Diagnostics, Biomarkers, and the Tumor Microenvironment

#2979

Boosting replication and penetration of telomerase-specific replicative virus by paclitaxel induces synthetic lethality in peritoneal metastasis of gastric cancer.

Toshihiro Ogawa,1 Satoru Kikuchi,1 Wataru Ishikawa,1 Hiroshi Tazawa,1 Motoyasu Tabuchi,1 Shinji Kuroda,1 Kazuhiro Noma,1 Masahiko Nishizaki,1 Shunsuke Kagawa,1 Yasuo Urata,2 Toshiyoshi Fujiwara1. 1 _Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan;_ 2 _Oncolys BioPharma, Inc., Tokyo, Japan_.

Background: Peritoneal metastasis is the most frequent form of distant metastasis and recurrence in gastric cancer and its prognosis is extremely poor. A promising result in phase III trial using intraperitoneal (i.p.) paclitaxel (PTX) for the peritoneal metastasis of gastric cancer has been reported in recent year; however, it is not sufficiently effective to eradicate disseminated gastric cancer. In this study, we investigated whether novel i.p. oncolytic virotherapy could be synthetically lethal with PTX for the peritoneal metastasis of gastric cancer.

Methods: OBP-401 (TelomeScan) is a green fluorescence protein (GFP)-expressing attenuated adenovirus with oncolytic potency that is driven by telomerase promoter. We assessed its synergistic effect with PTX using human gastric cancer cell lines (GCIY and KATO III) and xenograft peritoneal metastasis model. The mechanism underlying the synergistic antitumor effect was evaluated by in vitro viral replication, induction of mitotic catastrophe, and in vivo viral penetration into disseminated nodules.

Results: OBP-401 synergistically suppressed the viability of human gastric cancer cells in combination with PTX. PTX was synthetically lethal with OBP-401 by enhancing viral replication ability in cancer cells and the combination therapy increased mitotic catastrophe induction, resulted in accelerating autophagy and apoptosis. Peritoneally disseminated nodules were selectively visualized as GFP-positive spots by the i.p. administration of OBP-401 in the orthotopic human gastric cancer peritoneal dissemination model. And, PTX enhanced the penetration of OBP-401 deeply into the disseminated nodules. Moreover, non-invasive in vivo imaging system (IVIS) demonstrated that the combination therapy of i.p. OBP-401 administration with PTX significantly inhibited tumor growth of peritoneal metastasis and the amount of malignant ascites.

Conclusions: Intraperitoneal virotherapy with PTX may be a promising treatment strategy for the peritoneal metastasis of gastric cancer. Clinical trials to obtain the proof of concept are warranted in peritoneally disseminated gastric cancer patients.

#2980

The role of USP7 and USP7 inhibitor in HER2+ breast cancer treatment.

Jia Yu, Bo Qin, Judy Boughey, Matthew Goetz, Liewei Wang. _Mayo Clinic, Rochester, MN_.

INTRODUCTION: Breast cancer is the most common invasive cancer and the second leading cause of cancer death in women. Approximately 20% of breast cancer tumors overexpress human epidermal growth factor receptor 2 (HER2), which is associated with an aggressive clinical phenotype and with a poorer prognosis. The development of trastuzumab, an immunoglobulin G monoclonal antibody directed against HER2, has changed the treatment paradigm for this disease. However, approximately 15-20% still does not respond to anti-HER2 therapy. Furthermore, despite advances in the management of metastatic HER2-positive breast cancer, response rate varies greatly, with most patients eventually succumbing to their disease. It is crucial to develop additional therapeutic strategies to improve outcomes for HER2 positive patients, especially those with tumors that do not respond to anti-HER2 therapy.

METHODS and RESULTS: Using HER2+ breast cancer cell lines, we found that an USP7 inhibitor, P5091, dramatically sensitized cancer cells to HER2 inhibitor, trastuzumab treatment. To confirm it in vivo, we tested the drug combination in BT474 xenografts and found P5091 and trastuzumab synergistically suppressed tumor growth. Furthermore, using trastuzumab resistant HER2+ breast cancer cell line, we found that USP7 inhibitor, P5091, can dramatically re-sensitize trastuzumab-resistant BT474 cell line to trastuzumab treatment both in vitro and in vivo. More importantly, using patient derived xenograft (PDX) HER2+ models, we confirmed the synergistic combination effect of P5091 and trastuzumab. Mechanistically, USP7 inhibitor, P5091 led to significantly increased degradation of all four HER family members (HER1-4). When we knocked down USP7 by RNA interference or small hairpin RNA (shRNA), we observed similar substantial decrease in HER family member proteins.

CONCLUSIONS: We have demonstrated the feasibility of using USP7 inhibitor P5091 to sensitize HER2+ breast cancers to trastuzumab treatment. These results provide a rationale for the use of combination therapy of USP7 inhibitors and HER2 inhibitors to treat HER2+ breast cancer. This combination therapy might also be effective in other cancers with upregulated HER signaling, such as GI cancer, lung cancer or ovarian cancer.

#2981

Targeting solid tumor acidic microenvironment with an alphalex PARP inhibitor.

Vishwas Paralkar,1 Robert J. Aiello,1 Dan Marshall,1 Johanna Csengery,1 Patricia Bourassa,1 Qing Zhang,1 Brett S. Robinson,1 Lori Lopresti-Morrow,1 Jane Bechtold,1 Laurie Tylaska,1 Timothy Paradis,1 Gregory Slaybaugh,2 Hanna Visca,2 Anna Moshnikova,2 Dhammika Weerakkody,2 Oleg Andreev,2 Yana K. Reshetnyak,2 Donald Engelman,3 Ranjit Bindra,4 Peter Glazer,4 Per Hellsund1. 1 _Cybrexa Therapeutics, New Haven, CT;_ 2 _University of Rhode Island, Kingston, RI;_ 3 _Yale University, New Haven, CT;_ 4 _Yale School of Medicine, New Haven, CT_.

Poly (ADPribose) polymerase inhibitors (PARPi) have shown great promise in the treatment of cancer, however, their current clinical use has been largely limited to homologous recombination-deficient (HRD) tumors. While these drugs are efficacious as monotherapies, durable responses beyond 12 months are uncommon and their activity against HRD-negative tumors is limited. These findings have prompted great interest in combining PARPi's with chemotherapy to increase the duration of response in HRD-positive tumors and expand the activity of these drugs against HRD-negative tumors which comprise a much greater fraction of the total number of cancer cases each year. While such combinations are highly active against tumors independent of HRD status, they are also extremely cytotoxic against bone marrow cells. As such, dose reductions ranging from 5- to 20-fold are required when PARPi's are combined with chemotherapy which has resulted in favorable safety profiles but limited efficacy. Tumor-targeting strategies could overcome this efficacy barrier but the technologies developed thus far are limited by numerous issues, including: (a) lack of universal, non-saturating tumor-targeting mechanisms which limit their use to specific tumor types and their corresponding antigens, (b) inability to deliver therapeutically relevant levels of drug(s) directly into tumor cells, and (c) insufficient tumor penetration leading to sub-optimal tumor exposure. Herein, we report the development of alphalex, a novel tumor-targeted drug delivery platform which allows the efficient and selective delivery of PARPi's into tumors with significant normal-tissue sparing. Tumor-targeting is achieved using a pH- sensitive peptide which forms an alpha-helix in cellular membrane under low pH conditions associated with the tumor microenvironment. Under these conditions, the peptide translocates across cancer cell membranes to deliver the PARPi directly into the cytoplasm. We demonstrate that alphalex peptide-based conjugates can be combined safely and effectively with both cytotoxic chemotherapies and radiation therapy (RT) and that this approach can be used to selectively kill both HRD-positive and –negative tumors with significant sparing of the bone marrow. The safety and efficacy of the approach was demonstrated in both engineered and patient-derived xenografts (PDXs) in vivo using two FDA-approved PARPi's across two independent laboratories. These data highlight an entirely new approach to apply PARPi's against solid tumors independent of HRD status. Furthermore, our approach can be applied to a diverse range of DNA repair inhibitors for potent and selective chemo/radio-sensitization in a tissue-agnostic manner.

#2982

Monomerization of ALK fusion proteins as a therapeutic strategy in ALK rearranged cancers.

Noriko Hirai, Takaaki Sasaki, Yoshinobu Ohsaki. _Asahikawa Medical Univ., Asahikawa, Japan_.

Introduction: Since the discovery of the anaplastic lymphoma kinase (ALK) and echinoderm microtubule-associated protein like-4 (EML4) fusion protein, ALK tyrosine kinase inhibitors have been shown to have great anti-tumor activities against ALK rearranged non-small cell lung cancers (NSCLC). As previously reported, the EML4-ALK fusion proteins induce constitutive phosphorylation of ALK via the trimeric coiled-coil domain within EML4. ALK F1174L mutations known in neuroblastoma, can induce autophosphorylation without dimerization. We hypothesize that monomerization of ALK fusion proteins decreased tumorigenesis via competitive dissociation of the trimeric coiled-coil domain of EML4.

Methods: We established Ba/F3 cells stably expressing wild-type (wt) ALK intracellular domain or F1174L mutant (mu) with using iDimerize system (Takara Bio). Using this system, monomer or dimer of ALK fusion proteins were controlled by adding AP20187 (Takara Bio). These Ba/F3 cells were subcutaneously injected into BALB nu/nu mice followed by intraperitoneal administration of AP20187 or Mock intermittently. A chemically synthesized peptide composed of amino acid sequences of coiled-coil domain within EML4, was administered for H3122 (EML4-ALK) or A549 (KRAS) cells and the viabilities were analyzed after 24 hours. In order to demonstrate the specificity of the peptide, a synthesized peptide in which two amino acids were substituted was also used as a control. Each peptide was solubilized in DMSO and the peptide concentration was optimized.

Results: We successfully established the Ba/F3 stably expressing fusion-ALKwt, fusion-ALKmu under continuous administration of AP20187. Subsequently, ALK proteins were depolymerized by withdrawal of AP20187. The number of cells expressing ALKwt were decreased while the same of ALKmu were increased. The protein expression of phosphorylation of ALK or downstream survival signaling proteins were weakened after 24hours from AP20187 withdrawal. In fusion-ALKwt cells, tumor xenograft was developed in every mice with AP20187 (n=6) administration, while tumor was not developed in Mock controls (n=6). In fusion-ALKmu cells, tumor xenograft was developed in all mice with or without AP20187 administration (n=12). In vitro assay, cell growth of H3122 was 40% decreased by adding coiled-coil peptides, while increased cell growth was observed in control peptide groups and A549 cells.

Conclusion: Monomerization of ALK intracellular domain induced cell death and suppressed tumorigenesis. The coiled-coil peptide specifically suppressed the activity of EML4-ALK cell line. Monomerization of ALK fusion proteins via coiled-coil peptide is a promising therapeutic strategy for ALK rearranged NSCLC.

#2983

LpMab-23: an anti-podoplanin cancer-specific antibody for a prognostic marker of oral cancer.

Takashi Miwa,1 Shinji Yamada,2 Mika K. Kaneko,2 Yoshikazu Furusawa,1 Masato Fukui,1 Yukinari Kato2. 1 _ZENOAQ RESOURCE CO., LTD., Koriyama, Japan;_ 2 _Tohoku University Graduate School of Medicine, Sendai, Japan_.

Background

Podoplanin (PDPN) is a specific ligand of C-type lectin-like recepotor-2 (CLEC-2), and is involved in cancer metastasis. The physiological PDPN function has been discussed in many normal tissues, including lymphatic endothelial cells, pulmonary type I alveolar cells, and renal podocytes. Herein, we aimed to produce cancer-specific anti-PDPN monoclonal antibodies (mAbs). Furthermore, we investigated whether the reactivity of cancer-specific anti-PDPN mAbs might be a prognostic marker of oral cancer.

Materials and methods

We immunized mice with LN229/PDPN cells, and screened mAbs, which react with PDPN-expressing cancer cell lines, such as glioblastoma cell lines and lung squamous cell carcinoma cell lines and do not react with PDPN-expressing normal cells, including primary lymphatic endothelial cells and renal epithelial cell lines in flow cytometry. Furthermore, we investigated the reactivity of anti-PDPN mAbs by immunohistochemistry against oral cancers. Anti-tumor activities by mouse-human chimeric LpMab-23 (chLpMab-23) were examined using mouse xenograft models of oral cancer cells. Finally, we analyzed the association between the reactivity of anti-PDPN mAbs and clinical/pathological features of oral cancers.

Results

We established LpMab-23, a cancer-specific anti-PDPN mAb. LpMab-23 reacted with PDPN-expressing cancer cell lines in flow cytometry. In contrast, LpMab-23 recognized PDPN-expressing normal cells very weakly. LpMab-23 reacted only with PDPN-expressing cancer cells, not with lymphatic endothelial cells in oral cancer tissues by immunohistochemistry. The epitope mapping of anti-PDPN mAbs revealed that LpMab-23 recognized a cancer-specific glycopeptide including Thr55/Ser56. chLpMab-23 revealed high ADCC and anti-tumor activities against oral cancers. The Kaplan-Meier curves of the five-year new metastasis-free survival rate (nMFS) were significantly lower in LpMab-23-positive patients than in the patients with LpMab-23-negative ones.

Conclusions

A cancer-specific anti-PDPN mAb (LpMab-23) and its mouse-human chimeric mAb (chLpMab-23) were successfully established. LpMab-23 is advantageous in anti-tumor activities. LpMab-23-positive cases could be a useful predictor of poor prognosis for oral cancer.

#2984

Design and characterization of bispecific engineered toxin bodies for targeted cancer therapy.

Aimee Iberg, Garrett L. Cornelison, Caleigh Howard, Paul Amador, Steven Rivera, Michael Jamaleddine, Garrett L. Robinson, Erin K. Willert. _Molecular Templates Inc., Austin, TX_.

Engineered Toxin Bodies (ETBs) are a distinct class of targeted immunotoxins in development as anti-cancer therapeutics by Molecular Templates. ETBs drive a potent and targeted response mediated by antibody-like binding, induced internalization, and enzymatic ribosomal inhibition via the delivery of a Shiga-like toxin subunit A (SLTA) that has been proprietarily modified to avoid innate and adaptive immune recognition. MTEM has a pipeline of scFv targeted ETBs, including its clinical lead MT-3724. MT-3724 targets CD20 for treatment of B-cell lymphoma and has shown promising signs of activity in heavily pretreated patients. The novel MOA inherent to ETB therapeutics allows for activity in relapsed/refractory settings, as previously acquired resistance mechanisms should not hinder efficacy. To further expand the therapeutic benefit of the platform, MTEM is characterizing ETBs that are targeted through multiple binding domains. Bispecific ETBs that target two epitopes on the same receptor, or two distinct cell surface molecules both expressed on cancer cells, may allow for enhanced activity profiles. These possibilities include: (i) activity in the presence of a competitive binding protein (ii) sustained activity when one target molecule is shed or downregulated, (iii) synergistic binding events to increase overall potency, and (iv) increased specificity towards cancer over normal tissue. MTEM has performed in vitro experiments to explore the pairing of binding domains into a single fusion protein, resulting in two targeting arms and a single SLTA subunit. Included in these efforts are the design and characterization of ETBs harboring a single domain antibody derived from camelids, or VHH domains, paired in tandem with other VHH domains or with scFv binding domains to produce both bispecific and biparatopic ETBs. Bispecific ETBs have been generated to engage a variety of target combinations, relevant to both solid and hematologic cancer treatment. In vitro characterization studies conducted include cellular cytotoxicity assays and on-cell binding assays. Bispecific ETBs highlighted in this presentation demonstrate functionality of each targeting arm within a single ETB molecule. In addition, we have shown that one arm can bind target and elicit cytotoxicity even when the other target is blocked. Furthermore, we are learning that some bispecific combinations are cooperative and achieve activities in vitro not observed with the parent single-target ETBs from which they were derived. MTEM is exploring therapeutically relevant target combinations towards the development of a bispecific clinical lead. By pairing the biology and potency of ETB-mediated killing with the expanded targeting possibilities afforded by bispecific molecules, we aim to address unmet medical needs in cancer therapy.

#2985

**Identifying uptake and biodistribution of liposome nanoparticles associated with secretes phospholipase A** 2 **proteins and PLA** 2 **receptors within a prostate cancer.**

Ahmed S. Alnaim,1 Matthew W. Eggert,1 Ben Nie,1 Nhat D. Quanch,2 Shanese L. Jasper,1 Joshua Davis,1 Grafton S. Barnett,1 Brian S. Cummings,2 Peter R. Panizzi,1 Robert D. Arnold1. 1 _Auburn University, Auburn, AL;_ 2 _University of Georgia, GA_.

Exploiting differences in tumor pathophysiology can enhance the delivery and antitumor activity of chemotherapeutics encapsulated in drug carriers. Long-circulating nanoparticle drug carriers, such as pegylated-sterically-stabilized liposomes (SSL), can stably entrap drug, alter drug disposition, improve antitumor activity and minimize toxicity. However, control of their drug-release kinetics has limited their clinical potential. Secretory phospholipases A2 (sPLA2) are excreted and over expressed in a variety of tumors, e.g., up to 22-fold in prostate. These enzymes degrade phospholipids preferentially and have been hypothesized as targets to control drug release from lipid-based nanoparticles, such as liposomes. While they have shown activity in preclinical models, the clinical performance of these formulations has been limited. The goal of these studies was to gain insights into how sPLA2-targeted liposome formulation (SPRL) alter drug release and uptake, specifically examining the role of sPLA2 and its membrane receptor (PLA2R1). Studies were performed using a metastatic-derived human prostate adenocarcinoma cells (PC-3) and a PLA2R1 knock-down variant (PC-3-PLA2R-KD) while varying supplementation of sPLA2 enzyme isoforms (IIA, X, V). Liposomes (SSL & SPRL) were made as described previously and included DiR, a non-exchangeable near-infrared fluorescent dye. In vitro uptake and deposition of

various formulations was determined using flow cytometry and fluorescence microscopy by measuring DiR and doxorubicin (fluorescent anticancer agent) entrapped within some of the formulations. Biodistribution and circulation times were determined non-invasively on an IVIS Lumina XRMS and iThera Multispectral Optoacoustic Tomography (MSOT) systems in Athymic mouse xenograft models of prostate cancer. Liposomes formulations, SPRL and SSL were 120±1 and 118±2 nm. Circulation half-life and tumor deposition was similar for both formulations, suggesting the enhanced pharmacological activity is related to release/uptake after drug extravasation.. In-vitro studies showed that PC-3-PLA2R-KD cells had greater DiR fluorescence of both formulations than wild PC-3 after 48 hr. Moreover, SPRL had greater uptake than SSL formulation (p<0.05) beyond 24 hr. In-vivo studies showed greater uptake of SSL and SPRL in the PC-3-PLA2R-KD tumors. Overall these data suggest that PLA2R1 plays an important role in the performance of SSL and SPRL formulations. Further studies are needed to gain insights into the clinical significance and to determine if presence of PLA2R1 can be used to identify specific patient populations that would respond to treatment with SPRL.

#2986

Study of genetic dependencies associated with 1q-amplified multiple myeloma.

Mairead Reidy, Romanos Sklavenitis-Pistofidis, Daisy Huynh, Karma Ziad Salem, Jihye Park, Siobhan Glavey, Salomon Manier, Irene Ghobrial. _Dana Farber Cancer Institute, Boston, MA_.

Multiple myeloma is a malignancy of terminally differentiated plasma cells with a strikingly heterogeneous genomic landscape. Genetic alterations including t(4;14), and t(14;16) translocations alongside amplification of chromosome 1q21 are particularly associated with a poor outcome. Amplification of the long arm of chromosome 1 (1q) is among the most frequent copy number alterations encountered, with a confirmed adverse effect on survival. Gene expression profiling has identified a minimal common amplified region between 1q21 and 1q23 as a probable target of the amplification event, however the actionable gene dependencies in that region have not been explored. In this study, we employ a large number of inhouse and publicly available CRISPR, shRNA and drug screens in an effort to characterize the genetic dependencies of 1q-amplified myeloma and discover drugs that target them. Ultimately, we hope to propose a tailored therapeutic strategy for patients with 1q-amplified multiple myeloma.

We employed a combination of publicly available (Project Achilles, Dependency Map) and in-house CRISPR and RNAi screens to identify differential dependencies of 1q+ MM. All data were pre-processed and analyzed consistently. Genes were filtered for evidence of both differential dependency and differential expression in 1q+ cell lines. A frequentist approach, prioritizing genes by the number of datasets they hit in, was combined with a gene-wise weighted linear model approach. Genes that hit in at least two datasets and were confirmed by the linear model were taken into consideration for Hallmark pathway analysis with GSEA. Cell cycle, c-Myc, mTOR/PI3K, DNA repair, p53, protein secretion and degradation all constitute major addictions in 1q+ MM and suggest differential sensitivity to corresponding compounds.

We searched for differential drug sensitivities utilizing the Drug Repurposing Library as well as a CMap-guided screen. We identified as hits several compounds targeting both the ubiquitin and the cell cycle pathway, including PI3 kinase, PARP, NAMPT and GSK-3 inhibitors which act primarily through a G2/M block thus validating the dependencies discovered in our datasets.

In conclusion, here we employed a combination of multiple in-house and publicly available CRISPR, shRNA and drug screens, in the largest to date effort to characterize and target the genetic dependencies of 1q-amplified multiple myeloma. Cell cycle and the ubiquitin pathway came up as strong dependencies, while the drugs that target them were indeed shown to preferentially kill 1q-amplified myeloma cell lines. Thus, for the first time, our results suggest that patients with 1q-amplified myeloma might benefit from genetically tailored treatment involving cell cycle and ubiquitin inhibitors or a combination. As 1q amplification is one of myeloma's few frequent alterations, this discovery has the exciting potential to affect change in a large number of patients.

#2987

Generation and characterization of a panel of monoclonal antibodies against the tumor biomarker Thymidine Kinase 1 for research, clinical and therapeutic applications.

Edwin J. Velazquez, Taylor D. Brindley, Gajendra Shrestha, Rachel A. Skabelund, Corbin M. Lee, Zachary D. Ewel, Eliza E. Bitter, Michelle H. Townsend, Kelsey B. Bennion, Kai Li Ong, Kiara V. Whitley, Richard A. Robison, Scott K. Weber, Kim L. O'Neill. _Brigham Young University, Provo, UT_.

Our research explores the potential of a panel of monoclonal antibodies targeting the tumor proliferation biomarker Thymidine Kinase 1 (TK1) for both clinical and therapeutic applications. Cancer biomarkers have become a critical component of precision medicine. TK1 is a well-known tumor proliferation biomarker that is released into the serum, is up-regulated in malignant tissues and can be found on the cell membrane of several cancer types. Notwithstanding the versatility of TK1 as a tumor biomarker, there are a limited number of available antibodies for the detection and quantification of TK1. Moreover, to the date TK1 antibody-based therapies are not being tested in the clinical setting. Thus, the generation of more sensitive TK1 antibodies could increase the availability, accuracy and options of current TK1-based diagnostics and antibody-based immuno-cell therapies. Six peptide sequences across the TK1 molecule were selected. The antibodies were generated using hybridoma technology. Seventeen clones were evaluated in ELISA with a calibration dose-response curve. The calibration curves showed R squares ranging from 0.9738-0.9980 with a 10-15 %CV. The limit of detection and quantification (LOQ, LOD) were obtained. The clones 3B2E11, 9C10, 8G2, 5F7G11, 3B4 and 3G7 showed the lowest values, being 3B2E11 the most sensitive with a LOD of 18.6 ng/ml and a LOQ of 64 ng/ml. Western blot data showed specific binding to recombinant TK1 and to multiple forms of TK1 in cell lysates, and serum samples for 14 of the 17 clones. Although, differences in the binding patterns were found in cell lysates. Flow cytometry was performed to analyze TK1 surface expression. Antibodies 8G2, 3B4 and 57G11 showed consistent binding across 4 cancer cell types in a similar percentage to the commercial TK1 antibody (Abcam91651) with a maximum percentage of binding in lung of (95.6%) followed by prostate (72.2%), colon (62.4%) and breast (49.1%). No significant binding for either the commercial or the custom TK1 antibodies was found on normal mono nuclear cells (MNC). The clones 8G2 and 3B4 were selected for testing their potential as therapeutic agents in antibody-dependent cell-mediated cytotoxicity (ADCC) experiments. Around 50% and 42% increased killing of A549 cells was observed with antibodies 8G2B and 3B4 respectively 48 hrs. after adding the antibodies when compared with isotype controls (p <0.05). Based on dose response curves 2.5 ug/ml for the clone 8G2B and 5-7 ug/ml for the clone 3B4 were the minimum concentrations required to show significant specific cell death in A549 cells. The antibodies developed have shown capacity for detection and quantification of TK1 in serum and on the membrane of cancer cells. Moreover, our in vitro ADCC experiments provide more evidence that membrane associated TK1 has potential as an immunotherapeutic target.

#2988

Quantifying the Uptake of Metal Based Cancer Therapy Drugs Using Single Cell ICP-MS.

Chady Stephan,1 Ruth Merrifield,1 Stefan Wilhelm2. 1 _PerkinElmer Inc, Woodbridge, Ontario, Canada;_ 2 _University of Oklahoma, OK_.

Metallic based cancer therapy drugs have been around for several years, the most widely used being platinum-based drugs, however these come with severe side effects due to the non-specific targeting of these drugs. Recently a number of nanoparticle-based cancer therapeutics have been approved for clinical use or are currently under development. Advantages that engineered nanoparticles may offer over conventional small molecule drugs include: (i) prolonged circulation time in the body; (ii) reduction of nonspecific cellular uptake along with undesirable off-target and other side effects; and (iii) improvement in cellular interactions through specific cancer cell targeting moieties.

The therapeutic effect of cancer treatment is related to the amount of drug that interacts with each individual cancer cell. Traditional drug research techniques, such as conventional inductively coupled plasma mass spectrometry (ICP-MS), have been limited to cell ensemble measurements, which require homogenization of a given cell population for quantitative analysis. All cells within this population are assumed to be similar and therefore assumed to interact with the same amount of drug, however, recent studies demonstrate that cell populations are heterogeneous, and differences exist even for cells from the same cell population and cell line. For example, gene expression measurements based on homogenized cell populations are misleading as they only provide averaged results and do not account for the small but critical changes occurring in individual cells such as size, protein levels, and expressed RNA transcripts. These variations are key aspects when answering previously unsolvable questions in cancer research, stem cell biology, immunology, developmental biology, and neurology.

To overcome these limitations, PerkinElmer developed Single-Cell (SC) ICP-MS, which allows the rapid analysis of a large number of individual cells rather than a cell population as a whole or only a few cells. This allows for the quantification of the metal mass in individual cells, resulting in a histogram of the population showing not only the most frequent and mean masses of drug in the population but also the distribution throughout the population. Here we will show results for both cisplatin uptake and surface modified gold nanoparticle uptake into cancer cells. The first resulting in a wide distribution in the amount of platinum measured per cell over time with differences in resistant and non-resistant cancer cells. While the latter shows the ability of this technique to quantify the number for modified Au nanoparticles per cell as well as the number of cells containing the drug.

#2989

An all-in-one transcriptome-based assay to identify therapy-related biomarkers in lung cancer.

Klaas Kok, Jiacong Wei, Anna A. Rybczynska, Pei Meng, Martijn M. Terpstra, Anthonie J. van der Wekken, Jeroen T. Hiltermann, Ed Schuuring, Harry J. Groen, Anke van den Berg. _UMCG, Groningen, Netherlands_.

Background. In the past decade, treatment of advanced stage lung cancer patients guided by somatic aberrations has become routine practice. Different molecular tests are being applied to detect all targetable mutations and fusions. However, in most cases tissue biopsies are small, hampering multiple independent diagnostic tests. To optimize diagnostic testing we developed an all-in-one transcriptome-based assay.

Methods. We have developed a targeted next generation sequencing protocol that uses total RNA as input and is based on the Single Primed enrichment Technology (SPET). We included 11 cell lines, four frozen biopsies, 12 pleural effusion samples and 32 FFPE samples with in total 41 known mutations (including EGFR n=15; KRAS n=11; BRAF n=2; PIK3CA n=3 ), 21 fusion genes (including ALK n=15; ROS n=3) and 3 cases of MET exon14 skipping.

Results. We confirmed presence of 32 out of 41 mutations, 19 out of 23 fusions and all three cases of exon skipping by our assay. Besides confirming the fusions, we were also able to identify the fusion gene partner for all detected fusion transcripts. For the samples for which we failed to detect the mutations or fusions, the read depth of the target region was less than four, indicating low expression, low tumor content or insufficient unique reads. Independent RNA-based ddPCR on six unconfirmed mutations were all positive, albeit with low numbers of mutant droplets in some cases. One of three undetected fusions was positive in a NanoString fusion gene detection assay. For one of two negative cases, the fusion was most likely false positive by FISH, as this patient was FISH-break positive for both ALK and RET, which is never reported before.

Conclusions. This study proved feasibility of this targeted all-in-one transcriptome-based assay for simultaneous detection of mutations and fusions even in relatively small FFPE tissue biopsies. Moreover, we were able to detect the fusion partner genes in all positive cases. We expect that for routine diagnostic testing using an enrichment for tumor cell-rich areas of recently prepared FFPE blocks, the success rate of the all-in-one transcriptome approach will reach similar sensitivity as currently used diagnostic tests.

#2990

Individualized functional approach to tailoring acute myeloid leukemia therapy.

Shruti Bhatt, Binyam Yilma, Elyse Olesinski, Holly Zhu, Mark Murakami, Vineeth Kumar Murali, Sophia Adamia, David M. Weinstock, Jacqueline S. Garcia, Anthony Letai. _Dana-Farber Cancer Inst., Boston, MA_.

The major focus of drug development in AML has shifted to targeted agents because, despite initial sensitivity to conventional chemotherapy, durable remissions are limited. The clinical implementation of effective targeted therapies still lags due to the lack of predictive biomarkers. To make the best use of existing therapy and to identify new targeted therapies we adopted a broadly applicable functional approach to precision medicine called "dynamic BH3 profiling" (DBP). DBP measures early death signaling induced by short-term drug exposure. Increased cell death signaling is reflected by increased mitochondrial sensitivity (priming) to standardized BH3 peptides mimicking pro-apoptotic proteins.

To develop a personalized therapeutic strategy for AML using DBP, we utilized 17 patient-derived xenografts (PDX) established from de novo, primary refractory or relapsed (R/R) patients. Human myeloblasts from xenotransplanted mice were exposed to 30 targeted and 3 standard of care drugs to determine mitochondrial responses via DBP. Unsupervised clustering of ex vivo DBP responses segregated PDXs into two major clusters, where treatment naïve PDXs clustered distinctly from R/R PDXs. Most drugs induced priming in only selective PDXs, including kinase inhibitors, epigenetic modifiers, and a SMAC mimetic. In contrast, BH3 mimetics and CDK9 inhibitors showed activity across a majority of PDXs. Next, we validated the ability of DBP to predict in vivo responses of single-agent birinapant (SMAC mimetic), JQ-1 (BRD-4 inhibitor), quizartinib (FLT-3 inhibitor), and venetoclax (BCL-2 inhibitor) across 6 AML PDX models, prioritized based on their greatest range of priming responses. The models that showed increased ex vivo priming via DBP also showed greatest in vivo responses, indicating that DBP can rank PDXs according to their drug sensitivities (AUC of ROC 0.87, p<0.005). By comparing the predictive power of DBP to other precision medicine tools such as genomics, we found that DBP was able to accurately predict quizartinib activity in PDXs with WT FLT-3, which are categorized as unresponsive based on genomics. To test the applicability of DBP in assigning therapies in the relapsed setting, we induced resistance to PDX models to single agents via long-term in vivo selection. Myeloblasts of relapsing clone showed reduced baseline mitochondrial priming and loss of sensitivity to most of the agents. This suggested that acquired resistance to single agents selects for apoptosis-refractory clones which then drive a pan-drug resistant phenotype. Finally, we applied this approach to humans, showing that the pretreatment mitochondrial apoptotic priming determined by DBP identifies responders to lenalidomide plus MEC combination therapy in the phase 1 trial of R/R AML patients. In summary, our results suggest that mitochondria-based measurements may serve as a precision medicine tool to guide therapy for a heterogeneous population.

#2991

Targeting glutamine metabolism leads to terminal differentiation in acute myeloid leukemia (AML).

Robert Leone,1 Im-Meng Sun,1 Roshan Chikarmane,1 Radhika Viswanathan,2 Sanjeda Patwari,2 Matthew Arwood,1 Gabriel Ghiaur,1 Jonathan Powell1. 1 _Johns Hopkins Hospital, Baltimore, MD;_ 2 _Brooklyn College, New York, NY_.

The ability of All Trans Retinoic Acid (ATRA) to induce terminal differentiation in Acute Promyelocytic Leukemia (APL) has revolutionized treatment for this deadly subset of AML. Unfortunately, identifying targets that induce differentiation in non-APL cases of AML has met with limited success. Our lab has been interested in the role of glutamine metabolism in cellular differentiation programs. As such, we have investigated the degree to which glutamine contributes to the maintenance of an undifferentiated state in AML. Herein, we report that in vitro incubation with the glutamine blocking agent, 6-diazo-5-oxo-L-norleucine (DON) robustly induces differentiation in the human AML cell line, U937. DON treatment induces phenotypic and molecular changes indicative of differentiation, including increased granularity and vacuolization, and upregulation of differentiation markers CD11b and CD14. The differentiation of these cells was confirmed by their highly abrogated growth in methylcellulose-based colony-forming assays. Differentiated AML cells were not exposed to DON during colony-forming assays, suggesting an irreversible change in cellular differentiation triggered by short-term pretreatment. We have demonstrated that DON-induced cellular differentiation is active in other AML cell lines as well. Importantly, the differentiating effect of DON can be abrogated by the addition of a cell permeable form of α-ketoglutarate (αKG), dimethyl-2-ketoglutarate (DMK). αKG is a key metabolite in glutamine metabolism and is also a critical cofactor for a range of demethylase enzymes involved in epigenetic remodeling. Indeed, we have noted a robust increase in histone methylation marks, such as H3K27Me3, during DON-induced differentiation. Further, we have observed a dramatic increase in the transcription of critical transcription factors (CEBPA, FLT3, MYB, SOX4) twenty-four hours after DON treatment, a time point immediately preceding observed phenotypic differentiation changes. In light of these findings, experiments are currently underway to use DON differentiation therapy as a means of treating mouse models of AML. Taken as a whole, these data support the use of glutamine blockade as a novel differentiating therapy in AML. Given the remarkable curative potential of differentiation therapy and the poor survival rates of adults with AML (particularly older patients), our findings represent a particularly novel approach to treating this deadly disease.

#2992

**Cellular pharmacodynamics of mitochondrial one-carbon metabolism-targeting 5-substituted pyrrolo[3,2-** d **]pyrimidine antifolates.**

Aamod Dekhne,1 Khushbu Shah,2 Gregory S. Ducker,3 Md. Junayed Nayeen,2 Jade M. Katinas,4 Jennifer Wong,4 Arpit Doshi,2 Xun Bao,1 Hasini Kalpage,1 Jenney Liu,1 Seongho Kim,1 Adrianne Wallace-Povirk,1 Changwen Ning,1 Carrie O'Connor,1 Zhanjun Hou,1 Lisa Polin,1 Jing Li,1 Maik Hüttemann,1 Joshua D. Rabinowitz,5 Charles E. Dann,4 Aleem Gangjee,2 Larry Matherly1. 1 _Wayne State University, Detroit, MI;_ 2 _Duquesne University, Pittsburgh, PA;_ 3 _University of Utah, Salt Lake City, UT;_ 4 _Indiana University, Bloomington, IN;_ 5 _Princeton University, Princeton, NJ_.

Folate-dependent one-carbon metabolism (1CM) is compartmentalized in the mitochondria and cytosol and generates a number of metabolites critical to tumor propagation. Folates are taken up by the plasma membrane facilitative transporters, reduced folate carrier (RFC; major tissue transporter) and proton-coupled folate transporter (PCFT; narrow physiological niche, but commonly expressed in solid tumors), and then transported into mitochondria via the mitochondrial folate transporter (MFT; SLC25A32). Although drug-targeting of cytosolic 1CM remains a clinical mainstay for a variety of cancers, development of clinically-useful agents targeting mitochondrial 1CM remains elusive. Of particular pharmacological interest is the mitochondrial 1CM enzyme, serine hydroxymethyltransferase2 (SHMT2). SHMT2 expression correlates with the oncogenic phenotype in a host of different cancers and, overall, SHMT2 is the fifth-most differentially expressed metabolic enzyme in cancer versus normal tissues. Despite its unequivocal oncogenic importance and therapeutic potential, there are no clinically relevant inhibitors of SHMT2. In this study, we characterized cellular pharmacodynamics of novel 5-substituted pyrrolo[3,2-d]pyrimidine antifolates (AGF291, AGF320, and AGF347) which show in vitro antitumor efficacy toward H460 lung, HCT-116 colon, and MIA PaCa-2 pancreatic cancer cells. Inhibition of mitochondrial SHMT2 and cytosolic 1CM at the purine nucleotide biosynthesis enzymes glycinamide ribonucleotide formyltransferase (GARFTase) and/or 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFTase) by this series was established by in vitro targeted metabolomics in H460, HCT-116, and MIA PaCa-2 cells and in vitro cell-free assays with purified enzymes. By depleting SHMT2-derived formate, these compounds potentiate their own direct inhibition of GARFTase and AICARFTase. Depletion of adenine nucleotide pools in vitro by all compounds led to inhibition of mTOR signaling to S6K1 in HCT116 cells. Subcellular fractionation of MIA PaCa-2 and MFT-null and human MFT-transfected glyB Chinese hamster ovary cells confirmed synthesis of polyglutamyl forms of AGF347 in both cytosol and mitochondria with mitochondrial uptake of AGF347 in part mediated by MFT. Treatment by all compounds decreased the cellular GSH/GSSG ratio, indicating depleted ability to combat oxidative stress. In vivo, AGF347 demonstrated potent antitumor efficacy against MIA PaCa-2 xenografts in SCID mice (n=5) with median tumor growth delay (T-C) in 4 mice >38 days and 1 of 5 tumor-free survivors (cured). In vivo metabolomics on tumor xenografts confirmed inhibition of serine catabolism and purine biosynthesis. Collectively, our studies establish the exceptional therapeutic potential of inhibitors dual-targeting mitochondrial and cytosolic 1CM.

#2993

Impaired PARP1 DNA repair defines chemo-sensitivity in IDH1 mutant cell.

Chunzhang Yang, Yanxin Lu, Yang Liu, Mark Gilbert, Jing Wu. _Center for Cancer Research, National Cancer Institute, Bethesda, MD_.

INTRODUCTION: Mutations in isocitrate dehydrogenase (IDH1/2) are the most prevalent genetic deficiency in lower grade gliomas. IDH-mutated in glioma exhibits better clinical outcomes with longer patient survival, as well as greater sensitivity to chemotherapy. In the present study, we explored the molecular mechanisms that determine the chemo-sensitivity in IDH1-mutated cells, and seek for potential therapeutic strategy by targeting PARP/BER DNA repair pathway.

METHODS: We investigated transcriptomic profile from WHO grade II/III glioma based on their IDH1/2 mutation status. We established IDH1-mutated cells and investigated the alterations in nicotinamide adenine dinucleotide (NAD+), Poly (ADP-ribose) polymerase (PARP)-associated DNA repair and DNA damage in these cells in respond to temozolomide (TMZ). Moreover, we evaluated a PARP inhibitor olaparib and its synergistic effect on TMZ-associated cytotoxicity.

RESULTS: Our results showed that the IDH1-mutated cells are more vulnerable to genotoxic agent, which recapitulate the disease phenotype in IDH1-mutated glioma. TMZ treatment resulted in over 20-fold increase of cell death in IDH1-mutated cells compared with wild type counterpart. IDH1-mutated cells exhibited over 1.3-fold DNA damage and over 1.42-fold cellular apoptosis under TMZ treatment. Mechanistically, IDH1-mutated cells exhibited compromised NAD+ metabolism, as well as concomitant PARP/BER DNA repair pathway. IDH1-mutated cells produced 83.8% less poly (ADP-ribose) polymer (pADPR), an important substrate for PARP-associated DNA repair, during TMZ treatment, suggesting their incompetence to maintain genomic integrity due to decreased availability of NAD+. Targeting the PARP-associated DNA repair pathway using olaparib, an FDA-approved PARP inhibitor, remarkably potentiated TMZ-induced cytotoxic effects by 2.16-fold in IDH1-mutated cells.

CONCLUSION: Our findings demonstrate that metabolic defects in IDH1-mutated cells affect PARP-associated DNA repair pathway via NAD+ depletion, and therefore prompt the sensitivity to chemotherapies. Targeting PARP-associated DNA repair pathway suggests a novel therapeutic avenue for IDH1-mutated gliomas.

#2994

In vivo targeting of mesothelioma's molecular signature with nanoparticle functionalized with nucleolin-binding peptide.

Nuno A. Fonseca,1 Vera Moura,2 Fabiana Colelli,3 Daniela Pesce,3 Giacomo Signorino,3 Laura Focareta,3 Alessandra Fucci,3 Francesco Cardile,3 Claudio Pisano,3 Sérgio Simões,4 João Nuno Moreira4. 1 _Center for Neurosciences and Cell Biology, Univ. of Coimbra; TREAT U, Coimbra, Portugal;_ 2 _TREAT U and Center for Neurosciences and Cell Biology, Univ. of Coimbra, Coimbra, Portugal;_ 3 _Biogem – MIR, Via Camporeale, Ariano Irpino, Italy;_ 4 _Center for Neurosciences and Cell Biology; Fac Pharmacy; Univ. of Coimbra, Coimbra, Portugal_.

Mesothelioma is a malignant tumor of the mesothelium, often associated with asbestos exposure, with an overall survival of only 8.8 months. A combination of cisplatin and pemetrexed has been used as an adjuvant treatment to surgical resection or in patients who have inoperable disease. This combination remains as the current standard of care, regardless the sub-type of mesothelioma, with only a 9% 5-year survival rate. Herein, it is hypothesized that a novel targeted treatment, based on a doxorubicin (DXR)-containing nanoparticle functionalized with the nucleolin-binding F3 peptide (named PEGASEMP), will have significant a therapeutic impact against the most aggressive subtype of mesothelioma (biphasic). The hypothesis relies on nucleolin deregulated overexpression in cancer and endothelial cells from tumor blood vessels. Human biphasic mesothelioma cells were stably transduced with luciferase-reporter gene, and orthotopically injected intrapleurally into female immunocompromised mice. Three weeks (Early Stage) and 4 weeks (Advanced Stage) after cell injection, animals were randomly allocated to different treatment groups: vehicle, PEGASEMP at 5.6 or 7 mg of DXR/kg alone (q7dx5w); cisplatin at 4.0 mg/kg alone or combined with PEGASEMP at 5.6 mg of DXR/kg (q7dx5w); and the standard of care, a combination of cisplatin at 4.0 mg/kg (q7dx5w) plus pemetrexed at 100.0 mg/kg (q2dx3x5w). Bioluminescence was monitored weekly with IVIS Spectrum In Vivo Imaging system. Differential gene expression upon PEGASEMP treatment of advanced stage tumors was also performed in comparison with non-treated animals using the GeneChip Human Transcriptome Array 2.0. In the early stage of development, PEGASEMP, at 7.0 mg/kg, inhibited tumor growth by 2,713-fold relative to standard of care, which translated into a 1223-fold decrease in tumor burden. At an advanced stage of tumor development, the combination of PEGASEMP and cisplatin enabled 107-fold tumor growth inhibition, enabling a 55-fold reduction in tumor burden, relative to standard of care. In both cases, no decrease of the body weight was observed. Furthermore, 75% (146) gene transcripts were found to be deregulated (while the remaining 25% were upregulated), involving the downregulation of cell division, including ontologies such as cell cycle or chromatin organization and assembly. Specifically, downregulation of topoisomerase 2, cyclin B1 or Ki67 transcripts, part of the described molecular signature of mesothelioma, was observed and was consistent with the mechanism of action of doxorubicin. Overall, the novel mechanism of action associated with PEGASEMP, enables a significant benefit in terms of efficacy and safety in the treatment of biphasic mesothelioma, as compared with the current standard of care, affecting the molecular signature of the disease and thus supporting future clinical evaluation.

#2995

Thymoquinone attenuates breast cancer cell resistance to paclitaxel via targeting tumor associated stem cell population.

Hanan A. Bashmail,1 Aliaa A. Alamoudi,1 Gehan A. Hegazy,2 Ghada M.A. Agabnoor,1 Abdulwahab Noorwali,1 Ahmed M. Al-Abd3. 1 _King Abdulaziz University, Jeddah, Saudi Arabia;_ 2 _National Research Centre, Giza, Egypt;_ 3 _Gulf Medical University, Ajman, United Arab Emirates_.

Breast cancer remains the most common type of cancer in females, and remains to be one of the most leading causes of death worldwide. This led to increasing the interest to use adjuvant anti-cancer therapy to improve the efficacy of in-clinical use chemotherapeutic agents. Thymoquinone (TQ) is a naturally occurring bioactive compound which showed cumulative evidences of anticancer properties. Herein, we assessed the potential chemomodulatory effect of TQ as an adjuvant therapy in combination with paclitaxel (PTX) against breast cancer cells. Cytotoxic profiles of TQ alone or in combination with PTX were evaluated in MCF7 and T47D breast cancer cell lines. TQ showed cytotoxic effect against MCF7 and T47D cells with IC50's of 64.93±14 µM and 165±2 µM, respectively. In MCF7, TQ did not change PTX's IC50, and the combination index was indicative of antagonism (CI-value = 1.3). Similarly in T47D, combination of TQ with PTX increased PTX's IC50 from 90±8 nM to 140±20 nM with CI-value of 1.5 indicating antagonism. However, it was found that TQ completely depleted PTX resistant fractions in both cancer cell lines under investigation. Moreover, apoptotic and autophagic cell death due to combination of PTX with TQ were significantly increased in T47D compared to PTX treatment alone by 5.6 and 1.7 folds, respectively. In MCF7, combination of TQ with PTX significantly increased autophagic cell death compared to PTX treatment alone. Furthermore, tumor associated stem cell clone (CD44(+)/CD24(-)/(low) was assessed by flow cytometry in MCF7 and T47D cells. TQ alone significantly decreased CD44+/CD24\- tumor associated stem cell clone by 12.4±0.8%. In addition, combination of PTX with TQ further depleted CD44+/CD24- cell clone by 32.3±0.08%. Finally, TQ alone significantly downregulated TWIST-1, and upregulated SNAIL2 gene expression as an evidence to decreased cell resistance to PTX. In conclusion, TQ enhances PTX anticancer profile against breast cancer cells via depleting tumor associated stem cells clone rather than potentiating its direct cell killing effect.

#2996

Systemic depletion of L-methionine with an engineered human enzyme for the treatment of melanoma.

Carly S. Wilder, Achinto Saha, George Georgiou, Everett Stone, John DiGiovanni. _University of Texas at Austin, Austin, TX_.

Metastatic melanoma is an aggressive form of cancer responsible for the majority of skin cancer related deaths. While treatment for metastatic melanoma has improved in recent years with the introduction of targeted therapies and immunotherapies, the five-year survival rate for stage IV melanoma remains only 15-20%. To address the need for alternative options for melanoma treatment, we have begun exploring use of an engineered human enzyme called methionine-gamma-lyase (hMGL). Many cancers, including melanoma, have a high requirement for methionine in comparison with non-cancerous cells. The hMGL enzyme works to exploit this difference by degrading extracellular L-methionine resulting in cancer cell starvation. The hypothesis tested in this research is that the systemic depletion of L-methionine using hMGL will provide significant benefits for melanoma treatment alone or in combination with other therapeutic agents. Treatment of several melanoma cell lines (A375, Hs294T, Skmel28, and B16F10) with various concentrations of hMGL demonstrated that L-methionine depletion reduced survival and led to a G2 cell cycle arrest. To investigate the mechanism of action, analyses of melanoma cells treated with hMGL have shown that signal transducer and activator of transcription 3 (STAT3) tyrosine phosphorylation is significantly reduced. Additionally, hMGL treatment leads to activation of the uncharged tRNA amino acid sensing pathway as shown by an increase in p-eIF2α and Sestrin 2 and an increase in autophagy shown by a decrease in p-ULKSer757 and an increase in cleaved LC3B. Because L-methionine depletion affects levels of glutathione through reduction of cysteine synthesis, preliminary experiments were conducted to evaluate hMGL treatment combined with a thioredoxin reductase inhibitor. This combination, targeting two different intracellular antioxidant pathways, resulted in synergistic inhibition of survival in multiple melanoma cell lines. The current data support the hypothesis that L-methionine depletion using hMGL may have therapeutic efficacy either alone or in combinations for melanoma treatment. Research funded by CPRIT grant RP180590.

#2997

15-hydroxyprostaglandin dehydrogenase induced differentiation in colon cancer cells is regulated via Gli1.

Shakti R. Satapathy,1 Lubna M. Mehdawi,1 Roger Olsson,2 Anita Sjolander1. 1 _Lund University, Malmo, Sweden;_ 2 _Lund University, Lund, Sweden_.

Prostaglandin E2 (PGE2) is a crucial regulator of colon cancer (CC), an inflammation associated cancer. Tumor suppressor 15-hydroxyprostglandin dehydrogenase (15-PGDH) is a PGE2 degrading enzyme but is often downregulated in CC. Interestingly, CC-patients with high levels of Cysteinyl leukotriene receptor 2 (CysLT2R) exhibit good prognoses. Previously, we have reported differentiation promoting role of 15-PGDH in CC cells when induced by LTC4, the CysLT2R ligand. However the mechanism is unclear. Hedgehog (Hh) signalling is involved in embryogenesis and development but hyper activated in colon carcinogenesis. LTC4-induced activation of 15-PGDH downregulated both mRNA and protein expression of GLI1 in HT-29 and Caco-2 colon cancer cells. Moreover, absence of Gli1 significantly reduced the differentiation promoting ability of 15-PGDH, with no change in the differentiation markers Sucrase isomaltase, Mucin-2 and Protocadherin-24. In addition, no noticeable change in the mRNA expression of WNT-specific target AXIN-2 coupled with restoration of the proliferation marker cMYC was observed. In HT-29-derived colonospheres depletion of mRNA expression of CC-CSC markers ALDH1A1 and DCLK1 remained regulated by Gli1. Elevation in Mucin-2 expression in zebrafish xenograft with HT-29 was also noted to be Gli1 dependent. GLI1 and MUCIN-2 mRNA expression in normal mucosa and matched tumor in colon cancer patient also exhibited higher co-relation along with concomitant elevation in DCLK1 expression in tumor in compared to normal mucosa. Tumor microarray (TMA) with low expression of CysLT2R and 15-PGDH exhibited higher Gli1 expression with no trace of Mucin-2. Therefore, restoring the expression of 15-PGDH in CC through activation of CysLT2R signalling and targeting Hh-Gli signalling would open new therapeutic possibilities with minimal collateral damage to the normal tissue.

#2998

Co-targeting bulk tumor and CSCs in clinically translatable TNBC patient-derived xenografts via combination nanotherapy.

Andrew Sulaiman, Sara El-Sahli, Sarah McGarry, Lisheng Wang, Suresh Gadde. _University of Ottawa, Ottawa, Ontario, Canada_.

Triple negative breast cancer (TNBC) accounts disproportionally for the majority of breast cancer related deaths throughout the world. This is largely attributed to lack of a specific therapy capable of targeting both bulk tumor mass and cancer stem cells (CSC) as well as appropriate tumor animal models to accurately evaluate treatment efficacy for the clinical translation. Thus, development of effective and clinically translatable targeted therapies for TNBC is an unmet medical need. In this report, we developed a hybrid nanoparticles-based co-delivery platform containing both paclitaxel and verteporfin (PV-NPs) to target TNBC patient-derived xenograft (PDX) tumor and CSCs. MRI and IVIS imaging were performed on mice containing PDX tumors to assess tumor vascularity and selective accumulation of NPs. NF-kB, Wnt and YAP activities were measured by reporter assays. Mice bearing TNBC PDX tumor were treated with PV-NPs and controls; and tumors progression and CSC subpopulations were analyzed. Our MRI imaging studies indicated high vascularization of PDX tumors. IVIS imaging showed selective accumulation of NPs in PDX tumors. In comparison to control NPs and free-drug combination, PV-NPs significantly retarded tumor growth of TNBC PDX. PV-NPs simultaneously repressed NF-kB, Wnt and YAP that have been shown to be crucial for cancer growth, CSC development and tumorigenesis. In conclusion, NPs containing two clinically used drugs concurrently inhibited NF-kB, Wnt and YAP pathways and exhibited synergic effects on killing TNBC bulk tumor and CSCs. This combination nanotherapy evaluated with a PDX model may lead to an effective treatment of TNBC patients.

#2999

Hybrid complex between bispecific peptide and antibody for targeted cancer therapy.

Byeongjun Yu,1 Dobeen Hwang,2 Sangyong Jon,1 Junho Chung2. 1 _Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea;_ 2 _Seoul National University, Seoul, Republic of Korea_.

Antibodies have been used for cancer therapy for two decades, but they suffer from poor localization and penetration into solid tumors because of their large size. Peptides can be an alternative for treatment of solid tumors due to their small size. However, therapeutic use of peptides is limited because of their short plasma half-life. We present a new molecular platform based on a hybrid complex between bispecific peptide and antibody, designated as HyPEP-body, composed of bispecific peptides labeled with a hapten as a pharmacophore and an anti-hapten antibody. Two different peptides are joined together with linker and then, labeled with the hapten. The bispecific pharmacophore are bound to the anti-hapten antibody through antigen-antibody interaction. The pharmacophores which can be chemically optimized or replaced with other peptides are in charge of therapeutic function, while the antibody imparts long plasma half-life. As a model study, we constructed HyPEP-body that simultaneously binds extra domain B (EDB) and vascular endothelial growth factor (VEGF) with EDB binding peptide, VEGF binding peptide, cotinine and anti-cotinine antibody. Complexation with the antibody extended the plasma half-life of the bispecific pharmacophores. Furthermore, bispecific pharmacophores released from the antibodies distributed more widely in the tumor tissue than the antibodies. Finally, treatment of the HyPEP-body inhibited tumor growth in U87MG, human glioblastoma, xenograft mouse model. This versatile hybrid platform will enhance the value of peptide therapeutics and suggest an innovative solution to the development of bispecific antibodies that has suffered from productivity issues.

#3000

Impact of trastuzumab coating when prototyping immunoliposomes in breast cancer models: The more the merrier.

Anne Rodallec,1 Corentin Franco,2 Stéphane Robert,1 Guillaume Sicard,1 Sarah Giacometti,1 Ariel Savina,3 Fanny Bouquet,3 Bruno Lacarelle,1 Joseph Ciccolini,1 Philippe Poncelet,2 Raphaelle Fanciullino1. 1 _Aix-Marseille Univ., Marseille, France;_ 2 _Biocytex, Marseille, France;_ 3 _Institut Roche, Boulogne, France_.

Background: Improving drug delivery in oncology by developing antibodies targeted nanoparticles loaded with cytotoxics is a rising strategy in oncology. Determination of the optimal density of antibodies on nanoparticles surface remains an open question, mainly due to the difficulty in measuring accurately the actual level of coating. We have developed a stealth immunoliposome encapsulating docetaxel and harboring anti-Her2 trastuzumab. To better prototyping the nanoparticles, we have developed a method for the absolute quantitation of trastuzumab, so as to compare the performance of the immunoliposomes in terms of efficacy in breast cancer depending on the number of antibodies. Methods: Using flow cytometry we have developed a quick and quantitative assay to measure the amount of trastuzumab coated per docetaxel immunoliposome. Three batches of immunoliposomes exhibiting different densities of trastuzumab were synthesized using the standard thin-film method and a maleimide linker. Quality control was operated regarding size, docetaxel encapsulation and trastuzumab coating levels, including stability studies. In vitro and in vivo efficacy studies were performed on MDA-MB-453 used as a canonical model of Her-2+ human breast cancer cells. Results: The three batches of Immunoliposomes exhibited 330 ± 30 (batch1), 480 ± 110 (batch2) and 690 ± 80 (batch3) trastuzumab IgG molecules per liposome, respectively (p<0.01, One-Way Anova). In 2D apoptosis induction experiments, no difference was observed between free drugs and immunoliposomes, regardless of trastuzumab density. When shifting to 3D spheroids, higher antiproliferative efficacy was observed with batch2 as compared with other immunoliposomes or free drugs (+76%) or reference T-DM1 (+85%). Next, batch2 was further tested in mice bearing MDA-MB-453 xenografts. Immunoliposomes achieved a tumor reduction of 89 % when compared to free drugs and 66% as compared with T-DM1 (p<0.05, One-Way Anova). Conclusion: Our assay based on flow cytometry of submicron particles can accurately quantify the number of coated antibodies on single nanoparticles. In vitro studies demonstrated that maximal density of targeting agent on nanoparticles was not a prerequisite for maximal therapeutic effect. In vitro spheroids and in vivo studies in mice demonstrated higher efficacy of our immunoliposomes when compared to free trastuzumab docetaxel used in combo and antibody-drug conjugate T-DM1. Beyond the current project, this new quantitation method could be valuable when prototyping nanoparticles or conjugated drugs using monoclonal antibodies as targeting agent.

#3001

Isolation of large and diverse monoclonal antibody panels against difficult targets in oncology.

Joseph Rucker. _Integral Molecular, Philadelphia, PA_.

Although monoclonal antibodies (MAbs) are a well-established treatment approach in oncology, multipass membrane proteins are largely inaccessible as targets for MAb discovery due to their poor expression, membrane-dependent structure, small extracellular regions, and high sequence conservation between humans and rodents. We have developed a platform, MPS Antibody Discovery, that specifically addresses these challenges. We will present case studies of our successful isolation of large and diverse panels of MAbs, including functional (agonist/antagonist) MAbs, against oncology targets, including claudins, GLUT4, CXCR5, CCR7, and other chemokine receptors. Several key features of our MPS platform enabled our ability to isolate MAb panels against these targets. Antigen engineering allowed us to increase the expression levels of the target protein. Immunizing divergent animal species with a combination of DNA and Lipoparticles (high-concentration membrane proteins) allowed us to obtain high-titer immune responses against the native form of the membrane protein. Performing phage display with Lipoparticles allowed us to isolate diverse panels of MAbs with a success rate >95%. Our technology offers the potential for discovering MAbs against difficult targets in cancer, including neoantigens.

#3002

Bone-protective curcumin circulates as a pro-drug conjugate that is activated in bone by β-glucuronidase.

Andrew Kunihiro,1 Julia A. Brickey,1 jennifer B. Frye,1 Paula B. Luis,2 Claus Schneider,2 Janet L. Funk1. 1 _University of Arizona, Tucson, AZ;_ 2 _Vanderbilt University, Nasvhille, TN_.

Curcumin (CURC), a turmeric-derived dietary polyphenol, prevents osteoclast formation and bone resorption in murine models of arthritis, post-menopausal osteoporosis, and bone metastatic breast cancer despite being rapidly conjugated upon ingestion in both rodents and humans to form curcumin-glucuronide (G-CURC). However, recent studies from our laboratory demonstrate that aglycone CURC is enriched in bone relative to the circulation. Studies were therefore undertaken to compare the anti-resorptive effects of aglycone CURC vs G-CURC and to elucidate mechanisms responsible for enrichment of aglycone CURC in bone, including clinically relevant influences of age, sex, and reproductive status.

CURC dose-dependently inhibited RANKL-stimulated osteoclast formation in RAW264.7 cell cultures, while G-CURC was without effect. Likewise, CURC dose-dependently inhibited TGFβ-stimulated PTHrP secretion from MDA-MB-231 breast cancer cells, which readily form PTHrP-driven osteolytic bone metastases in vivo.

In wild type (WT) C57BL/6J mice, heparanase (HPSE) and β-glucuronidase (GUSB) were the only enzymes with reported deglucuronidation activity expressed in bone (Western). However, only GUSB was able to recognize G-CURC as a substrate, deconjugating G-CURC to form CURC (LC/MS). Consistent with these findings, tissue-specific levels of CURC were highest in bone (vs. heart, muscle, or kidney) and proportional to tissue specific GUSB enzyme activity.

To verify the role of GUSB in CURC bone enrichment, CURC metabolism and GUSB enzyme expression and activity were compared among mice with normal levels of GUSB (C57BL/6J) vs. partial (C3H/HeJ) or complete (mucopolysaccharidosis VII (mps/mps]) deficiencies in GUSB expression. As expected, partial (55% of WT) or complete deficiencies in GUSB expression significantly reduced CURC enrichment in bone.

While GUSB can be positively regulated by pharmacologic doses of androgens, neither male sex nor ovariectomy (OVX) altered GUSB activity (per protein) in bone relative to intact females, although a small but statistically significant increase in free curcumin was demonstrated in males (17%) and OVX females (10%). Age did not significantly change the enrichment of CURC in bone of 4 vs 28-week mice.

These findings suggest bio-inactive G-CURC acts as pro-drug that is converted to active CURC within bone, independent of sex, age or reproductive status, where it can inhibit the formation of bone resorbing osteoclasts or secretion of osteolytic PTHrP from breast cancer bone metastases.

#3003

Inhibiting the mitochondrial enzyme phosphatidylserine decarboxylase (PISD) reduces stemness and increases differentiation in acute myeloid leukemia (AML).

Mingjing Xu,1 Ayesh Seneviratne,1 Val A. Fajardo,2 Geethu E. Thomas,1 G. Wei Xu,1 Rose Hurren,1 S. Kim,1 Neil MacLean,1 Xiaoming Wang,1 Marcela Gronda,1 Danny Jeyaraju,1 Yulia Jitkova,1 David Sharon,1 Ahmed Aman,3 Rima Al-awar,3 Steven Chan,1 Mark D. Minden,1 Paul LeBlanc,2 Aaron D. Schimmer1. 1 _Princess Margaret Cancer Centre, Toronto, Ontario, Canada;_ 2 _Brock University, St. Catharines, Ontario, Canada;_ 3 _Ontario Institute for Cancer Research, Toronto, Ontario, Canada_.

Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by the accumulation of malignant myeloid cells that have arrested maturation. Most therapeutic regimens approved or under development are cytotoxics. An alternate, but less explored therapeutic approach, is to induce terminal differentiation of AML cells. Upon differentiation, AML cells cease to proliferate or die.

Phosphatidylserine decarboxylase (PISD) is a mitochondrial enzyme that converts phosphatidylserine (PS) to phosphatidylethanolamine (PE). Here, we explored the effects of inhibiting PISD on AML growth, stemness and differentiation.

Knockout of PISD by CRISPR reduced the growth and clonogenic growth of OCI-AML2 cells. The reported chemical PISD inhibitor, 7-chloro-N-(4-ethoxyphenyl)-4-quinolinamine (aka: MMV007285), reduced growth and viability of OCI-AML2 cells (IC50 = 4.741 μM) and TEX cells (IC50 = 4.868 μM). Using the 8227 primary AML cell culture model, we showed that inhibiting PISD induced cell death in the functionally defined stem cell fraction (CD34+CD38-). MMV007285 also preferentially inhibited the clonogenic growth of primary AML cells (n = 7) over normal hematopoietic cells (n= 3). Moreover, MMV007285 induced AML cell differentiation as evidenced by increased CD11b expression and staining for non-specific esterase.

Using high-performance thin layer chromatography (HPTLC), we found that inhibition of PISD with MMV007285 increased intracellular PS. To determine whether increased PS was functionally important, OCI-AML2 cells were treated with PS, resulting in reduced growth and clonogenic growth. Furthermore, PS supplementation targeted AML progenitor cells as it decreased engraftment of TEX cells in mice.

Mechanistically, inhibiting PISD induced differentiation and decreased stemness in AML by activating Toll-like receptor (TLR) signaling. Specifically, inhibiting PISD upregulated TLR4 and 8 expression and increased expression of cytokines downstream of TLR activation. We also showed that TLR activation was functionally important to induce AML differentiation.

Finally, we evaluated the effects of PISD inhibition in AML mouse models. MMV007285 (300 mg/kg/5 of 7 days orally for 10 days) decreased the growth of OCI-AML2 cells in SCID mice. Moreover, MMV007285 (150 mg/kg/5 of 7 days orally for 5 weeks) impeded the leukemic engraftment of primary AML cell in NOD/SCID mice without toxicity. Using secondary transplants, we showed that MMV007285 also targeted the leukemic stem cells.

Taken together, inhibition of PISD altered phospholipid metabolism, inhibited growth and stemness, and increased differentiation in AML cells. Our findings reveal a previously undescribed link between mitochondrial phospholipid metabolism and AML stemness and differentiation, highlighting a potential new therapeutic strategy for AML.

#3004

**Anticancer effects and associated molecular changes of** Carica papaya **against prostate cancer.**

Surya Pratap Singh,1 Sivapar V. Mathan,2 Arpit Dheeraj,2 Dhanir Tailor,2 Rana P. Singh,2 Arbind Acharya1. 1 _Banaras Hindu University, Banaras, India;_ 2 _Jawaharlal Nehru University, Delhi, India_.

Carica papaya (papaya), from the family of Caricaceae, is a perennial plant originating from the Southern part of Mexico. Carica papaya leaf extract (CPE) has been traditionally used to treat various diseases, including infectious diseases and cancer. However, the anticancer effects and molecular mechanism of CPE are elusive. The aim of our study is to examine the anticancer effects of aqueous leaf extract of Carica papaya against prostate cancer (PCa). We investigated the effect of CPE on LNCaP, DU145 and PC-3 PCa cells. CPE treatment (5, 10, 25µl/ml) significantly reduced the cell proliferation and induced cell death in PCa cells. Furthermore, we found that CPE induced G1, S and G1 as well as G2/M phase cell cycle arrest in LNCaP, DU145, and PC-3 cells, respectively. At molecular level, the cell cycle arrest was associated with decreased expression of CDK 4, cyclin D1, cyclin B1, and PCNA. CPE induced cell death was associatedwith depolarization of mitochondrial membrane potential, increase in ratio of Bax/Bcl2, cleavage of caspase-3 and cleaved -Poly(ADP-ribose) polymerase (PARP). CPE treatment also reduced mitochondrial fission and induced mitochondrial fusion by reducing the level of Drp1 protein. Further, we observed that CPE increased the expression of E-cadherin and decreased the expression of N-cadherin and vimentin. We checked the CPE toxicity in C57BL/6 male mice. We found that oral administration of CPE(0.25%, 0.5% and 1% v/v) in drinking water did not show any significant changes in body weight, water consumption and food intake as compared to control group. Collectively, CPE inhibited cell proliferation, induced cell death via apoptosis, mitochondrial fusion, epithelial marker and reduced mesenchymal markers.In vivo toxicity study with CPE treatment via drinking water was found to be non-toxic. These findings suggest that CPE has potential as anticancer therapeutic option for prevention and treatment of prostate cancer.

#3005

High density lipoprotein mimics inhibit prostate cancer exosome-mediated communication with myeloid cells.

Stephen E. Henrich, Kaylin M. McMahon, Michael P. Plebanek, C. Shad Thaxton. _Northwestern University, Chicago, IL_.

Introduction: Exosomes are 30-150 nm membrane-bound vesicles that mediate intercellular communication and are secreted in abundance by neoplastic cells. Tumor-derived exosomes are capable of transmitting pro-malignant signals to target cells at local or distant sites to enhance tumor invasiveness and promote metastasis. A sub-set of this pro-malignant intercellular communication is dependent upon target cell cholesterol homeostasis. Here, we show that prostate cancer exosome communication with myeloid cells can be inhibited by treatment with high density lipoprotein mimetic nanoparticles (HDL NPs), which reduce cholesterol in myeloid cells in a targeted fashion.

Methods: Exosomes were isolated by ultracentrifugation of conditioned media from enzalutamide resistant CWR-R1 cells and used for all experiments. In vitro assays were conducted using murine bone marrow macrophages obtained from culture of total murine bone marrow in M-CSF for 7 days. Confocal microscopy and flow cytometry were used for in vitro and in vivo uptake assays. Osteoclastogenesis assays were performed using a commercially available TRAP staining kit (Sigma-Aldrich). NF-kB signaling experiments were performed using a reporter human monocyte cell line (THP1-Dual). HDL NPs were synthesized using 5 nm gold nanoparticle templates, apolipoprotein A-1, and phospholipids.

Results: HDL NPs were found to inhibit the cellular uptake of prostate cancer exosomes in mouse bone marrow macrophages in vitro. Furthermore, HDL NPs inhibited exosome-induced osteoclastogenesis and exosome-induced monocyte NF-kB signaling. Finally, HDL NP-mediated inhibition of exosome communication was found to be dependent upon scavenger receptor type B-1 (SR-B1). SR-B1 was shown to be expressed ubiquitously in mouse bone marrow macrophages; and HDL NPs were unable to inhibit exosome communication in bone marrow macrophages derived from SR-B1-/- mouse bone marrow.

Conclusion: These data demonstrate that HDL NPs inhibit prostate cancer exosome communication with murine myeloid cells, as evidenced by cellular uptake, osteoclastogenesis, and NF-kB signaling. Moreover, SR-B1 is required for HDL NP inhibition of PCa exosome communication. In sum, these results indicate that target cell cholesterol homeostasis may be important for exosome-mediated signaling in prostate cancer, particularly in immune cells and in the bone microenvironment, and that HDL NPs are potent inhibitors of PCa exosome-mediated signaling.

#3006

A novel strategy of targeting opioidergic receptors to potentially reduce human breast cancer cell proliferation, colony formation, invasion, and migration.

Sengottuvelan Murugan, Vanisha Patel, Dipak K. Sarkar. _Rutgers Univ., New Brunswick, NJ_.

Breast cancer is the second most prevalent cancer after lung cancer among American women. The efficacy of current antitumor therapies is still quite limited. Endogenous opioid peptides, such as endorphin, which is involved in body stress controlling mechanisms, have been shown to enhance immune function and prevent cancer growth and progression in various animal models of cancers. Therefore, targeting the endogenous opioid peptides receptors in the regulation of cancer chemoprevention may offer new promise for the design of therapeutic strategies. Our previous studies have shown differential actions of mu-opioid receptor (MOR) and delta opioid receptor (DOR) on various cell types. In this study we tested the effects of a MOR antagonist naltrexone (NTX) and a DOR agonist (D-Ala2-,N-Me-Phe4,Gly-ol Enkephalin; DPDPE) on proliferation and invasion of human breast cancer cells of different molecular subtype such as T47D (Luminal A), MDA-MB-231 (triple negative, Claudin-Low) and MDA-MB- 468 (triple negative, basal A) in cultures. We used cytotoxicity, clonogenic, cell migration and cell invasion assays to identify effects of these drugs on cancer cells. We found that both NTX and DPDPE have significant cytotoxic effects on these breast cancer cells. Our work from clonogenic, cell migration and cell invasion assays showed that both NTX and DPDPE inhibit the formation of colony, migration and invasion of cancer cells in a concentration-dependent manner. These data suggest that naltrexone and DPDPE have anticancer efficacy in subtypes of human breast cancer cells. The anticancer effects varied depending on the concentration of the drugs and the subtype of breast cancer cells. (Supported by NIH grant R01 CA20863201).

#3007

Validation of an NGS panel assay for detection of hotspot cancer somatic mutations.

Wenzhi Li, Henry She, Xiaohua Tian, Margaret Neja, Adrian Harris, Yuhua Li. _Omega Bioservices, Norcross, GA_.

Background

The gene tests to predict whether cancers respond to specific targeted therapies or prediction of prognosis has quickly increased over the last decade. Next-generation sequencing (NGS) has become a powerful tool for high-throughput and sensitive gene mutation testing, which is required for development of targeted therapies, clinical trials, and further research. The aim of this study was to validate the AmpliSeq for Illumina Cancer Hotspot Panel v2 on the Illumina MiSeq platform and simultaneously analyze 2800 somatic mutations across the hotspot regions of 50 genes, including SNP and small INDEL, with known associations to cancer, as identified in the Catalogue of Somatic Mutations in Cancer (COSMIC) database. This panel enables the study of the genes associated with different cancer types, including the cancers of the lung, colon, breast, thyroid, oral, bladder, prostate, liver, kidney, ovarian and cervix, etc.

Methods and findings

We validated this assay using the genomic DNA extracted from human paraffin-embedded (FFPE) samples and peripheral blood with negative control DNA (NA12878), positive control DNA (HD701), blank control as well as the global proficiency testing samples. Following two lab scientists parallel NGS library preparation, sequenced on Miseq platform (2x150 PE) and analyzed using a pipeline centered on Illumina Basespace, the somatic mutations were called at an average number of reads coverage >2500X, variant frequency cutoff 1%. An integrated test report can be generated within five working days. Panel sequencing analysis showed that the mutations are 100% concordance with known mutations and consistent in four runs, between lab personal performances, different input DNA concentrations (1ng-100ng), etc. Briefly, 25 mutations were detected in the FFPE samples and 20 mutations detected in proficiency testing samples. The variant types, HGVS ID and frequency are 100% match with its standard results which are a prove of precision and sensitivity.

Conclusions

A sensitive, high-throughput, pan-cancer mutation panel for NGS analysis of cancer hot-spot mutations in 50 genes was highly repeatable and reproducible and validated for routine application in a gene testing lab.

#3008

PSMA glycosylation and aggressive prostate cancer progression.

Tanya C. Burch,1 Stephen S. Mackay,1 Ian O. Oduor,2 Joseph J. Otto,1 Raymond S. Lance,3 Dean A. Troyer,1 Oliver John Semmes,1 Julius O. Nyalwidhe1. 1 _Eastern Virginia Medical School, Norfolk, VA;_ 2 _Old Dominion University, Norfolk, VA;_ 3 _Sacred Heart Hospital, Spokane, WA_.

Introduction: Prostate cancer has a heterogeneous spectrum of clinical outcomes with phenotypes ranging from indolent asymptomatic cases to very aggressive metastatic and lethal forms. Our long term objective is to identify molecules and characterize mechanisms for PCa progression to aggressive lethal disease. PSMA, a membrane bound glycoprotein is significantly over-expressed in more aggressive and metastatic prostate carcinomas with a negative correlation with patient prognosis. The role of its glycans in disease progression and in the development of biomarkers for the disease and potential therapeutics are currently unknown. Aberrant glycosylation of glycoproteins has been observed in many malignancies, and these changes influence disease progression. Our rationale is that differential glycosylation of PSMA correlates with PCa aggressive disease progression. In this study, we characterize N-linked glycosylation profiles in disease stratified PCa cells and prostate proximal fluids show differential expression of specific PSMA conjugated glycan structures that correlates with aggressive phenotypes.

Methods/Results: PSMA was isolation from PCa cell lysates and post-DRE urines by column based affinity chromatography followed by SDS-PAGE and Western Blot and Coomassie staining. The fractionated PSMA protein band was excised and processed for proteomic and glycomic analysis. Isolated PSMA was trypsin digested and treated with PNGaseF and other glycosidases to cleave of N-linked glycans from the peptides. Isolated glycans were subjected to permethylation before MALDI-TOF-MS and high resolution, high mass accuracy and high sensitivity LC/MS/MS on an Orbitrap Fusion Lumos Tribrid MS instrument with multiple fragmentation capabilities (CID, HCD, ETD and EThcD) for glycan identification and quantitation. Validation of differential glycan expression was further performed by targeted glycan based MS and lectin based biochemical technologies.

Conclusions: Our preliminary studies show that there is differential expression of PSMA N-linked glycans with disease severity in prostate cancer cells and post DRE urine samples. Further analyses are currently underway using an extensive cohort of post DRE urine samples and additional studies are focusing on uncovering the mechanisms that are modulated by PSMA glycosylation that are responsible for PCa disease progression. These could assist in the development of novel diagnostic and therapeutic strategies for the disease. 

### Drug Resistance 4

#3009

FGFR1 overexpression ER positive breast cancer cells confer the cells resistant to Palbociclib via upregulation of RTK-ER signaling.

Yujie Shi,1 Zhikun Ma,2 Yudan Wu,2 Amanda B. Parris,2 Qiong Cheng,1 Lingfei Kong,1 Xiaohe Yang2. 1 _Henan Provincial People's Hospital, Zhengzhou, China;_ 2 _North Carolina Central University, Kannapolis, NC_.

Palbociclib is the first CDK4/6 inhibitor approved by the FDA for treating ER+/HER2- breast cancer. Palbociclib resistance is emerging as a clinical challenge. Identification of the factors that determine the sensitivity to Palbociclib is of pivotal significance. Fibroblast Growth Factor Receptor (FGFR) overexpression is frequently detected in breast cancer, which has been associated with poor prognosis. To investigate the impact of FGFR overexpression on Palbociclib resistance, we first established stable sublines of MCF7 and T47D cells that overexpress FGFR1, and examined their responses to Palbociclib. Data from MTT and clonogenic assays demonstrated that overexpression of FGFR1 rendered ER+ MCF7 and T47D cells resistant to Palbociclib significantly. Cell cycle analysis showed that the percentage of cells in S phase in Palbociclib treated MCF7/FGFR1 and T47D/FGFR1 cells was significantly higher than that of the control. FGFR overexpression also significantly attenuated Palbociclib-associated inhibition of cancer cell invasion. We further found that Palbociclib-induced inhibition of cancer cell stemness, as indicated by colony formation in soft agar and mammosphere assays, was significantly reduced in MCF7/FGFR1 and T47D/FGFR1 cells, as compared to control. The data indicate that FGFR1 overexpression in ER+ breast cancer cells are resistant to Palbociclib. We next examined the signaling of the key pathways in the paired cell lines treated with Palbociclib. We found that Palbociclib-induced inhibition of phospho-Rb, E2F, cyclin D3, cyclin B1, cdc-2, c-myc, and cdc-25 was significantly attenuated in MCF-7/FGFR1 and T47D/FGFR1 cells. The protein levels of p-ERK, p-AKT, and p-p38 in MCF-7/FGFR1 and T47D/FGFR1 cells were significantly higher than the control cells with/without Palbociclib treatment. The results also showed that ER alpha phosphorylation, estrogen response element (ERE)-luciferase activity and mRNA levels of ER target genes, including PS2, c-myc, E2F, FGF2 and FGFR2, in MCF-7/FGFR1 and T47D/FGFR1 cells were maintained at higher levels post Palbociclib, as compared to control. We then treated control and FGFR1 overexpressing MCF-7 and T47D cells with Palbociclib in combination with FGFR inhibitor, AZD 4547. The combined drug treatment resulted in remarkably synergistic effect on MCF-7/FGFR1 and T47D/FGFR1 cells, as indicated by MTT and clonogenic assays. Key markers in cell cycle regulation, RTK signaling and ER pathway were significantly inhibited in the cells treated with both Palbociclib and AZD 4547. Taken together, our results demonstrated that FGFR1 overexpression is a critical factor affecting Palbociclib sensitivity. The underlying mechanisms involve FGFR1 overexpression-associated RTK and ER signaling. Combination of Palbociclib with FGFR targeting agents may overcome FGFR-associated Palbociclib resistance.

#3010

Osteosarcoma cell lines display both shared and unique vulnerabilities to 140 targeted small molecules with RNA-seq revealing putative mechanisms.

Elliot J. Kahen, Darcy Welch, Jamie Teer, Andrew S. Brohl, Sean J. Yoder, Yonghong Zhang, Ling Cen, Damon Reed. _Moffitt Cancer Center, Tampa, FL_.

BACKGROUND: Osteosarcoma is the most common bone sarcoma in children, adolescents, and young adults. Osteosarcoma primarily arises through tumor suppressor inactivation, most typically due to TP53 attenuation mechanisms. Increasingly additional oncogenic and tumor suppressor pathways are being recognized in subsets of osteosarcoma. We hypothesized that novel insights into mechanisms of sensitivity and resistance for emerging targeted therapies could be elucidated by combining activity and genomic information across cell line models of osteosarcoma.

METHODS: In this study we evaluated 6 established osteosarcoma cell lines (U2-OS, MG-63, OS252, SAOS-2, 143B, MNNG) representing several TP53 and other tumor suppressor inactivation mechanisms with human osteoblasts as a control. We have obtained RNA-seq profiles detailing genomic variants, gene fusions, and expression of both protein-coding and miRNA genes for these cell lines. In addition to genomic changes, we evaluated 140 chemical probes and candidate drug molecules across these cell lines.

RESULTS: RNAseq identified TP53 changes in 5 of 6 cell lines with OS252, SAOS2 and MG-63 having intron 1 translocations and 143B and MNNG sharing a TP53 gain-of-function mutation. Variant calling from the whole exome sequencing data revealed additional putatively oncogenic mutations. Additionally, we observed differential gene expression patterns compared to control samples in genes associated with genomic stability, epigenetic regulation, and transcriptional activity. Differential sensitivities to several classes of small molecules were seen across the cell lines and were associated with genomic changes that warrant additional investigation such as: RAD51 pathway genomic findings associated with sensitivity to RS-1, disulfuram activity varying according to aldehyde dehydrogenase activity, and variable response to inhibitors of ATR and specific CDKs that are associated with the expression of their respective targets. Additional pathways of interest that demonstrate differential response to small molecules include histone/protein deacetylation, telomerase activity, protein translation, and others. Unique vulnerabilities associated with tumor suppressor or oncogenic changes specific to an individual cell line will be presented.

CONCLUSIONS: By characterizing response to small molecules in the context of gene expression, underlying pathway vulnerabilities may be determined for further testing suggesting potential biomarkers for basket trials. Other agents with shared sensitivities to agents across cell lines may be translated in a different manner.

#3011

Overcoming endocrine therapy resistance by drugs targeting YBX1 activation pathway in breast cancer.

Tomohiro Shibata,1 Kosuke Watari,1 Akihiko Kawahara,2 Tomoya Sudo,3 Yuichi Murakami,4 Eriko Tokunaga,5 Nami Yamashita,1 Eiji Oki,1 Yoshihiko Maehara,6 Jun Akiba,2 Yoshito Akagi,3 Maki Tanaka,7 Michihiko Kuwano,4 Mayumi Ono1. 1 _Kyushu University, Fukuoka, Japan;_ 2 _Kurume University Hospital, Kurume, Japan;_ 3 _Kurume University School of Medicine, Kurume, Japan;_ 4 _St. Mary's Institute of Health Sciences, Fukuoka, Japan;_ 5 _National Hospital Organization Kyushu Cancer Center, Fukuoka, Japan;_ 6 _Kyushu Central Hospital, Fukuoka, Japan;_ 7 _Kurume General Hospital, Kurume, Japan_.

[Background] Endocrine therapies effectively improve the outcomes of patients with estrogen receptor alpha (ERα)-positive breast cancer. However, the emergence of drug-resistant tumors is a serious challenge. Our previous studies have demonstrated that YBX1 plays pivotal roles in acquisition of endocrine therapy resistance through downregulation of ERα and upregulation of HER2/ErbB2 in breast cancer patients (Shibata et al., Cancer Res., 2017). Furthermore, many laboratories have consistently demonstrated that YBX1 expression is correlated with poor outcomes of breast cancer patients, but the mechanism underlying why YBX1 expression leads to a poor outcome has yet to be revealed. Herein, our present findings demonstrate the critical role of YBX1, and also novel approach to overcome resistance to endocrine therapy.

[Methods] We searched a TCGA database for top 500 genes that are positively or negatively correlated with YBX1 and with ESR1 in breast cancer patients. Furthermore, we established fulvestrant resistant breast cancer cell lines in which AKT/mTORC1/S6K signaling pathway is activated.

[Results] Based on our finding, YBX1 expression is consistently correlated with reduced expression of ERα and its effector genes, conferring breast cancer cells resistance to endocrine therapy. [1] The enhanced expression of YBX1 is negatively correlated with ESR1 and its effector genes in tumors, and also with poor outcomes in breast cancer patients (TCGA and patients in our hospital). [2] Enhanced expression of YBX1 and pYBX1 is closely correlated with recurrence and resistance to endocrine therapy in patients. [3] Breast cancer cells resistant to fulvestrant or tamoxifen showed markedly enhanced expression of pYBX1 and treatment with mTORC1 inhibitors almost completely overcame above resistance in vitro and in vivo. [4] Constitutive activation of YBX1 by the mutant construct induced resistance to fulvestrant, indicating that YBX1 phosphorylation is crucial for the acquired drug resistance. Enhanced expression of YBX1 and also pYBX1 is thus closely associated with endocrine therapy resistance, and also with malignant progression in breast cancer.

[Conclusion] Based on both basic and clinical findings, we will present our novel concept that activation of the oncogenic transcriptional activity by YBX1 phosphorylation is crucial for acquired resistance to endocrine therapy and also poor outcomes in breast cancer. The YBX1 activation by PI3K/AKT/mTOR and RAF/MEK/ERK signaling pathways could be useful candidates for development of overcoming drugs. We will discuss overcoming effects of mTORC1 inhibitors.

#3012

Single-cell transcriptomic characterization of luminal breast cancer cell lines with acquired resistance to the CDK4/6 inhibitor palbociclib.

Matteo Benelli,1 Giulia Boccalini,1 Dario Romagnoli,1 Martina Bonechi,1 Chiara Biagioni,1 Rachel Schiff,2 Carmine De Angelis,2 Angelo Di Leo,1 Ilenia Migliaccio,1 Luca Malorni1. 1 _Azienda USL Toscana Centro, Hospital of Prato, Prato, Italy;_ 2 _Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, TX_.

CDK4/6 inhibitors, such as palbociclib, target the cyclin-dependent kinases 4 and 6, essential for cell cycle regulation. CDK4/6 inhibitors are established treatment options in patients with endocrine receptor positive, HER2 negative metastatic breast cancer (BC).

We recently conducted a transcriptomic study of seven palbociclib resistant luminal BC cell lines, demonstrating a common transcriptional signature of resistance to palbociclib, including the over-expression of CCNE1 and under-expression of RB1. Genomic analysis based on ddPCR and whole exome sequencing (WES) demonstrated CCNE1 over-expression due to gene amplification in only two cell lines, and RB1 under-expression due to gene deletion in 3 out of 7 cell lines. This prompts the hypothesis that more complex molecular mechanisms of CCNE1 over-expression / RB1 under-expression may exist.

To improve the characterization of the molecular mechanisms underlying the resistance to palbociclib, we performed single-cell transcriptomic profiling (single-cell RNA-seq, scRNA-seq) of seven BC cell line resistant to palbociclib and their sensitive counterparts. Cells were isolated using the Bio-Rad ddSEQ single cell isolator and sequenced in order to obtain about 100,000 reads per cell. A total of 10,557 cells with at least 2,000 genes read per cell were selected for downstream analysis, corresponding to 5,116 palbociclib sensitive cells and 5,441 palbociclib resistant cells.

Considering the 5,000 most variable genes, dimensionality reduction analysis based on t-distributed stochastic neighbor embedding (tSNE) clearly segregated cells depending on their cell type, as expected based on previously generated bulk RNA-seq data. Using previously identified top cell-type specific differentially expressed genes between resistant and sensitive cell lines (absolute log2(fold-change) > 0.5), dimensionality reduction analysis correctly classified 98.2% of cells based on their sensitivity/resistant status, demonstrating the quality of our scRNA-seq data. Interestingly, we observed a small fraction (<1.5%) of misclassified sensitive cells showing resistant-like phenotypes. Co-expression analysis suggests modulation of key gene expression programs between sensitive and resistant cells, including E2F signaling. Further analyses to validate these data are ongoing and the results will be presented during the meeting.

This study represents, to our knowledge, the first attempt to generate a single-cell transcriptomic profiling of a large panel of luminal BC cell lines resistant to palbociclib. Results from this study might lead to the identification of new biomarkers of de-novo and acquired resistance to palbociclib, which would assist to better personalize treatment of patients with endocrine receptor positive BC.

#3013

Acquired resistance to immune checkpoint inhibition by melanoma phenotypic transformation.

Arnav Mehta,1 David Liu,2 Alvin Shi,3 Dennie T. Frederick,1 Li-Lun Ho,3 Ana B. Larque,1 Benchun Miao,1 Rumya S. Raghavan,1 Tatyana Sharova,1 John H. Shin,1 Nicola Rinaldi,3 Ivan A. Chebib,1 Manolis Kellis,3 Eliezer Van Allen,2 Nir Hacohen,1 Keith T. Flaherty,1 Genevieve M. Boland,1 Ryan J. Sullivan1. 1 _Massachusetts General Hospital, Boston, MA;_ 2 _Dana Farber Cancer Institute, Boston, MA;_ 3 _Massachusetts Institute of Technology, Boston, MA_.

Immune checkpoint blockade (ICB) has revolutionized the treatment of metastatic melanoma, however, cases of resistance have now begun to emerge. We report here a case of acquired resistance by melanoma phenotypic transformation in a patient treated on a Phase 1b/2 clinical trial (NCT02437136) with anti-PD1 (Pembrolizumab) and a histone deacetylase (HDAC) inhibitor (Entinostat). The patient presented to MGH with metastatic melanoma confirmed by core-needle biopsy of a liver lesion. A PET/CT scan one month after starting the trial revealed interval regression of liver and bone lesions, however, four months later the patient presented with disease progression. A tumor debulked from the L2 vertebral body at that time revealed the presence of a pleomorphic rhabdomyosarcoma. The patient was started on anti-CTLA4 (Ipilumimab) with further disease progression, and surgical specimens thereafter revealed either melanoma or rhabdomyosarcoma. Tumor tissue was evaluated for markers by immunohistochemistry. All tumors (5 specimens in total) were subjected to whole exosome sequencing (WES), RNA-sequencing (RNAseq) and epigenomic sequencing (ATACseq). WES revealed 844 mutations were shared between all tumors, suggesting a common ancestor including driver mutations in NRAS (G13D), NF1 (E1121* and W1314*) and the TERT2 promoter, among others. The pre-treatment melanoma biopsy had positive staining for S100 and MART-1, nuclear staining patterns for SOX10 and MITF, and were BRAF V600E negative. Importantly, 80% of the tumor cells had strong staining for PD-L1 expression and CD8 CTL infiltration was associated with 25% of the tumor. The post-treatment rhabdomyosarcoma biopsy was notable for diffusely positive staining for Desmin and MyoD1, and multifocal positivity for myogenin. The tumor stained negative for S100 and SOX10, and there was no PD-L1 staining and less than 5% tumor CD8 CTL infiltration. Gene expression analysis revealed distinct expression patterns between melanoma and rhabdomyosarcoma samples, with 150 differentially expressed genes implicated in processes such as antigen presentation, T cell activation, cell death and protein metabolism. In addition, each tumor was found to have a unique immune cell composition and T-cell receptor repertoire, suggesting an interplay between phenotypic transformation and immune response. Results from epigenomic sequencing are pending, but may shed light on the mechanisms of transformation leading to resistance. These data therefore elucidate a previously undescribed mechanism of acquired resistance to ICB through melanoma phenotypic transformation to a rhabdomyosarcoma. Whereas these tumors were genetically similar, they expressed distinct gene expression patterns suggestive of underlying epigenomic differences in the setting of an HDAC inhibitor.

#3014

Gene signatures identify quinolate phosphoribosyltransferase as a key mediator of acquired resistance to Panobinostat and Bortezomib in glioma, and NAD+ biosynthesis as a targetable pathway to reverse treatment resistance.

Esther P. Jane, Daniel R. Premkumar, Sameer Agnihotri, Max Myers, Ansuman Chattopadhyay, D. Lansing Taylor, Mark Schurdak, Andrew Stern, Ian F. Pollack. _Univ. of Pittsburgh, Pittsburgh, PA_.

Resistance of tumor cells to the induction of apoptosis is an important reason for the failure of anticancer treatments in patients with gliomas. Several factors working in concert have been implicated as sources of this treatment resistance; therefore, innovative therapeutic approaches that conspire to attack key tumor vulnerabilities are needed. HDAC and proteasome inhibitors are two classes of agents that have shown some benefit in the clinic, but patients often rapidly manifest intrinsic or acquired resistance mechanisms that limits their individual efficacy. We demonstrate that the combination of the HDAC inhibitor panobinostat and the proteasome inhibitor bortezomib synergistically induces apoptosis of human glioma cell lines. However, acquired resistance is observed even with this initially effective combination. To examine the mechanism of this resistance, we performed RNA sequencing and pharmacological screening of resistant compared to sensitive cells. Based on these studies, we present evidence that quinolinic acid phosphoribosyltransferase (QPRT), an enzyme catalyzes a rate determining step in de novo NAD+ biosynthesis, provides a critical adaptive survival mechanism that allows cancer cells to evade an initially effective therapeutic combination. We identified 1004 genes that had a significant change between panobinostat and bortezomib- resistant cells versus inhibitor naïve control cells. Furthermore, pathway analysis revealed that the experimental regimen significantly altered metabolic pathways. By silencing QPRT (using siRNA) we demonstrated that we can overcome resistance, thus suggesting QPRT as a crucial crossroad for cancer cell survival and as a new anticancer target. We also showed that treatment of panobinostat and bortezomib resistant cells with FK866, niraparib, selisistat, epacadostat, gemcitabine, 5-fluoruracil, and methotrexate significantly increased cell death highlighting the importance of NAD+ and folate pathway inhibitors in resensitizing the resistant cells. Together, targeting QPRT or NAD+ consuming enzymes hold promise for eliminating recurrent disease in glioma.

#3015

**IMiDs and BET inhibitors target distinct pathways of** MYC **dysregulation by super-enhancers in multiple myeloma.**

Daniel L. Riggs,1 Camille Herzog,2 Victoria M. Garbitt,1 Niamh Keane,3 Courtney J. Hillukka,1 Zachary J. Hammond,1 Julia E. Wiedmeier,1 Seth J. Welsh,1 Shulan Tian,4 Huihuang Yan,4 Ranjan Maity,5 Nizar Bahlis,5 Paola Neri,5 W Michael Kuehl,6 Marta Chesi,1 P Leif Bergsagel1. 1 _Mayo Clinic Arizona, Scottsdale, AZ;_ 2 _Harvard School of Dental Medicine, Boston, MA;_ 3 _University of Ireland, Galway, Ireland;_ 4 _Mayo Clinic Rochester, Rochester, MN;_ 5 _University of Calgary, Calgary, Alberta, Canada;_ 6 _NCI, Bethesda, MD_.

MYC dysregulation, the most common genetic aberration in multiple myeloma, is frequently due to the translocation of super-enhancers to the MYC locus. Several drugs target proteins that are enriched at many of these enhancers including BRD4 (BET inhibitors, BETi) and Ikaros (IMiDs), although their mechanism of action remains poorly understood. Here we present a characterization of the responses to these drugs in a collection of over sixty myeloma cell lines having a diversity of MYC rearrangements. We found that the anti-proliferative effects of these drugs significantly correlated with changes in MYC protein levels, consistent with both drugs targeting MYC expression. Despite this common target, there was no statistically significant correlation between the individual BETi and IMiD responses, suggesting that they act through different mechanisms. Of those lines having extremes of sensitivity or resistance, there were two major groups (BETiS/IMiDS and BETiS/IMiDR), a smaller group of four lines resistant to both drugs individually (BETiR/IMiDR) and only one line was BETiR/IMiDS. In the BETiR/IMiDR group, resistance to BETi was mediated by a BRD4-independent mechanism as BRD4 was efficiently released from the MYC-associated enhancers. In all three of BETiR/IMiDR cell lines that we examined, treatment with BETi and IMiD together abolished proliferation and down-regulated MYC, consistent with parallel BRD4- and Ikaros-dependent pathways driving MYC expression. These resistant lines all expressed high levels of the transcription factor ETV4 and knocking out its gene sensitized a line to each drug individually. Thus ETV4 appears to be necessary for the parallel pathways driving MYC expression. There were nine lines in the BETiS/IMiDR group. Sensitivity to BETi in these lines could be explained by either low ETV4 expression or by BETi repressing Ikaros levels (which was only observed in BETiS lines, suggesting that BRD4 drives IKZF1 expression in these lines). Thus, in ETV4-containing lines, BETi sensitivity is due to the simultaneously targeting of the BRD4- and Ikaros-dependent pathways. IMiD resistance likely was due to several reasons. In one cell line, OCIMY5, IMiD had little effect on Ikaros levels, likely due to the previously reported low levels of Cereblon. Seven of the eight remaining lines expressed high levels of either ETV4, or the other potential super-enhancer binding factors IRF4 or RUNX1. In the eight BETiS/IMiDS cell lines, IMiD strongly reduced both Ikaros and Aiolos protein levels, which likely caused IMiD sensitivity. As with the BETiS lines described above, the lines in this group either lacked ETV4 or BETi repressed Ikaros levels. In conclusion, by examining drug response in a collection of genetically annotated myeloma cell lines we have been able to identify factors that contribute the broad range of responses to BETi and IMiDs in myeloma cells.

#3016

Dicer overexpression contributes to chemoresistance in colorectal cancer via up-regulating a set of miRNAs.

Li Jyuan Lin, Liang-Yi Hung. _National Cheng Kung University, Tainan, Taiwan_.

Chemotherapy has a good success rate in the treatment of colorectal cancer; however, recurrence of colorectal cancer is still frequent due to acquired resistance. Aurora-A, a cell cycle-regulated kinase, plays an important role in colorectal cancer development and drug resistance. Dicer, one of the key enzymes of the microRNA (miRNA) biogenesis pathway, may be involved in chemoresistance through regulating the expression of miRNAs. According to the TCGA database, the expression levels of Dicer and Aurora-A were increased in colorectal cancer, which may contribute to chemoresistance. Our results showed that the protein levels of Dicer and Aurora-A are increased in oxaliplatin resistant cell lines. Knocked-down expression of Dicer or Aurora-A can enhance drug sensitivity in oxaliplatin-resistant cells; in contrast, overexpression of Dicer or Aurora-A increased drug resistance in parental drug sensitive cells. By next-generation sequencing, we found that the expression of a set of miRNAs is increased in oxaliplatin resistant cells. Dicer overexpression enhances the expression of those miRNAs in drug sensitive parental cells. Clinical evaluation further confirmed the increased expression of those miRNA in the plasma of CRC patients with a poor response to oxaliplatin. Our results suggest that those miRNAs may act as biomarkers to predict oxaliplatin response in colorectal cancer by detecting their plasma miRNAs. The molecular mechanism of those miRNAs in regulating the drug response is currently under our investigation.

#3017

Role of long noncoding RNA H19 in driving enzalutamide resistant neuroendocrine prostate cancer.

Neha Singh, Virginie Olive, Ritu Pandey, Jin Song, Jeremiah Bearrs, Sathish K. Padi, Koichi Okumura, Andrew S. Kraft, Ritu Pandey. _University of Arizona, Tucson, AZ_.

INTRODUCTION: Approximately one-fourth of the prostate cancer (PCa) patients relapsing androgen deprivation therapy (ADT) have been shown to develop neuroendocrine prostate cancer (NEPC). One of the primary mechanisms attributed to this resistance is the lineage switching of epithelial cells to neuroendocrine (NE) phenotype making them independent of androgen receptor (AR) pathways for survival. Molecular basis essential to this lineage switch requires further elucidation. Thus, we aim to understand key mechanistic pathways that drive the development and maintenance of NEPC, and to investigate if the inhibition of pathway can re-sensitize to ADT in NEPC.

METHODS: Bioinformatics analysis was performed with existing databases to compare prostate adenocarcinoma (PCA) vs NEPC. Molecular features of NEPC have been shown to depict RB1 loss in 70-90% and TP53 loss in 56-67% of the cases. Thus, we established prostate organoid cultures from P53fl/fl/Rbfl/fl mouse and infected them with Cre-lentivirus for combined loss of TP53/Rb1. Various PCA and NEPC cell lines and patient derived organoids were used to study the role of the long non-coding RNA H19 in NEPC. Organoid growth were assessed with Incucyte and invasive capacity was measured by Matrigel invasion assay.

RESULTS: Bioinformatic analysis of exisiting human PCa data sets demonstrate that H19 is one of the most highly expressed genes in NEPC and parallels the expression of NE markers. Indeed, we confirmed that H19 expression was high in various NEPC cell lines and NE patient derived organoids. H19 was also found to be markedly induced in mouse prostate organoids and LNCaP cells after TP53/Rb1 loss. Importantly, overexpression of H19 induced NE genes while suppressing the genes involved in AR signaling in LNCaP and primary PCa patient derived organoids and inversely, knockdown (KD) of H19 in LNCaP and mouse prostate organoids with TP53/Rb1 loss reduced the expression of NE gene. Interestingly, the KD of H19 in these cells re-induced the luminal phenotype, consequently restoring their sensitivity to ADT. These findings strongly implicate H19 in driving the NE lineage switch. The KD of H19 in these organoids led to a regression of tumor growth and invasiveness, suggesting H19 is required in malignant states. H19 KD decreases stem cell genes (SOX2, OCT4) via methylation of their promoters, and levels of EZH2, which methylates 'Lys-9' (H3K9me) and 'Lys-27' (H3K27me) of histone H3 and represses transcription of the target genes. H3K27me3 levels, which are high in NEPC, was reduced in the H19 KD, suggesting H19 works in an epigenetic regulation to induce NEPC phenotype.

CONCLUSION: These data highlight H19 as a novel regulator in controlling the lineage switch to NEPC, tumor growth, and the sensitivity to hormone blockade, suggesting that H19 regulation will be important for developing treatments specifically aimed for this highly aggressive form of prostate cancer.

#3018

Mechanisms of tyrosine kinase inhibitor resistance in chronic myeloid leukemia.

Anna M. Eiring,1 Rebecca Ellwood,2 Carme Ripoll Fiol,2 Robert K. Hills,3 Georgios Nteliopoulos,2 Alistair Reid,2 Dragana Milojkovic,2 Jane Apperley,2 Jamshid Sorouri-Khorashad2. 1 _Texas Tech University Health Sciences Center at El Paso, El Paso, TX;_ 2 _Imperial College London, United Kingdom;_ 3 _Oxford University, United Kingdom_.

Tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1 have turned chronic myeloid leukemia (CML) from a fatal to a chronic disease. Despite improved survival, resistance is a clinical problem, and TKIs do not target the quiescent CML leukemic stem cell (LSC), meaning that patients must be treated for life at a high economic burden and sometimes with significant side effects. TKI resistance is frequently characterized by mutations in the BCR-ABL1 kinase domain, but they explain only ~50% of clinical TKI failure. The remaining patients have BCR-ABL1-independent resistance, defined as survival despite BCR-ABL1 inhibition. We have previously reported a gene expression signature predictive of TKI failure in CD34+ cells from chronic phase CML patients (McWeeney et al. 2010). This gene expression signature demonstrated significant overlap with signatures of blast phase CML (Zheng et al. 2006), suggesting that similar biological processes may be driving TKI resistance and disease progression. We expanded our analysis, and also found significant overlap between the expression profiles of TKI resistance and quiescent CML LSCs reported by Graham et al. (p=4x10-14) and Cramer Morales et al. (p=7x10-6). These data suggest that there is a core of genes whose expression is consistently associated with multiple scenarios of BCR-ABL1-independent resistance. Our previous work has shown that BCR-ABL1-independent resistance is largely driven by STAT3, and that targeting STAT3 in combination with TKIs restores sensitivity in TKI-resistant CML stem and progenitor cells (Eiring et al. Leukemia 2015). Unexpectedly, gene set enrichment analysis revealed that our gene expression signature predictive of TKI failure (McWeeney et al. 2010) does not reveal a STAT3 transcriptional signature. Similarly, RNA sequencing data on TKI-resistant K562-R cells, which are resistant to TKIs but lack BCR-ABL1 kinase domain mutations, revealed that, while TKI resistance was not associated with a STAT3 transcriptional signature (p=1.0), it was correlated with a signature reminiscent of TNFα signaling via NFκB (p=0.024). Nucleocytoplasmic fractionation revealed higher levels of total- and phospho-NFκB in the nucleus of K562-R versus K562-S cells, and in CD34+ progenitors from TKI-resistant CML patients (n=3) compared to TKI responders (n=2) and normal individuals (n=2). These data indicate that NFκB may be driving the gene expression signature associated with BCR-ABL1-independent resistance, and suggest non-canonical functions for STAT3 that go beyond its traditional role as a transcription factor.

#3019

Dynamic kinome profiling of EGFRvIII-driven murine astrocyte models of glioblastoma reveals targets for dual kinase inhibitor therapy.

Erin Smithberger,1 Abigail K. Shelton,1 Madison K. Butler,2 Alex R. Flores,1 Ryan E. Bash,1 Steven P. Angus,1 Noah Sciaky,1 Harshil D. Dhruv,3 Gary L. Johnson,1 Michael E. Berens,3 Frank B. Furnari,4 C. Ryan Miller1. 1 _University of North Carolina, Chapel Hill, NC;_ 2 _National Cancer Institute, Rockville, MD;_ 3 _Translational Genomics Research Institute, Phoenix, AZ;_ 4 _University of California San Diego, San Diego, CA_.

Glioblastoma (GBM) is an aggressive brain tumor with few effective treatments. Epidermal growth factor receptor (EGFR) is frequently amplified and mutated in GBM, leading to trials of several EGFR tyrosine kinase inhibitors, but none have proven successful. One potential reason for failure is acquired resistance, particularly acute, adaptive responses in the kinome. To study this adaptive resistance mechanism, we used RNA-seq and multiplex inhibitor bead/mass spectrometry (MIB-MS) to analyze transcriptomes and kinomes of genetically-engineered murine astrocytes with genotypes commonly seen in human GBM. We previously showed that 38% (86 of 228) of the expressed kinome varied among a panel of genetically diverse murine astrocytes harboring Cdkn2a deletion (C) plus Pten deletion (CP), wild-type human EGFR (CE) or EGFRvIII (CEv3) overexpression, or both overexpressed EGFRvIII and Pten deletion (CEv3P). Pairwise genotype comparisons revealed multiple differentially activated kinases, including Pdgfrb, Fgfr2, Lyn, Ddr1, and several Ephrin family members. We further investigated these potential targets for dual therapy with EGFR TKI by examining the transcriptional response of cultured astrocytes at 4, 24, and 48 hours after 3 μM afatinib. Afatinib induced no kinome changes in C and only 3 kinases (Fn3k, Prkg2, and Syk) were altered in CP astrocytes. Despite similar baseline gene expression profiles, CE astrocytes overexpressing wild-type EGFR responded significantly differently than C astrocytes without. Five kinases (Dclk1, Epha3, Epha7, Fgfr3, and Prkg1) were induced, while 14 were repressed. Six were similarly repressed in CEv3 (Bub1, Nek2, Pask, Plk4, Prkcb, and Vrk1). Whereas the kinase transcriptome response was blunted in C, CP, and CE astrocytes, afatinib induced altered expression of significantly more kinases in CEv3 (82) and CEv3P cells (49). One particularly attractive target in CEv3 astrocytes was Epha4, which afatinib induced >40-fold. Dual inhibition of EGFRvIII and Epha4 kinases may thus provide an opportunity for more effective targeted therapy.

#3020

Natural & synthetic glucocorticoids induce therapy resistance in breast cancer in vitro.

Kristina Andrijauskaite, Michael J. Wargovich. _UT Health San Antonio, San Antonio, TX_.

BACKGROUND: Research indicates that chronic psychological stress, mediated by the excessive release of glucocorticoid (GC) cortisol, may play a role in cancer progression and patient survival. Thus, synthetic GCs, such as dexamethasone, are widely used in conjugation with cancer therapy due to their ability to induce apoptosis. Surprisingly, recent evidence suggest that GCs can induce therapy-resistance in solid tumors, although the precise mechanisms are not well defined. Therefore, there is an urgent need for both clinical and detailed mechanistic studies elucidating the role of GCs in cancer treatment. The goal of this study was to investigate the role of natural and synthetic GCs on resistance to cytotoxic therapy in breast cancer in vitro.

METHODS: We either pre-treated or co-treated human breast cancer cells (MCF7, MDA-MB-231) with physiological and therapeutic doses of cortisol and dexamethasone (0.1-10 µM) alone or in combination with chemotherapy drug Fluorouracil (5FU) for 48-96 hours. We performed cell viability, proliferation, cell cycle and apoptosis assays. Thus, we used the glucocorticoid receptor (GR) antagonist (RU-486) to assess the role of GC receptor signaling. The expression of GR was confirmed by western blots.

RESULTS: Our results indicate that GCs alone or in combination with 5FU enhanced cell proliferation, especially in MDA-MB-231 cells (p<0.01). Furthermore, GCs also modulated cell cycle and apoptosis. Finally, RU-486, which antagonizes the action of GR, restored the apoptosis sensitivity of the GCs treated cells.

CONCLUSIONS: Our study suggests that in contrast to the pro-apoptotic effect of GCs in lymphoid cells, co-treatment of GCs protected breast cancer cells from 5FU-induced apoptosis. Given our results, it is difficult to conclude whether the primary detrimental effect of GCs is on cell viability and apoptosis. We cannot rule out the possibility that GCs my enhance resistance to chemotherapeutic drugs by modifying inflammation, angiogenesis or cell migration. Therefore, our current studies are aimed at investigating the possible pathways mediating GCs induced resistance to therapy in vitro. Our ultimate goal is to elucidate the molecular mechanisms of the glucocorticoids mediated inhibition of chemotherapy-induced cell death thereby proposing the ways of reversing this process. The literature suggest that around 35-45 % of cancer patients experience significant level of stress which manifests in elevated production of natural GC cortisol. Furthermore, given that GCs are widely accepted as an adjuvant treatment during chemotherapy or radiotherapy for reducing side effects, it is critical to investigate the conditions upon which they exert either pro-apoptotic or anti-apoptotic action in different cancer types. Although more data is needed, our findings imply careful consideration for the profligate use of GCs combination therapy in the treatment of cancer patients.

#3021

The role of survivin splice variants in pancreatic ductal adenocarcinoma Gemcitabine resistance.

Janviere Kabagwira, Amber Gonda, Mei Li Kwong. _Loma Linda University, Loma Linda, CA_.

Pancreatic Ductal Adenocarcinoma (PDAC) is a highly lethal disease with poor prognosis and an increased chemoresistance. Dysfunction in alternative splicing has been associated with chemoresistance in many cancers. Alternative splicing of survivin mRNA generates six splicing isoforms some of which are associated with cancer progression and chemoresistance. Survivin is a member of the inhibitors of apoptosis (IAPs) family and a cell cycle-associated oncoprotein overexpressed in almost all cancers. Interestingly, some of these survivin splice variants are proapoptotic (2α and 2β) while others are antiapoptotic (survivin, Δex3, 3α and 3β). It has been reported that increased levels of survivin 2β and 3β play a role in chemoresistance in ovarian cancers and other tumors, but this has not been studied in pancreatic cancer. Our recent RT-qPCR data also show an overexpression of all survivin splice variants in chemoresistant PDAC cell lines compared to the chemo-sensitive cell line. We, therefore, hypothesize that increased expression of survivin splice variants 2β and 3β contribute to chemo-resistance in PDAC. To test this hypothesis, we repeatedly selected the resistant cells from the normally chemosensitive MiaPaca2 cell line after exposure to gemcitabine. We then assessed the levels of survivin splice variants in the new cell line (gemcitabine-resistant MiaPaca2). Using the Alamar blue assay, we confirmed that Panc1 and gemcitabine-resistant MiaPaca2 are more resistant to gemcitabine compared to MiaPaca2 cells. We also observed a statistically significant overexpression of survivin 2β and 3β in gemcitabine-resistant cell lines compared to the more sensitive MiaPaca2 cells. To further test our hypothesis, we will knockdown survivin 2β and 3β in Panc1 and gemcitabine-resistant MiaPaca2, and/or overexpress these splice variants in MiaPaca2 cells then assess response to gemcitabine. The outcomes of this study might create new biomarkers for predicting the clinical outcomes of gemcitabine treatment and generate new therapeutic targets for pancreatic cancer.

#3022

Dissecting rapamycin resistance in a Tsc2-null rat leiomyoma cell line developed in a murine xenograft.

Natalia Filippidou, Mathildi Valianou, Daniel L. Johnson, John J. Bissler, Aristotelis Astrinidis. _UTHSC, Memphis, TN_.

Despite the identification of mTOR hyperactivation as the main biochemical defect downstream of TSC1/TSC2 inactivation in Lymphangioleiomyomatosis (LAM) and Tuberous Sclerosis Complex (TSC), and the approval of rapamycin and rapalogs for the treatment of the related lesions, these drugs are cytostatic and continued treatment is required for clinical benefit. The latter raises the possibility of acquired drug resistance over long-term use. To explore the mechanisms leading to rapamycin resistance in LAM/TSC, we xenografted SCID mice with ELT3 cells (Tsc2-null derived from an Eker rat uterine leiomyoma), explanted a tumor that was not responsive to rapamycin, and generated cell line ELT3-245. The cell growth of ELT3-245 is not inhibited by rapamycin in vitro. SCID mice inoculated with ELT3-245 form palpable tumors significantly faster, compared to ELT3 cells, respond very poorly to rapamycin at the initial stages of treatment, but soon become non-responsive. In addition, these cells form macroscopically visible metastases to the lungs of ELT3-245 tumor-bearing mice. We performed global gene expression profiling, comparing ELT3-245 to ELT3 cells, and found that the rapamycin-resistant cells have decreased expression of genes associated with epithelial cells and increased expression of genes associated with mesenchymal-like characteristics. Relative gene expression of Myc, Egfr, Akt2, Jun, and Ocln were assessed by RT-qPCR in ELT3 vs ELT3-245 after 24h of treatment with 20 nM rapamycin or DMSO. Important discrepancies include a 2.8-fold increased expression of Egfr under rapamycin treatment in ELT3 cells but not in ELT3-245; however, ELT3-245 had a 15-fold higher expression of Egfr prior to treatment, compared to ELT3. In addition, ELT3-245 cells strikingly increase expression of Myc when challenged with rapamycin, compared to ELT3. Similarly, Akt2 is increased considerably under rapamycin treatment in ELT3-245 and is already higher prior to treatment compared to ELT3. Finally, Ocln expression was at least 100-fold reduced in ELT3-245, compared to ELT3, both under rapamycin treatment and no-treatment conditions. These results suggest that Myc, Egfr, Akt2, and Ocln are among the genes highly relevant to acquired rapamycin resistance in this Tsc2-null rat leiomyoma cell line and point toward the pathways that need to be interfered with in order to overcome it.

#3023

Clonal expansion of TP53 mutated cells is associated with chemoresistance in acute myeloid leukemia.

Bowen Yan, Yi Qiu. _Univ. of Florida College of Medicine, Gainesville, FL_.

Chemoresistance is a major burden for the treatment of many cancers, including acute myeloid leukemia (AML). AML is a clonal disease and chemoresistant cells may either evolved from the expansion of a subclone of the primary clone or from the clone gained additional mutations during therapy. We have generated chemoresistant cell lines from AML patient sample derived cell lines, OCI-AML2 and MV4-11, to study the chemoresistant mechanism. Interestingly, we found that both established resistant lines have TP53 mutations (Y220C or R248W) but not in the parental cell lines by Sanger sequencing. However, high resolution sequencing result shows that residual TP53 mutation is present in both parental cell lines. These small population of cells with TP53 mutation was expanded under the selection by chemotherapy and eventually became a dominant clone in chemoresistant lines. TCGA AML patient sample sequencing data also shows, there're around 3% of patients have residual TP53 mutation reads. However, since the reads are low, these patients are considered TP53 wild type conventionally. Importantly, these patients have worse outcome of survival as compared to the TP53 wild type patients, indicating the residual TP53 mutation may play an important role on chemoresistance in patients. Consistent with TP53 mutation in chemoresistant cells, Gene Set Enrichment Analysis (GSEA) from RNA-seq data shows p53 target genes are repressed in resistant cells. The cyclin-dependent kinase inhibitor p21 is one of p53 target genes and is down regulated in chemoresistant cells. Knocking down of p21 in parental cells increased cell chemoresistance, indicating p21 gene repression contributes to chemoresistance. Data from TCGA also shows patients with lower p21 expression have worse outcome of survival. In chemoresistant cell line, there is reduced p53 recruitment at p21 promoter, leading to failure of p21 activation. The epigenetic drug Romidepsin can elevate histone acetylation at p21 promoter region, reactivates p21 gene expression and induces cell death. Furthermore, GSEA from RNA-seq data shows Romidepsin can reactivate genes that repressed by p53 mutation. Our data indicate that TP53 mutation is directly linked to chemoresistance. Residual TP53 mutation in patients was neglected previously, however, it can be an important driver for clonal evolution and chemoresistance in AML. In addition, epigenetic drug Romidepsin can be a potential therapeutic drug to prevent chemoresistance development especially for patients with TP53 mutation or residual TP53 mutation.

#3024

Transcriptional and epigenetic regulation of resistance markers in cetuximab sensitive HNSCC cells.

Luciane T. Kagohara, Fernando Zamuner, Michael Considine, Genevieve Stein-O'Brien, Thomas Sherman, Daria A. Gaykalova, Elana J. Fertig. _Johns Hopkins University, Baltimore, MD_.

Acquired resistance is pervasive in cancers treated with targeted therapies. The understanding of resistance mechanisms and the timing of the driver molecular changes are crucial for alternative interventions while tumors are sensitive. In this study, we focus on cetuximab, the only targeted therapeutic approved to treat head and neck cancers (HNSCC). We and others have shown that signatures of resistance arise in early sensitive states of treatment, including notably EMT and TFAP2 activation. Because TFAP2 regulates both EMT genes and compensatory growth factor receptors and resistance is associated with epigenetic alterations, we hypothesized that epigenetic regulation of TFAP2 is a master regulator of future mechanisms of resistance.To test our central hypothesis, we treated three HNSCC cell lines (SCC1, SCC6 and SCC25) with CTX for 5 days, a time frame that cells are known to be sensitive. We performed RNA-seq and ATAC-seq to understand the global molecular changes induced by CTX. To verify the role of TFAP2A in cell growth and EMT control in response to CTX, we used siRNA for gene silencing and measured cell proliferation and migration ability (scratch assay). In order to identify a potential combination therapy, we also treated the cells with JQ1, a bromodomain inhibitor with a major impact in delaying acquired CTX resistance.RNA-seq analysis shows that CTX sensitive cells up-regulate growth factor receptors and EMT genes. Chromatin changes were also evident in the same cells. Enrichment of accessible chromatin areas for genes from the TFAP2A and EMT pathways suggests epigenetic regulation of CTX resistance associated genes as an early response to therapy. In both, RNA-seq and ATAC-seq analysis, we observed alterations in different sets of genes for each of the cell lines, indicating that resistance signatures vary in the cell models as observed in patients. TFAP2A plays a pivotal role in cell growth. Lack of TFAP2A results in lower proliferation rates in untreated cells and a synergistic effect is observed with CTX, JQ1 and CTX + JQ1 therapies. TFAP2A loss reflected in increased migration ability in SCC1 and SCC25, while in SCC6 we observed less cell motility. However, in all cell lines EMT markers were up-regulated and suggests EMT development, due to down-regulation of TFAP2A, is a cell type and time dependent mechanism with specific cell types requiring more time to translate the transcriptional changes.Overall, we demonstrate that resistance-associated genes in HNSCC sensitive cells are epigenetically regulated. These mechanisms are also cell type and time dependent reflecting observations from patients' cohorts. We show that TFAP2A regulates growth factor receptors and EMT genes in CTX sensitive cells and could be a potential target for drug development. We also show that the CTX + JQ1 combined therapy is a potential alternative and more effective approach while patients are sensitive to therapy.

#3025

Investigation of AKT3 in the resistance of non-small cell lung cancer to EGFR-TKI.

Shih-Hsiang Huang,1 Jin-Yuan Shih,2 Ching-Chow Chen1. 1 _National Taiwan Univ. College of Medicine, Taipei, Taiwan;_ 2 _National Taiwan University Hospital, Taipei, Taiwan_.

Lung cancer is the leading cause of cancer deaths worldwide, and EGFR-TKI is the first-line treatment for non-small cell lung cancer (NSCLC) harboring EGFR activation mutation. However, most patients develop acquired resistance around 12 months. Two resistant cells, HCC827/IR (IRESSA resistance) and H1975/AR (AZD9291 resistance) exhibiting EMT phenotypes were generated to investigate the molecular and cellular characteristics of the EGFR-TKI acquired resistance.

The expression and activation of EGFR were reduced in both resistant cells. Upregulation of AXL and FGFR1 were found in HCC827/IR but not H1975/AR cells. HCC827/IR (without T790M) and H1975/AR (without C797S) showed "off target resistance". Activation of AKT (p-AKT) in both parental cells was more sensitive to EGFR-TKI than that in resistant cells. RNA-seq revealed that AKT3 was upregulated in both resistant cells. High expression of AKT3 was predicted to correlate with the poor survival of lung adenocarcinoma patients. Knockdown of AKT3 in HCC827/IR cells inhibited cell migration and increased the protein expression of E-cadherin, as well as inhibited S phase population to reduce cell proliferation. Knockdown of AKT3 in H1975/AR cells enhanced AZD9291-induced inhibition on AKT activation (p-AKT). Immunoprecipitation of AKT3 in both resistant cells demonstrated its involvement in AKT activation, and AKT3 activation (p-AKT3) was not sensitive to inhibition by gefitinib and AZD9291 in resistant cells. We also found that protein but not mRNA of AKT3 was upregulated in gefitinib-treated HCC827/IR and AZD9291-treated H1975/AR cells. Therefore, AKT3 might serve as a predictive biomarker for EGFR-TKIs therapy, and its upregulation might be one of the mechanisms for EGFR-TKIs acquired resistance. In addition, combination of AZD9291 with AKT inhibitors elicited synergistic inhibition on cell viability of H1975/AR cells.

This work was supported by the National Health Research Institutes (NHRI)- NHRI-EX107-10707BI.

#3026

Rigosertib (RIG) modulates MAPK and hematopoiesis signaling and synergizes Azacitidine (AZA) altering viral mimicry pathway in myelodysplastic syndrome (MDS).

Stella M. Melana, Richa Rai, Shyamala C. Navada, Rosalie Odchimar-Reissig, Erin P. Demakos, E. Premkumar Reddy, Lewis R. Silverman. _Icahn School of Medicine at Mount Sinai, New York, NY_.

MDS is a heterogeneous stem cell disorder characterized by hyperproliferative bone marrow (BM) and peripheral blood cytopenias involving one or more lineages. AZA, a hypomethylating agent (HMA) is considered 1st line therapy for higher-risk disease. About 50% of MDS patients (pts) respond to AZA, with a median response of 14-24 month (Silverman Cancer Med 9th ed). For pts responding to AZA, most either relapse or progress with worsening BM failure and have a median survival of 4-6 months. Both primary and secondary resistance remains a significant challenge and results in poor survival. We previously reported that RIG, a ras-mimetic identified as a novel anticancer drug, inhibits cell cycle progression, induces apoptosis and acts as a HDAC inhibitor with chromatin modifying activity. In a Phase I/II study the combination of RIG+AZA produced an 85% overall response rate in pts who were HMA naive and 62% in HMA failures (Navada EHA 2017). The ability to reverse the resistance phenotype is a novel observation with clinical implications. In this study, we investigated the effect of AZA and RIG alone or in sequential combination (RIG/AZA; AZA/RIG) (SC) on MDS-L cell line to identify the mechanism of action of the drugs. QPCR array was used to identify the differential gene expression profile. Maximum gene alteration was observed (±2 fold change) on treatment with RIG alone, AZA/RIG and RIG/AZA with differential expression of 20 (14up+6dn), 22 (16up+6dn) and 21 (16up+5dn) genes, respectively. However, AZA alone showed minimal effect with dysregulation, the expression of only 2 genes (1up+1dn). Further, gene expression with 1.5 fold change was used for biological and functional analysis. Interestingly, hematopoiesis cell lineage and JAK-STAT signaling were the pathways most affected in RIG alone, and both the SCs. MAPK signaling is impacted in cells treated with RIG alone and AZA/RIG. This suggests that RIG plays a crucial role in modulating these pathways. However, cytokine-cytokine receptor interactions were impacted only in cells treated with both SCs. RIG-I like receptor signaling was specifically observed only in RIG/AZA. This pathway along with Toll-like receptor (TLR) signaling in RIG/AZA suggests the involvement of viral mimicry. Moreover, TLR signaling dysregulation with AZA also suggests association with viral mimicry and impacts the cell cycle pathway. These results indicate that RIG either alone or in combination acts via the MAPK signaling pathway and impacts hematopoietic signaling. In contrast, AZA had no impact on these signaling pathways. This suggests that RIG in combination with AZA affects important signaling pathways impacting hematopoiesis and may explain the synergy seen in MDS pts. Further study is needed to understand the mechanism of resistance to AZA in MDS patients and identify the potential target for reversal of drug resistance.

#3027

Pharmacological regulation of the ABCG2 multidrug transporter in A549 non-small cell lung cancer cells.

Daniella Kovacsics, Nikolett Mészáros, Borbála Tihanyi, Zsolt Matula, Adrienn Borsy, Edit Szabó, György Várady, Tamás Orbán, Ágota Apáti, Ábel Fóthi, Anna Brózik, Balázs Sarkadi. _MTA-TTK, Budapest, Hungary_.

The notoriously drug resistant non-small cell lung cancer (NSCLC) is a leading cause of cancer-related deaths and the ABCG2 multidrug transporter was shown to be involved in NSCLC drug resistance. Direct drug interactions with this transporter have been extensively studied, while a potential modulation of ABCG2 expression by drugs or hormones is less explored. To study the drug-dependent regulation of ABCG2 expression, we used CRISPR-Cas9 and homologous recombination-mediated DNA repair to generate fluorescent lung cancer reporter cells with preserved endogenous regulatory regions. In A549 lung adenocarcinoma cells, which have two wild type copies of the ABCG2 gene, we replaced the initial coding region of ABCG2 with a green fluorescent protein (eGFP)-coding sequence. In addition, since our previous studies demonstrated that an N-terminally eGFP-tagged ABCG2 protein is properly localized and functional, we have generated A549 cells in which the native ABCG2 coding region was modified to express an eGFP-tagged ABCG2 protein. The eGFP knock-in cells sensitively reported ABCG2 transcriptional regulation, while the cells expressing an eGFP-ABCG2 fusion protein allowed the assessment of the full, functional transporter. Using the engineered A549 cells, we found that several drugs, including anti-cancer medications, glucocorticoids, HDAC inhibitors and hypoxia-mimicking agents, caused a significant induction of ABCG2 expression. In these experiments, an intronic regulatory element was found to play a key role in the specific glucocorticoid induction of ABCG2. We suggest that our transgenic reporter cell lines, amenable to high-throughput studies, can be used to investigate the induction of ABCG2-based drug resistance in cancer and predict potential drug-drug interactions. Understanding how the generation of drug-tolerant persister cells occurs via ABCG2 expression may help the selective elimination of these multidrug resistant cancer cells. Supported by the NKFIH-OTKA grant K115375.

#3028

STAT3 represses miR-145-5p to exacerbate HER3 expression for surviving EGFR-TKIs in lung cancers.

Chun-Chia Cheng,1 Ya-Wen Chiang,2 Ken-Hong Lim,2 Jungshan Chang,3 Ai-Sheng Ho,4 Yi-Fang Chang2. 1 _National Health Research Institute, Taipei, Taiwan;_ 2 _MacKay Memorial Hospital, Taipei, Taiwan;_ 3 _Taipei Medical University, Taipei, Taiwan;_ 4 _Cheng Hsin General Hospital, Taipei, Taiwan_.

HER3 exerts resistance against epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), resulting in tumor relapse in lung cancers. Previously, we have demonstrated that EGFR induces HER3 overexpression and that contributes to the formation of cancer stem-like tumorspheres. However, the cellular mechanism of EGFR in regulating HER3 expression was obscure. We hypothesized that EGFR-downstream STAT3 participates in HER3 expression since STAT3 contributes to cancer stemness and surviving EGFR-TKIs. First, RNAseq was used to uncover the potential genes involving in formation of lung cancer HCC827-derived stem-like tumorspheres. EGFR-positive lung cancer cell lines (HCC827, A549, H1975) were individually treated with a panel containing 172 compounds which targeting to stem cell-associated genes in order to searching potential agents against EGFR-positive lung cancers. In addition, gene knockdown and RNAseq were used for investigating the molecular mechanism of STAT3 on regulating tumor progression and survival of lung cancers. We found that BBI608, a STAT3 inhibitor, was a potential therapeutic agent specifically reducing the cell viability of EGFR-positive lung cancers. Interestingly, the inhibitory effects caused by BBI608 were similar with that derived from YM155, an ILF3 inhibitor, both compounds reduced G9a-mediated HER3 expression. We, furthermore, demonstrated that STAT3 upregulated G9a to silence miR-145-5p for exacerbating HER3 expression in this study. In conclusion, this study figured out the potential cellular function of STAT3 in EGFR-positive lung cancers. We also evaluated that BBI608 was potential to eradicate EGFR-positive lung cancers and demonstrated that STAT3 regulated expression of HER3, indicating that STAT3 was a reliable therapeutic target against EGFR-TKI-resistant lung cancers.

#3029

Targeting Annexin A4 with antisense oligonucleotides improves platinum resistance of ovarian cancer.

Satoshi Nakagawa,1 Reisa Kakubari,1 Shinya Matsuzaki,1 Yutaka Ueda,1 Kosuke Hiramatsu,2 Satoshi Serada,2 Minoru Fujimoto,2 Tadashi Kimura,1 Tetsuji Naka2. 1 _Osaka University, Suita,Osaka, Japan;_ 2 _Kochi University, Nangoku, Kochi, Japan_.

[Introduction] Ovarian cancer (OvCa) is one of the main causes of gynecologic cancer death in many countries and the number of patients is increasing. The standard treatment for advanced OvCa is cytoreductive surgery and adjuvant chemotherapy. The key drug for OvCa chemotherapy is platinum. However more than 70% of advanced stage OvCa relapse within 5 years, and the major issue to treat them is platinum resistance. We previously reported that Annexin (Anx) A4 protein was associating with chemoresistance to platinum-based cancer drugs. Nucleic acid therapeutics is promising therapy to target molecule existing intra cell membrane. The aim of this study is to evaluate the usefulness of antisense oligonucleotides (ASOs) and toovercome the platinum chemoresistance with ASOs by suppressing the expression of Anx A4 in cancer cells.

[Methods] Cell line: To assess AnxA4 ASOs, two human ovarian cancer cell lines, RMG-1 and OVISE, which have platinum resistance and strongly express Anx A4, were used in each experiment. Anx A4 ASOs: We selected targeting Annexin A4 mRNA sequence in silico and made 16 ASOs. To enhance the stability, 2', 4'-bridged nucleic acid were inserted in both 3'and 5'ends. And we analyzed suppression of Annexin A4 in ovarian clear cell cancer cell lines in vitro using real time PCR and western blotting. Platinum resistance verification: In vitro, cells were transfected with ASOs using lipofectamine 2000 and were exposed to various concentrations of cisplatin (0 - 100 μM) for 72 hr. Then, drug concentrations resulting in a 50% inhibition of cell growth (IC50values) were calculated. In vivo, we used ICRnu/nu mice xenografted subcutaneously with RMG-I and OVISE cells. Intraperitoneal injection of cisplatin 3mg/kg after intratumoral administration of ASO 1mg/kg each twice a week were given to xenograft mice.

[Result] Of the 16 types of antisense oligonucleotides, we focused on 2 type of ASO, #7, #9 which had stronger knockdown effect for Anx A4 expression. In RMG-I cell, IC50value of Anx A4 ASO transfected cells were 6.42 µM in compared with 11.12 µM in control ASO transfected cells. (p=0.012) In OVISE cells, IC50value was also significantly higher in transfected Anx A4 ASO cells. (10.30 µM, 4.66 µM, p<0.0001) Significantly more amount of platinum had accumulated in Anx A4 ASO transfected cells in compared with control ASO transfected cells and no treatment cells.In vivo, tumor growth of mice treated with cisplatin+Anx A4 ASOs were significantly inhibited compared to cisplatin + control ASOs in both the RMG-1 and OVISE cell lines xenograft mice.

[Conclusion] By transfection of ANXA4 ASO, platinum resistance has been improved both in vitro and in vivo. Targeting ANX A4 with nucleic therapeutics could be an option for platinum resistant ovarian cancer.

#3030

HSD3B1 mediated anti-androgen resistance in prostate cancer requires specific androgen precursors.

Cameron M. Armstrong, Chengfei Liu, Wei Lou, Alan P. Lombard, Christopher Evans, Allen C. Gao. _UC Davis Medical Ctr., Sacramento, CA_.

Resistance to anti-androgens, such as enzalutamide (Enza) or abiraterone (Abi), develops within 24 months of initial exposure in most prostate cancer (PCa) patients. Commonly, dysregulated androgen signaling is a key feature of resistant disease. Previous studies demonstrate that androgen synthesis is upregulated in Enza and Abi resistance. HSD3B1 is steroidogenic enzyme which contributes to androgen synthesis and is associated with PCa progression. This study aims to determine the role of HSD3B1 in promoting Enza and Abi resistance in PCa.

Enza resistant (C4-2B MDVR) and Abi resistant (C4-2B AbiR) C4-2B PCa cells were generated by chronically exposing parental C4-2B cells to increasing Enza or Abi concentrations for >12 months. Cells were maintained in 20 µM Enza or 10 µM Abi thereafter. Microarray, RNA-seq, and rtPCR were used to determine differences in gene expression between parental and anti-androgen resistant cells and confirmed by Western blot. HSD3B1 expression was knocked down in C4-2B MDVR and C4-2B AbiR cells using shRNA and cell number was determined in media containing FBS, charcoal dextran stripped FBS (CDFBS), or CDFBS supplemented with 100 nM pregnenolone (P5), 100 nM DHEA, or 10 nM DHT in the presence and absence of 20 µM Enza or 10 µM Abi. PSA secretion was determined by ELISA and PSA-luciferase activity was measured by reporter assay.

C4-2B MDVR and C4-2B AbiR cells have increased HSD3B1 expression compared to parental C4-2B cells. This correlates to an increase in intracrine androgens in C4-2B MDVR cells as determined by LC-MS. Knockdown of HSD3B1 in C4-2B MDVR resensitized cells to Enza in FBS, CDFBS+DHT and CDFBS+P5 conditions as determined by a reduction in cell number and PSA secretion and/or PSA-luciferase activity in response to Enza. In the C4-2B AbiR cells, inhibition of HSD3B1 re-sensitized cells to treatment with Abi in FBS, CDFBS, and CDFBS+P5 conditions with the greatest effects seen in the FBS and CDFBS+P5 conditions. Supplementation with P5, but not DHEA, was able to induce PSA-luciferase activity and cell growth in C4-2B MDVR and C4-2B AbiR cells and this could be blocked by knockdown of HSD3B1.

HSD3B1 overexpression in C4-2B MDVR and C4-2B AbiR cells contributes to Enza and Abi resistance and targeting this enzyme could be a viable strategy to improve anti-androgen response in PCa cells. HSD3B1 activity is reliant on select androgen precursors, such as pregnenolone, indicating preference towards a specific androgen synthesis pathway by HSD3B1 in mediating anti-androgen resistance.

#3031

Diverse microenvironmental agonists promote a multi drug tolerant persister phenotype CLL and MCL.

Kallesh D. Jayappa, Vicki L. Gordon, Konrad J. Cios, Christopher G. Morris, Puja C. Arora, Timothy P. Bender, Michael E. Williams, Craig A. Portell, Michael J. Weber. _University of Virginia School of Medicine, Charlottesville, VA_.

Responses to ibrutinib (IBR) or venetoclax (VEN) in Chronic Lymphocytic Leukemia (CLL) or Mantle Cell Lymphoma (MCL) are often partial. We reported synergistic toxicity for the IBR+VEN combination in CLL/MCL ex vivo and initiated an IBR+VEN trial in MCL (NCT02419560). However, we noted variable de novo resistance even to IBR+VEN ex vivo, and this has been found in patients (Tam et al., 2018). We also reported that microenvironmental agonists (CpG-ODN, sCD40L, and IL10; "agonist mix") rapidly induce de novo resistance to IBR+VEN in CLL/MCL (Jayappa et al., 2017).

Here we show that microenvironmental agonists induce multi-drug tolerance to diverse pro-apoptotic drugs (bendamustine, fludarabine, vincristine, inhibitors of Mcl-1, Bcl-xL, and Bcl-2, and BAX activator) including IBR+VEN in CLL PBMCs (N=10). This is mediated by a pre-mitochondrial apoptosis blockade, as BAX activation failed in these cells. CLL cells with an activation phenotype (CD5+/19+/69+) in treatment naive patient PBMCs (N=3) also showed tolerance to several drugs, indicating that multi-drug tolerant cells pre-exist in vivo.

To identify extrinsic inducers of drug tolerance, we tested stromal cells, and agonists for TLRs, NOD1/2, CD40, and IL10R for induction of IBR+VEN tolerance. Variable levels of tolerance were noted in most CLL/MCL PBMCs (N=15) cultured with the TLR9 agonist CpG-ODN, sCD40L, or Jurkat cells expressing CD40L. IL10 and HUVEC induced modest levels of drug tolerance in a few CLL/MCL PBMCs, and TLR1/2, TLR7, and NOD1/2 agonists were effective only in MCL. Prior exposure to CpG-ODN enhanced the drug tolerance/proliferation response to sCD40L and vice versa in CLL/MCL (N=8), predicting mutually reinforcing interactions of agonists in vivo. CLL cells exposed to CpG-ODN/CD40L upregulated cognate receptors and/or NFkB proteins downstream, suggesting a mechanism for mutual reinforcement.

The agonist mix induced NFkB-dependent over-expression of anti-apoptotic proteins Mcl-1 and Bcl-xL. Consistently, multi-drug tolerance was rescued with NFkB inhibitors (BMS345541, MI-2, bortezomib) or drug combinations that inhibit multiple anti-apoptotic proteins in CLL cells exposed to agonist mix or CD5+/19+/69+ cells without agonists. By analyzing non-cancer (CD5+/19-) cells in patient PBMCs, bortezomib and MALT1 inhibitor MI-2 were selectively toxic to cancer cells, showing their value for clinical testing.

In summary, diverse microenvironmental agonists, mainly agonists of TLR9 and CD40, induce multi-drug tolerance in CLL/MCL, which is mediated by NFkB dependent over-expression of multiple anti-apoptotic proteins. Inhibitors of NFkB or drug combinations targeting apoptotic proteins overcame multi-drug tolerance. Thus, durable responses may require drug combinations targeting multiple apoptotic proteins, convergent pathways that induce multi-drug tolerance, and/or agents activating non-apoptotic death.

#3032

**Acquired resistance to osimertinib in patients with** de novo EGFRT790M **-positive non-small cell lung cancer (NSCLC).**

Ha-Ram Park,1 Yusoo Lee,1 Tae Min Kim,2 Soyeon Kim,3 Chan-Young Ock,2 Miso Kim,2 Bhumsuk Keam,2 Yoon Kyung Jeon,2 Dong-Wan Kim,2 Dae Seog Heo2. 1 _Seoul National University Cancer Research Institute, Seoul, Republic of Korea;_ 2 _Seoul National University Hospital, Seoul, Republic of Korea;_ 3 _Biomedical Research Institute, Seoul National University, Seoul, Republic of Korea_.

Background: Epidermal growth factor receptor (EGFR) T790M mutation is well known as primary and acquired resistance mechanisms to the 1st and 2nd generation EGFR tyrosine kinase inhibitors (TKIs). However, a de novo and clonal EGFR T790M mutation is rarely observed in 0.8-2% of patients with EGFR-mutant NSCLC. The 3rd generation EGFR TKIs including osimertinib are expected to be effective against de novo, clonal EGFRT790M-positive NSCLC. However, the efficacy and resistance mechanisms of osimertinib have been limited in patients with de novo and clonal EGFRT790M-positive NSCLC.

Methods: The seven (1%) of 690 patients with EGFR-mutant NSCLC were identified as having de novo and clonal EGFRT790M mutations between December 2002 and July 2014 in Seoul National University Hospital. Three with de novo EGFRL858R/T790M mutation received osimertinib at a dose of 80mg or 160mg once daily (NCT01802632) and two showed acquired resistance to osimertinib. Fresh tumor samples were obtained before and after treatment with osimertinib and analyzed by whole-exome and -transcriptome sequencings as well as droplet digital polymerase chain reaction (ddPCR). MET amplification was determined by fluorescence in situ hybridization (FISH) in formalin-fixed paraffin-embedded tumor tissues.

Results: Three patients with de novo EGFRL858R/T790M mutation received osimertinib and two (LC1 and LC2) showed acquired resistance to osimertinib at 16 and 30 months, respectively (Table 1). As of November 2018, the LC3 patient received osimertinib without disease progression. Novel MTOR L1433S mutation within FAT domain that was not seen at initial tumors (ploidy 2.3) was clonally acquired at osimertinib-resistant tumors (ploidy 2.4) in the LC1 patient. In addition, a novel and clonal MGA V124D mutation within T-box was found at osimertinib-resistant tumors (ploidy 5.9) of the LC2 patient. MET copy number was 14 with high fpkm level (721.9) at osimertinib-resistant tumors in the LC2 patient. Average MET gene copies per nucleus by FISH were 2.74 at initial tumors and 5.25 at osimertinib-resistant tumors in LC2 patient, resulting in MET amplification as a resistance mechanism. Subclonal MGA V124D mutation was found at initial tumors of the LC2 patient using ddPCR. The Ba/F3 systems confirmed that novel mutants were oncogenic.

Conclusions: Novel MTOR and MGA mutations were functional and clonally acquired as resistance mechanisms to osimertinib in patients with de novo EGFRL858R/T790M-mutant NSCLC. In addition, MET amplification contributed to acquired resistance to osimertinib. Taken together, acquired resistance mechanisms of osimertinib might be mainly mediated by alternative pathway activation in de novo EGFRT790M-positive NSCLC.

#3033

Uncovering adaptive mechanisms of resistance to targeted therapy in brain metastasis.

Sally J. Adua,1 Minghui Zhao,1 Paul Smith,2 Darren A. Cross,3 Don X. Nguyen1. 1 _Yale University, New Haven, CT;_ 2 _AstraZeneca, Gothenburg, Sweden;_ 3 _AstraZeneca, Cambridge, United Kingdom_.

Background

A subset of lung adenocarcinoma (LUAD) can be effectively treated with EGFR tyrosine kinase inhibitors (TKIs). However, the incidence of brain metastasis increases in patients that relapse after front-line treatment, underscoring the central nervous system (CNS) as a unique sanctuary site for persistent disease. We sought to perform an integrated examination of the cellular and molecular causes of resistance to targeted therapies in brain metastasis.

Results

The efficacy of osimertinib, a brain penetrant third generation TKI, was studied in mice using two different EGFR mutant LUAD models. In the H1975 (EGFR L858R/T790M) model of second-line osimertinib treatment, subcutaneous tumors achieved complete response rates while H1975 brain metastases continued to grow despite strong drug penetrance into the CNS. Similarly, in the PC9 BrM4 (EGFR ex19del) model of first-line osimertinib treatment, most responding animals developed osimertinib resistance preferentially in the brain following significant regression of multi-organ metastatic lesions. Importantly, tumor cells isolated from progressing brain metastases did not exhibit resistance in vitro. However, these cells exhibited significantly enhanced resistant capacity in comparison to controls when serially transplanted into the brain demonstrating both that this resistant phenotype is selected for and that exposure to the brain metastatic niche is a requirement for drug tolerance in vivo.

We utilized our recently optimized transcriptomic approach, referred to as Brain Metastasis Xenograft-RNA Sequencing (BMX-seq), to comprehensively distinguish the transcriptome of tumor versus stroma in intact drug resistant brain lesions. Accordingly, our analysis reveals that the stroma of drug resistant brain metastasis is characterized by activation of proinflammatory pathways implying that resistant cells induce and/or co-opt stromal inflammatory responses for their own benefit. Reciprocally, we identified stromal induced alterations in the expression of cytoskeletal and interferon response genes in drug resistant tumor cells. Interestingly, most of these genes are induced by the brain tumor microenvironment (TME) independently of drug treatment suggesting that the brain metastatic niche can precondition tumor cells for ensuing drug resistance.

Conclusions

Though advances have been made in the brain penetrating abilities of targeted therapies, acquired resistance in this unique metastatic niche still develops. Our results suggest that specific interactions between the tumor cells and stromal cells are required for the manifestation of this adaptive resistant phenotype. Furthermore, transcriptomic profiling has revealed potential regulators and molecular drivers that mechanistically explain how the brain TME preconditions metastatic cells for resistance.

#3034

TP53 **mutant cell lines selected for resistance to MDM2 inhibitors retain growth inhibition by MAPK pathway inhibitors but a reduced apoptotic response.**

Chiao-En Wu,1 Tsin Shue Koay,2 Yi-Hsuan Ho,2 Penny Lovat,3 John Lunec2. 1 _Division of Hematology-Oncology, Department of Internal Medicine, Chang Gung Memorial Hospital at Linkou, Taoyuan, Taiwan;_ 2 _Northern Institute for Cancer Research, School of Medicine, Newcastle University, Newcastle Upon Tyne, United Kingdom;_ 3 _Dermatological Sciences, Institute of Cellular Medicine, Newcastle University, Newcastle Upon Tyne, United Kingdom_.

Emergence of resistance to molecular targeted therapy constitutes a potential limitation to clinical benefits in cancer treatment. Cross-resistance commonly happens with chemotherapeutic agents but might not with targeted agents. In the current study, TP53 wild -type cell lines with druggable MAPK pathway mutations (BRAFV600E (WM35) or NRAS Q61K (SJSA-1)) were compared with their TP53 mutant sublines (WM35-R, SN40R2) derived by selection for resistance to MDM2/p53 binding antagonists. The continued presence of the druggable MAPK pathway targets in the TP53 mutant (TP53MUT) WM35-R and SN40R2 cells was confirmed. Trametinib and vemurafenib were tested on the paired WM35/WM35-R and SJSA-1/SN40R2 cells and similar growth inhibitory effects on the paired cell lines was observed. However, apoptotic responses to trametinib and vemurafenib were greater in WM35 than WM35-R, evidenced by FACS analysis and caspase 3/7 activity, indicating that these MAPK inhibitors acted on the cells partially through p53-regulated pathways. SiRNA mediated knockdown of p53 expression in WM35 replicated the same pattern of response to trametinib and vemurafenib as seen in WM35-R, confirming that p53 plays a role in trametinib and vemurafenib induced apoptosis. In contrast, these differences in apoptotic response between WM35 and WM35-R were not seen with the SJSA-1/SN40R2 cell line pair. This is likely due to p53 suppression by overexpressed MDM2 in SJSA-1. In conclusion, the TP53MUT cells selected by resistance to MDM2 inhibitors nevertheless retained growth inhibitory but not apoptotic response to MAPK pathway inhibitors.

#3035

Collagen hydroxylation promotes TNBC chemoresistance through stabilizing HIF1á.

Gaofeng Xiong, Rachel Stewart, Jie Chen, Tianyan Gao, Timothy Scott, Luis Samayoa, Kathleen O'Connor, Andrew Lane, Ren Xu. _Univ. of Kentucky Markey Cancer Ctr., Lexington, KY_.

Collagen prolyl 4-hydroxylase (P4H) expression and collagen hydroxylation in cancer cells are necessary for breast cancer progression. Here, we show that P4H alpha 1 subunit (P4HA1) protein expression is induced in triple-negative breast cancer (TNBC). By modulating alpha ketoglutarate (α-KG) and succinate levels P4HA1 expression reduces proline hydroxylation on hypoxia-inducible factor (HIF) 1α, enhancing its stability in cancer cells. Activation of the P4HA/HIF-1 axis enhances cancer cell stemness. Inhibition of P4HA1 sensitizes TNBC to the chemotherapeutic agent in xenografts and patient-derived models. We also show that increased P4HA1 expression correlates with short relapse-free survival in TNBC patients who received chemotherapy. These results suggest that P4HA1 promotes chemoresistance by modulating HIF-1-dependent cancer cell stemness. Targeting collagen P4H is a promising strategy to inhibit tumor progression and sensitize TNBC to chemotherapeutic agents.

#3036

BRD4 degrader and inhibitor of beta catenin-TCF7L2 are synergistically active against human AML cells resistant to BET inhibitor.

Dyana T. Saenz,1 Warren C. Fiskus,1 Taghi Manshouri,1 Christopher P. Mill,1 Steve Horrigan,2 Raffaella Soldi,3 Joseph D. Khoury,1 Sunil Sharma,3 Srdan Verstovesek,1 Kapil N. Bhalla1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _BetaCat Pharma, Houston, TX;_ 3 _TGen, Phoenix, AZ_.

Treatment with bromodomain and extra-terminal protein inhibitor (BETi) reduces in vivo AML cell burden, inducing clinical remissions in AML. Yet, resistance to BETi treatment develops uniformly. Here, following at least 10 exposures of secondary (s) AML control (parental) SET2 and HEL92.1.7 cells to 1.0 µM of the BETi OTX015 for 48 hours followed by full recovery, we generated BETi persister-resistant (BETi-P/R) SET2-P/R and HEL-P/R cells. These cells showed > 10-fold resistance to OTX015 and cross-resistance to other BETis. Compared to the controls, SET2-P/R and HEL-P/R cells lacked additional genetic alterations or altered levels of TRIM33, SPOP, DUB3 or phosphorylated BRD4 (previously described mechanisms of BETi-resistance). However, SET2-P/R and HEL-P/R cells demonstrated significantly higher nuclear levels of β-catenin, the transcription factor TCF7L2 and adaptor protein TBL1X (TBL1), associated with increased expression of TCF7L2 targets, including c-Myc, Cyclin D1, TERT and Survivin. ATAC-Seq and ChIP-Seq (H3K27Ac mark) analyses showed significant gain of peaks and active enhancers in HEL-P/R over HEL92.1.7 cells, with enrichment of STAT5, MYC, PU.1 and GATA2 binding sites, as well as newly gained peaks in the enhancers of JAK1/2, RUNX1, PU.1, MYC, BCL2L1 and CTNNB1. RNA-Seq analysis showed significant increase/decrease in mRNA expressions (340/247), with induction of gene-sets involving MYC/MAX, STAT5, NFkB and TCF7L2 targets. QPCR and Western analyses confirmed increase in the mRNA and protein levels of TCF7L2, c-Myc, Survivin and PIM1 in HEL-P/R over HEL92.1.7 cells. Also, confocal microscopy demonstrated increased binding of β-catenin with TBL1 and TCF7L2 in the nucleus in BETi-P/R sAML cells. β-catenin inhibitor BC2059, which disrupts binding of nuclear β-catenin with TBL1 and TCF7L2 and depletes β-catenin levels, exerted similar lethality in BETi-P/R sAML and control sAML cells. Both shRNA-mediated knockdown of BRD4 and BRD4-PROTAC (proteolysis-targeting chimera) ARV-771 (Arvinas, Inc.), which degrades BRD4/3/2, induced similar level of apoptosis in BETi-P/R and control sAML cells. Co-treatment with ARV-771 and BC2059 synergistically induced lethality in BETi-P/R sAML cells as well as in patient-derived, CD34+ sAML BPCs (combination indices < 1.0). This was associated with marked attenuation of c-Myc, TCF4, Survivin, CDK6, PIM1 and Bcl-xL levels. Also, compared to each agent alone, in vivo treatment with ARV-771 (30 mg/kg SQ daily x 5, per week) and BC2059 (30 mg/kg IP BIW, per week) for 3 weeks, significantly reduced sAML burden and improved survival of NSG mice engrafted with HEL-P/R cells (p < 0.01). Thus, increased levels and activity of β-catenin-TCF7L2-MYC axis is mechanistically responsible for BETi-P/R, and co-targeting with BRD4 degrader and β-catenin-TCF7L2 inhibitor is a promising therapeutic strategy against BETi-P/R sAML BPCs.

#3037

Stromal cell activation of MAPK signaling pathways mediates resistance to MERTK inhibition in acute myeloid leukemia.

Katherine A. Minson,1 Eleana Vasileiadi,1 Madeline G. Huey,1 Xiadong Wang,2 Stephen V. Frye,2 H. Shelton Earp,2 Deborah DeRyckere,1 Douglas K. Graham1. 1 _Emory University, Atlanta, GA;_ 2 _University of North Carolina at Chapel Hill, Chapel Hill, NC_.

MERTK is a TAM family (TYRO-3, AXL, MERTK) receptor tyrosine kinase that is aberrantly expressed in >80% of primary acute myeloid leukemia (AML) samples. MERTK inhibition mediated by the small molecule tyrosine kinase inhibitor (TKI), MRX-2843, induced apoptosis in leukemia cell cultures and prolonged survival in mouse xenograft models of AML, but was not curative. In these models, treatment efficiently reduced peripheral disease burden but was less effective in the bone marrow, suggesting a role for the bone marrow microenvironment in therapeutic resistance. To evaluate the role of the bone marrow stromal niche in resistance to MERTK inhibition by MRX-2843, Kasumi-1 and OCI-AML5 AML cell lines were cultured with the fibroblast-like Hs27 cell line and induction of apoptosis and cell death was measured by flow cytometry after treatment with MRX-2843 or vehicle. Co-culture with Hs27 cells significantly reduced AML cell death in response to MRX-2843 compared to leukemia cells alone (e.g. Kasumi-1 31.9% vs 61.2%, p<0.05). To identify parallel signaling pathways that may be activated under cytokine rich bone marrow stromal conditions and mediate resistance to MRX-2843, a unbiased phospho-kinase array was performed to identify targets differentially activated by co-culture and/or MRX-2843. Kasumi-1 cells were treated with MRX-2843 in the presence or absence of stromal co-culture, then cell lysates were incubated with human phospho-kinase arrays and proteins were quantitated by densitometry. Five kinases were differentially inhibited by >1.5-fold in the presence or absence of stromal cells, including numerous MAPK pathway components - p38α, ERK1/2, EGFR, TOR, and HSP27. Changes in ERK1/2 phosphorylation (pERK) were confirmed by immunoblot in Kasumi-1 and OCI-AML5 cell cultures. In the absence of co-culture MRX-2843 potently inhibited pERK at doses as low as 100nM, while in co-culture even a 3-fold higher dose did not fully inhibit pERK, consistent with a potential role in resistance. To determine whether the observed upregulation and persistent activation of ERK1/2 has functional significance, Kasumi-1 cells were treated with MRX-2843 and the MEK1/2 TKI pimasertib in the presence or absence of stromal co-culture. Treatment with MRX-2843 or pimasertib alone did not significantly affect survival of AML cells in co-culture (31.3% MRX-2843, 28.1% pimasertib vs 22.7% vehicle, p=ns), but treatment with a combination of MRX-2843 and pimasertib resulted in significant induction of apoptosis compared to vehicle or single TKIs (56.7%, p<0.05). Similar results were observed in OCI-AML5 cell cultures and in combination with the MEK1/2 inhibitor PD0325901. Together these results indicate a critical role for stromal cell-mediated activation of ERK1/2 in resistance to MRX-2843 and demonstrate the utility of translational strategies targeting both MERTK and MAPK pathways to overcome resistance.

#3038

Implication of DYRK1B Kinase in ovarian cancers and utilization of DYRK1B Inhibitors as a novel therapeutic strategy for ovarian cancer.

Alexandra Kuznetsova,1 Arianna Damiani,2 Lita De Leon,2 Michael Frid,1 Menelik Duey,3 Jason Law,2 Olga Potapova,2 Maria Vilenchik1. 1 _Felicitex Therapeutics Inc., Natick, MA;_ 2 _Cureline Inc., Brisbane, CA;_ 3 _University of Delaware, DE_.

Majority of tumors consist of two populations of cancer cells: actively proliferating and those in the reversible dormant or quiescent state, which contribute to the resistance of human tumors to chemotherapies. Conventional chemotherapeutic agents have limited efficacy in ovarian adenocarcinoma patients: while the appropriate chemotherapeutic agent kills proliferating cancer cells, dormant cancer cells survive the treatments and result in recurrent disease. The presence of dormant cancer cells is not revealed by standard H&E staining and morphological tumor analysis. CDK inhibitor p27 is known to be a marker of the dormant state. DYRK1B kinase (serine/threonine-protein kinase dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1B) is associated with survival of many types of cancers and was shown to play a key role in maintaining the cancer cells in quiescent state by stabilizing p27 and by inducing the breakdown of cyclin D isoforms. In this study, we investigated the functional and therapeutic relevance of DYRK1B in ovarian carcinomas. We have conducted an immunohistochemical study of 70 human ovarian tumors (35 primary/treatment-naive and 35 recurrent/standard chemotherapy resistant ones) and 20+ cases of normal and benign ovarian tissues for expression of DYRK1B, p27 and Ki-67. All FFPE tissue samples have been obtained from the consented patients and under the IRB-approved protocols clinical network, groups have been normalized for age and the disease type/stage. High levels of DYRK1B and p27 proteins expression in malignant treatment-naïve tumors and their significant correlation indicates the presence of dormant cancer cells mixed up with the proliferating ones within patient's tumors. This effect was detected regardless of the tumor histological subtype (endometrial, serous, mucinous, etc.) and/or stage. While all advanced stage tumors (Stage IIIC and IV) overexpress both markers of dormancy; the early stage tumors (Stage I-II) also presented a higher number of cases with greatly positive scores. Tissue microarray and immunohistochemistry analysis in the literature showed that higher expression levels of DYRK1B correlated with a worse prognosis. We suggest that specific immunostaining of DYRK1B in tumor and endothelial cells can be explored as a predictive risk factor of time to progression/death in patients with primary ovarian tumors. Inhibition of DYRK1B kinase activity with FX9847, specific and selective DYRK1B inhibitor, inhibited ovarian cancer cell growth and induced apoptosis. Moreover, combination of FX9847 with chemotherapeutic agents such as taxanes or vinca alkaloids demonstrated an increased anti-cancer effect on ovarian cancer cells. Together, these findings suggest that DYRK1B is critically involved in the survival of ovarian carcinoma providing a new rationale for their treatment with the kinase inhibitors.

### New Target Identification

#3039

**Targeting carcinoembryonic antigen cell adhesion molecule 6 (** CEACAM6 **) reshapes the tumor-stroma in pancreatic ductal adenocarcinoma (PDA).**

Ritu Pandey,1 Muhan Zhou,1 Shariful Islam,1 Baowei Chen,1 Paul Langlais,2 Anup Srivastava,2 Larry S. Cooke,1 Eric Weterings,1 Daruka Mahadevan1. 1 _Univ. of Arizona Cancer Ctr., Tucson, AZ;_ 2 _University of Arizona, Tucson, AZ_.

Background: CEACAM6, a cell adhesion receptor of the Ig-like superfamily, interacts with other CEACAMs (1, 5, 7 & 8) and is over-expressed in human cancers including PDA. Over-expression of CEACAM6 causes resistance to anoikis - regulated apoptosis induced by inadequate or inappropriate cell-matrix interactions, promoting a TGFβ-mediated invasive phenotype. CEACAM6 as a predictive biomarker has not been fully investigated with regard to its potential as a candidate therapeutic target in PDA. We report a detailed analysis of CEACAM6 by expression profiling in several pancreatic tumor types, stromal cells, cell lines and mechanistic studies supporting CEACAM6 as a therapeutic target, when inhibited impacts several hallmarks key to PDA pathogenesis.

Methods: RNAseq, expression arrays and clinical data was analyzed from The Cancer Genome Atlas (TCGA), International Cancer Genome Consortium (ICGC) and GEO using R (v3.4.3). Expression profiles were analyzed across PDA subtypes, stroma types and overall survival. CRISPR/Cas9 knockout (KO) of CEACAM6 in HPAF-II cells was investigated by quantitative proteomics and genomics with corroborative signaling pathway analysis. A therapeutic monoclonal antibody scFv-Fc (IgG) targeting CEACAM6 was tested in an orthotopic mouse PDA model.

Results: CEACAM6 is over-expressed in ~90% of primary and metastatic PDA samples, ~10-20 fold higher vs. normal cells. Basal and classical subtypes and activated stroma over-express CEACAM6, compared to low or normal stroma. TCGA dataset shows that PDA patients with high CEACAM6 expression have a poor prognosis vs. low expression. In addition, CEACAM6 over-expression is associated with mutant KRAS and immune suppression elaborated by low cytolytic T-cell activity. Correlation studies show that CEACAM6 is associated with genes involved in cell junctions and membrane integrity. HPAF-II cells with CEACAM6 KO showed changes to cell adhesion-extracellular matrix, transmembrane activity, increase in immune signaling and changes to enzymes of mitochondrial activity with significantly decreased mitochondrial membrane potential and ATP production. In addition, CEACAM6 KO cells have a significantly decreased growth rate with reduced Src, Akt and Fak signaling. The scFv-Fc has anti-PDA activity as a single agent in an orthotopic mouse model.

Conclusions: CEACAM6 over-expression is a stroma-associated poor prognostic marker and a novel therapeutic target in PDA. The scFv-Fc targeting CEACAM6 shows promising anti-PDA activity and is being developed to enhance anoikis and restore host anti-tumor immunity.

#3040

The expressional effects of RBBP6 in breast cancer cells are p53-dependent.

Pontsho Moela,1 Lesetja Motadi,2 Marcia Lekganyane2. 1 _University of Pretoria, Pretoria, South Africa;_ 2 _Northwest University, Potchefstroom, South Africa_.

The incidence rate of breast cancer has increased beyond that of lung cancer, making it the most common malignancy among women. Breast tumor progression is partly because of p53 inactivation by overexpressed that possess p53 binding domain. RBBP6 forms a member of ubiquitous regulatory proteins because its RING finger-like domain has an E3 ligase activity. The overexpression of RBBP6 in several malignancies makes it a potential target in cancer management. However, there is no evidence on whether the effect of RBBP6 on cell growth and apoptosis is cell line dependent, more especially in breast cancer cell lines that have distinct p53 expression profiles. Therefore, the aim of this study was to evaluate the RBBP6 effects on cell growth and apoptosis in breast cancer cell lines with different p53 expression profiles. RBBP6 expression was successfully manipulated using gene silencing and protein overexpression techniques in MCF-7 and MDA-MB-231 cell lines. Transfected cells were co-treated with anti-cancer agents followed by apoptosis detection using confocal microscopy and flow cytometry, which was further confirmed by caspase 3/7 activity and quantification of apoptotic genes. RBBP6 was overexpressed in breast cancer tissues that were classified as stage 3 and 4 while in stage 1 it was expressed but at much lower levels. The wt. p53-expressing MCF-7 cell line was more susceptible to apoptosis induction as opposed to the mt. p53-expressing MDA-MB-231. RBBP6 silencing led to a significant accumulation of p53 expression in MCF-7 as compared to MDA-MB-231. Co-treatment with GABA and camptothecin seemed to sensitize the cells to apoptosis induction. These data suggest that RBBP6 silencing triggers significant levels of intrinsic apoptosis and over-expression appears to promote cell proliferation in wild-type p53-expressing MCF-7 rather than in MDA-MB-231 cells. In conclusion, the effect of RBBP6 on cell proliferation and apoptosis induction in breast cancer seem to be cell line dependent based on p53 status.

#3041

CD72 as a potential new therapeutic target in diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL).

Erika Von Euw, Eileen Taschereau, Da Ming Ou, Monica Mead, Sarah Larson, Dennis Slamon. _UCLA, Los Angeles, CA_.

Cluster of differentiation 72 (CD72) is a 45 kDa type II transmembrane glycoprotein expressed primarily on the surface of B cells. In a search for a novel lymphoma specific target, public datasets were leveraged to assess mRNA expression. We investigated CD72 mRNA expression in cell lines (data from CCLE), patients (data from TCGA) and normal tissues (data from GTEx). The absence of CD72 in normal tissue and the presence in B cells and non-Hodgkin lymphomas (B-NHL) made CD72 a potential target for the treatment of B-NHL, mainly DLBCL and MCL.

CD72 expression on the cell surface was tested by FACS in 15 DLBCL and 5 MCL cell lines. Of these, 12 DLBCL and 4 MCL were positive for CD72, while 3 DLBCL and 1 MCL showed no CD72 expression. In all cell lines tested, the cell surface expression of CD72 correlated with mRNA expression.

To evaluate the potential of CD72 as an ADC target, 4 cell lines were treated in vitro with three commercial monoclonal anti-CD72 primary antibodies (clones J4-117, 3F3 and BU40), and MMAF-conjugated secondary antibody. The cell lines used were RL (DLBCL, CD72+), SCC-3 (DLBCL, CD72-), Jeko-1 and Mino (both MCL, CD72+). The cell growth inhibition after 96 hrs of treatment for for all control conditions; anti-CD72 primary antibody, IgG isotype, MMAF secondary antibody alone or combined with IgG isotype; tested in all cell lines, CD72+ or CD72- ranged from -3 to +4%. Conversely, anti-CD72 primary antibodies plus MMAF secondary antibody had marked growth inhibition effect ranged from 24 to 80% on the three CD72+ cell lines and no effect on cell growth in CD72- cell line, suggesting internalization of CD72 and growth inhibition dependent on the presence of CD72 on the cell surface.

This is the first study demonstrating a potential role for CD72 as a novel target for DLBCL and MCL. These data suggest that CD72 may be a candidate for an ADC approach in DLBCL and MCL. | |

|

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

Cell line | % growth inhibition anti-CD72 + MMAF 2°

|

Anti-CD72 clone J4-117 | Anti-CD72 clone 3F3 | Anti-CD72 clone BU40

SCC-3 (CD72-) | -3 | 1 | -5

RL (CD72+) | 50 | 33 | 30

Jeko-1 (CD72+) | 43 | 30 | 24

Mino (CD72+) | 77 | 80 | 66

#3042

**Splicing repressor** HNRNPC **is an indispensable and 'druggable' target in acute myeloid leukemia.**

Vindhya Vijay,1 Amy Meacham,1 Lauren Katzell,1 Aaron Winer,1 Jesse Terrell,1 Vincent Archibald,1 Jodi Bubenik,1 Alberto Riva,1 Jon Boatwright,1 Cristina Tognon,2 Jeffrey Tyner,2 Brian Druker,2 Christopher Cogle1. 1 _University of Florida, Gainesville, FL;_ 2 _Knight Cancer Institute, Oregon Health & Science University, Portland, OR_.

Refractory and relapsed disease is the greatest challenge in acute myeloid leukemia (AML), and we have shown that blood vessels serve as sanctuary sites for AML. Using a high throughput screening assay mimicking AML in the vascular niche, we screened 31 million compounds and identified a hit compound 2470-51 that selectively killed AML cells and CD34+CD38-CD123+ AML stem cells, while sparing bone marrow-derived endothelial cells, normal hematopoietic stem and progenitor cells (HSPC) as well as CD4+ T lymphocytes from healthy volunteers. In vivo AML patient-derived xenograft modeling further validated the efficacy of 2470-51 as a selective anti-leukemic agent compared to cytarabine (conventional control). We performed quantitative proteomics combining ITRAQ differential protein expression analysis, label-free shotgun analysis and SPR imaging to identify heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNPC) as the target of 2470-51. HNRNPC depletion in AML cell lines THP1, K562, MV411 and HL-60 significantly decreased cell proliferation, viability and clonogenic capacity of the AML cells. In contrast, normal mesenchymal/fibroblastic cells, endothelial cells and normal HSPCs from human umbilical cord blood specimens were unaffected by HNRNPC depletion, indicating the selective dependence of AML cells on hnRNPC. Furthermore, clinical studies using TCGA AML datasets showed significant survival advantage associated with lower HNRNPC expression. RNA-seq analyses revealed a distinct gene expression pattern suggesting widespread inhibition of Myc transcriptional targets. Using differential alternative splicing and differential transcript expression analyses, we discovered significant alternative splicing of MAX after HNRNPC depletion, resulting in MAX transcript isoforms that lacked Myc-interacting domains ("inactive" MAX). These Myc-interacting domains are necessary for the obligate heterodimerization of Myc and Max and are critical for Myc transcriptional activation. We further analyzed splicing landscapes of 578 AML patients and 33 healthy controls and identified substantial mis-splicing of mRNA in AML patients, despite the absence of somatic gene mutations of splicing factors, when compared to the healthy controls. Moreover, we found significant overexpression of "inactive" MAX isoforms in the healthy controls, while AML patients overexpressed fully functional MAX isoforms, suggesting that MAX splicing may have an important functional role in AML. Collectively, our studies show significant RNA splicing changes in AML and an essential role of HNRNPC in AML in contrast to normal hematopoietic and stromal cells where HNRNPC is dispensable. We present a pharmacologic agent for targeting HNRNPC and Myc-Max as a molecular mechanism of action. Our data indicate that hnRNPC is a critical factor in AML and inhibiting this splicing repressor may represent a new therapeutic strategy.

#3043

Examination of NMT1 and NMT2 as independent prognostic markers and novel drug targets in adult acute myeloid leukemia.

John R. Mackey, Aishwarya Iyer, Megan C. Yap, Zoulika Zak, Krista Vincent, Erwan Beauchamp, Lynne M. Postovit, Joseph Brandwein, Luc G. Berthiaume. _University of Alberta, Edmonton, Alberta, Canada_.

Myristoylation is required for biological activity of >200 intracellular proteins. N-myristoyltransferases (NMTs) transfer the fatty acid myristate to N-terminal glycine residue; there are two isoforms, NMT1 and 2. In acute myeloid leukemia (AML), upregulation of Lyn and Src, important myristoylated proteins, contribute to cell survival and proliferation. The specific roles of NMT1 and NMT2 are unknown in this context. The relationships among NMT1/NMT2 expression and acute AML patient outcomes were studied using RNA-sequencing and microarray cohorts with over 350 patients. We found that high NMT1 and low NMT2 expression were associated with reduced overall and event-free survival in adult AML, which was independent of other prognostic markers on multivariate analysis. High NMT1, but not NMT2, expression was associated with proliferative gene sets in AML cell lines, indicating potential for distinct isozyme substrates. Given these results, we examined NMT1 and NMT2 levels in AML cell lines and AML patient blast cells using Western blotting and flow cytometry. We determined that NMT2 expression varied greatly among patients, but was markedly reduced in most cases, while NMT1 expression was relatively preserved. A potent small molecule NMT inhibitor, PCLX-001, preferentially induced apoptosis and reduced viability in NMT2-deficient AML cell lines cultured in vitro compared with normal lymphocytes and peripheral blood mononuclear cells. PCLX-001 also killed freshly isolated human AML blasts ex vivo with an IC50 of ~170nM regardless of their mutational background. In a murine AML xenograft model, subcutaneously delivered PCLX-001 monotherapy demonstrated dose-dependent anticancer activity and produced complete remissions after five daily 50 mg/kg doses. NMT expression provides independent prognostic information to refine existing clinical stratification, and NMT inhibition is a promising novel therapeutic strategy for AML.

#3044

The role of GPR110 in tumorigenicity, tumor cell dissemination, and cell cycle regulation in HER2+ breast cancer.

Raksha Bhat,1 Lanfang Qin,2 Carmine De Angelis,3 Suhas Vasaikar,2 Hariprasad Thangavel,1 Noor AbdulKareem,1 Bing Zhang,2 Rachel Schiff,2 Meghana V. Trivedi1. 1 _University of Houston, Houston, TX;_ 2 _Baylor College of Medicine, Houston, TX;_ 3 _University of Naples Federico II, Naples, Italy_.

Identification of novel drug targets to overcome anti-HER2 therapy resistance is an unmet need. Since G protein-coupled receptors (GPCRs) are known to cross-talk with the HER superfamily, it is possible that some GPCRs may signal to modulate the HER2 pathway. We have identified GPR110 (also known as ADGFR1, Adhesion G Protein-Coupled Receptor F1) as a potential target in HER2+ breast cancer (BC) based on its expression in tumorigenic and anti-HER2-resistant cells. We have also shown that GPR110 knockdown in HER2+ BC cells inhibits colony formation in soft agar assay, mammosphere formation, and invasion/migration in trans-well assay, suggesting its potential role in tumorigenesis and tumor cell dissemination. To further characterize the role of GPR110 in HER2+ BC, we generated stable cell lines of BT474 and SKBR3, two HER2+ BC cell line models, that overexpress GPR110 in Doxycycline (Dox)-inducible manner. GPR110 overexpression enhanced colony formation in soft agar assay, mammosphere formation, and number of Aldefluor+ tumorigenic cells, substantiating its role in tumorigenesis. In addition, Dox-induced GPR110 overexpression increased invasion/migration potential of clones in trans-well assay, further supporting its role in cancer cell dissemination. To understand the mechanism of GPR110-mediated tumorigenesis and tumor cell dissemination in HER2+ BC, we carried out transcriptomic and proteomic analysis of selected cell lines (with or without Dox) using RNAseq and RPPA respectively. This analysis uncovered a previously unanticipated role of GPR110 in cancer cell regulation and maintaining quiescence. Overexpression of GPR110 by Dox treatment led to downregulation of various cell cycle pathways and targets such as E2F targets, G2M checkpoint pathway and MYC targets at the transcriptomic level. Validation of RNA-Seq and RPPA candidates demonstrated lower number of proliferative Ki67+ cells and reduced NF-kB staining, and inhibited STAT3 phosphorylation. In support of these results, GPR110 overexpression also led to cell cycle arrest at G0/G1 phase, when analyzed by the DNA content analysis with propidium iodide using flow cytometry. Ongoing in vivo studies will further elucidate the exact and overall role of GPR110 in HER2+ BC. In summary, our results demonstrate a previously uncovered role of GPR110 in tumorigenesis and metastasis as well as cell cycle regulation in HER2+ BC.

#3045

PEPT1 as a tumor promoter and novel drug target to treat pancreatic cancer.

Bradley Schniers, Yangzom Bhutia. _Texas Tech University Health Science Center, Lubbock, TX_.

Pancreatic ductal adenocarcinoma (PDAC) is the most lethal of all cancers. Gemcitabine is currently used as a first line therapy but with a very low success rate. With this projection in mind, it is imperative to discover a more effective treatment for PDAC. Our lab works on the Peptide Transporter 1 (PEPT1)/SLC15A1. PEPT1 is typically expressed in the small intestine, kidney, and bile duct and transports a wide array of di- and tri-peptides and peptide-like drugs. Literature evidence has shown PEPT1 to be upregulated in some PDAC cell lines. Our aim here was to first corroborate the literature evidence, then investigate if PEPT1 is a tumor promoter and finally understand the mechanistic aspect of its upregulation. Using Real-time PCR and Western blotting, we checked the expression of PEPT1 mRNA and protein, respectively in pancreatic cancer cell lines vs. normal pancreatic cell lines. It was interesting to note that PEPT1 was selectively and significantly upregulated only in cancer cells. We further corroborated the data using patient-derived xenografts (PDXs) and tumor samples from PDAC patients. Additionally, we performed radiolabeled glycylsarcosine (3H-Gly-Sar) uptake to check the functionality of PEPT1. The results of 3H-Gly-Sar uptake correlated with the protein expression in the cancer cells. To further investigate if PEPT1 could be a tumor promoter we performed a CRISPR/Cas9 mediated knockout of PEPT1. AsPC-1 was used as the model cell line. Following PEPT1 knockout, we injected AsPC-1/Control and AsPC-1/PEPT1-knockout cells in athymic nude mice and monitored the tumor growth. Interestingly, we found that the absence of PEPT1 significantly reduced the tumor growth suggesting its role as a tumor promoter. It is known that tumor cells generate large amounts of lactic acid by accelerating aerobic glycolysis. This metabolic switch leads to cellular acidification, but tumor cells circumvent this obstacle by releasing lactate and H+into the extracellular medium. Since PEPT1 is a proton-coupled transporter we hypothesized that lactate regulates its expression. To test this, we performed RT-PCR to check the expression of Pept1 in Gpr81/wildtype and Gpr81/knock-out intestinal samples. Since GPR81 is a lactic acid receptor we hypothesized if its activation by lactate would in turn activate PEPT1. Surprisingly, we found that lactate/GPR81 complex actually regulates PEPT1 expression. Further, we found that lactate also increases the expression of MMP-1, which will then break down the extracellular matrix protein collagen into large peptides. These peptides could be further hydrolyzed into dipeptides by DPP-IV/CD26, which could be the mechanism to generate dipeptide substrates for PEPT1 and thereby couple the process to amino acid nutrition for pancreatic cancer cells. In summary, PEPT1 promotes pancreatic cancer and therefore could be used as a drug target to treat PDAC. However, the molecular mechanisms needs further elucidation.

#3046

Targeting N-myristoylation in B cell lymphomas as a therapeutic strategy.

John R. Mackey,1 Erwan Beauchamp,1 Megan C. Yap,1 Aishwarya Iyer,1 Maneka A. Perinpanayagam,1 Krista M. Vincent,1 Abass M. Al-Momany,1 Ryan J. Heit,1 Jacky Y. Sim,1 Raymond Lai,1 Wei-feng Dong,1 Manikandan Lakshmanan,2 Anandhkumar Raju,2 Vinay Tergaonkar,2 Soo Yong Tan,2 Soon Thye Lim,3 Lynne M. Postovit,1 Kevin D. Read,4 David W. Gray,4 Paul G. Wyatt,4 Luc G. Berthiaume1. 1 _University of Alberta, Edmonton, Alberta, Canada;_ 2 _Institute of Molecular and Cell Biology, Proteos, Singapore;_ 3 _National Cancer Centre Singapore, Singapore;_ 4 _University of Dundee, Dundee, United Kingdom_.

Treatment of aggressive lymphoma is toxic, expensive, and a substantial proportion of patients relapse and die. There is an urgent need for more effective treatments. Myristoylation is required for biological activity of >200 intracellular proteins. N-myristoyltransferases (NMTs) transfer the fatty acid myristate to N-terminal glycine residue; there are two isoforms, NMT1 and 2. Since they are critical to intracellular signaling, NMTs are potential anti-cancer targets. We tested a novel potent pan-NMT inhibitor, PCLX-001, in B cell lymphoma cell lines. In vitro assays included cell viability, immunoblotting, and metabolic labeling of lymphoma cell lines. Immunohistochemistry was performed on formalin fixed paraffin embedded lymphoma specimens from patients. In vivo experiments included cell line derived murine xenografts and a patient derived mouse xenograft treated with increasing concentrations of PCLX-001. PCLX-001 selectively killed lymphoma cells, while sparing normal cells in vitro and in 3 mouse xenograft models, eradicating tumors in two of these models including a patient-derived xenograft from a R-CHOP refractory lymphoma patient. While NMT2 is overexpressed in some cancers, loss of NMT2 expression is common in numerous cancers and occurs at the highest prevalence in lymphomas, where it is independently linked to a worse prognosis. This NMT2 suppression occurred through epigenetic mechanisms and may account for lymphoma sensitivity to NMT inhibition. The global myristoylation of lymphoma cell proteins, including that of the protein tyrosine kinase oncogene Src, is profoundly inhibited by PCLX-001. Loss of Src myristoylation is accompanied by loss of Src activity and may account for loss of prosurvival signals causing lymphoma cell death. Targeting NMT2 deficient B cell lymphoma with a pan-NMT inhibitor suppresses the residual NMT1 function provides a novel, selective, and effective therapeutic strategy.

#3047

Connective tissue growth factor: Novel therapeutic target for non-small cell lung cancer.

Priyanka Chaudhary,1 Marie-Liesse Labat,2 Guangfang Wang,1 Mark Onaitis1. 1 _University of California San Diego (UCSD), La Jolla, CA;_ 2 _Walter and Eliza Hall Institute of Medical Research, Parkville, Australia_.

Lung cancer is the leading cause of cancer deaths worldwide. Non-small cell lung cancer (NSCLC), which accounts for 85% of all lung cancer cases, includes adenocarcinoma, squamous cell lung carcinoma and large-cell lung carcinoma. Despite recent advances in targeted therapies for adenocarcinoma, they are effective in only a small percentage of patients while there are no targeted therapies available for squamous cell lung cancer. Therefore, it is critical to identify novel intervention strategies against NSCLC. In the present study, we have identified connective tissue growth factor (CTGF) as a possible therapeutic target for NSCLC. CTGF is an extracellular-matrix (ECM) associated protein, which is involved in cellular processes like cell adhesion, migration, proliferation, differentiation, survival, apoptosis and senescence. CTGF has been associated with both oncogenic as well as tumor suppressive activities in context of different cancer types. The function of CTGF in lung cancer pathogenesis remains controversial. We performed immunohistochemistry on tissue microarray containing 60 cases, which clearly suggested that CTGF expression increases in lung carcinoma. More specifically, among other subtypes, squamous cell lung cancer cases expressed more CTGF compared to their matched normal tissues. To determine the functional role of CTGF in NSCLC, we employed both genetic and pharmacologic approaches to inhibit CTGF activity. Our results clearly demonstrated that CTGF inhibition by both lentiviral knockdown as well as monoclonal antibody against CTGF (FG-3019) caused marked inhibition in cell proliferation, colony formation and xenograft growth in immunodeficient mice. We have also found that CTGF knockdown causes cell cycle arrest and apoptosis. Furthermore, we tested the efficacy of FG-3019 along with drugs like cisplatin and gemcitabine in patient-derived xenograft (PDX) model of squamous cell lung cancer. Our results demonstrated that CTGF inhibition enhances the effects of gemcitabine chemotherapy in PDX model of squamous cell lung carcinoma. These results clearly suggested that CTGF has a profound effect on lung tumorigenesis, as its inhibition not only causes attenuation in tumor growth but also potentiates the chemotherapeutic sensitivity of commonly used drugs for NSCLC. We have also elucidated that tumor hypoxia and Yap signaling are responsible for CTGF up-regulation in lung carcinoma. Overall, our study underscores the significance of CTGF inhibition in non-small cell lung carcinoma and offers the potential use of FG-3019 for its treatment.

#3048

Targeting protein deglycosylation as a newly identified vulnerable point in melanoma.

Victor J.T. Lin,1 Ashwini Zolekar,1 Nigam M. Mishra,1 Yin Ying Ho,2 Hamed S. Hayatshahi,1 Abhishek Parab,3 Rohit Sampat,1 Xiaoyan Liao,4 Peter Hoffmann,5 Jin Liu,1 Kyle A. Emmitte,1 Yu-Chieh Wang1. 1 _University of North Texas Health Science Center, Fort Worth, TX;_ 2 _The University of Adelaide, Adelaide, Australia;_ 3 _Purdue University, West Lafayette, IN;_ 4 _University of Rochester Medical Center, Rochester, TX;_ 5 _University of South Australia, Adelaide, Australia_.

As an enzyme that removes N-glycans from glycopeptides, N-glycanase 1 (NGLY1) deglycosylates denatured glycoproteins and allows proteasome-mediated protein degradation to efficiently occur. Although NGLY1 is known for this pivotal function, information regarding the responses of human cancer and normal cells to NGLY1 suppression is limited. Here, we examined how NGLY1 expression affects viability, tumor growth, and responses to therapeutic agents in melanoma cells and an animal model. Molecular mechanisms contributing to NGLY1 suppression-induced anticancer responses were revealed by systems biology and chemical biology studies. Using computational and medicinal chemistry-assisted approaches, we established novel NGLY1-inhibitory small molecules. Compared with normal cells, NGLY1 was upregulated in melanoma cell lines and patient tumors. NGLY1 knockdown caused melanoma cell death and tumor growth retardation. Targeting NGLY1 induced pleiotropic responses, predominantly stress signaling-associated apoptosis and cytokine surges, which synergize with the anti-melanoma activity of chemotherapy and targeted therapy agents. Pharmacological and molecular biology tools that inactivate NGLY1 elicited highly similar responses in melanoma cells. Unlike normal cells, melanoma cells presented distinct responses and high vulnerability to NGLY1 suppression. Our work demonstrated the significance of NGLY1 in melanoma cells, provided mechanistic insights into how NGLY1 inactivation leads to eradication of melanoma with limited impact on normal cells, and suggested that targeting NGLY1 represents a novel anti-melanoma strategy with the opportunity for a broad therapeutic window.

#3049

Hornerin as a novel therapeutic target for pancreatic cancer.

Julien Dimastromatteo, Kimberly A. Kelly. _University of Virginia, Charlottesville, VA_.

Background and aim - Through our functional proteomics screening efforts, we recently identified hornerin (HRNR) as a protein expressed on the cell surface of both cancer and cancer blood vessels. Hornerin is a member of the S100 family of proteins, a group of calcium binding proteins involved in the maintenance of calcium homeostasis, as well other fundamental cellular processes and signaling cascades. Several family members have been implicated in a variety of cancers, with evidence demonstrating that altered expression of S100 proteins was associated with tumor progression and prognosis. Therefore, we hypothesized that HRNR may represent an important therapeutic target for cancer.

Method - De-identified human tumor microarray specimens underwent standard immunohistochemistry (IHC) procedure to determine HRNR expression in various type of cancer. Standard westernblot were also used to assess HRNR expression in eleven epithelial PDAC cell lines. Analysis of HRNR expression in cellular compartments was performed after differential centrifugation of PDAC cell lysates. Cell surface HRNR expression was further validated by immunofluorescence on non-permeabilized cells. Additionally, cells from 4 different renal carcinoma (RCC) PDX models were infected with human HRNR shRNA lentiviral particles in order to silence HRNR expression. Those cells were then injected subcutaneously into immunocompromised nude mice and assessed for tumor growth. Molecular mechanism of HRNR was evaluated via western blot. Effect of blocking HRNR on cancer in vitro and in vivo using a HRNR specific antibody was determined.

Results - Based on IHC scoring, HRNR expression has been observed in twenty-one different types of cancer among which are PDAC and renal cancer. Expression of HRNR in tumor cells and on the cell surface of cancer was confirmed through cell fractionation and immunofluorescence. Validation of HRNR as an important cancer therapeutic target was demonstrated through siRNA knockout studies. PDX models of renal cancer that were devoid of HRNR expression were unable to form tumors in vivo. As members of the S100 family have been shown to activate signaling pathways important for proliferation and migration, we hypothesized that HRNR would be able to activate proteins in these signaling cascades. Western blot analysis of cells treated with recombinant HRNR revealed the activation of AKT and ERK and the stabilization of EGFR. Therefore, we evaluated the efficacy of a HRNR targeted antibody as a therapeutic. Treatment with the HRNR targeted antibody resulted in a decrease in cell proliferation and complete cell death that was HRNR specific.

Conclusion - HRNR plays an important role in cancer and represents a novel therapeutic target.

#3050

Multimeric IgM antibodies targeting DR5 are potent and rapid inducers of tumor cell apoptosis and cell death in vitro and in vivo.

Beatrice Wang, Tasnim Kothambawala, Ling Wang, Avneesh Saini, Ramesh Baliga, Angus Sinclair, Bruce Keyt. _IGM Biosciences, Mountain View, CA_.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its functional receptors, death receptor 4 (DR4) and death receptor 5 (DR5) are attractive anti-tumor targets because of their ability to directly induce apoptosis in cancer cells. Though several agonistic IgG antibodies targeting DR5 have demonstrated evidence of preclinical efficacy, little evidence of clinical efficacy was observed likely due to insufficient receptor crosslinking in the tumor microenvironment. We have developed a novel multivalent IgM antibody targeting DR5 that effectively clusters the receptor and rapidly induces apoptosis and tumor cell cytotoxicity. In Colo205 colorectal tumor cells, anti-DR5 IgM induced caspase activation was detected within 30 minutes and by 2 hours nearly all cells had undergone apoptosis, demonstrating a more rapid and greater magnitude of apoptotic induction than anti-DR5 IgG. In an in vitro cytotoxicity screen of solid and hematologic tumor cell lines, an IgM antibody targeting DR5 ranged from 100-fold to greater than 10,000-fold more potent compared to IgG targeting DR5, and IgM was potent on cell lines resistant to IgG crosslinking mediated cell death. In vivo, anti-DR5 IgM induced tumor eradication in the Colo205 model, tumor regression in the IgG-resistant colorectal HCT15 model, and significantly extended overall survival in the B-cell leukemia model Nalm-6 even though dosing was only within the first week of study. In all in vivo studies the IgM was significantly more efficacious than IgG targeting DR5. The DR5 targeting IgM antibody was also efficacious in large established Colo205 tumors up to 600 mm3 in volume and in colorectal patient-derived xenograft (PDX) models as well. When administered in conjunction with standard of care chemotherapy Irinotecan, anti-DR5 IgM induced durable tumor regression in the HCT15 colorectal carcinoma model with 14 out of 30 animals from 2 separate studies tumor free at the end of study (day 71); no animals dosed with IgG were tumor free. In a less sensitive pancreatic model BxPC3, combination of standard of care Gemcitabine with anti-DR5 IgM resulted in an additive effect, while the IgG plus Gemcitabine efficacy was more modest and not statistically significant compared to Gemcitabine alone. Taken together, these results demonstrate that efficient IgM clustering of DR5 is significantly more potent in vitro and in vivo than IgGs and support the development of a human IgM antibody therapeutic targeting DR5 with the potential to treat both solid and hematologic tumors.

#3051

Targeting cdk12/13 re-sensitizes trastuzumab resistant her2+ breast cancers.

Francesca Vena,1 Max Aceti,1 Simon Bayle,1 Victor Quereda,1 Andrii Monastyrskyi,2 William Roush,2 Derek Duckett1. 1 _Moffitt Cancer Center, Tampa, FL;_ 2 _Scripps Research Institute, Jupiter, FL_.

Overexpression or amplification of the human epidermal growth factor receptor 2 (erbB2), which encodes for a receptor tyrosine kinase often referred to as HER2, occurs in about 20% breast cancer patients and is associated with aggressive disease and poor clinical outcome. The anti-HER2 therapy trastuzumab (a monoclonal antibody targeting HER2) has proven highly effective, increasing relapse free and overall survival for HER2 positive breast cancer patients. Nevertheless, acquisition of therapeutic resistance occurs frequently in advance disease. Recent studies have demonstrated that HER2 co-amplified genes may play a role in mechanisms of disease resistance. Cyclin dependent kinase 12 (CDK12) is among the genes that have been reported to be amplified in addition to HER2, and thus we aimed to investigate whether targeting CDK12 could be exploited as a therapeutic target and enhance and/or restore sensitivity in HER2 positive inhibitor resistant models of breast cancer.

We have developed an in-house, highly selective, potent, and orally bioavailable CDK12/13 inhibitor, which effectively blocks proliferation and induces apoptosis in a panel of breast cancer cell lines and acts in synergy with trastuzumab in HER2 treatment resistant cell lines. Furthermore, our lead compound significantly slows tumor growth in trastuzumab resistant patient derived xenograft (PDX) models (as a single agent) and importantly, provokes greater efficacy in combination with trastuzumab. We are beginning to identify the signaling pathways underlying HER2 positive treatment resistance and how inhibition of CDK12/13 thwarts these resistance mechanisms. Accordingly, we anticipate that the development of CDK12/13 inhibitors holds promise as an anti-breast cancer strategy and future treatment option for HER2 positive breast cancer patients with recurrent disease.

#3052

MGMT inhibition is associated with MAPK pathway inhibition and enhances Raf, MEK, ERK inhibitors and restores meaningful Temozolomide activity in melanoma.

George C. Bobustuc, Amin B. Kassam, Dmitry Bosenko, Deborah L. Donohoe, Richard A. Rovin, Santhi D. Konduri. _Aurora Health Care, Milwaukee, WI_.

Braf and MEK inhibition leads to limited survival gains in melanoma. In this research we show that, in vitro, MGMT controls all MAPK pathway effectors. We show that triple lock - upstream and downstream, along the MAPK pathway - effectively restores durable Braf and MEK inhibitor activity and significantly sensitizes melanoma to Temozolomide. The advantage of a multiple lock approach on the MAPK pathway is substantiated by the lack of signaling cross talk. We briefly discuss how this simple MGMT based regulatory paradigm could be immediately exploited in the care of melanoma patients. We also show how this strategy was exploited in two, surviving, metastatic (to include CNS disease) melanoma patients who had failed Braf and MEK inhibition (2 and 5 years ago) who now show stable or negligible residual disease burden while continuing on an intermittent, low treatment density, combination regimen

#3053

Development of novel cell-based bioassays for the development of biologics targeting LAG-3 and PD-1xLAG-3.

Jamison Grailer, Denise Garvin, Jim Hartnett, Frank Fan, Mei Cong, Zhi-jie Jey Cheng. _Promega Corp., Madison, WI_.

Programmed cell death protein 1 (PD-1) and Lymphocyte-activation gene 3 (LAG-3) are immune checkpoint receptors that bind to PD-L1/PD-L2 or MHCII, respectively, on antigen presenting cells or tumor cells. PD-1 or LAG-3 activation results in the inhibition of T cell receptor (TCR) signaling and reduced T cell activation. However, co-blockade of PD-1 and LAG-3 leads to a synergistic enhancement in T cell responses, suggesting functionally divergent inhibitory signaling mechanisms between the two receptors. Preclinical studies suggest that blocking LAG-3 re-activates the immune system to kill cancer cells, and co-blockade of both PD-1 and LAG-3 lead to a synergistic increase in tumor control and survival in preclinical models. Current methods for measuring the potency of LAG-3 and PD-1xLAG-3 blocking antibodies measure cytokine production from human PBMCs upon Staphylococcal Enterotoxin B (SEB) superantigen stimulation. These protocols are tedious, highly variable, and require the use of highly toxic superantigen. Herein, we describe the development of reporter-based bioassays for the development of LAG-3 and PD-1xLAG-3 combination-targeted biologics. Engineered T effector cell lines expressing LAG-3 alone or in combination with PD-1 are activated in an antigen-dependent manner by APC cell lines. The assays are specific for LAG-3 or PD-1xLAG-3-targeted biologics and are robust as demonstrated by Design of Experiments analyses. In addition, the assays can be used to measure the relative potencies of test samples compared with reference sample in a detection range of 50% to 200% and show appropriate precision, accuracy and linearity. The assays can detect potency changes for heat-stressed samples, and are tolerant to human serum, demonstrating the potential to be further developed into neutralizing antibody (NAb) assays. Therefore, these bioassays can serve as valuable tools for the development of biologics targeting LAG-3 and PD-1xLAG-3.

#3054

Cell surface targets in head and neck cancer.

Maria Kondratyev,1 Aleksandra Pesic,1 Azin Sayad,1 Troy Ketela,1 Natalie Stickle,1 Carl Virtanen,1 Jason Moffat,2 Laurie Ailles,1 Marianne Koritzinsky,1 Brad Wouters1. 1 _University Health Network, Toronto, Ontario, Canada;_ 2 _University of Toronto, Toronto, Ontario, Canada_.

HNSCC is 6th most common malignancy in the world. Despite advances in diagnosis and treatment, the survival rates remain low due in large part to metastatic disease. The underlying biology associated with metastatic disease and poor outcome in HNSCC remains unclear. Importantly, metastatic cells acquire new properties that permit them to invade surrounding tissues and seed metastasis at distant sites. While these acquired properties contribute to aggressiveness of metastatic cancer and interfere with success of therapies, they can also potentially be exploited to target metastatic cells selectively, sparing toxicity in normal tissues. We used functional genomic technologies to identify new potential therapeutic targets for advanced disease in HNSCC. These targets were identified by conducting whole genome shRNA screens in matched sets of cell lines derived from primary tumors and their respective metastatic sites, with the goal of identifying genes that become essential for cell survival only following metastasis. Since hypoxia is an important attribute of aggressive and therapy resistant subpopulations of HNSCC tumor cells, we also aimed to identify genes that became essential when cells are exposed to hypoxia. We are particularly interested in the identification of contextual synthetic lethal oncogenes expressed on the cell surface, as those are easily targetable by therapeutic antibodies. To identify these targets, we performed high-throughput flow cytometry screening that enables evaluation of 370 validated cell surface antibodies. Cell surface targets differentially expressed in metastatic lines included CECAM and CCR6 that were previously reported to be implicated in metastasis and tumor progression as well as Thy1, a known marker of stem cells involved in regulation of cell adhesion. Cell surface targets induced under hypoxic conditions across the cell lines included CA9, an enzyme that is known to regulate pH in hypoxic cells and be associated with tumor progression, as well as CD338, CD264 and CD312, that were previously associated with stemness in a few models of cancer. Interestingly, the described proteins were also found to be differentially essential in the shRNA screens, highlighting their functional importance in tumor progression and hypoxia survival. We are currently investigating the role of these proteins in HNSCC metastasis utilizing our unique collection of matched pairs of HNSCC lines from multiple patients. Moreover, we are utilizing our pipeline of patient derived HNSCC xenografts to test the effect of knocking down the described genes in patient tumors. We are also testing the expression of selected hits in histological sections of patient tumorsthe 400-patient TMA by immunohistochemistry looking for correlation with tumor grade, aggressiveness, levels of hypoxia as well as presence/absence of metastasis in the patient.

#3055

Genome-wide CRISPR/Cas9 screen for the identification of novel YAP1/TAZ modulators.

Jan Naujoks, Lisette Potze, Anna Anurin, Julia Kuehnlenz, Ralf Lesche, Atanas Kamburov, Ekaterina Nevedomskaya, Andreas Steffen, Martin Lange, Barbara Nicke. _Bayer AG, Berlin, Germany_.

Aberrant activation of the Hippo pathway effectors YAP1/TAZ promotes cell proliferation and tumorigenesis. To identify novel regulators of YAP1/TAZ as a possible means to treat cancer, we established a novel, FACS-based screening system monitoring YAP1/TAZ activity in MDA-MB-231 breast cancer cells. Using these cells, we performed a pooled genome-wide CRISPR/Cas9 knockout screen. We identified approximately 50 genes potentially activating YAP1/TAZ with functions in the Actin Cytoskeleton signaling, p53 signaling, cell polarity or ER stress, amongst others. Moreover, we identified about 30 potential targets which when knocked out induce activity of YAP1/TAZ. The list of hits included genes known to affect the YAP1/TAZ activity such as AJUBA, LATS2 and TEAD, demonstrating the validity of the screen. Functional validation of the novel potential YAP1/TAZ modulators will aid to the further understanding of YAP1/TAZ biology and may open the door to new therapeutic avenues for targeting YAP1/TAZ in cancer.

#3056

Development of an integrated CRISPR interference system targeting ΔNp63 to treat lung and esophageal squamous cell carcinoma.

Masakazu Yoshida,1 Etsuko Yokota,1 Tetsushi Sakuma,2 Nagio Takigawa,1 Toshikazu Ushijima,3 Takashi Yamamoto,2 Yoshio Naomoto,1 Takuya Fukazawa,1 Tomoki Yamatsuji1. 1 _Kawasaki Medical School, Okayama, Japan;_ 2 _Hiroshima University, Hiroshima, Japan;_ 3 _National Cancer Center Research Institute, Tokyo, Japan_.

TP63 encodes two different transcripts that have opposite functions in transcriptional control. One transcript encodes TAp63 that is functionally similar to the tumor suppressor TP53. The other transcript, ΔNp63, lacks the transcription-activating domain of TAp63 and has been proposed to function as a potent oncogene in squamous cell carcinomas. In this study, we have developed an integrated CRISPR interference system to selectively suppress ΔNp63 and we have termed this system CRISPRiΔNp63. We engineered the CRISPRi using tandemized guide RNA expression cassettes to target the 50 to 100 bp downstream region of the transcription start site of ΔNp63 and we used inactivated Cas9 linked to the transcription repression module Krüppel-associated box repressor domain. The plasmid vector harboring CRISPRiΔNp63 repressed ΔNp63 transcriptional activity in lung and esophageal squamous cell carcinoma cells that express ΔNp63. Moreover, an all-in-one adenoviral vector containing tandemized gRNAs and the dCas9/KRAB expression cassette, Ad-CRISPRiΔNp63, suppressed ΔNp63 expression in squamous cell carcinomas cells. Ad-CRISPRiΔNp63 effectively decreased cell proliferation and colony formation and induced apoptosis in lung and esophageal squamous cell carcinoma cells in vitro. Moreover, the all-in-one vector significantly inhibited tumor growth in a lung squamous cell carcinoma xenograft mouse model in vivo. These results indicate that ΔNp63 suppression by CRISPRiΔNp63 might be an effective strategy for the treatment of lung and esophageal squamous cell carcinoma.

#3057

Synergistic antiproliferative activity between anticancer drugs and RAD51 inhibitors IBR2 and IBR120.

Peter J. Ferguson, Morgan Black, Rene Figueredo, Mark D. Vincent, James Koropatnick. _London Regional Cancer Program, London, Ontario, Canada_.

The inherent genetic instability of cancer cells and the dependence of many tumor types on oncogenic drivers contribute selectivity of anticancer agents against tumor cells. However, that selectivity is limited, and toxicity to normal cells remains a major limitation to the success of chemotherapy. Aiming to increase selectivity by exploiting cancer cell genetic instability, we demonstrated that the small molecule IBR2 (an inhibitor of the DNA repair protein RAD51) enhanced cytotoxicity of numerous anticancer drugs, including agents that do not directly target DNA (J Pharmacol Expt Ther, 364: 46-54, 2018). Given the potential importance of combining IBR2 with anticancer treatment to increase therapeutic index, IBR2 was tested in combination with other untested agents against a panel of tumor cell lines. In addition, studies were expanded to include IBR120 [(R)-3-(2-(benzylsulfonyl)isoindolin-1-yl)-1h-indole], a new small molecule RAD51 inhibitor that is reportedly more selective against cancer cell lines (Euro J Med Chem 96: 196-208, 2015). Four-day drug exposures were conducted in 96-well plates. Relative cell density, determined using vital stains (alamarBlue©, neutral red), was reported as a percent of the fluorescence/absorbance of control cultures. Cell lines were representative of tumors from breast (MCF-7, SK-BR-3), prostate (LNCaP, DU145), colon (HT-29), stomach (N87), skin (SK-MEL-5), and lung (A549b, H1650, H1975), including osimertinib-resistant derivatives of the non-small-cell lung cancer (NSCLC) cell lines. The chemotherapy agents included inhibitors of epidermal growth factor receptor (EGFR) family (osimertinib, afatinib, lapatinib), Bcr-Abl (imatinib), B-raf (vemurafenib), multiple tyrosine kinases (regorafenib), sex steroid receptors (4-OH-tamoxifen, enzalutamide), and microtubules (vincristine). Both IBR2 and IBR120 decreased the concentration of drugs that inhibited proliferation by 50% (IC50) by up to 90%, indicating synergy between the agents. However, there were a small number of combinations in which no synergy was observed, indicating selectivity for this interaction. In the NSCLC cell lines, IBR2, IBR120, osimertinib, gefitinib, and afatinib decreased the cellular content of RAD51 by over 80% within 24 h (western blot with antibody 3C10), at concentrations at or lower than their IC50 value. The contribution of this decrease in RAD51 to synergy is being investigated. Studies are ongoing to determine the effect of IBR120 on growth of explants of the NSCLC cell line H1650 in mice, after which the combination of IBR120 and osimertinib will be tested in vivo. The ability of IBR2 and IBR120 to enhance antiproliferative activity of a wide variety of anticancer agents and their potential selectivity for cancer cells make possible their future use as systemic therapy potentiators to improve clinical outcomes.

#3058

Recharacterizing the FDA approved drug pyrvinium pamoate as a clinically relevant HuR inhibitor in pancreatic ductal adenocarcinoma.

Christopher W. Schultz, Teena Dhir, Samantha Z. Brown, Saswati Chand, Wei Jiang, Grace A. McCarthy, Alex O. Haber, Charles J. Yeo, Austin Goetz, Avinoam Nevler, Oloruntoba Bolaji, Jonathan R. Brody. _Thomas Jefferson, Philadelphia, PA_.

HuR is an RNA binding protein involved in a coordinated cellular survival response to stressors. Upon insults such as chemotherapy, HuR translocates from the nucleus to the cytoplasm where it binds the 3'UTR of target mRNAs. HuR's interaction with target mRNAs leads to the upregulation of target genes and ultimately treatment resistance. This is particularly relevant in the case of pancreatic ductal adenocarcinoma (PDA). PDA is highly resistant to radiotherapy and standard chemotherapy such as FOLFIRINOX or gemcitabine/nab-paclitaxel. Using a tumor microarray (TMA), we found 79% of patient tumor samples (n=70) were positive for active cytoplasmic HuR, while little to no cytoplasmic localization was detected in normal tissue. In addition, HuR CRISPR knockout cell lines have a xenograft lethal phenotype. The aim of our current study is to target HuR by re-purposing the anti-helminthic, FDA approved small molecule pyrvinium pamoate (PP) to inhibit HuR's translocation and sensitize PDA cells to concurrent therapies. PP has been shown in bladder cancer to inhibit the translocation of HuR in vitro and in vivo. We have reproduced this in multiple PDA cell lines and have shown impressive drug potency with IC50s as low as 38nM in 2D cultures of PDA cell lines and PDX lines and 16nM in a 3D mouse PDA organoid model. We have demonstrated that inhibition of HuR translocation is likely to occur through secondary effectors AMPK and CDK1. We have also demonstrated that PP's inhibition of HuR function may be through direct inhibition of target binding. In comparison to other published HuR inhibitors PP inhibits the binding of HuR to targets more potentlt with nanomolar IC50's. We confirmed this work through HuR RNA Immunoprecipitation experiments and determined that PP inhibited the ability of HuR to bind target mRNA. We generated HuR deficient CRISPR lines to and demonstrated that lack of HuR sensitizes PDA cells to various therapeutics, an effect which is exacerbated in physiologically relevant low glucose settings. We next demonstrated that PP can synergize with several therapeutics including the CDK4/6 inhibitors Abemaciclib and Palbociclib in PDA cells and that this synergy is increased in low glucose setting. This synergistic effect is ameliorated in HuR deficient CRISPR cell lines, indicating that PP achieves this synergistic potential through the inhibition of HuR. We performed targeted phosphoproteomics and found that PP robustly inhibited critical mTOR pathway members as well as validating previous reports that it can inhibit the WNT pathway. Finally, we have demonstrated that PP has a dose dependent effect on PDA tumor growth in vivo with IP and PO dosing regimens. This work supports the recharacterization of PP as a potentially effective therapeutic agent for the treatment of PDA. Early phase clinical trials of PP in human subjects are being planned for 2019.

#3059

**Identification and validation of an Aspergillus** nidulans **secondary metabolite derivative as an inhibitor of the Musashi1-RNA interaction.**

Lan Lan,1 Jiajun Liu,1 Amber Amith,1 Carl Appelman,1 Xiaoqing Wu,1 Ke Li,1 Anuradha Roy,1 Ragul Gowthaman,1 John Karanicolas,2 Amber Somoza,3 Clay Wang,3 Berl Oakley,1 Roberto De Guzman,1 Kristi Neufeld,1 Liang Xu1. 1 _Univ. of Kansas, Lawrence, KS;_ 2 _Fox Chase Cancer Center, Philadelphia, PA;_ 3 _University of Southern California, CA_.

RNA-binding protein Musashi-1 (MSI1) is a key regulator of several stem cell populations. It is involved in tumor proliferation and maintenance, and it regulates target mRNAs at the translational level. The known mRNA targets of MSI1 include Numb, APC and P21WAF-1, key regulators of Notch, Wnt signaling and cell cycle progression, respectively. In this study, we aim to identify small molecule inhibitors of MSI1-mRNA interaction thus blocking the growth of cancer cells with high levels of MSI1. Using a fluorescence polarization (FP) assay, we screened small molecules from several chemical libraries for those that disrupt the binding of MSI1 to its consensus RNA binding site. Two clusters of hit compounds have been identified, one of which is composed of derivatives of secondary metabolites from Aspergillus nidulans. The top hit, Aza-9, from this cluster was further validated by surface plasmon resonance and nuclear magnetic resonance which revealed that Aza-9 binds to MSI1 directly, in the RNA binding pocket. Next, we tested whether Aza-9 has anti-cancer potential in cells and whether such activities work through MSI1. Due to poor cellular uptake, we used PEGylated liposomes to facilitate Aza-9 entry into the cells. Aza-9-lipo inhibits colon cancer cell proliferation, induces apoptosis and autophagy, and down-regulates Notch/Wnt signaling in colon cancer cell lines. In conclusion, we identified a series of potential lead compounds for inhibiting MSI1 function while establishing a framework for identifying small molecule inhibitors of RNA binding proteins using FP-based screening methodology.

#3060

YAP1 induces STAT3 phosphorylation & combined blockade enhances chemotherapeutic efficacy.

Masahiro Shibata,1 Akira Ooki,1 Yoshikuni Inokawa,1 Evgeny Izumchenko,1 Enrico Munari,2 Giuseppe Bogina,2 Edward Gabrielson,1 Anju Singh,3 Mohammad Obaidul Hoque1. 1 _Johns Hopkins Univ. School of Medicine, Baltimore, MD;_ 2 _Sacro Cuore Don Calabria Hospital, Negrar, Verona, 37024, Italy;_ 3 _Johns Hopkins University School of Public Health, Baltimore, MD_.

Recent studies have identified transcriptions factors YAP1 and STAT3, as therapeutic targets in cancer, but efforts targeting either YAP1 or STAT3 pathway have met with limited success. Our study is focused on understanding the cross-talk between YAP1 and STAT3 signaling, impact of this cross-talk on lung tumorigenesis, drug resistance and blocking this pathway as a potential therapeutic strategy in lung adenocarcinoma (LUAD). Mechanistically, we demonstrate that YAP1 promotes the phosphorylation of STAT3 through IL-6 upregulation. In clinical LUAD samples, a positive correlation between YAP1 and phosphorylated STAT3 protein as well as an association between YAP1 and IL-6 mRNA was observed. Pre-clinically, genetic and pharmacologic dual inhibitions of YAP1 and STAT3 showed decreased cell growth and increased sensitivity to chemotherapy through the attenuation of cancer-stemness like features. Although verteporfin and S3I-201 were employed as YAP1 and STAT3 inhibitors respectively, verteporfin was found to suppress not only YAP1 but also STAT3, and their combination induced synergistic cytotoxic effects. Furthermore, addition of verteporfin and S3I-201 to standard-of-care chemotherapy regimen significantly inhibited tumor growth in patient-derived xenograft mouse models and attenuated in vivo expansion of malignant stemness. These findings provide the conceptual framework for combined targeting of YAP1 and STAT3 in LUAD.

#3061

Role of ID4 (inhibitor of DNA binding protein 4) in FKBP52-AR pathway.

Shravan Kumar Komaragiri. _Clark Atlanta University, Atlanta, GA_.

Introduction: Inhibitor of DNA binding/differentiation protein 4 (ID4), acts as a dominant negative regulator of basic helix loop helix (bHLH) family of transcription factors. In normal prostate epithelial cells, ID4 collaborates with androgen receptor (AR) and p53 to exert its tumor suppressor activity. Previous studies have shown that ID4 promotes tumor suppressive function of AR whereas loss of ID4 results in tumor promoter activity of AR through selective interaction with FKBP52. Pharmacological inhibition of FKBP52, by MJC13, attenuated the tumor growth, weight, and volume of LNCaP cells lacking ID4(L(-)ID4) xenografts. The L(-)ID4 cells also gains CRPC phenotype through FKBP52-mediated AR signaling. In this study we investigated the role of ID4 in pharmacological inhibition of FKBP52 with GMC1 (Initial hit molecule by in-silico screening with the most potent inhibition of FKBP52) in regulating the AR pathway.

Experimental procedures: GMC1 drug treatments in LNCaP(-)ID4 and CW22RV1 generated xenograft tumors were further analyzed for tumor volume and tumor weight. We also performed immunohistochemistry on GMC1 treated xenografts to determine AR, PSA, FKBP51 and FKBP52 protein expression. Our in vivo studies with GMC1 drug treatments in castrated male SCID mice injected with LNCaP-ID4 and CW22RV1 cells showed decreased tumor size and volume when compared to untreated mice tumors.

Results: IHC results on GMC1 treated LNCaP(-)ID4 and CW22RV1 generated xenograft tumors showed no change in ID4 expression with decrease expression of AR, PSA, FKBP51, FKBP52 and KI67 when compared to untreated xenograft tumors. These results suggest that inhibition of FKBP52-regulated AR activity (via GMC1) demonstrated a significant reduction in the tumor volume of subcutaneous xenografts in vivo. Overall, ID4 appears as a potential tumor suppressor gene that selectively regulates AR activity through direct interaction with FKBP52, and its loss, promotes CRPC through FKBP52‐mediated AR signaling.

Conclusions: In conclusion, we demonstrate that loss of ID4 in LNCaP cells elevates FKBP52 levels, transcriptionally potentiates AR activity which in turn then leads to progression of androgen-independent prostate cancer. GMC1 is a novel inhibitor that inhibits the binding of FKBP52 to AR thereby prevents the AR transcriptional hyperactivity in PCa

Acknowledgement: These studies were supported by DOD, Grant # W81XWH-17-1-0437

#3062

PLK1: a new therapeutic target in mucinous ovarian carcinoma.

Roberta Affatato,1 Rosaria Chilà,1 Monica Lupi,1 Valentina Restelli,1 Mark Erlander,2 Laura Carrassa,1 Giovanna Damia1. 1 _IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy;_ 2 _Trovagene Oncology, San Diego, CA_.

Epithelial ovarian cancer (EOC) is the most lethal gynaecological cancer. Amongst the five histotypes (high-grade serous, clear-cell, endometrioid, mucinous, and low-grade serous), mucinous epithelial ovarian cancer (mEOC) is a rare subset and represents approximately 3% of EOC. Beside these different histotypes are unique clinical entities with a different prognosis and response to therapy, they are still treated as a homogeneous group with a similar chemotherapeutic approach (platinum-paclitaxel combination), to which however mEOC is generally poor responsive. New specific and active therapeutic strategies are needed for mEOC.

To find new therapeutic options for mEOC, we have performed a high-throughput screening using a siRNA library directed against 719 human protein kinases in the mEOC MCAS cell line. After two independent screenings and validation experiments with specific esiRNAs, Polo-like kinase1 (PLK1), a mitotic serine/threonine kinase, was identified as the most significant kinase whose downregulation interfered with both cell proliferation and survival. Both PLK1 siRNA and nanomolar concentrations of commercially available inhibitors against PLK1 (onvansertib (NMS-P937) and volasertib) inhibited cell growth in other two additional mEOC cell lines (EFO-27 and JHOM-1). In addition, in the mEOC cell lines PLK1 inhibition was able to induce apoptosis and to block cells in the G2-M phase of the cell cycle. We then evaluated in vitro combinations between the PLK1 inhibitors and different chemotherapeutic drugs used in mEOC, including cisplatin and paclitaxel used in front line therapy in all the available mEOC cell lines. No synergism could be found in any of the cell lines, when both PLK1 inhibitors were combined with cisplatin. On the contrary, strong synergism could be observed in two out the three mEOC cell lines investigated when onvansertib and volasertib were combined with both paclitaxel and eribulin. On the basis of the strong in vitro synergistic effect between onvansertib and paclitaxel, this combination was tested in vivo in nude mice transplanted with MCAS cells. Onvansertib as single agent was inactive, while paclitaxel at the dose used was active. Interestingly enough, the combination of the two drugs was well tolerated and was much more active than paclitaxel single agent, with both a stronger tumor regression and more sustained tumor growth inhibition that translated in a longer survival time. These data suggest that onvansertib and paclitaxel could represent a new active therapeutic option in mEOC.

#3063

Over expression of protein kinase Nek1 and its modulation by Thioridazine in prostate cancer.

Vibha Singh, Praveen K. Jaiswal, Hari K. Koul, Xiuping Yu, Arrigo Benedetti. _LSU Health Science Center Shreveport, Shreveport, LA_.

Introduction: Prostate Cancer (PCa) is one of the most common urological malignancies in men in the United States. We have identified NIMA kinase NEK1 through a proteomic approach, that interacts and co-localizes with Tousled Like kinases (TLKs) and has a role in the DDR, upstream of ATR and Chk1. In the present study we have checked the NEK1 activity in PCa patient samples and tested Thioridazine (THD), an anti-psychotic drug and inhibitor of TLKs in inhibiting the activity of NEK1 in human and mice derived prostate cancer cell lines and xenografts derived from LNCaP cells.

Material & Methods: We have used tissue microarray (TMA) derived from patient samples and respective benign control. We have used a human prostate cancer cell lines: LNCaP, C4-2, C4-2b, 22Rv1, DU145 and PC3 and normal prostate cell line RWPE-1 as well as TRAMP-C2 cell lines derived from TRAMP mice. Clonogenic activity was measured by colony formation assay. Immunoblotting and immunohistochemistry was done for TLKs, NEK1, p-ATR, p-Chk1, cell cycle and apoptosis related protein in PCa cell lines and tumor tissues from xenografts. Xenografts derived from LNCaP and LNCaP-NEK1 mutant were used in present study. We have used THD (alone) or in combination with the anti-androgen drug bicalutamide in PCa cell lines as well as in xenografts mice model.

Results: We found a significant over expression of NEK1 activity as measured by p-NEK1 with PCa samples as compare to benign control (p<0.001) and also correlated with high Gleason score patients. Treatment with THD significantly impaired the colony formation efficiency in human PCa cells in vitro (even of androgen independent lines) as well in TRAMP-C2 cells. THD treatment halts the PCa cells in subG0 phase and affects the cyclin D1, p27, p21 levels and leads to apoptosis with induction of cleaved PARP and Caspase-3 activity. Further p-ATR and p-Chk1 axis was inhibited by treatment with THD and in combination with bicalutamide. Combination therapy of THD and Bicalutamide remarkably inhibited the tumor growth in LNCaP xenografts model while tumor growth in LNCaP xenografts with NEK1 mutant were inhibited by Bicalutamide treatment alone. Following DNA damage, addition of the THD impaired ATR and Chk1 activation, that leads to TLK1>NEK1>ATR>Chk1 pathway activation in PCa.

Conclusions: Our data indicated that NEK1 is over-expressed in PCa patients and its activity can be suppressed by treatment with THD (that inhibits its activating kinase, TLK1) in vitro and in vivo.

#3064

Epigenetic manipulation can sensitize AML cells to differentiate with ATRA.

Kalsi Heimdal, Bikalpa Ghimire, Edjay Ralph Hernandez, Heidi J. Gill Super. _Minot State University, Minot, ND_.

Human acute myeloid leukemia (AML), the most common type of acute leukemia, has approximately eight subtypes, many of which have poor prognosis. Most of these subtypes are associated with specific, recurrent chromosome translocations. These translocations result in fusion genes, which encode oncoproteins that block differentiation and promote proliferation of immature cells. The Myeloid Lymphoid Leukemia gene (MLL) is frequently involved in these translocations, and is considered a driver of the AML. Differentiation promoting drugs, such as all-trans-retinoic acid (ATRA) are an attractive alternative to cytotoxic chemotherapy, but few types of AML, other than acute promyelocytic leukemia (APL), respond to ATRA. We hypothesize that specific genes must be activated or inhibited in AML for drugs like ATRA to induce differentiation, and that gene activation or inhibition may be the result of specific epigenetic modification. We also hypothesize that AML with different genetic alterations may respond differently to specific epigenetic inhibitors. Our initial studies have focused on two MLL-driven AML cell lines, MV4;11 and THP-1, showing translocations t(4;11) and t(9;11), respectively, and one non-MLL related AML cell line, U937. MV4;11, THP-1, and U937 were treated with specific epigenetic modifiers, including tranylcypromine (TCP), an inhibitor of histone demethylase KMD1A/LSD1, N-acetyl-dinaline (CI-994), a general histone deacetylase inhibitor, and 3-deazaneplanocin A (DZNep), a S-adenosylhomocystein hydrolase inhibitor that depletes EZH2 and thus its associated H3K27me3 activity. Evidence for differentiation was noted in a variety of assays, including reduced cell proliferation as measured directly and by MTT assay, upregulation of myeloid-specific cell surface markers such as CD11b as measured by fluorescence-activated cell sorting (FACS), myeloid-related nuclear morphological changes noted with cytospin analysis of cells, and decreased AML-associated gene expression with qPCR. All three drugs seemed to sensitize U937 and THP-1 cells to differentiate when treated with ATRA. MV4;11 cells were sensitized to differentiate by CI-994 and DZNep with or without ATRA, although there was no indication of CD11b upregulation. However, MV4;11 was not sensitized by TCP to differentiate with or without ATRA. These experiments suggest epigenetic inhibitors may increase sensitivity to ATRA differentiation therapy, but since the two MLL cell lines responded differently the response may still be dependent on the specific MLL partner gene driving the AML.

#3065

Targeting androgen receptor and c-FLIP greatly reduces proliferation and invasion in castration resistant prostate cancer.

Akanksha Udhav Salunkhe, Harish C. Pal, Farrukh Afaq. _Univ. of Alabama at Birmingham, Birmingham, AL_.

Prostate cancer (PCa) is the most common cancer in males with an estimated 164,690 new cases and 29,430 deaths in the United States in 2018. Androgen receptor (AR) plays a vital role in the growth and function of normal prostate, however it is also a principal driver of prostate cancer (PCa). Androgen deprivation therapy, or blockage of androgen at the level of AR, remains the treatment options for this disease. Nevertheless, emergence of androgen resistance limits their therapeutic usefulness, and most patients with PCa progress from androgen-dependent status to invasive castration-resistant PCa (CRPCa). CRPCa continues to express AR and depends on functional AR signaling for growth. Studies have shown that c-FLIP, an anti-apoptotic protein, reduces the efficacy of AR-targeted therapy. In addition, the expression of c-FLIP correlates with progression to CRPCa. Currently, the management of CRPCa poses a great challenge, and there is no beneficial therapeutic treatment. Therefore, finding natural agents that can disrupt AR function, especially in conjunction with other small molecules that target c-FLIP, could dramatically improve the benefits of therapeutic intervention for CRPCa. We found that fisetin, a flavonoid, interacts with the ligand binding domain of the AR, and vorinostat, a HDAC inhibitor, increases the acetylation of Ku70 by disrupting the interaction of endogenous c-FLIP and Ku70 resulting in degradation of c-FLIP. We next determined whether fisetin in combination with vorinostat could offer therapeutic advantage against CRPCa. Treatment of CRPCa cells with fisetin and vorinostat in combination more effectively reduced cell growth and colony formation than individual agents. Combination treatment also resulted in increased apoptosis as revealed by cleaved PARP and modulation in Bcl2 family proteins. Furthermore, combination treatment more effectively reduced the protein expression of HDAC6, c-FLIP, and procaspase 8 than individual agents. Elevation of AR/AR signaling and induction of HDAC6 expression play an important role in cell migration and invasion by inducing epithelial-to-mesenchymal transition (EMT). We found that combination treatment more effectively reduced the invasion of CRPCa cells and inhibited EMT as revealed by decreased expression of mesenchymal marker proteins (N-cadherin, vimentin and fibronectin), and increased expression of E-cadherin, an epithelial marker protein. In summary, our findings demonstrate that combination treatment (fisetin plus vorinostat) exhibits more potent anti-proliferative, pro-apoptotic, and anti-invasive effects in CRPCa.

#3066

The expression of carcinoembryonic antigen-related cell adhesion molecule 6 and 8 in breast cancer.

Erina Iwabuchi, Yasuhiro Miki, Kiyoshi Takagi, Yoshiaki Onodera, Yukiko Shibahara, Takanori Ishida, Takashi Suzuki, Hironobu Sasano. _Tohoku University, Sendai, Japan_.

Carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 6 is a protein reported to be involved in tumor cell proliferation in breast cancer. The presence of intratumoral CEACAM8 positive neutrophils has been reported as an independent prognostic factor in both renal cell and hepatocellular cancer. In addition, CEACAM6 and 8 form a heterodimer; however, the possible interactions of these two proteins remain unclear in breast cancer. Therefore, in this study we first immunolocalized CEACAM6 and 8 in surgical pathology specimens of breast cancer. We examined 109 breast cancer and 20 metastatic breast cancer patients in this study. We then established MCF-7 cells (CEACAM6 positive) stably transfected with CEACAM8 and analyzed the interaction between CEACAM6 and CEACAM8 using proximity ligation assay (PLA) and further evaluated cell proliferation, invasion, and adhesion. Results of immunohistochemical analysis revealed that the number of CEACAM6 positive breast cancer cells was significantly higher in patients with low histological grade and stage also in CEACAM8 positive cells. The number of CEACAM6 and 8 double positive breast cancer cells was significantly higher in patients with low stage. In addition, CEACAM6 and 8 interaction was detected using the PLA through high expression of both in MCF-7 cells. Furthermore, their co-expression promoted cell adhesion of MCF-7 cells to the monolayer of endothelial cells but suppressed both cell invasion and proliferation. In metastatic breast cancer, CEACAM6 status was significantly higher in bone metastases than in lung metastases. To the best of our knowledge, this is the first study demonstrating the expressions of CEACAM6 and 8 in breast cancer. Results of our present study also indicated that both CEACAM6 and CEACAM8 could be involved in less aggressive subtypes of breast cancer possibly trough inhibition of vascular invasion and proliferation. In addition, results indicated that CEACAM6 was involved in breast cancer metastases to the bone but further studies are required to clarify the clinical significance of their interactions in breast cancer patients. 

### Novel Antitumor Agents 1

#3067

OHM-581, a dual JAK2-BET inhibitor for the treatment of myelofibrosis and other hematologic malignancies.

Ram S. Upadhayaya,1 Raghava Reddy Kethiri,2 Stephan W. Morris,1 H. Michael Shepard,1 Samuel R. Saks,1 Michael J. Weickert1. 1 _Ohm Oncology, Austin, TX;_ 2 _Laxai Life Sciences, Hyderabad, India_.

Ruxolitinib is the only JAK2 inhibitor approved for myelofibrosis (MF). Although JAK2 inhibitor therapy improves splenomegaly and systemic symptoms, it does not significantly reduce the MF clone or alter the natural history and survival in most patients. The novel small molecule OHM-581 not only inhibits JAK2, but also BET (bromodomain and extra terminal) proteins such as BRD4. Dual JAK2 and BET inhibition using other molecules has shown superior efficacy in murine MF models compared with JAK2 inhibition alone including amelioration of fibrosis, synergistic induction of apoptosis of primary patient, post-MF secondary acute myeloid leukemia (sAML) blasts, and significant improvement of the survival of mice engrafted with human sAML cells. OHM-581 is being developed for the treatment of MF and other hematologic malignancies to leverage these advantages of dual JAK2-BET inhibition.

Results: In vitro biochemical assays show OHM-581 to inhibit BRD4 BD1 and BD2 with IC50s of 189 and 116 nM, respectively, and JAK2 with an IC50 of 7.1 nM (JAK1=244 nM; JAK3=76 nM). No significant hERG liability was seen. OHM-581 displays significant anti-proliferative activity against multiple liquid cancer cell lines. Notably, the GI50 values for MV4-11 (AML, MLL fusion+), HEL 92.1.7 (AML, JAK2V617F+) and UKE-1 (AML, JAK2V617F+) are 32, 87 and 140 nM, respectively. Consistent with its mechanism of action, OHM-581 robustly down-regulates cMYC expression (cMYC mRNA down-regulation IC50=18.6 nM compared with 41.4 nM for BET inhibitor JQ1) and JAK2 signaling. Moreover, the compound triggers apoptosis in MV4-11 cells (~7-fold increase in Caspase 3/7 activity at 24-h). OHM-581 does not reflect an efflux liability, the efflux ratio being nearly 1.8. OHM-581 is soluble in PBS pH7.4 at 26 µM and metabolically stable with nearly 51%, 67% and 88% remaining in mouse, rat and human liver microsomes, respectively, after 30 min. OHM-581 exhibits an oral bioavailability of ~40%. As compared to the small molecule fedratinib, which is also being developed for MF, OHM-581 does not inhibit thiamine uptake. Dose proportionality with a greater than proportional increase in both Cmax and AUC at doses up to 60 mg/kg, and a saturation of Cmax at doses of 60 mg/kg and higher, are observed for OHM-581. In vivo efficacy studies using an MV4-11 xenograft model demonstrate robust tumor inhibition (~75-90%) following doses of 30 and 60 mg/kg po BID for 20 days.

Conclusion: OHM-581 is a novel dual inhibitor of JAK2 and BET that has potential as a therapeutic agent for MF and other hematologic malignancies. In addition to IND-enabling studies, current OHM-581 development activities are focused on further proof-of-concept analyses using preclinical models of MF and other myeloproliferative neoplasms, with results to be reported.

#3068

Synergistic effect of BCL-2 inhibitor APG-2575 and CDK9 inhibitor in acute myeloid leukemia and DLBCL preclinical tumor models.

Douglas D. Fang, Ran Tao, Qiuqiong Tang, Shoulai Gu, Guoqin Zhai, Jiajun Li, Qixin Wang, Xu Fang, Na Li, Dajun Yang, Yifan Zhai. _Ascentage Pharma (Suzhou) Co., Ltd, China_.

Effective treatments are urgently needed for elderly acute myeloid leukemia (AML) and relapsed or refractory diffuse large B-cell lymphoma (DLBCL) patients as only 10-25% of the population respond to standard therapies and resistance often occurred. Concurrent overexpression of anti-apoptotic BCL-2 gene and proto-oncogene MYC are frequently found in AML and DLBCL and this molecular characteristic is associated with poor prognosis of patients. CDK9 controls non-ribosomal transcription of genes including MYC and MCL-1. Dysregulation in the CDK9 pathway had been reported in AML and DLBCL, making it an attractive target for the combination with BCL-2 inhibitor to achieve more effective therapy.

To test the above hypothesis, in this study, we applied the combination of APG-2575 with a CDK9 inhibitor (alvocidib or dinaciclib) to preclinical tumor models. APG-2575 is a novel, orally bioavailable BH3-mimetic that selectively inhibits BCL-2, but not BCL-xL or MCL-1. After the treatment, synergistic effect of the combination treatment was demonstrated in mice bearing xenograft tumors of human AML or DLBCL. Specifically, in xenograft tumor models derived from human DLBCL cell line U2932, the treatment with APG-2575, alvocidib, or dinaciclib single agent for 22 days exhibited antitumor activity with T/C values of 20-50% (T/C value, average tumor volumes in treatment group vs. the vehicle control group). The combination treatment achieved substantial antitumor activity with T/C value of 5%. All animals in the combination groups showed either partial or complete tumor regression whereas none in the single arms.

Similar synergistic antitumor activity of the combination treatment was consistently observed in the myelodysplastic syndrome SKM-1 and AML OCI-AML-3 xenograft models. Taken together, our data suggest that APG-2575 and CDK9 inhibition combination has a potential therapeutic application in treatment of patients with AML and DLBCL.

#3069

Efficacy of RP4010, a calcium release-activated calcium (CRAC) channel inhibitor, in preclinical models of gastrointestinal cancers.

Irfana Muqbil,1 Rachel Sexton,2 Gabriel Mpilla,2 Anteneh Tesfaye,2 Steve Kim,2 Philip A. Philip,2 Amro Aboukameel,2 Srikant Viswanadah,3 Kumar V. Penmesta,3 Ramzi M. Mohammad,2 Asfar S. Azmi2. 1 _University of Detroit Mercy, Detroit, MI;_ 2 _Wayne State Univ., Detroit, MI;_ 3 _Rhizen Pharmaceuticals SA, La Chaux-de-Fonds, Switzerland_.

Background: Calcium Ca(2+) signals and the underlying Ca(2+) channels and transporters are known to influence development, growth, and metastasis of many cancers. Calcium release-activated calcium (CRAC) channel proteins, STIM and Orai, have been shown to play a role in cancer progression and metastasis. Here we investigate the impact of RP4010, a CRAC channel inhibitor, in GI [pancreatic ductal adenocarcinoma (PDAC), gastric cancer (GC) and pancreatic neuroendocrine tumors (PNETs)] cellular and patient derived tumor models.

Methods: GI cancer cell lines [3 PDAC and one normal human pancreatic ductal adenocarcinoma (HPDE); 3 GC cells and 2 PNET cell lines] were exposed to RP4010 in the presence or absence of standard of care drugs such as gemcitabine, nab-paclitaxel, or everolimus. Cell growth inhibition was evaluated using MTT. Apoptosis was determined by Annexin V FITC and 7AAD assays. Alterations in markers associated with the CRAC channel pathway were evaluated using RT-PCR and Western Blotting. In vivo efficacy was evaluated in gastric cancer sub-cutaneous xenograft as well as a hyaluronan positive PDAC patient derived xenograft in the presence or absence of chemotherapeutic agents.

Results: CRAC channel inhibition by RP4010 resulted in cancer cell selective growth inhibition (IC50s in the low micro M range). Growth inhibition was concurrent with apoptosis at similar doses. RP4010 synergized with gemcitabine and nab-paclitaxel in PDAC; nab-paclitaxel and docetaxel in GC (CI<1), and significantly enhanced the activity of everolimus in PNETs. RT-PCR and Western Blotting revealed that RP4010 caused a reduction in NFATC1, NFAT2, Akt, mTOR and related markers associated with CRAC channel pathway signaling. The combination treatment led to superior suppression of several CRAC channel regulated genes. RP4010 (given at 50 mg/kg daily for 4 weeks) as a single agent reduced tumor weight/volume in a hyaluronan positive (stroma +ve) PDAC PDx model. Anti-tumor activity of RP4010 was enhanced in the presence of gemcitabine-nab-paclitaxel (RP4010 50 mg/kg orally+50 mg/kg i.v once a week for 3 weeks and 30 mg/kg nab-paclitaxel once a week for 3 weeks). Residual tumor tissue analysis to capture CRAC channel pathway inhibition in vivo is currently ongoing.

Conclusion: Our studies indicate that RP4010, a novel CRAC channel inhibitor could be a viable therapeutic option in GI cancers that need further clinical evaluation. A multi-center Phase I/IB dose-escalation trial evaluating the safety and efficacy of RP4010, a CRAC channel inhibitor in patients with relapsed or refractory Lymphomas is currently ongoing in USA (ClinicalTrials.gov Identifier: NCT03119467).

#3070

Discovery of novel potent allele-selective KRAS-G12C covalent inhibitors stemming from DNA-encoded library.

Gary McCort,1 Rosalia Arrebola,1 Loreley Calvet,1 Baptiste Ronan,1 Fabrice Vergne,1 Francis Duffieux,1 Alexey Rak,1 Isabelle Meaux,1 David Papin,1 Florence Fassy,1 Cecile Delorme,1 Magali Matthieu,1 Jean-Paul Nicolas,1 Christophe Marcireau,1 Valerie Steier,1 Pierre-Yves Abecassis,1 Valerie Czepczor,1 Heather A. Thomson,2 Christopher D. Hupp,2 J.P. Guilinger,2 Ying Zhang,2 Anthony D. Keefe,2 John W. Cuozzo,2 Julie Liu,2 Laurent Debussche1. 1 _Sanofi, Vitry-sur-Seine, France;_ 2 _XChem Pharmaceuticals, Waltham, MA_.

From a screening of 27 106 covalent DNA-encoded compound library, a novel KRAS G12C chemical series has been identified. Original hit series compound displayed low µM covalent binding activity to KRAS G12C under GDP form associated with a k(inact)/Ki of 1,03 M-1.s-1, a stable electrophilic covalent warhead (T1/2 in 5mM GSH > 24h), +12°C stabilization Delta Tm in DSF assay and 3 µM IC50 value in GEF assay, while no detectable covalent binding to KRAS G12C under GTP form and to KRAS WT or KRAS G12D under GDP form. From 3 µM, it also induced significant pERK inhibition in H358 KRAS-G12C but not in A549 KRAS G12S NSCLC cell lines. Unique binding mode to SII pocket was demonstrated by Xray crystallography. Multi-parametric and structural biology guided chemical optimization yielded potent KRAS G12C lead compounds which combine k(inact)/Ki > 500 M-1.s-1, Delta Tm=20°C, 10 nM range pERK IC50 values with correlated direct KRAS G12C selective covalent modification. They also exhibited 100 nM range KRAS G12C allele-specific anti-proliferative activity and triggered significant apoptosis induction. In vivo treatment of mice bearing H358 tumour xenografts through oral route with optimized candidates showed a marked and prolonged inhibition of both pERK and DUSP6 mRNA as well as consistent direct KRAS-G12C covalent modification as evidenced by LC-MS in tumor samples. Altogether, these data demonstrate the power of DNA-encoded library approach to deliver potential drug candidates with selective cysteine-targeted irreversible mechanism of action on challenging oncology targets such as KRAS.

#3071

**Repotrectinib demonstrates promising activities in** ROS1 **wild-type and solvent-front mutant lung cancer patients-derived preclinical models.**

Dong Hwi Kim,1 Seok-Young Kim,1 Hyeong Seok Joo,1 You Won Lee,2 Han Na Kang,1 Min Hee Hong,2 Hye Ryun Kim,2 Byoung Chul Cho,2 Miran Yun1. 1 _Jeuk, Seoul, Republic of Korea;_ 2 _Division of Medical Oncology, Yonsei Cancer Center, Yonsei University College of Medicine, Seoul, Republic of Korea_.

The ROS1-rearranged non-small-cell lung cancer (NSCLC) is currently treated with ALK/ROS1 tyrosine kinase inhibitor (TKI) crizotinib as a first-line agent. However, resistance invariably develops and subsequent therapeutic option for overcoming them is limited. Therefore, we investigated the antitumor activity of several ROS1-TKIs in ROS1-positive patient-derived preclinical models.

In this study, we established 6 patient-derived cells (PDCs) from patients with different types of ROS1 fusion partners as follows: treatment-naïve (YU1078, YU1082 and YU1083), crizotinib-resistant G2032R (YU1079) and crizotinib- or entrectinib- primary resistant (YU1081 and YU1085). Sanger sequencing and whole-exome sequencing were then conducted to determine the characteristics. The in vitro and in vivo antitumor activities of ROS1-TKIs in these PDCs were evaluated by comparing the survival, expression assay, duration of tumor re-emergence and ability to penetrate the blood-brain barrier (BBB).

Repotrectinib potently inhibited cell proliferation and ROS1-downstream signaling pathways in YU1078 (CD74-ROS1). In YU1078-derived xenograft models, repotrectinib induced the marked tumor regression and delayed the duration of tumor re-emergence following drug withdrawal compared with crizotinib and entrectinib. Interestingly, both YU1081 (TPM3-ROS1) and YU1082 (SLC34A2-ROS1) with cross primary resistance to other clinically available ROS1-TKIs exhibited high sensitivity to repotrectinib in the concentration range of 100~200 nM. Immunoblot analysis demonstrated that repotrectinib completely suppressed the phosphorylation of ROS1 and STAT3. Notably, repotrectinib showed the selective and highly potency in vitro and in vivo against solvent-front mutant G2032R conferring crizotinib resistance. In G2032R-mutant xenograft models, the response of repotrectinib was still maintained until the end of the experiment (120 days) after drug withdrawal, but loratinib- or cabozantinib-treated tumors rapidly re-emerged. Moreover, repotrectinib induced profound tumor regression in brain metastasis model with excellent brain/plasma and tumor/brain area under the concentration-time curve value. As clinical proof of concept, the potent systemic and intracranial activity of ropotrectinib has been observed in patients who had progressed on prior TKIs or TKI-naïve patients, who were enrolled on-going phase 1/2 clinical trial (NCT03093116).

Our finding indicated a superior antitumor activity of repotrectinib in both wild-type and mutant ROS1 fusion, providing a rationale that repotrectinib may be an effective subsequent or upfront treatment option for patients with ROS1-positive NSCLC who have relapsed on the available TKIs as well as TKI naïve, including patients with progressive CNS metastases.

#3072

GFH018, a novel TGF-βRI inhibitor, for the treatment of advanced solid tumors.

Gang Hu,1 Rong Zhao,1 Fusheng Zhou,1 Jiong Lan,1 Biao Zheng,1 Qiang LU,1 Jianyu Lu,2 Lifang Wu,2 Fei Sun,2 Lihong Hu,2 Shuhua Han,3 Haijun Tong,3 Yuanfeng Xia,3 Wei Li,3 Liping Ma,3 Ying Chen,3 Charles Ding,3 Chichung Chan,3 Shuhui Chen3. 1 _GenFleet Therapeutics, Shanghai & Zhejiang, China; _2 _Wuxi AppTec, Wuhan, China;_ 3 _Wuxi AppTec, Shanghai, China_.

Background: The transforming growth factor-β (TGF-β) signaling pathway has been shown to play an important role in various cellular processes including growth inhibition, cell migration, tumor invasion, epithelial-mesenchymal transition, and immune-suppression. TGF-βs are often overexpressed in various types of cancers, and the anti-TGF-β therapy has been pursued intensively for cancer treatment.

Results: GFH018 is a potent, selective inhibitor of TGF-βRI with a kinase inhibition activity IC50 of 70.5 nM. Our studies demonstrated that GFH018 could inhibit TGF-β-induced SMAD phosphorylation and migration of tumor cells. GFH018 is a potent immune regulator in that it could inhibit in vitro Treg cell induction and reverse M2 phenotype to M1, leading to increased pro-inflammatory cytokine production. In addition, GFH018 exhibited a significant inhibitory effect on in vitro angiogenesis formation assay. Importantly, GFH018 has shown promising anti-tumor efficacy in multiple in vivo mouse syngeneic models as a monotherapy as well as in combination with an anti-PD-(L)1 antibody. Following oral administration, GFH018 demonstrated fast absorption, and high oral bioavailability in preclinical animal species including mouse, rat, and dog. Results from the toxicology studies in rat and dog have also supported further development of GFH018.

Conclusion: GFH018 has the potential to be developed as a monotherapy or combinational therapy with checkpoint inhibitors for advanced solid tumor patients. The first-in-human clinical trial is scheduled in 2019.

#3073

Development of novel BMI1 inhibitors targeting glioblastoma stem-like cells.

Michelle Chadwick,1 Eric Huselid,1 Monica Bartucci,1 Michele Patrizii,1 Kelly Jara,1 Monal Mehta,1 John Gilleran,2 David Augeri,2 Hatem Sabaawy1. 1 _Rutgers Cancer Institute of New Jersey, New Brunswick, NJ;_ 2 _Rutgers, The State University of New Jersey, Piscataway, NJ_.

Glioblastoma multiforme (GBM) are the most common and deadly primary brain tumors. GBMs are thought to derive from neuroglial stem or progenitor cells. Although surgery, radiotherapy and concomitant or alternating chemotherapy followed by anti-angiogenic therapy are still the mainstay of treatment, individually tailored strategies based on tumor-intrinsic dominant genomic, transcriptomic, epigenetic and antigenic tumor profiles may ultimately improve outcome. GBM recurrence is thought to be caused by a dynamically changing population of slow-cycling stem-like cells that are capable of evading current therapeutics which spare the radio/chemoresistant cells. Due to the extreme heterogeneity of these tumors, the likelihood of success of new therapeutic strategies for GBM relies on utilizing tailored combinations of targeted genetic-, epigenetic- and/or immune-modulating therapies. Targeting epigenetic polycomb repressor complex 1/2 (PRC1/2) and their key drivers BMI1 and EZH2 is highly attractive to target GBM stem-like and progenitor cells. Since PRC2 activity is dependent on PRC1 chromatin association, targeting BMI1 is a priority for GBM. BMI1 is strongly upregulated in most GBM subtypes, and we and others have demonstrated that reducing BMI1 levels results in a decrease in tumor cell self-renewal and eliminate the stem-cell like phenotypes in other tumor types. We have therefore developed novel small molecule BMI1 inhibitors, named RU-A compounds, for GBM therapy. First, we demonstrate that these RU-A compounds could eliminate BMI1 at low nanomolar conc. We further determined using modeling and luciferase assays that RU-A compounds exert effects on the BMI1 RNA, suggesting that RU-A compounds might bind to a pocket within the BMI1 untranslated region. Strikingly, RU-A compounds demonstrate complete ablation of self-renewal of GBM stem-like cells in serial clonogenic, sphere assays, and GBM organoids, and sensitize GBM cells to chemotherapy. We demonstrate the anti-GBM and reduction of tumor initiation activities of these inhibitors in a series of patient-derived xenografts and patient derived orthotopic mouse models of GBM. Due to the high potency of these compounds, when injected in mice, there were significant reductions in intratumor BMI1 associated with elimination of stem cell-like phenotypes, and no hematopoietic toxicity or neurotoxicity detected. These novel RU-A compounds by altering the stem-like populations of GBM cells offer effective targeted agents that could be utilized alone or combined with other targeted and immune modulatory agents in clinical trials for GBM patients.

#3074

**Broad spectrum activity of the BET inhibitor GSK 1324726A in hematologic and solid cancer cell lines** in-vitro **and determination of associated predictive biomarkers.**

Heinz-Herbert Fiebig,1 Gerhard Kelter,2 Hans R Hendriks,3 Vincent Vuaroqueaux1. 1 _4HF Biotec GmbH, Freiburg, Germany;_ 2 _Charles River, Discovery Services, Freiburg, Germany;_ 3 _Hendriks Pharmaceutical Consulting, Purmerend, Netherlands_.

The BET family of bromodomain proteins (BRD2, BRD3, BRD4, and BRDT) has emerged as a promising new cancer target for small-molecule drug discovery. GSK 1324726A (I-BET726) is a highly selective inhibitor of BET family proteins that binds BRD2, BRD3 and BRD4 with IC50 of 41 nM, 31 nM, and 22 nM, respectively. In the present study, we aimed to identify sensitive tumor types for GSK 1324726A and predictive biomarkers for response or resistance. GSK 1324726A was tested in vitro in a 2D monolayer assay in 274 human tumor cell lines of 36 tumor types (50 liquid and 224 solid cancer cell lines). Antitumor activity was then tested in vivo in 5 of the most sensitive tumor cell lines transplanted sc in nude mice. Drug sensitivity results in vitro were correlated with molecular data of the tumor models, including whole exome mutations, gene copy number variations and gene expression profiles.

GSK 1324726A showed a moderate anti-tumor potency (median IC70 6,6 µM, range 0.03 - >30 µM) and a pronounced selectivity in a wide range of blood and solid cancer cell lines. AML, ALL, multiple myelomas and B-cell lymphomas were 1 log more sensitive than the most sensitive solid tumors. Taking 10% of the median IC70 of all tumors as cut off (0.66 µM) 5/11 AML, 4/9 ALL, 5/11 MM and 7/13 B-NHL were sensitive. Among the solid tumor panel cervix uterus and prostate models were sensitive (2/5) followed by breast, soft tissue, CRC, ovarian cancers and melanomas (between 17 and 40%). In-vivo experiments of the most sensitive tumors are under way. The MTD of GSK 1324726A (po for 15 days) was 15 mg/kg/day as it effected a slight body weight loss of 4% and no lethality.

Evaluation of tumor cell lines for molecular parameters associated with response was carried out separately for hematologic and solid cell lines. For the leukemias, lymphomas and myelomas about 100 genes were significantly associated with response to GSK 1324726A at the transcriptome level (intersection of Spearman and Limma tests, adjusted p values <0.05). JAG1 was identified as the most significant gene among them. JAG1 is known to interact with receptors in the Notch signaling pathway and regulate cell fate decisions. Solid tumor models did not show a strong predictive gene pattern. Integrative analysis with significant genomic and transcriptomic parameters is currently ongoing to develop a molecular predictor of response to GSK 1324726A. Extending evaluation of the compound in vivo and continuing investigation of predictors of response are underway.

#3075

Antitumor activity of M8891, a potent and reversible inhibitor of methionine aminopeptidase 2.

Manja Friese-Hamim, Olga Bogatyrova, Maria J. Ortiz, Dirk Wienke, Timo Heinrich, Felix Rohdich, Klaus Duecker, Christoph Schultes, Bayard Huck, Frank T. Zenke, Andree Blaukat. _Merck KGaA, Darmstadt, Germany_.

N-terminal cleavage of methionine by methionine aminopeptidases is a crucial step in the maturation of proteins during protein biosynthesis. MetAP2, one of the two methionine aminopeptidases expressed in mammalian cells, has been identified as the target of the highly potent and irreversible inhibitor fumagillin to which antiangiogenic and antitumoral properties have been ascribed. This and other findings support the rationale to develop MetAP2 inhibitors for cancer therapy. The fumagillin derivative TNP-470 has been evaluated in several clinical trials, but development was discontinued following demonstration of unfavorable pharmacokinetics and side effects likely attributed to the reactivity of the compound. We have developed M8891, an orally bioavailable, potent, and reversible inhibitor of MetAP2. M8891 inhibited growth of primary endothelial cells as well as patient-derived tumor cells and demonstrated antiangiogenic and antitumoral activity in mouse models. Accumulation of an unprocessed MetAP2 substrate, methionylated elongation factor 1 α, Met-EF1a, was observed upon treatment with M8891 in vitro as well as in vivo confirming that MetAP2 can be effectively inhibited with this compound. In combination with sunitinib, M8891 demonstrated strong and durable antitumor activity in a cohort of patient-derived renal cancer xenografts at well-tolerated exposure levels. Predictive biomarker hypotheses generated from CRISPR sensitivity/resistance screens as well as analyses of molecular profiling data from PDX tumors are currently being evaluated in further nonclinical studies. A dose-escalation phase 1 study in patients with advanced solid tumors is currently ongoing.

#3076

Preclinical development of OP-1250, an oral complete estrogen receptor (ER) antagonist that shrinks ER-positive breast tumors in xenograft models.

Leslie Hodges-Gallagher, Richard Sun, David C. Myles, Cyrus L. Harmon, Peter J. Kushner. _Olema Pharmaceuticals, San Francisco, CA_.

Fulvestrant (FASLODEX) is both the most effective estrogen receptor antagonist and the most effective endocrine therapy for estrogen receptor positive (ER+) metastatic breast cancer (MBC). Despite this superiority, fulvestrant must be delivered by intramuscular injection and its efficacy may be limited by poor drug exposure. Thus, an oral compound that shares fulvestrant's pharmacodynamic virtues with improved drug exposure might be both more effective and more convenient than fulvestrant in treating ER+ MBC.

Fulvestrant is both a selective estrogen receptor degrader (SERD) and a complete estrogen receptor antagonist (CERAN), so named for its ability to shut off both the AF-1 and AF-2 transcriptional activation functions of the ERalpha. In contrast, selective estrogen receptor modulators (SERMs) such as tamoxifen efficiently shut off AF-2 signaling (turned on by estradiol), but incompletely antagonize AF-1, a function that is turned on by multiple cell signaling pathways and shown to play a role in the development of endocrine resistance. We have developed a set of novel and sensitive cell culture assays that distinguish between CERANs and SERMs. In our assays, several SERDs under clinical investigation, including AZD9496 and GDC-0810, were identified as SERMs that exhibited ER-mediated agonism on genes dependent on AF-1 activity. Further, these SERM/SERDs stimulate cell proliferation of CAMA-1 breast cancer cells even in the absence of estrogen. Since these assays may model clinical breast cancer progression, in which tumors become less dependent on estrogen and more sensitive to AF-1 signaling mediated by SRC family kinases, IGFs, and FGFs, we predict that SERM/SERDs will be inferior in preventing and treating resistance compared to CERAN/SERDs.

Guided by these assays, we have developed the CERAN/SERD OP-1250, which blocks AF-1 activity, is a potent inhibitor of breast cancer cell proliferation, and degrades ERalpha; both in vitro and in vivo. Oral administration of OP-1250 yields high and even drug exposure in animal studies.

One important mechanism of resistance to endocrine therapy is the emergence of activating mutations in ESR1, the gene that encodes ERalpha, that allow for estrogen-independent tumor growth. OP-1250 blocks AF-1 activity in wild type ERalpha and in the five constitutively active ER variants most commonly seen in patients. In one of the most common ESR1 mutations, Y537S, OP-1250 completely blocks AF-1 function whereas the SERM/SERDS AZD9496 and GDC-0810 activate AF-1 mediated transcription.

In preclinical studies OP-1250 shrinks patient-derived xenograft tumors driven by Y537S, suggesting that it has potential to be a superior compound to treat ER+ MBC, especially in patients whose tumors have activating mutations ins ESR1. We expect to bring OP-1250 into the clinic in the middle of 2019.

#3077

Preclinical studies of SR-8314, a highly selective ENPP1 inhibitor and an activator of STING pathway.

Alexis Weston, Trason Thode, Ruben Munoz, Sherin Daniel, Raffaella Soldi, Mohan Kaadige, Haiyong Han, Hariprasad Vankayalapti, Sunil Sharma. _Translational Genomics Research Institute (TGen), Phoenix, AZ_.

Purpose: STING (stimulator of interferon genes) plays an important role in innate immunity by activating type I interferons in response to cytosolic nucleic acid ligands such as cyclic dinucleotides (CDNs). In recent years, STING has become an attractive therapeutic target for cancer immune therapy and hydrolysis resistant CDNs have been developed as a new class of cancer therapeutics. These CDNs have been shown to possess potent preclinical efficacy but early results from phase I trials have been disappointing. Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1 or NPP1) is natural antagonist of STING pathway. ENPP1 constitutively hydrolyzes 2′3′-cGAMP, a CDN that is the natural ligand for STING. Previously, we reported that SR-8291, a highly selective ENPP1 inhibitor, induces STING activity and demonstrates anti-tumor activity in B16F10 melanoma and CT26 colorectal models. In this study, we extend these findings and report the discovery of SR-8314, an analog of SR-8291 that shows improved physiochemical and developability properties.

Methods: With the application of computational techniques (ICM, Maestro), human NPP1 homology model was built by utilizing the crystal structure of mouse NPP1 (PDB: 4GTW). Series of docking simulations on built in ligands were performed within the substrate binding pocket and that led to the identification of lead NPP1 inhibitor SR-8314. Binding of SR-8314 to ENPP1 was evaluated using thermal shift assay. Activity of recombinant human ENPP1 using ATP as a substrate was measured using Cell Titer Glo (Promega) reagent. IRF-Lucia reporter activity, RT-PCR and Western Blotting were performed to evaluate the effect of SR-8314 in 2′3′-cGAMP primed THP1 dual reporter cells (Invivogen). In vivo efficacy studies of intraperitoneally-dosed SR-8314 and SR-8291 were performed in a syngeneic murine tumor model. Tumor T cell infiltration, tumor and plasma pharmacokinetics were assessed by flow cytometry and mass-spectrometry, respectively.

Results: SR-8314 has higher binding affinity towards ENPP1 and it potently inhibits ENPP1 activity with a Ki value of 0.079µM. A significant increase in gene expression of IFNβ, ISG15 and CXCL10 along with an increase in the secretion of IFNβ was observed in SR-8314 treated THP1 cells. We show anti-tumor activity as well as an increase in CD3+, CD4+ and CD8+ T cells in both SR-8314 and SR-8291 treated tumors. In addition, there was a decrease in tumor associated macrophages in SR-8314 treated tumors.

Conclusions: In summary, we show SR-8314 as a potent inhibitor of ENPP1 that promotes STING activity in vitro. SR-8314 displays promising anti-tumor activity and has ideal candidate properties that warrant further evaluation.

#3078

Tesetaxel, a novel, oral taxane, crosses intact blood-brain barrier (BBB) at therapeutically relevant concentrations.

Joyce James, Kevin Tang, Thomas Wei. _Odonate Therapeutics, Inc, San Diego, CA_.

Background: Central nervous system (CNS) metastases in cancers such as breast and lung cancer are common and associated with poor outcomes. The taxanes paclitaxel (P) and docetaxel (D) do not significantly cross the BBB (brain to plasma exposure of only 1% and 8%, respectively), making them ineffective in treating CNS metastases. Tesetaxel (T) is a novel, orally administered taxane currently in a Phase 3 study in patients with advanced breast cancer (aBC; NCT03326674). Several properties make it unique, including: oral administration with a low pill burden; a long (~8-day) terminal plasma half-life in humans, allowing infrequent dosing; no history of hypersensitivity reactions; significant activity against chemo-resistant tumors; and, unlike P and D, minimal efflux by the P-glycoprotein pump, a key element of the BBB. The in vivo tissue distribution of tesetaxel was previously investigated in mice treated with 14C-T at 4 mg/kg and sacrificed between 1-168 hours. We now report 2 additional studies in dogs and monkeys. Methods: Dogs and monkeys were dosed at 0.6 mg/kg and 1 mg/kg of 14C-T, respectively, and tissue distribution was assessed at 336 hours. These doses equate to ~44% of the 27 mg/m2 dose in the ongoing Phase 3 study. In vivo radioactivity concentrations in the cerebrum were compared to plasma concentrations and mean tumor GI50 (concentration resulting in 50% tumor growth inhibition) determined by MTT assay in 23 tumor cell lines. Results: The absolute brain concentrations of T in dogs and monkeys far exceeded both the respective plasma concentrations and the tumor GI50 (Table 1). Conclusions: In dogs and monkeys, T brain concentrations far exceed its tumor GI50 for a sustained period when administered at therapeutically relevant concentrations. The inclusion criteria for the ongoing Phase 3 study of T in patients with aBC allows for CNS metastases.

Table 1: Tesetaxel CNS Levels Far Exceed Human GI50a in Dogs and Monkeys

---

|  | 14C-T Radioactivity Concentration

(ng eq./g or mL) b | Cerebrum Concentration/

Tumor GI50 c

|

N | Cerebrum

(Mean ± SD) | Plasma

(Mean ± SD) | Cerebrum/

Plasma

|

Dog d | 3 | 10.9 ± 4.0 | 0.9 ± 0.1 | 12x | 18x

Monkey e | 3 | 6.5 ± 3.8 | 0.9 ± 0.2 | 7x | 11x

a Concentration of drug required to inhibit growth by 50%

b 14 days after dosing

c Mean GI50 for T across 23 tumor cells lines = 0.6 ng/mL

d Single dose of 0.6 mg/kg (equivalent to 44% of a human dose of 27 mg/m2)

e Single dose of 1 mg/kg (equivalent to 44% of a human dose of 27 mg/m2)

#3079

Pharmacodynamic evaluation of the novel SUMOylation inhibitor TAK-981 in a mouse tumor model.

Allison J. Berger, Sharon Friedlander, Omid Ghasemi, Stephen Grossman, Erik Koenig, Eric Lightcap, Michael Milhollen, Pooja Shah, Gary Shapiro, Vaishali Shinde, Keli Song, Kristina Xega, Agatha Zawadzka, Dennis Huszar. _Takeda Pharmaceutical Company Ltd., Cambridge, MA_.

TAK-981 is a small molecule inhibitor of the SUMOylation enzyme cascade which is currently in a Phase I clinical trial (NCT03648372). TAK-981 selectively inhibits the activity of the SUMO activating enzyme, forming a covalent adduct between TAK-981 and SUMO, thereby decreasing levels of SUMOylated proteins. A central feature of pharmacological inhibition of SUMOylation is production of type I IFNs in immune cells, promoting both innate and adaptive immune responses.

TAK-981 demonstrates antitumor activity, including complete regression (CR) of some tumors, in immune-competent BALB/c mice bearing syngeneic A20 lymphoma tumors. This model was utilized to investigate 1) target engagement and pathway inhibition, 2) modulation of IFN-regulated genes in tumor and blood, 3) dependence of antitumor activity on Type I IFN signaling and adaptive immunity, and 4) changes in immune cell infiltrates in tumors.

IHC analysis of tumors showed TAK-981-SUMO adduct formation and decrease of SUMOylated protein levels after a single IV dose of 7.5 mg/kg TAK-981, as measured by assays for TAK-981-SUMO adduct and SUMO2/3 conjugates, respectively. IFN-regulated genes including CXCL10, IFIT1, and ISG15 are coordinately upregulated 4-8 hrs post-dose in tumor and in blood, as demonstrated by Nanostring, and plasma levels of IP-10 (CXCL10) increase. Single cell RNAseq experiments on dissociated tumors demonstrated upregulation of IFN-regulated genes in T cells and monocytes in the tumor microenvironment.

TAK-981-mediated antitumor activity is fully dependent on Type I IFN signaling; administration of a neutralizing antibody against the IFN alpha/beta receptor 1 (IFNAR) before dosing TAK-981 results in the loss of antitumor activity against A20. TAK-981-mediated antitumor activity is also dependent on the adaptive immune system, as demonstrated by two experiments in mice lacking T cells (BALB/c Rag2 KO mice, and mice treated with an anti-CD8 antibody to deplete T cells). Antitumor response was diminished in both settings, with no CRs achieved in either model. Flow cytometric analysis of immune infiltrates in A20 tumors grown in BALB/c mice indicated that TAK-981 response is accompanied by an increase in activated T cells in the tumor microenvironment as well as increased NK cells, potentially supporting a role for both innate and adaptive immunity in the MOA.

Evaluation of pharmacodynamic biomarkers in Phase I clinical development will be informed by these preclinical studies.

#3080

Activity of pan-FGFR inhibitor rogaratinib and PI3K inhibitor copanlisib in preclinical urothelial bladder cancer models.

Isabel S. Jerchel, Pascale Lejeune, Rita Lampignano, Alexander Walter, Ralf Lesche, Atanas Kamburov, Dominik Mumberg, Peter Ellinghaus, Oliver Politz, Sylvia Gruenewald. _Bayer AG, Berlin, Germany_.

Rogaratinib is a potent small molecule pan-FGFR inhibitor that leads to downregulation of MAPK and PI3K signaling (1). In a recent Phase I study rogaratinib demonstrated higher ORR in locally advanced or metastatic urothelial bladder cancer (UBC) with FGFR mRNA overexpression in patients with PIK3CA and RAS wildtype than in those harboring PIK3CA or RAS mutations (2). In UBC, about 25% of tumors contain mutated PIK3CA of which 75% represent hot spot mutations E545K or E542K (3). Copanlisib is a potent pan class I PI3K inhibitor with predominant activity against the α and δ isoforms being approved for treatment of relapsed follicular lymphoma (4). We evaluated the activity of rogaratinib in cellular and in vivo efficacy studies in bladder cancer models in monotherapy and in combination with copanlisib. Cell proliferation and viability in response to rogaratinib, copanlisib or the combination of both was studied in rogaratinib-sensitive UBC cell lines like RT112 as well as in a recombinant RT112-PIK3CAE545K cell line. Rogaratinib potently inhibited cell proliferation with IC50 values in the double nM range. Copanlisib's potency varied between 20 and 900 nM. In combination, rogaratinib and copanlisib increased cell death and were synergistic as shown by their proliferation combination indices for the absolute IC50 and IC80. Expression of the PIK3CAE545K hot spot mutant in RT112 cells decreased the anti-proliferative efficacy of rogaratinib but did not change the sensitivity to copanlisib. In vivo, rogaratinib led to tumor regression in the FGFR1-expressing JMSU1 xenograft and strongly inhibited tumor growth in the FGFR3-driven RT112 model. Copanlisib did not show significant inhibition of tumor growth in monotherapy in either model. In the JMSU1 model the combination was not superior to rogaratinib alone at maximal tolerated doses. In the RT112 as well as in the RT112-PIK3CAE545K model, the combination significantly improved anti-tumor activity. In a PDX model with FGFR3 overexpression and a PIK3CAH1047R hot spot mutation rogaratinib did not inhibit tumor growth significantly while copanlisib displayed significant anti-tumor effects. The combination of both drugs reduced tumor growth compared to either monotherapy group. In conclusion, in UBC tumor models overexpressing FGFR with differing sensitivities to rogaratinib, its anti-tumor activity could be further enhanced when combined with copanlisib and suggests that PIK3CA mutations may play a role in reduced sensitivity of UBC to FGFR inhibition. These promising results warrant further development of rogaratinib in monotherapy and in combination. Clinical trials with rogaratinib are currently recruiting (NCT03517956, NCT03410693, NCT03473756).

References:

Jerchel et al. Cancer Res. 2018; 78: Abs. 4781.

Joerger et al. JCO 2018; 36: Abs. 4513.

Platt et al. Clin. Cancer Res. 2009; 15:6008.

Markham, A. Drugs. 2017; 77:2057.

#3081

DZD9008, an oral, wild type selective EGFR inhibitor for the treatment of non-small-cell lung cancer with Exon20 insertion and other activating mutations.

Yan Xu, Lin Zhang, Lifang Zhu, Yingchun Wang, Mei Wang, Zhenfan Yang. _Dizal Pharmaceuticals Co., Ltd, Shanghai, China_.

Background: Several EGFR TKIs have been approved for the treatment of non-small cell lung cancer (NSCLC) with L858R mutation, Exon 19 deletion, and T790M mutations. However, for patients with uncommon mutations, such as EGFR or HER2 Exon20ins, there lacks safe and effective therapy. All Exon20ins inhibitors under clinical development have a negative selectivity profile, inhibiting wild type EGFR more potently than mutant ones. Consequently, severe wild type EGFR-driven toxicities have been observed in patients. DZD9008 is an oral, potent, irreversible, wild type-selective EGFR TKI against EGFR or HER2 Exon20ins and other mutations. The present study shows anti-tumor activity of DZD9008 in tumor cell lines and xenograft models.

Materials and Methods: The enzyme assay was performed by incubating DZD9008 with recombinant enzymes at 2 mM ATP concentrations. The cellular activity of DZD9008, including phosphorylation of EGFR or HER2 and cell proliferation, was evaluated in a panel of cell lines expressing wild type EGFR, mutant EGFR or HER2, using MSD assay and CellTiter-Glo assays. DZD9008 in vivo anti-tumor activity was evaluated in both cell line-derived (CDX) and patient-derived xenograft (PDX) models, carrying EGFR single or double mutations, Exon20ins mutations, and wild type EGFR.

Results: The enzymatic IC50 of DZD9008 in mutant EGFR ranges from 0.4 to 2.1 nM. DZD9008 downregulated pEGFR with IC50 ranging from 1 to 22 nM in a panel of tumor cell lines expressing EGFR L858R, Exon19del, L858R/T790M, various Exon20ins or uncommon mutations. Similar activity against pHER2 was observed in tumor cells with HER2 Exon20ins mutation, with IC50 at 7 nM. In contrast, DZD9008 was less potent in modulating pEGFR in tumor cells expressing wild type EGFR, with IC50 greater than 80 nM. In cell proliferation assays, DZD9008 suppressed cell proliferation with GI50 of 1 to 60 nM in tumor cells carrying EGFR L858R, Exon19del, L858R/T790M, various Exon20ins or uncommon mutations. In CDX and PDX models carrying EGFR Exon19del single mutation, L858R/T790M double mutations, and PDX models harboring G719S/L861Q or Exon20ins, DZD9008 induced dose dependent tumor growth inhibition and regression. Good PK/PD relationship was established across these tumor models.

Conclusion: DZD9008 is a potential EGFR TKI for NSCLC patients with EGFR or HER2 Exon20ins and other activating mutations.

#3082

Targeting hypoxic pancreatic cancer cells with glucose conjugated lactate dehydrogenase inhibitor NHI-Glc-2.

Btissame El Hassouni,1 Rocco Sciarrillo,1 Valentina Edith Gómez,1 Mina Maftouh,1 Giulia Mantini,1 Christian M. Vonk,1 Carlotta Granchi,2 Niccola Funel,2 Filippo Minutolo,2 Godefridus J. Peters,1 Elisa Giovannetti1. 1 _Amsterdam UMC, Vrije Universiteit Amsterdam, Medical Oncology, Cancer Center Amsterdam, Amsterdam, Netherlands;_ 2 _Università di Pisa, Pisa, Italy_.

Pancreatic ductal adenocarcinoma (PDAC) is an abysmal disease with a 5-year survival rate of merely 8%. The tumor microenvironment of PDAC is one of the factors contributing to drug resistance. More specifically, the hypoxic tumor core and the metabolic switch to aerobic glycolysis (the Warburg effect), contribute to the lack of drug response. Therefore, we investigated the effect of several novel lactate dehydrogenase (LDH-A) inhibitors (N-Hydroxyindole-based LDH-A inhibitors, NHI-1 and NHI-2, and the glucose conjugate NHI-Glc-2) in PDAC cells in vitro and in vivo, in combination with the standard drug gemcitabine. For this purpose we used our primary PDAC cancer cell cultures, tested growth inhibition with the SRB chemosensitivity assay, used 3D cultures and established an in vivo orthotopic bioluminescent model. Additionally, LDH-A enzyme activity inhibition by NHI-Glc-2 was assessed by spectrophotometry. LDH-A is overexpressed in PDAC and its expression is correlated with the prognosis of metastatic PDAC. The glucose transporter 1 (GLUT-1) is also overexpressed in PDAC, which would enable an increased uptake of NHI-Glc-2 by the tumor cells. LDH-A mRNA expression and enzyme activity were about 2-fold higher under hypoxic conditions. NHI-1, NHI-2 and NHI-Glc-2 were 4-15-fold more effective under hypoxic conditions compared to normoxia, but gemcitabine was 10-20-fold less active under hypoxia. NHI-1 showed a synergistic effect with gemcitabine in hypoxic PANC-1 and LPC006 cells (combination index 0.14 ± 0.06 and 0.29 ± 0.53, respectively). NHI-Glc-2 inhibited PDAC cell growth in micromolar range under hypoxic conditions and also showed a synergistic effect with gemcitabine. In a 3D spheroid culture (with a hypoxic core), NHI-Glc-2 disrupted the spheroid integrity. Moreover, in an orthotopic PDAC model NHI-Glc-2 showed a more pronounced inhibition (almost complete) of tumor growth compared to gemcitabine. NHI-Glc-2 also showed a favorable pharmacokinetics with a peak plasma concentration of 26 µM at 4 hr, which is higher than the IC50. In conclusion, LDH-A is a viable target in PDAC, and novel LDH-A inhibitors offer an innovative therapeutic tool. Remarkably, the LDH-A inhibitors NHI-1 and NHI-2 increased the effect of gemcitabine under hypoxic conditions, while the glucose conjugated NHI-Glc-2 showed an improved uptake possibly because of the increased GLUT-1 expression, leading to a pronounced in vivo effect.

#3083

A novel hedgehog signaling inhibitor for targeting pancreatic ductal adenocarcinoma.

Atul Khire, Yi Zhong, Kalpana Rajanala, Christine Iacobuzio-Donahue, Marilyn D. Resh. _Memorial Sloan Kettering Cancer Center, New York, NY_.

Sonic Hedgehog (Shh) signaling is deregulated in pancreatic cancer and can drive tumor onset and progression. Previous attempts to block Shh signaling used Smoothened (Smo) inhibitors, which target canonical Shh signaling. These approaches met with limited success, due to development of resistance, and contributions from non-canonical, Smo-independent signaling pathways that operate within tumor epithelial cells. Here, we show that Hedgehog acyltransferase (Hhat), the enzyme responsible for palmitoylation of Shh, is a novel target for inhibition of Shh signaling in human and mouse pancreatic cancer cells and tumors. TDI-003410, a small molecule inhibitor of Hhat recently developed by our group, reduced proliferation of AsPC1 cells, a human pancreatic cancer cell line, in vitroand in subcutaneous xenografts in mice in vivo. The mechanism is via induction of a G2/M cell cycle arrest leading to apoptosis. We next used cell lines isolated from tumors formed in KPC (K-RasG12D/p53R172H) mice, a genetically engineered mouse model of pancreatic cancer.TDI-003410 inhibited the growth of KPC cell linesand also reduced Gli1 levelsin vitro. Neither LDE225, a Smo inhibitor, nor SAG, a Smo activator, had any effect on KPC cell proliferation or Gli1 levels in vitro. In orthotopic xenografts using KPC cells implanted into the pancreas, TDI-3410 treatment significantly reducedin vivotumor burden. No significant changes in CD31 or Trichrome (collagen) stainingwith TDI-3410 were detected in KPC orthotopic tumor tissue. However, Gli-1 levels in tumor tissue from TDI-3410 treated mice were significantly reduced compared to the vehicle controls. These findings provide proof of concept for the potential therapeutic efficacy of Hhat inhibition in pancreatic cancer. The concept of using an Hhat inhibitor is unique: Hhat is a novel target that hits upstream, directly at hedgehog ligands. This approach can inhibit both canonical and non-canonical (Smo-independent) as well as autocrine and paracrine Shh signaling.

#3084

**BI-907828: A novel, potent MDM2 inhibitor, inhibits GBM brain tumor stem cells** in vitro **and prolonged survival in orthotopic xenograft mouse models.**

Hema A. Luchman,1 Danielle Bozek,1 Xiaoguang Hao,1 Dorothea Rudolph,2 Sophia Blake,2 Jörg Rinnenthal,2 Samuel Weiss1. 1 _Univ. of Calgary, Calgary, Alberta, Canada;_ 2 _Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria_.

Glioblastoma (GBM) is characterized by an aggressive clinical course, therapeutic resistance and striking molecular heterogeneity. GBM remains incurable with a median survival of 12-15 months post-surgery even with standard of care chemotherapy and radiation. The tumor suppressor p53 (TP53) is frequently mutated in cancer and along with its downstream effectors is inactivated in more than half of all cancers. The remaining 50% of tumors have TP53 wild-type status. However, TP53 function is frequently attenuated in these cancers by other mechanisms including amplification and overexpression of its key negative regulator MDM2. Inhibition of the protein-protein interaction between p53 and MDM2 is still a novel concept for cancer therapy. MDM2 inhibitors are designed to restore and maintain p53 activity in p53 wild-type tumors. BI-907828, currently in Phase I clinical trials, is a novel and potent MDM2 inhibitor with good oral bioavailability and pharmacokinetic properties. We investigated the efficacy of BI-907828 in p53 wild-type GBM patient-derived brain tumor stem cell (BTSC) lines with different amplification status of MDM2. BI-907828 potently reduced proliferation and viability in BTSC lines with IC50 values in the picomolar range. All BTSC lines, including lines resistant to the standard of care for GBM, temozolomide (TMZ), were sensitive to the MDM2 inhibitor irrespective of the copy number (CN) status of the MDM2 gene. Consistently, induction of cell death by BI-907828 was demonstrated in a BTSC line with an MDM2 gene amplification, as well as in another line with wild-type MDM2 CN status. PK-PD studies in SCID mice, orthotopically xenografted with BTSC lines, demonstrated that a single dose of BI-907828 resulted in strong activation of target genes. Human CDKN1a and GDF15 mRNA levels were significantly increased in brain tumor tissue in both the MDM2-amplified and non-amplified orthotopic models, suggesting that BI-907828 is able to pass the blood-brain-barrier and demonstrates on-target MDM2 inhibition in vivo. Efficacy of BI-907828 was assessed in Kaplan Meier survival studies as monotherapy or in combination with TMZ in two orthotopic BTSC xenograft models with MDM2 amplified or wild-type CN status, respectively and both with p53 wild-type status and promoter methylation of the MGMT gene. Long-term treatment with BI-907828 as monotherapy or in combination with TMZ was well tolerated in both these orthotopic BTSC xenograft models for several months. BI-907828 treatment demonstrated striking improved efficacy over vehicle control animals in both models. There was also significant survival benefit for the combinatorial treatment with TMZ in the MDM2 wild-type CN model over monotherapies alone. BI-907828 thus provides an extremely promising new therapeutic option for patients with primary brain tumors.

#3085

A novel salicylanilide derivative inhibits castration-resistant prostate cancer via endoplasmic reticulum stress-triggered PERK autophagic signaling pathway.

Chia-Ling HSIEH, Hsu-Shan Hung, Kuan-Chou Chen, Teigi Saka, Chih-Ying Chiang, Shian-Ying Sung. _Taipei Medical University, Taipei, Taiwan_.

Background and Purpose: Metastatic castration-resistant prostate cancer (CRPC) is currently incurable. This study is to extend pharmacological value of our synthesized salicylanilide derivative, that has previously testified for its osteoclastogenesis activity, by exploring its additional cytotoxic properties and underlying mechanism in CRPC cells.

Experimental Approach: LCC03, a 5-(2′,4′-Difluorophenyl)-salicylanilide derivatives, was chemically synthesized and examined for cell growth inhibition in a serial of CRPC cell lines. Possible mechanisms involved in the anticancer activity was elucidated based on the changes in pathway-related protein expression and cellular events, together with antagonists for functional validation. The therapeutic efficacy and safety of LCC03 for the treatment of bone metastatic CRPC was evaluated in an experimental animal model.

Key Results: LCC03 dose-dependently suppressed proliferation and retarded cell cycle progression in CRPC cells. The classical autophagy features including autophagsomes formation and LC3-II conversation were dramatically shown in LCC03-treated CRPC cells, and it was associated with the suppressed AKT/mTOR signaling pathways, a major negative regulator of autophagy, Moreover, an expanded morphology of the ER, increased expression of the ER stress markers GRP78 and PERK, and eIF2α phosphorylation were observed. Blockage of autophagy and PERK pathways using small molecule inhibitors or shRNA knockdown reversed LCC03-induced autophagy and cell death, thus indicating that the PERK-eIF2α pathway contributed to the autophagy induced by LCC03. Furthermore, treatment of tumor-bearing mice with intraperitoneal administered LCC03 suppressed the growth of CRPC xenografts in mouse bone without systemic toxicity.

Conclusion and Implications: LCC03 induces cellular responses toward ER stress-induced autophagy via the PERK-eIF2α pathway that impairs proliferation and cell cycle progression and exhibits cytotoxicity against CRPC cells. The dual action of 5-(2′,4′-difluorophenyl)-salicylanilide on both the osteoclasts and the tumor cells strongly indicates that LCC03 is a promising anticancer candidate for preventing and treating metastatic CRPC.

#3086

**Discovery of a** first-in-class **TEAD inhibitor which directly inhibits YAP/TAZ-TEAD protein-protein interaction and shows a potent anti-tumor effect in malignant pleural mesothelioma.**

Ayumi Kaneda, Toshihiro Seike, Takeshi Uemori, Kensuke Myojo, Kensuke Aida, Tomohiro Danjo, Takahiro Nakajima, Daisuke Yamaguchi, Tomoko Hamada, Yoshiro Tsuji, Kaori Hamaguchi, Mai Yasunaga, Nobumasa Otsubo, Hideyuki Onodera, Yoichi Nishiya, Michihiko Suzuki, Junichi Saito, Toshihiko Ishii, Ryuichiro Nakai. _Kyowa Hakko Kirin Co., Ltd., Sunto-gun, Shizuoka pref., Japan_.

Objective: Hippo signaling pathway is known to regulate organ development. In Hippo signaling pathway, YAP or TAZ works as a transcriptional co-activator and forms a transcriptional complex with TEAD. In several cancers, upstream factors in Hippo pathway are inactivated by genetic alterations. When the upstream factors are inactivated, TEAD is activated and forms a complex with YAP/TAZ resulting in enhancement of cell proliferation, drug resistance and so on. In the activation process, S-palmitoylation of TEAD is necessary for binding to YAP/TAZ. Malignant pleural mesothelioma (MPM) is one of cancer types which have genetic alterations in Hippo pathway genes. Although YAP/TAZ-TEAD inhibitor should be an ideal drug for MPM therapy, there are only a few reports about YAP/TAZ-TEAD inhibitor and the efficacy and selectivity are not sufficient. In this study, we succeeded to synthesize a small molecule TEAD inhibitor, K-975, and evaluated its mechanism of action and anti-tumor effect against MPM.

Materials/methods: Inhibitory activity of K-975 on YAP/TAZ-TEAD protein-protein interaction (PPI) was evaluated in surface plasmon resonance (SPR) and co-immunoprecipitation assay. The effect of K-975 on palmitoylation status of TEAD was also evaluated. The three-dimensional structure of YAP-binding domain of TEAD1 in complex with K-975 was determined by X-ray crystallography. Anti-tumor effect of K-975 was evaluated by using MPM cell lines. Furthermore, using a derivative of K-975, 2 week-toxicity studies in rats and monkeys were performed.

Results: K-975 inhibited YAP-TEAD and TAZ-TEAD PPI in NCI-H226 cells, a human MPM cell line. Also, K-975 inhibited palmitoylation of TEAD. The crystal structure revealed that K-975 directly bound to cysteine residue in YAP-binding domain of TEAD1. This cysteine residue is highly conserved in TEAD family and known as a site of S-palmitoylation. K-975 inhibited the cell proliferation of NCI-H226 with GI50 of about 20 nmol/L. K-975 also induced a change of gene expressions similar to that induced by YAP knockdown. In vivo experiments, K-975 strongly suppressed the tumor growth in several s.c. xenograft models and showed a significant survival benefit in an orthotopic xenograft model. However, 2 week-toxicity studies of a K-975 derivative with optimized bioavailability showed some pathological findings which suggested the renal toxicity.

Conclusion: We synthesized a first-in-class drug which directly binds to TEAD protein and inhibits YAP/TAZ-TEAD PPI. K-975 showed a strong anti-tumor effect in pre-clinical MPM models. Although the renal toxicity might cause some difficulty in clinical use, we believe that a K-975 derivative has a possibility to become an effective drug candidate for MPM therapy.

#3087

KZR-8834: A novel, small molecule inhibitor of Sec61-dependent protein secretion with anti-tumor activity.

Eric Lowe,1 Janet L. Anderl,1 Andrea R. Fan,1 Jing Jiang,1 Henry W. Johnson,1 Christopher J. Kirk,1 Dustin McMinn,1 Tony Muchamuel,1 Jack Taunton,2 Jennifer A. Whang1. 1 _Kezar Life Sciences, South San Francisco, CA;_ 2 _University of California, San Francisco, San Francisco, CA_.

The majority of secreted and transmembrane (TM) proteins, including growth factors and TM oncogenes, require translocation through Sec61 into the ER for further processing. Sec61, therefore, represents a unique drug discovery opportunity through blockade of typically "undruggable" targets and modulation of protein homeostasis. Multiple inhibitors of protein secretion have been described that directly target Sec61 and have anti-tumor activity but lack adequate pharmaceutical properties and/or tolerability to be considered clinical candidates. We aimed to discover novel, selective inhibitors of Sec61 with increased tolerability as a potential treatment for various malignancies.

Sec61 inhibitors were identified using HEK293 cell lines stably expressing secreted or TM proteins of interest fused to a luciferase reporter. Selective anti-tumor activity was assessed via HEK293 reporter cell viability in comparison to tumor cell lines with known levels of sensitivity to published Sec61-modulating compounds. An initial screen resulted in KZR-4152 (4152), which blocked secretion of several target proteins but had no impact on tumor cell viability. Following a medicinal chemistry campaign, KZR-8834 (8834) was identified, which exhibited a 60-fold increase in potency against target proteins relative to 4152. On-target activity of 8834 was demonstrated via protein secretion assays in cells overexpressing a previously described Sec61 mutant with resistance to published compounds. In addition, 8834 did not affect the viability of HEK293 cells or endotoxin- or TCR-stimulated PBMCs.

8834 induced cell death in H929 multiple myeloma cells in vitro (IC50 = 98 nM) with rapid activation (≤8 hours) of caspase 3/7 via dual activation of caspases 8 and 9. When assessed in a panel of 346 human cancer cell lines across 25 different tumor types, head and neck squamous cell carcinoma, acute lymphocytic leukemia, non-Hodgkin's lymphoma, melanoma, multiple myeloma, and prostate cancer were among the most sensitive tumor types. Weekly and twice-weekly administration of 8834 was effective in an H929 mouse xenograft model at doses that resulted in >90% tumor growth inhibition without significant body weight loss or clinical signs of toxicity. Anti-tumor activity was >50% at doses 1/3rd of the maximum tolerated dose, suggesting a broad therapeutic index. 8834 was also effective in the B16F0 melanoma model, showing tumor growth inhibition similar to that of anti-PD1 therapy.

KZR-8834 is a novel, small molecule inhibitor of Sec61-dependent translocation of multiple Sec61 client proteins. 8834 exhibits a broad anti-tumor profile in vitro and therapeutic activity in multiple mouse tumor models, demonstrating a potential new therapy for the treatment of malignant diseases.

#3088

Farnesyl/geranylgeranyl transferase dual inhibitor thwarts mutant KRas-driven patient-derived pancreatic tumors.

Aslamuzzaman Kazi,1 Shengyan Xiang,1 Hua Yang,1 Liwei Chen,1 Perry Kennedy,1 Muhammad Ayaz,1 Steven Fletcher,2 Christopher Cummings,3 Harshani Lawrence,1 Francisca Beato,1 Ya'an Yang,4 Michael P. Kim,4 Andrea Delitto,5 Patrick Underwood,5 Jason B. Fleming,1 Jose Trevino,5 Andrew D. Hamilton,3 Said M. Sebti1. 1 _Moffitt Cancer Center, Tampa, FL;_ 2 _University of Toronto, Ontario, Ontario, Canada;_ 3 _Yale University, New Haven, CT;_ 4 _University of Texas MD Anderson Cancer Center, Houston, TX;_ 5 _University of Florida, Gainesville, FL_.

Although mutant KRas is a significant driver of pancreatic oncogenesis and resistance to therapy, there are no KRas inhibitors available for these patients. Farnesyltransferase inhibitors (FTIs) were developed as potential anticancer drugs because KRas requires farnesylation for its membrane localization and cancer-causing activity. However, KRas becomes geranylgeranylated and active when cancer cells are treated with FTIs. In this study, we designed a Ras C-terminal mimetic dual Farnesyl/geranylgeranyltransferase-1 (GGT-1) inhibitor, FGTI-2734, to overcome the geranylgeranylation-dependent resistance to FTIs. Immunofluorescence, cellular fractionation, and gel shift assays showed that FGTI-2734, but not the selective FTI-2148 and GGTI-2418, inhibited membrane localization of KRas in mt KRas pancreatic, lung, and colon human cancer cells. FGTI-2734 inhibited the growth in mice of mt KRas-dependent but not -independent human tumors, indicating its selectivity for mt KRas-driven cancers. Importantly, FGTI-2734 inhibited the in vivo growth of xenografts derived from four pancreatic cancer patients with mt KRas (two G12D, and two G12V) tumors. In addition, FGTI-2734 was highly effective at inhibiting, in three-dimensional co-cultures with chemotherapy resistance-promoting pancreatic stellate cells, the viability of primary and metastatic mutant KRas (G12D, G13D, and G12V) tumor cells derived from 8 pancreatic cancer patients. Finally, FGTI-2734 suppressed oncogenic pathways mediated by Akt, mTOR, and cMyc while upregulating p53 and inducing apoptosis in patient-derived xenografts in vivo. Thus, the development of this novel dual FT and GGT-1 inhibitor overcomes a major hurdle in KRas resistance, thwarting the growth of patient-derived mutant KRas-driven xenografts from pancreatic cancer patients, and as such it warrants further advanced preclinical and clinical studies.

#3089

Evaluation of highly potent and selective single-molecule dual EGFR/PI3K inhibitors in preclinical models of adult and pediatric high-grade glioma.

Yusha Y. Sun,1 Cavan P. Bailey,1 Trever R. Carter,1 Christopher E. Whitehead,2 Judith S. Sebolt-Leopold,3 Joya Chandra1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _Mekanistic Therapeutics, Ypsilanti, MI;_ 3 _University of Michigan, Ann Arbor, MI_.

High grade gliomas (HGG), including glioblastoma (GBM) and diffuse intrinsic pontine glioma (DIPG), carry poor prognosis with median survival rates under 15 months post diagnosis. Due to dysregulation of the RTK/PI3K pathways within these tumors, including RTK pathway amplifications, targeted kinase inhibition has been considered a promising strategy to improve patient outcomes. However, many single-agent inhibitors of EGFR or PI3K have shown limited response in gliomas due to poor blood-brain barrier (BBB) penetrance and activation of compensatory signaling. The design of BBB-penetrant novel therapeutics that target multiple kinases is crucial for overcoming these obstacles. Single-molecule multikinase inhibitors may decrease resistance, present a single pharmacokinetic dosage profile, and reduce risks of multi-agent toxicities, supporting this strategy over dual drug approaches. A panel of inhibitors exploiting known binding modes of structurally-related ATP binding site inhibitors of EGFR/PI3K were synthesized and characterized, of which six promising candidates were chosen for in vitro study. The interaction of the inhibitors with common drug efflux proteins were probed to predict BBB penetrance. Of the compounds, MTX-241 was least likely to act as a substrate for efflux proteins such as P-glycoprotein. Treatment of MTX-241 in three human U87 GBM lines, some of which overexpress wild-type or vIII EGFR, three patient-derived DIPG lines, and two glioma stem cell (GSC) lines, one of which is radioresistant, showed strong cytotoxic potency measured by growth inhibitory activity, with IC50s in the <10 µM range. MTX-241 was significantly more potent than clinically relevant inhibitors targeting EGFR/RTKs (gefitinib, lapatinib, dasatinib, imatinib) or PI3K (alpelisib, idelalisib). Notably, selectivity of MTX-241 towards these HGG cell lines in comparison to normal human astrocytes (NHA) was observed, with NHA nearly insensitive to MTX-241 even at >100 µM. We found significant inhibition of p-EGFR and p-Akt in these cell lines compared to DMSO controls. Further, we identified a unique glycolytic-suppressing activity of MTX-241 in the U87/DIPG lines, to a greater extent than treatment with gefitinib or alpelisib. Additionally, as the clinically relevant HDAC inhibitor vorinostat upregulated EGFR activity, we examined the combination of MTX-241 and vorinostat, which significantly reduced proliferative abilities, achieving synergistic combination indices. MTX-241 and vorinostat treatment in the NHA model was not synergistic, highlighting the selectivity for HGG tumor models. Our data suggests that a dual inhibitor of EGFR and PI3K, alone or in combination with an HDAC inhibitor, represents a viable therapeutic strategy in adult/pediatric HGG. Future studies will focus on evaluation of in vivo efficacy in tumor-bearing mouse models.

#3090

In vivo **characterization of AMG 510 - a potent and selective KRAS** G12C **covalent small molecule inhibitor in preclinical KRAS** G12C **cancer models.**

Karen Rex,1 Anne Y. Saiki,1 Ji-Rong Sun,1 Tyler Holt,1 Neelima Koppada,2 Brian A. Lanman,1 Laurie P. Volak,1 Robert S. Foti,2 Victor J. Cee,1 J. Russell Lipford,1 Jude Canon1. 1 _Amgen Inc., Thousand Oaks, CA;_ 2 _Amgen Inc., Cambridge, MA_.

Somatic activating mutations of RAS family members are tumor driver mutations found in an estimated 21% of all cancers. Oncogenic KRAS mutations at residues G12, G13, and Q61 represent the most common RAS mutations found in solid malignancies. The prevalence of KRAS p.G12C tumors is ~13% of lung adenocarcinoma (including NSCLC), 3% of colorectal carcinoma (CRC), and 1% to 2% of numerous other solid tumors, representing an unmet medical need. We have developed AMG 510, an orally bioavailable, covalent inhibitor of KRASG12C with potent biochemical and cellular activity, and robust in vivo efficacy. AMG 510 inhibited SOS-catalyzed nucleotide exchange of recombinant mutant KRASG12C/C118A but had minimal effect on KRASC118A, which is wildtype at position 12. In cellular assays, AMG 510 covalently modified KRASG12C and inhibited KRASG12C signaling as measured by phosphorylation of ERK1/2 (p-ERK) in all KRAS p.G12C-mutant cell lines tested but did not inhibit p-ERK in cell lines with various other KRAS mutations. AMG 510 also selectively impaired viability of KRAS p.G12C mutant cell lines but did not affect cell lines with other KRAS mutations. In vivo pharmacodynamic assays demonstrated dose- and time-dependent inhibition of KRASG12C signaling in human pancreatic and NSCLC tumor xenografts. Covalent modification of KRASG12C by AMG 510 was measured by mass spectrometry and correlated with p-ERK inhibition in tumors. AMG 510 significantly inhibited the growth of KRAS p.G12C xenografts and resulted in tumor regression. Combination treatment of AMG 510 with standard-of-care and targeted agents demonstrated enhanced tumor growth inhibition compared to either single agent. In a syngeneic model of KRAS p.G12C mutant cancer, AMG 510 treatment significantly inhibited tumor growth and caused regression. AMG 510 is currently in Phase 1, first-in-human clinical trials for patients with advanced solid tumors harboring a KRAS p.G12C mutation.

## CLINICAL RESEARCH

### Immune Infiltration and Immune Therapy

#3091

Effects of melanoma cell-derived exosomes in melanoma patients' plasma on immune cell functions.

Priyanka Sharma,1 Brenda Diergaarde,1 Soldano Ferrone,2 John M. Kirkwood,1 Theresa L. Whiteside1. 1 _University of Pittsburgh, Pittsburgh, PA;_ 2 _Massachusetts General Hospital and Harvard Medical School, Boston,, Boston, MA_.

Background: Exosomes are a subset of small (30-150nm) extracellular vesicles (EVs) of the endocytic origin that mediate intercellular communication in health and disease. Exosomes are actively produced by tumor cells, circulate freely in body fluids and carry messages that alter functions of normal cells in the tumor microenvironment (TME), including immune cell functions. The ability of melanoma exosomes to reprogram immune cell functions might contribute to disease progression.

Methods: To evaluate effects of melanoma cell-derived exosomes (MTEX) on human immune cell subsets, we isolated exosomes from plasma of 12 patients with melanoma and separated MTEX from non-MTEX by immune capture using anti-CSPG4 mAb on beads. These exosome fractions, and exosomes from plasma of 6 normal donors used as controls (NC), were studied by on-bead flow cytometry to quantify their surface protein profiles and in co-incubation assays with immune cell subsets for functional attributes. Phenotypic and functional profiles of MTEX and non-MTEX were compared.

Results & Conclusion: MTEX were enriched (p<0.005)( in FasL and TRAIL, induced apoptosis of CD8+T cells, down-regulated CD69 expression levels on T cells and inhibited their proliferation (all p<0.0005). MTEX also interfered with NK cell functions (p<0.001). Non-MTEX were enriched (P<0.005) in co-stimulatory proteins, CD40L, OX40L, OX40 and, similar

to exosomes of NC, were not immunosuppressive. Thus, MTEX, present in excess in plasma of patients with metastatic melanoma, mediate immune suppression and contribute to immune escape of the tumor.

#3092

Activation of evolutionarily young transposable elements and the host innate immune system are linked to clinical response to 5-azacitidine.

Hitoshi Ohtani,1 Andreas D. Ørskov,2 Alexandra S. Helbo,3 Linn Gillberg,2 Minmin Liu,1 Wanding Zhou,1 Johanna Ungerstedt,4 Eva Hellstrom-Lindberg,4 Weili Sun,5 Gangning Liang,6 Peter A. Jones,1 Kirsten Grønbæk2. 1 _Van Andel Research Inst., Grand Rapids, MI;_ 2 _Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark;_ 3 _Novo Nordisk Foundation Center for Stem Cell Biology, Copenhagen, Denmark;_ 4 _Karolinska Institute, Stockholm, Sweden;_ 5 _City of Hope National Medical Center, Duarte, CA;_ 6 _University of Southern California, Los Angeles, CA_.

The DNA methyltransferase inhibitors 5-azacitidine (AZA) and 5-aza-2-deoxycytidine (DAC) have been approved for the treatment of higher-risk myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML), and acute myeloid leukemia (AML). A more comprehensive understanding of the molecular changes in patients treated with AZA is needed to help explain why only about 50% of these patients have clinical responses. We examined gene expression profiles in a total of 150 RNA samples taken before, during and after treatment with AZA, in three cohorts of patients with hematological cancers (40 patients total; 15 non-responders, 25 responders). We show that activation of the innate immune system is linked to clinical response to AZA treatment. We also measured the induced expression of transposable elements (TEs) to determine whether this was a potential trigger for the immune response. Both responders and non-responders showed robust upregulation of TEs however responders showed the statistically significant upregulation of specific classes of evolutionarily young transposable elements (TEs). These include short and long nuclear elements (SINEs and LINEs) and endogenous retroviruses (ERVs). Moreover, the upregulation of type I interferon signaling pathway genes in responders was significantly higher than non-responders, but not cancer testis antigens (CTAs) or tumor suppressor genes with the exception of CDKN2A and CDKN2B. Therefore, we suggest that induction of specific subsets of silenced evolutionarily young TEs may be a key to the success of epigenetic therapy in hematological cancer patients.

#3093

Prognostic value of novel immune basal subtypes in muscle invasive bladder cancer.

Woonyoung Choi,1 Debasish Sundi,2 I-ling Lee,3 Arlene Siefker-Radtke,3 Colin Dinney,3 Bogdan Czerniak,3 Seungchan Kim,4 David McConkey1. 1 _Johns Hopkins School of Medicine, Baltimore, MD;_ 2 _Ohio State University, Columbus, OH;_ 3 _MD Anderson Cancer Center, Houston, TX;_ 4 _Prairie View A &M University, Prairie View, TX_.

Using gene expression profiling we previously reported the existence of 3 molecular subtypes (basal, p53-like and luminal) characterized by distinct gene expression patterns and clinical outcomes in muscle-invasive bladder cancer (MIBC). Among the subtypes, basal tumors were associated with advanced stage at presentation and shorter survival in the absence of neoadjuvant chemotherapy (NAC). Our recent analysis identified two novel immune subtypes within the basal tumors (basal immune signature enriched - "BIE" and basal immune signature suppressed - "BIS") using immune infiltration markers that are known to have prognostic value in multiple solid tumors. The analysis revealed that BIE had significantly improved survival outcomes while BIS was associated with the worst survival outcomes among all subtypes in TCGA MIBC data (n=408). Survival outcomes in BIE tumors were validated in an independent cohort (GSE48075, p <0.05). The characterization of the immune landscape in our novel subtypes using TCGA's recent pan-cancer immunogenomics data showed that the BIE subtype was significantly enriched with 'INF -γ dominant' immune subtype out of the 6 TCGA immune subtypes (wound healing, IFN-γ dominant, inflammatory, lymphocyte depleted, immunologically quiet and TGF-β dominant, p < 0.05). Since immune activation has been associated with response to chemotherapy in other tumor types, we explored the potential significance of the novel immune subtypes using public data consisting of patients who received NAC followed by radical cystectomy (GSE52219 and GSE69795). In these cohorts, chemotherapy was active as measured by pathological downstaging rates (≤ pT1 at cystectomy). Based on pathological downstaging, BIE was the most responsive to chemotherapy while BIS and p53-like tumors were resistant to chemotherapy (p < 0.05). We also used the recently developed CIBERSORT tool to estimate stromal cell infiltration in the NAC cohort. The results predicted that the BIE subtype was highly enriched with CD8+ T cells and NK cells (p <0.001). Taken together, our results reveal the existence of novel immune subtypes within basal tumors with distinct molecular features and survival outcomes in MIBC. The clinical implications of these subtypes will be further evaluated.

#3094

Reclassification of pancreatic adenocarcinoma with distinct fibroblast and immune characteristics and therapeutic targets.

Pawan Poudel,1 Cindy Neuzillet,2 Hemant Kocher,3 Anguraj Sadanandam1. 1 _The Institute of Cancer Research, London, United Kingdom;_ 2 _Beaujon University Hospital, Paris, France;_ 3 _Barts Institute, London, United Kingdom_.

Pancreatic ductal adenocarcinoma (PDA) with overall poor prognosis is characterized by an abundant desmoplastic stroma comprised of extracellular matrix proteins producing cancer-associated fibroblasts (CAFs). PDA treatment is still primarily based on the use of cytotoxic chemotherapies in unselected patients with modest benefit. To define biomarkers of potential treatment prediction, we (Collisson and Sadanandam; PDAssigner) defined three cancer and four intrinsic CAF (pCAFassigner; first classification to our knowledge) subtypes using PDA patient tumors and primary CAF cultures, respectively. While other groups defined up to four cancer subtypes, there exists additional unrevealed heterogeneity at the level of cancer, stromal and immune cell interactions that defy systematic classification of PDA due to small sample sizes.

To circumvent this issue and to better characterize heterogeneity along with cellular phenotypes, we successfully applied signatures from our pCAFassigner and heterocellular subtypes (representing different cell types/phenotypes of colorectal cancer) along with CIBERSORT to clinical PDA (~757) samples. Interestingly, this analysis identified a subset of our published quasi-mesenchymal (QM)-PDA subtype samples with either increased stem/stroma or inflammatory (not immunogenic subtype) heterocellular characteristics. The stem-like subtype showed variable enrichment of intrinsic CAF (pCAFassigner) subtype characteristics with distinct tumor promoting capabilities (as assessed using CAF:cancer co-culture system) and immune cell interactions. Hence, QM-PDA subtype samples with stem-like characteristics were further split into four stroma-enriched sub-subtypes with significant (p<0.05; log-rank test) differences in patient prognosis. In contrast, the other stem-like QM-PDA subtype patients were enriched for adaptive immunity and may benefit from B cell therapy. On the other hand, inflammatory-enriched QM-PDA subtype samples contained increased macrophages and highly invasive CAFs leading to poorest patient prognosis. These inflammatory subtype patients may respond well to immune therapies targeting macrophages. While all PDA samples are considered to have uniformly high stroma, our study demonstrates that the stromal content is highly variable depending on the sub-subtypes leading to potential personalized therapy responses targeting immune and/or stromal compartments in this devastating cancer type.

#3095

Aneuploidy profiles in hepatocellular carcinoma and their impact on tumor progression and immune features.

Roger Esteban-Fabró,1 Laia Bassaganyas,1 Sara Torrecilla,1 Agrin Moeini,1 Sebastià Franch-Expósito,1 Maria Vila-Casadesús,2 Ferran Nadeu,1 Daniela Sia,3 Itziar Salaverria,1 Laia Cabellos,1 Roser Pinyol,1 Jordi Camps,1 Vicenzo Mazzaferro,4 Vicenzo Mazzaferro,4 Josep M Llovet1. 1 _Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Hospital Clínic, Universitat de Barcelona, Barcelona, Spain;_ 2 _Bioinformatics Unit, CIBEREHD, Barcelona, Catalonia, Spain, Barcelona, Spain;_ 3 _Icahn School of Medicine at Mount Sinai, Tisch Cancer Institute, New York, NY;_ 4 _Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy_.

Introduction: Aneuploidy is a cancer hallmark that includes broad somatic copy-number alterations (SCNAs), being whole chromosome- or arm-level events, or smaller focal SCNAs. Pan-cancer studies suggest that tumor broad and focal SCNAs are linked to distinctive molecular/clinical traits, and broad SCNAs may potentially interfere with tumor immune infiltrates. However, the impact of SCNA genomic loads in hepatocellular carcinoma (HCC) is still unresolved. Here we have assessed broad and focal SCNA burdens in HCC to unveil associations with clinic-molecular characteristics and immune cell profiles.

Method: The study includes 520 paired tumor/adjacent surgically resected HCC samples: 150 of a training cohort (HEPTROMIC) and 370 of a validation cohort (TCGA). Tumor ploidy and SCNA segments were extracted from SNP array data using ASCAT and SAASCNV. We applied the CNApp tool (bioinfo.ciberehd.org/CNApp) to extract a Broad SCNA Score (BCS) and Focal SCNA Score (FCS) to assess broad and focal SCNA loads of each sample. Broad and focal alterations were defined as those spanning ≥50% and <50% of a chromosome arm, respectively. Subsequently, the scores were integrated with a) gene expression data, b) clinic-pathological data, and c) the composition of the tumor immune infiltrate, determined using the Immunophenoscore.

Results: HCC tumors characterized by a low BCS (25% of Heptromic, 15% of TCGA) were associated with the HCC Immune class and up-regulation of genes related to inflammation, active infiltrate signaling, antigen presentation and cytolytic activity (FDR<0.1, p<0.05). Conversely, tumors with high BCS (25% in Heptromic, 45% in TCGA) were linked to polyploidy and TP53 loss of function, were enriched in proliferation and DNA repair gene signatures and presented up-regulation of genes from immune suppressor cells and cytokines. On the other hand, while tumors with high FCS (25% in Heptromic, 49% in TCGA) were associated with TP53 loss of function, up-regulation of genes related to proliferation and progenitor cells and gene expression signatures suggesting increased tumor aggressiveness. Conversely, low-intermediate FCS tumors displayed higher β-catenin pathway activity, with enrichment in CTNNB1 mutations. FSS was not associated with immunity.

Conclusions: Broad are more informative than focal SCNA burdens in terms of molecular features and immune status of HCC tumors. Those tumors characterized by chromosomal stability (low broad SCNAs loads) are enriched in antitumor immune response and antigenicity traits and therefore might correspond to those tumors responding to checkpoint inhibitors.

#3096

Host antitumor immune response activated by Dual POLA1-HDAC11 inhibitors endowed with a large spectrum of antitumor activity.

Claudio Pisano,1 Lucio Merlini,2 Sergio Penco,3 Raffaella Cincinelli,2 Nadine Darwiche,4 Mario B. Guglielmi,1 Ilaria La Porta,1 Maddalena Pizzulo,1 Federica Prosperi,1 Giacomo Signorino,1 Fabiana Colelli,1 Francesco Cardile,1 Laura Focareta,1 Alessandra Fucci,1 Egildo L. D'Andrea,1 Assunta Riccio,1 Sabrina Dallavalle2. 1 _Biogem, Centro Ricerche, Ariano Irpino (AV), Italy;_ 2 _University of Milan, Milan, Italy;_ 3 _Ronzoni Institute for Chemical and Biochem Research, Milan, Italy;_ 4 _American University of Beirut, Beirut, Lebanon_.

Small molecule modulators of chromatin modifying enzymes became the focus of drug discovery efforts, because of their direct antitumor effects, such as apoptosis and cell-cycle arrest, in addition to their indirect immunomodulation properties (Peedicayil, 2012; West and Johnstone, 2014; Falkenberg and Johnstone, 2014). Recent studies have shown that inhibition of HDACs or POLA1 activity exerts anti-proliferative effects and also potentiates the immune response by activating type I Interferon (Medon et al., 2017; Starokadomskyy et al, 2016). To exploit a potential synergy, hybrid molecules targeting simultaneously POLA1 and HDACs, were designed. Here, we present in vitro and in vivo data concerning the antitumor activity of new dual POLA1-HDAC11 inhibitors. Among a library of screened molecules, MIR002 and MIR144 showed an antiproliferative activity at nanomolar concentrations on a panel of human tumor cell lines. In vitro functional assays revealed that these molecules potently inhibit POLA1 and specifically act on HDAC11 that recently emerged as regulator of several immune cells differentiation, pointing out its immunomodulatory role. Mechanistically, MIR002 and MIR144 induce acetylation of Histone H4 and alpha-Tubulin, canonical targets of HDACs, and p53-K382, as well. Activation of p53 leads to p21 increase and cell cycle arrest in the G1/S boundary. These compounds could be even more effective in treating human cancers when they are appropriately combined with other chemotherapeutics, such as Cisplatin. Indeed, the combination of MIR002 or MIR144 with Cisplatin, revealed a synergistic interaction (Combination Index < 1) on NCI-H460 and H460-R9A (ST1926-resistant sub-line) cell growth. The in vivo oral administration of MIR002 or MIR144 showed a potent antitumor activity, with a Tumor Growth Inhibition comprised between 60 and 100% in a series of human and murine solid and hematological tumors (human MM473 and MM432 Pleural Mesothelioma, H460 and H460R9A NSCLC, A2780-Dx and A2780-DDP ovarian carcinomas, NB-4 Acute Promyelocitic leukemia and Murine EL-4 Lymphoma) xenografted in nude and immunocompetent mice. The strongest antitumor effect of MIR144 was observed in immunocompetent mice rather than in nude mice suggesting that an immunomodulatory effect, along with the direct antiproliferative activity, contributes to tumor inhibition. Further analysis confirmed that MIR144 antitumor activity involves the induction/activation of several components of the host immune system, both at humoral and cellular levels. The large spectrum of antitumor activity, together with the high tolerability observed, open the possibility for their clinical investigation in different population of cancer patients.

#3097

Investigating an aggressive molecular subtype of stage I lung adenocarcinoma: Evidences for the acquirement of stem-like and immune evasion phenotype.

Valentina Melocchi, Elisa Dama, Roberto Cuttano, Tommaso Colangelo, Paolo Graziano, Fabrizio Bianchi. _IRCCS Casa Sollievo della Sofferenza Foundation, San Giovanni Rotondo, Italy_.

Lung cancer early diagnosis is effective in reducing the risk of metastatic disease and improve overall survival. However, a significant fraction of patients (~20%) with early stage disease (Stage I) experience tumor recurrence and metastatic disease. Recent advances in high-throughput genomic screening allowed an in-depth molecular profile analyses of lung cancer, with particular emphasis for the identification of prognostic gene expression signatures. Indeed, we recently described the existence of an aggressive lung adenocarcinoma (LUAD) molecular subtype (Stage I), namely C1-LUAD, with a peculiar expression and mutational profile more resembling advanced metastatic cancer. Using an integrative approach relying on computational biology and lung cancer experimental models, we now discovered that this C1-subtype is characterized by stem-like and immune evading phenotype. In particular, gene-set enrichment analysis (GSEA) revealed that 'C1-LUAD' tumors are enriched in stem cell (SC) expression signatures (q-value<0.05). Notably, the gene network characterizing the C1-LUAD subtype was downstream to known transcriptional modulators involved in SC biology. In line with this, we found several iPS/Stem and EMT biomarkers overexpressed in C1-LUAD and in a panel of C1-LUAD-like lung cancer cells. Furthermore, forced overexpression of HOXB7 gene, a biomarker of C1-LUAD and a member of the Antp homeobox family, promotes the expansion of lung sphere-forming cells and reprogramming into iPS cells. Lastly, we performed an analysis of the Immune Subtypes and ImmunoPhenoScore in C1-LUAD tumors coupled with tumor mutational burden (TMB) and intra-tumor genetic heterogeneity (ITH) profile, which revealed: i) a predominance of the tumor permissive 'C1 wound healing' subtype (i.e. high Th2/TAMs, low CD8+Tc); ii) a reduction of the 'C3 inflammatory' subtype; iii) an increased ITH; and iv) a reduction of neoantigen burden. These are hallmarks of cancer immunoediting process which ultimately favor tumor cell escape. In conclusion, we identified an aggressive subtype of LUAD characterized by a stem-like and immune evasion phenotype. C1-LUAD can be identified by qPCR analysis using a panel of 10 genes, previously validated in a large cohort of FFPE samples. Our findings can contribute to improve survival of lung cancer patients by anticipating diagnosis of aggressive Stage I tumors and may pave the way for developing more effective therapeutic strategies.

#3098

Maximizing the sensitivity and utility of tissue biopsy by analyzing RNA and incorporating multiple immunotherapy markers.

Christopher Kasbek, Cai Chen, Nouran Almalki, Jillian Cortese, Jun Huang. _Admera Health, LLC, South Plainfield, NJ_.

Next-generation sequencing panels are becoming more widely accepted as diagnostics for guiding personalized medicine in oncology. Some weaknesses of current NGS panels include: a lack of prediction of the patient's response to the recently approved PD-1 checkpoint inhibitors, low sensitivities of fusion variant detection due to the complexity of the translocation events that can occur at the DNA level, and the need for matched normal sample. We are developing a 371-gene panel that uses both DNA and RNA from a single non-matched FFPE sample to guide targeted and immunotherapy decisions. Fusions are identified in RNA, while copy number variation, tumor mutation burden (TMB), microsatellite instability (MSI), HLA typing, single-nucleotide variants, and small insertion/deletion variants are identified in DNA. All exons plus UTR regions of 371 genes are covered at a mean depth of >500X. To validate the TMB test, whole-exome sequencing data from 376 colon adenocarcinoma samples was analyzed with our pipeline using a 4-fold cross-validation design. TMB-high vs. TMB-low was called with 95% accuracy, compared to 90% accuracy of a competitor's panel. For MSI, the canonical five mononucleotide-repeat sites are interrogated with a limit of detection of 2-5%. RNA samples with Dv200 >10% can be used to identify fusion variants. RNA is converted to cDNA and hybridized with a mixture of probes containing ≈300 well-known fusion junctions and full exon sequences of 37 of the 371 genes which are relevant to fusions. This design leads to improved fusion detection compared to the standard DNA detection, while allowing for the identification of novel fusion junctions. Gender and common SNP's in DNA and RNA are used to prevent sample misidentification. The accuracy and comprehensiveness of this panel should maximize benefit to the patient.

#3099

Characterization of bone marrow tumor associated macrophages in patients with chronic lymphocytic leukemia treated with Ibrutinib.

PAOLO STRATI, ELLEN SCHLETTE, LUISA SOLIS TOTO, DANIELA DUENAS, IGNACIO WISTUBA, JAN BURGER. _MD Anderson Cancer Center, HOUSTON, TX_.

Introduction. Complete remission (CR) is observed in 9-26% of patients with chronic lymphocytic leukemia (CLL), with virtually no patient converting to CR beyond 2 years of treatment. Residual bone marrow (BM) disease is typically observed in patients not achieving CR, increasing interest about the specific effects of ibrutinib on the CLL BM microenvironment. Ibrutinib markedly increases the number and function of T cells, and suppresses regulatory B-cell function. However, it may induce M2 polarization of tumor-associated macrophages (TAM), as suggested by limited studies conducted on peripheral blood.

Methods. In order to characterize the specific effect of ibrutinib on the CLL BM microenvironment, we identified patients with previously untreated CLL, receiving frontline single agent ibrutinib for more than 2 years, for whom BM biopsy samples were available at baseline and after 1 and 2 years of treatment. All samples were analyzed by immunohistochemistry and the number of positive cells per high power field (HPF)(200X) was assessed; CD68 was used as a general TAM marker, and CD163, PD-L1 and arginase-1 as M2 markers. Negative BM samples, collected as part of lymphoma staging, were used as normal controls.

Results. Thirteen patients were included in the study; 85% had unfavorable FISH abnormalities (including deletion 11q and deletion 17p) and 77% had unmutated IGHV. After > 2 years of treatment, 3 (23%) patients had achieved CR, and 10 (77%) partial remission (PR), characterized by residual BM disease.

BM samples were available for 11 patients at baseline, 12 patients at 1 year of treatment, and 9 patients at 2 years of treatment. PD-L1 and arginase-1 were negative in BM samples from all patients at all time points and in all 3 normal BMs. The median number of CD68-positive cells/HPF was 3 (range, 3-5) at

baseline, 5 (range, 3-20) at 1 year, and 5 (range, 3-10) at 2 years; no differences in CD68 expression was observed between patients who achieved PR and those who achieved CR. All patients with normal BM had five CD68-positive cells/HPF. The median number of CD163-positive cells/HPF was 5 (range, 5-15) at baseline, 15 (range, 10-30) at 1 year, and 20 (range, 15-20) at 2 years; a more pronounced increase in CD163-positive cells was observed among patients who achieved PR as compared to patients who achieved CR. All patients with normal BM had approximatively ten CD163-positive cells/HPF.

Conclusions. Ibrutinib increases BM CD68 (TAM) in patients with CLL, reaching a plateau after 1 year of treatment, and BM CD163 (TAM M2), with no plateau observed at 2 years of treatment. The increase in TAM M2 is more pronounced in patients who do not achieve CR after > 2 years of treatment, representing a potential therapeutic target for consolidation strategies. Additional M2 markers, including CSF1R, and digital analysis are being applied, and results will be presented at the meeting.

#3100

Blinatumomab is safe and effective in relapsed and MRD positive B-ALL CD19+ patients: The bologna compassionate program experience.

Giovanni Martinelli,1 Stefania Paolini,2 Claudio Cerchione,1 Alessandra Santoro,1 Valentina Robustelli,2 Simona Soverini,2 Caterina De Benedittis,2 Enrica Imbrogno,1 Andrea Ghelli Luserna Di Rora,2 Sarah Parisi,2 Chiara Sartor,2 Giovanni Marconi,2 Silvia Lo Monaco,2 Maria Chiara Abbenante,2 Maria Chiara Fontana,2 Antonella Padella,1 Anna Ferrari,1 Giorgia Simonetti,1 Elena Tenti,2 Federica Frabetti,1 Francesca Volpato,2 Samantha Bruno,2 Fabiana Mammoli,1 Maria Teresa Bocchicchio,1 Carmen Baldazzi,2 Cristina Papayannidis2. 1 _IRST IRCCS, Meldola (FC), Italy;_ 2 _University of Bologna, Bologna, Italy_.

Adult B-ALL patients still have a dismal prognosis, due to a high incidence of relapse even after allogenic SCT. Safety and efficacy of Blinatumomab, an anti CD3-CD19 Bite antibody, has been demonstrated both in MRD positive patients and in relapsed/refractory (R/R) setting. To evaluate safety profile and efficacy of Blinatumomab, obtained through a compassionate use, in a cohort of 18 adult patients affected by MRD+ or R/R B-ALL treated at Bologna University. From March 2015 to December 2017, 18 patients received Blinatumomab at the standard dosage (9 mcg/d x 7 days, 28 mcg/d x 21 days) in 28-days courses. All the patients were hospitalized to receive the first course of therapy. The following courses, based on the good safety profile of the compound, were administered in outpatient setting. 18 patients (M/F = 10/8; median age 43, range 18-73) have been treated. Philadelphia (Ph) chromosome was detected in 8/18 patients. 10 patients were MRD+ (5 Ph pos and 5 Ph neg); E2A-PBX1 and MLL-AF4 rearrangements were found in two patients. 8 patients had a R/R disease (3 Ph pos and 5 Ph neg). Median WBC count before starting therapy with Blinatumomab was 5400/mmc (range 500-76500). All the patients had previously received many lines of therapy (median 4, range 1-7). In 4 cases an alloTMO was already performed, and two patients had received two transplants. 12/18 patients were referred to us by other Italian Institutions. All the patients received at least one course of Blinatumomab. In one case three courses were administered; an elderly patient is actually receiving the fifth course. Globally, 32 courses of therapy have been administered (median 2, range 1-5). Bone marrow evaluations, including cytogenetics, molecular biology and immunophenotyping analysis were performed at the beginning of every course of therapy in order to assess patients' disease status. MRD evaluation was assessed through BCR-ABL fusion transcript quantitative analysis in Ph pos ALL patients and Ig rearrangment in Ph negative patients. Monitoring of adverse events was periodically performed. 16/18 patients are evaluable for response, at least to one cycle (one patient died during the first course, one patient is still receiving the first course). 9/16 (56%) patients obtained a CR (7/9 MRD+ and 2/7 R/R). In 7/9 (78%) responders patients a molecular CR was reached, (in 6 patients after the first course, in one case after the second one). 5 responders proceeded to alloBMT and are actually alive in CR (median follow-up after transplant 240 days). In terms of toxicity, one patient developed a grade IV neurological event (mental confusion, tremor), which completely resolved after a transient drug withdrawal. Our results confirm the high rate of response and to Blinatumomab in a poor patients' population, and the good management profile of the compound.

#3101

Functional analysis of HDAC11 in plasma cell development and multiple myeloma survival.

A G M MOSTOFA, Jason Brayer. _Moffitt Cancer Center and Research Institute, Tampa, FL_.

Though HDAC11 expression is confirmed in B cells and plasma cells (PC), its functions in these cells remain largely unknown. In this study, we attempted a functional analysis of HDAC11 in PC development along with its pro-tumorigenic function in multiple myeloma (MM) cells. Mouse models, including a transgenic mouse strain expressing eGFP under the regulation of the HDAC11 promoter (Tg-HDAC11-eGFP), and also an HDA11-deficient mouse (B6.HDAC11-/-) were studied to establish the importance of HDAC11 in PC biology. Pharmacologic inhibition of HDAC11 in MM cell lines was accomplished by using elevenostat (ES), a new HDAC11-selective inhibitor. Post-translational modifications and subcellular localization changes induced by ES exposure were assessed by western blotting of fractionated cell lysates, while co-immunoprecipitation (Co-IP) and proximity ligation assays (in situ PLA) were used to identify a binding partner for HDAC11. Studies in Tg-HDAC11-eGFP mice revealed that HDAC11 expression in B cell lymphopoiesis was minimally detectable prior to B cell activation but demonstrated strong induction upon maturation into a PC. Consistent with this, PC development was markedly impaired in the absence of HDAC11. Furthermore, HDAC11-selective inhibitor ES showed significant cytotoxic potential, measured by CCK-8 assay, in different MM cell lines and primary MM cells that express moderate to high level of HDAC11, with IC50 values ranging 0.6-2.0 µM. Apoptotic cell death was confirmed via detection of activated caspase-3 and annexin/propidium iodide staining by flow cytometry. Consistently, MM cell lines expressing null/very low level of HDAC11 were insensitive to ES. Moreover, combining ES with proteasome inhibitors bortezomib and carfilzomib resulted in significant synergistic effects. Furthermore, ES-treated cells showed a time-dependent alteration in the subcellular localization of HDAC11, it gradually disappeared from the nuclear fractions with simultaneous upregulation in cytoplasmic fractions. Inhibition of HDAC11 also caused downstream suppression of several pro-tumorigenic factors in MM cells, including IRF4 and c-Myc. Moreover, a novel interaction between HDAC11 and IRF4, an essential regulator of PC differentiation and MM survival, was identified by using PLA and Co-IP. HDAC11 dynamically interacts with IRF4 which can be induced by LPS and IL-6 stimulation and inhibited by ES, indicating the involvement of HDAC11 in the IRF4-mediated regulatory circuit. We also identified changes in the extent of IRF4 acetylation upon genomic or pharmacological induction/inhibition of HDAC11. Overall, we observe that targeted inhibition of HDAC11 can impair MM cell survival and overcome acquired resistance to proteasome inhibitors. Furthermore, we identify IRF4 as a nuclear binding partner of HDAC11 and propose this interaction as a candidate mechanism regulating PC maturation and survival.

#3102

The Chilean gastric cancer task force: Final report.

Mauricio P. Pinto,1 Ignacio N. Retamal,1 Maria Loreto Bravo,1 Matias Muñoz-Medel,1 Miguel Cordova-Delgado,1 Diego Romero,1 Maria Jose Maturana,1 Nathaly De La Jara,1 Javiera Torres,1 Manuel Espinoza,1 Carlos Balmaceda,1 Matias Freire,2 Valentina Garate-Calderon,2 Javier Caceres-Molina,2 Gonzalo Sepulveda-Hermosilla,2 Rodrigo Lizana,2 Liliana Ramos,2 Fernando Crovari,1 Ricardo Armisen,2 Alejandro Corvalan,1 Gareth I. Owen,1 Marcelo Garrido1. 1 _Pontificia Universidad Catolica de Chile, Santiago, Chile;_ 2 _Centro de Excelencia en Medicina de Precision (CEMP), Santiago, Chile_.

Background: Globally, gastric cancer (GC) ranks as the third cause of cancer related mortality. Like most malignancies, GC is characterized by its heterogeneity. In Chile, GC is the leading cause of cancer death claiming >3,000 deaths/year. Given its high heterogeneity an effective GC patient stratification may improve clinical outcomes. Here, we describe the results of the Chilean Gastric Cancer Task Force (GCTF) a descriptive cross sectional study (ClinicalTrials.gov identifier: NCT03158571, registered May 18, 2017) reporting clinical, genomic and protein expression data in a cohort of 224 patients.

Methods: Demographic and basic information was obtained from 224 Chilean patients. We assessed p53, p16, HER2, and PDL1 expression and markers of Microsatellite Instability (MSI) by Tissue Microarray (TMA). We also assessed Epstein Barr Virus (EBV) status in a subset of 90 patients. Using Next Generation Sequencing (NGS), we profiled 143 cancer-related genes in a subset of 116 patients.

Results: Patients were predominantly male (63.4%) staged III/IV (39.3 and 21.9%, respectively). Tumors were preferentially located at the stomach body (38.4%). Median overall survival (OS) was 39.0 months. By TMA analysis, 26.7% of patients were PDL1+, 13.3% were EBV+, and 13.3% MSI+. 36.7% were p16+, 13.3% HER2+3 and 56% possessed p53 overexpression. NGS confirmed TP53 as the most frequently mutated gene, followed by PI3KCA, VHL, PTEN and KRAS, together with alterations in numerous other actionable genes.

Conclusions. Our data in Chilean GC patients demonstrate EBV+ is present at a higher proportion than reported in other geographical regions. In line with the published literature in South America, most patients were males staged III/IV. As anticipated, molecular analysis confirmed p53 as the most frequently altered gene in our cohort. A high number of further genomic alterations within actionable genes may allow the use of precision medicine within the Chilean population. 

### New Discoveries in Childhood Cancer

#3103

Intraparenchymal delivery of chemokines and immunomodulators to eliminate pediatric brain tumor cells.

Eric S. Nealy,1 Cole A. DeForest,1 James Olson2. 1 _University of Washington, Seattle, WA;_ 2 _Fred Hutch, Seattle, WA_.

Pediatric brain tumors are among the leading causes of cancer-related death in children. These malignancies tend to occur in locations of the brain where surgery and radiation can permanently impair a patient's quality of life. Migratory cells from high grade brain tumors can invade nearby, inaccessible areas of the brain where they may grow unchecked and ultimately lead to patient death. This project seeks to tailor the microenvironment around the tumor cavity to promote the recruitment and elimination of remnant brain tumor cells by microglia and macrophages found in the brain. Our data suggests gradients of classical immune cell chemokines, like CCL2, are also potent chemotactic signals for a variety of patient-derived brain tumor types. Monoclonal antibody blockade of CD47, a cell-surface "don't eat me" ligand often over-expressed on tumor cells, is effective at promoting the elimination of tumor cells in close-proximity to murine macrophages in vitro. We engineered user-programable hydrogel depots capable of month-long molecule release and implanted them into tumor-bearing mouse brains. Hydrogels incorporated with CCL2 and CD47mAb demonstrated statistically significant co-recruitment of tumor and immune cells and evidence of macrophage-mediated tumor cell death compared to PBS-incorporated gels. These results suggest proper stimulation of immune cells within the brain could be an effective means to eliminate pediatric brain tumor cells without causing structural damage to vital nervous tissues. Our data may be the first steps towards clinical trials for children with incompletely resected brain tumors.

#3104

A high prevalence of chromosomal translocations as drivers in high-risk pediatric solid cancers.

Laura B. Corson,1 Alma Imamovic-Tuco,1 Gianna R. Strand,2 Deirdre Reidy,2 Duong Doan,2 Mark A. Applebaum,3 Rochelle Bagatell,4 Brian D. Crompton,5 Steven G. DuBois,6 Julia L. Glade Bender,7 AeRang Kim,8 Theodore W. Laetsch,9 Lobin A. Lee,2 Neal I. Lindeman,10 Laura E. MacConaill,2 Margaret E. Macy,11 Luke Maese,12 Seth Pinches,13 Navin Pinto,14 Amit J. Sabnis,15 Eliezer M. Van Allen,1 Susan I. Vear,16 Daniel A. Weiser,17 Catherine M. Clinton,6 Katherine A. Janeway,6 Alanna J. Church13. 1 _Dana-Farber Cancer Institute and Broad Institute, MA;_ 2 _Dana-Farber Cancer Institute, Boston, MA;_ 3 _University of Chicago Comer Children's Hospital, Chicago, IL;_ 4 _Children's Hospital of Philadelphia, Philadelphia, MA;_ 5 _Dana-Farber Cancer Institute, Broad Institute, and Boston Children's Hospital, MA;_ 6 _Dana-Farber Cancer Institute and Boston Children's Hospital, Boston, MA;_ 7 _Columbia University Medical Center, New York, NY;_ 8 _Children's National Medical Center, Washington, DC;_ 9 _UT Southwestern Medical Center, Dallas, TX;_ 10 _Brigham and Women's Hospital, Boston, MA;_ 11 _Children's Hospital Colorado, Denver, CO;_ 12 _Primary Children's Hospital, University of Utah, Salt Lake City, UT;_ 13 _Boston Children's Hospital, Boston, MA;_ 14 _Seattle Children's Hospital, Seattle, WA;_ 15 _University of California San Francisco, San Francisco, CA;_ 16 _Nationwide Children's Hospital, Columbus, OH;_ 17 _Children's Hospital at Montefiore, Bronx, NY_.

The GAIN iCat2 Project is a collaboration between Dana-Farber/Boston Children's Cancer and Blood Disorder Center and eleven pediatric oncology centers across the United States to sequence relapsed, metastatic, difficult-to-diagnose, and high-risk extracranial solid tumors from 825 patients. The goals are to gain a better understanding of the genomic events in pediatric cancers and determine the clinical impact of matched targeted therapy. Tumor samples are sequenced on one of four gene panels performed in CLIA certified, CAP accredited laboratories, most often utilizing OncoPanel at the Center for Advanced Molecular Diagnostics, Brigham Women's Hospital. This panel assesses SNVs and CNVs in 447 cancer-associated genes and interrogates intronic regions of 60 genes frequently involved in oncogenic translocation. For undifferentiated sarcomas and tumors in which oncogenic drivers are not identified by the gene panel, whole exome sequencing or RNA sequencing for fusion detection may be done. Interpretation of genomic results, including potential implications for diagnosis and hereditary risks, as well as assessment of possible matched targeted therapies and suitable trials are summarized in a report to the primary oncology provider.

An interim analysis of tumors from the first 275 patients enrolled who have OncoPanel results was performed to assess genomic alterations most prevalent in this group of pediatric cancers. 50% (137/275) have structural alterations in their tumors with over half of these (74/137) harboring an oncogenic fusion that is the main, or only identified, driver of the cancer. These include fusions pathognomonic for diseases such as Ewing sarcoma, alveolar rhabdomyosarcoma, synovial sarcoma, desmoplastic small round cell tumors, mesenchymal chondrosarcoma, low grade fibromyxoid sarcoma, and NUT midline carcinoma. Other cases showed recurrent disruption of key tumor suppressors, such as TP53 intron 1 translocations in osteosarcoma. Lastly, more generalized, key, cancer-driving fusions were seen with rearrangements involving BRAF, NOTCH, and NTRK. In addition to aiding in diagnosis, identification of fusions has led to targeted therapy recommendations for many patients. SNVs and CNVs also helped clarify diagnoses, especially in the case of DICER1 and SMARCB1 alterations, and identified potential targeted therapies to consider for relapsed patients. Although patient recruitment is ongoing, this study shows promise for advancing our understanding and treatment of pediatric cancers and highlights the critical importance of incorporating techniques for fusion detection in tumor profiling.

#3105

Serial profiling of ctDNA identifies clinically actionable genomic evolution in high-risk neuroblastoma.

Kristopher R. Bosse,1 Samantha Buongervino,1 Maria Lane,1 Adam Hyman,1 Maria Gemino-borromeo,1 Jennifer Saggio,1 Alana Fitzsimmons,1 Brady Forcier,2 Anne Murphy,2 John Wick,2 Matthew Cooke,2 Jennifer Webster,2 Russell Madison,2 Alley Welsh,2 Vincent A. Miller,2 Siraj M. Ali,2 John M. Maris,1 Yael P. Mosse1. 1 _Children's Hospital of Philadelphia, Philadelphia, PA;_ 2 _Foundation Medicine, Inc., Cambridge, MA_.

Background: Analysis of circulating tumor DNA (ctDNA) provides a noninvasive method to profile tumor-associated genomic aberrations, heterogeneity and evolution.

Methods: Peripheral blood from 5 newly diagnosed and 33 relapsed high-risk neuroblastoma patients was serially profiled for ctDNA (n=1-7 samples/patient; total n=122 samples) with the FoundationACT assay that utilizes hybrid capture-based genomic profiling of 62 genes. Samples were sequenced to a median unique coverage depth of at least 3168x and variants were evaluated and compared with temporally-matched tumor sequencing and imaging evaluations.

Results: Ninety-three percent (114/122) of peripheral blood samples yielded suitable cell-free DNA for sequencing. ctDNA was detected in 68% (78/114) of samples (maximum somatic allele frequency [MSAF] >0; median MSAF=0.53%; range MSAF 0-79%). At least 1 pathogenic genomic alteration (genomic short-variant or amplification) was found in 54% (61/114) of samples (range 1-6 alterations). Fifty-four percent (13/24) of detected ctDNA genomic-short variants were not present in temporally matched tumor samples at diagnosis or relapse (collected within 3 months of each other; n=23 patients), including pathogenic variants in ALK, TP53, TERT, NF1, FLT3, PTPN11, and PIK3CA. There was 100% concordance between the detection of MYCN (6/6) and ALK (3/3) amplification in paired ctDNA/tissue samples. For example, a newly diagnosed patient with stage 4 neuroblastoma had MYCN amplification noted on both tumor and ctDNA sequencing, however had 3 separate ALK mutations (R1275Q, F1245L, and F1174L) uniquely identified in ctDNA. Twenty-eight patients had multiple ctDNA samples sequenced (range 2-7 samples) and 50% (14/28) had alterations emerge or regress across serial samples. For example, pathogenic variants in ALK, BRCA2, NRAS, PTEN, TP53, CDKN2A, PTPN11, ABL1, CDH1, MET, ERRFl1 and ERBB2 appeared in subsequent ctDNA sequencing that were not present in the initial ctDNA or tumor sequencing. Overall, serial ctDNA profiling identified additional pathogenic variants in driver cancer genes beyond that derived from tumor sequencing in 45% (17/38) of cases, including identifying targetable ALK-RAS-MAPK pathway alterations uniquely in ctDNA in 21% (8/38) of cases. For the 18 patients that had 3 or more ctDNA samples, 33% (6/18) developed ctDNA unique ALK-RAS-MAPK pathway mutations during therapy. Finally, in most cases ctDNA profiling was complementary to standard imaging surveillance, however in 3 cases rising ctDNA allele frequencies occurred prior to clinical or imaging signs of disease relapse; additional correlation of ctDNA data and disease evaluations is ongoing.

Conclusions: Sequencing of ctDNA from neuroblastoma patients identified clinically actionable tumor-associated genetic aberrations emerging under the selective pressure of standard and targeted therapies.

#3106

Targeting vitamin D signalling in metastatic neuroblastoma.

Yagnesh Ladumor,1 Bo Kyung Alex Seong,1 Robin Hallett,2 Teresa Adderley,2 Yingying Wang,2 Lynn Kee,2 David Kaplan,2 Meredith Irwin2. 1 _University of Toronto, Toronto, Ontario, Canada;_ 2 _Hospital for Sick Children, Toronto, Ontario, Canada_.

Background: Neuroblastoma (NB) is the most common extra-cranial solid tumor and the most frequent cause of cancer-related deaths in children. More than half of patients with NB have metastases and their survival is <50%, and only 5% after relapse. Currently, there are no therapies that specifically target metastatic NB and there is a lack of NB models that recapitulate the sites and burden of metastases observed in patients.

Methods: To study metastatic NB, we have developed a mouse model using in vivo selection of SKNAS cells that metastasize following intra-cardiac injection. We isolated subpopulations from bone and brain with enhanced metastatic properties and compared their gene expression profiles to those of the parental SKNAS cells. Using Connectivity Map (CMap) analyses, we identified drugs that were predicted to change the gene expression in the metastatic subpopulations to a profile that is similar to that of parental cell lines.

Results: Calcipotriol, a synthetic analogue of vitamin D, was identified from the CMap analysis to be a potentially selective agent for metastatic NB cell lines. In comparison to the parental cells, treatment of the metastatic subpopulations with calcipotriol resulted in reduced proliferation. Furthermore, calcipotriol sensitivity was reduced in metastatic cells with a knockout of vitamin D receptor (VDR) suggesting its effect is on-target. In contrast to the parental cells, following calcipotriol treatment metastatic subpopulations did not exhibit an increase in protein levels of CYP24A1, the enzyme that metabolizes vitamin D, suggesting a potential mechanism for the differential sensitivity of parental and metastatic cells. Calcipotriol treatment also reduced levels of hippo pathway effectors, YAP and TAZ, which we previously reported to play a role in mediating the metastatic phenotype of the isolated subpopulations. Additionally, metastatic cells that were pre-treated with calcipotriol showed reduced migration in a transwell assay whereas the migration potential of parental cells was not affected. Furthermore, RASSF2, an upstream regulator of the hippo pathway, was identified to be upregulated in a VDR-dependent manner after calcipotriol treatment in the metastatic subpopulations indicating a possible mechanistic link for the effects of calcipotriol on the hippo pathway in these metastatic cells.

Conclusions: Calcipotriol was identified to be more effective against metastatic NB subpopulations in vitro and this may be in part due to defects in CYP24A1 induction in these metastatic cells. Our data also suggests a novel link between VDR, the hippo pathway and metastasis in NB. Further experiments are required to determine the role of VDR, mechanism of action of calcipotriol in selectively inhibiting growth of metastatic NB cells.

#3107

A scientific task force to generate proof-of-concept data packages for clinical trials in pediatric cancers: The hepatoblastoma example.

Carolina Armengol,1 Roland Kappler,2 Liqin Zhu,3 Nikolai Timchenko,4 Dina Kats,5 Charles Keller,5 Lisa Howard,6 Christophe Grosset,7 Delphine Nicolle,8 Olivier Déas,8 Martina Pigazzi,9 Laurence Brugières,10 Charlotte Mussini,11 Louise Galmiche-Rolland,12 Christophe Chardot,12 Sophie Brancereau,11 Jean-Gabriel Judde,8 Stefano Cairo8. 1 _Germans Trias i Pujol Research Institute (IGTP), Badalona, Spain;_ 2 _Dr. von Hauner Children's Hospital-LMU, Munich, Germany;_ 3 _St. Jude Children's Research Hospital, Memphis, TN;_ 4 _Cincinnati Children's Hospital, Cincinnati, OH;_ 5 _Children's Cancer Therapy Development Institute, Portland, OR;_ 6 _Macy Easom Cancer Research Foundation, Perry, GA;_ 7 _University of Bordeaux, Bordeaux, France;_ 8 _XenTech, Evry, France;_ 9 _Pediatric Research Institute (IRP), Padova, Italy;_ 10 _Gustave Roussy Institute, Villejuif, France;_ 11 _Bicêtre Hospital, Le Kremlin Bicêtre, France;_ 12 _Necker Enfants Malades Hospital, Paris, France_.

Identification of new treatments for relapsing pediatric cancer is an unmet clinical need and a societal challenge. Hepatoblastoma (HB) is a rare tumor that constitutes the most frequent form of childhood liver cancer. Current improvement in medical care allows 80% of children to survive this disease, but no second line treatment is available at relapse, so that 20% will not survive. Patient population small size and very low mutation rate besides beta-catenin activating mutation, which is at present undruggable, make HB an orphan disease. Very few anti-cancer drugs evaluated for adult diseases are investigated in the pediatric population, with 12 drugs approved for adult cancers every year against 6 drugs approved in childhood cancer since 1978. The need of incentives to encourage pharmaceutical industry to invest in this field has been well received by the European and US medicine agencies, and recent modification to regulations on adult and pediatric oncology drug development are expected to induce pharmaceutical companies to test their new compounds in the pediatric setting. Parallel to this initiative, it is imperative that the scientific community develops research tools and scientific rational to assist drug development as well as repositioning of drugs already approved in adult cancers. To this aim, in collaboration with the International Childhood Liver Tumour Strategy Group (SIOPEL) and the network of hospitals in the Paris area (AP-HP), XenTech has launched in 2010 the development of a panel of HB patient-derived xenografts (HB-PDXs). To date, 26 HB-PDXs have been established from 80 engraftments from patients at surgery after chemotherapy or at relapse. As these models were established from tumor cells that either evaded chemotherapy or promoted local and distant metastases, they present a biological surrogate of the recurrent disease. From these PDXs, 9 cellular models have been established to allow the exploration of the functional mechanisms of tumor growth and resistance to treatment, as well as to facilitate drug screening. This project is receiving worldwide participation from the HB scientific community including clinicians, academic researchers and parents associations. Thanks to this joint effort all the models are now fully characterized at the molecular level, PDXs pharmacological profiling has been done and is currently ongoing for many of these models, and PDXs and cellular models are being used from a growing number of collaborators to perform 3D culture, to improve our knowledge on the HB biology and to identify new drugs. The ambition of this project is to generate a reference preclinical platform that will be used for research purposes by the academic community and as a testing site for biotechs and pharmaceutical companies to accelerate the identification of anti-cancer therapies for children with aggressive HB.

#3108

Inhibition of the mTOR and MAPK pathways suppresses growth, induces apoptosis, and decreases vascularity in pediatric low grade glioma.

Antje Arnold, Lauren Harris, Ming Yuan, Fausto Rodriguez, Charles Eberhart, Eric Raabe. _Johns Hopkins University School of Medicine, Baltimore, MD_.

Pediatric low-grade glioma (pLGG) is the most common brain tumor in children. We and others have recently identified constitutive activation of the mTOR and MEK-pathway in pLGG. This led us to investigate the antitumor activity of TAK228, a second generation mTORC1/2-inhibitor, and the FDA approved MEK-inhibitor, trametinib, in mono- and combination therapy in pLGG. We examined antitumor activity in five different patient-derived pLGG cell models treated with TAK228, trametinib, and combination: JHH_NF1_PA1NF1mut, BT66_SV40BRAF:KIAA1549, BT40BRAFV600E, Res186PTEN-/- and Res259CDKN2A-/-. In all cell lines, as measured by western blot (WB), trametinib treatment led to suppression of phospho-ERK at low levels (1-5nM). Similarly, TAK228 treatment led to inhibition of mTORC1 and mTORC2 in the low nanomolar range. Treatment with TAK228 or trametinib reduced cell proliferation in a dose and time dependent manner (MTS-assay). The combination treatment exerted a synergistic effect at 5-20nM in JHH_NF1_PA1, Res186, and Res259 cells (by Chou-Talay method). The BT66 cell line had a 70% reduction in cell growth with 10nM TAK228 (p<0.001 by 1-way ANOVA), but not in combination, or mono-treatment with trametinib in clinically relevant dose range. The combination of TAK228 and trametinib increased apoptosis by up to 127% (p<0.001) in Res186, Res259, and JHH_NF1_PA1 cells as measured by cleaved caspase 3 immunocytochemistry. We tested trametinib and TAK228 against the mutant BT40BRAFV600E patient-derived xenograft cell line in immunodeficient mice. The combination of TAK228 with trametinib showed greater antitumor activity than that of either mono-treatment in vivo. BT40 tumor growth was significantly decreased in combination compared to vehicle or either agent alone, (p<0.01). Immunohistochemistry (IHC) imaging showed a significant decrease in proliferation marker Ki67 with combination treatment (p<0.0001), and trametinib mono-treatment (p=0.0466), compared to TAK228 and vehicle. TAK228 and combination treatment decreased the mTORC1 phosphorylation of S6 ribosomal protein (WB & IHC). Combination treatment strikingly reduced the vascularization of the tumor after combination treatment analyzed by VEGF protein expression (WB, p=0.0076), compared to the vehicle control, and mono-treatment. IHC confirmed a reduction in vascularization by CD31 staining. Our study provides evidence that pLGG-derived cell lines in vitro and in vivo are sensitive to mTORC1/2 kinase inhibition and MEK inhibition. Combination treatment with TAK228 and trametinib had a significant anti-tumor activity in vivo shown in survival rate, decreased tumor size, and reduced vascularity. These results provide the first strong rationale for combination therapy with TAK228 and trametinib for clinical consideration in pLGG in the near future.

#3109

Protein stability screen to identify compounds destabilizing EWS-FLI1 in Ewing sarcoma.

Gloria Pedot, Felix K. Niggli, Beat W. Schaefer. _University Children's Hospital Zurich, Zurich, Switzerland_.

Ewing sarcoma is an aggressive pediatric bone and soft tissue tumor driven by the expression of a fusion oncoprotein named EWS-FLI1, which acts as an oncogenic transcription factor. Tumor cells are strictly dependent on continuous expression of the fusion protein, since downregulation of EWS-FLI1 inhibits tumor growth. Therefore, interference with the fusion oncoprotein turnover is critical for the modulation of tumor cell proliferation and survival. We demonstrated previously that EWS-FLI1 is predominantly a proteasomal substrate with high turnover mediated by poly-ubiquitination at one specific lysine. This study provided novel insights into the crucial importance of targeting EWS-FLI1 stability as novel strategy for the treatment of Ewing sarcoma. Hence, we aimed at identifying compounds that destabilize EWS-FLI1 with subsequent reduction of tumor cell growth by performing a screen of 2'486 FDA-Approved drugs and 204 novel targeted compounds in a Ewing sarcoma reporter cell line. To this, we adopted a Global Protein Stability approach as novel read-out (Global protein stability profiling in mammalian cells, Science, 2008), which relies on a reporter construct expressing two fluorescent dyes from one mRNA to monitor changes in stability of EWS-FLI1 by high-throughput flow cytometry. This drug screen identified two main enriched classes of inhibitors that significantly destabilize EWS-FLI1. Validation of these results and characterization of their mechanism of action is currently under way. We conclude that the study of EWS-FLI1 turnover represents a novel approach to identify new effective drugs that can be used as monotherapy or in combination with other drugs as novel treatment opportunities in Ewing sarcoma.

#3110

Shorter naïve T cell telomere length is associated with thyroid subsequent malignant neoplasm: A report from the Childhood Cancer Survivor Study (CCSS).

Maria M. Gramatges,1 Geraldine Aubert,2 Elmira Hariri,2 Yan Chen,3 John A. Whitton,4 Wendy M. Leisenring,4 Michael A. Arnold,5 Joseph P. Neglia,6 Yutaka Yasui,7 Leslie L. Robison,7 Gregory T. Armstrong,7 Smita Bhatia8. 1 _Baylor College of Medicine, Houston, TX;_ 2 _Repeat Diagnostics, Vancouver, British Columbia, Canada;_ 3 _University of Alberta, Edmonton, Alberta, Canada;_ 4 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 5 _Nationwide Children's Hospital, Columbus, OH;_ 6 _University of Minnesota, Minneapolis, MN;_ 7 _St. Jude Children's Research Hospital, Memphis, TN;_ 8 _University of Alabama at Birmingham, Birmingham, AL_.

Introduction: Reduced blood telomere content has been associated with an increased risk for subsequent malignant neoplasms of the thyroid (thyroid SMN) in survivors of childhood cancer (PMID 24277454). Here, we further investigate this association by examining telomere length (TL) in leukocyte subsets. Methods: Survivors were enrolled to the CCSS, a multicenter, retrospective cohort of 5-year + survivors of childhood cancer. Cases were survivors with thyroid SMN, and matched (1:1) to survivor controls without SMN by primary diagnosis, year of primary diagnosis (decade), chemotherapy (yes/no), radiation field, and follow-up time (exceeding time to SMN for the case). Stem cell transplant recipients were excluded. Absolute TL was determined from viably frozen leukocytes (lymphocytes, naïve T, memory T, B, and NK cells) by telomere flow cytometry fluorescence in situ hybridization (telomere flow FISH, Repeat Diagnostics), and transformed to age-adjusted percentiles based on age at sample collection. For each leukocyte subset, we used McNemar's test to compare frequency of very low (VL, ≤1st age-adjusted percentile) or low (L, >1st to ≤10th percentile), and a paired t-test to compare age-adjusted TL between cases and controls. Odds ratios were determined by conditional logistic regression. Results: Of the 52 matched pairs identified, 46 pairs (92 survivors) had sufficient cell recovery for flow FISH: primary diagnoses included Hodgkin lymphoma (20 pairs), acute lymphoblastic leukemia (13 pairs), CNS tumors (7 pairs), neuroblastoma (2 pairs), non-Hodgkin lymphoma (3 pairs), and kidney tumor (1 pair). All survivors had age-adjusted TL below the population median. Cases had shorter age-adjusted TL than controls in 4 out of 5 leukocyte subsets: lymphocytes (p=0.04), naïve T cells (p=0.02), B cells (p=0.01), and NK cells (p=0.01). Naïve T cells had an overrepresentation of VL TL (cases with VL TL in 9/46 pairs vs. controls with VL TL in 2/46 pairs, p=0.04). The odds of L or VL naïve T cell TL was significantly higher in cases compared with controls (OR=2.8, 1.11-7.19, p=0.03), an observation that did not change after adjusting for age at diagnosis. Conclusions: Survivors of childhood cancer had shorter age-adjusted leukocyte TL than the general population. This negative deviation was more pronounced among survivors with thyroid SMN than in survivors without SMN, which may reflect a differential risk among survivors for excess premature aging in the hematopoietic compartment. The effect of SMN treatment on TL is unlikely to be a factor, as treatment for thyroid SMN is primarily surgical. In cancer-naïve populations, VL lymphocyte TL is a sensitive and specific indicator of underlying defects in telomere maintenance. In survivors of childhood cancer, VL TL in naïve T cells may reflect an association between SMN risk and defects in T cell-mediated cancer surveillance.

#3111

Zero Childhood Cancer: A comprehensive precision medicine platform for children with high-risk cancer.

Emily V. Mould,1 Loretta Lau,2 Greg Arndt,3 Paulette Barahona,1 Mark J. Cowley,1 Paul Ekert,4 Tim Failes,3 Jamie Fletcher,1 Andrew Gifford,5 Dylan Grebert-Wade,1 Michelle Haber,1 Alvin Kamili,1 Amit Kumar,6 Richard B. Lock,1 Glenn M. Marshall,2 Chelsea Mayoh,1 Murray Norris,1 Tracey O'Brien,2 Dong Anh Khuong Quang,4 Patrick Strong,1 Alexandra Sherstyuk,1 Toby Trahair,2 Maria Tsoli,1 Katherine Tucker,5 Meera Warby,5 Marie Wong,1 Jinhan Xie,1 David S. Ziegler,2 Vanessa Tyrrell1. 1 _Children's Cancer Institute, Sydney, Australia;_ 2 _Kid's Cancer Centre, Sydney Children's Hospital, Sydney, Australia;_ 3 _ACRF Drug Discovery Centre for Childhood Cancer, Lowy Cancer Research Centre, Sydney, Australia;_ 4 _Murdoch Children's Research Institute, Melbourne, Australia;_ 5 _Prince of Wales Hospital, Sydney, Australia;_ 6 _Peter MacCallum Cancer Centre, Melbourne, Australia_.

Molecular genomics analyses aim to identify subsets of patients harboring actionable aberrations as a pathway to improved targeted treatment selection. However, recent pan-cancer analyses of the molecular landscape of pediatric cancers1,2 have emphasized the stark contrast with adult cancers, with low mutation rates, distinct mutated genes and a prevalence of structural rearrangements suggesting that genomic analyses alone have limitations for translation into clinical benefit. The Zero Childhood Cancer (ZCC) program aims to assess the feasibility of precision medicine to identify targeted therapeutic agents for patients with high-risk (HR) pediatric malignancies (expected survival <30%). We combine comprehensive molecular profiling analysis [whole genome sequencing (tumor, germline DNA), deep sequencing of a 386 cancer associated gene panel, whole transcriptome (RNASeq), methylation profiling] with in vitro high-throughput drug screening (124 compound library, single agent) and patient-derived xenograft (PDX) drug efficacy testing. Results are curated and recommendations made by a national Multidisciplinary Tumor Board. Recommendations consist of targeted therapy, change of diagnosis or genetics referral for a germline cancer predisposition gene mutation. The national multicenter prospective trial (PRISM) opened in September 2017 at all 8 pediatric oncology centers around Australia, following the successful completion of a 2-year pilot feasibility study. PRISM has enrolled 131 patients to date (35% central nervous system tumors, 29% sarcoma, 13% leukemias/lymphomas, 6% neuroblastoma, 17% other rare or unknown cancers). The unique ZCC testing platform has resulted in at least one recommendation being issued for 67% of patients. Fifteen % of patients have a reportable germline cancer predisposition. We have developed an analytical pipeline to interrogate and cross-validate the full range of variants, structural abnormalities and mutational signatures identified in pediatric cancers and incorporate the molecular data with in vitro and in vivo drug sensitivity data where possible. The highest yield of reportable variants is derived from the integrated analysis of WGS and RNASeq; unique to ZCC compared to other pediatric precision medicine programs internationally. ZCC demonstrates the feasibility of a comprehensive precision medicine platform to identify treatment recommendations in HR pediatric cancer patients. The national trial is planned to run for 3 years, recruiting ~400 patients. In addition, ZCC is partnering nationally and internationally to conduct parallel research studies in immunoprofiling, liquid biopsy, psychosocial impact of precision medicine, health economics and health implementation. 1. Gröbner et al. Nature. 2018; 555(7696):321-327. 2. Ma et al. Nature. 2018; 555(7696):371-376.

#3112

Prospective immune phenotyping of neuroblastoma children at diagnosis reveals specific immune defects related to the aggressiveness of the disease.

Sandrine Susini,1 Isabelle Rochet,2 Estelle Verronèse,1 Christine Bardin,1 Chantal Rigal,1 Cécile Conter,3 Perrine Marec-Bérard,3 Christophe Bergeron,3 Audrey Lardy-Cleaud,1 Séverine Neymarc,1 Séverine Metzger,1 Jean-Louis Stephan,4 Dominique Plantaz,5 Christophe Caux,6 Christine Ménétrier-Caux,6 Aurelien Marabelle7. 1 _Centre Léon Bérard, Lyon, France;_ 2 _Institut d'Hématologie et d'Oncologie Pédiatrique, Centre Léon Bérard, Lyon, France;_ 3 _Institut d'Hématologie et d'Oncologie Pédiatrique, Centre Léon Bérard, Lyon, France;_ 4 _Centre Hospitalier et Universitaire de Saint-Etienne, Saint-Etienne, France;_ 5 _Centre Hospitalier et Universitaire de Grenoble, Grenoble, France;_ 6 _Centre de Recherche en Cancérologie de Lyon, Lyon, France;_ 7 _Gustave Roussy, INSERM U1015, Université Paris Saclay, Villejuif, France_.

CONTEXT: Neuroblastoma is the most common extracranial solid tumor in children. Patients with low- and intermediate-risk neuroblastoma (LIRNB) have favorable prognosis and an excellent five-year survival rate of more than 90%. However, in the case of high-risk neuroblastoma (HRNB; ~50% of cases), the prognosis of treatment remains unfavorable and the five-year survival rate remains under 40% despite aggressive multi modal therapy.

QUESTION: We wanted to know if there was a defect in immune surveillance that could explain part of the aggressive phenotype of HRNB.

METHODS: We performed a prospective multi-center study on thirty two new cases of neuroblastoma at all stages. We performed a wide phenotyping of immune cells by flow cytometry in fresh whole blood, and fresh whole bone marrow at diagnosis of children diagnosed for neuroblastoma. Cell counts were performed in order to be able to reason in absolute values (G/L) rather than only percentages. RESULTS: Blood and bone marrow from 20 LIRNB and 12 HRNB were prospectively analyzed. We found that specific subsets of circulating immune cells such as myeloid dendritic cells BDCA-1+, plasmacytoid dendritic cells, Teff/Treg CD4+ T-cells, were decreased in HRNB blood compared to LIRNB groups as opposed to other subsets (e.g iNKT, g9d2 T-cells,⋯). More surprisingly, these differences were not due to defects in cell production as there was no difference found for the same cell populations in the bone marrow between LIRNB and HRNB children.

CONCLUSION: Specific immune cell defects are found in the blood from High risk neuroblastoma children that are not present in the blood of low & intermediate risk neuroblastoma children. These defects are not due to bone marrow production impairment. These results suggest that high risk neuroblastoma disease generates an impairment in the immune homeostasis of children. Gene expression analysis of cytokine/chemokine from these neuroblastoma tumors is underway (NCT01295762).

#3113

**Regulation of TOR complex 1 by the BRAF** V600E **Mutant determines response and development of resistance to MEK inhibition by trametinib in glioma models.**

Fuyang Li. _UT Health San Antonio, San Antonio, TX_.

Pediatric Low-Grade glioma (PLGG) is the most common brain tumor in children. Current treatment protocols, particularly those involving high dose radiation therapy are associated with neurocognitive and neuroendocrine dysfunction. Genomic studies have revealed that PLGG are characterized by activation of the MAPK cascade. Recently, the value of selumetinib, an allosteric inhibitor of MEK1/2 has been demonstrated in PLGG. However, drug resistance will inevitably limit the efficacy. Trametinib is a more potent MEK1/2 inhibitor with better brain penetration than selumetinib. Here we have attempted to identify the mechanism/s of the resistance to trametinib that PLGG developes, and to explore combination treatments that may retard or prevent emergence of trametinib resistance in animal models.

In BT-40 (childhood anaplastic astrocytoma with BRAFV600E mutation) xenografts, trametinib caused complete tumor regressions, followed by tumor regrowth at the end of treatment. Trametinib treatment rapidly induced pSTAT3(Y705), that was blocked by the JAK1/2 inhibitor, ruxolitinib. Agaist BT-40 xenografts, ruxolitinib slightly enhanced the antitumor response to trametinib. The combination of trametinib with rapamycin was more active in each xenograft line than either single agent alone and in both subcutaneous and intracranial BT-40 models. In BT-40 xenografts, resistance to trametinib was consistently induced within 3 or 4 6-week cycles of treatment similar to that for the trametinib + ruxolitinib combination. In contrast, resistance to combined trametinib and rapamycin was not developed. Rapamycin prevented emergence of trametinib resistance, but did not reverse resistance to trametinib, once acquired. Inhibition of MAPK signaling by trametinib was similar in parental and trametinib-resistant BT-40 xenografts, however, trametinib reistant tumor cells failed to undergo apoptosis. Cells freshly isolated from trametinib resistant BT-40 xenografts had a higher apoptotic threshhold, whereas cells isolated from BT-40 xenografts after 3 cycles of trametinib + rapamycin, remained equally sensitive to parental BT-40 cells, as determined by BH3 profiling. This study reveals a potential new therapeutic use of rapamycin, preventing emergence of resistance to trametinib. Potentially, addition of rapamycin to MEK inhibitor therapy children with BRAF-driven low-grade glioma may be of value.

#3114

Correlation between response to atezolizumab and PD-L1 tumor expression in pediatric and young adult patients enrolled in the phase I/II iMATRIX-atezo study.

Katherine E. Hutchinson,1 Minlei Liao,1 Mufiza Farid-Kapadia,2 Francis Donaldson,3 Nga Wan Rachel Tam,1 Lingyan Helen Fu,1 Meghna Das Thakur,1 C. Michel Zwaan,4 Birgit Geoerger,5 Lynley V. Marshall,6 Tanya Trippett,7 Gianluca Rossato8. 1 _Genentech Inc, South San Francisco, CA;_ 2 _F. Hoffmann-La Roche Ltd, Mississauga, Ontario, Canada;_ 3 _F. Hoffmann-La Roche Ltd, Welwyn Garden City, United Kingdom;_ 4 _Erasmus MC-Sophia Children's Hospital & Princess Máxima Center, Rotterdam/Utrecht, Netherlands; _5 _Gustave Roussy Cancer Centre, Villejuif, France;_ 6 _The Royal Marsden NHS Foundation Trust and The Institute of Cancer Research, London, United Kingdom;_ 7 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 8 _F. Hoffmann-La Roche Ltd, Basel, Switzerland_.

Introduction The iMATRIX-atezo trial (NCT02541604) assessed the safety and pharmacokinetics (primary objectives) and preliminary activity (secondary objective) of atezolizumab in pediatric and young adult patients with refractory/relapsed solid tumors, including Hodgkin's lymphoma (HL) and non-Hodgkin's lymphoma (NHL). Atezolizumab targets PD-L1 expressed by tumor cells leading to an enhanced anticancer T-cell response. The purpose of this analysis was to investigate the association between PD-L1 expression and response to atezolizumab in patients participating in the iMATRIX-atezo trial.

Methods Patients aged <30 years received atezolizumab every 3 weeks until loss of clinical benefit. Biomarkers were analyzed across cohorts and by response to atezolizumab, with a minimum follow up of 6 months. Response was assessed by RECIST, modified International Neuroblastoma Response Criteria, Response Criteria for Malignant Lymphoma, and RANO. Pretreatment tissues were examined by immunohistochemistry for: T/B/NK cell markers, tumor-infiltrating lymphocytes (TILs) and PD-L1 expression in tumor cells [TC] and immune cells [IC]. Blood samples were analyzed for T/B/NK cell constitution by flow cytometry. Tumor mutational burden (TMB) was determined by FoundationOne® next-generation sequencing.

Results Eighty-seven patients received ≥1 dose of atezolizumab. Of 11 patients with high PD-L1 expression (TC2/TC3/IC2/IC3) and response data, four had a partial response (PR; two with HL, one with NHL, one with a rhabdoid tumor), one had stable disease (SD) and six had progressive disease (PD). Of 52 patients with low or no PD-L1 expression (TC1/TC0/IC1/IC0) and response data, no responses were observed. Though numerically limited, PD-L1 expression and response were significantly correlated (p=0.0006, Fisher's exact test). HL (8/9 patients) and NHL (2/3 patients) cohorts had the greatest proportion of patients with high baseline PD-L1. Levels of CD8+ (T cells), CD20+ (B cells) and stromal TILs at baseline were significantly associated with patients' response status (p<0.05, Mann-Whitney U test). High PD-L1 expression correlated with elevated expression of other tissue-based immune biomarkers, except NK cells. Blood biomarkers did not correlate with tumor-based counterparts. No tumors were TMB high; all were <16 mutations/megabase.

Conclusions High PD-L1 expression compared with low or no expression was associated with a greater response to atezolizumab, notably in patients with HL and NHL. Despite small sample sizes and limited responses, these biomarker data support adult studies citing the association between PD-L1 and response to atezolizumab; data suggest that PD-L1 may also be a predictor of atezolizumab response in pediatric and young adult patients.

#3115

High-capacity adenoviral vectors with controlled expression of interleukin 12 as a new strategy against pediatric osteosarcoma.

Marta Zalacain,1 Virginia Laspieda,1 Naiara Martinez-Vélez,1 Lucía Marrodán,1 María Buñuales,2 Marisol Gonzalez-Huarriz,1 Maider Varela,1 Montserrat Puigdelloses,1 Manuela Gonzalez-Aparicio,2 Marta María Alonso,1 Rubén Hernandez,2 Ana Patiño-García1. 1 _University Clinic of Navarra, Pamplona, Spain;_ 2 _Center for Applied Medical Research, Pamplona, Spain_.

Osteosarcoma (OS) is the most frequent and aggressive primary bone tumor in children and young adults, with a high propensity to metastasize to the lungs. Despite therapeutic efforts, the survival rate for metastatic or relapsed patients in under 30%. For this reason, we need new strategies to halt the progression of primary bone tumor and impede the metastatic stage. Interleukin-12 (IL12) is a cytokine implicated in a variety of immune activities including the stimulation of T and natural killer (NK) cells and the production of high levels of IFNɣ. In addition, this process triggers an anti-angiogenic response leading to T reg inhibition and tumor death. This cytokine has shown significant antitumor activity in several animal models and clinical trials. The aim of this work was to evaluate the antisarcoma effect of a high-capacity adenovirus vector carrying the mouse IL12 (mIL12). This vector harbours a mifepristone (RU486)-inducible system for controlled expression of IL12 (HCA-EFZP-IL12) in order to improve the treatment of OS and reduced the systemic toxicity associated with IL12. The murine K7M2 OS cell line was injected intratibially in Balbc mice to induce bone tumor. HCA-EFZP-IL12 was administered intratumorally (two doses 2 days apart) in the orthotopic model with mifepristone induction during two weeks. Blood samples were collected 10 h after the first RU486 induction; mIL12 protein level was measured by ELISA, and tumor volumes were measured using a calliper. At the end of the experiment we collected tibias, lungs and livers for immunohistochemical studies. Our results showed a reduction of local tumor growth and also a significant extended overall survival. Importantly, the treatment led to long-term survivors free of disease. We performed a re-challenge experiment to assess the generation of antitumor memory and we are currently waiting for those results. Immunohistochemical analyses showed an increase in CD3, mainly CD4 and CD8 and a decrease of T-reg cells. Moreover, splenocytes from treated mice produced significant higher amounts of IFNɣ than those of control mice. In summary, the role of the microenvironment and the potent antitumor immune response mediated by controlled expression of IL12 by an HC-Ad equipped with a drug-inducible system can be a real therapeutic option for primary pediatrics OS.

#3116

Targeting fibroblast growth factor receptors in rhabdomyosarcoma.

Nagjie Alijaj,1 Sandrine Moutel,2 Maxim Gray,1 Maurizio Roveri,1 Gabriele Manzella,1 Marco Wachtel,1 Franck Perez,2 Beat Schäfer,1 Michele Bernasconi1. 1 _University Children's Hospital Zurich, Zurich, Switzerland;_ 2 _Institut Curie, Paris, France_.

Rhabdomyosarcoma (RMS) is an aggressive pediatric soft tissue sarcoma and is classified into two main histopathological subtypes: embryonal RMS (eRMS), characterized by different genomic changes or alveolar RMS (aRMS), driven by the oncogenic fusion protein PAX3-FOXO1. The significant toxicity associated with conventional chemotherapies represents a major complication in pediatric oncology. To improve current therapies, we adopted two different strategies targeting fibroblast growth factor receptors (FGFR) in RMS.

FGFR1-4 are a family of transmembrane receptor tyrosine kinases. Their activation upon binding of fibroblast growth factors (FGF) triggers pro-survival and proliferative signals.

Our goal is to deliver drugs specifically to the tumor site by taking advantage of FGFR4 overexpression in RMS. To this end, we will covalently link FGFR4 specific nanobodies to the surface of liposomal vincristine in order to actively target RMS cells. We have established the optimal conditions to formulate liposomes loaded with vincristine. Following nanobody phage display selection on recombinant FGFR4 we focused on the top scoring candidates. Flow cytometry analysis on FGFR4-expressing versus FGFR4 knock-out RMS cell lines showed receptor-specific binding of three nanobodies. In activation assays with the FGFR4 specific growth factor FGF19, we demonstrated that the three binding candidates also blocked downstream ERK activation in RMS cells. We will now assess their theranostic potential on drug-loaded and fluorescently labeled nanovesicles on RMS tumor cells in vitro and in xenografts in vivo.

Surprisingly, we observed change in cell morphology followed by cell death upon exposure to FGF2 in a subset of cultured cells established from eRMS patient-derived xenografts. Inappropriate expression of FGFRs and FGF signaling is implicated in tumor progression and therefore our findings appear contradictory. Dose-response experiments have shown that FGFR inhibition with small molecule inhibitors completely rescued FGF2 toxicity. In contrast, however, we detected high expression levels of FGFR1, 2 and 4 as well as activating mutations of FGFR4 in FGF2-sensitive eRMS cells. Therefore, our results are of upmost clinical relevance since genetically-based drug selection could lead to an inappropriate treatment inducing tumor promoting conditions. Hence, our second goal is to further unravel the molecular mechanism underlying the toxic effect of FGF-2 in a subset of eRMS tumors to avoid potentially harmful treatments.

In summary, we have identified FGFR4 specific nanobodies that bind to the receptor and block downstream signaling in RMS cells. Active drug delivery of liposomal vincristine to the tumor site has the potential to enhance the therapeutic impact and decrease side effects. Moreover, we discovered a toxic effect of FGF2 in a subgroup of eRMS patient derived xenograft cells which might open new avenues for treatment.

#3117

Delta-24-RGD/DNX-2401: Oncolytic virotherapy for pediatric high grade glioma and DIPG.

Naiara Martinez-Velez,1 Marc Garcia-Moure,1 Virginia Laspidea,1 Maider Varela,1 Oren Becher,2 Candelaria Gomez-Manzano,3 Juan Fueyo,3 Ana Patiño,1 Ricardo Diez-Valle,1 Sonia Tejada-Solis,1 Marta M. Alonso1. 1 _Clinic University of Navarra, Pamplona, Spain;_ 2 _Northwestern University, Chicago, IL;_ 3 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Pediatric High Grade Glioma (pHGG), including Diffuse Intrinsic Pontine Glioma (DIPG) are the most common and aggressive pediatric brain tumor. Despite great strides in the knowledge of their genetic make-up and new therapies, the outcome for children affected with these malignancies remains dismal. Therefore, it is critical to implement new therapeutic approaches that improve the survival and quality of life of these kids. Delta-24-RGD (DNX-2401 in the clinic), is a tumor specific oncolytic adenovirus that has been tested in preclinical and clinical studies in adult high grade glioma, demonstrating its safe profile and certain degree of efficacy. The objective of this work was to evaluate the safety and the antitumor effect of DNX-2401 in pHGG and DIPGs. Delta-24-RGD showed a potent antitumor effect in a battery of pHGG (N=5) and DIPGs (N=6) cell lines (IC50 ranging from 1 to 50 MOIs) that was mediated by an effective replication. Moreover, Delta-24-RGD administration to mice bearing pHGG and DIPG tumors resulted in a significantly increase in the overall survival, and in some cases leading to 50% of long-term survivors. Clinical trials with DNX-2401 have shown that the immune response triggered in a subset of glioma patients is the responsible for the durable responses observed. Therefore, we assessed viral efficacy in two immunocompetent models bearing mice DIPG tumors. Delta-24-RGD, administered in mice brainstem, was well tolerated and no signs of toxicity were observed. Importantly, Delta-24-RGD treatment resulted in a significant survival benefit in immunocompent DIPG orthotopic models. Tumor examination showed that Delta-24-RGD also promoted an increase in T-cell infiltration (CD3+, CD4+ and CD8+; p<0.001) within the tumor. mRNA expression levels of IFNg, CD8a and CD4 were also increased in treated tumors (p<0.001). In addition, esplenocytes from Delta-24-RGD treated mice significantly increase the IFNg production (p<0.0001) when were co-cultured with DIPG tumor cells. In summary, Delta-24-RGD administration triggers an immune response against DIPG tumors These encouraging preclinical results allowed us to propose a phase 1 clinical trial for naïve DIPGs to evaluate the safety and feasibility of injecting this virus into the pons (NCT03178032). DIPG patients undergo a tumor biopsy and immediately after DNX-2401 is infused intratumorally, using the same biopsy route. Up to date 6 patients have been treated with the virus (with up to 3x1010 viral particles in 1mL). The biopsy and the virus injection were well tolerated by the patients and they were home 3 to 4 days after the surgery. In the next months will have more information regarding the possible effect of the virus. We believe that the information acquired through this study could serve to develop or improve new therapies in the battle against DIPG and constitute a paradigm change in the treatment of this devastating tumor.

#3118

Obesity as a risk factor for pediatric acute lymphoblastic leukemia: A report from the Children's Oncology Group.

Taumoha Ghosh, Michaela Richardson, Justin Ryder, Logan Spector, Lucie Turcotte. _University of Minnesota, Minneapolis, MN_.

Introduction: Increases in the incidence of both obesity (a risk factor for many adulthood cancers) and acute lymphoblastic leukemia (ALL) in childhood have been observed over the past three decades, thus we sought to identify whether obesity may be an unrecognized risk factor for childhood ALL.

Methods: Demographics, anthropometrics and disease characteristics from children and young adults (aged 1-30 years) diagnosed with ALL between 2004-2017 and treated on Children's Oncology Group (COG) frontline treatment protocols with available pre-treatment anthropometric data (n=4775, AALL17D2) were compared to National Health and Nutrition Examination Survey (NHANES) controls (n=30,107). Individuals were classified as underweight, normal weight, overweight, or obese, per standard CDC age- and sex-based pediatric and adult definitions for body mass index (BMI). Multivariate logistic regressions were performed, adjusting for sex, race/ethnicity, age, socioeconomic status and obesity status, to assess associations between BMI classification and ALL. Additional models were performed stratifying by ALL disease characteristics.

Results: ALL patients (72% B-ALL, 28% T-ALL) were more likely to be male (62%), 58% were non-Hispanic white, 9% non-Hispanic black and 24% identified as Hispanic. Among ALL patients, 5% were underweight, 58% normal weight, 17% overweight and 20% obese. Using normal weight as reference, BMI above normal weight classification was associated with ALL diagnosis (overweight, OR=1.10, 95% CI 1.00-1.20, p=0.046; obese, OR=1.15, 95% CI 1.05-1.25, p=0.002), as was underweight (OR=1.74, 95% CI 1.48-2.03, p=<0.0001). When stratified by sex, the associations with overweight and obesity were only observed in males (ptrend<0.0001), and when stratified by ALL immunophenotype, associations with overweight and obesity were only observed in B-ALL (ptrend<0.0001). Obesity was also associated with moderate to high levels of ALL central nervous system (CNS) involvement (CNS2, OR=1.30, 95% CI 1.08-1.57, p=0.006; CNS3, OR=2.28, 95% CI 1.24-4.17, p=0.005).

Conclusions: This is the first study, to our knowledge, to show that pre-treatment overweight or obesity is associated with ALL, specifically among males and B-cell immunophenotype. Furthermore, ALL CNS involvement was associated with obesity. This study also confirmed the known association between underweight and ALL. Although the associations between BMI status and newly diagnosed ALL may be secondary to detrimental physiologic effects of ALL (i.e. underweight at time of diagnosis), they also suggest a role for inflammation, environmental exposures, or other genetic susceptibility in ALL pathogenesis. Further analyses are needed to elucidate whether other ALL disease characteristics, such as cytogenetics, may be associated with pre-treatment childhood BMI.

#3119

Novel measures of somatosensory impairment in chemotherapy-exposed pediatric cancer patients.

Jessica M. Holst-Wolf, Juergen Konczak, Lucie Turcotte. _University of Minnesota, Minneapolis, MN_.

Introduction: The magnitude and timeline of somatosensory impairments due to chemotherapy-induced peripheral neuropathy are not well understood due to a lack of objective measures with appropriate resolution. Here, we have updated a methodology for measuring proprioceptive acuity and developed a novel measure of haptic function, appropriate for adult and pediatric populations, to measure somatosensory impairment in chemotherapy-exposed pediatric cancer patients and identified relationships between chemotherapy-related somatosensory impairment and cumulative chemotherapy exposure.

Methods: To map the development of proprioceptive acuity, 308 typically developing (TD) children (ages 5-17 years) and 26 adults (ages 18-25 years) performed a forearm position matching task with a bimanual manipulandum. Haptic acuity (discrimination) or sensitivity (detection) thresholds were measured using curvature perception assessments in 99 and 56 children respectively (ages 6-17 years). Healthy adults completed both haptic assessments (n = 27, ages 19-25 years). These brief proprioceptive and haptic assessments were utilized to characterize somatosensory impairment in 15 individuals treated with vinca alkaloid-containing chemotherapy regimens for non-central nervous system (CNS) pediatric cancers (ages 6-25 years). Normative quantiles were calculated for the proprioceptive and haptic measures from children and adults in the control group to use as comparison for the individuals exposed to chemotherapy. Regression analysis were used to determine if the somatosensory measures were correlated with treatment variables such as cumulative dosage of chemotherapeutic agents.

Results: 7 of 15 cancer survivors exhibited elevated proprioceptive precision measures and 11 of 15 exhibited at least one elevated haptic function measure. Elevated measures are above the 75th percentile of the age matched normative cohort indicating more variability in limb position sense and higher haptic discrimination and detection thresholds. A multiple linear regression model of cumulative dosage of chemotherapeutic agent types predicted 80% of the variability in the haptic discrimination thresholds (adjusted r-squared = 0.80). Greater doses of plant alkaloids and anti-tumor antibiotics were associated with higher haptic discrimination thresholds.

Conclusion: This study demonstrated proof-of-concept for identifying somatosensory deficits in individuals treated with chemotherapy for non-CNS pediatric cancers. The haptic assessment has sufficient resolution to correlate with cumulative chemotherapy exposure. These brief, objective, clinically relevant, somatosensory assessments can identify somatosensory deficits in pediatric cancer patients with known or suspected chemotherapy-associated dysfunction and may be used to follow progression and resolution of symptoms over time.

#3120

The novel MEK inhibitor binimetinib decreases cell growth and induces apoptosis in AT/RT.

Shubin Shahab, Jeffrey Rubens, Charles Eberhart, Eric Raabe. _Johns Hopkins University, Baltimore, MD_.

Atypical teratoid/rhabdoid tumors are a class of malignant CNS tumors disproportionately affecting children with limited therapeutic options. Our group has previously demonstrated that the MAP kinase pathway is altered in a large proportion of these tumors and the MEK inhibitor selumetinib decreases cell growth and promotes apoptosis of AT/RT cells. One of the major drawbacks of selumetinib is its poor blood-brain barrier penetration. Binimetinib is a novel MEK inhibitor currently in pediatric phase II clinical trials for low grade glioma that is thought to have improved brain penetration. Here we demonstrate nanomolar levels of binimetinib successfully decreases cell proliferation and induces apoptosis (2-3 fold induction of cleaved Parp) in AT/RT cell lines, suggesting binimetinib could offer a potential avenue for treating these deadly tumors.

#3121

**Osteopontin-c is overexpressed and mediates cell adhesion and proliferation in leukemia cell line with** KMT2A-AFF1 **.**

Ana Clara Santos da Fonseca Bastos,1 Júlio César Santoro,1 Caroline Barbieri Blunck,1 Luciana Bueno Ferreira,1 Maria do Soccro Pombo-de-Oliveira,1 Mariana Emerenciano,1 Etel R. Gimba2. 1 _INCA, Rio de Janeiro, Brazil;_ 2 _UFF/INCA, Rio de Janeiro, Brazil_.

Osteopontin (OPN) primary transcript suffers alternative splicing generating at least five OPN splicing isoforms (OPN-SI) named OPNa, OPNb, OPNc, OPN4 and OPN5. In solid tumors, total OPN (tOPN) expression has been correlated to several cancer features. However, OPN-SI roles in hematological malignancies are still under investigation. A hallmark of B-cell acute lymphoblastic leukemia (B-ALL) is the recurrence of specific gene rearrangements associated with prognostic risk stratification. Patients harbouring the KMT2A-AFF1 fusion gene are included into poor prognostic subgroup, with an overall survival rate of 50% and increased risk of extramedullary sites involvement. In B-ALL, it has been reported that tOPN support tumor dormancy and cell survival in response to chemotherapeutic drugs. However, the specific roles of OPN-SI have not been addressed. The aim of this study was to analyze the expression of OPN-SI in B-ALL samples and then investigate the role of the most overexpressed OPN-SI on modulating cellular and molecular aspects of B-ALL presenting KMT2A-AFF1. We found that OPNc is overexpressed in relation to OPNa and OPNb in patients with KMT2A-AFF1. In addition, OPNc transcript levels were associated with poor prognostic features, such as central nervous system infiltration, white blood cell counting and the high-risk group at diagnosis. In response to OPNc silencing in B-ALL cell lines, we found increased proliferation rates, as opposed to decreased cell adhesion properties over matrigel matrix. Our data evidenced that overexpressed OPNc in B-ALL with KMT2A-AFF1 is correlated to poor prognostic features and may modulate the leukemic phenotype by the decrease of cell proliferation while promoting cell adhesion. Further work should be conducted in order to evaluate OPNc putative application as an additional risk-stratification and prognostic marker for B-ALL and how OPNc could control leukemia development and progression.

#3122

**A functional** POT1 **variant and risk of thyroid subsequent malignant neoplasm: A report from the Childhood Cancer Survivor Study.**

Melissa A. Richard,1 Philip J. Lupo,1 Lindsay M. Morton,2 Yutaka Yasui,3 Michael A. Arnold,4 Joseph P. Neglia,5 Lucie M. Turcotte,5 Wendy M. Leisenring,6 Stephen J. Chanock,2 Joshua N. Sampson,2 Gregory T. Armstrong,3 Leslie L. Robison,3 Smita Bhatia,7 Maria M. Gramatges1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _National Cancer Institute, Bethesda, MD;_ 3 _St. Jude Children's Research Hospital, Memphis, TN;_ 4 _Nationwide Children's Hospital, Columbus, OH;_ 5 _University of Minnesota, Minneapolis, MN;_ 6 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 7 _University of Alabama at Birmingham, Birmingham, AL_.

Purpose: Reduced telomere content has been associated with an increased risk for subsequent malignant neoplasms of the thyroid (thyroid SMN) in survivors of childhood cancer (PMID 24277454). However, variation in nine common single nucleotide polymorphisms (SNPs) that influence leukocyte telomere length was not associated with thyroid SMN (PMID 30377209). Here, we report results of a candidate gene approach investigating associations between thyroid SMN and genes functionally related to telomere maintenance.

Methods: Genome-wide SNP data were generated using the Illumina HumanOmni5Exome array in 5,324 5-year survivors of childhood cancer enrolled to the Childhood Cancer Survivor Study (CCSS), and imputed to the 1000 Genomes reference haplotypes. Of these 5,324 survivors, 117 developed thyroid SMN. We mapped 4,290 variants to 18 genes involved in telomere maintenance (TERC, TERT, RAD50, NHP2, POT1, TERF1, NBN, TPP1, MRE11A, TINF2, NOP10, PARN, ACD, TERF2, RAP1 (TERF2IP), WRAP53, CTC1, and RTEL1) and used RegulomeDB to annotate each variant by its projected functional impact. Time-to-event Cox regression was used for thyroid SMN, censored by date of any SMN, death, or last follow-up. For each functional SNP, hazard ratios (HR) were estimated, adjusting for sex, primary cancer diagnosis, neck radiation exposure (yes/no), alkylating agent exposure (yes/no), and thyroid nodules.

Results: Our analysis included 103 SNPs with a RegulomeDB score ≤ 2, signifying high likelihood for affecting transcriptional regulation. After Bonferroni correction (α=0.000485), an imputed variant in an intronic region of POT1 (Protection of Telomeres 1), rs58722976 (CCSS minor allele frequency = 0.2%), was associated with risk for thyroid SMN (adjusted HR=6.1, 95% CI: 2.4, 15.5, p=0.0001) and was present in 3 cases and 14 controls.

Conclusions: Using a candidate gene approach, we observed an association between an intronic regulatory POT1 variant and risk for thyroid SMN in survivors. POT1 is a highly conserved gene encoding a key component of the shelterin complex, which protects telomere ends against DNA damage recognition and facilitates telomerase-mediated telomere elongation. The ENCODE Consortium identifies rs58722976 as a strong enhancer and DNase in multiple tissues, including the hematopoietic compartment. ENCODE ChIP-Seq data suggest that rs58722976 genotypes also affect protein binding of RAD21, SMC3, and CTCF, components of cohesin and a co-localizing protein that play key roles in maintaining genomic integrity. Germline variants in POT1 have been described in familial glioma, melanoma, colorectal cancer, chronic lymphocytic leukemia, and non-TP53 familial cancer syndromes. The results of this study suggest that intronic variation in POT1 may affect key protein binding interactions related to defects in telomere maintenance and affecting genomic integrity.

#3123

Targeting SHP2 and RAS MAPK pathway in neuroblastoma.

Ivette Valencia-Sama,1 Teresa Adderley,2 Lynn Kee,2 Gabriella Christopher,2 Yoshihito Kano,1 Michael Ohh,1 Meredith Irwin2. 1 _University of Toronto, Toronto, Ontario, Canada;_ 2 _The Hospital for Sick Children, Toronto, Ontario, Canada_.

Introduction: Neuroblastoma (NB), the most common pediatric extra-cranial solid tumor, recurs in >50% of patients who present with metastases. Strategies to treat relapsed NB, which is often fatal, include targeting relapse-specific signaling pathways. Recently, sequencing studies have revealed that 78% of mutations detected in relapse samples are predicted to activate the RAS-MAPK pathway, suggesting there may be opportunities to pharmacologically target this pathway to treat recurrent NB.

Experimental Design: Our lab previously reported that the PTPN11-encoded tyrosine phosphatase SHP2 is an activator of the RAS pathway, and pharmacologic inhibition of SHP2 inhibited glioblastoma growth in vivo. To determine whether inhibiting SHP2 in NB would impact tumor growth we treated a panel of NB cells with diverse genetic backgrounds, including mutations in NRAS, with different SHP2 inhibitors (SHP099 and II-B08) alone and in combination with additional drugs that target RAS-MAPK signaling.

Results: In comparison to NB cell lines harboring endogenous NRAS mutations (NRASmt), cells with NRAS wild-type (NRASwt) were more sensitive to SHP2 inhibitors. In addition, NRASwt cells engineered to overexpress NRASmt were more resistant to SHP099 than cells with endogenous or overexpressed NRASwt, as demonstrated by higher levels of proliferation, increased survival, and diminished apoptosis. Furthermore, SHP099 effectively inhibited SHP2 in both NRASwt and NRASmt cells, but only failed to inactivate the downstream RAS effector ERK1/2 in NRASmt cells, suggesting that SHP099 treatment alone is ineffective in cells harboring NRAS mutations. To determine whether SHP099 resistance in NRASmt cells could be overcome with combination strategies, sensitive and resistant NB cells were treated with SHP2 inhibitors alone or in combination with other RAS-MAPK pathway inhibitors (vemurafenib, trametinib and ulixertinib). Interestingly, in comparison to SHP099 or II-B08 alone, all three combinations significantly reduced survival and IC50 values in both NRASwt and NRASmt cells. Using the Bliss independence model we determined all three combinations were synergistic.

Conclusions: Our studies demonstrate that NRAS mutations are associated with resistance to SHP2 inhibitors, and that in certain tumors, based on the genetic status of RAS-MAPK-SHP2 signaling components, combinations of drugs targeting this pathway could be effective strategies for relapsed NB.

#3124

SNAI2 **function in embryonal RMS.**

Silvia Pomelo,1 Prethish Sreenivas,2 Berkley Gryder,1 Long Wang,2 Baxi Kunal,2 Nicole Hensch,2 Eleanor Chen,3 Peter Houghton,2 Rossella Rota,4 Javed Khan,1 Myron S. Ignatius2. 1 _National Cancer Institute, National Institutes of Health, Bethesda, MD;_ 2 _UT Health San Antonio, San Antonio, TX;_ 3 _University of Washington, Seattle, WA;_ 4 _Ospedale Pediatrico Bambino Gesù Research Institute, Rome, Italy_.

Rhabdomyosarcoma (RMS) is a pediatric malignancy of the muscle and a key feature of the histology is muscle cells blocked in differentiation despite robust expression of diagnostic muscle differentiation factors MYOD1 and Myogenin. Thus, there are mechanisms operating in tumors that block myogenic differentiation. We previously defined roles in differentiation, self-renewal and growth for a NOTCH1/SNAI1/MEF2C pathway in Embryonal RMS, the major RMS subtype driven predominantly by Ras signaling. However, we observed that SNAI1 knockdown did not result in as robust differentiation as in NOTCH1 shRNA knockdown cells. We hypothesized that SNAI1 and SNAI2 function might be redundant in ERMS. Analysis of SNAI2 expression in primary tumors and cell lines finds that indeed SNAI2 is highly expressed in RMS and ERMS tumors typically express higher SNAI2 compared to SNAI1. To address SNAI2 function, we knocked down SNAI2 using 2 independent shRNAs and assessed effects on differentiation, self-renewal and growth in ERMS RD, SMS-CTR and JR1 cells. Knockdown of SNAI2 both in stable and transient experiments resulted in robust differentiation (10 fold increase) as assessed by differentiated myosin MF20 expression in RD, JR1 and SMS-CTR cells p<0.001). This increase in differentiation was associated with increased expression differentiation genes including MYOD1, MYOGENIN, MEF2C, MEF2D and differentiated myosins and a loss of precursor gene PAX7 as assessed by quantitative RT-PCR and protein expression. SNAI2 knockdown RD and JR1 cells also formed significantly fewer rhabdospheres (p<0.01). Finally, SNAI2 knockdown with 2 independent shRNAs resulted in significantly smaller and more differentiated tumors when xenografted subcutaneously in vivo in SCID mice. Since SNAI2 is a known DNA binding transcriptional repressor, we performed ChIPseq for SNAI2 and H3K27acetyl in SMS-CTR and RD cells coupled with RNAseq to define direct and indirect SNAI2 regulated genes. Our ChIPseq results identified the known SNAI2 DNA binding motif, however additionally we find that SNAI2 chromatin binding significantly enriched for myogenic E box elements bound by MYOD1, E2A and Myogenin. Additionally, SNAI2 binding was more significantly associated with the muscle differentiation program. Given the differential roles of MYOD1 and SNAI2 on gene activation vs. gene repression, we hypothesized that SNAI2 by competing with MYOD1 at terminally differentiated genes maintains early cell cycle effects of MYOD1 but blocks terminal differentiation. Analysis of MYOD1 expression in SNAI2 knockdown cells finds a redistribution of MYOD1 binding from cell cycle to more differentiated muscle genes and is associated with a concomitant exit from the cell cycle and robust differentiation. In summary, SNAI2 is a robust driver of ERMS differentiation and in vivo growth. High SNAI2 expression competes with MYOD1 at terminally differentiated genes blocking differentiation and exit from the cell cycle in ERMS.

### Novel Strategies for Biomarker Identification and Use in Cancer 1

#3125

SOX2 controls the tumor initiation and development and acts as a prognostic predictor of oral squamous cell carcinoma.

ZIHAO WEI, FENGYING YIN, PENG DENG, XUEFENG ZHANG, JING DENG, JING LI, LIANG XIE, QIANMING CHEN. _Sichuan University, Chengdu, China_.

Background: Sex determining region Y-BOX2 (SOX2), a regulator of the self-renewal ability of stem cells, plays a compelling role in initiation and development of cancer. However, little is known about the role and network controlling of SOX2 in oral squamous cell carcinoma (OSCC) development.

Method: In this study, we conducted a retrospective cohort of 229 OSCC patients from two independent Chinese centers from May 2003 to August 2012. SOX2 expression in these samples was determined by immunostaining. Univariate and multivariate analyses were performed. Then we generated SOX2-creERT2 mice that expressing an inducible Cre recombinase under the control of a minimal promoter of SOX2, along with a tdTomato reporter. These mice were exposed to low dose 4NQO in the drinking water either for 8, 12 or 16 weeks. We further investigate the network controlling of SOX2 by ChIP assay. Then we detected the expression of SOX2 in response to AFF4 knockdown and overexpression by western blot, respectively. Furthermore, we used wound-healing, transwell, sphere formation and limiting dilution assay to confirm whether SOX2 expression was regulated by AFF4.

Results: In the multiple cohorts, high SOX2 protein expression was negatively correlated with the overall survival for OSCC patients. Impressively, we found chemotherapy only improved outcomes of patients with low SOX2 expression, but very limited effects to high SOX2 expression patients. Meanwhile low SOX2 expression patients had significantly better survival with rather than without chemotherapies. Lineage tracing study in vivo suggested that SOX2 positive cells play an important role in the carcinogenesis of OSCC as epithelial stem cells. Increasing SOX2 positive cells and their daughter cells was a major trigger in the initiation of OSCC. Mechanistically, we found SOX2 was regulated by AFF4, the core component of Super elongation complex (SEC) to promote the proliferation, migration and invasion of OSCC cells. SOX2 expression changed in parallel with AFF4 expression in response to depletion and overexpression of AFF4, respectively. More importantly, the inhibited proliferation, migration, invasion and ALDH activity induced by knockdown of AFF4 in OSCC cells were rescued by overexpression of SOX2.

Conclusion: Collectively, our findings indicate SOX2 acts as a prognostic predictor and controls the tumor initiation and development in oral squamous cell carcinoma. SOX2 may serve as a potential target of therapies for patients with OSCC.

#3126

A novel biomarker signature in predicting chemoresistance in colorectal cancer: Potential application in chemotherapy.

Saiprasad Gowrikumar,1 Kristina Pravoverov,1 Caroline Selisteda,1 Kiran D. Bastola,2 Steven Chen,1 Joshua J. Smith,3 Mary K. Washington,4 Amar B. Singh,1 Punita Dhawan1. 1 _UNMC, Omaha, NE;_ 2 _UNO, Omaha, NE;_ 3 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 4 _Vanderbilt University, Nashville, TN_.

Despite advances in the cytotoxic and targeted therapy, resistance to chemotherapy remains one of the greatest challenges in long-term management of metastatic colorectal cancer, which eventually contributes to patient death as tumors accumulate means of evading treatment. We have recently demonstrated that expression of the tight junction protein claudin-1 increases while claudin-7 expression decreases with human colon cancer (CRC) progression and metastasis. Epithelial-mesenchymal transition (EMT) and cancer stem cells (CSC) are critically implicated in cancer metastasis and chemoresistance. Taking this to account, we performed a computational assessment of 250 patient datasets from two different cancer centers (Vanderbilt and Moffitt) and identified the gene sets whose expression increased proportionally with claudin-1 expression and decreased with claudin-7, and vice versa, and correlated these gene sets it with chemoresistance, EMT and CSC markers. We identified a resulting 23-gene set biomarker signature, out of these gene clusters. Some of the important candidates based on literature and their function in colorectal cancer which we focused on were SLC6A6, PIK3CA, ASAP1, TMEM and E2F2. This signature was then validated using the TCGA database. To further evaluate functional relevance of this biomarker signature, we developed oxaliplatin resistant DLD-1 and HT29 colon cancer cells. The chemoresistance of these cells were confirmed by determining the IC50 values DLD (Parental) (4.5μM) and HT29 (Parental) (10.08 μM), after exposing them to increasing conc. of clinically relevant dose of oxaliplatin DLD (Oxaliplatin-R) (16.35 μM) and HT29 (Oxaliplatin-R) (20.05 μM). To our interest, we observed a significant upregulation of SLC6A6, PIK3CA, ASAP1, TMEM and downregulation of E2F2 in DLDOxaR and HT29OxaR cells compared to parental cells. Moreover, DLDOxaR and HT29OxaR cells possessed significantly increased expression of EMT markers such as α-SMA, vimentin, Snail and Slug along with enrichment of the stem cell markers like CD133, CD44 and Aldh1, colony and sphere forming ability. A concomitant decrease in E-cadherin characterized these cells. A similar increase in the expressions of SLC6A6, PIK3CA, ASAP1, TMEM and decrease in E2F2 was observed in colon cancer mouse models as well as chemoresistant patient samples. Taken together, we propose a new predictive biomarker signature which may offer insights into identifying new therapies required to overcome the acquired resistance of colon cancer towards Oxaliplatin and uncover potential molecular pathways involved in treatment failure to help guide therapeutic alternative.

#3127

**Multicenter study evaluating the** ROS1 **status in lung adenocarcinoma using the novel SP384 immunohistochemistry clone. Towards a new algorithm for** ROS1 **status assessment in routine.**

Véronique Hofman,1 Isabelle Rouquette,2 Sandra Lassalle,1 Simon Heeke,3 Jean-Christophe Sabourin,4 Nicolas Piton,4 Julien Mazières,5 Jean-Michel Vignaud,6 Clémence Yguel,6 Anne Laure Lepage,7 Frédéric Bibeau,7 Elodie Long-Mira,1 Katia Zahaf,1 Hugues Begueret,8 Jonathan Benzaquen,1 Michel Poudenx,9 Charles-Hugo Marquette,1 Marius Ilie,1 Paul Hofman1. 1 _CHU de Nice, Nice, France;_ 2 _CHU Toulouse, Toulouse, France;_ 3 _University Côte d'Azur, Nice, France;_ 4 _Charles Nicolle Hospital, Rouen, France;_ 5 _CHU Toulouse Larrey Hospital, Toulouse, France;_ 6 _CHU Nancy, Nancy, France;_ 7 _Normandie Université, Caen, France;_ 8 _CHU Bordeaux, Pessac, France;_ 9 _Centre Antoine Lacassagne, Nice, France_.

Background: The detection of a ROS1 rearrangement in advanced or metastatic lung adenocarcinoma (LUAD) lead to a targeted treatment with tyrosine kinase inhibitors with improved progression free survival (PFS) and overall survival (OS) of the patients. Thus, it is mandatory to screen systematically for the ROS1 rearrangement in this patient population. ROS1 rearrangements can be detected using fluorescence in situ hybridization (FISH), however ROS1 immunohistochemistry (IHC) can be used as a screening test since is largely available, easy, rapid to perform, and cost-effective. However, some false positive and negative IHC results are observed when using the D4D6 clone leading to confirmatory ROS1 FISH in IHC positive samples.

Patients and methods: We evaluated the sensitivity and specificity of anti-ROS1 SP384 (Ventana, Tucson, AZ) and D4D6 (Cell Signaling, Danvers, MA) antibodies in a multicenter population of 336 LUAD cases enriched for ROS1 FISH positive cases (n=51) provided from 6 French molecular pathology platforms. Three senior lung pathologists independently scored the SP384 slides as positive or negative around a cutoff of staining in >30% tumor cells at a ≥2+ intensity level, and for the D4D6 clone around a cutoff of ≥2+ intensity level in any tumor cells. Inter-reader precision between pathologists was assessed. Results were correlated to the PFS and the OS of patients treated with crizotinib.

Results: Sensitivity and specificity rates were 100% (95%CI 93.2-100%) and 99.31% (95%CI 97.51-99.92%) for the SP384 clone, and 90.6% (95%CI 79.34-96.87%) and 99.65% (95%CI 98.05-99.99%) for the D4D6 clone, respectively. Inter-reader agreement was 97.5% (95%CI 94.8-99.7) for the SP384 clone and 86.3% (95%CI 91.3-95.6) for the D4D6 clone. Overall, when compared to ROS1 FISH analysis, the SP384 clone had an accuracy of 99.4%, while D4D6 clone of 98.2%. Using log-rank test, we observed that LUAD with positive ROS1 SP384 status had a longer PFS than those characterized by the D4D6 clone (P=0.021).

Conclusions: Interpretation above a cutoff of >30% tumor cells with staining at a ≥2+ intensity level, the ROS1 SP384 clone demonstrates superior sensitivity to D4D6 clone, while preserving similar specificity rate for the detection of ROS1 rearrangements. The presented data provide evidence that the SP384 clone may be used for effective stratification prior to confirmation with orthogonal methods.

#3128

miR-9 expression regulates and predicts the response to EGFR inhibitors in head & neck squamous cell carcinoma.

Francesca Citron,1 Gian Luca Rampioni Vinciguerra,1 Giuseppe Fanetti,1 Ilenia Segatto,1 Barbara Belletti,1 Andrea Vecchione,2 Giovanni Franchin,1 Gustavo Baldassarre1. 1 _CRO-Aviano National Cancer Institute, IRCCS, Aviano, Italy;_ 2 _University of Rome "La Sapienza", Rome, Italy_.

Introduction. Most Head & Neck Squamous Cell Carcinoma (HNSCC) patients are diagnosed with a locally advanced disease. Radiotherapy (RT) plus anti-EGFR monoclonal antibodies (Cetuximab - CTX) represents an effective combination therapy for locally advanced HNSCC patients. However, the 5-year overall survival is still 45%, mainly due to the appearance of loco-regional recurrences, suggesting that the identification and validation of predictive biomarkers of CTX activity is urgently needed to identify patients at high-risk of recurrence, who will benefit more from this therapeutic strategy. We recently validated a 4-microRNAs (miRs) signature, related to Epithelial to Mesenchymal Transition (EMT) and able to stratify HNSCC patients at high-risk of recurrence development. Among these 4 miRs, miR-9 was the only up-regulated in primary tumors from patients who developed recurrence within 2-year follow-up. Here we evaluated whether miR-9 expression had a functional role in HNSCC onset, progression and response to therapies.

Methods. miR-9 modified (overexpressing and silenced) HNSCC cells were generated, characterized for their growth and response to radio-, chemo- and targeted-therapies (i.e.Cisplatin, Paclitaxel, 5-Fluorouracil and CTX) in vitro and in vivo. Preclinical evidences were confirmed in a cohort of primary HNSCC samples by intercrossing the expression of miR-9 and selected target genes with patients' clinical variables and response to therapy.

Results. Biochemical and biological in vitro and in vivo experiments showed that high miR-9 expression triggers EMT and increases tumor initiating properties in HNSCC cells. Interestingly, miR-9 silenced HNSCC cells displayed a higher sensitivity to RT and CTX, but not to chemotherapy, when compared to controls. Using an in vivo model of HNSCC, we observed that intra-tumor injection of anti-miR-9 improves the efficacy of RT alone and remarkably in combination with CTX. Mechanistically, we demonstrated that in HNSCC cells, EGFR activation triggers miR-9 expression that promotes the transcription of SP1, establishing a positive forward loop between EGFR activation and SP1 transcription. Accordingly, in primary HNSCC miR-9 expression strongly correlates with the one of SP1 and EGFR. More importantly, in a cohort of HNSCC patients treated with RT plus CTX, high miR-9 expression acts as an efficient predictive biomarker of intrinsic resistance.

Conclusion. Altogether by integrating wet-lab and clinical data, we provide strong evidences indicating that in HNSCC a subpopulation of miR-9 expressing cells is intrinsically resistant to RT plus CTX therapy, confirming its potential prognostic value. Since we set up an assay to easily and quantitatively evaluate miR-9 expression in diagnostic tumor biopsies, we propose that it could be used to personalize the treatments for this group of patients, avoiding unfaithful toxic therapies.

#3129

Investigating the importance of prostate tumor location and symmetry in a racially diverse, military cohort.

William Gesztes,1 Grant Williams,2 Jennifer Cullen,1 Allen Burke,3 Denise Young,1 Justin Mygatt,4 Yongmei Chen,1 Huai-Ching Kuo,1 Shiv Srivastava,1 Kevin Rice,4 Inger Rosner,4 Isabell Sesterhenn5. 1 _Center for Prostate Disease Research (CPDR), Rockville, MD;_ 2 _Uniformed Services University of the Health Sciences, Bethesda, MD;_ 3 _The Joint Pathology Center, Silver Spring, MD;_ 4 _Center for Prostate Disease Research, Department of Surgery, Uniformed Services University of the Health Sciences and the Walter Reed National Military Medical Center, Bethesda, MD;_ 5 _Center for Prostate Disease Research (CPDR) and The Joint Pathology Center (JPC), Silver Spring, MD_.

Introduction and objective

Prostate cancer (CaP) tumor location (TL) and symmetry (TS), may help forecast CaP outcomes in radical prostatectomy (RP) patients. The study aim was to examine associations between CaP TL and TS with disease pathology and progression, including biochemical recurrence (BCR)-free survival especially whether such associations differ across race.

Methods

This retrospective cohort study included 1140 patients enrolled at the Walter Reed National Military Medical Center who underwent RP between 1993 and 2008. TL and TS were recorded for index tumors (i.e, largest/highest grade) in whole-mounted RP specimen sections. TLs were divided into coronal versus axial planes. For coronal plane, TL was sub-divided into anterior (A), antero-lateral (AL), lateral (L), postero-lateral (PL), posterior (P) and diffuse (D) (includes A, AL, L, PL and P). The axial plane locations included base (B), mid (M) and apex (A). Kaplan Meier (KM) estimation curves were used to model BCR-free and DM-free survival.

Results

Mean age at diagnosis was 59.3 years, 27.1% were African-American (AA) and 76.7% were non-symmetric (NST). According to KM analysis patients with NST experienced significantly worse BCR-free survival (p=0.003) with no racial differences found (CA: p=0.02, AA: p=0.04). Tumor location (coronal plane) distribution by race was: A and AL tumors (CA: 9.6%, 7.5% vs. AA: 9.5%, 5.5% respectively), D tumors (CA: 6.9% vs. AA: 9.4%, respectively), L tumors (CA: 9.3% vs. AA: 6.5%, respectively), P and PL tumors (CA: 21.2%, 45.6% vs. AA: 22.7%, 46.3%, respectively) (p=0.36). In the axial plane exclusively A or B tumors are rare (CA: 3.1%, 1.3% vs. AA: 2.6%, 0%, respectively) while exclusively M tumors are more frequent (CA: 27.9% vs. AA: 23%, respectively). Tumors spanning two axial location categories MA and BM tumors were (CA: 27.6%, 15.6% vs. AA: 34%, 8.4%, respectively). Tumors spanning three axial location categories BMA were (CA: 21.8% vs. AA: 30.7%, respectively) (p<.0001). No racial differences in BCR-free survival were observed for D (CA: p<.0001, AA: p=.07) or BMA (CA p<.0001; AA p=0.0004) tumors. The share of tumors with nuclear grade III in D or BMA location categories was (10.5% and 7.2%, respectively; p<.0001) while the difference in NST and ST was (4.2% vs. 0.8%, p=.0002).

Conclusions

In this longitudinal, racially diverse cohort of RP patients, TL and TS were informative features in predicting BCR. However, this study did not find evidence of racial differences in these associations with the exception of axial tumor location. These findings have implications for treatment stratification at time of RP.

#3130

Using digital pathology based "IO Scorecards" to describe relationships between PD-L1 expression and CD8 positive immune cell infiltration.

Charles Caldwell, Will Paces, Jeni Caldara, Bharathi Vennapusa, Joseph S. Krueger. _Flagship Biosciences Inc, Westminster, CO_.

Several studies have shown that the location and expression of infiltrating immune cells in patient tumors can better identify which patients are more likely to respond to anti- PD-1/PD-L1 therapy. In particular, immunohistochemistry-based studies have shown that the spatial location of PD-L1 expression has particular biological relevance, as PD-L1 expression in the tumor cells or immune cells in the tumor syncytium, tumor microenvionment (TME), or tumor-stroma boundary all describe differential PD-L1 biology.

Here, we use Flagship's digital pathology platform (cTA®) to investigate IHC based PD-L1 and CD8 staining patterns in Non-Small Cell Lung (NSCLC) and Urothelial Carcinoma (UC) tissue biopsies. The cTA platform creates thousands of per-cell Biofeatures™ derived from the digital pathology images of the IHC stained tissue, and applies Artificial Intelligence (AI) to the data to summary score endpoints for patient and cohort classification. In this approach, each tissue's IO landscape is represented using an "IO Scorecard", which summarizes the IHC biomarker data in a summary score which captures a comprehensive analysis of the tissue sample. The AI-determined scorecard models can be used to monitor changes before and after drug treatment and/or create predictive models for patient response outcomes.

In this study, NSCLC and Urothelial Carcinoma samples were sectioned and stained using either the FDA-approved Dako 22C3 or SP263 PD-L1 IHC assays. Serial sections of each tissue specimen were also stained for CD8 expression. The cTA process detected all cells, assigned them to the tumor or TME compartments, and recorded the Biofeatures™ data which characterized PD-L1 or CD8 staining in the Tumor, Tumor/TME margin, or TME compartments. The method was validated by its ability to reproduce pathologist scoring for PD-L1 and CD8. Using the accepted data, we used the AI system to create novel Scorecard based patient classifications which describe differential PD-L1 status, CD8 status, and PD-L1/CD8 status combined. The AI Scorecard approach demonstrated that certain PD-L1 staining Biofeatures™ may also predict the CD8 status of a tumor, suggesting that additional CD8 staining may not be necessary to understand important expression patterns pertaining to cytotoxic T-cells.

In summary, we demonstrated how the "IO Scorecards" are able to classify patients into differential immune status cohorts using a novel AI based scoring system, which relies only on PD-L1 IHC staining, by creating a comprehensive, contextual profile of PD-L1 staining that does not require additional CD8 IHC staining to characterize the impact of cytotoxic T-cells in a tissue sample.

#3131

Novel multi-gene classifier for prediction of relapse-free survival after FU-based adjuvant chemotherapy for stage III colon cancer: A biomarker study of the ACTS-C trial, a randomized controlled study.

Toshiaki Ishikawa,1 Kenta Murotani,2 Megumi Ishiguro,1 Eiji Nakatani,3 Hiroyuki Uetake,1 Shigeyuki Matsui,4 Kenichi Sugihara5. 1 _Tokyo Medical & Dental University, Graduate School of Medicine and Dentistry, Tokyo, Japan; _2 _Kurume University, Graduate School of Medicine, Fukuoka, Japan;_ 3 _Foundation for Biomedical Research and Innovation at Kobe, Hyogo, Japan;_ 4 _Nagoya University, Graduate School of Medicine, Aichi, Japan;_ 5 _Tokyo Medical & Dental University, Tokyo, Japan_.

Background & Purpose: The ACTS-CC trial is a phase III study that have demonstrated the efficacy of S-1 as adjuvant chemotherapy for stage III colon cancer by evaluating its non-inferiority to UFT/LV. As an additional study, gene expression analysis of 5-FU metabolizing enzymes and folate metabolizing enzymes was performed prospectively to identify predictive and/or prognostic biomarkers. We analyzed the correlation between these gene expression levels and relapse free survival (RFS), and then constructed the scoring system to predict the efficacy of each regimen.

Methods: Among 1535 patients enrolled in the trial, total RNA was extracted from 798 formalin-fixed, paraffin-embedded specimens. Gene expression levels of 11 enzymes (TS, DPD, TP, OPRT, FPGS, GGH, DHFR, MTHFR, MTHFD, FOLRA, GART) related to 5-FU and folic acid metabolism were analyzed by quantitative real-time reverse transcription - TaqMan PCR. Impact of the mRNA expression level of each gene on RFS was investigated. A multivariate Cox regression with elastic net penalization was employed using treatment-by-gene interaction terms extracted by Tian's transformation (Tian L, et al.; J Am Stat Assoc. 109, 2014). To evaluate the predictive accuracy of this scoring system method, a double 10-fold cross-validation was performed to stratify all patients based on the cross-validated score. Finally, the prediction algorithm was applied to the entire cohort data to build the scoring system for use in future patients.

Results: FPGS and GGH significantly correlated with RFS (HR; 1.289 [95%CI; 1.054-1.577], p= 0.014 and HR; 0.908 [95%CI; 0.832-0.991], p= 0.031) and TP seemed to correlate with RFS (HR; 0.906 [95%CI; 0.816-1.007], p= 0.068). In the S-1 treated group, TP and FPGS significantly impacted on RFS (HR; 0.853 [95%CI; 0.731-0.995], p= 0.043 and HR; 1.591 [95%CI; 1.212-2.089], p< 0.001). In the UFT/LV treated group, there was no significant correlation between the gene expression levels of 5-FU/ folate metabolizing enzymes and RFS. In the cross-validation analysis with the penalized Cox regression scoring method, it was demonstrated that a third part of patients with the lowest cross-validated scores had improved RFS by receiving S-1 treatment. The final scoring system developed using the entire patient cohort was composed of TS, TP, and FPGS.

Conclusions: Gene expression level of FPGS, GGH and TP were correlated with the prognosis of stage III colon cancer. The predicting scoring system using the gene expression level of TS, TP and FPGS seemed to be useful for the selection of oral fluoropyrimidine. S-1 might be effective for patients with the low-score colon cancers.

#3132

Immune regulatory gene expression and clinical outcome in the NeoALTTO trial.

Cinzia Solinas,1 Pushpamali De Silva,1 David Venet,2 Soizic Garaud,1 Chunyan Gu-Trantien,3 Florentine Hilbers,4 Evandro de Azambuja,5 Olena Werner,6 Lorena De la Peña,4 Amylou Dueck,7 Serena Di Cosimo,8 Istvan Lang,9 Jens Huober,10 Sherko Küemmel,11 Carsten Denkert,12 Roberto Salgado,13 Christos Sotiriou,2 Martine Piccart-Gebhart,5 Debora Fumagalli,4 Karen Willard-Gallo1. 1 _Molecular Immunology Unit, Institut Jules Bordet and Université Libre de Bruxelles, Bruxelles, Belgium;_ 2 _Breast Cancer Translational Research Laboratory, Institut Jules Bordet and Université Libre de Bruxelles, Bruxelles, Belgium;_ 3 _Institute for Medical Immunology, Université Libre de Bruxelles, Bruxelles, Charleroi, Belgium;_ 4 _Breast International Group, Bruxelles, Belgium;_ 5 _Department of Medical Oncology, Institut Jules Bordet and Université Libre de Bruxelles, Bruxelles, Belgium;_ 6 _Novartis Pharma AG, Basel, Switzerland;_ 7 _Mayo Clinic, Scottsdale, AZ;_ 8 _Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy;_ 9 _National Institute of Oncology, Budapest, Hungary;_ 10 _Universitätsfrauenklinik Ulm, Ulm, Germany;_ 11 _Breast Unit, Kliniken Essen-Mitte, Essen, Germany;_ 12 _Institute of Pathology, Charité Universitätsmedizin Berlin, Berlin, Germany;_ 13 _Department of Pathology, GZA-ZNA, Antwerpen, Belgium_.

Background: The level of immune suppression in an individual breast cancer (BC) patient is relevant for having a major benefit from treatments that act via or are directed to the immune response, such as anti-HER2 agents and immune checkpoint molecules, respectively. The link(s) between the tumor immune microenvironment and treatment responses has been investigated in HER2-positive BC. This study analyzed the association between baseline expression of genes regulating T cell activities (PD1, CTLA4, LAG3, TIM3, PDL1, PDL2, FOXP3, IL10, TGFβ (1-3), FOXP1 and other related genes) and pathologic complete response (pCR) rates in the NeoALTTO trial. Methods: NeoALTTO randomized early-stage HER2-positive BC patients to neoadjuvant trastuzumab (T), lapatinib (L) or T+L for 6 weeks followed by 12 weeks of concurrent paclitaxel. Anthracyclines were given after surgery (FEC). Patients with RNA sequencing data from their baseline tumor samples (N=254 patients out of 455 from the original cohort) were included in the current study. The primary endpoint, pCR, was defined as ypT0/is ypN0. Logistic regression was used for analysis of pCR. Cox regression univariate and multivariate (adjusted for clinicopathological parameters and treatment arms) analyses were performed. Results: In the total cohort analyzed, high PD1 (odds ratio, OR: 1.4, P=0.03), PDL2 (OR: 1.4, P=0.04), CTLA4 (OR: 1.4, P=0.03) and TGFβ3 (OR: 1.4, P=0.02) expression was associated with an increased probability of achieving pCR at the multivariate analysis. ERBB2 (HER2) expression was associated with pCR in the three arms (T: OR: 2.7, P=0.02; L: OR: 2.3, P=0.01; T+L: OR: 5, P<0.001). Additionally in the T+L arm, PAM50 HER2-enriched subtype (OR: 2.9, P=0.03), as well as immune genes PD1 (OR: 2.1, P=0.01), PDL1 (OR: 1.8, P=0.03) and CTLA4 (OR: 2; P=0.01), were also associated with pCR. Conclusions: In the NeoALTTO population examined, PD1, PDL2, CTLA-4 and TGFβ3 expression at diagnosis were all associated with improved pCR rates after anti-HER2 treatment. The effect of these treatments seemed to be dependent on HER2 expression levels, which was particularly relevant in the T+L arm. These observations confirm previous findings that link immune infiltrates to higher pCR rates, demonstrate the clinical significance of the PD-1 pathway and additionally show that CTLA-4 and TGFβ3 could also be important in early stage HER2-positive BC. The association of tumors linked with immune regulatory gene expression with positive responses to neoadjuvant anti-HER2 agents suggests that they act in a way that reinvigorates the antitumor immunity in the face of tumor-mediated suppression. Patients whose tumors highly express these genes may be good candidates for immunotherapy before the start or after the neoadjuvant treatment when residual disease is detected at surgery, although these hypotheses require further confirmation.

#3133

Leukocyte telomere length, cancer incidence and all-cause mortality among Chinese adults: Singapore Chinese health study.

Hamed Samavat,1 Hung N. Luu,1 Renwei Wang,1 Aizhen Jin,2 Woon-Puay Koh,2 Jian-Min Yuan1. 1 _Univ. of Pittsburgh, Pittsburgh, PA;_ 2 _Duke-NUS Medical School Singapore, Singapore City, Singapore_.

Background: Telomeres play a key role in the chromosomal maintenance and stability. To date, very few studies have investigated the association between leukocyte telomere length with cancer incidence and all-cause mortality, particularly among Asian population.

Methods: Relative telomere lengths in genomic DNA from peripheral blood samples were quantified using a validated quantitative real-time PCR among 26,540 middle-aged or older Chinese adults. Hazard ratios (HRs) and 95% confidence intervals (CIs) of incident cancer and deaths by quintiles of telomere length were calculated using the Cox proportional hazards regression method with adjustment for potential confounders.

Results: During 13.2 years of follow-up, 4,353 individuals developed cancer and 2,660 participants died of cancer. Participants with the longest telomeres had significantly 16% higher risk of developing cancer compared to those with the shortest telomeres after controlling for established risk factors (95% CI: 1.06, 1.28; Ptrend= 0.0003). We found similar pattern of results for lung adenocarcinoma (HRQ5 vs. Q1=2.89; 95% CI: 1.95, 4.27), pancreatic cancer (HRQ5 vs. Q1=2.36; 95% CI: 1.29, 4.32), kidney cancer (HRQ5 vs. Q1=3.23; 95% CI: 1.47, 7.53), and genitourinary system cancers including kidney, renal pelvis, ureter, and bladder (HRQ5 vs. Q1=1.56; 95% CI: 1.02, 2.39). In contrast, longer telomeres were associated with lower risk of overall death (HRQ5 vs. Q1=0.93; 95% CI: 0.86, 1.00), and deaths due to ischemic heart disease (HRQ5 vs. Q1=0.85; 95% CI: 0.70, 1.02), respiratory disease (HRQ5 vs. Q1=0.76; 95% CI: 0.64, 0.91), non-cancer (HRQ5 vs. Q1=0.80; 95%: 0.73, 0.88) and other diseases (HRQ5 vs. Q1=0.77; 95% CI: 0.63, 0.94).

Conclusion: The findings of this cohort study support the hypothesis that longer telomere length may be a risk factor for cancer and cancer-related deaths, but might have opposing impact on overall survival.

Funding Sources: National Institutes of Health (NIH) (R01 CA144034 and UM1 CA182876); NIH/NCI T32CA186873; National Medical Research Council, Singapore (NMRC/CSA/0055/2013); NIH (P20CA210300).

#3134

CCN3 is a prognostic biomarker and functional mediator of prostate cancer bone metastasis.

Matthew Dankner,1 Veronique Ouellet,2 Laudine Desreumaux-Communal,2 Estelle Schmitt,2 Dru Perkins,1 Matthew G. Annis,1 Veronique Barres,2 Christine Caron,2 Anne-Marie Mes-Masson,2 Fred Saad,3 Peter M. Siegel4. 1 _McGill University, Montreal, Quebec, Canada;_ 2 _Centre Hospitalier de l'Université de Montréal/CRCHUM, Montreal, Quebec, Canada;_ 3 _Centre Hospitalier de l'Université de Montréal/CRCHUM, University of Montréal, Montreal, Quebec, Canada;_ 4 _Goodman Cancer Research Centre, Montreal, Quebec, Canada_.

Background: Prostate cancer commonly metastasizes to the bone, resulting in pathological fractures and poor prognosis. CCN3/NOV (Nephroblastoma overexpressed) is a secreted protein with a known role in breast cancer metastasis to bone. However, in prostate cancer, CCN3 has been ascribed conflicting roles; some studies suggest that CCN3 promotes prostate cancer metastasis while others argue a tumor suppressor role for CCN3 in this disease. Indeed, in the latter context, CCN3 has been shown to sequester the androgen receptor (AR) and suppress AR signaling. The C-terminal domain of CCN3 is thought to mediate many the protein's pro-metastatic functions given its role in binding to growth factors and promoting dimerization of CCN family members. We hypothesize that the CCN3 CT domain is required to promote osteolytic PC bone metastasis and that CCN3 represents a prognostic biomarker in primary PC tumors to predict recurrence to bone.

Methods: CCN3WT and CCN3∆CT were overexpressed in LNCaP C4-2 cells. The role of CCN3 was assessed with in vitro proliferation, migration and invasion assays, and in vivo through intracardiac injection in male Nude mice (Nu/Nu). Ex vivo µCT scans were performed on murine bone metastasis specimens. CCN3 expression was assessed in two unique tissue microarrays (TMA) comprising over 1500 human primary prostate tumor using different anti-CCN3 antibodies with immunohistochemistry and immunohistofluorescnece, respectively.

Results: While CCN3WT and CCN3∆CT had little effect in vitro on cell proliferation, migration or invasion, intracardiac injection of CCN3WT resulted in increased incidence of bone metastasis compared to empty vector control and CCN3∆CT. Ex vivo µCT revealed decreased bone mineral density in bones from mice injected with CCN3WT cells compared to empty vector control and CCN3∆CT expressing LNCaP C4-2 cells. In both TMAs studied, high CCN3 expression in tumor epithelium correlated with increased risk of biochemical relapse and bone metastasis at 5 years and 15 years post- surgical resection, respectively.

Conclusion: CCN3 requires its C-terminal domain for its bone metastatic function, and CCN3 is correlated with aggressive disease biology in primary prostate cancer specimens. These findings point to CCN3 protein expression as a biomarker that can be useful in predicting prostate cancer aggressiveness and recurrence to bone, while providing clarity on CCN3's functional role as a mediator of prostate cancer bone metastasis.

#3135

Genomewide expression profiling identifies a novel miRNA-based signature for the detection of peritoneal metastasis in gastric cancer.

Tadanobu Shimura,1 Shusuke Toden,1 Raju Kandimalla,1 Yuji Toiyama,2 Yoshinaga Okugawa,2 Mitsuro Kanda,3 Hideo Baba,4 Yasuhiro Kodera,3 Masato Kusunoki,2 Hiroki Hori,5 Ajay Goel1. 1 _Center for Gastrointestinal Research; Center from Translational Genomics and Oncology, Baylor Scott & White Research Institute and Charles A. Sammons Cancer Center, Baylor University Medical Center, Dallas, TX; _2 _Department of Gastrointestinal and Pediatric Surgery, Division of Reparative Medicine, Institute of Life Sciences, Mie University Graduate School of Medicine, Mie, Japan;_ 3 _Department of Gastroenterological Surgery, Nagoya University Graduate School of Medicine, Nagoya, Japan;_ 4 _Department of Gastroenterological Surgery, Graduate School of Medical Science, Kumamoto University, Kumamoto, Japan;_ 5 _Department of Pediatrics, Mie University Graduate School of Medicine, Mie, Japan_.

Purpose: Even though peritoneal metastasis (PM) in patients with gastric cancer (GC) has long been recognized to associate with poor survival, currently there is a lack of availability of biomarkers for its robust diagnosis. Availability of such biomarkers will facilitate more accurate identification of PM in GC patients, and could be clinically transformative as it will permit a timely intervention leading to reduced mortality associated with this malignancy. Recent advances in next generation sequencing technologies for an in-depth genomic and epigenomic profiling of various malignancies has paved the path for identification of previously unrecognized biomarkers. One such molecular substrates includes microRNAs (miRNAs), which are 18-25 nucleotides long, single-stranded noncoding RNAs, and act as post-transcriptional gene repressors. Although expression of specific miRNAs is frequently dysregulated in various human cancers including GC, their clinical significance as potential biomarkers for detecting PM has not been evaluated in large, adequately powered, independent patient cohorts. Accordingly, in this study, we undertook a comprehensive effort to identify and establish a novel miRNA-based signature for diagnosing presence of PM in GC patients.

Experimental design: We performed a genomewide, systematic biomarker discovery by analyzing miRNA expression profiles in primary tumors from GC patients with and without PM, followed by independent testing and validation in multiple patient cohorts of 354 patients, and establishment of a miRNA-signature for the diagnosis of PM in patients with advanced GC.

Results: Five miRNAs (miR-30a-5p, -134-5p, -337-3p, -659-3p, and -3917) were identified during the initial discovery phase; three of which (miR-30a-5p, -659-3p, and -3917) were significantly overexpressed in the primary tumors from PM-positive patients in the testing cohort (p=0.002, 0.04 and 0.007 respectively), and robustly distinguished patients with vs. without PM (AUC=0.82). Furthermore, high expression of these miRNAs was also associated with poor prognosis (HR=2.18, p=0.04). The efficacy of the combination miRNA-signature was successfully validated in an independent patient cohort (AUC=0.74). Finally, this miRNA signature in combination with the macroscopic Borrmann's type score offered a significantly superior diagnostic accuracy in all three cohorts (AUC=0.87, 0.76, 0.79, respectively), and led to the establishment of a risk-prediction nomogram for the diagnosis of PM in GC patients.

Conclusions: Using a genomewide transcriptomic profiling biomarker discovery and validation approach, we have established a novel miRNA-signature that robustly identifies presence of peritoneal metastasis in gastric cancer patients, which might lead to improved survival outcomes in patients suffering from this malignancy.

#3136

Discovery of low abundance colorectal cancer related biomarkers by the ADAPT Biotargeting System.

Tassilo Hornung,1 Jelena Zarkovic,1 Michelle Kassner,1 Matthew Rosenow,1 Mark R. Miglarese,1 Günter Mayer,2 Michael Famulok,2 David B. Spetzler1. 1 _Caris Life Sciences, Phoenix, AZ;_ 2 _University of Bonn, Bonn, Germany_.

Disease biomarkers play an essential role in disease-diagnostics and in monitoring their responses to therapies. Identification of low to medium abundance biomarkers is one of the biggest challenges in the field. We have developed Adaptive Dynamic Artificial Poly-ligand Targeting (ADAPT), a method in which libraries of single-stranded oligodeoxynucleotides (ssODNs) are enriched for sequences that bind to targets existing in samples in various amounts. Pull-downs with enriched libraries followed by LC-MS/MS allows for the enrichment and identification of low abundance biomarkers that cannot be identified by conventional proteomics.

A highly diverse library of 1011 ssODNs was subjected to multiple rounds of positive selection against formalin-fixed paraffin-embedded (FFPE) colorectal cancer tissue with negative selection against adjacent non-cancer tissue of the same specimen. An enriched library of ~3x106 ssODNs that bound preferentially to cancer tissue was obtained and used in combination with LC-MS/MS to identify biomarkers related to colorectal cancer. Using this approach, a total of 14 proteins were identified in cancer but not in non-cancer tissue lysates. Their identification was only possible due to their enrichment by binding of the immobilized ssODN library to the cancer sample since the same proteins were undetectable by conventional proteomics. Immunohistochemical staining (IHC) of multiple colorectal cancer cases verified that several of the identified target proteins, including nucleophosmin (NPM1) and synaptotagmin-like protein 2 (SYTL2), showed higher expression in cancer tissue compared to adjacent non-cancer tissue.

Our data indicate the potential of the ADAPT platform to profile small differences between cancer affected and unaffected tissue and provide a novel approach for cancer biomarker discovery.

#3137

DNA damage response and repair pathway alteration and its association with tumor mutation burden and platinum-based chemotherapy in small cell lung cancer.

Sehhoon Park,1 Hayoon Lee,2 Boram Lee,2 Jong-Mu Sun,1 Woong-Yang Park,2 Jin Seok Ahn,1 Myung-Ju Ahn,1 Se-Hoon Lee,1 Keunchil Park1. 1 _Samsung Medical Center, Seoul, Republic of Korea;_ 2 _Samsung Advanced Institute of Health Science and Technology, Sungkyunkwan University, Seoul, Republic of Korea_.

Background: Impairment in DNA damage response and repair (DDR) pathway is known as a predictive biomarker of platinum sensitivity in many cancer types. In the era of cancer immunotherapy, DDR alteration is re-emphasized as a predictive biomarker of immune-checkpoint inhibitor due to its positive correlation to tumor mutation burden (TMB).

Patients and methods: Target gene sequencing (381 genes) was conducted from 100 extensive disease (ED) and 66 limited disease (LD) small cell lung cancer (SCLC) patients. DDR-related genes were pre-defined by literature review, and mutations were classified as double-strand breaks (DSB, n=82): homologous recombination (n=54), non-homologous end joining (NHEJ, n=19), and Fanconi anemia (FA, n=32); or single-strand breaks (SSB, n=31): mismatch repair (MMR, n=19), base excision repair (BER, n=7), and nucleotide excision repair (NER, n=6). Based on the mutation profile, correlation of TMB and survival outcomes were analyzed.

Results: Compared to patients with an intact DDR pathway (n=70), a higher TMB was observed in patients with homologous recombination (P<0.001), NHEJ (P=0.026), MMR (P<0.001), BER (P=0.046), NER (P=0.017), DSB (P<0.001) and SSB (P<0.001). Survival analyses based on TMB level showed no predictive or prognostic values in ED patients. In LD patients, prolonged progression-free survival (PFS, hazard ratio [HR] 0.497, P=0.015) to platinum compound and overall survival (HR 0.383, P=0.010) were observed in those with TMB above median. Individual DDR pathway alteration showed no survival benefit in ED patients receiving platinum-based chemotherapy. In LD patients, those with mutations in the FA pathway had shorter PFS (HR 2.048, P=0.036) to initial treatment.

Conclusions: DDR pathway alterations, both DSB and SSB, in SCLC have a positive correlation with high TMB. However, it has demonstrated limited value in prediction of platinum efficacy.

Tumor mutation burden and its correlation with DNA damage related gene sets

---

|

FA | |  | HR | |

|

(-) n=131 | (+) n=32 | P | (-) n=112 | (+) n=54 | P

Low TMB | 69 (52.7%) | 14 (43.8%) | 0.479 | 65 (58.0%) | 20 (37.0%) | 0.018

High TMB | 62 (47.3%) | 18 (56.2%) | |

47 (42.0%) | 34 (63.0%)

|

|

NHEJ | |  | BER | |

|

(-) n=147 | (+) n=19 | P | (-) n=156 | (+) n=7 | P*

Low TMB | 78 (53.1%) | 7 (36.8%) | 0.277 | 80 (51.3%) | 3 (42.9%) | 0.716

High TMB | 69 (46.9%) | 12 (63.2%) | |

76 (48.7%) | 4 (57.1%)

|

|

MMR | |  | NER | |

|

(-) n=147 | (+) n=19 | P* | (-) n=157 | (+) n=6 | P*

Low TMB | 81 (55.1%) | 4 (21.1%) | 0.007 | 81 (51.6%) | 2 (33.3%) | 0.437

High TMB | 66 (44.9%) | 15 (78.9%) | |

76 (48.4%) | 4 (66.7%)

|

|

DSB | |  | SSB | |

|

(-) n=84 | (+) n=82 | P | (-) n=135 | (+) n=31 | P

Low TMB | 50 (59.5%) | 35 (42.7%) | 0.044 | 76 (56.3%) | 9 (29.0%) | 0.006

High TMB | 34 (40.5%) | 47 (57.3%) | |

59 (43.7%) | 22 (71.0%)

|

#3138

SMARCA4 **mutations in** KRAS **-mutant lung adenocarcinoma: A multi-cohort analysis.**

LIANG LIU, Tamjeed Ahmed, Willam Jeffrey Petty, Stefan Grant, Jimmy Ruiz, Thomas W. Lycan, Umit Topaloglu, Ping-Chieh Chou, Lance D. Miller, Gregory A. Hawkins, Martha A. Alexander-Miller, Stacey S. O'Neill, Bayard L. Powell, Ralph B. D'Agostino, Reginald F. Munden, Boris Pasche, Wei Zhang. _Wake Forest Baptist Comprehensive Cancer Center, WINSTON SALEM, NC_.

KRAS is a key oncogenic driver in lung adenocarcinoma (LUAD). Chromatin-remodeling gene SMARCA4 was co-mutated with KRAS in LUAD; however, the impact of SMARCA4 mutations on clinical outcome has not been adequately established. This study sought to shed light on the clinical significance of SMARCA4 mutations in LUAD. The association of SMARCA4 mutations with survival outcomes was interrogated in 4 independent cohorts totaling 564 patients: KRAS-mutant patients with LUAD who received non-immunotherapy treatment from 1) The Cancer Genome Atlas (TCGA) and 2) the MSK-IMPACT Clinical Sequencing (MSK-CT) cohorts; and KRAS-mutant patients with LUAD who received immune checkpoint inhibitor-based immunotherapy treatment from 3) the MSK-IMPACT (MSK-IO) and 4) the Wake Forest Baptist Comprehensive Cancer Center (WFBCCC) immunotherapy cohorts. Of the patients receiving non-immunotherapy treatment, in the TCGA cohort (n=155), KRAS-mutant patients harboring SMARCA4 mutations (KS) showed poorer clinical outcome (P=6e-04 for disease-free survival (DFS) and .031 for overall survival (OS), respectively), compared to KRAS-TP53 co-mutant (KP) and KRAS-only mutant (K) patients; in the MSK-CT cohort (n=314), KS patients also exhibited shorter OS than KP (P=.03) or K (P=.022) patients. Of patients receiving immunotherapy, KS patients consistently exhibited the shortest progression-free survival (PFS; P=.0091) in the MSK-IO (n=77), and the shortest PFS (P=.0026) and OS (P=0.0014) in the WFBCCC (n=18) cohorts, respectively. Therefore, mutations of SMARCA4 represent a genetic factor that lead to adverse clinical outcome in lung adenocarcinoma treated by either non-immunotherapy or immunotherapy.

#3139

EphB4-EphrinB2 receptor-ligand are downstream effectors and novel targets of PTEN deficient prostate cancer.

Grace X. Li,1 Binyun Ma,1 Valery G. Krasnoperov,2 Imran Siddiqi,1 Akash Sali,1 Gangning Liang,1 Inderbir S. Gill,1 Jacek K. Pinski,1 David I. Quinn,1 Sarmad Sadeghi,1 Parkash S. Gill1. 1 _University of Southern California, Keck School of Medicine, Los Angeles, CA;_ 2 _Vasgene Therapeutics Inc., Los Angeles, CA_.

Background EphB4 is upregulated in prostate cancer in over half the cases, and correlates with stage and survival. EprhinB2, the ligand for EphB4 has not been well studied. Functional studies show EphB4 provides a survival signal through the PI3K/PTEN/Akt/pS6 pathway. A therapeutic agent, soluble EphB4-albumin fusion protein (sEphB4) which blocks bidirectional signaling is currently in clinical development. We thus wished to conduct detailed investigation into the role of EphB4 and EphrinB2 in genetic mouse model of prostate cancer, in tumor initiation and progression, by knockout of EphB4 and by pharmacological agent (sEphB4).

Methods We studied the expression of Ephrinb2 in 180 samples of human prostate cancers and normal prostates using highly characterized, specific EphrinB2 mAb. We next used genetically engineered mouse model of prostate cancer condition deletion of PTEN in prostate epithelium, crossed with transgenic mouse line expressing luciferase. To test the role of EphB4-EphrinB2, we generated Floxed allele of Ephb4 to conditionally delete in prostate epithelium. PTENf/f, EphB4f/f and luciferase were crossed and imaged over time. Secondly, PTENf/f mice were treated with sEphB4 in prevention, established tumor and castration resistant prostate cancer (CRPC) cohorts. Prostate cell lines C4-2B, PC3 and 22RV1 were used to analyze the role of EphB4 in regulation of PI3K pathway and androgen receptor (AR).

Results EphrinB2 is expressed in 50% of human prostate cancers, but not the normal tissues. PTEN null mouse prostate tumors had elevated EphB4 and EphrinB2, but not other family members. Conditional deletion of EphB4 in the context of PTEN deletion in prostate epithelium abolished tumor formation and caused inhibition of the PI3K pathway. Treatment with sEphB4 precluded tumor development and induced tumor regression in both early prostate cancer and CRPC mice. PI3K/AKT/pS6 activity nearly complete declined in the treatment group. Mechanistic studies using EphB4 knock down showed downregulation of PI3K alpha, beta, not gamma and delta. Most surprisingly, AR levels were markedly reduced in sEphB4 treated tumors. Further studies in vitro showed decline in AR with EphB4 knock down, which could be rescued with ectopic expression of PI3K beta, but not other PI3K isoforms. These changes were recapitulated in a patient with late stage CRPC treated in a single patient IND for sEphB4.

Conclusions EphB4-EphrinB2, a receptor-ligand pair are expressed in prostate cancer and induced by loss of PTEN or activation of PI3K pathway. Genetic and pharmacological intervention validates the potential role as a downstream effectors through downregulation of PI3K signaling and AR. Drugs that can reduce AR levels and PI3Kβ inhibitors are potential candidates for prostate cancer therapy and sEphB4 holds therapeutic potential in prostate cancer.

#3140

A prospective, double-blinded clinical study using atomic force microscopy for fast diagnosis and subtyping of low and high-risk breast cancers.

Rosemarie Burian,1 Ahmed Jizawi,1 Gabriel Zihlmann,2 Tobias Appenzeller,2 Philipp Oertle,2 Christian Raez,1 Roderick Y. Lim,2 Serafino Forte,3 Sophie Dellas,1 Simone Muenst,1 Tatjana Vlajnic,1 Ellen Obermann,2 Maria Ricci,4 Kevin Grimm,4 Ferdinand Niedermann,4 Marko Loparic,4 Marija Plodinec2. 1 _University Hospital Basel, Basel, Switzerland;_ 2 _University of Basel, Basel, Switzerland;_ 3 _Kantonsspital Baden, Baden, Switzerland;_ 4 _ARTIDIS AG, Basel, Switzerland_.

Mechanical properties and physical interactions of cells and their microenvironment that occur at nanometre scale play a critical role in cancer progression and metastatic dissemination (https://physics.cancer.gov). Nanomechanical profiling of breast tissue enables fast and precise cancer diagnosis. Detecting extreme viscoelastic (soft) cancer cells in biopsies provides a direct biomarker of cancer aggressiveness (Plodinec et. al., 2012). In an ongoing single centre clinical trial conducted in a routine clinical setting we distinguish benign from cancerous breast lesions within 2.5 hours using an atomic force microscope (AFM)-based method known as ARTIDIS. ARTIDIS employs a micro-fabricated 20nm-sharp tip which indents 10,000 locations/sample to measure the stiffness of cellular and matrix structures. In a comprehensive web-based platform we collect, verify and analyze these 2.54 millions of AFM indentations in relation to over 100 clinical parameters /patient using artificial intelligence. In our interim analysis of core needle and vacuum biopsies (N=254), 34 samples were classified as B1 (normal); 127 as B2 (benign); 12 as B3 (uncertain malignant potential); 0 as B4 (suspicious); 18 as B5a (ductal carcinoma in situ), and 58 as B5b (malignant). The diagnostic AFM ROC curve of B1 and B2 lesions vs. all B5 lesions (CI 95%; 100% sensitivity, 90% specificity, AUC = 0.98) demonstrated ARTIDIS outstanding ability to detect cancer in samples with >80% neoplastic tissue. An analysis including lesions with <5% neoplastic tissue, (CI 95%; 96% sensitivity, 74% specificity, AUC = 0.91) showed similarly outstanding results. The secondary comparison of B3 vs. B2 lesions could inform clinical practice as these overlapped significantly (CI 95%; 50% sensitivity, 88% specificity, AUC = 0.712) suggesting low malignancy potential; comparing B3 to B5a (CI 95%; 89% sensitivity, 83% specificity, AUC = 0.842) might be clinically relevant as B3 lesions similar to B5a lesions could be considered higher risk. Comparing Luminal A to Luminal B samples (CI 95%; sensitivity 83%, specificity 70%, AUC = 0.77) suggested a potential of ARTIDIS to distinguish a more aggressive Luminal B subtype with positive nodal status in 12 out of 23 patients. The results demonstrate the ability of ARTIDIS to differentiate benign from malignant breast lesions within 2.5 hours in a routine clinical setting. The secondary endpoint results suggest that we are able to subclassify breast lesions into specific subtypes. In particular, luminal B cancers exhibit nanomechanical profiles that could be associated with better or worse prognosis as confirmed by the post-AFM positive nodal status. The final analysis (n=508) will determine clinical utility for primary endpoint and indications for future studies on nanomechanical characterization of breast cancer for prediction and treatment optimization.

#3141

The relationship between BRAF/NRAS/KIT genomic driver mutations and anti-PD1 immunotherapy responses in patients with advanced melanoma.

April A.N. Rose,1 Susan M. Armstrong,1 David Hogg,2 Marcus Butler,2 Anthony M. Joshua,3 Danny Ghazarian,4 Suzanne Kamel-Reid,4 Ayman Al Habeeb,4 Mary Anne Chappell,2 Kendra Ross,2 Eitan Amir,2 Phillippe L. Bedard,2 Lillian Siu,2 Anna Spreafico2. 1 _University of Toronto, Toronto, Ontario, Canada;_ 2 _Princess Margaret Cancer Center, Toronto, Ontario, Canada;_ 3 _Kinghorn Cancer Center, Sydney, Australia;_ 4 _University Health Network, Toronto, Ontario, Canada_.

Background: Anti-PD1 immunotherapy, when used alone or in combination (combo) with anti-CTLA4, prolongs overall (OS) and progression-free survival (PFS) of patients (pts) with advanced melanoma, compared to single-agent (SA) anti-CTLA4. Combo immunotherapy is more toxic than SA anti-PD1, therefore it is of interest to identify patients most likely benefit from SA anti-PD1 to spare these pts the toxicity of combo immunotherapy. We asked whether genomic driver mutations (mts) in BRAF, NRAS or KIT could predict anti-PD1 responses in melanoma pts.

Methods: We preformed a single-center retrospective analysis of 292 melanoma pts who received SA or combo anti-PD1 immunotherapy at Princess Margaret Hospital between 2012-17. BRAF/NRAS/KIT mts and other covariates were abstracted from chart review. Multivariable Cox models were used to assess differences in OS/PFS.

Results: We identified 209 and 83 melanoma patients who received SA-anti-PD1 or combo immunotherapy, respectively. Pt characteristics were: mean age 59 yrs; 56% male; 77% cutaneous/unknown primary 23% uveal or mucosal; 17% brain metastases; 34% stage M0/M1a/M1b, 66% stage M1c/M1d, 51% BRAF/NRAS/KIT mt. Among pts receiving SA anti-PD1, presence of a BRAF, NRAS, or KIT mt was an independent predictor of poor PFS (HR 1.84; 95% CI 1.21-2.81) and OS (HR 1.93; 95% CI 1.17-3.22) in multivariate models. Other negative predictors of poor survival were: elevated LDH, multiple previous lines of therapy, 3 or more sites of metastasis, and uveal or mucosal histology. Combo immunotherapy was independently associated with longer OS compared to SA anti-PD1 in BRAF/NRAS/KIT mt pts (HR 0.43; 95% CI 0.20-0.90), but not in BRAF/NRAS/KIT wild-type pts (HR 1.34; 95% CI 0.46-3.90).

Conclusions: In this retrospective study, the presence of a BRAF NRAS or KIT mt was an independent predictor of poor PFS/OS in melanoma pts treated with SA anti-PD1. Combo immunotherapy prolonged survival vs SA anti-PD1 in BRAF/NRAS/KIT mt melanoma, but not in BRAF/NRAS/KIT wild-type melanoma. These data suggest that BRAF/NRAS/KIT wild-type melanoma pts may derive less added benefit from combo immunotherapy (vs single agent anti-PD1) compared to pts with BRAF/NRAS/KIT mutations. Validation analyses are on-going.

#3142

**A** CD274, PDCD1LG2, CD8A, **and** IRF1 **multiplex in a closed system RT-qPCR panel and immunotherapy outcome in metastatic melanoma.**

Swati Gupta,1 Leena McCann,2 Yvonne G.Y. Chan,2 Edwin W. Lai,2 Pok Fai Wong,1 James W. Smithy,3 Jodi Weidler,4 Brian Rhees,2 Michael Bates,4 Harriet M. Kluger,5 David L. Rimm1. 1 _Department of Pathology, Yale University School of Medicine, New Haven, CT;_ 2 _Oncology Research and Development, Sunnyvale, CA;_ 3 _Department of Medicine, Brigham and Women's Hospital, Boston, MA;_ 4 _Medical and Scientific Affairs and Strategy, Oncology, Sunnyvale, CA;_ 5 _Department of Internal Medicine (Medical Oncology), Yale University School of Medicine, New Haven, CT_.

Background: In melanoma, there is no companion diagnostic test to predict response to programmed cell death 1 (PD-1) axis immune checkpoint inhibitor therapy (ICI). In the adjuvant setting, only 1 in 5 patients may benefit from ICI, so biomarker is needed to select those that may or may not benefit. Candidate techniques for the assessment of predictive markers include immunohistochemistry (IHC), multiplex fluorescence, genome sequencing, and RNA expression profiles. Here we test a new 4-gene research use only (RUO)* prototype mRNA expression profile on the GeneXpert closed system using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) for association with clinical benefit after treatment with ICI in metastatic melanoma patients.

Methods: Pretreatment formalin-fixed paraffin-embedded (FFPE) tissue sections from melanoma patients treated with anti-PD-1 therapy (pembrolizumab, nivolumab, or ipilimumab plus nivolumab) between 2011-17 were selected from the Yale Pathology archives. FFPE sections were macrodissected to enrich for tumor for quantitative assessment of CD274 (PD-L1), PDCD1LG2 (PD-L2), CD8A, and IRF1 by RT-qPCR multiplex mRNA panel. Multiplex panel transcript levels were correlated with clinical benefit (CR/PR/SD); disease outcomes (progression-free survival, PFS and overall survival, OS); and protein levels assessed by quantitative immunofluorescence (QIF). Median values for each marker were used to define high versus low mRNA or protein expression groups. This study was approved by Yale Human Investigation IRB protocol ID 9505008219.

Results: Inter-transcript regression was observed among all four markers with R2 ranging from 0.20 to 0.51. Transcript levels were significantly higher in CR/PR/SD than in PD for CD8A (p = 0.0001) and IRF1 (p = 0.0019). PFS was strongly associated with high CD274 (p = 0.0046), PDCD1LG2 (p = 0.0039), CD8A (p = 0.0002), and IRF1 (p = 0.0030) mRNA expression. Similar associations were observed for OS with high CD274 (p = 0.0004), CD8A (p = 0.0030), and IRF1 (p = 0.0096) mRNA expression. Multivariate analyses revealed significant associations with OS independent of age, sex, stage, mutation, treatment, and prior ICI for CD274 (HR = 0.30), CD8A (HR = 0.40) and IRF1 mRNA (HR = 0.36). Similar PFS association with CD8A (HR = 0.39) and IRF1 (HR = 0.48) parameters were observed by multivariate analyses. Nonlinear exponential relationship was observed between transcript and protein levels for CD8A (R2 = 0.66) and IRF1 (R2 = 0.40).

Conclusions: Although tested in only a single melanoma cohort, CD274, CD8A and IRF1 mRNA levels show promising associations with outcome. The turnaround time of the test (2h) and easy standardization of the platform makes this an attractive approach for further study in the search for predictive biomarkers for ICI. *for Research Use Only - not approved or reviewed by any regulatory body

#3143

Predicting treatment response using patient derived organotypic cancer spheroids.

Carley M. Sprackling,1 Jeremy D. Kratz,1 Peter F. Favreau,2 Mohammad R. Karim,2 Christopher P. Babiarz,1 Cheri A. Pasch,1 Amani A. Gillette,1 Linda Clipson,1 Kristina A. Matkowskyj,1 Jens C. Eichoff,1 Kayla K. Lemmon,1 Hannah K. Houtler,1 Mark E. Burkard,1 Devon Miller,1 Melissa C. Skala,2 Dustin A. Deming1. 1 _University of Wisconsin-Madison, Madison, WI;_ 2 _Morgridge Institute for Research, Madison, WI_.

Background: There are limited clinical tools for predicting the effectiveness of cancer therapies. We aim to prospectively predict patient treatment response using patient-derived organotypic cancer spheroids (PDOCS) as an in vitro model which recapitulates the genetic characteristics and 3D organization of the patient's tumor. Using optical metabolic imaging (OMI) to analyze single cells, we can determine heterogeneous subpopulations in response to drug treatment. Further clinical validation of these techniques and analysis methods are needed before clinical translation.

Methods: Tissue biopsies and gross tissue resections were acquired through the University of Wisconsin Precision Medicine Molecular Tumor Board (IRB#UW15068) and UWCCC TSB Biobank. Next-generation sequencing (NGS) from the biopsies was performed to determine molecular profiling. In alignment with the patient's treatment course, PDOCS were treated with physiologic doses of chemotherapy or targeted therapy. Treatment response was evaluated by measuring change in diameter in conjunction with optical metabolic imaging (OMI) using a multiphoton microscope to measure the fluorescence and redox ratio of NAD(P)H and FAD as an indication of cellular metabolism. Diameter changes between control and treatment groups were compared using Glass's delta; resistance to therapy was indicated by a Glass's delta score of below 1.5. The optical redox ratios determined by OMI were compared using Glass's delta, and resistance was indicated below 0.5. Clinical response was measured using RECIST v1.1 standard response assessment criteria.

Results: PDOCS were successfully isolated from colorectal (CRC), lung, gastrointestinal stromal tumor (GIST), ovarian, and breast cancers. These biopsies were all obtained in the treatment refractory setting. PDOCS were generated for seven patients and treated with the same pharmacologic treatment as the patient from which the PDOCS were generated. Multiple treatments were able to be tested both in vitro and clinically for a subset of patients. Treatments included: 5-fluouracil, oxaliplatin, gemcitabine, paclitaxel, olaparib, panitumumab, osimertinib, fulvestrant, and palbociclib. In this cohort, two treatments resulted in stable disease and seven treatments resulted in disease progression. Change in spheroid diameter correlated with clinical treatment outcomes with an effect size (Glass's delta) threshold of 1.5. OMI predicted response for all patients imaged with an effect size threshold of 0.5 which correlated with the size change analyses. Treatment heterogeneity of OMI was observed in many of the samples.

Conclusions: In this largely prospective cohort of patients across disease types, changes in PDOCS size and OMI indices predict treatment benefit for individual patients. Studies on a larger scale are needed to further validate these findings.

#3144

Deep learning-based predictive biomarker for adjuvant chemotherapy in early-stage hormone receptor-positive breast cancer.

Soo Youn Cho,1 Eun Yoon Cho,1 Kyunghyun Paeng,2 Geunyoung Jung,2 Sarah Lee,2 Sang Yong Song1. 1 _Department of Pathology and Translational Genomics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea;_ 2 _Lunit Inc., Seoul, Republic of Korea_.

Introduction: Predictive value of adjuvant chemotherapy for patients with early-stage hormone receptor-positive breast cancer has been suggested by 21-gene expression assay, although its cost-effectiveness has not been well-defined. We have developed the deep learning-based H&E image analyzer named Lunit SCOPE, identifying and quantifying various histologic components from H&E-stained whole slide images We hypothesized that cell proportions analyzed by Lunit SCOPE would be a potential prognostic and predictive biomarker of adjuvant chemotherapy in early-stage hormone receptor-positive breast cancer.

Method: We have collected clinical data and H&E slides from de-identified 2,915 early breast cancer patients in Samsung Medical Center, retrospectively. The 898 patients with hormone receptor-positive, T1b ~ T3 and N0 ~ N1mi have been selected to analyze the predictive value of adjuvant chemotherapy. Deep learning-based H&E image analyzer, Lunit SCOPE, has been trained by 1,191 H&E-stained whole slide images from another breast cancer patient cohort. In the whole slide image, biological and histological components such as cancer epithelium, cancer stroma, normal, fat, necrosis, lymphocyte, fibroblast, and other cells, have been annotated by over 10 pathologists. The outputs of Lunit SCOPE are the ratio of cancer epithelium, cancer stroma, normal, necrosis and fat in a whole slide image and intratumoral tumor infiltrating lymphocyte (TIL) and stromal TIL density. The recurrence score (RS) based on the output of Lunit SCOPE has been determined by using multivariate cox regression analysis for disease-free survival (DFS) in the patients without adjuvant chemotherapy.

Result: Recurrence score (RS) was proportional to the cancer stroma ratio and stromal TIL density, but inversely proportional to intratumoral TIL density. When the RS cutoff was 0.913, 21.3% (191 out of 898) of patients were classified as high risk group (RS > cutoff). Among those without adjuvant chemotherapy, high risk group presented poor DFS (hazard ratio [HR] 4.23, 95% confidence interval [CI] 1.87-9.59, P = 1.73 x 10-4) and overall survival (OS, HR 4.95, 95% CI 1.39-17.6, P = 6.07 x 10-3) than the low risk group. Adjuvant chemotherapy did not prolong OS in patients with low risk group (HR 1.08, 95% CI 0.38-3.12, P = 0.885). However, interestingly, in those with high risk by Lunit SCOPE, adjuvant chemotherapy prolonged DFS (HR 0.35, 95% CI 0.15-0.86, P = 0.0161) and OS (HR 0.22, 95% CI 0.05-0.95, P = 0.0254), reflecting RS by Lunit SCOPE would be a significant predictive biomarker of adjuvant chemotherapy.

Conclusion: Deep learning-based H&E image analyzer, Lunit SCOPE, was possible to analyze the prognosis of breast cancer. Especially, only high risk patients of RS by Lunit SCOPE had survival benefit from adjuvant chemotherapy, which needs to be validated in clinical trials.

#3145

Detection of premature aging among adolescent and young adult osteosarcoma and Ewings sarcoma survivors.

Laurence H. Baker, Denise Reinke, Philip Boonstra, Erin Peregrine Antalis. _Univ. of Michigan, Ann Arbor, MI_.

BACKGROUND Bone sarcoma survivors are at high risk for multiple chronic diseases at a young age, which can be characterized as premature aging. Bone sarcoma survivors have the highest cumulative incidence of multiple serious chronic conditions according to analysis of the Childhood Cancer Survivor Study 1. Cardiovascular biomarkers can be used to detect CAD, and enable early intervention in asymptomatic patients.

METHODS

Patients are seen annually at the Sarcoma Survivorship Clinic at the University of Michigan and includes high risk patients over the age of 18 who are at least two years free of disease after treatment completion. Clinical data includes: Blood Pressure, BMI, lipid profile, high sensitivity C-Reactive Protein, and imaging.

RESULTS

30% of patients aged 18-39 exhibit elevations in the cardiovascular biomarker hsCRP, in addition to the traditional biologic markers of elevated lipids, hypertension, and BMI at their most recent clinic visit (Table 1). A subset of patients independently evaluated by 2 nuclear cardiologists also show evidence of coronary artery calcifications (CAC), which is considered pathognomonic of CAD. 40% have been diagnosed with 2 or more of the following chronic diseases: dyslipidemia, obesity, renal insufficiency, anxiety and/or depression, pulmonary disease, cardiovascular disease, thyroid dysfunction, hypertension, liver disease, and anemia.

CONCLUSION

The overall cardiac risk profile among high risk bone sarcoma survivors is characteristic of premature aging. Examining the underlying mechanism of premature aging is essential in this population. Proposed investigation includes alteration of immune pathway as effected by diet, the microbiome, metabolomics, and cellular senescence.

REFERENCES:

1. Gibson TM et al: Temporal patterns in the risk of chronic health conditions in survivors of childhood cancer diagnosed 1970-99: a report from the Childhood Cancer Survivor Study cohort. The Lancet Oncology 2045:1-12, 2018

Characteristics of bone sarcoma survivors, aged 18-39 (n=20)

---

Chronic Disease Diagnosis | %

1 | 50

2 | 25

3+ | 15

Cardiovascular biomarkers

|

hsCRP ≥ 2.0 | 30

BMI ≥ 30 | 15

Cholesterol ≥ 200 mg/dL | 12

Hypertension ≥130/80 | 15

Glucose ≥ 100 mg/dL | 20

CAC>0 | 15

#3146

MFG-E8: A novel prognostic marker for gastric cancer.

Sai Ge, Jing Gao, Lin Shen. _Peking University Cancer Hospital & Institute, Beijing, China_.

Gastric cancer is one of the leading causes of cancer-related death all over the world. Milk fat globule-epidermal growth factor (EGF)-factor VIII (MFG-E8) was found to be highly unregulated in a variety of cancers, could induce phagocytosis of apoptotic cells and a significant factor in immune systems. However, its prognostic potential has not been evaluated in gastric cancer (GC). In our former proteomic study, MFG-E8 was found significantly overexpressed in gastric cancer tissues comparing with paired non-cancerous gastric tissues. This study aimed to analyze the association between the expression of MFG-E8 in GC tissues and clinical outcomes in GC patients. In this study, 3 independent cohorts (GSE15460, n = 192; TCGA, n = 380 and ACRG, n = 300) were used to assess MFG-E8 as a biomarker, Kaplan-Meier survival analysis showed that its overexpression was associated with poor prognosis of GC with good discriminative ability in 3 independent cohorts (GSE15460, P <0.001; TCGA, P <0.001 and ACRG, P <0.001). Multivariate analysis further confirmed its prognostic significance (GSE15460, P =0.028; TCGA, P <0.001 and ACRG, P =0.012). These results suggest MFG-E8 may become a potential prognostic factor for GC.

#3147

A novel next generation sequencing strategy for detecting microsatellite instability in colorectal and gastric cancer.

Wenyun Li,1 Fengmei Pi,2 Xiaoxia Liang,1 Taiyuan Cao,1 Yiheng Lin,1 Xiaohui Zhai,1 Mengli Huang,3 Hao Qin,3 Chan Gao,3 Shangli Cai,3 Xianrui Wu,1 Jian Xiao,1 Ping Lan1. 1 _The Sixth Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China;_ 2 _Nanobio Delivery Pharmaceutical Co., Ltd, Columbus, OH;_ 3 _3D Medicines Inc., Shanghai, China_.

Background: Microsatellite instability (MSI) has become a critical predictive and prognostic biomarker in multiple cancers. The mainstream MSI-detecting techniques, immunohistochemistry (IHC) and polymerase chain reaction (PCR), may have trouble profiling an expanded panel if tissue samples are not available in adequate quantity, depriving patients of treatment options, especially for those with advanced disease. Next generation sequencing, in contrast, allows for massively parallel examination of numerous targets in one single test. In this work, we developed a novel NGS-based assay to detect MSI in colorectal and gastric cancer (CRC/GC) and clinically validated its performance in comparison with IHC and PCR.

Methods: Surgical or biopsy specimens collected from 104 CRC and 15 GC patients were examined for MSI status using IHC, PCR and NGS employing a novel algorithm. The concordance rates between the three techniques were assessed using z-test.

Results: Of the 119 patients enrolled, 53.8% had metastases. IHC tests identified deficient MMR protein expression in 40 (33.6%) cases (dMMR), seven of which were microsatellite stable (MSS) using both PCR and NGS. A discrepancy was also observed for one MMR-proficient (pMMR) case where NGS and IHC consistently produced negative results (pMMR/MSS), while PCR reported a MSI-high (MSI-H) status. The NGS method demonstrated 97.1% (33/34) sensitivity and 100% (85/85) specificity compared to PCR,and 82.5% (33/40) sensitivity and 100% (79/79) specificity compared to IHC. The concordance rate was 99.1% between NGS and PCR, and 94.1% between NGS and IHC. Of the 33 dMMR/MSI-H patients, 30.3% harbored germline defects, suggestive of Lynch Syndrome. In addition, in line with previous reports, the dMMR/MSI-H subset showed a significantly higher median tumor mutational burden (TMB) than the pMMR/MSS patients (P=0.0039).

Conclusion: Our NGS-based approach is reliable and robust for MSI status determination in CRC/GC. 

### Tumor Markers to Assess the Biology and Clinical Course of Cancer 1

#3148

Identification and establishment of an 18-gene redox signature for recurrence prediction in colorectal cancer.

Priyanka Sharma,1 Jasjit K. Banwait,1 Luis Bujanda,2 Ajay Goel1. 1 _Baylor Scott & White Research Institute and Charles A. Sammons Cancer Center, Dallas, TX; _2 _Gastroenterology Department, Instituto Biodonostia, Universidad del País Vasco (UPV/EHU), Centro de Investigación Biomédica en Red de Enfermedades Hepaticas y Digestivas (CIBERehd), San Sebastián, Spain, San Sebastián, Spain_.

Purpose: Colorectal cancer (CRC) is the third most common cancers worldwide. The immediate clinical challenge is the identification of high-risk CRC patients, particularly stage II and III, who are genuine candidates for adjuvant chemotherapy, while sparing the low-risk patients from the toxicity and expense of such treatment modalities. According to ASCO guidelines, the high-risk stage II CRC patients are currently defined by specific clinicopathological risk factors. However, the prognosis of patients, even with similar clinicopathological features varies significantly; highlighting their inadequacy in identifying true high-risk CRC patients. Accumulating evidence shows that redox adaptation provides survival benefit to cancer cells and enhances resistance to conventional chemotherapy. Therefore, in this study we performed a comprehensive evaluation of the clinical potential of redox-related genes as recurrence prediction biomarkers in CRC patients.

Experimental Design: We undertook a rigorous biomarker discovery effort (GSE33113, n=90), followed by validation of a redox gene signature for recurrence prediction in two datasets (GSE39582, n=461 and GSE14333, n=185) of stage II and III CRC patients. The 18-gene signature discovered from the genome wide discovery effort was subsequently validated in an independent in-house clinical cohort (n=128) by qRT-PCR assays. Univariate and multivariate analyses were performed using CoxPH regression by including various clinicopathological features.

Results: A systematic analysis of 312 redox-related genes in three public datasets of 736 CRC patients led to the identification of an 18-gene panel for predicting recurrence free survival (RFS) in stage II and III CRC patients. This gene signature could successfully discriminate high vs. low risk patients in the discovery cohort (AUC=0.88, HR=17.0, p<0.0001), as well as two validation cohorts (GSE39582: AUC=0.70; HR=3.1, p<0.0001; GSE14333: AUC=0.63; HR=3.8, p<0.0001). Subsequent validation in an independent CRC clinical cohort (n=128) confirmed that this gene signature was significantly associated with RFS (AUC=0.81; HR=9.3, CI=3.8 to 22.5, p<0.0001). Univariate analysis identified tumor stage, tumor infiltration, and TNM stage as significant predictors of RFS, and multivariate analysis further confirmed that our gene signature was a significant predictor of RFS in stage II/III CRC patients (p<0.0001). A risk-assessment model by combining the 18-gene signature and the three clinical factors exhibited an even superior prediction for RFS (AUC = 0.877; HR=10.34, CI=4.2 to 25.6, p<0.0001).

Conclusions: We identified and established a novel 18-gene redox signature for the prediction of tumor recurrence in high-risk stage II/III CRC patients, which has the potential for translation into the clinic for improved risk-assessment and stratification in CRC patients.

#3149

Prognostic impact of PD-L1 expression in correlation with neutrophil-to-lymphocyte ratio in squamous cell carcinoma of the lung.

Kazue Yoneda, Taiji Kuwata, Masataka Mori, Masatoshi Kanayama, Ayako Hirai, Yuko Tashima, Naoko Imanishi, Koji Kuroda, Yoshinobu Ichiki, Fumihiro Tanaka. _Univ. of Occupational And Environmental Health, Kitakyushu, Japan_.

Aim: To investigate the prognostic impact of programmed death-ligand 1 (PD-L1) expression on tumor cells in correlation with neutrophil-to-lymphocyte ratio (NLR) in early-stage squamous cell carcinoma of the lung, as PD-L1 is a potent regulator of cancer immunity and NLR is a potential surrogate of immune status.

Patients and methods: Eighty-three patients with completely resected pathologic stage I lung squamous cell carcinoma were retrospectively reviewed. Tumoral PD-L1 expression was evaluated with immunohistochemistry, and its prognostic impact was analyzed in correlation with NLR.

Results: Forty-three patients (51.8%) had tumor with positive PD-L1 expression (percentage of tumor cells expressing PD-L1, ≥1%). There was no significant correlation between PD-L1 expression and NLR. PD-L1-positivity failed to provide a significant prognostic impact (overall survival [OS] rate at 5 years, 70.1% in PD-L1-negative patients versus 53.0% in PD-L1-positive patients; P=0.117). Among NLR-low (<2.2) patients, however, PD-L1-positivity was significantly correlated with a poor prognosis (OS rate at 5 years, 86.0% in PD-L1-negative patients versus 46.1% in PD-L1-positive patients; P=0.020). In contrast, among NLR-high (≥2.2) patients, PD-L1-positivity provided no prognostic impact (P=0.680). When NLR status and tumoral PD-L1 status were combined, "NLR-low and tumoral PD-L1-negative" was a significant and independent factor to predict a favorable recurrence-free survival (hazard ratio, 0.237 [95% confidence interval, 0.083 to 0.674]; P=0.007) and OS (hazard ratio, 0.260 [95% confidence interval, 0.091 to 0.745]; P=0.012).

Conclusions: The prognostic impact of PD-L1 expression on tumor cells was distinct according to NLR. "NLR-low and tumoral PD-L1-negative" patients showed a favorable prognosis.

#3150

Impact of interleukin-1 alpha and EGFR expression on recurrence and survival outcomes in head and neck squamous cell carcinomas.

Andrean L. Simons,1 Anand Rajan,1 Katherine Gibson-Corley,1 Georgina Ofori-Amanfo,1 Patrick Ten Eyck,1 Madelyn Espinosa-Cotton,1 Sandra Schmitz,2 Joseph Coppock,3 Steven Sperry1. 1 _University of Iowa, Iowa City, IA;_ 2 _St Luc University Hospital, Brussels, Belgium;_ 3 _University of Virginia, Charlottesville, VA_.

Interleukin-1 alpha (IL-1α) is a pleiotropic cytokine involved in inflammation and immune response and is upregulated in many solid tumors including head and neck squamous cell carcinomas (HNSCCs). Although IL-1α expression is generally associated with poor prognosis, the implications of the subcellular localization of IL-1α expression in patient outcomes are poorly understood. This study is aimed at investigating the clinical relevance of immunohistochemical IL-1α expression in HNSCCs. Tissue microarrays (TMAs) containing human papillomavirus (HPV)-positive and HPV-negative HNSCCs were analyzed for IL-1α and epidermal growth factor receptor (EGFR) expression by immunohistochemistry. Nuclear and cytoplasmic IL-1α and EGFR expression scores were correlated with clinicopathological parameters and patient outcomes. IL-1α expression was observed in the nuclear and/or cytoplasm compartments in 98% of evaluable tumors and 78% of tumors expressed IL-1α in both compartments. A higher percentage of tumors with combined high nuclear and moderate cytoplasmic IL-1α expression were observed in HPV-negative tumors compared to HPV-positive tumors. In HPV-negative tumors a combined EGFR-negative and high nuclear IL-1α expression profile was associated with a low risk of tumor recurrence and favorable overall survival compared to all other EGFR/IL-1α expression profiles. Lastly, a higher frequency of tumors with combined high nuclear and moderate cytoplasmic IL-1α expression was observed in cetuximab monotherapy responders compared to non-responders. Altogether, IL-1α in combination with EGFR expression may be a strong indicator of risk of recurrence in HPV-negative HNSCCs and warrants further study as a tissue biomarker for predicting HNSCC patient outcomes.

#3151

Quantitative measurement of Siglec-15 expression in non-small cell lung cancer and its association with PD-L1, B7-H4 and tumor infiltrating lymphocytes.

Maria Toki,1 Jon Zugazagoitia,1 Mehmet Altan,2 Linda Liu,3 Nikita Mani,4 Yuting Liu,1 Konstantinos Syrigos,5 Lieping Chen,6 Solomon Langermann,3 Roy Herbst,7 David Rimm1. 1 _Department of Pathology, Yale University School of Medicine, New Haven, CT;_ 2 _Department of Thoracic/Head &Neck Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX; _3 _NextCure, Inc., Beltsville, MD;_ 4 _NorthWestern University, The Feinberg School of Medicine, Chicago, IL;_ 5 _Department of Medicine, University of Athens, School of Medicine, Sotiria General Hospital, Athens, Greece;_ 6 _Department of Immunobiology, Yale University School of Medicine, New Haven, CT;_ 7 _Department of Medicine, Yale University School of Medicine, New Haven, CT_.

Introduction: Siglecs are sialic acid-binding transmembrane receptors that regulate the functions of innate and adaptive immune system through the recognition of glycan ligands. Siglec-15 has been recently described as an immune suppressive molecule that shares molecular structure with PD-L1, but its role in NSCLC is still unknown. In this study, we determined Siglec-15 expression in three retrospective NSCLC cohorts and evaluated its association with mutation status, major clinicopathologic characteristics and survival.

Experimental procedures: We used multiplexed automated quantitative immunofluorescence (QIF) to develop a validated assay for Siglec-15 measurement and used it to assess Siglec-15 expression and its association to major clinicopathologic variables and survival in three NSCLC cohorts of over 600 patients. Additionally, we investigated the correlation of Siglec-15 expression with tumor-infiltrating lymphocytes, PD-L1, B7-H3 and B7-H4 tumor expression.

Results: In our study, Siglec-15 tumor positivity was found in 12.5%, 13.7% and 22.8% of NSCLC cohort patients. Siglec-15 expression was higher in EGFR mutant and EGFR/KRAS wild type tumors compared to KRAS mutants but was not associated with any other major clinicopathological characteristics. Siglec-15 followed a mutually exclusive pattern of expression with PD-L1, B7-H3 and B7-H4 (co-expression in 3.1%, 13.2% and 7.7% of the patients respectively), while high levels were not correlated with lymphocyte infiltration or T-cell activation, suggesting a different upregulation mechanism not mediated by IFN-gamma.

Conclusions: Siglec-15 is a protein expressed with high frequency in NSCLC. Co-expression of Siglec-15 with PD-L1, B7-H3 and B7-H4 is relatively rare, suggesting that Siglec-15 has distinct and non-redundant features compared to other B7 family members.

#3152

A tumor suppressor-regulated cell cycle derived gene signature is prognostic of recurrence risk in prostate cancer.

Joshua M. Corbin,1 Constantin Georgescu,2 Sandra Thibivilliers,1 Zachary Webb,1 Yan Zhao,1 Jan Koster,3 Kar-Ming Fung,1 Adam Asch,1 Jonathan Wren,2 Maria Ruiz-Echevarria1. 1 _Oklahoma University Health Sciences Center, Oklahoma City, OK;_ 2 _Oklahoma Medical Research Foundation, Oklahoma City, OK;_ 3 _University of Amsterdam, Amsterdam, Netherlands_.

Prostate cancer (PCa) is a complex heterogeneous disease, with the majority of cases remaining indolent and 10% of cases progressing to lethality. While some of this clinical heterogeneity can be explained by traditional clinicopathological factors, molecular profiling studies signal an extraordinary genomic variability, which may lead to diverse clinical outcomes. The identification of molecular subclasses of PCa has the potential to guide the prediction of clinical outcomes, the discovery and design of innovative prognostic biomarkers, and the development of novel therapeutics. To this end, we investigated genes associated with TMEFF2, an androgen-regulated tumor suppressor gene with exceptionally heterogeneous expression in PCa. Low levels of TMEFF2 mRNA significantly (p<0.0001) correlate with reduced disease-free survival (DFS) in patients from the Memorial Sloan Kettering Cancer Center (MSKCC) dataset. Using RNA interference, we identified a panel of 11 TMEFF2 regulated cell cycle related genes (TMCC11), with strong prognostic value. TMCC11 expression stratified radical prostatectomy (RP) patients on the risk of recurrence, served as an independent indicator of poor prognosis, and improved the prognostic value of standard clinicopathological markers in four geographically different patient cohorts (n= 834 samples). The prognostic ability of TMCC11 panel exceeded previously published oncogenic gene signatures (p=0.00017). This study provides evidence that the TMCC11 gene signature is a robust prognostic marker for PCa, reveals the value of using highly heterogeneously expressed genes, like TMEFF2, as guides to discover prognostic indicators, and suggests the possibility that low TMEFF2 expression marks a distinct subclass of PCa.

#3153

Prostatic acid phosphatase (PAP) predictive effect for prostate cancer in a population-based study: The renewal of PAP.

Fubo Wang, Huan Xu, Yinghao Sun. _Changhai Hospital, Shanghai, China_.

Objective: Use prostatic specific acid phosphatase (PAP) to characterize the disease progression and median survivals of patients with prostate cancer (PCa) based on the analysis in a population-based study from Surveillance, Epidemiology, and End Results (SEER) database.

Materials and Methods: PCa patients with completed PAP results were selected from the SEER database of the National Cancer Institute. Mann-Whitney Sum test was utilized to compare the statistical significance for measurement data and ranked data, which were stratified by age, race, TNM Classification of Malignant Tumors (TNM), pathological grades, number of tumors, PAP and survival duration. Logistic analysis was performed to identify predictors of the presence of invasion and metastases. Cox regression was utilized to analyze the factors associated with all-cause mortality and prostate-cancer specific mortality. Moreover, survival curve was also present.

Results: In total, there are 5184 PAP+ patients and 3161 PAP- patients involved in this study. The Mann-Whitney Sum test showed that slightly greater tumor size(p=0.03), elevated lymphatic (p=0.005) and distant (p<0.001) metastases rate, higher pathologic grade(p<0.001), localized tumor number (p<0.001), shortened survival months (p<0.001) were observed in the PAP+ group compared with the PAP- group. In the Multivariable Logistic Regression, invasion and metastases Hazard Ratio (HR) were elevated significantly (p<0.001) in the PAP+ individuals. In the survival analysis, PAP- patients experience the prolonged median survival. In the post-surgical patients, the survival months were still longer in PAP+ patients compared with the negative ones (p<0.001), though surgery prolonged the survival months of both groups. Survival months stratified by localized, invasion and metastases situations were analyzed. In the three stratified sub-groups, survival duration is significantly decreased in the PAP+ individuals in the localized PCa group (P<0.001**) and the metastases group (P=0.013).

Conclusions: The findings of this study provide population-based estimates of the PCa progress and prognosis for patients with different PAP results, which may suggest a renewed period for the PAP.

#3154

Tumor expression of Activin A is associated with clinical outcomes in patients with colorectal cancer.

Nobuya Daitoku, Yuji Miyamoto, Yuki Sakamoto, Kojiro Etoh, Yukiharu Hiyoshi, Yohei Nagai, Masaaki Iwatsuki, Takatsugu Ishimoto, Yoshifumi Baba, Shiro Iwagami, Naoya Yoshida, Hideo Baba. _Department of Gastroenterological Surgery, Graduate School of Medical Sciences, Kumamoto university, Kumamoto, Japan_.

Background:

Activin A is a member of the transforming growth factor β (TGF-β) superfamily. Activin A shows an extensive variety of biological activities, including mesoderm induction, bone remodeling, muscle atrophy. Activin A is also related to Smad 2/3 mediated cancer development and metastasis. The overexpression of Activin A has been reported in several types of cancers and high expression of Activin A leads to poor prognosis. We examined the relationship between tumor expression of Activin A and clinical outcomes in patients with colorectal cancer.

Methods:

AACR Annual Meeting 2019

We analyzed the association between tumor expression of Activin A and clinicopathological features and patient's prognosis in 157 primary colorectal cancers, which were curatively resected between 2008 and 2012. The expression level of Activin A was measured in tumor tissue by quantitative RT-PCR. Patients were categorized into the high or low groups based on median expression value of Activin A and Kaplan-Meier survival analyses were performed to display difference in overall survival (OS) and relapse-free survival (RFS) between two groups. The between-group differences were assessed by the log-rank test and stepwise multivariate Cox regression analysis was used to calculate hazard ratios (HRs) and 95% confidence intervals (CIs) for OS.

Result:

RT-PCR analysis showed that expression of Activin A was significantly higher in tumor areas compared with that in normal (p<0.001). Activin A was highly expressed in the rectal cancer (p=0.015). Other clinicopathological factors were not significantly correlated with Activin A expression. Patients with Activin A high-expressed colorectal cancers showed a significantly shorter OS and RFS than those with activin A low-expressed tumor (5-year OS: Low 79.2% vs High 63.8%, p=0.037). High expression of Activin A is an independent predictor of poor prognosis in patients with colorectal cancers after curative surgery (Low vs High, HR=5.558, 95%CIs=2.229-15.17,

AACR Annual Meeting 2019

p=0.003).

Conclusion:

We concluded that high Activin A expression in tumor is associated with poor prognosis in colorectal patients.

#3155

Multiple biomarker quantification as absolute and continuous variables in 1059 FFPE breast cancer specimens using QDB method.

Jiandi Zhang,1 Fangrong Tang,1 Guohua Yu,2 Yunyun Zhang,1 Jiahong Lv,1 Wenfeng Zhang1. 1 _Quanticision Diagnostics, Inc, Durham, NC;_ 2 _Yantai Yuhuangding Hospital, Affliated hospital of Qingdao University, Yantai, China_.

Purpose: Although immunohistochemistry (IHC) based tissue biomarker assessment has become an integral part of solid tumor diagnosis and prognosis, the inherent problems with IHC, including its subjectivity and inconsistency, significantly limit its usage to its full potentials in clinical practice. The results from IHC analysis are also presented as discrete variables, unable to reflect the broad range distribution of biomarkers at protein level. Absolute quantification of tissue biomarkers for daily practice is needed to provide more accurate and reliable assessment of biomarkers at protein level.

Methods: Quantitative Dot Blot (QDB) method was used to measure several biomarkers including ER, PR, Ki67, Her2, p53, PCNA in 1059 FFPE breast cancer tissues. Total tissue lysates were extracted from 2X15μm FFPE slices for QDB measurement using antibodies well validated for IHC analysis (IHC antibodies), including 4B5 and EP3 for Her2, MIB and UMAB107 for Ki67, SP1 for ER, 1E2 for PR, DO-7 for p53, and PC10 for PCNA. Purified recombinant proteins were used as protein standard to achieve absolute quantification of these biomarkers. The results were validated indirectly with provided IHC/FISH results, and the correlation analysis were performed with other clinicopathological parameters using Spearman rank (rho) and/or Pearson (r) correlation analysis.

Results: QDB method was able to measure these biomarkers objectively and consistently with intra and inter-CV below 15% from three independent experiments, each in triplicate. Both Her2 and Ki67 levels were measured with two independent IHC antibodies with highly consistent results (r>0.96). When converted Her2 levels into dichotomous variables using ROC analysis and provided IHC score, QDB achieved concordance with FISH at 94.2%. When analyzed as absolute and continuous variables, the p53, Ki67 and PCNA show strong correlation among themselves, with Ki67 and PCNA at rho=0.67, p=0.0000. Age was found to be positively associated with ER (p<0.0001), negatively associated with PR (p<0.01). Histological grade was best correlated with Ki67 (p<0.0001), followed by PCNA (p<0.0001), p53 (p<0.0001) and Her2 (p< 0.005) and negatively associated with ER levels (p<0.0001). Both Ki67 and PCNA was found to be positively associated with PR (p<0.001), but not ER.

Conclusions: This is the first large scale study with so many biomarkers measured as absolute and continuous variables simultaneously in FFPE specimens. QDB method was proved to be objective, consistent and in high throughput format to meet the daily need of clinical diagnosis and prognosis. It can be easily standardized to circumvent the limitations associated with current methods, and open path for database-based diagnosis and prognosis in the near future. The adoption of this method may have direct impact on precision medicine, especially on the field of targeted therapies.

#3156

Pathway Specific Functional Biomarkers for the Early Detection of Liver Cancer.

Kirti Shetty,1 Chu-Xia Deng,2 Wilma S. Jogunoori,2 Richard Amdur,2 Linda S. Resar,3 Patricia Latham,2 Raja Mazumder,2 Anelia Horvath,2 Bao-Ngoc Nguyen,2 Shulin Li,4 Xifeng Wu,5 Herbert Yu,6 Linda L. Wong,6 Jon White,2 Sylvia Silver,2 Asif Rashid,5 Vikas Kundra,5 Xin Wei Wang,7 Lopa Mishra2. 1 _University of Maryland Medical Center, Baltimore, MD;_ 2 _The George Washington University, Washington, DC;_ 3 _The Johns Hopkins University School of Medicine, Baltimore, MD;_ 4 _The University of Texas MD Anderson Cancer Center, Washington, DC;_ 5 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 6 _University of Hawaii Cancer Center, Honolulu, HI;_ 7 _National Cancer Institute, Bethesda, MD_.

In its early stages, HCC is curable; once advanced, HCC is associated with high mortality. HCC is associated with alterations in key driving pathways, including the Wnt pathway, TGF-β pathway, the p53 pathway, and pathways that regulate Myc activity. Members of the TGF-β superfamily regulate liver inflammation and can have tumor-suppressing or tumor-promoting activities that have been demonstrated to play key roles in liver and GI cancers through mouse models and human genomics. Yet, specific populations that are targetable with altered TGF-β remain unclear. Based upon our functional preclinical models and pilot studies in human liver disease samples, our hypothesis that aberrant expression of TGF-β pathways can lead to early detection of HCC and risk stratification. Methods and Results: Through preclinical studies that integrate analysis of human genomic data from The Cancer Genome Atlas (TCGA), mouse models, and human tissue/cell line studies, we sought to determine whether specific high-risk populations may be identified through a functional TGF-β pathway related biomarkers alterations. We observe the following: • Altered expression of TGF-β-Smad signaling genes is common in HCC (40%) • Somatic mutations in at least one gene encoding a member of the TGF-β-Smad pathway are common in HCC (38%) with the SMAD3 adaptor, β2SP (encoded by SPTBN1) having the highest frequency of mutation (6%), regardless of etiology, HBV or HCV infection or non-alcoholic steatohepatitis (NASH) • Disruption of the TGF-β-Smad pathway is associated with dysregulation of potentially targetable oncogenes that include MDM2, Telomerase, IGF2 and others. • TGF-β-Smad pathway activity clusters HCC patients into 4 groups. • HCC patients with a signature indicative of "inactivated" TGF-β-Smad signaling had shorter survival times than HCC patients with a signature indicative of "activated" TGF-β-Smad signaling reflecting a tumor suppressor role of TGF-β signaling in HCC. • TGF-β-Smad pathway activity correlates with genes in the DNA damage response and sirtuins. • Analysis of liver samples from normal subjects, patients with alcohol-induced cirrhosis, and alcohol-associated HCC showed that in the cirrhotic tissue TGF-β-Smad3 signaling and FANCD2 were highest and that in the HCC tissue these were lowest, suggesting a loss of this tumor-suppressing pathway in HCC • Mice deficient in either sirtuin activity (Sirt6-/-) or compromised TGF-β signaling (Sptbn+/-/Smad3+/-) are susceptible to liver injury, steatosis and spontaneously develop HCC • TGF-β-Smad pathway activity correlates with genes associated with hepatic fibrosis, immune cells, and the tumor microenvironment. Conclusions: Our data indicate that TGF-β-Smad members, particularly Smad3 and β2SP, and markers of DNA repair processes, particularly FANCD2 are potentially useful biomarkers of the loss from tumor-suppression by TGF-β signaling, which may be an early indicator of HCC.

#3157

Cyclin E1 overexpression identifies patients with greater benefit from bevacizumab in platinum sensitive recurrent ovarian cancer.

Adriana Regina G. Ribeiro, Marcella M. Salvadori, Louise de Brot, Graziele Bovolim, Henrique Mantoan, Felipe Ilelis, Mariana R. Alves, Nayra Soares Amaral, Solange M. Sanches, Joyce Lisboa, Elizabeth S. dos Santos, Ronaldo Pereira, Fabricio S. Castro, Joao Paulo S. Lima, Andrea P. Guimaraes, Glauco Baiocchi, Alexandre Andre B. Da Costa. _AC Camargo Cancer Ctr., Sao Paulo, Brazil_.

Introduction: Bevacizumab is an antiangiogenic agent approved to treat ovarian cancer in combination to chemotherapy. The relative benefit of bevacizumab in ovarian cancer patients seems to be greater the more the disease becomes platinum resistant. Cyclin E1 overexpression is a marker of platinum resistance. In this study we aimed to evaluate the benefit of bevacizumab in platinum sensitive recurrent ovarian cancer and to test if the benefit changes according to platinum-free interval (PFI) and cyclin E1 expression.

Methods: We retrospectively evaluated data from patients with platinum sensitive recurrent ovarian cancer treated with CT plus bevacizumab (Bev group) and CT alone (CT group) at a tertiary cancer center in Brazil from 2005 to 2017. The two groups were paired according to histology, platinum free interval and number of previous treatment lines. Cyclin E1 expression was evaluated by immunohistochemistry in tissue microarray. Progression-free survival (PFS) was compared between the groups with log rank test and cox regression.

Results: 124 patients were included, 62 in each group. Bev group and CT group were well balanced regarding histology (high grade serous carcinoma 94% in Bev group, 93% in CT group), PFI (PFI > 12 months in 36.2% in Bev group, 39.3% in CT group) and number of previous treatment lines (one previous chemotherapy in 55.7% in Bev group, 60.0% in CT group). Median age and median PFI were 56.2 years old and 59.2 years old, and 9.7 months and 10.5 months, in the Bev and CT groups, respectively. All patients were treated with platinum doublets with paclitaxel, gemcitabine or liposomal doxorubicin except for one patient treated with cisplatin plus bevacizumab and one patient treated with liposomal doxorubicin. Median PFS (mPFS) was 19.5 months for the Bev group vs. 16.0 months in the CT group (p = 0.150). Patients with a PFI > 12 months showed a mPFS of 20.0 months vs. 15.5 for patients with a PFI < 12 months (p=0.029). Patients with cyclin E1 overexpression showed a mPFS of 15.8 months vs. 19.7 months for those without cyclin E1 overexpression (p=0.05). Benefit of bevacizumab was present only in the subgroup of patients with PFI < 12 months (mPFS 18.6 versus 10.4 months, p=0.002) and in the subgroup of patients with cyclin E1 overexpression (mPFS 16.3 versus 7.0 months, p=0.010).

Conclusions:Markers of resistance to chemotherapy such as cyclin E1 overexpression and PFI identify patients with the greatest benefit of bevacizumab. This data could help to discern between maintenance treatment options in the era of PARP inhibitors and anti-angiogenics.

#3158

Prediction of overall survival in urothelial cancer patients using tumor sizes and baseline risk factors: longitudinal modeling approach for durvalumab and durvalumab + tremelimumab.

Mei Tang,1 Yu Jiang,1 Han Si,1 Yanan Zheng,2 Chen Gao,1 Guozhi Gao,1 Natasha Angra,3 Shaad Abdullah,1 Brandon Higgs,1 Lorin Roskos,1 Rajesh Narwal1. 1 _MedImmune, Gaithersburg, MD;_ 2 _MedImmune, South San Francisco, CA;_ 3 _Astrazeneca, Gaithersburg, MD_.

Background: Durvalumab [D] is a human mAb that binds to PD-L1 and blocks its interaction with PD-1 and CD80. Tremelimumab [T] blocks the inhibitory effects of CTLA-4, and therefore enhances T-cell activation. The objectives of this analysis were to develop a model linking overall survival (OS) to baseline risk factors and changes in tumor size during treatment to identify key factors impacting response to D or D +T.

Methods: The analysis dataset included UC patients from two clinical trials: Study 1108 (D 10 mg/kg Q2W; n=201) and Study 10 (D 20 mg/kg Q4W + T 1 mg/kg Q4W for 4 doses, followed by D 20 mg/kg Q4W alone; n=168). Longitudinal tumor size data were analyzed using a nonlinear mixed effect model with key parameters describing tumor growth, tumor killing, and delay in immune response. Subsequently, a parametric survival model was developed to link baseline risk factors and predicted percent change in tumor size at week 8 to OS.

Results: Tumor kinetic model adequately described the longitudinal tumor size data from UC patients. Baseline tumor size (p<0.01) and PD-L1 status (p<0.01) were identified as significant covariates for tumor killing rate. The most influential factor associated with faster tumor growth was liver metastasis (p<0.01), while higher hemoglobin levels (p<0.01) were associated with decreased tumor growth rate. Based on parametric survival modeling, liver metastasis (~34% decrease in OS, p<0.0001), albumin (~ 1-fold increase in OS per 1g/dL increase, p<0.0001), and percent change in tumor size at week 8 (~52% increase in OS with 30% tumor shrinkage at week 8, p<0.0001) were found to be significant and clinically relevant predictors of OS.

Conclusions: The parametric survival model coupled with tumor kinetic model adequately described clinical outcomes in UC patients treated with D or D+T and enabled identification of key factors potentially impacting response to immune therapy in UC. This approach can be a useful tool for guiding patient selection/enrichment strategies and optimizing trial designs for immuno-oncology (IO) therapies. Further validation and prospective evaluation of this model may be conducted in other IO trials.

#3159

Do molecular markers differentiate between sessile serrated adenoma/polyps and hyperplastic polyps.

Hassan Ashktorab,1 Saman Azam,1 Taraneh Tarjoman,1 Priyanka Kanth,2 Edward Lee,1 Mehdi Nouraie,3 Nazli Atefi,1 Babak Shokrani,1 Adeyinka Laiyemo,1 Ajay Goel,4 Mark W. Hazel,5 Ruoxin Yao,5 Angela Snow,5 Deborah Neklason,5 Don Delker,5 Hassan Brim1. 1 _Howard University Hospital, Washington, DC;_ 2 _Huntsman Cancer Institute, Salt Lake City, UT;_ 3 _University of Pittsburgh, Pittsburgh, PA;_ 4 _Baylor Scott & White Research Institute and Charles A. Sammons Cancer Center, Dallas, TX; _5 _University of Utah, Salt Lake City, UT_.

Background: A significant proportion of colorectal cancers, as much as up to 30%, develop through the alternate serrated pathway. The precursor lesions in this pathway comprise of sessile serrated adenomas or polyps (SSA/SSPs) and hyperplastic polyps (HPs), both with a varying risk-profile associated with them for progression into colorectal cancer. Although both types of polyps can be identified histologically, the clinical challenge for gastroenterologists and pathologists remain for risk-prediction as to which of these polyps have a higher likelihood for subsequent development and progression into colorectal cancer. These data highlight the imperative need for the development of more specific and objective markers that can adequately differentiate between the two types of colonic polyps.

Aim: The aim of our study was to determine a variety of molecular biomarkers that may distinguish SSA/Ps and HPs, and to establish biomarker profiles that associate with low vs. high-risk SSA/Ps.

Methods: We conducted a retrospective study of all colonoscopies (n=12,085) performed at the Howard University Hospital between January 2010 to December 2015; of which 83% were conducted in patients of African American (AA) descent (n=10,027). Among AAs, pathology reports confirmed 4,070 patients with polyps, including 252 with SSA/Ps. Gene expression and mutation frequency profiles were analyzed in a total of 47 patients which included 62 specimens (29 SSPs, 26 HP, 3 tubular adenomas (TA) and 4 normal colonic tissues). From a panel of 51 candidate transcripts, we validated 4 RNA markers (MUC6, FSCN1, SEMG1, and TRNP1) using qRT-PCR. MSI and BRAF mutations were also analyzed. CIMP analysis was performed for the aberrant methylation of CACNA1G, IGF2, NEUROG1, RUNX, SOCS and MLH1. The frequency of gender, age groups, anatomic location, clinical/pathological symptoms and reason for colonoscopy in SSA/P patients was analyzed. The median age range for SSA/P diagnosis was between 50 to 64 years.

Results: MUC6, SEMG1, TRNP1, and FSCN1 expression was significantly higher in SSA/Ps vs. HPs (P < 0.05); with corresponding fold differences of 37.2 10.7, 5.8 and 2.5, respectively. BRAF mutations were found in 55.6% of SSA/Ps as opposed to 12.0% of HPs (P < 0.05). The frequency of CIMP was higher in SSA/Ps and correlated with BRAF mutation, while the degree of MSI was more prevalent in HP (P > 0.05). SSA/P lesions were distal (67%).

Conclusion: Our results show that MUC6 and SEMG1 expression and BRAF mutation have the strongest correlation with SSA/Ps in comparison to HPs. In addition, SSA/Ps were predominantly distal in location. These are novel and distinguishing features compared to the published literature in non-AA populations and may help explain why MSI and CIMP, usually linked to proximal lesions, are not optimal molecular biomarkers in AA patients with such serrated lesions.

#3160

Stromal markers of activated tumor associated fibroblasts predict poor survival and are associated with necrosis in non-small cell lung cancer.

Jordi Alcaraz,1 Josep-Lluís Carrasco,1 Laura Millares,2 Iuliana-Cristiana Benchea,1 Francisco-Javier Fernández,1 Anabel Martínez,3 María José Pajares,4 Julio Sánchez de Cos,5 Ramon Rami-Porta,6 Luis Seijo,7 Josep Ramírez,8 Noemí Reguart,8 Esther Barreiro,3 Eduard Monsó2. 1 _Univ. of Barcelona, Barcelona, Spain;_ 2 _Hospital Parc Taulí de Sabadell, Sabadell, Spain;_ 3 _IMIM-Hospital del Mar-CIBERES, Barcelona, Spain;_ 4 _Center for Applied Medical Research, Pamplona, Spain;_ 5 _Hospital San Pedro de Alcántara, Cáceres, Spain;_ 6 _Hospital Mutua de Terrassa-CIBERES, Barcelona, Spain;_ 7 _Fundación Jímenez Díaz-CIBERES, Madrid, Spain;_ 8 _Hospital Clínic de Barcelona, Barcelona, Spain_.

Tumor associated fibroblasts (TAFs) are essential contributors of the progression of non-small cell lung cancer (NSCLC). Most lung TAFs exhibit an activated phenotype characterized by the expression of α-SMA and fibrillar collagens. However, the prognostic value of these activation markers in NSCLC remains unclear. To address this question we conducted a retrospective multicentric study of the prognostic value of the standard markers of activated fibroblasts. For this purpose, we conducted a quantitative image analysis of α-SMA immunostaining and picrosirius red staining of fibrillar collagens imaged by bright-field and polarized microscopy, respectively, using tissue microarrays with samples from 220 surgical patients, which elicited a percentage of positive staining area for each marker and patient. Kaplan-Meier curves showed that all TAF activation markers were significantly associated with poor survival, and their prognostic value was independent of TNM staging as revealed by multivariate analysis, which elicited an adjusted increased risk of death after 3 years of 129% and 94% for fibrillar collagens imaged with bright-field (p = 0.004) and polarized light (p = 0.003), respectively, and of 89% for α-SMA (p = 0.009). We also found a significant association between all TAF activation markers and tumor necrosis, which is often indicative of hypoxia, supporting a pathologic link between tumor desmoplasia and necrosis/hypoxia. Our findings identify patients with large histologic coverage of fibrillar collagens and α-SMA+ TAFs to be at higher risk of recurrence and death, supporting that they could be considered for adjuvant therapy. Moreover it supports that antifibrotic drugs aiming to target tumor fibrosis may be an effective therapeutic approach to improve survival in NSCLC.

#3161

Novel evidence for Trefoil Factor 3 as an oncogenic mediator of disease progression, and a potential predictor for neoadjuvant chemotherapy in colorectal cancer.

Ying Long,1 Tsuyoshi Ozawa,2 Hanhua Li,1 Weijie Pan,1 Wenhao Weng1. 1 _Yangpu Hospital, Tongji University School of medicine, Shanghai, China;_ 2 _Teikyo University School of Medicine, Tokyo, Japan_.

Colorectal cancer (CRC) is a major cause of morbidity and mortality throughout the world. The increased incidence rates of CRC places a considerable burden on individuals and society in Asian countries. Although surgical resection remains the standard treatment for CRC patients, accumulating evidences showed advanced patients derived an additional benefit from molecular targeted agents. Therefore, there is a great need to identify novel cellular targets in CRC that can be exploited for therapeutic benefit. Trefoil factor 3 (TFF3), is a member of a family of small secretory proteins characterized by three intrachain disulfide bonds constituting the trefoil motif. TFF3 was initially found as a mediator of mucosal healing and play a critical role in epithelial restitution. Recent studies showed that TFF3 may contribute to tumorigenesis. However, its role in CRC remains unclear. Herein, the protein expression of TFF3 was measured in colorectal adenoma polyps (PLP, n=20), cancer tissues (n=20) and matched adjacent healthy tissues (n=20) by immunohistochemical (IHC) assay. We further evaluated serum and urine level of TFF3 from healthy volunteers (n=40), PLP patients (n=40) and CRC patients (n=40) by using ELISA assay. To investigate the functional or mechanistic role of TFF3 in CRC, we conducted several in vivo and in vitro approaches. The IHC showed that TFF3 was frequently overexpressed in cancer tissues (12/20, P<0.0001), while scarcely detected in PLP (2/20) or healthy tissues (3/20). In addition, serum or urine level of TFF3 was increased in CRC patients, compared to PLP patients or healthy volunteers (P=0.00032). Intriguingly, we found patients with lower serum TFF3 level are more sensitive to neoadjuvant chemotherapy than those with higher TFF3 level (with 3.4 fold increase, P<0.0001). With regard to biological function of TFF3, we found overexpression of TFF3 in CRC cell lines HCT-116 and SW480 significantly enhanced cell viability, invasion, and colony formation capacity. Subsequent mice model validated its oncogenic role in CRC, which TFF3 treatment remarkably enhances xenograft growth. In conjunction with its higher serum level in chemoresistant patients, we found overexpression of TFF3 endows HCT-116 cells with 5'-Fluorouracil resistant phenotype. Mechanistically, we observed overexpression of TFF3 is sufficient to activate Akt and Autophagy pathways in HCT-116 and SW480 cells, confirming its correlation with malignant clinical and biological phenotype. In conclusion, we identified TFF3 as a novel diagnostic marker in CRC and a promising predictor for neoadjuvant chemotherapy. Targeting TFF3 may provide an effective strategy for improving treatment of this malignancy.

#3162

Prognostic value of tumor mutational burden using a 409 gene NGS panel in cancer patients with advanced stage recurrent or treatment refractory disease.

Richard K. Yang, Peng Wang, Fatima Z. Jelloul, Mark J. Routbort, Scott Kopetz, Kenna R. Shaw, Jack J. Lee, Jiexin Zhang, Hui Chen, Keyur P. Patel, Raja Luthra, Russell R. Broaddus. _UT MD Anderson Cancer Center, Houston, TX_.

Tumor Mutational Burden (TMB) is a promising biomarker for prediction of response to immune checkpoint blockade (ICB). It is uncertain whether ICB has prognostic value outside of ICB therapy. The CMS400 next generation sequencing panel (NGS) is a 409 gene, 15,992 amplicon, and 1.745 Mb panel instituted during 2014-2015 and run for 556 cancer patients who had been consented for participation within a prospective molecular pathology biomarker trial (PA14-0099). All patients had advanced or recurrent solid tumor malignancies that were refractory to at least one line of systemic therapy prior to enrollment. Survival time was calculated from time of NGS-tested tissue collection. TMB was calculated by dividing reported mutations (RM) by 1.745Mb, the genetic footprint of the NGS panel. Subtraction of germline single nucleotide polymorphisms was performed for each patient. GraphPad Prism 7.03 software was used to calculate p values and to plot Kaplan-Meier survival curves. One hundred seven patients (19.2%) received ICB. When stratified by reported mutations (RM: 0, 1, 2, 3, 4-5, 6-7, 8-9, 10-18, and >19), a statistically significant decrement of overall survival was seen with increasing TMB in patients not treated with ICB (Table 1, p<0.0001). Also, in patients treated with ICB, significantly increased overall survival was seen on the extreme ends of the TMB spectrum (Table 1, p=0.0249). In contrast, stratification by the top four histologic diagnoses (Colorectal ADCA [n=94] - 34.2 months, breast ductal ADCA [n=44] - 26.6 months, gynecologic high grade serous [n=39] - 45.6 months, and lung ADCA [n=30] - 31.5 months) did not show difference in overall survival (p=0.332). We report here a novel molecular phenomena showing that high TMB in patients with advanced cancers is associated with worse survival. This negative impact of high TMB can be reversed by treatment with ICB. These effects of ICB were seen across a broad spectrum of cancer types.

Median Survival Stratified by Tumor Mutational Burden and ICB Treatment Status

---

|

All Pts | All Pts | ICB Treated | ICB Treated | No ICB | No ICB | |

|

Reported Mutations | # of Pts | Median Survival (Months) | # of Pts | Median Survival (Months) | # of Pts | Median Survival (Months) | p-value (Log-rank) | Hazard Ratio of ICB Therapy | 95% CI of HR of ICB Therapy

0 RM | 71 | 50.7 | 19 | 58.8 | 52 | 48.5 | 0.319 | 0.705 | 0.368 - 1.34

1 RM | 85 | 50.9 | 12 | 62.0 | 73 | 47.4 | 0.339 | 0.684 | 0.345 - 1.36

2 RM | 94 | 33.9 | 19 | 28.0 | 75 | 33.9 | 0.865 | 0.951 | 0.535 - 1.69

3 RM | 88 | 30.5 | 19 | 51.2 | 69 | 28.9 | 0.189 | 0.704 | 0.428 - 1.16

4-5 RM | 93 | 30.8 | 11 | 41.0 | 82 | 30.4 | 0.539 | 0.815 | 0.442 - 1.50

6-7 RM | 49 | 28.4 | 11 | 27.3 | 38 | 28.55 | 0.623 | 0.827 | 0.401 - 1.71

8-18 RM | 56 | 23.95 | 8 | 28.8 | 48 | 23.1 | 0.549 | 1.252 | 0.553 - 2.84

>19 RM | 20 | 41.3 | 8 | 102.3 | 12 | 24.2 | 0.0023 | 0.180 | 0.0599 - 0.543

Total | 556 | 35.8 | 107 | 47.9 | 449 | 33.4 | 0.0049 | 0.707 | 0.567 - 0.882

p-value (Log-rank) | |

<0.0001 | |

0.0249 | |

<0.0001 | |

|

#3163

The role of early response genes (ERG's) as a biomarker of response to Wee1 targeted therapies.

Victoria L. Dunne,1 Niamh McGivern,2 Kienan I. Savage,1 Nuala McCabe,2 Richard Kennedy2. 1 _Queens University Belfast, Belfast, United Kingdom;_ 2 _Almac Diagnostics, Craigavon, United Kingdom_.

Introduction: WEE1 kinase is a key component in maintaining the G2/M cell cycle checkpoint for pre-mitotic DNA repair, and is overexpressed in several cancer types. Novel therapeutics are currently being developed to target WEE1 kinase in cancer, however, to date no predictive biomarkers have been approved to aid patient stratification and clinical trial design. To address this, we employed a siRNA screening to identify tumour suppressor genes (TSGs) whose loss mediates sensitivity to WEE1 inhibition.

Experimental procedures: U2OS cells were reverse transfected with a customised siRNA library containing 3 independent siRNAs targeting 178 tumour suppressor genes and 24 hours later treated with either DMSO control or MK-1775 (Wee1 Kinase Inhibitor). Cell viability was measured using a cell titer-glo luminescent Cell Viability Assay 72 hours post-treatment. Hits were selected based on robust z-score analysis. Those genes with 2 or more targeted siRNAs demonstrating a robust z-score of ±1 median absolute deviation (MAD) were taken forward for validation studies. Sensitive hits were selected on a z-score of <-1 and resistant hits were selected on a z-score of >1. siRNA knockdown of WEE1 was performed in multiple human cancer cell lines and confirmed by western blotting and RT-q-PCR. Basal expression levels of phosphorylated WEE1, total WEE1, FOS and JUNB were assessed by western blotting.

Results: Consistent with previously published findings, the siRNA screen demonstrated that loss of BRCA2 conferred increased sensitivity to WEE1 inhibition (Aarts et al. 2015). The siRNA screen also identified an additional 12 TSGs whose loss mediated sensitivity and 14 TSGs whose loss mediated resistance to WEE1 kinase inhibition. Interestingly, we found that loss of two early response genes, FOS and JUNB conferred resistance to WEE1 inhibition. FOS and JUNB interact to form the AP1 heterodimer, and previous published work has demonstrated the presence of an AP1 binding motif on the WEE1 promoter (Kawasaki et al. 2003). Using publically available gene expression data (TCGA) we have shown a significant correlation between expression of WEE1 with FOS and JUNB in multiple cancer types.

Conclusions: Using a TSG siRNA screen, we have identified that loss of JUNB and FOS confers resistance to the WEE1 inhibitor MK1775. Future studies will investigate the mechanisms by which the loss of these genes affects response to WEE1 inhibition, and will also investigate the utility of these genes as predictive biomarkers for response to WEE1 inhibition in clinical samples, thereby aiding patient stratification.

#3164

Identifying Bcl-2 family protein dependencies in tumors using dimerization specific antibody biomarkers as a method for predicting response to apoptosis inducing therapies.

Bahriye Karakas,1 Sae Rin Jean,1 Max Narovlyansky,1 Sonia Kumar,1 Stephen Gillies,1 Stan Krajewski,2 Bora Lim,3 Michael Cardone1. 1 _Eutropics, Cambridge, MA;_ 2 _Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA;_ 3 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Here, we present a novel method for detecting heterodimer complexes consisting of anti-apoptotic and pro-apoptotic Bcl-2 family proteins as indicators of cancer cell apoptotic priming state. The readout of Bcl-2 family complex-specific biomarkers is seen to identify specific survival dependencies in leukemia and breast cancer cells suggesting utility for guiding cancer treatments.

Methods: Engineered immunogens that recapitulate conformation-specific epitopes induced during binding of the Bcl-xL/Bim protein complex were made. Mice were immunized with selected immunogen and the resulting monoclonal antibodies that exclusively bound to the conformation-induced epitope were identified. One Heterodimer Specific Bcl-xL Bim (HSBXB) was chosen. The selective binding of HSBXB was confirmed using ELISA, FP (fluorescence polarization), IF (immunofluorescence) in Bim or Bcl-xL knockdowns, Bcl-xL knock out cell line, as well as immunohistochemistry (IHC) in formalin-fixed paraffin-embedded (FFPE) patient tissue. Correlation of HSBXB measurements to BH3 profiling readouts from the Bcl-xL restricted Hrk BH3 containing peptide was explored, including shifts in signals after treatment with Bcl-xL-specific BH3 mimetic compounds.

Results: We have demonstrated the dependency of the HSBXB signal on both Bcl-xL and Bim protein levels in in vitro and ex vivo experiments. Multiplexed two-color IHC staining in cell blocks comprised of breast cancer and AML cell lines showed that the measurement of the ratio of heterodimer (Bcl-xL/Bim) to unbound protein (Bcl-xL) signal ([HSBXB]/[Bcl-xL]) aligned with flow-based BH3 profiling readouts. The [HSBXB]/[Bcl-xL] readout was also taken in AML and CLL patient samples using flow cytometry. A strong correlation (p=0.003) was seen between the [HSBXB]/[Bcl-xL] ratio and the Hrk readout of BH3 profiling assay. The [HSBXB]/[Bcl-xL] readout also shifted following treatment of cells with Bcl-xL specific BH3 mimetics. Further, the dynamic range of the [HSBXB]/[Bcl-xL] signal ratio was demonstrated across triple-negative breast cancer FFPE samples. The [HSBXB]/[Bcl-xL] readout potentially provides a functional biomarker for cancer cell sensitivity to a wide range of Bcl-xL-targeted compounds in solid tumors.

Conclusion: This work provides a basis for further studies into the Bcl-xL dependence as a predictive biomarker for Bcl-xL targeted therapies, and/or therapies that are resistant in Bcl-xL dependent cells. IHC based measurement of a heterodimer complex offers easier access in comparison to other assay platforms, thus harbors a potential for use in clinical settings. Additional antibodies targeting Bcl-2 family complexes as predictive biomarkers for Mcl-1 dependence in solid and liquid tumors have been identified.

#3165

Identification of candidate biomarkers for aggressive prostate cancer using targeted proteomics and FFPE tissue samples with outcomes data.

Yuqian Gao,1 Yi-Ting Wang,1 Hui Wang,1 Denise Young,2 Jennifer Cullen,2 Yingjie Song,2 Yongmei Chen,2 Athena Schepmoes,1 Gyorgy Petrovics,2 Thomas Fillmore,1 Tujin Shi,1 Wei-Jun Qian,1 Richard Smith,1 Sudhir Srivastava,3 Jacob Kagan,3 Albert Dobi,2 Inger Rosner,2 Karin Rodland,1 Isabell Sesterhenn,4 Shiv Srivastava,2 Tao Liu1. 1 _Pacific Northwest National Laboratory, Richland, WA;_ 2 _Walter Reed National Military Medical Center, Bethesda, MD;_ 3 _National Cancer Institute, Bethesda, MD;_ 4 _Joint Pathology Center, Silver Spring, MD_.

Introduction: Although many (~40%) of the screen-detected prostate cancers are indolent (Gleason Score <= 6) and pose minimal risk for progression, advanced stage prostate cancer is a lethal disease with 5-year survival rates around 29%. The challenge is to identify biomarkers for early detection of aggressive disease, when the cancer is still organ confined. Such markers would be also used to better select patients with indolent and low risk cancers for active surveillance.

Material and Methods: To identify a panel of protein markers that could predict prostate cancer progression, we developed ultra-sensitive, high-pressure, high-resolution separations coupled with intelligent selection and multiplexing-selected reaction monitoring (PRISM-SRM) assays for 52 protein markers. Candidate protein markers were identified and selected from existing prostate cancer genomics data sets and validated lists of known prostate cancer drivers. The PRISM-SRM assays used heavy isotope-labeled synthetic peptides as internal standards for quantitative proteomics analysis. Study comprised of a prostate cancer patient cohort with organ confined primary tumors (N=338) presenting following post-surgery features: 53 (15.7%) metastatic progression, 124 (36.7%) biochemical recurrence (BCR), and 161 (47.6%) no progression after more than ten years of follow-up after radical prostatectomy. Index tumor region for each case was scraped from representative 10-m sections of formalin-fixed paraffin embedded (FFPE)-whole-mounted prostatectomy specimens and processed for PRISM-SRM analysis.

Results: Overall, PRISM-SRM analysis of the FFPE tissue samples enabled the detection of 42 (80.8%) out of 52 biomarker candidates; in comparison regular LC-SRM without the front-end chromatographic enrichment could detect only 21 (40.4%) of these candidates at the protein level. Kruskal-Wallis testing was used for statistical evaluation of the PRISM-SRM results and comparison of relative protein levels between the "no progression", BCR and "metastatic progression" groups. Several prostate differentiation/androgen receptor signaling related proteins (FOLH1, PSA and NCOA) and tumor progression-related proteins (TGFB1, CCND1 and SPRC) had significantly different expression levels between the three groups, and showed initial promise in predicting progression to invasive cancer, BCR and metastasis.

Conclusion: Ultra-sensitive targeted proteomics can be used to select and verify performance of early prognostic markers based on the analysis of prostatectomy specimens. The top performing markers appeared able to predict progression from organ confined cancer to BCR and metastasis.

#3166

**Evaluation of genetic modulation of** ACVR1 **(aka** ALK2 **) kinase gene as a clinical biomarker of the ACVR1 inhibitor TP-0184.**

Mark L. Wade, C Lars Mouritsen, Yuta Matsumura, Breeann V. Bryan, David J. Bearss, Steven L. Warner. _Tolero Pharmaceuticals, Inc., Lehi, UT_.

The receptor kinase ALK2, the product of the ACVR1 gene, is a member of the bone morphogenic protein (BMP) receptor family and a type I receptor of the greater TGFβ family. Germline mutations in ACVR1, and specifically the R206H manifestation, are a driving factor in the development of fibrodysplasia ossificans progressiva (FOP). Additionally, the childhood brain tumor, diffuse intrinsic pontine glioma (DIPG), is also associated with mutations in the ACVR1 gene including R206H, but accounts for approximately only 25% of DIPG. In adult cancers, mutations in the ACVR1 gene are observed, yet their role is less established. TP-0184 is a small molecule inhibitor of ACVR1 which inhibits the kinase activity of wild-type and mutant forms of ALK2. We examined the genetic make-up of ACVR1 mutations across large data sets to evaluate which mutations may portend benefit with TP-0184 treatment. Using publicly available sequence databases (e.g. cBioPortal, Cosmic, TCGA) we searched for ACVR1 mutations in tissues of cancer patients. Results were compared to the known mutations of ACVR1 in the literature associated with various cancers or demonstrate gain-of-function activity. Multiple mutations were found within ACVR1. The FOP community has done extensive work to identify and understand mutations that can drive aberrant activation of ACVR1. These mutations include L196P, R206H, Q207E, R258S, G328E/R, and G356D to name a few. ACVR1 mutations identified in adult cancers include many of these FOP mutations, but also demonstrate more diversity in their ACVR1 mutational profile with fewer "hotspots" and many mutations of unknown significance. For example, 8.49% (45/530) of endometrial cancer patients in the TCGA database have ACVR1 mutations, yet these are represented by 52 different mutations. The most common ACVR1 mutation in endometrial cancer is the bona fide R206H mutation, but it only makes up 0.9% of the 8.49% of ACVR1 mutations in endometrial cancer. If all ACVR1 mutations of known significance are considered, 2.45% of endometrial cancers have validated gain-of-function mutations in ACVR1. The remaining ACVR1 mutations in endometrial cancer are of unknown significance. Similar results were observed in other types of cancer such as melanoma, prostate and breast cancer and in other publicly available databases. The results from surveying these rare mutations in multiple cancer types will be presented. Since the BMP and TGFβ pathways are frequently dysregulated in cancers, the importance of any mutation that regulates ACVR1 activity in cancer may be an important regulator of tumorigenesis and a target of ACVR1 inhibitors. A Phase I trial with TP-0184 is in progress and will assess the correlation of ACVR1 mutations and compound clinical activity (Clinical trial information: NCT03429218). Further studies are in progress to assess the consequences and causal impact of ACVR1 mutations in cancer.

#3167

Improving and standardizing TMB assay performance.

Matthew G. Butler,1 Yves Konigshofer,1 Lequan Nguyen,1 Shikha Kalotra,1 Omo Clement,2 Russell K. Garlick,2 Bharathi Anekella1. 1 _SeraCare Life Sciences, Inc., Gaithersburg, MD;_ 2 _SeraCare Life Sciences, Inc., Milford, MA_.

Determining and using the most effective and safest treatment is of great importance in cancer disease management. Checkpoint inhibitors that target immune regulatory molecules such as PD-1, PD-L1, and CTLA-4, have successfully improved PFS and OS - but only in some patients and for some cancers. In the case of PD-1 and PD-L1 inhibitors, it is believed that PD-L1 expression by a solid tumor allows it to escape attack from the immune system and that by inhibiting the PD-L1/PD-1 interaction immune evasion is no longer possible. This may then cause some of the mutations that give rise to expressed neoantigens to act as targets for T cells and allow for the gradual elimination of the tumor. Since the risk for developing autoimmune adverse events is not insignificant with the use of checkpoint inhibitors, determining which patients are likely to respond favorably to their use is imperative. Similarly, expanding approved uses to new indications also benefits from the identification of populations that are most likely to show improvement in PFS and OS - generally, through clinically-validated biomarkers.

Current indications for the use of PD-1 and PD-L1 inhibitors rely on cancer type and several biomarkers. One of these is the expression (or level of expression) of PD-L1 on the tumor. Another - usually in colorectal cancer - is the presence of microsatellite instability (MSI), which indicates that DNA is not being copied with high fidelity and resulting mutations may lead to the emergence of potential neoantigens. While MSI can be assessed by the PCR amplification of several loci, the presence or absence of neoantigens cannot, and they can also be created in the absence of MSI. This has given rise to a potential biomarker - tumor mutational burden (TMB) - that is an assessment of the number of relevant mutations in a tumor. TMB measurement is challenging since different targeted next generation sequencing (NGS) panels look at different regions and percentages of the genome and use different criteria as to what constitutes a relevant mutation. Presently, there is poor correlation between different TMB assays at mutation levels that may be relevant for a companion diagnostic where patients may be denied treatment.

Here, we describe the characterization of a panel of reference materials for the assessment, harmonization, and improvement of TMB measurements by NGS assays. The panel is comprised of cancer cell lines and their SNP-matched normals. DNA from unfixed cells was used to establish a baseline truth set of somatic mutations and germline SNPs, and DNA from FFPE cells was used to determine how TMB assessment is affected by fixation artifacts - especially, at lower variant allele frequencies. We present data from high coverage whole exome sequencing - the current gold standard in TMB assessment - and from targeted NGS panels that are more typical of what may be used clinically. We show that accurate measurements at what may be clinically relevant TMB cutoffs remain a challenge.

#3168

Methods for assessment of the "adenosine fingerprint" in clinical trials of AB928.

Daniel DiRenzo, Devika Ashok, Amy E. Anderson, Akshata Udyavar, Joanne B. Tan, Irene M. Luu, Kristen Zhang, Jenna L. Jeffrey, Lisa Seitz, Manmohan R. Leleti, Stephen W. Young, Jay P. Powers, Matthew J. Walters. _Arcus Biosciences, Inc., Hayward, CA_.

BACKGROUND: The tumor microenvironment (TME) contains high levels of immunosuppressive adenosine (ADO), which activates the A2aR and A2bR receptors on immune cells, leading to an ineffective anti-tumor response. Ecto-5'-nucleotidase (CD73) and tissue non-specific alkaline phosphatase (TNAP) are primarily responsible for the conversion of extracellular adenosine monophosphate (AMP) to ADO and exhibit both membrane-bound and secreted forms. We have previously shown that AB928, a dual A2aR/A2bR antagonist, rescues the immunosuppressive effects of ADO in experimental tumor models. Herein, we describe the development of assays to measure the expression and activity of adenosine-generating enzymes in human tumor samples and peripheral blood. These assays are being used to define an "adenosine fingerprint" that identifies tumor types and patients most sensitive to adenosine inhibition by AB928.

METHODS: CD73 and TNAP immuno-histochemistry (IHC) and mRNA analysis were performed on sections of formalin fixed paraffin embedded (FFPE) tumor tissue. Circulating levels of CD73 were quantified with an in-house developed ELISA, and AMP-ase enzymatic activity in serum was determined using an AMP-GloTM (Promega) assay. Gene expression data were extracted from The Cancer Genome Atlas (TCGA) and expressed as a ratio of log2 counts per million per sample.

RESULTS: TCGA data identified non-small cell lung (NSCLC), renal clear cell, triple-negative breast, ovarian, colorectal, and gastro-esophageal cancers as tumors that highly express the adenosine producing enzymes CD73 or TNAP. To confirm these gene expression patterns, IHC assays for both CD73 and TNAP were developed using normal and tumor human tissue. IHC for CD73 was strongest in NSCLC (54.3 +/- 11.2 µm2) and colorectal (22.5 +/- 8.1 µm2) adenocarcinomas, whereas prostate (1.0 +/- 0.3 µm2) cancer exhibited the weakest staining. In contrast, TNAP staining was strongest in ovarian cancer and NSCLC adenocarcinoma, whereas gastric and colorectal adenocarcinomas showed very little TNAP staining. Therefore, CD73 and TNAP IHC broadly recapitulate the gene expression patterns found in TCGA. A CD73-specific ELISA was developed using human cancer patient blood which established a range of circulating CD73 protein levels (2-8 ng/mL) and showed a strong correlation between plasma and serum levels (r2 = 0.94). To measure adenosine-generating enzyme activity in peripheral blood, the AMP-Glo biochemical assay was performed and showed strong concordance with the CD73 ELISA in cancer patient serum (r2 = 0.72).

CONCLUSIONS: These assays provide a detailed picture of the adenosine-generating capacity in the local TME as well as the peripheral activity and levels of CD73/TNAP to better identify patients that may benefit from adenosine inhibition.

#3169

Development of an engineered T cell receptor-based system for the rapid detection of cancer biomarkers.

Joseph D. Kittle,1 Joel Lwande,2 M Russell Williams,3 Shengwen Liang,2 Kyle McQuaid,2 Melissa Frenchmeyer,2 Yuanyuan Tang,2 Allison Neese,2 Jiangzhou Hua,2 Charles McBrairty3. 1 _Kittle Consulting, Athens, OH;_ 2 _Molecular Technologies Laboratories, Athens, OH;_ 3 _Fundamental Solutions Corporation, Easton, PA_.

Biomarkers play a central role in cancer management by facilitating detection and characterization and are key to the development of personalized therapeutic strategies. We have developed a novel Surface Programmable Activation Receptor (SPAR) diagnostic platform which uses T cell-based technology to achieve the rapid and sensitive identification of cancer biomarkers. SPAR cells are T cells modified to express both an engineered T-cell receptor (TCR) and the luminescent reporter aequorin. Jurkat T cells are first transfected with the aequorin expression vector pEF1-Aeq introduced via random insertion, and stable transfectants selected using G418. The TCR complex is functionally modified via the gene fusion of enhanced monoavidin (eMA) with the CD3ε subunit of the human T cell receptor complex to form eMA-CD3ε and introduced as a homozygous insert into Jurkat/pEF1-Aeq T cells via electroporation, using CRISPR/Cas9 targeting to replace endogenous CD3ε. The modified TCR complex efficiently binds biotinylated target detection molecules (TDMs), which target specific biomarkers via an engineered protein binding domain and a biotinylated protein tag, and act as a ligand to program the SPAR TCR. Simultaneous binding of the TDM to the target and to the modified TCR causes receptor aggregation. Aggregation signals are amplified via the native T-cell signal cascade and result in rapid calcium release from the endoplasmic reticulum. Increased calcium concentrations activate aequorin, triggering the emission of detectible light. Initially developed for programmable pathogen detection, the system has demonstrated reliable and specific detection of the lymphoma marker CD19. Cultured human Burkitt's lymphoma (Raji B) cells abundantly expressing the CD19 surface receptor were combined with SPAR cells programmed with a biotinylated anti-CD19 antibody as TDM. Binding of the anti-CD19 SPAR cells to cell surface CD19 resulted in TCR aggregation, signal transduction, and the generation of reporter signal. Repeat studies conducted in the presence of blood demonstrated equivalent sensitivity. We achieve similar results using the human melanoma markers GD2 and CD133, and mouse melanoma markers TRP1 and CD44 in cell lines known to express these markers. In these studies, a variant of the SPAR system was used to screen commercial monoclonal antibodies to identify those with biologically relevant activity against the cell surface biomarkers, thus demonstrating the utility of this system in optimizing useful antibody/ target combinations without requiring target protein purification. With optimized TDMs, no significant light signal could be detected with K562 cells used as control. The SPAR diagnostic platform is a rapid and effective method for the detection of known cancer biomarkers and may provide a direct screening tool for the optimization of interacting proteins used to guide cell therapies.

#3170

BRAF **expression is associated with poor survival in colorectal cancers independent of** BRAF **(V600E) mutation.**

Mingli Yang,1 Michael J. Schell,2 Timothy J. Yeatman1. 1 _Gibbs Cancer Ctr. and Research Inst., Spartanburg, SC;_ 2 _Moffitt Cancer Ctr. and Research Inst., Tampa, FL_.

The RAS pathway is known to be commonly activated and a key determinant of colorectal cancer (CRC) biology, with mutational activation of RAS/RAF genes observed in ~50% of CRC patients. The relationship between gene mutation and expression is, however, poorly understood. We analyzed how the expression of RAS/RAF genes vs. their respective mutations impacted outcomes. The relationship to other rarely mutated RAS/RAF genes (HRAS, ARAF, RAF1 (i.e. c-RAF)) was also examined in these tumors. While KRAS expression was positively associated with KRAS mutations (r=0.26, p<0.0001), BRAF expression was, unexpectedly, negatively correlated with BRAF (V600E) mutation (r= -0.18, p=0.0001). Thus, a substantial number of patients express relatively high levels of BRAF absent of BRAF mutations. Additional analysis indicates that lower BRAF expression was significantly correlated with MSI or the CMS1 subtype that is strongly associated with BRAF (V600E). Intriguingly, despite negative association with mutant BRAF (V600E) that is well-established to be associated with poor prognosis, higher BRAF expression was also associated with poor overall survival, as indicated by Kaplan-Meier survival analysis of BRAF expression by quartiles in either 458 CRCs (Logrank test for trend p=0.0262) in 406 WT BRAF CRCs (p=0.0102). Note that the expression of all other five RAS/RAF genes (HRAS, KRAS, NRAS, ARAF and RAF1) was not associated with survival. Further analysis with the gene expression signatures measuring RAS pathway activation reveals that unlike BRAF (V600E) (or KRAS mutations or KRAS expression), BRAF expression appeared to be negatively correlated with RAS pathway activation signature scores. Taken together, these data suggest that higher BRAF expression in CRC patients may activate an "alternative" pathway to the canonical pathway directed by mutant BRAF (V600E) as yet another means to deliver tumor progression.

#3171

Delta-like protein 3 expression in Merkel cell carcinoma.

Hao Xie,1 Kumiko Isse,2 Yan Sun,2 Johanna Ramoth,2 Dorothy M. French,2 Laura R. Saunders,2 Aaron S. Mansfield1. 1 _Mayo Clinic, Rochester, MN;_ 2 _AbbVie Stemcentrx LLC, South San Francisco, CA_.

Purpose: Delta-like protein 3 (DLL3) is being developed as a predictive biomarker to identify small cell lung cancer (SCLC) patients for treatment with the DLL3-targeting antibody-drug conjugate rovalpituzumab tesirine (Rova-T). Merkel cell carcinoma (MCC) is an aggressive neuroendocrine carcinoma of the skin, associated with Merkel cell polyomavirus. Given the neuroendocrine features of SCLC and MCC, we sought to evaluate DLL3 expression, its association with common clinicopathological factors, and its prognostic role in MCC.

Methods: A total of 65 formalin-fixed and paraffin-embedded MCC cases were consecutively identified from an institutional biospecimen repository and were cut at 5 µm. Each case was stained for DLL3 protein with SC16.65 mouse monoclonal antibody at 3 µg/mL with the FLEX+ system (DAKO). Slides were then scanned at 20X with AT2 (Leica/Aperio) and read out for percentage of positive tumor cells with 1+, 2+, and 3+ intensity side-by-side with isotype control (IgG2a). Any subcellular staining pattern (membranous, cytoplasmic or punctate) was counted as overall positive. Polyomavirus status was determined by immunohistochemistry (IHC) using an antibody that binds to MCPyV large T-antigen (sc-136172, Santa Cruz Biotechnology, Inc.). A generalized additive model was used to visualize the trend between common clinical factors and overall survival (OS). A multivariable Cox proportional hazards model was applied to assess the association between DLL3 expression and OS. The association between DLL3 expression and other clinicopathological factors was evaluated with median and t tests.

Results: A total of 67 patients were included with a median age of 74 years. Thirty-six (55%) had stage I/II disease; 29 (45%) had stage III/IV disease. Thirty-five (52%) patients died from MCC. Among the 65 cases with successful DLL3 stain, the median H-score of DLL3 expression was 60 (interquartile range: 30-100). Fifty-eight cases (89%) had ≥1% tumor cells positive for DLL3 expression with any intensity, of which the median DLL3 expression was 50% (interquartile range: 25-70%). Thirty-four cases (52%) had ≥50% tumor cells positive for DLL3 expression with any intensity. Older age significantly predicted shorter overall survival (p = 0.03). However, higher AJCC stage did not predict shorter OS (p = 0.3). H-score of DLL3 expression was not associated with AJCC stages (p = 0.4). However, higher H-score of DLL3 expression was associated with higher polyomavirus nuclear expression (p = 0.02) when it was dichotomized to 0/1+ and 2+/3+. In a multivariable Cox regression analysis, H-score of DLL3 expression did not predict OS of patients with MCC (p = 0.8).

Conclusions: DLL3 overexpression is very common in MCC by IHC. DLL3 expression was significantly associated with Merkel cell polyomavirus expression but was not prognostic for OS in this cohort. The high levels of DLL3 expression in a subset of MCC may potentially be used to select patients to receive Rova-T.

#3172

**Prognostic and therapeutic implication of programmed death ligand 1 (PD-L1), microsatellite instability, and** PIK3CA **gene in lipoarcoma.**

Hyo Song Kim,1 Hyae Min Jeon,2 Jae Seok Lee,3 Soo Hee Kim,2 Young Han Lee,1 Hyuk Hur,1 Sung Hoon Kim,1 Seung Hyun Kim,1 Hei-Cheul Jeung4. 1 _Yonsei Cancer Center, Seoul, Republic of Korea;_ 2 _Pathology Center, Seegene Medical Foundation, Seoul, Republic of Korea;_ 3 _Department of Pathology, Samsung Changwon Hospital, Sungkyunkwan University School of Medicine, Changwon, Republic of Korea;_ 4 _Yonsei University College of Medicine, Seoul, Republic of Korea_.

Background: With the promising efficacies with personalized approach, we tested clinically relevant molecular target programmed death ligand 1 (PD-L1), microsatellite Instability (MSI), and PIK3CA molecules in liposarcoma to provide practical guide.

Methods: A consecutive series of 74 liposarcoma patients who underwent curative resection between 2005 and 2016 were assessed using immuno-molecular panels: PD-L1 (22C3 pharmDx assay) and mismatch repair proteins (MLH1, PMS2, MSH2, and MSH6) by immunohistochemistry (IHC), MSI using PCR, and PIK3CA mutation/amplification using pyrosequencing and fluorescence in situ hybridization.

Results: For all the cases, PD-L1 was positive only in tumor infiltrating lymphocytes (TILs), while no PD-L1 expression was found on tumor cell and 23% were tumors with PD-L1+ TILs. PD-L1+ TILs was significantly predominant in abdominal site (P=0.004) and had significantly longer overall survival than PD-L1- TILs (5 year OS 84.4% vs 60.8%, P=0.007). Regarding the MSI status, 2 well differentiated liposarcoma showed MSI [MSI-H (n=1) and MSI-L (n=1)]. For concordance with mismatch repair protein using IHC, only 1 out of 8 with concurrent loss of MLH1, MSH6, and PMS2 showed MSI-H (D2S123, D17S250). Activating PIK3CA mutation was detected in 7 patients [9.5%, exon 9 (n=4) and exon 20 (n=3) mutation] and 6 cases were PD-L1-TIL group. PIK3CA copy number gain were detected in 18 (24.3%) cases and significantly associated with PD-L1+TIL tumors (P=0.045).

Conclusions: With our comprehensive immuno-moleuclar panel, liposarcoma need to be categorized based on the molecular genomic subtype and this may facilitate the development of successful future clinical trials using molecular agents.

#3173

Clinical significance of Fanconi anemia complementation group E(FANCE)DNA repair-related gene expression in hepatocellular carcinoma.

Junichi Takahashi,1 Takaaki Masuda,1 Yosuke Kuroda,1 Akihiro Kitagawa,1 Yushi Motomura,1 Kensuke Koike,1 Dai Shimizu,1 Shotaro Kuramitsu,1 Atsushi Fujii,1 Miwa Noda,1 Kuniaki Sato,1 Yusuke Tsuruda,1 Hajime Otsu,1 Hidetoshi Eguchi,1 Keishi Sugimachi,2 Masaki Mori,3 Koshi Mimori1. 1 _Kyushu University Beppu Hospital, Beppu-shi, Japan;_ 2 _National Hospital Organization Kyushu Cancer Center, Fukuoka, Japan;_ 3 _Kyushu University, Fukuoka, Japan_.

Background : Hepatocellular carcinoma (HCC) is one of the most threatening malignancies because of the limited availability of radical therapeutic options. Thus, identification of prognostic biomarkers as well as molecular therapeutic targets of HCC should be very important for HCC patients. Fanconi anemia complementation group E (FANCE) is a DNA repair-related gene and it's deletion is one of causes of Fanconi anemia. Recent studies reported that FANCD2 which is activated by FA complex involving FANCE shows high expression in HCC and expression of FANCD2 is in direct proportion to the grade of malignancy of HCC (Anticancer Res, 2017). And other studies reported that FANCE shows high expression in breast cancer (J Mol Biol, 2015). However clinical significance of FANCE expression in HCC is unknown.

Objective : To clarify the clinical significance of FANCE expression in HCC.

Material and method: Firstly, we assessed the relation between mRNA expression of FANCE and prognosis using large scale HCC gene data sets (The Cancer Genome Atlas; TCGA, Gene Expression Omnibus; GEO, and European Genome-phenome Archive; EGA). Secondly, the mRNA expression of FANCE was measured in 72 surgically resected HCC in our hospital during the period from 2000 to 2004 by RT-qPCR (normalized by internal control GAPDH), and we compared the expression of FANCE in between tumors and normal tissues. Thirdly, we assessed the associations between expression of FANCE and clinicopathological factors. Finally, we investigated the localization of FANCE by immunohistochemical staining data of THE HUMAN PROTEIN ATLAS.

Result: In large scale HCC gene data sets and our samples, tumor tissues have higher mRNA expression of FANCE than normal tissues(t test. p<0.001). The high expression of FANCE was significantly associated with poor prognosis (Kaplan-Meier method, Log rank test. p<0.05). In the clinicopathological analysis, FANCE expression was not associated with any clinicopathological factors except age (p<0.05). THE HUMAN PROTEIN ATLAS showed that FANCE is expressed in the nuclei of HCC cells.

Discussion: FANCE was overexpressed in HCC cells, and the high expression of FANCE was associated with poor prognosis. So high expression of FANCE could be a prognostic biomarker in HCC. Now we are performing knockdown experiment for FANCE to clarify the biological significance of FANCE expression in HCC.Conclusion: FANCE could be a prognostic biomarker in HCC.

#3174

A novel prognostic model for tongue squamous cell carcinoma based on the characteristics of tumor and its microenvironment: iBD score.

Xiqiang Liu. _Sun Yat-sen Univ., Guangzhou, China_.

Aims: Tumor budding and invasive depth can predict survival of patients with tongue squamous cell carcinoma (TSCC), while the prognostic value of tumor microenvironment (TME) remains unknown. Here, both characteristics of tumor and its microenvironment were examined and a novel prognostic model has been proposed.

Methods: A total of 246 patients with TSCC were included. Using H&E stained sections, pathological parameters of tumor and the TME were assessed. Inflammatory response (i), tumor budding (B), and invasive depth (D) were combined as iBD score. The association between these variables and the patient survival was determined.

Results: Both tumor budding and inflammatory status were independent variables for predicting overall survival (OS) and disease-free survival (DFS) of TSCC patients. Invasive depth was correlated with differentiation, T classification, lymph node metastasis, clinical stage, and recurrence (P< 0.05). The novel iBD model was strongly correlated with T classification, lymph node metastasis, clinical stage, and recurrence, and showed clear distinction of score 0, 1 and 2. High iBD score had a strong association with reduced OS and DFS (P< 0.01).

Conclusions: iBD scoring model is strongly associated with lymph node metastasis and recurrence in TSCC and could be a promising survival predictor for TSCC patients.

#3175

Detection of tumor T-cell clones in mediastinal lymph nodes is associated with lower risk of tumor progression.

Julie A. Rytlewski,1 Mark P. Rubinstein,2 Chadrick E. Delinger,2 Barry Gibney,2 Erik C. Yusko,1 Catherine Sanders,1 John M. Wrangle,2 Kathryn Lindsey2. 1 _Adaptive Biotechnologies, Seattle, WA;_ 2 _Medical University of South Carolina, Charleston, SC_.

Background: The presence of tumor metastases in standard of care lymph node biopsies during surgical resection is used to help determine clinical prognosis. Given the importance of T cells in mediating anti-tumor responses, we sought to assess the clinical significance of T-Cell Receptor (TCR) usage in matched hilar and mediastinal lymph nodes from non-small cell lung cancer (NSCLC) patients.

Methods: TCR-Beta chains were quantified by the immunoSEQ® Assay (Adaptive Biotechnologies) in primary early-stage NSCLC tissue, matched hilar lymph nodes (proximal), and matched mediastinal lymph nodes (distal) for 27 patients, who were treated with surgical resection. Patients were selected such that half experienced recurrence and half remained recurrence-free over the 30+ months of patient follow-up.

Results: T-cell clonality in resected tumors is significantly correlated (Spearman's Rho = 0.52, p = 0.0005) but poorly concordant (Pearson's R2 = 0.14, p = 0.05) with hilar and mediastinal lymph node clonality. The clonality of hilar and mediastinal lymph nodes is both significantly correlated and moderately concordant (Spearman's Rho = 0.7, p << 0.01; Pearson's R2 = 0.44, p = 0.0001). 70-80% of top T-cell clones in tumors were detected in at least one lymph node, and the 100 most abundant tumor clones comprised a median 8.4% and 7.5% of the hilar and mediastinal lymph node repertoires of progression-free subjects, and 7.6% and 6.4% of subjects with tumor progression. Aside from the traditional lymphvascular invasion assessment by histopathology and PET SUVs, the abundance of the top 100 tumor clones and the fraction of top 100 tumor clones detected in the mediastinal lymph node were both independently predictive of early-stage NSCLC progression-free survival (Cox Likelihood p = 0.031 and 0.017, respectively). A 10% increase in the abundance of top 100 tumor clones was associated with a 2.3x increase in risk for progression, suggesting that greater tumor repertoire diversity is favorable in the surgical resection setting. In contrast, for every 10 tumor clones detected in the mediastinal lymph node, the risk of progression decreased by 0.32x, suggesting that increased T-cell trafficking between the tumor and more distant lymph nodes is a favorable prognostic biomarker.

Conclusion: The representation of top tumor T-cell clones is fairly similar in hilar and mediastinal lymph nodes. However, the detection of more top tumor clones in the mediastinal lymph nodes is strongly associated with less risk of progression after surgical tumor resection. This biomarker is likely a surrogate for more robust T-cell trafficking between the tumor and lymph circulation and may be a novel hallmark of better patient outcomes.

Disclaimers: For Research Use Only. Not for use in diagnostic procedures.

#3176

Biomarker identification using xenograft mouse model based clinical trial simulation and artificial intelligence data analytics.

M Afshar,1 Francis Bichat,2 Olivier Duchamp,2 A Etcheto,1 Damien France,2 M Kindermans,1 Caroline Mignard,2 F Parmentier,1 Hery Ratsima2. 1 _Ariana Pharmaceuticals, Paris, France;_ 2 _Oncodesign S.A., Dijon Cedex, France_.

The growing number of anti-cancer drugs available at different stages of clinical development combined with the broadening potential use of combination therapy further complexifies the early identification of companion markers, markers of synergy as well as novel indications for existing and new drug combinations. Well characterized patient derived xenograft mouse models (PDX), combined with Artificial Intelligence tools that can integrate and analyze the broad range of generated data can help address this challenge. PDX experiments can providing an opportunity to simulate a clinical assessment using multiple mice models.In this study, we developed a PDX platform combined with the KEM® Artificial Intelligence data analytics, that is based on Formal Concept Analysis, to simulate a clinical trial and identify biomarkers of response. The platform was tested on colon cancer patient derived PDX. Respectively mRECIST response and survival of respectively 21 and 26 PDXs against Oxalipaltin combined with 5-Fluorouracil and folinic acid (Folfox) was experimentally assessed against a placebo, simulating a clinical trial-like setting with 2 arms. Biomarkers of response (mRECIST) and survival were identified using KEM®, combined with statistical modelling (Cox survival-modelling). 24 candidate biomarker genes were identified including PGAP3, ERBB2, NOTCH2, WDR70, and ZNF227. Alone or combined, these biomarkers are significantly linked to an increase or decrease of the survival PDX (p ranging from 2.2e-10 to 0.048, odd-ratio ranging from 0.12 to 10.00), with the potential to be used as inclusion or exclusion biomarkers. This work demonstrates the ability of a combined PDX / Artificial Intelligence platform to simulate clinical trials and identify biomarkers of drug efficacy and synergy, thus fostering the design of precision medicine clinical trials.

#3177

KRAS gene status in gastric signet-ring cell carcinoma patients and act as biomarker of MEK inhibitor.

Nandie Wu,1 Ying Huang,1 Xiangshan Fan,2 Yang Yang,1 Qin Liu,1 Lixia Yu,1 Rafael Rosell,3 Baorui Liu,1 Jia Wei1. 1 _The Comprehensive Cancer Centre of Drum Tower Hospital, Medical School of Nanjing University & Clinical Cancer Institute of Nanjing University, Nanjing, China; _2 _Pathology Department, Affiliated Drum Tower Hospital to Medical School of Nanjing University, Nanjing, China;_ 3 _Catalan Institute of Oncology, Hospital Germans Trias i Pujol, Badalona, Spain_.

Background: Gastric cancer is the fifth most common cancer worldwide and the third leading cause of cancer-related death, particularly in China. Signet-ring cell carcinoma (SRCC) has specific epidemiology and oncogenesis in gastric cancer. Mutations in KRAS are detected in many types of human tumors and have been proved to be associated with development and progression of cancer. In this study, we sought to explore the role of KRAS in signet-ring cell carcinoma.

Patients and methods: Genomic data from 393 GC samples from The Cancer Genome Atlas (TCGA) was extracted to analyze mutation status of KRAS. Sanger sequencing for KRAS was applied in 234 FFPE SRCC patient samples. Immunohistochemical(IHC) and Fluorence in situ hybridization(FISH) were performed to detect KRAS expression level and KRAS amplification status in FFPE samples. Patients were evaluated in terms of survival and clinicopathological characteristics, as well as KRAS mutation and expression status in TCGA and in our cohort, respectively. We also explored KRAS mutation and expression level in 4 SRCC cell lines. Drug inhibition rate of MEK/mTOR inhibitors was evaluated by cell viability assay.

Results: Thirty-one patients (0.079%) and thirty-seven GC patients (0.094%) in TCGA harbored point mutations and copy number variation (CNV), respectively. Patients with KRAS point mutations and CNV showed higher mRNA level compared to non-mutant cases (P = 0.003 and P < 0.001). No survival difference was observed between KRAS different gene status in TCGA cohort. In our 234 in-house SRCC samples, 15 samples harbored KRAS mutations. We further did analysis on the 75 patients which had enough FFPE samples. Eight patients showed KRAS mutations and two patients showed KRAS amplification. The median overall survival (OS) was 12.5 months for patients with KRAS mutation, and 19.5 months for patients without KRAS mutation (P = 0.045). The majority of SRCC patients are positive with KRAS detected by IHC, which is higher than our intestinal cohort (80% vs 38.6%, P<0.001). However, no difference of overall survival was observed between different KRAS expression level. We further explore the correlation between KRAS status and drug sensitivity in 4 SRCC cell lines. SNU601 and SNU668, which harbored KRAS mutation, were hypersensitive to MEK inhibition. However, mTOR inhibitor showed no preference on SRCC cell lines in terms of KRAS status.

Conclusion: These data demonstrate the status of KRAS gene in SRCC and uncover the therapeutic potential for targeting of these tumors through MEK inhibition in SRCC.

## IMMUNOLOGY

### Adoptive Cell Therapy 3

#3178

Comparative study of methods and platforms for T-cell receptor repertoire.

Mukta Dutta, Mariya Smit, Heather Collins, Anjali Malge, Steve Anderson, Corey Braastad. _Covance Genomics Laboratory, Redmond, WA_.

High-throughput sequencing technologies facilitate in-depth sequencing of T-cell receptor (TCR) repertoires to gain insights into human adaptive immunity. These studies can help decipher the diversity of T-cell receptors, relevant to physiological and disease conditions, and potential therapy responses. A variety of TCR kits and programs have been developed for the identification of V, D and J gene segments, complementarity-determining region 3 (CDR3) sequence extraction and clonality analysis. However, there is still a deficit of systematic comparative studies to assist in the selection of an optimal platform. Here, we present a detailed comparison of 4 state-of-the-art TCR platform on samples with different complexities (ranging from cancers to immune disorders) by taking into account aspects such as clonotype detection [unique V(D)J combination] and CDR3 identification. Our results provide new insights into the effect of method selection on analysis results, and will consequently inform users in the selection of an appropriate analysis method for the desired application(s).

#3179

**Design and activity of 2** nd **generation, dual-targeted CAR T cell factories against ccRCC.**

Yufei Wang,1 Eloah Rabello Suarez,1 Matthew Chang,1 Rebecca Jennings,2 Sabina Signoretti,2 Quan Zhu,1 Wayne Marasco1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Brigham and Women's Hospital, Boston, MA_.

Clear cell renal cell carcinoma (ccRCC) is the major type of RCC which is among the 10 most common cancers in both men and women. Chimeric Antigen Receptor (CAR) T cells have proven to be a powerful, clinically translatable immunotherapy for hematologic malignancies. However, these results have not been translatable to solid tumors due to inefficient homing of CAR T cells, the suppressive tumor microenvironment, and on-target off-tumor toxicities resulted from the sharing of CAR T targeting epitopes on healthy tissues. To combat the suppressive microenvironment, immune checkpoint blockade has shown promising effect on antitumor response by restoring the local antitumor immunity. CAR T cell factories were designed to empower CAR T cells through the secretion of human anti-immune checkpoint inhibitor monoclonal antibodies (mAbs) locally at the tumor site. Our results show a dramatic improvement in CAR T killing of ccRCC in vitro and in vivo by reversing CAR T cell and tumor infiltrating lymphocyte (TIL) exhaustion.

CAIX is an ideal target for ccRCC therapy and used as a CAR target for the first clinical trial. However, it led to adverse side effects (ADEs) due to CAIX expression on the bile duct. Therefore, it is crucial to develop a CAR with elevated efficacy and safety (limited on-target off-tumor effect). To achieve that, the 2nd targeting scFv was introduced in the CAR T cell factory together with anti-CAIX scFv to enable the CAR to target two unique antigens simultaneously. By IHC staining of ccRCC patient samples, we found that target B is an ideal target to be utilized as the 2nd target since it is highly expressed on ccRCC and co-expressed with CAIX.

Our 27 billion-member human scFv-phage display library was panned against the antigen expressing skrc-59 ccRCC cells to identify novel scFvs. Their binding kinetics (Kon/Koff) were then measured and scFvs with desirable kinetics were evaluated for their ability to bind with antigen expressing cells. Ideal candidates were cloned into vectors where anti-target B and anti-CAIX scFvs were combined in different permutations by changing the order of the two targeting scFvs with various linkers connected to a costimulatory domain (CD28, 41BB) and an activating domain (CD3). Primary T cells isolated from PBMCs were transduced to express dual CAR and were tested against different cell lines in vitro. For further evaluation in vivo, a humanized orthotopic ccRCC mouse model was established by injecting luciferized ccRCC cells under the kidney capsule of NSG-SGM3 mice with a reconstituted human immune system.

In summary, utilizing a dual CAR T discovery platform, we generated a series of CARs with different scFvs, linkers, and hinges. By combining the best dual CAR with immune checkpoint blockade as payload, we propose that lead 2nd generation CAR T cell factory candidates will be discovered that should mitigate against ADEs on normal tissues and hold great promise for the treatment of ccRCC.

#3180

Targeting peptide-loaded-DC CIK cells induce a specific antitumor response.

Yimin Zhu, Xueyuan Cui, Cuijuan Liu. _Suzhou Inst. of Nano-Technology and Nano-Bionics, CAS, Suzhou, China_.

Currently, immunotherapy has become an effective alternative therapeutic approach for cancers. Cytokine-induced killer (CIK) cells have a higher proliferation rate, increased efficacy with few side-effects, and non- MHC-restricted killing after co-culturing with dendritic cells (DCs). However, the specificity and efficacy of DC-CIK treatment still need to be improved. In our study, the antitumor effects of CIK cells co-culturing with DCs pulsed with non-cell derived targeting peptides, which could specifically bind to certain tumor cells, were evaluated. Our results indicated that targeting peptide-loaded DCs could enhance the differentiation and cytotoxicity of CIK cells. CIK cells, which were treated with specific targeting peptide-loaded DCs, could effectively and specifically kill tumor cells in vitro and in vivo, as long as tumor cells were pre-treated with these targeting peptides. Moreover, the CIK cytotoxic effect of this type acquired a certain properties of memory and resistance to the tolerance. In conclusion, targeting peptides could guide DC-CIK more effectively and specifically kill tumor cells and this non-cell derived targeting peptide-loaded-DC CIK may work as a novel means for cancer therapy.

#3181

A novel, bioluminescent assay for the selective detection of target cell killing in mixed cultures.

Brock Binkowski, Aileen Paguio, Christopher Eggers, Braeden Butler, Michael Beck, Frank Fan. _Promega Corporation, Madison, WI_.

Efforts to develop and commercialize T cell immunotherapies would benefit from sensitive and easy-to-use assays to monitor target cell killing. We have developed an approach to selectively measure the death of target cells mediated by CAR/TCR-T cells or other types of effector cells. The method relies on the release of a HiBiT-tagged protein from target cells following cell lysis. Once released, HiBiT, an 11 a.a. peptide tag, binds to cell-impermeable Large BiT (LgBiT), a 17.6 kDa protein, to reconstitute NanoBiT Luciferase. In the presence of furimazine substrate, the luminescent signal is proportional to the amount of target cell killing, and cell lysis can be quantified at a single time point using Maximum Release and Spontaneous Release controls. The assay is compatible with a wide range of target cells per well (e.g. 100-500,000 cells/well), and low levels of Spontaneous Release allow the sensitive detection of low levels of target cell lysis (e.g. 1-5%). Using a modified format, target cell killing can be measured continuously in a bench-top luminometer for 24 hours or more. We demonstrate these approaches using commercially available CAR-T cells and target cells stably expressing a HiBiT-tagged protein via random integration of plasmid DNA or viral transduction. We also demonstrate the use of a thaw-and-use format to facilitate MOA-based lot release assays. The approach provides simple, non-radioactive assay formats for selectively monitoring target cell killing in mixed culture experiments.

#3182

Directed screening TCRs against Ras mutations in mCRC patients.

Jijun Yuan. _Shanghai Genbase Biotechnology Co., Ltd., Shanghai, China_.

Ras gene is involved in many signaling pathway to regulate cell proliferation, metabolism and motility, for example MAPK and PI3K pathway. Ras mutation is constitutively active to drive uncontrolled cell proliferation that results in malignant transformation. Ras mutation frequently occurs in human malignancy, for example pancreatic cancer (98%), colorectal cancer (45%) and lung cancer (31%). Drug development targeting Ras mutation was not succeed despites decades of effort in small molecule fields.

T cell receptor (TCRs) can recognize processed and presented neoantigens on human leukocytes antigen (HLA). Ras G12D mutation was report as neoantigen and could be recognized by tumor infiltrating lymphocytes restricted by HLA-C0802 in mCRC patients. We assume that human TCR repertoire encompassing specific TCRs against Ras mutation restricted by other HLAs in Chinese patients.

We design a novel synthetic presentation mRNA construct which harboring all pathogenic Ras mutations. The construct significantly enhanced Ras mutation peptides presentation on HLAs (>60%) compared with conventional used construct. We developed a directed screening method to identify T cell clone against Ras mutation restricted by Chinese pop HLAs in tumor infiltrating lymphocytes and sequenced TCRs by using 10xGenomics. We identified several TCRs that showed effector function against Ras mutation in China mCRC patients for further developing TCR-T cell therapy.

#3183

**Initial safety of AFP** **SPEAR T-cells in patients with advanced hepatocellular carcinoma.**

Lipika Goyal,1 Matthew Frigault,1 Tim Meyer,2 Lynn G. Feun,3 Jordi Bruix,4 Anthony El-Khoueiry,5 Petr Hausner,6 Bruno Sangro,7 Theodore T. Pierce,1 Elliot Norry,8 Sulabha Ranganathan,8 Rafael G. Amado,8 Richard S. Finn9. 1 _Massachusetts General Hospital Cancer Center, Boston, MA;_ 2 _University College London, London, United Kingdom;_ 3 _Sylvester Comprehensive Cancer Center, Miami, FL;_ 4 _University Hospital of Barcelona, BCLC group, Hospital Clínic, Barcelona, Spain;_ 5 _University of Southern California, Los Angeles, Los Angeles, CA;_ 6 _University of Maryland, MD;_ 7 _Clinica Universidad de Navarra and CIBEREHD, Pamplona, Spain;_ 8 _Adaptimmune, Philadelphia, PA;_ 9 _UCLA Jonsson Comprehensive Cancer Center, Los Angeles, CA_.

Background: Genetically engineered affinity-enhanced autologous SPEAR T-cells (AFPc332T-cells) directed towards the HLA-A*02-restricted AFP peptide FMNKFIYEI are being tested in an ongoing Phase 1 trial to evaluate safety and antitumor activity in patients with hepatocellular carcinoma (HCC) (NCT03132792).

Methods: This is a first-in-human study in HCC patients not amenable to transplant, resection, or loco-regional therapy and failed/intolerant/refused standard of care treatment. Patients must be HLA-A*02:01+ or 02:642+. Patients must have AFP expression by immunohistochemistry at ≥1+ in ≥20% HCC tumor cells or serum AFP ≥400 ng/ml and ≤5% IHC AFP in non-cancerous liver tissue. Up to 24 patients will be enrolled using a modified 3+3 design. Lymphodepletion is with fludarabine 20 mg/m2/day and cyclophosphamide 500 mg/m2/day on days -7 to -5. The initial transduced cell dose is 0.1×109 cells; additional doses are 1×109 and 5×109. Cohort expansion will occur at maximum tolerated dose and may allow doses up to 10×109 transduced cells. Dose-limiting toxicities (DLTs) are adjudicated by a Safety Review Committee.

Results: As of 21Sep18, 2 patients were treated with 0.1×109 AFP SPEAR T-cells. Both had cytopenias related to lymphodepleting chemotherapy. Neither experienced cytokine release syndrome or SAEs during initial hospitalization. Liver chemistries show no AFPc332T-related hepatotoxicity. AFPc332T-cells were detected in both patients. One patient had grade 1 cognitive disturbance on day 8. This patient had SAEs of biliary obstruction at week 9 treated with stenting, and abdominal pain at week 12; neither was considered related to AFPc332T. Post-treatment imaging shows stable disease at week 12 by RECIST v1.1. Serum AFP was 12665 ng/ml at baseline, 29616 ng/ml at week 2, and 16,489 at week 12. Week 8 tumor biopsy showed diffuse tissue necrosis with cholestasis suspicious for necrotic tumor cells. No viable tissue was present. Immunostaining for CD3 showed numerous T-cells and T-cell aggregates within the necrotic tissue. The 2nd patient had no SAEs reported; post-treatment imaging is pending.

Conclusions: AFP SPEAR T-cells at the 0.1×109 cell dose show no evidence of on target or off target toxicity in the first 2 patients. No protocol defined DLTs were reported. Current data support continued investigation of AFPc332T-cells. Updated data will be presented.

#3184

Targeting multiple solid tumor types with anti-CD70 allogeneic CAR-T cells.

Mary Lee Dequeant, Sushant Karnik, Zinkal Padalia, Minh Thu Tham, Tony Ho, Julie Carson, Jonathan A. Terrett. _Crispr Therapeutics, Cambridge, MA_.

CD70 (CD27 ligand) is highly expressed in multiple hematologic malignancies, such as non-Hodgkin lymphoma, multiple myeloma, and chronic lymphocytic leukemia, and is uniquely highly expressed in the solid tumor type clear cell renal cell carcinoma (ccRCC). Malignancies showing high CD70 expression have been the subject of multiple clinical trials evaluating anti-CD70 agents, such as antibodies with enhanced antibody-dependent cell-mediated cytotoxicity and antibody drug conjugates (ADCs). The focus on high expression indications seems to be a requirement, particularly for ADCs. We have shown previously that CTX130, a CRISPR/Cas9 gene-edited allogeneic anti-CD70 CAR-T, is potently cytotoxic against even the lowest expressing RCC cells (e.g. ACHN). With this in mind, we evaluated CD70 expression in other solid tumor indications, including pancreatic, lung, and ovarian cancers, and assessed the cytotoxic activity of CTX130 against cell lines derived from these tumor types. These studies show that high expression is not a requirement for potent activity for CAR-T cells as it appears to be for CD70-targeted ADCs. Further, these data highlight a translational medicine route for CTX130 in many solid tumor indications.

#3185

CAR-T cells can eradicate human uveal melanoma and immune-therapy resistant malignant melanoma in IL-2 transgenic NOD/SCID IL2 receptor gamma knockout mice.

Elin Forsberg,1 Mattias Lindberg,1 Henrik Jespersen,1 Samuel Alsén,1 Roger Olofsson Bagge,1 Marco Donia,2 Inge Marie Svane,2 Ola Nilsson,1 Lars Ny,1 Lisa Nilsson,1 Jonas Nilsson1. 1 _Sahlgrenska Cancer Center, Gothenburg, Sweden;_ 2 _Center of Cancer Immunotherapy, Herlev, Denmark_.

Immune therapies including checkpoint blockade and adoptive T cell transfer show great promise for the treatment of melanoma, with long-term effects in some patients. However, not all patients respond to current immune therapies and are in need of other treatment strategies. For example, around half of the patients with metastatic malignant melanoma will not be cured with available therapies today. For metastatic uveal melanoma (a rare melanoma of the eye) available immune therapies have less effect, and there is currently no approved therapy for these patients. Our objective is to find novel immune therapies with the potential to treat immune-therapy resistant melanoma, with the use of chimeric antigen receptor T cells (CAR-T). So far, no CAR-T therapy is approved for use in solid tumors. We have previously developed a humanized mouse model, where tumor cells and T cells from the same patient are grafted in IL-2 transgenic NOD/SCID IL2 receptor gamma knockout (NOG) mice, and found that responses in the mouse model correlated to responses in the corresponding patients in a clinical trial of adoptive T cell transfer (Jespersen et al, Nature Communications, 2017). In order to test the potential for CAR-T therapy in immune-therapy resistant melanoma in this mouse model, we used TCGA to determine the expression in melanoma biopsies of targets for commercially available CAR-T cells. Interestingly, we were able to use this model to treat immune-therapy resistant malignant melanoma and uveal melanoma patient-derived xenografts with human CAR-T cells and obtain curative responses, but only in IL-2 transgenic mice and not in regular NOG mice. These findings prove potential for the use of CAR-T cell therapy in immune-therapy resistant melanoma, and indicate the utility of this mouse model in the translation of CAR-T therapies. We are currently working on the development of CAR-T cells derived from patient-specific T cells.

#3186

A novel combination therapy for treating advanced drug resistant acute lymphoblastic leukemia.

Yorleny M. Vicioso,1 Rose Beck,1 Herman Gram,2 Abhishek Asthana,1 keman Zhang,1 Derek Wong,1 John Letterio,1 Reshmi Parameswaran1. 1 _Case Western Reserve University, Cleveland, OH;_ 2 _Novartis Institutes, Basel, Switzerland_.

Purpose: Drug resistance and relapse are two major problems in Acute Lymphoblastic Leukemia (ALL) as these patients do not respond to chemotherapy. We observed BAFF-R expression on drug resistant and relapse B-ALL patient cells. We wanted to explore the in vivo therapeutic efficacy of a BAFF-R antibody, optimized for antibody dependent cellular cytotoxicity (ADCC), against drug resistant ALL cells.

Experimental design: ADCC assay was performed using primary human natural killer (NK) cells isolated from normal donors or ALL patients. NK cell mediated cytotoxicity was measured using Calcein-AM assay. Immunocompromised NSG mice were used for in vivo xenograft model of ALL.

Results: We found that anti-BAFF-R antibody enhanced NK cell mediated killing of drug resistant ALL cells in vitro. Early treatment with anti-BAFF-R antibody and NK cells significantly reduced disease burden in xenograft ALL models and increased overall mice survival. However, in advanced disease, the efficacy of NK cells to mediate ADCC is reduced. ALL cells are known to produce TGF beta, that causes NK cell dysfunction. We found that ALL cells produce TGF-β and co-culturing ALL cells with NK cells lead to a decrease in CD16 expression on NK cells, which is reversible by addition of EW-7197, a potent TGF-beta receptor I inhibitor. We found that EW-7197 treatment improved anti-BAFF-R antibody mediated ADCC of drug resistant ALL cells, in vivo, even if treatment is started late.

Conclusion: Combination treatment using BAFF-R antibody, NK cells and EW-7197 is a potential strategy to treat advanced drug resistant ALL patients with BAFF-R expressing blasts.

#3187

Engineering a new generation of cell therapies for solid tumor oncology using the SQZ platform.

Kelan A. Hlavaty, Matthew G. Booty, Scott Loughhead, Katarina Blagovic, Alfonso Vicente-Suarez, Defne Yarar, Howard Bernstein, Armon Sharei. _SQZ Biotechnologies, Watertown, MA_.

Effective T cell priming is crucial to induce anti-tumor CD8+ T cell responses and requires the efficient presentation of antigen on major histocompatibility complex class I (MHC-I) by antigen presenting cells (APCs). Previous efforts using dendritic cells to prime CD8+ T cell responses have proven difficult due to limited cell availability in the blood and challenges delivering antigen to the APC cytosol, a necessary step for MHC-I presentation and CD8+ T cell activation. To overcome this limitation, we deliver antigen directly to the cytosol of target APCs using the microfluidics-based SQZ platform. SQZ uniquely facilitates antigen loading into both professional and unconventional APCs, including B cells, T cells, and heterogenous populations of cells, which can be easily obtained directly from the blood. Protein and peptide antigens are delivered using SQZ to each of these APCs effectively, leading to efficient presentation of immunogenic epitopes on MHC-I. Here, we demonstrate that murine SQZ-APCs can stimulate antigen-specific CD8+ T cell responses in vitro and in vivo as measured by expansion of antigen-specific T cells and production of IFNγ. In the TC-1 tumor model for HPV-associated cancers, antigen-loaded SQZ-APCs have strong anti-tumor effects both prophylactically and therapeutically. Following therapeutic immunization, the anti-tumor responses correlate with an increase in antigen-specific CD8+ tumor infiltrating lymphocytes compared to untreated mice. In addition, compared to a traditional subcutaneous peptide vaccine, SQZ-APCs elicit a five-fold greater intratumoral CD8+ T cell response and drive significantly more tumor growth inhibition. Importantly, this SQZ-enabled cancer cell therapy translates to human B cells, T cells, and heterogenous populations of cells engineered to function as APCs. When a peptide is delivered to the cytosol using SQZ, all of these primary human cells activate antigen-specific CD8+ T cell responses in vitro by stimulation of IFNγ from antigen-specific CD8+ T cell responders. In comparison to cells incubated in the presence of peptide antigen, SQZ-APCs stimulate a 10-fold increase in IFNγ production from antigen-specific CD8+ responder T cells (n=13 donors). Finally, the SQZ process has been scaled to engineer human SQZ-APCs in preparation for clinical trials with a throughput of greater than 4 billion cells SQZ'd per minute. Collectively, these findings highlight the significant clinical potential of the SQZ platform to engineer potent APCs for a new generation of cancer cell therapies.

#3188

Transimmunization prevents recurrence and reprograms the immune milieu in a mouse model of ovarian cancer.

Ayesha B. Alvero, Douglas Hanlon, Mary Pitruzzello, Renata Filler, Eve Robinson, Olga Sobolev, Roslyn Tedja, Alessandra Ventura, Richard L. Edelson, Gil Mor. _Yale Univ. School of Medicine, New Haven, CT_.

Introduction: Targeting recurrent disease is vital in the improvement of survival in ovarian cancer patients. Transimmunization (TI) is a modification of extracorporeal photochemotherapy, which is a dendritic cell (DC)-based vaccination, currently used in cutaneous T cell lymphoma but has never been tested in gynecologic tumors. The objective of this study is to determine if TI can induce a tumor-specific immune response capable of preventing recurrence in ovarian cancer.

Methods of study: Ovarian cancer cells expressing mCherry fluorescent protein were injected i.p. in C57bl/6 mice to mimic the establishment of recurrent disease. These cells (TKO cancer cells) were isolated from spontaneously formed ovarian tumors from p53LSL-R172H/+Dicerflox/floxPtenflox/flox Amhr2cre/+ mice. Tumor burden was quantified using mCherry fluorescence ROI area. TI vaccination was conducted as follows: (1) cancer cells were treated with 8-MOP and exposed to UVA to induce apoptosis; (2) PBMCs were collected from tumor-bearing mice, mixed with apoptotic cancer cells, and circulated through the TI chamber to induce functional DC from monocytes; (3) cells collected were incubated overnight to facilitate antigen uptake and presentation and injected i.p. Treatment groups were as follows: (1) PBS control, n = 20 ; (2) TI TKO, n =10 (i.e. using apoptotic TKO cells); and (3) TI YUMM, n = 10 (using apoptotic mouse melanoma YUMM1.7 cells as antigen-unrelated control). Immune milieu was characterized by IHC on tumors and FACS analysis using cells isolated from peritoneal lavage.

Results: Eighty percent of mice in the TI TKO group were disease free compared to 35% in the PBS control group and 40% in the TI YUMM group. In addition, TI TKO vaccination (p < 0.0001), but not TI YUMM vaccination (p = 0.1079) induced a statistically significant decrease in tumor kinetics compared to PBS control. IHC analysis showed significantly higher levels of infiltrating CD8+ (p = 0.0037) and CD4+ T cells (p = 0.0079) in TI TKO group compared to PBS group. Similarly, mice in the TI TKO group have higher i.p. levels of CD8+/CD44+/CD62L+ central memory T cells (p = 0.004), higher levels of CD8+/CD44+/CD62L- effector memory T cells (p = 0.03), lower levels of CD8+/CD44-/CD62L+ naïve T cells (p = 0.02), and lower levels of CD11b+/Gr-1high myeloid-derived suppressor cells (p = 0.01) compared to PBS control group. Levels of these cells in TI YUMM group were not significantly different from the PBS control.

Conclusion: We demonstrate for the first time that TI vaccination with autologous cancer cells can prevent recurrence and modify the immune phenotype in ovarian cancer. TI efficacy depends on the tumor antigen source, confirming TI as an antigen-specific DC-based immunotherapy. Our results highlight the value of TI in ovarian cancer and its potential application to other i.p. cancers.

#3189

CAB-CAR-T: A novel conditionally active biologics approach to minimize on-target off-tumor effects in adoptive immunotherapy.

Jianfang Hu,1 Benjamin Lopez,1 Tiffany Lam,1 Anirban Kundu,2 Timothy Mayall,1 Gregory Schreiber,3 Farzad Haerizadeh,1 James Onuffer,1 Gregory Frost3. 1 _F1 Oncology, Inc, San Diego, CA;_ 2 _Exuma Biotechnology SEZC, Georgetown, Cayman Islands;_ 3 _F1 Oncology, Inc, West Palm Beach, FL_.

Adoptive cellular therapy (ACT) using chimeric antigen receptor (CAR) modified T cells has demonstrated promising antitumor effects in hematologic malignancies leading to the recent approval of two CAR-T products targeting the B cell receptor associated protein CD19, however the safe development of CAR-T therapies for solid tumor malignancies remains less advanced in part due to the lack of precision in targeting solid tumor antigens. Identification of clinically viable tumor restricted target antigens while avoiding on-target off-tumor toxicities in normal tissue presents a significant challenge in the solid tumor CAR-T therapeutic scenario. The altered glycolytic pathway of the Warburg effect within the tumor microenvironment (TME) drives lactic acid production leading to an opportunity to develop novel CARs with functional TME dependent switching ability. The resulting acidic extracellular pH is a major feature of the TME and this unique property represents one novel mechanism by which target specificity may be achieved. Here we describe a novel Conditionally Active Biologics approach in adoptive immunotherapy termed CAB-CAR-T. By genetically engineering combinations of amino acid mutations into the antibody variable antigen recognition domains (scFv) of CAR constructs, we demonstrate that TME restricted target antigen engagement can be engineered into chimeric antigen receptors. The described CAB-CARs display minimal antigen dependent activity at the physiological pH of normal tissue. Under TME conditions (low pH) the affinity of CAB-CAR scFv domains against their antigen increases, such that tumor target recognition and CAR-T activity become pH dependent. Thus CAB-CAR-T cells display reversible "AND logic gate" properties, requiring both antigen presence and TME conditions for activity. Comparisons of target cell killing under TME permissive and physiologic pH conditions in a cellular impedance based kinetic killing assay demonstrate that CAB-CAR-T cells efficiently lyse antigen positive target cells at pH 6.7 (TME) with minimal activity at pH 7.4 (normal tissue). pH profiles of CAB-CAR-T were examined through buffering the culture medium down from physiologic pH in 0.1 pH increments, and the data demonstrated that the cytolytic activity of CAB-CAR-T incrementally increased as extracellular pH was decreased compared to control T cells. Similar pH dependence was observed for other measures of CAB-CAR-T activation including the early T cell surface activation marker CD69, the degranulation marker CD107a and cytokine release (IL-2 and IFNγ). Conditionally active chimeric antigen receptor T cells (CAB-CAR-T) harness unique properties of the tumor microenvironment to provide an opportunity to develop safer CAR-T therapeutics for tumor antigen targets also expressed on healthy tissues by minimizing on-target off-tumor activity.

#3190

Standardization and clinical implementation of liquid biopsy assays - IMI's CANCER-ID.

Klaus Pantel,1 Leon W. Terstappen,2 Nicolò Manaresi,3 Harry J. Groen,4 Ed M. Schuuring,5 Ellen Heitzer,6 Michael Speicher,6 Bjørn Naume,7 Jon Amund Kyte,7 Thomas Schlange,8 for the IMI CANCER-ID consortium. 1 _University Cancer Center Hamburg/Eppendorf, Hamburg, Germany;_ 2 _University of Twente, Enschede, Netherlands;_ 3 _Menarini/Silicon Biosystems, Bologna, Italy;_ 4 _University of Groningen and University Medical Center Groningen, Groningen, Netherlands;_ 5 _University Medical Center Groningen, Groningen, Netherlands;_ 6 _Medical University of Graz, Graz, Austria;_ 7 _University of Oslo, Oslo, Norway;_ 8 _Bayer AG, Wuppertal, Germany_.

The Innovative Medicines Initiative (IMI) project CANCER-ID (www.cancer-id.eu) is a 5 year (2015-2019) international public-private partnership of currently 40 partners from 14 countries with the aim to evaluate technologies for Circulating Tumor Cell (CTC), circulating free tumor DNA (ctDNA), microRNA (miRNA) and exosome enrichment, isolation and analysis. At the core of CANCER-ID's activities are establishment of harmonized best practice protocols from patient sample collection, pre-analytical sample handling, sample and bioinformatics analyses down to the actionable information guiding patient selection and personalized treatment. CANCER-ID is furthermore testing and supporting development of standards for liquid biopsy as well as clinical implementation of liquid biopsy based protocols in the clinical setting. This includes interaction with regulatory bodies in Europe (EMA Innovation Task Force) and the US (FDA Public-Private Partnership liaison) to support future approval of liquid biopsies in multi-centered worldwide clinical studies.

During the clinical validation phase of the project, clinical-ready liquid biopsy protocols have been implemented in an observational study on the potential predictive value of monitoring treatment response towards Immune Checkpoint Inhibition (ICI) in 180 NSCLC patients at the UMC Groningen, The Netherlands, as well as in two ICI-chemotherapy combination studies in Triple-Negative Breast Cancer and Luminal B-type breast cancer, respectively, run by the University of Oslo, Norway (ALICE NCT03164993 and ICON NCT03409198). Within both studies, blood has been collected at baseline and at follow-up visits for ctDNA and CTC analysis, including technical evaluation of CTC PD-L1 protein expression. The aim is to assess whether the allelic frequency of mutations identified by plasma NGS as a potential measure for Tumor Mutational Burden or the number of PD-L1–positive/overall CTCs at different time points is indicative of treatment success. The studies aim at providing data to assess whether clinical predictive information could be inferred from baseline number of detected mutations and PD-L1 expressing CTCs. Preliminary data of these analyses will be presented.

As a follow-up activity of the IMI CANCER-ID program, the European Liquid Biopsy Society (ELBS) is currently being established by Prof. Pantel at UKE Hamburg, Germany. The ELBS will be open to all interested liquid biopsy stakeholders worldwide as a platform for scientific exchange, further efforts to standardize technologies and protocols in the field as well as for the initiation of new basic and clinical research projects with the aim to make liquid biopsies an integral part of clinical studies and patient care.

This work is supported by IMI JU & EFPIA (grant no. 115749, CANCER-ID). Samples from patients and healthy volunteers, respectively, were collected under signed informed consent.

### Combination Immunotherapies 2

#3191

FT516, an off-the-shelf engineered NK cell therapeutic product for universal anti-tumor targeting strategy in combination with monoclonal antibodies.

Ryan Bjordahl,1 Huang Zhu,2 Paul Rogers,1 Svetlana Gaidarova,1 Moyar Q. Ge,1 Robert Blum,2 Frank Cichocki,3 Jode Goodridge,1 Helen Chu,1 Greg Bonello,1 Tom Lee,1 Brian Groff,1 Ramzey Abujarour,1 Bruce Walcheck,3 Jeffrey Miller,3 Dan Kaufman,2 Bahram Valamehr1. 1 _Fate Therapeutics, San Diego, CA;_ 2 _University of California, San Diego, San Diego, CA;_ 3 _University of Minnesota, Minneapolis, MN_.

Monoclonal antibody (mAb) treatment is an effective therapeutic strategy for many cancer types, with significant opportunity to optimize natural killer (NK) cell and mAb interaction to improve antibody-dependent cellular cytotoxicity (ADCC). NK cells are critical mediators of ADCC, where they recognize and kill malignant cells coated with antibody through the Fc receptor CD16. However, NK cell function is often impaired in cancer patients, which limits the induction of ADCC in mAb therapy. To enhance ADCC in combination with commercialized mAb therapies, we have developed FT516; a novel, off-the-shelf NK cell immunotherapeutic engineered to uniformly express a high-affinity, non-cleavable version of CD16 (hnCD16).

FT516 is manufactured from a renewable master induced pluripotent stem cell (iPSC) line with the potential to generate hundreds to thousands of doses of allogeneic NK cells uniformly expressing hnCD16 (hnCD16 iNK cells) per manufacturing run. In an in vivo xenograft model of disseminated lymphoma, FT516 reduced tumor burden below the limit of detection at day 28 after transplant when delivered in combination with rituximab, which was significantly more potent than peripheral blood NK cells (p = 0.03). This was attributed to enhanced CD16-mediated activation of FT516, as observed through improved calcium flux and enhanced activation of signaling pathways such as ERK (p = 0.016), LAT (p = 0.0007), and ZAP70 (p = 0.0003), leading to enhanced ADCC and cytokine production. FT516 also maintained tumor cell specificity, with preferential targeting of K562 leukemia cells when presented with a mixture of K562 and normal PBMC targets (p < 0.0001).

We also explored strategies to further engineer therapeutic function to enhance FT516 efficacy. Combined expression of hnCD16 with an IL-15/IL-15ra fusion construct enhanced the persistence of iNK cells and allowed survival of up to 8 weeks in vivo without exogenous cytokine (p < 0.0001), with correlation to improved efficacy.

In vitro modeling of FT516 with daratumumab demonstrated ADCC against multiple myeloma (MM) targets. However, as reported, daratumumab induced NK cell fratricide through binding of CD38 on NK cells. To rescue daratumumab-mediated fratricide, we specifically deleted CD38 at the iPSC level and demonstrated that fratricide was undetectable in hnCD16 CD38-/- iNK cells (<1% vs. 35% for peripheral blood NK). By avoiding fratricide, hnCD16 CD38-/- iNK cells had improved persistence and efficacy against MM cells in vitro and in a disseminated xenograft model of MM. These data support clinical development of FT516 in combination with rituximab for the treatment of B cell malignancies and demonstrate a targeting platform that enables further modifications to address challenges related to efficacy, safety, and persistence of allogeneic adoptive immunotherapies.

#3192

Activation of p53 in the tumor microenvironment by MDM2 inhibitor APG-115 synergizes with PD-1 blockade independently of p53 status of tumor cells.

Douglas D. Fang,1 Qiuqiong Tang,1 Yanhui Kong,1 Qixin Wang,1 Jiaxing Gu,1 Xu Fang,1 Peng Zou,2 Tao Rong,1 Jingwen Wang,1 Dajun Yang,1 Yifan Zhai1. 1 _Ascentage Pharma (Suzhou) Co., Ltd, Suzhou, China;_ 2 _Oncology & Immunology Unit, WuXi Apptec (Suzhou) Co., Ltd, Suzhou, China_.

Blockade of the checkpoint inhibitor programmed death 1 (PD-1) has gained big success in cancer therapy. However, the response rate of anti-PD1 agents remains low. Molecularly targeted agents offer selectivity and high tumor response rates, but patients develop resistance to these drugs inevitably. Combinations of targeted agents and immunotherapy provide new opportunities to improve cancer treatments. Recent studies found that p53 activation in the myeloid linage suppressed alternative (M2) macrophage polarization and attenuates tumor development and invasion, leading to the hypothesis that p53 activation may further augment antitumor immunity elicited by anti-PD-1 therapy. APG-115 is an orally active, selective, small molecule inhibitor of the MDM2-p53 protein-protein interaction. APG-115 acts as an antitumor agent by activating the p53 tumor suppressor in p53 wild-type tumors. However, its role in regulating immune responses remained unknown. In this study, we investigated the role of APG-115 in immune modulation both in vitro and in vivo. Enhanced antitumor activity was first demonstrated in p53 wild-type MH-22A, p53 mutant MC38, and p53 knockout (p53-/-) MH-22A syngeneic tumor models after the combination treatment of APG-115 and anti-PD-1 antibody. Despite differential changes in tumor-infiltrating leukocytes, including an increase in cytotoxic CD8+ T cells in the p53 wild-type tumors and an increase in proinflammatory M1 macrophages in the p53 mutant tumors, the combination treatment consistently reduced immunosuppressive M2 macrophages in the tumor microenvironment regardless of p53 status of tumor cells. In addition, in vitro, the treatment of bone marrow-derived macrophages with APG-115 resulted in activation of p53 and p21 gene expression, as well as a decrease in M2 macrophages population and reduction of c-MYC and M2-related gene expression. Moreover, enhanced M1 macrophage polarization in the spleen was also observed in naïve mice treated with APG-115. Furthermore, APG-115 increased production of multiple proinflammatory cytokines, including IFN-γ, TNF-α, IL-2 and IL-6, in stimulated T cells. Collectively, for the first time, our findings suggest that p53 activation by a pharmacological MDM2 inhibitor enables reversal of immunosuppressive tumor microenvironment and enhance antitumor immunity independently of p53 status of tumors. Specifically, in complementary to PD-1 blockade that predominantly activates cytotoxic CD8+ T cell populations, APG-115 primarily targets tumor-associated macrophages. Collectively, our data provide a rationale for applying the combination of APG-115 plus PD-1 blockade to a broader patient population with p53 mutant tumors. Accordingly, a clinical trial of APG-115 in combination with pembrolizumab in metastatic melanoma patients regardless of p53 status has been initiated in the USA.

#3193

Combining immunotherapies: A strategy for improving CD8 T cell-mediated antitumor responses.

Aaron E. Fan,1 Hussein Sultan,2 Takumi Kumai,3 Esteban Celis1. 1 _Augusta University, Augusta, GA;_ 2 _Washington University School of Medicine, St. Louis, MO;_ 3 _Asahikawa Medical University, Asahikawa, Japan_.

Adoptive cell therapy (ACT) of retrovirally transduced (RV) CD8 T cells is a powerful technique that has shown promise in tumor eradication in cancer patients. However, some major barriers to current methods are that ACT is expensive, time consuming, and requires harmful and toxic adjunct procedures. Our laboratory has demonstrated the use of TriVax, a potent peptide vaccination strategy that dramatically expands ACT cell populations and bypasses the necessity for adjunct procedures. The purpose of this project was to enhance current methods of ACT+TriVax by testing an antigen-specific antitumor response of RV CD8 T cells and if it could be improved with constitutively active STAT5 (CA-STAT5) expression, a protein activated downstream several cytokine pathways that have been shown to play a role in increasing CD8 T cell persistence and resistance to apoptosis. We tested the hypothesis that CA-STAT5 in CD8 T cells enhances an antitumor effect by increasing T cell persistence and efficacy.

Our results show that TriVax administration selectively expanded frequencies of the ACT cell population expressing gp100-TCR in both blood and spleen. When co-transduced with CA-STAT5, an even higher fold expansion of antigen-specific cells was observed. +CA-STAT5 T cells were able to expand more robustly than -CA-STAT5 T cells upon repeated antigen stimulation (vaccine boost), demonstrating nearly 4000-fold increases in antigen-specific CD8 T cells. +CA-STAT5 T cells also seemed to persist longer in vivo over time, and they expressed lower levels of surface PD-1. Using B16F10 melanoma, ACT+TriVax of these cell populations into tumor-bearing mice demonstrated a powerful antitumor effect, leading to tumor regression in treated groups. CA-STAT5 seemed to recapitulate similar antitumor effects our laboratory observed previously with combinatorial anti-PD-L1 treatment or IL2/anti-IL2 mAb complexes (IL2Cx), suggesting a potential role for STAT5 in resisting the PD-1/PD-L1 inhibitory pathway. When compared to tumor-bearing mice treated with ACT+TriVax with anti-PD-L1 antibody, our approach using +CA-STAT5 T cells generated a comparable antitumor response. Altogether, these results demonstrate that RV CD8 T cells expressing gp100-TCR and CA-STAT5 are capable of antigen-dependent expansion in response to TriVax. CA-STAT5 plays a role in increasing T cell proliferation and persistence, as well as increasing efficacy through mechanisms that seem to indicate resistance to PD-1/PD-L1 inhibition.

#3194

A new small allosteric activator of P2RX7 triggers potent anti-tumor immunity and synergizes with anti-PD-1 to induce complete tumor regression in mice.

Laetitia Douget,1 Jonathan Benzaquen,1 Serena Janho Dit Hreich,1 Thierry Juhel,1 Xavier Dezitter,2 Germain Homerin,3 Nicolas Renault,2 Alina Ghinet,3 Regis Millet,2 Sahil Adriouch,4 Paul Hofman,1 Valerie Vouret-Craviari1. 1 _IRCAN, Nice, France;_ 2 _Institut de Chimie Pharmaceutique Albert Lespagnol (ICPAL), Lille, France;_ 3 _Yncréa Hauts-de-France, Lille, France;_ 4 _Universite Rouen, Rouen, France_.

Durable tumor regression induced by immunotherapies, such as checkpoint blockade, is achieved in a minority of patients with metastatic lung cancer or melanoma. This suboptimal outcome could be overcome by using immunotherapy combinations. Here we develop a new positive allosteric modulator of the purinergic P2RX7 receptor that activates both innate and adaptive immune cells to control tumor growth and improve mice survival in syngeneic tumor models. Adoptive transfer experiments in p2rx7 deficient animals revealed that this pharmacological P2RX7 activator triggers its anti-tumor efficacy by stimulating P2RX7 expressing immune cells rather than targeting tumor cell death. Mechanistically, our molecule stimulates P2RX7 expressing dendritic cells to generate IL-18 that leads to the production of IFN-γ by NK and CD4+ T cells within the tumor microenvironment and consequently to the up regulation of MHC-I and PDL-1 expression on tumor cells. By increasing LLC tumor immunogenicity our molecule improves efficacy of anti-PD-1 treatment. Complete tumor regression is reached in 80% of tumor bearing mice treated with a combination of our drug and anti-PD-1, whereas only 12% of mice respond to anti-PD-1 alone. The maximal anti-tumor efficacy promotes functionally relevant memory of CD8+ T cell populations. These results demonstrate for the first time the capacity of a P2RX7 positive modulator to control tumor burden by triggering potent anti-tumor immune responses.

#3195

Intratumoral electroporation of plasmid IL-12 and CXCL9 with membrane-bound anti-CD3 elicits robust anti-tumor immunity.

Mia Han, Anandaroop Mukhopadhyay, Bianca Nguyen, Kurt Sakurada, Jack Lee, David A. Canton, Christopher G. Twitty. _OncoSec Medical Incorporated, San Diego, CA_.

Preclinical and clinical studies have demonstrated that plasmid IL-12 (tavokinogene telseplasmid) delivered intratumorally via electroporation (TAVO) induces local expression of IL-12p70, converting immunologically excluded tumors into inflamed immunogenic lesions, which is fundamental to generating objective responses in both treated and untreated distant tumors. Recent optimization of electroporation parameters and plasmid design has yielded a significant increase in intratumoral expression and anti-tumor efficacy in preclinical models. To further develop this platform, longitudinal biomarker data from TAVO clinical trials was interrogated to identify key immunological components associated with an effective therapy. This analysis revealed that the composition of TAVO-inflamed lesions, specifically the frequency of CD8+ TIL and the chemokines necessary for their maintenance relative to suppressive intratumoral immune subsets, coincided with clinical responses. We therefore developed a plasmid that encodes both IL-12 and the effector T cell chemokine, CXCL9, which, when transfected into a tumor, are functionally active both in vitro as well as in vivo, yielding abscopal responses and increased survival. To further amplify this technology, a strategy was developed to engage the highly relevant and recently described bystander non-tumor reactive T cells in the tumor microenvironment (TME). To accomplish this pan-T cell amplification, a separate therapeutic plasmid encoding membrane-bound anti-CD3 scFv (145-2C11) was used to decorate transfected tumors with the potent polyclonal T cell stimulator. Membrane expression of anti-CD3 in vivo was confirmed by immunoblot and flow cytometry. Combination of IL-12/CXCL9 with anti-CD3 delivered by electroporation (collectively, "SPARK") increased both antigen-specific and polyclonal T cells responses, demonstrated by in vivo CTL and proliferation assays. Finally, SPARK upregulated the transcription of key immunological genes, leading to regression of both treated and untreated tumors, which heightens its ability to reshape the TME and drive systemic anti-tumor immunity. In conclusion, this data supports a model whereby IL-12 in concert with CXCL9 inflames the lesion, leading to a brisk T cell infiltrate, which in sequence with anti-CD3 stimulation, drives a broad yet robust systemic T cell response. SPARK represents a significant advancement in cytokine-based immunotherapy.

#3196

Combination immunotherapy with PD-1 and CXCR4 blockade activates antitumor immunity against pancreatic neuroendocrine tumors.

Xiuyun Jiang, Kevin Sullivan, Kevin Labadie, Sara K. Daniel, David Seo, Arezou Abbasi, Teresa Kim, Raymond Yeung, Venu Pillarisetty. _Univ. of Washington, Seattle, WA_.

Introduction: Although pancreatic neuroendocrine tumors (PNET) are less common and less deadly than pancreatic adenocarcinoma, they remain a considerable source of morbidity and mortality. The cases increasingly discovered due to advanced cross sectional imaging and the majority of PNETs are malignant when diagnosed. Immunotherapy for PNET has not yet been successfully demonstrated, although we and others have previously shown that they often contain robust T cell infiltrates. We hypothesized that combined immunotherapy could reactivate endogenous antitumor activity in organotypic tumor slice cultures of human PNET.

Methods: Human PNET tumors were collected from the operating room and 250μm slices were cut using a vibratome and place on 0.4μm pore size membrane inserts (6 tumors). Slice culture survival testing was conducted using immunohistochemistry (IHC), the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay, or live immunofluorescence imaging. To test the combining immunotherapeutic effects, slices were treated with a PD-1 blocking monoclonal antibody or isotype control, with or without the CXCR4 inhibitor, AMD3100. For time-lapse live imaging, slices were stained with fluorescently conjugated antibodies for CD8 and EpCAM, as well as a reagent that binds to activated caspase 3/7 enzymes to indicate induction of apoptosis. The live slice was first imaged alone to serve as a non-treated control, then anti-PD1 antibody and AMD3100 were applied, and the slice imaged immediately afterward to monitor response after treatment.

Results: We confirmed that cultured slices maintain their baseline morphology and architecture over 9 days in culture. The MTT assay showed stable metabolic activity over the same period. The demonstration of the live and dynamic microenvironment was performed first via live multicolor IF including PNET cells (EpCAM+) and immune cells (both CD45+ and CD4+ cells) within the microenvironment. IHC demonstrated 10% or 12% increased of cleaved-Caspase-3+ cells with combined PD-1 and CXCR4 blockade, as compared to control, after 2 days or 7 days in culture. To confirm the role of cytotoxic T cells, live stained slices were imaged before and after addition of PD-1 blockade and AMD3100. We demonstrated that caspase activation was increased in EpCAM+ cells proximal to CD8+ T cells immediately following combination drug treatment. Furthermore, CD8+ cells proximity to EPCAM\+ cells (<20 µm) was significantly increased after addition of PD-1 blockade and AMD3100. There was no difference in the proportion of apoptotic EpCAM+ cells that did not have a CD8+ cell nearby, suggesting that apoptosis induction is specifically due to cytotoxic T cell function.

Conclusion: Our study demonstrates that combination PD-1 and CXCR4 blockade enhances CD8+ T cell migration and antitumor activity in human PNET tumor slice cultures.

#3197

BI-907828, a novel and potent MDM2-p53 antagonist, acts synergistically in a triple combination with anti-PD-1 and anti-LAG-3 antibodies in syngeneic mouse models of cancer.

Dorothea Rudolph, Ulrike Weyer-Czernilofsky, Markus Reschke, Martina Sykora, Jörg Rinnenthal, Sophia Blake, Gabriela Gremel, Andreas Wernitznig, Andreas Gollner, Norbert Kraut, Jürgen Moll. _Boehringer Ingelheim RCV GmbH & Co KG, Wien, Austria_.

MDM2-p53 antagonists block the interaction between the Tumor Protein p53 and MDM2, its key negative regulator, and represent a new therapeutic concept for cancer therapy. MDM2-p53 antagonists are designed to restore p53 activity in TP53 wild-type tumors. Several MDM2-p53 antagonists are currently being evaluated in early clinical development. BI-907828 is a novel and potent MDM2-p53 antagonist with optimized drug-like properties that has shown efficacy in human tumor xenograft models at daily low oral dose as well as intermittent high dose schedules. Recent preclinical studies in syngeneic mouse models of cancer have demonstrated that BI-907828, apart from its direct tumor-targeting activity, also has immunomodulatory activity shown to contribute to efficacy. Single-agent BI-907828 induced anti-tumor immunological memory that controlled tumor growth in a re-challenge study. Moreover, a dual combination with an anti-mouse PD-1 checkpoint inhibitor resulted in synergistic efficacy in a syngeneic mouse model of cancer (AACR 2018, abstract 4866). Here we present data for the triple combination of BI-907828 with two checkpoint inhibitors. In syngeneic mouse tumor models (Colon-26 and B16-F10), the combination of BI-907828 with tool antibodies against mouse PD-1 and mouse LAG-3 shows high response rates of 50-90% with tumor regressions observed for even very large tumors. Moreover, efficacy of the triple combination is superior to each single agent and all dual combinations. An antibody-mediated depletion study suggests a contribution of CD8+ T cells but not of CD4+ T cells to full efficacy of the triple combination. FACS analysis of tumors isolated from Colon-26 tumor-bearing mice indicates that treatment with the triple combination leads to expansion of tumor-infiltrating CD8+ T cells. In summary, BI-907828 is a novel, potent, orally bioavailable MDM2-p53 antagonist that shows synergistic efficacy in a triple combination with antibodies targeting the immune checkpoints PD-1 and LAG-3 in syngeneic mouse models of cancer. BI-907828 is currently under evaluation in a Phase I clinical study (NCT03449381).

#3198

Therapeutic potential of MET inhibitor combined with immune checkpoint blockade in pancreatic cancer.

Mei Gao,1 Miranda Lin,1 Yachao Yang,2 Joseph Kim1. 1 _University of Kentucky, Lexington, KY;_ 2 _Shandong Weihai Municipal Hospital, China_.

Novel Combination Therapeutic Targeting of the Immune Checkpoint PD-1 and c-MET in Pancreatic Cancer

Introduction: Therapeutic regimens incorporating immune checkpoint inhibitors (ICIs) have improved survival in many cancers, but have had little impact in pancreatic ductal adenocarcinoma (PDAC). We previously discovered that these ICIs directly target autonomously expressed and functional PD-1 on PDAC cells. We sought to investigate downstream effectors of PD-1 signaling in PDAC cells as potential novel therapeutic targets.

Methods: We performed a Phospho Explorer Array, which measures the levels of over 200 phospho-specific antibodies and their corresponding total proteins, to determine activated signaling pathways following exposure of PDAC cells to PD-L1 (the ligand for PD-1). Upon detecting upregulation of PD-1 mediated MET signaling, we verified MET pathway activation by exposing PDAC cells (MIAPaCa-2 and PANC-1) to PD-L1 and measuring levels of phosphorylated MET by immunoblotting. Then, we tested whether the MET small molecule inhibitor blocked PD-1 mediated MET signaling. Finally, PDAC patient-derived organoids (PDOs) were utilized to test the cytotoxicity of ICIs alone or in combination with cabozantinib.

Results: The phospho-protein array revealed 10-fold upregulation of MET activity followed exposure of PDAC cells to PD-L1. Western blot assay verified increased levels of MET phosphorylation that varied in a time-dependent manner following PD-L1 treatment in both PANC-1 and MIAPaCa-2 cell lines. Pre-treatment of PDAC cells with cabozantinib completely blocked PD-1/PD-L1 mediated MET phosphorylation. By CellTiter-Glo assay the IC50 of cabozantinib was calculated in MIAPaCa-2 and PANC-1 cells as 9.5 µM and 8.6 µM, respectively. Combining cabozantinib with clinical grade anti-PD-1 monoclonal antibodies nivolumab and pembrolizumab revealed synergistic cytotoxicity in PDAC cells and PDOs.

Conclusion: In these preliminary studies, we report PD-1 activation of the MET pathway and synergy killing of PDAC cancer models with novel combination therapy of cabozantinib and ICIs in PDAC. This positive data warrants further exploration of the interaction of MET with the PD-1/PD-L1 axis and its role in PDAC progression.

#3199

Targeting LHRH-R to improve immune response in ovarian cancer.

Mark S. Kim, Shaolin Ma, Carola Leuschner, Sanghoon Lee, Robert L. Coleman, Anil K. Sood. _UT MD Anderson Cancer Ctr., Houston, TX_.

Background: While immune checkpoint inhibitors have revolutionized the treatment of multiple cancers, such therapies have had limited efficacy in ovarian cancer. We hypothesized that a lytic peptide could stimulate local immune response and enhance efficacy of checkpoint blockade. Here, we tested the effects of a synthetic lytic peptide targeting the luteinizing hormone receptor (LHRH-R), EP-100, on immune therapy in ovarian cancer models.

Methods: We carried out series of in vitro (MTT assay, immunoblot analysis, cytokine array, and immune profiling assay) and in vivo (orthotopic mouse model) experiments to determine the biological effects of EP-100 monotherapy and its combination with checkpoint blockade, α-PD-L1.

Results: We found that LHRH-R positive murine ovarian cancer cells (ID8, IG10, IF5, and 2C12) were sensitive to EP-100 and were specifically killed at low micromolar levels through LHRH-R. The in vivo syngeneic mouse models (ID8 and IG10) demonstrated that EP-100 reduced tumor volume, tumor weight, and ascites volume as a single agent. The greatest effects on tumor volume reduction and ascites volume were observed with the combination of EP-100 and α-PD-L1 antibody, indicating a potentially synergistic effect in vivo. Immune profiling of tumors showed that the population of CD8+ T cells, NK cells, dendritic cells, and macrophages were significantly increased in tumor and ascites treated with α-PD-L1, EP-100 and in the combination group. However, monocytic myeloid suppressor cells, B cells, and regulatory T cells were decreased in tumors treated with α-PD-L1, EP-100 and in the combination group. We found similar effects of EP-100 combination with α-PD-L1 antibody in ascites from each group. These data indicate that EP-100 promotes a tumor microenvironment that is favorable for immune response. In vitro cytokine arrays revealed that EP-100 induced IL-1α, IL-33, CCL20, VEGF, and LDLR secretion. Among them, we validated increasing Il-33 level following EP-100 treatment in vivo and in vitro and determined the specific biological role of CD8+ T cell activation with IL-33 gene silencing using siRNA and Cas9-CRISPR approaches. In addition, we found that CD8+ T cell expressed a low level of LHRH-R and had minimal direct effects from EP-100. Conclusion: These data demonstrate strong efficacy of EP-100 and checkpoint blockade therapy. Our results provide new directions to enhance the efficacy of immune therapies for ovarian cancer.

#3200

**Immunotuning CD8** + **T cells with BCAT1 inhibition to increase the efficacy of anti-PD-1 therapy and to eradicate moderately immunogenic tumors.**

Adonia E. Papathanassiu,1 Dong-Wook Kim,2 Kwon-Sik Park,2 Jeong Hun Ko,3 Jacques V. Behmoaras3. 1 _Ergon Pharmaceuticals, LLC, Washington, DC;_ 2 _University of Virginia, Charlottesville, VA;_ 3 _Imperial College London, London, United Kingdom_.

Activation and differentiation of adaptive and innate immune cells require metabolic reprogramming involving diverse metabolic processes. Understanding these processes affords the opportunity to influence and ultimately re-direct the function and fate of these cells. Here, we present data suggesting that exposure of newly activated human CD8+ T cells to a small molecule inhibitor (ERG245; 50-400 μM) of BCAT1 (the cytosolic isoform of the enzyme responsible for the first step in the catabolism of leucine, isoleucine, and valine) resulted in inhibition of T cell proliferation, inhibition of IFNγ and Granzyme B production, and upregulation of PD-1. The antiproliferative effect, as determined by CFSE dilution, required inhibition of BCAT1 during the first 24 hrs of T cell activation and it was reversible upon early withdrawal of the inhibitor. Early withdrawal of ERG245 reversed the observed immunosuppression, ultimately giving rise to CD8+ T cells with higher proliferative capacity and increased cytotoxicity compared to untreated control cells. Exposure of CD8+ T cells to ERG245 resulted in a distinct metabolic phenotype including reduced concentrations of α-ketoglutaric acid (αKG), and lower levels of trimethylation of the lysine 4 (K4) and lysine 27 (K27) sites of histone 3 (H3), which implies that BCAT1 inhibition has epigenetic effects on human CD8+ T cells by modulating αKG-dependent histone demethylases. Similar observations were made with CD8+ T cells, isolated from the spleens of Bcat1 KO mice. In vivo, ERG245 (given at 5 mg/kg ip, bid at days 0, 1 and 2 of treatment), dramatically increased the efficacy of an anti-PD-1 antibody (clone RMP1-14; given at 10 mg/kg at days 0, 4, 7, and 11 of treatment) in the CT26 colon cancer model. Specifically, the combination of ERG245 and anti-PD-1 eradicated established tumors within a week of treatment initiation (cure rate of >80%), whereas the monotherapies (ERG245 or anti-PD-1 alone) did not improve the disease outcome. The data suggest that the temporal exposure of moderately immunogenic tumors to BCAT1 inhibition sensitizes the tumors to checkpoint inhibition.

#3201

Rigosertib, a Ras mimetic, inhibits melanoma cell viability and synergizes with anti-PD1 to promote anti-tumor immune responses.

Chi Yan,1 Premkumar E. Reddy,2 Ann Richmond3. 1 _Vanderbilt University, Nashville, TN;_ 2 _Icahn School of Medicine at Mount Sinai, New York, NY;_ 3 _Tennessee Valley Healthcare System, Nashville, TN_.

Activating mutations in BRAF or NRAS are present in 40% and 21% of melanoma patients, respectively, leading to enhanced cell survival and proliferation. Rigosertib (RGS) is a non-ATP-competitive small molecule RAS mimetic that has the potential to block RAS-RAF-MEK-ERK and PI3K-AKT-mTOR signaling pathways and interfere with CRAF interaction with PLK1 and consequently its centrosomal localization. Here, we demonstrate that RGS inhibits the cell viability at µM levels of human (including A375/SKMel2/SKMel5/HS294T) and murine (including B16F10 and YUMM2.1/3.3/4.1/5.2) melanoma cell lines with a variety of somatic mutational backgrounds. We discovered that RGS treatment immediately (<15mins) and constantly (up to 24hrs) suppresses PI3K-AKTT308 and mTORC2-AKTSer473 phosphorylation. Using the murine melanoma cell line YUMM3.3 (BrafV600E/wtCdkn2-/-), we showed that RGS monotherapy, elevated the production of mitochondrial reactive oxygen species, promoted cellular apoptosis, suppressed mitosis in vitro, and inhibited tumor growth in C57BL/6 mice. The optimal in vivo dose of RGS (300mg/kg), which exhibited >50% inhibition of tumor volume and tumor weight, was well tolerated in mice. RGS-treated tumors exhibited an inflammatory tumor microenvironment (TME) with enrichment of dendritic cells and CD45-MHCII+ cells, elevation in frequency and activation of both CD4+ and CD8+ T cells and NK cells, but a decrease in the level of tumor-infiltrating macrophages. Of note, treatment with RGS plus αPD-1 checkpoint blockade synergistically inhibited tumor growth by ~70%. The RGS + αPD-1 combination treatment, but not the monotherapies, reduced the frequency of exhausted PD-L1+LAG3+TIM3+ CD8+ T cells at the tumor sites, as well as in the tumor-draining lymph nodes. Conclusion: These results suggest that RGS, which is a Ras mimetic, may be used in combination with anti-PD-1 immunotherapies to enhance anti-tumor immunity and optimize the treatment of melanoma. This combination therapy warrants a clinical study.

The authors sincerely thank Onconova Therapeutics, Newtown, PA 18940 for kindly supplying Rigosertib for this work.

#3202

A probody drug conjugate targeting CD166 (ALCAM) enhances preclinical antitumor activity of a probody therapeutic targeting PD-1.

Erwan Le Scolan, Tiffany Tse, Michael Krimm, Will Garner, Hikmat Assi, Jennifer Razo, Laurie Wong, Kenneth Wong, Victoria Singson, Jennifer Leong, Linnea Diep, Jennifer Richardson, Siew Schleyer, Dylan Daniel, Marcia Belvin, Michael Kavanaugh. _CytomX Therapeutics, South San Francisco, CA_.

Immune checkpoint blockade therapies have been shown to induce potent and durable anti-tumor immunity in many cancer types. Nevertheless, not all patients benefit from immunotherapy, and immune-related adverse events remain a problem. Recently, it has been demonstrated that Antibody Drug Conjugates (ADCs) are not only capable of killing cancer cells but also can act to induce the immunogenic cell death of tumor cells as well as directly activate dendritic cells. These results provided a rationale to combine ADCs with immunotherapy to enhance the potential of immune checkpoint blockade therapies in a broader population of patients.

CytomX Therapeutics has developed a new class of antibodies called Probody™ therapeutics, designed to widen the therapeutic window by minimizing binding to target in healthy tissue while being specifically activated in the tumor microenvironment (TME) by tumor-associated proteases. Probody technology has been evaluated in preclinical studies in several antibody formats, with efficacy and increased safety windows observed for Probody therapeutics targeting the PD-1 pathway, Probody drug conjugates (PDCs) targeting highly expressed tumor antigens, and T-cell engaging bispecific Probody therapeutics. Here we extend our evaluation of the Probody platform to the combination of CX-2009, an investigational PDC targeting human CD166, with an investigational Probody therapeutic targeting PD-1.

To evaluate the anti-tumor activity of PDC CX-2009 in a syngeneic mouse model, human CD166 was overexpressed on the surface of the CT-26 murine colon carcinoma cell line. The combination treatment of CX-2009 with a surrogate mouse anti-PD-1 Probody molecule significantly inhibited tumor growth in human CD166 positive CT-26 tumor-bearing mice as compared to CX-2009 or anti-PD-1 Probody molecule alone. Tumor rejection is partially dependent on CD8+ T cells as illustrated by the evidence of a CD8+ memory T cell response in a re-challenge assay, and a reduced activity of CX-2009 alone or in combination with a mouse anti-PD-1 Probody molecule after CD8+ T cell depletion. The immunogenic potential of CX-2009 was further evaluated in multiple in vitro assays using human cancer cells and human PBMCs. In contrast to its cytotoxic activity towards CD166+ tumor cells, CX-2009 spares T cells and may enhance T cell priming.

These preclinical data demonstrate the potential utility of a combination of PDC CX-2009 with a Probody therapeutic targeting the PD-1 pathway. Generally, these data highlight the potential to combine ADCs or PDCs with immune checkpoint blockade therapies.

PROBODY is a trademark of CytomX Therapeutics, Inc.

#3203

Intratumoral STING activation normalizes tumor vasculatures and synergizes with anti-angiogenic therapy to enhance cancer immunity.

Hannah Yang, Hyojoong Kim, Joo Hoon Kim, Hong Jae Chon, Chan Kim. _CHA Bundang Medical Center, Seongnam, Republic of Korea_.

Purpose: The stimulator of interferon genes (STING) signaling pathway is a critical link between innate and adaptive immunity, and induces anti-tumor immune responses. STING is expressed in vasculatures, but its role in tumor angiogenesis has not been elucidated. Here we investigated STING-induced tumor vascular remodeling and the potential of STING-based combination immunotherapy.

Experimental Design: We examined the endothelial STING expression pattern and clinical implications in human malignancies. STING-induced vascular remodeling was studied using STING-deficient or wild-type mice. Implanted LLC tumors and transgenic MMTV-PyMT breast tumors were intratumorally injected with STING agonists (cGAMP or RR-CDA), with or without anti-VEGFR2 antibody and/or immune checkpoint inhibitors. The tumor microenvironment was evaluated by histologic and immune profiling analyses.

Results: Endothelial STING expression was correlated with enhanced T-cell infiltration, reduced lymphovascular invasion, and prolonged survival in human colon and breast cancer. Intratumoral STING activation normalized tumor vasculatures, as shown by increased pericyte coverage and intact basement membrane, which was mediated by upregulation of vascular stabilizing genes (e.g., Angpt1, Pdgfrb, and Col4a) and endothelial-lymphocyte interaction genes. These effects were dependent on type I interferon signaling and CD8+ T cells. STING activation also induced M1-like macrophage polarization within tumors. Notably, STING-based immunotherapy was maximally effective when combined with VEGFR2 blockade and/or immune checkpoint blockade (αPD-1 or αCTLA-4), leading to complete regression of immunotherapy-resistant tumors.

Conclusions: Our data show that intratumoral STING activation can normalize tumor vasculature and the tumor microenvironment, providing a rationale for combining STING-based immunotherapy and anti-angiogenic therapy.

#3204

Pre-immunization of donor lymphocytes with GITR agonistic antibody enhances antitumor immunity in autologous hematopoietic stem cell transplantation.

Kenta Narumi, Marina Henmi, Chihiro Shibasaki, Aya Hirata, Yukihiro Mizoguchi, Kazunori Aoki. _National Cancer Center Research Institute, Tokyo, Japan_.

Lymphopenia-induced homeostatic proliferation (HP) of T cells following autologous hematopoietic stem cell transplantation (HSCT) skews the T-cell repertoire by engaging tumor-associated antigens, and induces an antitumor immunity. However, the cure by autologous HSCT alone is difficult in case of solid cancers. Glucocorticoid-induced tumor necrosis factor receptor (GITR) is well-known immune-checkpoint molecules. GITR-GITR ligand interaction can provide a co-stimulatory signal to both CD4+ and CD8+ naïve T cells, enhancing proliferation and effector function. Since the tumor-reactive lymphocytes preferentially proliferate during the condition of HP, due to the synergic effect with encountering their cognate antigens in tumor-bearing host, we hypothesize that administration of GITR agonistic antibody (Ab) could increase tumor-responsive T cells in the graft and induce a strong antitumor immunity in HSCT recipients. First, we confirmed that the intraperitoneal administration of GITR Ab into CT26 subcutaneous tumor model mice significantly increased the number of IFN-γ+ lymphocytes in response to CT26 cells in the spleens and regional lymph nodes. Second, to examine the antitumor effect of HSCT, the bone marrow cells and lymphocytes were harvested from CT26 tumor-bearing mice as donor cells, and infused into the lethally-irradiated recipient mice with CT26 inoculation (autologous HSCT model). In the HSCT recipients infused with primed lymphocytes by GITR Ab, the CT26 tumor growth was markedly suppressed compared with the recipients infused with non-primed lymphocytes, and tumors disappeared in all recipient mice infused with primed lymphocytes. An ELISpot assay showed that the number of IFN-γ+ spots in response to CT26 cells was significantly increased in HSCT recipient mice infused with GITR Ab-primed and non-primed donor lymphocytes as compared to wild type mice until 4 weeks after HSCT; while the frequency in recipients with primed lymphocytes was markedly elevated compared with that in mice with non-primed lymphocytes at 2 weeks after HSCT. At present, autologous HSCT is clinically practiced after intensive chemotherapy in patients with lymphomas and solid cancers such as neuroblastoma and sarcoma, the infusion of hematopoietic stem cells and pre-immunized lymphocytes after high dose chemotherapy is an attractive and feasible clinical introduction of HSCT-mediated immunotherapy. Although the subcutaneous tumors of NHOS murine osteosarcoma cells were resistant for HSCT, the infusion of GITR Ab-primed lymphocytes significantly induced stronger antitumor effect than the non-primed lymphocytes did. In conclusion, the combination of HSCT with pre-immunization by GITR Ab can induce a strong antitumor immunity. This therapeutic strategy deserves an evaluation in future clinical trial for solid cancers.

#3205

Superiority of response to carboplatin vs cisplatin in combination with pembrolizumab for head and neck squamous cell carcinoma predicted using ex vivo platform, CANscript.

Vidushi Kapoor,1 Munisha Smalley,2 Amit Verma,3 Saravanan Thyiagarajan,1 Biswanath Majumder,1 Nandini Basak,1 Abhishek Basu,1 Pradeep Kar,1 K.s. Sabitha,4 D.C. Doval,5 Aaron J. Goldman2. 1 _Mitra Biotech, Woburn, MA;_ 2 _Harvard Medical School, Cambridge, MA;_ 3 _Max Super Speciality Hospital, India;_ 4 _Kidwai Memorial Institute of Oncology, India;_ 5 _Rajiv Gandhi Cancer Institute & Research Centre, India_.

Background: Immunotherapy is a developing paradigm in the treatment of head and neck squamous cell carcinoma (HNSCC). An ongoing clinical trial (KEYNOTE-048) examines the efficacy of combining a PD-1 checkpoint inhibitor (pembrolizumab) with carboplatin or cisplatin (pt) and 5-fluorouricil (5-FU) vs. 5-FU+pt+cetuximab (EXTREME). Early evidence from this trial suggests overall survival is improved with the combination of pembrolizumab compared to EXTREME. However, no evidence has yet been reported around carboplatin vs. cisplatin in these regimens.

Methods: Here, with informed consent under IRB, fresh tumor biopsies were obtained from a randomly-selected cohort of HNSCC patients diagnosed with metastatic or locally advanced disease (N=45). We then employed CANscriptTM, a clinically-validated ex-vivo tumor platform that can predict clinical efficacy of agents using a proprietary algorithm, termed M-Score. The following regimens were tested in CANscript: (i) pembrolizumab as single agent, (ii) combination of pembrolizumab + Pt + 5-FU and (iii) cetuximab + Pt + 5-FU regimen. Importantly, Pt was run as either carboplatin or cisplatin in the combination regimens. In addition, PDL-1, CD4 and CD8 status was quantified for every patient by immunohistochemistry.

Results: Consistent with the recently reported data from KEYNOTE-048, we predicted, based on M-Score, that single agent pembrolizumab is inferior to either EXTREME or the pembrolizumab chemotherapy combination regimens. Interestingly, however, when samples were analyzed based on the type of platinum used (Carboplatin vs. Cisplatin) we determined that greater clinical response is achieved with the combination of Pembro/Carbo/5-FU (45.5% or 10/22) vs. Pembro/Cis/5-FU (21.7% or 5/23). In contrast, we predicted EXTREME regimen alone results in similar response rates regardless of the addition of Carbo or Cis (31.8% vs. 30.4%, respectively). Upon further interrogation we observed changes in immunohistochemical staining of PD-L1, CD8 and CD4 before vs. after treatment with either Cis or Carbo, but did not find a correlation between PD-L1 status at baseline with response. In addition, we found unique patterns of immunological response in the carbo vs. cis arms, which may explain why pembrolizumab performed better.

Conclusions: These results demonstrate the utility and highlight the ability of CANscript system to profile clinical response of immunotherapy. More importantly, our evidence that discrete differences exist between carboplatin and cisplatin could help future design of combination chemotherapy-immunotherapy regimens.

#3206

Host immunity following near infrared photoimmunotherapy is enhanced with PD-1 checkpoint blockade to eradicate established highly antigenic tumors.

Tadanobu Nagaya, Jay Friedman, Yasuhiro Maruoka, Fusa Ogata, Shuhei Okuyama, Paul E. Clavijo, Peter L. Choyke, Clint Allen, Hisataka Kobayashi. _National Institutes of Health, Bethesda, MD_.

Near infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer treatment that employs a targeted monoclonal antibody-photo-absorber conjugate (APC). Following antibody localization of the APC to a tumor cell surface antigen, NIR light is used to induce highly selective cytolysis. Extensive pre-clinical evidence demonstrates that NIR-PIT is effective at inducing tumor cell lysis using a number of different antibody-APC conjugates and NIR-PIT targeting EGFR with a cetuximab-APC conjugate has shown promising results in phase 2 clinical evaluation for the treatment of recurrent head and neck squamous cell carcinoma. NIR-PIT induces rapid, necrotic cell death that yields innate immune ligands that activate dendritic cells, consistent with immunogenic cell death. Yet, NIR-PIT treatment of syngeneic tumors in wild-type mice has mostly failed to induce durable regression of established tumors, suggesting the presence of one or more mechanisms of resistance to formation of meaningful anti-tumor immunity. In this study, we hypothesized that NIR-PIT could induce anti-tumor immunity that was being restricted by the PD-1/PD-L1 signaling axis, and that PD-1 could reverse innate immune resistance to induce durable, effective anti-tumor immune responses. Using a CD44-targeting APC, we demonstrated the ability of PD-1 immune checkpoint blockade to significantly enhance antigen-specific anti-tumor immunity induced by NIR-PIT in multiple syngeneic tumor models (MC38, LLC, and MOC1 cancer cell line). In two of three models, NIR-PIT monotherapy halted tumor growth, enhanced dendritic cell tumor infiltration, and induced de novo tumor antigen-specific T-cell responses absent at baseline. The addition of PD-1 blockade appeared to reverse adaptive immune resistance, resulting in both enhanced pre-existing tumor antigen-specific T-cell responses and enhanced de novo T-cell responses induced by NIR-PIT. Enhanced immune responses appeared to correlate with shared tumor antigen expression, suggesting that antigenicity is a major determinant of response to combination NIR-PIT and PD-1 blockade. Combination treatment induced complete rejection of MC38 tumors treated with NIR-PIT as well as untreated, distant tumors. Accordingly, tumor antigen-specific T-cell responses were measured in both treated and untreated tumors, validating the development of systemic anti-tumor immunity. Cured mice resisted tumor challenge, indicating the presence of systemic immune memory. Cumulatively these results demonstrate reversal of adaptive immune resistance following induction of innate and adaptive immunity by NIR-PIT, resulting in high rates of tumor rejection and/or significant tumor growth control in highly antigenic syngeneic models of cancer.

#3207

Preclinical development of first-of-kind dual-targeted off-the-shelf CAR-NK cell product with engineered persistence for an effective treatment of B cell malignancies.

Jode Goodridge,1 Sajid Mahmood,1 Huang Zhu,2 Svetlana Gaidarova,1 Robert Blum,2 Ryan Bjordahl,1 Frank Cichocki,3 Hui-Yi Chu,1 Greg Bonello,1 Tom Lee,1 Brian Groff,1 Karl-Johan Malmberg,4 Bruce Walcheck,3 Jeffrey Miller,3 Dan Kaufman,2 Bahram Valamehr1. 1 _Fate Therapeutics, San Diego, CA;_ 2 _University of San Diego, CA;_ 3 _University of Minnesota, Minneapolis, MN;_ 4 _University of Oslo, Oslo, Norway_.

The unprecedented success of chimeric antigen receptor (CAR) and monoclonal antibody (mAb) -based immune-therapies has provided a clear indication that a system as complex as human immunity can be harnessed, even enhanced, toward a growing number of hematological cancers. Here we describe pre-clinical progress to develop a multi-functional induced pluripotent stem cell (iPSC)-derived natural killer (iNK) cell platform that combines engineered longevity with CAR and mAb-based modalities to leverage the intrinsic polyfunctionality of NK cells. As frontrunners of immune surveillance, NK cells employ a diverse array of germline encoded receptors in distinct combinations, which engage multiple signaling pathways to deliver potent effector responses that can be directed toward tumor cells, drive rapid proliferation, and pave the way for recruitment of adaptive immunity. Specific engagement of multiple signaling pathways was achieved in iNK cells through design of an NK cell-centric CAR combining the transmembrane domain of activating receptor NKG2D with intracellular signaling domains of 2B4 and CD3ζ. Recombining an anti-CD19 scFv onto this signaling platform, CAR modified iNK cells produced specific in vitro recognition of CD19+ B cell lymphoma cells in short term and long term cytotoxicity assays (84% vs 40% clearance of tumor cells at 60H, p<0.001). Further introduction of a fusion receptor consisting of Interleukin-15 (IL15) with IL15 receptor α, enabling autonomous IL15 stimulation, greatly improved iNK longevity and functional persistence in animal models. Moreover, iNK cells modified with IL15 fusion receptor showed enhanced functional maturation including KIR expression and effector molecules such as granzyme B ( ≥2 fold). While iNK cells with anti-CD19 CAR delayed tumor progression in vivo prior to relapse, iNK cells engineered with anti-CD19 CAR and IL15/IL15 receptor were curative against B cell lymphoma, (p<0.002). Expression of CAR and IL15 fusion receptor was then combined with a third modality, a high affinity CD16a receptor modified to prevent proteolytic cleavage (hnCD16). These multifunctional iNK cells demonstrated enhanced directed cytotoxicity in vitro in combination with rituximab against CD19+ targets (>99% vs 90% clearance of tumor cells) and CD19- targets (>99% vs 50% clearance of tumor cells by iNK with anti-CD19 CAR alone, p<0.0001), revealing a unique opportunity to combine CAR with a universal targeting modality to mitigate antigen escape and address heterogeneity in the tumor population by a multi-node targeting strategy. The resulting product, FT519, is designed to provide a flexible, potent and persistent engineered immune cell that utilizes the intrinsic versatility of NK cells to enable a highly effective combination therapy in a single, standardized, scalable, off-the-shelf platform.

#3208

Using T cell engineering plus triple checkpoint blockade to enhance the efficacy of adoptive immunotherapy in ovarian cancer.

Kristin G. Anderson, Madison G. Burnett, Valentin Voillet, Edison Y. Chiu, Breanna M. Bates, Nicolas M. Garcia, Raphael Gottado, Philip D. Greenberg. _Fred Hutchinson Cancer Research Ctr., Seattle, WA_.

Over 20,000 women are diagnosed with ovarian cancer in the United States annually, and over half will die within 5 years. Outcomes have changed little in the last 20 years, highlighting the need for therapy innovation. One promising new strategy employs immune T cells engineered to target proteins uniquely overexpressed in tumors; such T cell immunotherapies have the potential to control tumor growth without toxicity to healthy tissues. In considering candidate immunotherapy targets, we focused on mesothelin (MSLN), which contributes to invasive progression and malignancy in ovarian cancer but has limited expression in healthy cells. We showed that T cells engineered to express a human or mouse MSLN-specific high-affinity T cell receptor (TCRMSLN) can kill human patient-derived ovarian cancer cell lines or the murine ID8 cell line, respectively. In a disseminated ID8 tumor model, adoptively transferred TCRMSLN T cells preferentially accumulated within established tumors, delayed ovarian tumor growth, and significantly prolonged mouse survival. However, our data also revealed that the ovarian tumor microenvironment (TME) limits engineered T cell persistence and cancer cell killing.

To identify immunosuppressive features active in both the human and murine ovarian TME, we performed gene expression analyses. Deep transcriptome profiling confirmed similar gene expression signatures in human cancers and in the preclinical ID8 model. Among these, RNA sequencing detected PD-L1, Galectin-9 and Galectin-3, ligands for CD8 T cell-expressed PD-1, Tim-3 and Lag-3 'checkpoint' receptors, respectively. We also measured PD-L1, Galectin-9 and Galectin-3 expression in human and mouse ovarian cancers by flow cytometry and immunohistochemistry, and multiplex immunohistochemistry of human ovarian tumors confirmed the presence of endogenous CD8 T cells expressing one, two or all three inhibitory receptors. Moreover, flow cytometry revealed that TCRMSLN-transduced T cells increase expression of the inhibitory receptors in ID8 tumors relative to cells in the spleen as early as three weeks after transfer, in association with decreased production of anti-tumor cytokines.

Based on our results, we hypothesized that we could overcome engineered T cell suppression via inhibitory receptor ligation. We treated tumor-bearing mice with TCRMSLN T cells plus anti-PD-1, anti-Tim-3 and/or anti-Lag-3 checkpoint-blocking antibodies, targeting up to three inhibitory receptors simultaneously. Triple checkpoint blockade dramatically increased anti-tumor cytokine production by intratumoral TCRMSLN T cells. As many solid tumors both overexpress MSLN as well as PD-1, Tim-3 and Lag-3, the use of multi-checkpoint blockade with engineered T cells has real potential to also enhance the efficacy of engineered adoptive T cell therapy against other malignancies.

#3209

BLZ945 and anti-PD-1 combination immunotherapy modulates the immune landscape in pancreatic ductal adenocarcinoma.

Chelsie K. Sievers, Philip B. Emmerich, Hanna R. Rainiero, Connor Maloney, Rosabella Pitera, Cheri A. Pasch, Linda Clipson, Kristina A. Matkowskyj, Fotis Asimakopoulos, Dustin A. Deming. _University of Wisconsin, Madison, WI_.

Background: Immune therapies have shown great promise for some cancers. To date, pancreatic ductal adenocarcinomas (PDAC) have been largely resistant to immunotherapeutic approaches in part due to their exclusion of tumor infiltrating lymphocytes. The mechanisms by which this exclusion occurs are not well-characterized but are thought to involve both innate and adaptive immune responses.

Methods: The primary objective of this study was to investigate how dual anti-PD-1/anti-CSF1R therapy changes the tumor immune cell niche and tumor growth in an allograft version of the KPC mouse model of PDAC (Pdx-1-Cre; LSL-KrasG12D; LSL-Trp53 R172H). Anti-PD1 therapy in the form of monoclonal antibody from BioXCell® was dosed twice weekly via intraperitoneal injections at 200µg/mouse. Anti-CSF1R in the form of the small-molecule inhibitor BLZ945 from Selleckchem® was dosed daily via oral gavage at 200 mg/kg. Human THP-1 cells differentiated and polarized to macrophages were used for viability and gene expression analyses.

Results: In B6 recipient mice, the combination anti-PD-1 plus BLZ945 resulted in reduced percent tumor volume change (11% ±17) versus vehicle treated controls (121% ±174) (p=0.02). Flow cytometry of the spleens of tumor-bearing mice demonstrated an increase in conventional dendritic cells with combination treatment compared to controls (15.3 ±2.3% vs 8.3 ±0.6%, respectively; p=0.02). Mice lacking conventional dendritic cells (B6.BATF3-/-) had an increased percent tumor volume change (84% ±78) compared to B6 wild type mice (11% ±16) (p=0.01) when receiving combination treatment. IHC staining of allografted tumors revealed a trend towards increased infiltrating CD8+ T cells/hpf in B6 wild type mice treated with anti-PD-1 or combination therapy (21±24/hpf) compared to control treated mice (11±18/hpf). A similar trend was observed in B6.BATF3-/- mice (2.9±4.4/hpf vs 0.5±0.50/hpf); however, the magnitude was greatly reduced compared to B6 wild type mice. Interestingly, BLZ945 treated tumors did not show evidence of statistically significant reduced macrophage recruitment in vivo (268 vs 241, p=0.16). Macrophage viability in vitro was also unaffected by BLZ945 treatment (fold change in viability 0.03, p=0.6). Preferential changes in macrophage polarization in vitro were observed, with significant reductions in M2 polarization markers Arg-1, YM-1, and CD206 (p= 0.0005, 0.0029, and 0.012, respectively).

Conclusions: The combination of an anti-PD-1 agent in combination with inhibition of the macrophage-predominant cytokine, anti-CSF1R, slows PDAC tumor growth in a conventional dendritic cell-dependent manner. Further characterization of the interplay between innate and adaptive immune cells upon immunotherapy treatment should be investigated to increase the efficacy of these therapeutics.

#3210

A potential immunotherapeutic approach for the treatment of osteosarcoma.

Marlene Hennessy,1 Souri Felix,2 Andrew Wahba,2 Saul Kivimae,1 Takahiko Miyama,1 Mariella Cabrera,3 Maria Gabriela Segura,4 Jonathan Zalevsky,1 Willem Overwijk,1 Nancy Gordon2. 1 _Nektar Therapeutics, San Francisco, CA;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 3 _Lincoln Medical and Mental Health center, NY;_ 4 _Children's Memorial Hermann, UT Health Science Center, Houston, TX_.

Purpose: Survival of osteosarcoma (OS) patients has remained stagnant for the past 30 years thus the need for new therapeutic strategies.

NKTR-214 is a novel cytokine therapy that provides sustained activation of the IL-2 pathway with a bias to the IL-2 receptor CD122 (IL-2Rbeta gamma) so that it can selectively expand and proliferate CD8+ T cells. Compared to recombinant human IL-2, NKTR-214 demonstrated superior therapeutic benefit in a melanoma mouse model and was well-tolerated. Because systemic IL-2 has shown limited efficacy against OS, we hypothesize that NKTR-214 could potentially offer increased therapeutic benefit.

Methods: The K7M2/K7M3 disseminated and orthotopic OS mouse models were used. Three doses of NKTR-214 were given intravenously every 9 days. In the K7M2 disseminated model a prophylactic or therapeutic approach was taken where treatment was started right after tumor injection or when there was evidence of lung metastases. In the K7M3 orthotopic model, treatment was started once tumor was established (day 20) or 10 days after tumor injection. Bone tumor size was assessed by serial plain X-rays. Immunohistochemistry was performed to assess histologic tumor response (bone and lung) as well as evidence of immune cell infiltrate. Tumor immune infiltrate was analyzed by flow cytometry.

Results: We demonstrated therapeutic effect of NKTR-214 as a single agent. In the disseminated model, NKTR-214 administered every 9 days i.v. prophylactically resulted in an increase in the median survival of mice with OS lung metastases (21 days untreated vs. 51 days NKTR-214). Survival was similar when therapeutic treatment was started after tumor nodules were apparent in the lung and continued every 9 days (51 days vs 52.5 days). In the orthotopic model, we demonstrated NKTR-214 efficacy against K7M3 primary bone tumor and the development of pulmonary metastases. The total tibia tumor area in the NKTR-214-treated group was significantly lower than in the untreated group (p<0.01). Likewise, the total lung metastases tumor area as well as the number of micrometastases was significantly lower in the treated as compared to the untreated group (p<0.009). We also demonstrated increase in the number of CD8+ and CD4+ T cells in mice treated with NKTR-214 single agent as compared to the untreated group. This data provides rationale for the use of NKTR-214 alone or in combination with checkpoint inhibitors or adoptively transferred NK cells or T cells as a potential effective therapy for OS.

Conclusion: In conclusion, our findings demonstrate that NKTR-214 is effective in controlling the growth of primary OS, regrowth of tumor after amputation and inhibition of lung metastasis formation. These pre-clinical studies provide the basis for potential translation of NKTR-214-based immunotherapeutic regimens for OS into the clinic.

#3211

MTL-CEBPA combined with radiofrequency ablation and immunotherapy enhances immunological anti-tumour response in an HCC mouse model.

Mikael H. Sodergren,1 Kai-Wen Huang,2 Vikash Reebye,1 Cheng-Ean Chee,3 Dimitris Zacharoulis,4 Robert Habib,5 David Blakey,5 John Rossi,6 Nagy Habib1. 1 _Imperial College, London, United Kingdom;_ 2 _National Taiwan University Hospital, Taipei, Taiwan;_ 3 _National University Cancer Institute, Singapore, Singapore;_ 4 _University Hospital Larissa, Larissa, Greece;_ 5 _Mina Therapeutics, London, United Kingdom;_ 6 _Beckman Research Institute of City of Hope, Duarte, CA_.

The transcription factor CEBPA (CCAAT/enhancer-binding protein alpha) is recognised for its antiproliferative effects. MTL-CEBPA is a small activating RNA drug which upregulates gene expression of CEBPA and amongst other effects causes and immunomodulatory effect on peripheral granulocytes. Radiofrequency ablation (RFA) is standard treatment for some tumour types such as liver cancer and induces modulation of both innate and adaptive immune systems. To investigate any synergistic effect of MTL-CEBPA with RFA and immune checkpoint inhibition we initiated a reverse translation experiment, where syngeneic BNL hepatocellular carcinoma tumour cells were injected in the two opposite flanks of immunocompetent BALB/c mice (n=8 in each group). Treatments for hepatoma bearing mice included: 1) RFA on one flank (day 0), 2) Anti-PD-1 inhibition immunotherapy (RMP1-14 antibody, BioXCell, West Lebanon, NH, USA at 200 μg IV/mouse/dose on days 0, 2 & 5) and 3) MTL-CEBPA (3mg/kg IV/mouse/dose on days 0, 2 & 5) as well as combinations of all 3 interventions. We found that tumour control on the opposite flank was augmented by addition of RFA and most significant in the triple combination group (RFA + anti-PD1 + MTL-CEBPA) in which 2/8 animals showed a complete response and 5/8 a partial response. This was also the only group that showed a statistically significant increase in CD8+ (Cytotoxic) as well as CD49b+/CD45+ (Natural Killer) tumour infiltrating lymphocytes. These data suggest a clinical role for combination treatment with checkpoint blockade, RFA and MTL-CEBPA through synergistic priming of the immune tumour response, enabling RFA to have a pronounced anti-tumour abscopal effect.

#3212

Patient-level pharmacodynamics of response to combined ipilimumab and nivolumab for gastric cancer using a human autologous ex-vivo platform, CANscript.

Vidushi Kapoor,1 Munisha Smalley,1 Nandini Pal Basak,2 Abhishek Basu,2 Manjusha Biswas,2 Manas Kumar Mandal,3 Pinaki Roy,3 Pradip K. Majumder,1 Aaron Goldman1. 1 _Mitra Biotech, Woburn, MA;_ 2 _Mitra Biotech, Bangalore, India;_ 3 _Nil Ratan Sircar Medical College and Hospital, Kolkata, India_.

Background: Combined cancer immunotherapy is an emerging paradigm for the treatment of cancer. PD-L1, the cognate receptor for the programmed death receptor 1 (PD-1) is expressed in up to 40% of gastric/gastroesophageal junction (GEJ) tumors. Despite this, patients show modest responses to PD-1 inhibitors. A recent clinical trial, Checkmate-032, demonstrated superior efficacy when PD-1 inhibitor, Nivolumab, was combined with CTLA-4 inhibitor, Ipilimumab. Despite this, biomarkers that predict clinical response remain poorly understood.

Methods: Here, with informed consent under IRB, fresh tumor biopsies were obtained from a randomly-selected cohort of gastric/GEJ patients (N=39). We then employed CANscriptTM, a clinically-validated human autologous ex-vivo tumor platform to interrogate the pharmacodynamics and prognostic efficacy (M-Score) of nivolumab +/- ipilimumab. PDL-1 status was determined for every patient by immunohistochemistry, flow cytometry was performed to analyze intratumor CD4+ and CD8+ T-cell profiles before and after treatment, and RNA seq. gene expression was performed to understand mechanistic changes under drug pressure.

Results: We predicted, based on M-Score, that single agent nivolumab results in an overall response rate (ORR) of 10%, which closely matched previous single agent results from Checkmate-032 (10%). In contrast, we determined the combination of ipi+nivo resulted in a response rate of 20%, a finding consistent with reported clinical data (Checkmate-032; ORR 18%). Interestingly, we determined that only 28% of patients who responded to the ipi+nivo combination were predicted to respond to the monotherapy, suggesting the increased efficacy of the combination. RNA seq revealed unique features of adaptive immunity in the subset of patients who were predicted to respond to the combination immunotherapy, while PD-L1 status was a less-important biomarker to predict response.

Conclusions: These results demonstrate the utility of the CANscript system to profile clinical response of immunotherapy. Our efforts may better inform clinical trial design and patient stratification before therapy to improve the potential for response.

#3213

Combination of anti-IL-6 and anti-PD-L1 antibodies synergistically reinvigorates cancer immunity by activating both CTL and Th1 cells in mice.

Yasuharu Nishimura,1 Koji Fujieda,1 Azusa Miyashita,2 Satoshi Fukushima,2 Tokunori Ikeda,3 Yosuke Kubo,2 Satoru Senju,1 Hironobu Ihn,2 Hiroyuki Oshiumi,4 Hirotake Tsukamoto4. 1 _Department of Immunogenetics, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan;_ 2 _Department of Dermatology and Plastic Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan;_ 3 _Department of Clinical Investigation, Kumamoto University Hospital, Kumamoto University, Kumamoto, Japan;_ 4 _Department of Immunology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan_.

We previously demonstrated that Th1 differentiation of tumor-specific CD4+ T cells was attenuated in tumor-bearing or aged mice in an IL-6-dependent manner (Nat. Comm. 6: e6702, 2015, Cancer Immunol. Res. 1: 64, 2013). Furthermore we found that accompanied by the systemic increase of soluble IL-6 receptor (sIL-6R) in cancer patients, inhibition of IL-6 trans-signaling mediated through the sIL-6R restored the Th1 responses, their helper activity toward CD8+ T cells, and anti-tumor activity in tumor-bearing mice (Cancer Res. 77: 2279, 2017). The systemically increased sIL-6Rs were mainly produced by myeloid cells. Moreover, cMaf-deficient CD4+ T cells were resistant to Th1 suppression and impairment of T cell-mediated anti-tumor immunity induced by IL-6/sIL-6R indicating that c-Maf activity was responsible for Th1 suppression. Myeloid cell-derived sIL-6R was also possibly associated with Th1 suppression and c-Maf expression in head and neck cancer patients. These results suggest that targeting a pro-inflammatory cytokine, IL-6 enhanced the tumor-specific Th1 responses and subsequent anti-tumor effects. However, IL-6 blockade in turn up-regulated the expression of immune-checkpoint molecule, PD-L1 on melanoma cells. This PD-L1 induction was canceled in IFN-γ-deficient mice or mice depleted of CD4+ T cells, suggesting an important role of CD4+ T cell-derived IFN-γ in the PD-L1 induction in tumor-bearing hosts. On the other hand, in some patients with melanoma, anti-PD-1 antibody, Nivolumab treatment increased the systemic level of IL-6, which were associated with their poor clinical responses. This PD-L1 blockade-evoked IL-6 induction was also observed in melanoma-bearing mice (Cancer Res. 78: 5011, 2018). Considering the mechanistic linkage between IL-6 and PD-1/PD-L1 signals, we found that PD-1/PD-L1 blockade prompted PD-1+macrophages to produce IL-6 in tumor microenvironment. Depletion of macrophages in melanoma-bearing mice revealed that macrophages functioned as a source of IL-6 during PD-L1 blockade, which was responsible for the defective Th1 response. Furthermore, combined blockade of the mutually regulated-immunosuppressive activities mediated by IL-6 and PD-1/PD-L1 signals enhanced the infiltration of IFN-γ-producing CD4+ T cells in tumor tissues, and exerted a synergistic anti-tumor effect, whereas PD-L1 blockade alone did not promote Th1 response. Collectively, these findings suggest that IL-6 is considered to be a rational immunosuppressive target to overcome a narrow therapeutic window of anti-PD-1/PD-L1 therapy. [ This research was financially supported by the JSPS KAKENHI grant nos. 26430165 and 18K07325 to HT, nos. 15H04311 and 16H06498 to YN, and the P-CREATE from the AMED, Japan to YN and HT. ]

#3214

In situ vaccination with CCL21-modified dendritic cells (CCL21-DC) combined with checkpoint blockade in murine models of NSCLC.

Raymond J. Lim, Ramin Salehirad, Bin Liu, Rui Li, Linh M. Tran, Kostyantyn Krysan, Stephanie Ong, Zi L. Huang, David B. Shackelford, Sherven Sharma, Steven M. Dubinett. _UCLA, Los Angeles, CA_.

Studies that target the PD-1/PD-L1 axis of immune checkpoints have demonstrated clinical responsesin approximately 20% of non-small cell lung cancer (NSCLC) patients.However, majority of the patients do not benefit from the initial treatment.Mechanistic studies reveal that responses to PD-1/PD-L1 blockade are associated with high tumor mutational burden (TMB) andincreased CD8+T cell tumor infiltration and high baseline tumor PD-L1 expression. Furthermore, recent studies have identified Stk11/Lkb1 loss as a major driver of primary resistance to PD-1 blockade in inKRAS-mutant lung adenocarcinoma (LUAC), a major subtype of NSCLC. One potential approach to enhance the effectiveness of checkpoint inhibitors is to enhance tumor antigen presentation and tumor-specific T cell immune responses by in situvaccination, taking advantage of the full repertoire of available tumor antigens. In preclinical and clinical studies, we have demonstrated that CCL21-secreting dendritic cells (CCL21-DC) have the capacity to induce the infiltration of DC and T cells into the TME and to promote tumor-specific T cell activation both locally and systemically. This response, however, was accompanied by the upregulation of PD-L1 expression in the tumor. We hypothesize that in situvaccination with CCL21-DC could restore tumor antigen presentation and T lymphocyte infiltration into the tumor, thereby sensitizing non-responsive NSCLC tumors to checkpoint blockade. To test this hypothesis, we first established novel genetically-engineered murine models (GEMMs) of lung cancer that bear the common driver mutations (KPL; KrasG12D;Tp53-/-;Lkb1-/-) and varying mutational loads to accurately reflect the clinical disease. This was achieved by exposing KPL cells in vitroto the tobacco carcinogen N-methyl-N-nitrosourea (MNU) with various duration. Using these models, we demonstrated that (1) KPL with higher TMB is more sensitive to anti-PD-1 monotherapy as compare to low TMB, consistent with previous studies; (2) Intratumoral (IT) administration of CCL21-DC in combination with anti-PD-1 resulted in significantly enhanced anti-tumor response compared to either monotherapy alone. Further work is underway to evaluate the efficacy of the combination therapy with IT CCL21-DC and PD-1 inhibition in multiple murine models of NSCLC. In addition, mechanistic studies will be performed to understand the anti-tumor effect mediated by the CCL21-DC and anti-PD-1 combination therapy, including immune phenotyping of TME and functional studies of DC and T cells. As we prepare to initiate our clinical trial that combines IT CCL21-DC with PD-1 inhibition in advanced NSCLC patients with low baseline PD-L1 expression, we anticipate these preclinical models will serve as a platform to enhance our understanding of the molecular mechanisms of response and resistance to immunotherapy.

#3215

STAT3 antisense oligonucleotide (ASO) reverses immunosuppression and enhances cytotoxic cell function to enhance PDL1 blockade.

Srimathi Srinivasan,1 Yang Xu,1 Theresa Proia,1 Nanhua Deng,1 J. Carl Barrett,1 Alexandra-Chloe Villani,2 Patricia E. McCoon1. 1 _AstraZeneca R &D Boston, Waltham, MA; _2 _Massachusetts General Hospital, Charlestown, MA_.

Danvatirsen (AZD9150) is a therapeutic antisense oligonucleotide that selectively targets human STAT3, a ubiquitously expressed transcription factor and master regulator of immune suppression in the tumor microenvironment (TME). Danvatirsen has shown clinical benefit alone and combined with durvalumab (anti-PDL1) in Phase 1/2 clinical studies. To further our mechanistic understanding, we conducted (1) immunophenotyping, (2) ex vivo stimulation/cytokine assays, and (3) single-cell RNA sequencing (scRNASeq) analysis using murine surrogate drugs at three timepoints (following one (1W), two (2W), or three (3W) weeks of mouse surrogate STAT3 ASO, vehicle, or control ASO treatment alone, or anti-PDL1 alone or combined with STAT3 ASO (combo) in W2 and W3) in Balb/c mice bearing syngeneic CT26 tumors. Consistent with clinical data and previous findings (Ref 1, 2), tumor growth inhibition was observed in both monotherapy (STAT3 mean TGI 56%; anti-PDL1 mean TGI 54%) and combo treated (mean TGI >100%) groups. Consistent with the robust anti-tumor activity, significant (p<0.05) immunophenotypic changes were observed following 3W of STAT3 ASO or combo treatment. Fewer changes were observed with anti-PDL1 monotherapy. As reported previously, M-MDSC decreased (60%) and neutrophil/G-MDSC increased (8-fold) in both STAT3 ASO and combo treated at 3W. We also discovered significant changes in NK, NK T, and CD11b NK cells, which increased >5-fold after 3W combo treatment. Both STAT3 ASO and combo enhanced expression of GZB in CD11b NK cells, supporting their role in NK cytotoxicity. Combo treatment also increased cross-presenting CD8+CD103+ DCs (4-fold) and inflammatory DCs (3-fold). Further, CD8+ cells had reduced levels of T cell exhaustion marker TIM3 and increased CD69 and GZB in the combo, suggesting enhanced CD8 T cell effector function.

Although few immunophenotypic changes were observed following 2W of treatment, preliminary data on cytokines (IFNγ, TNFα, IL2) at 2W suggest cytokine changes precipitate the immunophenotypic changes seen at 3 weeks.

These findings expand our understanding of the mechanism by which STAT3 ASO treatment as monotherapy and in combination with anti-PDL1 modifies the TME to produce significant anti-tumor effects. The results are in accord with previously reported increases in IFNg and Type I IFN signatures and decreases in a suppressive gene signature in paired pre- and on-treatment biopsies from danvatirsen-treated patients (Ref 2) and suggest STAT3 ASO treatment modulates both innate and adaptive immune responses. Studies are ongoing to perform further subtyping of drug-elicited cell population changes using single cell RNA Sequencing (results to be presented).

References

1. Woessner et al (2017) Cancer Research 77(13 Supplement):3684-3684

2. Cohen et al (2018) Annals Oncology 29, Supplement 8: viii372-viii399.

#3216

CKD-516, a novel vascular disrupting agent, enhances anticancer activity of anti-PD-1 antibody in SMAD4-deficient colon cancer model.

Soo Jin Kim,1 Hark Kyun Kim,2 Keun Ho Ryu,1 Chung Il Hong1. 1 _Chong Kun Dang Pharmaceuticals, Yongin, Republic of Korea;_ 2 _National Cancer Center, Goyang, Republic of Korea_.

CKD-516 is a potent vascular disrupting agent (VDA), selectively acting on tumor vessels. A clinical study of CKD-516 combined with irinotecan is undergoing in colorectal cancer. There have been exponential gains in immune-oncology (I-O) in recent times through the development of immune checkpoint inhibitors (ICIs). ICIs demonstrated durable response and some patients achieved disease control for several years. However, there are still critical unmet medical needs for the combination therapies due to limited response rate of ICIs. The first step of the cancer-immunity cycle is dendritic cells (DCs) maturation which is necessary for the anticancer immunity. It is well known that mature DCs play critical roles in priming immune responses in cancer patients. We found that CKD-516 is able to induce DC maturation through Rho signaling pathway in DCs. A tumor suppressor gene, SMAD4 is associated with several cancers including stomach cancer, colorectal cancer, and pancreas cancer. The loss of SMAD4 is a poor prognostic biomarker in these cancer types and its mutation is related to poor clinical outcome on immunotherapies. A SMAD4-deficient mouse colorectal cancer cell line (3349LM) was primary cultured from a spontaneous intestinal adenocarcinoma formed in a Villin-Cre;Smad4(F/F);Trp53(F/F) mouse. CKD-516 showed a synergistic effect with anti-PD-1 antibody in 3349LM syngeneic model. In addition, 3349LM is also a MSS type cancer. Thus, CKD-516 displayed a therapeutic potential for several cancer patients who do not respond to immunotherapy. In summary, CKD-516 is a novel VDA with immune boosting effect. In SMAD4 deficient cancer model, CKD-516 has shown synergistic effects in combination with PD-1 antibody. Therefore, these data suggest that CKD-516 potentiates the anticancer activity of immunotherapy.

#3217

Preclinical evaluation of the recombinant dendritic cell growth factor CDX-301 (Flt3L), and AST-008, a TLR9 agonist SNA.

Lawrence J. Thomas,1 Li-Zhen He,2 Lauren E. Gergel,1 Eric M. Forsberg,1 Elizabeth Q. Do,1 James M. Boyer,1 April R. Baronas,1 Mallary Rocheleau,1 Kathleen M. Borrelli,1 Anna Wasiuk,2 Jeffrey Weidlick,2 Henry C. Marsh,1 Bart R. Anderson,3 SubbaRao Nallagatla,3 Richard Kang,3 Ekambar R. Kandimalla,3 Tibor Keler2. 1 _Celldex Therapeutics Inc., Needham, MA;_ 2 _Celldex Therapeutics Inc., Hampton, NJ;_ 3 _Exicure, Inc., Skokie, IL_.

Recent studies have highlighted the critical role of CD103+/CD141+ dendritic cells (DCs) in antitumor immunity. CDX-301 is a soluble, recombinant human FLT3 ligand (Flt3L). Flt3L is a hematopoietic cytokine which stimulates the proliferation and differentiation of various blood cell progenitors, including CD103+/CD141+ DCs. CDX-301 is in clinical development for multiple cancers and may hold significant opportunity for synergistic development in combination with other immunotherapies, in particular toll-like receptor 9 (TLR9) agonists that are known to promote maturation and activation of DCs. AST-008 is a TLR9 agonist oligonucleotide in a spherical nucleic acid (SNA) format and is in clinical development for multiple cancers in combination with pembrolizumab. SNAs are densely packed, radial arrangements of oligonucleotides around a nanoparticle core. SNAs have increased cellular uptake, nuclease stability, and affinity to targets compared with linear oligonucleotides. AST-008 induces potent TH1-type immune responses in vitro, in mice and non-human primates, and has shown potent antitumor activity as a monotherapy and enhanced checkpoint inhibitor activity in several murine tumor models. AST-008 increases tumor-infiltrating lymphocytes, interferon-inducible gene expression, and activation and expansion of CD8+ T cells with reduced T-regulatory cells in the tumor microenvironment. We examined the effects of the combination of CDX-301 and a murine version of AST-008, muAST-008, in mice on DCs and on antitumor efficacy in a tumor model. Mice were implanted with MC38 murine colon adenocarcinoma tumor cells (day 0) and animals were treated with either CDX-301 (5 μg, days 2-8 i.p.) only, muAST-008 only (1 mg/kg or 3 mg/kg, days 9, 16, 23 and 30 p.t.), or the combination of CDX-301 (5 μg, days 2-8) with muAST-008 (1 mg/kg or 3 mg/kg, days 9, 16, 23 and 30). Treatment with CDX-301 and muAST-008 showed an additive effect in retarding tumor growth and prolonging survival. To help understand the mechanisms involved in the antitumor effects, animals were treated with a similar course of CDX-301 and muAST-008 and spleens were harvested for flow cytometry evaluation 2 days after the last CDX-301 dose. For these studies, we also included a second DC activating agent, an agonist anti-CD40 mAb FGK45.5 (50 μg i.p. together with the last dose of CDX-301) among the combinations. We observed a significant increase in the percentage of CD103+CD8\+ cDCs by the addition of muAST-008 to CDX-301 treatment. In addition, muAST-008 led to the up-regulation of activation markers on dendritic cells, which was markedly enhanced when combined with CD40 activation. These data demonstrate that muAST-008 leads to systemic activation of CDX-301 expanded DCs, leading to more potent anti-tumor immunity and support the potential of combining CDX-301 and AST-008 in augmenting the immunotherapy of cancers.

#3218

Anti-PD1 and -CTLA-4 combination in vivo enhances DC-based tumor-associated mitochondria antigen immunotherapy efficacy.

Renzo F. Perales-Linares, Stefano Pierini, Mireia Uribe, Sergei Pustylnikov, Francesca Costabile, Silvia Beghi, Andrea Facciabene. _University of Pennsylvania, Philadelphia, PA_.

Somatic mitochondria DNA (mtDNA) abnormalities have been identified in various types of human cancers. Previously, we generated tumor protective immunity under prophylactic and therapeutic approaches employing a murine kidney tumor model system and dendritic cell (DC)-based vaccination containing RENCA-derived mitochondria enriched lysates. Our results shown that tumor-associated mitochondria antigens (TAMAs) are targetable antigens for cancer immunotherapy (PMID: 26378078). Preliminary data also revealed increased endogenous PD-L1 mRNA expression levels in RENCA tumors of treated mice versus healthy kidney tissue. To investigate the impact of PD1/PD-L1 axis antagonism, Balb/C mice were challenged with RENCA cells at day 0 followed by TAMA-vaccination at day 3 and concurrent administration of anti-PD1 (aPD1) 5 times every 3 days. Interestingly, the aPD1-TAMA treatment resulted in reduced tumor growth, greater T CD4+ and T CD8+ cell infiltration, and lower intratumoral-MDSC as compared to the TAMA vaccine alone. In addition, we observed higher T cell activation from the aPD1-TAMA group upon co-culture with bone marrow-derived dendritic cells exposed to RENCA mitochondria lysate, suggesting that the inhibition of PD1 also enhanced the immunogenicity of the TAMA vaccine. Further analysis of the tumor environment revealed that the PD1-PD-L1 axis antagonism correlated with an increment of intratumoral CTLA4 expression and recruitment of T regulatory (Treg) cells. We introduced a third arm of treatment including a CTLA4 antagonist antibody in combination with aPD1 and TAMA vaccine in vivo. Strikingly, the tumor progression was greatly impacted causing tumor rejection in the majority of tumor-bearing animals, sustained tumor volume reduction in the others, a decrease in Treg cell recruitment, and concomitant increment of T CD8+ cell infiltration and activation. Altogether, the combination of check point inhibitors in vivo significantly improved the efficacy of the TAMA immunotherapy by remodeling the tumor microenvironment, reducing the immunosuppressive potential, and facilitating the infiltration of effector T cells into the tumor.

#3219

In vivo **efficacy and safety evaluation of anti-human PD-1 and CD40 mAbs using double humanized PD-1/CD40 mouse model.**

Yanan Guo. _Biocytogen, Beijing, China_.

In recent years, monoclonal antibodies have been successfully used in clinical trials to block or activate key mediators of immune checkpoint pathways, including CTLA4, PD-1, PD-L1, CD40 and others. Combination therapy can promote antigen release and T cell priming, T cell activation and homing, which helps to overcome tumor immune-evasive mechanisms and maximize efficacy, ultimately benefitting most patients. However, along the IO drug development process, in vivo efficacy models have always been a rate-limiting step, especially to test combination therapy using two IO antibodies.

The transmembrane protein receptor CD40 is a member of the tumor necrosis factor (TNF) receptor super family and is involved in co-stimulation of immune cells. CD40 is expressed by antigen-presenting cells (APCs) including dendritic cells (DCs), B-cells, macrophages, and monocytes. It has the ability to "wake" DCs to prime effective cytotoxic T-cell responses. Thus, effective anti-CD40 antibodies provide an ideal therapy alternative in combination with other IO antibodies.

To evaluate the in vivo efficacy and safety (immune-related adverse events) of CD40 and PD-1 combination antibodies, we developed a double humanized B-hPD-1/hCD40 mouse model. In this model, mouse PD-1 gene exon 2, and CD40 gene exons 2-7 were replaced with their human counterparts. Murine colon cancer MC38 cells were subcutaneously implanted into homozygous B-hPD-1/hCD40 mice. We compared the effect among single hPD-1 antibody Keytruda, single hCD40 antibody Selicrelumab and combination of the two.

Combination treatment of Selicrelumab and Keytruda show higher inhibitory effects than single antibody treatments. The blood chemistry assays showed that the CD40 antibody had no toxicity effects compared with the control group. Based on these findings, we conclude that the B-hPD-1/hCD40 double-humanized mouse model is a powerful tool for in vivo efficacy and safety evaluation of hPD-1 and hCD40 antibodies for combination therapy.

#3220

Enhancing breast cancer immunotherapy via IONP-mediated photothermal therapy.

Hongwei Chen, Xin Luan, Hayley Paholak, Joseph Burnett, Nicholas Stevers, Kanokwan Sansanaphongpricha, Miao He, Alfred Chang, Qiao Li, Duxin Sun. _Univ. of Michigan, Ann Arbor, MI_.

Despite immunotherapy by harnessing host's own immune system to fight against cancer has elicited promising durable responses in metastatic melanoma and lung cancer patients, its efficacy in treating breast cancer remains to be low. In this work, we demonstrate that sequential photothermal therapy (PTT), mediated with systemically administered stealthy iron oxide nanoparticles (IONPs), can significantly enhance checkpoint blockade-based immunotherapy for breast cancer. Our data suggests that the combination therapy when combining IONP-mediated PTT with anti-CTLA-4 therapy results in a synergistic antitumor effect to completely inhibit established 4T1 murine breast tumor growth in BALB/c mice, while control treatments, including the antibody alone or IONP-mediated PTT alone, fail to do so. Our data further reveal that the enhanced antitumor effect may be attributed by the effective regulation of the suppressive tumor immunity by IONP-mediated PTT. Our flow cytometry data suggests that CD4+FoxP3+ regulatory T-cells (Tregs) are effectively depleted via IONP-mediated PTT, with a significant reduction of the Treg percentage from 42.2 ± 15.8% (control) to 11.9 ± 6.5% (p < 0.05). Our data further suggest that IONP-mediated PTT significantly reduces suppressor-cell-attractive cytokine granulocyte-colony stimulating factor (G-CSF) secretion, which is the driving force for Treg recruitment. Upon effective regulation of suppressive tumor immunity, the proportion of CD8+ T-cells in tumor tissues is significantly increased from 5.6 ± 2.1% (control) to 37.5 ± 6.9% (p < 0.001) following IONP-mediated PTT. Interestingly, our data suggest that Treg elimination and CD8+ T-cell activation can only be achieved through sequential PTT, not a single dose of PTT, suggesting that the suppressive contribution from the newly responding immune cells following initial antitumor treatment, and the sequential PTT may provide an effective way to not only deplete primary resident suppressive cells, but also newly responding ones. Our data further suggests that the combination therapy against primary tumors elicits a systemic immune rejection of cancer cells at distal sites, while this dramatic immune response is lost when CD8+ T-cells are depleted using anti-CD8 antibody, suggesting the immune rejection triggered by IONP-mediated PTT is through CD8+ T-cell-mediated immune responses. Our data also suggests that the majority of the combination treatment-cured mice can reject re-challenged 4T1 cancer cells, indicating memory T-cell immune surveillance. In conclusion, this study may provide an effective and translational nanomedicine-based strategy to effectively regulate suppressive tumor immunity and turn "cold" breast tumors into "hot", thereby to significantly enhance checkpoint blockade-based breast cancer immunotherapy.

### Immune Checkpoints 1

#3221

T-cell receptor sequencing for pharmacodynamic and response biomarkers of checkpoint blockade.

Simon Papillon-Cavanagh (co-lead author), Alice M. Walsh (co-lead author), Zhenhao Qi, Megan Wind-Rotolo, Abdel Saci, Parul Doshi, Radu Dobrin, Joseph Szustakowski. _Bristol-Myers Squibb, Princeton, NJ_.

Background: Immunomodulatory cancer drugs such as anti-programmed death 1 (PD-1) and anti-cytotoxic T-lymphocyte antigen 4 (CTLA4) antibodies have shown significant clinical benefits across multiple indications. However, not all patients benefit from checkpoint blockade (CB) treatment strategies, highlighting the need for predictive biomarkers of response. Features of the T-cell receptor (TCR) repertoire have been reported to correlate with CB treatment and response (1). Here, we performed a comprehensive, retrospective analysis of TCR repertoires from different tissues (blood and tumor) at different time points (baseline and on treatment). To the best of our knowledge, our study, comprising a total of 558 samples across 3 tumor types (non-small cell lung cancer [NSCLC], renal cell carcinoma [RCC], and melanoma), is the largest analysis of TCR-sequencing data in CB clinical trials to date.

Methods: We performed TCR sequencing on formalin-fixed paraffin-embedded tissue samples or peripheral blood samples using immunoSEQ Assays (Adaptive Biotechnologies Corp.) to profile the immune repertoire of patients from 3 clinical trials of nivolumab (anti-PD-1) or nivolumab + ipilimumab (anti-CTLA4) (NCT02041533, NCT01358721, NCT01621490). Repertoire clonality and clonal expansion statistics were computed, compared, and tested for their association with clinical activity.

Results: We characterized the TCR repertoire from peripheral blood at baseline for 263 patients (209 NSCLC, 54 RCC) with matched on-treatment profiles for 49 patients (49 RCC). In addition, we profiled tumor TCR repertoires at baseline and on treatment for 127 patients (54 RCC, 73 melanoma) at time points predefined in each study's protocol. Our analyses show that clonal expansion was accompanied by a comparable contraction of other clones and thus was not directionally enriched upon CB treatment. Moreover, comparison of baseline and on-treatment repertoire clonality did not reveal a significant shift following CB treatment. Results were consistent in blood and tumor tissue and across response groups. Baseline peripheral blood TCR clonality was not associated with best overall response or progression-free survival.

Conclusions: Our results highlight that TCR repertoires are highly dynamic and that the currently established metrics are difficult to interpret or implement clinically as pharmacodynamic or response biomarkers. The variability in TCR clonality modulation upon CB treatment underscores the challenge of selecting the adequate on-treatment time point. Thus, our comprehensive analysis highlights the need for a better understanding of the TCR repertoire dynamics and development of methods to identify and functionally annotate tumor-specific TCRs.

Reference:

1. Riaz N et al. Cell 2017;171:934-49

#3222

The sharing of T cell clones in peripheral CD8+PD-1+ T cells with TILs is a novel biomarker predicting the efficacy of anti-PD-L1 therapy.

Jiefei Han,1 Zhijie Wang,1 Yuqi Wang,2 Si Chen,2 Hua Bai,1 Jianchun Duan,1 Jie Wang3. 1 _Cancer Hospital Chinese Academy of Medical Science & Peking Union Medical College., Beijing, China; _2 _Geneplus-Beijing Institute, Beijing, China;_ 3 _State Key Laboratory of Molecular Oncology, Department of Medical Oncology, National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China_.

Background: The optimal biomarker to select suitable patients for immune checkpoint blockades (ICBs) therapy remains to be well established. The dynamic variations of molecular alterations during ICBs treatment need to be better understood. Here,we explored a novel biomarkerbased on T cell receptor (TCR) repertoire (TcR) and traced the variations during PD-L1 blockade delivery.Methods:Patients treated with anti-PD-L1 clinical trials were enrolled in this pilot study (n=21). Next-generation sequencing (NGS) was performed to assess molecular tumor burden index (mTBI) based on a 1021 gene panel, for genomic DNA from tumor tissue and cfDNA. Multiple PCR and NGS on the CDR3 region of TCR beta chain was used to reflect the distribution of T cell clones in TILs and peripheral CD8+PD1+ T cells. We applied gene altation data combined with each patient's major histocompatibility complex (MHC) class I haplotype in a neoantigen prediction platform that evaluates binding affinities of somatic peptides to class I MHC, antigen processing, and self-similarity. Neoantigen peptides were synthesis and used to stimulate Peripheral blood mononuclear cell(PBMC)from the same patient. The expression of OX-40 and 4-1BB were anylisied by flow cytometry to determine activity change of stimulated T cells.

Results: A number of T cell clones (1-24) in TILs were shared in peripheral CD8+PD1+ T cells. The total ratio of shared clones to top 100 clones in TILs ranged from 0.00% to 31.35%, while the mean ratio of each shared clone was 1.24% (0.00% to 3.18%). Patients responding to anti-PD-L1 therapy showed higher mean ratio of shared clonesthan non-responding ones (1.76% vs. 0.85%; P= 0.012), our further analysis showed the progression free survival (PFS) is also longer in patients with higher mean ratio of shared clones (P=0.022). Intriguingly, the ratio of shared clones to total CD8+PD1+ T cell clones of peripheral blood showed synchronized change consistent with mTBI and tumor burden. PBMC stimulated by neoantigen peptide expressed higher OX-40 and 4-1BB than control. Further TCR sequencing performed on stimulated T cells showed consistency with shared clone at baseline.

Conclusion: The high mean ratio of shared clones in TILs is a potential and promising biomarker to predict the efficacy of ICBs therapy and reflect the target T lymphocytes variations during the treatment, which warrants the further validation in large cohort.

#3223

Overcome LKB1 mutated cancer resistance to anti-PD1 treatment.

Jiehui Deng,1 Aatish Thennavan,2 Yuanwang Pan,1 Igor Dolgalev,1 Ting Chen,1 Heather Silver,1 Matthew Harris,1 Val Pyon,1 Fei Li,1 Chelsea Lee,1 Aristotelis Tsirigos,1 Eli Rothenberg,1 Charles M. Perou,2 Kwok-Kin Wong1. 1 _NYU Langone Medical Center, New York, NY;_ 2 _Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC_.

KRAS/STK11 (LKB1) mutant lung cancers are a subtype of cancer that respond poorly in the clinic to immunotherapies with shorter progression-free survival and overall survival comparing with other KRAS mutant lung cancer patients. Interestingly, this group of patients have high tumor mutational burden (TMB) comparable with other KRAS mutant lung cancers, which usually is an indicator for better response to anti-PD1 treatment. By using genetically engineered mouse model (GEMMs), we found that LKB1 loss of function can cause high level of nonsynonymous mutations, as a consequence of both replication dependent and independent DNA repair machinery dysfunctions. This LKB1 mutant dependent increased mutational load might further synergize with smoking mutagen exposures. When LKB1 function is defective, the neoantigens are not properly presented to T cells, which is required for proper T cell activation and effector T cells expansion. This will lead to insensitivity to anti-PD1 antibodies in high TMB tumors and tolerance to neoantigens produced by cancer cells. We further demonstrated that using a small molecule drug that impacts on the LKB1 downstream effector pathway, we can reverse this process through activating innate immunity in tumors, which will restore the antigen presentation functions. Combinational treatment of this small molecule with anti-PD1 can reverse the tumor progression in vivo, through releasing the suppressive tumor infiltrating lymphocytes (TILs) in LKB1 mutant lung cancers. Our study not only helps to further our understanding of the mechanism that contributes to immune evasion in high TMB tumors, but also provides a potential solution for LKB1 mutant cancers to overcome resistance to anti-PD1 treatment in the clinic.

#3224

Copper homeostasis: A new player in anti-tumor immune response.

Florida Voli,1 Luigi Lerra,1 Kathleen Kimpton,1 Federica Saletta,2 Sylvie Shen,3 Giuseppe Cirillo,4 Maria Kavallaris,1 Orazio Vittorio1. 1 _Children's Cancer Institute, Randwick, NSW, Australia;_ 2 _Kids Research Institute - The Children's Hospital at Westmead, Westmead, NSW, Australia;_ 3 _Kids Cancer Centre - Sydney Children's Hospital, Sydney, NSW, Australia;_ 4 _University of Calabria, Rende, Italy_.

Immunotherapy has shown great potential for treating aggressive cancers and it is becoming the fourth and newest pillar of cancer therapy complementing surgery, cytotoxic therapy, and radiotherapy. In particular, immune checkpoint inhibitors targeting the PD-1/PD-L1 axis have shown extraordinary clinical efficacy in several types of cancer.

This is because tumor cells express molecules, such as the Programmed Death Ligand 1 (PD-L1), to prevent immune cells activity (immune-evasion). The immune checkpoint protein Programmed Death receptor 1 (PD-1) expressed by lymphocytes instructs T-cells not to attack any tumor cell expressing PD-L1. Several therapies anti PD-1/PD-L1 have been approved by FDA, but concerns have been raised about their long term efficacy and safety. Therefore, there is a need for better understanding of the biology and the mechanisms regulating PD-1/PD-L1 axis, to develop different approaches to target this pathway. Drugs modulating the transcriptional and post-transcriptional regulation of PD-L1 could represent new therapeutic strategies for increasing the efficacy and reducing side effects of the current anti PD-L1 antibodies.

Copper transporter 1 (CTR-1) and copper levels are elevated in tumors and the use of copper targeting agents is currently under intense investigations. It has been also reported that copper plays a major role in the immune-system, but its activity is unclear.

In this study we demonstrated that copper plays a key role in the expression of PD-L1 in cancer cells. Tissue microarrays from neuroblastoma (NB) and glioblastoma patients showed a significant correlation between CTR-1 and PD-L1 expression (p=0.00014 and p=0.012 respectively). In vitro experiments showed that downregulation of CTR-1 caused a decrease of intracellular copper which in turn led to a downregulation of PD-L1 expression in cancer cells. On the other hand, addition of copper into the media clearly induced PD-L1 upregulation. RNA-seq analysis revealed specific pathways and candidate genes associated with tumor copper homeostasis and PD-L1 expression. Consistently, Dextran-Catechin (DC) and TEPA, drugs reducing copper, were able to downregulate PD-L1 expression in tumors. In vivo studies showed that copper lowering drugs prolonged mice survival, and ex vivo immunohistochemistry staining confirmed the downregulation of CTR1 and PD-L1 expression. In addition, 24h and 48h of DC treatments showed an increase of tumor-infiltrating CD4+ and CD8+ lymphocytes and activated Natural Killer cells (NK) cells in NB immune-competent mouse model. In conclusion, there is a strong association between PD-L1 expression and intracellular copper levels. Copper dysregulating agents reduce PD-L1 in vitro and in vivo, highlighting the possibility to enhance tumor immune surveillance by targeting intracellular copper levels. This study shows the potential utility of copper targeting drugs to improve anti-cancer immunotherapies.

#3225

Enhancement of anti-PD1 and anti-CTLA4 efficacy by NBTXR3 nanoparticles exposed to radiotherapy.

Yun Hu,1 Ping Zhang,2 Audrey Darmon,2 Maria Angelica Cortez,1 Sébastien Paris,2 James Welsh1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _Nanobiotix, Paris, France_.

The use of checkpoints inhibitors (CPI) has radically changed the medical practices for cancer treatment. Unfortunately, the activity of CPI depends on a preexisting anti-tumor immune response, limiting their use to a small percentage of patients. It is crucial to propose solutions to prime an effective anti-tumor immune response and convert CPI non-responder patients to responders. Recent preclinical/clinical studies have reported that radiotherapy (RT) acts as an efficient modulator of tumor immunogenicity. RT can set in motion processes facilitating tumor recognition by the immune system. Unfortunately, RT rarely generates a sustained anti-tumor immunity and reduction of metastases burden outside the irradiated area - a phenomenon called 'abscopal effect' - is hardly obtained after RT and the toxicity to healthy tissues limits the maximum dose of irradiation delivered to patients. NBTXR3 is composed hafnium oxide nanoparticles (HfO2-NP) designed to increase energy dose deposition from inside the cancer cells. The size, shape and surface charge of HfO2-NP allow strong interactions with cancer cells and persistence within the tumor mass after a single intra-tumor administration during the whole RT treatment. The high electron density of HfO2-NP increases interaction probability with ionizing radiations (when compared to tumor tissues with low electron density), resulting in the enhancement of tumor destruction, compared to RT alone. The recent results of phase III in locally advanced Soft Tissue Sarcoma patients demonstrated the significant superiority and clinical benefits of intratumorally injected HfO2-NP activated by RT to treat cancer compared to RT alone, validating the first-in-class mode of action of NBTXR3. In addition, preclinical studies have reported that HfO2-NP activated by RT can induce an anti-tumor immune-response and an abscopal effect. Here, we explored the ability of RT-activated NBTXR3 to increase the efficacy of anti-PD1 or anti-CTLA4 using abscopal assay in immunocompetent mice. In a first assay, mice were subcutaneously injected with 344SQP (mouse lung cancer) cells on both flanks. Then, right tumors were injected with HfO2-NP (or vehicle) and irradiated (or not), while left tumors remain untreated. Some groups of mice received injections of anti-PD1. The same approach was used with CT26 (mouse colorectal cancer) cells, except that mice received anti-CTLA4. For the 344SQP model, tumor growth analysis revealed that NBTXR3+RT and anti-PD1 treatment allows a better tumor control on both sides, compared to other conditions. For CT26 model, NBTXR3+RT and anti-CTLA4 treatment led to a better tumor growth control on both sides, compared to other conditions. These results suggest that NBTXR3 activated by RT could potentiate the anti-PD1 and anti-CTLA4 efficacy, opening new opportunities for the treatment of patients by combination of NBTXR3+RT+CPI.

#3226

The IRF8-osteopontin-CD44 axis functions as an immune checkpoint to control CD8+ T cell activation and tumor immune evasion.

John D. Klement,1 Amy V. Paschall,1 Priscilla S. Redd,1 Mohammed L. Ibrahim,1 Chunwan Lu,1 Dafeng Yang,1 Esteban Celis,1 Scott I. Abrams,2 Keiko Ozato,3 Kebin Liu1. 1 _Augusta University, Augusta, GA;_ 2 _Roswell Park Cancer Center, Buffalo, NY;_ 3 _NICHD, Bethesda, MD_.

Despite breakthroughs in immune checkpoint inhibitor (ICI) immunotherapy, not all human cancers respond to ICI immunotherapy and only fraction of patients with responsive tumors have a durable response to current ICI immunotherapy. This clinical conundrum suggests that additional immune checkpoints may exist, particularly in cancers resistant to current ICI immunotherapy, such as colorectal cancer. We report here that interferon regulatory factor 8 (IRF8) deficiency led to impairment of cytotoxic T lymphocyte (CTL) activation in a peptide vaccine model and allowed allograft transplant tumor tolerance. These effects were associated with upregulation of the CTL surface marker CD44. However, analysis of chimeric mice with competitive reconstitution of wild type and IRF8 KO bone marrow cells as well as mice with IRF8 deficiency only in T cells indicated that IRF8 plays no intrinsic role in CTL activation. Instead, IRF8 functioned as a repressor of osteopontin (OPN), the physiological ligand for CD44 on T cells, in CD11b+Ly6CloLy6G+ myeloid cells and OPN acted as a potent T cell suppressor. In vitro stimulation of CTLs in the presence of OPN resulted in decreased expression of activation markers CD69 and CD25 and inhibited proliferation and interferon gamma (IFNg) secretion. Expression of OPN was found to be upregulated in both myeloid cells and colon epithelial cells following silencing of IRF8 expression. IRF8 bound to the Spp1 promoter, which encodes OPN, to repress OPN expression in colon epithelial cells. Correspondingly, human colon carcinoma cells exhibited decreased IRF8 and increased OPN expression. These increased OPN levels inhibited human PBMC proliferation and IFNg secretion. The elevated expression of OPN in human colon carcinoma was correlated with decreased patient survival. Our data indicates that myeloid and tumor cell-expressed OPN acts as a novel immune checkpoint to suppress T cell activation and confer host tumor immune tolerance. Blockade of this checkpoint may expand the pool of patients who may benefit from ICI immunotherapy.

#3227

Differential expression of T cell co-inhibitory receptors: KLRG1 alignment with cytotoxicity and adaptive resistance mechanisms.

Steven A. Greenberg,1 Evan Thompson,2 Stefano V. Gulla2. 1 _Brigham and Women's Hospital, Boston, MA;_ 2 _Abcuro, Inc., Newton, MA_.

Introduction: KLRG1 is a lymphocyte co-inhibitory, or immune checkpoint, receptor expressed predominantly on late-differentiated effector and effector memory CD8+ T and NK cells.

Methods: We studied KLRG1 expression in human blood and tumor samples from available genomic datasets.

Results: KLRG1 is differentially expressed from CTLA-4 and PD-1, with predominant expression on cytotoxic CD8 T and NK cells over CD4 T cells. Within the CD8+ T cell population, KLRG1 expression, unlike CTLA-4 and PD-1 expression, is linked to greater antigen-driven differentiation states, with increased expression on CD45RO+CCR7- T effector memory (TEM) and CD45RA+CCR7- T effector memory RA (TEMRA) cells compared to CD45RA+CCR7+ naïve T cells (TN) and CD45RO+CCR7+ central memory T cells (TCM). The cytotoxic potential of CD8+ T cells, as assessed by the presence of cytokine and cytotoxic molecules IFNg, TNFa, perforin and granzyme B, is aligned with KLRG1, but not CTLA-4 or PD-1, expression. In single cell RNAseq analyses, KLRG1+ TILS accounted for 16-48% of CD8+ TILS, a frequency similar to that of PD-1+ TILS, in renal cell carcinoma, hepatocellular carcinoma, melanoma, ovarian cancer, HNSCC, and astrocytoma. KLRG1 expression was numerically increased post-treatment in 20/21 (95%) of datasets, statistically significant in 10/21 (48%), in response to a range of treatments including radiotherapy, chemotherapy, endocrine therapy, and immunotherapies (including ipilimumab, nivolumab, and pidilizumab), over periods of time ranging from 1-25 weeks.

Conclusions: In human blood and tumor samples, KLRG1 expression is aligned with cytotoxic T and NK cell differentiation, and upregulated in human tumor samples after a variety of therapies, potentially contributing to adaptive resistance. The upregulation of KLRG1 could contribute to limited efficacy and adaptive resistance that develops with current immunotherapies, and suggests KLRG1 blockade may work efficaciously, including as a neo-adjuvant therapy.

#3228

Immune checkpoint blockade and autoimmune diseases: Development of a mouse model of colitis induced by anti-CTLA-4.

Edwige Nicodeme, Florence Blandel, Valerie Boullay, Yannick Saintillan, Gael Krysa, Anne-Benedicte Boullay, Jean-Jacques Tousaint, Robin Artus, Laure Levenez, Jeremy Odillard, Ingrid Jacquet, Olivier Duchamp, Fabrice Viviani. _Oncodesign S.A., Dijon Cedex, France_.

Immunotherapies prime or activate patient's immune system to fight disease and has recently been a source of registered and promising new cancer treatments with major beneficial clinical outcomes in several cancers. However, by increasing the activity of the immune system, therapies as such targeting the immune checkpoint blockade can have profound inflammatory side effects, termed immune-related adverse events, with particular organs affected such as gastrointestinal tract, endocrine glands, skin and liver. In patients treated with the anti-CTLA-4 antibody, Ipilimumab, the overall incidence of diarrhea and colitis has been reported as 32.8%. This side effect is also observed in patients treated with anti-PD-1 therapy. The reproduction of such aspects of immune checkpoint inhibitors side effects in preclinical animal models would serve to dig into deciphering mechanisms involved, but also to evaluate combination therapies and modalities to reduce and manage such incidence with potential beneficial translation in clinical practice. We thus first ought to establish a checkpoint blockade-related autoimmune mouse model of colitis, using female C57BL/6 mice which were tested to determine their response to orally administered dextran sulfate sodium (DSS) combined to multiple injections of anti-CTLA-4 antibody or an isotype control antibody. After administration of DSS in drinking water for 7 days, mice that received an anti-CTLA-4 antibody showed no difference in body weight loss and Disease Activity Index (composite of body weight loss, stool consistency and presence of blood in stools), when compared to mice that received DSS plus the isotype control antibody. Histological sections analysis from the colons of mice culled at Day 10 confirmed that the combined treatment did not change the colitis score.However, when looking at later time points corresponding to the usual recovery phase of colitis, the effect of anti-CTLA4 treatment was clearly revealed by a sustained body weight loss and the persistence of a high Disease Activity Index, while the isotype control-treated animals slowly returned to baseline values similar to untreated animals. At sacrifice on Day 19, these in life findings were confirmed by a decrease in colon length (- 0.94 cm), an increase in colon weight (+ 231 mg) and a high mucus colon production in the anti-CTLA4-treated group versus the isotype control-treated group. Histological analysis of colons, and a more in depth characterization of the immune infiltrates will be presented.

#3229

Radiation and chemotherapies result in increasing neoantigen intratumor heterogeneity and resistance to nivolumab in a patient with pleomorphic lung cancer.

Xiaohua Hong,1 Yuting Liu,1 Yiying Liu,2 Yue Hu,1 Qifan Yang,1 Di Wu,1 Kai Zhang,1 Yongchao Li,2 Jinsong Yang,1 Jiaqian Wang,2 Jun Liu,3 Li Liu1. 1 _Department of Thoracic Oncology, Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China;_ 2 _YuceBio, Shenzhen, China;_ 3 _Department of Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China_.

BACKGROUND:Immune checkpoint inhibitors (ICIs) have significantly changed the landscape of cancer treatment, but the response rate of non-small-cell lung carcinoma (NSCLC) to ICIs is less than 30%. Tumor mutational burden (TMB) is the most promising predictive biomarker for NSCLC immunotherapies. However, some NSCLC patients with high-TMB have little response to ICIs. The neoantigen intratumor heterogeneity (ITH), which determined by the ratio of subclonal neoantigen in the tumor, has been verified to be a potential therapeutic biomarker for immunotherapies in some cancer types. Pulmonary pleomorphic carcinoma, a rare subtype of pulmonary sarcomatoid carcinoma, has been shown to respond remarkably to ICIs, but the biomarkers for ICIs in pulmonary pleomorphic carcinoma have not been fully proven. CASE PRESENTATION:A 57 years old female pulmonary pleomorphic carcinoma patient, who underwent surgery, chemotherapy, anti-angiogenic therapy, radiotherapy, and anti-PD1 therapy, with disease relapse at the last, was included in this study. Primary and relapse tumor samples were collected for gene sequencing by a target sequencing panel which covered 811 cancer genes, during ICIs treatment. Peripheral bloods were collected for ctDNA and T cell receptor sequencing every two months. The results showed that the TMB (>10 muts/Mb) was high, but the clonal neoantigen load (1.33 neos/Mb) was low. The neoantigen ITH was increased after chemotherapy and radiotherapy with a score at nearly 0% in the primary tumor but increased to 85.72% in relapse tissues. The clonal neoantigen ratio detected by ctDNA was increased ~0.8 fold, while the high-frequency clonotypes of TCR to neoantigens dropped during ICIs treatment. CONCLUSIONS:Our results indicated that cytotoxic chemotherapy and radiotherapy can induce the subclonal neoantigens production and increase the neoantigen ITH, thus might negatively regulating patients' response to ICIs therapy. These results highlight that the neoantigen ITH might be an important auxiliary to TMB in predicting patient response to immune therapies. More studies and clinical trials are needed to further validate these results.

#3230

Safety and efficacy of immune checkpoint inhibitors (ICIs) in patients with HIV, hepatitis B, or hepatitis C viral infections.

Neil J. Shah,1 Ghassan AL-Shbool,2 Matthew Blackburn,1 Michael Cook,1 William J. Kelly,1 Anas Belouali,1 Sebastian Ochoa,1 Bradley S. Colton,1 Jeevan Puthiamadathil,1 Michael T. Serzan,1 Alice R. Knoedler,1 Stephen V. Liu,1 Michael J. Pishvaian,1 Mahsa Mohebtash,2 Subha Madhavan,1 Aiwu R. He,1 Michael B. Atkins,1 Geoffrey T. Gibney,1 Chul Kim1. 1 _Georgetown University, Washington, DC;_ 2 _Washington Hospital Center, Washington, DC_.

Background: Immune checkpoint inhibitors (ICIs) have become standard of care for many malignancies. However, patients (pts) with human immunodeficiency virus (HIV), hepatitis B (HBV), and hepatitis C (HCV) were excluded from many clinical trials. Therefore, the safety and efficacy of ICI therapy in these patient populations is not well characterized.

Methods: We performed a retrospective analysis of pts with HIV, HBV, or HCV treated with ICI therapy at five MedStar Health hospitals from January 2011 to April 2018. The incidence of immune-related adverse events (irAEs) per CTCAE4.03, objective response rate (ORR) per RECIST v1.1, changes in viral status and CD4 T-cell counts during treatment were analyzed.

Results: A total of 50 pts were identified; (21 HIV, 4 HIV/HCV, 1 HIV/HBV, 15 HBV, 22 HCV, and 3 HBV/HCV) treated with anti-PD-(L)1 monotherapy (43) or in combination with ipilimumab (1) or chemotherapy (6). In the HIV group (21), the median age was 62 (29-85), 52% were male, 33% white, and 67% African American (AA). Tumor types included non-small-cell lung carcinoma (NSCLC; n=12), Hodgkin's lymphoma and anal carcinoma (2 each), head and neck cancer, colorectal cancer, renal cell carcinoma (RCC), Burkitt's lymphoma, and hepatocellular carcinoma (HCC) (one each). Any grade irAEs was 24% (5), and grade ≥ 3 irAEs was 10% (hepatitis and pneumonitis). Four pts had elevated HIV viral loads at baseline; 2 of 2 assessed had decreased levels after starting ICI therapy without changes in their antiretroviral therapy. Nine pts maintained an undetectable HIV viral load during ICI therapy. CD4 T-cell counts improved in 5 pts and decreased in 6 pts during ICI therapy. The ORR in response-evaluable pts for the entire group (16) was 25% (1 CR, 3 PR, 4 SD, and 8 PD). In the HBV/HCV cohort (34), median age was 62 (37-77), 71% were male, 24% white, and 50% AA. Tumor types included HCC (16), NSCLC (10), RCC (3), and head & neck, gastric and small cell lung cancer, one each. Any grade irAEs were noted in 50% (17) and grade ≥3 in 26% [colitis (2), hepatitis (3), pneumonitis, diabetic ketoacidosis, neurological weakness and rash, one each]. Of 15 HBV pts, 4 pts had detectable, and 8 pts had undetectable viral load before initiation of ICI. Among 4 pts with detectable viral load, no transaminitis was observed. Of 22 HCV pts, 9 were previously treated, 11 were untreated and no data available in 2. Among 4 pts with pre- and post-viral loads, 2 untreated pts' viral loads showed small improvement and other 2 previously treated pts' viral loads remained undetectable. The ORR in response-evaluable pts for the entire group (27) was 22% (6 PR, 7 SD, and 14 PD) and HCC pts (13) treated with anti-PD-(L)1 monotherapy was 23% (3 PR, 3 SD, and 7 PD).

Conclusion: ICI therapy was not associated with any new safety signal in pts with HIV, HBV, or HCV infection with no evidence of viral reactivation. Prospective trials are needed to validate the above findings.

#3231

Oncolytic immunotherapy with PD-1 blockade and telomerase-specific oncolytic adenovirus in osteosarcoma.

Koji Demiya,1 Hiroshi Tazawa,2 Yusuke Mochizuki,1 Miho Kure,1 Joe Hasei,3 Toshiyuki Kunisada,4 Yasuo Urata,5 Toshifumi Ozaki,1 Toshiyoshi Fujiwara2. 1 _Department of Orthopaedic Surgery, Okayama University, Graduate School of Medicine, Dentistry and Ph, Okayama City, Japan;_ 2 _Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama City, Japan;_ 3 _Department of Sports Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama City, Japan;_ 4 _Department of Medical Materials for Musculoskeletal Reconstruction, Okayama University, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama City, Japan;_ 5 _Oncolys BioPharma, Inc., Tokyo, Japan_.

Background: Osteosarcoma (OS) is the most frequent primary malignant tumor of bone in children and adolescents. Although immune checkpoint inhibitor, anti-programmed death protein 1 (PD-1) antibody, has dramatically improved the clinical outcome in some cancer patients, OS patients are less sensitive to PD-1 blockade due to poor immune responses. Recently, oncolytic virotherapy has been shown to stimulate the immune system through induction of immunogenic cell death (ICD). We recently developed a RGD fiber-modified telomerase-specific oncolytic adenovirus OBP-502, which can enter into tumor cells by binding to cell surface integrin and induce oncolytic cell death in a telomerase-dependent manner. In this study, we assessed the in vitro and in vivo antitumor efficacy of combination therapy with PD-1 blockade and OBP-502 in OS cells.

Methods: We used 2 murine OS cell lines, K7M2 and NHOS. The expression of PD-L1, coxsackie and adenovirus receptor (CAR), and integrin on the cell surface was analyzed by flow cytometric analysis. We analyzed the in vitro antitumor effect of OBP-502 using XTT assay and western blot analysis. Virus-induced ICD was assessed by analyzing the level of extracellular ATP and high-mobility group box protein B1 (HMGB1). To evaluate the therapeutic potential of oncolytic immunotherapy, we investigated the in vivo antitumor effect of combination therapy with anti-PD-1 antibody and OBP-502 using a subcutaneous K7M2 xenograft tumor model. Moreover, the number of tumor-infiltrating CD8+, CD4+ and Foxp3+ T cells was analyzed by immunohistochemistry.

Results: Flow cytometric analysis demonstrated that K7M2 and NHOS cells had the expression of PD-L1 and integrin, but not CAR. XTT assay showed that OBP-502 efficiently suppressed the viability of K7M2 and NHOS cells in a dose-dependent manner. Western blot analysis revealed that OBP-502 induced the apoptosis-related cell death in K7M2 and NHOS cells. ELISA assay demonstrated that OBP-502 significantly increased the level of extracellular ATP and HMGB1 in K7M2 and NHOS cells. In vivo experiment using a subcutaneous K7M2 xenograft tumor model showed that combination of anti-PD-1 antibody and OBP-502 significantly reduced tumor growth compared to mock treatment or monotherapy. Immunohistochemical analysis revealed that OBP-502 significantly increased the number of tumor-infiltrating CD8+ T cells compared to mock treatment or monotherapy. By contrast, OBP-502 did not affect the number of CD4+ and Foxp3+ T cells in tumor tissues.

Conclusion: These results suggest that oncolytic immunotherapy with PD-1 blockade and telomerase-specific oncolytic adenovirus is a promising antitumor strategy to promote the therapeutic potential of PD-1 blockade in OS through stimulation of antitumor immune response.

#3232

Pharmacokinetic analysis of anti-PD-1 and PD-L1 antibodies and evaluation of their anti-tumor effects.

Hiroto Hatakeyama, Taiki Kurino, Reiko Matsuda, Hiroyuki Suzuki, Ayu Terui, Tomoya Uehara, Yasushi Arano, Akihiro Hisaka. _Chiba Univ., Chiba, Japan_.

Objective: Recently anti-PD-1 antibodies (aPD-1 Abs), anti-PD-L1 (aPD-1) Abs have been approved. However, the difference between both Abs in pharmacokinetics and anti-tumor effects have not been fully understood. In this study, we analyzed the difference between both Abs in blood concentration, biodistribution and degradation in tumor-bearing mice by using aPD-1/PD-L1 Abs labeled with radioisotopes (In-111/I-125) and evaluated the relationship between PK and therapeutic effects.

Method: Abs were labeled with In-111 via chelate agents, and labeled with I-125 through covalent bond. Tumor bearing mice were prepared by s.c. inoculation with mouse colon cancer MC38 cells or mouse breast cancer MM48 cells. The labeled Abs were intraperitoneally injected into tumor-bearing mice. Tumors and organs were harvested at several time points after the injection, and radio activities in organs were measured by a gamma counter. The accumulation of Abs were expressed as % of injected dose/g organs. Because In-111 tends to be accumulated in organs due to poor permeability and I-125 was eliminated from organs rapidly due to high permeability, the ratio of I-125 and In-111 could reflect the degradation of Abs after cellular uptake. In pharmacological studies, Abs were intraperitoneally injected into tumor-bearing mice at doses of 50 to 200 μg (2.5 to 10 mg/kg) at day 5, 8, and 12 after tumor-inoculation. Tumor volume was evaluated to evaluate tumor progression.

Result and Discussion: aPD-1 Ab showed anti-tumor effect in both MC38 and MM48 tumor models in a dose-dependent manner. On the other hand, aPD-L1 Abs showed lower anti-tumor effect in MC38 models , and negligible effect in MM48 tumor bearing mice at tested doses. According to PK studies, it was observed that aPD-L1 Abs were largely accumulated in normal tissues, especially in the spleen, liver, and kidney, and degraded rapidly compared with aPD-1 Abs, resulting that the blood concentration and distribution in tumors of aPD-L1 Abs tended to be low. Moreover, aPD-L1 Abs showed more rapid degradation in both tumors than aPD-1 Ab.

Conclusion: aPD-L1 Ab showed less anti-tumor effect in tested tumor models due to less distribution and faster degradation in tumors than aPD-1 Ab. Collectively, the PK of aPD-1/PD-L1 Abs which target the same axis were not equivalent and the selectivity of expression of target molecules in both normal tissues and tumors should be considered to optimize their therapeutic efficacy.

#3233

Novel CTLA-4 antibodies of potent antitumor activity were verified in humanized mouse models.

Yuelei Shen,1 Jian Ni,2 Benny(Yi) Yang,1 Tian Gan,1 Chaoshe Guo1. 1 _Biocytogen, Beijing, China;_ 2 _Eucure, Beijing, China_.

CTLA-4 (cytotoxic T-lymphocyte-associated protein 4), is a member of the immunoglobulin superfamily. It is expressed in T cells upon activation and transmits an inhibitory signal to T cells. It is also constitutively expressed in regulatory T cells (Tregs) which is associated with their immunosuppressive phenotype. CTLA-4 is homologous to the co-stimulatory protein CD28, and both receptors bind to the same ligands, CD80(B7-1) and CD86(B7-2). Importantly, cancer cells can be recognized and destroyed by the host's immune system. In this setting, CTLA-4 functions as a brake to dampen anti-tumor T cell responses, which promote cancer progression.

In this context, we are exploring antibodies that are as effective or exceed the effects of the approved CTLA-4 antibody. Antibodies blocking CTLA-4 are expected to subvert T cell inhibition. Moreover, regulatory T cells residing in the tumor microenvironment are sensitive to anti-CTLA-4 antibody since they have higher expression levels of CTLA-4 compared to activated T cells. Yervoy, the first FDA-approved CTLA-4 antibody, protects a fraction of cancer patients when used alone or in combination with other drugs. Here we report two humanized antibodies whose efficacy is equivalent or exceed that of Yervoy using the pharmacological efficacy evaluation platform established at Biocytogen. Potent inhibition and biological activity were further demonstrated by a series of tests in vitro. In conclusion, we successfully discovered two antibodies that exhibit superior anti-cancer activity in vivo with our humanized CTLA-4 mouse model. Downstream clinical evaluation of these novel antibodies is needed.

#3234

PD-1 is intrinsically expressed by lung cancer cells with stemness features inhibited by PD-1 blockade.

Ramona Rotolo,1 Valeria Leuci,1 Chiara Donini,1 Martina Sanlorenzo,2 Igor Vujic,3 Giovanni Medico,4 Francesca Vita,4 Loretta Gammaitoni,5 Luisella Righi,6 Chiara Riganti,7 Elisa Vigna,1 Lorenzo D'Ambrosio,1 Giovanni Grignani,5 Giorgio V. Scagliotti,6 Silvia Novello,6 Massimo Aglietta,1 Dario Sangiolo1. 1 _University of Torino, Candiolo Cancer Institute FPO-IRCCS, Candiolo, Italy;_ 2 _Comprehensive Cancer Center, Institute of Cancer Research, Medical University of Vienna, Vienna, Austria;_ 3 _The Rudolfstiftung Hospital, Vienna, Austria;_ 4 _University of Torino, Italy;_ 5 _Candiolo Cancer Institute FPO-IRCCS, Candiolo, Italy;_ 6 _University of Torino at San Luigi Gonzaga Hospital, Orbassano, Turin, Italy;_ 7 _University of Torino, Department of Oncology, Torino, Italy_.

Purpose: Aim of this study is to explore the intrinsic expression, functional role and therapeutic modulation of PD-1 receptor in Non Small Cell Lung Cancer (NSCLC) cells. We previously reported (Sanlorenzo M et al. Clinical Cancer Res 2018) that PD-1+ melanoma cells may sustain disease relapse following treatment with BRAF/MEK inhibitors. We hypothesized that PD-1+ tumor cells may characterize a "stem-like" compartment also in NSCLC, sustaining chemo-resistance and disease relapse with potential therapeutic implications.

Experimental procedures: The expression of PD-1 by NSCLC cells was explored by flow cytometry, western blot (WB) and RT-PCR. Its presence and role in lung cancer stem cells (CSC) was explored by sphere formation essays. CSC were further visualized by tumor-engineering with a lentiviral CSC-detector vector (LV-CSC) encoding eGFP under control of the OCT4 stem gene promoter. Selective PD-1 blockade and PD-1 stimulation with soluble ligand (PD-L1) were used to study the functional role and therapeutic modulation of intrinsic PD-1 in NSCLC.

Results: PD-1 is intrinsically expressed by a small subset of NSCLC cells with stemness features. We found PD-1 consistently expressed on the membrane of a small tumor cell fraction (2% ± 0.3) within 6 NSCLC cell lines (H1975, EBC-1, H23, H820, HCC827), including a primary patient-derived NSCLC culture (SL1). Data were confirmed by RT-PCR and WB. Viable PD-1+ tumor cells were significantly enriched in NSCLC spheres generated in stem conditioned cultures, compared with the monolayer controls (10% [4-36] vs 2% [1-5] P<0,0001, n=6). The levels of PD-1 and stem gene OCT4 RNA comparably increased in NSCLC spheres (4.5 vs 4 fold, n=5). The tumor-intrinsic expression of PD-1 was confirmed by data mining in 67 adenocarcinoma (Affimetryx RNA value 4.348 [3.882-6.361]) and 28 squamous lung carcinoma (RNA value 4.338 [3.995-5.178]) cell lines (CCLE). The formation of NSCLC spheres was significantly inhibited (-30%±2, n=6 P=0.0004) by anti-PD-1 blocking antibody (100μg/μl), while enhanced (25% ± 2, n=3, P= 0.007) by soluble PD-L1 (50 μg/μl). Similar results were confirmed by selective PD-1 RNA-interference that revoked the pro-tumorigenic effect of soluble PD-L1. In vitro treatment with Cisplatin (IC50 dose) led to a relative enrichment of PD-1\+ (2.5±0.3 fold, n=4) cells and OCT4+ CSC (3.5±0.3 fold, n=4). The sequential PD-1 inhibition significantly delayed NSCLC cell recovery (-45%±9.8, n=4) and sphere formation after cisplatin.

Conclusions: PD-1 is intrinsically expressed by NSCLC cells with stemness features and mediates pro-tumorigenic activity. PD-1+ NSCLC cells are enriched after chemotherapy and may be inhibited by selective PD-1 blockade. We report a new, intrinsic, expression pattern of PD-1 in NSCLC, providing rationale to explore a lymphocyte-independent activity of anti-PD1 antibodies.

#3235

**Activation-inducible TNFR family receptor (AITR) signaling mediates the polarization of CD4** + **T cells into Th1, Th2, and Th17, converts Treg to Teff and eradicates solid tumors.**

Byoung S. Kwon, Seung J. Lee, Young H. Kim, Joong W. Lee, Sun H. Hwang, Dass S. Vinay. _Eutilex Co.,Ltd., Seoul, Republic of Korea_.

AITR (activation-inducible TNFR family receptor, human GITR), an inducible costimulatory receptor of human T cells, is expressed constitutively on Treg and inducible on Teff upon antigen engagement. AITR co-stimulates T cell activation and recruits certain members of TRAF family upon interaction with its ligand. We examined whether the stimulation of distinct regions of extracellular domain of AITR can generate signals that promote different subsets of CD4+ T cells. We found that crosslinking AITR by agonistic anti-AITR Abs (A27, A41, and A35), that recognize three distinct regions of the extracellular domain, polarize CD4+ T cells into Th1, Th2 and Th17, respectively. The subset polarization evoked by these antibodies was the result of recruitment of specific TRAFs, phosphorylation of distinct STATs and expression of Th subset fate-determining master regulatory genes. Stimulation with A27 recruited TRAF-1 and -2, activated STAT-1, -4 and JNK1/2, and induced transcription factor T-bet. On the other hand, activation with A35 recruited TRAF-6, activated STAT-3 and p38 MAPK, and induced RORγt. In contrast, A41 recruited TRAF-3 and -5, activated STAT-5, -6 and ERK1/2, and produced GATA-3. Remarkably, A27 induced the conversion of CD25+Foxp3+ Treg cells to IFN-γ-producing Th1 cells with a concomitant suppression of Foxp3 and TGF-β expression both in healthy individuals as well as cancer patients. A35 also, but not A41, suppressed the expression of Foxp3 and induced IL-17 in Treg cells. A27 alone, but not others produced a strong anti-tumor effect in a human tumor xenograft model of humanized mice in an IFN-γ- and Treg conversion-dependent mechanism. Taken together, the data that AITR regulates Th subset polarization and eradicates solid tumors can be a target for broad-spectrum immune check point therapeutics against cancers and autoimmune diseases.

#3236

T-cell intrinsic mechanisms of resistance to PD-1 checkpoint blockade.

Duane Moogk,1 Lin Wang,1 Kaitao Li,2 Zhou Yuan,2 Jeffrey Weber,1 Iman Osman,1 Cheng Zhu,2 Michelle Krogsgaard1. 1 _NYU School of Medicine, New York, NY;_ 2 _Georgia Institute of Technology, Atlanta, GA_.

Although much clinical progress has been made in harnessing the immune system to recognize and target cancer, there is still a significant lack of an understanding of how tumors evade immune recognition and the mechanisms that drive tumor resistance to both T cell and checkpoint blockade immunotherapy. Our objective is to understand how tumor-mediated signaling through inhibitory receptors, including PD-1, combine to affect the process of T cell recognition of tumor antigen and signal activation in melanoma patients. The broader goal of the project is to understand the basis of resistance to PD-1 blockade and potentially identify new molecular targets to enable T cells to overcome dysfunction mediated by multiple inhibitory receptors (IR). Our biophysical measurements have shown that that the activities of TCR-proximal signaling components affect T cell antigen recognition and sensitivity at the earliest stages of T cell stimulation. We have shown that T cell antigen recognition is influenced by PD-1 and other inhibitory receptors via Shp-1/2 by targeting CD28 and Lckby directly suppressing TCR-pMHC-CD8 binding. Phospho-proteomics and flow cytometry-based analysis of melanoma patient-derived T cells from PD-1 blockade responders and non-responders identified additional mediators, signaling components and pathways associated with blockade resistance. CRISPR/Cas9 mediated genome editing was utilized to determine if resistance is mediated by the continued signaling of multiple IRs by perturbing IR signaling in mouse models of PD-1 blockade.Targeting these interactions and understanding the basis of resistance to PD-1 blockade would potentially allow identification of novel biomarkers of resistance or new molecular targets to enable T cells to overcome dysfunction during PD-1 checkpoint blockade.

#3237

Combinatorial immune checkpoint inhibitor therapy in anaplastic thyroid cancer.

Sanjukta Chakraborty,1 Rachana R. Maniyar,1 Sina Dadafarin,1 Ghada Ben Rahoma,1 Sarnath Singh,1 Augustine Moscatello,2 Jan Geliebter,1 Raj K. Tiwari1. 1 _New York Medical College, Valhalla, NY;_ 2 _Westchester Medical Center, Valhalla, NY_.

Anaplastic thyroid cancer(ATC) is a rare but extremely aggressive form of endocrine malignancy that accounts for only 1-2% of total thyroid cancer cases but responsible for 20-30% of annual mortality from thyroid cancer in the USA. Genetic lesion landscape in ATC is not conducive to multiple targeted therapies. BRAF mutation is one of the dominant genetic lesions observed in 30-40% of ATC cases and 98% of them are BRAFV600E positive. Due to the limited therapeutic efficacy of BRAFV600E inhibitor vemurafenib, identification of novel therapeutic candidates would provide a viable alternative. Immunecheckpoint therapies have showed huge promise in recent years but are relatively underexplored in ATC patients. To this end, 4 ATC cell lines 8505C, T238, SW1736 and HTh74; 3 PTC cell lines TPC-1, BCPAP and K1 and 1 FTC cell line CGTH-W-1 were screened for expression of 29 immune-checkpoint molecules by qRT PCR with or without 10μM vemurafenib treatment for 24 hrs. Initial screening revealed a differential expression level of the transcripts among the cells. Vemurafenib treatment further up regulated expression of CD160, HVEM, BTLA, TIM3 and galectin9 in cell lines positive for BRAFV600E mutation (8505C, SW1736, BCPAP) and the ones harboring an additional PIK3CA mutation (T238, K1). This observation was further confirmed by immunocytochemistry. CD160 was not detected in any of the cell lines except for K1, TPC-1 and NTHY at protein level. Flow cytometry confirmed presence of BTLA, HVEM and TIM3 on the surface of these tumor cells which would enable them to engage their cognate ligands on T cells and suppress antitumor immune response. Western blots confirmed upregulation of BTLA, HVEM, TIM3 and galectin 9 in response to vemurafenib in both BRAFV600E positive cell lines and HTh74. Expression of these molecules were also validated by immunohistochemistry in patient samples. In an effort to evaluate the functional activity of these immunecheckpoint molecules, co-culture studies were done. Both HVEM and BTLA showed immunomodulatory capability and were capable of redirecting T cell differentiation towards a suppressive phenotype. Preclinical studies with combination of BRAFV600E or PI3K inhibitor and antagonistic antibodies against these molecules are currently underway. This study has identified two novel immune checkpoint molecules in ATC and might provide viable therapeutic targets.

#3238

SIRPa blockade reinvigorates myeloid cells in the tumor microenvironment and reverses T-cell exclusion.

Vanessa Gauttier,1 Sabrina Pengam,1 Justine Durand,1 Kévin Biteau,2 Mélanie Néel,2 Sophie Conchon,2 Dominique Costantini,1 Bernard Vanhove,1 Nicolas Poirier1. 1 _OSE Immunotherapeutics, Nantes, France;_ 2 _CRTI - UMR1064, Nantes, France_.

Cancer immunotherapies by immune checkpoint blockade (CKI) has shown great therapeutic efficacy by helping the immune system to recognize and attack cancer cells. However, a significant percentage of patients do not respond or develop resistance. T-cell exclusion from tumor core, mediated by myeloid cells and fibroblast colonizing the tumor micro-environment, has been described as an important factor of resistance to checkpoint inhibitors. Myeloid cells represent one of the most abundant immune cell types in many solid tumors and are often associated with a poor outcome. Interaction of SIRPalpha (SIRPá), expressed by myeloid cells, with the ubiquitous receptor CD47 is an important immune checkpoint of the innate response, involved in the regulation of macrophages and dendritic cells functions (e.g. phagocytosis, antigen presentation). Here we evaluated the impact of selective SIRPá blockade on the function of myeloid cells from tumor micro-environment.

In vivo, in an orthotopic and syngeneic hepatocellular carcinoma (HCC) mouse model, anti-SIRPá antagonist monoclonal antibody in combination with an adaptive immune checkpoint inhibitor (anti-PD-L1) dramatically enhanced overall survival and lead to complete remission in up to 60% of immunocompetent mice (p<0.001; n=11/18). A robust memory immune response developed since a second tumor challenge after drug elimination was always rejected (p<0.01). Transcriptional analysis of tumor at early stage (10 days after treatment) using Nanostring technology revealed a significant enrichment of a multiple chemokine signature in animals that received anti-SIRPá and anti-PD-L1 combination. Histological analysis of HCC tumors revealed that anti-PDL1 mAb alone increased T-cell infiltrates, but T-cell remained excluded from tumor core. In contrast, tumor nodules were strongly reduced, and T-cell significantly enriched in tumor nest in combination with the SIRPá antagonist (p<0.01). In vitro, we next observed that CD47-induced signals inhibited secretion of chemokines by immature human macrophages. This inhibition could be reversed by a selective anti-SIRPá antagonist mAb. Ex-vivo, selective anti- SIRPá antagonist mAbs significantly increased the chemokine transcriptomic expression signature of human myeloid cells purified from ovarian cancer ascites or of HCC tumor explant fragment maintained in culture for 48 hours. Finally, we found in vivo in a humanized mice model of transmigration that SIRPá blockade significantly increased human T cell migration in microenvironment locally enriched by human macrophages.

In conclusion, we showed that selective SIRPá antagonist mAbs synergized with T-cell CKI by modifying the tumor microenvironment, in particular the chemokine secretion by mouse or human myeloid cells which contributes to limit T cell exclusion and favors efficient and robust anti-tumor immune responses.

#3239

Co-blockade of innate and adaptive immune checkpoints with SIRPαxPD-L1 bispecific antibodies for immunotherapy.

Ugur Eskiocak, Wilson Guzman, Thomas Daly, Allison Nelson, Pearl Bakhru, Jason Lajoie, Sara Haserlat, Samantha Ottinger, Lucy Liu, Amanda Oliphant, Alan Leung, Girish Hemashettar, Rachel Rennard, Benjamin Wolf, Monia Draghi, Michael Schmidt, Robert Tighe. _Compass Therapeutics, Cambridge, MA_.

Blockade of inhibitory checkpoint pathways, such as PD-1/PD-L1 and CTLA-4, has provided significant benefit to subsets of patients and changed the cancer therapy landscape. These checkpoint inhibitors promote adaptive T cell-mediated anti-tumor immunity while signaling regulatory protein alpha (SIRPα)/CD47 axis represents an innate specific checkpoint that has become an attractive target for immunotherapy. CD47 is expressed by virtually all cells, providing an anti-phagocytic "don't eat me" signal through its interaction with the inhibitory immune receptor SIRPα which is expressed on myeloid cells. Blockade of the CD47-SIRPα interaction has been shown to promote phagocytosis and antigen presentation. Bispecific antibodies are emerging as an attractive therapeutic modality capable of simultaneously engaging two targets with a single drug to improve efficacy. Here we describe the discovery and characterization of a series of fully human, tetravalent common light chain SIRPαxPD-L1 bispecific antibodies.

Novel PD-L1 monoclonal antibodies (mAbs), used as building blocks, demonstrated equivalent activity to approved PD-L1 mAbs both in vitro and in vivo. The CD47 binding domain of SIRPα is known to be highly polymorphic, with allelic variants differing by as many as 13 amino acids. Novel, affinity optimized SIRPα mAbs, used as building blocks, bound to all tested polymorphic variants with higher specificity towards SIRPα expressed on myeloid cells as opposed SIRPγ expressed on T cells. In vitro studies showed that, the SIRPα mAbs enhanced antibody-dependent cell mediated phagocytosis (ADCP) and increased antigen presentation by dendritic cells. In vivo, the combination of the SIRPα mAbs and the Trp1 targeting TA99 mAb significantly reduced tumor burden in the B16F10 lung metastasis model without inducing hematological toxicities commonly associated with anti-CD47 antibodies.

Compass Therapeutics' common light chain discovery platform enables the rapid assembly of individual Fabs into multispecific constructs with comparable manufacturability and stability profiles as conventional mAbs. Using this platform, we have generated a series of SIRPαxPD-L1 bispecific antibodies in various orientations and isotypes and identified bispecific antibodies with superior activity (5-10 fold) compared to combination of the individual mAbs in phagocytosis assays in vitro. We are currently performing deep characterizations of these novel SIRPαxPD-L1 bispecifics to identify lead candidates that optimally boost tumor antigen presentation to T cells through SIRPα blockade, while simultaneously releasing T cells from PD-1/PD-L1 mediated suppression. This novel approach holds the potential to improve therapeutic outcomes for cancer patients.

#3240

EOS884448, a high affinity fully human antibody directed against TIGIT, mediates in vitro anti-tumor activity through multiples mechanisms of action involving activation of intratumor effector cells and depletion of regulatory T cells.

Julia Cuende,1 Virginie Rabolli,1 Florence Nyawouame,1 Marjorie Mercier,1 Angela Pappalardo,2 Lucile Garnero,1 Julie Dechanet-Merville,2 Gregory Driessens,1 Catherine Hoofd1. 1 _iTeos Therapeutics, Gosselies, Belgium;_ 2 _Université of Bordeaux, Bordeaux, France_.

T cell Immunoreceptor with Ig and ITIM domains (TIGIT) is a T cell co-inhibitory receptor recently described as a key checkpoint driving tumor cell immunosuppression. It is predominantly expressed on regulatory CD4+ T cells (Tregs), CD8+ T cells and NK cells from healthy individuals but further upregulated in cancer patients where it frequently co-expresses with exhaustion markers such as PD-1. TIGIT cognate receptors are members of the poliovirus receptors, among which CD155 has the highest affinity for TIGIT. They are expressed on antigen presenting cells but also on tumor cells which provides a strong rationale for blocking TIGIT as a therapeutic approach to reverse T or NK cell dysfunction linked with cancer progression. This anti-tumor mode of action of an aTIGIT antibody can be further enhanced with the selection of a FcγR interaction enabling isotype to allow for the potential depletion of TIGIT+ Tregs, described as highly immunosuppressive.

EOS884448 was selected among antagonist anti-TIGIT antibodies, using a yeast display library of fully human antibodies and characterized for their binding and anti-tumor properties. EOS884448 displays a subnM affinity to primary T cells from healthy donors and cancer patients and potently prevents binding of TIGIT ligands. High affinity binding to cynomolgus TIGIT allows for its direct use in GLP Tox studies in non-human primates.

EOS884448 potently prevents TIGIT activation and restores pro-inflammatory cytokine release in presence of CD155 ligand. This mode of action was assessed first in an IL-2 driven reporter-based system and also with primary T cells from healthy individuals or patients suffering from different solid tumor indications. Importantly, EOS884448 shows superior potency compared to antibodies from other anti-TIGIT programs in which sequences have been published.

Interestingly, EOS884448-mediated restoration of cytokine release activity in presence of TIGIT ligand was observed both for conventional and non-conventional T cells.

When evaluated with different isotype formats, EOS884448, as an hIgG1, shows preferential depletion of Tregs over memory CD4+ and CD8+ T cells. This key finding supports the use of an isotype with high affinity for activatory FcγR in cancer patients who could benefit from a drug acting simultaneously through depletion of highly suppressive Tregs and restoration of T and NK cell effector functions within tumor.

#3241

Novel anti-CD40 antibodies demonstrate anti-tumor activity in humanized mouse models.

Chaoshe Guo,1 Benny(Yi) Yang,1 Jian Ni,2 Yanan Guo,1 Tian Gan1. 1 _Biocytogen, Beijing, China;_ 2 _Eucure, Beijing, China_.

CD40 (Cluster of Differentiation 40) is a member of the TNF-receptor superfamily. CD40 is a co-stimulatory protein found primarily on antigen presenting cells, including B cells, macrophages and dendritic cells. Its ligand, CD40L (CD154) is predominantly expressed on T helper cells, whose engagement activates antigen presenting cells and induces a variety of positive downstream effects. Whereas most immune checkpoint receptors are expressed on the surface of T cells, CD40 stands out as it is expressed by antigen presenting cells and controls the proper activation of these cells. In turn, CD40 activation exerts a significant impact on T cell-mediated activity against cancer cells. Several pharmaceutical companies are pursuing anti-CD40 therapies for treating hematological and solid cancers.

Therefore, it is imperative to develop novel CD40 antibodies to boost cancer patients' own immunity in light of other successful immune checkpoint modulators. We asked whether we could find suitable anti-CD40 reagents that successfully combat developed tumors in preclinical settings. We designed a powerful screening strategy based on Biocytogen's in vivo drug screening platform. Candidate antibodies were generated by immunizing wild-type mice with recombinant hCD40 protein. Hybridomas were then screened by high-throughput flow cytometry. Instead of examining the immune modulating activity in cell culture, we tested these antibodies in B-hCD40 mice bearing MC38 tumors and monitored the tumor growth. Through this straightforward screening strategy, several clones stood out by their unparalleled benefits. These clones were further selected for humanization. Furthermore, we identified that one clone works in concert with Keytruda in the dual humanized model of h-CD40 and h-PD1. In conclusion, we uncovered CD40 antibody candidates with the most potent anti-tumor activity which are promising as tools for cancer treatment in the preclinical setting.

#3242

Triple checkpoint blockade targeting PD-1, TIM-3, and LAG-3 reinvigorates ovarian cancer-infiltrating T cells by increasing T cell polyfunctionality and effector function.

Johanna K. Kaufmann,1 Brianna Flynn,2 Kevin Morse,2 Maria C. Speranza,1 Jing Zhou,2 Sridhar Ramaswamy,1 Sean Mackay,2 Kevin G. Coleman1. 1 _TESARO Inc, Waltham, MA;_ 2 _IsoPlexis, Branford, CT_.

Co-expression of immune checkpoint receptors (ICRs) PD-1, TIM-3, and LAG-3 characterizes chronically activated and exhausted tumor-infiltrating T cells (TILs), suggesting their targeting may have applicability for the treatment of multiple cancer types. We previously reported improved tumor control in various syngeneic and humanized mouse models when treated with a combination of TSR-042 (αPD-1), TSR-022 (αTIM-3), and TSR-033 (αLAG-3) as compared to single or double combinations. Here, we are characterizing TILs from ovarian cancer tissues and their functional response to triple combination treatment. Immune profiling using flow cytometry confirmed expression of all three ICRs on TILs isolated from primary resections of ovarian cancer. Ex vivo re-stimulation of immune infiltrates with S. aureus enterotoxin B in presence of ICR-targeting antibodies led to increased secretion of IFN-γ and IL-2 when treated with TSR-042. Notably, triple combination of TSR-042, TSR-022, and TSR-033 further amplified cytokine release, indicating more effective TIL reinvigoration. To further understand the differential effects of triple combination treatment over PD-1 blockade, we analyzed αCD3/αCD28-stimulated and antibody-treated ovarian cancer TILs on a single cell level using a microfluidic IsoCode chip technology that allows for parallel detection and quantification of 32 secreted proteins from live single cells. Combining the amount of each protein secreted by polyfunctional T cells (co-secreting two or more proteins per cell) with the frequency of such cells results in a measurement of polyfunctional strength (PSI), a unique IsoCode-enabled metric that has been associated with improved response to ICR inhibition. TSR-042 increased the PSI of CD4+ and CD8+ TILs 1.4 and 1.5-fold over isotype control treatment. Importantly, triple combination treatment was able to significantly increase the PSI of both subsets by 2.9 and 3.7-fold, respectively (p < 0.001). For CD8+ TILs, this increase was mainly driven by an increase in the frequency of polyfunctional subsets, while for CD4+ TILs, the absolute amounts of secreted cytokines had a larger impact. Interestingly, both classical effector cytokines like Granzyme B and IFN-γ as well as other secreted factors like chemoattractant factors, implicated in the recruitment of multiple immune cell subsets to tumor tissue, contributed to T cell polyfunctionality. Taken together, triple ICR blockade targeting PD-1, TIM-3, and LAG-3 reinvigorated ovarian cancer TILs more effectively than PD-1 inhibition alone. This was mediated by increasing T cell polyfunctionality, which has been associated with improved anti-tumor activity and response to ICR inhibition. This data further supports the concept of triple combination checkpoint blockade as a treatment option for ovarian cancer.

#3243

A CTLA4 antibody with enhanced properties.

Leehee Weinberger,1 Ariel Stanhill,1 Aviv Boim,1 Karin Meyer,1 Orith Leitner,2 Hedva Hamawi,2 Ami Navon2. 1 _Tikcro Technologies, Rehovot, Israel;_ 2 _Weizmann Institute of Science, Rehovot, Israel_.

Background: Immune therapy has been viewed in recent years as a major new path in oncology greatly due to the pioneering success in clinical practice of Yervoy (Ipilimumab), a CTLA-4 monoclonal antibody. To date, only a handful of immune modulator antibodies are U.S. FDA approved. CTLA-4 antibody mechanism of action of is not fully resolved and addresses Fc dependent elimination of tumor associated T regulatory cells as well as blocking of CTLA4-CD80/CD86 ligand interaction. We present a new CTLA-4 monoclonal antibody (TikAb) that demonstrates superior properties both in-vitro and in-vivo.

Methods: Based on the crystal structure of CTLA-4:CD80 and CTLA-4:CD86, a mimitope (a synthetic mimetic of the CTLA-4 surface interacting with CD80/CD86) was synthesized and used to immunize humanized mice. Known procedures for fusion and monoclonal antibody generation were performed and clones were selected based on FACS binding to CTLA-4 and CD80/86 interaction blocking. Functional T cell activation was performed and human CTLA-4 knock-in mice were used in a MC38 colon tumor model to evaluate TikAb efficacy in-vivo.

Results: TikAb, a full human antibody, demonstrates high binding affinity towards CTLA-4 expressing cells of 6-8-fold increased affinity over Ipilimumab, as was determined by cell-based assay (FACS) and by SPR (Biacore). Blocking of CTLA-4 ligands CD80 and CD86 was also significantly increased relative to Ipilimumab. TikAb binding towards endogenous CTLA-4 expressing cells (activated CD4+ T-cells) was also observed and was improved compared with ipilimumab. CTLA-4 epitope for TikAb binding differs from the reported epitope of Ipilimumab on CTLA-4, by the differential binding of TikAb towards CTLA-4 of different species. The same difference in the binding epitope was also observed following a comparison to numerous other pre-clinical and early stage CTLA-4 antibodies. Synergetic activation of T-cell activation is observed upon a combined treatment of PBMC cells with TikAb and Nivolumab (a PD-1 monoclonal Ab). In-vivo tumor models of MC-38 colon cells in CTLAh/h mice showed a dose dependent reduction of tumor size and greater reduction of tumor size and a significant increase in survival rates compared with Ipilimumab. Re-challenge of TikAb surviving mice with tumor cells showed immunological memory and complete recovery without further treatment.

Conclusions: We present TikAb - a new full human CTLA4 antibody with enhanced properties in values determined in-vitro and in-vivo. These observation support further examination of TikAb as a therapeutic candidate to treat cancer which may allow a lower treatment dose with an improved efficacy to peers.

#3244

Improved Fc-mediated effector functions by an anti-CTLA-4 multivalent Fc agent.

Laura Rutitzky, Daniel F. Ortiz, Jonathan C. Lansing, Julia Brown, Joseph Paquette, Kevin Garofalo, Josephine D'Alessandro, Naveen Bhatnagar, Maurice Hains, Abhinav Gupta, Stan Lee, Radouane Zouaoui, Jason Wang, John Schaeck, Salvatore Marchese, Robin Meccariello, Nathaniel Washburn, Kimberly Holte, Carlos J. Bosques, Anthony M. Manning. _Momenta Phamaceuticals Inc, Cambridge, MA_.

Numerous therapeutic monoclonal antibodies (mAbs) rely on antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC) Fc effector functions to deplete target cells and achieve clinical efficacy. It has been previously shown that adding multiple Fc domains to Abs or afucosylating the Fc domain increases Fc effector functions. We have leveraged a proprietary Fc multimerization technology (SIF; selective immunomodulator of Fc receptors) to identify potential novel products designed to improve the immune system's elimination of tumor and other pathogenic cells. These agents utilize the valency effect of Fc multimerization to increase the binding to Fc receptors and complement, enhancing immune-mediated cytotoxicity mechanisms. CTLA-4 is a clinically validated immune checkpoint inhibitor exemplified by the human IgG1 therapeutic mAb ipilimumab. CTLA-4 is induced upon activation on T cells and constitutively expressed on T regulatory cells (Tregs). Based on data from mouse models of cancer and clinical studies, the proposed mechanisms of action of anti-CTLA-4 mAbs, including ipilimumab, are to block the interaction of CTLA-4 with its ligands CD80 and CD86 resulting in T cell activation and to induce Fc-mediated ADCC of CTLA-4+ cells, mainly intratumoral Tregs. Therefore, we have produced an anti-CTLA-4 multivalent IgG1 Fc agent termed anti-CTLA-4 SIFbody with the purpose of enhancing binding to Fc gamma receptors (FcγRs) and complement and increasing Fc-mediated elimination of intratumoral Tregs. Data from avidity binding to low affinity FcγRs showed more than 100-fold increase with anti-CTLA-4 SIFbody as compared to ipilimumab. In in vitro functional assays using CTLA-4 transfected target cells and primary human effector cells, anti-CTLA-4 SIFbody showed more than 10-fold increase in potency in ADCC and more than 5-fold increase in ADCP as compared to ipilimumab. Anti-CTLA-4 SIFbody induced 80% cell lysis by CDC, whereas ipilimumab failed to show any activity. More importantly, we generated in vitro expanded Tregs with suppressive function and showed that anti-CTLA-4 SIFbody induced significant enhanced ADCC on these cells as compared to ipilimumab and to an afucosylated anti-CTLA-4 mAb. ADCP was also significantly increased on Tregs. Unexpectedly, Tregs were resistant to anti-CTLA-4 SIFbody induced CDC. Notably, the blocking activity of the SIFbody F(ab)s was not altered by the multivalent Fc structure as shown by similar CTLA-4/CD80 and CD86 blockade and induction of IL-2 production upon antigen stimulation of PBMC. In conclusion, these data demonstrate that our Fc multimerization technology applied to an anti-CTLA-4 mAb significantly improves Fc-dependent immune-mediated cytotoxicity and suggest that anti-CTLA-4 SIFbody may represent an optimized novel product to deplete intratumoral Tregs and enhance anti-tumor activity.

#3245

Durvalumab induces an NK cell response associated with clinical benefit of patients with advanced NSCLC.

Maria Libera Ascierto, Yashaswi Shrestha, Qu Zhang, Jixin Wang, Han Si, Lydia Greenlees, Rebecca Halpin, Ikbel Achour, Zachary Aaron Cooper, Rajiv Raja, Shaad Abdullah, Katie Streicher, Brandon Higgs. _Medimmune, Washington, MD_.

Introduction: The role of CD8 cells in determining clinical outcome to programmed death ligand-1 (PD-L1) blocking treatments has been well characterized, however, the contribution of NK cells is not well understood. This is partly due to the paucity of NK cell-specific markers that can identify NK cells in the tumor microenvironment (TME). We developed an NK cell-specific transcriptional signature to estimate the NK cell abundance in the TME. This signature, together with NK-chemokines shown to modulate the priming of adaptive immunity1 were investigated in patients with advanced non-small cell lung cancer (NSCLC) treated with a PD-L1 inhibitor, durvalumab.

Methods: Peripheral blood mononuclear cells (PBMCs) and Fluorescence-Activated Cell Sorted (FACS) NK/ CD8 populations from three heathy donors were subjected to single cell RNA sequencing (scRNAseq, 10X Genomics) and transcriptome analysis (Affymetrix), respectively. Fresh frozen tumor biopsies from 97 NSCLC were profiled with RNA sequencing prior to durvalumab treatment; 29 of these had paired tumors procured 29 days following treatment with durvalumab. Kaplan Meier (KM) analyses were performed to identify predictive effects of the NK cell-specific signature. Clinical trial:1108/NCT01693562

Results: Transcripts over-expressed in sorted NK relative to CD8 cells were first identified (p <0.01; fold >3) and intersected with 28 mRNAs up-regulated in the NK cell cluster determined by scRNAseq, providing an 8 gene NK cell-specific transcriptional signature defined as MEDI-NK. MEDI-NK correlated with NK signatures recently described2, and included chemokines shown to induce an effective NK- response1. When evaluated in TCGA, higher expression of MEDI-NK was associated with good prognosis (Overall Survival, OS) of patients with melanoma and breast cancer (p value =0.03 and =0.001, respectively).

At baseline, MEDI-NK was highly correlated with the previously identified IFNγ signature3 and was associated with Progression Free Survival (PFS p value < 0.02) of NSCLC patients treated with durvalumab. Following treatment with durvalumab, the increased expression of MEDI-NK and of additional genes leading to NK-priming of adaptive immunity1 was observed to be associated with patients' overall survival (OS p value <0.01). Similar findings were not observed prior to durvalumab treatment.

Conclusions: Using single cell analysis, an NK cell-specific signature was developed to better define the role of NK cells in anti-PDL1 therapy. The increased expressions of the NK cell-specific signature and of genes leading to NK-cell priming of adaptive immune response were associated with clinical benefit to durvalumab.

References:

1. Böttcher JP et al, Cell, 2018.

2. Barry KC et al, Nature Medicine, 2018

3. Higgs B et al, Clinical Cancer Res, 2018

#3246

Dynamics of T-cell checkpoint receptor profiles during melanoma progression.

Jarem Edwards, Annie Tasker, Inês Pires da Silva, Camelia Quek, Benjamin M. Allanson, Robyn P. M Saw, John F. Thompson, Alexander M. Menzies, Umaimainthan Palendira, James S. Wilmott, Georgina V. Long, Richard Scolyer. _The University of Sydney, Sydney, Australia_.

Background A myriad of novel monoclonal antibody based immunotherapies targeting co-stimulating and co-inhibitory receptors have entered clinical trials in melanoma with the aim of increasing response rates and overcoming resistance to standard anti-CTLA-4 and anti-PD-1 immunotherapy. However, little is known about the abundance, co-expression and immune cells enriched for each specific drug target in the various stages of melanoma progression. Therefore, we sought to assess the relative abundance of checkpoint receptors and their expression during melanoma disease progression, as well as the immune cells enriched for each of these molecules.

Methods - Multiplex immunofluorescence staining for immune checkpoint receptors (ICOS, GITR, OX40, PD-1, TIM-3, and VISTA) was performed on 95 melanoma biopsies from 41 melanoma patients, including patient matched biopsies for primary, regional lymph node or distant metastases. - Mass cytometry was performed on leukocytes isolated from 18 treatment-naïve melanoma tumors to explore immune subsets enriched for many of the checkpoint receptors currently targeted in clinical trials.

Results & Conclusions GITR and OX40 were the least abundant checkpoint receptors in melanoma (p<0.001), with less than 1% of intra-tumoral T cells expressing either marker. TIM-3 and VISTA were mostly expressed on non-T cell populations, with TIM-3 enriched on dendritic cells. Tissue resident T cells (CD69+ CD103+ CD8+) represented a population highly enriched for TIGIT (>70%) and other co-inhibitory receptors but not co-stimulatory receptors. The proportion of GITR+ T cells decreases from primary melanoma (>5%) to patient matched lymph node (<1%, p=0.04) and distant metastases (<1%, p=0.0005). This data will underpin future clinical trial design and provide a rationale for combining these molecules in the clinic.

#3247

Dynamic clonality of T cell receptor differentiate atypical progression in NSCLC patients treated with PD-1/PD-L1inhibitors.

Jiefei Han,1 Zhijie Wang,1 Yuqi Wang,2 Si Chen,2 Hua Bai,1 Jianchun Duan,1 Jie Wang1. 1 _Cancer Hospital Chinese Academy of Medical Science & Peking Union Medical College., Beijing, China; _2 _Geneplus-Beijing Institute, Beijing, China_.

Background:The advent of immune checkpoint inhibitors (ICI) has radically changed the paradigm of care for patients with non-small cell lung cancer (NSCLC). However, response patterns of tumors treated with immunotherapies may differ compared with conventional chemotherapeutic agents or targeted therapies, and accurate assessment of the response can be radiologically challenging. Most of the current immune-related response criteria are designed to identify pseudoprogression but not hyperprogressive disease (HPD). The objective of the current analysis was to explore the biomarkers which can distinguish different patterns of responses of in ICI-treated NSCLC patients.Methods:Among patients with NSCLC treated with ICI at our Institute, at least 2 CT scans before ICI therapy (baseline and the most recent scan before baseline) and 1 CT scan during treatment were mandatory for radiological evaluation. The tumor growth rate (TGR) was calculated from the sum of the largest diameters of the target lesions as per RECIST version 1.1 to evaluate the percentage increase in tumor volume per month. HPD was defined as delta TGR exceeding 50%. Pseudoprogression was defined as initial progression, followed by complete response or partial response or stable disease. Flow cytometry was used to obtain CD3+CD8+ T cells from peripheral blood mononuclearcells. Immunosequencing of the CDR3 regions of TCRb chains was performed. TCR clonality is compared pre-therapy to initial evaluation. Results:A total of 51 patients were included in our study. By the time of first radiological assessment,11 cases were defined as Responders (21.6%), 16 patients with Stable Disease (31.4%), 24 cases defined as Progressors (47.1%).Six patients meet the criteria for HPD. During the following treatment, 3 of 24 progressors were confirmed to have pseudoprogression. The degree of TCR clonality change ranged from 2.17 to 0.64 times in all patients. Clinical response is significantly correlated with increased repertoire clonality of CD3+CD8+ T cells (P = 0.01). Among three kinds of disease progression, pseudoprogression showed an increase in TCR clonality than true progression patients (1.47 vs. 0.79, P=0.01). Interestingly, the decrease of TCR clonality is greater in patients developed typical PD than in HPD (-0.19 vs. -0.05, P=0.03). Our further analysis showed that Patients who demonstrated RECIST-PD with an increased clonality had a longer median PFS compared to patients with a declined clonality (8.9 versus 2.4 months, p = 0.01).Conclusion: The results demonstrate that the change in clonality of CD3+CD8+ T cell receptor can differentiate atypical patterns of progression in patients with NSCLC treated with ICI. Results of this TCR test performed at regular intervals during ICI treatment reflect tumor biology and have potential as a powerful biomarker to predict long-term response.

#3248

B7-H3, an immune checkpoint protein is overexpressed in AML and the blocking monoclonal antibodies enhance NK cell-mediated apoptosis in AML cells.

Stanley Ly,1 Bin Yuan,1 Sabrina Grimm,2 Michael Andreeff,1 Hans-Jörg Bühring,2 Venkata Lokesh Battula1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _University Clinic of Tubingen, Germany_.

Acute myeloid leukemia (AML) is the most common and aggressive acute leukemia found in adults. Immune checkpoint inhibition has led to important clinical advances in cancer therapy in recent years due to superior cure rates compared with standard therapy. We hypothesize that B7-H3 (CD276) an immune checkpoint protein is overexpressed in AML cells and targeting B7-H3 activates immune cells against AML cells. We analyzed B7-H3 expression in peripheral blood (PB) and bone marrow (BM) mononuclear cells from AML patients (n=65) and healthy donors (n=10) at MD Anderson Cancer Center. Cell surface expression analysis by flow cytometry revealed that the cells of ~60% of the patients were positive for B7-H3 and its expression was 2- to 3-fold higher in AML cells than in healthy donor cells. B7-H3 expression is relatively higher in CD34+ AML cells than in CD34- AML cells (p<0.01). In contrast, no difference was observed between CD34+ and CD34- cells from healthy donors. The Cancer Genome Atlas RNA sequencing data revealed that patients with high B7-H3 expression had significantly lower overall and disease-free survival durations than did patients with low B7-H3 expression (p=0.024). To investigate the role of B7-H3 in immunomodulation, we stably knocked down B7-H3 in OCI-AML3 and co-cultured them with or without human PB-derived NK cells at a 2:1 ratio and measured apoptosis induction in AML cells by annexin-v binding approach. We found that knockdown of B7-H3 induced NK cell-mediated apoptosis in AML cells 3-fold compared to control AML cells. These data indicate that inhibition of B7-H3 in AML cells enhances NK cell-mediated apoptosis in AML cells. To target B7-H3, we have generated four monoclonal antibodies: B1, B2, B3 and B4 (codenamed to protect IP). To investigate whether these novel anti-B7-H3 monoclonal antibodies are able to block B7-H3 immunomodulatory function and activate NK cells, we performed a co-culture experiment with GFP-expressing OCI-AML3 cells and PB-derived NK cells in the presence or absence of anti-B7-H3 antibodies. Apoptosis induction was measured by real-time annexin-v binding using IncuCyte live cell imaging system. The addition of anti-B7-H3 monoclonal antibodies, B1, B2, B3 and B4 at 25μg/ml enhanced NK cell-induced apoptosis 3-fold in OCI-AML3 cells. These data indicate that anti-B7-H3 antibodies block the immunomodulatory function of B7-H3 and induce NK cell-mediated apoptosis in AML cells. In vivo testing of these antibodies against AML-PDX models is currently ongoing. In conclusion, we found that B7-H3 is overexpressed in AML cells and its expression is associated with bad prognosis in AML patients. Knockdown or antibody-mediated blocking of B7-H3 enhanced NK cell-induced apoptosis in AML cells. These data indicated that B7-H3 is a novel immune-checkpoint protein in AML and patients could potentially benefit from anti-B7-H3 therapies.

#3249

a-TIGIT mediates antitumor activity through multiple mechanisms of action involving activation of intratumor effector T cells and depletion of regulatory T cells.

Julie Preillon,1 Sofie Denies,1 Diane Jamart,1 Matthew J. Carter,2 Mark S. Cragg,2 Gregory Driessens1. 1 _iTeos Therapeutics, Gosselies, Belgium;_ 2 _University of Southampton, Southampton, United Kingdom_.

T cell Immunoreceptor with Ig and ITIM domains (TIGIT) is a co-inhibitory receptor expressed by lymphocytes, preferentially CD8+ T cells, NK, as well as regulatory T cells (Treg). TIGIT can be bound by several ligands including CD155 and CD112 that are frequently expressed by tumor cells. In cancer, TIGIT expression is upregulated on conventional T cells and even more on regulatory T cells. Co-expression with exhaustion markers such as PD-1 is common, giving a strong rationale for blocking TIGIT as a therapeutic approach for reversing T or NK cell dysfunction linked with cancer progression.

The mechanism of action of novel a-TIGIT monoclonal antibody (mAb) was characterized in vivo in the murine CT26 tumor model. This model mimics the human situation based on expression of TIGIT ligands on tumor cells and infiltration by effector and regulatory T cells that express higher levels of TIGIT compared to splenocytes. Two different isotypes (mIgG2a and mIgG1) of a-TIGIT mAb were used to evaluate anti-tumor efficacy, with only the mIgG2a, deleting isotype able to induce strong antitumor efficacy. The importance of Fc gamma receptors (FcγR) for the antitumor efficacy of a-TIGIT activity was further demonstrated by the use of mice KO for activatory FcγR or by depletion of NK cells which also resulted in the loss of a-TIGIT activity.

The anti-tumor efficacy of a-TIGIT mAb was associated with an increased activity of conventional CD8+ and CD4+ T cells but also with a preferential depletion of Treg within the tumor microenvironment. This activity also correlated with full occupancy of TIGIT receptor in mice.

As it has been shown that TIGIT is expressed on tumor cells in certain hematological cancers, we also established a model of EL4 tumor with engineered TIGIT expression. In this model, a-TIGIT demonstrates a strong antitumor effect that strictly depends on the isotype of the mAb.

Finally, the activity of a-TIGIT was tested in less immunogenic models including of pancreatic PancO2 cancer where it demonstrated some tumor growth delay as a single agent and very strong combination potential with other immune checkpoints.

In summary, these results show that antitumor mediated efficacy of a-TIGIT mAb depends upon target binding and also on interaction with FcγR. These findings support the clinical evaluation of a-TIGIT mAb able to engage those receptors.

#3250

B cell-produced IL-27 up-regulates PD-L1 expression in the tumor microenvironment to promote breast cancer development.

Hui Yan, Suryavathi Viswanadhapalli, Daniel Chupp, Maria Fernandez, Shuai Wu, Jingwei Wang, Justin Moroney, Julia Taylor, John Im, Carlos Rivera, Yiliao Luo, Junhao Liu, Gangadhara Sareddy, Paolo Casali, Ratna Vadlamudi, Zhenming Xu. _UTHSC San Antonio, San Antonio, TX_.

The host immune response can be evaded by tumor cells, e.g., through upregulation of the PD-L1/PD-1 immune checkpoint, and - paradoxically - even hijacked to promote cancer progression. Here we showed that B lymphocytes, which can produce anti-tumoral antibodies/autoantibodies in breast cancer (BCa), played an important role in promoting murine BCa development, as B cell-deficient μMT mice displayed virtually abrogated tumor growth from transplanted syngeneic ERα\+ breast adenocarcinoma cells, concomitant with significantly reduced PD-L1 expression and much increased cell death in residual tumors. In BCa that grew in wildtype mice and in tumor-associated ectopic lymph nodes, B cells were the major source of IL-27, which is the IL-27p28/EBI3 heterodimer and a pleotropic cytokine with both pro- and anti-inflammatory functions. Deficiency in IL-27 expression specifically in B cells also significantly reduced the BCa growth, indicating that the effect of B cells in BCa development was mainly through IL-27 production. This was effectively induced in B cells primed by TLR ligands, including those that would be released during uncontrolled cell death in the tumor microenvironment, and stimulated by CD154 and IL-21, as likely expressed by activated tumor-infiltrating CD4+ T cells. IL-27 in turn triggered JAK-dependent STAT1 and STAT3 phosphorylation in BCa cells, enhanced PD-L1 gene expression and promoted growth of these cells. In addition, among the 2040 genes compiled by the Ingenuity Pathway Analysis as involved in BCa tumorigenesis, those encoding PD-L1, IFN-γ and Hif-1α (cytokine and transcription factor known to induce PD-L1, respectively), IDO-1, chemokine receptor CXCR3 and its ligand CXCL10 were specifically upregulated in B cells by IL-27, which also induced an overall gene signature similar to what induced by IFN-γ, reflecting the activation of STAT1 and STAT3 by both cytokines. In humans, the two genes encoding IL-27 receptor subunits were highly expressed in the breast tissue, at levels comparable to those in lymphoid organs. As shown by The Cancer Genome Atlas (TCGA) and The Kaplan Meier Plotter datasets, the higher IL27p28 expression was associated with worse survival in BCa patients overall, who displayed increased circulating IL-27 levels as compared to healthy subjects, and in ER+ BCa patients after endocrine therapies. Indeed, IL-27 supported human BCa cell growth in the presence of tamoxifen, suggesting a role of B cell-produced IL-27 in the acquisition of drug resistance by BCa. Thus, TLR-fueled IL-27 induction in B cells reinforces PD-L1 expression in the tumor environment to drive BCa cell-intrinsic growth mechanisms and generation of PD-L1+ B regulatory cells involved in the immune checkpoint, leading to BCa progression and possibly anti-estrogen therapy resistance. 

### Novel Immunomodulatory Agents 1

#3251

**Fibroblast activation protein (FAP)-selective delivery of CD40 agonistic DARPin** ® **molecule for tumor-localized immune activation.**

Nicolo Rigamonti, Anja Schlegel, Sophie Barsin, Jonas Schwestermann, Susanne Mangold, Yvonne Kaufmann, Christof Zitt, Niina Veitonmäki, Victor Levitsky, Clara Metz. _Molecular Partners AG, Zurich Schlieren, Switzerland_.

CD40 is a co-stimulatory molecule belonging to the tumor necrosis factor receptor superfamily which can activate both innate and adaptive immune system, making it an interesting target for tumor immunotherapy. Systemic administration of agonistic CD40 antibodies (Ab) has shown signs of activity in cancer patients, but dose-limiting toxicity impaired the clinical efficacy. New approaches are therefore needed to increase the therapeutic index of CD40-targeting molecules and achieve better clinical outcomes. Here, we report an alternative approach designed to activate CD40 specifically in the tumor microenvironment (TME), and not systemically, in order to increase efficacy and reduce systemic toxicity. This novel approach is based on a bispecific DARPin® molecule, targeting CD40 and fibroblast activation protein (FAP) alpha, intended to induce immune activation only when clustered by binding to FAP-expressing cells in the TME. The bispecific FAP x CD40 DARPin® molecule was tested in a reporter assay and in additional cell assays using primary human 1) B cells, 2) macrophages and 3) dendritic cells. These studies demonstrated CD40 activation only in the presence of FAP-positive, but not with FAP-negative cells, confirming a mechanism of action strictly dependent on FAP-mediated cross-linking. A surrogate mouse-specific FAP x CD40 DARPin® molecule (mFAP x CD40) was generated and tested in similar in vitro assays and showed FAP-dependent activation of CD40 and comparable results as the human construct. In vivo experiments, performed in tumor-free mice, showed a comparable half-life between mFAP x CD40 and an anti-mouse CD40 Ab (clone FGK45). However, mFAP x CD40, in contrast to the FGK45 Ab, did not increase the serum level of IL-6, supporting a mode of action that is dependent on FAP-mediated crosslinking of CD40 receptor. In additional studies mFAP x CD40 was active and inhibited the growth of FAP+ tumors. There were no signs of toxicity with mFAP x CD40 in contrast to the FGK45 Ab which resulted in body weight loss. In conclusion, we have generated bispecific agonist FAP x CD40 DARPin® molecules able to activate the CD40 pathway with a targeting (FAP)-dependent mechanism of action.

#3252

TAK-981: A first in class SUMO inhibitor in Phase 1 trials that promotes dendritic cell activation, antigen-presentation, and T cell priming.

Mithun Khattar, Keli Song, Stephen Grossman, Kristina Xega, Xingyue He, Neeraja Idamakanti, Dennis Huszar. _Takeda Pharmaceuticals, Cambridge, MA_.

SUMOylation is a reversible post-translational modification that regulates protein function by covalent attachment of the small ubiquitin-like modifier (SUMO) protein to protein substrates. TAK-981 is a novel, selective small molecule inhibitor of the SUMOylation enzymatic cascade, which has demonstrated induction of potent anti-tumor immunity in preclinical models. In this study, we evaluated changes in immune cell composition and states after TAK-981 treatment in preclinical models. In both mouse bone-marrow and human peripheral blood mononuclear cell derived dendritic cells (DCs), TAK-981 treatment ex vivo induced markers of activation and maturation including CD40, CD80 and CD86, as well as increased secretion of inflammatory cytokines like IP-10, MCP1, MIP-1α, MIP1β, IFNα and IFNβ. These effects were reversed by an interferon alpha receptor (IFNAR) blocking antibody, demonstrating dependence of TAK-981 induced pharmacodynamic effects on Type I interferon signaling in DCs. In vivo, a single sub-cutaneous injection of TAK-981 in naïve Balb/c mice at the brachial lymph nodes induced activation of DCs, measured as significant increases in CD40 and CD86 expression. Concomitantly, an increase in expression of the early lymphocyte activation marker CD69 was observed on T cells and NK cells, demonstrating pleiotropic effects of TAK-981 on immune cells. Increased expression of CD69 was also observed in purified human T and NK cells treated with TAK-981 ex vivo. To investigate if enhanced activation of DCs by TAK-981 leads to improved cross-presentation of exogenous antigens, we challenged naïve C57BL/6 mice with chicken-ovalbumin (OVA) protein alone or in combination with TAK-981. Interestingly, an increase in the frequency of Kb-SIINFEKL tetramer positive CD8 T cells was observed in the draining lymph nodes overnight, and in the spleen on day 14, after treatment with TAK-981+OVA, suggesting that TAK-981 promotes cross-presentation of SIINFEKL, the class-I MHC epitope of OVA, to cognate antigen-specific CD8 T cells. We also observed an increase in the frequency of CD8α+ DCs loaded with the peptide 'SIINFEKL' on H-2Kb (class-I MHC of C57BL/6 mice), providing direct evidence for enhanced antigen-cross presentation. Similar findings were observed with TAK-981 administered either sub-cutaneously or via the therapeutic route of intra-venous injection in the above OVA immunization model. These results suggest a mechanism by which TAK-981 may promote anti-tumor immune responses in mice via enhanced cross-presentation of exogenous antigens released by dying tumor cells, leading to priming and activation of antigen reactive cytotoxic T cells. Currently, TAK-981 is being evaluated in Phase 1 clinical trials in adult patients with metastatic solid tumors or lymphomas (ClinicalTrials.gov Identifier: NCT03648372).

#3253

Next-gen STING-agonist like BCG confers enhanced immunogenicity and antitumor efficacy in vitro and in vivo.

Alok K. Singh, Monali Praharaj, Gregory A. Joice, Takahiro Yoshida, Max Kates, David McConkey, William R. Bishai, Trinity J. Bivalacqua. _Johns Hopkins Medical Institution, Baltimore, MD_.

INTRODUCTION: Stimulator of interferon genes (STING), a cytosolic sensor of cyclic dinucleotide (CDN; c-di-AMP and c-di-GMP) activates type I interferons (IFN I) and pro-inflammatory cytokines. Recombinant Bacillus-Calmette Guerin (BCG) overexpressing CDN (STING-agonist) remains unexplored in non-muscle invasive bladder cancer (NMIBC). We hypothesize that intravesical recombinant BCG is more efficacious than BCG for NMIBC. We constructed a novel STING-agonist-like recombinant BCG Pasteur and Tice strains (rBCG-disA-OE) carrying plasmids that harbors di-adenylate cyclase (disA) gene to test this hypothesis.

METHODS: WT and rBCG-disA-OE were tested for immunotherapeutic potential in WT and STING-deficient murine macrophages, to ascertain STING-dependent IFN I and pro-inflammatory cytokines activation. Similar experiments were conducted in RT4, J5637, and NBT2 cell lines. Fisher 344 rats intravesically received 4 doses of 1.5 mg/kg N methyl N nitrosurea (MNU) to induce NMIBC. Intravesical instillation of WT- and rBCG-disA-OE was administered weekly x 6. Histologic tumor grade, tumor involvement index, gene expression (q-PCR) as well as systemic immune responses (ELISA) were assessed. Growth and virulence of rBCG-disA-OE in BALB/c and SCID mice were tested using aerosol infection protocol.

RESULTS: rBCG-disA-OE Pasteur and TICE strains overexpressing STING-agonist were potent inducers of IFN I in STING-dependent manner in murine macrophages. IFN I induction by rBCG-disA-OE increased TNF-α (P=0.01), IL-6 (P=0.04) and IL-1β (P=0.004) in bladder cancer cells and murine macrophages. Intravesical instillation of rBCG-disA-OE in MNU-rats resulted into significantly (P=0.004) lower tumor involvement index accompanied by a potent induction of IFN I signaling, M1 macrophage associated cytokines (Nos2, P=0.05 & IL-6 P=0.05) and chemokines (CCL2, P=0.048 & MCP-1, P=0.05). rBCG-disA-OE prevented invasive cancer when compared WT-BCG. BALB/c mice infected with rBCG-disA-OE showed significantly less lung bacillary burden (P=0.005) suggesting strain attenuation. SCID-mice infected with rBCG-disA-OE had prolonged survival.

CONCLUSION: We demonstrate STING-agonist like rBCG-disA-OE is superior to WT-BCG for induction of macrophage cytotoxicity and activation of IFN I signaling. rBCG-disA-OE demonstrated enhanced antitumor activity and cytokine production in a pre-clinical model of NMIBC suggesting an alternative intravesical agent in BCG unresponsive population.

#3254

A novel TGFβRI inhibitor suppresses tumor progression by increasing T-cell infiltration into tumor.

Xu Zusheng,1 Yangtong Lou,1 Qiang Zhao,2 Wei Wang2. 1 _Shanghai Yingli Pharmaceutical Co., Ltd, Shanghai, China;_ 2 _Shanghai ChemPartner Co., Ltd, Shanghai, China_.

TGF-beta (TGF-β) pathways, despite serving as tumor suppressor in early stage of carcinogenesis, have shown to be one of the key tumor-promoting factors in later-stage tumor progression. TGF-β receptor type 1 (TGFβRI) inhibitors have shown great potentials in their anti-tumor and anti-metastasis efficacy in preclinical studies and some of them are being tested in the clinical settings. A specific TGFβRI inhibitor YL-13027 was developed at Yingli Pharmaceutical. Our previous data have shown that YL-13027 significantly inhibited the T cell differentiation into Treg and the secretion of IL-10 in vitro, as well as tumor growth in immune-competent mice with CT26 tumors. Our further studies indicated that the anti-tumor efficacy of YL-13027 was largely mediated by T cells. More importantly, YL-13027 synergistically enhanced the anti-tumor efficacy of anti-PD-L1 antibody in CT26 model. YL-13027 displayed significant anti-tumor efficacy in other syngeneic mouse tumor models tested including orthotopic murine breast carcinoma 4T1 model in BALB/c mice and murine hepatocellular carcinoma Hepa1-6 model in C57BL/6 mice. YL-13027 is well tolerated by the animals with no treatment-related toxicity observed in these efficacy studies. Analyses of tumors treated with YL-13027 revealed that YL-13027 significantly increased the number of tumor-infiltrating lymphocytes in tumors. These data suggest that YL-13027, a novel TGFβRI inhibitor, represents a promising and safe immune modulator and shows great potential as a cancer immuno-therapeutics.

#3255

A novel small molecule inhibitor of AhR suppresses the polarization and activity of M2 macrophages.

Candy Garcia, Hadia Lemar, Christina Galang, James Joseph, Marcos Gonzalez-Lopez, Jeffrey Hager, Michael P. Dillon, Fred J. Aswad. _Ideaya Biosciences, South San Francisco, CA_.

Tumor associated macrophages, mainly represented by the M2-like phenotype, are a highly immunosuppressive macrophage subset that exert a pro-tumorigenic role in a variety of cancer types. Understanding the factors that promote M2 macrophage differentiation and activity is of critical importance to unlocking the potential of anti-tumor immune therapy. Thus, we investigated the role of the AhR pathway in M2 macrophages. We found the Aryl Hydrocarbon Receptor (AhR) highly expressed in in vitro polarized human M2 macrophages derived from peripheral blood mononuclear cells. Stimulation of AhR with small molecule agonists TCDD or kynurenine resulted in a significant enhancement of the suppressive activity of M2 macrophages on anti-CD3/CD28 stimulated CD4 T cell proliferation and IFNγ production. Inhibition of AhR using a novel, highly potent and selective AhR inhibitor (IDE-AhRi-1) fully inhibited the suppressive activity of M2 macrophages on T cells. Surprisingly, stimulated CD4 T cells co-cultured with IDE-AhRi-1 exposed M2 macrophages had an enhanced ability to proliferate and produce IFNγ compared with stimulated T cells in the absence of M2 macrophages. This suggests that IDE-AhRi-1 not only inhibits the suppressive mechanisms of M2 macrophages, but also boosts their ability to generate pro-inflammatory responses that enhance T-cell activity. Further studies to elucidate the AhR-driven molecular mechanisms responsible for this activity are underway. To find out whether AhR driven M2 polarization and activity is evident in human cancer, we analyzed TCGA-derived RNA sequencing data from an array of solid tumor indications. In a subset of cancer indications, high AhR activity correlated with key M2 macrophage and immuno-regulatory signature genes. Surprisingly, expression of CYP1B1 also correlated with expression of TDO2 mRNA, but not IDO1 mRNA. These finding establish AhR as a key modulator of M2 macrophages and demonstrate a correlation between AhR activity and tumor associated macrophages in human tumor samples. Furthermore, we demonstrate that IDE-AhRi-1 can potently suppress M2 macrophages and propose IDE-AhRi-1 as a novel approach for potentiating anti-tumor immunity.

#3256

RTX-IL-12, an allogeneic red cell therapeutic expressing IL-12, exhibits potent in vitro and in vivo activity and favorable safety profile.

Anne-Sophie Dugast, Enping Hong, Maegan Hoover, Arjun Bollampalli, Douglas C. McLaughlin, Omkar Bhate, Timothy J. Lyford, Torben Straight Nissen, Christopher L. Carpenter, Thomas J. Wickham, Sivan Elloul. _Rubius Therapeutics, Cambridge, MA_.

Recombinant IL-12 is a potent cytokine that has held significant promise as an immunotherapeutic. In preclinical studies, recombinant IL-12 has impressive anti-tumor activity; however toxicity and a narrow therapeutic window halted development in humans. To address these limitations, Rubius Therapeutics has developed genetically engineered red cell therapeutics (RCTs) with cell surface expression of IL-12 alone (RTX-IL-12) or in combination with co-stimulatory or other cytokine molecules. These cells present ligands in their native form that potently activate and expand both T and NK cells through potent cell-cell interactions. On-target, off-tissue toxicity of RCTs may be limited due to their biodistribution, which is restricted to the vascular system. Based on its reported role in promoting a TH1 response and proliferation of cytotoxic NK and CD8 T cells, RTX-IL-12 activity was evaluated in vitro and demonstrated proliferation of human CD8 T cells (2-3 fold), Th1 differentiation of naïve CD4 T cells, IFNγproduction (6-11 fold increase over control), as well as NK cell activation and cytotoxicity against K562 targets (2-5 fold increase over control). To evaluate in vivoimmune response, anti-tumor activity and safety, a mouse surrogate red cell therapeutic, mRBC-IL-12, was developed by chemically conjugating recombinant Fc-IL-12 to mouse red blood cells. This surrogate overcame the rapid clearance of human red cells in mice. Administration of mRBC-IL-12 (1X109cells) to C57Bl6 mice that received B16F10 melanoma cells IV, showed a 52% decrease in the number of lung metastases and was associated with increased proliferating and cytotoxic CD8 and NK cells in the lungs (p=0.0002). Administration of mRBC-IL-12 (1X109cells) to mice bearing B16F10 and MC38 subcutaneous models exhibited 82% and 80% tumor growth inhibition, respectively. When combined with anti-PD1 treatment, mRBC-IL-12 (1X109cells) efficacy was improved in these two models and showed 86% and 85% tumor growth inhibition, respectively. Efficacy was accompanied by an increase in survival (p=0.0004 B16F10, p=0.0003 MC38 ) and the infiltration of M1 macrophages (p=0.007) into the tumor. Mice treated with the highest feasible dose of mRBC-IL-12 displayed no significant body weight loss and 3-10-fold lower serum IFNg levels compared to soluble rec. Fc-IL-12. By combining IL-12 with IL-15TP or 4-1BBL on the same red cell, tumor growth was strongly inhibited and in some cases improved over mRBC-IL-12 alone (TGI 46% and 63% in B16F10 SC, TGI 83%, 77% in MC38 and 78%, 70% in B16F10 IV). In summary, the data demonstrate that sequestering IL-12 in the vasculature through expression on red cells drives significant reductions in tumor growth, while improving the tolerability profile of the cytokine,supporting further testing in cancer patients.

#3257

Activation of CD137 using multivalent and tumor targeted Bicyclic peptides.

Kristen E. Hurov,1 Punit Upadhyaya,1 Jessica Kublin,1 Xueyuan Zhou,1 Julia Kristensson,2 Rachid Lani,3 Gemma Mudd,2 Katerine van Rietschoten,2 Frank An,1 Johanna Lahdenranta,1 Liuhong Chen,2 Gavin Bennett,2 Kevin McDonnell,1 Peter Park,1 Nicholas Keen1. 1 _Bicycle Therapeutics, Lexington, MA;_ 2 _Bicycle Therapeutics, Cambridge, United Kingdom;_ 3 _Abreos France, Marseille, France_.

CD137 (4-1BB/TNFRSF9) is a costimulatory T-cell receptor belonging to the TNF receptor superfamily. Agonistic anti-CD137 antibodies have shown potent, often curative anti-tumor activity in preclinical models. Two human anti-CD137 antibodies, urelumab and utomilumab are currently undergoing clinical testing. Urelumab has shown several single-agent, partial responses, but its use has been hampered by hepatoxicity, while utomilumab has shown little or no single agent activity.

Bicycles® are a new class of drugs - fully synthetic, constrained bicyclic peptides that combine the attributes of antibodies, small molecules, and peptides by delivering high affinity, selectivity, and rapid clearance. Their small size (1.5-2 kDa) delivers advantages in tumor penetration, and renal elimination may avoid the liver and GI toxicity often associated with other drug modalities, including certain antibodies. We hypothesized that fully synthetic Bicycle CD137 agonists with rapid clearance, minimal liver exposure and no Fc receptor interaction may induce CD137 mediated anti-tumor activity while avoiding liver toxicity.

A high affinity lead BCY3814 (KD ~30 nM) that binds to the CD137 ligand-binding site was identified. CD137 activation requires receptor crosslinking, thus multivalent binding would be expected to recapitulate the action of the natural trimeric ligand. We envisioned that CD137 agonism could be achieved directly by using multimeric CD137 Bicycles or in a tumor targeted fashion with bispecific or "heterotandem" Bicycles. The synthetic simplicity and highly modular nature of the Bicycle® platform enabled us to rapidly explore both formats.

To generate a "pure" CD137 agonist we synthesized >50 different multimeric variants of BCY3814 with chemical linkers of various lengths and rigidity and using different sites of attachments, while maintaining a compact size (<15 kDa). Tumor targeted CD137 agonists were generated as heterotandems, whereby BCY3814 is conjugated to a tumor antigen targeting Bicycle. In this design, the CD137 Bicycle only induces CD137 agonism after the molecule binds to a tumor cell with high receptor expression.

We discovered Bicycle multimers that exhibit a range of potencies in a cell-based CD137-dependent reporter assay, activate human T cells in vitro as indicated by increased cytokine release, and show biological activity in vivo. Bicycle heterotandems targeting Nectin-4 and EphA2 exhibited highly potent and tumor cell specific activity in both the cell-based reporter assay and the human T cell assay. Selected CD137 multimers and heterotandems are being tested further in humanized mouse models for T cell activation, anti-tumor activity, and liver safety. These molecules are promising, novel cancer immunotherapy candidates and importantly, they pave the way for development of synthetic agonists of other TNF receptors that can be targeted to the local tumor microenvironment.

#3258

THOR-707, a novel not-alpha IL-2, elicits durable pharmacodynamic responses in non-human primates and efficacy as single agent and in combination with anti PD-1 in multiple syngeneic mouse models.

Ingrid B. Joseph, Lina Ma, Jerod L. Ptacin, Carolina E. Caffaro, Hans R. Aerni, Kristine M. San Jose, Michael J. Pena, Robert W. Herman, Yelena Pavlova, David B. Chen, Ken Bragstad, Shukuan Li, Jasmine Nguyen, Laura K. Shawver, Lilia K. Koriazova, Marcos E. Milla. _Synthorx, Inc., La Jolla, CA_.

Aldesleukin is a recombinant form of IL-2 approved for metastatic melanoma and renal cell carcinoma that induced complete, durable remissions in certain patients. Yet, its use is infrequent because of vascular leak syndrome (VLS), a severe dose-limiting adverse event stemming from the engagement of the high affinity IL-2 receptor (IL-2R) alpha chain in type 2 innate lymphoid cells, eosinophils and vascular endothelial cells. THOR-707 is a site-directed, singly pegylated form of IL-2 completely lacking IL-2R alpha chain engagement yet retaining normal binding to the intermediate affinity IL-2R beta-gamma signaling complex expressed by natural killer (NK) and CD8+ T tumor-killing cells. We studied THOR-707 pharmacokinetics (PK) and pharmacodynamics (PD) in non-human primates (NHP) to evaluate peripheral biomarkers of immune cell activation (expansion of NK, CD8+ T and CD4+ regulatory T (Treg) cells and the induction of pSTAT5 and the proliferation marker Ki67). Here we show that in NHP, THOR-707 elicits the expansion of peripheral CD8+ T cells and the persistence of that response with different administration regimes. We also show that in the mouse syngeneic colon tumor model CT-26, THOR-707 induced Ki67 and the expansion of peripheral NK and CD8+ T cells. Within the tumor, THOR-707 promoted an increase in the numbers of infiltrating tumor-killing NK and CD8+ T cells without expansion of suppressive CD4+ Treg cells. Immunohistochemical (IHC) analysis showed that in THOR-707 treated animals, CD8+ T cells were mostly seen around the edges of the tumor and at areas of necrosis. Evaluation of T cell receptor (TCR) clonality revealed that treatment with THOR-707 increased intra-tumoral T cell diversity compared to the untreated animals. THOR-707 induction of CD8+ T cell tumor infiltration resulted in single agent dose-dependent anti-tumor efficacy, and additive efficacy in combination with PD-1 checkpoint inhibitor, increasing the survival of CT-26 tumor-bearing mice over either group alone. The pharmacodynamics, T cell infiltration and clonality, and efficacy of THOR-707 in two additional syngeneic models, have also been studied. Filing of an investigational new drug application for THOR-707 is expected in the second quarter of 2019 and thereafter initiation of a Phase 1/2 clinical trial in multiple tumor types as a single agent and in combination with an immune checkpoint inhibitor.

#3259

heterodimeric IL-15 monotherapy results in complete regression of EO771 murine breast tumors through cDC1-lymphocyte interactions and induction of antitumor immunity.

Dimitris Stellas, Sevasti Karaliota, Vasiliki Stravokefalou, Bethany A. Nagy, Barbara K. Felber, George N. Pavlakis. _National Cancer Institute at Frederick, Frederick, MD_.

Introduction: IL-15 is a cytokine important for the maintenance, proliferation and activation of lymphocytes, including CD8+ T and natural killer (NK) cells. The native form of IL-15 is a heterodimer either embedded in the plasma membrane or released in a soluble form. Several preclinical cancer models have supported the anti-tumor activity of heterodimeric IL-15, which is presently in clinical trials. We have produced heterodimeric IL-15 (hetIL-15) and also fusions to the Fc fragments (hetIL-15FC) of human, macaque or mouse antibodies. We assessed the effects of hetIL-15 monotherapy after systemic and locoregional administration in the proximity of EO771 orthotopically implanted breast tumors.

Experimental procedures: We evaluated the therapeutic efficacy of hetIL-15 and hetIL-15FC immunotherapy in the murine EO771 triple negative breast cancer orthotopic model using both syngeneic C57BL/6 mice and RAG-1 immunodeficient mice. We monitored the effects of hetIL-15 treatment on Tumor Infiltrating Lymphocytes (TILs), lymphoid organs and blood by Flow cytometry and Immunohistochemisty (IHC). Using Seahorse technology, we also evaluated the metabolic profile of the CD8+ T cells, reflecting their activation status.

Results: Monotherapy with hetIL-15FC peritumorally in the area of mammary pad resulted in complete tumor regression in half of the treated mice. hetIL-15 treatment resulted in an increased survival accompanied with decreased or completely eradicated metastatic disease, in C57BL/6 mice. Significant tumor delay was also observed after peritoneal injection of hetIL-15 in C57BL/6 as well as in RAG-1 mice with hetIL-15FC, but complete tumor regression was not achieved. hetIL-15FC peritumoral administration reshaped the tumor microenvironment and caused an increased infiltration of NK cells and CD8+ T cells with effector phenotype. Both cytotoxic T cells and NK were activated and proliferating, as shown by Flow, including Granzyme B and Ki67 staining. hetIL-15FC treatment resulted in increased tumor infiltration of both CD8+ and CD4+ T memory cells and provided strong protection to these mice against subsequent re-challenges with EO771 tumor.

Furthermore, peritumoral hetIL-15FC administration resulted in an increase of intratumoral conventional Dendritic cells (cDC1), suggesting a mechanism for the intratumoral attraction of lymphocytes.

Conclusion: Monotherapy with hetIL-15FC locoregional administration resulted in complete regression of EO771 primary breast cancer tumors, activating both cytotoxic arms of the immune system, and providing persisting protective immunity. Mechanism of function of hetIL-15 is through cDC1 and lymphocyte interactions that lead to the reciprocal attraction of cDC1 and activated cytotoxic lymphocytes into the tumors and the generation of long-term anti-tumor immunity.

#3260

**Engineered red-cell therapeutics (RCT) as artificial antigen presenting cells promote** in vivo **expansion and anti-tumor activity of antigen specific T cells.**

Xuqing Zhang, Shamael R. Dastagir, Naren Subbiah, Mengyao Luo, Vikram Soman, Sneha Pawar, Douglas C. McLaughlin, Nicholas Bayhi, Viral Amin, Torben Straight Nissen, Christopher L. Carpenter, Thomas J. Wickham, Tiffany F. Chen. _Rubius Therapeutics, Cambridge, MA_.

T cell-based therapies have demonstrated efficacy in a small subset of cancers; however, they have the potential to proliferate uncontrollably and manufacturing these therapies at scale has proven difficult. To address this limitation, Rubius Therapeutics has genetically engineered red cells to create allogeneic artificial antigen presenting cells (RCT-aAPCs) that express MHC class I loaded with a tumor specific antigen, together with costimulatory molecules that recapitulate normal APC-T cell interactions. These RCT-aAPC cells are designed to expand and activate tumor-specific T cells already present within the patient, thus eliminating the need to individually manufacture patient-derived T cells. As a proof of principle, red cells were engineered to express mouse MHC class I H-2Kb loaded with OVA 257-264 peptide and murine 4-1BBL. These cells induced in vitroT cell proliferation of OVA antigen-specific OT1 cells, whereas red cells expressing only MHC I or 4-1BBL did not induce proliferation. The RCT-aAPC expanded OT1 cells demonstrated an activated phenotype with increased CD44 expression, secretion of both IFNγ and IL2, as well as antigen-specific tumor killing of EG7.OVA tumor cells. To test in vivo efficacy, a mouse surrogate RCT-aAPC was created using murine red blood cells chemically conjugated with H-2Kb OVA and the m4-1BBL molecule. CellTrace Violet (CTV)-labeled OT1 cells were adoptively transferred into B6 Cd45.1 mice followed by intravenous dosing of the RCT-aAPC several hours later. Significant OT1 proliferation was observed 3-4 days post-dosing as measured by CTV dilution. Administration of a second RCT-aAPC dose at this time drove >200-fold expansion of OT1 cells with a memory-like phenotype in the peripheral blood and secondary lymphoid organs. Using a similar dosing strategy, administration of RCT-aAPC to mice bearing EG7.OVA tumors caused 60% tumor growth inhibition by Day 7 after dosing, which corresponded with the increased expansion of the OT1s. Treatment with RCT-aAPC significantly prolonged survival compared to the control group (p-val = 0.0024). After interacting with RCT-aAPC, antigen-specific T cells, traffic to the lymph nodes and tumor as demonstrated by OT1 presence at these sites. Based on the proof of concept using a murine system, human RCT-aAPCs expressing [human] 4-1BBL and [human] HLA-A2 loaded with an HPV E7 peptide were developed to expand and activate HPV E7-specific T cells. These RCT-aAPC cells activated TCR signaling in primary HPV E7-specific T cells as measured by upregulation of Nur77 expression and in engineered HPV E7-specific TCR Jurkat lines, measured using an NFAT luciferase reporter assay. Further validation of RCT-aAPC is ongoing and will be the focus for future clinical development in patients with HPV-positive cancers.

#3261

**EOS100850, a non-brain penetrant highly selective A** 2A **receptor antagonist, uniquely maintains high potency within the adenosine rich tumor microenvironment.**

Erica Houthuys, Paola Basilico, Margreet Brouwer, Theo Deregnaucourt, Michel Detheux, Gregory Driessens, Bruno Gomes, Xavier Leroy, Joao Marchante, Reece Marillier, Jakub Swiercz, Stefano Crosignani. _iTeos Therapeutics, Gosselies, Belgium_.

High levels of extracellular adenosine in the tumor microenvironment promote tumor immune evasion. We and others have shown that adenosine, predominantly through the A2A receptor, suppresses innate and adaptive immune cell responses leading to suppression of antitumor immunity. We demonstrated that A2A receptor antagonists initially designed for Parkinson's disease but repurposed for immuno-oncology dramatically loose potency in a high adenosine environment. We therefore developed EOS100850, an A2A receptor antagonist specifically designed as a potent, highly selective, non-brain penetrant orally administered agent to treat a wide range of tumor types. EOS100850 has been discovered via structure-based drug design followed by rational medicinal chemistry to improve drug-like properties. EOS100850 demonstrated nanomolar affinity and high selectivity for human A2A receptors, with pico- to nanomolar potency on human and mouse A2A receptors. Assays were performed on A2A-overexpressing HEK293 cells and primary human T cells, using conditions mimicking normal physiological and tumor-like microenvironments. In these assays, EOS100850 potency was not affected by increasing extracellular adenosine concentrations. A unique feature of this compound is the remarkable long residence time on human A2A receptors after binding, resulting in a prolonged inhibition of A2A receptor signaling at a wide range of adenosine concentrations (up to 1000 µM). Furthermore, unlike competitor A2A receptor antagonists, EOS100850 reversed with nanomolar potency A2A receptor-dependent inhibition of cytokine secretion by T cells in tumor-like assay conditions. EOS100850 potency to inhibit A2A receptor-mediated CREB phosphorylation in T cells in human and mouse whole blood was in the low nanomolar range.One of the major metabolites of EOS100850, EOS100612, identified both in vitro and in vivo, has been synthesized and was found to be active on A2A receptors. Similar to EOS100850, EOS100612 displayed pico- to nanomolar potency, high selectivity and long residence time on A2A receptors in various in vitro assays.In conclusion, EOS100850 represents a novel non-brain penetrant best-in-class A2A receptor antagonist. It remains highly potent in environments characterized by high adenosine concentrations, such as the TME, maximizing the potential intra-tumor effect and further minimizing the risk of collateral CNS toxicity.

#3262

Discovery of a series of novel toll-like receptor 7 agonists for systemic immunotherapy of cancer.

James R. Appleman, Stephen E. Webber. _Primmune Therapeutics, San Diego, CA_.

While ICPIs (immune checkpoint inhibitors) have fundamentally changed the practice of cancer therapy for tumors arising from many different tissues, ways to increase both response rate and durability are critically needed. On theoretical grounds, combining stimulators of innate immunity with activators of adaptive immunity should lead to better outcomes. Consistent with this hypothesis, encouraging results have been obtained with intratumoral injections of TLR9 agonists in combination with ICPIs in treatment-naïve patients and in patients that have failed previous ICPI therapy. However, there are substantial limitations inherent in any drug that must be administered intratumorally, particularly where repeated administration is preferred. We therefore have invented a novel series of TLR7 agonists that engage the same elements of the immune system as TLR9 agonists while having the advantages of oral delivery and safe systemic engagement of the immune system. The ability to successfully achieve such a profile while maintaining a reasonable therapeutic window was previously demonstrate by Anadys Pharmaceuticals with ANA773. This drug candidate was evaluated in healthy volunteers, chronic HCV and HBV patients and treatment-experienced solid tumor patients. Based upon our analysis of desirable pharmacologic features of ANA773 and challenges faced by molecules like 3M-852A, where no therapeutic window could be identified, we have derived a critical set of unusual target properties that are evaluated in our testing cascade:

\- Intrinsic potency for TLR7 activation in a defined range that maintains ability to vary degree of engagement with concentration

\- Specificity for TLR7 and, in particular, a lack of activity at TLR8

\- Defined target profile of cytokine and chemokine induction in human peripheral blood mononuclear cells

\- Efficient delivery in primates to systemic circulation by prodrugs that intrinsically lack TLR7 agonist activity themselves

\- PK volume of distribution ~ 1 L/kg

\- Relatively short PK t1/2 with substantially more long-lived pharmacodynamic response

We hypothesize that a molecule with the above criteria can be dosed QOD continuously over a 24-month period to appropriately engage innate immunity that is well-tolerated by the patient and increases response rate and durability.

From our original starting point - a relatively weak TLR7 agonist with no oral bioavailability - we have invented a novel series of molecules meeting our target profile. We are currently engaged in evaluation of three leading candidates in advanced models with the objective of selecting one to proceed into IND-enabling studies.

#3263

Checkpoint-targeted IL15/IL15Rα-Fc fusions for preferential TIL expansion.

Matthew J. Bernett, Rajat Varma, Suzanne Schubbert, Christine Bonzon, Rumana Rashid, Liz Bogaert, Kie Liu, Kendra N. Avery, Irene W. Leung, Nicole Rodriguez, Sung-Hyung Lee, Yoon Kim, Connie Ardila, Nargess Hassanzadeh-Kiabi, Juan E. Diaz, Michael Hedvat, Seung Y. Chu, Umesh S. Muchhal, Gregory L. Moore, John R. Desjarlais. _Xencor, Inc., Monrovia, CA_.

The therapeutic potential of IL2Rβ/γc binding human cytokines IL2 and IL15 has been well established in animal models and human trials. However, natural cytokines are extremely potent and have evolved for local activity at very low concentrations. Consequently, as potential drugs given systemically, they suffer from low tolerability and very fast clearance that limits therapeutic window. Higher drug concentration and prolonged exposure are desirable in order to allow lymphocyte activation and proliferation at the tumor site, but this can be difficult to achieve due to dose-limiting toxicities associated with this axis. IL15 functions as a stabilized heterodimeric complex with membrane-bound IL15Rα on the surface of monocytes and DCs, and this IL15/IL15Rα complex is presented in trans to lymphocytes expressing IL2Rβ and γc. We hypothesized that we could selectively target tumor-reactive TILs by combining a reduced potency IL15/IL15Rα heterocomplex with Fab-based checkpoint(CP)-targeting arms to bias binding and activation to CP-positive TILs, potentially improving therapeutic index. We designed a format in which a single-chain IL15/IL15Rα was attached to one side of a heterodimeric Fc-region and CP-targeted Fab attached to the other. Fc-fusions were tuned for optimal selectivity by engineering amino acid substitutions in IL15 - at the IL2Rβ or γc interface - that reduced receptor affinity and overall in vitro potency. Fab regions targeting immune checkpoints, including PD1, were engineered for optimal affinity. In vitro proliferation of lymphocytes in normal human PBMCs, or PBMCs stimulated with sub-optimal concentrations of anti-CD3 to induce checkpoint expression, was monitored by counting Ki67+ cells after incubation with Fc-fusions for 4 days and by measuring signaling in a STAT5 phosphorylation assay. In vivo activity was evaluated using a huPBMC-NSG mouse model by measuring the extent of human leukocyte expansion. Targeted IL15/IL15Rα-Het-Fc were produced with favorable yield and purity. The Fc-fusions showed enhanced proliferation of lymphocytes stimulated for induced checkpoint expression in vitro, whereas a control protein with a non-targeting Fab region showed dramatically reduced activity. Treatment of huPBMC-NSG mice with targeted IL15/IL15Rα-Het-Fc promoted significantly enhanced T cell engraftment. Moreover, CP-targeted IL15/IL15Rα combined productively with an anti-PD1 antibody to promote additional T cell expansion. These results demonstrate that CP-targeted IL15/IL15Rα-Het-Fc show a promising profile of selective IL15 delivery to TIL with minimal peripheral activity.

#3264

A novel TLR8 agonist induces immune responses and tumor regression.

Yuxun Wang, Heping Yang, Huanping Li, Shuda Zhao, Yin Zhang, Renjie Huang, Jinmiao Wu, Yikun Zeng, Panpan Zhang, Xingzhong Zhang, Longsheng Wang, Guangliang Fu, Zhiheng Wu, Panhu Zhu, Hui Xu, Yaqiao Gao, Pei Wang, Daxin Gao. _Denovo Pharmatech, Shanghai, China_.

Toll like receptors (TLRs) have been a recent focus of drug discovery for cancer immunotherapy. TLRs play a crucial role in bridging innate and adaptive immunity. TLR8 is distinguished from other TLRs by its functions in reversing regulatory T (Treg) cell and myeloid derived suppressor cell (MDSC)'s immune suppression effects. Treg and MDSCs induce an immunosuppressive microenvironment that is a major cause of failed tumor immunotherapy. Therefore, activation of TLR8 can exert potent immune-mediated anticancer activity. We discovered a novel TLR8 selective agonist DN052 and demonstrated that DN052 clearly differentiated from motolimod, the only TLR8 agonist drug candidate in clinical development. DN052 was more potent than motolimod and exhibited better PK profiles and more strongly induced immune responses in the in vivo monkey and ex vivo human PBMC studies. However, it has been challenging to characterize TLR8 agonists in vivo partially due to the phylogenetic specificity of TLR8 and its diminished activity in rodents. We developed two new in vivo approaches using mouse syngeneic and mouse xenograft tumor models. Our in vivo studies showed that DN052 strongly suppressed tumor growth in a dose-dependent manner as a single agent and combination with the chemotherapeutic agent or PD-1 monoclonal antibody further enhanced the efficacy of the single agents in several tumor models. Remarkably, DN052 caused complete tumor regression as a single agent or in combination in some of the immune-competent tumor-bearing mice. GLP toxicity studies in rats and monkeys showed that DN052 had favorable safety profiles. Taken together, DN052 is a novel Best-in-Class TLR8 selective agonist for immunotherapy as a single agent or in combination with other immuno-oncology agents or chemotherapeutics for broad cancer indications. DN052 warrants further clinical development.

#3265

NKTR-255, a polymer-conjugated IL-15 enhances anti-tumor NK cell responses and synergizes with monoclonal antibodies to provide long-term survival in human lymphoma model.

Takahiro Miyazaki, Saul Kivimäe, Rhoneil Pena, Peiwen Kuo, Marlene Hennessy, Murali Addepalli, Neha Dixit, Wildaliz Nieves, Sara Sheibani, Mekhala Maiti, Laurie VanderVeen, Joanna Wilczek, Loui Madakamutil, Jonathan Zalevsky. _Nektar Therapeutics, San Francisco, CA_.

Background: IL-15 is a cytokine that activates and provides survival benefit to NK cells. Exploiting the therapeutic value of native IL-15 has been challenging due to its unfavorable pharmacokinetic properties and tolerability. NKTR-255 is a polymer-conjugated human IL-15 that retains binding affinity to the alpha subunit of IL-15 receptor and exhibits reduced clearance to thereby provide a sustained pharmacodynamics response. NKTR-255 has potential for providing an enhanced immunotherapeutic effect when combined with monoclonal antibodies that mediate tumor killing by antibody dependent cellular cytotoxicity (ADCC). Here we investigate the pharmacological properties of NKTR-255 on NK cells and the therapeutic effect of NKTR-255 when combined with tumor-directed monoclonal antibodies in a B cell lymphoma model.

Methods: KHYG-1 cells (human NK cell line) were used to measure phosphorylated STAT5 (pSTAT5) and cell proliferation. Human PBMCs were stimulated with NKTR-255 and/or daratumumab for in vitro NK cell characterization. In the cytotoxic assay, mice received single IV doses of 0.3 mg/kg of NKTR-255 and splenic NK cells were co-cultured with YAC-1 cells (mouse T lymphoma cell line) to measure cytotoxic function. In the Daudi lymphoma model, 1x107 Daudi cells were inoculated IV on Day 0. NKTR-255 (0.03, 0.1 or 0.3 mg/kg SC) was administered on Days 14, 21 and 28 and antibody treatment was administered on Day 14 (daratumumab 0.5 mg/kg IP) or on Days 14 and 17 (rituximab 40 mg/kg IP). Survival rate was determined by onset of hindlimb paralysis as a surrogate parameter.

Results: NKTR-255 dose-dependently induced pSTAT5 and proliferation in KHYG-1 cells (EC50 values for pSTAT5: 0.2 ng/ml, proliferation: 5 ng/ml). NKTR-255 also resulted in enhanced pSTAT5 and NKG2D surface expression on human primary NK cells. In addition NKTR-255 increased NK cell degranulation in co-culture experiments with U266 cells (myeloma cell line) with/without daratumumab (anti-human CD38 antibody), as determined by enhanced CD107a surface expression. In vivo pretreatment with NKTR-255 resulted in sustained cytotoxic function of NK cells in both ex vivo and in vivo studies. Finally, NKTR-255 synergistically provided long-term survival benefit in a dose-dependent manner when administered with daratumumab or rituximab (anti-human CD20 antibody) in the Daudi B cell lymphoma model.

Conclusions: NKTR-255 is an immune stimulator of NK cells that provides a dose-dependent effect in the proliferation and activation of NK cells. This property of NKTR-255, when administered with daratumumab or rituximab, translates into enhanced therapeutic efficacies of the antibodies in a B cell lymphoma model. These results indicate that combining NKTR-255 with a tumor-directed antibody having an ADCC mechanism can provide a synergistic effect for treating cancers.

#3266

Distinct immune stimulatory functions of anti-human VISTA antibodies are determined by the antibody isotype.

Sven Mostböck,1 Helen Wu,2 Timothy Fenn,2 Christina Taubert,3 Gregory Vladimer,3 Anne Vogt1. 1 _Boehringer Ingelheim RCV, Vienna, Austria;_ 2 _Boehringer Ingelheim Pharmaceuticals, Inc, Ridgefield, CT;_ 3 _Allcyte, Vienna, Austria_.

The clinical success of immune modulatory antibodies in oncological indications has demonstrated the importance of harnessing the immune system for the treatment of cancer. VISTA (PD-1H) is an Ig superfamily cell surface molecule mainly expressed on myeloid cells, but also to some extent on NK cells and T cells. Substantial published data supports the view that VISTA-binding antibodies or administration of VISTA itself can provide an inhibitory signal to T cells though the mechanism of action remains unclear including whether this activity is mediated directly or indirectly. We present here novel findings regarding the impact of different IgG isotypes on the activity of anti-human VISTA antibodies with immune stimulatory effects on primary human immune cells. The immune stimulatory effects of IgG antibodies were found to differ between variants with an identical antigen-binding domain but with different IgG isotypes (IgG4 and IgG1). First, we observed that both variants increased ongoing immune reactions, while the IgG1-variant was additionally able to activate resting immune cells. In a mixed leukocyte reaction (MLR) with healthy donor PBMCs, the ongoing immune reaction was effectively augmented by both, anti-VISTA-IgG4 and anti-VISTA-IgG1 isotype antibodies, as observed by increased levels of cytokines, e.g. TNF. However, in an experimental culture system of unstimulated PBMC of single healthy donors, only VISTA-IgG1 isotype antibodies induced increased levels of activation markers, e.g. upregulation of HLA-DR on resting myeloid cells. Second, we showed that Fc-receptor binding was a necessary interaction for immune activation as preventing the Fc-gamma-receptor binding to either of the two isotypes abolished the effects. The effects observed were specific as non-VISTA-binding isotype control mAbs were inactive. We then investigated the effects of anti-VISTA-IgG4 isotype antibodies on cells from AML patient samples by pharmacoscopy, a novel microscopy analysis method able to robustly quantify cell-cell proximity as a measure of cellular interactions. Here, VISTA-IgG4 isotype antibodies augmented interactions between multiple immune cell populations as well as between immune cells and AML cancer cells. This augmented interaction was abolished by blockade of Fc-gamma-receptor interactions, confirming our previous results. In summary, the immune stimulatory effects of anti-VISTA antibodies are defined by the antibody isotype and interaction with Fc-gamma-receptors, highlighting the importance of understanding these interactions when designing immune stimulatory antibody therapeutics for IO applications.

#3267

Cancer immunotherapy by use of FANA ASO therapy to silence Foxp3, impair Treg function and promote anti-tumor immunity.

Wayne W. Hancock,1 Liqing Wang,1 Veenu Aishwarya2. 1 _CHOP/U Penn, Philadelphia, PA;_ 2 _AUM LifeTech, Inc., Philadelphia, PA_.

There is an inverse correlation between tumor and draining lymph node infiltration by Foxp3+CD4+CD25+ Treg cells and clinical outcomes in carcinomas of the breast, liver, stomach, ovary, pancreas, and lung, as well as melanomas and glioblastomas. Tregs suppress tumor responses by host CD8 and CD4 T cells, B cells, NK cells, macrophages and dendritic cells. There are currently no effective ways to target Tregs, since toxins (e.g. IL-2-diptheria toxin) and cyclophosphamide have only transient effects with rebound and may also affect conventional T cells, use of CD25 monoclonal antibodies have confounding effects given IL-2 receptor (CD25) expression by activated T cells, and given the phenomenon of "peripheral" conversion (iTreg development) at the tumor site that can allow Tregs to evade therapy. These problems led us explore use of FANA antisense oligonucleotides (ASO) to suppress expression of Foxp3 mRNA. FANA ASO are highly specific and are taken up by cells without use of any delivery agents, conjugates or formulations, making them attractive compounds for cancer drug development. Various Foxp3 sequence-specific or scrambled FANAs were designed, and fluorescently-labeled FANA tested by addition to Foxp3+ Tregs in vitro; these studies showed successful uptake and both cytoplasmic and nuclear localization. FANAs were further screened by assessing effects on Treg suppressive function, and those resulting in 40-50% reductions were assessed for effects on expression of Foxp3 mRNA (qPCR) and protein (Western blots). We next tested the in vivo efficacy of Foxp3 FANA by intravenous or intraperitoneal injection, either daily or twice/week, of Foxp3 FANA, followed by isolation of Tregs from lymphoid tissues. Selected fluorescently-labeled FANA showed Treg uptake, and unlabeled FANA led to >75% reduction in Foxp3 protein expression (flow cytometry, Western blots), and marked reductions in Treg suppressive function. Lastly, we compared the effects of scrambled vs. specific Foxp3 FANA ASO on TC1 lung tumor growth in syngeneic C57BL/6 mice. Compared with mice receiving scrambled FANA ASO, mice receiving specific Foxp3 FANA ASO showed impaired tumor growth, with 50% showing complete tumor elimination (p<0.0001), and markedly reduced Foxp3+CD4+ Treg infiltration (p<0.01). Our data indicate that FANA oligos can modulate Foxp3 expression and Treg function in vitro and in vivo, such that their use may provide an important new approach to cancer immunotherapy.

#3268

Costimulatory T-cell engagement by PRS-342, a GPC3/4-1BB bispecific molecule, leads to activation of T-cells and tumor growth inhibition in a HCC humanized mouse model.

Birgit Bossenmaier, Corinna Schlosser, Rachida Siham Bel Aiba, Christian Barthels, Benjamin Weiche, Timo Eichner, Michelle Yegres, Shane A. Olwill. _Pieris Pharmaceuticals GmbH, Freising, Germany_.

Background: Increasing evidence shows that 4-1BB is a key costimulatory immunoreceptor and a highly-promising therapeutic target in cancer. Current antibody-based approaches showed immune cell activation not only in tumor tissues, but also in the periphery, which is associated with dose-limiting on-target toxicity. To overcome this limitation, we generated PRS-342, a GPC3/4-1BB bispecific molecule based on the Anticalin technology. This molecule is designed to promote 4-1BB clustering by bridging 4-1BB-positive T cells with GPC3-expressing tumor cells. GPC3 is an oncofetal protein with high tumor selectivity and high expression in not only hepatocellular carcinomas, but also in a variety of other tumors with high medical need.

Methods: Anticalin therapeutics are 18 kD proteins derived from human lipocalins. We utilized phage display to generate an Anticalin protein binding to 4-1BB with high affinity and specificity. The PRS-342 bispecific construct was generated by genetic fusion of a 4-1BB-specific Anticalin protein to a humanized high affinity GPC3-targeting monoclonal antibody with an engineered IgG4 backbone.

Results: We show that the bispecific molecule PRS-342 retains its ability to bind both targets (4-1BB and GPC3) with similar affinity to the parental building blocks and is capable of binding both targets simultaneously. Biophysical characterization of PRS-342 reflect its good drug-like properties. Using in vitro assays based on mixed culture of human pan-T cells and GPC3-expressing tumor cell lines, PRS-342 induced T-cell costimulation leading to increase production of IL-2 with EC50 in the sub-nanomolar range. In contrast, monospecific binding to GPG3 or 4-1BB by benchmarks or single building blocks was not able to activate T cells in this assay. These data demonstrate the ability of PRS-342 to bind both targets simultaneously, which is necessary for clustering of 4-1BB. PRS-342 was also evaluated for activity in a HepG2 mouse xenograft engrafted with human PBMCs with results supporting its differentiated MoA compared to relevant benchmark controls.

Conclusion: PRS-342 was designed to elicit 4-1BB costimulatory effects in a tumor-localized manner. Here we report potent T-cell activation that is strictly dependent on the presence of GPC3-positive tumor cells. Collectively our in vitro and in vivo data support the continued development of PRS-342.

#3269

Discovery and characterization of E7766, a novel macrocycle-bridged STING agonist with pan-genotypic and potent antitumor activity through intravesical and intratumoral administration.

Kuan-Chun Huang, Atsushi Endo, Shannon McGrath, Dinesh Chandra, Jiayi Wu, Dae-Shik Kim, Diana Albu, Christy Ingersoll, Karen Tendyke, Kara Loiacono, Thomas Noland, David Verbel, Chi Zhang, Ming-Hong Hao, Mark Matijevic, Vaishali Dixit, Renee R. Hukkanen, Janna Hutz, John Wang, Frank Fang, Xingfeng Bao, Donna Kolber-Simonds, Muzaffar Akram, Nadeem Sarwar. _Eisai AiM Institute, Andover, MA_.

Introduction: We report discovery and characterization of E7766, a structurally novel STING agonist, as a potential immunotherapy for solid cancers through intratumoral (IT) administration and for Bacillus Calmette-Guerin (BCG) unresponsive non-muscle invasive bladder cancer (NMIBC) through intravesical (VE) administration.

Methods: E7766 was designed and synthesized to optimize the potency of binding to dimerized STING proteins of different genetic isoforms. The compound was extensively and comparatively characterized in a variety of biochemical, molecular and cellular, in vivo, ex vivo, and primary human tumor and cellular studies for potency, mechanisms and translational biomarkers. Novel preclinical models to mimic orthotopic NMIBC and deep lesion metastasis were developed, and co-crystalization with recombinant proteins of genetic variations was performed.

Results: E7766, a novel Macrocycle-Bridged STING Agonist, showed highly specific and potent agonist activity in both human and mouse STING. In human PBMCs, E7766 demonstrated potent and consistent activity across seven tested human STING genotypes (IC50, 0.15-0.79 μM). By contrast, a reference cyclic dinucleotide STING agonist showed weaker potency and substantial variability across genotypes (IC50, 1.88 μM - >50 μM). Co-crystal structures indicated a structural basis for the superior interactions of E7766 with STING proteins compared with conventional cyclic dinucleotide STING agonists. Intravesical administration of E7766 to a preclinical orthotopic mouse bladder cancer model mimicking the BCG-unresponsive NMIBC demonstrated a dose-dependent and curative activity without serious adverse effects. The anti-tumoral activity was associated with a robust induction of IFNβ, CXCL10 and other downstream effectors of STING pathway inside the bladder cavity. In addition, single IT administration of E7766 to a subcutaneous (SC) tumor in mice bearing dual CT26 tumors in liver and SC lesion cured 90% of animals without recurrence for over 8 months. Those tumor-free animals rejected re-challenge of the same tumor cells in the absence of CD8+ T cells or NK cells, indicating the presence of a highly effective immune memory response following treatment with E7766 independent of either cell population alone.

Conclusions: E7766 is a structurally novel and highly potent STING agonist with pan-genotypic activity, demonstrating curative anti-tumoral activity in murine models of BCG-unresponsive NMIBC and of metastatic tumors in deep lesions. Clinical investigation of E7766 is under discussion.

#3270

Mechanism of action of a novel agonist TNFR2 antibody that induces co-stimulation of T cells and promotes robust anti-tumor immunity.

Ross B. Fulton, Adam Camblin, James F. Sampson, Jennifer Richards, Christina Wong, Alexander Koshkaryev, Lia Luus, Yang Jiao, Lihui Xu, Violette Paragas, Maja Razlog, Marco Muda, Eric M. Tam, Daryl C. Drummond, Andreas Raue. _Merrimack Pharmaceuticals, Cambridge, MA_.

TNFR2 is a member of the TNF receptor superfamily that is upregulated upon T cell activation and is highly expressed by tumor-infiltrating effector and regulatory T cells. We investigated TNFR2 levels on T cells in syngeneic mouse tumor models and in secondary lymphoid tissues by flow cytometry. Non-regulatory T cells in the spleen and lymph nodes expressed little TNFR2 whereas Tregs constitutively expressed intermediate levels. In contrast, tumor-infiltrating effector T cells expressed high levels of TNFR2 with Tregs expressing the highest levels. To investigate TNFR2 as a therapeutic target, we generated a novel monoclonal antibody specific to murine TNFR2 and investigated its mechanism of action. Antibodies against murine TNFR2 were generated by screening a human antibody-display library or by rabbit immunization. Antibodies were assessed for affinity, ability to compete with TNFα and for developability. A select number of antibodies were expressed as murine IgG2a and evaluated for activity in multiple syngeneic mouse tumor models. The mechanism of action of the most active clone, Y9, was investigated further. In vitro, Y9 stimulation of purified T cells from healthy mice caused increased proliferation and effector function, indicating that Y9 acts as an agonist and can provide co-stimulation. In vivo, Y9 treatment of mice with established tumors resulted in complete tumor clearance across a variety of models. Using CRISPR knockout cell lines, we showed that Y9 activity did not depend on TNFR2 expression on tumor cells. However, it required T cells as it showed no activity in nude mice. The activity of Y9 on immune cells was further confirmed by its decreased activity in mice depleted of NK or CD8+ T cells. Unlike the proposed Treg-depletion mechanism for other co-stimulatory therapeutic antibodies, depletion of Tregs is not the primary mechanism of action of Y9 treatment. Instead, decreased TNFR2 and other co-inhibitory receptor surface expression was observed following treatment. Y9 activity depended on FcγR binding as demonstrated by the lack of activity of an antibody variant with mutations preventing FcγR binding. We showed further that FcγR binding facilitated enhanced agonist activity by comparing activity of Y9 variants with different Fc isotypes and in FcγR knockout mice. We present a novel anti-TNFR2 antibody that exhibited pronounced anti-tumor in vivo activity in our mouse models with co-stimulation of tumor-specific T cells as its dominant mechanism of action. A corresponding human anti-TNFR2 antibody (MM-401) has been identified and is being developed as a potential novel treatment option for cancer patients.

#3271

A systemically administered, conditionally active TLR8 agonist for the treatment of HER2-expressing tumors.

Kara Moyes, Ty Brender, Sean W. Smith, Hengyu Xu, Ben Setter, Li-Qun Fan, Rebecca Brunette, Justin Killebrew, Phil Tan, Craig Coburn, Peter Baum, Valerie Odegard. _Silverback Therapeutics, Seattle, WA_.

Clinical development of systemically administered myeloid cell agonists has been hindered by acute toxicities due to peripheral activation of the targeted cell types. Intratumoral administration, the route of delivery typically used for innate immune/myeloid cell agonists, is limited by tumor accessibility and a dependence on abscopal responses. Here, we describe an example of a new composition class, termed ImmunoTAC, that allows for systemic delivery of an immune modulator with activity localized to tumor sites and secondary lymphoid structures. Agonism of TLR8 in human myeloid cells drives anti-tumor immunity by inducing direct macrophage killing of tumor cells, repolarizing suppressive myeloid cell populations to a pro-immunogenic phenotype, and inducing dendritic cells to drive tumor-specific CTL responses. SBT6050 is an ImmunoTAC comprised of a novel, potent TLR8 agonist conjugated to a HER2-directed monoclonal antibody. SBT6050 is designed to activate human monocytes and macrophages only in the presence of HER2pos tumor cells with moderate (2+ by IHC) or high (3+ by IHC) expression levels. This activity is dependent upon the ability of the Fc domain of the antibody to engage Fcγ receptors on the surface of the myeloid cells. Additionally, SBT6050 potently activates conventional dendritic cells that, in turn, drive a Th1/CTL program in T cells. Systemic delivery of a SBT6050 surrogate in mice shows robust single agent efficacy in tumor models intrinsically resistant to checkpoint blockade, such as the previously characterized HER2+ CT26 syngeneic model. The intratumoral immune response is characterized by robust activation of tumor-associated myeloid cells, infiltration of neutrophils, persistent increases in local cytokine and chemokine production, decreases in Treg infiltrate, and the generation of a potent, neo-Ag specific anti-tumor CTL response. Mice cured with single-agent treatment are resistant to tumor rechallenge, demonstrating induction of long-lived immunological memory. In contrast to observations with small molecule TLR8 agonists, ImmunoTAC does not induce peripheral cytokine production or associated CRS-like toxicity in mice, consistent with the localized activity of the molecule. Silverback's lead candidate, SBT6050, is currently in preclinical development for patients with moderate or high HER2-expressing tumors. More broadly, the data presented here describe a novel therapeutic modality that allows for systemic administration of immune modulators with tissue-localized activity.

#3272

RTX-212, an allogeneic red cell therapeutic expressing 4-1BBL and IL-15TP, exhibits potent in vitro and in vivo activity and a favorable safety profile.

Anne-Sophie Dugast, Shannon McArdel, Maegan Hoover, Enping Hong, Shannon Curtis Leonard, Arjun Bollampalli, Douglas C. McLaughlin, Jennifer Mellen, Torben Straight Nissen, Christopher L. Carpenter, Thomas J. Wickham, Sivan Elloul. _Rubius Therapeutics, Cambridge, MA_.

Recombinant agonists that activate co-stimulatory and cytokine receptors have shown limited clinical activity perhaps due to 1) toxicity; 2) a need for coordinated activation of co-stimulatory and cytokine pathways; or 3) the failure of agonist antibodies to recapitulate signaling by endogenous ligands. To address these limitations, Rubius Therapeutics developed genetically engineered red cells with cell surface expression of both co-stimulatory and cytokine ligands, which present ligands in their native form through cell-cell contact to potently activate and expand both T and NK cells. On-target, off-tissue toxicity may be limited due their biodistribution, which is restricted to the vascular system. IL-15 is known to promote NK survival and CD8 T cell memory and 4-1BB agonists are known to promote T cell prolioferation and survival. Rubius Therapeutics has developed RTX-212, an allogeneic red cell therapeutic genetically engineered to co-express 4-1BBL and an IL-15/IL-15Rαfusion (IL-15TP). In vitro assays demonstrated that the combination of both ligands on RTX-212 expanded both memory CD8 T cells (2.5-fold) and NK cells (10-15-fold), in the absence of TCR stimulation. RTX-212 further induced dramatic proliferation of CD8 T cells and CD4 T cells in the presence of TCR stimulation with increased IFNγsecretion. In addition to the synergistic effects of 4-1BBL and IL-15TP, each molecule provided complementary functions that expanded the activity of RTX-212 beyond RTX-4-1BBL or RTX-IL-15TP alone. IL-15TP uniquely activated NK cytotoxicity and ADCC, while 4-1BBL uniquely stimulated CD4 and CD8 T cell proliferation and production of IFNγ. To evaluate in vivo immune responses, anti-tumor activity and safety, a mouse surrogate therapeutic, mRBC-212, was developed where recombinant Fc-IL-15-sushi and m4-1BBL were chemically conjugated to mouse red blood cells. This surrogate overcame the rapid clearance of human red cells in mice. Intravanous administration of mRBC-212 to C57Bl6 mice that received B16F10 melanoma cells IV, showed a 66% decrease in the number of lung metastases compared to control mice (p=0.0001) and was associated with a significant increase in NK cell infiltration into the lungs (p=0.02). In a CT26 tumor model, mRBC-212 treated mice exhibited 55% tumor growth inhibition, which was accompanied by a 1.7-fold increase in the tumor infiltration of proliferating and cytotoxic CD8 T cells. Mice treated with the highest feasible dose of mRBC-212 showed no change in serum transaminases, infiltration of CD8 T cells and macrophages to the liver or liver inflammation score compared to agonistic 4-1BB antibodies treated mice. Taken together, these data indicate that RTX-212 has the potential to be an effective therapy with an improved safety profile compared to 4-1BB agonist antibodies and IL-15 agonists, supporting its clinical development.

#3273

CMP-001, a virus-like particle containing immunostimulatory CpG-A, for treatment of peritoneal carcinomatosis of gastrointestinal and pancreatic cancers.

Ann M. Miller, Caitlin Lemke-Miltner, Sue Blackwell, Ann Tomanek-Chalkley, Kristen Coleman, George Weiner, Carlos Chan. _University of Iowa, Iowa City, IA_.

Peritoneal carcinomatosis is a common form of metastasis occurring in approximately 10% of gastrointestinal and pancreatic cancers with life expectancy often less than 6-12 months. Due to the limited options for treatment of peritoneal carcinomatosis, there is a significant need for novel therapeutic strategies. We explored the therapeutic potential of CMP-001, a novel virus-like particle composed of the Qβ bacteriophage capsid protein encapsulating an immunostimulatory CpG-A oligodeoxynucleotide (CpG-A ODN) in this cancer. CpG-A ODN is a known activator of toll-like receptor 9 (TLR9), which is expressed by human and murine plasmacytoid dendritic cells (pDCs). Initial studies evaluated the effect of CMP-001 in vitro on cells obtained from the ascitic fluid of patients with peritoneal carcinomatosis. These studies demonstrated that pDCs are present in the ascitic fluid and produce IFN-α in response to CMP-001 stimulation. A mouse model of peritoneal carcinomatosis was used to further explore the therapeutic potential of CMP-001. C57BL/6 mice were challenged intraperitoneally (i.p.) with Panc02 mouse pancreatic cancer cells. On days 5, 9, and 13 post-tumor challenge mice were treated i.p. with either saline or 100 μg CMP-001. Mice treated with CMP-001 had a median survival of 35 days compared to mice treated with saline alone with a median survival of 28 days (n = 10 per group, p = 0.028). Examination of the immune response demonstrated CMP-001 induced an influx of dendritic cells (CMP- 001 8.99% ± 0.95 vs saline 4.57% ± 0.7, p = 0.0015), NK cells (CMP-001 0.26% ± 0.052 vs saline 0.087% ± 0.0085, p = 0.006), and CD4+ T cells (CMP-001 3.29% ± 0.85 vs saline 0.95% ± 0.097, p = 0.0168) into the ascitic fluid. In addition, CMP-001 induced a significant increase in antigen-experienced, effector and memory, CD11ahiCD44hi CD4+ T cells (CMP-001 54.55% ± 6.52 vs saline 28.12% ± 3.68, p = 0.0028) and CD11ahiCD8αlo CD8+ T cells (CMP-001 58.49% ± 4.47 vs saline 23.04% ± 2.86, p < 0.0001) in the ascites. These data demonstrate that CMP-001 treatment activates pDCs in the peritoneal fluid of patients with carcinomatosis. In addition, CMP-001 stimulates a robust immune response in the peritoneal cavity of mice with peritoneal carcinomatosis. This robust immune response likely contributes to the enhanced survival and decreased disease progression in these mice. Collectively, these promising preclinical results suggest that CMP-001 may have potential as an immunotherapy for the treatment of patients with peritoneal carcinomatosis and is worthy of further evaluation.

#3274

LTX-401 as a novel antitumor and immunotherapeutic agent in an experimental liver cancer model.

Brynjar Mauseth,1 Ketil Camilio,1 Ji-Hua Shi,2 Øystein Rekdal,1 Baldur Sveinbjørnsson,1 Pål-Dag Line2. 1 _Lytix Biopharma, Oslo, Norway;_ 2 _Institute of Clinical Medicine, Oslo, Norway_.

Liver cancer is estimated to be the third most common cause of cancer-related mortality worldwide in both sexes. Hepatocellular carcinoma (HCC) is the most prevalent type of primary liver cancer in which potential curative surgical options are limited and overall survival poor. Hence, there is an unmet need for new and improved therapies. LTX-401 is a novel oncolytic compound with potent antitumor properties. Owing to its amphiphatic nature, LTX-401 may affect the integrity of cancer cell membranes and induce rapid cell death. Transmission electron microscopy indeed revealed a necrotic cell death phenotype accompanied by massive vacuolization of the cytoplasm and dilated mitochondria. Additionally, LTX-401 induces features of immunogenic cell death (ICD) as seen by the extracellular release of ATP and HMGB1, as well as the mitochondrial DAMP cytochrome C. The antitumor effects of LTX-401 has been explored in an experimental model of hepatocellular carcinoma (JM1 model) using immunocompetent rats. When injected into subcutaneously established lesions, the majority of animals went into remission followed by systemic protective immune responses. Additionally, LTX-401 could affect the growth of distal tumor deposits simulating metastases, hence indicating immune-mediated abscopal responses. Intratumoral treatment of orthotopic liver tumors resulted in cure of approximately half of the animals treated, which later displayed protection against subcutaneous tumor (re)challenge. Splenocytes harvested from immunized animals produced significantly larger amounts of IFN-y when stimulated with tumor cells ex vivo, indicating the presence of tumor-specific (memory) T cells. Histological examinations revealed that LTX-401 caused massive hemorrhagic tumor necrosis, both in subcutaneous and intrahepatic compartments, followed by increased infiltration of T cells. Taken together these results indicate that oncolytic compounds generate an immunogenic tumor environment and may be therapeutically exploited as a novel antitumor and immunotherapeutic approach in HCC.

#3275

Inhibition of ER-stress factor C/EBP homologous protein (Chop) with LNAplus™ antisense-oligonucleotides to improve immunotherapy of cancer.

Richard Klar,1 Yu Cao,2 Eslam Mohamed,2 Sven Michel,1 Monika Schell,1 Lisa Hinterwimmer,1 Stefanie Raith,1 Paulo Rodriguez,2 Frank Jaschinski1. 1 _Secarna Pharmaceuticals GmbH & Co. KG, Planegg, Germany; _2 _H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL_.

The microenvironment generated by tumor cells and suppressive stromal cells creates unfavorable conditions for effector immune cells in order to escape anti-tumor immunity. Besides suppressive pathways like for example the CD39/CD73 axis, mechanisms including nutrient starvation, hypoxia, exposure to high levels of reactive oxygen species and acidosis contribute to the suppression of tumor-reactive immune cells. We have recently discovered that the ER-stress response, in particular the C/EBP homologous protein (Chop) plays an important role in the suppression of tumor-exposed T cells. Furthermore, it is upregulated in activated T cells in vitro and may hamper the efficacy of adoptive T cell therapies. As Chop is a transcription factor it falls into the category of "difficult to drug" and therefore represents an optimal target for antisense-oligonucleotides. In order to revert Chop-induced suppression of T cell activity, we designed locked nucleic acid (LNA) ASOs with specificity for mouse or human Chop using our Oligofyer™ bioinformatics system. Knockdown on mRNA level was investigated in cancer cell lines and in T cells in vitro and the most potent ASOs were selected for further experiments. Downstream effects of Chop knockdown were investigated by mRNA expression analysis and flow cytometry. We furthermore investigated the effect of ex vivo Chop knockdown on the efficacy of adoptively transferred Pmel-specific T cells (recognized target: gp10025-33 peptide) in a B16 melanoma model. Treatment of cells with selected Chop-specific ASO leads to potent knockdown of Chop in vitro. In accordance with observations made in T cells derived from Chop-knockout mice, we observed an increase in T cell-associated transcription factor (Tbet), Interferon-gamma (IFN-γ) and Granzyme B (GZMB) expression in T cells that have been treated with a Chop ASO compared to control oligo treated cells. Strikingly, this translated into improved tumor control by Pmel-specific T cells. We furthermore observed an increase in IFN-γ producing tumor-infiltrating T cells and increased frequency of IFN-γ producing T cells in gp100 restimulated splenocytes from animals that received Chop ASO-treated Pmel-specific T cells. Of note, the impact of Chop knockdown was comparable to the effects that we observed in T cells derived from Chop knockout animals. Taken together, we herein show that Chop is a highly promising novel target in immunotherapy and can effectively be targeted by LNAplus™ ASOs. As shown in a model of adoptive T cell therapy, Chop ASOs have a high potential to optimize the efficacy of T cell therapies, such as chimeric antigen receptor (CAR)- or T cell receptor (TCR)-transgenic T cells. We are currently investigating the effect of systemic Chop ASO treatment in murine tumor models to expand the spectrum of applications of this innovative therapeutic tool.

#3276

**Damage-associated molecular pattern (DAMP) induction by 15-deoxy, Δ** 12,14 **-prostaglandin J** 2 **-ethanolamide: Examination of tumor selectivity, dendritic cell activation and oxidative stress in melanoma.**

Ahmed Elhassanny, Rene Escobedo, Daniel Ladin, Rukiyah Van Dross. _East Carolina University, Brody School of Medicine, Greenville, NC_.

Melanoma is the most lethal form of skin cancer in the United States. Current trends show that melanoma incidence has been increasing for the last 30 years. Chemotherapeutic agents for advanced stage metastatic melanoma have limited efficacy. Although the use of immune-modulating agents such as checkpoint inhibitors has significantly improved therapeutic outcomes, these agents can cause life-threatening autoimmune side effects. Therefore, immunotherapeutic agents that can preferentially eliminate melanoma cells are needed. Agents that induce the release of damage-associated molecular patterns (DAMPs) can stimulate antitumor immunity. DAMPs are produced by damaged or dying cancer cells and include signals such as expression of cell surface calreticulin (ecto-CRT) and the secretion of ATP. DAMPs can recruit and activate immune cells such as dendritic cells which in turn stimulate tumor-specific cytotoxic T cells to kill cancer cells in a process referred to as immunogenic cell death. The elicitation of DAMPs depends on endoplasmic reticulum stress (ER) and oxidative stress. Our group found that our patented agent, 15-deoxy, Δ12,14-prostaglandin J2-ethanolamide (15dPMJ2), inhibits melanoma tumor growth. In addition, 15dPMJ2 induces DAMPs in melanoma cells via an ER stress dependent mechanism. The purpose of this study was to investigate the selectivity of DAMP induction in melanoma by 15dPMJ2, the effect of 15dPMJ2-treated cells on the activation of bone marrow-derived dendritic cells (BMDCs) and the role of oxidative stress in this process. To determine if 15dPMJ2 causes tumor-selective activation of BMDCs, we first examined ecto-CRT and the release of ATP in B16F10 melanoma cells and Melan-A non-tumorigenic melanocytes. Our results showed that 15dPMJ2 significantly increased DAMP expression in tumorigenic compared to non-tumorigenic melanocytes. Importantly, we found that 15dPMJ2-treated B16F10 cells induced maturation of BMDCs as manifested by an increase in the expression of MHCII, CD80 and CD86. However, 15dPMJ2-treated Melan-A cells did not activate BMDCs. In this study, we also tested the role of oxidative stress in 15dPMJ2-induced DAMPs. Our results showed that the antioxidant Trolox significantly decreased 15dPMJ2 induced ecto-CRT and ATP release in B16F10 cells. However, Trolox did not inhibit the activation of BMDCs by 15dPMJ2-treated B16F10 cells. These results suggest that oxidative stress plays an important role in 15dPMJ2-induced DAMPs, but it is not required for the BMDC activation. In conclusion, 15dPMJ2 can selectively induce DAMPs in melanoma which results in the activation of BMDCs. Hence, 15dPMJ2 is a potential small molecule immunotherapeutic for melanoma.

#3277

IACS-9779, a development candidate that inhibits 2,3-dioxygenase (IDO) activity by blocking heme incorporation into IDO apoenzyme.

Faika Mseeh,1 Matthew M. Hamilton,1 Joseph R. Marszalek,1 Norma E. Rogers,1 Connor A. Parker,1 Simon S. Yu,1 Zhen Liu,1 Naphtali J. Reyna,1 Timothy McAfoos,1 Brett W. Virgin-Downey,1 Paul G. Leonard,1 Jason B. Cross,1 Ningping Feng,1 Angela L. Harris,1 Andy M. Zuniga,1 Keith Mikule,2 Martin Tremblay,2 Yongying Jiang,1 Mikhila Mahendra,1 Jihai Pang,1 Qi Wu,1 Quanyun Xu,1 Timothy P. Heffernan,1 Philip Jones,1 Richard T. Lewis1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _Tesaro Inc., Waltham, MA_.

Increased expression of IDO1 is believed to create a tumor microenvironment that is immunosuppressive. In the course of our research directed at identifying potent and selective inhibitors of IDO1, we identified a class of compounds that inhibited IDO1 activity in a cellular context, but not in isolated enzymatic assays. We have conducted detailed mechanistic studies and shown that these molecules inhibit IDO1 by binding to the apo-enzyme, thus preventing the incorporation of the heme-cofactor into the active site of the holo-enzyme.

Through an extensive medicinal chemistry campaign, we optimized a series of orally bioavailable, highly potent and selective inhibitors of IDO1 that possess excellent pharmacological properties. For several lead molecules, pharmacokinetic (PK) - pharmacodynamic (PD) relationships were established in whole blood and SKOV3 xenograft assays. The inhibition of IDO1 in a human whole-blood assay correlated well with the suppression of tumor kynurenine (KYN) that was observed in SKOV3 xenografts. At plasma concentrations of 3 µM, IACS-9779 supressed tumor KYN levels by 90%. IACS-9779 was well tolerated with excellent in vivo PK properties across multiple preclinical species, and a human PK prediction consistent with a low daily dose needed for full suppression of KYN production via IDO1.

#3278

**EOS100850 potently restores adenosine A** 2A **receptor-dependent suppression of T cell function in the adenosine rich tumor microenvironment.**

Erica Houthuys, Paola Basilico, Veronique Bodo, Margreet Brouwer, Michel Detheux, Gregory Driessens, Bruno Gomes, Annelise Hermant, Catherine Hoofd, Florence Lambolez, Xavier Leroy, Reece Marillier, Chiara Martinoli, Marjorie Mercier, Florence Nyawouame, Shruthi Prasad, Ariane Scoumanne, Stefano Crosignani. _iTeos Therapeutics, Gosselies, Belgium_.

Adenosine is a potent immunosuppressive metabolite that is often found elevated in the extracellular tumor microenvironment (TME). We determined concentrations of extracellular adenosine in different patient-derived xenografts (PDX) from 7 different histological types, in which extracellular median adenosine concentrations were shown to range from 0.5 to 45 µM, with the overall median adenosine concentration being about 4.5 µM. Adenosine concentration in the non-tumorous subcutaneous space in mice was measured at about 0.5 µM. Adenosine in the TME is generated mainly by the concerted action of the ectonucleotidases CD39 and CD73. The expression of these enzymes across various cancer types was evaluated by flow cytometry in dissociated human tumor biopsies. CD73 and CD39 were strongly expressed by multiple tumor-infiltrating T cell types. Tumor-associated myeloid cells mostly expressed CD39 and high frequencies of EpCAM\+ tumor cells strongly expressed CD73. These data strongly suggest that adenosine levels could be further increased compared to non-immune-infiltrated PDX.Adenosine activates 4 G protein-coupled receptor subtypes, of which the adenosine A2A receptor in particular suppresses innate and adaptive immune cell responses leading to suppression of anti-tumor immunity. Among the 4 adenosine receptors, we confirmed the A2A receptor as the main adenosine receptor expressed in CD4\+ and CD8\+ T cells, natural killer cells, monocytes, and dendritic cells. Stimulation of these immune cell subsets further increased A2A receptor expression. A2B receptor was expressed at very low levels in stimulated CD4\+ and CD8\+ T cells and in monocytes and immature DCs. A1 and A3 receptors were hardly detected in these subsets of immune cells. EOS100850, a highly potent and selective A2A receptor antagonist, was characterized in various in vitro functional assays. A2A receptor activation by a selective agonist suppressed priming of mouse OVA-specific OT1 T cells and subsequent antigen-specific CD8\+ T cell cytotoxicity in a co-culture assay of effector CD8+ T cells and target cancer cells. This A2A receptor-mediated immune suppression was potently and dose-dependently reversed by EOS100850. In a mixed lymphocyte reaction between human dendritic cells and T cells, EOS100850 blocked the adenosine-dependent inhibition of T cell proliferation and secretion of IFNg, TNFa and IL-2 in a dose-dependent manner. In conclusion, extracellular adenosine as well as adenosine pathway components were strongly present across multiple tumor types, and across multiple tumor-associated cell types. In addition, EOS100850, a highly potent A2A receptor antagonist, reversed A2A receptor-mediated suppression of T cell priming, cytotoxicity, cytokine production and proliferation.

#3279

**All-** trans **-retinoic acid (ATRA) markedly augments anti-tumor immunity.**

Lu Huang, Zibo Chen, Leila Williams, Yulong Chen, Yunfei Wang, Masanori Kawakami, Jason Roszik, Patrick Hwu, Ethan Dmitrovsky, Weiyi Peng, Xi Liu. _UT MD Anderson Cancer Ctr., Houston, TX_.

All-trans-retinoic acid (ATRA) engages diverse actions on populations of immune cells. These include effects on regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), Th17 and other immune cells. Prior work found that ATRA can induce anti-tumor immunity through MDSCs and CD8+ T cells and pro-tumor immunity by increasing Treg cells. In this study, we explored how ATRA modulated anti-tumor immunity in solid tumors. To do this, syngeneic lung cancer and colon cancer models were independently developed by optimizing subcutaneous injections of ED1SQ4 lung cancer and MC38 colon cancer cells into immune-competent mice. ATRA-treatment markedly repressed both syngeneic lung cancer and colon cancer growth as compared to vehicle-treated mice. To exclude the possibility that ATRA directly suppressed growth of cancer cells, ED1SQ4 and MC38 cells were independently implanted subcutaneously into athymic mice in order to allow these lung and colon cancer xenografts to grow. Notably, ATRA-treatment did not reduce the growth of these respective xenograft tumors. Intriguingly, ATRA did not suppress in vitro proliferation of either ED1SQ4 or MC38 cells. This implicated ATRA as engaging mechanisms that affect the tumor microenvironment. To learn if the observed anti-tumorigenic activity was mediated by regulation of T cell immunity, CD4+ or CD8+ T cell populations were depleted in syngeneic mice before in vivo ATRA-treatment. CD4+ and CD8+ T cell depletion from syngeneic mice at least partially reversed the tumor suppressive activity of ATRA. Flow cytometry assays conducted on intratumoral immune cells revealed that ATRA-treatment decreased the CD8+ T to Treg cellular ratios while increasing the ratios of CD8+ T to Treg cells. Cytokine and chemokine assays were performed on these syngeneic cancers as well as on peripheral blood harvested from ATRA or vehicle-treated syngeneic mice in order to identify mediators of the observed anti-tumorigenic effects. Findings will be presented. Taken together, this study uncovered a previously unrecognized role for ATRA in augmenting immunotherapy. These preclinical immunotherapy findings can be translated into the cancer clinic.

#3280

TTFields induces immunogenic cell death and STING pathway activation through cytoplasmic double-stranded DNA in glioblastoma cells.

Dongjiang Chen, Nagheme Thomas, David D. Tran. _University of Florida, GAINESVILLE, FL_.

Glioblastoma (GBM) is the most common and deadliest malignant brain cancer in adults despite aggressive chemoradiotherapy. Tumor Treating Fields (TTFields) was recently approved in combination with adjuvant temozolomide chemotherapy for newly diagnosed GBM patients. The addition of TTFields resulted in a significant improvement in overall survival. TTFields are low-intensity alternating electric fields that are thought to disturb mitotic macromolecules' assembly, leading to disrupted chromosomal segregation, integrity and stability. In many patients, a transient stage of increased peritumoral edema is often observed early in the course of TTFields treatment followed subsequently by objective radiographic responses, suggesting that a major component of therapeutic efficacy by TTFields may be an immune mediated process. However, the mechanism underlying these observations remains unclear. Here we report results on a panel of GBM cell lines treated with TTFields at the clinically approved frequency of 200 kHz using an in vitro TTFields system. Our data showed 24 hrs TTFields-treated GBM cells had a significantly higher rate (19.9% vs. 4.3%, p=0.0032) of micronuclei structures released into the cytoplasm as a result of TTFields-induced chromosomal instability. Nearly 40% of these micronuclei were co-localized with two upstream dsDNA sensors: absent in melanoma 2 (AIM2)andInterferon (IFN)-inducible proteinCyclic GMP-AMP synthase (cGAS), compared to absence of co-localization in untreated cells. TTFields-activated micronuclei-dsDNA sensor complexes led to i) induction of pyroptotic cell death, as measured by a specific LDH release assay, and through AIM2-recruited caspase1 and cleavage of pyroptosis-specific Gasdermin D; and ii) activation of STING pathway components including Type I IFNs and pro-inflammatory cytokines downstream of the NFκB pathway. GBM cell-specific shRNA depletion of either AIM2 or STING or both in a co-culture experiment of bone marrow cells or splenocytes with supernanants obtained from knockdown GBM cells was able to reverse the inducement of immune cells. These results provide compelling evidence that TTFields function as an activator of the immune system in GBM cells, and a strong rationale for combining TTFields with immunotherapy aimed at augmenting an anti-tumor immune response such as immune checkpoint inhibitors.

## EPIDEMIOLOGY

### Factors Influencing Cancer Outcomes

#3281

The association between body mass index (BMI) and risk of pancreatic cancer depends on age at BMI assessment.

Eric J. Jacobs,1 Christina C. Newton,1 Alpa V. Patel,1 Victoria L. Stevens,1 Farhad Islami,1 W. Dana Flanders,2 Susan M. Gapstur1. 1 _American Cancer Society, Atlanta, GA;_ 2 _Emory University, Atlanta, GA_.

Importance: Pancreatic cancer rates in the United States have steadily increased since the early 2000's despite declines in smoking. This increase is not easily explained by population increases in body mass index (BMI) given the relatively weak association between BMI and pancreatic cancer risk typically observed in epidemiologic studies. Most of these studies, however, assessed BMI in older adulthood, which may be less strongly associated with pancreatic cancer than BMI earlier in adult life.

Objective: To examine the association between BMI and pancreatic cancer mortality by age at BMI assessment.

Design: Prospective cohort study.

Setting: Cancer Prevention Study II (CPS-II), a nationwide study of cancer mortality enrolled in 1982 and followed through 2014.

Participants: 963,317 US adults aged 30 to 89 years at enrollment.

Exposure: BMI calculated from height and weight reported at enrollment.

Main outcomes and measures: A total of 8,354 participants died of pancreatic cancer during follow-up. Hazard ratios (HRs) for BMI were calculated using multivariable proportional hazards regression models. Population attributable fractions by birth cohort were calculated using HRs from CPS-II and BMI distributions from nationally representative National Health and Examination Surveys (NHANES).

Results: HRs declined with increasing age at BMI assessment. HRs per 5 BMI units among those aged 30-49, 50-59, 60-69, and 70-89 years at assessment were 1.25 (95% confidence interval (CI) 1.18-1.33), 1.19 (95% CI 1.14-1.23), 1.14 (95% CI 1.08-1.21) and 1.13 (95% CI 1.02-1.26), respectively (p-trend = 0.005). The prevalence of obesity in early middle age is substantially higher in more recent US birth cohorts than in earlier ones. Therefore, based on a HR of 1.25 per 5 BMI units at age 45 years, we estimate that 28% of pancreatic cancer deaths in the US among those born from 1970-74 will be attributable to BMI levels > 25 kg/m2, nearly twice the equivalent percentage in those born in the 1930s.

Conclusions and Relevance: BMI before age 50 may be more strongly associated with pancreatic cancer risk than BMI at older ages. These results underscore the importance of preventing excess weight gain before middle age for reducing rates of this highly fatal cancer.

#3282

BMI and other factors in relation to overall survival of female breast cancer in China.

Yunqiu Dong,1 Franzel J. B. van Duijnhoven,2 Lu Wang,1 Ming Wu,3 Yun Qian,1 Ellen Kampman2. 1 _Wuxi Center for Disease Control and Prevention, Wuxi, China;_ 2 _Wageningen University, Netherlands;_ 3 _Jiangsu Provincal Center for Disease Control and Prevention, China_.

The aim of this study is to determine the relationship between body mass index(BMI) at diagnosis and various factors including the epidemiologic and clinical factors on survival of female breast cancers. We analysed the data of 653 patients in any stage from the Wuxi Breast Cancer Cohort. Cox Proportional Hazard regression models were conducted to compare the overall survival(OS) outcomes among BMI groups, and adjusted by covariates which yielded a >10% change in the hazard ratios(HRs) estimate, including age, stage, menopause status, physical activity in work. There were significant differences among BMI groups in age(P=0.000), WHR(P =0.000), physical activity in work(P =0.019),red meat intake(P =0.029), oil intake(P =0.000),tumor stage(P =0.021),menopause status(P =0.001). Overweight(OW,BMI=24 to 27.9kg/m2) and obesity(OB, BMI≥28kg/m2) showed better OS comparing with normal weight(NW, BMI=18.5 to 23.9kg/m2)(respectively ,adjusted HRs=0.45, 95%CI=0.15 to 1.34, adjusted HRs=0.59,95%CI=0.21 to 1.69), meanwhile underweight(UW, BMI<18.5kg/m2) showed a poorer OS(adjusted HRs=4.65,95%CI=0.93 to 23.37). There was no significant relationship between the BMI and some other factors in OS. In conclusion, the findings of our current study indicate that BMI might play a role in the prognosis of female breast cancer in China, but the evidence was not strong enough.

#3283

Associations of change in BMI before surgery with disease-free and overall survival in colorectal cancer patients: Results from the ColoCare Cohort.

Jennifer Ose,1 Biljana Gigic,2 Tengda Lin,1 Juergen Boehm,1 Petra Schrotz-King,3 Petra Schrotz-King,3 Sheetal Hardikar,1 Caroline Himbert,1 Martin Schneider,4 Alexis Ulrich,4 Cornelia M. Ulrich1. 1 _Univ. of Utah Huntsman Cancer Inst., Salt Lake City, UT;_ 2 _University of Heidelberg, Germany;_ 3 _National Center for Tumor Diseases, Heidelberg, Germany;_ 4 _University of Heidelberg, Heidelberg, Germany_.

BACKGROUND Body weight in colorectal cancer patients may change substantially before and after diagnosis and treatment. The prognostic significance of pre-surgery weight loss is still unclear. We investigated the association of pre-surgery weight loss with clinical outcomes in colorectal cancer patients.

METHODS We used prospectively collected data from n=148 newly diagnosed colorectal cancer patients (stage I-IV) from the ColoCare Cohort. Data on body mass index (BMI; (kg)/ m2) 1 year before surgery and at the time of surgery were collected. We calculated BMI differences between these time points. We applied Cox proportional hazard models to investigate associations of BMI change (continuous), biological sex (male, female) and tumor stage (I, II, III, IV) and neoadjuvant chemo- and/or radiotherapy (no/yes).

RESULTS After 24 months of follow-up n=22 patients were deceased and n=19 patients had a recurrence. Pre-surgery change in BMI was associated with poorer overall and disease-free survival: for every loss in one unit in BMI patients had a 47% increase in risk of overall death: Hazard Ratio (HR): 1.47; 95% confidence interval (CI) (1.21-1.76), p=0.0001 and a 44% increase in risk for either death or recurrence: HR: 1.44; 95% CI (1.19-1.73), p=0.0002.

CONCLUSIONS Pre-surgery weight change is, independently of tumor stage, age, biological sex and neoadjuvant therapy, associated with long-term clinical outcomes in colorectal cancer survivors.

#3284

Adiposity, muscle mass and delays and dose reductions on adjuvant, taxane-based chemotherapy for breast cancer.

Elizabeth M. Cespedes Feliciano,1 Valerie Lee,1 Wendy Y. Chen,2 Carla M. Prado,3 Shlomit S. Shachar,4 Stacey Alexeeff,1 Bette J. Caan1. 1 _Kaiser Permanente Northern California, Oakland, CA;_ 2 _Dana Farber Cancer Institute, Brigham and Women's Hospital, Harvard Medical School, Boston, MA;_ 3 _University of Alberta, Edmonton, AB;_ 4 _Rambam Health Care Campus, Haifa, Israel_.

Introduction: Low muscle mass and excess adiposity are thought to increase the risk of chemotherapy toxicity, leading to dose reductions or delays. Yet, few studies of body composition and chemotherapy examine patients with breast cancer; prior studies have been small and looked mainly at toxicities. Here, we evaluate whether adiposity or muscle mass and radiodensity (a measure of intramyocellular lipid accumulation) are associated with risk of dose reductions or delays among nonmetastatic breast cancer patients receiving adjuvant taxane-based chemotherapy, and whether dose reductions are associated with cancer-specific survival.

Methods: Our study included 1,403 patients with stage II-III breast cancer receiving taxane-based chemotherapy at Kaiser Permanente. We evaluated body composition from clinically-acquired CT scans at diagnosis. We defined dose reductions from infusion records as relative dose intensities <0.85 (delivered v. planned dose) while on taxane therapy, and dose delays as treatments received more than 3 days later than scheduled. We defined neuropathy from diagnosis codes and hematologic toxicities from laboratory values during chemotherapy. Logistic regression models adjusted for age and dosing body surface area (BSA). Cox proportional hazards models for cancer-specific survival adjusted for age, BSA, body composition and tumor characteristics.

Results: Mean (standard deviation [SD]) age at diagnosis was 53 (10) years. Higher visceral adiposity was associated with a 20% increased risk of dose reductions and a 17% increased risk of dose delays on taxane-based chemotherapy (odds ratios of 1.20; 95%CI:1.02-1.42 and 1.17; 95%CI:0.99-1.39 per SD, respectively). Higher muscle radiodensity (indicating lower intramyocellular lipid infiltration, i.e., leaner muscle) was associated with a 13% lower risk of dose reductions and a 16% lower risk of dose delays (odds ratios of 0.87; 95%CI:0.75-1.00 and 0.84; 95%CI:0.72-0.98 per SD, respectively). Muscle mass and subcutaneous adiposity were not associated with dose reductions or delays, though lower muscle mass did increase risk of hematologic toxicity. Women who experienced dose reductions on taxane-based chemotherapy had a 37% increased risk of dying from breast cancer relative to those with higher relative dose intensities (hazard ratio 1.37; 95%CI:1.01-1.85; median follow-up 6 years, 203 breast cancer deaths).

Conclusions: Excess visceral adiposity and lower muscle radiodensity were associated with dose reductions and delays among breast cancer patients receiving taxane-based chemotherapy, reducing the efficacy of these life-saving therapies: women who experienced dose reductions were at higher risk of dying from breast cancer. Body composition information assessed from clinically-acquired CT scans may help identify patients for supportive interventions to mitigate toxicity.

#3285

Body mass index, weight loss, and progression and mortality in metastatic colorectal cancer: Results from CALGB/SWOG 80405 (Alliance).

Brendan Guercio,1 Sui Zhang,2 Alan P. Venook,3 Fang-Shu Ou,4 Donna Niedzwiecki,5 Heinz-Josef Lenz,6 Federico Innocenti,7 Hanna Sanoff,7 Michelle R. Mahoney,4 Bert H. O'Neil,8 James E. Shaw,9 Blase N. Polite,10 Howard S. Hochster,11 James N. Atkins,12 Richard M. Goldberg,13 Robert J. Mayer,2 Charles D. Blanke,14 Charles S. Fuchs,15 Jeffrey A. Meyerhardt2. 1 _Brigham & Women's Hospital, Boston, MA; _2 _Dana-Farber/Partners CancerCare, Boston, MA;_ 3 _University of California, San Francisco, San Francisco, CA;_ 4 _Alliance Statistics and Data Center, Mayo Clinic, Rochester, MN;_ 5 _Duke Department of Biostatistics and Bioinformatics, Duke University, Durham, NC;_ 6 _USC Norris Comprehensive Cancer Center, Los Angeles, CA;_ 7 _Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC;_ 8 _Simon Cancer Center, Indiana University School of Medicine, Indianapolis, IL;_ 9 _Virginia Commonwealth University, Richmond, VA;_ 10 _Pritzker School of Medicine, University of Chicago, Chicago, IL;_ 11 _New York University Cancer Institute, New York, NY;_ 12 _Southeast Clinical Oncology Research (SCOR) Consortium, Winston-Salem, NC;_ 13 _West Virginia University Cancer Institute, Morgantown, WV;_ 14 _SWOG and Oregon Health & Science University, Portland, OR; _15 _Yale Cancer Center, Yale School of Medicine, New Haven, CT_.

Background: In non-metastatic colorectal cancer, a body mass index (BMI) paradox is observed where overweight and early obese patients experience improved outcomes compared to patients with normal, low, or morbidly obese BMI. The influence of obesity on patients with advanced or metastatic colorectal cancer (mCRC) is relatively unexplored.

Methods: We conducted a prospective BMI companion study in CALGB (now Alliance for Clinical Trials in Oncology)/SWOG 80405, a phase III mCRC treatment trial. BMI was measured at trial registration. Primary and secondary endpoints were overall and progression-free survival, respectively. To minimize confounding by poor and rapidly declining health, we used Cox proportional hazards regression to adjust for known prognostic factors, comorbidities, physical activity, and weight loss, and excluded individuals with low BMI <21 from statistical tests for trend. We also examined self-reported weight loss over the six months prior to trial enrollment as an independent predictor of patient outcome.

Results: In this sub-study of a phase III mCRC trial, BMI was recorded for all 2,323 patients enrolled. Following adjustment for confounders, there were no significant associations between BMI and overall or progression-free survival (excluding BMI <21, Ptrend with increasing BMI = 0.12 and 0.42, respectively). Conversely, weight loss prior to treatment was associated with shorter overall and progression-free survival; compared to individuals with weight change <5%, individuals with weight loss >20% experienced an adjusted hazard ratio of 1.46 for all-cause mortality (95% confidence interval 1.15 to 1.85, Ptrend <.0001) and of 1.27 for disease progression or death (95% confidence interval 1.04 to 1.56, Ptrend = .009).

Conclusion: In this prospective study of patients with mCRC, BMI at time of first-line chemotherapy initiation was not associated with patient outcome. Weight loss prior to study entry was associated with increased risk of patient mortality and disease progression.

#3286

Breast cancer risk factors and survival by tumor subtypes: A pooled analysis from the breast cancer association consortium studies.

Anna Morra,1 Audrey Y. Jung,2 Sabine Behrens,2 Rose Yang,3 Heather Eliassen,4 Michelle Holmes,4 Montserrat Garcia-Closas,3 Marjanka K. Schmidt,1 Jenny Chang-Claude,2 on behalf of the Breast Cancer Association Consortium. 1 _Netherlands Cancer Institute, Amsterdam, Netherlands;_ 2 _German Cancer Research Center (DKFZ), Heidelberg, Germany;_ 3 _National Cancer Institute, Rockville, MD;_ 4 _Harvard Medical School, Boston, MD_.

Background: Breast cancer (BC) is a heterogeneous disease with differing risk factors, prognosis and response to treatment strategies. Lifestyle and personal factors have been shown to influence BC prognosis. It is unknown whether lifestyle factors are differentially associated with prognosis according to BC subtypes.

Methods: Analyses were based on 102,823 BC patients from 58 studies participating in the Breast Cancer Association Consortium. Cox regression models were used to assess associations between prediagnosis lifestyle and reproductive BC risk factors—including age at menarche, age at first full-term pregnancy, parity, breastfeeding, time since last full-term birth, BMI, oral contraceptive (OC) use, use of estrogen and progesterone therapy (EPT) and cigarette smoking- and 10-year overall survival (OS) and BC survival in all patients and by tumor subtypes. We used two tumor classifications: by estrogen receptor (ER) status; and by invasive subtypes based on ER, progesterone receptor, the human epidermal growth factor receptor 2 (HER2), and grade. Multiple imputation of risk factors and clinico-pathological variables was performed simultaneously. All analyses were stratified by study and adjusted for age at diagnosis, tumor size, nodal status, tumor grade (except for the luminal B HER2- subgroup), and systemic treatment. Multiple testing was accounted for using Bonferroni correction.

Results: There were a total of 13,953 total deaths and 6,893 BC deaths over 10 years. The most significant associations were observed between OS and the following risk factors: parous vs nulliparous (HR (95%CI): 0.84 (0.78,0.89), P=1.4E-07); OC use vs never use (0.82 (0.78,0.87), P=1.2E-11); EPT use vs never use (0.64 (0.58,0.70), P=1.5E-19); BMI > 30 kg/m2 vs 18.5-25.0 kg/m2 in postmenopausal women (1.25 (1.17,1.34), P=1.9E-10 and 1.34 (1.18, 1.53), P=4.6E-06 in premenopausal women); cigarette smoking vs nonsmokers (1.37 (1.28,1.47), P=2.1E-19); time since last full-term birth < 5 years vs ≥ 10 years (2.09 (1.78, 2.44), P=1.3E-19). Associations with BC survival were similar to those for OS but weaker. We observed no differential associations by tumor subtype. The strongest evidence of subtype differential effects was for time since last full-term birth < 5 years vs ≥ 10 years for luminal A (3.00 (2.25,3.98)) vs luminal B-HER2- (1.69 (1.27,2.24)), HER2+ enriched (1.40 (0.96,2.05)), and triple negative (1.49 (1.15,1.93)), respectively. Multivariable models are being constructed to account for the interplay between risk factors.

Conclusion: Our results suggest that BC risk factors influence survival after diagnosis. The effect appears to be stronger when OS is considered as endpoint, which may be due to power or associations of BC risk factors with other diseases. The data did not support a strong differential effect of BC risk factors on prognosis by tumor subtype.

#3287

Survival disparities for second primary malignancies diagnosed among childhood cancer survivors: A population-based assessment.

Austin L. Brown, Vidal M. Arroyo, Jennifer Agrusa, Michael E. Scheurer, Maria M. Gramatges, Philip J. Lupo. _Baylor College of Medicine, Houston, TX_.

Background: Curative therapy places childhood cancer survivors at increased risk of second primary malignancies (SPMs). However, there have been few population-based attempts to characterize differences in outcomes between SPMs in childhood cancer survivors and comparable de novo first primary malignancies (FPMs).

Methods: We extracted clinical and demographic information from childhood cancer survivors who developed SPMs and from individuals with comparable de novo FPMs using the Surveillance, Epidemiology, and End Results (SEER) 1973-2015 database. Hazard ratios (HR) and 95% confidence intervals (CI) were estimated with Cox proportional hazards models comparing overall survival (OS) between individuals with and without a history of childhood cancer. OS was evaluated overall and within specific cancers diagnosed in ≥50 childhood cancer survivors. Models accounted for potential confounders, including sex, race, age, decade of diagnosis, histology, and disease stage.

Results: Compared to individuals with FPMs (n=1,332,203), childhood cancer survivor with a similar SPM as the FPMs (n=1,409) experienced poorer OS (HR=1.86, 95%CI: 1.72-2.02) after accounting for age, sex, race, and decade of diagnosis. Estimated five-year overall survival for SPMs diagnosed in survivors of childhood cancer was 61.7% (95% CI: 58.9-64.4), compared to 77.4% (95% CI: 75.0-79.5) among de novo FPMs propensity score-matched on confounding factors. A history of childhood cancer remained a poor prognostic factor for all specific cancer evaluated, including: breast (HR=2.07, 95%CI: 1.63-2.62), thyroid (HR=3.59, 95%CI: 2.08-6.19), acute myeloid leukemia (HR=2.38, 95%CI:1.87-3.05), brain (HR=2.09, 95%CI:1.72-2.55), melanoma (HR=2.57, 95%CI: 1.55-4.27), bone (HR=1.88, 95%CI:1.37-2.57), and soft tissue sarcoma (HR=2.44, 95%CI: 1.78-3.33). Survival disparities were most pronounced in survivors diagnosed with a childhood cancer before age 10 (HR=2.83, 95%CI: 2.46-3.25), diagnosed with a SPM within 10 years of the childhood cancer (HR=2.61, 95%CI: 2.29-2.99), and exposed to radiotherapy during childhood (HR=2.13, 95%CI: 1.92-2.37).

Conclusion: Compared to individuals without a prior cancer diagnosis, survivors of childhood cancer with a SPM experience inferior outcomes. These survival disparities persist across cancer types, therapeutic exposures, and clinical factors.

#3288

Ovarian cancer survivors' views of factors that influenced their exceptional survival: A qualitative study.

Aliya Alimujiang,1 Lilah Khoja,1 Ashley Wiensch,1 Malcolm C. Pike,2 Penelope Webb,3 Anne Chase,4 Jean Richardson,5 Celeste Leigh Pearce1. 1 _University of Michigan School of Public Health, Ann Arbor, MI;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 3 _QIMR Berghofer Medical Research Institute, Brisbane, Australia;_ 4 _Patient advocate, CA;_ 5 _University of Southern California, CA_.

Introduction Although most women with high-grade serous ovarian cancer (HGSC) die within five years, approximately 15% will survive for 10 or more years. The factors contributing to this exceptional survival are currently unknown, but are thought to be a complex interaction between clinical, genetic, immunologic, and lifestyle factors. The purpose of this study is to qualitatively explore factors that may influence exceptional ovarian cancer survival.

Methodological Approach Four focus groups, one each in Los Angeles (California), Ann Arbor (Michigan), New York (New York) and Edmonton (Alberta, Canada), were conducted. Women previously diagnosed with HGSC who have survived 5 or more years were invited to participate. Physical activity, diet, meditation, prayer, treatment, integrative medicine, and side effects were explored in the semi-structured focus group interviews. All four sessions were audiotaped and transcribed. The transcriptions were coded by two individuals using grounded theory. The coded transcripts were analyzed using Dedoose.

Findings Of the 26 women who participated, 24 were diagnosed with HGSC. Two women, one with a stage III, low-grade mucinous tumor and another with a MMMT carcinosarcoma, did not meet our inclusion criteria, but were not asked to leave. Among the 24 women with HGSC, 19 have survived 10 or more years, two 8-9 years and three 5-7 years; all but one woman had stage III or IV disease. Three overarching themes were uncovered: (a) Survivors were highly motivated to improve lifestyle factors, including but not limited to fitness and diet, and had, in fact, done so; (b) Survivors had a strong life purpose, which manifested as positivity, taking charge, and self-advocacy; and (c) Survivors were able to draw on strong support systems, which included family, friends, support groups, faith, and healthcare workers.

Conclusion HGSC long-term survivors have varying experiences with their cancer, but share motivation and persistence, strong life purpose, and strong support systems. This study sheds light on how the specific behaviors and attitudes of patients may contribute to their long-term survival with HGSC. Focus groups are preparatory to further prospective studies that will determine whether short term and long term survivors differ on these characteristics. Our results highlight the need for more research to gain better understanding of the role that life purpose and support systems play in survival with HGSC.

#3289

Time to diagnosis of second primary cancers among survivors of smoking-related malignancies.

Eric Adjei Boakye,1 Nosayaba Osazuwa-Peters,1 Min Jee Lee,2 Sabha Ganai,2 Maggie Wang,1 Matthew C. Simpson,1 Wiley D. Jenkins2. 1 _St. Louis Univ., Saint Louis, MO;_ 2 _Southern Illinois University School of Medicine, Springfield, IL_.

Background: Despite steady declines in cigarette smoking prevalence, approximately one-third of cancer deaths in the United States are associated with smoking. This growing population of cancer survivors is at increased risk of developing second primary cancers (SPCs). We explored the incidence of SPC by latency period among survivors of the most commonly diagnosed smoking-related malignancies.

Methods: The current study utilized Surveillance, Epidemiology, and End Results data (2000-2015) concerning diagnoses with a primary malignancy from 10 smoking-related cancer sites (lung and bronchus, head and neck, esophagus, urinary bladder, liver, stomach, kidney and renal pelvis, acute myeloid leukemia, uterine cervix, and colorectal). SPC was defined as the first subsequent primary cancer occurring at least 2 months after first cancer diagnosis. SPC risks were quantified using standardized incidence ratios (SIRs) stratified by latency period (<1 year, 1-5 years, 5-10 years, 10+ years).

Results: A cohort of 1,895,307 patients was identified and 132,635 (7.0%) developed SPC. Overall risk of SPC for all primary smoking-related malignancy was significantly elevated for all latency periods compared with the general population. Almost all smoking-related cancer sites presented with a significant increase in the risk of SPC relative to the general population (SIR range 1.09-3.38) within the first 5 years (with the exception of acute myeloid leukemia cancer) and individual cancer risk declined with increasing latency (Table).

Conclusion: We found that 1-in-12 survivors of smoking-related primary malignancies developed an SPC. For most such malignancies, the risk of SPC was higher within 5 years after the primary diagnosis and declined with increasing latency. As cancer survivors transition back to primary care management in the years following cancer cure/remission, a better understanding of SPC risk and associated factors is needed to inform surveillance and prevention guidelines.

Risk of second primary cancers for smoking-related malignancies

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<1 year | |

1-5 years | |

5-10 years | |

10+ years

|

Index smoking-related cancer | Observed SPC | SIR (95% CI) | Observed SPC | SIR (95% CI) | Observed SPC | SIR (95% CI) | Observed SPC | SIR (95% CI)

All smoking-related cancers | 30,652 | 1.81 (1.79, 1.83) | 62,587 | 1.45 (1.44, 1.46) | 31,205 | 1.35 (1.33, 1.36) | 8,191 | 1.23 (1.20, 1.26)

Lung and bronchus | 5,707 | 1.26 (1.23, 1.29) | 11,360 | 1.59 (1.57, 1.62) | 5,205 | 2.09 (2.03, 2.14) | 1,106 | 2.03 (1.91, 2.15)

Urinary bladder | 9,204 | 3.38 (3.31, 3.45) | 13,361 | 1.58 (1.55, 1.61) | 6,245 | 1.30 (1.27, 1.33) | 1,586 | 1.17 (1.11, 1.23)

Head and neck | 3,130 | 2.01 (1.94, 2.08) | 8,953 | 2.00 (1.96, 2.04) | 4,994 | 1.98 (1.93, 2.04) | 1,308 | 1.78 (1.69, 1.88)

Kidney & renal pelvis | 3,487 | 2.49 (2.41, 2.58) | 6,299 | 1.42 (1.39, 1.46) | 3,243 | 1.26 (1.22, 1.30) | 818 | 1.16 (1.09, 1.25)

Uterine cervix | 483 | 2.20 (2.01, 2.41) | 1,066 | 1.54 (1.45, 1.65) | 673 | 1.29 (1.19, 1.39) | 262 | 1.17 (1.04, 1.32)

Colon & rectum | 6,808 | 1.39 (1.36, 1.43) | 18,003 | 1.18 (1.16, 1.20) | 9,445 | 1.03 (1.01, 1.06) | 2,765 | 0.99 (0.95, 1.02)

Liver | 336 | 1.24 (1.11, 1.38) | 543 | 1.23 (1.13, 1.34) | 156 | 1.06 (0.90, 1.24) | 57 | 1.09 (0.82, 1.41)

Esophagus | 435 | 1.12 (1.02, 1.23) | 824 | 1.39 (1.29, 1.49) | 336 | 1.48 (1.33, 1.65) | 75 | 1.29 (1.02, 1.61)

Stomach | 679 | 1.09 (1.01, 1.17) | 1,555 | 1.26 (1.20, 1.33) | 657 | 1.17 (1.08, 1.26) | 166 | 1.14 (0.97, 1.33)

Acute myeloid leukemia | 172 | 1.11 (0.95, 1.29) | 267 | 1.14 (1.01, 1.28) | 124 | 1.09 (0.90, 1.30) | 50 | 1.27 (0.94, 1.67)

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#3290

Risk factors for post-radiotherapy pain in a multi-racial ethnic population of breast cancer patients: a prospective study.

Eunkyung Lee,1 Shannon Snyder,1 Edward Ip,2 Jean L. Wright,3 Jennifer J. Hu4. 1 _University of Central Florida, Orlando, FL;_ 2 _Wake Forest School of Medicine, Winston-Salem, NC;_ 3 _Johns Hopkins University, Baltimore, MD;_ 4 _University of Mimai, Miami, FL_.

Cancer and/or treatment-related pain is a critical quality of life issue for cancer survivors. In a prospective longitudinal study of 1,000 breast cancer patients (enrolled during 2011-2013), we examined risk factors for pain among women undergoing adjuvant radiotherapy (RT). A pain score was assessed as a mean of four pain severity items (i.e., pain at its worst, least, average, and now) from the Brief Pain Inventory with an 11-point numeric rating scale (0-10) at baseline and post-RT. The study sample consists of 623 whites, 280 black/African Americans, 64 Hispanic whites, and 33 others. The mean age was 58.1 ±10.8 years and the mean body mass index (BMI) was 30.7 ±7.0 kg/m2. The mean pain score was 1.5 ±1.0 (baseline) and 2.5 ±1.8 (post-RT). In multivariable linear regression analysis (n= 863), post-RT pain score was significantly associated with baseline pain score (β= 0.39 per 1 point increment, p< 0.0001), age (β= -0.03 per 1 year increment, p< 0.001), BMI (β= 0.03 per 1 kg/m2 increment, p= 0.004), current smoking status (β= 0.57, p= 0.022), conventional RT fractionation (β= 0.48, p= 0.033), and pain medication use (β= 1.85, p< 0.0001). Further stratified analysis showed that post-RT pain score was associated with baseline pain score, age, BMI, and mastectomy among patients who did not use pain medication (n= 625); however, it was associated with baseline pain score and conventional RT fractionation among patients who did use pain medication (n= 238). Altogether, baseline pain score is the most significant and robust risk factor for post-RT pain, and conventional RT fractionation is a significant risk factor post-RT pain only among those who experienced more pain. These risk factors may help identify individuals at-risk for post-RT pain; thus, this information may help guide these high-risk individuals in their RT decision-making processes, such as considering a hypo-fractionated RT schedule.

#3291

Non-alcoholic fatty liver disease and increased risk of mortality in NHANES III: Results after 27 years follow-up.

Christian S. Alvarez, Barry I. Graubard, Jake E. Thistle, Jessica L. Petrick, Katherine A. McGlynn. _NCI, Rockville, MD_.

Background: Non-alcoholic fatty liver disease (NAFLD) is the most common liver disorder in the US. It encompasses a range of conditions from hepatic steatosis to cirrhosis and liver cancer. Prior studies of the association between NAFLD and mortality have been limited by fairly short follow-up times, proxy measures of NAFLD and relatively few outcome events. Thus, the current study examined the association of NAFLD with all-cause and cause-specific mortality in the Third National Health and Nutrition Examination Survey (NHANES III) conducted in 1988-1994, with mortality follow-up through 2015.

Methods: The analysis included 12605 individuals aged 20-74 years who underwent a hepatic/gallbladder ultrasound examination in NHANES III. NAFLD was defined as mild to severe hepatic steatosis detected by ultrasound in the absence of high alcohol consumption. Individuals were followed up for mortality by linkage to the National Death Index. Cox proportional hazard models were used to estimate the hazard ratios (HR) and 95% confidence intervals (CI) for all-cause and cause-specific mortality by NAFLD status as well as by a fibrosis score after adjustment for age, gender, race/ethnicity, education, physical activity, smoking, moderate alcohol consumption and body mass index.

Results: The prevalence of NAFLD in the study was 33% and a total of 3,843 deaths occurred. More than 50% of the deaths were due to either cardiovascular disease (CVD) (34.2%) or cancer (18.7%). In addition, there were 83 (2.2%) deaths related to liver disease, including liver cancer. The risks of all-cause and cause-specific mortality were higher among persons with NAFLD than among persons without NAFLD: all-cause mortality [HR:1.22, 95%CI:1.10,1.36]; CVD [HR:1.12, 95%CI:0.96,1.31]; cancer [HR:1.34, 95%CI:1.04,1.72]; liver disease [HR:3.03, 95%CI:1.58,5.82]; kidney disease [HR:2.22, 95%CI:1.09,4.51]; diabetes [HR:2.55, 95%CI:1.48,4.39]. The risks of mortality from all-causes [HR:1.52, 95%CI:1.11,2.07], and from liver disease [HR:6.08, 95%CI:1.79,20.66] were markedly higher among persons with NAFLD with elevated liver enzymes than among persons without NAFLD. Among persons with NAFLD, a higher liver fibrosis score was significantly associated with increased risks of all-cause and liver disease mortality compared to those with lower fibrosis score: [HR:1.59, 95%CI:1.09,2.31] and [HR:17.08, 95%CI:4.08,71.56], respectively.

Conclusions: Persons with NAFLD had an increased risk of all-cause and certain types of cause-specific mortality, independent of sociodemographic and lifestyle risk factors after 27 years of follow-up. Elevated liver enzymes as well as a higher fibrosis score among persons with NAFLD further increased the risk of all-cause and liver disease mortality. Persons with NAFLD should be closely monitored to prevent disease progression and reduce the risk of mortality in the US population.

#3292

Prevalence and socio-behavioral determinants of psychological distress among cancer patients and survivors in the United States, 2010-2015.

Gopal K. Singh,1 Lihua Liu,2 Audrey L. Chai,2 Alexander Ung,2 Michelle Allender1. 1 _U.S. Department of Health & Human Services, Rockville, MD; _2 _Univ. of Southern California, Los Angeles, CA_.

Background: Cancer patients face many unique physical and mental health challenges that directly affect their cancer outcome and quality of life. As cancer treatment improves, the prevalence of long-term cancer survivors increases. However, the psychological well-being of cancer survivors has been largely understudied, which may lead to missed opportunities to improve cancer survivorship.

Methods: Using 2010-2015 National Health Interview Surveys (N=194,757), we examined the prevalence of psychological distress among 17,657 adult cancer patients and survivors and identified the socio-demographic and behavioral correlates in the United States. Differentials in serious psychological distress (SPD, defined by a 6-item scale) and factor-based psychological distress scores (a composite index with a mean of 100 to measure levels of psychological distress) were analyzed by multivariate linear and logistic regression models.

Results: The prevalence of SPD among cancer patients was 4.7% (95% CI: 4.2, 5.1), compared with 3.3% (95% CI: 3.1, 3.4) for the general population without a cancer history or diagnosis. The SPD prevalence was significantly higher among patients diagnosed with stomach, ovarian, cervical, and uterine cancers than the non-cancer population (age-adjusted odds ratio: 3.2, 3.1, 2.9, 2.7, respectively). Patients diagnosed with lung, colorectal, and thyroid cancer also reported significantly higher levels of psychological distress. SPD prevalence and distress index scores were substantially higher among American Indian/Alaska Native, Hispanic, and non-Hispanic (NH) Black cancer patients, compared with NH whites with cancer. Prevalence and levels of psychological distress were significantly greater among cancer patients with low education and income levels, non-professional occupations, unmarried status, functional limitation, and among smokers and physically inactive patients.

Conclusions: Our findings underscore the importance of addressing the psychological well-being of cancer patients and survivors, particularly among the high-risk groups, in order to achieve the best treatment outcome and quality of life among the increasing number of cancer survivors.

#3293

Serum and intraprostatic lipidome level shift during statin use among prostate cancer patients.

Paavo V. Raittinen,1 Kati Niemistö,2 Seppo Auriola,3 Pauliina Ilmonen,1 Teemu J. Murtola2. 1 _Aalto University, Espoo, Finland;_ 2 _University of Tampere, Tampere, Finland;_ 3 _University of Eastern Finland, Kuopio, Finland_.

Prostate cancer patients using cholesterol-lowering statins have 30 % lower risk of prostate cancer death compared to non-users. The effect is attributed to the inhibition of the mevalonate pathway which is active in prostate cancer cells. Statin intervention also causes lipidome level shift in the serum. However, it is unknown whether statin intervention also affects intraprostatic lipidome (IPL) as well.

We studied the lipidome level shift among Finnish males diagnosed with prostate cancer and scheduled for radical prostatectomy in a randomized, placebo-controlled double-blind clinical trial. Total number of participants was 86 where 41 were given placebo and 45 were treated daily with 80 mg of atorvastatin (AS) for a median of 27 days preceeding the surgery. The serum lipidome (SL) level was measured before and after the intervention using mass spectrometer, whereas the IPL level was measured after the surgery. SL contains 213 lipid aggregates, while the IPL contains 4,652 single-molecule lipids. Furthermore, we investigated if the baseline lipidome level or the shift during intervention displays relationship with tumor Gleason grade, PSA level, Ki67 proliferation marker level in the tissue, and / or intraprostatic inflammation.

The relationship was studied using supervised random forest classification (RFC), and linear regression (LR) adjusted for age and body mass index. RFC robustness with respect to the intrinsic randomization was measured by repeating RFC 100 times. The 4,652 IPL molecules were reduced to 101 after limiting analysis to ones demonstrating statistically significant difference between placebo and AS group. The statistically significant difference was tested with t-test and Mann-Whitney test depending on the sample distribution.

The SL difference before-after intervention separates the two groups with extremely low RFC error of 8.1 – 9.3 %. This indicates that the intervention has systematically altered the SL. The RFC error for the IPL was 29 – 37.7 % and does not separate the placebo and AS group as well as in the serum. Thus, the AS effect on IPL is not as strong as in the serum, although a suggestion of differing lipid profiles by treatment arm was observed. One serum lipid, Glycoproteinacetylsmainlya1acidglycoprotein (GP), did show a clear linear relationship with the PSA level change; however, it was independent of the AS intervention. Baseline SL did not predict high-grade prostate cancer, Ki67 level, or inflammation level in general, according to RFC or LR.

AS intervention displays a clear effect on SL level, but only a modest effect on the IPL. Our finding suggests the AS affects the lipids in the prostate as well. GP serum lipid aggregate shows linear relationship with the PSA level change but it is independent of AS intervention. IPL does not correlate with the tumor Gleason grade or Ki-67 proliferation activity.

#3294

Urinary estrogen metabolites and long-term all-cause and cause-specific mortality following breast cancer diagnosis: A population-based study.

Tengteng Wang,1 Patrick B. Bradshaw,2 Sarah J. Nyante,1 Hazel B. Nichols,1 Patricia G. Moorman,3 Geoffrey C. Kabat,4 Susan L. Teitelbaum,5 Alfred I. Neugut,6 Marilie D. Gammon1. 1 _The University of North Carolina at Chapel Hill, Chapel Hill, NC;_ 2 _University of California, Berkeley, CA;_ 3 _Duke University, Durham, NC;_ 4 _16 Bon Air Ave., New Rochelle, NY;_ 5 _Icahn School of Medicine at Mount Sinai, New York, NY;_ 6 _Columbia University, New York, NY_.

Background: Estrogen metabolites play a role in breast cancer development. Previous studies have particularly focused on the two competing metabolism pathways which yield metabolites 2-hydroxyestrone (2-OHE1) and 16-hydroxyestrone (16-OHE1). 2-OHE1 has been shown to have antiestrogenic effects, but 16-OHE1 has strong estrogenic and even genotoxic activity. No study has investigated their biologically plausible role in predicting prognosis/mortality among women diagnosed with breast cancer.

Methods: In the Long Island Breast Study Project, spot urine samples were obtained from 687 women diagnosed with first primary breast cancer (shortly after diagnosis) in 1996-1997. Urinary concentrations of estrogen metabolites 2-OHE1 and 16-OHE1 were measured using enzyme linked immuno-assay. Vital status was determined by the National Death Index through December 31, 2014; 244 deaths (84 breast cancer-specific and 80 cardiovascular diseases-specific) were identified. We used multivariable-adjusted Cox proportional hazards regression model to estimate hazard ratios (HRs) and 95% confidence intervals (95% CIs) for all-cause, breast cancer and cardiovascular diseases mortality as related to the two individual metabolites and their ratio (2-OHE1/16-OHE1). Multiplicative interactions with menopausal hormone therapy, body mass index, menopausal status, and breast cancer treatments were evaluated with likelihood ratio tests.

Results: During a median follow-up of 18 years, urinary concentration of the 2-OHE1/16-OHE1 ratio (> median of 1.8 vs. ≤ median of 1.8) was associated with reduced risk of all-cause mortality (HR=0.74, 95% CI=0.56-0.98) among women with breast cancer. This inverse association with the 2-OHE1/16-OHE1 ratio was also observed for breast cancer mortality (HR=0.73, 95% CI=0.45-1.17) and cardiovascular diseases mortality (HR=0.76, 95% CI=0.47-1.23), although the 95%CIs included the null. The 2-OHE1/16-OHE1 ratio-mortality associations did not significantly differ by menopausal hormone therapy, body mass index, and menopausal status at the time of urine collection (Pinteration >0.05). Consistent patterns of association were not observed between the individual metabolites and mortality outcomes.

Conclusion: To our knowledge, our study represents the first population-based epidemiologic evidence suggesting that the urinary concentration of the 2-OHE1/16-OHE1 ratio measured shortly after breast cancer diagnosis may be associated with improved overall mortality for breast cancer survivors. Future investigation is necessary to confirm our findings and to further understand the underlying biological mechanisms for estrogen metabolism–mortality relationships following breast cancer diagnosis.

#3295

GPRASP1:A novel potential biomarker for neuroendocrine carcinoma.

Minggang Xiong,1 Fuhao Wang,1 Haiyen E. Zhau,2 Xin Huang,1 Leland Chung,2 Jian Zhang,1 Yi Lu1. 1 _Southern University of Science and Technology, Shenzhen, China;_ 2 _Cedars-Sinai Medical Center, Los Angeles, CA_.

Neuroendocrine differentiation has been observed to correlate with the progression and prognosis of prostate cancer (PCa), gastric cancer and pancreatic cancer, but the mechanisms remain unclear. GPRASP-1, a G protein coupled receptor-associated sorting protein 1, is known as a significant modulator of lysosomal sorting, and functional down-regulation of G- protein coupled receptors by interacting with Beclin2. The GPRASP-1 was originally considered as a serum tumor biomarker for breast cancer and later it was confirmed as a ubiquitous tumor marker among lung, liver and brain cancers. Based on its function as a tumor biomarker, we conducted serum ELISA analysis of GPRASP-1, and correlated results with RNAseq and gene copy numbers among a large cohort of PCa patients. We found overexpression and gene amplification of GPRASP-1 in 24 of 77 patients (31%) with neuroendocrine PCa (NEPCa). The patients with elevated serum GPRASP-1 level had a higher risk of metastasis to liver, lung and lymph nodes. Furthermore, immunohistochemical analysis for GPRASP-1 showed a strong staining of the pathological section from pancreatic neuroendocrine carcinoma patients and their liver metastases. Knocking down of the GPRASP-1 in a NEPCa PC3 cell line showed dramatic downregulation of TACR family, Tachykinin Receptor, which is known to be involved in the neuroinflammation and neurotransmitter transport and cancer proliferation. Our results offer a novel link among GPRASP-1, neuroendocrine differentiation and castration resistance in human PCa. We proposed serum GPRASP-1 can serve as a novel biomarker differentiating cancers with and without neuroendocrine differentiation. (Supported by NSFC projects 81773146; Guangdong Provincial Key Laboratory of Cell Microenvironment and Disease Research, 2017B030301018); JCYJ20170412152943794, JCYJ20170412154619484, JCYJ20170307105128101, JCYJ2017030711041760)

#3296

Tumor antigens Fetuin-A and Secreted Protein Acidic and Rich in Cysteine (SPARC) Autoantibodies as diagnostic and prognostic biomarkers in prostate cancer.

Shyh-Han Tan,1 Andy Martinez,1 Anshu Rastogi,1 Wei Huang,1 Sreedatta Banerjee,1 Lakshmi Ravindranath,1 Denise Young,1 Amina Ali,1 Indu Kohaar,1 Yongmei Chen,1 Jennifer Cullen,1 Gyorgy Petrovics,1 Albert Dobi,1 David G. McLeod,1 Jacob Kagan,2 Sudhir Srivastava,2 Isabell A. Sesterhenn,3 Inger L. Rosner,1 Shiv Srivastava,1 Alagarsamy Srinivasan1. 1 _Center for Prostate Disease Research, Uniformed Services University of the Health Sciences and the Walter Reed National Military Medical Center, Bethesda, MD;_ 2 _National Cancer Institute, Bethesda, MD;_ 3 _Joint Pathology Center, Silver Spring, MD_.

Introduction: Improvements in blood-based biomarkers for distinguishing between indolent and aggressive prostate cancer are critical in enhancing the management of the disease. To address this, we have focused on the quantification of autoantibodies (AAbs) against tumor antigens present in the sera of patients. We have selected SPARC and Fetuin-A (also known as Alpha 2-HS Glycoprotein [AHSG]) for analysis as they are shown to be highly expressed at late stages of prostate cancer. The objectives of this study are: 1) To measure AAbs against SPARC and Fetuin-A in the sera of prostate cancer patients; 2) To determine whether there is a correlation between levels of SPARC and Fetuin-A AAbs in serum, disease and race status.

Methods: Sera from prostate cancer patients and healthy controls were evaluated for AAbs against SPARC and Fetuin-A by using recombinant full-length proteins as substrates in an enzyme-linked immunosorbent assay (ELISA) assay. Sera from 117 Caucasian American (CA) n= and 111 African American (AA) prostate cancer patients with Gleason grades 6-10, and healthy controls (CA, n=52; AA, n=45) were analyzed in addition to sera from a biopsy cohort (n=99). The specificity of AAbs against the respective target proteins was confirmed by immunoblot analysis.

Results: SPARC AAbs were detected in the sera, with significantly lower levels in both CA (p<0.0001; AUC=0.80), and AA prostate cancer patients (p<0.0001; AUC=0.82), compared to healthy controls. AAbs against Fetuin-A were significantly lower in prostate cancer patients in comparison to controls (p<0.0001; AUC=0.96). The range of AAb reactivity to SPARC and Fetuin-A was similar in both CA and AA prostate cancer patients. The results from biopsy cohort showed lower SPARC AAbs in cancer positive (n = 49) in comparison to cancer negative (n = 42) cases, and healthy controls.

Conclusions: In this study, we established the presence of AAbs against SPARC in prostate cancer patient serum for the first time. More importantly, we observed highly significant differences between prostate cancer patient (low) and controls (high) sera, across different ethnic groups, similar to AAbs noted against Fetuin-A. These data support the further evaluation of SPARC and Fetuin-A AAbs as promising serum biomarkers for prostate cancer.

#3297

Prediagnostic circulating concentrations of vitamin D binding protein and survival among colorectal cancer patients.

Chen Yuan,1 Mingyang Song,2 Brian M. Wolpin,1 Jeffrey A. Meyerhardt,1 Shuji Ogino,1 Bruce W. Hollis,3 Andrew T. Chan,4 Charles S. Fuchs,5 Kana Wu,2 Molin Wang,2 Stephanie A. Smith-Warner,2 Edward L. Giovannucci,2 Kimmie Ng1. 1 _Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA;_ 2 _Harvard T.H. Chan School of Public Health, Boston, MA;_ 3 _Medical University of South Carolina, Charleston, SC;_ 4 _Massachusetts General Hospital and Harvard Medical School, Boston, MA;_ 5 _Yale Cancer Center, New Haven, CT_.

Higher total 25-hydroxyvitamin D [25(OH)D] levels are associated with an improvement in survival among colorectal cancer (CRC) patients, but the relationships between plasma vitamin D binding protein (VDBP), bioavailable or free 25(OH)D, and CRC survival remain unknown. In two prospective cohort studies, the Health Professionals Follow-Up Study and the Nurses' Health Study, we examined the association between prediagnostic plasma levels of VDBP, bioavailable 25(OH)D, and free 25(OH)D and survival among 604 participants diagnosed with CRC between 1991 and 2011. Plasma 25(OH)D and VDBP were directly measured, while bioavailable and free 25(OH)D were calculated using a validated formula based on total 25(OH)D, VDBP, and albumin levels. Cox proportional hazards models were used to estimate hazard ratios (HRs) for overall and CRC-specific mortality, adjusted for other prognostic markers and potential confounders. During the follow-up, there were 279 deaths, 177 of which were due to CRC (63%). Higher VDBP levels were associated with a significant improvement in overall and CRC-specific survival (Ptrend=0.005 and 0.02, respectively). Compared to patients in the lowest quartile, those in the highest quartile of VDBP had a multivariable-adjusted HR of 0.61 (95% confidence interval [CI], 0.42-0.89) for overall mortality and 0.56 (95% CI, 0.35-0.92) for CRC-specific mortality. The results remained similar after further adjustment for total 25(OH)D levels. In contrast, no association with overall or CRC-specific mortality was observed for bioavailable or free 25(OH)D levels. In conclusion, higher prediagnostic plasma VDBP levels were associated with improved survival among CRC patients. The clinical utility of VDBP as a prognostic marker warrants further exploration, as well as research into underlying mechanisms of action.

#3298

Oncotype DX recurrence score: Implications for disparities in receipt of chemotherapy and breast cancer-specific mortality in Georgia.

Lindsay J. Collin,1 Brittany Crawford,2 Ming Yan,1 Renjian Jiang,1 Kevin Ward,1 Mylin Torres,3 Keerthi Gogineni,3 Preeti Subhedar,3 Lauren E. McCullough1. 1 _Emory University, Atlanta, GA;_ 2 _University of South Carolina, Columbia, SC;_ 3 _Emory University School of Medicine, Atlanta, GA_.

Background: Among women diagnosed with stage I-IIIa, node negative or positive (1-3), hormone receptor positive (HR+) and HER2 negative breast cancer (BC), the Oncotype DX recurrence score is used to guide chemotherapy treatment decisions. There is limited evidence on differences in the distribution of recurrence scores among non-Hispanic Black women (NHB), and whether this could contribute to the well-documented racial disparities in BC-specific mortality. The objective of this study was to evaluate the role of Oncotype DX recurrence score on racial disparities in BC-specific mortality between black and white women in Georgia.

Methods: In this study, patients were identified from the Georgia Cancer Registry. We included 4562 non-Hispanic White (NHW) and 1353 NHB women with an initial diagnosis of stage I-III HR+ breast cancer in Georgia (2010-2014), with a corresponding Oncotype DX recurrence score from Genomic Health, Inc. Logistic regression was used to estimate the odds of chemotherapy receipt between NHB and NHW women by Oncotype DX recurrence score (low [<18], medium [18-30], and high [≥31]). Cox proportional hazard regression was used to calculate the hazard ratios (HRs) and 95% confidence intervals (CIs) comparing BC-specific mortality rates by both race and recurrence score.

Results: Compared with NHW breast cancer patients, NHB women were more likely to be classified with a high recurrence score (11% vs. 7%), and less likely to be classified with a low risk of recurrence (53% vs. 59%). In the age-adjusted models, NHB women with a high recurrence score were less likely to receive chemotherapy (OR=0.60, 95%CI 0.35, 1.04), however there was no difference in receipt of chemotherapy between NHB and NHW women with low or medium recurrence risk scores. After adjusting for age, we observed that NHB women with a low recurrence score had 3.05 times the hazard of BC-specifc mortality (95%CI 1.29, 7.04) compared to NHW women. Among women with a medium or high recurrence score we did not observe a racial disparity in BC mortality (HRhigh vs. low=1.49, 95%CI 0.51, 4.37).

Conclusion: Our results indicate variation in receipt of chemotherapy by race, particularly among NHB women with a high Oncotype DX score. However, we observed the most pronounced racial disparity in BC-specific mortality among women with low Oncotype Dx recurrence scores. These findings should be replicated in larger studies with robust numbers of NHB women. Additional research is needed to understand differences in chemotherapy treatment decisions and validation of the Oncotype DX recurrence score cut points in diverse populations.

#3299

Socio-demographic and lifestyle factors associated with the neutrophil-to-lymphocyte ratio: A systematic evaluation of the United States National Health and Nutrition Examination Survey.

Rachel Howard,1 Aaron Scheiner,2 Kathleen M. Egan1. 1 _H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL; _2 _Rutgers University, New Brunswick, NJ_.

Background:

The neutrophil-to-lymphocyte ratio (NLR) is an established marker of subclinical inflammation, and a high NLR is associated with poorer clinical outcomes in cancer patients. Despite this, factors that influence the magnitude of NLR independently of disease are poorly understood. Here, we systematically assess the influence of socio-demographic and lifestyle factors on NLR to identify a) potential confounders and effect modifiers in the relationship between NLR and cancer outcomes, and b) possible targets for intervention in patients with high NLR at diagnosis.

Methods:

Based on a total participant cohort of 48,023 adults with available NLR data, we identified 20 factors of interest relating to patient demographics, socioeconomic status and lifestyle that were included in the National Health and Nutrition Examination Survey (NHANES) between 1999 and 2016. Multivariable regression analysis identified factors significantly associated with the magnitude of NLR after adjusting for comorbidities and medications. Interactions between factors of interest were evaluated, and effect modification by demographic characteristics including age, sex and race was quantified.

Results:

Seven demographic and lifestyle factors were found to be significantly and independently associated with NLR: age, sex, race, marital status, physical activity, smoking history and alcohol use. Females, under 60s and non-Hispanic blacks demonstrated significantly lower NLR than males, over 60s and non-Hispanic whites. Participation in vigorous activity for at least ten minutes at a time was associated with decreased NLR, as was never-smoker status. Moderate drinking was also associated with a decreased NLR as compared to complete abstinence from alcohol.

Conclusions:

Multiple demographic and lifestyle factors are independently associated with elevated NLR. For an accurate assessment of the prognostic power of the NLR in the clinical setting, these factors should be routinely adjusted for in studies of the association between NLR and cancer outcomes. The present study reiterates that physical activity may have the potential to improve clinical outcomes by way of reducing systemic inflammation.

#3300

Pre-diagnosis neutrophil-to-lymphocyte ratio and lung cancer mortality.

Laurie Grieshober,1 Stefan Graw,2 Matt J. Barnett,3 Mark D. Thornquist,3 Gary E. Goodman,3 Chu Chen,3 Devin Koestler,2 Carmen Marsit,4 Jennifer Doherty1. 1 _Huntsman Cancer Institute, Salt Lake City, UT;_ 2 _University of Kansas Medical Center, Kansas City, KS;_ 3 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 4 _Emory University, Rollins School of Public Health, Atlanta, GA_.

The neutrophil-to-lymphocyte ratio (NLR) is a marker of systemic inflammation that has been reported to be associated with smoking status as well as survival outcomes from chronic diseases including lung cancer. Most prior studies have examined NLR measured in blood collected at diagnosis, which may therefore reflect disease-related inflammation. Although blood cell type counts, and therefore NLR, cannot be quantified in stored samples, algorithms have been developed to estimate blood cell type counts based on lineage-specific DNA methylation patterns across the genome as methylation-derived NLR (mdNLR). We hypothesize that the inflammatory profile reflected by pre-diagnosis mdNLR may be associated with lung cancer mortality. To test this hypothesis, we examined mdNLR and lung cancer-specific mortality, overall and by histotype, among 293 cases from the Beta Carotene and Retinol Efficacy Trial (CARET) of heavy smokers (≥20 pack years). We used the ratio of predicted neutrophil and lymphocyte proportions derived from DNA methylation signatures in whole blood samples collected on average 4.1 years prior to diagnosis, to estimate mdNLR, which was discretized as quartiles ranging from lowest to highest; i.e. low to high levels of systemic inflammation. We fit Cox proportional hazards models adjusted for age, sex, race (white vs non-white), smoking status, intervention arm, asbestos exposure, and pack years smoked to examine the association between mdNLR lung cancer-specific mortality. We also adjusted for time between blood draw and diagnosis and included early versus late stage as a stratification variable. mdNLR was not associated with mortality for all cases combined, nor squamous cell carcinoma (N=100) or small cell lung cancer (N=59). In contrast, for adenocarcinoma (N=122), we observed a statistically significant linear trend (p=0.02) for increasing quartiles of mdNLR and adenocarcinoma-specific mortality, with a hazard ratio (HR) of 2.03 (95% Confidence Interval (CI): 1.03-4.03) comparing the highest (Q4) to lowest (Q1) mdNLR quartiles. Though subgroup analyses were sparse, we observed associations of larger magnitude (Q4 compared to Q1) for those who were younger at blood draw (HR=4.70, 95% CI: 1.73-12.76) and at diagnosis (HR=6.09, 95% CI: 1.98-18.72), as well as those with smoking histories lower than the median of 53 pack years (HR=2.91, 95% CI: 1.03-8.22) and those in the active intervention arm (HR=3.32, 95% CI: 1.11-9.93). Our findings suggest that an inflammatory response prior to diagnosis as indicated by higher mdNLR levels may be associated with mortality in heavy smokers who go on to develop lung adenocarcinoma. Since indicators of survival may be used to guide treatment decisions and gauge response to treatment, further research into the association between mdNLR and lung adenocarcinoma-specific survival is needed to understand its potential clinical utility.

#3301

Predictors of disease progression and treatment response among lung cancer patients treated with immunotherapy.

Matthew B. Schabath, Ilke Tunali, Jhanelle E. Gray, Robert J. Gillies. _H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL_.

Background: Immune-checkpoint blockades provide durable responses and improved long-term survival in a subset of advanced non-small-cell lung cancer (NSCLC) patients. However, predictive markers of response to immune-checkpoint blockades are a significant unmet clinical need. The objective of this study was to identify clinical predictors of disease progression and treatment response among NSCLC patients treated with immune-checkpoint blockades.

Methods: Pre-treatment/baseline predictors included patient demographics, clinical data, driver mutations, blood chemistry, and hematology data. Using stepwise backward elimination and Classification and Regression Tree (CART) analyses, parsimonious models were developed to identify the most predictor factors associated with progression-free survival (PFS) and hyperprogressive disease (HPD) defined as patients that exhibited a greater than two-fold increase in tumor growth rate in less than 2 months.

Results: This analysis included 228 NSCLC patients treated with single agent or double agent immunotherapies. Univariable analyses identified 15 covariates significantly associated with PFS which was reduced to 3 covariates by backward elimination. Among these 3 covariates, CART analyses revealed three patient subgroups based on those who had prior systemic treatment (PST) and baseline absolute neutrophil count (ANC). Patients without PST and low ANC had significantly (log-rank p-value < 0.001) improved 36-month PFS (22.8%; Hazard Ratio [HR] = 1.0) compared to patients without PST and high ANC (13.6% 36-month PFS; HR = 2.37) or patients with PST irrespective of ANC (11.3% 36-month PFS; HR = 2.18). For the HPD analyses, HPD patients had significantly worse survival compared to patients who had PD without HPD (median survival = 3.2 months vs. 8.4 and 0% 36-month PFS vs. 25.3%, respectively; p < 0.001). In multivariable analysis with HPD versus non-HPD as the dependent variable, the full model with two clinical covariates yielded an Area under Receiver Operating Characteristic (AUROC) of 0.783 whereas the parsimonious model included only Royal Marsden Hospital (RMH) prognostic score with an AUROC of 0.712.

Conclusion: Because of the complexity in objective immunotherapy response and acquired resistance, there is a pressing challenge to identify which patients are least likely to respond. The models identified in this study have potential important translational implications to identify highly vulnerable NSCLC patients that experience poor outcomes and hyperprogressive disease in the immunotherapy setting.

#3302

Cardiovascular and thromboembolic diseases as a cause of death in multiple myeloma patients: A population based study.

Muhammed Khaled Elfaituri,1 Sara Morsy,2 Amr Ehab El-Qushayri,3 Hazem Abdelkarem Faraj,1 Minh-Duc Nguyen Tran,4 Amr Ebied,5 Amr Ebied,5 Nguyen Tien Huy6. 1 _University of Tripoli, Tripoli, Libyan Arab Jamahiriya;_ 2 _Tanta University, Tanta, Egypt;_ 3 _Minia University,, Minia 61519, Egypt;_ 4 _Univesity of Medicine and Pharmacy at Ho Chi Minh City, Ho Chi Minh City 70000, Viet Nam;_ 5 _Egyptian National Blood Transfusion Services, Giza, Egypt;_ 6 _Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-852, Japan_.

Introduction: Multiple myelomas (MM) is characterized by the neoplastic proliferation of plasma cells producing a monoclonal immunoglobulin. Despite the remarkable improvement in survival rates with different treatment approaches; multiple myeloma patients are at higher risk of cardiovascular and thromboembolic events. Therefore, we aim to identify high-risk patients for cardiovascular mortality and provoke awareness among physicians for choosing appropriate therapy for better survival rates in those patients.

Methods Surveillance, Epidemiology, and End Results Program database used for this work. Kaplan Meier survival curves were constructed to compare the survival probabilities. Furthermore, Cox regression analysis was conducted to detect the most important factors determining the survival time. The significant variables were used to construct nomogram and its fit was assessed using c-index.

Results The cohort included 55991 multiple myeloma patients with 4510 deaths by cardiovascular and thromboembolic events. There was a significant difference in age between both groups (P-value < 0.001). There was a significant difference between both groups regarding the treatment choices, type of radiation, race and marital status. SEER database identified four diseases as a cause of death in multiple myeloma: Aortic aneurysm and dissection (n = 39), atherosclerosis (n = 88), cerebrovascular diseases (n = 1861) and diseases of the heart (n = 3799). Kaplan Meier survival curves revealed a significant difference in the survival probability between the multiple myeloma grades (P < 0.001). We found that hypertension with multiple myeloma has the best median survival (median =1, 95% CI (0.133,1) and the worst survival was in patients diagnosed with an aortic aneurysm and dissection (median =0.09, 95% CI (0.03,0.25). Cardiac causes of death had less survival than other reported causes of death. The log-rank test found significantly different median survival between different cardiac causes (p<0.0001). Cox proportional hazard ratio regression revealed that gender [Male (HR = 1.39, SE = 0.03, P-value = 0.005], age (HR = 1.07, SE = 0.0015, P-value = 0.005), marital status (HR = 0.81, SE = 0.064, P-value = 0.0014), grade and surgery were significant risk factors for death with thromboembolic and cardiovascular diseases. These variables were used in nomogram. The c-index of the nomogram was [0.72, 95%CI (0.71 - 0.73)]. The prediction error (OOB) of the tree was 0.10. We found that random forest model had less survival over time than cox regression.

Conclusion: The mortality from by cardiovascular and thromboembolic events in MM was associated with several factors. Our considerations and further investigations of them is needful. It may aid in identifying the patients who can get the uppermost benefits of cardiovascular and thromboembolic prophylaxis and treatment measures.

#3303

Development and initial validation of a prognostic score based on structured data from EHRs.

Daniel Backenroth, Josh D. Haimson, Neal J. Meropol, Shrujal S. Baxi. _Flatiron Health, New York, NY_.

Introduction

Real world data (RWD) is increasingly used to supplement clinical trial data. Prognostic scores (PS) such as ECOG performance status (ECOG) are important for matching patient populations and assessing treatment impact, yet such PS are often missing from patient records or are embedded in physician narratives. An accurate PS that does not rely on physician assessment or data entry would have broad utility. We therefore undertook development of a PS based solely on structured data from RWD across numerous cancer diagnoses (the structured PS). We then sought initial validation of the structured PS in patients treated with checkpoint inhibitors (CIT).

Methods

Using the Flatiron Health electronic health record-derived database, we applied LASSO regression to develop the structured PS to predict for overall survival (OS) after starting any first-line (1L) cancer therapy. Data included laboratory values and vital signs from between 30 days before and 15 days after start of treatment as well as age. The structured PS was developed from ~39K patients with multiple myeloma, chronic lymphocytic leukemia or one of 8 solid tumor malignancies. We evaluated the structured PS using concordance, i.e., how often the score correctly predicts the relative OS of two randomly selected patients, on ~24K patients, comparing to a baseline based on age and ECOG, when available. We then measured the association of OS with the structured PS at start of 1L CIT in patients with five tumor types where 1L CIT is common.

Results

The concordance of the structured PS was 66%-73%, on average 6% higher than that of age and ECOG.

Table 1 shows by cancer type # of patients and median OS from 1L CIT for quartiles of the structured PS (NR=not reached).

Conclusion

A structured PS can be an important pan-tumor variable in investigations of OS using RWD, including for patients treated with first-line CIT.

OS from 1L CIT by quartiles of structured PS

---

Disease | n | Q1 median (95% CI) in months | Q2 median (95% CI) in months | Q3 median (95% CI) in months | Q4 median (95% CI in months)

Head and neck | 370 | 4 (3-5) | 9 (7-12) | 14 (12-Inf) | NR (NR-Inf)

Melanoma | 1635 | 5 (4-6) | 20 (15-26) | 33 (22-Inf) | NR (NR-Inf)

Non-small cell lung cancer | 4171 | 4 (3-4) | 9 (8-10) | 14 (13-16) | 24 (21-29)

Renal cell carcinoma | 277 | 5 (3-7) | 23 (12-Inf) | 20 (13-Inf) | NR (28-Inf)

Urothelial cancer | 612 | 2 (2-3) | 6 (4-7) | 9 (7-13) | NR (15-Inf)

#3304

CNS metastases in prostate cancer: an analysis of SEER database.

Lin Mei, Taha Al-Juhaishi, Andrew Poklepovic, Asit Paul. _Virginia Commonwealth University, Richmond, VA_.

Metastases to central nervous system (CNS) from various solid tumors, such as lung cancer (40-50%), breast cancer (15-25%) and melanoma (5-20%), are relatively common and confer a poor prognosis. CNS metastases from prostate cancer are uncommon. Due to the rarity of this disease and lack of studies, little is known about the clinical and pathological characteristics as well as outcome of these patients. CNS metastases also have therapeutic implications in terms of targeting hypothalamic-pituitary axis for androgen deprivation as well as CNS penetration of systemic chemotherapy agents. The objective of this study is to analyze clinicopathologic features and outcome of prostate cancer patients with CNS metastases in Survival, Epidemiology, and End Result (SEER) database. Between 2010 and 2015, a total of 23,776 patients with metastatic prostate were identified. Among them, 23,468 cases were adenocarcinoma and 308 cases were prostate cancer with neuroendocrine differentiation. A number of variables, including age, race, prostate-specific antigen (PSA) level, Gleason score, and prior radiotherapy, site of metastases were extracted. Primary outcome were overall survival (OS) and disease specific survival (DSS). CNS metastases were identified in 208 (0.89%) and 11 (3.57%) patients with adenocarcinoma and neuroendocrine tumor, respectively. Majority of patients (56.2%) were 65 years or older. Median OS and DSS were 13 months and 14 months, respectively. In comparison to patients with lymph nodes metastases only (n=8,103), the median OS and DSS were 40 months and 58 months, respectively. In patients with bone dominant metastasis (n=14,407), the median OS and DSS were 27 months and 34 months, respectively. Multivariate analysis identified age was an adverse predictive factor in patients with CNS metastasis. Race, high Gleason score (≥9), high PSA level (≥98ng/ml) and prior radiotherapy were not associated with OS and DSS. In conclusion, CNS metastases are more common in neuroendocrine tumor than in adenocarcinoma of prostate, and it confers poor survival outcome irrespective of histological subtypes. OS and DSS were worse in patients with prostate cancer and CNS metastases compared to those with lymph node metastases only or bone-dominant metastases.

#3305

Cancer mortality in children surviving congenital heart surgery: A study from the pediatric cardiac care consortium.

Amanda S. Thomas,1 Logan Spector,2 Lazaros K. Kochilas3. 1 _Emory University School of Medicine, Atlanta, GA;_ 2 _University of Minnesota, Minneapolis, MN;_ 3 _Emory University School of Medicine and Childrens Healthcare of Atlanta, Atlanta, GA_.

Introduction: Children with congenital heart defects (CHD) are a unique population with abbreviated survival compared to the general population. Case-control studies have suggested an elevated risk of childhood cancer in children with CHD. This has not been evaluated in prospective studies nor has risk of cancer in adult survivors of CHD. This study describes mortality from cancer among survivors of CHD in the Pediatric Cardiac Care Consortium (PCCC).

Methods: We performed a retrospective cohort study of individuals who underwent surgery for CHD at 0-21 years of age in the PCCC during 1982-2003. Patients surviving their first CHD surgery (n= 35,998) were linked to the National Death Index (NDI) through 2014. Cancer mortality was compared to that in the general population using standardized mortality ratios (SMRs) matched on age, year, and sex.

Results: Seventy patients were identified as having an underlying cause of death as cancer and surviving a CHD operation in the PCCC (males, n=38). Of these, 17 (24.3%) had a diagnosis of Trisomy 21. Deaths occurred over a median follow-up of 8.81 years (IQR: 5.9-15.8) post hospital discharge with median age at death of 13.5 years (IQR: 7.0-20.0). Overall adjusted SMRs for deaths relating to cancer were 2.75 (95% CI: 2.11-3.40) and increased for those with a chromosomal abnormality (2.31 to 6.76). Decrease in mortality was observed the older the patient was at CHD surgery (neonate to adolescence: 5.03 to 1.65 respectively). Increased mortality in surgically severe CHDs (SMR: 4.75; 95% CI: 2.26-7.23) was seen.

Conclusions: The PCCC-NDI linked database leverages the largest cohort of children with CHD in the U.S. We found an increased risk of death from cancer, especially in those with chromosomal abnormalities. This may demonstrate an increased risk of cancer, but could indicate higher risk of death from cancer for children who previously had CHD. To resolve these possibilities we will link to cancer registries to obtain incident disease. | N (%) | SMR | 95% CI

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

OVERALL | 70 | 2.75 | (2.11-3.40)

Lesion Group

Left-to-Right Lesions

Impaired Systemic Flow

Impaired Pulmonary Flow

Complex Lesions

Anomalous Pulmonary Venous Return

Transposition Physiology

Other

Single Ventricle | 36 (51.4)

13 (18.5)

7 (10.0)

3 (4.3)

2 (2.9)

2 (2.9)

4 (5.7)

3 (4.3) | 2.82

2.71

2.52

2.07

5.92

2.74

2.38

3.74 | (1.90-3.75)

(1.23-4.18)

(0.65-4.39)

(0.0-4.42)

(0.0-14.12)

(0.0-6.55)

(0.05-4.71)

(0.0-7.98)

Age at Surgery

Neonate

Infant

Child

Adolescent | 12 (17.2)

17 (24.3)

22 (31.4)

19 (27.1) | 5.03

3.14

3.59

1.65 | (2.19-7.88)

(1.65-4.63)

(2.09-5.10)

(0.91-2.40)

Chromosomal Abnormality

Yes

No | 17 (24.3)

53 (75.7) | 6.76

2.31 | (3.55-9.97)

(1.69-2.94)

Severity

Moderate

Mild

Severe

N/A | 28 (40.0)

22 (31.4)

14 (20.0)

6 (8.6) | 2.99

2.21

4.75

1.91 | (1.88-4.10)

(1.29-3.13)

(2.26-7.23)

(0.38-3.43)

#3306

Prognostic value of clinical signs at admission in patients with primitive liver cancer at university hospital of Yopougon: Retrospective cohort study.

Djivede Renaud Jutor Tchibozo. _University Hospital of Cocody, ABIDJAN, Côte D'Ivoire_.

Introduction: The primitive liver cancer (PLC) is common in Ivory Coast and has a late diagnosis. The PLC has a bad prognosis and few studies have assessed the predictive role of the clinical signs at admission, namely abdominal pain (AD), edematous-ascitic syndrome (OSA), febrile ascites (AF), and Digestive hemorrhage (HD) on the prognosis of patients with PLC .

Objective: To determine the prognostic role of clinical signs in the admission of hospitalized patients for PLC at University Hospital of Yopougon.

Patients And Methods: A retrospective cohort study of 126 patients (average age : 48.2 years, men = 71.4%) hospitalized for PLC at University Hospital of Yopougon from 2012 to 2016 was conducted in the Medicine and Hepato-Gastroenterology Department. For each patient the socio-demographics, clinicals, biologicals and endoscopics data and survival time were collected. These parameters were analyzed by

Kaplan Meier methods and Cox proportional hazards with a significance set at 5%. The risk is expressed in terms of chance ratio (HR) with the confidence interval.

Results: The patients were admitted for DA (32.5%), SOA (50%), AF (7.1%) and HD (10.3). The median duration of follow-up was 0.28 months. The mortality rate was 38.9% (49 of 126 patients) during follow-up. It was 12.7% for AD, 16.7% for SOA, 4.8% for AF and 4.8% for HD. The median overall survival was 0.8 (95% CI: 0.5-1.4) months. It was 0.7 (95% CI: 0.5-2) months for AD, 1.1 (95% CI: 0.4-1.1) months for OSA, 0.3 (95% CI: 0.21-3.6) months for AF and 1.1 (95% CI: 0.6-16.2) months for HD. In multivariate analysis, the predictive factors for mortality were: male sex (HR = 2.12, 95% CI: 1.08-4.17, p = 0.03), AF (HR = 4.51; 95% CI: 1.30 - 15.70, p = 0.02) and the presence of esophageal varicose (HR = 3.43, 95% CI 1.78-6.62, p <0.0001).

Conclusion: Patients suffering from PLC have a higher risk of death due to the presence of febrile ascites or esophageal varicose on admission.

#3307

Real-world outcomes of first-line pembrolizumab monotherapy for PD-L1-positive (TPS ≥50%) metastatic non-small cell lung cancer (NSCLC).

Vamsidhar Velcheti,1 Sheenu Chandwani,2 Xin Chen,2 M. Catherine Pietanza,2 Thomas Burke2. 1 _New York University, Scarsdale, NY;_ 2 _Merck & Co., Inc., Branchburg, NJ_.

Introduction Pembrolizumab monotherapy was approved in October 2016 for first-line (1L) treatment of metastatic NSCLC with PD-L1 tumor proportion score (TPS) ≥50% based on KEYNOTE-024 (KN024) trial results. A subsequent trial, KN042, confirmed overall survival (OS) benefit with pembrolizumab monotherapy over platinum doublet chemotherapy in the same population. Our aim was to investigate effectiveness of 1L pembrolizumab monotherapy in real-world settings in the US for patients clinically similar to those in KN024 and KN042 with PD-L1 TPS ≥50%.

Methods This retrospective study used Flatiron Health's nationally representative database derived from electronic health record (EHR) data. We identified patients (≥18 years) with histologically confirmed stage IV NSCLC, PD-L1 TPS ≥50%, no documented EGFR/ALK aberration, and ECOG performance status (PS) of 0-1 who initiated 1L pembrolizumab monotherapy after December 1, 2016. Patients with <6-months follow-up or participating in clinical trials were excluded. Structured and unstructured data, curated via technology-enabled abstraction, were used to determine real-world tumor response (rwTR), real-world progression (rwP), which we used to determine real-world progression-free survival (rwPFS), and OS.

Results Of 186 eligible patients, 48% were male; the median age was 72 years (Table). Complete and partial tumor responses were recorded for 12 (6.5%) and 74 (39.8%) patients, respectively, with rwTR of 46.2% (95% CI 38.9-53.7%). Median rwPFS was 7.0 months; median OS was not reached (Table).

Conclusions In real-world oncology practice, patients prescribed 1L pembrolizumab monotherapy for PD-L1 TPS ≥50% metastatic NSCLC tended to be older and included fewer men than in clinical trials, even after limiting to key trial eligibility criteria. Benefits observed were consistent with phase 3 trial findings, supporting the effectiveness of pembrolizumab in real-world settings.

Real-world outcomes with 1L pembrolizumab monotherapy for PD-L1-positive metastatic NSCLC

---

|

Patients N=186

Characteristics

|

Age, median (range), yr | 72 (46-84)

Male sex, n (%) | 90 (48)

Positive smoking history, n (%) | 169 (91)

Squamous histology, n (%) | 46 (25)

ECOG PS 1, n (%) | 111 (60)

Brain metastases, n (%)† | 22 (12)

Outcomes

|

Follow-up, median (range), months* | 11.5 (6.0-17.9)

|

rwPFS | OS

Events, n (%) | 110 (59) | 62 (33)

Median (95% CI), months | 7.0 (5.6-8.4) | NR (15.1-NR)

Rate at 12 months, % (95% CI) | 34.6% (26.8-42.5) | 63.6% (55.2-70.9)

* Follow-up time from pembrolizumab initiation to data cutoff.

† No data available on pretreatment of brain metastases.

NR, not reached. Data cutoff: May 31, 2018

#3308

Impact of residency training programs on outcomes of tumor lysis syndrome: A study from national inpatient data.

Aakash Desai,1 Neel Patel,2 Hardik Sonani,3 Harsh Parmar,1 Aswanth Reddy1. 1 _University of Connecticut, Hartford, CT;_ 2 _Herbert Wetheim College of Medicine, Hartford, CT;_ 3 _Ichan School of Medicine Mount Sinai, New York, NY_.

Background: Limited evidence is available on the effect of residency training on outcomes of

tumor lysis syndrome (TLS)

Objective: Our study measured the difference in outcomes in terms of inpatient length of stay (LOS), cost and mortality in patients with TLS between teaching and non-teaching hospitals.

Design/Methods: We used the National Inpatient Sample (NIS) dataset, a national representative weighted sample of all US hospital discharges, from 2009 to 2014 to identify the diagnosis of TLS (ICD-9 code 277.88) in cancer patients. Data was matched and weighted for purpose of analysis. Hospitals were classified as teaching if they had one or more Accreditation Council for Graduate Medical Education (ACGME) approved residency programs, were a member of the Council of Teaching Hospitals (COTH) or had a ratio of full-time equivalent interns and residents to beds of .25 or higher. Categorical and continuous variables were tested using Chi-square test and Student t-test respectively. Multivariate hierarchical regression was used for the analysis of primary outcomes of interest.

Results: We identified a total of 12,362 cases of TLS from 2009 to 2014. Among them, 6116 (49.47%) were admitted to non-teaching and 6246 (50.52%) to teaching hospitals. Both samples were identical in terms of baseline patient demographics. Patients in both samples were largely comparable in terms of comorbidities (non-teaching vs teaching): Obesity (10.2% vs 9.5%, p=0.166), liver failure (5.7% vs 5.1%,p=0.176), renal failure (20.2% vs 20.8%,p=0.406), coagulopathy (33.7% vs 33%,p=0.387) except for hypertension (46% vs 47.9%, p=0.04) which was more significant in the teaching hospital population.

New onset renal failure requiring dialysis was found in 16.6% of the total population with equal distribution among both the samples (16.9% vs 16.3%, p=0.415). Seizures (1.7% vs 0.9%, p=<0.01) occurred more commonly in the non-teaching population while cardiac dysrhythmias (22.6% vs 27%, p<0.01) were significantly higher in the teaching population.

Even though both the populations were comparable, the mean LOS (≈11.04 days vs ≈14.67, p <0.01) and cost (35,130.86$ vs 40,967.65$, p<0.01) were significantly higher at teaching hospitals with mean difference of 3.63 days and 15,168.47$ respectively. However, the mortality rate was higher in teaching hospitals (22.8% vs 24.7%,p= 0.014), but did not reach statistical significance on regression analysis (OR: 1.096, p=0.345)

Conclusion: There is no overall mortality benefit for TLS patients treated at teaching hospitals despite having a significantly increased length of stay and cost.

With poor overall outcomes for TLS patients and with increasing use of anti-neoplastic agents, early treatment and involvement of specialists is recommended. Further prospective trials are needed to determine an ideal cost effective strategy to address this oncologic emergency.

#3309

Healthcare resource utilization in follicular lymphoma patients treated with maintenance rituximab in the Veterans Health Administration.

Ahmad S. Halwani, Catherine Li, Kelli M. Rasmussen, Vikas Patil, Christina Yong, Zachary Burningham, Brian C. Sauer. _University of Utah School Medicine, Salt Lake City, UT_.

OBJECTIVES: Examine healthcare resource utilization (HCRU) in follicular lymphoma (FL) patients treated with or without maintenance rituximab (MR).

METHODS: FL patients treated in the Veterans Health Administration (VHA) between 2006-2014 were identified by linking information from the VA Cancer Registry System (VACRS) and the Corporate Data Warehouse. HCRU was defined as the total number of outpatient visits (OPV), inpatient visits (IPV), and the number of emergency department visits (EDV) during follow-up time, which was approximately four years. To avoid immortal time bias, a prescription time distribution matching method was performed, followed by an inverse probability weighted regression analysis to account for variation in the follow-up period. The observational period was partitioned into four-yearly intervals. The observed HCRU value was weighted by the inverse of the probability of not being censored by the end of the yearly interval. A generalized linear model with Poisson distribution and log link was performed for each of the four intervals while adjusting for the covariates. Using a 95% bootstrap confidence interval, the change of HCRU was summed across the four-yearly intervals to obtain the cumulative change of HCRU between FL patients who received MR or did not receive MR.

RESULTS: From 2006-2014, 2,290 FL patients were diagnosed at VHA. After excluding patients with a prior cancer, documented grade 3b or stage I disease, 905 MR-eligible patients remained, of which 319 (35%) received MR and 586 (65%) did not receive MR. When compared with non-MR patients, MR patients were slightly more likely to have a mean increase in OPV (0.69%; CI: 0.44%-0.94%) and a mean decrease in IPV (14.83%; CI: 16.09%-13.58%). There was no significant change in the number of EDV between the MR treated and non-MR treated patients.

CONCLUSIONS: This nationwide real-world study suggests that on average FL patients treated with MR have fewer IPV and more OPV than non-MR patients.

#3310

Association between molecular characteristics of serrated polyps and subsequent advanced colorectal neoplasia.

Xinwei Hua,1 Polly Newcomb,1 Jessica Chubak,2 Rachel Malen,1 Rebecca Ziebell,2 Aruna Kamineni,2 Lee-Ching Zhu,2 Melissa Upton,3 Hana Newman,1 Sheetal Hardikar,4 Andrea Burnett-Hartman5. 1 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 2 _Kaiser Permanente Washington Health Research Institute, Seattle, WA;_ 3 _University of Washington, Seattle, WA;_ 4 _Huntsman Cancer Institute, University of Utah, Salt Lake City, UT;_ 5 _Kaiser Permanente Colorado Institute for Health Research, Denver, CO_.

Purpose: Molecular characteristics, including BRAF mutation, MLH1 methylation, and CpG island methylator phenotype (CIMP), help identify aggressive subtypes of colorectal cancer (CRC), but their role is unclear for precursor lesions, especially in serrated polyps. Hyperplastic polyps (HPs) are serrated polyps that were considered to have no malignant potential, but a subset of HPs has been reclassified as sessile serrated polyps/adenomas (SSP/As), and SSP/As are now considered CRC precursors. In this study, we evaluated the association between molecular markers of serrated polyps and subsequent advanced colorectal neoplasia.

Methods: Study subjects included Kaiser Permanente Washington members who were 20-75 years old, received an index colonoscopy between 1/1/98-12/31/07 with a diagnosis of HPs or SSP/As and were free of synchronous conventional adenomas, inflammatory bowel disease, familial CRC syndromes, and prior or prevalent CRC. All eligible participants received ≥1 subsequent endoscopy or a CRC diagnosis before 1/1/13. These subsequent endoscopy pathology reports and clinical biopsies were reviewed for advanced colorectal neoplasia, defined as CRC, conventional adenomas ≥10 mm, with ≥20% villous components, with high-grade dysplasia, or SSP/As with nuclear dysplasia. Incident CRC cases were also identified through linkage to the SEER registry. Polyps from index colonoscopies were assayed for BRAF mutation (V600E), MLH1 methylation, and CIMP (using an 8-marker panel). We used generalized estimating equations with a binomial distribution, logit link, and independent correlation structure to calculate adjusted odds ratios (ORs) and 95% confidence intervals (CIs) for advanced colorectal neoplasia, comparing subgroups of serrated polyps with different molecular characteristics.

Results: We included 733 subsequent endoscopies among 553 individuals with index serrated polyps (420 HPs and 133 SSP/As). The prevalence of BRAF mutation, MLH1 methylation, and CIMP-high among index serrated polyps were 51%, 2%, and 4% respectively. Compared to those without MLH1 methylation, those with MLH1 methylated serrated lesions were more likely to have advanced colorectal neoplasia at a subsequent endoscopy, but the association was not statistically significant (adjusted OR = 1.95; 95% CI: 0.46-8.35). When restricted to index SSP/As, we observed a stronger association with MLH1 methylation (adjusted OR = 4.66, 95% CI: 1.06-20.51). BRAF and CIMP were also not statistically significantly associated with advanced colorectal neoplasia, even among those with SSP/As at the index colonoscopy.

Conclusion: There was no association between the molecular characteristics of serrated polyps and subsequent advanced colorectal neoplasia. However, among index SSP/As, we observed a strong association between MLH1 methylation and subsequent advanced neoplasia.

## PREVENTION RESEARCH

### Clinical Prevention, Early Detection, and Interception 1

#3311

Harnessing protease activity profiling for the early diagnosis of pre-malignant pancreatic cysts.

Francesco Caiazza,1 Sam L. Ivry,1 Jeremy M. Sharib,1 Giselle M. Knudsen,2 Tyler York,1 Matthew Ravalin,1 Katrin Jaredeh,1 Anthony J. O'Donoghue,3 Kimberly S. Kirkwood,1 Charles S. Craik1. 1 _UCSF, San Francisco, CA;_ 2 _Alaunus Biosciences, San Francisco, CA;_ 3 _UCSD, San Diego, CA_.

Pancreatic cysts are increasingly incidentally detected by cross-sectional abdominal imaging. The majorities of these cysts remain asymptomatic and never progress to cancer; however, 10-68% of cysts (depending on type) are potential precursor lesions to pancreatic ductal adenocarcinoma (PDAC), and should be resected if they harbor high-grade dysplasia or invasive cancer (HGD/IC). Clinical decision making relies largely on radiographic and clinical features, augmented by analysis of cyst fluid collected by endoscopic ultrasound with fine- needle aspiration (EUS-FNA). Unfortunately, with current clinical guidelines, distinguishing benign (non-mucinous) from pre-malignant (mucinous) cysts remains a challenge, therefore 80% of patients with pancreatic cysts eventually undergo pancreatic surgery, which is associated with high morbidity and mortality. Half of these surgeries are unnecessary, as the cysts were eventually determined to be benign or harboring low-grade dysplasia. Thus, there is an urgent unmet need for improved diagnostic tools to stratify patient risk. In a recent study, we used a global protease activity profiling technology to identify the aspartyl protease gastricsin as promising biomarker for differentiating mucinous from non-mucinous cysts. Analysis of gastricsin activity using a simple, fluorescence-based assay was 95% accurate for classifying mucinous cysts, significantly outperforming current diagnostic standards such as CEA. Here we performed a blinded validation study, using an independent cohort of N=234 cyst fluid samples from multiple clinical sites. Collaborating with the NCI Early Detection Research Network (EDRN), we are also performing a comparative analysis of our assay against four other investigational diagnostic tests developed at other institutions. In parallel, we have expanded our initial global protease activity profiling of cyst fluid samples to discover additional differentiating activities that could improve the accuracy of our current diagnostic assay. We show here that aminopeptidase activity is also increased in mucinous cysts; using a combination of proteomic analysis and biochemical assays, we identified a lysosomal serine protease that was increased more than 3-fold in mucinous cysts, an observation that may be related to the increased reliance on lysosomal function for nutrient scavenging that is observed with pancreatic intraepithelial neoplasia-derived pancreatic cancer. Moreover, this protease is primarily associated with mucinous cysts that harbor HGD/IC (89% sensitivity and 40% specificity). Inclusion of this additional activity in our current diagnostic assay may improve early detection and treatment of these high-risk pancreatic cysts.

#3312

Appropriate follow-up for patients with suspicious lung cancer screening findings: Lessons learned from Federally Qualified Health Centers.

Lesley Watson, Megan M. Cotter, Robert A. Smith, Katherine Sharpe. _American Cancer Society, Atlanta, GA_.

Background: Low-dose CT (LDCT) lung cancer screening is associated with improved outcomes in high-risk adults, but uptake remains low and such medical advances are often not available in low-resource areas. The American Cancer Society (ACS) launched a pilot program focused on establishing effective processes to refer and screen patients for lung cancer in under-resourced areas in West Virginia and Tennessee. ACS partnered with two Federally Qualified Health Centers (FQHCs) and accredited screening facilities to refer and screen patients, and to identify critical facilitators, barriers, and lessons learned in implementing LDCT and moving patients through the screening continuum.

Methods: Annual site visits in 2017 and 2018 captured data on implementation, progress, and lessons learned. ACS evaluators conducted 47 key informant interviews with staff from both study sites, including navigators, clinical staff, and administrators. Interviews were recorded and transcribed verbatim. Evaluators used transcripts and project notes to conduct a thematic analysis to assess factors associated with effective implementation and improved outcomes.

Results: Participants shared a wealth of insight on program implementation, including lessons learned about forming successful partnerships, personnel and resource requirements, determining screening eligibility, and conducting shared decision-making. One key area where site teams had to overcome implementation challenges was in determining appropriate follow-up testing for patients with suspicious or borderline suspicious findings (L-RADS 3 or 4). Some referring primary care providers were confused by existing clinical guidelines, unsure of when to order LDCT versus chest-CT, and felt ill-equipped to determine the optimal follow-up tests. There was confusion about the difference between billing follow-up exams as "screening" versus "diagnostic." Program leaders investigated these and other matters and came to consensus on the most practical, logical solutions. One study site also initiated a lung nodule team to discuss suspicious findings in-depth. This practice allowed the team to review clinical history and gain consensus around appropriate diagnostic testing for individual patients, and its implementation went well enough for the team to recommend it to ACS as a potential best practice for future programs.

Conclusions: By identifying challenges in conducting follow-up testing after LDCT and successful means of overcoming these challenges, this pilot study can inform practitioners in means of overcoming challenges that may enable underserved populations to move successfully through the lung cancer screening continuum. In so doing, this study may promote further reduction in cancer health disparities.

#3313

Plasma levels of MMP7 are significantly increased in patients with colorectal cancer compared to benign disease colon pathology.

HMC Shantha Kumara,1 Dasuni N. Gamage,1 Neil Mitra,1 Carl S. Winkler,1 Sandhu K. Jaspreet,1 Xiaohong Yan,1 Vesna Cekic,1 Hiromichi Miyagaki,2 Nipa D. Gandhi,1 Richard L. Whelan1. 1 _Mount Sinai West Hospital, New York, NY;_ 2 _Osaka Rousai Hospital, Osaka, Japan_.

Introduction: MMP-7 (matrilysin), a matrix metalloproteinase (MMP) family member, is expressed in epithelial cells and some cancers. MMP7 is capable of activating pro-collagenases and digesting extracellular matrix (ECM) proteins. MMP7 supports connective tissue remodeling and regulates intestinal host defense and innate immunity. Increased MMP7 expression is associated with inflammation, tumorgenesis, and metastasis. MMP7 regulates neutrophil recruitment at sites of neoangiogenesis. MMP7 is capable of activating pro-MMP-1,-2,and-9, and osteopontin. Preoperative (preop) colorectal cancer patient's plasma levels of MMP2, MMP3 and osteopontin have been shown to be higher than benign disease patient's preop levels. MMP7 overexpression is reported in many malignancies (colorectal cancer (CRC) included). This study's purpose was to compare PreOp plasma MMP7 levels in CRC and benign colon disease (BCP) patients (pts).

Method: Pts undergoing colorectal resection for CRC or BCP who were prospectively enrolled in an IRB approved tissue/data bank for whom plasma samples were available were studied. Clinical, demographic and pathological data were reviewed. Plasma MMP7 levels were determined via ELISA in duplicate and reported as median +95%CI (ng/ml). Expression levels were determined in tumors and paired normal colon tissues in a subgroup of study pts by QRT-PCR. The candidacy of MMP7 as a diagnostic marker for CRC was validated by the receiver operating characteristic (ROC) curve and area under the ROC curve (AUC) results. The Mann-Whitney test was used for statistical analysis.

Results: A total of 120 CRC (72% colon, 28% rectal) and 120 BCP pts (adenoma 33%, diverticulitis 52%, other 15%) were studied. The male: female ratios in both groups were similar but CRC pts were older. The CRC stage distribution was stage 1,30%; stage 2,27%, stage 3,33%, stage 4,10%. The median plasma MMP7 level was significantly higher in the CRC group than in the BCP group(3.69,CI: 3.39,4.07vs 2.79 ,CI: 2.51,3.2;P=< 0.001). The AUC for the ROC curve (AUC) for plasma MMP7 in association with a CRC diagnosis was 0.698 (sensitivity 48%, specificity 80%). All CRC tumors tested showed elevated MMP7 expression vs. paired normal tissue.

Conclusion: The median PreOp plasma MMP7 level was 33% higher in CRC vs. BCP group. The AUC results suggest MMP7 may have value as a CRC prognostic marker, perhaps, in combination with other protein markers. High MMP7 levels may be related to tumor vascularization and inflammation associated with the cancer. Further studies that assess larger populations are needed to further assess preop MMP7 levels.

#3314

Normal breast tissue at risk for cancer development: A breast cancer initiating role for mammary adipocytes.

Taekyu Kang,1 Christina Yau,1 Stephen Benz,2 Gregor Krings,3 Roman Camarda,1 Jill E. Henry,4 Mark Powell,5 Christopher C. Benz1. 1 _Buck Inst. for Research on Aging, Novato, CA;_ 2 _Nantomics LLC, Culver City, CA;_ 3 _University of California, San Francisco, San Francisco, CA;_ 4 _Indiana University Simon Cancer Center, Indianapolis, IN;_ 5 _Buck Institute for Research on Aging, Novato, CA_.

Adipocytes are the predominant cell population in the normal breast and while recent attention has pointed to adipocyte-tumor cell crosstalk as a driver of breast cancer biology there have been few reports on the potential role of adipocytes in driving breast cancer initiation. Because normal breast tissue studies have invariably used reduction mammoplasty, benign biopsy or cancer-adjacent tissues, we studied random breast core biopsy samples donated by 145 healthy, parous, non-obese, white women (median age = 45, range 27-66 y) without any history of breast cancer. Using questionnaire data to calculate future breast cancer risk (Gail scores), we compared digitized microscopic breast tissue (H&E) images with whole genome transcriptome profiling (RNAseq) from FFPE-extracted RNA. We used unsupervised hierarchical clustering of 1487 genes (normalized, median centered, log2-scaled RSEM values) to identify 32% of normal samples with an "Active" (vs. 68% "Inactive") transcriptome phenotype previously associated with later-life risk of death from breast cancer. Despite slightly lower BMI values, donors with the Active transcriptome phenotype showed significantly higher Gail scores as well as higher mammary adipocyte nuclei counts (median 80% vs. 60%, p=2.3e-6). Tissue resident leukocytes were uncommon but Active transcriptome tissues expressed significantly altered immune modules enriched in TGFβ, interferon and macrophage gene signatures (including single gene increase in CD68) and depleted (relative to Inactive samples) of CD8+ T-cell and serum response/inflammation/wound healing signatures. Active samples were not enriched in cell senescence, SASP or DNA damage response gene signatures but were enriched in an autophagy-to-senescence-transition (AST) signature with increased CAV1 (caveolin-1, p=2.7e-12) and BNIP3 (Bcl2 interacting protein-3, p=4.7e-05) expression, genes that also regulate lipoprotein digestion/mobilization and adipocyte remodeling. Strongest among significant associations linking Active with adipocyte-enriched normal breast samples were increases in two adipokine growth factors, IGF-1 (p=2.2e-16) and FGF2 (p=3.0e-11), the adipokine (resistin) receptor CAP1 (p=0.04) recently linked to poor breast cancer outcomes, and a cAMP-dependent pro-lipolytic signature (p=0.01) known to drive breast cancer progression which, in these samples, correlated positively with average adipocyte area values. Altogether, the collective histologic and molecular features characterizing the normal breast tissue of >30% of healthy parous and non-obese women with increased predicted breast cancer risk seem to implicate a dysregulated mammary adipocyte microenvironment similar to but distinct from that associated with established breast tumors, that precedes microscopic and clinical evidence of breast tumorigenesis.

#3315

Workplace program that offers annual fecal immunochemical testing improves adherence to colorectal cancer screening guidelines.

Carmen H. Tong, Anita Satish, Maren S. Fragala, Lance A. Bare, Charles E. Birse. _Quest Diagnostics, San Juan Capistrano, CA_.

Background: Although regular screening reduces the incidence and mortality of colorectal cancer, ~ 35% of eligible adults remain non-adherent to screening guidelines. We asked if a workplace screening program, using a non-invasive fecal immunochemical test (Insure FIT), would improve adherence to guidelines.

Methods: An employer-sponsored program provided eligible employees (age 50-75 years) the opportunity to participate in colorectal cancer screening. InSure FIT collection kits were mailed annually to: (i) newly eligible employees, (ii) new hires, (iii) those who had participated the previous year and (iv) those who requested testing. Employees returned kits by mail for quantitative detection of human hemoglobin in fecal samples. Those with positive test results were contacted by phone and mail with a recommendation to contact their physician for follow up diagnostic testing. Using a limited data set of health plan claims, we identified those employees, with continuous medical coverage over the 5-year study period (2013-2017), who were adherent to current screening guidelines (colonoscopy any year or FIT every year). Colonoscopy procedures were identified in professional services and outpatient event files using current procedural terminology (CPT) codes.

Results: Approximately a third (32.7%; 1,858/5,686) of eligible employees participated in the program. Some 42.7% (794/1,858) of participants underwent colonoscopy over the 5-year study period. An additional 15.9% (296/1,858) were tested with InSure FIT all 5 years. In total 58.7% (1,090/1,858) of participants were adherent to guidelines (colonoscopy any year or annual FIT). In comparison, only 34.6% (1,323/3,828) of non-participants were adherent to guidelines. | |

---|---|---

|

Participants | Non-Participants

Total | 1,858 | 3,828

Mean age, y (SD) | 55.9 (4.2) | 55.7 (4.3)

Female, n (%) | 1,314 (70.7%) | 2,470 (64.5%)

Adherent to screening guidelines, n (%) | 1,090 (58.7%) | 1,323 (34.6%)

Conclusion: Participants in an employer-sponsored FIT program had 24% higher adherence to guidelines for colorectal cancer screening compared to non-participants.

#3316

Screening blood tests and cancer detection in Li-Fraumeni syndrome.

Leatrisse Oba,1 Jennifer T. Loud,1 Maria I. Achatz,2 Sharon A. Savage,1 Payal P. Khincha1. 1 _NIH/NCI, Rockville, MD;_ 2 _Hospital Sírio e Libanês, São Paulo, Brazil_.

Li-Fraumeni syndrome (LFS) is an autosomal dominant cancer predisposition syndrome, caused primarily by germline pathogenic TP53 variants and associated with a very high lifetime cancer risk which reaches approximately 100% by age 60 years. Individuals with LFS are also at significant risk of developing multiple primary cancers. Current comprehensive cancer screening recommendations in LFS include annual whole-body MRI, brain MRI, breast MRI (for women), and bloodwork (complete blood count, erythrocyte sedimentation rate, lactate dehydrogenase level) every 3-4 months for the detection of hematological malignancies. Additionally, children undergo 3-4 monthly abdominal ultrasound and hormonal testing (serum progesterone, testosterone, DHEAS,dehydroepiandrostenedione, androstenedione, alpha fetoprotein and beta-human chorionic gonadotropin levels) for detection of adrenocortical carcinomas and germ cell tumors. While this screening is effective in early cancer detection, it adds to the psychosocial burden of LFS.

We evaluated the benefit of 4-monthly blood work in 139 participants of the National Cancer Institute's IRB-approved longitudinal cohort screening study (NCT01443468, http://lfs.cancer.gov) between 2012 and October 2018. All participants underwent annual screening at the NIH Clinical Center; 3-4 monthly bloodwork and pediatric ultrasounds were coordinated with their healthcare providers. The cohort included 89 females (64%) and 50 males (36%). Twenty-six patients (19%) were of children at enrollment, and 113 (81%) were adults. The median age at baseline screening was 35 years (range 1-68), with a median annual screening of 4 visits (range 1-7). The median follow-up time was 35 months (range 3-72) and median number of quarterly screening bloodwork was 9 (range 1-18). Seventy-two of 1248 total blood tests (6%) were missed due to non-compliance or medical reasons. Of the 139 patients, 33 (24%) developed 45 screen-detected or interval cancers during their study participation. Of the 45 cancers detected, only one (2%), a colorectal cancer, was detected with screening bloodwork due to a down-trending hemoglobin that led to colonoscopy. No hematological malignancies, adrenocortical carcinomas or germ-cell tumors were detected as a result of blood screening. One female participant was diagnosed with polycythemia vera after identification of persistent thrombocytosis. One adult male was diagnosed with iron-deficiency anemia requiring iron infusions due to persistent anemia. Eight additional abnormal tests required follow-up bloodwork, all of which were within the normal range.

Our data suggest that quarterly screening blood tests may not independently add to the benefit of radiological screening in LFS. Longitudinal follow-up and detailed analyses are underway to determine the appropriate screening interval in individuals with LFS.

#3317

Development of a lung nodule cohort with integrated clinical, molecular and imaging biomarkers.

Sanja Antic,1 Travis Osterman,1 Aneri Balar,1 Dhairya Lakhani,1 Rina Nguyen,1 Sara Block,1 Kimberly Fileds,1 Brandon Winston,1 Anel Muterspaugh,1 Yuankai Huo,2 Riqiang Gao,2 Joseph Leader,3 David Wilson,4 Viswam Nair,5 Robert Gillies,6 Matthew Schabath,6 Chirayu Shah,7 Bennett Landman,2 Pierre Massion1. 1 _VUMC, Nashville, TN;_ 2 _Vanderbilt University, Nashville, TN;_ 3 _University of Pittsburgh, Pittsburgh, PA;_ 4 _UPMC, Pittsburgh, PA;_ 5 _University of South Florida, Tampa, FL;_ 6 _Moffitt Cancer Center, Tampa, FL;_ 7 _VA Nashville, Nashville, TN_.

Background: We are facing an epidemic of indeterminate pulmonary nodules (IPN). Current diagnostic strategies lack accuracy such that the management of IPNs 6-30 mm leads to an unacceptable rate of invasive biopsies and of missed opportunities for cure. Based on the critical need of evaluating candidate biomarkers across heterogeneous populations, we intended to assemble a large cohort of fully annotated IPNs from five collaborative institutions.

Methods: Standard operating procedures (SOPs) were developed to capture subject demographics and assign consistent identifiers across clinical, bio-specimens, and imaging data. REDCap database was created for clinical, imaging and biospecimen data capture. Sample collection, processing, storage and shipping SOPs were created and shared among institutions to assure for consistency and quality accuracy. DICOM files, demographic and clinical data, blood and tissue samples were collected prospectively through IRB approved studies at each institution (VUMC, Nashville VAMC, Moffitt Cancer Center, and UPMC). Imaging studies and specimens were de-identified locally using custom JavaScript program in a secure web browser and assigned as specific identifier. De-identified thin slice, non-contrast chest CT studies were tested for quality control and transmitted to an imaging repository (eXtensible Neuroimaging Repository-XNAT) that can be mined by all collaborators.

Results: To date, a cohort of 845 subjects, 507 (60%) males and 338 (40%) females, with lung nodules was assembled. 36 % are current smokers, 56 % former smokers and 8% never smokers, with an average of 46 pack year smoking history. Clinical data including risk prediction models such as the Mayo and PLCO m2012 are reported. Pathological confirmation of nodules is available for 322 benign and 444 malignant nodules. The cohort 283 lung adenocarcinomas, 71 squamous cell carcinomas, 53 small cell carcinoma, 17 non-small cell lung cancer, 9 carcinoid, and 79 subjects considered benign based on CT follow without growth. Serum, plasma and peripheral blood monocyte related DNA is available on all. All diagnostic chest CT are available in our thoracic imaging repository (XNAT) in a de-identified format.

Conclusions: We assembled a unique cohort of incidental and screening detected lung nodules prospectively enrolled at four institutions for which full clinical data capture, chest CT DICOM files and blood specimens were collected. This repository allows the derivation and independent validation of candidate molecular and imaging biomarkers for the management of IPNs. This work is supported by UO1 152662, UO1CA186145 and UO1CA196405

#3318

Development of a cfDNA based trans-omics approach for early cancer detection.

Yuying Wang,1 Heng Zhao,2 Kaijian Ling,3 Jianchao Zheng,1 Zhilong Li,1 Shuangshuang Chen,1 Jia Li,1 Chichuan Liu,1 Guanghui Yang,1 Hongpo Zhou,1 Jiaxi Peng,1 Lili Ye,4 Liuhong Zeng,4 Jianlong Sun,1 Ruijingfang Jiang,1 Li Deng,3 Yanzhou Wang,3 Kezhong Chen,2 Zhiqing Liang,3 Fan Yang,2 Taiping Shi,1 Mingzhi Ye1. 1 _BGI Genomics, Shenzhen, China;_ 2 _Peking University People's Hospital, Beijing, China;_ 3 _The First Hospital Affiliated to AMU, Chongqing, China;_ 4 _BGI-Guangzhou Medical Laboratory, Guangzhou, China_.

Circulating tumor DNA (ctDNA) in plasma of cancer patients provides valuable information about the cancer and also holds great promise for non-invasive early cancer detection. Nevertheless, since ctDNA is diluted by circulating cell-free DNA (cfDNA) of noncancerous origins, its detection poses significant challenge, especially during early stage of cancer. Previous studies on early cancer detection have mostly focused on single feature of the ctDNA, namely either cancer driver gene mutations or alterations in the methylome. We have developed a set of experimental and computational tools to measure both genetic and epigenetic signals from ctDNA using next-generation sequencing, aiming to improve its detection. Briefly, cfDNA extracted from cancer patients and healthy individuals were used for targeted deep sequencing as well as targeted bisulfite sequencing. More specifically, duplex-UMI libraries were prepared in order to suppress errors introduced by sequencing and PCR artifacts. Libraries were subsequently captured using a custom designed panel targeting cancer driver gene mutations. Bisulfite sequencing libraries were prepared from single strand cfDNA following bisulfite conversion and captured using a panel targeting gene promoter regions. These methods ensure higher quality of the cfDNA libraries and more sensitive ctDNA detection. A subset of samples also had matched tumor tissue samples sequenced using targeted sequencing and WGBS to validate mutations as well as changes of CpG methylation level found in plasma. All sequencing runs were conducted on the MGIseq platform developed by BGI genomics. To this end, we have developed classification models based on these data using machine learning approaches. Optimal model achieves a sensitivity of 52% in early stage lung cancer, and a sensitivity of 83.3% in ovarian cancer, while holding specificity > 99%. These results hold great potential to be further explored for early cancer detection application. We are currently expanding the study to additional cancer types, especially the ones with high incidence rate and/or poor survival rate in China, such as colorectal cancer and liver cancer.

#3319

Rare cell isolation by enhanced microfluidic filter using wing-shaped micro posts.

Nanda Kasani, Manjunath Yeriswamy, Guangfu Li, Jussuf Kaifi, Jae W Kwon. _University of Missouri Columbia, Columbia, MO_.

Purpose: Study of effective isolation of rare cells using specially designed microfluidic filter.

Design: A novel microfluidic filter chip is proposed to enhance the isolation efficiency on rare cells, and has been successfully tested with micro beads and A549 cell lines using our unique designs of micro-posts. While most of the other filtration techniques have serious issues of channel clogging and prolonged processing, we have successfully accomplished outstanding improvements from such problems by utilizing inertial displacement through curved structures, built-in bypassing routes, and dedicated trapping zones. A solution containing polystyrene micro beads of 9.9 μm in diameter was successfully separated at the isolation efficiency of 96% with a high flow rate of 1ml/min. Additional experiments were also carried out, with A549 cell-lines in a PBS solution to see the filtration feasibility on bio-particles. Our device may offer an effective and reliable label free separation technique without channel blockage issues. By far, we have studied two design configurations called Model I and Model II. (Model II is an improved version of Model I).

Results: Capture efficiency of 96% was recorded from Model II for microbeads at the flowrate of 1ml/min. The capture efficiency of 85% was marked from Model I for microbeads at the flowrate of 1 ml/min. The relationship between the flowrate and capture efficiency has also been studied as shown in Table.1

Conclusion: Our studies demonstrate the excellence of our microfluidic devices with a capture efficiency of 96% and exhibits potential capabilities of isolating circulating tumor cells from peripheral blood of cancer patients.

Table 1: Flow rate vs Filtering efficiency of microbeads and A549 cells  | |  | |

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

Flow rate (ml/min) | Model - I Filtering efficiency % (Standard Error Mean) | Model II Filtering efficiency % (Standard Error Mean)

|

Microbeads | A549 cells | Micro beads | A549 cells

1 | 85 (±4.5%) | 78 (±4.0%) | 96 (±3.4%) | 92 (±4.0%)

1.5 | 80 (±4.0%) | 71 (±3.5%) | 90 (±3.1%) | 85 (±3.4%)

2 | 73 (±3.5%) | 65 (±3.1%) | 84 (±2.8%) | 80 (±3.0%)

2.5 | 56 (±2.6%) | 48 (±2.5%) | 80 (±2.3%) | 73 (±2.2%)

3 | 45 (±2.2%) | 38 (±1.8%) | 70 (±2.0%) | 65 (±1.5%)

#3320

Dietary inflammatory potential in relation to the gut microbiome among persons at varied risk of colorectal neoplasia.

Jiali Zheng,1 Kristi L. Hoffman,2 Nitin Shivappa,3 Jonathan Busquets,4 Jiun-Sheng Chen,1 Samir Hanash,1 Susan Schembre,5 James Hébert,3 Joseph Petrosino,2 Peng Wei,1 Carrie R. Daniel1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _Baylor College of Medicine, Houston, TX;_ 3 _University of South Carolina, Columbia, SC;_ 4 _Rice University, Houston, TX;_ 5 _University of Arizona, Tucson, AZ_.

Purpose: Diet modulates gut microbiome composition and systemic inflammation—both intermediary factors in the development of colorectal neoplasia. We investigated the association of total dietary inflammatory potential, as assessed by the literature-derived dietary inflammatory index (DII®), with gut microbiota diversity and composition, microbial gene pathways, and circulating markers.

Methods: This epidemiological study was comprised of 36 cancer-free colonoscopy patients (79% positive for precancerous polyps) and 65 healthy volunteers from the medical center community (N=101). Participants completed dietary assessments and provided stool samples for 16S rDNA sequencing >1 month after colonoscopy/polyp removal or antibiotic use. In a subset (n=47), we conducted whole genome shotgun (WGS) sequencing and collected a fasting blood sample. Energy-adjusted (E)-DII score was calculated for each subject and categorized into tertiles. The association between E-DII and fecal microbiota alpha- and beta-diversity was examined. Linear discriminant analysis Effect Size (LEfSe) was used to identify differentially abundant taxa by E-DII level. Multiple linear regression models were used to examine associations controlled for confounders. Results from the least absolute shrinkage and selection operator (LASSO) method and Spearman's correlation were used to select and evaluate associations of bacterial species with E-DII, their functional pathways, and circulating markers.

Results: We observed large between-person variation in Bacteroidaceae and Enterobacteriaceae, two dominant bacteria in the overall sample. Neither alpha- nor beta-diversity significantly differed across E-DII levels. Ruminococcus torques, Acidaminococcus intestine, and Clostridium leptum were most abundant in the most pro-inflammatory diet group, while Akkermansia muciniphila was enriched among subjects with the most anti-inflammatory diet. In the WGS subset, Luteimonas mephitis was inversely associated with E-DII, circulating Lipocalin-2 (r=-0.34; P=0.02), and plasminogen activator inhibitor-1 (r=-0.29; P=0.05). Akkermansia muciniphila appeared to be inversely correlated with monocyte chemoattractant protein-1 (r=-0.26; P=0.09). Two microbial pathways inversely associated with E-DII, AMP-activated protein kinase (AMPK) and carbon metabolism, were also inversely correlated with C-peptide (AMPK: r=-0.35, P= 0.02; carbon: r=-0.30, P=0.04).

Conclusions: Dietary inflammatory potential was associated with differential composition of specific microbes, but not overall diversity of the gut microbiome. Species associated with E-DII were similarly associated with circulating inflammatory biomarkers. Microbial AMPK signaling and carbon metabolism pathways may underlie diet-microbiota interactions that modulate systemic inflammation.

#3321

Effect of aspirin on cancer events in the healthy elderly: Results from the ASPREE trial.

Andrew T. Chan,1 Peter Gibbs,2 Suzanne G. Orchard,3 Jessica E. Lockery,3 Andrew Haydon,3 Rory Wolfe,3 Robyn L. Woods,3 Finlay Macrae,4 John Zalcberg,3 Galina Polekhina,3 Mark R. Nelson,5 Christopher M. Reid,6 Brenda Kirpach,7 Anne M. Murray,7 Ellen Richmond,8 Leslie Ford,8 Asad Umar,8 John J. McNeil,3 for the ASPREE Investigator Group. 1 _Massachusetts General Hospital, Boston, MA;_ 2 _Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia;_ 3 _Monash University, Melbourne, Australia;_ 4 _University of Melbourne, Melbourne, Australia;_ 5 _University of Tasmania, Hobart, MA;_ 6 _Curtin University, Perth, Australia;_ 7 _Hennepin Health Research Institute, Minneapolis, MN;_ 8 _National Cancer Institute, Bethesda, MD_.

Background: Compelling evidence largely derived from middle-aged adults (<70 years old) demonstrates that aspirin reduces risk of cancer, particularly colorectal cancer (CRC) over long-term follow-up. Recently, the ASPirin in Reducing Events in the Elderly (ASPREE) study, a placebo-controlled randomized trial of daily low dose aspirin (100 mg) in older adults, showed a surprising increase in the secondary endpoint of all-cause mortality, primarily due to excess cancer deaths (McNeil, NEJM 2018). We now report detailed analysis of cancer events in ASPREE during the treatment phase.

Methods: 19,114 Australian (N=16,703) and US (N=2,411) participants aged 70+ years (US minorities 65+ years) were recruited in 2010-14. Previous history of cancer was not an exclusion; however participants were required to be in good health, free of major disease and expected to survive 5+ years. Participants reported cancer events at annual study visits and an expert panel reviewed medical records to adjudicate. Cox proportional-hazards models were used in intention-to-treat analyses to compare aspirin and placebo groups.

Results: After a median 4.7 years, there were 981 cancer events in the aspirin arm and 952 in the placebo arm. There was no statistically significant difference between groups for overall incident cancer (HR=1.04, 95% CI 0.95-1.14), non-metastatic cancer (HR=1.00, 95% CI 0.89-1.11), or hematological cancer (HR=0.98, 95% CI 0.73-1.30). A non-significant excess of participants in the aspirin group (N=194) developed incident metastatic cancer compared with the placebo group (N=166) (HR=1.18, 95% CI 0.96-1.45). Among participants with available staging data (~65%), no difference between the aspirin and placebo groups was observed for stage 1 (HR=0.98, 95% CI 0.76-1.25), stage 2 (HR=0.87, 95%CI 0.68-1.12), stage 3 (HR=1.02, 95% CI 0.75-1.39) or stage unknown (HR=1.03, 95% CI 0.90-1.19) cancers. However, aspirin was associated with a HR of 1.20 (95% CI 1.00-1.43) for stage 4 cancers. Among participants with stage 4 cancer, those in the aspirin group had a HR of 1.30 (95% CI 1.03-1.62) for death compared to the placebo group. No statistically significant differences between groups were observed according to cancer type.

Conclusions: After 4.7 years, the increase in cancer deaths associated with the initiation of aspirin in healthy elderly adults was not accompanied by a significant increase in overall incident cancer. The excess of metastatic cancers and death in participants with stage 4 cancer in the aspirin group support the need for further investigation of the possibility that aspirin may adversely affect short-term outcomes among elderly participants with undiagnosed cancers (e.g. tumors prevalent at the time of enrolment or early incident tumors). Additional follow-up of the ASPREE cohort to examine a long-term "legacy" effect of aspirin on cancer incidence is warranted.

#3322

Chronology and risk-dependence of age-related remodelling of oesophageal epithelia.

Akira Yokoyama,1 Nobuyuki Kakiuchi,1 Tetsuichi Yoshizato,1 Yasuhito Nannya,1 Hiromichi Suzuki,1 Yasuhide Takeuchi,1 Yusuke Shiozawa,1 Yusuke Sato,1 Kosuke Aoki,1 Soo kim,1 Yoichi Fujii,1 Kenichi Yoshida,1 Keisuke Kataoka,1 Masahiro M. Nakagawa,1 Yoshikage Inoue,1 Tomonori Hirano,1 Yuichi Shiraishi,2 Kenichi Chiba,2 Hiroko Tanaka,2 Masashi Sanada,3 Shinya Ohashi,1 Shin'ichi Miyamoto,1 Shigeru Tsunoda,1 Koshi Mimori,4 Sachiko Minamiguchi,1 Satoru Miyano,2 Hideki Makishima,1 Manabu Muto,1 Seishi Ogawa1. 1 _Kyoto UNIV., Kyoto, Japan;_ 2 _Institute of Medical Science, The University of Tokyo, Tokyo, Japan;_ 3 _Nagoya Medical Center, Nagoya, Japan;_ 4 _Kyushu University Beppu Hospital, Beppu, Japan_.

Clonal expansion in aged normal tissues has been implicated in cancer development. However, its chronology and risk-dependence are poorly understood. Esophageal squamous cell carcinoma (ESCC) is a predominant esophageal cancer among Asian populations and substantially affected by heavy smoking and drinking, likely through a 'field effect'. To elucidate the role of these lifestyle risks on ESCC development, we investigated clonal expansion in physiologically normal esophageal epithelia (PNE) using multiple microscale sampling, as small as 0.2 mm2 in size, followed by an unbiased detection of somatic mutations with whole exome sequencing. Mutations were detected in most of PNE samples (151/157), where none of the mutations were shared between samples collected >10 mm apart. The number of mutations and their allele frequency increased with age, suggesting age-related clonal expansion in PNE (ARCE), which was significantly promoted by heavy smoking and drinking. Mutations were dominated by age-related patterns and a still poorly-defined, 'esophagus-specific signature, as well as a COSMIC 16-like signature. The latter has recently been related to alcohol drinking and was enriched in high-risk samples, which was confirmed by whole genome sequencing of single cell-derived colonies. As many as 10 genes were significantly mutated or positively selected in ARCE. Among most commonly affected genes were NOTCH1, TP53, FAT1, PPM1D, NOTCH2, and NOTCH3, which substantially differed from those in ESCC, showing prominent over-representation of NOTCH1, PPM1D, FAT1 and NOTCH2, and significant underrepresentation of TP53, NFE2L2, and CDKN2A were significantly underrepresented, suggesting different mechanisms of positive selection between ARCE and ESCC. Driver mutations were detected more frequently and in higher numbers in high-risk PNE samples than low-risk ones, with accentuated NOTCH1, TP53 and PPM1D mutations. Analyses of densely collected micro-scale samples (0.2 mm2) disclosed fine structure of ARCE with its chronological history. Driver-mutated clones emerge multifocally from early childhood as early as <2 years and accompanying their own phylogenetic structures, increase their number and size with aging, ultimately replacing almost entire oesophageal epithelia in the extreme elderly. In conclusion, remodelling of oesophageal epithelia by driver-mutated clones is an inevitable consequence of normal aging, impacting cancer development depending on lifestyle.

#3323

The feasibility for detecting hereditary genetic findings of familial gastric cancer.

Sadaaki Nishimura. _Osaka City University Graduate School of Medicine, Osaka City, Japan_.

Background: Multiplex gene panel tests using next-generation sequencing has currently been clinically available for various cancer patients including gastric cancer in Japan. The original purpose of the Multiplex gene panel tests is to find actionable genetic alterations which might correlate with promising therapeutic antitumor drugs. Accordingly, secondary genetic findings as germline mutation which is associated with hereditary disease may also be detectable by the Multiplex gene panel tests. It has been reported that familial cancers represent around 5%‒10% of all cancer patients. Gastric cancer is one of popular cancers in Japan. The prediction of patients with familial gastric cancer (FGC) before the multiplex gene panel tests might be important for the gene consultation for the patients and their family.

Objective: The aim of study is to clarify the characteristic clinico-pathologic features of FGC to predict the possibility of hereditary gastric cancer.

Patients and Methods: A total of 2301 gastric cancer patients who received gastrectomy at our department was enrolled in this study. We classified FGCs into 2 categories by modification of International Gastric Cancer Linkage Consortium (IGCLC) which excluded pathogenic types, as follows, group A: gastric cancer in an individual under the age of 40; group B: two gastric cancer cases in a family regardless of age. In contrast, the most gastric cancers were sporadic, which was classified as group C. The clinicopathological factors including E-cadherin and p53 expression by immunostaining, was retrospectively compared among 3 groups.

Results: One hundred and fifty-two cases (6.6%) of 2301 patients were classified as FGCs (group A: 71 cases; group B: 81 cases), whereas the remaining 2149 patients were classified as group C (93.4%). Female (43.4%) and diffuse-type gastric cancer (57.2%) was significantly (p<0.001) more frequent in FGCs than that (30.5% and 42.5%) in group C. The significant difference (p<0.05) of clinicopathological variables between group A (n=71) and B (n=81) was found in the rates of female (52.1% vs 35.8%), diffuse-type (83.1% vs 34.5%), and T4 status (38.0% vs 17.3%), respectively. There was also significant difference (p<0.05) in the rates of female (52.1% vs 30.5%), diffuse-type (83.1% vs 42.5%) and Borrmann type 4 (15.5% vs 6.8%) between group A and C. No significant difference was found in clinicopathological variables between group B and C. No statistical correlation of E-cadherin loss between 3 groups. In this study, 2 cases were found in which p53 expression was positive for all cells.

Conclusion: Young age (<40 years) and female patients with diffuse-type gastric cancer might have a high risk for FGC. It is necessary to take into consideration of hereditary gastric cancer when the multiplex gene panel test is performed for the young female gastric cancer patients with diffuse-type cancer.

#3324

Non-invasive detection of aberrant DNA methylation in colorectal cancer by multiplex methylation specific PCR.

Lili Ye,1 Chunting Zheng,1 Yuan Jie,2 Bin Li,1 Yuan Li,3 Kunling Hu,1 Liuhong Zeng,1 Yuying Wang,4 Mao Mao,4 Peirong Ding,3 Taiping Shi,4 Mingzhi Ye4. 1 _BGI-Guangzhou Medical Laboratory, Guangzhou, China;_ 2 _The Fifth Affiliated Hospital, Southern Medical University, Guangzhou, China;_ 3 _Sun Yat-sen University Cancer Center; State Key Laboratory of Oncology in South China, Guangzhou, China;_ 4 _BGI Genomics, Shenzhen, China_.

Colorectal cancer (CRC) is a leading cause of morbidity and mortality worldwide, with an increasing prevalence. The 5-year relative survival rate for advanced stages is very low, with only 14% for stage IV. However, that of the early stage of CRC is about 90%. Therefore, it is important to screen CRC in the early stage. Epigenetic alterations linked to the carcinogenesis of CRC have been shown to occur earlier and more frequently than genetic alterations in CRC. Here, our study aims to develop a CRC screening methodology by applying the multiplex methylation specific PCR (MMSP) assay to detect CRC-specific methylation biomarkers, which has been pre-selected from public databases and subsequently validated in CRC samples of the Chinese population. Experimentally, cell free DNA (cfDNA) is extracted from ~4ml peripheral blood plasma. Following bisulfite conversion of the cfDNA, MMSP were applied to amplify and detect CRC-specific methylated CpG sites within the cfDNA. From the results of 28 CRCs and 52 control specimens, a sensitivity of 85.7% and the a specificity of 92.3% was achieved. Current standard and most widely used method of detecting CRC is colonoscopy screening. However, colonoscopy requires bowel preparation, and the sedation for patients is a complex and time-consuming procedure with high cost. Additionally, this invasive method also increases the possibility of infection and complications and patient compliance still largely remains a problem. Our non-invasive MMSP assay therefore provides an alternative non-invasive screening option that is cost-effective, meeting the urgent demand of early CRC detection. In addition, because colonoscopy screening currently has low adherence in Chinese population, our method holds great potential for increasing CRC screening rate in China.

#3325

EGFR mutation and protein expression analysis in sinonasal inverted papilloma and squamous cell carcinoma.

Virginia N. Cabal,1 Marta Menéndez,2 Sira Potes-Ares,1 Blanca Vivanco,3 Laura Suárez-Fernández,1 Cristina Riobello,1 Rocío García-Marín,1 Fernando López,2 José Luís Llorente,2 Mario Hermsen1. 1 _ISPA, Oviedo, Spain;_ 2 _Otolaryngology HUCA, Oviedo, Spain;_ 3 _Pathology HUCA, Oviedo, Spain_.

Introduction Inverted sinonasal papilloma has been demonstrated to be precursor to a subset of sinonasal squamous cell carcinoma and to carry frequent mutations in EGFR exon 20. The aim of this study was to evaluate EGFR mutation and protein expression as risk marker for malignant transformation of inverted papillomas.

Experimental procedures The total number of samples studied was 41 inverted papillomas (ISP) and 32 squamous carcinomas (SCC). We defined patients with one single ISP, with multiple ISP (ISP-ISP) or ISP with malignant transformation (ISP-transformed). In addition, we classified SCC related with ISP (SCC-ISP) and those without relation (SCC de novo). EGFR exon 20 was amplified by PCR and analyzed by Sanger sequencing using the ABI PRISM 3100 and 3730 Genetic Analyzer, (Applied Biosystems, Foster City CA). Immunohistochemistry was performed on an automatic staining workstation (Dako Autostainer Plus; DakoCytomation, Glostrup, Denmark) using the antibody anti-pEGFR clone D7A5 (Cell Signaling Technology, Cambridge, UK). Results were evaluated by two experienced investigators (BV and MM).

Results We found EGFR exon 20 mutations in 62% (8/13) ISP, 58% (7/12) ISP-ISP, 63% (10/16) ISP-transformed, 54% (7/13) SCC-ISP and 5% (1/19) SCC de novo. Protein expression of pEGFR was detected in 56% (5/9) ISP, 75% (6/8) ISP-ISP, 47% (7/15) ISP-transformed, 42% (5/12) SCC-ISP and 71% (10/14) SCC de novo. We observed an inverse correlation between EGFR exon 20 mutation and pEGFR expression (p=0.034). Overall survival was significantly better for SCC-ISP compared with SCC de novo (2-year survival 54% and 29% respectively; p=0.030).

Conclusions EGFR exon 20 mutations occurred in a high frequency, both for cases with single or multiple ISP and for ISP-transformed, making it a characterizing genetic abnormality for ISP in general. This result also means that the presence of a EGFR exon 20 mutations is not of value as a risk marker for malignant progression. Among the SCC, EGFR exon 20 mutations were notably less and pEGFR expression more frequent in SCC de novo compared to SCC-ISP, suggesting that the EGFR signaling pathway is important in both types of SCC, albeit activated in a different manner. Patients with SCC-ISP had a more favorable clinical course, however, neither EGFR exon 20 mutations nor pEGFR expression demonstrated prognostic value. Nevertheless, EGFR exon 20 mutations can be targeted with specific inhibitors and may be of value for adjuvant therapy for SCC as well as ISP that are difficult to manage.

#3326

Mucositis, candidiasis, and associations with the oral microbiome in treatment naive patients with oropharyngeal cancer.

Christine M. Pierce,1 Stephanie Hogue,1 Shirlene Paul,1 Bo-Young Hong,2 Wildson Vieira da Silva,1 Maria F. Gomez,1 Anna R. Giuliano,1 Jimmy J. Caudell,1 George M. Weinstock2. 1 _Moffitt Cancer Center, Tampa, FL;_ 2 _Jackson Laboratory for Genomic Medicine, Farmington, CT_.

Purpose: Oropharyngeal cancer (OPC) incidence continues to increase dramatically in the US. Accumulating evidence suggests that oral microbiota (communities of microorganisms that reside in the oral cavity) may influence cancer treatment-related toxicities. We sought to examine the composition and diversity of oral microbiota in OPC patients prior to treatment, and identify associations between oral microbiota and subsequent development of oral mucositis (inflammation of mucous membranes) and candidiasis (fungal infection with Candida yeast).

Methods: 60 newly diagnosed, treatment naïve, OPC patients were recruited from Moffitt Cancer Center (2015-2017). Patients provided mouthwash-based oral gargles before starting chemoradiation or radiation. DNA was isolated using the QIAGEN DNeasy PowerSoil kit, and the V1-V3 region of the bacterial 16S rRNA gene was sequenced. An operational taxonomic unit table was generated and Ribosomal Database Project Classifier used to assign taxonomy. The development of oral mucositis (no/mild vs. severe) and candidiasis (no vs. yes) during treatment was abstracted from medical records. Comparisons of bacterial relative abundance (Mann Whitney U), alpha diversity (Chao1, Shannon, and Simpson), and beta diversity (Bray-Curtis) were performed in R and its extension (Phyloseq).

Results: Of 60 OPC patients, 65% were stage IV, 48% tonsillar and 42% base of tongue, and 40% never smokers. Pre-treatment levels of genus Klebsiella and class Gammaproteobacteria were significantly greater in the oral cavity of patients who had no/mild mucositis vs. those who developed severe mucositis (p<0.04). Genera Atopobium and Mogibacterium, as well as families Coriobacteriaceae and Clostridiales Incertae Sedis, were significantly more abundant in patients who did not develop oral candidiasis compared to those who did (p<0.04). Oral bacterial alpha diversity (intra-subject) was higher in patients who developed vs. did not develop severe mucositis, and lower in patients who developed vs. did not develop candidiasis during treatment. Analyses of beta diversity (inter-subject) showed that the microbial community structures were not significantly different by mucositis or candidiasis development.

Conclusions: Microbiome profiling may hold promise as a prognostic biomarker of treatment-related toxicities. Several potentially predictive taxa were identified to be differentially abundant in OPC patients who subsequently developed oral mucositis or candidiasis. Future analyses will evaluate the role of oral microbial resilience (the rate of recovery after disturbance) using oral specimens collected 3 mo. after treatment. Translational research focused on understanding how oral microbiota influence local immune and inflammatory responses may aid in the development of microbiota-based interventions to minimize adverse events.

#3327

HPV vaccination among adults at high-risk for HIV infection: A public health priority.

Lisa T. Wigfall,1 Shalanda A. Bynum,2 Jessica Wells,3 Jennifer K. McGee-Avila,4 Nikita S. Wagle5. 1 _Texas A &M University, College Station, TX; _2 _National Institutes of Health, Center for Scientific Review, Bethesda, MD;_ 3 _Emory University, Atlanta, GA;_ 4 _Rutgers, The State University of New Jersey, New Brunswick, NJ;_ 5 _Texas A &M University Health Science Center, College Station, TX_.

This study describes HPV vaccination rates among individuals who engaged in HIV infection high-risk behaviors (injection drugs and/or high-risk sexual behaviors) because HIV/HPV co-infection increases HPV-associated cancer risk.

Data from the 2016 Behavioral Risk Factor Surveillance System (BRFSS) survey were used to describe HPV vaccination rates among HIV infection high-risk adults aged 18-36 years. We included: females aged 27-36 years for whom HPV vaccination was recommended in 2006; heterosexual males aged 23-29 years and gay/bisexual males aged 18-33 years for whom HPV vaccination was recommended in 2009. Stata/SE 15.1 was used to perform chi-square tests.

Of 486,303 adults who completed the 2016 BRFSS survey, only 3.39% (n=16,507/486,303) had used injection drugs and/or engaged in high-risk sexual behaviors. Among these 16,507 HIV infection high-risk adults, only 416 (2.52%) had complete data for all variables. About one fourth of gay/bisexual males aged 18-33 years had initiated the 3-dose HPV vaccine series (25.68%). Similarly, about one fourth of heterosexual females aged 18-36 years had completed the 3-dose HPV vaccine series (25.01%). Only 10.91% of heterosexual males aged 18-29 years had initiated the 3-dose HPV vaccine series. Age, sex, and sexual orientation differences in HPV vaccination rates were statistically significant (p=0.0047). All transgender men/women and gender-nonconforming individuals were unvaccinated (p=0.4138). While racial/ethnic differences in HPV vaccination rates were not statistically significant (p=0.0630), almost all non-Hispanic Blacks (96.09%) were unvaccinated. Although both HPV vaccine initiation (11.91%) and completion (15.72%) rates were higher among high-risk adults who had been tested for HIV, these differences were not statistically significant (p=0.0750).

In 2016, adult HPV vaccination recommendations included all females and gay/bisexual males aged 18-26 years, and heterosexual males aged 18-21 years. The Advisory Committee on Immunization Practices considers people living with HIV infection a special population of interest because of their increased HPV-associated cancer risk. That said, it was alarming that HPV vaccination rates were low among HIV infection high-risk adults in our study, including gay/bisexual males who have the highest HPV-associated cancer risk. For example, anal cancer risk among gay/bisexual males is exacerbated by HIV/HPV co-infection. Additionally, it was also alarming that almost all non-Hispanic Blacks in our study were unvaccinated, especially given the disproportionate burden of HIV/AIDS among this racial/ethnic minority group. Taking known HIV/AIDS and HPV-related cancer disparities among racial/ethnic and sexual/gender minority populations into account, it is essential that increasing HPV vaccination rates among unvaccinated HIV infection high-risk adults remain a public health priority.

#3328

Prevalence and distribution of high risk human papillomavirus types in invasive cervical cancer in South West Nigeria.

Nnamdi Orah, Adekunbiola Banjo. _Lagos University Teaching Hospital, Idi Araba, Nigeria_.

This study was carried out to determine the prevalence and distribution of human papillomavirus types in invasive cervical cancer in Nigeria. Paraffin embedded tissue blocks of all archival cervical cancers diagnosed in three laboratories from South-West Nigeria between 2010 and 2014 were analyzed for the presence of HPV DNA ,187 out of 285 samples were considered appropriate for HPV detection after histological evaluation. Of these, 160 (85.6%) were positive for HPV DNA. The five most common types identified as single types among HPV positive cases were HPV16 (46.9%), HPV18 (19.4%), HPV45 (11.9%), HPV35 (5.0%) and HPV31 (3.1%). Others were HPV33, 39, 51, 52, 56, 58, 59, 66 and 68. HPV16 and 18 in single/multiple infections accounted for 69.4% of the samples. Multiple infections were detected in 4.4%. All the adenosquamous and neuroendocrine carcinomas tested positive for HPV, while 86.1% and 66.7% of the squamous cell and the adenocarcinomas were positive respectively. These results are in consonance with reports from all other parts of the world that HPV 16 and 18 account for almost 70% of cervical cancers, supporting data that effective vaccination against these 2 types will reduce the cervical burden in South West Nigeria.

#3329

Lung cancer screening initiative and identification of novel blood biomarkers for early detection of lung cancer.

Adijan Kuckovic,1 Joseph Berei,1 Shylendra Sreenivasappa,2 Joseph Ross,1 Sandra Martell,3 Connie Vitali,1 William Schulz,4 Neelu Puri1. 1 _Univ. of Illinois College of Medicine Rockford, Rockford, IL;_ 2 _OSF Saint Anthony Center for Cancer Care at Rockford, Rockford, IL;_ 3 _Winnebago County Health Department, Rockford, IL;_ 4 _SwedishAmerican, Rockford, IL_.

In lung cancer, 81% of patients detected are late-stage, since early-stage patients are generally asymptomatic. Winnebago County has a 14% higher mortality rate for lung cancer when compared to the national rate; hence, to improve early detection of lung cancer in the Winnebago County, we implemented low-dose computed tomography (LDCT) screening. Currently, LDCT is used to detect lung cancer at early-stages, and the CDC has proposed new guidelines for early screening of lung cancer in individuals between 55-77 years of age with a 30 pack/year smoking history. Using circulating DNA from lung cancer patients and examining changes in methylation patterns in genes that are expressed in lung cancer patients can be used for early detection. Epidermal growth factor receptor (EGFR) is shown to be expressed in lung cancer patients' serum in all stages; hence, it can be used as a potential biomarker for early detection. To improve early detection of lung cancer in the Winnebago County, we educated physicians/smokers about LDCT screenings to promote early detection of lung cancer. We evaluated the number of LDCTs performed and recorded the number of lung cancer cases detected in the Winnebago County between June 2015 to October 2017. To explore EGFR as a promising biomarker, plasma was separated from whole blood and collected. Circulating DNA was extracted with the MagMax cell-free circulating kit and bisulfite converted. Identification of percent methylation patterns in the EGFR promoter region in CpG islands was obtained after next generation sequencing (NGS) followed by bioinformatics analysis. In our study, 1,116 LDCT screenings occurred in the Winnebago County. Lung cancer was diagnosed in 19 patients, out of which 11 patients (57.8%) were early-stage patients. We detected 45.4% of patients screened that were present with nodules compared to 24.2% of patients in the National Lung Cancer Screening Trial. We observed that 1.70% of patients referred for LDCT screening were diagnosed with lung cancer, compared to 0.87% of patients in the National Lung Cancer Screening Trial. This may be due to the higher incidence of smoking in Winnebago County (18%) compared to the nation (15.5%). Our screening study had 96.3% false positives, which is comparable (96.4%) to the results obtained in the National Lung Cancer Screening Trial. Using NGS, methylation in the promoter region of EGFR was studied using degenerate EGFR primers. In the promoter region of EGFR, our analysis showed that there are regions of hypermethylation (68-86%) in three CpG islands in sequences at the beginning of the promoter (8-241 bases). However, there was hypomethylation (6-21%) at four CpG island further downstream (375-689 bases from the beginning of the promoter). Currently we are studying the methylation patterns in normal subjects to determine whether these regions which could be used as predictive biomarkers for early detection.

#3330

Circulating HPV DNA as a biomarker for metastatic cervical cancer detection, genotyping and monitoring.

Zhigang Kang, Sanja Stevanović, Christian Hinrichs, Liang Cao. _National Cancer Inst., Bethesda, MD_.

Purpose: Circulating cell-free (cf) human papillomavirus (HPV) DNA is a unique tumor marker for metastatic cervical cancers. We developed a method to genotype and quantify blood circulating HPV DNA in patients with metastatic cervical cancer for patient selection, treatment monitoring, as well as informing data on an experimental T cell therapy.

Patients and Methods: we developed a digital droplet (dd) PCR method for HPV genotyping and quantification with cfDNA. In a retrospective study, HPV cfDNA was measured in serum samples from nine metastatic cervical cancer patients received tumor-infiltrating lymphocyte (TIL) immunotherapy. cfDNA data were aligned with the tumor HPV data, drug treatment, and clinical outcome. In a HPV screening, the genotyping and DNA copy number of HPV cfDNA from 47 cervical cancer patients were measured and the allele frequencies were determined in relative to total genomic cfDNA of each patient.

Results: The ddPCR assay is highly sensitive, specific, and capable of accurate quantification of both HPV16 and HPV18 cfDNA. In the clinical validation, we detected HPV cfDNA from 19/19 (100%) patients with HPV-positive metastatic and recurrent cervical cancer but not in any of the 45 healthy blood donors. The HPV genotype harbored in the patients' tumors was correctly identified in 87/87 (100%) sequential patient serum samples. Our data showed the use of HPV cfDNA for pharmacokinetic studies, effectiveness on an investigational therapy, and patient monitoring. In three patients who experienced objective responses, a transient HPV cfDNA peak was detected 2-3 days after TIL immunotherapy and a persistent clearance of HPV cfDNA was observed in only two patients with a complete response (CR). Among the HPV positive cases, the median HPV cfDNA allele frequency is above 30% of genomic single copy genes in these cervical cancer patients.

Conclusions: We developed and validated a highly sensitive and specific HPV cfDNA genotyping and quantification method. As a promising non-invasive tumor marker, HPV cfDNA may have value in detecting the efficacy of therapeutic agents and monitoring cervical cancer patients in remission. The cfDNA based HPV genotyping is currently being evaluated for patient selection in an experimental T-cell therapy against a specific HPV antigen. HPV cfDNA may also be a potential biomarker for early diagnosis of cervical cancer.

#3331

Association of lifestyle and clinical characteristics with receipt of radiotherapy treatment among women diagnosed with DCIS in the National Institutes of Health-AARP Diet and Health Cohort Study.

Maeve Mullooly,1 Diana R. Withrow,2 Rochelle E. Curtis,2 Shaoqi Fan,2 Linda M. Liao,2 Ruth M. Pfeiffer,2 Amy Berrington de Gonzalez,2 Gretchen L. Gierach2. 1 _Royal College of Surgeons in Ireland, Dublin, Ireland;_ 2 _National Cancer Institute, Rockville, MD_.

Introduction: The long-term risks and benefits of breast radiotherapy for the treatment of ductal carcinoma in situ (DCIS) remain unclear. A recent study using data from the Surveillance, Epidemiology and End Results (SEER) registries showed that DCIS-associated radiotherapy treatment significantly increased risks of second non-breast cancers. To help disentangle those observations and understand whether breast cancer risk factors are related to radiotherapy treatment decision making, we aimed to identify factors associated with receipt of radiotherapy treatment for DCIS.

Methods: Accordingly, among 1,628 women with DCIS who participated in the large prospective National Institutes of Health (NIH)-AARP (formerly American Association of Retired Persons) Diet and Health Cohort Study, that were diagnosed between study entry and follow up (through 31st December 2011) and eligible for inclusion, we examined associations between demographic, lifestyle and clinical factors and receipt of radiotherapy treatment. Odds ratios and 95% confidence intervals (CIs) were estimated from multivariable logistic regression models, to determine associations.

Results: Among women diagnosed with DCIS, 45% (n=730) received radiotherapy treatment. No relationships were observed between receipt of radiotherapy and lifestyle factors including smoking status. The strongest associations were observed for clinical factors. Receipt of radiotherapy was associated with a more recent diagnoses period (2005-2011 versus 1995-1999; OR=1.60, 95%CI: 1.14, 2.25), diagnosis within eastern (Michigan/New Jersey) versus western US states, (OR=1.44, 95%CI: 1.07, 1.93), poorly than well-differentiated tumours (OR=1.69, 95%CI: 1.16, 2.46) and receipt of endocrine therapy (OR=3.37, 95%CI: 2.56, 4.44).

Discussion: Clinical characteristics of the DCIS and temporal trends in radiotherapy use were the strongest determinants of radiotherapy treatment within this population. Receipt of radiotherapy was largely unrelated to lifestyle factors suggesting that the previously observed associations in SEER between radiotherapy treatment and risk of non-breast cancers were likely not confounded by lifestyle factors including smoking status. Thus, these findings are relevant for understanding the aetiology of subsequent secondary cancers among the increasing population of DCIS survivors. The lack of association observed for breast cancer family history and prior breast biopsy suggest that these risk factors do not influence radiotherapy clinical decision making.

#3332

Clonal nuclear and mitochondrial genetic alterations in smoker lung cancer patients and their histologically normal appearing follow-up biopsies.

Julie V. Philley,1 Kayla Johnston,2 Hirendra N. Banerjee,2 David E. Griffith,1 Santanu Dasgupta1. 1 _UT Health Science Ctr. at Tyler, Tyler, TX;_ 2 _Elizabeth City State University, Elizabeth City, NC_.

Understanding the cascade of molecular events leading to metastatic progression of lung squamous cell carcinomas (LSCC) is critical to intervene with appropriate treatment options. In this era of precision medicine, identification of key genetic alterations through cutting edge technologies such as high-resolution single nucleotide polymorphism arrays and next generation sequencing have revolutionized cancer therapeutics and surveillance impacting overall survival. The present study investigated resected primary cancer tissues and matched bronchoscopically abnormal follow up mucosal biopsies from 4 LSCC patients with an aim to characterize clonal molecular genetic changes therein. A total of 25 samples including matched normal lymph node as control from these subjects were analyzed on Affymetrix high-resolution 250K SNP array (NspI) platform. Copy number loss and copy neutral loss (LOH) and genomic amplifications were measured in these samples and clonal molecular alterations were also assessed. We observed clonal copy number and copy neutral loss in various potential tumor suppressor genes (TSGs) including GPC5, LTBP1, SOX6, IRF2, LRP8, GSTT1, DAPK1, CACNA1C, DNAH8, CDKAL1, PPP2R2B, CDH4, MAL1D1, PTPRT, NRG2 and FMNL2. Notably, homozygous deletion was observed in one patient in 10q11.1-10q11.22 chromosomal region. The genes effected due to homozygous deletion includes: MARCH8, ANUBL1, FAM21C, CTGLF1, PTPN20A, PTPN20A, PTPN20B, FRMPD2L1, FRMPD2L2, SYP15, GPRIN2, PPYR1 and ANXA8. Clonal amplification (3-10 fold) of molecules that primarily function in mitochondria was also noted in primary cancer and corresponding mucosal biopsies of the majority of these subjects. This includes SUPV3L1, PDK2, MDH1, VDAC2, LONP1, NDUFA11, and OGG1, key molecules, which regulate normal mitochondrial function and homeostasis. Thus, molecular profiling of histologically normal appearing biopsies utilizing high-resolution SNP arrays identified primary cancer associated novel nuclear and mitochondrial genetic alterations in resected lung cancer patients who were heavy smokers. Presence of LSCC associated clonal genetic alterations in the histologically normal appearing mucosal biopsies suggests for possible treatment refractory secondary tumor evolution from the respiratory epithelium harboring the necessary molecular aberrations. Periodic navigation through molecular profiling of the resected LSCC patients and further functional characterization of the identified molecules could lead to the development of new monitoring and treatment strategies.

Acknowledgement: Supported by UTHSCT, Willett Foundation and ECSU-NIH-MARC award.

#3333

Potential of kava in reducing lung cancer risk, tobacco use, and associated disparities.

Chengguo Xing,1 Yi Wang,1 Naomi Fujioka,2 Sreekanth Narayanapillai,1 Junxuan Lu3. 1 _Univ. of Florida, Gainesville, FL;_ 2 _Univ. of Minnesota, Minneapolis, MN;_ 3 _Penn State University, Hersey, PA_.

Tobacco use is the primary risk factor for lung cancer, the leading cause of cancer deaths in the US. Quitting, however, is challenging due to the addictive nature of nicotine. Thus, preventing lung carcinogenesis in conjunction with smoking cessation may be necessary. Racial disparities also exist - African American (AA) smokers, with the same level of tobacco consumption, are at the highest risk of developing lung cancer. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one key carcinogen in tobacco for human lung cancer risk and its differential uptake in AA and CA smokers may contribute to the disparity. AA smokers also have the lowest success rate in quitting. Kava is a daily beverage to help people relax and improve the quality of sleep. It is commercially available in US as a dietary supplement. Epidemiological data suggest that kava consumption may reduce cancer risk. Its relaxing property may help reduce tobacco dependence/use. Our preclinical data showed that kava completely blocked NNK induced tumorigenesis in A/J mice with enhancing NNK detoxification and reducing DNA damage as the potential mechanism. Building upon these, a pilot pre- and post- one-week kava trial was performed among smokers (n = 21). The results showed that kava was well-tolerated with high compliance and there were no signs of adverse effects when rigorous safety measures were implemented. Excitingly, the results suggested that one-week kava intake helped smokers 1) reduce tobacco use; 2) reduce the amounts of urinary DNA adducts; and 3) increase urinary excretion of NNAL. Specifically, urinary Total Nicotine Equivalent (TNE), the sum of nicotine N-oxide, total nicotine, cotinine, and 3-HO-cotinine, was used to estimate tobacco use and one-week kava use led to a 29.8% reduction in TNE (p < 0.001). Urinary 3-methyladenine (3-mA), a NNK-dependent DNA adduct biomarker, was used to estimate NNK-induced DNA damage and one week kava used led to a 33.6% reduction (p = 0.014). Lastly, urinary total NNAL was used as a biomarker for NNK excretion and one-week kava use led to a 95% increase (p = 0.002). Plasma cortisol of smokers, a stress biomarker, decreased by > 40% (p<0.01) after kava use, which may contribute to the reduction in tobacco use. The 21 participants include 13 CA, 7 AA and one American Indian. The impact of kava was also analyzed between AA and CA smokers. The reductions in urinary TNE and 3-mA were more pronounced in AA smokers while the increase in urinary NNAL was higher in AA smokers as well. Consistently, the reduction in plasma cortisol was greater in AA than CA smokers. The results from this pilot trial indicate kava's potential to reduce lung cancer risk via reducing tobacco use, enhancing NNK/NNAL detoxification, and reducing DNA damage. The results also strongly suggest kava's unique potential to address the associated racial disparities. Future rigorous trial is needed to confirm these observations.

#3334

Predictive therapeutic value of NGS with liquid biopsy in metastatic NSCLC.

Silverio Tomao, Monica Verrico, Luigi Rossi, Iacobelli Stefano. _University of Rome, Rome, Italy_.

Analysis of circulating tumor cells ( CTCs) and circulating cell-free DNA (cfDNA) , detected in peripheral blood of patients, is an extraordinary novel strategy to identify and study NSCLC patients, in order to obtain prognostic and predictive molecular informations with a simple , non invasive and sensitive method. Liquid biopsy therefore offers a new source of cancer cells and cancer-derived materials in order to test more personalized treatment. With two PCR-based next generation sequencing (NGS) approaches we evaluated 45 patients with not squamous NSCLC ; in eighteen pts ( 40 % ) almost one genomic mutation was detected among 56 different genes analyzed with NGS. Median age of patients was 69 years ( 55-78) and 14 (73%) were males. Altogether, 11 genes were mutated with a panel of 56 genes analyzed (19,6% ) . The most frequent mutations occurred in KRAS, TP53, MET,KDR, KIT, SMAD4 and MET genes. Several patients expressed many mutations and the most frequent detected concerned gen c-Kit ( 22%). Other mutations concerned the following genes : TP53 ( 16 % ), MET/KDR. / SMAD4/KRas ( 44%). Other genomic alterations concerned the following genes: JACK3 (5%); APC(5%); ERBB4(5%); CTNNB! (5%); EGFR (5%). Altogheter the response to therapy was higher among patients with mutation of Smad 4 gene and lower in patients with mutations of KIT gene. All side effects occurred with target therapy was categorized according to the Common Terminology Criteria for Adverse Events, 4.0 version. An increase of gastroenteric (G3/G4) and hematologic toxicity (G3/G4) was observed in patients with Kit mutations. In our study this correlation was observed mainly during treatment with Tyrosine Kinase Inhibitors (TKIs ).Evaluating Progression Free Survival (PFS) in all the patients recruited in the study, a median PFS of 12.5 , 11.5 , 4 and 9 months was observed respectively in patients with mutations of KIT , SMAD4 , TP53 and MET. In this study, all the genetic alterations analyzed and highlighted were related to the response to chemotherapeutic treatments and possible tyrosine kinase inhibitory treatments in terms of PFS and toxicity.

### Science and Health Policy 1

#3335

Innovative mathematical model links TRPV6 genotype to high triple negative breast cancer mortality rate in African-American females.

Constance B. Hilliard. _University of North Texas, Denton, TX_.

INTRODUCTION: The mortality rate for African-American (AA) females is 4 times that of Whites due to the former's higher susceptibility to Triple Negative Breast Cancer (TNBC). This study suggests that the African/ancestral TRPV6 calcium ion channel may be more calcium-absorbent than the European/derived TRPV6 variant. The 800 mg. daily calcium intake of AA women is twice that of their low-osteoporotic Niger-Kordofanian ancestors. This maladaptation may expose AA females' mammary tissue to free calcium ions in excess of biological need. Ca2+ flooding appears to trigger over-expression of TRPV6 transcript, which becomes a biomarker for estrogen receptor (ER)-negative breast tumors. Therefore, this study introduces a Genetic-Population Specific Mathematical Model (GPs Mathematical Model), which identifies the ancestral TRPV6 calcium ion channel in AA females as a novel therapeutic target for TNBC and suggests TNBC-avoidant dietary guidelines for this ethnic population. .

METHODS: The GPs Mathematical Model correlates ratios of DNA ancestry and calcium intake levels per genetic population. An interactive map based on data from 74 countries provided global calcium intake. Retrospective data sets on the genetic ancestry of 5,269 self-described African Americans, 8,663 Latinos, and 148,789 European Americans were correlated to the 1000 Genomes Project data.

RESULTS: This Model identifies a normal range of dietary calcium consumption for AAs. The admixture ratio of this population is on average, 0.74 Niger-Kordofanian DNA; 0.24 Northern European DNA; 0.01 Native American DNA. Nutritional studies indicate that the average daily calcium intake for healthy Niger-Kordofanians, Northern Europeans and Native Americans are 200 to 400 mg, 1000 to 1200 mg, and 1100 mg. respectively or 0.75 (NK DNA Ancestry (200,400) + 0.24 NE DNA Ancestry (1000, 1200) + 0.01 NA DNA Ancestry(1100) = a range from 401 to 599, averaged to 500 mg/calcium/day. This Model shows that the calcium intake of AA females is 160% to 240% higher than the theoretical value derived from this ethnic group's genetic ancestry.

SUMMARY: This Genetic Population-Specific Mathematical Model suggests that the higher calcium intake of AA females relative to their genetic ancestors may create hyper-reactivity of ancestral TRPV6 leading to TNBC.

CONCLUSION: Dietary calcium intake in excess of biological needs may trigger AA females' high susceptibility to "TRPV6-expressing" Triple Negative Breast Cancer. The Genetic Population-Specific Mathematical Model provides an AA set-point of 500mg/d for dietary calcium intake, which is 0.375 lower than this ethnic population's average consumption of 800 mg/calcium/day. The use of TRPV6 inhibitors should be tried in order to control the disease. In addition, a reduction in dietary calcium may have a prophylactic effect on this population's high susceptibility to TNBC.

#3336

Changes in prescriptions for breast cancer medications after Medicaid expansion.

Johanna Catherine Maclean,1 Michael T. Halpern,2 Steven C. Hill,3 Michael F. Pesko4. 1 _Temple University, Philadelphia, PA;_ 2 _Temple University College of Public Health, Philadelphia, PA;_ 3 _Agency for Healthcare Research and Quality, Rockville, MD;_ 4 _Georgia State University, Atlanta, GA_.

Introduction: As of summer 2018, 34 states have expanded Medicaid eligibility under the Affordable Care Act. While the Medicaid expansions decreased rates of being uninsured among women with breast cancer and increased early breast cancer detection, it is unknown whether expansions increased receipt of medications used to prevent and treat breast cancer. This study examines differences over time in receipt of two types of breast cancer hormonal therapies (tamoxifen and aromatase inhibitors) and associated payments for these medications in states that did vs. did not expand Medicaid during the period 2011-2017.

Methods: The study's data source is the Medicaid State Drug Utilization Database (SDUD). This data set, compiled by the Centers for Medicaid and Medicare (CMS), is administrative data submitted by state Medicaid programs. The data comprise outpatient prescription medications that are covered under the Medicaid Drug Rebate Program for which Medicaid serves as a third-party payer; this includes aggregate numbers of prescriptions and associated payments for individuals enrolled in both fee for service and managed care Medicaid programs. Both branded and generic prescriptions for three aromatase inhibitors (anastrozole, exemestane, and letrozole) were included in the study's analyses. Analyses used differences-in-differences and event study models (controlling for state characteristics) to compare changes in Medicaid expansion states to changes in non-expansion states before vs. after expansion.

Results: Initial regression analyses indicate that prescriptions for all hormonal therapy medications increased by 27% (p<0.05) and prescriptions for aromatase inhibitors (branded and generics combined) increased by 29% (p<0.05) in expansion states relative to non-expansion states. Prescriptions for tamoxifen and for generic aromatase inhibitors only also increased significantly. Post-expansion, both total payments and Medicaid payments for all hormonal therapies increased by 13% (p<0.01) in expansion states relative to non-expansion states. The similar increases for total and Medicaid payments suggest that state Medicaid programs, not patients, financed the increased hormonal therapy prescription payments.

Conclusions: Our findings indicate that states that expanded Medicaid with the ACA experienced increased prescriptions for breast cancer hormonal therapies relative to states that did not expand Medicaid; this effect of Medicaid expansion increased over time. The increased prescriptions were financed by Medicaid, not by patients.

#3337

A racially/ethnically diverse 3D PDX model of prostate cancer.

Lindsey K. Sablatura,1 Kristin M. Bircsak,2 Peter Shepherd,3 Rick Kittles,4 Pamela E. Constantinou,3 Anthony D. Saleh,2 Nora M. Navone,3 Daniel A. Harrington5. 1 _Rice University, Houston, TX;_ 2 _Mimetas US, Gaithersburg, MD;_ 3 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 4 _City of Hope, Duarte, CA;_ 5 _UTHealth, Houston, TX_.

Prostate cancer (PCa) incidence and mortality rates in African American men are double that of any other race/ethnicity in the United States. Thorough understanding of the biological factors that contribute to this long-standing cancer health disparity (CHD) is required to improve the major public health concern and close this gap. However, few models exist that can compare racially-diverse specimens directly and provide a platform for dissecting the impact of ancestry-dependent factors on disease pathway selection and drug susceptibility. Both the conventional 2D culture of clonal human cell lines and the purely rodent-based in vivo models fail to reflect the heterogeneity of human tumors, often leading to inaccurate prediction of in vivo tumor response in patients, and confounding researchers' ability to detect potentially subtle biological factors that may contribute to prostate CHD. PCa patient-derived xenografts (PDXs) offer substantially greater fidelity to original patient tumors but are non-adherent and ultimately non-viable in extended in vitro 2D culture. Therefore, a population-based PCa platform which accurately mimics the three-dimensional (3D) tumor microenvironment (TME) is urgently needed.

We have employed MIMETAS' OrganoPlate®, a high throughput microfluidic culture platform containing 40-96 individual tissue chips, for ex vivo 3D culture of multiple racially/ethnically diverse PCa PDXs (African American, Caucasian, Hispanic) developed at MD Anderson Cancer Center (the MDA PCa PDXs series). MDA PCa PDX tumors were reconstituted from single-cell digestates into multicellular clusters, suspended within HyStem® hyaluronic acid hydrogel precursor solutions, and loaded into the OrganoPlate®. PDXs were maintained in 3D either as monocultures, as cocultures with bone marrow-derived stromal fibroblasts, or as tricultures with endothelial cell blood vessel mimics under continuous perfusion. High-content fluorescence imaging identified retention of stable, viable cultures for at least 7 days. Positive immunofluorescent staining for human nuclear antigen (HNA) confirmed that nearly 100% of encapsulated PCa cells were of human origin. For each PCa model developed, appropriate expression of phenotypic prostate-specific antigen (PSA) and androgen receptor (AR) was maintained over the life of the culture. PCa cultures were treated with various chemotherapeutic drugs and viability was monitored to generate dose response curves for comparison to clinical data. This engineered "tumor-on-a-chip" will better predict patient responses and, by incorporating PCa cells from patients with diverse ancestries, support CHD research.

#3338

Understanding the effect of racial disparities on patients response to dietary intervention during cancer therapy.

Sylvester Jusu, Kaitlyn Dykes, Kevin Ko, Tiziana DeAngelis, Edith Mitchell, Nicole Simone. _Thomas Jefferson University, Philadelphia, PA_.

Purpose:

African American (AA) women have a lower incidence of breast cancer however, their mortality rate is significantly higher compared to Caucasian (C) women. It is postulated that this is in part, due to the increased prevalence of chronic metabolic syndromes such as obesity and diabetes in the AA population. Also, cytotoxic therapies such as radiation, have decreased efficacy in obese and diabetic patients compared to normal weight counterparts. In this study we investigated whether caloric restriction (CR) during radiation, could affect metabolic differences to affect this disparity. We hypothesize that CR may better benefit AA patients via modulation of inflammatory pathway to improve cancer outcomes.

Methods:

Thirty five patients (16 AA/16C) with Stage 0 or 1 breast cancer were accrued from 2013-2016 and enrolled on an IRB approved study (NCT01819233). Baseline caloric intake was assessed by food diaries for 7-10 days prior to RT simulation and patients were counseled to decrease their total caloric intake by 25% starting after RT simulation. Patients underwent CR for 10 consecutive weeks: 2 weeks of CR alone, 6 weeks of CR and RT to a dose of 60Gy, and 2 weeks of CR alone after RT. Patients were evaluated before and after the 10 weeks for biometric properties, quality of life metrics and serum markers.

Results:

Compliance with diet between AA and C and cohorts was comparable and while the total cohort lost an average of 9.21 ± 1.27 lbs, the C cohort had a greater decrease in weight of 11.67 ±2.56 lbs compared to the AA patients who lost an average of 7.33 ±1.62 lbs. To determine if the AA patients had a molecular benefit to diet, oncomiR-21, a regulator of inflammation was evaluated. miR-21 increased by 200 fold in control patients treated with RT without diet. The C patients on CR had a dampened upregulation with a miR-21 that only increased 3.2 ± 0.02 fold, while the AA patients had a significant change with an upregulation of 1.9 ± 0.55, p<0.001. A 200 analyte protein array analysis showed AA patients had a unique protein profile with 15.42 % down-regulated analytes compared to 10.45 %. AA patients showed significant reduction in pro-inflammatory markers including IL-1, IL-6, EGFR, bNGF, FGF-4, TGF-b and BMP 5 and BMP-7 (p<0.05).

Conclusions:

Though AA patients experienced less weight loss compared to their C counterparts, the molecular benefit experienced was significant in terms of the decreased inflammatory response, skin toxicity and decrease in migration/invasion pathways. Further investigation using diet to narrow the breast cancer outcome disparity experienced by our AA patients is warranted.

#3339

Ethnic disparity in ovarian malignancy tumor markers: MIA and ROMA.

Charles Dunton,1 Herbert Fritsche,2 Rowan Bullock3. 1 _Lankenau Medical Center, Broomall, PA;_ 2 _Aspira Labs, TX;_ 3 _Vermillion, CT_.

Objective: Review and analyze serum values of Risk of Ovarian Malignancy Algorithm (ROMA) and Multivariate Index Assay (MIA) in subgroups of women who underwent surgery for adnexal masses to determine sensitivity in different ethnic populations.

Methods: Serum samples from 179 women diagnosed with ovarian malignancy were analyzed for ROMA and MIA results. Biomarker data was obtained from previous prospective studies that validated the MIA test. Of these, 167 women were Caucasian (C) and 12 African-American (AA) in the MIA testing. Sample for ROMA included 161 C and 11 AA. Sensitivity (true positive rate) for preoperative test results were calculated using DTCompair package of the R programming language. In premenopausal women, a risk of ovarian malignancy algorithm (ROMA) value equal to or greater than 1.14 indicates a high risk of finding epithelial ovarian cancer. In premenopausal women, MIA values of greater than 5.0 are associated with greater risk of malignancy.

In postmenopausal women, a ROMA value equal to or greater than 2.99 indicates a high risk of finding epithelial ovarian cancer.

In postmenopausal women, MIA values of greater than 4.4 are associated with greater risk of malignancy.

Results: Primary ovarian malignancy was diagnosed in 179 cases (167 C/12 AA).

Sensitivity testing results are seen for each subgroup and for MIA levels and CA125 results:

Primary Ovarian Malignancy: Caucasian MIA 97.0%, ROMA 89.4%, AA MIA 75.0% ROMA 45.5%.

Sensitivity of ROMA in AA premenopausal women was 50% and in postmenopausal women 33.3 %.

Conclusion: Our results demonstrate that ROMA in both C andAA women with adnexal masses have lower sensitivity for detection of malignancy than does MIA. It is more pronounced in AA women. Implementation of MIA in evaluation of adnexal masses will increase sensitivity of detection of malignancy compared to CA125 testing, with most marked results in AA women.

Key Words: Ethnicity, CA125, and Multivariate Index Assay

Sensitivity of MIA vs. ROMA Ovarian Malignancy

---

Race | Test | StatusMenopause | Number | Sensitivity

Caucasian | MIA | All | 167 | 97.0

Caucasian | MIA | Pre | 43 | 95.3

Caucasian | MIA | Post | 124 | 97.6

Caucasian | ROMA | All | 161 | 89.4

Caucasian | ROMA | Pre | 40 | 92.5

Caucasian | ROMA | Post | 121 | 88.4

African America | MIA | All | 12 | 75

African America | MIA | Pre | 9 | 77.8

African America | MIA | Post | 3 | 66.7

African America | ROMA | All | 11 | 45.5

African America | ROMA | Pre | 8 | 50.0

African America | ROMA | Post | 3 | 33.3

#3340

Lung cancer stigma: A ten year look at public attitudes about lung cancer.

Maureen Rigney,1 Eleni Rapsomaniki,2 Jennifer C. King1. 1 _Lung Cancer Alliance, Washington, DC;_ 2 _AstraZeneca, Gaithersburg, MD_.

Background: Lung cancer is the leading cause of cancer death in the United States in both men and women (ACS Facts and Figures, 2018). The presence of lung cancer stigma is well documented (Chapple et al, 2004; Chambers et al, 2012; Marlow et al, 2015) and has been shown to impact the care and treatment of lung cancer survivors (Tod et al. 2008; Carter-Harris et al 2014). In 2008, a large survey of over 1000 members of the general population revealed that most participants felt lung cancer was principally caused by external factors, that it was preventable, and that lung cancer patients were at least partly to blame for their illness (Weiss et al. 2014; 2017). We replicated the survey to understand whether perceptions have changed over the last decade.

Methods: 1001 members of the general public were surveyed with the identical survey instrument from 2008 survey along with three additional questions at the end. The survey was carried out by phone and online between June 6 and July 26, 2018. Statistical analysis was performed comparing 2008 and 2018 datasets using paired t-tests if normally distributed or Mann-Whitney U tests for continuous data and Chi-squared or Fisher's exact test for categorical data.

Results: General awareness about lung cancer has significantly improved over the last decade with 94% of the public reporting familiarity with lung cancer (p<.001). Sixty five percent of respondents reported increased visibility of lung cancer in the media. Despite the increased visibility of the disease, stigma remains a critical problem. There were no statistically significant changes in the proportion that felt that patients are at least partly to blame for their illness (56% in 2018). Sixty-eight percent of respondents felt that patients experience the same or more stigma than 10 years ago. In addition, despite the increased awareness there was no increase in the proportion of people who donate or volunteer time to lung cancer organizations and no increase in those selecting lung cancer as the type of cancer they believe should receive more research support.

Conclusions: After a decade of research progress in lung cancer, these data show that lung cancer awareness has been considerably elevated. Unfortunately, they also indicate that the stigma surrounding the disease remains pervasive. This work underscores the need to address stigma with proactive multilevel approaches (Hamann, 2018).

#3341

Rural and urban disparities in colonoscopy use persisted despite cost-sharing reduction among Medicare beneficiaries.

Min Jee Lee, Wiley Jenkins, Eric Adjei Boakye, Sabha Ganai. _Southern Illinois University, Springfield, IL_.

Background: Due to passage of the Patient Protection and Affordable Care Act (ACA), Medicare began waiving Part B deductibles and eliminating coinsurance for all colonoscopies in 2011. As rural populations have significantly lower income than urban, there is hope the cost reduction would decrease rural screening disparities. We thus examined rural-urban colonoscopy use pre-/post-ACA implementation.

Methods: We used Behavioral Risk Factor Surveillance System data (2008-2016) to examine colonoscopy utilization for two years pre-ACA (2008 and 2010) and three years post-ACA (2012, 2014, and 2016) with stratification by rural/urban residence. Multivariate logistic regression was used to examine the differences in screening likelihood across rural and urban groups, while controlling for other factors such as age, sex, marital status, race/ethnicity, household income, educational attainment, gastroenterologist availability, exercise, smoking, and years.

Results: Of the 302,941 eligible Medicare beneficiaries, 203,426 (67.2%) received a colonoscopy. Colonoscopy receipt increased from 62.8% pre-ACA to 70.2% post-ACA years (p<.001). Across rural-urban areas, we found a significant increase in the receipt of colonoscopy from pre- to post-ACA (p<.001). The receipt of colonoscopy was higher among urban residents (69.2%) compared to rural residents (63.5%, p<.001). After adjusting for covariates, urban residents were more likely to receive colonoscopy both pre-ACA (OR=1.11, 95% CI: 1.06-1.15) and post-ACA (OR=1.11, 95% CI: 1.06-1.16).

Conclusions: Despite cost-sharing reduction of Medicare coverage for colonoscopy, rural and urban differences in colonoscopy use persisted over time among Medicare beneficiaries. Although the receipt of colonoscopy increased over time, the gap between rural and urban populations has remained.

Colonoscopy Use Before and After Implementation of The ACA

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|

PreACA | |  | |

Post ACA | |

|

Ruality | % | OR | 95% | CI | % | OR | 95% | CI

Rural | 59.4 | 1 | |  | 66.6 | 1 | |

Urban | 64.9 | 1.11 | 1.06 | 1.15 | 72.1 | 1.11 | 1.06 | 1.16

#3342

A novel indigenous knowledge system based approach to study cancer health disparities in rural population of North East India.

Lekhika Pathak,1 Bidisha Pal,2 Tutumoni Baishya,3 Anupam Sarma,1 Bikul Das2. 1 _KaviKrishna Laboratory, IIT-Guwahati Campus, Guwahati, India;_ 2 _Thoreau Lab for Global Health, Lowell, MA;_ 3 _KaviKrishna Telemedicine Center, Sualkuchi, India_.

Background: India's North East is an economically deprived and politically unstable region inhabited by diverse groups of indigenous people. The incidence of cancer (mainly oral) is unusually high in the region. Through our KaviKrishna telemedicine care (www.kavikrishnalab.org) with collaboration of Thoreau Lab for Global Health (www.thoreaulab.org) we have initiated a longitudinal study in the greater Sualkuchi (50,000 population) area to identify different elements that underlie cancer health disparities. Specially, we wanted to develop an indigenous knowledge system (IKS) based approach (1) as an analytical tool to study cancer health disparities. IKS may be defined as a knowledge-emergence mechanism as a result of non-linear communication between an individual and his/her culture (1).

Methods: An IKS-based experimental approach was developed as follows. First, we identified 45 cancer patients (mostly oral cancer, age group- 30-65 years; 60% male) living in Sualkuchi. Second, we mapped out each patient's social network/support system through regular home visit, interviews, focused group discussion, and visits to attending doctors. Third, one group of patients (n= 30 out of 45 patients) and relatives were empowered with basic knowledge about their disease and the available treatments needs through regular focused group meetings. After three months, we repeated interviews/group discussion to find whether they communicated their knowledge/experience to generate an IKS-based system (1). The data were then fed into the IKS-network analysis tool, which we developed partly, based on hub-system (3) and thematic network based approach (3). 10 patients from urban area served as a control population.

Results: We found that 40 out of 45 patients find it extremely difficult to navigate the complex cancer care system due to lack of communication with treating oncologists/surgeons as compared to urban patients. 15 patients discontinued follow up visit mainly for this reason alone. 25 patients showed evidence of communicating through a pre-existing IKS-based system. Surprisingly, these 25 patients were positively responding to our care services/group discussion and becoming emotionally and psychologically strong towards managing their self-care.

Conclusion: Our study indicates that lack of communication is a major cause of cancer health disparity. An IKS based approach may enhance communication, and contribute to cancer care in rural India.

1. Bikul Das. Globalization and Emerging Opportunities of Indigenous Culture. 2003. (www.academia.edu/ 7882695).2. Anita Kothari et al. Using an integrated knowledge translation approach to build a public health research agenda. Healthy Research Policy and Systems, 2014. (PMID: 24475759).3. Jennifer Attride-stirling: Thematic networks: an analytic tool for qualitative research. 2001. Qualitative Research.

#3343

The Florida women's cancer study: Breast cancer presentation among African American and Afro Caribbean women in south Florida, a 10-year cohort.

Priscila Barreto Coelho,1 Matthew Schlumbrecht,2 Danielle Cerbon,3 Carlos Parra,4 Judith Hurley,2 Sophia George2. 1 _Jackson Memorial Hospital, Miami, FL;_ 2 _Sylvester Comprehensive Cancer Center, Miami, FL;_ 3 _Universiy of Miami, Miami, FL;_ 4 _University of Miami, Miller School of Medicine, Miami, FL_.

Introduction: The Black population in the US constitutes about 4 million immigrants. Of this, 50% is from the Caribbean region. Jamaica is the largest contributing country, followed by Haiti and Trinidad and Tobago. Florida has the second largest Caribbean population in the USA. Breast cancer is the main cause of cancer death among females responsible for 14%-30% of cancer deaths in the Caribbean; this is up to two times higher than the USA. Little is known about the molecular subtypes of breast cancer and the demographics of Black Caribbean Immigrants. Objectives: Study the demographics of BC patients in the African diaspora, determine similarities and differences in cause, subtype and outcome of women with breast cancers in African American (AA) and Afro-Caribbean (AC) immigrants.

Methods: Approved by the Ethics Committee of the University of Miami IRB. Patients treated for breast cancer between 2006 to 2016 were included. Abstracted data included sociodemographic factors, genetic testing results, and treatment histories.

Results: A retrospective US-based cohort of 1369 women (self-identified as black), diagnosed with BC - the Florida Women's Cancer Study (FLWCS), at Sylvester Comprehensive Cancer Center and Jackson Memorial Hospital in Florida. This cohort contains data from 624 (46%) African-American (AA) women and 507 (37%) AC women diagnosed with breast cancer between 2006-2016. Ninety per cent (n=1232) of the cohort is of non-Hispanic ethnicity.FLWCS country distribution includes is composed of Haiti (18.3%), Jamaica (6.5%), Bahamas (3.1%), Cuba and Dominica Republic (2.8% each), Trinidad and Tobago (1%) and other nationalities from the Organization of Eastern Caribbean States (Antigua, St. Kitts and Nevis, Anguilla, Dominica, St. Lucia and Grenada) at 2.5%. ACwomen living in Miami were diagnosed at a younger age (53.7 years versus 54.9 years), than AA women.Twenty-eight percent of the AA women were premenopausal compared with 32% of the AC women. The AA women had a higher BMI, 32.1 vs 29.8 (p=0.0001), a lower proportion of HER2 positive breast cancer of 17.6% versus 23% (p=0.027), and more children 3.1 versus 2.8 (p=0.023), than the AC women. The rate of HER2 overexpression in the Caucasian population is12% while HER2 positivity was seen in 26.2% Jamaican women, 25.8% in Haitian women and 26.5% in Cuban women (p=0.043) respectively. Less than 5% of the cohort underwent genetic testing with less than 1% having a pathogenic germline mutation.

Conclusion: African American (AA) and AC women have many similarities there are significant differences in terms of ancestral diversity, inherited genetic mutations, environmental exposures and access to medical care. Thus, it is imperative to gain an understanding of the causes of cancer in AC women in their own countries in order to better serve those immigrant populations once they reach the US.

#3344

Effect of anticoagulation on the cancer stage at time of diagnosis of endometroid adenocarcinoma in a cohort of postmenopausal patients.

Ghadear Shukr, Tra Pham, Thomas Buekers. _Henry Ford Hospital, Detroit, MI_.

Objectives: We sought to determine whether postmenopausal women on chronic anticoagulation were diagnosed with endometrial cancer at an earlier stage.

Methods: A retrospective case-control study of women at a tertiary care medical center with a diagnosis of Endometrioid Adenocarcinoma on chronic anticoagulation at time of diagnosis in comparison with patients not on anticoagulation was performed. Patients with personal history of cancer, including breast cancer, non-postmenopausal bleeding, and non-Endometrioid Adenocarcinoma histopathology were excluded. Anticoagulation was defined as use of Enoxaparin, Warfarin, Apixaban, Rivaroxaban or Clopidogrel. Indications for anticoagulation included atrial fibrillation, deep vein thrombosis/pulmonary embolism, or stroke. The primary outcome was Endometrioid Cancer stage (I, II, III/IV and X). The secondary outcomes were duration of bleeding to presentation and presentation to diagnosis. Statistical analysis was performed using Wilcoxan-Rank Sum Test and Cochran-Armitage Trend Test.

Results: 25 postmenopausal patients met the inclusion criteria for being treated in the last 10 years. 100 controls not on anticoagulation (non-AC) were identified for a 4:1 comparison to obtain a 2-sided alpha level of 0.05. Results were divided into 3 groups: 1) All patients, 2) Non-white patients, 3) White patients. Subjects were controlled for age (P<0.001) and BMI (P<0.049). The results indicated that non-AC patients were more likely to be diagnosed at Stage 1 when compared to the AC patients in all 3 categories. The trend was not statistically significant for the non-white patients (p=0.133), but was statistically significant in the overall population (p<0.001) and White patients (p<0.001). No statistical significance was detected for duration from bleeding to presentation and presentation to diagnosis between the AC and non-AC groups. However, median days from bleeding to presentation are considerably larger for the non-AC than AC (Group 1: 42 vs 14 days, Group 2: 75 vs 33 days Group 3: 30 vs 14 days, respectively).

Conclusion: Non-anticoagulated patients had an earlier cancer stage at diagnosis. Although anticoagulated patients may present earlier, they have advanced cancer stage. These findings should prompt immediate evaluation of patients on chronic anticoagulation to optimize oncologic management and prognosis.

#3345

Making the link between breastfeeding and breast cancer risk reduction among Hispanic women of childbearing age.

Karoline Sondgeroth,1 Rosalba Ruiz-Holguin,2 Joe Padilla,1 Rebeca Ramos,2 Rebecca Palacios1. 1 _New Mexico State University, Las Cruces, NM;_ 2 _Alliance of Border Collaboratives, El Paso, TX_.

Background/Purpose. Breast Cancer (BC) is the most commonly diagnosed cancer and the second leading cause of cancer related deaths for women in Texas (TX). In the border county of El Paso, TX, the breast cancer incidence and mortality rates are higher for Hispanic women relative to all races combined. Evidence indicates that breastfeeding can be protective against BC by (1) limiting breast cells' ability to act abnormally, (2) lowering estrogen levels, and (3) promoting healthier lifestyle choices. Research also indicates that the longer a woman breastfeeds, the lower her risk of developing BC. Unfortunately, among all infants born in TX in 2015, only 48% were exclusively breastfed for up to 3 months and only 24.1% were exclusively breastfed for up to 6 months. The goal of the Breastfeeding Breast Cancer Connection Program (BFBCCP) is to increase intent to breastfeed among Hispanic women of childbearing age (18-44 years) by providing a brief education intervention focused on the importance of breastfeeding and BC prevention.

Methods. Study participants included 50 Hispanic women of childbearing age living at the El Paso Housing Authority in El Paso, TX. The study consisted of an intervention group (n=25) and a control group (n=25). The control group received only educational brochures about breastfeeding, whereas, the intervention group received an educational presentation about the importance of breastfeeding and BC prevention. Measures assessed in this study included: 1) knowledge of breastfeeding and BC 2) attitudes towards breastfeeding in the workplace, and 3) behavioral intentions to breastfeed. All measures were assessed via a pre and post self-report survey in the participants preferred language.

Results/Findings. Overall, Hispanic women participating in the BFBCCP learned more about the importance of breastfeeding, particularly as it relates to BC prevention, reported greater levels of intent to breastfeed, and reported more positive attitudes towards breastfeeding in the workplace compared to the control group. Given their high BC incidence and mortality rates, Hispanic women in the TX border region would benefit from interventions like BFCCP that promote exclusive breastfeeding for a duration of six mos. to one year as a means of reducing their BC risk. The next step is to assess the efficacy of the BFCCP in increasing breastfeeding completion and duration among pregnant Hispanic women.

#3346

The role of insurance on mammogram adherence among Latina ethnicity and citizenship status.

Roman B. Johnson. _University of Alabama at Birmingham, Homewood, AL_.

Introduction: Cancer screening researchers have long observed that Latinas are less likely to be mammogram adherent, but little is understood how this varies by citizenship status.

Methods: Logistic regression analysis. The sample size was weighed according to CDC specifications. There were 1002 latina women in the sample. The 2016 National Health Interview Survey was utilized.

Results: Compared to naturalized Latina women, both U.S. citizen and non-citizen Latina women are less likely to be mammogram adherent (p<0.05). Insurance status attenuated the relationship between Latina ethnicity and mammogram adherence. For those who have a private insurance and other insurance, they were significantly more likely to be mammogram adherence (p<0.05).

Findings: Cancer screening researchers benefit from disaggregating Latina ethnicity by citizenship status to develop more tailored interventions regarding mammogram adherence in this population.

#3347

Young onset colorectal cancer patients are diagnosed with advanced disease after multiple misdiagnoses.

Ronit I. Yarden, Kim L. Newcomer, Never Too Young Advisory Board, Colorectal CancerAlliance. _Colorectal Cancer Alliance, Washington, DC_.

Colorectal cancer (CRC) is the second leading cause of cancer-related death among males and females in the US. Despite a decrease in overall incidence and mortality, there has been a rapid and alarming increase of CRC diagnosis among young adults (20-49 years old). The Colorectal Cancer Alliance is a patient advocacy organization with a mission to raise awareness for colorectal screening and prevention, provide patient and family support and fund innovative research to eradicate CRC. The Colorectal Cancer Alliance conducted an annual comprehensive survey of young onset patients and survivors, administered over social media, to track the self-reported clinical, psychosocial, financial and quality of life experiences of this often overlooked, group. The survey was completed by 1195 living patients and survivors. The majority of participants (57%) were diagnosed between the ages of 40 and 49, a third were diagnosed between the ages 30-39 (33%) and about 10 percent were diagnosed before the age of 30. Only 8% of the respondents were diagnosed with Lynch syndrome although about 30% reported some family history. According to the American Cancer Society, most CRC patients older than 50 years old are diagnosed in early stages of the disease. Conversely, our survey revealed that most of the young-onset patients and survivors (71%), were diagnosed at advanced stages (stage III and stage IV), which subjected them to aggressive therapies and a substantial decrease in quality of life including neuropathy, anxiety, clinical depression, and sexual dysfunctions. Most of the patients (63%) and survivors waited 3-12 months before visiting their doctor, partially because they did not recognize their symptoms as CRC related. Moreover, even when visiting their doctors, most patients indicated that they were initially misdiagnosed. Sixty-seven percent of the respondents reported having seen at least two physicians, and some more than four physicians, before being diagnosed correctly with CRC. Of the thirty-three percent of patients and survivors who have seen only one physician prior to their diagnosis, 17% claimed they were initially misdiagnosed (~5 percent of overall patients). Medical providers most commonly misdiagnosed patients as suffering from hemorrhoids and inflammatory bowel disease instead of CRC. Overall, our survey indicates that medical professionals and young adults need to be aware of the increasing incidence rate of young-onset CRC, the signs and symptoms, and the importance of timely screening when those symptoms are present, regardless of age. Yet, 50% of physicians did not explain to the patients' family members about their elevated risk of the disease and their need for screening 10 years prior to patients' age at diagnosis or by the age of 40.

#3348

Top 10 living with and beyond cancer research priorities.

Feng Li,1 Adrienne Morgan,2 Angela McCullagh,3 Anne Johnson,4 Ceinwen Giles,5 Diana Greenfield,6 Graeme Crawford,7 Jacqui Gath,2 Jane Lyons,8 Jervoise Andreyev,9 Jonathan Tobutt,10 Julia Tugwell,3 Karen Robb,11 Laura Cove-Smith,12 Lindsey Bennister,13 Natalie Doyle,14 Nicolas Lee,15 Rebecca Nash,15 Richard Simcock,16 Richard Stephens,3 Sabine Best,17 Susan Moug,18 Kristina Staley,19 Sandra Regan,20 Patricia Ellis,21 Stuart Griffiths,1 Ian Lewis1. 1 _National Cancer Research Institute, London, United Kingdom;_ 2 _Independent Cancer Patients' Voice, London, United Kingdom;_ 3 _National Cancer Research Institute Consumer Forum, London, United Kingdom;_ 4 _Velindre NHS Trust, Cardiff, United Kingdom;_ 5 _Shine Cancer Support, London, United Kingdom;_ 6 _Sheffield Teaching Hospitals NHS Foundation Trust, Sheffield, United Kingdom;_ 7 _Bangor Health Centre, Northern Ireland, Bangor, United Kingdom;_ 8 _Cancer 52, London, United Kingdom;_ 9 _United Lincolnshire Hospitals NHS Trust, Lincolnshire, United Kingdom;_ 10 _Kirkwood Hospice, Huddersfield, United Kingdom;_ 11 _Transforming Cancer Services Team in London, London, United Kingdom;_ 12 _The Christie NHS Foundation Trust, Manchester, United Kingdom;_ 13 _MQ, London, United Kingdom;_ 14 _The Royal Marsden NHS Foundation Trust, London, United Kingdom;_ 15 _Macmillan Cancer Support, London, United Kingdom;_ 16 _Brighton and Sussex University Hospital Trust, Brighton, United Kingdom;_ 17 _Marie Curie, London, United Kingdom;_ 18 _Royal Alexandra Hospital Paisley, Glasgow, United Kingdom;_ 19 _TwoCan Associates, United Kingdom;_ 20 _NIHR Oxford Health Biomedical Research Centre, Oxford, United Kingdom;_ 21 _James Lind Alliance, Southampton, United Kingdom_.

More and more people are living with the consequences of cancer and its treatment (living with and beyond cancer), yet the level of relevant research is low compared to other types of cancer research in the UK. NCRI aims to increase the level of research in this area and to ultimately improve the lives of those affected by cancer. Undefined research priorities in this broad area has been a barrier to research. The 2015 NHS Independent Cancer Taskforce report also recommends defining research priorities and to enable this research to happen. To address this barrier the NCRI has undertaken a James Lind Alliance Priority Setting Partnership (PSP) to identify priorities that matter most to people affected by cancer and the health and social care professionals.A PSP consists of patients and carers, health and social care professionals. PSPs have several stages and begin with a UK-wide survey to gather questions about uncertainties in living with and beyond cancer. Once the results were analysed, an interim exercise takes place to further prioritise the uncertainties. The last stage is a final workshop where partners debate and finally arrive at a top 10 list of shared uncertainties.The living with and beyond cancer PSP received 3500 questions submitted by people affected by cancer and healthcare professionals. Through a 18-month established rigorous process, the questions are prioritised down to the Top 10 living with and beyond cancer priorities for research in June 2018. This is the first time that clear research priorities have been identified in this area. They are the most impactful research questions that will help improve the lives of people affected by cancer. The Top 10 uncertainties will be publicised widely to ensure that researchers and those who fund research really understand what matters to people affected by cancer. The top uncertainties will be promoted to many research organizations and relevant funders in the UK. We anticipate they will directly influence future research.

#3349

Collected experiences of young-onset colorectal cancer caregivers.

Kimberley L. Newcomer, Ronit Yarden, Never Too Young Advisory. _Colorectal Cancer Alliance, Washington, DC_.

The Colorectal Cancer Alliance launched a survey for caregivers of young-onset colorectal cancer (CRC) patients and survivors. A caregiver of young-onset CRC is an unpaid or paid member of a person's social network who helps them with activities of daily living. Caregivers who participated in our survey (n=427) are diverse: in age, gender, and racial/ethnic group. The majority of them were between the ages of 30-39. The caregivers reported they faced many challenges including lack of resources and information on young-onset disease and the course of the disease progression. Also, many caregivers indicated they had difficulty understanding the different treatment options offered by the physicians as well as the risk and long-term side-effects associated with these aggressive treatments. Only 40 percent of providers talked to patients about genetic testing and explained to the patient's family member their elevated risk of the disease and the associated need for timely screening. The majority of caregivers (59%) reported that their loved one experienced changes in their ability to perform expected social tasks, including those of a spouse, child rearer, friend, or worker. Some caregivers also mentioned the loss of sexuality, depression, pain, despair, lack of sleep, sadness, and stress, along with a loss of faith and hope, which may cause additional strain on their relationships. Thirty-two percent of caregivers reported insufficient psychosocial (66%) and financial support (44%) to effectively care for their patients. Participants also indicated that resources such as transportation, child care, household maintenance, prevention, and surveillance information for family members are limited. Overall, about one in three caregivers reported they were missing 24 hours or more of work each month to care for their loved one. A caregiver stated she needed, "Emotional and basic help around the house with her children and someone responsible enough to stay in the house to understand what we were going through as a family." Taken together, our survey indicates that additional resources are needed to improve the ability for caregivers to manage everyday tasks, potentially helping caregivers feel more organized and in control. The ability of caregivers to care for themselves and use tools to care for their loved ones will reduce emotional and physical demands involved with caregiving that can cause strain and burnout. The Alliance will use these survey results to learn about and track the self-reported medical, psychosocial, and quality of life experiences of this often overlooked group.

#3350

Not letting the cancer take any more than it had from me **:** **Latina mothers surviving cancer in the Paso del Norte border region.**

Clara Reyes,1 Rebecca Palacios,1 Karoline Sondgeroth,1 Frances Lewis2. 1 _New Mexico State University, Las Cruces, NM;_ 2 _University of Washington School of Nursing, Seattle, WA_.

Cancer survivorship research has largely focused on non-Latino white participants, with fewer studies exploring survivorship in multi-ethnic groups. The goal of this study was to explore the experiences of Latina mothers living with cancer in the southwest U.S.-Mexico border region. Diagnosed mothers were asked to describe their (i) challenges to coping with cancer, (ii) facilitators to coping with cancer, and (iii) types and sources of social support for coping with their cancer. Nine Latina mothers, most of whom reported non-localized cancer diagnoses, participated in focus groups or individual interviews in a pilot study. We conducted a secondary analysis of the verbatim transcripts using inductive content analysis adapted from a grounded theory framework. Coding to consensus, systematic peer debriefing, and maintaining an audit trail protected trustworthiness of study results. The core construct Not letting the cancer take any more than it had from me, is grounded in three domains reflecting their challenging position as Latina mothers under the age of 50 living with various types and stages of cancer in a resource-challenged border region. The first domain, Having the most difficult time of my life, was composed of three categories that covered their struggles with cancer treatment (i.e. feeling like treatment is worse than the disease, being scared to look in the mirror, and feeling very alone). The second domain, Figuring out how to live day-by-day, was composed of two categories that captured the impact of cancer on their homes and their need for instrumental support (i.e. being especially challenging on my family and home life and needing other things). The last domain, Giving me the strength to fight and carry on, was composed of the remaining six categories that revealed how the mothers garnered all the resources available to them to physically and mentally overcome the stress of cancer and its treatment (getting support and compassion from medical staff, friends, family, their children, and other cancer patients; trusting God; and not letting the cancer stop them). Latina mothers under the age of 50 living with diverse cancer types and stages on the U.S.-Mexico border described the complexity of factors influencing their survivorship experience and the strategies they used to move forward. Results highlighted the importance of assessing cancer survivorship experiences across cancer types, age groups, regions, and diverse racial/ethnic groups. These findings can be used to develop targeted approaches to improve the quality of survivorship among Latinas.

#3351

Overall survival prediction of glioblastoma patients combining clinical factors with texture features extracted from 3-D convolutional neural networks.

Weiwei Zong, Joon Lee, Chang Liu, James Snyder, Ning Wen. _Henry Ford Health System, Troy, MI_.

Objective:

We aim to predict overall survival (OS) in Glioblastoma treated with gross total resection (GTR) using pre-operative MRI images.

Methods:

A cohort of 87 GBM patients (59 patients for training and 28 patients for validation) who underwent GTR was analyzed using multi-institutional data from brain tumor segmentation (BraTS) challenge 2018 (Menze BH, et al. TMI 2015; Bakas S, et al. NSD 2017).

Each patient consisted of a series of pre-surgical MR images including T1 pre contrast, T1-Contrast Enhanced, T2 and Flair images. A group of experienced radiologists delineated edema, tumor core and enhanced tumor for each testing patient using these image sequences. A 2D U-net was trained to segment these structures on the validation cohort.

A 3D CNN model with orthogonalized random filters was used to learn images features from the three segmented subregions including texture, size, location, etc. Global maximum pooling was performed on intermediate convolutional layers to obtain representative image features for each patient.

Since mid-term survivors (6-18 months) outnumbered short (<6 months) and long term (>18 months) survivors for a large margin, the MR images from both short and long term survivors were augmented

with random rotations to balance the number of patients among three cohorts of patients. The extracted image features was then fed into an RBF-kernel based L-2 norm regression algorithm (Huang GB, IEEE SMC, 2012) to predict patient's OS.

Results:

The average [standard deviation] of dice similarity coefficient (DSC) for the whole tumor, enhanced tumor, and tumor core contours were 0.882[0.080], 0.712 [0.294], and 0.769 [0.263], respectively for the validation cohort. Parameters of regression algorithm was optimized using leave one out cross validation.

One convolutional layer was used in the CNN archietecture due to limited training samples. The model performance deteriorated when using deeper layers. The best architecture to classify patients into short, mid and long term survivors was one convolutional layer with 30 filters. The prediction accuracy was 64.3%, and the spearman'r sank correlation was 0.395.

The model performance was slightly improved by including clinical factors such as age, tumor location, ratio of the whole tumor size to the entire brain etc. The Spearman's rank correlation coefficient was increased to 0.432 while the accuracy maintained the same.

Conclusions:

We developed a 3D CNN model followd with kernel regression to extract image features from pre-operative MR images and predict OS of GBM patients after GTR. These signatures have shown potential values as biomarkers to predict OS.

#3352

Cost-effectiveness of current and potential serum based colorectal screening strategies: Can a serum based test do better.

Ali Jalali,1 Richard Nelson,2 Raminder Nirula2. 1 _University of Utah, Salt Lake City, UT;_ 2 _University of Utah School of Medicine, Salt Lake City, UT_.

Background: Despite efficacy of colorectal cancer (CRC) screening, recent trends in screening rates have not improved. Furthermore, national society recommendations have not prioritized a single screening modality. Both flexible sigmoidoscopy (Flex-Sig) and guaiac fecal occult blood test (gFOBT)) have been recommended. Studies suggest that noninvasive tests may improve screening rates, however, compliance remains modest for such tests. A recent study found that health system transition from gFOBT to a fecal immunochemical test (FIT) moderately improved test compliance rates. However, FIT is an expensive substitute for gFOBT and sensitivity of fecal based tests for precursor lesions in the colon remain inferior to structural examinations. Serum-based blood tests (SBT) may provide higher compliance rates, but necessary accuracy for such a test to be a dominant or cost-effective screening strategy over current recommendations have not been determined. This study analyzed the cost-effectiveness of multiple CRC screening strategies and estimated minimum test characteristics of a hypothetical SBT to be a preferred screening strategy over current modalities.

Methods: A Markov microsimulation model was developed to analyze the costs and effects, (quality-adjusted life-years, QALYs) of CRC screening following a hypothetical cohort of 10,000 individuals at age 50 until death. Our model considered three strategies: 1) initial colonoscopy followed by Flex-Sig every 5 years, 2) initial colonoscopy with yearly gFOBT, and 3) initial colonoscopy with yearly FIT. An initial colonoscopy with a hypothetical yearly SBT was also examined at a range of test sensitivities. A long-term payer perspective was assumed for professional and facility costs and lifetime costs of cancer treatment. Model probabilities and utility values were obtained from the literature or calculated from National Vital Statistics Reports.

Results: Annual gFOBT was the least costly strategy ($3,242) while annual FIT was eliminated through extended dominance. Flex-Sig was the costliest strategy at $4,667 but had the highest expected QALYs at 19.68. The incremental cost-effectiveness ratio (ICER) of Flex-Sig relative to gFOBT was $35,615. Our secondary analysis demonstrated that a minimum joint sensitivity of 70% and 60% is required for an SBT at 80% and 100% compliance rates, respectively, to achieve extended dominance of both Flex-Sig and FIT by SBT and gFOBT. ICER's of SBT ranged from $12,984 to $4,779 in these sensitivity ranges compared to gFOBT with expected QALYs as high as 19.96. Total individual costs in the model varied from $365 to $111,535.

Conclusion: With improved sensitivity and high compliance rates, annual screening for CRC via a non-invasive SBT is a cost-effective approach compared to structural examinations and currently recommended gFOBT and FIT.

#3353

Four vs six cycles of docetaxel and cyclophosphamide (TC) in early stage triple-negative breast cancer.

Aparna Basu, Hadi A. Mohammed, Sharmeen Mahmood, Vrushali Dabak, Randa Loutfi. _Henry Ford Health Systems, Detroit, MI_.

Background: ABC trials established the use of non-anthracycline containing regimen, docetaxel and cyclophosphamide (TC) in the adjuvant setting in early stage breast cancer. In clinical practice, TC is commonly used in Stage I Triple-Negative Breast Cancer (TNBC). However, no specific recommendations exist in literature, regarding the number of cycles that can be used. i.e. TC4 vs TC6. Our aim, was to determine if TC4 is non-inferior to TC6 when used as adjuvant therapy in early stage TNBC.

Methods: We retrospectively reviewed 77 patients who were diagnosed with early stage TNBC between 2007 to 2017, at our institution who had received either TC4 or TC6 as adjuvant therapy. The number of cycles the patients received were based on provider preference. The two groups (TC4, TC6) were compared in regard to stage of cancer at diagnosis based on AJCC 7thedition, grade of adverse events, recurrence and death from breast cancer recurrence.

Results: Out of 77 patients, based on T stage, 25 (32.5%) were T1b, 38(49.4%) were T1c, 13(16.9%) were T2 and 1(1.3%) was T3. All patients were node negative. 53(68.8%) received TC4 and 24(31.2%) received TC6. Regarding side effects, adverse of any grade were seen in 42(79.2%) patients who received TC4 and 23(95.8%) in patients who received TC6(p=0.091). Adverse events which were grade 3 or higher were seen in 7(15.9%) in TC4 group and 3(13%) in the TC6 group (p=1.000). Recurrence in the TC4 group was seen in 4 patients (7.5%) and 3(12.5%) patient in the TC6 group (p=0.259). Death due to breast cancer recurrence was seen in 1 patient (1.9%) in the TC4 group and 1(4.1%) patient in the TC6 group.

Conclusions: In this limited series, TC4 appears to be equally effective to TC6, with fewer adverse events of any grade. However, a longer follow up and a larger patient base is required to be studied for a more definitive conclusion.

#3354

Patterns of care for patients with non-operable T1-4 N+ M0 non-small cell lung cancer in the US and outcomes with radiation or chemotherapy monotherapies.

Ellen Kim,1 Megan E. Daly,2 Kenneth Westover,3 James Murphy,4 Timur Mitin5. 1 _Vanderbilt University Medical Center, Nashville, TN;_ 2 _UC Davis Comprehensive Cancer Center, CA;_ 3 _UT Southwestern Medical Center, TX;_ 4 _UC San Diego, CA;_ 5 _Oregon Health and Science University, OR_.

Background Standard management of unresectable N+ M0 non-small cell lung cancer (NSCLC) is concurrent chemoradiation (CRT). However, some patients may receive only radiation therapy (RTmono) or chemotherapy (CTmono) as monotherapies. The primary aim of this study was to investigate the patterns of care in the US and analyze the outcomes achieved with monotherapies.

Methods Patients in the National Cancer Database (NCDB) diagnosed in 2004-2013 with N+ M0 NSCLC who did not undergo surgery and received monotherapy with either high dose radiation alone (RTmono) or chemotherapy alone (CTmono) were studied. Patient characteristics and overall survival (OS) were compared with descriptive statistics (chi-square, Kruskal-Wallis, logistic regression), Kaplan-Meier and stratified Cox models, and restricted mean survival times (RMST); all used alpha of 0.05.

Findings 74,867 patients received CRT (10,915, 15%), CTmono alone (34,978, 47%), RTmono (2,396, 3%), or no treatment (26,578, 36%). In a multivariable model, RTmono was associated with non-Medicare insurance, academic or research facility, Western geography, lower T and N stages, and older age. CTmono had better RMST compared to RTmono: at 3 years, average survival was 17.9 vs 16.1 months (difference 1.8, 95% CI 1.3-2.3); at 5 years, average survival was 21.6 vs 18.7 months (difference 2.9, 95% CI 2.2-3.6). In stratified Cox models, improved survival was associated with female gender, White race, private insurance, metropolitan location, lower Charlson comorbidity score, lower T and N stage, smaller tumor, younger age, and later year of diagnosis.

Interpretation Despite the established benefits of CRT, only 15% of non-operable N+ M0 NSCLC US patients received definitive CRT. Future clinical studies should focus on patients not receiving concurrent CRT, as outcomes with monotherapies (RTmono or CTmono) are suboptimal.

#3355

**Challenges in the implementation of molecular diagnostic testing for non-small cell lung cancer** **.**

Pearl A. Campbell,1 Kednapa Thavorn,1 Bryan Lo,2 Ali Karimnezhad,1 Theodore J. Perkins,1 Robin Urquhart,3 Suzanne Kamel-Reid,4 Harmanjatinder Sekhon,2 David J. Stewart1. 1 _Ottawa Hospital Research Institute, Ottawa, Ontario, Canada;_ 2 _Eastern Ontario Regional Laboratories Association, Ottawa, Ontario, Canada;_ 3 _Dalhousie University, Halifax, Nova Scotia, Canada;_ 4 _Toronto General Hospital/Research Institute (UHN), Toronto, Ontario, Canada_.

Next generation sequencing (NGS) has been used to catalogue genetic mutations in cancer. Recent studies employing NGS have identified specific genetic mutations that reliably predict therapeutic success with targeted treatment in many forms of cancer, and particularly in non-small cell lung cancer (NSCLC). Importantly, patients with oncogenic driver mutations have better tumor control with targeted agents than with chemotherapy, while those lacking such a mutation derive more benefit from chemotherapy. To detect actionable mutations, all patients with metastatic disease must be tested. Mutation assays are generally developed using tissues derived from surgical samples. However, for many patients with metastatic NSCLC the only tissue available is from fine needle aspirates (FNAs). Given the limited number and heterogeneity of cells found in FNAs and the expanding number of clinically actionable mutations, the development and implementation of testing strategies that rapidly and accurately define driver mutations in NSCLC remains a challenge.

Our project focuses on the identification of best methods (pre-analytical, analytical, and bioinformatic) to identify driver mutations in lung FNAs to standardize targeted NGS testing for NSCLC. The overarching goal of this project is to develop a strategy for Canada-wide implementation of the developed test. As a first step in this process, our team organized a stakeholder meeting to: A) Identify potential individual and/or system level challenges and barriers to implementation of standardized protocols for molecular oncology diagnostics; B) Outline guidelines and strategies to overcome identified challenges and barriers; and C) Initiate a research project to further study the barriers and facilitators of implementing Canada-wide diagnostic testing strategies for personalized cancer care. For this presentation, we will outline key challenges that impact implementation of the new test, including tumor characteristics (cellularity, heterogeneity); cost and reimbursement issues; required turn around times; bioinformatic requirements; testing strategy (technical limitations of test, panel size); technical staffing and infrastructure requirements; and barriers to implementation of the test into routine standard of care. Finally, we will present a preliminary workflow map, which builds upon the Lung Cancer Pathway Maps provided by Cancer Care Ontario (https://www.cancercareontario.ca/en/pathway-maps/lung-cancer) and addresses these barriers and explores various scenarios for implementation of new testing strategies.

#3356

Working together to put kids first: Outreach strategies driving collaborative research, data sharing and cross-disease analysis to accelerate discoveries in pediatric cancer and structural birth defects.

Tatiana S. Patton,1 Robert Moulder,1 Erin Alexander,1 Donna Vito,1 Jonathan Waller,1 Colleen Gaynor,1 Sarah Thomas,1 Bailey Farrow,1 Joseph Yamada,2 Kim Cullion,2 Danyelle Winchester,3 Angela Waanders,1 Allison Heath,1 Pichai Raman,1 Adam Resnick,1 Jena Lilly1. 1 _The Children's Hospital of Philadelphia, Philadelphia, PA;_ 2 _Ontario Institute of Cancer Research, Ontario, Canada;_ 3 _NIH Common Fund, MD_.

The Gabriella Miller Kids First Pediatric Research Program launched the Kids First Pediatric Data Resource Center (DRC) in 2017 as a collaborative, pediatric research effort with the goal of understanding the genetic causes of and links between childhood cancer and structural birth defects. The DRC is charged with developing data-driven platforms that integrate large amounts of genomic and clinical data, empowering the collaborative discovery, engagement, and necessary partnerships that are crucial for progress in our biological understanding of diseases, enabling rapid translation to personalized treatments for patients and accelerating discovery of genetic causes and shared biologic pathways within and across these conditions. The DRC is comprised of 3 cores including the Data Resource Portal Core, Data Coordination Core and the Administrative & Outreach Core (AOC). The AOC brings together researchers, physicians, and patient and foundation advocates to support collaborative research and data sharing to accelerate discoveries. The AOC specific aims are to employ outreach strategies including print, web, social media, in-person presentations, conferences, videos, webinars, e-newsletters, surveys, communication strategies, and reports to support accelerated discoveries. The AOC is committed to learning from the childhood cancer and birth defect communities. By capturing, synthesizing, and prioritizing unmet needs for development of the Kids First DRC portal, website, and materials, the AOC engages with researchers, clinicians, foundations, and patient advocates in the childhood cancer and structural birth defect communities. In its first year, the AOC partnered with 32 foundations to launch the Kids First DRC Portal and support data sharing throughout the research community. Key findings during the first six months of requirements gathering revealed the following unmet needs: a) Increase understanding of the disease types, research projects, and the investigators that are a part of the Kids First community b) Highlight the need for cross-disease analyses including structural birth defects and childhood cancers and c) Promote education on the data sharing, agreements, data availability and accessibility. The AOC, gathered pertinent user requirements, conducted educational activities, and engaged prospective users of the researcher community resulting in over 200 users and 22,000 portal views since launch and will continue to use feedback from the research community to further inform the development of the Kids First DRC tools and materials to meet the goals of the program.

#3357

The fibrolamellar registry: A model for the study of rare diseases.

Michelle Desmond,1 Julie Latone,1 Siobhan Lett,1 Rachael D. Migler,1 Elana P. Simon,1 Sanford M. Simon2. 1 _The Fibrolamellar Registry, New York, NY;_ 2 _Rockefeller Univ., New York, NY_.

Advances in genomics and proteomics have enabled more precise characterizations of tumors with the consequence that many cancers are being segregated into smaller categories. With this larger number of categories, more cancers are being categorized as rare. The downside to such categorizations is that the reduced numbers of patients in each category makes it difficult to gather enough information about each cancer. We are a group of patients and caregivers who have joined together to form a repository for patient-shared data and reports in an IRB-approved, non-profit medical registry for the rare and usually lethal childhood liver cancer, fibrolamellar hepatocellular carcinoma (FLC). Since the Fibrolamellar Registry is patient-run and patient-owned, we have the trust of the patient community that the records will not be sold for profit. This has enabled us, in our first two years, to gather detailed medical records, scans and tests from over 140 patients. With input from scientists and clinicians who study FLC, we have written 600 questions of specific interest to this disease. Most of our patients have also opted to allow these medical records to be shared with a tissue FLC repository that already has samples from 110 patients. We have initiated two different collaborations with the clinical-research community to explore the records and our patients have been answering additional questions when pertinent to a particular study. Our ability to gather so many records for what is a rare cancer comes from our ability to involve patients from across institutions and across the globe. We are working with other patient groups and we feel that the Fibrolamellar Registry could be a model for gathering and organizing data for many rare diseases.

#3358

Cost of treatment for colorectal cancer in central of Vietnam.

BInh Thang Tran,1 Hoang Lan Nguyen,2 Thi Thanh Binh Nguyen2. 1 _National Cancer Center, Seoul, Republic of Korea;_ 2 _Hue University of Medicine and Pharmacy, Hue, Viet Nam_.

Introduction: Colorectal cancer is one of three cancer, along with breast cancer, cervical cancer, which are prioritised in the National Cancer Control Program in Vietnam. Nine out of ten Vietnamese patients diagnosed with colorectal cancer are in advance stage (stage 3 and 4). Therefore, we aim to estimate the direct medical cost of a 5-year treatment course for those with primary colorectal cancer in central Vietnam.

Methods: Data will retrospectively be retrieved from medical records at the Hue Central Hospital (Regional cancer registry in central ad highland) between 2010 and 2017 were analyzed. A direct cost analysis will proceed from the health care payers' perspective. Various direct medical cost categories will obtain for a 5-year treatment course for patients with colorectal cancer. Costs, in US dollars, discounted at a 3% rate, will convert to 2017 after adjusting for inflation. For each cost category, the mean, standard deviation, median, and cost range will be computed. The relationship between costs and the stage, age at diagnosis, and the health insurance coverage of the patients will be conducted. Since this study is an ongoing project and data will be conducted to accomplish by February 2019. The results will be presented on the poster.

Results: Initial results from the analysis of the cost will be presented, and analytic issues will be discussed, including an approach that will help describe the situation on the burden of cancer treatment among Vietnamese patients.

CONCLUSION: We assume that regarding our result, the Government subsidization of public hospital possibility charges lowered the direct medical costs of a 5-year treatment course for primary colorectal cancer in Vietnam. In addition to the cost of treatment, the long treatment course may influence by out-of-pocket payments for patients without health insurance.

#3359

Multiple objective analysis of first line medicine with non-small cell lung cancer.

Yueh-Fu Fang,1 Min-Chia Lee2. 1 _Chang Gung Memorial Hospital, Taipei, Taiwan;_ 2 _National Central University, Taoyuan, Taiwan_.

Background

The standard first line treatments of lung cancer are chemotherapy and targeted therapy. Taiwan launch National Health Insurance since 1995 and Taiwanese can receive medical treatment in a low price. However, National Health Insurance facing bankruptcy so it is important to find which treatment provide good medical effect and low medical cost. We will use analytic hierarchy process (AHP) to combine medical effect and medical cost.

Patients and Methods

We had analyzed the Database of Lung Cancer from Taiwan National Health Insurance Database (NHIRD). The database included the coding of diagnoses, examinations and treatments from 1996 to 2013 in the patients who were diagnosed with lung cancer in 1996 to 2013. The Analytic Hierarchy Process (AHP) is a theory of measurement through pairwise comparisons. It relies on the judgements of experts to get the weight of the pairwise comparison. And it uses the pairwise comparison to derive priority scales. It scales that measure intangibles in relative terms. The methods of survival analysis are Kaplan-Meier method and Life table method.

Results

The aim of the questionnaire for AHP is to get the weights between each criteria or subcriteria. The weight of medical effect is about 71%, and the weight of medical cost is approximately 29%. According to the result of questionnaire, medical effect is much more important than medical cost. There are four part of medical cost. The weight is about 23% in average medical cost in first line. The weight is about 22% in average medical cost related to lung cancer in first line. The weight is approximately 26% in average medical cost per day after receiving first treatment. The weight is about 29% in average medical cost related to lung cancer per day after receiving first treatment. The average progression survival day is 106.86 days. The overall survival day is 262.68 days. The average time of the emergency is 2.15. The average medical cost in first line is 4655.38 new Taiwan dollars per day. The average medical cost related to lung cancer in first line is 4545.92 new Taiwan dollar per day. The average cost after receiving the first treatment is 3369.75 new Taiwan dollar per day. The average cost related to lung cancer after receiving first treatment is 3262.38 new Taiwan dollar. KPI of chemotherapy with Cisplatin or Carboplatin is higher than thatn of chemotherapy without Cisplatin or Carboplatin. For those who only use chemotherapy medicine, the highest KPI is not fixed on a certain medicine. In addition, Alimta is the most expensive one so that the lowest KPI is usually on Alimta.

Conclusion

This is the first time not only using the survival analysis but also using a new method to evaluate the efficiency of a treatment or drug. The future work is to develop a new method based on this analytic model.

### Science and Health Policy 2 / Regulatory Science and Policy

#3360

The 4D Nucleome NIH approach to microscopy metadata and calibration to increase data fidelity and reproducibility.

Davd Grunwald,1 Mathias Hammer,1 Farzin Farzam,1 Colton Hormann,1 Carlas Smith,2 Maximiliaan Huisman1. 1 _UMass Medical School, Worcester, MA;_ 2 _TU Delft, Delft, Netherlands_.

Fluorescence microscopy has become a more and more sensitive and versatile tool for many branches of science, thanks to many advances in fluorescent labelling as well as microscope technology and image processing. As we continue to push the limits of what is technically possible, the quality of data obtained through fluorescence microscopy is increasingly determined by factors that are often not be readily visible in the image: the image acquisition settings, microscope properties and data-processing steps often contribute significantly to the experimental outcome and therefore need to be known and understood for proper interpretation and comparison. While keeping notes on microscopy experiments should be relatively unchallenging, as the microscope is a machine equipped with a limited number of known parts and settings. Nevertheless, to this date no widely adopted set of metadata descriptors to be recorded or published with imaging data exists. Metadata automatically recorded by microscopes from different companies vary widely and pose a substantial challenge for microscope users to create a good faith record of their work. Similarly, the complexity and aim of experiments using microscopes varies leading to different reporting requirements from the simple description of a sample to the need to document the complexities of sub-diffraction resolution imaging in living cells and beyond. However, there are certain crucial pieces of information that simply are not captured in even the most rigorous and precise routines for record-keeping and calibration, as they simply cannot be measured without the aid of external devices which can be costly, cumbersome and complicated. Here we present a tiered system of guidelines for describing and documenting microscopy experiments and a comprehensive list of metadata key-value pairs that should be recorded for each tier. We further present an inexpensive, easy-to-use calibration device that allows the user to measure excitation power and perform basic detector calibration routines. In doing so, the "MetaMax" tool provides crucial meta-data to evaluate potential photo-toxicity and allows current and future model-based data processing tools to get as much quantitative information as possible out of the images.

#3361

A prescription for new trial designs for drug development focused on the neoadjuvant setting: Save lives, resources, and time.

Andreas Karlsson,1 Yiwey Shieh,2 Andre Dempsey,2 Christina Yau,2 Angela Dmichele,3 Doug Yes,4 Laura van't Veer,2 Nola Hylton,2 Martin Eklund,5 Laura Esserman2. 1 _Karolinska, Stockholm, Sweden;_ 2 _UCSF, San Francisco, CA;_ 3 _University of Pennsylvania, Philadelphia, PA;_ 4 _University of Minnesota, Minneapolis, MN;_ 5 _Karolinska Institutet, Stockholm, Sweden_.

Background: Traditionally, new drug combinations are first tested for safety and efficacy in the stage 4 setting. The I-SPY 2 TRIAL has focused on bringing promising new combinations into the phase 2/3 high-risk setting once safety is established. I-SPY 2 is an adaptive neoadjuvant platform trial for women with stage 2/3 breast cancer at high risk for early recurrence. In this setting, complete pathologic response (pCR) is the primary endpoint. We and others have demonstrated that pCR is highly predictive of distant recurrence free survival at 3 years. In I-SPY 2 the hazard rate is 0.2 (Confidence Interval 0.11-0.37), regardless of subtypes or agent. Agents that extend progression free survival in the metastatic setting, can be lifesaving when given at an earlier stage setting.

Methods: The health economic benefits of achieving pCR were investigated using a Markov model describing the risk of recurrence, progression and death. The risk of recurrence was informed by I-SPY 2 and the risk of cancer progression and the associated treatment costs were based on literature values. We assumed constant recurrence rate without accounting for subtype specific rates in this first version of the model.

Results: For each additional patient with pathologic complete response a net monetary benefit of $170,000 (Credible Interval 95% 100,000—250,000) and a gain of 4 life-years (CrI 95% 2—6) was predicted. The ICER for pCR is -$45,000. This represents the aggregate benefit across all I-SPY cancers. Modeling accounting for cancer subtype is in process and will be presented also.

Conclusion: A short term endpoint, response to chemotherapy in the neoadjuvant setting, which is highly correlated with long term outcome, provides the opportunity to focus drug development on improving that endpoint. Enabling more patients to achieve a pCR would provide enormous benefit in terms of lives and resources saved. The economic modeling, along with the findings from I-SPY 2 suggests that we should shift drug development to the high risk early setting and evolve drug designs to improve every patient's outcome. In I-SPY 2, we are shifting our platform trial design to increase every patient's chance to achieve pCR. Platform adaptive trials in the early stage neoadjuvant setting are ideal for advancing the field and maximizing benefit for patients.

#3362

Factors driving industry participation in the collaborative I-SPY2 platform trial.

Jurr M. van Ramshorst,1 Laura J. van 't Veer,2 Dave Mandelkern,3 Laura J. Esserman,2 Daniel Dornbusch3. 1 _VU University, Amsterdam, Netherlands;_ 2 _UCSF, San Fransisco, CA;_ 3 _Quantum Leap Healthcare Collaborative, San Francisco, CA_.

Background: Platform trials enable faster, less expensive, and more efficient clinical readouts by evaluating multiple products and combinations. This study explores the willingness of pharmaceutical companies to put experimental drugs into a platform trial, and examines the drivers and barriers to their participation.

Methods: The I-SPY2 (Investigation of Serial studies to Predict YourTherapeutic Response with Imaging And molecular anaLysis2) pre-competitive collaborative platform trial was used as a case study to elucidate the motivation and constraints of the pharmaceutical industry. Rogers' Diffusion of Innovation theory (ISBN 0029266718) was used as theoretical framework, primary interviews were conducted with 14 executive level representatives from 9 different pharmaceutical companies, and data was analyzed in ATLAS.ti to derive the influence factors. The I-SPY2 neo-adjuvant breast cancer platform trial is a partnership of 10 pharmaceutical and 5 biotech companies, alongside 18 academic institutions, patient advocates and the FDA, under the sponsorship of the non-profit Quantum Leap Healthcare Collaborative. I-SPY2 has entered 15 investigational agents of which 10 have completed evaluation and 5 are still ongoing.

Results: Two factors were critical drivers for whether pharmaceutical companies were willing to put experimental drugs into a platform trial: the registration status of the agent and whether or not it was the primary indication for treatment. Drugs were more likely to be considered for these types of trials if other registration trials were completed or in progress and if the primary indication for the agent was in a different cancer organ type (e.g. not breast). Four types of perspectives positively influenced pharmaceutical company to participation: a gain in efficiency of drug evaluation, the opportunity for knowledge generation, innovativeness of the trial design, and the expertise and reputation of the consortium. Primary barriers that reduced the chance of participation included a loss of control over the evaluation of the drug and the organizational and governance structure. No significant differences were identified between company size and experience in a therapeutic area. Finally, regulatory guidelines specified by the FDA and EMEA as well as payer engagement were the most significant influence factors which could increase pharmaceutical company's future participation in platform trials.

Conclusion: This is the first study to examine the drivers and barriers to pharmaceutical company participation in platform trials and provides insight for why innovative companies participate in these groundbreaking studies. The FDA has expressed strong interest in promoting platform trials because of their efficiency and knowledge generation. Working to integrate a registration component as part of adaptive platform trials is likely to be a key driver of successful future adoption.

#3363

Mapping cancer: Employing geographic information systems (GIS) to explore co-authorship networks.

Monica A. Gallagher, Jeremy L. Warner. _Vanderbilt University, Nashville, TN_.

Introduction: The systemic treatment of cancer has evolved over the past 70 years through incremental knowledge gained via clinical trials (CTs). As a result of the efforts of large numbers of individuals and institutions, prognosis has improved markedly for many cancer types. Many CTs are organized through cooperative group structures, and increasingly through pharmaceutical industry or investigator-initiated mechanisms. One means to understand the complex organizational processes involved is through analysis of the geospatial relationships of cancer researchers. Here, we examine the co-authorship networks formed as a result of publication of CT results.

Methods: We utilized the knowledge embodied in HemOnc.org, which contains a large number of references to randomized CTs, as well as to prospective non-randomized CTs with practice-changing implications. Author names from HemOnc.org's references were merged and normalized to MEDLINE names, when available. Author affiliation information was programmatically extracted from MEDLINE; these data were highly variable and required extensive post-processing. We then used the ggmap package in R to obtain geographic coordinates from the Data Science Toolkit API. As a pilot, we mapped the geolocations of nine Vanderbilt-Ingram Cancer Center (VICC)-affiliated authors and their co-authors, and describe some characteristics of this subgroup of the larger network.

Results: Our dataset consisted of 4,105 publications with 24,976 authors (median 14 authors/pub., interquartile range [IQR] 10-19). Affiliation could be determined for 24,934 of 61,681 author entries (40.4%). The VICC subset of nine authors had 658 geolocatable co-authors (individual range 34-155). These co-authors were geolocated to 200 cities in 28 countries; the most common co-affiliates were Memorial Sloan Kettering Cancer Center in New York (MSKCC), Dana-Farber Cancer Institute in Boston, MA (DFCI), and Bristol-Myers Squibb in Princeton, NJ (BMS). The median co-author distance from Nashville, TN was 1332 km (IQR 1047-6745 km).

Conclusion: This pilot work is the first, to our knowledge, to use geolocation to describe the relationships of cancer clinical trialists. Given that VICC is a member of the ECOG-ACRIN Cancer Research Group, it is unsurprising that the most common co-affiliates are also ECOG-ACRIN sites (MSKCC, DFCI). VICC had a large role in the nivolumab trials, explaining the BMS connection. While this analysis reveals a dense geospatial network, it also indicates opportunities for underrepresented areas; e.g., only one co-author is from South America and there are none from Africa or the Indian subcontinent. In summary, this pilot work demonstrates the feasibility of geolocating authors of pivotal CTs in cancer. Future work will include imputation of missing affiliations, geospatial network analysis, and examination of author impact as a function of time and affiliation.

#3364

A study to assess the critical result reporting of the patients diagnosed with cancer at B&C Medical College and Teaching Hospital.

Sujan Sharma. _B &C Medical College and Teaching Hospital, Birtamode, Nepal_.

Objective: This critical value can occur while performing panels of tests at laboratory by different chemistry or blood analysers with varieties of principles. The main objective of the study was to study the process of critical result reporting and to know the way of communication and documentation done for critical value in the laboratory, ICUs and the wards.Methods: This study was prospective and non-experimental was conducted at B&C Medical College and Teaching Hospital from 14.04.2018 to 14.05.2018 . Total 60 critical values samples were included. The data was collected by means of observing the critical values of inpatient and the process of reporting from the laboratory to the respective wards and ICUs.Results: Out of 60 samples included in our study, there was 100% communication to concerned treating units. For the confirmation of critical value repeat test was done in 68% of cases. In 75% of cases clinicians did follow up. Recording of a critical report in lab was done in 96% of cases and almost all of the critical values 98% were immediately reported to the respective wards by technical staffs. There was no communication in 1.6% of cases to treating units by technical staff. Majority 78% of critical values were communicated by respective wards and ICUs nurses to concerned doctors.Conclusion: Critical value can occur while performing panels of tests at laboratory and reporting such values to treating clinicians or respective wards or ICUs could help heath care providers for effective treatment of the patients and their adequate care.

#3365

Pediatric trials and labeling information for newly approved cancer therapies with targets potentially relevant to pediatric cancers.

Thomas J. Hwang,1 Liat Orenstein,2 Steven Dubois,3 Florence Bourgeois2. 1 _Harvard Medical School, Boston, MA;_ 2 _Boston Children's Hospital, Boston, MA;_ 3 _Dana-Farber Cancer Institute, Boston, MA_.

Objective: Few new therapies have been approved for pediatric cancers. In 2017, Congress enacted the Research to Accelerate Cures and Equity (RACE) for Children Act, which requires pediatric studies for adult cancer drugs with a molecular target relevant to a pediatric cancer. The pediatric study requirements will apply to new drugs approved by the FDA beginning in 2020. To inform the implementation of this new law, we evaluated pediatric trials and labeling information available for cancer drugs approved by the FDA.

Methods: We identified new adult cancer drugs approved by the FDA from January 2007 to December 2017 using the Drugs@FDA database. New formulations, drugs approved only for pediatric cancers, and imaging and contrast agents were excluded. For each drug, the potential pediatric relevance of the molecular target was determined using the Pediatric Molecular Target List published by the FDA in August 2018. Information on pediatric clinical trials, enrollment, and start and end dates was obtained from ClinicalTrials.gov as of September 2018. Drug labels were examined for pediatric efficacy, safety, or PK/PD and dosing data. A trial potentially open to children was defined as a study for which any participants < 18 years of age were eligible. We also examined pediatric studies, defined as trials for which the midpoint of the eligible age range was < 21 years.

Results: Among the 78 adult cancer drugs approved between 2007 and 2017, 61 (78%) had targets considered potentially relevant for pediatrics under the RACE Act. At the time of approval, 4 (5%) drugs had any pediatric labeling information. As of September 2018, after a median follow-up of 5.1 years (IQR: 2.9-7.0 y) from the date of first FDA approval, 17 (22%) had any pediatric information, and 8 (10%) had a pediatric indication. For these 17 drugs, the median time from first approval to addition of any pediatric information was 1.5 years (IQR: 1.1-4.6 y). Overall, 362 trials identified on ClinicalTrials.gov (total planned enrollment: 57,827 participants) were potentially open to children for 67 of the 78 drugs (86%), including 57 drugs (93%) with targets on the Pediatric Molecular Target List. For these 67 drugs, the earliest planned end date for trials open to children was a median of 3.3 years after first approval (IQR: 0.2-5.5 y). There were 171 solely pediatric studies for 56 (72%) drugs, and the earliest planned trial end was a median of 4.2 years (IQR: 2.3-5.9 y) after first approval.

Conclusions: Less than a quarter of FDA-approved cancer drugs include information on use in children, and most clinical trials open to children are not expected to be completed until several years after FDA approval. Under the RACE Act, most new drugs for adult cancers are likely to be subject to pediatric study requirements, indicating that timely completion of such trials may substantially increase the pediatric data available for cancer therapies.

#3366

SEER-CAHPS: A national population-based data resource to evaluate patient-centered cancer care.

Michael T. Halpern,1 Michelle Mollica,2 Lisa M. Lines,3 Julia Cohen,3 Erin E. Kent2. 1 _Temple University College of Public Health, Philadelphia, PA;_ 2 _National Cancer Institute, Rockville, MD;_ 3 _RTI International, Waltham, MA_.

Background: Delivery of patient-centered cancer care includes a focus on the preferences and values of individuals with cancer. SEER-CAHPS is a new data resource developed by the National Cancer Institute (NCI) linking cancer registry data from NCI's Surveillance, Epidemiology and End Results (SEER) program with Medicare claims and patient experience of care collected by the Medicare Consumer Assessment of Healthcare Providers and Systems (CAHPS®) survey. We report on the unique data available in SEER-CAHPS for population-based assessments of patient experience and discuss several studies of patient-centered care conducted using these data.

Methods: SEER-CAHPS includes SEER cancer registry data from 1973-2013 (including type/stage of cancer at diagnosis, patient sociodemographic characteristics, and mortality), Medicare fee-for-service (FFS) claims data from 2002-2015, and Medicare CAHPS survey responses from 1997-2015 (including patient-reported health status and experience of care ratings). CAHPS data in SEER-CAHPS include global ratings of overall care, personal doctor, specialist, health plan, and prescription drug plan and composite ratings of Doctor Communication, Care Coordination, Getting Needed Care, and Getting Care Quickly. The data also contain optional survey weights to account for the CAHPS sampling design.

Results: SEER-CAHPS currently includes 249,474 individuals with a history of cancer documented in SEER. Individuals enrolled in both FFS Medicare (36,284 with a CAHPS survey prior to cancer diagnosis, 64,642 with a survey after diagnosis) and Medicare Advantage (70,378 with a survey before cancer diagnosis, 78,170 with a survey after diagnosis) are included. The database also includes 805,124 CAHPS respondents without cancer from SEER regions (FFS: 326,476; MA: 478,648). These data allow assessments of the impacts of patient sociodemographic and clinical characteristics, treatment patterns, and self-reported general and mental health status on patient experience of care. SEER-CAHPS has been used to examine factors associated with experiences in individuals diagnosed with cancer during their last year of life; experiences of cancer survivors; and experiences of dually eligible (Medicare-Medicaid) cancer patients. A recent analysis focused on associations of their experiences of care with receipt of guideline-concordant follow-up care among people with colorectal cancer. Online information (https://healthcaredelivery.cancer.gov/seer-cahps/) provides details on variables in SEER-CAHPS, instructions for obtaining these data, and other researcher resources.

Conclusions: SEER-CAHPS is an important population science resource for assessing treatment patterns, unmet needs, and other factors to enhance patient-centered cancer care.

#3367

Treatment patterns in patients with cancer-associated venous thromboembolism in the US: A real world retrospective database analysis.

Jennifer D. Guo,1 Patrick Hlavacek,2 Tayla Poretta,1 Gail Wygant,1 Daniel C. Lane,1 Magdaliz Gorritz,3 Xin Wang,3 Chi-Chang Chen,3 Xianying Pan,1 Lisa Rosenblatt1. 1 _Bristol-Myers Squibb Company, Lawrence Township, NJ;_ 2 _Pfizer, New York, NY;_ 3 _IQVIA, Plymouth Meeting, PA_.

Background: Cancer patients have higher risk of venous thromboembolism (VTE) due to the cancer-induced hypercoagulable state or activation of the coagulation cascade by chemotherapy. The 2018 National Comprehensive Cancer Network guidelines recommend low molecular weight heparin (LMWH) to treat cancer-associated VTE and direct oral anticoagulants (DOACs) in some circumstances. This study aimed to understand VTE treatment patterns in a cancer-associated VTE population during hospitalization and post-discharge.

Methods: Patients with cancer-associated VTE and their respective treatments were identified in IQVIA's hospital Charge Data Master database from 7/1/15 to 4/30/18. Patients were followed for 1 month after VTE hospitalization. Post-discharge therapy was obtained from outpatient medical and pharmacy claims data.

Results: A total of 5,920 cancer patients hospitalized for VTE (54% female; mean age 65.9 [SD=13.0] years) were included. Lung (20%) and breast (13%) cancers were the most common cancer types. Average length of stay was 5.3 days (SD=4.1). 5,366 (91%) patients were treated during the hospital stay; LMWH (51%), DOACs (15%) and warfarin (12%) were the most common inpatient treatments. Within 30 days after discharge, the most common treatments were DOACs (26%) and LMWH (16%); 2,858 (48%) had no observed treatment in the outpatient setting. After discharge, 1,272 (21%) patients remained on the same therapy and 1,436 (24%) switched to a new treatment. Among those patients initiating DOACs (N=865) during hospitalization, 46% remained on DOACs after discharge.

Conclusions: In US hospital and outpatient settings, this real world study showed that LMWH treatment and treatment with DOACs were the most common initial inpatient therapies for cancer-associated VTE. DOACs were the most common post discharge treatment. Further investigation of patients without continued treatment within 30 days of discharge is warranted.

Distribution of post-discharge VTE therapy by initial VTE observed during index hospitalization

---

Initial VTE therapy during hospitalization(n=5,920) | Post-Discharge VTE Therapy

LMWH(n=918) | Heparin(n=12) | Warfarin(n=560) | DOAC(n=1,561) | Thrombolytic Agent (n=11) | No post-discharge VTE treatment(n=2,858)

LMWH n=3,040; 51.4% | 648 (21.3%) | 6 (0.2%) | 213 (7.0%) | 678 (22.3%) | 6 (0.2%) | 1,489 (49.0%)

Heparin n=566; 9.6% | 48 (8.5%) | 2 (0.4%) | 34 (6.0%) | 166 (29.3%) | 1 (0.2%) | 315 (55.7%)

Warfarin n=685; 11.6% | 80 (11.7%) | 2 (0.3%) | 242 (35.3%) | 86 (12.6%) | 2 (0.3%) | 273 (39.9%)

DOAC n=865; 14.6% | 25 (2.9%) | 0 (0.0%) | 13 (1.5%) | 400 (46.2%) | 1 (0.1%) | 426 (49.2%)

Thrombolytic agent n=210; 3.5% | 36 (17.1%) | 1 (0.5%) | 13 (6.2%) | 50 (23.8%) | 1 (0.5%) | 109 (51.9%)

No initial VTE treatment n=554; 9.4% | 81 (14.6%) | 1 (0.2%) | 45 (8.1%) | 181 (32.7%) | 0 (0.0%) | 246 (44.4%)

* Patients with no treatment within 30 days after hospital discharge were censored at 30-days post-dishcarge

#3368

Prevalence and significance of drug-drug interactions among patients with lung cancer.

Sawsan Rashdan,1 Hui Yang,2 Tri Le,1 David Hsieh,1 Carlos A. Alvarez,2 David Gerber1. 1 _UT Southwestern, Dallas, TX;_ 2 _Texas Tech University HSC | School of Pharmacy, Dallas, TX_.

IMPORTANCE: Significant drug-drug interactions may exclude patients from clinical trials and complicate routine clinical care. Prevalence and significance of drug-drug interaction (DDI) among cancer populations is unknown.

OBJECTIVE: To determine the rate of all potential drug interactions as well as the rate of major DDIs among individuals newly diagnosed with lung cancer in a national cohort.

DESIGN, SETTING, AND PARTICIPANTS: We performed a retrospective cross-sectional study of adult patients in the United States Veterans' Affairs (VA) medical system diagnosed with lung cancer between 2003 and 2016. Data were obtained from the VA Corporate Data Warehouse. Lists and categorization of potential DDIs were obtained from the Flockhart table provided by Indiana University School of Medicine. A list of commonly prohibited medications that are associated with major DDI, was determined after 11 clinical trials protocols of tyrosine kinase inhibitors in lung cancer treatment were reviewed.

MAIN OUTCOMES AND MEASURES: The primary endpoint was the prevalence of exposure to all potential DDI during the three months leading up to and including the date of lung cancer diagnosis. The secondary endpoint was the prevalence of exposure to major DDIs (prohibited medications).

RESULTS: Overall, 280 068 patients were included in the study. Mean age was 70 years, 98% were male, and 72% were white. Overall, 55.9% of patients were prescribed medications associated with potential DDI, and 5.1% of patients were prescribed medications with major DDIs that would be prohibited in

certain clinical trials. Among the 20 most commonly prescribed drugs associated with potential DDI, only two (gemfibrozil and phenytoin) were associated with major DDI.

CONCLUSIONS AND RELEVANCE: Medications with potential DDI are commonly prescribed to patients with lung cancer. However, only about 5% of patients are prescribed medications with major DDIs that would be prohibited in certain clinical trials. Further studies to determine the true clinical risk of all potential DDIs are warranted.

#3369

NCI's Office of Data Sharing: Promoting broad & equitable policies and processes to improve cancer care.

Freddie L. Pruitt, Sylvia Shabaya Gayle, Nina Ghanem, Anna V. Mencarelli, Anthony R. Kerlavage, Vivian Ota Wang, Jaime M. Guidry Auvil. _National Cancer Institute, Rockville, MD_.

Data sharing increases our understanding of factors that influence health and diseases by enabling additional research questions from secondary data users, improving statistical power through combining multiple data sources, facilitating reproducibility and validation of research results, and supporting innovation with the development of research tools and methodologies.

The NCI recently established an Office of Data Sharing (ODS), whose goal is to promote broad and equitable data sharing policies and processes to improve cancer knowledge and care. ODS advocates for open-access and broad data sharing policies to enable reproducibility, secondary use, knowledge sharing and innovation. ODS key priorities include: 1) coordinating the interpretation and implementation of the NCI and NIH Data-Sharing policies across the NCI, 2) streamlining data-access and -submission processes for NCI- and/or NIH-supported data repositories, 3) advocating for the proper balance of open-access, open-source, open-data-sharing policies while respecting the needs of research and participant communities.

The NIH and NCI Data Sharing policies facilitate scientific progress by encouraging data access and sharing of genomic, phenotype, environmental, and behavioral data. Data access and sharing management, stewardship, and operating procedures are coordinated through a NIH governance structure of senior leadership and staff across NIH Institutes/Centers (ICs). NIH Data Access Committees (DACs) ensure data access based on shared principles and procedural standards of data management, security, privacy, and research participant protections.

In efforts to improve data access efficiency, the ODS consolidated The Cancer Genome Atlas, intramural and extramural NCI DACs into a centralized operation, the NCI DAC. In comparison to other ICs, the NCI DAC receives the largest volume of data access requests (DARs) for access to controlled-tier level data. Specifically, the NCI DAC received 9214 DARs in 2017, representing 30% of all DARs received by NIH with a mean review time of 53 days, in comparison to the combined average of 35 days for all other NIH DACs. The NCI DAC oversees 216 studies registered in dbGaP, with 93 general research use (GRU) and 22 health, medical, biomedical (HMB) consent groups that allow data to be used for broader research uses. The centralized NCI DAC implemented data access review procedures for all DARs and expedited processes for projects requesting access to datasets with GRU and HMB data use limitations. These expedited procedures have produced a 26.5-fold decrease in time-to-decision from 53 to 2 days for researchers requesting access to NCI datasets.

Additionally, ODS is committed to addressing health disparities ethics and is developing an Ethical, Economic, Legal, and Social Implications program that will address policies, practices, and ethical issues that arise from data sharing. 

## BIOINFORMATICS AND SYSTEMS BIOLOGY

### Applications of Cancer Computational Biology 1

#3370

Chromatin-informed inference of transcriptional programs in gynecologic and basal breast cancers.

Hatice Ulku Osmanbeyoglu,1 Fumiko Shimizu,2 Angela Rynne-Vidal,3 Tsz-Lun Yeung,3 Petar Jelinic,4 Samuel C. Mok,3 Gabriela Chiosis,2 Douglas A. Levine,4 Christina Leslie2. 1 _UPMC Hillman Cancer Center, Pittsburgh, PA;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 3 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 4 _New York University Langone Medical Center, New York, NY_.

Epigenomic data on transcription factor occupancy and chromatin accessibility can elucidate the developmental origin of cancer cells and reveal the enhancer landscape of key oncogenic transcriptional regulators. We develop a computational strategy called PSIONIC (patient-specific inference of networks informed by chromatin) to combine cell line chromatin accessibility data with large tumor expression data sets and model the effect of enhancers on transcriptional programs in multiple cancers. We generated a new ATAC-seq data set profiling chromatin accessibility in gynecologic and basal breast cancer cell lines and applied PSIONIC to 723 patient and 96 cell line RNA-seq profiles from ovarian, uterine, and basal breast cancers. Our computational framework enables us to share information across tumors to learn patient-specific TF activities, revealing regulatory differences between and within tumor types. Many of theidentified TFs were significantly associated with survival outcome in basal breast, uterine serous and endometrioid carcinomas. To validate one PSIONIC-derived prognostic TF, we performed immunohistochemical analyses in 31 uterine serous tumors for ETV6 and confirmed that the corresponding protein expression pattern was also significantly associated with prognosis. Moreover, PSIONIC-predicted activity for MTF1 in cell line models correlated with sensitivity to MTF1 inhibition, showing the potential of our approach for personalized therapy.

#3371

Mapping the evolution of T cell states during DLI response and resistance using single-cell data and computational tools.

Elham Azizi,1 Pavan Bachireddy,2 Vinhkhang N. Nguyen,3 Shuqiang Li,3 Donna S. Neuberg,3 Robert J. Soiffer,3 Jerome Ritz,4 Edwin P. Alyea III,5 Dana Pe'er,1 Catherine J. Wu3. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _Broad Institute of MIT and Harvard; Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA;_ 3 _Dana-Farber Cancer Institute, Boston, MA;_ 4 _Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA;_ 5 _Harvard Medical School, Boston, MA_.

Donor lymphocyte infusion (DLI) is a standard of care and potentially curative immunotherapy for relapsed leukemia after allogeneic hematopoietic stem cell transplant (allo-SCT). Despite low response rates for many leukemias, chronic myelogenous leukemia (CML) has historically exhibited an exquisite DLI sensitivity, and we previously reported that durable response to DLI was associated with reversal of exhaustion of bone marrow (BM) -infiltrating T cells (Bachireddy et al., Blood 2014). Critical questions remain, however, regarding the exact transcriptional states of those T cell subtypes mediating exhaustion, anti-leukemia responses, and resistance to DLI.

To map evolving phenotypic T cell states in situ at single cell resolution, we profiled viable cells isolated from cryopreserved BM mononuclear cells from a median of 3 timepoints before and after DLI from 12 patients with relapsed CML after allo-SCT, including 6 long-term responders to DLI (R's) and 6 nonresponders (NR's), using single cell RNA sequencing (scRNA-seq). Using our computational tools for processing and analyzing scRNA-seq data (Azizi et al., Cell 2018), we detected 381,462 cells in total derived from 43 unique patient-timepoints that met our quality metrics.

Because DLI's anti-leukemic efficacy derives in large part from T cell activity, we sought a more refined characterization of T cells using our tool Biscuit (Azizi et al., Cell 2018) to merge, normalize and cluster T cells. We observed a marked increase in the number of T cell clusters in post-DLI samples compared to matched pre-DLI samples (p<0.001). Both R and NR cases exhibited increases in phenotypic volume induced by DLI (p<1x10-6), suggesting DLI induces multiple, independent gene expression components in both clinical outcomes. However, at both pre- and post-DLI timepoints, phenotypic volumes in R cases were higher than that of NR cases.

Comparing T cell exhaustion between R vs NR cases, we confirmed increased T cell exhaustion signatures in R-pre T cells. Using factor analysis techniques we found that anergy, dysfunction and tolerance are shared factors driving a subset of T cells that are enriched in NRs, suggesting multiple forms of T cell dysfunction in DLI resistance. We found that the clusters dominated by post-DLI R T cells were characterized by greater diversity of T helper subsets (Th1, Tfh, Th2, Th9, and Th22) and enrichment for exhaustion, type I and II IFN pathways, proinflammatory gene sets and CD8 T cell activation. Clusters dominated by NR T cells displayed increases in Th17 and Treg signatures, anergy and tolerance.

These data suggest that (1) pretreatment T cell phenotypic diversity may be important for DLI response; (2) that DLI increases such diversity differently in R's than in NR's; (3) while T cell subsets exhibit some overlap pre-therapy, responders and non-responders become increasingly dissimilar post therapy.

#3372

Cell-free DNA (cfDNA) fragment length patterns of tumor- and blood-derived variants in participants with and without cancer.

Earl Hubbell, Tara Maddala, Oliver Venn, Eric Scott, Susan Tang, Archana Shenoy, Alex Aravanis. _GRAIL, Inc., Menlo Park, CA_.

Previous studies on transplanted tissue or single cancers indicated that cfDNA variant fragment lengths reflect their respective source. The Circulating Cell-free Genome Atlas (NCT02889978) study provides an opportunity to examine cfDNA variant fragment lengths across tumor types and describe the nature of cfDNA variants derived from different sources.

Blood samples (N=1406) were evaluated from participants with (n=845) and without (n=561) cancer; cancer samples included 339 breast, 118 lung, 69 prostate, 45 colorectal, 27 uterine, 26 pancreas, 26 renal, 24 esophageal, 22 lymphoma, 19 head/neck, and 17 ovarian (113 remaining samples represented cancers with ≤15 samples each). cfDNA and genomic DNA from white blood cells (WBC) were subjected to a high-intensity targeted panel (507 genes, 60000X) with error-corrected sequencing; 533 samples also had matched tumor biopsy tissue subjected to whole-genome sequencing (30X).

Somatic single-nucleotide variants (SNVs; that passed noise filters) were identified and classified using the sequencing results into one of four categories: tumor biopsy-matched (TBM; present in cfDNA and biopsy), WBC-matched (WM; present in cfDNA and WBC), non-matched (NM; low probability [P<0.01] of being WBC-derived), or ambiguous (AMB; unidentifiable source). Fragment lengths of reference and SNV alleles were recorded. A statistical model based on fragment lengths was built to predict the likelihood that an SNV belonged to a WBC-like source without using the WBC sequencing results.

A total of 21604 SNVs were identified. The proportion of SNVs from each category were: 4% TBM, 68% WM, 19% NM, and 8% AMB. The number of samples (non-mutually exclusive) that had each SNV category were 152 TBM, 1338 WM, 499 NM, and 761 AMB.

Across categories, the median (SD) length of fragments containing the reference allele was 167 (16.3). Median (SD) fragment lengths of TBM, WM, NM, and AMB were 156 (22.2), 169 (14.8), 158 (20.8), and 165 (17.8), respectively. AMB and WM median SNV fragment lengths were similar to that of the reference allele, suggesting that fragment length shifts are minimal in SNVs derived from clonal hematopoiesis (CH). Fragment lengths of TBM and NM SNVs were similar; further, most NM SNVs came from cfDNA samples in the cancer cohort, suggesting that NM SNVs may be tumor-derived. As expected in a population with a median (SD) age of 61 (12.2), most SNVs occurred in the WM category.

The prediction model distinguished TBM from WM SNVs with an AUC of 0.87. However, at a specificity of 98% (to match filtering based on WBC sequencing), false-negative rates were 35% (TBM) and 52% (NM).

Together, these data suggest that source prediction based on fragment length alone is less robust than source assignment using individual-matched WBC sequencing, highlighting the importance of accounting for CH-derived SNVs when using targeted cfDNA-based approaches for cancer detection.

#3373

Identification of KRAS membrane bound states using an integrated computational and experimental approach.

Andrew G. Stephen,1 Animesh Agarwal,2 Angel E. Garcia,2 Gnana S. Gnanakaran,2 Jeevapani Hettige,2 Christopher Neale,2 Timothy Travers,2 Harsh Bhatia,3 Peer-Timo Bremer,3 Tim Carpenter,3 Jim Glosli,3 Helgi Ingolfsson,3 Piyush Karande,3 Felice Lightstone,3 Tomas Oppelstrup,3 Liam Stanton,3 Shiv Sundram,3 Xiaohua Zhang,3 Debsindhu Bhowmik,4 Arvind Ramanathan,4 Christopher Stanley,4 Debanjan Goswami,1 Gulcin Gulten,1 Frantz Jean-Francios,1 Dhirendra Simanshu,1 Tommy Turbyville,1 Rebika Shrestha,1 Que Van,1 Frank McCormick,5 Dwight Nissley,1 Fred Streitz,3 Constance Agamasu1. 1 _Frederick National Laboratory for Cancer Research, Frederick, MD;_ 2 _Los Alamos National Laboratory, NM;_ 3 _Lawrence Livermore National Laboratory, San Jose, CA;_ 4 _Oak Ridge National Laboratory, TN;_ 5 _University of California San Francisco, CA_.

Driver mutations in KRAS occur in almost 30% of human tumors, primarily in pancreatic, colorectal and lung tumors. These mutations result in increased cell proliferation and survival predominantly mediated through the MAPK signaling pathway. MAPK signal transduction is initiated by the interaction of RAF kinase with active RAS at the plasma membrane. The precise molecular details of this process are currently unknown. The Frederick National Laboratory for Cancer Research has partnered with the Department of Energy to harness high-performance computing and experimental data to generate models and hypotheses of how KRAS engages with RAF kinase at the plasma membrane to initiate signal transduction. The initial phase of this work has focused on identifying membrane bound states of KRAS. We have used a variety of biophysical approaches (including NMR, protein foot-printing and neutron reflectivity) to investigate the structural orientation of KRAS at the membrane. In addition, large scale coarse-grained simulations of membrane bound KRAS spanning the millisecond time scale, have been completed. Three predominant membrane bound KRAS states were observed computationally: an exposed state (where switch 1 is available for RAF binding), an occluded state (where switch 1 is unavailable for RAF binding) and a transition state (where helix 5 is perpendicular to the membrane). These three states are also identified in the experimental data. Cumulatively, experimental and computational data predict KRAS exists in a dynamic equilibrium on the plasma membrane, interconverting between 3 states on the nanosecond time scale. The experimental data indicates the most populated conformation of KRAS is the transition state. Future efforts will address the significance of these three states for RAF interaction and signal transduction. This in depth understanding of RAS activation of RAF and the MAPK pathway is critically important for developing effective therapeutic interventions for cancers harboring mutant RAS.

#3374

Assessing the impact of neoantigen load on checkpoint blockade efficacy.

Zeynep Kosaloglu-Yalcin, Angela Frentzen, Ashmitaa Logandha Ramamoorthy Premlal, Jason Greenbaum, Alessandro Sette, Bjoern Peters. _La Jolla Institute for Immunology, La Jolla, CA_.

Mutations acquired during oncogenesis can result in so-called neoantigens, i.e. mutation-bearing peptides that have the potential to be bound by HLA molecules and recognized by T cells. Neoantigens were suggested to be crucial for the outcome of immune checkpoint therapies and accordingly, the neoantigen load of a tumor is often being used to predict therapy outcome. Current approaches utilize HLA binding predictions to rank peptides, assuming that neoepitopes are ranked towards the top. The neoantigen load of a given tumor is then usually defined as the number of peptides binding above a certain threshold. Using this approach some reports found a very strong correlation of neoantigen load and response to checkpoint blockade therapy while others found a better correlation when mutational load was used instead. However, comparing results between studies is hard because they used different tools and thresholds to identify mutations, and applied different criteria to prioritize their set of mutated peptides. We sought to overcome this lack of uniformity in re-analyzing data from several published checkpoint blockade therapy studies to consistently define the mutational burden and neoantigen load, and correlate them with therapy outcomes

We obtained sequencing data from checkpoint blockade studies deposited in NCBI dbGaP and run all datasets through our in-house mapping and variant calling pipelines. Neoepitope candidates were predicted and scored using our previously published pipeline. We applied different scoring schemes to define the neoantigen load which were based on HLA binding predictions with varying thresholds and different metrics used (i.e. predicted IC50 vs. percentile rank). We found that the exact definition of neoantigen load has a major impact on how well this metric performs in predicting checkpoint blockade therapy outcome. Furthermore, we implemented a likelihood score which is based on data of known immunogenic neoantigens and describes the likelihood of a predicted neoantigen to be immunogenic. Based on our preliminary analysis to date, we found that the cumulative likelihood score of a patient is a good predictor of checkpoint blockade therapy outcome, outperforming tumor mutational burden and predicted neoantigen load in several cohorts.

#3375

leveraging protein dynamics to identify mutational hotspot communities in cancer driver genes.

Sushant Kumar, Declan Clarke, Mark Gerstein. _Yale University, New Haven, CT_.

Large-scale exome sequencing of tumor genomes has enabled the identification of cancer driver genes using recurrence analysis and clustering-based approaches. Some of these methods also employ 3D protein structural data to identify missense mutational hotspots in many cancer-associated genes. However, in determining such mutational clusters in protein structures, a majority of these approaches overlook the role of protein dynamics. In this work, we present a novel framework to identify cancer driver genes using a more sensitive search of mutational hotspot communities on protein structures by incorporating models of protein dynamics. We applied our method to the TCGA pan-cancer atlas missense mutation catalog to identify cancer genes with hotspot mutational clusters within their associated PDB structures. Overall, our analysis predicts one or more mutational hotspot within the resolved structures of known as well as novel cancer driver genes. Ontological enrichment analysis implicates the roles of genes with predicted hotspots in vital biological processes, including cell differentiation, cell proliferation, signal transduction, kinase activity, and nucleotide binding. Furthermore, a comparison between our approach with previous 3D structure-based cancer hotspot detection methods suggests that the including protein dynamics significantly improves the identification of additional putative driver genes. In summary, our proposed framework predicts novel cancer driver genes and provides mechanistic insight into cancer progression through the lens mutational hotspots.

#3376

The secondary genomic architecture of melanoma.

Jake Conway,1 Amaro Taylor-Weiner,1 Saud AlDubayan,2 Brendan Reardon,2 Felix Dietlein,2 David Liu,2 Eliezer M. Van Allen2. 1 _Harvard Medical School, Boston, MA;_ 2 _Dana-Farber Cancer Institute, Boston, MA_.

Introduction: While targeted- and immuno-therapies have revolutionized the treatment of melanoma, a significant proportion of patients develop resistance to therapy and lack targetable tumor drivers. Thus, there remains a critical need for the discovery of novel tumor drivers to identify additional therapeutic targets. Melanoma has been divided into 4 genomic subtypes based on the presence of mutations in the three most frequently mutated, mutually exclusive, driver genes: BRAF, NRAS, NF1, and triple wild-type (TWT), and, in particular, no significantly mutated genes (SMGs) have been identified in TWT tumors. We hypothesized that uniform genomic analysis of expanded cohorts would provide power to identify driver genes altered at low frequencies, identify novel biological targets and therapeutic vulnerabilities unique to genomic subclasses, and specifically enable discovery of additional molecular properties unique to TWT melanoma.

Methods: We aggregated 1,013 melanoma tumor and matched germline samples that passed joint quality control metrics and performed harmonized genomic analysis. Mutational significance analysis was performed using MutSigCV2 and OncodriveFML, and candidate genes filtered using TCGA expression data. Mutational signature analysis was performed using deconstructSigs, and validated in a separate cohort of melanomas. DESeq2 and edgeR were used to perform differential expression analysis.

Results: We identified a set of 38 high-confidence drivers in melanoma (MutSigCV2, q < 0.1; OncodriveFML, q < 0.1), 21 of which were in either the COSMIC Cancer Gene Census or OncoKB. Several of these genes (e.g. ARID1A, CDK4) have not been considered significantly mutated or previously reported in melanoma. Further, mutational significance analysis within each subtype identified 63 BRAF, 73 NRAS, 26 NF1 and 11 TWT SMGs (MutSigCV2, q < 0.1), and revealed that secondary driver genes are rarely shared between the subtypes. Mutational signature analysis determined that a subset of melanomas (7%) have homologous recombination (HR) deficiency (signature 3), and this signature is significantly enriched in TWT melanomas (21.6%, p = 1.21 x 10-10, Fisher's). ATM, APLF and SMARCD1 expression were significantly correlated with signature 3 contribution (p < 0.05), and significantly differentially expressed between signature 3 and non-signature 3 tumors in TWT melanoma (q < 0.05).

Conclusion: Through uniform analysis of the largest melanoma whole-exome-sequenced cohort to date, we identified a set of high confidence driver genes, many of which were not previously associated with melanoma, as well as secondary driver genes unique to each genomic subtype. Mutational signature analysis identified the enrichment of HR deficiency in TWT melanomas, which appears to be facilitated through downregulation of ATM and may have immediate translational implications for patient-stratification of therapies that target HR deficient melanomas.

#3377

T-cell receptor repertoire profiling using an augmented transcriptome.

Eric Levy, Pamela Milani, Sean M. Boyle, Shujun Luo, Gabor Bartha, Charles Abbott, Rena McClory, Robin Li, John West, Richard Chen. _Personalis, Inc., Menlo Park, CA_.

Immunotherapy is growing as one of the most promising therapeutic approaches in clinical oncology practice. This brings with it an increasing need for comprehensive immuno-genomic profiling of tumors to better understand the interaction with the immune system. This includes profiling of the T and B-cell receptor repertoires (TCR/BCR), which has traditionally not been feasible with an exome/transcriptome platform.

To address these challenges, we developed ImmunoID NeXT, an augmented, immuno-oncology optimized exome/transcriptome platform designed to provide comprehensive information regarding the tumor and tumor microenvironment (TME) from limited FFPE tumor biopsies, including the TCR alpha, beta, gamma and delta chains and BCR heavy and light chains. We show how this platform accurately profiles abundant clones, and can be applied to understand the diversity and activity of the adaptive immune system.

We characterize the performance of ImmunoID NeXT at profiling TCR beta from RNA. We analyze the reproducibility of clones identified using replicates of PBMC and FFPE samples, and assess the concordance of top clones from a standalone TCR sequencing approaches to ours. Then, we test LOD by diluting well-characterized clonal T-cell line samples into PBMCs.

We also analyze patient-derived FFPE tumors to understand the profiles of tumor-infiltrating immune repertoires. First, we compare TCR beta profiling with IHC quantification of CD3+ cells in both tumor and adjacent normal tissues. Finally, to better understand both B and T cell infiltration, we profile the intra-sample heterogeneity of BCR and TCR in a set of tumors.

Between replicates of PBMC samples, abundances for shared clones have high concordance (R2>0.98). We observe strong concordance of the abundances for shared clones between adjacent curls of a tumor FFPE sample (R2>0.87), showing that our approach is robust to degraded FFPE samples. Compared to a standalone approach, we identify over 93% of the top 1000 clones, with highly concordant abundances (R2>0.94). Assessing LOD in dilutions of T cell lines into a PBMC sample, we are able to identify clones present at over 0.00032% RNA by mass.

In our analysis of T-cell infiltration, we find a significant T-cell population in normal tissues. We also compare TCR read counts detected by ImmunoID NeXT in tumor and normal tissues with IHC results. Finally, we find substantial inter-sample variations in the number of TCR and BCR clones in tumors.

ImmunoID NeXT has been designed to enable sensitive detection of abundant TCR and BCR clones in addition to comprehensive biomarkers from exome/transcriptome data. We demonstrate that our platform is reproducible, sensitive, and concordant with the top-abundance clones derived from targeted TCR methods, as well as feasible with FFPE samples. Finally, we highlight how immune repertoire results from ImmunoID NeXT can be used to gain understanding about the immunological composition of the TME.

#3378

Systematic pharmacogenomic analysis of large patient derived xenografts data.

Arvind Singh Mer,1 Wail Ba-alawi,1 Petr Smirnov,1 Yi Xiao Wang,1 Ben Brew,2 Ming-Sound Tsao,1 David Cescon,3 Anna Goldenberg,2 Benjamin Haibe-Kains1. 1 _Princess Margaret Cancer Centre University Health Network, Toronto, Ontario, Canada;_ 2 _SickKids Research Institute, Toronto, Ontario, Canada;_ 3 _University Health Network, Toronto, Ontario, Canada_.

One of the key challenges in cancer precision medicine is finding robust biomarkers of drug response. Patient-derived tumor xenografts (PDXs) have emerged as reliable preclinical models since they better recapitulate tumor response to chemo- and targeted therapies. However, the lack of standard tools poses a challenge in the analysis of PDXs with molecular and pharmacological profiles. Efficient storage, access and analysis is key to the realization of the full potential of PDX pharmacogenomic data. To address this, we have developed Xeva (XEnograft Visualization & Analysis), an open-source software package for processing, visualization and integrative analysis of a compendium of in vivo pharmacogenomic datasets. The Xeva package follows the PDX minimum information (PDX-MI) standards and can handle both replicate-based and 1x1x1 experimental designs. We used Xeva to characterize the variability of gene expression and pathway activity across passages. We found that only a few genes and pathways have passage specific alterations (median intraclass correlation of 0.53 for genes and positive enrichment score for 92.5% pathways). For example, activity of the mRNA 3'-end processing and elongation arrest and recovery pathways were strongly affected by model passaging (gene set enrichment analysis false discovery rate [FDR] <5%). We then leveraged our platform to link the drug response and the pathways whose activity is consistent across passages by mining the Novartis PDX Encyclopedia (PDXE) data containing 1,075 PDXs spanning 5 tissue types and 62 anticancer drugs. We identified 87 pathways significantly associated with response to 51 drugs (FDR < 5%), including associations such as erlotinib response and signaling by EGFR in cancer pathways and MAP kinase activation in TLR cascade and binimetinib response. Among the significant pathway-drug associations, we found novel biomarkers based on gene expressions, Copy Number Aberrations (CNAs) and mutations predictive of drug response (concordance index > 0.60; FDR < 0.05). Xeva provides a flexible platform for integrative analysis of preclinical in vivo pharmacogenomics data to identify biomarkers predictive of drug response, a major step toward precision oncology.

#3379

Assessment of tumor mutation burden using a novel panel design strategy utilizing highly mutated genomic regions.

Amrita Pati, Daniel Klass, Zhongyun Huang, Melissa Loyzer, Alex Lovejoy. _Roche Sequencing Solutions, Pleasanton, CA_.

Background

Tumor Mutation Burden (TMB) has grown in importance as a predictive biomarker for predicting response to check-point immunotherapy in several cancer disease areas such as non-small cell lung cancer (NSCLC) and melanoma. Although TMB derived from analysis of WES data is the current gold standard measure of the number of neoantigens in the system, WES is often cost-prohibitive as a routine practice for clinical interpretation. Targeted panels offer a more flexible and cost-effective option, but TMB estimates based on these subsets of the exome can be more variable and often disagree with each other. Current best practices for TMB panels advocate for panels with sizes of 1 Mb with high correlation between panel-derived TMB and WES TMB demonstrating effectiveness of a panel at assessing TMB accurately.

Methods

We present a systematic approach to designing panels for detecting TMB effectively while minimizing cost of sequencing, based on our Surveillance panel design algorithm. Our approach relies on the prioritization of highly recurrent non-driver mutations present in large cohorts (such as TCGA) for disease areas of interest. Such an approach greatly boosts the number of observations of short variants using a targeted panel while still meeting expectations outlined within current best practices such as a high correlation with WES-derived TMB, and high PPA and NPA with WES-derived TMB. Our NGS based workflow combines whole genome library preparation, hybrid capture target enrichment, high-throughput sequencing and a proprietary analysis algorithm that utilizes short exonic variants that are likely to be somatic.

Results

Using a combination of in silico data and clinical cohorts, we demonstrate the effectiveness of the above approach in designing panels for the assessment of TMB. Results show high correlations between panel-derived TMB and WES-derived TMB at panel sizes as low as 450Kb while highlighting the steady decrease in variability of the TMB score as panel size increases. We apply this panel and WES analysis to clinical NSCLC and CRC samples to demonstrate that recommendations from current best practices are met in terms of PPA and NPA with WES-derived TMB and high correlation with WES-derived TMB.

#3380

Methylome analysis of non-alcoholic fatty liver disease-associated hepatocellular carcinoma reveals etiology-specic pathway perturbations.

Qiong Wu. _The Chinese University of Hong Kong, Hong Kong, China_.

Chronic hepatitis B infection (HBV) and non-alcoholic fatty liver disease (NAFLD) have been shown to induce aberrant DNA methylation that may contribute to the development of hepatocellular carcinoma (HCC), which is one of the most common liver cancers. Most previous studies on DNA methylation in HCC have focused on altered methylation at gene promoters and CpG islands/shores. However, it has been increasingly recognized that DNA methylation in other genomic regions, including distal enhancers and gene bodies, could also play important roles in gene regulation in cancers. In our whole-genome bisulfite sequencing (WGBS) analysis of 10 human HBV-associated tumors and 10 human NAFLD-associated tumors, methylation states at all CpG sites in the genome of HCC tumors are comprehensively studied. To see how the methylomes of these two groups of HCC samples are related to other liver-related normal and pathogenic samples, we collected published WGBS data for HCC tumor, tumor adjacent and normal liver samples. Global hypermethylation of NAFLD-HCC has been identified compared with HBV-HCC and differentially methylated regions in enhancer and gene body regions also have been detected. Different methylation patterns for HCC pathogenesis with different etiologies have been detected and may offer potentially more specific therapeutic targets. This project is supported by Collaborative Research Fund C4017-14G and the Focused Innovations Scheme 1907309.

#3381

Investigating the structural aspects that confer differential immunogenicity in tumoral T cell epitopes.

Eduardo C. Antonio,1 Marcelo A. Bragatte,1 Gustavo F. Vieira2. 1 _Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil;_ 2 _Universiade La Salle, Canoas, Brazil_.

The search for what characteristics define an epitope as either an immunogenic or a non-responsive target for immunotherapy has eluded researchers in this field for years. Several studies demonstrate that certain positions in the peptide sequences, the MHC anchor residues, have a preferential composition of amino acids (allelic motifs), being those epitopes more likely to display a better immunogenic response. First of all, not all MHC ligands are immunogenic, considering that we have unnumbered self-epitopes being continuously presented in the cell surfaces. Still, additional steps on the antigen processing pathway are missing in regular in silico epitope prospection. In this specific work, we will test an additional element, central in our hypothesis, that alterations in tumor protein sequences result in a structural change that shifts the electrostatic surface of the pMHC molecules, pivotal for TCR recognition and the initiation of an immunogenic response.

Previously tested tumoral epitope sequences, associated with differential immune responses in cancer, were recovered. Despite the fact that the sequences were very similar, they triggered responses that were considerably different, and currently, there is no well-established explanation of why they conspicuously differ in immunogenic aspects to each other. Here we propose that structural changes in the TCR interaction surfaces of pMHCs, due to amino acids shift, can lead to modifications which can influence in the immunological synapses formation.

The sequences were submitted to an in silico modeling process to generate pMHC models through the Docktope tool (http://tools.iedb.org/docktope). These pMHC models will then be used to generate images of their electrostatic surfaces, looking for qualitative differences that can indicate the distinct responses triggering. Additionally, the complexes will be subjected to a clusterization process along immunogenic complexes stored in the Crosstope Database (www.crosstope.com). This databank is composed of pMHCs structures containing immunogenic epitopes recovered from the Immune Epitope Database (IEDB). In this sense, we will be able to possibly verify if immunogenic tumor epitopes are more similar to their pathogens immunogenic counterparts.

The presented novelty is related to the reliable modeling of the previously studied tumor sequences in MHC receptors, which allow us to investigate not only if a sequence is immunogenic or not, but the analysis of the process genesis from a structural point of view.

We believe that through structural investigation of tumoral T cell epitopes and their non-immunogenic variants, it will be possible to extract and understand the signs and, possibly, the regions where changes impact in a critical way immunogenicity abrogation or enhancement.

#3382

A pan-cancer analysis reveals high frequency genetic alterations in mediators of signaling by the TGF-β superfamily.

Kazufumi Ohshiro,1 Sobia Zaidi,1 Anil Korkut,2 Jian Chen,2 Shuyun Rao,1 Shoujun Gu,1 Wilma Jogunoori,1 Bibhuti Mishra,1 Rehan Akbani,2 Lopa Mishra1. 1 _George Washington Univ., Washington, DC;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Background: TGF-β/SMAD signaling is a crucial, often contradictory regulator in multiple stages of liver disease that include inflammation, cirrhosis and development of HCC as well as other cancers. The context-specific role of this pathway in treatment strategies has yet to be clarified. Therefore, understanding the multiple context-specific roles of the pathway across broad cancer types is critical towards deciphering the complexities of the pathway.

Methods: We followed our previous analysis of HCCs, by extending and examining TGF-β pathway across 33 TCGA tumor types and 9125 samples to address this question. We focused on 43 core genes that encode components that regulate signaling by the TGF-β superfamily with 50 target genes collectively identified through a consensus among TCGA network members. In addition, we extended our analyses to functional studies in mouse mutants and human cell lines with alterations of TGF-β signaling.

Results: Focusing on 43 core TGF-β pathway genes, we found at least one of them was genomically altered in 39% of samples (mutations: 24%, homozygous deletions: 10%, or amplifications: 14%). We observed the highest alteration frequencies with hotspot mutations, 65% of which were in liver and GI cancers. We identified hotspots in 6 genes, with new discoveries in TGFBR2 and BMP5. Interestingly, with all 6 hotspot mutations we observed increased expression of TERT, HMGA2, IL6, MMP9, COL1A1/1A2/3A1, MYC, and FOXP3. Surprisingly, CDH2, and ALDH1A1expression levels were markedly reduced in liver and GI cancers. Alterations in the core genes correlated positively with expression of metastasis-associated genes, and poor patient survival. Epigenetic silencing and miRNA expression were associated with limited activity of the pathway in a cancer dependent manner. Using proteomics data, elevated TGF-β pathway activity showed positive correlation activity of DNA damage repair and EMT pathways (R=0.24, p < 0.0001), while the cell cycle and apoptosis pathways showed strong negative correlation (R= -0.3, and -0.15, p < 0.0001). Functional analyses reveal that disruption of TGF-β leads to increased sensitivity to cisplatin and other DNA cross linking agents as well as radiation.

Conclusions: Our data suggest that TGF-β superfamily indices when combined with specific genes, such as HMGA2 and TERT, may represent strong prognostic markers, and targets in some cancer types such as HCC. This study provides a rich resource and broad molecular perspective that could guide future functional and therapeutic studies of the diverse set of cancer pathways mediated by TGF-β superfamily. In addition, when the pathway is disrupted, epithelial cells are more susceptible to transformation and invasion, potentially identifying specific populations that are more sensitive to chemotherapy such as cisplatin and 5FU, as well as radiation therapy.

#3383

EPIC: MHC-I epitope prediction integrating mass spectrometry derived motifs and tissue-specific expression profiles.

Weipeng Hu,1 Si Qiu,2 Youping Li,1 Geng Liu,3 Xiuqing Zhang,1 Leo J Lee4. 1 _BGI-Shenzhen, Shenzhen, China;_ 2 _BGI-Shenzhen, SHENZHEN, China;_ 3 _GenoImmune Therapeutics Co., Ltd, Wuhan, China;_ 4 _University of Toronto, Toronto, Ontario, Canada_.

Background: Accurate prediction of epitopes presented by human leukocyte antigen (HLA) is crucial for personalized cancer immunotherapies targeting T cell epitopes. Mass spectrometry (MS)profiling of eluted HLA ligands, which provides unbiased, high-throughput measurements of HLA associated peptides resulting from in vivo cellular processing, can be a highly valuable training set to build predictive models of HLA binding. In addition, gene expression profiles measured by RNA-seq data in a specific cell type could significantly improve the positive predictive value (PPV) of epitope presentation prediction. Although large amount of high-quality mass spectrometry data of HLA-bound peptides is being generated in the last few years, few of them provide matching RNA-seq data, which makes incorporating gene expression into epitope prediction difficult. Here, we aim to develop a publicly available prediction tool incorporating both sources of information, and demonstrate its superior performance over existing methods.

Methods: We obtained public HLA peptidome datasets with matching RNA-seq data of twelve cell lines derived from multiple tissues. We used these MS HLA ligand data to build Position Score Specific Matrixes (PSSMs) for five HLA-I alleles across these cell lines. We then used logistic regression to model the relationship among PSSM score, gene expression, peptide length distribution and whether the peptide could be presented in each of the twelve cell lines, and compared the feature weights among them.

Results: We found that the feature weights across different HLA-I alleles and cell lines were close to each other, suggesting that there is a universal relationship between PSSM score and gene expression across different cell lines that could be applied to epitope presentation prediction for multiple alleles in diverse tissues. When we replaced the cell-line-specific weights with universal weights summarized from all the cell lines, the logistic regression model's predicted power for each cell line only dropped slightly and still substantially outperformed predictions based on PSSM scores alone. Based on such a finding, we applied the universal feature weights to more than 180,000 unique HLA ligands collected from public HLA peptidomics datasets, and presented an Epitope Presentation Integrated prediCtion (EPIC) model for 66 HLA alleles. EPIC was substantially better than other popular methods, including MixMHCpred, NetMHCpan (v4.0), and MHCflurry, when evaluated on independent HLA eluted ligand datasets, with an average 0.1%PPV of 53.58%, compared to 40.50%, 40.20%, 29.81%, and 25.57% achieved by MixMHCpred, NetMHCpan (EL), NetMHCpan (BA), and MHCflurry, respectively.

Conclusion: By integrating MS and expression data, EPIC is superior to currently available methods in predicting epitope presentation for the 66 common HLA alleles that our models were built on.

#3384

Tumor growth dynamics in EGFR mutation-positive non-small cell lung cancer patients with gefitinib, a Bayesian approach.

Hong Yan,1 James Dunyak,1 Helena Edlund,1 Diansong Zhou,2 Nidal Al-Huniti2. 1 _AstraZeneca, Lexington, MA;_ 2 _AstraZeneca, Waltham, MA_.

Objectives: Tumor size dynamics has been considered as a significant predictor for patient's survival in solid tumor. Drug treatment may have a delay effect on tumor size and drug resistance might be developed after certain period of treatment. The objective of this analysis was to develop generalizable tumor size dynamic model to capture both time delay and loss of drug effect due to resistance.

Methods: Clinical data from IPASS Phase 3 study (NCT00322452) of gefitinib in non-small cell lung cancer (NSCLC) patients were used to develop a tumor dynamic model in a Bayesian framework using STAN. A total of 130 NSCLC patients on gefitinib treatment arm were included in the analysis. Different tumor size models including a three-clone model: sensitive cells (s), a priori resistant cells (r) and cells with acquired resistance (q) were tested. The model was verified with 10-fold cross validation approach and posterior predictive checks.

Results: More complex model such as considering sensitive, priori resistant and acquired resistant tumor dynamics could describe the data well. The loss of drug effect was successfully described by a log-normal density function. Based on 10-fold cross validation information criteria, the model provided a better characterization of drug resistance and time delay compared to the exponential decay model. The 4 MCMC chains converged to a common distribution for each parameter and the data was generally within the 95% prediction interval of the posterior predictive checks.

Conclusion: Tumor growth dynamic were evaluated using different bayesian models. The final model described both time delay and loss of drug effect due to resistance. The simulated individual tumor dynamic data could be potetntially utilized to establish the relationship with overall survival.

#3385

Comparison of genomic biomarkers identified by the whole exome, RNASeq and whole genome sequencing pipelines developed for the PDMR.

Li Chen,1 Rajesh Patidar,1 Biswajit Das,1 Chris Karlovich,1 Tomas Vilimas,1 Corinne Camalier,1 Vivekananda Datta,1 Shahanawaz Jiwani,1 William Walsh,1 Palmer Fliss,1 Sean McDermott,1 Justine N. McCutcheon,1 Amanda Peach,1 Michelle Ahalt-Gottholm,2 Carrie Bonomi,1 Kelly Dougherty,1 John Carter,1 Sergio Y. Alcoser,2 Tiffanie Chase,1 Raymond Divelbiss1,1 Marion Gibson,1 Kelly Hedger,1 Candace Mallow,1 Chelsea McGlynn,1 Malorie Morris,1 Marianne Radzyminski,1 Howard Stotler,1 Jesse Stottlemyer,1 Debbie Trail,1 Yvonne Evrard,1 Melinda G. Hollingshead,2 Mickey Williams,1 James H. Doroshow3. 1 _Leidos Biomedical Research Inc., Frederick, MD;_ 2 _National Cancer Institute at Frederick, Frederick, MD;_ 3 _National Cancer Institute, Bethesda, MD_.

Background: The National Cancer Institute (NCI) has developed a Patient-Derived Models Repository (PDMR; www.pdmr.cancer.gov) of patient-derived xenografts (PDXs) with clinical annotation and comprehensive genomic characterization using whole exome sequencing (WES) and RNASeq. An in-house data analysis pipeline has been developed and validated to call germline and somatic variants and to perform transcriptional profiling in these models. There is a need to incorporate additional biomarkers into standard data analysis pipeline, including loss of heterozygosity (LOH), microsatellite instability (MSI), structure variants (SVs)/fusions and copy number variation (CNV) for identifying appropriate PDX models for preclinical drug studies. Validation of the methods used for the assessment of these and other genomic biomarkers is a crucial aspect in the development of the PDMR data analysis pipeline.

Methods: WGS, WES and RNASeq were conducted on 58 PDX samples and genomic biomarkers were derived from different assays. For LOH calling, a set of ~800,000 heterozygous SNPs was first constructed from a population level genomic database (gnomAD) and a specific list of ~3000 highly heterozygous SNPs from a previous study. LOH regions were detected using Runs of Homozygosity (BCFtools/RoH) based on the genotypes of ~800,000 SNPs. Finally, percent of genomic LOH was calculated as the percent of eligible LOH regions in the whole genome. For MSI calling. mSINGS was used to assign a microsatellite instability score based on the fraction of unstable microsatellite loci. Gene fusions were detected using Tophat-fusion and Fusion-catcher from RNASeq data and Manta from WGS. CNVs were derived from WGS and WES using CNVkit.

Results: Genomic biomarkers derived from WES and RNASeq were highly concordant with the ones derived from WGS. Specifically, we found 1) the percent of genomic LOH was highly correlated between WGS and WES across 52 samples with R2=0.99, where LOH% ranged from <1% to ~50% and specimens within the same models had consistent data; 2) a strong concordance rate (91%) of MSI score was observed between WGS and WES across 49 samples; 3) clinically and diagnostically relevant structural variants/fusions (e.g. FGFR3-TACC3 and EWSR1-FLI1) detected from RNASeq data can be detected and validated from WGS data; and 4) CNV genomic profiles were highly correlated between WGS and WES and amplifications/deletions in clinically relevant genes were consistently detected by the two assays.

Conclusions: We observed excellent consistency between WGS, WES and RNASeq data in the assessment of percent of LOH, MSI score, SVs/fusions and CNVs. Our data analysis pipeline can accurately call genomic biomarkers from WES and RNASeq data, which facilitates the molecular characterization and prioritization of PDMR models for preclinical drug treatment.

#3386

Acute myeloid leukemia stem cell heterogeneity and RNA clonal expansion during disease progression and relapse.

Lindsay C. Stetson,1 Dheepa Balasubramanian,1 Anne Roe,1 Susan Pereira Ribeiro,1 Xuan Xu,1 Slim Fourati,1 Ashish Sharma,1 Samuel Li,1 Yogen Saunthararajah,2 Jaroslaw Maciejewski,2 Thomas LaFramboise,1 Jill Barnholtz-Sloan,1 Rafick-Pierre Sekaly,1 David Wald1. 1 _Case Western Reserve Univ., Cleveland, OH;_ 2 _Cleveland Clinic Foundation, Cleveland, OH_.

Acute myeloid leukemia (AML) is the most common type of acute leukemia affecting adults and is responsible for the largest number of leukemia related deaths. Despite the increase in remission rates, treatment outcomes remain variable and most AML patients succumb to their disease due to relapse. It is hypothesized that AML relapse results from the persistence of the relatively quiescent AML stem cells that escape chemotherapy. A permanent cure for AML will require targeting these cells that are responsible for initiating and maintaining the leukemic clonal hierarchy. Little is currently known about the heterogeneity of leukemic stem cells and how they evolve during AML disease progression. AML exhibits extremely few DNA mutations; therefore, it is likely that RNA based changes also play crucial roles in the development and progression of AML. We have optimized a method to robustly and cost-effectively perform single cell RNA sequencing on AML stem cells. We completed a study of serial diagnosis/relapse samples from six AML patients, which revealed strong evidence of the evolution of RNA based changes in AML stem cells. The RNA cluster groups we identified were independent from DNA based clonal evolution. We found that RNA expression was heterogeneous and that a small population of AML stem cells highly influenced the expression of the bulk transcriptome, masking the expression patterns present in the majority of single stem cells. Notably, the expression of drug targets (CD33, HDACs, c-KIT, Ras, PLKs, TET1, SYK, UBA1) exhibited high heterogeneity and sparse expression among some single AML stem cells, potentially negatively impacting the drugs' effectiveness in eradicating this cell population. We used a parallel consensus clustering approach to identify RNA clonal groups. While each patient studied had a distinct pattern of RNA clonal evolution, we observed that progression was characterized by the post-treatment loss of the major diagnosis clonal group combined with the expansion of a minor diagnosis clone to a relapse dominant clone. We identified distinct clonal groups in each pilot study patient and tracked their evolution in a pseudotime analysis. Disease progression in was characterized by a significant up regulation of cytokine mediated signaling particularly in the IRF2/7/8/9, CXCR4, STAT3/5/6, and CCL5. This was combined with a significant downregulation in apoptosis/p53 and a global downregulation in metabolism (glycolysis, oxidative phosphorylation, and fatty acid synthesis) during disease progression. These changes were not significant when looking at pooled AML stem cells or when analyzing paired diagnosis/relapse bulk total AML samples in several publicly available datasets, revealing the power of single cell sequencing to uncover relevant biology. We confirmed our findings using high-density flow cytometry on an independent set of fifty AML patients.

#3387

**Comprehensive detection and analysis of mutant-** SF3B1 **splice pattern.**

Samar Alsafadi,1 Dorine Bellanger,1 Michele Cornella,1 Erik Lehnert,2 Sergio Roman-Roman,1 Marc-Henri Stern,1 Tatiana Popova1. 1 _Institut Curie, PSL Research University, Paris, France;_ 2 _Seven Bridges Genomics, Cambridge, MA_.

Aberrant alternative splicing is emerging as a frequent and important process in cancer. Splice aberrations are mainly associated with missense mutations within genes encoding splice factors involved in 3′-splice site recognition. SF3B1, U2AF1, SRSF2 and ZRSR2 are the most frequently mutated genes encoding splicing factors in tumors. Interestingly, these mutations are mutually exclusive and each leads to a distinct aberrant splice pattern.

Based on RNA-seq analyses of 74 primary tumors of uveal melanoma, we previously defined the aberrant splice pattern of SF3B1 hotspot mutations. In order to further characterize the catalogue of pathogenic mutations in SF3B1 that induce the aberrant splice pattern, we used a Sequence Bloom Tree (SBT) constructed from RNA-seq data for a total of 11,350 different tumors from the TCGA (Collaborative project with Seven Bridges Cancer Genome Cloud) to rapidly screen for samples consistent with the aberrant splice pattern.

Based on this analysis, 75 tumors from different tissues of origin presented with the mutant-SF3B1 pattern, including 70 tumors harboring SF3B1 mutations: 48 cases of the hotspot mutations (21 tumors with SF3B1R625, 6 with SF3B1K666 and 21 with SF3B1K700) and 22 cases harbored non-hotspot SF3B1 mutations. No mutation or deletion in SF3B1 was detected in 5 tumors. Taking into account that the threshold of scores defining the significant presence of the aberrant splice pattern varies between tumor types, we proceeded to an in cellulo validation of a set of newly described mutations of SF3B1 and confirmed the induction of the aberrant splice pattern.

Overall, our study highlights the potential of splice patterns as a biomarker in absence of detectable splice related mutations and sheds light on an efficient, large-scale computational approach to screen clinical cases for splice patterns.

#3388

Tumor suppressor genes may promote tumorigenesis through allele specific expression.

Evan Clayton. _Georgia Institute of Technology, Atlanta, GA_.

Cancer has long thought to be a disease which develops from de novo cancer driver mutations in oncogenes and tumor suppressor genes (TSGs). The purpose of our study is to examine how TSGs contribute to tumorigenesis. Accordingly, we studied loss-of-function (LoF) mutations in TSGs within 2504 individuals from 1000 Genomes Project (1KGP) and 233 patients across four different cancer types from The Cancer Genome Atlas (TCGA). We found a large fraction of 1KGP individuals were carriers of heterozygous LoF mutations in TSGs. However, compound heterozygosity of LoF mutations in at least one TSG was only found in 20% of our TCGA patients. Further, analysis of allele specific expression (ASE) in these tumors identified several TSGs where the mutant allele is overexpressed relative to the reference allele. This evidence of ASE suggests TSGs have the potential to drive tumorigenesis in the heterozygous condition, if the reference allele is sufficiently repressed.

#3389

Analysis of the hepatocellular carcinoma transcriptome reveals dysregulation in pathways implicated in immunotherapy efficacy.

Wei Tse Li, Christine O. Honda, Yuanhao Qu, Thomas K. Honda, Omar A. Saad, Jessica Wang-Rodriguez, Weg M. Ongkeko. _University of California San Diego, CA_.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related mortality in the world, but current treatment options are very limited in efficacy for patients who are diagnosed late, which often occurs because of limitations in current screening methods. Immunotherapy has emerged in recent years as arguably the most effective treatment for advanced HCC, but the failure of a large percentage of patients to respond to immunotherapy remains the ultimate obstacle to successful treatment. A phase I clinical trial with CTLA-4 checkpoint blockade has only achieved a 17.6% partial response rate in patients with advanced HCC, while another clinical trial using the PD-1 checkpoint inhibitor nivolumab has reported 18% partial response. Etiology-associated dysregulation of immune-associated (IA) genes may be able to explain the development of this differential clinical response. Using large-scale RNA-sequencing data from The Cancer Genome Atlas (TCGA), we identified immune-associated genes potentially dysregulated by alcohol or viral hepatitis B in HCC. Thirty-four dysregulated IA genes exhibiting significant correlation of gene expression to survival data and clinical variables were identified. To investigate the molecular mechanism for their dysregulation and explore the possibility of genome-based patient stratification, we profiled the correlation of all genomic alterations in HCC patients to IA gene expression using the information theory-based algorithm REVEALER. We also studied gene expression regulators and identified multiple microRNAs implicated in HCC pathogenesis that can potentially regulate these IA genes' expression. Finally, we validated the dysregulation of several genes by alcohol, including the downregulation of NDRG2 and SOCS2, in vitro using HCC cell lines. Our study identified several potentially key pathways, including the IL-7 pathway, TNFRSF4 (OX40)- NF-κB pathway, and IL-10 pathway, that may be targeted in HCC immunotherapy treatments as well as possible sources of dysregulation for pathways known to be implicated in HCC immunotherapy efficacy. Lastly, we present microRNAs as promising therapeutic targets for dysregulated IA genes because of their extensive regulatory roles in the cancer immune landscape.

#3390

RNA interactome topology perturbation analysis reveals candidate driver mutations in cancer.

Yongsheng Li,1 Daniel J. McGrail,2 Juan Xu,3 Junyi Li,3 Song Yi,1 Nidhi Sahni4. 1 _The University of Texas at Austin, Austin, TX;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 3 _Harbin Medical University, Harbin, China;_ 4 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Identification of the driver genomic variants is important for precision therapy of human cancer. Interaction between RNA-binding proteins (RBPs) and RNA plays important roles in regulating cellular function. Decoding genome-wide protein-RNA regulatory networks as well as how cancer-related mutations impair RNA regulatory activities in cancer will help prioritizing candidate driver mutations.

Here, we first explored the genetic alteration patterns of RBPs and found that candidate driver mutations are likely to occur on the surface of RBPs. This observations indicate that the driver mutations may perturb the RBP regulatory network in cancer. We then constructed protein-RNA interactome networks by integration of target binding screens and expression profiles. Regulatory network analysis highlights regulatory principles among interacting RBPs. In addition, somatic mutations selectively target functionally important genes (cancer genes, core fitness genes or conserved genes) in cancer. These regulatory patterns were further validated using independent data. A computational method (MERIT) and a web-based user friendly resource were further proposed to analyze the RBP-gene regulatory network perturbation induced by mutations across cancer types. Pan-cancer analysis also suggests that cancer cells selectively target "vulnerability" genes to perturb protein-RNA interactome that is involved in cancer hallmark-related functions. Based on the expression of perturbed RBP and target genes, we identified three subtypes of liver cancer with different survival rates. Specifically, we experimentally validated four pairs of RBP-gene interactions perturbed by mutations, which play critical roles in cell proliferation.

Taken together, our results provide a valuable resource for characterizing somatic mutation-perturbed protein-RNA regulatory networks in cancer, yielding valuable insights into the genotype-phenotype relationships underlying human cancer, and potential biomarkers for precision medicine.

#3391

Dissecting intratumoral cell-cell interactions in myeloid reprogramming by single cell RNA-seq.

Qianqian Song, Gregory A. Hawkins, Leonard Wudel, Ping-Chieh Chou, Elizabeth Forbes, Ashok K. Pullikuth, Liang Liu, Guangxu Jin, Lou Craddock, Umit Topaloglu, Gregory Kucera, Stacey O'Neill, Edward A. Levine, Peiqing Sun, Kounosuke Watabe, Yong Lu, Martha A. Alexander-Miller, Boris Pasche, Lance D. Miller, Wei Zhang. _Wake Forest University School of Medicine, Winston-Salem, NC_.

Tumor-infiltrating myeloid cells are the most abundant leukocyte population within tumors. Molecular cues from the tumor microenvironment promote the differentiation of immature myeloid cells toward an immunosuppressive phenotype. However, the in situ dynamics of the transcriptional reprogramming underlying this process are poorly understood. Therefore, we applied single cell RNA-seq (scRNAseq) to computationally investigate the cellular composition and transcriptional dynamics of tumor and adjacent normal tissues from 4 early-stage non-small cell lung cancer (NSCLC) patients. Our scRNA-seq analyses identified 11,485 cells that varied in identity and gene expression traits between normal and tumor tissues. Among these, myeloid cell populations exhibited the most diverse changes between tumor and normal tissues, consistent with tumor-mediated reprogramming. Through trajectory analysis, we identified a differentiation path from CD14+ monocytes to M2 macrophages (monocyte-to-M2). This differentiation path was reproducible across patients, accompanied by increased expression of genes (e.g. MRC1/CD206, MSR1/CD204, PPARG, TREM2) with significantly enriched functions (Oxidative phosphorylation and P53 pathway) and decreased expression of genes (e.g. CXCL2, IL1B) with significantly enriched functions (TNFa signaling via NF-kB and inflammatory response). Our analysis further identified a co-regulatory network implicating upstream transcription factors (JUN, NFKBIA) in monocyte-to-M2 differentiation, and activated ligand-receptor interactions (e.g. SFTPA1-TLR2, ICAM1-ITGAM) suggesting intratumoral mechanisms whereby epithelial cells stimulate monocyte-to-M2 differentiation. Overall, our analysis identified the prevalent monocyte-to-M2 differentiation in NSCLC, accompanied by an intricate transcriptional reprogramming mediated by specific transcriptional activators and intercellular crosstalk involving ligand-receptor interactions.

#3392

**Genomic features of** Helicobacter pylori **-associated gastric cancer and its enriched pathways.**

Claudia Machicado, Claudina Sancho, Maria Teresa Castromonte. _Universidad Peruana Cayetano Heredia, Lima, Peru_.

Gastric cancer (GC) is the third most common cancer. One of the risk factors of this lethal disease is the chronic infection by Helicobacter pylori, a Gram-negative bacteria that inhabits the gastric mucosa. During the H. pylori infection the host cell suffers multiple changes, including modifications in gene expression, that lead to dysplasia and cancer. Both cell proliferation and apoptosis are affected by the bacterial but the precise mechanism is unknown. Additionally, H. pylori is able to extract cholesterol from the cell membrane thus modifying the cholesterol homeostasis. Here we hypothesized that H. pylori (+) GC patients have different genomic features compared to H. pylori (-) GC. Our aim was to determine differences in gene expression, point mutation and copy number alterations (CNA) between H. pylori (+) and H. pylori (-) GC patients. We analyzed 470 genes that comprised 17 KEGG pathways including cholesterol homeostasis, apoptosis and others. The TCGA dataset was consulted to identify those differentially expressed genes (DEGs) by using UALCAN and UCSC Xena. The point mutations and CNAs (amplifications and deletions) were recognized through cBioportal. Gene ontology was determined by EnrichR and networking was elucidated using String and Cytoscape. Our findings showed a total of 55 DEGs when comparing H. pylori (+) and H. pylori (-) GC samples (p-value < 0.05). Those genes are involved in cholesterol homeostasis, cell cycle control, apoptosis, invasion, DNA damage, and Notch and TNF-β pathways. Four genes including ACOX1, CASP9, NODAL and SMAD2 were downregulated in H. pylori (+) GC samples whereas upregulated in H. pylori (-) GC samples. Some DEGs were associated with Overall Survival (p<0.05) including Myo5B, ABCA12 and LPL (cholesterol homeostasis); ADAM10 (Notch pathway); BMP4 (TGF-β pathway); and ERBB4. The networking and enriched pathways of H. pylori (+) GC samples were different from H. pylori (-) GC patients. In the other hand, the point mutations in p53 were found distributed through its three main domains in H. pylori (-) GC patients whereas the tetramerization motif was not affected in H. pylori (+) GC patients. Similarly, point mutations in ARID1A, MUC16 and TTN affected all of the conserved domains in H. pylori (-) GC patients whereas no alteration was found in the H. pylori (+) GC patients. Some differences were also noticed between the study groups related to CNAs such as the deletion/amplification of DMD in H. pylori (+)/H. pylori (-) GC patients, respectively. In overall our findings show that gene expression, point mutations and CNAs are differentially affected due to the bacterial infection in GC patients. Of interest, besides the classical pathways of cell proliferation and apoptosis, the presence of H. pylori seems to influence in other biological activities in the cell host such as cholesterol homeostasis through changes in gene expression.

#3393

Altered immune response in the transcriptome of patients with lung cancer.

Kahkeshan Hijazi,1 Julian Lel,1 Ehab Billatos,1 Elizabeth Moses,1 Christopher S. Stevenson,2 Matthew V. Lorenzi,3 Gang Liu,1 Joshua D. Campbell,1 Yusuke Koga,1 Jiarui Zhang,1 Fenghai Duan,4 Helga Marques,4 Marc E. Lenburg,1 Avrum E. Spira,1 Jennifer Beane1. 1 _Boston University School of Medicine, Boston, MA;_ 2 _Johnson & Johnson Lung Cancer Initiative, London, United Kingdom; _3 _Janssen Research & Development, London, United Kingdom; _4 _Brown University, Providence, RI_.

Introduction The immune system is critical to surveying and eradicating abnormal cells, but tumor cells develop ways to escape immunosurveillance and induce an immunosuppressive state. We previously developed and validated gene expression (GE) signatures measured in the normal airway-epithelial brushings in patients undergoing bronchoscopy for suspicion of lung cancer (LC). In this study, we seek to understand if the immunosuppressive environment extends to the airway field of injury via profiling of endobronchial biopsies from the central airway containing a broader range of cell types, including immune cells.

Methods Endobronchial biopsies from normal-appearing regions of the central airway were collected from ever smokers undergoing workup of indeterminate pulmonary nodules (7-30 mm in diameter) suspicious for LC at military and VA hospitals within the DECAMP consortium. Initially, total RNA from the biopsies (n=44, discovery-set) were isolated and sequenced. Reads were aligned to hg19 using STAR and gene level counts were quantified with RSEM. Poor quality samples were removed using FASTQC and RSEQC. Differential GE associated with cancer status was identified using edgeR, adjusting for smoking-status, COPD and sample quality. RNA from additional endobronchial biopsies (n=49, validation-set) were isolated and preprocessed similarly. Genes differentially expressed with LC status in the discovery-set were tested in the validation-set using gene set variation analysis (GSVA). Functional enrichment of cancer associated genes was explored using Enrichr. Comparison of cancer signatures identified in previously published LC studies was investigated using GSEA. CIBERSORT, xCell, TIMER software and single-cell RNA sequencing data generated from airway brushings were used to deconvolute the immune cell content of the bulk biopsy samples.

Results We identified a GE signature associated with LC which was significantly and concordantly enriched in the validation set of biopsies and two previously published studies of LC-associated GE in airway brushings. Genes decreased in LC patient biopsies were enriched for genes involved in immune-related pathways, including cytokine interactions, the inflammatory response and neutrophil degranulation. Computational deconvolution and comparison with single-cell RNAseq data predicts a decrease in neutrophils in the airway of LC patients.

Conclusion We identified LC-associated GE alterations in smokers presenting with indeterminate pulmonary nodules. Down-regulated genes in LC subjects are strongly associated with immune system function, specifically neutrophil biology. Subjects with LC appear to have an immunosuppressive environment directed towards myeloid cell populations, and this could have implications for the future development of immunoprevention therapies.

#3394

Diagnosis of intrahepatic metastasis (IM) and multi-entric (MC) carcinogenesis by novel mutational signature in patients with multiple and recurrent hepatocellular carcinomas.

Pinghua Yang,1 Shilei Bai,1 Yanpeng Zhang,2 Zhihao Xie,1 Zhangjun Cheng,1 Jie Yang,2 Jun Li,1 Jiaqian Wang,2 Zhengqing Lei,1 Yong Xia,1 Kui Wang,1 Zheng Li,1 Baohua Zhang,1 Xuying Wan,1 Mengchao Wu,1 Feng Shen1. 1 _Eastern Hepatobiliary Surgery Hospital, Shanghai, China;_ 2 _YuceBio, Shenzhen, China_.

Hepatocellular carcinoma is a common solid tumor worldwide and represents the third leading cause of cancer death. Patients with hepatocellular carcinoma (HCC) have poor prognosis because of frequent multi-centric (MC) and intrahepatic metastasis (IM). The discrimination between MC and IM is profoundly important for interpreting tumorigenesis and treatment decision making. However, it is difficult to correctly estimate by histological. Previous studies suggest that HBV integration sites, mitochondrial D-loop mutations, microsatellite loss of heterozygosity (MSI LOH) may provide more accurate information. Paired two tumor lesions and adjacent non-tumor tissues from 10 HBV-position patients were collected for whole genome sequencing. The proportion of shared somatic SNV among the tumor lesions was used to separate MC and IM groups. 4 of 10 patients with shared mutation rate higher than 10% (15.89-49.48%) were identified as IMs. 6 patients with shared mutation rate lower than 10% (0.33-1.97%) were identified as MCs. We also used shared HBV integration rate, mitochondrial D-loop, MSI LOH, diameter ratio to identify IM and MC. The coincidence rates between shared mutation rate and shared HBV integration rate, mitochondrial D-loop, MSI LOH, diameter ratio is 88.89%, 77.78%, 88.89%, and 90%, respectively. It suggests that the diameter ratio of multiple HCCs provide a no-invasion method in clonality analysis. Finally, we developed a panel covering 1M regions to replace whole-genome sequencing to identify IM and MC. We validated the shared mutation rate of target panel region in another 19 whole-genome sequencing HCC patients with multiple tumor lesions from ICGC. A

consistent rate of 94.74% in shared mutation rate between target panel region and whole genome region, indicated that for patients with resectable multiple HCCs, sequencing-based molecular diagnosis using somatic single nucleotide variation information showed higher sensitivity compared to previous techniques. And a smaller size of target panel contains adequate information is more cost-effective approach for clinical decision making.

#3395

TargetTranslator: Big data identifies non-canonical targets for high risk neuroblastoma.

Elin Almstedt,1 Caroline Wärn,1 Ramy Elgendy,1 Neda Hekmati,1 Emil Rosén,1 Ida Larsson,1 Rebecka Jörnsten,2 Cecilia Crona,1 Sven Nelander1. 1 _Uppsala Univ., Uppsala, Sweden;_ 2 _Chalmers University of Technology, Gothenburg, Sweden_.

Despite the many advances in the molecular characterization of neuroblastoma, effective treatments for high-risk patients are currently lacking. Using publically available data, integrated computational analysis offers new opportunities to uncover druggable subgroups. In the TargetTranslator project, we have combined state-of-the-art Big Data techniques to identify new targets in high-risk subgroups of neuroblastoma. Starting with clinical or genomic risk factors, TargetTranslator builds a consensus molecular signature across cohorts. This signature is scored against a massive amount of data from (i) childhood tumor biobanks (TARGET, R2), (ii) drug profiling data from cancer cell models (NIH-LINCS), and (iii) drug targets and pathways (STITCH, STRING, MSIGDB). As output, the system provides ranked lists of compounds, molecular targets and pathways for each risk factor, as well as an aggregated probability score. In a proof-of-concept study, we used TargetTranslator to recommend treatments for high-risk neuroblastoma subgroups, including COG high-risk, MYCN amplification, 11q deletion, and signatures of differentiation and ALK activation. Depending on risk group stratification, we detected between 21 and 680 substances (p<0.001), most of which were strongly associated to approximately 10 key targets. In addition to confirming a key role for the PI3K/mTOR and MAPK pathways, we detected high scoring compounds in non-canonical pathways, including neurotransmission, GLI/SMO, ROCK and PKA. Experimental validation of 11 predicted drugs in patient-derived neuroblastoma lines confirmed our signatures and reduced viability. We also identified four new compounds that significantly (p<0.05) suppressed MYCN protein levels (ROCK inhibitor fasudil, CNR2 agonist GW405833, CDK inhibitor AZD5438, lovastatin) at concentrations non-toxic in zebrafish embryos. By integrating data from patients, drugs, and drug-protein networks, we establish a new computational pipeline that predicts protein targets in specific patient subgroups. The TargetTranslator introduces a way to bridge drug effects with patient data and will be available as a user-friendly web tool on www.targettranslator.org in 2018.

#3396

Structural modeling and functional characterization of caspase inhibitor and tumor oncogene survivin 2B isoform: Implications in ligand binding and targeted drug design.

Eric H. Lee,1 Chen-Shing Chen,1 Ly Le2. 1 _Loma Linda University Medical Center, Loma Linda, CA;_ 2 _Ho Chi Minh International University, Ho Chi Minh City, Viet Nam_.

Survivin is a protein expressed in a variety of human tissues whose function is to inhibit apoptosis. It achieves this by indirectly blocking caspase 3- 7- and 9- activation, thereby retarding or preventing programmed cell death. Survivin has been found to be highly expressed and specific in cancer tissues, but is absent in terminally differentiated cells, thus making it an attractive drug target.

The structural basis through which survivin interacts with its neighboring proteins to inhibit apoptosis and promote cell growth is not well understood. Survivin is not believed to bind directly to caspases; instead, intermediary proteins such as XIAP, SMAC, and DIABLO are believed to first bind with survivin's "BIR" domain and then bridge its binding to caspases.

Unfortunately at the present, small molecule inhibitors against wild-type survivin have not demonstrated consistent anti-tumor activity to reach viability. The recent discovery of functional splicing variants for survivin (exon insertions or deletions) has drawn focus into whether these isoforms confer increased tumor resilience or aggressiveness, and whether inhibitors should specifically be designed for these targets. Specifically, the deltaEx3 (exon3 frameshift), 2B (intron 2 ins. of 69bp), 2alpha (intron 2 197bp tail ins.), and 3B (intron 3 165bp ins.) splice isoforms have been correlated with poor prognosis in AML, astrocytoma, breast, lung, renal, colorectal, brain, thyroid, bladder, prostate, sarcoma, and ovarian cancer.

To date, no structural model, either crystal or NMR, has been elucidated for any of the survivin splice variants, nor has the full survivin molecule been solved in complex with its known ligands. We therefore employed a combination of homology modeling and molecular dynamics (MD) simulations to characterize and predict the three dimensional structure for the survivin's most ubiquitous isoform, the 2B isomer, in which an alternative exon 2 termed "2B" includes a 69bp insertion. In addition, we characterized a model for survivin 2B complexed with SMAC/DIABLO, demonstrating for the first time in atomic level detail that the exon 2B insertion sequence plays a crucial role in stabilizing the survivin-SMAC/DIABLO interface involving both a hydrophobic interface protecting the binding pocket from solvent intrusion and also a conserved receptor-ligand network of stabilizing hydrogen bonds. By identifying the key residues and interactions on survivin's BIR domain, we therefore can predict exposed regions which represent drug targets to competitively bind the BIR domain and prevent survivin 2B from initiating its functional cascade that inhibits apoptosis of malignant cells. The hypotheses generated from this study would naturally lend itself toward future high throughput studies for targeted small molecular inhibitors.

#3397

The mechanism of PI3K activation at the atomic level.

Mingzhen Zhang, Hyunbum Jang, Ruth Nussinov. _National Cancer Institute at Frederick, Frederick, MD_.

PI3Ks are a family of lipid kinases in the PI3K/Akt/mTOR pathway that regulates cellular processes. PI3Kα harbors the frequent somatic oncogenic driver mutations conferring gain of function. It is an obligate heterodimer with the regulatory p85α subunit and catalytic p110α subunit. Here, we determine the PI3Kα activation mechanism by nSH2 release at the atomic resolution. The PIP2 substrate binding site is far from ATP and the membrane binding surface is buried in the inactive PI3Kα, raising the question of how catalysis is executed. Our results show that release of nSH2 domain from PI3Kα triggers significant conformational change in p110α, exposing kinase domain for membrane interaction. The C-lobe of kinase domain shows the structural rearrangement to reduce the distance between the ATP and the substrate binding site, offering an explanation to how phosphoryl transfer is executed. This explains how oncogenic mutations promote PI3Kα activation by facilitating nSH2 release and offers an innovative, PI3K isoform-specific drug discovery principle that prevents substrate phosphorylation by targeting isoform-specific residues in the substrate binding site.

#3398

Interaction of STAT proteins with genistein: A computational analysis.

Sneha Govardhanagiri,1 Shipra Reddy Bethi,1 Madhu Sudhana Saddala,2 Bassel F. El-Rayes,1 Ganji Purnachandra Nagaraju1. 1 _Emory University, Atlanta, GA;_ 2 _John Hopkins University, Baltimore, MD_.

Background: Signal transducer and activator of transcription (STAT) proteins are a seven-member family of cytoplasmic proteins. A conserved SH2 domain facilitates STAT initiation by allowing binding to phosphotyrosine motifs for STAT-receptor interactions and STAT dimerization. STAT proteins (STAT1, STAT2 and STAT3) are promising targets for anti-cancer drugs, because irregular facilitation of STAT initiation may result in cancer, inflammation and auto-immunity.

Methods: The crystal structure of the phosphotyrosine STAT1 and STAT3 dimers bound to DNA was obtained from a protein data bank (PDB), while STAT2 was modelled through Swiss-Model workspace by using 1YVL as a template. The stereochemical quality and integrity of STAT2 was tested by RAMPAGE server, ERRAT, ProSA, and Verify3D programs. The active sites were predicted using CASTp 3.0 with a new algorithm, which identifies and measures surface accessible pockets and interior inaccessible cavities on STAT1, STAT2, and STAT3. MD (molecular dynamics) simulation was conducted with STAT2 protein. Molecular Docking was used to predict the binding modes and approximate binding free energies of genistein to STAT1, STAT2 and STAT3 SH2 dimerization sites. Genistein was docked into the active site with AutoDock Vina in PyRx Virtual Screening tool.

Results: Genistein showed best binding energies. The most successful binding energies were -5.6 (STAT1), -6.7 (STAT2), -6.3 (STAT3) kcal/mol respectively. The binding interactions of this compound with the active site of STAT proteins suggested that amino acid residues (Tyr631, Leu639, Tyr640, Glu643, His647, Met648, Gln649, Ile659, Met660 and LYS16) play a crucial role in anti-cancer activity.

Conclusion: These observations increase the understanding of the functional role of STAT proteins in cancer and allow better development for additional druggable STAT inhibitors with high potency, specificity and outstanding bioavailability.

#3399

Uncovering hidden sources of transcriptional dysregulation arising from inter- and intra-tumor heterogeneity.

Bahman Afsari,1 Leslie Cope,1 Daria A. Gaykalova,1 Donald Geman,2 Sidharth Puram,3 Loyal A. Goff,2 Alexander Favorov,1 Elana Judith Fertig1. 1 _Johns Hopkins Sidney Kimmel Comp. Cancer Ctr., Baltimore, MD;_ 2 _Johns Hopkins University, Baltimore, MD;_ 3 _Washington University, St Louis, MO_.

Introduction: This study develops an innovative computational framework, Expression Variation Analysis (EVA), to model transcriptional dysregulation in cancer. Heterogeneity poses a major challenge in translational research. For example, inter-tumor heterogeneity limits the biomarker discovery and intra-tumor heterogeneity enables therapeutic resistance. Moreover, in some cancers driver mutations are insufficient to account for the widespread transcriptional variation responsible for these outcomes. Thus, new computational tools to model transcriptional variation are essential.

Methods: EVA is a unified computational framework to model transcriptional variation in cancer. Briefly, EVA quantifies transcriptional heterogeneity for one set of samples or cells from one phenotype using the expected dissimilarity between pairs of expression profiles. U-statistics theory can then quantify the statistical significance of the difference in transcriptional heterogeneity between phenotypes.

Results: We apply EVA to perform a comprehensive characterization of transcriptional variation in head and neck squamous cell carcinoma (HNSCC). At a pathway level, transcriptional variation in HNSCC tumors is higher than normal controls. Applying EVA to integrate ChIP-seq data with RNA-seq reveals that these pervasive transcriptional differences occur in enhancers. Similarly, applying EVA at a gene level to model splicing reveals more heterogeneity in transcript usage in tumor samples than normals. HPV- HNSCC tumors are unique in having mutations in genes that regulate the splicing machinery, and the HPV- tumors with these alterations have a greater number of dysregulated splice variants than those without. Nonetheless, the EVA analysis identifies a similar number of alternative splice variants in HPV+ as HPV- tumors suggesting an alternative mechanism of transcriptional heterogeneity in HPV+ disease. Adapting EVA to single cell data demonstrates that increased fibroblast composition is associated with greater variation in immune pathway activity in HNSCC. Moreover, we observe greater transcriptional heterogeneity in HNSCC primary tumors than lymph node metastasis consistent with a clonal outgrowth.

Conclusions: We demonstrate that the statistical framework from EVA enables differential heterogeneity analysis in HNSCC ranging from pathway dysregulation, splice variation, epigenetic regulation, and single cell analysis. This algorithm provides a critical framework to model the hidden multi-molecular mechanisms underlying the complex patient outcomes that are pervasive in cancer.

## MOLECULAR AND CELLULAR BIOLOGY / GENETICS

### Cancer Genomics 4

#3400

Comprehensive genomic profiling in Chinese patients with pulmonary sarcomatoid carcinoma.

Rong Shen,1 Fabo Qiu,2 Hao Zou,2 Lixin Li,3 Bin Yue,2 Zhihao Lu,4 Hanxia Han,5 Lijuan Chen,5 Lin Zhang5. 1 _Shandong Provincial Hospital Affiliated to Shandong University, Jinan, China;_ 2 _The Affiliated Hospital of Qingdao University, Qingdao, China;_ 3 _Qingdao Center University, Qingdao, China;_ 4 _Beijing Cancer Hospital, Beijing, China;_ 5 _OrigiMed, Shanghai, China_.

Background:Pulmonary sarcomatoid carcinoma (PSC) is a unique type of lung tumor with low incidence and poor prognosis. There is no standard treatment for advanced PSC patients. Most of them refer to NSCLC chemotherapy. The molecular characteristic of PSC was not as clear as other histological subtypes of NSCLC. Comprehensive genomic analysis of PSC would provide guidance for targeted and immune therapies.

Methods:Next generation sequencing (NGS) targeting 450 cancer genes was performed on FFPE and matched blood samples collected from 13 Chinese PSC patients (16 samples). Genomic alterations (GAs) including single nucleotide variations, short and long insertions and deletions, copy number variations, and gene rearrangements were analyzed. Microsatellite instability (MSI) status was assessed by NGS algorithms. Tumor tissues were analyzed for PDL1 expression by immunohistochemistry (IHC) staining. .

Results:The median age of PSC patients was 63 years (range 32-79), among them 10 were male and 3 were female. Average of 12.4 GAs /sample and 2.2 actionable GAs/sample were detected. The most frequently mutated genes were TP53 (10/13), PDGFRA (4/13), KRAS (3/13), MET (3/13), BAP1 (3/13), SPTA1 (3/13), FAT3 (3/13), LRP1B (3/13). In seven of the 13 patients (53.8%), two or more gene variants were co-existed, with up to five mutations in a single case. RB1 and KRAS alterations were found only in TP53-mut cases. The frequencies of the seven genes recommended by NCCN guidelines for NSCLC were MET (3/13), BRAF (2/13), EGFR (2/13), ALK (1/13), respectively. No RET, HER2 and ROS1 variations were found. Notably, the genomic alteration frequencies of MET and BRAF were higher compared with that in other NSCLC (MET: 4.9%, BRAF: 4.4%). MET exon 14 alterations including splice point mutation (c.3028+1G>C) and point mutation (D1010Y) were identified in two patients. BRAF and EGFR mutations occurred in PSC patients were all hotspot including V600E for BRAF and exon 19 deletion and L858R for EGFR. The variations of ALK include gene rearrangement and point mutation (A1200V). In addition, other potential therapeutic targets were also detected: PDGFRA (4/13), PIK3CA (2/13), PTEN (1/13), FGFR1 (1/13) and CDKN2A (1/13). MSI status was evaluated in 12 patients, and all the 12 patients were MSS. PD-L1 IHC assay was only performed in 4 available patients, and 3 patients were PD-L1 positive and 1 patient was PD-L1 negative. More pts will be collected and involved in the study.

Conclusion: Chinese PSC patients have multiple genomic alterations that can be targeted for treatments. The proportion of MET exon 14 mutations in PSC was higher, which was consistent with previous literatures. Therefore, the use of full-scale genome sequencing to explore potential therapeutic targets can help PSC patients benefit from potential targeted therapy and immunotherapy.

#3401

Genomic and transcriptome profile of 66 Chinese biliary tract cancer patient derived cell lines.

Feiling Feng,1 Qingbao Cheng,1 Bin Li,2 Liang Yang,1 Hua Dong,2 Bin Li,1 Dadong Zhang,2 Chang Xu,1 Xiaoya Xu,2 Yong Yu,1 Zishuo Chen,2 Zhizhen Li,1 Fugen Li,2 Zhiquan Qiu,1 Chen Liu,1 Xiaoqing Jiang1. 1 _Shanghai Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, China;_ 2 _3D Medicine Inc., Shanghai, China_.

Background: Biliary tract cancer (BTC) is an aggressive cancer with very poor prognosis. Currently there is no effective target therapy for BTC. To understand the genetic basis and look for potential drug targets of BTC, we sequenced the whole exome and transcriptome of 66 patient-derived primary cancer cells (PDC) from 23 unique patient samples.

Methods: We cultured 66 patient derived cell lines (PDC) passage range from P10 to P15 , from 23 BTC patients, with 1-9 PDC each patient. Both genomic DNA and total RNA were extracted from these cell lines, followed by WES and RNA sequencing on NovaSeq and HiSeq, respectively. Average sequencing coverage for WES is 139X and average data amount for RNA is 8.4Gbp. Variant calling was done by union set of Mutect and Pindel, with VAF cut-off 0.1 and filtered with a white variant list from COSMIC database. CNV was called from WES data by CNVKit with log2ratio cut-off 1.2. RNASeq data was mapped by STAR and fusion was calling by STAR-Fusion with at least 3 split reads.

Results: A median of 57 non-silent mutations were identified in Chinese BTC which was about 2 folds (57 vs. 30) of that in TCGA cholangiocarcinoma. Ethnic differences were found between Chinese and western population. The top four genes with frequency in Chinese BTC higher than TCGA cholangiocarcinoma were TP53 (56.5% vs. 14.3%), EPPK1 (26.1% vs. 2.9%), FRAS1 (13.0% vs. 2.9%), and ZFHX3 (13.0% vs. 2.9%), while ANKRD36C (4.4% vs. 20.0%), PBRM1 (8.7% vs. 22.9%), and TCHH (4.4% vs. 17.1%) were the top three genes with frequency lower than TCGA cholangiocarcinoma. We also found two previously reported cholangiocarcinoma related genes, PEG3 and GNAS, both harbored mutations in 3 of 23 patients (8.7%) in our data. However, they were not reported in TCGA cholangiocarcinoma. A median of 158 CNVs (range: 5-299) in gene level were identified, and the top three frequently mutated CNVs genes were LILRA3, SIRPB1, and GSTT1. Copy number loss of CDKN2A were found in 7 of 23 patients. Totally, 234 unique fusions were identified with 2-17 fusions per patient (median: 8). Among these fusions 5 of them (C15orf57-CBX3, SCARB1-UBC, SAMD5-SASH1, NCOR2-UBC, and FTH1-EIF5A) were reported to be associated with cancers. There were also 19 fusions found in TCGA pan-cancer datasets.

Conclusions: We built a comprehensive mutation landscape for Chinese BTC patients. Our results demonstrated ethnic differences between Chinese and TCGA Western cholangiocarcinoma patients. These PDC samples with profiled genetic information are good in vitro models for drug target discovery and validation for BTC patients.

#3402

Evaluation of laser microdissected primary breast tumors for RNA-Seq over bulk processing.

Praveen-Kumar Raj-Kumar,1 Lori A. Sturtz,1 Albert J. Kovatich,2 Brenda Deyarmin,1 Jeffrey A. Hooke,2 Leigh Fantacone-Campbell,2 Anupama Praveen-Kumar,1 Jianfang Liu,1 James Craig,1 Leonid Kvecher,1 Jennifer Kane,1 Jennifer Melley,1 Stella Somiari,1 Stephen C. Benz,3 Justin Golovato,3 Shahrooz Rabizadeh,4 Patrick Soon-Shiong,4 Richard J. Mural,1 Craig D. Shriver,5 Hai Hu1. 1 _Chan Soon-Shiong Institute of Molecular Medicine at Windber, Johnstown, PA;_ 2 _Clinical Breast Care Project, Murtha Cancer Center, Uniformed Services University /Walter Reed National Military Medical Center, Bethesda, MD;_ 3 _NantOmics, Culver City, CA;_ 4 _NantWorks, Culver City, CA;_ 5 _Murtha Cancer Center, Uniformed Services University/Walter Reed National Military Medical Center, Bethesda, MD_.

Introduction: RNA-Seq based gene expression profiling of breast tumor samples is widely used to subgroup patients and to identify gene signatures of prognostic value. However, tumor samples are highly heterogeneous, and so bulk processing of tumor tissue will consist of several different cell types. Here, we evaluated the advantage of using laser microdissected (LMD) breast tumors for RNA-Seq over bulk processing.

Methods: Patients for the in-house dataset were duly consented under an IRB-approved protocol of the Clinical Breast Care Project. A total of 118 primary breast tumors embedded in OCT (Optimum Cutting Temperature) were selected and processed by LMD. Total RNA and protein were extracted using the Illustra triplePrep kit. Paired-end RNA sequencing of 118 cases was performed using the Illumina HiSeq platform and the reads were preprocessed using a PERL-based pipeline involving PRINSEQ, GSNAP and HTSeq. The Cancer Genome Atlas (TCGA) primary breast cancer RNA-Seq data for 1097 samples was downloaded. Differential expression of genes (DEG) was assessed using DESeq2. Significance was described for DEG with fold change >2 and p-adjusted value of 0.05.

Results: A total of 24,518 genes with a mean expression of ≥ 10 raw counts across 118 tumor samples were identified in the in-house LMD dataset. In TCGA breast cancer RNA-Seq, 14,281 genes with a mean expression of ≥ 100 raw counts across 1097 tumor samples were identified. The conventional PAM50 classifier was used for intrinsic subtyping of in-house data, yielding 36 Basal-like, 14 HER2-enriched, 43 Luminal A, 22 Luminal B and 3 Normal-like calls. The provided PAM50 calls were used for TCGA which are 192 Basal-like, 82 HER2-enriched, 566 Luminal A, 217 Luminal B and 40 Normal-like calls. Within commonly expressed 13,165 genes, LMD and bulk processing exhibited approximately 40-78% non-overlap in significantly differentially expressed genes (SDEG) among the intrinsic subtypes. 21 unique stromal genes were present in SDEG unique to TCGA whereas there were only 5 SDEG unique to in-house dataset. Overall high positive correlation is observed among the stromal genes present in SDEG unique to TCGA suggesting strong stromal contribution in bulk processing. Pathway analysis of SDEG unique to LMD data suggested alterations in known cancer pathways (B-cell immune response, RNA metabolism and splicing, phagocytosis, and signaling components).

Conclusion: Analysis of The Cancer Genome Atlas breast cancer RNA-Seq data set (based on bulk processing tissue) suggested contribution of stromal signature genes and important differences from LMD specimens. Thus, tumor selection via LMD can result in better expression profiling by RNA-Seq which has the potential to uncover many cancer genes and pathways. The views expressed in this abstract are those of the author and do not reflect the official policy of the Department of Army/Navy/Air Force, Department of Defense, or U.S. Government.

#3403

Genetic analysis of pheochromocytoma.

Tatsuki Ogasawara,1 Yoichi Fujii,1 Masanori Fujimoto,2 Yusuke Shiozawa,1 Tetsuichi Yoshizato,1 Hiromichi Suzuki,1 Kenichi Yoshida,1 Yuichi Shiraishi,3 Hideki Makishima,1 Satoru Miyano,3 Tomoaki Tanaka,2 Seishi Ogawa1. 1 _Kyoto University, Kyoto, Japan;_ 2 _Chiba University, Chiba, Japan;_ 3 _Tokyo University, Japan_.

Backgrounds

Pheochromocytomas (PCs) are rare neuroendocrine tumors of the adrenal medulla characterized by excessive production of catecholamines. Although many are benign tumors, some patients show aggressive clinical courses. Despite improved understanding of gene mutations associated with PC, molecular pathogenesis is still unclear in up to 40% of the cases, particularly in those showing aggressive clinical pictures.

Materials & methods

To elucidate the molecular basis of PC, we investigated germline and somatic mutations, as well as copy number abnormalities in a total of 113 Japanese PC cases (JPN cohort), including 5 with aggressive tumors, using whole exome sequencing (WXS) of paired tumor/normal DNA (n=21), followed by targeted capture sequencing of known candidate driver genes (n=92). WXS data from The Cancer Genome Atlas (TCGA) database (n=144) was also analyzed to compare genetic characteristics between benign and aggressive cases with a higher statistical power (total; n=165, aggressive case; n=15).

Results

WXS of 21 Japanese PC cases disclosed 283 single nucleotide variants (SNVs) and 22 insertions and deletions with a median of 13 (range: 1-26) mutations per sample, predominantly showing age-related signatures. The landscape of genetic lesions was largely similar between JPN and TCGA cohorts. Both cohorts being combined, 36% of the cases had somatic mutations in one or more driver genes previously reported in PC. Mutations were most frequently observed in HRAS (12% of cases), followed by NF1 (9%), RET (5%), and EPAS1 genes (3%), suggesting a major role of alterations of RAS/mTOR and hypoxia pathways. Copy number abnormalities (CNAs) were detected in most cases, including del(1p), del(3q), del(17p), and del(22q). As many as 20% of the cases carried a germline variant, frequently detected in either RET, VHL, or NF1, confirming a strong genetic background in PC pathogenesis. EPAS1 mutation, MAML3 fusion gene, and a higher number of somatic mutations were associated with aggressive tumors. Of interest, two aggressive tumors harbored alterations in previously unreported genes, including a somatic PTEN mutation (frameshift) and a germline TP53 variant, respectively.

Conclusion

We disclosed a landscape of 113 PC cases, including a somatic truncating PTEN mutation and a germline TP53 mutation which were newly detected. Somatic EPAS1 mutations and MAML3 fusion genes could be associated with aggressive PC. Molecular pathogenesis is still unclear in 40% of all PC cases, for which a more unbiased analysis with whole genome sequencing is warranted.

#3404

Landscape and genomic correlates of ctDNA-based tumor mutational burden across six solid tumor types.

Katie Quinn, Elena Helman, Tracy Nance, Jennifer Yen, John Latham, Kristin Gleitsman, Ravi Vijaya-Satya, Carlo Artieri, Alex Artyomenko, Marcin Sikora, Darya Chudova, Richard B. Lanman, AmirAli Talasaz. _Guardant Health, Redwood City, CA_.

Background: Tumor mutational burden (TMB) has emerged as a predictive biomarker of response to immune checkpoint inhibitor (ICI) therapy. Current panel-based TMB algorithms aggregate signal from certain types of somatic variants (e.g. non-synonymous coding SNVs); however, delineating the contributions of these and other types of mutations may refine TMB calculation from gene panels. Moreover, early studies suggest other possible genomic correlates of patient outcome to ICI which may be complementary to TMB. Here, we explore the landscape of mutations comprising TMB and other genomic features correlating with TMB on a subset of several thousand late-stage plasma samples run on GuardantOMNITM (OMNI), a highly sensitive 500-gene cfDNA sequencing platform.

Methods: We developed a cfDNA-based TMB algorithm which is robust to variable tumor shedding levels and presence of clonal hematopoiesis. We assessed cfDNA-based TMB in over 1,000 plasma samples across six tumor types, including lung and prostate. We examine the contribution of nonsynonymous, synonymous, intronic SNVs, and indels to TMB score. We investigate correlations between TMB and additional genomic features, including chromosomal instability, loss of HLA-bearing chromosome 6p, microsatellite instability (MSI), and common oncogenic and resistance mutations.

Results: We found that the distribution of WES-calibrated TMB scores across this cohort of samples is consistent with TCGA, with median 10 mutations/Mb and upper-tertile of 14 mutations/Mb across tumor types. The number of non-synonymous coding SNVs per sample correlated highly with synonymous coding SNV and intronic SNV counts (Pearson's r > 0.7 for each). Including this additional signal in TMB calculation improves clinical sensitivity by up to 5%. In MSS samples, indels were highly correlated with SNVs, indicating that both likely arise from a similar underlying mechanism. We found no clear correlation between high TMB and chromosomal instability, with high TMB samples exemplifying a range of tumor ploidies. TMB association with oncogenic drivers is consistent with existing literature, with lower median TMB in EGFR-driven lung tumors (p < 0.01), but little to no correlation between TMB and KRAS or PIK3CA driver status, or STK11 loss of function (p > 0.05), suggesting these latter events could be independent clinical biomarkers to TMB.

Conclusions: Panel-based TMB scores can leverage synonymous and non-coding mutations to strengthen the signal of exome-wide mutation load. As more patient outcome data becomes available, TMB algorithms and orthogonal biomarkers of tumor genome immunogenicity will evolve further for improved guidance of patient response to immunotherapy. Sequencing panels with high sensitivity for TMB, via large panel space, and the ability to detect copy-number variations and MSI-status, will be important for biomarker development and clinical applications.

#3405

Integrated analysis of urothelial carcinoma.

Yoichi Fujii,1 Yusuke Sato,2 Hiromichi Suzuki,1 Tetsuichi Yoshizato,1 Kenichi Yoshida,1 Yuichi Shiraishi,3 Taketo Kawai,2 Tohru Nakagawa,2 Hiroaki Nishimatsu,4 Toshikazu Okaneya,5 Masashi Sanada,1 Hideki Makishima,1 Hiroyuki Aburatani,6 Satoru Miyano,3 Haruki Kume,2 Seishi Ogawa1. 1 _Kyoto Univ. Graduate School of Medicine, Kyoto, Japan;_ 2 _The University of Tokyo Hospital, Tokyo, Japan;_ 3 _Institute of Medical Science, The University of Tokyo, Tokyo, Japan;_ 4 _The Fraternity Memorial Hospital, Tokyo, Japan;_ 5 _Toranomon Hospital, Tokyo, Japan;_ 6 _Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan_.

[Introduction]

Accounting for 5-10% of all urothelial malignancies, upper urinary tract urothelial carcinoma (UTUC) is a relatively rare, whose molecular pathogenesis is poorly understood. We performed comprehensive genetic analysis in a large cohort of UTUC.

[Materials and Methods]

Surgical specimens of UTUC and matched normal samples were obtained from 209 patients with various stages who underwent nephroureterectomy, and were subjected to whole exome/RNA sequencing and methylation array.

[Results]

In total, we identified 33 significantly mutated genes (SMG) in UTUC, of which most frequently observed was the TERT promoter (53%), followed by KMT2D (46%), FGFR3 (42%), CDKN2A (42%), TP53 (35%), and RAS family genes (H-/K-/NRAS, 18%). More than 94% cases harbored either TP53, MDM2, FGFR3, or RAS alterations in a largely mutually exclusive manner. Dirichlet process clustering based on the SMG identified 3 distinct subgroups. Group 1 cases were frequently affected by TP53, MDM2, and CCND1 alterations with complex copy number alterations (CNAs) and characterized by high stage/grade and poor prognosis. Group2 tumors were characterized by FGFR3 and CDKN2A alterations and associated with lower stage/grade and favorable prognosis, while all cases in Group3 harbored RAS-family gene alterations, showing intermediate prognosis. Gene expression analysis using unsupervised clustering identified 5 clusters. These subtypes showed strong correlation with the mutation status; cluster 1 was enriched for FGFR3 mutations, while clusters 3-5 were dominated by TP53 mutations. Most of the RAS-mutated cases were classified into cluster 2. Cluster 1,2, and 5 showed high expression of luminal markers, while clusters 3 and 4 showed high expression of basal markers. Cluster 5 also had strong expression of p53-like and EMT markers, showing a worse prognosis than other 2 luminal (+) subtypes. Cluster 3 was characterized by strong expression of SCC and immune markers, including CD274 and PDCD1LG2, with frequent squamous differentiation. Finally, clustering analysis combining UBC from TCGA revealed a close association between UTUC and UBC expression subtypes: cluster 1 was similar to the 'luminal-papillary' and 'luminal', cluster 5 to luminal-infiltrated', and clusters 3 and 4 to 'basal-squamous'. Interestingly, cluster 2 was totally different from any of the TCGA subgroups, which might represent an expression profile unique to UTUC. Hierarchical analysis of methylation data revealed that approximately 60% of examined samples showed CpG island methylator phenotype (CIMP). Although there was no correlation between mutation/expression subgroups and methylation profile, KMT2D mutations were significantly enriched in CIMP (-) cases (p=0.0063).

[Conclusion]

UTUC showed a distinct genetic landscape, associated with various clinical features. Our findings of molecular characteristics in UTUC will contribute to the development of novel diagnostics and therapeutics.

#3406

Comprehensive genomic analysis of brain metastases from multiple cancer types.

Kazutaka Fukumura,1 Xizeng Mao,1 Xingzhi Song,1 Grant M. Fischer,1 Jie Yang,1 Erik P. Sulman,2 Michael A. Davies,1 Jianhua Zhang,1 Jason T. Huse1. 1 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _NYU Langone Health, New York, NY_.

Purpose: Brain metastases occur in approximately 8-10% of patients with cancer, and the incidence has increased over the past decades. The most common primary tumors responsible for brain metastases are lung cancer, melanoma, renal cell carcinoma (RCC), breast cancer and colorectal cancer, and the prognosis is still very poor with an overall 2-year survival rate of 8%. The precise mechanisms by which genomic and transcriptional abnormalities drive the formation of brain metastases remains unclear. Here, we conducted comprehensive genomic and transcriptional analysis with paired primary tumor tissue (or extracranial metastasis tissue) and brain metastasis tissue using whole-exome sequencing (WES), mRNA-Seq and global methylation profiling.

Methods: All patient samples were collected at the University of Texas MD Anderson Cancer Center. Frozen, paired brain metastasis tissue and primary tumor tissue (or extracranial metastasis tissue) and white blood cells were acquired from RCC (n=12), breast cancer (n=17), lung cancer (n=15) and cutaneous melanoma (n=14) patients. DNA and RNA were extracted from regions of frozen tissue with at least 70% viable tumor cells and peripheral blood leukocytes. Libraries for WES and mRNA-Seq were prepared and sequenced on the Illumina HiSeq4000 platform. For methylation profiling, DNA was subjected to bisulfite conversion and analyzed using Illumina Infinium MethylationEPIC Beadchip arrays.

Results: Genome-wide hypermutation due to POLE or POLD1 mutations was observed in one breast cancer patient and two lung cancer patients. Two of these cases acquired the hypermutation during development to brain metastasis. Somatic mutations or methylation of VHL gene were identified in 81.8% of RCC patients, and two patients had somatic VHL mutations in brain metastases only. Interestingly, Gene Set Enrichment Analysis revealed significant enrichment for hypoxia pathway transcripts in the RCC brain metastases relative to primary tumors. The most common alterations in breast and lung cancer patients were TP53 mutations with frequencies of 50.0% and 73.3%, followed by ERBB2 alterations (43.8%) in breast cancer patients and mutually exclusive alterations of EGFR (33.3%) and KRAS (26.7%) in lung cancer patients. Mutually exclusive alterations of NRAS (42.9%) and BRAF (42.9%) were also observed in melanoma patients. Gene expression and epigenetic analysis revealed characteristics of brain metastases depending on primary cancer types.

Conclusions: Comprehensive genomic analysis of brain metastases from four different cancer types revealed that brain metastasis tissue has unique genomic, transcriptional and epigenetic profiles according to histopathology groups. Therefore, the therapeutic strategies should be designed based at least in part on tumor histiogenesis.

#3407

Development of a chemokine signature identifying dMMR patients (pts) with poor prognosis.

Ying Wang,1 Rim S. Kim,1 Corey Lipchik,1 Ashok Srinivasan,1 Huichen Feng,1 Nan Song,1 Carmen J. Allegra,2 Patrick G. Gavin,1 Samuel A. Jacobs,1 Norman Wolmark,3 Peter C. Lucas,4 Katherine L. Pogue-Geile1. 1 _NSABP Foundation, Inc., Pittsburgh, PA;_ 2 _NSABP Foundation, Inc., and University of Florida Health, Gainesville, FL;_ 3 _NSABP Foundation, Inc., and Allegheny Health Network Cancer Institute, Pittsburgh, PA;_ 4 _NSABP Foundation, Inc., and University of Pittsburgh School of Medicine, Pittsburgh, PA_.

BACKGROUND: Colorectal cancer (CRC) pts with deficient mismatch repair (dMMR) tumors have greater immune cell infiltration than pts with proficient (pMMR) tumors and is thought to be partly responsible for the robust response of dMMR pts to PD-1/PD-L1 blockade. Not all pts with dMMR tumors respond to PD-1 blockade. Therefore, we conducted exploratory analyses to understand the complexity of the tumor immune microenvironment. We developed a gene expression signature predictive for MMR status and tested it for associations with prognosis.

METHODS: The one-sided Wilcoxon signed-rank test was used to select genes that were significantly over expressed in dMMR v pMMR tumors in a randomly selected discovery cohort in NSABP C-08 (N=500). Six chemokine genes were selected to build a predictive signature for MMR status because they were among the top 20 most significantly differentially expressed genes and were part of an immune chemo-attractant pathway. Addition of other genes did not improve prediction accuracy. The signature was tested in an independent cohort of C-08 (N=454) and in three other data sets: NSABP C-07 (N=1603), TCGA colon (N=619), and the NSABP Molecular Profiling Registry (MPR-1) (N=263). MPR-1 tissue samples were from primary tumors in pts who had recurred. Survival analyses were performed in C-07, C-08, and TCGA data, using Cox proportional hazards models. MMR status was determined for MPR-1 tissues with IHC using antibodies for MSH2, MLH1, PMS2, and MSH6.

RESULTS: Higher chemokine scores (CKS) were significantly associated with dMMR tumors in all three independent data sets composed of treatment-naïve early-stage CRC pts (P<0.01). However, in primary tumors of dMMR pts who developed recurrent disease, CKS was non-significantly lower than pts with pMMR tumors (P=0.072). As expected, high CKS was associated with a better prognosis in all three data sets, composed of early-stage CRC and was a more robust prognostic indicator in all 3 datasets than was MMR status. When CKS was combined with MMR status in early-stage pts, pts with high CKS had a better prognosis than pts with low CKS regardless of MMR status.

CONCLUSIONS: The CKS score has a strong association with MMR status in three different data sets and is associated with a better prognosis in early-stage CRC. In these pts, dMMR tumors had significantly higher CKS than pMMR tumors, but primary tumors from MPR1 pts who had all become metastatic showed a trend for association in the reverse direction. We conclude that dMMR primary tumors, which are likely to become metastatic, have a very different immune microenvironment than those that do not because the CKS is composed of chemo attractants for a variety of immune cells including NK, T, and B cells. Such a signature could be useful for prospectively identifying dMMR pts with early-stage CRC who are at high risk for relapse. SUPPORT: Genentech; sanofi; U10CA180868, UG1CA189867, U24CA196067, PA DoH; NSABP Foundation.

#3408

Molecular profiling in penile cancer in peruvian population: A retrospective analysis of clinical features and its association with molecular characteristics.

Carolina Belmar-Lopez,1 Bruno Muñante,2 Sandro Casavilca,2 Manuel Chumpitaz,2 Nelly Polo,2 Ebert Poquioma,2 Federico Valdez,1 Claudio Flores,3 Joseph Pinto,3 Henry Gomez2. 1 _OncoGenomics, Lima, Peru;_ 2 _Instituto Nacional de Enfermedades Neoplásicas, Lima, Peru;_ 3 _Oncosalud, Lima, Peru_.

BACKGROUND: Penile cancer (PC) is a rare malignancy in the developed world. The incidence is higher in less developed countries and is associated with poor survival due to the aggressiveness of the disease and lack of effective systemic therapies. Precision medicine is an emerging approach to more accurately predict which treatment strategy will be optimal.

METHODS: PC patients attending at INEN from 2006 to 2016 were included in the study. Tumors were re-staged according to the AJCC 8th. FFPE tumor samples were collected to assessed the presence of high-risk human papillomavirus (HPV) by qPCR. Next Generation Sequencing (NGS) was performed using the Ampliseq for Illumina cancer hotspot panel v2 target 2800 COSMIC mutation from 50 oncogenes and tumor supress genes. Kaplan-Meier estimation curves overall survival (OS) was applied and a p≤0.05 was considered statistically significant.

RESULTS: A total of 515 PC cases were diagnosed with a mean age of 60 (23-98) years. Most patients (80.95%) underwent tumor surgical treatment. The most frequent surgical procedure was partial penectomy (60.5%). Lymph node dissection was performed in 59.2% (247/417) of patients. Clinical stages were distributed, in I (6.4%), II (23.6%), III (22.7), IV (37.5). At diagnosis, 36.8% had spread regionally and 5.5% had distant metastases. All tumors were epidermoid histological type. Tumor size was ≤5cm (63.9%). Most tumors were invasive (91.9%) and moderately differentiated (63.8%). Positive lymph node was found in 61.9%; (153/247) and lymph extranodal in 52.9% (81/153). Chemotherapy was administered in 18.6%. The median follow-up period was 47.3 months (37.5-57.2), and 5-years OS was 32 months. Tumor surgical treatment and lymph node dissection were associated to OS (55.1% vs 7%) at 2-years and (46.5% vs 25%) at 5-years (ECIII: 40.7%; ECIV: 21.3%). HPV detection was performed in 327 samples. HPV DNA was detected in 84.1% (HPV16: 203/327; HPV18: 72/327) of PC samples. NGS was performed in 48 PC cases representing the spectrum of pathologic grades, stages, QT responses and HPV status. Nonsynonymous point mutations, stopgains/nonsense mutations, or indels were observed in 68% (34/50) genes. TP53 was mutated in 29 cases (69%) and was the most common genomic alteration. Most frequenly mutated genes were EGFR (28.6%), CDKN2A (30.9%), PI3KCA (59.5%), HRAS (61.2%), FBXW7 (21.4%), KDR (66.7%), SKT11 (42.8%) and SMAD4 (30.9%) of cases. No significant associations were present between mutation status for an individual gene and tumor grade, stage or histology.

CONCLUSION: High-risk HPV was associated with high indicence. Adecuate staging and clasification of locally adavanced or unresectable disease patients and comprehensive genomic profiling, findings of frequent mutations in peruvian PC, are necessary to select targets and to design effective personalized treatment options.

#3409

MSK-IMPACT Heme: Validation and clinical experience of a comprehensive molecular profiling platform for hematologic malignancies.

Ryan N. Ptashkin, Ryma Benayed, John Ziegler, Anoop Balakrishnan Rema, Justyna Sadowska, Iwona Kiecka, Caleb Ho, JinJuan Yao, Christine Moung, Kseniya Petrova-Drus, Khedoudja Nafa, Connie Batlevi, Martin Tallman, Ross Levine, Sergio Giralt, Anas Younes, Marc Ladanyi, Mike Berger, Ahmet Zehir, Maria E. Arcila. _Memorial Sloan Kettering Cancer Center, New York, NY_.

Background:

As the repertoire of molecular targeted therapies for hematologic malignancies continues to expand, so too does the opportunity for molecular profiling to inform treatment decisions. While mutations in certain genes, such as JAK2, MPL, MYD88 and BRAF have diagnostic utility, others such as FLT3, NPM1, IDH1, IDH2, DNMT3A, KIT and CEBPA have prognostic value. Here, we present the development and clinical experience of MSK-IMPACT Heme (Integrated Mutation Profiling of Actionable Cancer Targets for Hematologic malignancies), a comprehensive molecular profiling platform, utilizing hybridization capture and high coverage next generation sequencing of paired tumor and normal tissues.

Methods:

We designed custom DNA probes corresponding to all exons of 400 key oncogenes and tumor suppressor genes implicated in hematologic malignancies, including all genes that are targetable by approved and experimental therapies being investigated in clinical trials at our institution. The accuracy, precision, and sensitivity of MSK-IMPACT Heme was assessed on a validation set of 113 unique tumor samples with known SNVs and indels previously confirmed by orthogonal methods. We implemented a custom analysis pipeline to integrate the analysis of any number of normal samples with a given tumor and provide a reliable assessment of somatic alterations, even in post-transplant chimeric patients. The selection of matched nail, saliva, and/or blood tissue was determined at the time of test initiation as indicated by patient diagnosis and transplant history. The ability to detect somatic copy number alterations was demonstrated with samples previously characterized by SNP array platforms.

Results:

We sequenced 821 tumor samples, from 759 patients that represented over 50 tumor types to a mean depth of 758X. 429 patients were male (56.5%) and 20 cases were post allogeneic stem cell transplantation. The most common tumor types sequenced were Follicular lymphoma (11.9%), DLBCL (11.3%), and AML (11.0%). We identified 4,935 mutations from 732 samples. The most commonly altered genes were TP53, KMT2D, and CREBBP. Implementation of the MSK-IMPACT Heme workflow enabled the characterization of complex tumor specimens, including sorted cells and tumor samples from post-transplant chimeric patients. The joint utilization of matched patient and donor normal tissues enabled differentiation between somatic alterations and both host and donor derived common polymorphisms.

Conclusions:

The MSK-IMPACT Heme assay provides molecular profiling of hematologic malignancies with high accuracy and sensitivity. Paired analysis of tumors and patient and/or donor matched normal tissue samples enables the unambiguous detection of somatic alterations and the ability apply these data towards tumor classification, risk assessment, prognosis, disease monitoring, and treatment optimization.

#3410

Hodgkin and Reed-Sternberg cells genome-wide copy number alteration analysis at single cell level by high-throughput automated platforms.

Andrea Raspadori, Paola Tononi, Chiara Mangano, Marianna Garonzi, Claudio Forcato, Chiara Bolognesi, Genny Buson, Francesca Fontana, Gianni Medoro, Nicolò Manaresi. _Menarini Silicon Biosystems, Castel Maggiore, Bologna, Italy_.

Background: Classical Hodgkin Lymphoma (cHL) is generally highly curable with standard frontline therapies, although about 20% of the patients relapse or become refractory after initial treatment. cHL hallmark is the presence of morphologically characteristic malignant Hodgkin and Reed-Sternberg (HRS) cells that represent only a small fraction (about 1%) of the surrounding non-malignant environment. Genetic alterations of HRS cells are potentially a precious source of information to develop new treatments or prognostic biomarkers. In this perspective, low tumor cellularity, worsened by DNA degradation of FFPE samples, poses technical challenges to unravel malignant cells genetic alterations. Hereby we present new insights on purified HRS single cells obtained through highly automated platforms, providing precise observation of tumor genetic alterations.

Methods: FFPE tissue sections from 5 cHL patients were dissociated down to single cell suspensions. Cells were immunofluorescently labeled using anti-CD30-FITC and anti-PD-L1-PE antibodies. HRS cells, along with normal leukocytes, were selected on the basis of morphological and immunofluorescence criteria, and isolated using DEPArray™ NxT (Menarini Silicon Biosystems, MSB). Customized, high-throughput automated protocols were developed and implemented on STARLet liquid handler (Hamilton Life Sciences) to amplify isolated purified single cells genomic DNA and to generate genome-wide copy-number alterations (CNAs) profiles using Ampli1™ WGA and Ampli1™LowPass kits (MSB), respectively.

Results: More than 150 HRS cells were isolated from the 5 patient samples, from which CNA profiles were obtained. HRS cells presented extensive gains and losses across the whole genome, while leukocytes displayed flat profiles as expected. HRS cells clustered coherently with patients, revealing a high degree of heterogeneity of CNA profiles among different patients. However some commonalities across the patients genomes were identified. In particular, gains and amplification were detected in PD-L1, PD-L2 and JAK2 region (9p24), as well as gains and losses in regions where REL and other genes involved in NF-kB pathway map.

Conclusions: Leveraging on high throughput automated platforms and single cells isolation, the described method enabled cHL genome-wide genetic analysis at a single cell level, overcoming the intrinsic limitations of low-frequency of HRS and DNA degradation due to FFPE samples. Furthermore, unprecedented data on single HRS cells were described, opening up to a new approach to understand tumor diversity and to potentially develop personalized therapeutic strategies for cHL patients.

#3411

Genomic profiling of non-small cell lung cancer in Xuanwei, Yunnan Province.

Gang Guo,1 Tao Shou,2 Gaofeng Li,1 Hao Peng,2 Juan Zhao,3 Honglin Guo,3 Lin Zhang,3 Yunfei Shi4. 1 _The Third Affiliated Hospital of Kunming Medical University,Yunnan Tumor Hospital, Yunnan, China;_ 2 _First People's Hospital of Yunnan Province, Yunnan, China;_ 3 _OrigiMed, Shanghai, China;_ 4 _First Affiliated Hospital of Kunming Medical University, Yunnan, China_.

Background

Xuanwei, a rural county, is located in the northeast of Yunnan Province, China. According to a national retrospective survey on cancer mortality during 1973-1975, the highest mortality rate of lung cancer in Xuanwei was 5.3 times higher than that in rural areas nationwide. Age standardized mortality rates (ASMR) of lung cancer were 23.14/105 and 4.34/105 in Xuanwei and rural areas in China respectively. Characterization of genomic alterations(GAs) that drive patients (pts) disease development is critical in cancer management. However, the comprehensive genomic features of pts with non-small cell lung cancer (NSCLC) in Xuanwei have not been well understood.

Methods:

Next generation sequencing (NGS) targeting 37 lung cancer related driver genes was performed on FFPE and matched blood samples that collected from 85 NSCLC pts in Xuanwei. The pts included 50 males (59%) and 35 females (41%) with a median age of 54 years (range 36-78). GAs including single nucleotide variations (SNV), short and long insertions and deletions (Indel), copy number variations (CNV), and gene rearrangements were analyzed.

Results:

The histological subtypes of our cohort included LUAD(n=68, 80%), LUSC(n=9, 10.6%) and others(n=8, 9.4%).The most common GAs detected were TP53 (56%), EGFR (46%), KRAS mutation (25%), APC (8%), PIK3CA (8%), CDKN2A (7%), PTEN (6%), ALK (5%), CDK4 (5%), MTOR (5%), NTRK3 (5%) and PDGFRA (5%). In total, 74.1% of the pts had one or more actionable GAs. PI3K/mTOR pathway alterations including PTEN, MTOR,TSC1/2 and PIK3CA mutations were detected in 23.5% of the pts. Compared with OrigiMed unpublished data from larger Chinese NSCLC pts (EGFR 47.6%, ALK fusion 8.3%, KRAS 12.3%, n=1200), we identified similar frequency of EGFR mutation, while we found significant higher frequency of KRAS mutation(25%, n=85, P <0.001) and lower incidence of ALK fusion (1.2%, n=85, P =0.025) in Xuanwei cohort.

Comparing to OrigiMed unpublished data(as above), comprehensive genomic profiling (CGP) identified unique EGFR mutation profile in Xuanwei cohort that G719X occurred much more frequently (43.6% vs. 5.15%, P <0.001).

43.6%(17/39) EGFR-mutant pts harbored the uncommon mutation G719X, which could be identified alone (8%) or combined with other EGFR mutations (35.6%), the distribution of combined G719X mutations are G719X+S768I (15.4%),G719X+E709A(5.1%),G719X+G779C(5.1%),G719X+L838V(2.5%),G719X+L861Q(2.5%),G719X+S768I+N1107D(2.5%),G719X+T790M+V651L(2.5%).

Conclusions:

Comparing to the large cohort of Chinese pts with NSCLC, we identified higher mutation frequency of KRAS (P <0.001) and lower frequency of ALK fusion (P =0.025) in Xuanwei cohort. EGFR uncommon mutation G719X were identified in 43.6% of EGFR-mutant pts, but only accounted for 5.15% of EGFR-mutant pts in large Chinese NSCLC cohort. Therefore, the NGS based target sequencing assay revealed a unique pattern of driver gene mutations in Xuanwei cohort.

#3412

Genomic signature of trastuzumab neoadjuvant therapy predictive of patient survival in HER2-positive breast cancer.

Natalia Briones,1 Salvatore Facista,1 Rebecca Halperin,1 Paul Heaton,2 Daruka Mahadevan,3 Wiiliam Hendricks,1 Suwon Kim2. 1 _Translational Genomics Research Institute, Phoenix, AZ;_ 2 _University of Arizona College of Medicine-Phoenix, Phoenix, AZ;_ 3 _University of Arizona Cancer Center, Tucson, AZ_.

Although the adjuvant use of trastuzumab has significantly improved survival, therapy resistance still plagues HER2-positive breast cancer patients. A recent clinical trial study showed that tumors exposed to single dose trastuzumab have increased Immune index and PD-1 expression1, suggesting that trastuzumab not only blocks HER2 signaling but also alters the tumor immune microenvironment. In order to identify genomic signature(s) of tumors that correlate with trastuzumab-induced tumor immune response, we analyzed eight HER2-positive breast tumors samples from patients treated with 2 or more cycles of neoadjuvant trastuzumab. First, we assessed the tumor immune environment using immunohistochemistry (IHC) with a specific interest in PD-1, a T cell immune checkpoint marker. Second, we conducted DNA/RNA sequencing and identified gene mutations and expression signatures. Lastly, we used the gene signature to evaluate survival outcomes of breast cancer patients using the TCGA dataset. DNA sequencing detected that two tumors contained deleterious TP53 mutations, two tumors activating PIK3CA mutations, and one tumor a GATA1 inactivating mutation, closely reflecting the prevalent gene mutation frequencies previously reported in breast cancer. Unsupervised clustering of the RNA sequencing data revealed two distinct clades in this patient cohort. The pathway analysis showed the gene list that distinguished the two clades included inflammatory genes such as TNFα and CXCL10 as well as immune checkpoint genes, CTLA-4 and iCOS. IHC staining for immune cell markers was consistent with the RNA sequencing results showing the presence of a large number of T cells positive for CD4, CD8, or PD-1 in tumors expressing pro-inflammatory genes. Supervised clustering of the two clades identified a gene signature composed of 59 upregulated and 24 downregulated genes in the "pro-inflammatory" clade. The gene signature effectively stratified HER2 breast cancer patients in the TCGA dataset with a striking Hazard Ratio of 7.3 (n=182, p = 0.0026, 95% C.I. 2.01-27.23) and patients with the "pro-inflammatory" tumors had significantly favorable survival outcomes. This patient stratification by the gene signature was uniquely specific to HER2-positive breast cancer, indicating a functional relevance of the gene signature related to HER2 cancer biology and/or therapy. These results support the idea that trastuzumab elicits local immune response, which contributes to therapeutic efficacy. Furthermore, patients with tumors containing the "pro-inflammatory" gene signature may benefit from immune checkpoint inhibitor therapy in combination with trastuzumab.

1Varadan et al (2016) Clin Cancer Res 22(13):3249-99

#3413

CRIPAK genomic alterations are associated with indolent lung adenocarcinoma and predicts longer survival.

Jun Qian, Heidi Chen, Yong Zou, Pierre Massion. _Vanderbilt University Medical Center, Nashville, TN_.

Background: Multiple lung screening studies have shown a beneficial reduction in cancers specific mortality with annual low-dose computed tomography (LDCT) screening in high risk patients and resulted in a much improved probability of early stage disease at diagnosis. Nonetheless, still ~30% of such patients have a recurrence within 5 years following surgical resection. This begs the question what are the molecular determinants of cancer behavior between indolent and aggressive tumors.

Methods: A targeted deep sequencing was performed on the DNA samples of 21 adenocarcinoma in situ (AIS), 27 minimally invasive adenocarcinoma (MIA) and 54 fully invasive adenocarcinoma. These cancers were classified based on the tumor size (1.5 cm) as indolent (n=42) and aggressive (n=60) tumors. We analyzed the association between tumor aggressiveness and genomic alterations of these targeted genes including frequency of gene mutations, copy number changes, mutation signatures, tumor clonality as well as overall survival.

Results: Copy number loss of CRIPAK (chr4p16) and copy number gain of NOTCH1 were enriched in indolent tumors and chromosome 19q13 deletion was enriched in aggressive tumors. CRIPAK alterations including both mutations and copy number loss together were further significantly enriched in a subset of indolent tumors (16/25) that only consists of AIS/MIA. CRIPAK alterations were associated with longer survival in all population, after adjusting for histology and age (P=0.02). Interestingly, we found higher CRIPAK mRNA level was also significantly associated with better survival in a combined seven public lung adcarcinoma datasets (n=673, HR=0.37, P=1.1e-05), after adjusting for stage, gender and smoking history. In addition, both APOBEC and MMR mutation signatures were correlated with indolent tumors. Known driver genes such as KRAS, EGFR or TP53 mutations were not distinct between indolent and aggressive tumors, indicating an early role for these genes in the development of lung adenocarcinoma. Finally, we found that mutations of NCOR1, USP9X and NF1 were enriched in indolent tumors while mutations in GNAS and ATM were enriched in aggressive tumors.

Conclusion: Our study suggests the genomic alterations of the chromosome 4p16 harboring a novel cancer gene CRIPAK's deletions and mutations are predictive of indolent behavior, a finding that warrants further validation. This work was supported by UO1CA196405.

#3414

Identifying dysregulated lncRNAs and miRNAs in low grade glioma: Uncovering signaling pathways and novel therapeutic targets.

Stephen Carney, Felipe Nuñez, Pedro R. Lowenstein, Maria G. Castro. _University of Michigan, Ann Arbor, MI_.

A molecular subtype of low grade gliomas (LGG) is characterized by mutations in Alpha-Thalassemia Mental Retardation X-linked (ATRX), TP53 and isocitrate dehydrogenase 1 (IDH1R132H, mIDH1). Our lab has generated a genetically engineered mouse model that harbors the characteristic genetic lesions found in this molecular subtype of LGG. We determined that mouse neurospheres (NS) bearing these mutations showed downregulation of signaling pathways involved in oligodendrocyte differentiation. The production of 2-hydroxyglutarate (2HG) by mIDH1 induces histone and DNA hypermethylation, which has been shown to impair neural precursor cells' differentiation. We aim to investigate how the presence of mIDH1 impacts the epigenetic regulation of long non-coding RNAs (lncRNAs), microRNAs (miRNAs) and mRNAs in human mutant IDH1 glioma cell cultures obtained from patients at the time of surgery. We hypothesize that the dysregulation/remodeling of lncRNAs, miRNAs and mRNAs contributes to the invasiveness, differentiation status and stemness of glioma cells. Only a handful of lncRNAs have been studied in relation to their role in neural cells' lineage commitment and glioma stem cells' biology. We identified 127 differentially expressed (DE) lncRNA (FDR<0.01) in a human primary glioma cell culture SJGBM2, which has a ATRX mutation and was stably-transfected to express mIDH1. Of the 14 DE miRNAs identified, hsa-miR-210-3p and hsa-miR-4461, which have been linked to tumor invasiveness. The results from this work will reveal novel mechanistic pathways by which lncRNA/miRNAs and differentially expressed mRNAs impact downstream signaling pathways in LGG and uncover new therapeutic targets for this molecular glioma subtype.

SC is supported by the T32 Cancer Biology Training Grant, the work is supported by grants from NIH/NINDS to MGC and PRL.

#3415

Tumor fitness and immune exhaustion: Checkpoint vs endogenous settings.

Adrian Bubie, Nicholas Akers, Augusto Villanueva, Bojan Losic. _Icahn School of Medicine at Mount Sinai, New York, NY_.

Recently proposed tumor fitness measures based on neoepitope profiling and viral epitope similarity have led to treatment efficacy and immune response prediction models in the checkpoint setting for melanoma (SKCM) and small-cell lung cancer (NSCLC). In this work we test if these checkpoint based fitness measures are associated with either tumor-infiltrating T-cell activity or abundance, and also overall patient survival, in the endogenous setting for the SKCM (n=337) and NSCLC (n = 307) TCGA cohorts. We also investigated if epitopes arising from tumor viral co-factors in Hepatitis B liver cancer (HBV+ HCC) drove T cell activity or response compared to neo-epitope burden.

We find no statistically significant correlation between immune activity and response (as measured by tumor RNA-seq profiling) and the proposed neo-epitope checkpoint-based fitness measures, and similarly no significant survival effect. Further, we found firm evidence that tumor neoepitopes dominate HBV viral epitopes in putative immunogenicity and drive immune response in TCGA HCC (n = 178), and confirmed this trend in multi-regional data (12 patients, 72 samples). These results suggest that tumor-immune response and recruitment in SKCM and NSCLC, and their interplay with viral-cofactor HBV in HCC, are fundamentally driven by different factors in the endogenous setting.

Finally, we propose that tumor fitness may be partially encoded by a simple T-cell exhaustion expression signature, which we demonstrate is a significant predictor of patient survival for SKCM and NSCLC and may translate more widely into other tumor types in the endogenous setting.

#3416

APOBEC3C-H gene expression and APOBEC3-mediated RNA editing in breast cancer tumors are associated with immune response and improved outcome.

Mariko Asaoka,1 Santosh K. Patnaik,1 Eriko Katsuta,1 Takashi Ishikawa,2 Kazuaki Takabe1. 1 _Roswell Park Comprehensive Cancer Center, Buffalo, NY;_ 2 _Tokyo Medical University Hospital, Tokyo, Japan_.

APOBEC enzymes are strong mutagens in breast cancer. High APOBEC3B (A3B) expression in tumors is associated with poor prognosis, but clinical significance of the other APOBEC3s (A3A, C-H) is unclear. We investigated the associations of A3A and C-H with survival and tumor immune activity in 1091 primary breast cancer tumors of The Cancer Genome Atlas project. Tumor cytolytic activity, T cell receptor (TCR) diversity, and immune cell fractions were estimated from RNA sequencing data. Patients were divided into 3 equal groups by gene expression to compare high and low expressors. Cox, Spearman and t tests were used for survival, correlation and comparison analyses, respectively. Hallmark gene-sets were used for enrichment analysis. RNA editing was determined from whole exome and RNA sequencing data for 5,208 sites of 3,630 gene transcripts that are known to undergo APOBEC3-mediated RNA editing. Examination of 55 breast cancer cell-lines of Cancer Cell Line Encyclopedia showed that A3B and A3C represented 91% of A3 gene expression. Examination of Illumina Human Body Map showed that all APOBEC3s are expressed in leukocytes at a 4-450x higher level compared to breast. In patients, expression of A3B but not other APOBEC3s was higher in tumors compared to normal tissue (4.5x). A3C-H expression levels correlated positively with both leukocyte and lymphocyte fractions in tumor (r = 0.29-0.70 and 0.20-0.50, resp.). Expression of genes related to immune function like interferon response and complement activation was enriched in high A3C-H expressors, which also had significantly more CD4 and CD8 T cells, and TCR diversity (2.3-4.0x, 2.1-5.4x and 1.3-2.1x, resp.). Concordantly, for each of A3C-H, expression correlated with tumor immune cytolytic activity (r = 0.31-0.79), which was increased 3.1-7.9x in high expressors. A3B or A3A gene expression levels had no effect on overall survival (OS), but higher expression for each of A3C-H was significantly associated with improved OS (HR = 0.45-0.66). To obtain insights on any causative mechanism for the prognostic value of A3C-H expression, we examined APOBEC3-mediated C>U RNA editing in the tumors. RNA editing at a mean level of 6.3% (SD = 4.3%) was observed among tumors for 767 genes. Editing was not associated with tumor subtype or pathologic stage but tumors with high editing had 1.4-11x higher gene expression of A3A and C-H (P <0.05 for all). TCR diversity and immune cytolytic activity in tumors correlated with RNA editing (P <0.001 for both), and increased editing was associated with improved disease-free interval (P <0.05). Even though APOBEC3C-H are DNA mutators, their high expression in tumor is associated with a strong immune response and improved survival in breast cancer. This is also observed for APOBEC3-mediated RNA editing, suggesting that RNA editing of specific genes by APOBEC3s may be promoting immune activity against cancer.

#3417

Genomic analyses of clear cell papillary kidney cancers reveal large metabolic alterations and activation of the hypoxia pathway.

David Lindgren,1 Jonas Sjolund,1 Helen Nilsson,1 Borje Ljungberg,2 Martin Johansson,3 Hakan Axelson1. 1 _Lund University, Lund, Sweden;_ 2 _Umea University, Umea, Sweden;_ 3 _Gothenburg University, Gothenburg, Sweden_.

Tumors arising from the nephron of the kidney are collectively called renal cell carcinomas (RCCs). Recent high-throughput genomics endeavors, such as the TCGA initiative, have provided a roadmap for a deepened molecular understanding of these tumor types. With the aim to characterize RCCs that do not conform to the most common subtypes of RCC we scrutinized RNA-Seq transcriptomes of kidney tumor samples supplied by the TCGA project. Dimensionality reduction analyses revealed one defined a transcriptional subset of RCCs, which upon re-review of tissue histology could be classified as clear cell papillary RCC (ccpRCC). In line with previous analyses, samples were defined by overexpression of KRT7, CA9, CCND1 and GATA3 and harbored few genomic alterations as well as a limited number of gene mutations; only mTOR was found recurrently mutated. We observed upregulation of the Hypoxia inducible factor (HIF) pathway, possibly caused by VHLinactivation by gene promoter methylation in the majority of ccpRCCs. This may explain the morphological resemblance of ccpRCCs to VHL driven conventional clear cell RCCs. Further, the methylomes of ccpRCCs were distinct from other tumors and transcription factors specific for epithelial cells of the distal nephron were overexpressed, thus suggesting distinct tumor ontogeny of ccpRCCs compared to other renal tumors.

#3418

A NGS-based SNP array for the identification of human xenograft tumors, mouse homograft tumors, human and mouse cell lines, organoids.

Xiaobo Chen,1 Wubin Qian,2 Sheng Guo,2 Henry Li3. 1 _Crown Bioscience (Beijing) Inc, Beijing, China;_ 2 _Crown Bioscience (Suzhou) Inc, Suzhou, China;_ 3 _Crown Bioscience Inc, San Diego, CA_.

Transplanted tumor models, including xenograft tumors (e.g. patient derived xenograft, or PDX, human cell line derived tumors, mouse homograft (e.g. syngeneic tumors), as well as many cancer cell lines of both human and mouse origins are the key preclinical tools used in cancer researches, including drug efficacy assessment, drug target/biomarker discovery, as well as drug mechanism of actions and resistances, etc. The broad applications of these models in all different global laboratories at different times creates great risk of cross-contaminations (intra-/inter-species) as well as unintended de-identification. It is prudent to be able to track and QC tumors to ensure their authenticity by a simple and routine assays. The most common assay to identify human xenografts are Short Tandem Repeat (STR) test using multiple independently assorted STR loci. However, STR testing may suffer from several limitations, e.g. inadequate accuracy, especially for samples of kinship or close genetic background, inability to distinguish tumor of mouse origins as well as assessing mouse components in human xenografts. In addition, STR is also labor intensive, costly, low through-put, and ineffective for samples of low amount/poor quality. Single Nucleotide Polymorphisms (SNPs) based assays are increasingly used recently to replace STR testing with higher accuracy1-4. SNPs are genetic markers at single-nucleotide resolution. It is also amicable to automation, suitable for low amount/degraded samples with fragment length as short as only 60-80bp. Multiple SNPs can be simultaneously assayed by Next Generation Sequencing (NGS) technology. We developed a NGS-based SNP array for the identification of our libraries of xenografts, homografts, cell lines, etc. The assay is consisted of ~300 SNPs, 200 of which are used to authenticate different human origins, which were first constructed as unique fingerprints for every PDX from Whole-Exome Sequencing (WES) and RNAseq data, followed by that a sample is tested by the NGS-based SNP array to obtain its SNP fingerprint which is compared against all known fingerprints. The remaining 100 SNPs are used to infer gender and ethnicity, common viral infection, mouse content, mouse strain, homograft tumors, etc., as well as possible mycoplasmas contamination. Validation studies on multiple PDX models show that the array achieves near perfect results. In summary, we have established a high-throughput multi-purpose NGS-based SNP array that can be applied to the quality assurance of all the common cancer models.

#3419

Sex, mutations, and mortality in early stage melanoma.

Matthew R. Schwartz, Li Luo, Marianne Berwick. _University of New Mexico, Albuquerque, NM_.

Our goal is to investigate the relationship between sex, tumor mutation burden (TMB) and survival in untreated primary melanoma tumors, using data collected in an ongoing large P01 study on the Integration of Clinical and Molecular Biomarkers for Melanoma Survival (InterMEL). Here we present preliminary findings on sex, survival, and somatic TMB from 60 Stage II and Stage III primary melanoma tumors, none of which have received immunotherapy using the MSK IMPACT™ next generation sequencing assay. Our preliminary analysis includes 30 patients who died within 5 years (median survival of 31 months) and 30 patients who lived for more than 5 years since diagnosis (median follow-up of 86 months). We found the expected relationship between TMB and better survival in a univariate Cox regression analysis (HR=0.43, 95% CI=0.19 to 0.97, P=0.04), but no evidence of association between TMB and sex (Wilcoxon rank-sum test P=0.7) or any relationship between sex and survival (logrank test P=0.42).

Our study is the first to investigate the relationship between sex, tumor mutational burden, and mortality in an early stage primary cohort that has not received immunotherapy. In our small sample, we observed the expected protective effect of TMB on survival, but no evidence of gender differences in TMB or survival. This is surprising given the robust, consistent, and well-documented female survival advantage (Roh et al., 2015; de Vries et al., 2008), and merits further investigation. Our results are an important first step to increasing our understanding of the relationship between mutational burden, survival, and biological sex.

References

Roh, M.R., Eliades, P., Gupta, S., Grant-Kels, J.M., and Tsao, H. (2015). Cutaneous melanoma in women. Int. J. Womens Dermatol. 1, 21–25.

de Vries, E., Nijsten, T.E.C., Visser, O., Bastiaannet, E., van Hattem, S., Janssen-Heijnen, M.L., and Coebergh, J.-W.W. (2008). Superior survival of females among 10

538 Dutch melanoma patients is independent of Breslow thickness, histologic type and tumor site. Ann. Oncol. 19, 583–589.

#3420

Integrative tumor profiling beyond panel sequencing.

Corine K. Lau,1 Alena S. Harley,1 Paul Choppa,1 Janine Cooc,1 Ainura Kyshtoobayeva,1 Natalia Jun,1 Mark Dayrit,1 Wayne Delport,1 Mark Saldivar,1 Yop Jun,1 Raaj Trivedi,1 Alexandra E. Gylfe,1 Travis R. Lacey,1 Ezra Cohen,2 Kenneth Bloom,3 Eve Shinbrot1. 1 _Human Longevity, Inc., San Diego, CA;_ 2 _UCSD, San Diego, CA;_ 3 _Konica Minolta Precision Medicine, Aliso Viejo, CA_.

Introduction: Targeted cancer therapies rely on the identification of biomarkers specific to the tumors. Next generation sequencing (NGS) based genomic profiling has informed clinical decision making by identifying somatic alterations such as single nucleotide variants (SNVs), small insertion and deletion mutations (indels), structural variations, tumor mutation burden (TMB), and microsatellite instability (MSI) status. Here, we describe an integrative approach to characterize the genomic complexity of solid tumors using whole genome sequencing (WGS), whole exome sequencing (WES), whole transcriptome sequencing (RNAseq), and tumor only panel sequencing.

Methods: We report on WES for 84 paired tumor normal samples in a variety of tumor types including breast, colon, head and neck, melanoma, cervical, thyroid, glioblastoma, lung, pancreatic, prostate, appendiceal, cholangiocarcinoma, and kidney. A subset of these samples were also sequenced with high depth tumor only gene panel. WGS and RNAseq data were included in the analysis for additional 14 tumor normal pairs. Somatic alteration assessments included SNV, indels, MSI status, TMB, mutational signatures, copy number variations, gene fusions and structural variations.

Results: Analysis using tumor normal WES and RNAseq identified both clinically relevant genes as well as mutational processes. We found a high percentage of germline mutations were misidentified as somatic variants using tumor only panels, further highlighting the importance of paired tumor normal sequencing. In some cases, the putative driver gene or the variant in a cancer gene identified by WES was not included in panel sequencing. For example, mutations in FOXA1 gene were recently implicated as clinically relevant resistance and metastasis marker. Exome wide mutational signature analysis also identified BRCA (Cosmic sig 3) signature in tumors with no alterations in BRCA1/2 genes. WGS analysis and fusion gene detection revealed novel fusion genes as well as important structural alterations. Examples include a novel BRAF fusion in a cholangiocarcinoma devoid of other known driver mutations, a novel NTRK3 fusion partner in a glioblastoma tumor, and numerous tandem duplications in an ovarian cancer.

Conclusion: In contrast to tumor only gene panels, tumor and matched normal whole exome assay examines the entire coding portion of the genome without the limitations of a predefined gene list. This allows for detection of mutations in recently identified cancer drivers, ability to reliably distinguish somatic variants from clonal hematopoiesis or other germline variants, calculate genome wide mutational patterns including TMB, MSI, and identify the underlying mutational processes such as genomic signatures. The addition of WGS sequencing allows the identification of clinically relevant genomic rearrangements and novel structural variations.

#3421

Comparison of mutations in different subtypes of sarcomas and its clinical implications.

Xinghua Song,1 Zuping Lian,2 YanBin Xiao,3 Kunpeng Bu,4 Ye Qiu,4 Tao Ji,5 Dan Liu,6 Angen Liu,6 Kai Wang6. 1 _The First Affiliated Hospital of Xinjiang Medical University, Urumqi, China;_ 2 _Ruikang Hospital Affiliated to Guangxi University of Chinese Medicine, Nanning, China;_ 3 _The Third Affiliated Hospital of Kunming Medical University, Yunnan Tumor Hospital, Kunming, China;_ 4 _Affiliated Tumor Hospital of Guangxi Medical University. Nanning, China., Nanning, China;_ 5 _Peking University People's Hospital, Musculoskeletal Tumor Center, Beijing, China;_ 6 _OrigiMed, Shang Hai, China_.

Background:

Sarcoma is a malignant cancer that arises from transformed cells of mesenchymal origin. Sarcoma has the characteristics of low incidence and wide distribution of onset age. Sarcoma patients have poor prognosis and very limited selection of approved targeted drugs. Till now, the genomic features of sarcoma patients in China have not been well understood. Here we present the comprehensive genomic features of 202 sarcoma patients and compared mutations in different subtypes of sarcomas.

Methods:

Deep sequencing targeting 450 cancer genes was performed on FFPE and matched blood samples collected from 202 sarcoma patients. Based on the histology patients included 33 osteosarcoma (OS), 24 rhabdomyosarcoma (RMS), 20 liposarcoma (LPS), 19 fibrosarcoma (FBS), 16 leiomyosarcoma (LMS), 14 Ewing's sarcoma (ES), 11 hemangiosarcoma (HMS), 10 synovial sarcoma (SS), 9 chondrosarcoma (CDS), and 46 others in this study. Genomic alterations (GAs) including single nucleotide variants (SNVs), insertions and deletions, copy number variations (CNV) and fusions were assessed.

Results:

Based on the deep sequencing results, there are 1219 somatic mutations detected in all 202 sarcomas, average 6.0 mutations per sample. The most common GAs of LPS, ES and SS's are consistent with those mentioned in NCCN guidelines (MDM2 55%, EWSR1 78.6% and SS18 80%, respectively). OS, RMS and LMS shared the same most common GAs of TP53 (25%-50%). The second ranked common GAs were NCOR1 (24%), FRS2 (20.8%), ATRX (25%) genes, and the third ranked GAs were RB1 (24%), CDK4 (16.7%) and MAP2K4 (25%) genes. 161/202 patients (79.7%) harbored at least one actionable mutations which might benefit from FDA approved drugs or drugs were under investigation in clinical trials. All FBS, 93.8% LMS and 90.9% HMS have actionable mutations, however, only 10% SS and 35.7% ES have actionable mutations. 8 samples have CNV (including MDM2/4, EGFR and CCND1) which was related to hyper-progression of immunotherapy. All these 8 samples were bone tumor including 4 OS, 1 CDS, 2 ES and 1 fibrosarcoma of bone.

Conclusions:

Through the comprehensive genomic profiling, different types of sarcomas showed different characteristics of genomic alterations, and potential therapeutic targets may be identified for the patients. Further, NGS provides information on whether or not patients may have the potential hyper-progression of immunotherapy.

#3422

Label-free circulating tumor cells isolation platform for lung cancer EGFR mutation identification and colorectal cancer with KRAS mutation detection.

Hung-Chih Lin,1 Yu-Lin Yang,2 Chia-Hsun Hsieh1. 1 _Chang Gung Memorial Hospital, Taoyuan, Taiwan;_ 2 _Chang Gung Memorial Hospital, Keelung, Taiwan_.

Circulating tumor cell becomes an urgent issue in personalized cancer treatment studies. AS the liquid biopsy, many previous reports showed the number and the characteristic of CTC are very important for patient prognosis. In cancer therapy, specific gene mutation in primary tumor decides the therapy strategy and drug response. However, the patient who cannot perform an operation or take biopsy would become a risk factor for treatment and affect therapy outcome. Hence, drug treatable related gene mutation detection by CTC is necessary. Platforms for mutation detection in tissue were well established but less useful for CTC. Most of them were lower efficacy for rare cell or DNA. In order to solve this dilemma, scientists use next-generation sequence (NGS) or droplet digital PCR (ddPCR) for gene mutation detection. But, in most clinical situations, we only have to follow-up some specific gene mutation, instead of many mutant types. Thus, we eager to develop a fast and low-cost methodology for drug treatable related gene mutation detection by CTC. In this study, 4mL blood was collected by EDTA vacuum blood collection tube from 37 patients. Whole blood was processed with RBC lyse steps, then added CD45 and glycophorin A antibodies conjugated with the nanoparticle to remove leukocyte and remained RBC. Further, Genomic DNA was extracted from negative selection cell mixture. An ARMS-PCR (Amplification-refractory mutation system PCR) combined with a touchdown PCR program was used for EGFR mutations identification. We enrolled 31 lung cancer patients. Nineteen patients' CTC were matched with tissue mutation results (14 mutant and 5 wild-types), and 12 unmatched. In those 12 patients, 2 patients with mutant tissue but wild-type CTC due to their blood were drawn at disease-free status after long-term therapy, 4 patients with wild-type tissue but mutant CTC were still had tumor burden after therapy and need to more long-term follow-up. Overall, we have a mutation correlation rate of (19 + 2) / 31 = 68% between CTC and primary tumor. Furthermore, an LNA-PCR (Locked nucleic acid PCR) system was designed for colorectal cancer KRAS codon 12 and codon 13 mutations detection. In this platform, modified RNA was designed for blocked wild-type DNA amplification in PCR. We enrolled 6 patients with primary tumor mutant positive, 5 of 6 (83%) patients were also mutant positive in their CTC sample. In conclusion, an easy CTC isolation platform and PCR design could hugely help cancer patient mutation detection. Only less than 4 hours and 100 USD were needed for drug treatable gene mutation detection by CTC.

#3423

HER2 **amplification and microsatellite status distribution in Chinese gastric cancer patients.**

Jun Guo,1 Da Jiang,2 Wei Zhang,3 Ting Deng,3 Zhaojian Niu,4 Wenting Chen,5 Junping Shi,5 Shuirong Zhang,5 Lin Zhang5. 1 _Xingtai People's Hospital, Xingtai, China;_ 2 _The Fourth Hospital of Hebei Medical University, Shijiazhuang, China;_ 3 _Tianjin Medical University Cancer Institute and Hospital, Tianjin, China;_ 4 _The Affiliated Hospital of Qingdao University, Qingdao, China;_ 5 _OrigiMed, Shanghai, China_.

Background:Amplification and/or overexpression of human epidermal growth factor receptor-2 (HER2, also known as ERBB2) were present in 6.1-23.0% of gastric cancer. In breast cancer, amplification and overexpression of the HER2 are associated with poor prognosis. Meanwhile, MSI-H patients have an improved prognosis in gastric cancer. However, little is known about the association of these two indicators in gastric cancer.

Method:260 tumor tissues and matched normal from gastric cancer patients were included in this study to reveal HER2 amplification and microsatellite status distribution of Chinese patients with gastric cancer using deep next generation sequencing (NGS) targeting 450 cancer genes. Microsatellite status (MSS or MSI-H) were evaluated according to NGS algorithms.

Result:Male to female ratio was about 2:1 with a mean age of 60 years old. 241 tumors were primary tumors and 19 were metastases. MSI-H was detected in 16 samples (6.2%) and HER2 amplification was detected in 18 samples (6.9%), which were consistent with the previously reported incidences. The proportion of MSI-H in metastatic lesions was higher than that in primary lesions, but the difference was not statistically significant (10.5% vs. 5.8%, P=0.33). The detection of HER2 amplification in metastatic foci and primary tumors had a similar trend as that in MSI-H (15.8% for metastatic foci vs. 6.2% for primary tumors, P=0.13). Interestingly, our study revealed that HER2 amplification was mutually exclusive with MSI-H in both primary and metastatic lesions. MSI-H/HER2amp- , MSS/HER2amp+, and MSS/HER2amp- subgroups accounted for 5.8%, 6.2%, and 88% in primary cancers, while MSI-H/HER2amp-, MSS/HER2amp+ and MSS/HER2amp- subgroups accounted for 10.5%, 15.8% and 73.7% in metastasis, respectively.

Conclusion:We analyzed the distribution of HER2 amplifications and microsatellite status in Chinese patients with gastric cancer. The detection rate of HER2 amplifications and MSI-H was consistent with previous reports. Interestingly, our data showed that both HER2 amplifications and MSI-H have higher detection rate in metastases than that in primary lesions, and the two biomarkers were mutual exclusive in our cohort.

#3424

Consensus molecular subtype of triple negative breast cancer to implicate in chemotherapy response.

Jihyun Kim,1 Doyeong Yu,1 Jiyoon Noh,2 Wooyeong Jang,1 Hanna Yang,1 Youngmee Kwon,1 Keun Seok Lee,1 Sung Hoon Sim,1 Sun-Young Kong,1 In Hae Park,1 Charny Park3. 1 _National Cancer Center, Seoul, Republic of Korea;_ 2 _Korea Research Institute of Bioscience & Biotechnology, Daejun, Republic of Korea; _3 _National Cancer Center, Ilsandong-gu, Republic of Korea_.

Objective: The heterogeneity of triple negative breast cancer (TNBC) confers the difficulties in chemotherapy and induces poor outcome. Subtype classification of TNBC using gene expression profile could achieve to identify molecular markers to suggest therapeutic guidance.

Methods: In this study, we collected gene expression profiles of 957 TNBC patients from GEO and integrated into one meta-data using meta analysis. We identified subtypes using nonnegative matrix factorization (NMF) and explored for comprehensive characteristics of consensus molecular subtype (CMS) by investigating key pathway activity, tumor microenvironment, stemness and so on. In addition, we computed the drug response score (DRscore) from MSigDB chemical perturbation signature gene set and examined drug associations with key pathways of CMS. Drug candidates were validated from independent two data sets; our patients' expression profile (n = 38) and biobank TNBC organoids (n=64).

Results: We classified four different TNBC subtypes displaying gene expression patterns including mesenchymal-like (MSL), luminal-AR (LAR), immunomodulatory (IM), and stem-like (SL). MSL were activated pathways with epithelial-to-mesenchymal (EMT) and TGF-beta signaling whereas SL was up-regulation of cell cycle and WNT signaling pathway. The LAR subtype was activated of androgen and estrogen receptor pathway. Although metastatic TNBC generally shared equal activity of key pathways with primary, coagulation, toll-like receptors, TNF, and Jak-STAT signaling pathways were dysregulated in metastasis. Especially, SPP1 gene expression to induce metastasis was associated with poor prognosis. DRscores were discriminative in CMS of meta-data and biobank expression profile. We found subtype-specific 18 drugs as therapeutic candidates and these drugs were also correlated with key pathways. In a case of cisplatin, DRscores appear resistant in MSL while response in SL. Our patients' expression profiles were shown to consistent result as well as biobank.

Conclusion: These finding might facilitate understanding heterogeneity of TNBC. Our novel approach to explore drug response suggested functional intervention landscape of drug

response without in-vivo experiment. Taken together, we propose biomarkers for diagnosis and also suggest therapeutic strategy depending on CMS.

#3425

Mutations in DNA repair and microtubule assembly genes found in triple negative breast tumors treated with neoadjuvant chemotherapy.

Michael Grant,1 George Snipes,1 Rebecca Halperin,2 Paul Heaton,3 Suwon Kim2. 1 _Baylor Scott White Research Institute, Dallas, TX;_ 2 _Translational Genomics Research Institute, Phoenix, AZ;_ 3 _University of Arizona College of Medicine-Phoenix, Phoenix, AZ_.

Up to 20% of breast cancer is clinically defined as triple-negative breast cancer (TNBC) lacking estrogen, progesterone, and HER2 receptors. Because no targeted therapy is available, cytotoxic chemotherapy is the primary mode of adjuvant TNBC therapy, but is effective in ~30% patients. In order to determine molecular underpinnings of chemotherapy resistance in TNBC, we analyzed 12 TNBC patient tumor samples collected post neoadjuvant chemotherapy using DNA/RNA sequencing. DNA analysis showed two tumors contained TP53 inactivating mutations and two tumors PIK3CA activating mutations. In addition, a number of mutations in the DNA repair pathway genes were identified including XRCC1, XRCC3, and ATXR, suggesting that DNA damaging nature of chemotherapy agents may have enriched these mutations. In addition, we found mutations in the genes involved in microtubule assembly including BUB3 and APC2, suggesting that these gene mutations may confer resistance to taxol, an anti-microtubule chemotherapy agent. RNA sequencing revealed a true heterogeneous nature of TNBE in that unsupervised clustering did not identify any distinct gene signature correlative of chemotherapy resistance. We are currently evaluating phenotypes related to chemotherapy resistance using cells genetically engineered with the BUB3 or APC2 mutations.

#3426

Combining cell barcoding and CRISPR sgRNA libraries with targeted gene expression for single-cell genetic analysis of tumor metastasis.

Alex Chenchik, Paul Diehl, Mikhail Makhanov. _Cellecta, Inc., Mountain View, CA_.

Pooled lentiviral libraries of CRISPR sgRNAs to mediate genome-wide gene knockout have become an invaluable tool for uncovering the functional genetic drivers required for a biological response. Another type of pooled lentiviral library designed with unique DNA sequence tags have also been used to label large populations of cells with unique cell-specific barcodes which allows monitoring changes in sub-populations of cells with distinct phenotypes over time. We have combined cell barcodes with CRISPR sgRNA to construct libraries that enable the identification of multiple occurrences of a common phenotypic response from independent transductions of the same sgRNA effector. Specifically, we demonstrate how this sort of library can be used to identify genes whose activation promotes metastasis of tumors derived from MDA-MB-231 cells engrafted into mice after transduction with a barcoded CRISPR-activation (CRISPRa) sgRNA library. By incorporating multiple barcodes with each sgRNA effector, it is possible to see that, not only are cells in metastatic tumors more likely to have a particular sgRNA targeted to a certain gene, but also that independent transductions of that sgRNA sequence lead to multiple independent metastatic events. With multiple independent clones producing the same phenotype, it is possible to confidently isolate the sgRNA, and by implication, the increased activation of its gene target, as the primary cause of the metastatic phenotype.

#3427

Exome sequencing of primary papillary thyroid cancer and their distant metastases reveals the role of DNA methylation and transcriptional repression genes.

Tariq Masoodi, Abdul K. Siraj, Sarah Siraj, Saud Azam, Zeeshan Qadri, Wafaa N. Albalawy, Sandeep Kumar Parvathareddy, Saif S. Al-Sobhi, Fouad Al-Dayel, Fowzan S. Alkuraya, Khawla S. Al-Kuraya. _King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia_.

Distant metastasis is a rare occurrence in thyroid cancer, associated with dismal prognosis. The genomic repertoires of various solid malignancies have previously been reported but remain under-explored in metastatic Papillary Thyroid Cancer (PTC). Furthermore, whether distant metastases harbor distinct genetic alterations beyond those observed in primary tumors is unknown. We performed whole exome sequencing on 14 matched distant metastases, primary PTC tumors and normal tissues. Point mutations, copy number alterations, cancer cell fractions and mutation signatures were defined using state-of-the-art bioinformatics methods. All likely deleterious variants were validated by orthogonal methods. Genomic differences were observed between primary and distant metastatic deposits with a median of 62% (range 21% - 92%) of somatic mutations detected in metastatic tissues, but absent from the corresponding primary tumor sample. Mutations in known driver genes including BRAF, NRAS and HRAS were shared and preferentially clonal in both sites. On the other hand, likely deleterious variants affecting DNA methylation and transcriptional repression signaling genes including SIN3A, RBBP1 and CHD4 were found to be restricted in the metastatic lesions. Moreover, mutational signature shift was observed between the mutations that are specific or enriched in the metastatic and primary lesions. Primary PTC and distant metastases differ in their range of somatic alterations. Genomic analysis of distant metastases provides an opportunity to identify potentially clinically informative alterations not detected in primary tumors, which might influence decisions for personalized therapy in PTC patients with distant metastasis.

#3428

The landscape of germline mutations in Chinese patients with solid tumor.

Juming Li,1 Yongzhong Wei,1 Xiaoqian Chen,2 Angen Liu,2 Junping Shi,2 Jinwei Hu,2 Ming Yao2. 1 _Jiangsu Province Hospital, Jiangsu, China;_ 2 _OrigiMed, Shanghai, China_.

Background: Germline mutations harbored by cancer patients can be passed from parents on to offspring, which increases the tumor risk in offspring as well. Therefore, investigating the germline mutations in cancer patients has important clinical implications for treatment selection and familial cancer risk assessment.

Methods: FFPE tumor and matched blood samples were collected from 3645 Chinese patients with solid tumors for next generation sequencing analysis. The NGS panel targeted 450 cancer genes, 47 of these genes were tumor inheritance susceptibility genes that were mentioned by NCCN guidelines. Genomic alterations including single nucleotide variations (SNV), short and long insertions/deletions (Indel), copy number variations (CNV) and gene rearrangements and fusions were assessed.

Results:Germline mutations were identified in 241 of 3645 Chinese patients with solid tumor (6.6%). The patients with germline mutations included 137 males and 104 females with a median age of 54. Tumor types with higher frequency of germline mutations were: ovarian cancer (22.0%), breast cancer (13.4%), pancreatic cancer (9.9%), cholangiocarcinoma (8.6%), small intestinal cancer (8.6%), colorectal cancer (8.6%), soft tissue sarcoma (6.3%), uterine Cancer (5.9%), gastric cancer (5.8%) and bone cancer (5.1%). The distribution of germline mutations were 14.1% in BRCA1, 13.7% in BRCA2, 10.8% in SPINK1, 9.1% in ATM, 5.8% in RAD50, 4.6% in PALB2, 3.7% in BARD1 and FANCA, 3.3% in MSH6 and 2.9% in MLH1, respectively. The types of germline mutations were SNVs and short Indels, but no long Indels, CNV, gene rearrangements or fusions were observed. The percentage of patients with a family history was 27.4%, without family history was 52.7%, unknown was 19.9%.

Conclusions:Our data revealed that germline mutations occurred in 6.6% of Chinese solid tumor patients. Gynecological tumor, gastrointestinal tumor, bone and soft tissue tumor are the most common types of tumors that harbored germline mutations. The most common mutant genes were HRD (64.5%) and MMR (10.5%) related genes. More than 50% patients with germline mutations had no family history, which suggests the importance of germline mutations detection for all solid tumor patients to verify if there is a risk of genetic transmission to offspring. NGS based panel sequencing showed the advantage of identifying solid tumor patients with germline mutations, and provided the information of familial cancer risk assessment and guided the precise cancer treatment options.

#3429

Genetic analysis of pancreatic neuroendocrine neoplasms grade 3.

Nobuyuki Kakiuchi,1 Kenichi Yoshida,1 Yusuke Shiozawa,1 Akira Yokoyama,1 Keisuke Kataoka,1 Yoshikage Inoue,1 Yasuhide Takeuchi,1 Tomonori Hirano,1 Yoichi Fujii,1 Hiroo Ueno,1 Susumu Hijioka,2 Nobumasa Mizuno,3 Waki Hosoda,3 Yasushi Yatabe,3 Kenichi Chiba,4 Hiroko Tanaka,4 Yuichi Shiraishi,4 Satoru Miyano,4 Toshihiko Masui,1 Shinji Uemoto,1 Akihiko Yoshizawa,1 Hironori Haga,1 Norimitsu Uza,1 Hiroshi Seno,1 Yuzo Kodama,5 Seishi Ogawa1. 1 _Kyoto University, Kyoto, Japan;_ 2 _National Cancer Center Hospital, Kyoto, Japan;_ 3 _Aichi Cancer Center Hospital, Kyoto, Japan;_ 4 _Human Genome Center, the University of Tokyo, Japan;_ 5 _Kobe University Graduate School of Medicine, Kyoto, Japan_.

Pancreatic neuroendocrine neoplasms (PanNENs) are pancreatic tumors with neuroendocrine differentiation. PanNENs are classified into three grades according to the proliferating cell fraction as measured by Ki-67 or mitotic index. Among them, PanNEN grade 3 (PanNEN G3) shows the highest Ki-67 index > 20%, which is morphologically divided into two categories, well-differentiated (PanNET G3) and poorly differentiated (PanNEC) tumors. Although sharing some overlapping histological features, both tumors also differ from each other with respect of clinical characteristics. To date, the molecular basis of these difference and similarity are poorly understood. To understand their molecular pathogenesis, we performed comprehensive genetic analysis on PanNEN G3.

In total, we enrolled 25 PanNEN G3 cases, including 13 PanNET G3, 10 PanNEC, and 2 PanNEN G3 NOS, which were analyzed for somatic mutations. All tumors showed neuroendocrine markers such as synaptophysin or chromogranin A. The median Ki67 index of PanNET G3 was 30.5% (15-78%), which was lower than that of PanNEC (median 70.9%, range 44-100 %). Genomic DNA was extracted from microdissected tumors and paired normal tissues from formalin-fixed paraffin-embedded PanNEN G3 specimens guided by H&E stained sections and subjected to whole-exome sequencing (WES). Copy number alterations were also analyzed on the basis of sequencing data. All tumor specimens were centrally reviewed by the two expert pathologists and assigned to PanNET G3, PanNEC, or PanNEN G3 not otherwise specified (PanNEN G3 NOS).

WES analysis detected a total of 1,688 somatic mutations with a median of 45 (range 21-450)/sample. There was no clear difference in mutational burden between PanNET G3 and PanNEC. Combined with copy number alterations, mutations most frequently affected TP53 (40%), followed by MEN1(28%), SMAD4 (28%), KRAS (24%), and CDKN2A (20%). Frequently mutated in PanNET G1/2, MEN1, DAXX, and ATRX mutations were associated with well-differentiated morphology (n=7/7), while tumors with KRAS or RB1 mutations exhibited poorly differentiated histology (n=5/5). MEN1, DAXX, and ATRX mutations frequently co-occurred with mutations in mTOR pathway genes, such as NF1, TSC2, or DEPDC5 (q<0.001), suggesting that some PanNET G3 might evolve from PanNET G1/2 by acquiring mutations that activate the mTOR pathway. Whereas, in PanNEC, MEN1, DAXX, and ATRX had never been mutated and KRAS and RB1 are recurrently mutated. Therefore, PanNEC does not derive from PanNET and it occurs independently. In survival analysis of PanNEN G3, SMAD4 mutation as well as high grade pathology was significantly contributed to shorter overall survival.

Our results help understand a molecular basis of PanNEN G3, contributing to a better understanding and classification of PanNET G3 and PanNEC. 

### Cell Signaling 1

#3430

TGFBR2 mediated phosphorylation of BUB1 at Ser-318 is required for transforming growth factor-β signaling.

Shyam Nyati,1 Brandon Gregg,1 Jiaqi Xu,1 Grant Young,1 Lauren Kimmel,1 Nyati Mukesh,1 Dipankar Ray,1 Hongtao Yu,2 Alnawaz Rehemtulla1. 1 _Univ. of Michigan Medical School, Ann Arbor, MI;_ 2 _University of Texas Southwestern Medical Center, Dallas, TX_.

The Ser/Thr kinase BUB1 (budding uninhibited by benzimidazoles-1) is required for efficient signaling by the TGF-β receptor. Recruitment of BUB1 to the activated heteromeric TGF-β-receptor complex including SMAD2 and SMAD3, is required for ligand mediated canonical and non-canonical downstream cascades (Nyati et. al. 2015). In an effort to delineate the structure-function basis for this, we utilized in vitro kinase reactions followed by mass spectrometry to demonstrate that TGFBR2 phosphorylates BUB1 at Serine 318. To elucidate the functional significance of TGFBR2 mediated phosphorylation of BUB1, S318 was substituted with alanine (S318A) or aspartic acid (S318D). Co-immunoprecipitation studies of these phospho-deficient or phospho-mimicking BUB1 mutants with TGFBR1, and SMAD2 revealed that the S318A mutant interacted more efficiently to TGFBR1 and SMAD2 compared to the S318D BUB1. To investigate if the Ser318 residue was involved in mediating BUB1 interaction with TGF-β signaling components, we utilized deletion mutants of BUB1. Our findings reveal that the N-terminal (1-241) and C-terminal (482-723) domains of BUB1 independently interact with SMAD2, SMAD3, TGFBR1, or TGFBR2. The middle domain (241-482) having a S318A mutation interacted efficiently with TGFBR2 while the S318D mutant did not. Additionally, the 241-482 S318D mutant exhibited an increased interaction with SMAD2. Over-expression of 241-482 S318D resulted in a decrease in TGFBR1-TGFBR2 interaction as well as TGFBR1-SMAD2 interaction, suggesting that TGFBR2 mediated BUB1 phosphorylation at S318 maybe the trigger for the dissociation of the activated receptor complex, and therefore a key regulator of TGF-β signaling. Taken together, we show that TGFBR2 phosphorylates BUB1 at Ser318 and propose a model with the following steps: (i) BUB1 is recruited to TGFBR1-TGFBR2 complex in response to ligand, (ii) BUB1 participates in the recruitment of SMAD2/3 to the receptor, (iii) TGFBR2 phosphorylates BUB1 at S318, which triggers the disassembly of the activated complex.

#3431

PI3Kbeta regulates macropinocytosis in PTEN-null tumor cells.

Gilbert Salloum, Charles Jakubik, Anne R. Bresnick, Jonathan M. Backer. _Albert Einstein College of Medicine, Bronx, NY_.

Class I phosphoinositide 3-kinases (PI3Ks) have been previously implicated in the regulation of vesicular trafficking, including receptor-mediated endocytosis and macropinocytosis. Using NIH3T3 cells in which the genes for the PI3Kbeta or PI3Kalpha catalytic subunits were deleted by CRISPR/Cas9, we were unable to detect any defects in receptor-mediated endocytosis of [125I]-PDGF or in pinocytosis of Lucifer yellow. However, we observed a marked inhibition of macropinocytosis in PI3Kbeta knockout cells; deletion of the other major PI3K isoform in these cells, PI3Kalpha, had no effect. These studies were confirmed using isoform-selective inhibitors: only inhibition of PI3Kbeta blocked macropinocytosis. Similarly, in MDA-MB-231 breast cancer cells, HGF-stimulated macropinocytosis was markedly reduced by inhibition of PI3Kbeta, but not the other PI3K isoforms. Notably, expression of a mutant PI3Kbeta that cannot be activated by Gbeta-gamma, or treatment of WT cells with pertussis toxin, also inhibited macropinocytosis in MDA-MB-231 cells. A requirement for Gbeta-gamma binding to PI3Kbeta was also seen in primary macrophages from mice expressing WT or Gbeta-gamma-uncoupled PI3Kbeta, as both CSF-1 and C5a-stimulated macropinocytosis was inhibited by mutant PI3Kbeta. To define the mechanism by which PI3Kbeta regulates macropinocytosis, we measured circular dorsal ruffle (CDR) formation. Genetic ablation or inhibition of PI3Kbeta completely suppressed CDR formation in PDGF-stimulated NIH3T3 cells, whereas inhibition or ablation of other PI3K isoforms had no effect. While previous studies have suggested that Class I PI3Ks act late in macropinosome formation, just prior to macropinosome sealing, our data suggest that PI3Kbeta functions early in macropinocytosis, as it mediates CDR formation in a Gbeta-gamma-dependent manner. Given previous work showing a positive feedback loop involving PI3Kbeta and Rac1, we tested whether macropinosome formation was rescued by expression of CA-Rac1. CA-Rac1 expression potently stimulated macropinocytosis in MDA-MB-231 cells. Strikingly, this was completely blocked by expression of the Gbeta-gamma-uncoupled PI3Kbeta, and partially blocked by expression of a mutant PI3Kbeta that cannot bind Rac1. Finally, given the well established role of PI3Kbeta in the growth of tumor cells lacking the PTEN tumor suppressor, as well as recent studies showing that high rates of basal macropinocytosis can serve as a nutrient a uptake mechanism in PTEN null cells, we measured macropinocytosis in two PTEN-deficient tumor lines, BT549 (breast) and PC3 (prostate). Both lines showed elevated basal levels of macropinocytosis, which were specifically blocked by inhibition of PI3Kbeta. Our data support a highly specific role for PI3Kbeta in the regulation of macropinocytosis, which may contribute the role of PI3Kbeta in the growth of PTEN-null tumor cells.

#3432

Novel roles of LY6K in glioblastoma tumorigenesis.

Namratha G. Sastry, Tianzhi Huang, Angel Alvarez, Rajendra Pangeni, Xiao Song, Xuechao Wan, John Kessler, Ichiro Nakano, Bo Hu, Shi-Yuan Cheng. _Northwestern University, Chicago, IL_.

Glioblastoma (GBM) is the most malignant brain cancer, with extremely poor prognosis in patients. GBM tumors are characterized by distinct molecular subtypes, known as proneural (PN), classical, and mesenchymal (MES). Inherited heterogeneity and aberrant aggressiveness contribute to therapy resistance and frequent recurrence of malignant GBM tumors. In addition, GBM tumors also contain a small subpopulation of tumor-initiating or stem-like cancer cells (GSCs). Gene expression profiling studies from our laboratory showed that patient-derived GSCs can also be classified into two subtypes, PN and MES, that are phenotypically similar to clinical GBM. Among three thousand genes that are differentially expressed between PN and MES-like GSCs, Lymphocyte Antigen 6 Complex, Locus K (LY6K) was identified as one of the top differentially expressed genes. LY6K is a GPI-anchored protein from the LY6 family. Several members of the LY6 family have been implicated in human cancers, including breast, esophageal, and lung cancers. Moreover, the function of LY6K in GBM has not been reported. Here, we examined the roles of LY6K upregulation in GBM tumorigenesis and investigated the underlying mechanism of LY6K action. We hypothesized that high levels of LY6K in GSCs promotes GBM tumorigenesis. Based on our other preliminary data, we further hypothesized that LY6K functions by enhancing ERK activation, thus promoting radioresistance. To test our hypotheses, we first examined the role of LY6K in advancing GBM tumorigenic behaviors of GSCs. We performed in vitro and in vivo tumorigenicity assays and showed that the presence of LY6K significantly increases GSC cell growth and glioma sphere forming ability in vitro and promotes tumor formation in orthotopic brain xenograft mouse models. The underlying mechanism governing LY6K expression was found to be DNA promoter methylation. Moreover, irradiation strongly induces LY6K expression via demethylation of LY6K promoter in GSCs with otherwise undetectable levels of LY6K. Furthermore, we investigated the mechanism by which LY6K contributes to GSC tumorigenicity. We observed that there is a strong relationship between LY6K and ERK signaling in GSCs and U87 glioma cells. The presence of LY6K stimulates ERK activation and subsequently augments cell proliferation of GSCs. Lastly, we identified the GPI-anchor domain of LY6K as a key region in enhancing ERK activation and are currently examining the mechanistic properties of this domain. The mechanistic insights gained from our studies will advance current knowledge of aberrantly upregulated LY6K and ERK signaling enhancement in promoting GBM tumorigenicity.

#3433

IL11 promotes the PhIP/DSS-induced colon carcinogenesis.

Hong Wang,1 David Wang,2 Yuhai Sun,1 Xu Yang,1 Chung S. Yang1. 1 _Rutgers Univ., Piscataway, NJ;_ 2 _Ohio State University, Columbus, OH_.

Inflammation of the large intestine, such as colitis, is known to increase the risk of colorectal cancer in human. The colitis induced by dextran sodium sulfite (DSS) has been shown to promote carcinogen- and genetic-induced colon tumorigenesis in rodent models. In a CYP1A-humanized (hCYP1A) mouse colon cancer model that we developed to study dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine (PhIP), DSS-induced colitis was required for colon tumor development after the PhIP treatment. Nevertheless, the mechanism for DSS-induced colitis to promote carcinogenesis is not clear. To uncover the key cancer promotion factor induced by colitis is important in understanding the mechanism of colon cancer development. By investigating the gene expression profiles of PhIP/DSS-induced colon tumors and normal colon epithelium, we found that cytokine IL11 was significantly upregulated in all tumors. The elevated levels of IL11 are also found in the human colon cancer genomic data available in The Cancer Genome Atlas (TCGA), suggesting an important role of IL11 in human colon cancer. Consistently, the activation/phosphorylation of STAT3, a key downstream factor of IL11 signaling, was also positively identified in tumors but not the normal adjacent tissues. To investigate the role of IL11 signaling, we generated hCYP1A:IL11Rα1-/- mice for investigating the role of IL11 signaling in PhIP/DSS-induced carcinogenesis by crossing the IL11Rα1-/- mice with hCYP1A mice. These mice are infertile as the phenotype reported previously for the IL11Rα1 knockout mice, and need to be maintained through the breeding by the heterozygous. The experimental mice were maintained on the AIN93M diet. We found that the tumor incidence was reduced by 51.0% in hCYP1A:IL11Rα1+/- mice and 80.0% in hCYP1A:IL11Rα1-/- mice, suggesting that IL11 signaling plays critical roles in PhIP/DSS-induced colon carcinogenesis. By immunohistochemical staining for pSTAT3, we found the pSTAT3 staining in the colitis epithelial tissues induced by the DSS treatment in both heterozygous and homozygous mice were significantly reduced; whereas the tissues from the wildtype mice displayed strong positive staining in the nucleus. IL11 signaling could be a potential therapeutic target for colon cancer prevention. (supported by the John L. Colaizzi Chair Endowment Fund and GI Pilot Study from Rutgers Cancer Institute of New Jersey Cancer Center Support Grant P30CA072720)

#3434

Selective inhibition of mTORC2 by disruption of mLST8 scaffolding function.

Laura C. Kim,1 Yoonha Hwang,2 Wenqiang Song,2 Rebecca S. Cook,1 Jin Chen2. 1 _Vanderbilt University, Nashville, TN;_ 2 _Vanderbilt University Medical Center, Nashville, TN_.

Mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that acts in two distinct complexes, mTORC1 and mTORC2, and is dysregulated in many diseases including cancer. mLST8 is a shared component of both mTORC1 and mTORC2, yet little is known regarding how mLST8 contributes to assembly and activity of the mTOR complexes. We assessed mLST8 loss in a panel of normal and cancer cells, finding little to no impact on mTORC1 activity, nor on assembly of mTOR with the mTORC1 co-factor Raptor. However, mLST8 loss blocked mTOR association with the mTORC2 co-factors Rictor and Sin1, and impaired mTORC2 kinase activity, including its phosphorylation of AKT at S473. We identified two sites within mLST8 governing its interaction with mTOR, finding that simultaneous mutation of these sites together, but not individually, disrupted not only mTOR-mLST8 interactions, but also blocked mTOR assembly with Rictor and Sin1, thus disrupting mTORC2 activity. Similary, a single mutation on mLST8 with a corresponding mutation on mTOR interfered with mTORC2 assembly, without affecting mTORC1 assembly or activity. We further discovered a direct interaction between mLST8 and the NH2-terminal domain of the mTORC2 cofactor Sin1. Using PTEN-null prostate cancer xenografts, we found that mLST8 mutations disrupting the mTOR interaction motif inhibited AKT S473 phosphorylation, decreased tumor cell proliferation, and increased tumor cell death in vivo. Together, these data suggest that the scaffolding function of mLST8 is critical for assembly and activity of mTORC2, but not mTORC1, an observation which could enable therapeutic mTORC2-selective inhibition, while sparing the activity of mTORC1.

#3435

Targeting AKT activity inhibits MYCN and sensitizes neuroblastoma to chemotherapeutic agents.

Marion Le Grand, Kathleen Kimpton, Christine Gana, Emanuele Valli, Chelsea Mayoh, Maria Kavallaris. _Children's Cancer Institute, Randwick, Australia_.

MYCN-amplified neuroblastoma is one of the deadliest forms of childhood cancer and remains a significant clinical problem. Direct pharmacological inhibition of MYCN protein is challenging and one possible strategy to treat MYCN-amplified neuroblastoma patients is antagonizing proteins involved in its regulation. The AKT/GSK3β pathway directly regulates stabilization of MYCN, providing a therapeutic rational in MYCN-amplified neuroblastoma. In this study, we addressed the question of how AKT can play a pivotal role in MYCN-amplified neuroblastoma and whether small molecule AKT inhibitors can be promising anti-cancer compounds in children diagnosed from this cancer.

Bioinformatics analysis revealed that high gene expression of AKT1 and AKT2, but not AKT3 was correlated with MYCN amplification and was significantly associated with poor outcome in neuroblastoma patients. Using RNAi-mediated depletion of AKT isoforms or pharmacological inhibition, we then demonstrated that the total AKT activity inhibition rather than the expression of particular isoforms was necessary to cause a significant decrease in neuroblastoma cell proliferation. Our results showed that AKT activity inhibition led to a significant downregulation of MYCN expression through GSK3β regulation. These results demonstrated the potential of targeting AKT activity in MYCN-amplified neuroblastoma patients. Our in vitro and in vivo data revealed promising efficacy for the pan-AKT inhibitor perifosine combined with conventional cytotoxic drugs in MYCN-amplified neuroblastoma cell. Importantly, by genetically modulating MYCN expression (siRNA or plasmid vector), our results demonstrated that MYCN expression is required for perifosine chemosensitisation. This effect was linked to the combined actions of apoptosis activation and the downregulation of ABC transporter expressions.

Collectively, our data strongly support that combining the pan-AKT inhibitor perifosine, which is currently in clinical trial, with clinically-approved drugs for neuroblastoma patients could be a novel therapeutic strategy to indirectly target MYCN in neuroblastoma.

#3436

PRMT5 upregulation and its oncogenic cooperation with PI3K/AKT signaling in diffuse large B cell lymphoma.

Lixin Rui. _Univ. of Wisconsin School of Medicine & Public Health, Madison, WI_.

Protein arginine methyltransferase PRMT5, which regulates gene expression by symmetric dimethylation of histones and non-histone target proteins, is overexpressed and plays a pathogenic role in many cancers. A growing literature demonstrates a critical role of PRMT5 in tumorigenesis. PRMT5 expression is upregulated in various cancers, including diffuse large B cell lymphoma (DLBCL), the most common, aggressive form of non-Hodgkin lymphoma. PRMT5 upregulation is associated with Epstein-Barr virus (EBV) infection. Given that less than 10% of DLBCL is EBV-positive, the molecular mechanisms of PRMT5 overexpression in DLBCL are still largely unknown.

Here, we demonstrated increased expression of PRMT5 in DLBCL cell lines and in a tissue microarray of 104 DLBCL cases when compared with normal naïve B cells. Notably, the level of PRMT5 expression was also elevated in germinal center B cells, which experience antigen stimulation and give rise to DLBCL. In addition, PRMT5 expression in these DLBCL cases correlated with BTK expression, a key component of the B cell antigen receptor (BCR) signaling pathway and an effective drug target in DLBCL. We dissected the mechanisms of differential BCR signaling in regulating PRMT5 expression. Using the CRISPR/Cas9 genome editing technology or by specific pharmacological inhibitors, we discovered that two main BCR downstream signaling pathways, NF-kB and PI3K/AKT, contribute to PRMT5 overexpression. Specifically, the NF-kB member p65 and MYC, a downstream target of the PI3K/AKT signaling pathway, induced PRMT5 transcription. More importantly, inhibition of PRMT5 by its sgRNA or its specific inhibitor was lethal to all DLBCL cell lines tested but not to normal B cells. The antitumor effect of PRMT5 sgRNA or its inhibitor was further revealed in two DLBCL xenograft mouse models as well as in patient derived xenografts (PDX). Further RNA-Seq and biochemical analysis demonstrated that PRMT5 promotes cell cycle progression and activates PI3K-AKT signaling, suggesting a positive feedback regulatory mechanism to enhance cell survival and proliferation. To intervene in the positive feedback regulatory mechanism, we used the PRMT5 inhibitor GSK3326595 and the AKT inhibitor AZD5363, both of which are currently used in clinical trials for solid cancers. Indeed, our in vitro analysis in DLBCL cell lines demonstrated their synergistic cytotoxicity. We will further test the synergism of these two inhibitors in xenograft mouse models as well as in primary cancer cells.

Taken together, our study elucidates the mechanisms of PRMT5 upregulation and also demonstrates an oncogenic role for PRMT5 in DLBCL, suggesting targeting PRMT5 by a specific inhibitor or co-targeting with an AKT inhibitor as a potential therapeutic strategy for DLBCL patients.

#3437

Requirement for YAP1 signaling in myxoid liposarcoma.

Marcel Trautmann,1 Ya-Yun Cheng,2 Patrizia Jensen,2 Ninel Azoitei,3 Ines Brunner,2 Jennifer Hüllein,2 Mikolaj Slabicki,2 Ilka Isfort,1 Magdalene Cyra,1 Eva Wardelmann,1 Sebastian Huss,1 Bianca Altvater,1 Claudia Rossig,1 Susanne Hafner,3 Thomas Simmet,3 Anders Ståhlberg,4 Pierre Åman,4 Thorsten Zenz,5 Undine Lange,6 Thomas Kindler,6 Claudia Scholl,7 Wolfgang Hartmann,1 Stefan Fröhling2. 1 _Münster University Hospital, Münster, Germany;_ 2 _National Center for Tumor Diseases, Heidelberg, Germany;_ 3 _Ulm University Hospital, Ulm, Germany;_ 4 _Sahlgrenska Cancer Center, Gothenburg, Sweden;_ 5 _Zurich University Hospital, Zurich, Switzerland;_ 6 _Mainz University Hospital, Mainz, Germany;_ 7 _German Cancer Research Center, Heidelberg, Germany_.

Myxoid liposarcomas (MLS) account for 20% of malignant adipocytic tumors and are characterized by a high rate of local recurrence and development of distant metastases in approximately 40% of patients. Most MLS are driven by the FUS-DDIT3 fusion gene encoding an aberrant transcription factor. The mechanisms whereby FUS-DDIT3 mediates sarcomagenesis are incompletely understood, and strategies to selectively target MLS cells remain elusive. In this study, we employed genome-scale RNA interference (RNAi) screening to uncover that human mesenchymal stem cells engineered to express FUS-DDIT3 and MLS cell lines are dependent on YAP1, a transcriptional co-activator and central effector of the Hippo pathway involved in tissue growth and tumorigenesis. Analysis of a large cohort of primary MLS specimens (n=223) revealed that nuclear YAP1 expression was significantly more prevalent in MLS compared to other liposarcoma subtypes. In support of the concept that increased YAP1-mediated transcriptional activity represents an essential feature of MLS development, RNAi-based YAP1 depletion in cultured MLS cells resulted in suppression of cell viability, cell cycle arrest, cellular senescence, and induction of apoptosis accompanied by decreased YAP1 target gene expression, and YAP1-positive primary MLS tumors showed strong expression of YAP1 downstream effectors such as FOXM1 and PLK1. Mechanistically, FUS-DDIT3 promotes YAP1 transcription, nuclear localization, and transcriptional activity and physically associates with YAP1 in the nucleus of MLS cells, pointing to the coordinate establishment of gene expression programs that promote MLS tumorigenesis. Consistent with the hypothesis that a YAP1-directed therapeutic approach could represent a rational strategy to selectively target FUS-DDIT3-expressing MLS cells, pharmacologic inhibition of YAP1 activity with verteporfin suppressed cell viability and YAP1 target gene expression in MLS cell lines, and the growth-inhibitory effects of YAP1 knockdown or verteporfin treatment could be recapitulated in MLS cell line-based xenograft models. Collectively, our data identify dependence on aberrant YAP1 activity as specific liability of FUS-DDIT3-expressing MLS cells, and provide preclinical evidence that YAP1-mediated signal transduction represents a candidate target for therapeutic intervention that warrants further investigation.

#3438

PI3Kα/δ inhibitor, copanlisib is highly effective in ER-positive breast cancer.

Pradip De, Jennifer C. Aske, Casey Williams, Nandini Dey, Brian Leyland-Jones. _Avera Research Inst., Sioux Falls, SD_.

Purpose: Over 60% of ER+ breast cancer (BC) cases are associated with activation of the PI3K-AKT-mTORC1/C2 pathway. PIK3CA, the catalytic subunit of PI3K, is by far the most frequently mutated gene in luminal ER+ BC patients. It has long been known that hormone receptor and oncogene cooperation plays a key role in driving tumor development and that most hormone therapy-resistant BC depends on the PI3K pathway. Here, we present the results of a preclinical study of the PI3K α/δ (dominant) inhibitor copanlisib alone and in combination with letrozole in ER+ BC cell lines.

Method: Anti-proliferative, apoptotic, cell cycle, and intracellular signaling effects of copanlisib alone and in combination with letrozole were evaluated in a panel of ER +/PIK3CA mutated, and ER+/PTEN mutated breast cancer cell lines.

Results: 1) Copanlisib inhibited PI3K, mTOR and their downstream signaling molecules in ER+/PIK3CA or ER+/PTEN mutated BC cell lines; 2) interestingly, copanlisib also inhibited RAS-MAPK signaling in the earlier time points in ER+/PIK3CA mutated cells (T47D & MCF7) but not in ER+/PTEN mutated cells (CAMA1); 3) copanlisib caused a strong differential growth inhibition in ER+ BC cell lines as shown by 3D-ON-TOP clonogenic assay and real-time monitoring in an IncuCyte Zoom. Inhibition was greater when copanlisib was combined with letrozole; 4) administration of copanlisib induced cell cycle G0/G1 arrest and resulted in increased apoptosis in a dose-dependent manner; 5) unlike everolimus, copanlisib blocked HIF1α accumulation in hypoxic conditions and this blocking effect was reversed by prior treatment with the proteasome inhibitor carfilzomib, suggesting copalnisib enhanced proteasome-mediated HIF1α degradation; and 6) copanlisib also attenuated ER+ cell migration, an important phenotypic feature for metastasis, along with integrin-mediated RAC1 activation.

Conclusions: Copanlisib is highly effective (blocks proliferation, induces apoptosis, and inhibits PI3K and its downstream signaling targets) in ER+ BC cell lines (both PIK3CA and PTEN mutated). The addition of copanlisib to aromatase inhibitor might represent an improved treatment strategy for patients with hormone-resistant/relapse ER+ BC.

#3439

Sulindac sulfide as a gamma secretase modifier to target triple negative breast cancer.

Fokhrul Hossain,1 Deniz A Ucar,1 Samarpan Majumder,1 Margarite Matossian,2 Keli Xu,3 Yong Ran,4 Lisa Minter,5 Yaguang Xi,1 Matthew Burow,2 Todd Golde,4 Barbara Osborne,5 Lucio Miele1. 1 _LSUHSC, New Orleans, LA;_ 2 _Tulane University, New Orleans, LA;_ 3 _University of Mississippi Medical Center, Jackson, MS;_ 4 _University of Florida, Gainesville, FL;_ 5 _UMass Amherst, Amherst, MA_.

Triple negative breast cancer (TNBC) is defined as pathologically negative for estrogen receptor (ER-), progesterone receptor (PR-), and human epidermal growth factor receptor 2 amplification (HER2-). TNBCs are a heterogeneous group of clinically aggressive breast cancers with high risk of recurrence and metastasis, but the current treatment options remain limited. There is strong evidence supporting the involvement of Notch signaling in TNBC progression. Expression of Notch1 and its ligand Jagged1 correlate with poor prognosis. Notch inhibitors, Including Gamma Secretase Inhibitors (GSIs) are quite effective in preclinical models of TNBC. However the success of GSIs in clinical trials is limited by their intestinal toxicity and adverse immunological effects. Our overarching goal is to replace GSIs with agents that lack their systemic toxicity and adverse immunological effects. We identified Sulindac Sulfide (SS), the active metabolite of FDA-approved NSAID Sulindac, as a potential candidate to replace GSIs. SS has documented Gamma Secretase Modifier (GSM) activity, in addition to cyclo-oxygenase (COX) inhibition. We confirmed that SS inhibits Notch1 cleavage in TNBC cells, but not in murine T-cells. SS significantly inhibited mammospheres growth in all human and murine TNBC models we tested: 1) human MDA-MB-231 cells; 2) murine TNBC model C0321, from targeted conditional knockout of Lunatic Fringe (LFng-/-); and 3) Two TNBC patient-derived xenograft models, 2K1 and 4IC. In C0321 tumors, we found that SS had remarkable single-agent anti-tumor activity and virtually eliminated Notch1 expression in tumors. SS caused an increase in intra-tumoral CD11c+ dendritic cells, but decreased CD4 cells, which in this model are largely PD-1 positive (exhausted). CD8 cells were modestly increased. SS did not affect the numbers of tumor infiltrating macrophages or myeloid-derived suppressor cells (MDSC). However, SS blocked the immunosuppressive function of bone marrow-derived MDSC. RNA-Sequencing of SS-treated tumors revealed significant reduction of CXCL14, EGR1, HOXC6, MAGI2, NCAM1, APOE, CLU (a Wnt target), DTX4 (an E3-ligase positive regulator of Notch activation), and TGFB3 genes and upregulation of CCL17, EPCAM, FABP4, C4A, LTF, ZBTB16, INADL, and FGFR2 genes. Our data support further investigation of SS for the treatment of TNBC, with standard of care or with immunotherapy. Repurposing an FDA-approved, safe agent for the treatment of TNBC may be significantly easier and more cost-effective than developing unproven investigational agents.

#3440

Beta-2 adrenergic receptors role in tumor aggressiveness of MDA-MB-231 breast cancer cells.

Benedicte Rousseau, Ananya Tirupathur, Sengottuvelan Murugan, Dipak K. Sarkar. _Rutgers University, New Brunswick, NJ_.

Breast cancer is the world's leading cause of cancer mortality among women. Despite advances in both diagnosis and treatment a still large number of women die because of metastatic breast cancer. Research demonstrated a strong link between chronic stress and weakened immune system leaving the body prone to disease like cancer. Indeed, psychological stress has been shown to enhance tumor growth and progression. In addition, stress hormones, such as norepinephrine and epinephrine, are implicated in the growth, invasiveness and metastasis of cancer. Therefore, targeting the beta-adrenergic receptors in cancer chemoprevention may offer new promises for the design of therapeutic strategies. For that reason, we determine the cellular processes involved in beta-2 adrenergic receptor (ADRB2) mediated effects on human breast cancer cells in cultures. We particularly focused on the impact of the inhibition of ADRB2 on cell proliferation, migration and invasion of the triple negative breast cancer cell line MDA-MB-231. In order to determine the effects of loss of expression of the gene, we suppressed ADRB2 by knock down, using CRISPR technology. We also used pharmacologically suppression of ADBR by employing a general beta receptor blocker; propranolol. Cell proliferation, migration and invasion assays were performed to define a degree of aggressiveness to the cells phenotype after the knock down or pharmacological suppression of ADRB2 in comparison to control cells. Also, Western Blot analysis on characteristics proteins of the epithelial-mesenchymal transition (EMT) was used to further characterize the phenotype. PCR Array expression studies were done to analyze modifications of genes expression profile and signaling cascades in the ADRB2 knocked down cells. We found that the knocking down by CRISPR of the ADRB2 receptors or, pharmacologically suppressing ADRB2 reduces proliferation, migration and colony formation of MDA-MB-231 cells. Additionally, ADRB2 knock down increased expression of epithelial proteins (beta-catenin and E-cadherin) while decreased mesenchymal markers (vimentin and N-cadherin). The mRNA expression study using a PCR Array targeting cancer stem cell genes shows that the inhibition of ADRB2 significantly modifies signaling involved in cell movement, invasion, migration and proliferation such as Wnt/beta-catenin signaling, ILK signaling and Notch signaling. Within these signaling pathways, the expression of several genes such as ILK, MAP2K6, GSK3B and JUN seem to be critical as they are under-expressed. These data provide preliminary understanding of the possible connection between the beta-2 adrenergic receptor system and signaling pathways use by the tumor cells to grow and acquire a more aggressive phenotype. Also, some genes are identified as new targets that may be valuable for developing effective treatment of advanced breast cancer. (Supported by NIH grant R01 CA20863201)

#3441

A RPL39-SGK3 signaling pathway that may play a key role in breast cancer therapy resistance.

Dan Liu,1 Bhuvanesh Dave2. 1 _Baylor College of Medicine, Houston, TX;_ 2 _Netnoids Rx Laboratories LLC, Houston, TX_.

Breast cancer is the most common cancer in the United States, where 1 in 8 women are affected by it during their lifetime. In 2018, an estimated 266,120 new cases of invasive breast cancer are expected to be diagnosed in women in the U.S., along with 63,960 new cases of non-invasive (in situ) breast cancer. In order to identify novel targets and better understand the mechanism of survival of these cancers, we describe the mechanism of action of ribosomal protein like 39 (RPL39) as one potentially useful target for therapy resistance. RPL39 is over-expressed in breast cancer and regulated by nitric oxide signaling (NOS). In the present study we examine the detailed mechanism of action of RPL39 as it relates to treatment-resistant breast cancers and the role of SGK3 in modulating its signal transduction pathway. Methods and Results: Mass spectrometric analysis following immunoprecipitation of RPL39, in two breast cancer cell lines SUM159 and MDAMB231, indicated that proteins involved in translation, specifically the mTOR-dependent translational pathway, were part of the complex. In order to verify that the results we observed were due to targeting of the translational arm of mTOR signaling, we knocked down both the 4EBP-1 (translation) and S6K (cell size) arms of the mTOR pathway in three breast cancer cell lines (SUM159, MDAMB231 and MDAMB436). Analysis of these pathways by proliferation, apoptosis, sphere formation and western blotting in all three breast cancer cell lines demonstrated that the translation arm was significantly altered: self-renewal (p<0.05, Student's t test) and proliferation (p<0.05, student's t test) in a NOS signaling dependent manner. Additional bioinformatic analysis determined a potential role for SGK3 upstream of RPL39. SGK3 is an AGC kinase family member that is over-expressed in ER+ breast cancers. It functions downstream of PI 3-kinase and can activate the mTOR pathway. Conclusion: RPL39 gene may be modulated by the selective translational initiation complex which affects self-renewal and proliferation through SGK3 and NOS signaling. The significance of targeting SGK3 and RPL39 pathway allows a potentially novel therapeutic strategy for the treatment of breast cancer.

#3442

**Functional and molecular analysis of the somatic mutations in the NF-κB pathway inhibitor, CYLD,** **in nasopharyngeal carcinoma.**

Mingdan Deng, Wei Dai, Maria Lung. _The University of Hong Kong, Hong Kong_.

Background: NF-κB is an important signaling pathway for regulating multiple signaling cascades involved in cancer development such as inflammation, immune response, cell survival, and cell proliferation. Constitutive activation of the NF-κB pathway plays a key role in the development of NPC. Our previous whole-exome sequencing (WES) study in NPC biopsies has identified five mutations in CYLD, including R758Q. As R758X is a mutational hotspot in CYLD, it has been reported in multiple familial trichoepithelioma type 1, Brooke-Spiegler syndrome and familial cylindromatosis. Thus, we attempted to evaluate the functional effect of CYLD WT and R758Q in regulating NF-κB activity in NPC, which is crucial in NPC progression and development.

Methods: C17, C666-1 and NPC43 EBV-positive cell lines and HK1 were co-infected by CYLD wild-type (WT) and mutant constructs with a NF-κB specific dual luciferase promoter plasmid. CRISPR-CAS9 system was used to knock out CYLD. The functional role of CYLD was determined by MTT, CFA and wound healing assays. NF-κB downstream targets expression was evaluated by the method of RT-QPCR. Co-immunoprecipitation (Co-IP) experiments were performed with the Sigma Anti-FLAG M2 magnetic beads and the results were confirmed by Western blot analysis. Female athymic BALB/cAnN (nude) mice were utilized to investigate tumorigenicity. NPC cell lines were treated with cisplatin to check cell sensitivity. Results: The reporter assay showed that CYLD can downregulate NF-κB binding activities in comparison to the vector-alone (VA), but R758Q failed to inhibit the NF-κB binding activities. Over-expression of CYLD can suppress NF-κB downstream target genes expression. Knocking out of CYLD results in higher NF-κB downstream genes expression compared with VA, which confirmed that CYLD can suppress NF-κB activity. R758Q increases these genes expression compared with WT. CYLD can suppress cell growth rate, while R758Q promoted cell proliferation and migration compared to WT. Furthermore, WT CYLD can significantly suppress tumor growth rate, whereas R758Q has the same growth rate with VA, consistent with R758Q loss of WT function, causing tumor suppression. Moreover, CYLD can enhance cell sensitivity to cisplatin treatment compared to the VA.

Conclusions: These results suggest that CYLD can suppress NF-κB activity compared to VA, while R758Q lost WT function in NPC. CYLD can inhibit tumorigenesis compared to VA, whereas R758Q induces tumor growth compared to WT. The NPC cell line with CYLD WT is more sensitive to cisplatin treatment.

Acknowledgement: Research Grants Council Area of Excellence grant AoE-M06/08 to MLL.

#3443

Doublecortin-like kinase 1 (DCLK1) is novel Notch pathway signaling regulator in HNSCC.

Esther Channah Broner,1 Tejaswini Subbannayya,2 Alex Zhavoronkov,3 Mike Korzinkin,3 Artem Artemov,3 Ivan Ozerov,3 Ido Sloma,4 David Sidransky,1 Aditi Chatterjee,2 Evgeny Izumchenko1. 1 _Johns Hopkins University, Baltimore, MD;_ 2 _Institute of Bioinformatics, Bangalore, India;_ 3 _Insilico Medicine, Inc, ETC, Baltimore, MD;_ 4 _Champions Oncology, Baltimore, MD_.

Introduction: Despite advancements in the field, the 5 year survival rate of Head and Neck Squamous Cell Carcinoma (HNSCC) still hovers at 60%. DCLK1 has been shown to regulate epithelial-to-mesenchymal transition (EMT) as well as serving as a cancer stem cell marker in colon, pancreatic and renal cancer. Although it was reported that DCLK1 is associated with poor prognosis in oropharyngeal cancers, very little is known about the molecular characterization of DCLK1 in HNSCC.

Methods and Materials: In a quest for novel therapy targets for this disease, we performed an extensive and comprehensive quantitative phosphoproteomics scan on 10 HNSCC patient derived xenografts (PDXs) in order to find dysregulated phosphoproteins. We also performed a comprehensive transcriptome-based computational analysis on hundreds of HNSCC patients from TCGA and GEO databases. We used immunohistochemistry (IHC) to stain a tumor microarray (TMA) of 45 HNSCC samples. We have selected 6 cell HNSCC cell lines (JHU-011, JHU-022, JHU-029, FaDu, SCC22B and Cal27) that express DCLK1, and inhibited DCLK1 with LRRK2-in1 inhibitor and siRNA. We selected 1 HNSCC cell line with low DCLK1 expression, SCC25 and 2 normal keratinocyte cell lines, OKF6 and NOKSI, in which we overexpressed DCLK1. We assessed DCLK1 and active Notch1 expression in these cell lines by Western Blot and Immunohistochemistry (IHC). We evaluated metastatic characteristics with scratch assay, invasion assay, alamar blue proliferation assay, and colony formation assay.

Results: We found that DCLK1 is hyperphosphorylated in most of the HNSCC derived PDXs. In our comprehensive transcriptome-based computational analysis on, we found that along with EMT and metastasis pathways, high DCLK1 expression also correlates with NOTCH pathway signaling signatures in HNSCC. Since Notch signaling pathway has a recognized role in tumorigenesis in HNSCC tumors, we have focused on investigating the role of DCLK1 in Notch pathway regulation. Towards this end, we performed a series of in vitro experiments in a collection of HNSCC cell lines. Our analyses revealed DCLK1 inhibition both with LRRK2in1 and siRNA resulted in substantially decreased proliferation, invasion, migration, and colony formation. Furthermore, these effects paralleled downregulation of active NOTCH1, and its downstream effectors, Hey1, Hes1 and Hes5 in all cell lines tested. Additionally, overexpression of DCLK1 in HNSCC cell lines and normal keratinocytes leads to upregulation of Notch signaling as well as increased proliferation. IHC staining on 45 HNSCC tumors indicates that 45% of the samples are positive for DCLK1 compared to 90% DCLK1 negative staining in normal oral tissue.

Conclusion: Overall, our results demonstrate the novel role of DCLK1 in HNSCC as a regulator of the NOTCH signaling network and suggests its potential as a therapeutic target in HNSCC.

#3444

Identification of Interleukin-1 (IL-1) induced gene expression pattern in breast cancer (BCa) cells.

Afshan Fathima Nawas,1 Mohammed Kanchwala,2 Shayna Thomas-Jardin,1 Vanessa Anunobi,1 Ally Wong,1 Chao Xing,2 Nikki Delk1. 1 _UT Dallas, Richardson, TX;_ 2 _The University of Texas Southwestern Medical Center, TX_.

Breast cancer (BCa) and prostate cancer (PCa) are both hormone driven cancers with similar etiology and tumor microenvironment inflammation promotes BCa and PCa progression. Interleukin-1 (IL-1) is an inflammatory cytokine present in the tumor microenvironment and IL-1 is elevated in BCa and PCa patient tumor and/or blood serum and correlates with poor prognosis. We have shown that IL-1 represses the BCa and PCa therapeutic targets, Estrogen Receptor Alpha (ERa) and Androgen Receptor (AR), respectively, in PCa and BCa cell lines; yet the cells remain viable. Thus, IL-1 may promote treatment resistance and disease progression. The pro-survival protein Sequestome-1 (SQSMT1/p62) is also upregulated in both BCa and PCa cell lines exposed to IL-1, suggesting that BCa and PCa cells have evolved a shared response to IL-1 and hormone receptor loss. RNA sequencing of an IL-1-treated PCa cell line revealed an IL-1-modulated gene suite predicted to confer AR-independent tumorigenicity. We performed RT-qPCR in PCa cell lines for several select genes to confirm the RNA sequencing results and given the similar etiology and IL-1 regulation of ERa, AR, and p62 expression in BCa and PCa cell lines, we presumed that our select genes would be similarly regulated by IL-1 in BCa cells. However, outside of ERa, AR, and p62 expression, our select genes did not show similar IL-1 regulation in BCa cell lines, suggesting that IL-1 regulates a unique set of genes that could contribute to ERa-independent tumorigenicity in BCa cells. Therefore, to identify a BCa-specific IL-1-modulated gene suite, we performed RNA sequencing on an IL-1-treated ERα+ BCa cell line and compared gene expression changes with 1) basal gene expression in an ERa- BCa cell line and 2) our IL-1-modulated PCa-specific gene suite. Our bioinformatics analysis revealed pathways that are predicted to promote ERa-independent tumorigenicity in BCa cells and investigations are underway to demonstrate the functional significance of our gene expression data. Taken together, our studies provide insight into the mechanistic function of IL-1 in PCa and BCa resistance to hormone receptor-targeted therapies and tumor progression.

#3445

Mechanism, physiological and therapeutic implications of LGR-independent potentiation of WNT signaling by R-spondins.

Andres M. Lebensohn,1 Rajat Rohatgi2. 1 _National Cancer Institute, National Institutes of Health, Bethesda, MD;_ 2 _Stanford University School of Medicine, Stanford, CA_.

The WNT signaling pathway encodes positional information in animals, orchestrates patterning and morphogenesis during embryonic development, and promotes tissue renewal and regeneration in adults. Dysregulation of WNT signaling has been implicated in many diseases, including developmental malformations, skeletal and dental abnormalities, cardiovascular and neurodegenerative disorders, diabetes, and many types of cancer. Members of the R-spondin family of secreted growth factors have emerged as key regulators of WNT signaling strength. The R-spondin system is a vertebrate adaptation that may be related to the appearance of sophisticated stem cell compartments, which require exquisite control of WNT signaling. The four members of the family (R-spondin 1-4) can strongly potentiate responses to WNT ligands during development and in stem cells, but the mechanisms by which they transduce signals are not fully understood and the reasons why they produce different physiological effects are largely unknown. Unexpectedly, we discovered that R-spondins 2 and 3 can uniquely potentiate WNT signaling in cells lacking LGRs 4, 5 and 6, the principal R-spondin receptors. We determined the protein domains on R-spondins necessary and sufficient for this new mode of signaling, and showed that it is mediated by an alternative interaction with cell-surface heparan sulfate proteoglycans. This finding is transformative because LGRs 4-6 were thought to be required to transduce all R-spondin signals and hence determine their site of action. Indeed, these LGRs are highly expressed in many stem cell compartments whose maintenance depends on potentiation of WNT signaling by R-spondins. Instead our work shows that there are two modes of signaling by R-spondins, an LGR-dependent and an LGR-independent mode, and that different R-spondins may use distinct molecular mechanisms to transduce signals in various biological contexts. Supporting our findings, recent work from another group demonstrated that during limb development, R-spondin 2 signals through an LGR-independent mechanism. Since components of the R-spondin signaling module are commonly mutated in cancer, therapeutic targeting of LGR-independent signaling deserves evaluation. I will present the results demonstrating our conclusions and discuss their implications in physiology and disease.

#3446

STRAP is involved in mutated Apc-induced stemness and tumorigenesis in colon cancer.

Trung Vu. _University of Alabama at Birmingham, Birmingham, AL_.

Inactivation of the Apc gene is a critical early event in the development of sporadic colorectal cancer (CRC). Loss of its normal function leads to constitutive activation of β-catenin/T-cell factor 4 signaling and hence transcription of Wnt target genes. The signaling scaffold protein STRAP is upregulated in several cancers, where it promotes tumorigenicity and stemness. Here, we tested the effect of conditional deletion of Strap on intestinal tumor formation and growth by crossing ApcMin/+ mice with mice that are null for Strap in the intestinal epithelium, Strapfl/fl;Vil-Cre mice (StrapIEKO). We observed a dramatic decrease in the number of intestinal polyps in the ApcMin/+;Strapfl/fl;Vil-Cre mice (called StrapIEKO;ApcMin/+) compared to mice with intact Strap (Strapfl/fl;ApcMin/+). In an attempt to understand the mechanism of function of STRAP, we have observed that colon tumors from StrapIEKO;ApcMin/+ mice displayed a decrease in β-catenin nuclear localization and downregulation of Wnt targets such as c-Myc, AXIN2, and CCND1. Enteroids prepared from StrapIEKO;ApcMin/+ mice exhibited more budding compared to the ApcMin/+ enteroids, indicating the role of STRAP in stemness. STRAP knockdown in human CRC cells significantly diminished β-catenin/TCF4 transcriptional activity. Specifically, STRAP knockdown impaired the assembly of β-catenin/TCF4 complex and recruitment of the complex to the promoters of β-catenin target genes. Functional proteomic analyses of crypt cells from different genetic mice suggested that the MEK/ERK signaling pathway is suppressed in StrapIEKO;ApcMin/+ mice that is required for the effect of STRAP on Wnt/β-catenin signaling. We have shown that STRAP associates with MEK1/2 and promotes binding between MEK1/2 and ERK1/2 and subsequently induces the phosphorylation of ERK1/2. Given that MEK/ERK signaling is found to promote Wnt/β-catenin signaling, our results suggest that STRAP indirectly induce Wnt/β-catenin signaling through activating MEK/ERK pathway. Importantly, STRAP is found to be a novel target of Wnt/β-catenin

signaling as ChIP assays determined putative binding sites of β-catenin/TCF4 complex on Strap promoter. Finally, we showed that forced expression of STRAP in colon cancer organoids derived from patients with colon cancer can induce the growth of colon tumor organoids through promoting Wnt/β-catenin signaling. Altogether, these results suggest that STRAP is involved in mutated APC-induced tumor initiation through a novel feed-forward STRAP/MEK-ERK/β-catenin/STRAP regulatory axis.

#3447

APC truncating mutations in Middle Eastern population: Tankyrase inhibitor is an effective strategy to sensitize APC mutant CRC to 5-FU chemotherapy.

Sandeep K. Parvathareddy, Abdul K. Siraj, Rong Bu, Prateeshkumar Poyil, Tariq Masoodi, Divya Sasidharan Padmaja, Rica Concepcion, Rafia Begum, Maria A. Sabido, Roxanne Melosantos, Nasser Al-Sanea, Fouad Al-Dayel, Khawla S. Al-Kuraya. _King Faisal Specialist Hospital & Research Centre, Riyadh, Saudi Arabia_.

Colorectal Cancer (CRC) is the leading cause of cancer related death and the most common cancer affecting Saudi males. Mutation of the adenomatous polyposis coli (APC) gene is considered as the initiating step of transformation in familial and sporadic CRC. The prevalence and prognostic role of APC mutation spectra are not well established in Middle Eastern CRC. Next generation sequencing of 412 CRC tumors was performed to determine mutations in APC. Association with protein expression and clinico-pathological correlation was performed. APC truncating mutation is found at a frequency of 58.2% (240/412) and is significantly associated with superior overall survival, whereas no difference in survival between short truncating and long truncating APC was noted. To test the hypothesis that APC mutations might influence 5- Fluorouracil (5-FU) therapy, which is the first line chemotherapy used in CRC, we compared CRC cell lines and showed that those expressing truncated APC exhibit limited response to 5-FU, unlike cells which were APC wild type. In COLO-320 DM and HCT-15, APC mutated CRC cells, we were able to increase sensitivity of APC truncated cells to 5-FU by inhibiting Tankyrase (TNKs) which are members of PARP family, using XAV939. In conclusion, the study identified the prognostic value of APC truncating mutation in Middle Eastern ethnicity. In vitro data further showed that APC mutation can be used as a molecular biomarker to predict response to 5-FU. Furthermore, Tankyrase inhibitor XAV939 selectively targeting APC-mutated CRC cells can be an effective strategy to overcome 5-FU resistance in these cells.

#3448

Dasatinib attenuates Src pathway signaling and combined with veliparib and carboplatin potently inhibits tumor growth in a preclinical model of TNBC.

Yuliang Sun, Xiaoqian Lin, Jennifer C. Aske, Casey Williams, Mark Abramovitz, Brian Leyland-Jones. _Avera Cancer Institute, Sioux Falls, SD_.

Background: The combination of a poly ADP ribose polymerase (PARP) inhibitor with a DNA-damaging agent has shown promise in treating triple-negative breast cancer (TNBC); however, not all patients respond to this combination (Rugo HS et al. The New England Journal of Medicine. 2016). The Src protein kinase modulates multiple cancer cell properties and plays a key role in tumorigenic processes. However, Src inhibitors as single agents have shown limited effects in solid tumors (Finn RS et al. Clinical Cancer Research. 2011). In this study, we examined the antitumor effects of the Src inhibitor dasatinib, the PARP inhibitor veliparib, and the DNA-damaging agent carboplatin in TNBC cells in vitro and tumors in vivo to try to identify the combination with the most clinical potential.

Methods: The KRAS/BRAF-mutated TNBC cell line, MDA-MB-231, was used in this study. For in vitro studies, proliferation, cellular apoptosis, and 3D on-top clonogenic growth were measured, tube formation assays and western blot analysis were performed. For in vivo studies, a xenograft model was used to examine treatment responses, and immunohistochemical analysis was performed.

Results: Surprisingly, treatment with the combination of veliparib plus carboplatin led to an increase in Src phosphorylation. Importantly, addition of dasatinib attenuated Src overexpression induced by veliparib plus carboplatin and further inhibited the downstream signaling of Src, thereby enhancing the antitumor efficacy of veliparib plus carboplatin. In an MDA-MB-231 xenograft model, the triple combination of dasatinib with veliparib plus carboplatin showed greater tumor growth inhibitory effects compared with single agents or double combinations. No systemic toxicity was observed in mice treated with the triple combination.

Conclusions: Here, we provide novel evidence that veliparib combined with carboplatin drives Src activation in the preclinical model tested. Moreover, we provide provocative in vitro and in vivo data highlighting the potential for increasing therapeutic efficacy by combining dasatinib with veliparib and carboplatin in patients with KRAS/BRAF-mutated TNBC.

#3449

Tyrosine phosphatase activity is required for response to Src kinase inhibition in cholangiocarcinoma.

EeeLN Buckarma, Nathan Werneburg, Ayano Niibe, Gregory Gores, Rory Smoot. _Mayo Clinic, Rochester, MN_.

Background: Cancer of the biliary tract, cholangiocarcinoma (CCA), is increasing in incidence and has limited treatment options. In an attempt to further understand signaling pathways driving oncogenesis and impacting response to therapy we, and others, have identified altered activation of the transcriptional co-activator, Yes- associated protein (YAP) in CCA. Canonical regulatory pathways impacting YAP consist of serine kinases; however, more recently we have demonstrated a central role for tyrosine phosphorylation in regulating YAP function in CCA. Herein we explore the role of tyrosine phosphatases in regulating YAP tyrosine phosphorylation.

Methods: In silico analysis of the TCGA cholangiocarcinoma dataset was undertaken specifically evaluating tyrosine phosphatase levels. Molecular studies utilized the human CCA cell lines HuCCT-1 and KMCH as well as the murine CCA cell line SB1 (expressing a flag-tagged YAP). Baseline tyrosine phosphatase levels were assessed by RT-PCR and immunoblot. YAP-interacting phosphatases were identified by co-immunoprecipitation. Tyrosine phosphorylation was inhibited utilizing the multi-kinase inhibitor dasatinib. The pan-tyrosine phosphatase inhibitor sodium orthovanadate (Na3VO4) and the selective SHP1/2 inhibitor NSC87877 were utilized. Tyrosine phosphorylation was assessed by immunoblot. YAP transcriptional activity was evaluated by RT-PCR. Apoptosis was evaluated by caspase 3/7 assay.

Results: Multiple tyrosine phosphatases were noted to be expressed at lower levels in tumor versus normal adjacent liver in the TCGA RNAseq dataset. YAP-interacting tyrosine phosphatases identified by co-IP included PTPN1, 11, 23, and PTPRK. Profiling of tyrosine phosphatase levels by RT-PCR and immunoblot demonstrated higher levels in KMCH cells compared to HuCCT-1; notably the YAP-interacting phosphatase PTPN11 (SHP2) was elevated. Consistent with the anticipated function of the phosphatases, immunoblot demonstrated lower levels of tyrosine phosphorylated YAP (p-YAPY357) in KMCH cells compared to HuCCT-1. The role of SHP2 was further probed by incubation of KMCH and HuCCT-1 cells with NSC87877 which was associated with an increase in p-YAPY357 levels and YAP co-transcriptional activity. Conversely, incubation of HuCCT-1 cells with dasatinib rapidly decreased p-YAPY357 levels; and this effect was diminished by pre-incubation with either Na3VO4 or NSC87877. The effect on YAP tyrosine phosphorylation paralleled effects on apoptosis; incubation of HuCCT-1 cells with dasatinib lead to an apoptotic response as measured by caspase 3/7 assay, which was eliminated by pre-incubation with Na3VO4 or NSC87877.

Conclusions: Inhibition of the tyrosine phosphatase SHP2 is associated with elevated p-YAPY357 levels, elevated YAP co-transcriptional activity, and a blunted therapeutic response to dasatinib in cholangiocarcinoma cell lines.

#3450

Impact of BRCA1 on glucocorticoid signaling in fallopian tube epithelial cells.

Kasra Khalaj, Alexandra Kollara, Julia Hollingsworth, Vladimir Djedovic, Theodore J. Brown. _Lunenfeld-Tanenbaum Research Institute, Toronto, Ontario, Canada_.

Background: High-grade serous ovarian cancer (HGSC), the most predominant ovarian cancer histotype, originates in the fallopian tube epithelium (FTE). Women with germline BRCA1 mutations are at high risk for this cancer subtype. Our previous work indicates that FTE cells from BRCA1 mutation carriers (mtBRCA1) have increased EGFR and NFκB signaling compared to FTE from control patients, and that glucocorticoid receptor (GR) signaling may be impaired in BRCA1-deficient cells. Glucocorticoids are potent anti-inflammatory steroids that inhibit NFκB signaling. Studies using breast cancer cells report that BRCA1 increases microRNA miR-146 expression, which targets transcripts for EGFR and activators of NFκB. In this study, we examined the impact of BRCA1 deficiency on GR expression and signaling, and on miR-146 expression.

Methods: Primary FTE cells were derived from mtBRCA1 carriers or control patients. Immortalized human FTE OE6/7 cells were engineered to overexpress BRCA1. UWB1.289 and UWB1.289+BRCA1 HGSC cells were obtained from ATCC. Levels of GRα, GRβ (splice variant isoform that acts as a dominant-negative of GRα), inflammatory cytokines, and GR-regulated genes were quantified using qPCR and western blotting. miR146 was quantified using miR PCR assays.

Results: FTE and HGSC cells express both GR isoforms. GR mRNA levels in primary cultured FTE cells from mtBRCA1 carriers were similar to FTE cells from mtBRCA2 and control patients, although mtBRCA1 FTE cells had increased GRα:GRβ transcript ratios. In contrast, BRCA1 overexpression in OE6/7 cells and UWB1.1 289 cells increased the GRα:GRβ transcript ratio. Total GR protein levels were similar in UWB1.289 and UWB1.289+BRCA1 cells; however, BRCA1 expression associated with increased dexamethasone (DEX)-induced GR transactivation. GR target genes SGK1 and GILZ were upregulated by DEX similarly regardless of BRCA1 expression. TNFα was decreased in OE6/7 and UWB1.1 289 cells expressing higher levels of BRCA1 and was further decreased by DEX. miR-146 levels were elevated, rather than suppressed, in BRCA1-deficient cells, and correlated with decreased expression of targeted NFκB activators.

Conclusions: These data indicate GR signaling is affected by BRCA1 and that both GRα and GRβ are expressed in FTE cells. BRCA1 expression alters relative isoform expression ratios raising the possibility of altered response to glucocorticoids that could impact NFκB signaling. Furthermore, these studies indicate that mechanisms other than decreased miRNA-146 underlie increased NFκB and EGFR signaling in mtBRCA1 FTE cells.

#3451

Interpreting gene lists from -omics experiments.

Augustin Luna,1 Jeffrey V. Wong,2 Emek Demir,3 Igor Rodchenkov,2 Özgün Babur,3 Chris Sander,1 Gary D. Bader2. 1 _Harvard Medical School, Boston, MA;_ 2 _University of Toronto, Ontario, Canada;_ 3 _Oregon Health & Science University, Portland, OR_.

Understanding the mechanisms responsible for a cellular behavior often begins with observations of genes and gene products. Depending on the type of experiment, the number of resulting genes can be small, but increasingly, researchers are faced with many thousands of measurements, as in the case of transcriptomic or protein-DNA binding observations. Here, we describe ways to pair experimental results consisting of one or more genes with analysis tools with the overall aim being to make results more biologically interpretable. In certain cases, experimental approaches such as screens for essential genes can generate one or a few 'genes of interest' and there is a desire to understand their relationship to one another as well as discover links to additional, interesting genes. To this end, 'GeneMANIA' is a web tool that accepts gene names and returns a network visualization of related genes based on similarity in expression, localization, protein domains and those involved in physical interactions. Likewise, 'PCViz' is a web tool that displays a network of interactions drawn from Pathway Commons, a web resource for pathway and interaction knowledge. In cases where experiments generate a lengthy list of genes, for instance, transcriptomic measurements, there is a desire to understand their relevance to a phenotype of interest. Pathway enrichment analysis methods aim to summarize gene lists as pathways, which have a closer link to cell function. An online 'Guide' by Pathway Commons includes workflows that illustrate how to chain together software tools to identify pathways from the corresponding gene-level data then organize and summarize the pathway-level results in an interactive visualization known as an Enrichment Map. For those wishing to drill-down to individual pathways, Pathway Commons offers a set of web apps, including 'Search' that enables users to query by keyword and visualize ranked search results. Ongoing development of web apps aims to enhance the accessibility to pathways and integrate support for analysis and visualization of experimental data. The full complement of data, tools and resources offered by Pathway Commons in support of pathway analysis are described.

#3452

hnRNPUL1 regulation by ATR in DNA damage response.

Hui Zhang, PamelaSara E. Head, Duc M. Duong, Nicholas T. Seyfried, David S. Yu. _Emory University, Atlanta, GA_.

The DNA damage response (DDR) is a signaling network that recognizes damages to DNA and orchestrates a variety of DNA repair and cell cycle checkpoint pathways. The DDR is pivotal for cancer prevention. Ataxia Telangiectasia And Rad3-Related Protein (ATR), a protein kinase, functions as an essential transducer of the DDR signaling cascade. ATR primarily responds to single-stranded DNA (ssDNA) generated from double-strand break resection or at stalled replication forks. Depletion of ATR leads to cellular senescence and cancer related phenotypes. The mechanisms regarding how the ATR signal pathway is regulated, however, remain elusive. To discover novel DDR proteins regulated by ATR, we performed proteomic analysis to identify proteins that partner with ATR in response to DNA damage in cells. Our analysis revealed a network of proteins as potential ATR substrates, including Heterogeneous nuclear ribonucleoprotein U-like 1 (hnRPUL1), a protein functions in RNA metabolism. We validated that ATR interacts with hnRNPUL1. In addition, hnRPUL1 is phosphorylated on SQ/TQ sites in response to IR. ATR depletion suppresses the IR induced hnRPUL1 phosphorylation. Our results identify hnRNPUL1 as a novel ATR substrate, critical for the DNA damage response and provide insight into how DDR and RNA metabolism might work synergically in maintaining genomic stability and preventing cancer. Additionally, hnRNPUL1 phosphorylation by ATR may be targeted as an adjunct to improve the efficacy of ionizing radiation for cancer therapy.

#3453

CXCL10 contributes to aggressive disease progression in ING4-deficient breast cancer.

Emily Szeto, Suwon Kim. _University of Arizona, Phoenix, AZ_.

CXCL10 is a chemoattractant secreted by various cell types during inflammation for immune cells expressing its cognate receptor CXCR3. In breast cancer, CXCL10 has been associated with increased tumor lymphocytic infiltrates and poor patient prognosis. In addition, pharmacological inhibition of CXCR3 resulted in reduced lung metastases of mammary tumors in mice, suggesting CXCL10/CXCR3 signaling contributes to aggressive breast cancer. We previously identified CXCL10 as an NF-κB target gene repressed by Inhibitor of Growth 4 (ING4) in breast cancer cells. As ING4 deficiencies reported in 34% of breast tumors have been correlated with faster disease recurrence in patients, we investigated a functional interplay between CXCL10 and ING4. First, we assessed the clinical relevance of CXCL10, CXCR3, and/or ING4 expression levels using a public gene expression data set. Second, we genetically engineered T47D breast cancer cells to overexpress or delete the ING4 gene and determined cell phenotypes in the presence or absence of CXCL10 by utilizing cell migration assays and Western blot. Analysis of the GDS806 gene data set showed that patients with tumors expressing high levels of CXCL10 experienced significantly increased rates of disease recurrence while CXCR3 expression did not have a significant effect. The analysis also showed tumors expressing low levels of ING4 expressed higher levels of CXCL10, indicating an inverse expression pattern between the two genes. Moreover, patients with ING4-low/CXCL10-high tumors had a 4-fold faster recurrence rate compared to ING4-high/CXCL10-low tumor patients. These results indicated that high CXCL10 expression contributed to significantly accelerated disease recurrence compounded by low ING4 expression, suggesting a functional relationship between the two genes which may exacerbate tumor phenotypes. While CXCL10 did not affect growth rates of T47D cells with or without ING4, CXCL10 induced migration only in ING4-deleted cells. These results suggested that ING4 inhibited CXCL10 signaling. The western blot analyses showed no change in CXCR3 expression, but elevated phosphorylation of insulin-like growth factor 1 receptor (IGF1R) after CXCL10 treatment, suggesting CXCL10 may signal by activating IGF1R. Consistent with this idea, when cells were treated with an IGF1R inhibitor, Linsitinib (OSI-906), CXCL10-induced migration was attenuated in ING4-deleted cells. In conclusion, these results suggest that in order to promote an aggressive tumor phenotype, CXCL10 induces migration of ING4-deficient tumor cells in part by activating IGF1R. This study reports the first demonstration of the chemokine CXCL10 exerting a direct effect on breast cancer cells and puts forth the CXCL10/IGF1R signaling pathway as a potential therapeutic target for ING4-deficient aggressive breast cancer.

#3454

Nkx2-1 controls cancer progression by dampening ERK activity.

Michelle Mendoza, Kelley Ingram, Eric Snyder, Rediet Zewdu. _University of Utah, Salt Lake City, UT_.

The RAS/RAF/MEK/ERK MAPK pathway is hyper-activated in a significant fraction of carcinomas, including ~50% of human non-small cell lung cancer (NSCLC). A sweet-spot of ERK activity drives malignant transformation, while excessive signaling induces cellular toxicity and senescence. KRAS mutation in mice induces low ERK activation that requires additional mutations for high ERK activity and NSCLC progression (Heidorn, Milagre et al. 2010, Kamata, Hussain et al. 2010, Poulikakos, Zhang et al. 2010, Cicchini, Buza et al. 2017, Nieto, Ambrogio et al. 2017). The mechanisms by which tumors overcome ERK's homeostatic feedback mechanisms to acquire high ERK activity include Ras amplification (Cicchini Cell Reports 2017, Juntilla Nature 2010), but are otherwise un-resolved. The transcription factor NKX2-1/TTF-1 is downregulated in ~20% of lung adenocarcinomas and confers a worse clinical prognosis (Barletta, Loda 2009, Berghmans 2006). We tested if NKX2-1 is part of ERK's feedback mechanism and if its loss promotes tumor growth and metastasis. Using autochthonous mouse models and human NSCLC cell line xenografts, we show that NKX2-1 induces the ERK negative regulators DUSP6 and SPROUTY2. In cells with silenced NKX2-1, re-introduction induces DUSP6 and SPROUTY2, reduces ERK activation, and reduces tumorigenicity and metastasis. This regulation correlates with regulation of cell proliferation and migration in vitro. Thus, NKX2-1 silencing during NSCLC progression unleashes ERK hyperactivation and lung adenocarcinoma progression through the loss of ERK feedback inhibitors.

#3455

TNFAIP8 increases drug resistance and steatosis in liver cancer by inducing autophagy.

Suresh Niture, Maxwell A. Gyamfi, Deepak Kumar. _North Carolina Central Univ., Durham, NC_.

Tumor necrosis factor-α-inducible protein 8 (TNFAIP8) is a TNF-α inducible anti-apoptotic protein which facilitates tumor growth and progression in several cancers. The molecular mechanisms how TNFAIP8 modulates the progression of liver cancer remain elusive. The present study investigated the role of TNFAIP8 in the liver cancer cell growth, drug resistance/survival and cell steatosis. Tissue microarray data from patients demonstrated an increased expression of TNFAIP8 protein during development of different stages of liver cancer compared with normal liver tissues. To test the correlation between TNFAIP8 expression and liver cancer progression, we transiently/stably expressed TNFAIP8 protein in liver cancer cell lines. Expression of TNFAIP8 in liver cancer cells HepG2 and SK-Hep1 increased both cell growth/survival and drug resistance against anti-liver cancer drugs sorafenib and regorafenib by inhibition of apoptosis. In addition, TNFAIP8 knockdown sensitized tumor cell lines to sorafenib and regorafenib. Expression of TNFAIP8 modulates AKT/mTOR pathway and induced autophagy and thus increased drug resistance. Moreover, TNFAIP8 induced liver cancer cell steatosis by increasing the expression of L-FABP1, SCD1, ACC, and PPARγ proteins which involved in lipid synthesis. We also demonstrate that chronic EtOH induced steatosis in mice liver was associated with increased TNFAIP8 expression and autophagy suggesting that TNFAIP8 mediate EtOH induced steatosis. In addition, increased expression of TNFAIP8 and hepatic steatosis was observed in patients with a history of alcohol use compared with no history of alcohol use. Taken together our data suggested that TNFAIP8 increases drug resistance/survival, EtOH-induced cell steatosis and early liver cancer development by the induction of autophagy.

#3456

Distinct control of PERIOD2 degradation and circadian rhythms by the oncoprotein MDM2.

Xianlin Zou,1 Jingjing Liu,1 Tetsuya Gotoh,1 Anne M. Brown,1 Liang Jiang,1 Esther L. Wisdom,1 Jae Kyoung Kim,2 Carla V. Finkielstein1. 1 _Virginia Tech, Blacksburg, VA;_ 2 _Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea_.

The circadian clock relies on post-translational modifications to set the timing for degradation of core regulatory components and, thus, sets clock progression. Ubiquitin-modifying enzymes targeting clock components for degradation are known to mostly recognize phosphorylated substrates. A case in point is the circadian factor PERIOD 2 (PER2) whose phospho-specific turnover involves its recognition by β-transducin repeat containing proteins (β-TrCPs). Yet, the existence of this unique mode of regulation of PER2's stability falls short of explaining persistent oscillatory phenotypes reported in biological systems lacking functional elements of the phospho-dependent PER2 degradation machinery.

In this study, we challenge the phosphorylation-centric view that PER2 degradation enhances circadian rhythm robustness by i) identifying the PER2:MDM2 endogenous complex, ii) establishing PER2 as a previously uncharacterized substrate for MDM2, iii) revealing an alternative phosphorylation-independent mechanism for PER2 ubiquitin-mediated degradation, iv) pinpointing residues for ubiquitin modification, and v) establishing the importance of MDM2-mediated PER2 turnover for defining the circadian period length. Our results not only expand MDM2's suite of specific substrates beyond the cell cycle to include circadian components but also uncover novel regulatory players that likely impact our view of how other mechanisms crosstalk and modulate the clock itself.

#3457

Flubendazole targets STAT3 signaling and distant metasis in triple-negative breast cancer.

Eunhye Oh, Yoon-Jae Kim, Seojin Jang, Tae-Min Cho, Soeun Park, Jung Min Park, Minsu Park, Ji Young Kim, Jae Hong Seo. _Korea University, Seoul, Republic of Korea_.

Triple-negative breast cancers (TNBC) with higher cancer stem-like populations are reported to exhibit a more aggressive metastatic phenotype. In the present study, we investigated the effects of flubendazole, a candidate drug for repurposing and its novel mechanism of action with a focus on targeting STAT3 signaling and breast cancer stem-like traits in TNBC. The effect of FLU on TNBC cell lines in vitro was evaluated in terms of cell viability, breast cancer stem cells (BCSC)-like properties and expression of STAT3 signaling-related factors. An orthotopic injection model with BCSC-enriched population was used to examine the effect of flubendazole on tumor growth and metastasis in vivo. Our results showed that the BCSC-enriched populations harbored a higher Aldefluor-positive population and markedly elevated levels of CD49f and ALDH1A1 as well as higher STAT3 activation in vitro. Flubendazole attenuated STAT3 activation, as demonstrated by a significant reduction in phospho-STAT3 levels as well as subsequent downregulation of its downstream effector cyclin D1. Sphere-forming ability was significantly suppressed following two pharmacological STAT3 inhibitor, LLL12 or S3I-201 challenge. Treatment with interleukin-6 (IL-6) was observed to significantly increase the number and volume of mammospheres, while flubendazole abolished this effect. Bioluminescence in vivo imaging (BLI) revealed that the BCSC-enriched populations exhibited enhanced metastasis with higher STAT3 activation, while flubendazole administration inhibited tumor growth and lung and liver metastasis, coinciding with decreased MMP-2 and MMP-9 levels in circulating blood. To our knowledge, these findings are the first to report that flubendazole reduces BCSC-enriched tumor burden and metastasis via STAT3 inactivation, implying that flubendazole treatment may have application in addressing metastasis.

#3458

Investigation of oxidative stress-related gene signature in cancer-associated cachexia.

Sumeet Jain, Surendra K. Shukla, Aneesha Dasgupta, Pankaj K. Singh. _University of Nebraska Medical University, Omaha, NE_.

About eighty percent of pancreatic cancer patients demonstrate excessive loss of skeletal muscles with or without loss of adipose deposits, a condition known as cancer-associated cachexia. Severe complexities related to cancer-associated cachexia contribute to mortality in one-third of pancreatic cancer patients. Multiple factors, including loss of appetite, systemic inflammation, digestive enzymes, and hormonal imbalance may contribute to the development of cancer-associated cachexia. Poor understanding of the molecular mechanism is the key challenge for targeting cancer-associated cachexia. Hence, to facilitate the identification of new targets for ameliorating cancer-induced muscle loss, we undertook genomic studies. We evaluated differentially expressed gene signatures in muscle tissues of cachectic and non-cachectic human pancreatic cancer patients. To better model the mechanisms of cancer cachexia, we also compared the identified gene signatures to that of orthotopic and spontaneous mice models of pancreatic cancer cachexia. We observed significant alterations in multiple genes related to oxidative stress in muscle tissues from human patients with cachexia and mice cachexia models. We targeted oxidative stress regulatory genes by genetic manipulations or targeted oxidative stress with pharmacological approaches to evaluate the impact of oxidative stress in skeletal muscles on cancer-associated cachexia. Our results suggest that targeting the oxidative stress pathway prevents/reverts cancer-associated cachexia. Moreover, inhibition of oxidative stress also abolished cancer cell-conditioned medium-induced atrophy in C2C12-derived myotubes and diminished the expression of atrophy markers. Overall, our studies revealed the potential role of oxidative stress induced by cancer cells in muscle wasting. Future studies to target tumor-induced oxidative stress in muscle fibers may provide new therapeutic approaches to prevent cancer-associated cachexia.

#3459

PKD1 **regulates susceptibility to ulcerative colitis and colorectal cancer.**

Anna S. Nikonova, Anna Kiseleva, Ilya Serebriiskii, Sergei Grivennikov, Erica A. Golemis. _Fox Case Cancer Center, Philadelphia, PA_.

Compromised integrity of the epithelial barrier function of colon tissue associated with inflammatory bowel diseases (IBDs) or arising from tumor-elicited inflammation (TEI) are associated with and support formation of colorectal cancer (CRC). Apical tight junction (TJ) proteins, including multiple specific claudins, are critical in regulating TJ barrier function and paracellular permeability. Changes in TJ composition in the kidney are strongly linked to the etiology of autosomal dominant polycystic kidney disease (ADPKD), with ADPKD-inducing mutations in PKD1 causing non-leaky barriers that can withstand high hydrostatic pressure within renal cysts a hallmark of this common (1 in 500) inherited disease. Intriguingly, a large population study has found a decreased incidence of CRC in ADPKD patients. Based on these and other suggestive data, we hypothesized that loss of PKD1 would hinder development of ulcerative colitis and initiation of colitis associated cancer, based on reorganizing TJs and TEI in the colon. To evaluate the impact of Pkd1 loss on colon barrier function, we treated 10-12 week old wt or Pkd1fl/fl mice with tamoxifen-induced, Cre expression from the CreERT2 cassette for 5 days with 2.5% DSS in drinking water to induce acute colitis, compared to a no-DSS control group. For the wt cohort, treatment with DSS increased orally gavaged FITC-dextran detectable in serum versus controls. In contrast, no Pkd1-/- mice showed a similar DSS-dependent response to FITC-dextran, suggesting reinforced colon barrier function; further, histopathological assessment confirmed less DSS-induced damage in Pkd1fl/fl mice. Further, based on immunofluorescence (IF) analysis of tissue sections, claudins 4 and 7 associated with increased barrier function) strongly elevated in the colonic epithelium of Pkd1fl/fl versus wt mice, with the expressed proteins having consistently greater localization to cell junctions in Pkd1fl/fl versus wt mice. We also investigated the formation of CRC tumors, using a CDX2-ERT2/Cre model for tamoxifen-induced loss of Apc and/or Pkd1 in the colon to compare tumorigenesis in the Apcfl/fl, Apcfl/fl Pkd1fl/fl, and Apcfl/flPkd1fl/+ genotypes. We observed a highly significant progressive increase in the number and size of Apc mutation-induced tumors based on retention of the Pkd1 gene. Preliminary qRT-PCR analysis of these tumors shows that a Pkd1fl/fl genotype substantially increases CLDN4 in normal and tumor tissue, and decreases TNFα expression in tumors, suggesting reduced inflammation. This work and extended mechanistic characterization identifies PKD1 as a vital regulator of signaling systems already associated with colitis and CRC. Understanding the role of PKD1 and its effectors may provide useful information to genetic counselors assessing risk of IBD and CRC, and suggest therapeutic strategies.

### Deregulated Tumor Suppressors and Carcinogenesis

#3460

MPRIP-ALK, **a novel ALK rearrangement that responds to ALK inhibitor in non-small-cell lung cancer.**

Wenfeng Fang,1 Jiadi Gan,1 Feng Lu,2 Yangyang Deng,3 Liang Chen,2 Yunpeng Yang,1 Li Zhang1. 1 _Sun Yat-sen University Cancer Center, Guangzhou, China;_ 2 _Jinan University, Guangzhou, China;_ 3 _MyGene Diagnostics, Guangzhou, China_.

Oncogenic rearrangements of the anaplastic lymphoma receptor tyrosine kinase (ALK gene) have recently been described in 3% to 5% of lung adenocarcinomas. Kinds of fusion partners of ALK have been reported in non-small-cell lung cancer (NSCLC), with echinoderm microtubule-associated protein-like 4 (EML4)-ALK being the most prevalent one. Several tyrosine kinase inhibitors have been approved for treating patients with advanced-stage ALK-positive, NSCLC by Food and Drugs Administration (FDA).

Comprehensive genomic profiling performed by means of next-generation sequencing assay demonstrated a novel myosin phosphatase-Rho-interacting protein gene (MPRIP)-ALK fusion in our case, in which a translocation involving chromosomes 2 and 9 has taken place. The rearrangement was verified by reverse transcription reverse transcription polymerase chain reaction (RT-PCR) and Sanger sequencing. ALK immunohistochemistry demonstrated strong staining in patient's tumor sample as extracted from formalin-fixed paraffin-embedded (FFPE) sections.

We expressed MPRIP-ALK cDNA construct in non-cancer cell line Ba/F3 which showed IL-3-independent growth compared the growth stop of cells carrying empty vector, moreover, hyper-activation of the protein ALK, ERK, AKT were detected in Ba/F3 cells with MPRIP-ALK over-expressed. Furthermore, the colonies and activations of ALK downstream signaling pathways could be inhibited by crizotinib. The patient received standard chemotherapy on the first-line. She experienced progressive disease after 7 months, and commenced on crizotinib (250mg, twice per day) from March 2018. The patient showed significant improvement in cough, dorsalgia, and polypnea within one week. Partial response was gained after one month treatment and confirmed at the third month. She still remains on the crizotinib treatment without disease relapse up to now. In this study, we report a novel ALK rearrangement in a patient with NSCLC by RNA-seq. Our in vivo and in vitro studies have shown that the novel MPRIP-ALK fusion is a driver gene and can be targetable by crizotinib.

#3461

Characterizing the PTEN - p85alpha interaction.

Jeremy Marshall,1 Paul Mellor,1 Xuan Ruan,1 Dielle Whitecross,1 Stanley Moore,1 Deborah Anderson2. 1 _University of Saskatchewan, Saskatoon, Saskatchewan, Canada;_ 2 _Saskatchewan Cancer Agency, Saskatoon, Saskatchewan, Canada_.

Introduction: The phosphatidylinositol 3-kinase (PI3K) pathway plays a key role in regulating cell growth and cell survival and is frequently deregulated in cancer cells. p85α regulates the p110α lipid kinase, and also stabilizes and stimulates PTEN, the lipid phosphatase that downregulates this pathway. We set out to identify residues in both PTEN and p85α that mediate their interaction to better understand the regions important in mediating binding.

Experimental Procedures: We previously showed that the BH domain of p85α is sufficient to mediate binding to PTEN. In this work, a deletion analysis and point mutations were used to mutate each of PTEN and the p85α BH domain to identify residues important for binding, determined using a pull-down analysis. Mutations in the p85α BH domain and in PTEN that reduced their interaction were then used as input data and docking software was used to model possible interaction interfaces for the two the proteins. Further mutagenesis and follow-up binding experiments provided support for our model of the PTEN - p85α BH domain complex.

Results: We identified key residues responsible for mediating PTEN - p85α complex formation. Based on these experimental results, a docking model for the PTEN - p85α BH domain complex was developed that is consistent with the known binding interactions for both PTEN and p85α. This model involves extensive side-chain and peptide backbone contacts between both the PASE (R84, Q87, Y88, E91, E99) and C2 (R189, P190, Q219, C250, D252) domains of PTEN and the p85α BH domains with a buried surface area of 1211 Å2 (PTEN - bovine p85α BH) and 1366 Å2 (PTEN - human p85α BH). The p85α BH domain residues that directly contact PTEN in the two docking models were not identical, however both models implicated p85α residues E212, Q221, K225, R228, H234 and W237. The majority of these p85α BH domain residues were confirmed experimentally as important for PTEN binding. We also verified experimentally the importance of PTEN-E91 in mediating interaction with the p85α BH domain.

Conclusions: These results shed new light on the mechanism of PTEN binding and regulation by p85α.

#3462

The F-box protein FBXL16 regulates the stability of c-myc oncoprotein.

Marion Morel, Weiwen Long. _Wright State University, Dayton, OH_.

F-box proteins are major components of the SCF (SKP1-CUL1-F-box) E3 ubiquitin ligases as they are responsible for substrate recognition. In this complex, F-box proteins bind to SKP1 through the F-box motif to bring the ubiquitination machinery and mediate protein ubiquitination of the substrates. So far, 69 F-box proteins are identified in humans, and they fall into 3 families depending on their substrate recognition domains: FBXLs (Leucine-Rich Repeats or LRR), FBXWs (WD repeats) and FBXOs (Other domains). FBXL16 is a poorly studied F-box protein which consists of an N-terminal Proline-rich domain, an F-box motif and a C-terminal domain of Leucine-rich repeats. FBXL16 was first identified as a transcriptional target of E2F1 (Sato et al, 2010) and was then showed as a binding partner of PP2A (Protein Phosphatase 2A) and as a regulator of its phosphatase activity (Honarpour et al, 2014). Interestingly, FBXL16 was shown to be overexpressed in a number of cancers, particularly invasive breast carcinoma (Oncomine) indicating that FBXL16 may play important roles in cancers. In this study, by both knockdown and overexpression experiments, we found that FBXL16 promotes cancer cell migration. To our surprise, we observed that knockdown of FBXL16 in cancer cells resulted in a strong decrease of c-myc protein level. Importantly, we also found that FBXL16 binds to and stabilizes c-myc protein. Mechanistically, FBXL16 overexpression decreased c-myc protein ubiquitination. Taken together, our study demonstrates a positive role of FBXL16 in regulating c-myc protein stability by inhibiting its ubiquitination.

#3463

**Use of AACR Project GENIE, a clinicogenomic registry, to define the natural history of** AKT1 **E17KMutant ER+/HER2- metastatic breast cancer (MBC).**

Lillian M. Smyth,1 Quin C. Zhou,1 Celeste Yu,2 Eva Lepisto,3 Monica Arnedos,4 Michael Hasset,3 Michele L. Lenoue-Newton,5 Natalie Blauvelt,1 Semih Dogan,4 Christine Micheel,5 Chetna Wathoo,6 Hugo Horlings,7 Jan Hudecek,7 JJ Gao,1 Nikolaus Schultz,1 Andrew Zarski,1 Jocelyn Lee,1 Seth Sheffler-Collins,8 Ben H. Park,5 Charles Sawyers,1 Fabrice Andre,4 Mia Levy,5 Funda Meric-Bernstam,6 Philippe Bedard,2 Alexia Lasonos,1 Deborah Schrag,3 David Hyman,1 GENIE Consortium8. 1 _MSKCC, New York, NY;_ 2 _Princess Margaret Cancer Centre, Toronto, Ontario, Canada;_ 3 _DFCI, Boston, MA;_ 4 _Institut Gustave Roussy, Villejuif, France;_ 5 _Vanderbilt Ingram Cancer Center, Nashville, TN;_ 6 _MD Anderson Cancer Center, Houston, TX;_ 7 _Netherlands Cancer Institute (NKI), Amsterdam, Netherlands;_ 8 _AACR, Philadelphia, PA_.

Background: AKT1 E17K mutations are oncogenic events in ~4% of ER+ breast cancer (BC). AKT inhibitors (AKTi) have promising clinical activity in ER+ AKT1 E17K mutant (mut) MBC. Understanding the prognosis of AKT1 E17K mut BC is critical to development & regulatory review of therapies (tx) for pts harboring this rare mutation.

Methods: GENIE assembled AKT1 E17K mut cases (AKT+) & matched controls (AKT-) with ER+/HER2- MBC. Clinicopathological features, response to standard tx, clinical outcomes, & broader genomic profiles were evaluated. Controls were matched 2:1 with cases at the institutional level by year of genomic sequencing, birth year & histology type. Primary endpoint was overall survival (OS) from date of distant metastatic disease. Secondary endpoints included duration of treatment (DOT). OS estimates accounted for left truncation & censored surviving AKT+ patients (pts) who received an AKTi at the start of this tx.

Results: 455 pts (153 cases, 302 controls) were analyzed from 6 international centers. Cohorts were balanced by age at & year of diagnosis; race; stage; grade; & distribution of metastatic disease, with the exception of more frequent liver & lymph node metastases in AKT+ pts. Cohorts were balanced for primary & metastatic disease tx. Median prior lines of tx for metastatic disease was 4 (2 endocrine tx; 2 cytotoxic tx; ~30% with CDK4/6 & mTOR inhibitor exposure. Median follow-up was 35.8 months (mths). There was no significant difference in adjusted median OS of AKT+ cases vs AKT- controls (24.9 vs 29.9 mths, HR=1.01, 95% CI: 0.75-1.37, p=0.93). DOT comparisons & genomic enrichment analysis will be presented.

Conclusions: AKT1 E17K mut MBCs have similar clinicopathologic features & survival outcomes to AKT1 E17K WT tumors. GENIE can expeditiously define the natural history of rare genomic subpopulations, with implications for drug development in these genomically orphaned diseases.

Table: OS by AKT1 E17K Mutational Status | |

---|---|---

|

AKT1 Mut | AKT1 WT

Deaths | 62 | 144

Median OS, mths (95%CI) | 24.9 (18.4-35.7) | 29.9 (25.4-33.8)

3-Yr OS, % (95%CI) | 37.4 (26.1-48.7) | 39.9 (32-47.8)

5-Yr OS, % (95%CI) | 19.1 (10.3-30) | 17.9 (12.4-24.1)

#3464

CBAP: A novel rheostat molecule for regulation of TSC GAP activity and mTORC1 signaling in cancer cells.

Jong-Young J. Yen,1 Yun-Jung Chiang,1 Wei-Ting Liao,1 Shih-Hao Wang,1 Hsin-Fang Yang-Yen2. 1 _Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan;_ 2 _Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan_.

The PI3K-mTOR signaling pathway is one of the most frequently dysregulated signaling cascades in cancer. Understanding the molecular wiring of the PI3K-mTOR signaling network and its activation in cancer will improve our understanding of their contribution to cancer pathology and provide novel therapeutic strategies by targeting this network. Our previous works demonstrated that Common receptor Beta chain Associated Protein (CBAP) is required for Jurkat cell leukemogenesis and regulates Akt-dependent TSC2 phosphorylation and lysosomal dissociation of TSC complexes, and in turn enhances mTORC1 signalling and expression of c-MYC and HIF-1a in Jurkat T-ALL leukemic cells. In this report, we tackle the question how TSC/Rheb/mTORC1 pathway is regulated by CBAP via cell-based functional assay, immunoprecipitation and in vitro GTPase assay. Our data revealed that CBAP interacted with TSC2 via tuberin-binding domain and could compete for TSC2 binding with TSC1 and suppressed the GTPase activation activity of TSC1/2 complexes. Akt could also associate with TSC2 complexes and CBAP in both Jurkat and CBAP-knockout Jurkat cells, except that TSC1 could be detected within the complexes only in the absence of CBAP. Moreover, the Rheb-GAP activity of this Akt-associated TSC2 complexes was sensitive to the presence of CBAP and TSC1/2 GAP activity was strongly suppressed by recombinant CBAP proteins in the in vitro reconstituted assay. Finally, a peptide domain of CBAP interacting with TSC2 was mapped and disruption of CBAP-TSC2 interaction by overexpressing this peptide inhibits the Akt/Rheb signal axis and suppresses leukemia cell growth. In summary, our data revealed an important role of the interaction between CBAP and TSC2 proteins in promoting Rheb/mTORC1 signaling activity, and suggested a crucial role of overexpression of CBAP protein in tumor cell proliferation.

#3465

FANCD2 depletion suppresses tumor growth in esophageal squamous cell carcinoma.

Chan Lei,1 Zhuoyou Yu,1 Lvwen Ning,1 Mun-Yee Ko,1 Lidong Wang,2 Maria Li Lung1. 1 _The University of Hong Kong, China;_ 2 _Zhengzhou University, Zhengzhou, China_.

Introduction: Esophageal squamous cell carcinoma (ESCC) has a remarkably high incidence in Northern China. Our next-generation sequencing analysis on familial ESCC samples suggests that members of the Fanconi Anemia (FA) pathway play important roles in ESCC development. Fanconi anemia complementation group D2 (FANCD2) as a key player of FA pathway has multiple cellular functions. It is well-known for its role in DNA damage repair through the FA pathway. Patients with germline FANCD2 mutations are prone to tumor formation. Identifying roles of FANCD2 in ESCC development may enhance our foundation for future development of specific treatment regimens for this deadly disease.

Methods and Results: To study the function of FANCD2 in ESCC, we applied the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technique to inhibit FANCD2 protein expression by functional knock-out (fKO). As expected, we observed increased DNA damage in FANCD2 fKO cells as shown by Comet assay, consistent with the classic role of FANCD2 in genome integrity. We also observed that the DNA damage by irradiation takes longer to be repaired in FANCD2 fKO cells, indicating that FANCD2 enhances irradiation-induced DNA damage repair but is not necessary. Similarly, cells with FANCD2 fKO are more sensitive to irradiation, as indicated by cell cycle arrest at S/G2 phase.

Surprisingly, without inducing any DNA damage, FANCD2 fKO cells also show cell cycle arrest at S/G2 phase. The MTT cell proliferation assay also show that FANCD2 fKO reduces proliferation and colony-forming ability. Consistent with the in vitro data, FANCD2 fKO cells also form smaller tumors in vivo by the nude mouse tumorigenicity assay.

Conclusion: These results indicate that FANCD2 plays important roles in ESCC development and suggest a pro-oncogenic feature of wildtype FANCD2 in ESCC. Further investigation is ongoing to examine the functional role and molecular mechanisms of the oncogenic FANCD2, ultimately aiming at improving ESCC disease management.

Acknowledgement: We acknowledge the grant support from the Hong Kong Research Grants Council Collaborative Research Fund (C7031.15G) and the Asian Fund for Cancer Research to M.L.L.

#3466

The oncogenic roles of ANXA1 in gastrointestinal tract cancer.

Motonobu Saito. _Fukushima Medical University, Fukushima, Japan_.

Annexin A1 (ANXA1) is a calcium-dependent phospholipid-linked protein that is associated with anti-inflammatory effects, regulation of cellular proliferation, and apoptosis and, therefore, ANXA1 is associated with cancer development and metastasis. We have previously reported that ANXA1 was upregulated in gastrointestinal cancer and associated with venous invasion (P=0.023) and lymph node metastasis (P=0.042). We also performed in vitro cell experiments and revealed that elevated ANXA1 expression was induced resistance to 5-FU in the colon cancer cells. So next, we have evaluated the association between ANXA1 and cancer related genes, including p53 by immunohistochemical (IHC) staining in two independent gastric cancer cohorts (n=200 and n=220). The percentage of positive ANXA1 was significantly higher in p53 positive tumors compared with p53 negative tumors (P=0.023 and P=0.022, respectively). We also evaluated the association with ARID1A, the chromatin remodeler AT-Rich interactive 1A, that is frequently mutated in gastric cancer and is considered as a candidate therapeutic target by synthetic lethality. Although it was reported that the expression of ANXA1 was upregulated in ARID1A mutated breast cancer cases, significant association between ANXA1 and ARID1A expressions was not found in gastric cancer. We next performed IHC staining for ANXA1 and p53 in three human gastric cancer cell lines (MKN7, NUGU4, KATOIII). Positive staining for both ANXA1 and p53 was detected in cells with TP53 missense mutation. On the other hand, weak positive staining for ANXA1 and negative staining for p53 was found in NUGC4 cells with TP53 wild-type and KATOIII cells with TP53 truncating mutation. These results suggested that ANXA1 expression may be induced by aberrant p53 protein, resulting cancer progression and resistance to 5-FU. Our results also provide a possible strategy to overcome 5-FU resistance by modulating ANXA1 expression.

#3467

Inhibition of AURKA targets gastrointestinal cancer cells with activated KRAS by inhibiting RPS6KB1.

Zheng Chen,1 Lihong W. Bishop,2 Ahmed Gomaa,1 Albert C. Lockhart,1 Safia Salaria,3 Jeffrey Ecsedy,4 Kay Washington,3 Robert D. Beauchamp,3 Wael El-Rifai1. 1 _University of Miami, Miami, FL;_ 2 _Vanderbilt University, Nashville, TN;_ 3 _Vanderbilt University Medical Center, Nashville, TN;_ 4 _Takeda Pharmaceuticals International Co, Cambridge, MA_.

Background & Aims: Amplifications and mutations of KRAS play essential roles in resistance to chemotherapeutics in luminal gastrointestinal cancers. We investigated the regulation of ribosomal protein S6 kinase B1 (RPS6KB1) by AURKA and the effects of knockdown AURKA or its inhibitions using alisertib, an AURKA inhibitor, in human gastrointestinal cancer cells with amplified or mutant activated forms of KRAS.

Methods: We performed CellTiter-Glo luminescence and clonogenic cell survival assays using 10 upper gastrointestinal or colon cancer cell lines with KRAS mutations or amplifications to test the effects of alisertib, AURKA overexpression or knock down. To determine protein co-localization, we used proximity ligation in situ and immunoprecipitation assays. Nude mice with xenograft tumors grown from HCT116, SNU-601, SW480, or SNU-1 cells were given oral alisertib (40 mg/kg, 5 times/week) for 4 weeks. Tumor samples were collected and analyzed by immunoblots and immunohistochemistry. In addition, immunohistochemistry was performed using AURKA antibody on tissue microarrays containing 151 paraffin-embedded human colon tumors with adjacent normal and adenomas.

Results: AURKA knockdown or inhibition with alisertib reduced Alisertib reduced proliferation and survival of cell lines tested. In addition, we detected a decrease in the levels of phosphorylated RPS6KB1 (at T389), and increased levels of proteins that induce apoptosis including BIM, cleaved PARP, and cleaved caspase 3. Proximity ligation assay and immunoprecipitation indicated that AURKA co-localized and interacted with RPS6KB1, mediating RPS6KB1 phosphorylation at T389. We detected AURKA-dependent phosphorylation of RPS6KB1 in cell lines with mutations in KRAS, but not in cells with wild-type Ras. Administration of alisertib to mice with xenograft tumors significantly reduced tumor volumes (P < .001). These effects were associated with reduced phosphorylation of RPS6KB1 and Ki-67, and increased levels of cleaved caspase 3, in tumor xenograft tissues. The tissue microarrays demonstrated significant overexpression of AURKA in gastrointestinal tumor tissues compared with non-tumor tissues (P=.0003).

Conclusion: Our studies indicate that AURKA can phosphorylate RPS6KB1 in cancer cells with activated KRAS to promote cell proliferation and survival and growth of xenograft tumors in mice. AURKA inhibitors such as alisertib might slow the growth of gastrointestinal tumors with activation of KRAS.

#3468

Aberrant activation of gli1 and notch1 contributes to racial disparity in triple negative breast cancer progression.

Sumit Siddharth, Nethaji Muniraj, Sheetal Parida, Arumugam Nagalingam, Dipali Sharma. _Johns Hopkins School of Medicine, Baltimore, MD_.

Background and Aim: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by the absence of estrogen receptor, progesterone receptor and HER2 expression. Mortality from TNBC is significantly higher in African American (AA) women compared to European American (EA) women (5-year relative survival of 14% for AA compared to 36% for EA). Irrespective of stage at diagnosis, AA-TNBC is aggressive with higher metastasis and poorer survival than EA-TNBC; hence, it is imperative to understand the molecular determinants that drive aggressive progression of AA-TNBC. Overall this study aims to decipher the alterations in the molecular circuitry underlying racial disparity in TNBC progression.

Results: AA-TNBC cells (HCC1806 and HCC1569) exhibited increased growth and higher migration potential in comparison to EA-TNBC (Hs578t, BT549, HCC1937 and HCC1187) cells. AA-TNBC cells also exhibited higher expression of stemness factors, increased number of mammospheres and higher CD44+/CD49f+ population. To decipher the molecular mechanism underlying these functional differences, we analyzed RNA sequencing data of multiple AA and EA TNBC cell lines for self-renewal pathways and observed significantly higher levels of GLI1 in AA-TNBC cell lines while no significant alterations were observed in other pathway components. Further analysis of TCGA dataset revealed a positive correlation between GLI1 and Notch1 in AA-TNBC (Pearson coefficient = 0.308303) with a negative correlation in EA-TNBC (Pearson coefficient = -0.06695). Immunoblot analyses showed increased expression of components of GLI1 and Notch1 pathway (SHH, Jagged, NICD, Hes1 and FOXM1) in AA-TNBC compared to EA-TNBC cells. AA-TNBC cells showed increased nuclear localization of GLI1 and NICD as compared to EA-TNBC cells. We observed that GLI and NICD co-localize in AA-TNBC cells and their interaction was confirmed using co-immunoprecipitation assays. High expression of GLI1 and Notch1 correlated with poor overall survival in TNBC patients. Concomitant inhibition of GLI1 and Notch1 using respective small molecule inhibitors, GANT61 and DAPT, along with standard chemotherapeutic agents (Doxorubicin and Carboplatin) effectively inhibited AA TNBC growth and progression in mice; proliferation, migration and invasion of ex vivo tumor cells and downregulated CD44+/CD49f+ and ALDH1+ population in a synergistic manner. Combined treatment with GANT61+ DAPT+ Carboplatin effectively reduced stem cell frequency of AA-TNBC tumors in in vivo limiting dilution assay.

Conclusions: In conclusion, these results show that AA-TNBC cells are inherently aggressive with increased growth, migration and stemness potential. We found aberrant activation of GLI and Notch pathway and a crosstalk between GLI1 and NICD whose inhibition effectively inhibits AA-TNBC and sensitizes AA-TNBC to standard chemotherapy.

#3469

CYP1B1 induces cancer progression through regulation of TRAIL pathway and uPA-uPAR system.

Yeo-Jung Kwon, Young-Jin Chun. _Chung-Ang University, Seoul, Republic of Korea_.

Human CYP1B1 is known as a major metabolizer for estrogen and shows tumor-specific hyper-expression. To explore the role of CYP1B1 on progression of human cancer cells, we studied the effects of CYP1B1 in MCF-7, MDA-MB-231, and HeLa cells. CYP1B1 significantly induced invasion of cancer cells while strongly inhibited cancer cell apoptosis. Suppression of CYP1B1 by si/shRNA or treatment with TMS, a specific inhibitor for CYP1B1, markedly increased the protein expression level along with the level of release from cells of TNF-related apoptosis inducing ligand (TRAIL), a death ligand for cancer cells, whereas significantly decreased the expression of urokinase-type plasminogen activator receptor (uPAR) as well as uPA. We also found that CYP1B1 showed to inhibit the expression of key factors in TRAIL-induced apoptotic pathway and promote the inducers in uPA-uPAR metastasis system including integrin β1 and α5. Interestingly, uPAR overexpression only caused a significant increase of integrin β1 and α5 in protein levels, indicating protein degradation may play an important role in regulating integrin protein level. Surprisingly, CYP1B1 down-regulated p53 expression through MDM2 activation and nutlin-3a, an inhibitor for MDM2, blocked the promoting effects of CYP1B1 inducer, DMBA, on uPAR, which explains that CYP1B1 induced uPAR expression through regulation of MDM2-p53 system. Furthermore, we found that CYP1B1 suppresses TRAIL-related apoptosis through not only inhibition of TRAIL expression but also induction of DNA methylation on the promoter region of DR4, a receptor for TRAIL. Taken together, our data suggest that CYP1B1 promotes cancer cell metastasis via activating uPA-uPAR pathway which is one of the targets of p53 signaling and suppresses cancer cell apoptosis through inhibition of TRAIL-induced apoptosis by suppression of TRAIL release as well as post-translational regulation on DR4.

#3470

**The antagonistic functional duality of p21** WAF1/CIP1 **in cells derived from multicellular tumor spheroids.**

Viswanath Das, Narendran Annadurai, Dušan Holub, Marián Hajdúch. _Institute of Molecular and Translational Medicine, Olomouc, Czech Republic_.

The cyclin-dependent kinase inhibitor p21WAF1/CIP1 (p21) is a key mediator of p53-dependent cell cycle arrest after DNA damage, in addition to p53-independent mechanisms. Being one of the major transcriptional targets of p53 and due to its anti-cell proliferative activity, p21 was considered initially as a potent tumor suppressor. However, emerging studies now show the anti-apoptotic and pro-cell proliferative effects of p21, highlighting the oncogenic role of p21 in cancer. In particular, p21 results in genomic instability and the development of aggressive and chemo-resistant traits in a subset of highly proliferating tumor cells through p53-independent pathways. The cellular localization of p21 has been proposed to be critical either in promoting cell survival or in inhibiting cell growth. Herein, using a combination of gene knockout, cytotoxicity assay, immunofluorescence, immunoprecipitation, and mass spectrometry techniques, we study the consequence of nuclear and cytoplasmic localization of p21 on cell survival and drug response in cells derived from multicellular spheroids of multiple human cancer cell lines. Our study shows that cells derived from multicellular spheroids show an increased induction of p21 despite the reversal from three-dimensional to two-dimensional cultures. The p21-overexpressing, highly proliferative cells are significantly resistant to a number of standard anti-cancer agents. Further analysis shows that the majority of p21 in this subset of spheroid-derived cells is localized in the cytoplasm and forms a potential 'anti-apoptosome'-like complex with mitochondrial apoptosis-associated proteins. These findings add to the shifting paradigm on the oncogenic role p21 due to cellular mislocalization in tumor cells.

#3471

Mutual regulation of MDM4 and TOP2A in cancer cell proliferation.

Tao Liu, Hailong Zhang, Sha Yi, Lubing Gu, Muxiang Zhou. _Emory Univ. School of Medicine, Atlanta, GA_.

MDM4 and topoisomerase IIα (TOP2A) are important cancer-related factors. MDM4 acts as an oncoprotein promoting cancer progression by inhibiting tumor suppressor p53. As a DNA replication- and cell division-regulating enzyme, TOP2A is the main target of many anticancer therapy regimens, even though the exact role of TOP2A in cancer remains elusive. Herein we report that MDM4 and TOP2A bind to each other and are mutually upregulated at the post-translational level, leading to enhanced TOP2A catalytic activity, inhibition of p53, and increased tumor-cell proliferation. We demonstrate that the C-terminal region (CTR) of TOP2A binds to a unique sequence (residues 188-238) of MDM4, which contains an auto-inhibitory segment regulating MDM4-p53 interaction. TOP2A binding in turn activates MDM4 for p53 binding, resulting in enhanced inhibition of p53 and cancer cell proliferation. Conversely, binding of the MDM4 sequence to the CTR of TOP2A upregulates TOP2A protein expression. This in turn interferes with TOP2A DNA-decatenating activity. These results reveal novel functions of MDM4 and TOP2A as well as their interactions in oncogenesis, suggesting that inhibition of the MDM4-TOP2A interaction may represent a novel strategy in specifically and simultaneously targeting TOP2A and MDM4 for cancer treatment.

#3472

The role of mutant p53 R248W in oral squamous cell carcinoma.

Mayu Enaka, Masako Nakanishi, Yasuteru Muragaki. _Wakayama Medical University of Medicine, Wakayama City, Japan_.

TP53 is the most commonly mutated gene in oral squamous cell carcinoma (OSCC). It is known that mutant p53 has not only loss of function but gain of function. Cytokeratin 17 (K17) has been shown to promote tumorigenesis and aggressiveness in OSCCs and other various carcinomas. Although p53 reportedly induces K17 transcription by irradiation, the molecular interaction between p53 and K17 in OSCCs remains unknown. To address this issue, we investigated the relationship between p53 and K17 using SAS cells with nonsense mutation and Ca9-22 cells with R248W mutation in the TP53 gene. Western blot and immunohistochemistry showed higher K17 expression in SAS cells and virtually no K17 expression in Ca9-22 cells, suggesting negative correlation between p53 and K17 expression. ChIP analysis revealed that p53 bound specifically to the tentative p53 binding site in the promoter region of the K17 gene. Unexpectedly, however, overexpression of wild-type (wt) p53 did not suppress K17 expression in SAS cells, whereas knock down of mutant (mt) p53 significantly upregulated K17 expression in Ca9-22 cells, raising a possibility that only R248W mtp53 suppresses K17 expression. Furthermore, overexpression of wtp53 and R248Q mtp53 did not suppressed K17 expression in Ca9-22 cells with knock down of p53, whereas R248W mtp53 overexpression clearly showed suppression of K17. Taken together, R248W mtp53 acts as a direct transcriptional repressor of K17 in OSCCs, reducing aggressiveness of the carcinoma cells.

#3473

Comprehensive genetic screening in hereditary colorectal cancer.

Anna Rohlin,1 Frida Eiengård,1 Emma Mårtensson,1 Theofanis Zagoras,1 Samuel Gebre-Medhin,2 Margareta Nordling1. 1 _Biomedicine, Gothenburg, Sweden;_ 2 _Division of Clinical Genetics, Lund, Sweden_.

High-penetrant pathogenic variants in established genes associated with hereditary colorectal cancer (CRC) explain the disease in approximately 5-6% of cases. In as much as 20%-30% of all, including sporadic CRC cases, genetic factors are thought to play a significant role. Dependent on technical as well as limitations due to interpretation challenges, the high-penetrant pathogenic variants are gathered in the exonic regions of the genes. Contributions of pathogenic variants in genes, not traditionally associated with hereditary CRC have also recently been recognized. It is crucial for the patients to get a correct molecular diagnosis that allows for adequate follow-up since the majority of the syndromes include predisposition also for tumors in other organs. A comprehensive panel including high-penetrant susceptibility genes as well as genes not generally associated with hereditary CRC was used for the analyses. Sequencing of 50 kb upstream and downstream of all genes including intronic regions were performed. All SNVs and CNVs in exons were thoroughly evaluated concerning pathogenicity and also intronic and intragenic sequences were screened for the presence of possibly pathogenic variants. In addition, cDNA were analysed to detect variants in splice-sites as well as aberrations causing other effects on mRNA level. The study included 206 patients referred to the Cancer Genetics Counselling Clinic in Gothenburg, Sweden. The spectra of pathogenic variants in well-known high-penetrance predisposition genes as well as the contribution of pathogenic variants in low-penetrant genes were determined. The contribution of pathogenic variants also in "non-CRC" genes was examined. The findings include identification of previously unreported variants and putative causative variants in unexplained patients with hereditary CRC. An attempt to identify putative genetic variants that could explain the variability in cancer risk among carriers of specific variants in high-penetrance genes was also made.

#3474

Loss of function mutations in APOB and their clinical significance in hepatocellular carcinoma.

Gena Lee,1 Yun Seong Jeong,2 Min Jun Kwak,3 Kim DoWon,3 Ju-Seog Lee,2 Yim Sun Young4. 1 _Shadow Creek High School, Pearland, TX;_ 2 _UT MD Anderson Cancer Ctr., Houston, TX;_ 3 _University of Texas at Austin, Houston, TX;_ 4 _Korea University, Seoul, Republic of Korea_.

APOB is major lipid binding proteins of low-density lipoprotein (LDL) particles and responsible for carrying fat molecules to cells within all tissues. High levels of APOB are known to be related to heart disease and vascular disease. Interestingly, recent The Cancer Genome Atlas (TCGA) study revealed that APOB is one of frequently mutated genes in multiple cancers including melanoma, liver cancer, stomach, esophageal, head and neck, uterine, and lung cancers (from >20 to 10%). Majority mutations are considered to cause loss of function as they are truncation mutations, suggesting that APOB might have tumor suppressive activity. However, because of its physiological roles in circulating fat molecules in our body, APOB has never been studied in cancer-related setting and thus its clinical relevance or biological roles of APOB in cancer development is currently unknown. Here we show that loss of APOB in hepatocellular carcinoma (HCC) is significantly associated with poor survival of HCC patients by applying comparative genomics approach that integrate genomic data from mouse and human HCC tumors. We further show that loss of APOB leads to shifting balance of lipid metabolisms favoring for tumor growth.

For development of genomic signature reflecting hepatic APOB activity and test and validation of its association with prognosis, we used unsupervised approach combined with supervised prediction models by integrating gene expression data from mouse models and human HCC. When HCC patients were stratified into APOB-active and APOB-inactive subgroups, overall survival (OS) rate and recurrence free survival (RFS) rate of patients in APOB-inactive group is significantly lower than those in APOB-active (P < 0.001), indicating that APOB activity in HCC is significantly associated with prognosis of patients. This association was validated in 4 independent cohorts of HCC patients (n=88, 240, 242, and 371 in total of 941 patients). Gene network analysis revealed that many of the regulators involved in cell growth were activated in Apob-silenced livers. Many of the upregulated genes in Apob-silenced livers are direct downstream targets of Erbb2, Vegfa, and regulator of metastasis and cancer initiation cells Cd44. Consistent with activation of many tumor-promoting regulators, Tp53, a major tumor suppressor, was inactivated in Apob-silenced livers, suggesting that loss of Apob in the liver may provoke initiation of tumor development. More interestingly, proliferation of three liver cancer cells was significantly increased when expression of APOB was silenced by siRNAs, further suggesting functional roles of APOB in tumor development. For the first time, we showed that APOB inactivation has a clinical impact in HCC patients with significant alteration in regulators associated with tumorigenesis.

#3475

Suppression of inflammatory signaling by HOXA5 via modulation of the NF-KB pathway in breast cancer.

Priya Pai, Guannan Wang, Diana Ráez Rodríguez, Wayne Yu, Saraswati Sukumar. _Johns Hopkins University, Baltimore, MD_.

Background and Aims: HOXA5 is a tumor suppressor in breast cancer (BC) and transcriptionally regulates E-cadherin, CD24, progesterone receptor and p53. The goal of this study was to establish whether the loss of HOXA5 cooperates with common tumor suppressor genes and oncogenes in isogenic untransformed breast epithelial MCF10A sublines to induce tumorigenesis and to determine the mechanism thereof.

Materials and Methods: Isogenic MCF10A sublines with a single knock in mutation in p53 (R248W), or double knock in mutations in both HER2 (V777L) and PIK3CA (E545) generated by Cre recombinase mediated excision/insertion were used. Sanger sequencing was used to confirm genotype. Immunoblotting and qPCR was used to analyze gene expression. All-trans Retinal- ATAL (Sigma #R2500) (1 μM for 7 days) was used. For xenograft studies, 2 X 106 cells in 80% growth factor reduced Matrigel and 20% PBS were implanted subcutaneously. Agilent Human v2, 4x 44K Expression, two-color expression array was used (Biological replicates, n=3). Differentially expressed probes were sorted by p value (<0.05) and fold change (1.5 or greater).

Results: Stable knock down (KD) of HOXA5 using shRNAs in the V777L DKI cell line (HER2 V777L and PIK3CA E545K) caused epithelial to mesenchymal transition (EMT): spindle-like morphology with attendant alterations in N-cadherin, Slug, Vimentin, E-cadherin, increased invasion and migration. Overexpression of HOXA5 in the p53 R248W cell line resulted in an opposing phenotype. ATAL treatment of p53 R248W restored expression of HOXA5 and its targets. V777L DKI-HOXA5 knock down cells formed pre-invasive outgrowths upon subcutaneous injection in NSG mice. Expression array and ingenuity pathway analysis showed IL-22 signaling as a top differentially regulated pathway and TNF as top upstream regulator. Validation in multiple BC cell lines by qPCR and western blot showed that inflammatory transcripts IL-6, IL-8, IL1β, PTX-3 and COX-2 were up-regulated upon HOXA5 KD and reduced upon overexpression of HOXA5 or ATAL treatment. Treatment with NF-κB inhibitor BAY11-7085 partially abrogated the effect of HOXA5 KD, suggesting a NF-κB mediated regulation. Reduced p65 nuclear localization and increased IκB-α expression was observed upon HOXA5 overexpression. KD of HOXA5 increased NF-κB-mediated luciferase reporter activity.

Conclusions: KD of HOXA5 expression in the partially transformed V777L DKI cell line results in EMT, and transformation from normal ductal structures to preneoplastic nodules when implanted in NSG mice. Expression array of KD cells showed that NF-κB regulated inflammatory transcripts were perturbed. HOXA5 was found to suppress NF-κB mediated transcription, thus reducing levels of inflammatory cytokines and chemokines.

#3476

Evaluable antitumor activity in metastatic pancreatic adenocarcinoma with specific inhibitor of nuclear export based treatment.

Asfar S. Azmi,1 Yosef Landesman,2 Michael Kauffman,2 Sharon Shacham,2 Gabriel Mpilla,1 Amro Aboukameel,1 Steve Kim,1 Mandana Kamgar,1 Anteneh Tesfaye,1 Ramzi M. Mohammad,1 Philip A. Philip1. 1 _Wayne State Univ., Detroit, MI;_ 2 _Karyopharm Therapeutics Inc, Newton, MA_.

Background: Pancreatic ductal adenocarcinoma (PDAC) remains a deadly disease in urgent need of newer therapeutic modalities. Earlier we have shown that in PDAC, the over-expression of the nuclear exporter protein Exportin-1 (XPO1) leads to functional inactivation of tumor suppressor proteins (TSPs; FOXO3a, p27, Par-4 etc.) through mis-localization. We demonstrated that inhibition of XPO1 by CRISPR/Cas9 validated Selective Inhibitor of Nuclear Export (SINE) Selinexor and analogs restores the anti-tumor function of multiple TSPs leading to PDAC cell death and tumor inhibition in orthotopic models.

Methods: Here we evaluate the synergy between SINE compounds and standard of care gemcitabine-nab-paclitaxel in PDAC models in vitro, in vivo and in a Phase Ib/2 trial (NCT02178436).

Results: Selinexor and second generation SINE compound eltanexor synergized with gemcitabine (GEM) and nab-paclitaxel leading to suppression of PDAC growth, induction of apoptosis, and superior spheroid disintegration of PDAC derived cancer stem cells (CSCs). The observed synergy was due in part to enhanced nuclear localization of TSPs and suppression of both CSCs and epithelial-to-mesenchymal transition (EMT) markers. Label-Free quantitative (LFQ) proteome profiling with nuclear and cytoplasmic enrichment showed superior enhancement in nuclear protein fraction in combination treatment. The protein class with highest percent of nuclear retention were DNA binders. Selinexor and eltanexor as single agent (used at MTD) inhibited the growth of PDAC-CSC and two patient derived (Pdx) sub-cutaneous xenografts (p<0.01). In combination experiment, selinexor-GEM-nab-paclitaxel used at sub-MTD dose could significantly suppress the growth of PDx tumors. Molecularly, we observed down-regulation of CRM1 and target TSPs ex vivo. In a Phase 1b/2 study examining patients with metastatic pancreatic cancer, 9 patients were exposed to selinexor (60 mg oral) with GEM (1000 mg/m2 IV) and nab-paclitaxel (125 mg/m2 IV) once weekly (Mondays) for 3 weeks. Evaluable responses were observed in patients on this trial. 2 patients showed partial response (PR) and 2 had stable disease. Outstanding objective response was observed in 1 patient who demonstrated remission for 16 months and remained alive for 22 months. Remarkable and sustained reduction in CA19-9 levels were observed in the responding patient.

Conclusions: These results rationally fortify selinexor-gemcitabine-nab-paclitaxel as new and effective therapy for metastatic PDAC and strengthen our ongoing Phase II study.

#3477

ADGRB3 is epigenetically silenced in WNT-medulloblastoma and inhibits WNT signaling.

Debanjan Bhattacharya,1 Dan Zhu,1 Satoru Osuka,1 Saroja Narra Devi,1 Erwin G. Van Meir2. 1 _Emory University School of Medicine, Atlanta, GA;_ 2 _Emory University School of Medicine and Winship Cancer Institute, Atlanta, GA_.

Medulloblastoma (MB), is the most aggressive primary malignant brain tumor in children and are classified into four molecular subgroups. While some subtypes of MB show a favorable prognosis with treatment, still one third of patients succumb to this disease and the children who survive after therapy suffer from long-term neurocognitive and endocrine side effects of the conventional treatments. ADGRB3 (formerly called BAI3) is a member of the ADGRB1-3 subfamily of adhesion GPCR transmembrane proteins, which are highly expressed in the brain specially in cerebellum and hippocampal neurons. Our recent analysis of RNA-seq data from a published panel of medulloblastoma tumor samples and RT-PCR experiments with MB tumor samples showed that ADGRB3 mRNA expression was selectively repressed in WNT-MB tumor tissue compared to other three molecular subgroups and normal human cerebellar tissue. Using bisulfite sequencing and MS-PCR we have detected hypermethylation of the ADGRB3 promoter region exclusively in WNT-MB subgroup of MB tissues but not in the other three molecular subgroups and normal human cerebellar tissue. ChIP assays revealed enrichment of repressive methyl CpG binding protein MBD2 and the trimethylated histone H3K9me3 in the ADGRB3 promoter region of UW288-1 cells. Collectively, these indicates epigenetic silencing of ADGRB3 in WNT-MB via promoter hypermethylation and repressive histone modifications. We found that knockdown of ATP dependent chromatin remodeler protein Brg1 in MB cells can silence ADGRB3 expression through epigenetic reprogramming at the gene promoter. Lentiviral reconstitution of ADGRB3 in silent MB cells (UW288-1, PFSK-1) inhibited growth of these cells in culture and inhibited WNT signaling targets. ADGRB3 reconstituted UW288-1 MB cells when xenografted yielded significantly reduced tumor in immunocompromised mice compared to the parental cells. Pharmacological reactivation of ADGRB3 expression in silent WNT MB cells using our recently established MBD2 antagonist and an EZH2 inhibitor significantly reduced cell growth in vitro and inhibits some specific WNT signaling targets. We further identified a novel mechanism underlying ADGRB3 mediated regulation of WNT signaling by performing co-immunoprecipitation experiments. Altogether, our findings define an epigenetic mechanism for ADGRB3 silencing in WNT-MB and demonstrates a mechanism through which ADGRB3 restrains activation of WNT signaling involved in cerebellar transformation. Our findings highlight the potential of epigenetic reactivation of ADGRB3 as a less toxic therapeutic intervention for the children suffering from WNT-MB.

#3478

APC loss mediates doxorubicin response in breast cancer via changes in intracellular drug concentration and DNA repair pathways.

Casey Stefanski,1 Kaitlyn Keffler,1 Stephanie McClintock,1 Lauren Milac,1 Jenifer Prosperi2. 1 _University of Notre Dame, Notre Dame, IN;_ 2 _Indiana University School of Medicine-South Bend, South Bend, IN_.

Chemoresistance is one of the leading causes of breast cancer related deaths. Understanding the molecular basis for chemoresistance is essential for novel therapeutic advancement to improve patient outcome. The Adenomatous Polyposis Coli (APC) tumor suppressor is either mutated or hypermethylated in up to 70% of sporadic breast cancer; however, little is known about how APC loss contributes to chemoresistance. Using mammary tumor cells isolated from the ApcMin/+ mouse crossed to the Polyoma middle T antigen (PyMT) transgenic model, we demonstrated that APC loss decreased doxorubicin (DOX) induced apoptosis. DOX, a commonly used chemotherapeutic in breast cancer, inhibits topoisomerase IIa, resulting in double stranded DNA breaks, resulting in cell cycle arrest to allow repair or apoptosis. We made the novel observation that APC loss in MMTV-PyMT;ApcMin/+ cells activated signal transducer and activator of transcription 3 (STAT3) thereby increasing the expression of the drug efflux pump, multidrug resistance protein 1 (MDR1). Therefore, we hypothesized that APC loss prevents doxorubicin-mediated cell death through: 1) reduced intracellular DOX and 2) alterations in DNA damage and repair. To investigate the intracellular accumulation of DOX, we first used calcein incorporation assay and demonstrated that APC loss increased MDR1 activity, which was restored by an MDR1 inhibitor. In addition, MDR1 inhibition sensitized the MMTV-PyMT;ApcMin/+ cells to DOX-mediated apoptosis. To investigate the effect of APC loss on DNA damage repair pathways, we initially monitored damage recognition pathways after 24-hour DOX treatment. The MMTV-PyMT;ApcMin/+ cells exhibited decreased γH2AX and ataxia-telangiectasia mutated (ATM) phosphorylation following DOX treatment compared to controls, suggesting decreased DNA damage either through decreased intracellular DOX or enhanced DNA repair. Decreased phosphorylation of Chk1 and Chk2 was also observed in DOX-treated MMTV-PyMT;ApcMin/+ cells. Preliminary data to investigate if this decreased DNA damage was due to increased DNA damage repair, via monitoring γH2AX expression throughout treatment and recovery from DOX, suggest enhanced DNA repair in MMTV-PyMT;ApcMin/+ cells. Using the ATM inhibitor, we observed an increase in DOX sensitivity in MMTV-PyMT;ApcMin/+ cells. Alternatively, ATR inhibition did not affect DOX-mediated apoptosis. We will further investigate the efficiency of repair pathways through reporter plasmids and radiation induced damage to separate the drug transport alterations. Finally, we will test whether combination therapy of doxorubicin with an MDR1 inhibitor will reduce tumor burden in vivo. Taken together, APC loss mediates DOX resistance via reducing intracellular DOX and increasing DNA damage repair demonstrating the potential use of combination therapy to overcome chemoresistance.

#3479

**Functional significance of** ESR1 **fusions with diverse gene partners in endocrine therapy resistant breast cancer.**

Jonathan T. Lei,1 Xuxu Gou,1 Sinem Seker,1 Vaishnaivi Devorakonda,1 Kimberly R. Holloway,1 Adrian V. Lee,2 Dan R. Robinson,3 Matthew J. Ellis1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _University of Pittsburgh, Pittsburgh, PA;_ 3 _University of Michigan, Ann Arbor, MI_.

Emerging evidence suggests that ESR1 fusion transcripts that produce functional fusion proteins in estrogen receptor positive (ER+) breast cancer play a role in acquired endocrine therapy resistance. We recently reported two in-frame ESR1 fusion transcripts, ESR1-YAP1 and ESR1-PCDH11X, identified in patients with endocrine therapy resistant breast cancer. Both ESR1 fusions generated stable in-frame, fusion proteins that were transcriptionally active, driving endocrine therapy resistant proliferation and also promoting an epithelial-to-mesenchymal transition (EMT) transcriptional program leading to metastasis in experimental models. Here, we extend our studies by examining properties of additional ESR1 fusions with diverse partner genes, ESR1-DAB2, ESR1-GYG1, ESR1-SOX9, ESR1-ARNT2, ESR1-PCMT1, and ESR1-ARID1B, all identified in endocrine-refractory breast tumors, to better understand the role of ESR1fusion gene formation in endocrine therapy resistance. Structurally, ESR1-ARNT2, ESR1-PCMT1, and ESR1-ARID1B fusions retain the first 6 exons of ESR1, therefore lacking ESR1 exons encoding the ligand-binding domain (LBD) that endocrine therapies recognize, but are instead fused in-frame to C-terminal sequences from partner genes, which follows the same fusion pattern of previously identified ESR1-YAP1, ESR1-PCDH11X, ESR1-DAB2, ESR1-GYG1, and ESR1-SOX9 fusions. ESR1-ARNT2, ESR1-PCMT1, and ESR1-ARID1B constructs, along with ESR1-DAB2, ESR1-GYG1, and ESR1-SOX9 constructs were stably expressed in an ER+ breast cancer cell line, T47D, and all produced stable fusion proteins. ESR1-ARNT2 and ESR1-SOX9 promoted hormone-independent and fulvestrant-resistant cell proliferation that was sensitive to cyclin-dependent kinase (CDK) 4/6 inhibitors, palbociclib and abemaciclib. In addition, ESR1-ARNT2 induced hormone-independent activation of estrogen responsive genes (TFF1, GREB1, and PGR), and EMT genes (SNAI1 and VCAN) along with upregulation of Snail protein. These results indicate that ESR1-ARNT2 and ESR1-SOX9 have similar functional and pharmacological properties to our previously described ESR1-YAP1 and ESR1-PCDH11X fusions. These data further support a role for ESR1 fusions in driving not only endocrine therapy resistance but also activating the metastatic process. Ongoing studies are examining the ability of ESR1 fusions to induce EMT phenotypes and to determine structure function relationships of ESR1 fusions. Although the formation of ESR1 fusions likely confers resistance to all endocrine therapies that target the LBD, ESR1 fusion driven growth remained sensitive to CDK4/6 inhibitor treatment therefore providing rationale for testing ESR1 fusion detection in guiding treatment with CDK4/6 monotherapy.

#3480

TMEM30A **loss-of-function mutations drive lymphomagenesis and confer therapeutically exploitable vulnerability in B-cell lymphoma.**

Shannon Healy,1 Daisuke Ennishi,1 Ali Bashashati,1 Saeed Saberi,1 Christoffer Hother,1 Anja Mottok,1 Fong Chun Chan,2 Lauren Chong,1 Robert Kridel,3 Merrill Boyle,1 Barbara Meissner,1 Tomohiro Aoki,1 Katsuyoshi Takata,1 Bruce W. Woolcock,1 Elena Vigano,1 Libin Abraham,2 Michael Gold,2 Adele Telenius,1 Pedro Farinha,1 Graham Slack,1 Susana Ben-Neriah,1 Daniel Lai,1 Allen W. Zhang,1 Sohrab Salehi,1 Hennady P. Shulha,1 Derek S. Chiu,2 Sara Mostafavi,2 Alina S. Gerrie,1 Diego Villa,1 Laurie H. Sehn,1 Kerry J. J. Savage,1 Andrew J. J. Mungall,1 Andrew P. Weng,1 Marcel Bally,1 Ryan D. Morin,4 Gabriela V. Cohen Freue,2 Joseph M. Connors,1 Marco A. Marra,1 Sohrab P. Shah,1 Randy D. Gascoyne1,1 David W. Scott,1 Christian Steidl,1 Ulrich Steidl5. 1 _BC Cancer Agency, Vancouver, British Columbia, Canada;_ 2 _University of British Columbia, Vancouver, British Columbia, Canada;_ 3 _University Health Network, Toronto, Ontario, Canada;_ 4 _Simon Fraser University, Vancouver, British Columbia, Canada;_ 5 _Albert Einstein College of Medicine, Bronx, NY_.

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma subtype worldwide, accounting for 40% of all non-Hodgkin lymphomas. DLBCL presents as an aggressive disease requiring immediate treatment. Although significant improvement in outcome has been achieved, ~40% of patients still experience treatment failure. Here, we characterized the recurrent genetic alterations and transcriptomic signatures in diagnostic biopsies from a population registry-based cohort of 347 patients with de novo DLBCL uniformly treated with R-CHOP. This analysis revealed bi-allelic loss of function mutations of TMEM30A that were associated with favorable treatment outcome. TMEM30A is a chaperone protein, involved in maintaining the asymmetric distribution of phosphatidylethanolamine and phosphatidylserine, an integral component of the plasma membrane and "eat-me" signal recognized by macrophages. Using TMEM30A knockout systems by CRISPR genome editing techniques, we have functionally characterized this loss-of-function mutation in representative human and mouse DLBCL cell line models. We have discovered that TMEM30A loss is associated with increased B-cell signaling following antigen stimulation, including a two-fold increase in the diffusion rate of B-cell receptor (BCR) clustering, using high resolution Single Particle Tracking (SPT) technology. In addition, we have measured three-fold increase in chemotherapeutic drug accumulation in both knockout cell lines and randomly selected patient biopsies with TMEM30A biallelic loss. This observation was validated in a xenograft mouse model, which presented improved survival and limited tumor growth following vincristine treatment in mice injected with TMEM30A null DLBCL cell lines compared with native cell lines. This phenotype explains the improved prognosis observed in DLBCL patients following R-CHOP treatment. Furthermore, we have observed over two fold higher numbers of tumor-associated macrophages in B-cell lymphoma syngeneic mouse models with Tmem30a loss-of-function, prior to any form of treatment, suggesting the existence of "hot" and primed tumors. Our data highlight a multi-faceted role for TMEM30A and plasma membrane physiology in B-cell lymphomagenesis, and characterize intrinsic and extrinsic vulnerabilities of cancer cells that can be therapeutically exploited. Characterization of these mechanisms will address a missing link in the cancer field as related insights in lymphoma will outline therapeutic approaches that can be extended to cancer therapy in general.

#3481

Roles of tumor suppressor candidate 2 (TUSC2) in glioblastoma progression and gliomagenesis.

Tadas K. Rimkus. _Wake Forest University Health Sciences, Winston-Salem, NC_.

Tumor suppressor candidate 2 (TUSC2, also known as FUS1) was identified as a candidate tumor suppressor gene located in a region on chromosome 3p21.3 that undergoes allelic loss in lung and breast cancers. Loss of TUSC2 expression has been reported in various cancers and is associated with poor survival. Evidence to date indicates that TUSC2 behaves as a tumor suppressor in lung cancer; however, its role as a tumor suppressor in other tumor types has not been fully established. Since the mechanism for gliomagenesis is still unclear, we investigated the role of TUSC2 in the development and progression of glioblastoma (GBM), the most common and deadliest brain cancer in adults. Here, we found that forced TUSC2 expression suppressed neurosphere-forming capability of glioma stem cells, regardless of molecular subtypes. Forced expression of TUSC2 in GBM cell lines inhibited their ability to form colonies and neurospheres. To further determine whether TUSC2 plays a tumor suppressive role in GBM, we knocked down TUSC2 expression in TUSC2-expressing GBM cells using siRNA and CRISPR/Cas9, and found TUSC2 knockdown to significantly enhance neurosphere formation of GBM cells. Using an orthotopic GBM xenograft mouse model, we further observed that CRISPR/Cas9-mediated TUSC2 knockout significantly promoted the intracranial growth of GBM tumors. To gain inisghts into the mechanisms underlying TUSC2's tumor suppressive function in GBM, we conducted RNA-Seq using control and TUSC2-knockout GBM cell lines, and identified a number of genes whose expression was altered in response to TUSC2 loss. Ongoing studies are being conducted to validate idenified genes, and elucidate their involvement in gliomagenesis and GBM progression. Furthermore, we speculated that TUSC2 may interact with cellular proteins leading to tumor suppression. To test this hypothesis, we conducted protein interactome analysis using immunoprecipitaton followed by mass spectrometry in which cell lysates from GBM cells and human astrocytes (a cell-of-origin for GBM) were used. This study has identified approximately 40 proteins that differentially interact with TUSC2 in GBM cells versus human astrocytes. Roles of these TUSC2-interacting proteins in GBM suppression and gliomagenesis are being examined in onging studies. Finally, we have generated conditional TUSC2-knockout mice to further address the role that TUSC2 plays in gliomagenesis. Collectively, these findings support a novel role that TUSC2 plays in GBM progression and gliomagenesis, thereby advancing our understanding of GBM pathobiology.

#3482

ZBTB7A mediates the transcriptional repression activity of androgen receptor in prostate cancer cells.

Dong Han,1 Sujun Chen,2 Wanting Han,1 Shuai Gao,1 Jude Owiredu,1 Susan C. Patalano,1 Jill A. Macoska,1 Hansen H. He,2 Changmeng Cai1. 1 _University of Massachusetts Boston, Boston, MA;_ 2 _Princess Margaret Cancer Centre, Toronto, Ontario, Canada_.

Prostate cancer (PCa) is one of the most common cancers in men globally. While the initiation and development of PCa depend on the activity of androgen receptor (AR), a ligand-dependent transcription factor nuclear receptor, loss of expressions of the context-specific tumor suppressors are also critical events that facilitate the progression of PCa to a more malignant form, castration-resistant prostate cancer (CRPC). Zinc finger and BTB domain containing transcription repressors, such as ZBTB7A and ZBTB16, have recently been reported as tumor suppressors that play important functions to prevent the progression of PCa, and mutations and/or deep deletions of these genes can be found in over 5% of PCa patient samples. ZBTB7A, also known as LRF/POKEMON, consists of a protein-protein interacting BTB domain at N-terminus and DNA binding zinc fingers at C-terminus. Even though ZBTB7A has been identified as a proto-oncogene in some other cancer types, a recent study using a transgenic mouse model indicates that it functions as a tumor suppressor in PCa and loss of its expression can drive the development of aggressive invasive tumor in Pten-null prostate epithelial cells by bypassing the Pten-loss induced cellular senescence. In this study, using combined ChIP-seq and RNA-seq analyses in PCa cells, we have precisely mapped the binding sites of ZBTB7A and identified its directly regulated genes. Interestingly, the ZBTB7A-repressed genes were enriched for the activation function of E2F and MYC, suggesting that ZBTB7A may suppress their oncogenic activities in PCa cells. Since both ZBTB7A and ZBTB16 have been reported to negatively regulate AR signaling, we next determined how ZBTB7A chromatin binding in PCa cells globally impacts the transcriptional activity of AR. By co-analyzing the previous reported AR cistrome database in PCa cells, we showed that a significant portion of ZBTB7A binding sites overlapped with AR binding sites, particularly at promoter region. Using the unbiased Binding and Expression Target Analysis (BETA), we further showed that these ZBTB7A and AR overlapping sites were significantly associated with the repression activity of AR on gene transcription. By co-immunoprecipitation assays, we demonstrated that ZBTB7A can physically interact with AR and Rb, suggesting that ZBTB7A may be an additional component of the AR repressor complex. Moreover, we have also identified a subset of long non-coding RNAs (lncRNAs), as novel AR-repressed genes and showed that ZBTB7A contributes to the suppression activity of AR on a PCa-specific lncRNA, PCAT-1. Finally, we have also showed that ZBTB7A functions to suppress CRPC tumor growth in vitro and in vivo. Overall, our study has provided novel molecular insights for ZBTB7A function in PCa cells and demonstrated globally its critical role in mediating the transcriptional repression activity of AR.

#3483

The African-specific S47 variant of the p53 tumor suppressor gene alters cell metabolism.

Keerthana Gnanapradeepan,1 Subhasree Basu,2 Che-Pei Kung,3 Thibaut Barnoud,2 Madeline Good,2 William Quinn,1 Joyce Lee,1 Kathryn Wellen,1 Zachary Schug,2 Joseph Baur,1 Donna George,1 Maureen Murphy2. 1 _University of Pennsylvania Perelman School of Medicine, Philadelphia, PA;_ 2 _Wistar Institute, Philadelphia, PA;_ 3 _Washington University School of Medicine, St. Louis, MO_.

The TP53 gene, often referred to as the guardian of the genome, is the most frequently mutated gene in human cancer. This gene encodes the tumor suppressor p53, a master regulator of various processes such as programmed cell death, growth arrest and senescence. However, there is increasing evidence that highlights the role of p53 in tumor suppression with regards to maintaining metabolic homeostasis. Our lab has identified a polymorphic variant of p53 that encodes a serine residue instead of a proline at amino acid 47 (hereafter S47). This variant is most prevalent in African and African-American individuals, and mice and humans carrying this variant have increased cancer risk and impaired response to therapy. Using both mouse models and human cells, we have found that S47 cells have significantly altered metabolism compared to the WT counterpart. Specifically, S47 mice have increased weight and increased lean content compared to WT mice. These mice also show increased fitness in treadmill studies. Analyses of cell metabolism in S47 murine and human cells indicate that S47 cells show increased glycolysis and glycolytic flux, along with increased oxygen consumption rate on a Seahorse analyzer. The combined data support the premise that the S47 variant confers increased cancer risk but also increased metabolic fitness; the latter may explain the high rate of this variant in certain regions of Africa. By clearly elucidating the roles of WT p53 and the S47 variant in the context of metabolism, we hope to uncover new therapeutic avenues and ultimately enable more personalized medicine approaches for individuals who carry this variant.

#3484

Ras recruits oncogenic serine protease hepsin to disrupt mammary epithelial integrity.

Topi A. Tervonen, Shishir M. Pant, Denis Belitskin, Johanna Englund, Katja Närhi, Emmy Verschuren, Panu Kovanen, Juha Klefström. _University of Helsinki, Helsinki, Finland_.

Type II transmembrane serine protease hepsin is overexpressed and redistributed in clinical breast cancer samples, although the HPN gene is not a frequent target of cancer-specific genetic alterations. Here, we sought to identify cancer relevant upstream factors responsible for oncogenic deregulation of hepsin. We explored the effects of ectopic expression of major oncogenes and tumor suppressors on hepsin protein expression levels in non-malignant mammary epithelial cells, finding that HrasV12, KrasV12, ectopic wild-type Kras, endogenous mutant KrasD12 and E2F-1 induce the expression of active form of hepsin. Oncogenic Ras proteins also reduced expression levels of cognate hepsin inhibitor, HAI-1. Concomitantly, oncogenic Ras expression led to disappearance of desmoplakin and desmoglein 2 from desmosomal junctions, which correlated with mislocalization of hepsin from its predominant localization in desmosomes to cytosol. To explore the effects of mutated Ras expressed at endogenous levels we used lung tumors derived from lox-stop-lox (LSL)-KrasD12; p53-/- and LSL-KrasV12 mammary epithelial structures in ex vivo 3D cultures. In both cases, hepsin was highly expressed and predominantly localized to cytosol. Knockdown of hepsin by shRNA rescued HrasV12-induced epithelial integrity defects in mammary epithelial 3D culture and furthermore, MEK and ERK inhibitors prevented HrasV12-induced hepsin deregulation as well as desmosomal and basement membrane defects. Critical pathways downstream of MAPK pathway are being studied and will be discussed. These findings suggest a critical role for hepsin in mediating Ras-MAPK-mediated disruption of epithelial integrity during tumorigenesis.

#3485

PELP1 is a novel mediator of medulloblastoma progression.

Yiliao Luo,1 Gangadhara Reddy Sareddy,2 Uday P. Pratap,2 Mengxing Li,2 Junhao Liu,2 Prabhakar Pitta-Venkata,1 Suryavathi Viswanadhapalli,1 Xiaonan Li,2 Manjeet Rao,1 Rajeshwar Rao Tekmal,1 Ratna K. Vadlamudi2. 1 _UT Health Science Ctr. at San Antonio, San Antonio, TX;_ 2 _UT Health Science Ctr. at San Antonio, Helotes, TX_.

Background: Medulloblastoma (MB) is the most common and deadliest primary brain tumor in children that accounts for 15-20% of all pediatric brain tumors. Despite recent advances in multimodal treatment, the 5-year overall survival of MB patients is approximately 60-70%. Unfortunately, improved outcome have been associated with significant long-term toxicities. Identifying novel targets that drive MB progression is urgently needed. Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1) is a scaffolding protein that functions as a coregulator of several nuclear receptors. Oncogenic PELP1 signaling is implicated in the progression of several cancers including breast, ovarian, prostate, lung, pancreas and colon. However, its role in the progression of medulloblastoma remains unknown. Here, we examined the role of proto-oncogene PELP1 in the progression of MB.

Methods: The expression of PELP1 in tumor micro arrays was analyzed using validated PELP1 antibodies for immunohistochemistry. PELP1 expression was determined in vitro by western blotting. PELP1 knockdown cells were generated using PELP1 shRNA lentiviral particles or PELP1 siRNA. The effect of PELP1 knockdown or overexpression was studied using cell proliferation, colony formation and migration using established in vitro assays. Mechanistic studies were conducted using RNA-seq, RT-qPCR, immunohistochemistry, reporter gene assays and signaling analysis. Interaction of PELP1 with NF-κB was examined by immunoprecipitation. Mouse orthotopic xenografts models were used for preclinical evaluation of PELP1 knock down.

Results: Immunohistochemical analysis of MB tissue microarrays revealed that PELP1 is overexpressed in MB specimens compared to normal brain specimens. Knockdown of PELP1 using PELP1 specific siRNA or shRNA significantly reduced cell proliferation, cell survival, and cell migration of MB cell lines. RNA-seq analysis revealed that PELP1 knockdown significantly downregulated the pathways related to inflammation, angiogenesis and extracellular matrix. Further, gene set enrichment analysis (GSEA) confirmed that the PELP1-regualted genes were negatively correlated with NF-κB, extracellular matrix and angiogenesis. Mechanistic studies showed that PELP1 knockdown reduced the expression of NF-κB reporter gene activity and its target genes. Additionally, knockdown of PELP1 significantly reduced in vivo MB tumor progression in orthotopic models, and improved overall mice survival. IHC analysis demonstrated that the proliferation marker Ki67 and NF- κB targets were significantly downregulated in PELP1 knockdown tumors compared to controls.

Conclusions: Taken together, these results provide the evidence that PELP1 could be a potential therapeutic target for therapeutic intervention in medulloblastoma.

#3486

Combined inhibition of tumor suppressors PTEN and PP2A drives anoikis resistance and is associated with therapy relapse in prostate cancer.

Christian Rupp,1 Aleksi Isomursu,1 Anna Aakula,1 Andrew Erickson,2 Song-Ping Li,1 Amanpreet Kaur,1 Pragya Shah,3 Yuba R. Pokharel,4 Lloyd Trottman,5 Jan Lammerding,3 Antti Rannikko,2 Pekka Taimen,1 Tuomas Mirtti,2 Ilkka Paatero,1 Johanna Ivaska,1 Jukka K. Westermarck1. 1 _University of Turku, Turku, Finland;_ 2 _University of Helsinki, Finland;_ 3 _Cornell University, Ithaca, NY;_ 4 _University of Turku, Finland;_ 5 _Cold Spring Harbor Laboratories, Cold Spring Harbor, NY_.

Reactivation of tumor suppressor phosphatases may provide entirely novel opportunities for cancer therapy. Here, we discover clinically relevant functional co-operation between loss of activities of two human tumor suppressor phosphatases, PTEN, and PP2A. Analysis of prostate cancer tissue microarray material consisting of 358 patients treated primarily with radical prostatectomy revealed that overexpression of PP2A inhibitor protein PME-1 associates with significantly shorter time to therapy relapse in patients with PTEN-deficient PrCa. Further, PP2A inhibition by PME-1 overexpression in PTEN-deficient cell models inhibits apoptosis induction in anchorage-independent conditions (anoikis). PP2A reactivation by small molecules (SMAPs) was also found to inhibit viability of PTEN-deficient PrCa cells. Importantly, rather than regulating the well-known PP2A target pathways, PME-1 was found to physically associate with, and to regulate deformability of the nuclear lamina in PrCa cells. Mass spectrometry phosphoproteomics analysis identified several PME-1-regulated nuclear lamina constituents, and PME-1 deficient cells with compromised nuclear lamina were particularly vulnerable to apoptosis induction by mechanical stress. As a direct molecular target, Lamin A/C phosphorylation was found to be protected by PME-1-mediated PP2A inhibition under anoikis-inducing conditions. PME-1 inhibition in PrCa cells resulted in increased apoptosis in an in ovo tumor model, and PME-1-depleted cells had compromised long-term survival in zebrafish circulation. In summary we discover that PP2A reactivation by PME-1 targeting sensitizes PTEN-deficient PrCa cells to anoikis. Clinically, the results identify PME-1 as a novel candidate biomarker for increased relapse risk in PTEN-deficient PrCa, and indicate pharmacological PP2A activation as a novel potential therapeutic approach against circulating prostate cancer cells. At the general level, the results clearly emphasize the need for better understanding of phosphatases as key modulators of cancer progression.

#3487

NOTCH/HES5 signaling exhibits distinct pro- and anti-tumorigenic roles in liver carcinogenesis.

Sarah Luiken,1 Matthias Bieg,2 Angelika Fraas,1 Raisatun Sugiyanto,1 Benjamin Goeppert,1 Stephan Singer,1 Stefan Pusch,2 Arianeb Mehrabi,1 Thomas Longerich,1 Peter Schirmacher,1 Stephanie K. Roessler1. 1 _Heidelberg University Hospital, Heidelberg, Germany;_ 2 _German Cancer Research Center, Heidelberg, Germany_.

The NOTCH pathway is an evolutionary conserved signaling pathway that is known to play a pivotal role in physiological liver development and regeneration. Thereby, the NOTCH pathway controls cell fate decisions of bipotential liver progenitor cells promoting a biliary lineage rather than hepatic differentiation. Furthermore, aberrant NOTCH signaling is also a potential driver of liver inflammation, formation and progression of hepatocellular (HCC) and intrahepatic cholangiocarcinoma (iCCA). Previous studies mainly focused on the expression levels of the NOTCH receptors (NOTCH1-4) and their ligands (DLL1/3/4, JAG1/2). However, little is known regarding mutational effects within components of the Notch pathway and their relevance in liver cancer. In a whole exome sequencing approach of 54 human HCC samples, we discovered 19 amino acid-altering mutations in 15 different NOTCH pathway genes which were confirmed by Sanger sequencing to be somatic. In summary, these 15 mutations affected 25.9% of patients (14 out of 54 patients) and presented at least one mutation in one of the included NOTCH pathway components (NOTCH1/3/4, DLL1, DTX1/3/4, HES5, DVL2, LFNG, NUMB, NCSTN, DMXL2, GXYLT2). Consistently, 30% of HCC patients have been reported to contain a tumor-associated hyper-activated NOTCH pathway. Using multiple online tools to predict the effect of the mutations on protein function and tumor relevance, we observed that HES5-Arg31Gly mutation ranked highest. In order to test, whether HES5 is activated by NOTCH in HCC cells, we generated cell lines with inducible expression of NOTCH1 or NOTCH3 intracellular domain and found that the transcription factor HES5 but not HES1 is strongly activated in several HCC cell lines. Furthermore, induction of HES5 in an inducible Hep3B cell line resulted in increased expression of stem cell markers and epithelial-mesenchymal transition markers. In contrary, CCND1, HNF4alpha and HES1 were downregulated. Interestingly, HES5-Arg31Gly failed to modulate the expression of the here tested target genes. The loss of function of HES5-Arg31Gly may be partially explained by lower nuclear translocation of HES5-Arg31Gly protein. In vivo, mouse models using hydrodynamic transduction of transposons expressing MYC and HES5 indicated a tumor suppressive role of HES5, whereas, in AKT-driven liver tumorigenesis, HES5 exhibited oncogenic potential. Consistent with our in vitro experiments, AKT/HES5-driven murine tumors exhibited partial downregulation of HNF4alpha and upregulation of the cholangiocyte marker cytokeratin suggesting a transdifferentiation towards a cholangiocytic phenotype. Thus, we identified somatic mutations in NOTCH pathway genes in 25.9% of HCC patients. Our functional in vitro and in vivo analyses suggest that HES5 has oncogenic or tumor suppressive properties depending on the underlying oncogene or tumor subtype, respectively.

#3488

Loss of nuclear alpha-catenin is associated with race, aggressive disease, and chemoresistance in triple negative breast cancer.

Rania Bassiouni,1 Yunchi Li,1 Victoria David-Dirgo,2 Krystine Garcia-Mansfield,2 Ritin Sharma,2 Patrick Pirrotte,2 Nasreen Vohra,3 Sandeep Singhal,4 Kevin Gardner,4 John D. Carpten1. 1 _University of Southern California, Los Angeles, CA;_ 2 _Translational Genomics Research Institute, Pheonix, AZ;_ 3 _East Carolina University, Greenville, NC;_ 4 _Columbia University Medical Center, New York, NY_.

Triple negative breast cancer (TNBC) is a particularly aggressive and difficult-to-treat subtype of the disease. A known health disparity exists within TNBC: African American (AA) women are more likely to be diagnosed with and die from the disease. Our group previously reported homozygous deletions in the CTNNA1 gene, which encodes the protein alpha-catenin, in AA TNBC. We have undertaken a basic and translational research study to understand the mechanistic role and clinical impact of alpha-catenin loss in TNBC. To validate our findings of alpha-catenin loss in TNBC, we analyzed over 500 breast cancer patient samples by immunohistochemistry. We found loss of both nuclear and cytoplasmic alpha-catenin to be inversely correlated with survival in TNBC. However, loss of nuclear alpha-catenin was observed more frequently in tumors from AA patients, and corresponded to poorer survival in this group. While its cytosolic role has been well studied, little is known about nuclear alpha-catenin and its role in disease. To examine the nuclear function of alpha-catenin, we used CRISPR/Cas9-mediated gene editing to generate CTNNA1 knockout (KO) BT-549 and MB-MDA-436 cell lines. We also reintroduced CTNNA1 into MDA-MB-468 cells - a line derived from an AA woman with an endogenous deletion in CTNNA1. Using these models, we confirmed that all lines contained a pool of nuclear alpha-catenin. To identify binding partners, we performed co-immunoprecipitation of alpha-catenin from nuclear lysates, followed by mass spectrometry. Nuclear alpha-catenin was found to interact directly with ATR, a kinase critical to the DNA damage response (DDR). ATR mediates both the repair of DNA lesions and the G2/M cell cycle checkpoint to ensure that only cells with undamaged DNA may enter mitosis. We found high levels of activated ATR in KO cells, which corresponded with more efficient DNA repair after exposure to UV light. Loss of alpha-catenin also resulted in decreased sensitivity to the DNA-damaging chemotherapeutics cisplatin, carboplatin, doxorubicin, etoposide, and olaparib, several of which are approved for treatment of TNBC patients. Furthermore, alpha-catenin KO cells were more sensitive to inhibitors of ATR, as well as to inhibitors of the G2/M checkpoint proteins Chk1 and Wee1. Tellingly, the KO cells were less sensitive to inhibitors of ATM or DNA-PK, two regulators of alternate DDR pathways. This suggests that nuclear alpha-catenin likely plays a specific role in ATR-directed processes. In our studies, we have identified nuclear alpha-catenin as a tumor suppressor that affects TNBC's susceptibility to chemotherapy by playing a role in the DDR and the G2/M checkpoint. Our data suggest that loss of alpha-catenin is more common in AA patients, and is associated with poor prognosis. Therefore, CTNNA1 status may be important in determining appropriate therapeutic strategies for this subset of patients.

#3489

**A requirement for** STAG2 **in replication fork progression creates a targetable synthetic lethality with DNA repair factors in cohesin-mutant cancers.**

Gourish Mondal, Alan Ashworth, David A. Solomon. _University of California, San Francisco, San Francisco, CA_.

Cohesin is a multiprotein ring that is responsible for cohesion of sister chromatids and formation of DNA loops that enables enhancer-promoter interactions to regulate gene expression. Genomic analyses have identified that the cohesin subunit STAG2 is frequently inactivated by mutations in human cancer. However, the reason STAG2 mutations are selected for during tumorigenesis and strategies for therapeutically targeting mutant cancer cells are unknown. Here we show that STAG2 is essential for DNA replication fork progression, whereby STAG2 inactivation leads to replication fork stalling and collapse due to disruption of interaction between the cohesin ring and the replication machinery along with failure to establish SMC3 acetylation. As a consequence, we find that STAG2 mutation confers synthetic lethality with DNA double-strand break repair genes and increased sensitivity to select cytotoxic chemotherapeutic agents and ATR inhibitors. These studies identify a critical role for STAG2 in replication fork procession and elucidate a potential therapeutic strategy for cohesin-mutant cancers. 

### DNA Damage and Repair 3

#3490

Molecular profiling of MSI-high colorectal cancer (CRC) in Indian population.

Ashmala Naz,1 Vijaya Tourani,2 Satish Rao,3 Murali Dharan Bashyam1. 1 _Centre for DNA Fingerprinting and Diagnostics, Hyderabad, India;_ 2 _Care Hospital, Hyderabad, India;_ 3 _Kims Hospital, Hyderabad, India_.

Background: CRC is the 3rd most commonly diagnosed malignancy and 4th leading cause of cancer related deaths worldwide. Over two decades of extensive research from the West has revealed three major tumorigenesis pathways responsible for CRC viz. a) Chromosomal Instability (CIN) arising from aberrant activation of Wnt signalling, b) Microsatellite Instability (MSI) caused by inactivation of MisMatch Repair (MMR) genes and c) CpG Island Methylator Phenotype resulting in promoter DNA methylation induced inactivation of tumour suppressor genes. Previous results from our laboratory showed a significant proportion of Early Onset Sporadic Rectal Cancer (EOSRC) to be driven neither by aberrant Wnt signalling nor MSI. We also identified a significant proportion of MSI+ familial CRC not exhibiting loss either of the four major MMR genes indicating involvement of addition MMR or non-MMR genes. We therefore endeavoured to determine status of MSI in sporadic CRC samples from the Indian population. We are performing analysis of the status of other molecular features including CIMP, BRAF/KRAS/p53 mutation status, Wnt, etc. to obtain a wholesome molecular profile of sporadic CRC from the Indian population. Methodology: The microsatellite pattern in CRC tumours was determined using the five microsatellite marker panel as per the Bethesda guidelines. Expression status of MMR proteins (MLH1, MSH2, MSH6 & PMS2) was evaluated using immunohistochemistry. Mutations in BRAF (V600E) and KRAS (Codon 12 and 13) were identified by Sanger sequencing. CIMP status is being determined using the MethyLight technology as well as methylation specific PCR. Results: A comprehensive screen of close to 200 CRC samples revealed a significantly higher proportion of sporadic CRC tumors (41%; 50% of colonic and 31% of rectal cancer samples) exhibiting MSI as compared to 14% reported in The Cancer Genome Atlas (TCGA) data. Among MSI-high tumours, only 27% and 15% showed loss of MLH1 or MSH2 respectively, a significantly lower frequency compared to Caucasian data. Instability in the BAT25 microsatellite was significantly high in tumors harboring MLH1 loss. More importantly, a significant proportion of MSI+ CRC tumors were devoid of loss of either of the four major MMR genes. In addition, the BRAF V600E mutation, a known cause of MLH1 silencing, is absent in all but one MSI+ tumors. Work on determining status of CIMP and Wnt signalling is currently underway. Conclusions: Significantly higher proportion of sporadic CRCs from Indian population exhibit MSI but the underlying mechanism appears to be novel and distinct from those identified from the Caucasian population. Deep sequencing based studies are currently underway to determine molecular aberrations driving MSI in the Indian sporadic CRC.

#3491

Functional assessment of DNA damage repair defects and the anti-tumor immune response in high grade serous ovarian cancers using patient-derived organoids.

Sarah J. Hill,1 Patrick Lizotte,1 Neil S. Horowitz,2 Michael G. Muto,2 Michael J. Worley,2 Colleen M. Feltmate,2 Bose Kochupurakkal,1 Khanh T. Do,1 Panagiotis Konstantinopoulos,1 Marisa R. Nucci,2 Joyce F. Liu,1 Ursula A. Matulonis,1 Geoffrey I. Shapiro,1 Ross S. Berkowitz,2 Christopher P. Crum,2 Alan D. D'Andrea1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Brigham and Women's Hospital, Boston, MA_.

Patients with high grade serous ovarian cancer (HGSC) have limited additional therapeutic options beyond traditional carboplatin and paclitaxel. Immuno-oncologic (IO) agents have had limited effect, and despite the fact that 50% of HGSCs have genomic alterations in DNA damage repair genes, we still have no means of predicting which of these tumors actually harbor repair defects and will respond to these agents. Using patient-derived organoids which contain patient immune cells, we have developed functional assays to test the DNA damage repair capacity, anti-tumor immune response, and therapeutic vulnerability of HGSCs. These assays include testing for defects in the two key DNA damage repair pathways, homologous recombination (HR) and stalled replication fork protection, testing for activity and specificity of the immune cells in the cultures against the tumor cells when exacerbated by specific therapeutic combinations, and testing for therapeutic sensitivity to targeted and traditional chemotherapy agents and IO agents either alone or in rational combinations. In parallel, many of the tumors and organoids have undergone genomic and RNA sequencing, searching for relevant alterations to explain detected defects. Flow cytometry analysis of the parent tumors and short term (7-10 day) organoids reveal that organoids contain an immune milieu with IO receptor expression levels similar to the parent tumors. Upon treatment with IO agents alone or in combination with chemotherapeutic agents, we have found that specific IO receptor expression is altered, certain combinations lead to induction of cytokine expression that may repress an anti-tumor response, and that some combinations do not induce the expected cytotoxicity. The DNA damage repair functional assays have revealed that in HGSC, stalled fork protection defects are more common than HR defects, regardless of the repair gene mutational status of the tumors. Importantly, there is a wider array of therapies available to target these defects. For instance, organoids with unstable replication forks are more sensitive to ATR and CHK1 inhibitors. Organoids with stable forks are more sensitive to combinations of drugs which confer replication stress, such as the combination of a CHK1 inhibitor plus gemcitabine. Overall, the repair assays will allow for a better understanding of the types and mechanisms of repair defects present in tumors and a more accurate prediction of sensitivity to targeted agents. The immune functional assays will allow for a better mechanistic understanding of what response specific agents actually induce in immune and tumor cells and allow for better rational therapeutic pairings. Through assessment of a larger number of patients, we hope to demonstrate that these functional assays can have a clinical impact in rapidly predicting patient response.

#3492

A targeted semi-conductor based next-generation sequencing (NGS) test to characterize microsatellite instability in FFPE tumor samples.

Anelia Kraltcheva,1 Sameh El-Difrawy,2 Asha Kamat,2 Janice Au-Young,2 Simon Cawley,2 Seth Sadis3. 1 _Thermo Fisher Scientific, Carlsbad, CA;_ 2 _Thermo Fisher Scientific, South San Francisco, CA;_ 3 _Thermo Fisher Scientific, Ann Arbor, MI_.

Microsatellite instability (MSI) arises from defects in the mismatch repair (MMR) system and is associated with hypermutability of short DNA sequence repeats. Defects in MMR are observed in diverse cancer types but are common in colorectal, gastric and endometrial cancers. MSI testing is increasingly important for patient management. For example, in diverse cancer types, MSI is associated with favorable response to immune checkpoint inhibitors. The mainstay of MSI testing has been PCR/fragment analysis or IHC to monitor loss of expression of MMR proteins. Although generally robust, these methods are single biomarker tests that may consume limited biopsy samples. For this reason, MSI testing has been recently incorporated into NGS tests. However, targeted amplicon based NGS tests for MSI that consume limited sample input and provide fast turn-around time remain an unmet need. Herein, we describe a targeted NGS-based method to assess MSI that leverages Ion AmpliSeq™ or Ion AmpliSeq™ HD multiplex PCR and Ion GeneStudio™ S5 next-generation sequencing. The test is comprised of diverse microsatellite markers including mono- and di-nucleotide repeats that range from 10 to 40 bp. A novel algorithm was developed that leverages the unique signal processing properties inherent in semi-conductor sequencing and workflows were developed for tumor only samples as well as paired tumor-normal samples. The test provides results for individual microsatellites and generates an MSI score for the sample. The performance of the assay was verified over a large cohort of colorectal, gastric and endometrial cancer samples with MSI status independently assigned by orthogonal on-market tests. The MSI panel can be used by itself or integrated into larger targeted sequencing panels. MSI tests that leverage the inherent advantages of targeted semiconductor sequencing, low sample input and fast turn-around time, will support expanded research opportunities into the association of MSI with other targeted alterations and help elucidate the interaction of MSI, DNA repair defects, and the response to immune checkpoint inhibition.

#3493

Tumor treating fields (TTFields) significantly alters how tumor cells repair double stranded breaks using homeologous Alu sequences.

Maria E. Morales, Tiffany K. Kaul, Prescott L. Deininger. _Tulane University Cancer Center, Epidemiology Department, Tulane University, New Orleans, LA_.

Tumor Treating fields (TTfields) are an FDA-approved therapeutic modality that utilizes low intensity alternating electric fields. Although the therapy's primary mechanism seems to cause aberrant mitosis in dividing cells and subsequently cellular death, TTfields have also been reported to alter DNA repair pathways. Recent reports showed that chromosomal double strand breaks (DSBs) repair is delayed after TTfields treatment of glioma cells. DSBs are often repaired by error-prone pathways that can be influenced by proximity of repetitive sequences, such as Alu elements. Alu elements are known to contribute to genetic instability though homeologous recombination due to their sequence similarity and high density in the genome. We find that different tumor cells resolve the Alu heteroduplexes formed during DNA repair in different ways. To understand the molecular mechanisms underlying the biological effects of TTfields exposure, we examined mechanistic aspects of DNA repair using our previously published Alu/Alu recombination system in HEK293 and HeLa cells. Of note, our assay probes the recombination properties that occur in the cells that survive TTfields treatment, thus providing potential insights into therapeutic resistance. Our Alu/Alu recombination system is able to efficiently measure both the rate of Alu/Alu recombination (Single Strand Annealing-SSA), as well as Alt-Non Homologous End Joining (Alt-NHEJ) deletions that cause a deletion in the vicinity of the two Alu elements in the system. Sequence verification confirmed that most of the genetic deletions that occurred post TTfields treatment were the result of Alu/Alu recombination events that predominantly arose from repair by SSA in HEK293 and HeLa. This increase in SSA repair could be the result of SSA-intermediates that escaped heteroduplex rejection. These observations suggest the possibility that treatment of TTfields significantly alters how tumor cells repair DSBs using homeologous Alu sequences.

#3494

Centrobin is involved in the cellular response to DNA damage.

Na-mi Ryu, Jin Ki Jung, Seok Won Jang, Jung Min Kim. _Medical Research Center for Gene Regulation, Chonnam National University Medical School, Gwangju, Republic of Korea_.

DNA-repair proteins have been found to localize to the centrosomes and defects in these proteins cause centrosome over-duplication. Centrobin is a centriole-associated protein that is required for centriole duplication. In the present study, we investigated a possible role of centrobin in DNA damage response. Centrobin was phosphorylated in response to UV radiation and its phosphorylated form was detected only in the detergent / DNase 1-resistant fraction. UV-induced phosphorylation of centrobin was significantly decreased by ATR inhibition, but not by ATM inhibition. In addition, centrobin-depleted cells showed reduced survival rate after UV exposure compared with control cells. Moreover, centrobin knockdown in DR-GFP/U2OS cells had a significant reduction in DNA repair efficiency of homologous recombination (HR) and this defect was recovered by expression of wild-type centrobin, but not by a mutant form of centrobin. Taken together, these results suggest that centrobin may play a critical role in DDR and that ATR dependent phosphorylation of centrobin may be at least partially responsible for this function.

#3495

The CCT chaperonin is a regulator of mutagenic APOBEC3A activity.

Abby M. Green, Ariel S. Dineen, Katarzyna Kulej, Julia H. Szeto, Matthew D. Weitzman. _Children's Hospital of Philadelphia, Philadelphia, PA_.

APOBEC3 (A3) enzymes deaminate DNA cytosine bases and are implicated as the cause of a prevalent mutational signature found in cancer genomes. Mutation of the cellular genome is presumed to be an off-target activity of the enzymes. A3 enzymes normally function as part of the innate immune system by deaminating viral genomes to restrict infection. Basal expression of A3 enzymes is low in healthy tissues, but is increased by type I interferon signaling. Aside from interferon induction, the mechanisms regulating expression and activity of A3 enzymes are unknown. Thus the circumstances that allow for off-target activity of A3 on cellular DNA, leading to mutations and genome instability, cannot be predicted. We sought to determine protein interactors of A3 enzymes to identify potential regulators of expression and/or activity. Using a doxycycline-inducible expression system, we introduced A3 genes into isogenic cell lines. Each A3 enzyme was immunoprecipitated (IP) and lysates were analyzed by mass spectrometry (MS). MS analysis of A3A interactors revealed all 8 subunits of the TriC/CCT (TCP-1 ring complex or chaperonin containing TCP1) complex. The CCT complex is a molecular chaperone that assists in folding of many newly-synthesized proteins, preventing aggregation and facilitating protein function. Validation of MS results by A3A IP followed by immunoblotting confirmed interaction with CCT complex subunits. Endogenous A3A levels are increased significantly by interferon in peripheral blood mononuclear cells (PBMCs). We performed IP of CCT in PBMCs followed by immunoblot for A3A, which demonstrated an interaction between endogenous A3A and the CCT complex. To evaluate the impact of the CCT-A3A interaction, we used siRNA to knock down the CCT complex. Although CCT is an essential chaperonin, transient knockdown of individual subunits resulted in minimally decreased cell viability. However, knockdown of CCT subunits combined with induction of A3A expression led to significant decrease in cell proliferation and increase in cell death. To determine the mechanism by which CCT knockdown with A3A expression results in cytotoxicity, we performed an in vitro deamination assay on lysates from cells that were transfected with siRNA targeting CCT subunits and/or treated with doxycycline to induce A3A expression. Quantification of deamination activity demonstrated that CCT knockdown resulted in increased in A3A activity relative to expression levels. Finally, we evaluated cancer genome sequences in TCGA and found an enrichment of the mutational signature attributed to A3 deamination in cancers with deleterious mutations in CCT subunit genes. Together, these data suggest that the CCT complex regulates A3A activity, and disruption of CCT function results in increased A3A mutational activity.

#3496

Defective DNA damage repair leads to frequent catastrophic genomic events in murine and human tumors.

Manasi Ratnaparkhe,1 John K. Wong,1 Pei-Chi Wei,2 Mario Hlevnjak,1 Thorsten Kolb,1 Milena Simovic,1 Daniel Haag,1 Yashna Paul,1 Frauke Devens,1 Paul Northcott,1 David T. Jones,1 Marcel Kool,1 Anna Jauch,3 Agata Pastorczak,4 Wojciech Mlynarski,4 Andrey Korshunov,1 Rajiv Kumar,1 Susanna M. Downing,5 Stefan M. Pfister,1 Marc Zapatka,1 Peter J. McKinnon,5 Frederick W. Alt,2 Peter Lichter,1 Aurelie Ernst1. 1 _German Cancer Research Center, Heidelberg, Germany;_ 2 _Harvard Medical School, Boston, MA;_ 3 _University of Heidelberg, Heidelberg, Germany;_ 4 _University of Lodz, Poland;_ 5 _St. Jude Children's Research Hospital, Memphis, TN_.

Chromothripsis and chromoanasynthesis are catastrophic events leading to clustered genomic rearrangements. Whole-genome sequencing revealed frequent complex genomic rearrangements (n= 16/26) in brain tumors developing in mice deficient for factors involved in homologous recombination repair or non-homologous end-joining. Catastrophic events were tightly linked to Myc/Mycn amplification, with increased DNA damage and inefficient apoptotic response already observable at early postnatal stages. Inhibition of repair processes and comparison of the mouse tumors with human medulloblastomas (n=68) and glioblastomas (n=32) identified chromothripsis as associated with MYC/MYCN gains and with DNA repair deficiencies, pointing towards therapeutic opportunities to target DNA repair defects in tumors with complex genomic rearrangements.

#3497

SAMHD1 is a Novel Biomarker and Therapeutic Target for Radiation Therapy and PARP Inhibition in Breast Cancer.

Elizabeth Thompson. _Emory University, Atlanta, GA_.

SAMHD1 is a Novel Biomarker and Therapeutic Target for Radiation Therapy and PARP Inhibition in Breast Cancer

Ashley Schlafstein, Waaqo Daddacha, David S. Yu

Background/Introduction:

Patients with breast cancer are often treated by ionizing radiation (IR) and PARP inhibitors; however, the effectiveness of these treatments is often limited by lack of response and/or resistance. Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a deoxyribonucleotide triphosphate (dNTP) triphosphohydrolase with a well-established role in HIV-1 restriction by depleting dNTPs required for reverse transcription and replication. SAMHD1 is also dysregulated in breast and other cancers and in autoimmune disease. In addition to its well-established dNTPase activity, our lab has defined a novel role for SAMHD1 in DNA end resection to facilitate DNA double strand break (DSB) repair by homologous recombination (HR). Because of its role in DNA DSB repair and its link to breast cancer, we hypothesized that SAMHD1 could be used as a novel biomarker and target for breast cancer therapy.

Methods:

We queried the TCGA database for patients with breast cancer who were treated with or without IR to determine if there was an association of SAMHD1 expression level with clinical outcome. We also determined if targeting SAMHD1 for proteasomal degradation with novel virus like particles (VLPs) containing Vpx, a viral accessory protein, can sensitize breast cancer cells and tumors to DNA damaging agents.

Results:

In patients with breast cancer treated with IR, low SAMHD1 expression was associated with a statistically significant improvement in overall survival, while for those not treated with IR, no significant difference in association of SAMHD1 expression with outcome was observed. Breast cancer cells and tumors treated with VLPs containing Vpx were hypersensitive to IR and PARP inhibitor.

Conclusion:

Our results identify SAMHD1 as a novel biomarker for stratifying breast cancer patients for treatment with IR. In addition, targeting SAMHD1 with VLPs containing Vpx may be a novel therapeutic strategy for sensitizing breast cancer to IR, PARP inhibitors, and other agents that induce DNA DSBs.

#3498

Highly specific macrocyclic ATR inhibitors for the targeted treatment of a broad spectrum of cancers showing lack of anemia or neutropenia in pre-clinical animal models.

Sahithi Pamarthy,1 Dansu Li,1 Ekaterine Goliadze,2 Tina Gill,1 Lanqi Jia,1 Erin George,2 Laura R. Butler,1 Ryan L. Ragland,2 Michel Afargan,1 Fiona A. Simpkins,2 Eric J. Brown,2 Oren Gilad1. 1 _Atrin Pharmaceuticals, Ambler, PA;_ 2 _University of Pennsylvania, Philadelphia, PA_.

Ataxia Telangiectasia and Rad3-related (ATR) and its downstream effector Checkpoint Kinase 1 (CHK1) are central to the protection of stalled replication forks.Specific targeting of the ATR is synthetically lethal with multiple cancer-associated changes including oncogenic stress and defects in the DDR pathway and represents an emerging strategy to treat a broad spectrum of cancers. Atrin Pharmaceuticals has rationally designed a novel series of conformationally constrained macrocyclic ATR inhibitors with higher potency and selectivity than other ATR inhibitors currently in clinical development. An in-depth characterization of the ATRN series identified ATRN-119 as our lead compound with an in vitro enzyme IC50 of 20 nM and inhibition of ATR substrate CHK1 Ser345 phosphorylation in cells at an IC50 of 5 nM. ATRN-119 inhibits ATR in cells at much lower concentrations and demonstrates higher selectivity (>2000) for ATR over other closely-related PIK-kinases like ATM, DNA-PK and mTOR. Additionally, ATRN series have favorable ADME properties with increased water solubility and metabolic stability in human serum of up to 4 hours. Oral dosing of ATRN-119 showed significant antitumor effects in human pancreatic and colon cancer xenografts and in orthotopic ovarian Patient Derived Xenograft (PDX) tumors. Notably, hematological analysis of mice treated with daily oral dosing of ATRN-119 indicated no thrombocytopenia, anemia or neutropenia up to 4 weeks of treatment. Exploratory multiple high dosing toxicity studies in rats and dogs indicate significant exposure and good tolerability with lack of anemia or neutropenia. In a series of in-vitro cell viability assays, three dimensional organoid cultures and in-vivo combinationstudies, ATRN-119 showed significant synergism with various PARP inhibitors as well as restoration of PARPi sensitivity in PARPi resistant tumors. Our data suggests a new generation of highly potent and selective ATR inhibitors with favorable safety profile and a broad clinical therapeutic potential either as monotherapy, in combination with PARP inhibitors or as a synthetic lethal approach with key DDR mutations.

#3499

Dianhydrogalactitol (VAL-083) in combination with AZD1775 increases survival in diffuse intrinsic pontine glioma (DIPG), in vivo.

Anne Steino,1 Xiaodong Yang,2 Cassie Kline,2 Jeffrey Bacha,1 Dennis M. Brown,3 Sabine Mueller2. 1 _Delmar Pharmaceuticals Inc., Vancouver, British Columbia, Canada;_ 2 _University of California, San Francisco, San Francisco, CA;_ 3 _Delmar Pharmaceuticals Inc., Menlo Park, CA_.

OBJECTION: VAL-083 is a structurally unique bi-functional DNA targeting agent that readily crosses the blood-brain barrier and accumulates in brain tumor tissue. VAL-083 induces interstrand crosslinks leading to DNA double-strand breaks and S/G2 cell cycle arrest. By inhibiting the G2 checkpoint regulator kinase Wee1, AZD1775 allows a cancer cell with DNA damage to progress past the G2 checkpoint and into premature mitosis leading to cancer cell death. Herein we assess the activity of VAL-083 as single agent as well as in combination with Wee1 Kinase inhibitor AZD1775 in patient derived model systems of DIPG.

METHODS: DIPG derived cell lines SF8628 and NEM157 (H3.3K27) as well as SF10693 (H3.1K27M) and pediatric glioblastoma cell lines SF188 (H3.3K27 wildtype) were treated with increasing concentrations of single agent VAL-083 as well as in combination with AZD1775. To determine potential synergistic activity, we applied the Chou-Talalay method, which allows the quantitative determination of drug interactions by calculating a combination index (CI). In vivo activity of VAL-083 (3 mg/kg) as single agent as well as in combination with AZD1775 (60 mg/kg) was assessed in an orthotopic engraftment model of pediatric DIPG (SF8628).

RESULTS: The IC50 of VAL-083 as single agent ranged from 1µM to 10µM. VAL-083 exhibited synergistic activity in combination with AZD1775 in cell lines SF8628 and SF188 and additive effect in NEM157 with CI values ranging from 0.04 to 0.95, with CI < 1 indicating synergy. In vivo, treatment with VAL-083 as single agent and in combination with AZD1775 conferred significant survival benefit to mice with engrafted DIPG tumors compared to control as well as single agent treatment with AZD1775. The median survival for mice treated with VAL-083 alone was 54.5 days (p=0.0004, VAL-083 vs. control) and for the VAL-083/AZD1775 combination it was 62 days (p<0.0001, VAL-083/AZD1775 vs. control), compared to 44 days for control and 47 days for mice treated with AZD1775 alone (p=0.0839, AZD1775 vs. control).

CONCLUSION: Our present study highlights that the combination of VAL-083 and AZD1775 might be a promising new therapeutic strategy for children with DIPG. Ongoing studies continue to assess the in vivo activity in other DIPG models as well explore the underlying mechanism of action of the combination strategy.

#3500

Highly potent and selective ATM kinase inhibitor M4076: A clinical candidate drug with strong anti-tumor activity in combination therapies.

Thomas Fuchss, Ulrich Graedler, Kai Schiemann, Daniel Kuhn, Holger Kubas, Heike Dahmen, Astrid Zimmermann, Frank Zenke, Andree Blaukat. _Merck KGaA, Darmstadt, Germany_.

M4076 is an ATP-competitive inhibitor of the Ataxia telangiectasia mutated (ATM) kinase (IC50 < 1 nM), which targets tumor cell survival and growth by inhibiting double-strand break (DSB) repair as well as checkpoint control. DSB repair is crucial for survival of malignant tumor cells, especially under treatment with DNA damaging chemo- and radiotherapy. As such, the rationale of pharmacological inhibition of ATM is to increase and maintain the extent of unrepaired DNA damage generated by radio-, chemotherapy, and targeted therapies to drive tumor cells into cell death. We have developed an orally administered, sub-nanomolar potent & selective kinase inhibitor of ATM, M4076, for cancer therapy in combination with DNA damaging modalities. Here, we present its structure-activity relationship, its chemical structure (first-time disclosure) & physicochemical profile as well as its preclinical characterization using biochemical, cellular & human tumor xenograft models. M4076 sensitizes tumor cell lines to radiation therapy in vitro and strongly enhances the anti-tumor activity of ionizing radiation in vivo. These effects are due to the inhibition of ATM kinase activity as demonstrated by the inhibition of radiotherapy induced ATM autophosphorylation and CHK2 phosphorylation in xenograft lysates.

#3501

Entinostat enhances the efficacy of olaparib in preclinical models of ovarian cancer.

Vijayalaxmi G. Gupta,1 Yoskaly Lazo Fernandez,1 Katherine Roby,1 Harsh Pathak,1 Jeff Hirst,1 Andrew J. Wilson,2 Andrew Godwin,1 Dineo Khabele1. 1 _Univ. of Kansas Medical Center, Kansas City, KS;_ 2 _Vanderbilt University Medical Center, Nashville, TN_.

Introduction: Ovarian cancer is a rare but often fatal disease and a leading cause of gynecologic cancer death, in the United States. Poly ADP ribose polymerase inhibitors (PARPi) are promising new drugs that are most effective in patients with tumors with altered homologous recombination (HR) DNA repair genes, particularly BRCA1/2. To expand their clinical utility, PARPi are being tested in combination with other drugs. Our group has published that histone deacetylase inhibitors (HDACi) sensitize HR proficient BRCA wild-type ovarian cancer cells to PARPi. We have developed an investigator-initiated phase 1/2 clinical trial of the PARPi olaparib combined with the HDACi entinostat in patients with recurrent, HR proficient/BRCA wild-type ovarian cancer.

Objective: To test the effects of a novel combination of olaparib and entinostat in preclinical models of BRCA wild-type ovarian cancer.

Methods: HR proficient BRCA wild-type SKOV3 and ID8 ovarian cells were treated with Entinostat followed by Olaparib alone or the combination of Entinostat and Olaparib. Treated and control cells were analyzed for cell proliferation by sulforhodamine B assays, clonogenicity, and DNA damage by Comet assays. SKOV-3-luciferase cells were injected intraperitoneally into NOD-SCID mice. Mice were randomized into 4 treatment groups and treated with vehicle, entinostat, olaparib, or the combination. The mice were monitored for toxicity and by bioluminescence imaging (BLI). At sacrifice, tumor burden was quantified.

Results: In cell culture assays, entinostat significantly enhanced the sensitivity of SKOV3 and ID8 cells to Olaparib. Entinostat combined with olaparib led to synergistic reductions in cell proliferation and clonogenicity in SKOV-3 and ID8 ovarian cancer cells, compared to each drug alone. Evidence of DNA damage was enhanced by the drug combination. In the SKOV3-luc xenograft mouse model, a significant reduction in tumor burden in the group of mice treated with the combination of entinostat and olaparib, compared to each drug alone. There was no significant toxicity with drug treatment.

Conclusion: Entinostat sensitizes ovarian cancer cells to Olaparib in vitro and in vivo. Experiments in ID-luciferase and patient-derived xenograft models are ongoing. These preclinical models are powerful tools that will be used to discover markers and mechanisms of sensitivity and resistance to inform the parallel clinical trial.

#3502

Clinical characteristics and combined somatic mutation using target sequencing among breast cancer patients with germline BRCA mutation.

Sehhoon Park,1 Eunjin Lee,1 Seri Park,2 Sohee Lee,2 Joon Young Hur,1 Sang Eun Yoon,1 Kangkook Lee,1 Jang Ho Cho,1 Ji-Yeon Kim,1 Jin Seok Ahn,1 Young-Hyuck Im,1 Woong-Yang Park,1 Yeon Hee Park1. 1 _Samsung Medical Center, Seoul, Republic of Korea;_ 2 _Sungkyunkwan University, Seoul, Republic of Korea_.

Purpose: Although both gBRCA1/2 mutations are known to increases incidences of breast and ovarian cancer by abruption in homologous recombination pathway, gBRCA1 mutant mainly present with a molecular subtype of triple negative breast cancer (TNBC) and gBRCA2 with hormone receptor (HR) positive which we have little understanding regarding the underlying pathophysiology. In this study, we have comprehensively analyzed clinical characteristics and somatic mutation in gBRCA1/2 mutant patients.

Methods: Patients with gBRCA mutation tested (n=2720) in Samsung Medical Center between Jan 2007 to Oct 2018 were retrospectively reviewed. 386 patients were identified as gBRCA mutant and follow-up period less than 2 years (n=105), insufficient clinical data (n=22) and no surgical treatment (n=6) were excluded. Total of 259 patients, gBRCA1 (n=128), gBRCA2 (n=126) and both gBRCA1/2 mutation (n=5), were analyzed. Among the study population, 46 patients had deep target sequencing data with 40 patients also available for matched transcriptome outcome.

Results: Median age was 40 years old (range 23-68). Patients were mostly under pre-menopausal status (81.2%) with invasive ductal carcinoma pathology (86.2%). Initial stage at diagnosis were stage 0 (4.7%), 1 (30.3%), 2 (40.6%) and 3 (24.4%). A novel mutation was observed in 16.9% of the study population and frameshift (44.9%) and nonsense (31.5%) mutations were predominant. Molecular subtypes were as follows: HR-positive (n=125, 49.2%), HR/HER2 positive (n=10, 3.9%), HER2 positive (n=3, 1.2%) and TNBC (n=116, 46.7%). TNBC was observed mostly in gBRCA1 mutant patients (71.9% vs. 19.1%) and HR-positive in gBRCA2 mutant patients (76.2% vs. 22.7%). Sixty-seven patients (26.4%) experienced disease recurrence and median time to recurrence were 145.0 months (95% confidential interval 107.3-170.2). Looking into co-altered somatic mutation using target sequencing (n=45), notably, alteration in other DNA-damage response pathway-related genes, such as TP53 (60%), ATR (27%), CHEK2 (20%), MSH6 (20%), were identified. Evaluated for mutual exclusivity, only gBRCA2 was significantly (P<0.05) exclusive with CDH2, KDM5A, GNA, TGFBR2, MAP3K1, MLL3, TP53. Mean tumor mutation burden was 13.05 per mega bases with no difference between gBRCA1 and gBRCA2 mutant.

Conclusions: From our dataset, we have witnessed similar clinical characteristics but the difference in molecular subtype between gBRCA1/2 mutant. Despite the fact that both gBRCA1/2 mutations disrupt the homologous recombination pathways, this study provides early evidence regarding the hypothesis that difference in co-occurred somatic alteration between gBRCA1/2 lead to different carcinogenesis mechanism which consequences different molecular subtype. Detail analyses using paired transcriptome result will be presented in the poster.

#3503

Biomarkers for inhibitors of the replication stress response proteins WEE1 and ATR in triple negative breast cancer.

Violeta Serra,1 Cristina Cruz,1 Zhongwu Lai,2 Marta Castroviejo-Bermejo,1 Marta Palafox,1 Urszula M. Polanska,3 Gemma N. Jones,3 Anderson Wang,3 Filippos Michopoulos,3 Rachel Brough,4 Brian Dougherty,2 Elaine Cadogan,3 Susan Critchlow,3 Alejandra Bruna,5 J. Carl Barrett,2 Cristina Saura,1 Christopher J. Lord,4 Carlos Caldas,5 Joaquin Arribas,1 Judith Balmaña,1 Mark J. O'Connor3. 1 _Vall d'Hebron Inst. of Oncology, Barcelona, Spain;_ 2 _AstraZeneca, Waltham, MA;_ 3 _AstraZeneca, Cambridge, United Kingdom;_ 4 _Institute of Cancer Research, London, United Kingdom;_ 5 _Cancer Research UK, Cambridge, United Kingdom_.

Replication stress (RS) is a hallmark of cancer and has the potential to be exploited by selective DNA Damage Response inhibitors. During replication stress, the DNA polymerase is uncoupled from the replisome helicase activity resulting in extended regions of single strand DNA that lead to the initiation of a replication stress response (RSR). Two key regulators of the RSR are the ATR and WEE1 kinases. Here, we aimed to identify patient selection biomarkers for WEE1 and ATR inhibitors (WEE1i, ATRi) from a cohort of 37 patient-derived tumor xenografts (PDX) from ovarian or triple negative breast cancer, 20 of which harbored BRCA1/BRCA2 deleterious mutations. The antitumor response of the WEE1i AZD1775 and the ATRi AZD6837 was evaluated, and PDX tumors were characterized by exome sequencing, metabolomics, western blot and immunohistochemistry. AZD1775 treatment response in PDXs, measured as tumor regressions (≤-30% change in tumor volume), was observed in 28% of models tested. Exome sequencing identified an association between treatment response and concomitant alterations in RB1 and STK11 (LKB1), whose functional relevance was validated in an in vitro model. Immunoblots showed that WEE1i treatment resulted in downregulation of the ribonucleotide reductase subunit RRM2 and induction of an S-phase DNA damage response (DDR) in vivo. Metabolomics identified a profound change in the purine and pyrimidine synthesis pathways in WEE1i-sensitive PDXs and these changes appear functionally relevant since PDX ex vivo data demonstrate rescue of the WEE1i effects after addition of nucleosides. Another important association with WEE1i sensitivity was increased cyclin E expression. Together, these data suggest that early entry into S phase and an imbalance of origin firing to available dNTPs represent drivers of sensitivity to the WEE1i. Interestingly by contrast, ATRi-sensitive PDXs (representing 10% of the models) were characterized primarily by those tumors harboring ATM alterations, suggesting that synthetic lethality with this major DDR pathway is the primary mechanism of sensitivity to the ATR inhibitor in ovarian and triple negative breast cancer. This study therefore highlights differences between the drivers of sensitivity to inhibitors of these two important RSR proteins.

#3504

Mutation analysis of ovarian carcinoma patients presenting optimal response to neoadjuvant chemotherapy.

Etienne Rouleau,1 Elizabeth Santana dos Santos,2 Francilette Maela,1 Ludovic Lacroix,1 Aurélie Auguste,1 Patricia Pautier,1 Philippe Morice,1 Catherine Genestie,1 Alexandra Leary1. 1 _Institut Gustave Rousy, Villejuif, France;_ 2 _A.C.Camargo Cancer Center, São Paulo, Brazil_.

Background: Neoadjuvant chemotherapy (NAC) followed by interval debulking does not present inferior results to those of primary cytoreduction and offers the opportunity to evaluate chemo-sensitivity in vivo. Pathological response scores (CRS) have been shown to correlate with outcome with a complete or near-complete CRS (CRS3) predicting improved progression-free survival. Approximately 20% of ovarian cancers present BRCA1/2 mutations, which predict a better response to platinum salts. Our proposal is to determine the prevalence of BRCA mutations or other homologous recombination (HR) alterations among patients with a CRS3 after NAC.

Methods: We performed a retrospective analysis of clinical, pathological, and sequencing data of patients who experienced a complete or near-complete response to platinum based NAC. For patients with wild-type (WT) sequencing and with tumor samples available, somatic analysis based on Next Generation Sequencing (NGS) with a large panel of genes (BRCA1, BRCA2, ATM, BARD1, BRIP1, CCNE1, CDK12, CHEK2, PALB2,RAD51C, RAD51D, TP53), including specific non-coding regulatory regions of BRCA1, BRCA2 and RAD51C, was also performed. We present preliminary data on the 1st cohort analysed at the time of abstract submission.

Results: A total of 33 patients were identified who demonstrated CRS3 post-NAC. Tumors were in majority EC IIIc (57%) and grade II-III serous ovarian carcinoma (90%). Median overall survival (m OS) of the entire cohort was 55 months (IC95% 36-74). To date, germline and/or somatic analyses were available on 23 pts. The prevalence of pathogenic BRCA1/2 variants is higher than expected in this cohort of patients presenting a CRS3. In total, 7 of 23 patients (30%) had a germline (4) or somatic pathogenic (3) BRCA variant. In addition, among the 16 BRCA1/2 WT pts subjected to further HR NGS analysis, the following cases of alterations were identified: one ATM nonsense mutation (c.2465T>G; p.Leu822*), one pathogenic mutation of CDK12 gene (c.3G>A;p.Met1?), one likely pathogenic variant located on BRCT domain of BRCA1 (c.5165C>T p.Ser1722Phe), two 3'UTR BRCA2 variants of uncertain significance (VUS) (c.*14C>T; c.*72A>G), and one BRIP1 VUS (c.2932G>C, p.Gly978Arg). Further, one BRCA WT case presented an unexpected CCNE1 amplification.

Conclusion: HGOC patients presenting with a pCR are enriched for BRCA germline and/or somatic BRCA mutations. In addition, among the small subset of BRCA WT pCR tumors, a deleterious ATM and CDK12 mutation were identified. This prevalence can be underestimated in a context of pCR since somatic screening is impossible on the debulking material. Somatic BRCA1/2 and complete HR sequencing on the initial biopsy is recommended to identify additional pts with exclusive somatic mutations. More complete data on this cohort will be presented.

#3505

AZD7648: A potent and selective inhibitor of DNA-PK with pharmacodynamic and monotherapy anti-tumor activity .

Elaine B. Cadogan,1 Jacqueline H. Fok,1 Antonio Ramos-Montoya,1 Neil James,1 Valeria Follia,1 Mercedes Vazquez-Chantada,2 Paul Winjhoven,1 Lenka Oplustil O'Connor,1 Ankur Karmokar,1 Anna Staniszewska,1 Emma Dean,3 Simon J. Hollingsworth,4 Barry Davies1. 1 _Oncology, IMED Biotech Unit, AstraZeneca, Cambridge, UK, Cambridge, United Kingdom;_ 2 _Discovery Science, IMED Biotech Unit, AstraZeneca, Cambridge, UK, Cambridge, United Kingdom;_ 3 _TMU, IMED Biotech Unit AstraZeneca, Cambridge, UK, Cambridge, United Kingdom;_ 4 _Oncology Business Unit, AstraZeneca, Cambridge, UK, Cambridge, United Kingdom_.

DNA-dependent kinase (DNA-PK) is a nuclear serine/threonine protein kinase complex that is a key component of the non-homologous end joining (NHEJ) process. DNA-PK plays an important role in the cellular response to DNA damage through the detection and repair of DNA double strand breaks (DSB) and is a critical component of the DNA Damage Response (DDR). DSB can be induced by a range of agents, including chemotherapy, radiation or Poly ADP Ribose Polymerase (PARP) inhibitors such as olaparib, and thus a DNA-PK inhibitor is likely to sensitize to these agents. DNA-PK inhibitors may also be effective as monotherapy in tumors with high endogenous levels of DNA damage resulting from defects in other DNA repair pathways. We have developed a highly potent and selective inhibitor of DNA-PK, AZD7648 (pDNA-PK IC50 in A549 cells = 92 nM). AZD7648 shows broad growth inhibitory activity across a panel of 244 cancer cell lines (GI50 1.3 - 30 µM). Consistent with known synthetic lethal interactions with DNA-PK, AZD7648 shows a 10-13-fold greater growth inhibitory effect in FaDu head and neck and A549 non-small cell lung cancer cell lines with ATM knocked out (KO) by zinc finger nuclease or CRISPR respectively compared to their isogenic wild-type counterparts (WT). This growth inhibition is associated with increased levels of DNA damage as measured by micronuclei formation detected using high-content immunofluorescence (2-fold increase vs DMSO at 2 µM AZD7648). Moreover, a 6-fold increase in chromosomal breaks are detected in the ATM KO cells using metaphase spread analysis (mean breaks/cell: FaDu ATM KO = 1.7, ATM WT = 0.28) In vivo, monotherapy treatment with AZD7648 (75-100 mg/kg bid) inhibited tumor growth in a panel of 14 PDX and 2 xenograft models, derived from breast, lung, ovarian and head and neck cancers. This included models with and without loss of ATM. Treatment with AZD7648 resulted in dose-dependent inhibition of the phosphorylation of DNA-PK (S2056), RPA32 (S4/8) and nuclear γH2AX in FaDu ATM KO xenografts, where AZD7648 75mg/kg inhibited γH2AX and phosphorylation of DNA-PK (S2056) and RPA32 (S4/8) by 71, 98 and 95% respectively at 2 h after dosing. These data confirm that DNAPK inhibition using AZD7648 has potent pharmacodynamic and monotherapy anti-tumor activity in a range of pre-clinical models. This includes, but is not restricted to, models with engineered and endogenous loss of ATM.

#3506

AZD7648, a potent and selective inhibitor of DNA-PK, potentiates activity of the PARP inhibitor olaparib resulting in sustained anti-tumour activity in xenograft and PDX models.

Antonio Ramos-Montoya,1 Jacqueline H. Fok,1 Neil James,1 Valeria Follia,1 Mercedes Vazquez-Chantada,2 Paul Wijnhoven,1 Lenka Oplustil O'Connor,1 Ankur Karmokar,1 Anna Staniszewska,1 Emma Dean,3 Simon J. Hollingsworth,4 Barry Davies,1 Elaine B. Cadogan1. 1 _Oncology, IMED Biotech Unit, AstraZeneca, Cambridge, UK, Cambridge, United Kingdom;_ 2 _Discovery Science, IMED Biotech Unit, AstraZeneca, Cambridge, UK, Cambridge, United Kingdom;_ 3 _TMU, IMED Biotech Unit AstraZeneca, Cambridge, UK, Cambridge, United Kingdom;_ 4 _Oncology Business Unit, AstraZeneca, Cambridge, UK, Cambridge, United Kingdom_.

DNA-dependent kinase (DNA-PK) is a nuclear serine/threonine protein kinase complex and a key component of the non-homologous end joining (NHEJ) process. DNA-PK plays an important role in the cellular response to DNA damage through the detection and repair of double strand breaks (DSB). DSB can be induced by a range of agents, including chemotherapy and radiation. The PARP inhibitor olaparib has also been shown to induce DSB as a consequence of trapping PARP proteins at sites of damaged DNA. Therefore, we hypothesised that DNA-PK inhibitors may combine therapeutically with PARP inhibitors. AZD7648 is a highly potent and selective inhibitor of DNA-PK (pDNA-PK cell IC50 = 92 nM) The combination treatment of AZD7648 with olaparib for 10 - 12 days in vitro leads to at least 20% greater cell growth inhibition compared with either agents as monotherapies in a panel of cell lines with deficiencies in the ATM pathway (e.g. cells lacking ATM protein or where ATM substrates are not phosphorylated after exposure to DSB inducing agents). This effect is also seen in isogenic ATM knock-out (KO) FaDu head and neck and A549 non-small cell lung cancer cell lines. At concentrations of AZD7648 (0.6 - 2 µM) and olaparib (1 µM) that have monotherapy activity in the ATM KO cells but not in their wild-type counterparts (WT), the combination treatment enhanced the G2/M cell cycle arrest caused by olaparib and led to greater levels of micronuclei formation as detected using high-content immunofluorescece assays (mean per cell: FaDu WT = 0.1, FaDu ATM KO = 0.4). This was associated with a larger quantity of chromosomal aberrations in the ATM KO versus WT cells following combination treatment detected by metaphase spread analysis (mean per cell: FaDu ATM KO = 5.5, FaDu WT = 1.2). The same phenotype was observed in A549 ATM KO versus WT cell lines. In vivo, continuous dosing of AZD7648 (75 mg/kg bid) in combination with olaparib (100 mg/kg qd) inhibited the growth of FaDu WT tumours by ~60%. However, in the FaDu ATM KO tumours complete regressions were seen after 70 days of dosing and no re-growth was detected up to 220 days later. Additionally, in PDX models of breast, lung, ovarian and head and neck cancer this combination showed tumour growth inhibition (50-100%) in 13 models and regression in 5 models, only one of these five models being ATM pathway deficient. These data confirm that DNA-PK inhibition using AZD7648 enhances the efficacy of olaparib in vitro and in vivo, providing a clear rationale for its clinical investigation.

#3507

LKB1 deficiency and KEAP1/NRF2 pathway alterations as biomarkers of response for ATR and ATM inhibitors and other inhibitors of DNA damage response (DDR) in NSCLC.

Ana Galan-Cobo, Alissa Pottetee, John V. Heymach. _UT MD Anderson Cancer Center, Houston, TX_.

The serine/threonine kinase STK11 (LKB1) is the second most commonly altered tumor suppressor in NSCLC; however, there are currently no effective treatment strategies for this subset of tumors. KRAS-mutant LKB1 deficient tumors often also have alterations in KEAP1 or NRF2 gene, which activate the NRF2 pathway known to be involved in antioxidant response.

Inhibitors of ATM and ATR, two key proteins in the DNA damage response (DDR) pathway, are currently undergoing clinical testing but there are no biomarkers established for identifying which subgroups of patients are more likely to benefit from treatment. Here we have identified that alterations of LKB1, and the KEAP1/NRF2 pathway, are associated with enhanced response to ATM and ATR inhibitors (AMTi and ATRi) and other inhibitors of the DDR and may be useful biomarkers for predicting therapeutic response.

To investigate the impact of LKB1 loss and KEAP1/NRF2 pathway activation on response to DDR inhibitors (DDRi), we first tested the in vitro activity of ATM inhibitor in NSCLC murine cell lines with or without knock out of LKB1 and/or KEAP1. In these cells, the loss of LKB1 and/or KEAP1 significantly sensitize cells to ATMi AZD0156. In addition, we evaluated the activity of the ATRi AZD6738 in NSCLC cells with or without knockout of LKB1 and/or KEAP1. Cells deficient in LKB1 (KL) and/or KEAP1 (KLK/KK) were more sensitive to AZD0156 and AZD6738 than cells with intact LKB1 and KEAP1. Next, we investigated whether the activity of ATR and ATM inhibitors in KL, KK or KLK tumor cells could be enhanced by the addition of a PARP inhibitor (Olaparib). Although all NSCLC cells were resistant to the PARP inhibitor olaparib when used as a single agent, treatment of LKB1, KEAP1 or LKB1 plus KEAP1 deficient cells with the combination of olaparib plus ATM or ATR inhibitors significantly enhanced the antitumor cell activity of ATM or ATR inhibitors alone. We confirmed these data in an additional panel of LKB1 deficient NSCLC human cell lines (A549, H460 and H2030) treated with a broad spectrum of ATR and ATM inhibitors. In all human cell lines re-expression of LKB1 clearly reduced the sensitivity to ATR inhibition. LKB1 lost was also associated with sensitivity to PARP and ATM inhibitor, although these effects seemed to be less significant compared with ATR inhibitors.

Tumors with LKB1 deficiency or KEAP/NRF2 mutations are typically resistant to standard chemotherapy drugs and immunotherapy. Our data indicate that LKB1 and KEAP1/NRF2 loss significantly enhance the sensitivity to ATR and ATM inhibitors in vitro. Thus, we have identified that NSCLC tumors bearing STK11 or KEAP1/NRF2 mutations are highly sensitive to ATM or ATR inhibitors and that genes may serve as biomarkers for selecting appropriate patients for treatment alone or in combination treatments, such as PARPi or immunotherapy.

#3508

Combination of the Chk1 inhibitor (prexasertib) with a PI3K/mTOR inhibitor (LY3023414) induces synergistic anti-tumor activity in triple negative breast cancer (TNBC) models.

Wenjuan Wu, Greg Donoho, Philip Iversen, Jack Dempsey, Andrew Capen, Mark Castanares, Jennifer Stephens, Karsten Boehnke, Christoph Reinhard Reinhard, Aimee Lin. _Eli Lilly and Company, Indianapolis, IN_.

TNBC, the most aggressive subtype of breast cancer, is characterized by high level of genomic instability, defects in DNA damage response (DDR) and increased replication stress (RS). The current treatment options for TNBC are limited and new approaches are needed. Checkpoint kinase 1 (Chk1) is a key protein kinase that regulates the cell cycle, DNA damage and RS response, and has emerged as an attractive target for anticancer therapy. Prexasertib, an ATP-competitive inhibitor of Chk1 has demonstrated activity in vitro and in vivo across a variety of tumor histologies. Prexasertib is being evaluated in patients with TNBC in a Phase II study sponsored by NCI (NCT02203513). The preliminary results reported in 2016 showed modest single agent activity. PI3K/AKT/mTOR is a critical pathway with key roles on cancer cell survival, homologous recombination repair and drug resistance. Our previous study indicated the expression level of genes related to PI3K/AKT signaling is elevated in prexasertib resistant TNBC patient-derived xenograft (PDX) models. It is hypothesized that inhibiting both the Chk1 and PI3K/AKT pathway may improve prexasertib efficacy. In this study, we evaluated the combination effect of prexasertib with a PI3K/mTOR inhibitor (LY3023414, a selective dual inhibitor of PI3K and mTOR, which is being investigated in Phase 2 clinical trials) in TNBC in vitro and in vivo. The prexasertib/LY3023414 combination induced the synergistic or additive inhibition on cell proliferation in 10 of 12 TNBC cell lines. The prexasertib/LY3023414 combination enhanced DNA damage (γH2AX), replication stress (phospho-RPA32) and proliferation inhibition in MDA-MB-231 cells when compare with single agent treatment. In three TNBC xenograft models (HCC1806, HCC1187 and MX-1) LY3023414 induced 0%, 62% and 24% tumor inhibition; prexasertib induced 94%, 78% and 96% tumor inhibition; and the combination led to 35%, 61% and 21% tumor regression, respectively. The prexasertib/LY3023414 combination also increased the inhibition of both primary tumor growth and spontaneous lung metastasis in a MDA-MB-231 mammary fat pad orthotopic model when compared with the respective single agent activity. The efficacy of prexasertib/LY3023414 combination was further assessed in 38 TNBC PDX models, and the combination showed a synergistic effect in 8 of 38 models and an additive effect in 22 of 38 models. Overall, the combined inhibition of Chk1 and PI3K/mTOR pathway enhanced antitumor activity in TNBC models. Taken together, these data will inform the clinical development of potential combination of Chk1 inhibitor prexasertib with a PI3K/mTOR inhibitor in the treatment of TNBC patients. The safety of this combination is being assessed in an ongoing Phase 1b clinical trial (NCT02124148), which includes an expansion cohort focused on patients with TNBC.

#3509

An unexpected effect of low-dose arsenic on genomic integrity.

Hang Su, Meijun Long, Mi Young Son, Teresa C Marple, Paul Hasty, Chul Soo Ha. _University of Texas Health Science Center at San Antonio, San Antonio, TX_.

Background: Arsenic trioxide is currently used to treat acute promyelocytic leukemia and is known as a cytotoxic agent. Arsenic is also known as a carcinogen. However, a cytoprotective effect of low-dose arsenic (LDA) has also been observed in some studies. Our previous studies have found that pretreatment with LDA before DNA damaging agents such as chemotherapy and radiation therapy protects normal cells without impairing the cancer killing effects. We have demonstrated that intact p53 function is required in this process, and therefore, LDA does not protect cancer cells as most of them have mutated or dysfunctional p53 pathway. We have also reported that LDA helps protect the telomere from enhanced erosion by DNA damaging agents in ConA-activated normal human lymphocytes. As enhanced telomere shortening is associated with genomic instability, we decided to investigate the effect of LDA in genomic integrity.

Methods: Mouse embryonic stem cells (mESCs) with a mutator phenotype in DNA repair pathways (MSH2-/-, Rad51KA and Ku70null) and their wildtype counterparts were utilized for cell viability studies and HPRT minigene mutation assay. Cell viability under different doses of LDA (0-1000nM) and radiation (0-4Gy) was firstly examined by colony formation assay. DNA damage and DNA repair capacity were also measured by comet assay in MSH2-/- mESCs. TailDNA% was analyzed by OpenComet and used to describe the comet feature.

Results: Arsenic had little effect on the cell viability at doses ≤200nM in all tested cell lines. In MSH2-/- mESCs, there was no significant difference between the tailDNA% of cells treated with 200nM LDA (11.41±0.52%) and the control (10.82±0.49%) immediately after radiation. However, LDA treatment significantly decreased the tailDNA% 30 minutes after radiation (4.68±0.35% vs. 7.29±0.51%, Student's test P<0.0014). While HPRT mutation frequency was not detectable in radiation-treated Rad51KA, Rad51wt and MSH2wt mESCs, LDA decreased the fraction of 6-Thioguanine resistant cells after radiation in MSH2-/-, Ku70null and Ku70res mESCs, suggesting fewer radiation-induced mutants were formed after LDA treatment.

Discussion: Our data suggest that brief use of LDA enhances DNA repair activities and provides protection from radiation-induced mutations in mESCs. The inability to correctly repair DNA damage from chemotherapy and radiation therapy has been attributed to the development of therapy related secondary malignancy. Helping maintain genomic integrity with LDA during chemotherapy and radiation therapy may have a role in reducing therapy related secondary malignancy. Further work is in progress to test the potential use of LDA as a genome protector in vivo.

#3510

IDH1-R132H tumor cells are not robustly sensitive to PARP inhibition in a 2-HG dependent manner.

Elia Aguado-Fraile, Christine Hudson, Taryn Sleger, Sebastien Ronseaux, Rohini Narayanaswamy, Sung Choe, Bin Wu, Peter Kalev, Brandon Nicolay. _Agios Pharmaceuticals, Inc., Cambridge, MA_.

Mutations in the metabolic enzymes isocitrate dehydrogenase (IDH) 1 or 2 arise in a variety of malignancies and lead to the production of the oncometabolite (D)-2-hydroxyglutarate (2-HG). The recent approval by the FDA of mutant IDH1 and IDH2 (mIDH1/2) inhibitors in patients with mIDH1/2 relapsed/refractory acute myeloid leukemia (AML) underscores the clinical benefit of blocking production of 2-HG. Parallel investigations have indicated that IDH1/2 mutation leads to a 'BRCAness' phenotype and preferential sensitivity to PARP inhibition, ascribed to increased basal DNA damage and reduced capability of DNA damage repair. Significantly, these effects, and sensitivity to PARP inhibition, have been proposed to be 2-HG-dependent and thus potentially antagonistic with inhibition of mIDH1/2. In an effort to better understand the potential use of PARP inhibitors in mIDH cancers, we examined the effect of IDH mutations on the induction of DNA damage and regulation of DNA repair in IDH1-R132H mutant and wild type cell lines. We analyzed the levels of DNA damage by visualization of γH2AX by immunofluorescence and western blot and found, in agreement with previous work, that the presence of mutations in IDH1 correlates with higher basal DNA damage levels. However, in contrast to previous observations, we found that the reduction of 2-HG by treatment with mIDH1 inhibitors in vitro does not reverse this phenotype. Additionally, we observed that the presence of an IDH1 mutation provides reduction in homologous recombination efficiency, though not to the same extent as a cell lacking BRCA2. Furthermore, we failed to detect a heightened sensitivity to PARP inhibition alone in mIDH1 cells in cell-based assays as compared with the reduced viability observed for cells lacking BRCA2 when treated with PARP inhibitors. Similarly, we failed to observe any tumor growth inhibition in mIDH1 mouse xenografts treated with a PARP inhibitor. To account for this disconnect we examined large, publicly-available data sets to look for a 'BRCAness' genomic signature in AML, low grade glioma, and cholangiocarcinoma - all tumor types with a prevalence of IDH1 mutations. While we found a very minor fraction (<3%) of low-grade glioma carrying a mutational signature associated with DNA mismatch repair activity, we failed to find a 'BRCAness' signature in any of the three primary mIDH1 indications. Taken together, our data demonstrate that, while mIDH1 cells have increased basal levels of DNA damage, the reduction of 2-HG by selective mIDH1 inhibitors does not reduce the basal DNA repair deficiency in mIDH1 cells. Furthermore, the increase in DNA damage found in mIDH1 cells does not lead to heightened sensitivity to PARP inhibition in vivo.

#3511

Exploration of pre-clinical relationships between pharmacokinetics, pharmacodynamics and tumor volume for the novel DNA-PK inhibitor AZD7648.

Michael Davies,1 Joost de Jongh,2 Emma Dean,3 Jacquelline H. Fok,4 Frederick W. Goldberg,5 Neil James,4 Ankur Karmokar,4 Antonio Ramos-Montoya,4 Anna Staniszewska,4 Andy Sykes,1 Tamara van Steeg,2 Elaine Cadogan4. 1 _DMPK, Oncology, IMED Biotech Unit, AstraZeneca, Cambridge, United Kingdom;_ 2 _LAP &P Consultants BV, Leiden, Netherlands; _3 _TMU, Oncology, IMED Biotech Unit, AstraZeneca, Cambridge, United Kingdom;_ 4 _Bioscience, Oncology, IMED Biotech Unit, AstraZeneca, Cambridge, United Kingdom;_ 5 _Medicinal Chemistry, Oncology, IMED Biotech Unit, AstraZeneca, Cambridge, United Kingdom_.

AZD7648 is a potent and highly selective inhibitor of DNA-dependent protein kinase (DNA-PK) that has been nominated for clinical development. DNA-PK is a nuclear serine/threonine protein kinase complex involved in DNA damage repair, and a key component of the non-homologous end joining repair mechanism of double strand breaks (DSBs). This work aimed to explore the relationships between pharmacokinetics (PK), pharmacodynamics (PD) and xenograft tumor volume from pre-clinical studies, in order to define PD requirements for pre-clinical efficacy, and to estimate a target clinical dose for AZD7648 in combination with DSB-inducing agents such as olaparib or pegylated liposomal doxorubicin (PLD). A population-based modelling approach was used to explore the PK of AZD7648 in mice. The PK model was developed using data from full PK profiles (multiple longitudinal samples per mouse), and validated against terminal sample PK data. The potential influences of strain-dependence, time non-linearity, and interaction with olaparib on the pharmacokinetics of AZD7648 were investigated. Direct and indirect inhibition PD models were fitted to the responses of biomarkers describing target engagement (pDNA-PK) or proximal downstream effects (pRPA32 (S4/8) and γH2AX). A compartmental model accurately described AZD7648 PK in mice, with rapid absorption, dose-proportional PK across the range of doses tested, time-independent parameters and no effect of olaparib co-dosing on AZD7648 PK. The PD of proximal target engagement biomarkers were best described with an Imax model with very rapid turnover (<10 minutes), which showed there was negligible delay (due to tumor distribution or pharmacology) and effectively a direct relationship between systemic PK and xenograft biomarker inhibition. Across a number of FaDu ATM KO and BT474c xenograft tumor studies, the duration of cover over IC90 correlated with efficacy in combination with olaparib or PLD, demonstrating the importance of inhibiting DNA-PK for an extended duration in each dosing period. This result was applied to define a target level and duration of PD inhibition, and, when combined with predicted human PK behaviour, a target clinical dose for AZD7648 in combination with DSB-inducing agents to inform the clinical investigation of AZD7648.

#3512

**AZD7648, a potent and selective inhibitor of DNA-PK, potentiates the activity of ionising radiation and doxorubicin** in vitro **and causes tumour regression in xenograft models.**

Jacqueline H. Fok,1 Antonio Ramos-Montoya,1 Neil James,1 Mercedes Vazquez-Chantada,2 Valeria Follia,1 Ankur Karmokar,1 Anna Staniszewska,1 Lenka Oplustil O'Connor,1 Emma Dean,3 Simon J. Hollingsworth,4 Barry R. Davies,1 Elaine B. Cadogan1. 1 _Oncology, IMED Biotech Unit, AstraZeneca, Cambridge, United Kingdom;_ 2 _Discovery Science, IMED Biotech Unit, AstraZeneca, Cambridge, United Kingdom;_ 3 _IMED Biotech Unit, AstraZeneca, Cambridge, United Kingdom;_ 4 _Oncology Business Unit, AstraZeneca, Cambridge, United Kingdom_.

DNA-dependent kinase (DNA-PK) is a nuclear serine/threonine protein kinase complex that is a key component of the non-homologous end joining (NHEJ) process. DNA-PK plays an important role in the cellular response to DNA damage through the detection and repair of DNA double strand breaks (DSB). Cancer therapies such as ionising radiation (IR) or topoisomerase II inhibitors (doxorubicin) generate DSB which can be repaired by homologous recombination (HR) or non-homologous end-joining (NHEJ). It can therefore be hypothesised a DNA-PK inhibitor would potentiate the activity of these agents. We have developed a highly potent and selective inhibitor of DNA-PK, AZD7648, which inhibits IR-induced DNA-PK S2056 auto phosphoryalation with an IC50 = 92 nM in A549 non-small cell lung cancer (NSCLC) cells. AZD7648 is a potent radiosensitiser where treatment in combination with IR led to a concentration-dependent reduction of the colony survival capacity of A549 and H1299 NSCLC cells (DEF37 at 100 nM = 1.7 and 2.5, respectively). In A549 cells, AZD7648 (≥1 µM) in combination with 2Gy IR for 48 hours led to a significant accumulation of cells arrested in the G2/M of the cell cycle, a 4-fold increase in micronuclei formation, and 3-fold induction of γH2AX, pATM S1981 and 53BP1 foci formation compared with IR alone. AZD7648 was also found to combine synergistically with doxorubicin in a panel of ovarian and triple negative breast cancer (TNBC) cell lines in cell growth inhibition assays when applying the Loewe additivity model (synergy scores 4 - 35). In vivo the combination of AZD7648 with IR (5x 2Gy) induced tumour regression in H1299 and A549 NSCLC xenografts in a dose-dependent manner (84 and 11% regression respectively), while monotherapy treatment only achieved tumour growth inhibition. In these two models the increased activation by IR of three primary DNA-PK pharmacodynamic markers, pDNAPK (S2056), pRPA32 (S4/8) and γH2AX, was inhibited by AZD7648 treatment (70-90% inhibition 2 h after IR + AZD7648). Similarly, liposomal doxorubicin (2.5 mg/kg weekly) in combination with AZD7648 (37.5 mg/kg bid) induced tumour regressions in the BT474c ER+ breast cancer xenograft model and in a TNBC PDX model (63% and 33% regression respectively), while monotherapy treatments only achieved tumour growth inhibition. These data confirm that DNA-PK inhibition with AZD7648 enhances the efficacy of a range of DSB inducing agents in vitro and in vivo, providing a clear rationale for its clinical investigation.

#3513

**The CHK1 kinase inhibitor prexasertib (LY2606368) shows potent single-agent efficacy in** in vitro **and** in vivo **models of castrate-resistant prostate cancer.**

Ann McNulty, Greg Donoho, Jack Dempsey, Adem Abel, Jennifer Stephens, Ricardo Martinez, Damien Gerald, Carole Perruzzi, Marguerita O'Mahony, Christoph Reinhard, Aimee Lin, Wenjuan Wu. _Eli Lilly and Company, Indianapolis, IN_.

Reported genomic alterations in the DNA repair pathway in studies of castrate-resistant prostate cancer (CRPC) patients have prompted interests in targeted therapies related to the DNA damage response (DDR). CHK1 kinase plays a critical role in mediating the DNA damage checkpoint response by contributing to orderly cell cycle progression, regulation of the mitotic spindle-assembly checkpoint response and is essential for homologous recombination repair. The CHK1 kinase (CHK1) inhibitor prexasertib (LY2606368) is currently in clinical development. In this study we investigated the efficacy of prexasertib as single agent in CRPC in vitro cell lines and in vivo tumors. Prexasertib inhibited cell proliferation of prostate cancer cell lines showing the more consistent and potent inhibition in androgen-receptor positive (AR) + models (n=5, IC50 value ranges from 4.3 to 13.1 nM) while AR- cell lines had IC50 values ranging from 6.4 nM to 1000 nM (n=8). A sub-set of cell lines including VCAP, LNCaP, 22RV1 and PC3 underwent additional in vitro studies including cell cycle regulation and programmed cell death induced by prexasertib. A 24-hour treatment with 50 nM prexasertib increased S-phase populations in all cell lines (VCAP, LNCaP, 22RV1 and PC3) and sub-G1 populations in VCAP and 22RV1 cells. Live-cell imaging showed 50 nM prexasertib triggered caspase 3/7 induction by 30, 19, 15 and 10 fold change in VCAP, 22RV1, LNCaP and PC3, respectively when compared with control. Western blot studies characterized the activation of the DDR pathway signaling giving rise to a time- and concentration-dependent DNA damage response leading to induction of pCHK1 (S345), γH2AX, pRPA32(S4/8) and PARP cleavage. Importantly, in all three AR+ cell lines, prexasertib yielded both time- and concentration-dependent inhibition of AR-full length and AR-variant7 expression. In addition, prexasertib demonstrated similar single-agent activity in prostate cancer patient-derived organoid (PDO) models by inhibiting proliferation and increasing apoptosis. Finally, in vivo n=1 studies of six patient-derived xenograft (PDX) models which represent heavily pretreated mCRPC patients yielded single-agent prexasertib efficacy in 4/6 models. Similarly, xenograft models including 22RV1, LNCaP and PC3 also yielded single-agent efficacy compared to vehicle groups. On-going efforts continue to deepen the understanding of the mechanisms underlying the action of prexasertib and potential markers for drug response by using RNA/gene arrays. In conclusion, prexasertib yielded potent single-agent activity in preclinical studies including cancer cell lines, PDO, xenograft and PDX models of castrate-resistant prostate cancer and these data provide rationale of the development of the prexasertib for the treatment of CRPC.

#3514

RANBP9 presence affects levels of Tip60 and activated p53 in lung cancer cells in response to DNA damage.

Shimaa Soliman,1 Arturo Orlacchio,1 Anna Tessari,1 Marina Capece,1 Rosa Visone,2 Carlo Croce,1 Dario Palmieri,1 Vincenzo Coppola1. 1 _Ohio State Univ., Columbus, OH;_ 2 _Universita' G. D'Annunzio, Chieti, Italy_.

Lung cancer is the leading cause of cancer-related deaths. Despite recent success with targeted and immune-therapies, DNA-damaging agents, such as platinum-based drugs, still represent the first-line of treatment. However, side effects and drug resistance limit their efficacy and use. We recently demonstrated that RAN Binding Protein 9 (RANBP9) plays a critical role in the DNA-damage response (DDR) of lung cancer cells. However, how it affects the mechanisms and outcome of the DDR is not known. In this regard, p53 activity is crucial for cell cycle arrest or programmed cell death. In particular, acetylation of p53 at lysine 120 by the Tat-Interacting Protein of 60 KDa (Tip60) is dispensable for p53-mediated growth arrest but is essential for p21 induction and p53-dependent apoptosis. It has been reported that RANBP9 and Tip60 co-localize. Here, we investigate whether RANBP9 affects Tip60-dependent activation of p53 and its relevance in deciding cell faith in response to DNA damage. For our study we used H1299, A549 and HeLa cells. By CRISPR/Cas9, we generated RANBP9 wild type (WT) and knockout (KO) A549 clones. We also established KO clones stably re-expressing RANBP9-FLAG. Cell response to DNA damage was assessed upon treatment with Ionizing Radiation (IR) and/or Cisplatin (CDDP). Interaction between RANBP9 with Tip60 was assessed by immunoprecipitation of transfected HeLa cell lysates. We show that RANBP9 and Tip60 co-immunoprecipitate either by pulling-down RANBP9 or Tip60. Using different deletion mutants of Tip60, we found that a region encompassing the Zinc Finger and part of the Histone Acetyl-Transferase domain is required for the interaction with RANBP9. Further, the enzymatic-dead Tip60 retained its ability to interact with RANBP9. Using p53 null H1299 cells, we established that this interaction is independent of p53. However, RANBP9 depletion affects both total and activated p53 (p-p53s15) levels after DNA damage. Re-expressing RANBP9 in stable KO clones rescued p53 levels. Upon CDDP treatment, p53 protein levels were higher in the clones stably re-expressing RANBP9 compared to the KO ones. Upon IR or CDDP treatment, p21 protein levels were higher in the presence of RANBP9. Finally, we found that Tip60 levels were higher in CDDP-treated and untreated WT cells comparing to the KO ones. Our results indicate that RANBP9 and Tip60 constitutively interact. The decrease of p53 in RANBP9 absence suggests that RANBP9 might favor p53 acetylation by Tip60 upon DNA damage. Further investigations will elucidate how RANBP9 affects the levels and the functions of Tip60. Also, we will measure the levels of p53-AcK120 both in the WT and KO clones. If p53-AcK120 is impaired when RANBP9 is absent, RANBP9 KO cells might undergo cell death instead of cell cycle arrest. Altogether, our results shed light on a potential RANBP9-Tip60-p53 axis at play during the DDR. 

### High-Throughput Sequencing

#3515

Functional antigen presentation is required to interpret the tumor mutation burden (TMB) test.

Gustavo C. Cerqueira, Laurel A. Keefer, Donna Nichol, Julie R. Meyer, Nicholas C. Dracopoli. _Personal Genome Diagnostics, Baltimore, MD_.

Therapeutic response to checkpoint inhibitors requires a prior, suppressed immune response that is released by a monoclonal antibody blocking the interaction of the checkpoint receptors with their cognate ligands. The presence of this suppressed immune response can be detected by a variety of tests including tumor mutation burden (TMB), tumor infiltrating leukocytes, T-cell receptor clonality, circulating interferons and cytokines, and upregulation of the expression of the checkpoint ligands. Today, TMB has proven to be the most predictive biomarker of response to immunotherapy.

TMB is a surrogate marker of immune response. The test works by sampling a small region of the cancer genome to estimate the number of mutations/Mb of the cancer exome. A high TMB score is associated with better response to immunotherapy because it carries more somatic mutations and a higher chance of presenting an immunogenic neoepitope that will lead to CD-8+ T-cell mediated destruction. Restriction or loss of antigen presentation caused by mutations and loss of heterozygosity (LOH) of the beta-2-microglobulin (B2M) and HLA Class I genes has been shown to be a common means of evading CD-8+ T-Cell destruction. Consequently, the outcome for high TMB tumors will be dependent on their ability to present antigens, so that the mutation count will not matter in cells that are unable to present antigens in their HLA Class I/B2M complex.

We propose that TMB should be considered together with the ability of the tumor to present these putative neoantigens. To test this hypothesis, we used the PGDx elio™ Tissue Complete test (507 genes in 1.3 Mb) to measure TMB and antigen presentation in the same assay. We tested 212 cancer patients and showed that in FFPET samples with >20% tumor content, we could detect LOH of the MHC Class I and B2M genes with >90% accuracy compared to whole exome data from the same sample. These data confirmed our hypothesis that it was possible to measure TMB and evaluate the genes involved in antigen presentation in the same in silico analysis of a 507 gene next-generation sequencing (NGS) panel. We are currently using this combined analysis to measure TMB and somatic alterations of the antigen presentation complex in several different cancers and to test the hypothesis that adding neoantigen presentation capability to TMB scores will significantly improve prediction of response to a variety of immunotherapies.

#3516

A genomics model to predict immune-related adverse events in cancer patients treated with checkpoint inhibitors.

Emma C. Scott,1 Dickran Kazandjian,1 Luis Santana-Quintero,1 Tigran Ghazanchyan,1 Svetlana Petrovskaya,1 Yong Zhang,1 Amy Rosenberg,1 V. Ashutosh Rao,1 Jennifer L. Marte,2 Gideon M. Blumenthal,3 Marc R. Theoret,1 Richard Pazdur,3 James L. Gulley,2 Julia A. Beaver1. 1 _FDA-CDER, Silver Spring, MD;_ 2 _NIH-NCI, Bethesda, MD;_ 3 _FDA-OCE, Silver Spring, MD_.

The overall objective of this study is to use next-generation sequencing technology and bioinformatics to better inform the safety of immunotherapy treatment for cancer. Cancer patients commonly develop immune-related adverse events (irAEs) during and after treatment with checkpoint inhibitors. These irAEs can be serious or even fatal. Therefore, a biomarker for prediction of irAE development could have utility for heightened surveillance, personalized therapy decisions, and regulatory evaluation of drugs. Due to the similarity between irAEs and autoimmune diseases and the high heritability of autoimmune diseases, we hypothesized that certain patients could have a genetic predisposition for developing irAEs. To test this hypothesis, we conducted whole exome sequencing on an Illumina NextSeq to interrogate germline genomes of solid tumor patients (n=50) treated with an anti-PD-L1 antibody (NCT01772004). Relevant clinical data, such as adverse events and irAE classification, were provided for this retrospective analysis; twenty percent of patients (10/50) had irAEs. A preliminary germline genetic model of irAEs was constructed using short variant calls from this initial training set. This was generated using a proprietary algorithm that implements a Monte-Carlo simulation expansion of Fisher's regularized linear discriminant analysis (RLDA) in a multidimensional measurement system to create a model that maximizes separation between two groups. This model consists of 131 genes, each of which make a relatively small contribution to the overall signature. The ten genes with the highest contribution coefficients together account for 21% of the signature. Ingenuity Pathway Analysis (IPA) identified a network associated with infectious diseases, antimicrobial response, and inflammatory response that contains 21 interconnected genes from the signature. IPA also revealed that genes in the signature have a variety of molecular and cellular functions, the most significant of which are cell death and survival, cellular movement, and cell-to-cell signaling and interaction. This model has 100% sensitivity, specificity, and accuracy on the training set. Future directions will test the performance of this putative genomic model on a new dataset to assess the validity and utility of the model as a predictive biomarker to identify patients at risk for developing irAEs in response to checkpoint inhibition.

#3517

Establishing reference samples for universal benchmarking study of NGS technologies.

Li Tai Fang,1 Wenming Xiao,2 Somatic Mutation Working Group of the SEQC-II Consortium. 1 _Roche Sequencing Solutions, Belmont, CA;_ 2 _Center for Devices and Radiological Health, FDA, Silver Spring, MD_.

Regulatory authorities including the FDA are well aware of the crucial role Next Generation Sequencing (NGS) plays in precision medicine. However, the lack of thoroughly-characterized and community-validated reference samples creates a challenge for the development and review of NGS applications in precision oncology.

Over 200 members from over 60 institutions have formed the Somatic Mutation Working Group within the FDA-led Sequencing Quality Control Phase II (SEQC-II) Consortium to address this challenge. We have developed strategies to establish a community standard reference samples. We used multiple orthogonal sequencing technologies, sequencing replicates, sequencing centers, and bioinformatics analysis pipelines for mutation detection, thus minimizing biaes related to any particular sequencing platform, assay, or informatics software. Combining with targeted sequencing and microarray technologies, we established a high-confidence mutation call set (Gold Set). Here, we present our initial reference samples: a human triple-negative breast cancer cell line (HCC1395), and matched B lymphocyte derived normal cell line (HCC1395BL). The first public release of the Gold Set includes the fully phased germline variants, high-confidence somatic single nucleotide variants (SNV), small insertion and deletions (InDel), copy number variations (CNV), and structural variations (SV).

For variant discovery, we have performed whole genome sequencing (WGS) for each of the tumor-normal genomes to combined depths of 650X from 12 replicates on Illumina HiSeq sequencers, 380X from 9 replicates on Illumina NovaSeq sequencer, 1000X from 11 replicates on 10X Genomics platform, and 50X on PacBio sequencer. In addition, we have also performed Hi-C sequencing to 34X. For variant validation, we have performed WGS to 300X for seven different tumor purities in a tumor-normal titration series, whole exome sequencing (WES) to a total of 12000X from 14 replicates, AmpliSeq targeted sequencing to 1000X, RNASeq to 186 million reads, Affymetrix GeneChip array to 2.1 million probes, and Cytogenetic arrays to 4.3 million probes.

HCC1395 has a highly rearranged near-triploid genome with 66 chromosomes. The confidence levels of somatic mutations in the Gold Set were obtained based on the cross-institution and cross-aligner reproducibility of each mutation call. There are approximately 40,000 and 2,000 high-confidence somatic SNVs and InDels, with variant allele frequencies (VAF) ranging between 2% and 100%. The VAFs of both somatic and germline variants are consistent with our CNV calls. We will continuously accept input from the community to advance accuracy and completeness of our Gold Set for the reference samples in order to serve as a community standard well into the future.

#3518

SPARC related signaling genes in non-small cell lung cancer by RNA-sequencing transcriptome analysis.

Sajani S. Lakka, Bhavesh K. Ahir. _Univ. of Illinois at Chicago, Chicago, IL_.

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, accounting for about 80 % cases of lung cancer and is the leading cause of cancer-related death in the United States and worldwide. SPARC (secreted protein acidic and rich in cysteine), a multicellular non-structural glycoprotein is known to be involved in the regulation of cell adhesion, cell proliferation as well as in tumorigenesis and metastasis in various cancers including NSCLC. SPARC contributes to matrix remodeling and turnover and is involved in modulating the tumor microenvironment. Previous studies have suggested that SPARC can be used as a prognostic factor for NSCLC and represents a highly promising therapeutic modality. In this study we used high-throughput RNA-sequencing transcriptome profiling analysis to delineate pathways involved in SPARC signaling. We assessed differentially expressed genes, novel genes, biological processes and biological signaling pathways in SPARC expressed NSCLC cell line. We identified a total of 3,280 upregulated and 1,608 downregulated differentially expressed genes with known functions in SPARC expressed NSCLC cells. KEGG enrichment analysis indicated that the differentially expressed genes were involved in cellular metabolic processes, regulation of biological processes or biological signaling pathways like cell cycle, spliceosome, apoptosis and WNT signaling pathway. The RNA-seq analysis further revealed that genes involved in apoptosis were significantly altered after SPARC modulation to NSCLC cells. Co-transfection experiments using SPARC shRNA indicated that SPARC modulation induced apoptosis via regulating PI3K/AKT signaling pathway. In summary, this study uncovers SPARC dependent regulatory mechanisms in NSCLC.

#3519

High-throughput unbiased microwell-based single-cell CNV detection reveals tumor clonal subtypes in hepatocellular carcinoma .

Liang Wu,1 Yuzhou Wang,1 Miaomiao Jiang,2 Shiping Liu1. 1 _BGI-Shenzhen, Shenzhen, China;_ 2 _School of Biological Science and Medical Engineering, Southeast University, Nanjing, China_.

Single-cell genomics is critical for understanding cellular heterogeneity in cancer, but existing DNA amplification and library preparation methods are expensive and introduce coverage bias during sample pre-amplification. Based on a massively parallel 5184-microwell chip, we developed a high-throughput unbiased single-cell DNA library preparation approach up to 1800 cells for copy number variation (CNV) detection, which provides a comprehensive, scalable solution for revealing genome heterogeneity. Compared with existing methods, it is more efficient and cost-effective to construct a barcoded-single-cell library without pre-amplification in 2 days. Besides, it has significantly lower Median Absolute Pairwise Difference (MAPD:0.17±0.004) values which are used to evaluate the amplification biases and noises, than using MDA (multiple displacement amplification) method (MAPD: 0.79±0.23) or MALBAC (multiple annealing and looping-based amplification cycles) approach (MAPD : 0.24±0.002). We further successfully applied this approach to obtain single-cell CNV information of more than 500 cells from a tumor sample of hepatocellular carcinoma in one run. Using the unsupervised clustering, we identified at least two clonal subpopulations with a multitude of non-integer alterations across the genome. Overall, this is an unbiased, effective, high-throughput and low cost approach to figure out tumor heterogeneity and understand clonal evolution in tumor at the single-cell level.

#3520

Detection of low-frequency variants in highly degraded DNA and RNA samples.

Ariel Royall, Ushati Das Chakravarty, Katharine Dilger, Manqing Hong, Kevin Lai, Kristina Giorda, Keith Bryan, Yu Wang, Lynette Lewis, Scott Rose, Yu Zheng. _Integrated DNA Technologies, Redwood City, CA_.

Diagnostic tools based on next generation sequencing are fundamentally transforming clinical oncology. However, there is a lack of adequate library preparation strategies for highly degraded, clinically relevant samples, such as cell-free DNA (cfDNA) and formalin-fixed paraffin-embedded (FFPE) DNA. Due to the extreme heterogeneity of these sample types, targeted sequencing is often used to achieve deep coverage of genomic loci and enable detection of low-frequency variants. Commercially available protocols for library preparation require stringent size-selection to remove adapter-dimers, which reduces library complexity and variant detection power. Achieving high specificity can be challenging because low-frequency artifacts arise from a variety of sources, including DNA extraction, library construction, PCR, hybrid selection, and sequencing. These artifacts can be identified by "duplex sequencing", where strand-specific unique molecular identifiers (UMIs) are used to confirm the presence of an alteration on both strands of an input molecule. However, duplex sequencing typically delivers low conversion rates with degraded samples due to poor ligation efficiency and template loss during size-selection. Here, we present the IDT library preparation kit optimized for low-input and degraded samples. Our novel library construction chemistry relies on an engineered DNA ligase and proprietary duplexed sequencing adapters that prevent chimeras, suppress dimer-formation (negating the need for size-selection), and enhance variant calling sensitivity. We adapted the workflow for both DNA and RNA applications and demonstrated efficacy using diverse sample types. To assess sensitivity, we created libraries with varied inputs using mixtures of Genome in a Bottle gDNA (NA12878 and NA24385) and performed hybrid capture using a 52 kb custom panel targeting: single nucleotide variants (SNVs), copy number variants (CNVs), and gene fusions. When compared to commercially available methods, our approach yielded a 1.5- to 4-fold increase in library complexity with improved sensitivity to 0.25% variants using 1-25 ng of cfDNA, and 0.5% using 25-250 ng FFPE DNA. We also obtained 100% specificity using duplexed UMI correction, which removed all false-positive calls. RNA libraries were constructed from FFPE NGS reference standards to evaluate the detection of ALK, RET, ROS, NTRK1, and NTRK3 fusions and sequenced to an average target depth of 10,000X. Our method provides superior sensitivity and specificity for detection of low-frequency variants, even with highly degraded DNA.

#3521

Critical Assessment of CNV Calling Using Next Generation Sequencing.

Yun-Ching Chen,1 Fayaz Seifuddin,1 Cu Nguyen,2 Chunhua Yan,2 Qingrong Chen,2 Wenming Xiao,3 Mehdi Pirooznia,1 Daoud Meerzaman2. 1 _National Institutes of Health, Bethesda, MD;_ 2 _National Institutes of Health, Rockville, MD;_ 3 _U.S. Food and Drug Administration, Jefferson, AR_.

Tumor-derived Copy Number Variations (CNVs) are among the most important genomic aberrations in cancers. Since a CNV amplification is often associated with oncogene activation, and a CNV deletion is frequently attributed to the gene inactivation, an accurate detection of CNVs plays a crucial role in cancer diagnosis and treatment. In this study, using the next generation sequencing, we systematically interrogated somatic CNVs in paired tumor-normal cell lines to identify factors affecting detection reproducibility and accuracy. Whole-genome and whole-exome sequencing were carried out at six sequencing centers followed by processing with six CNV callers to evaluate their reproducibility. Different types of samples with varying input amount and tumor purity were processed using multiple library construction protocols and read depths. We compared the consistency of reference replicates, the effect of experimental, technical, and biological confounding factors, and benchmarked our callers using cytogenetic array. Our evaluation was based on two approaches; bin-based and gene-based approach. The bin-based comparison used a sliding window of 10 Kb, while gene-based comparison used segments at the gene level. Our evaluations indicate variations among CNV calls are mainly driven by callers and less by the sequencing centers. In addition to caller effect, PCA analyses also show site-specific effect, largely pairwise distances on the PC plot for pairs between different centers than for those within the same centers. Among confounding factors, namely FFPE, library concentration, and tumor purity with several sequencing depths, we report that tumor purity has a dominant and major effect in caller's performance. PCA analysis shows low tumor purity (<= 50%) has a large effect on CNV calls which are evidenced by low purity samples being away from reference samples on PC plots for all callers. Tumor purity <= 50% leads to severe missing calls and lower copy numbers in amplified regions while sequencing depth seems to affect the outcome. We illustrated that most of the other confounding factors such as WES, FFPE, and LBP are caller-specific. Comparing to other factors, the effect of low sequence depth seems to be minimal. FFPE also affects CNV calls by increased variability in several callers. Consensus call and slightly stringent cutoffs for calling amplification/deletion can produce high confidence calls. Comparing to cytogenetic array, we found high correlations between the array and WGS segments in some callers while other callers show large variability.

#3522

Enzymatic DNA repair enables high quality library preparation and accurate sequencing from highly damaged DNA inputs.

Margaret R. Heider,1 Lixin Chen,2 Chen Song,2 Luo Sun,2 Pingfang Liu,2 Laurence Ettwiller,2 Lauren Higgins,1 Eileen Dimalanta,2 Theodore Davis,2 Thomas Evans2. 1 _New England Biolabs, Inc., Ipswich, MA;_ 2 _New England Biolabs, Inc., Ipwsich, MA_.

High-throughput sequencing is key for clinical diagnostic applications such as disease mutation profiling and identifying pathogens with high taxonomic specificity. Preparation of high quality next-generation sequencing (NGS) libraries is critical for obtaining accurate patient data, but clinical scientists are often limited by input DNA quality. In cancer genomics, formalin-fixed, paraffin-embedded (FFPE) tissue from patient biopsy is a common source of DNA, but the extracted DNA is often poor in both yield and quality due to fixation- and storage-induced damage. Additionally, these damage-induced sequencing artifacts raise the background level of mutations, making it difficult to discern true, low frequency, disease-related variants from noise. Even relatively high-quality DNA inputs contain nicks and gaps limiting library yields and read lengths for long read sequencing platforms including Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PacBio).

We developed a second-generation DNA repair enzyme mix (V2) that efficiently repairs the most prevalent damage types found in FFPE DNA and further improves the quality and yield of NGS libraries through efficient nick and gap repair. Illumina library yield and quality were improved when DNA samples were treated with FFPE DNA Repair V2 before library preparation. Target enrichment and whole exome enrichment experiments showed that the V2 repair mix did not alter the overall frequency of variants identified, thus it did not introduce bias, but significantly improved the sequencing accuracy by reducing the number of false variant calls due to damage-induced sequence artifacts. True mutation frequencies were validated using droplet digital PCR (ddPCR). Repaired ONT libraries using DNA inputs of varying quality also showed improved library quality as well as greater read lengths. Therefore, enzymatic DNA repair prior to NGS library preparation is a critical first step for improving the quality and accuracy of short- and long-read sequencing libraries.

#3523

A high-throughput screening strategy for the identification of novel lymphoproliferative elements.

Laurence Jadin,1 Hiba Shaban,2 Anirban Kundu,3 Gregory Schreiber,2 Scooter Willis,2 Farzad Haerizadeh,1 James Onuffer,1 Gregory Frost2. 1 _F1 Oncology, San Diego, CA;_ 2 _F1 Oncology, West Palm Beach, FL;_ 3 _Exuma Biotechnology SEZC, Georgetown, Cayman Islands_.

Introduction - Chimeric Antigen Receptor (CAR) T-cell therapy is effective against certain leukemias and lymphomas and shows promise for other incurable malignancies. Considerable challenges remain however to expand this platform technology beyond transplant-oriented hospital care. Centralized manufacturing of genetically modified T cells, lymphodepleting chemotherapy and patient management of current CAR-T therapies are associated with significant costs and treatment complexity. As a first step to reduce this treatment complexity, the present study describes a high throughput combinatorial domain library screening method to identify synthetic lymphoproliferative elements capable of driving in vivo expansion and survival of CAR-T cells in a lymphoreplete host without the homeostatic proliferation signals generated by lymphodepleting chemotherapy.

Methods - High-diversity semi-rationally-designed combinatorial libraries of putative lymphoproliferative protein subdomains were DNA barcoded and assembled into a lentiviral vector co-expressing a ROR2-targeted CAR. Human PBMC were transduced with the library and cultured in vitro for several days. Purified cells were injected into mice bearing xenograft tumors modified to express the ROR2 antigen and compared to unmodified xenograft controls. The expansion rate of integrated cells was monitored weekly by quantitative PCR and, after 21 days of exposure, genomic DNA was isolated from blood, spleen and xenograft tumor tissues. Enriched barcodes were amplified using PCR and amplicons were subjected to HiSeq Next-Generation Sequencing. Barcode decoding was achieved using PacBio long read sequencing analysis to align full-length construct sequences with barcode quantitation.

Results - Using this approach, putative CAR-T cell driver candidates and common key protein subdomains were identified that support selective in vivo expansion and survival of human lymphocytes in a tumor-bearing mouse model.

Conclusion - Taken together, these results demonstrate that a high throughput combinatorial screening strategy with quantitative bioinformatics is a viable method for identifying protein domain combinations capable of selectively driving human CAR-T cells in vivo. These small synthetic combinatorial protein domains may facilitate lymphodepleting chemotherapy-free regimens and lower CAR-T cell doses in the future.

#3524

Whole-exome sequencing of esophageal squamous cell carcinoma identifies recurrent mutations in Chinese subjects.

Meng Liu,1 Lin Feng,2 Hai yin An2. 1 _Cancer Research Institution, Urumqi, China;_ 2 _State Key Laboratory of Molecular Oncology, Beijing, China_.

Background and Aims:

Epidemiological and etiological studies have shown that environmental and genetic factors play vital roles in esophageal carcinogenesis. However the genetic risk factors of esophageal squamous cell carcinoma (ESCC) still remain unclear. Here, we aimed to identify the recurrent mutations of ESCC in Chinese Uygur subjects from the China Xinjiang high-risk region by using a whole-exome sequencing method.

Materials and Methods:

We collected 50 Chinese Uygur patients with ESCC tumors and matched blood samples and adjacent normal oesophageal tissues (≥5cm from tumour site) at Xinjiang tumor hospital in China. Informed consent was obtained from each participant, and this study was approved by the Institutional Review Board of the Xinjiang tumor hospital. All the individuals underwent oesophagectomy and received no chemotherapy or radiotherapy before surgery. Whole-exome sequencing analysis (100bp) of DNA samples from 50 ESCC was performed on the Illumina HiSeq X Ten System.

Results:

We obtained 353,56 SNVs and 1157 indels in the exonic regions of 50 ESCC samples. Besides, we found the predominant mutations were the C>T and C>A transitions at TpCpW trinucleotide sites. And Three ESCC signatures were derived, which are highly similar to validated Signatrue-1, Signature-5 and Signature-13 in Cosmic. We identified four significantly mutated genes including TP53, CDKN2A, LGALS3 and C16orf3 (q<0.5). Moreover, the aberrant pathways including the receptor tyrosine kinases (RTK/RAS/ phosphoinositide-3 kinase (PI3K)), NOTCH, WNT, Hippo, TP53 and cell cycle were identified.

Conclusions:

We provided mutated genes and disrupted pathways in ESCCs. These findings might be used to specific treatments for esophageal squamous cell carcinoma in Chinese Uygur subjects.

Acknowledgements: This work was supported by the Research Fund of Key Laboratory of Xinjiang oncology (No. 2017D04006) and Outstanding Youth Science and technology training project fund of Xinjiang (No. 2017Q058).

#3525

Achieving reproducibility and accuracy in cancer mutation detection with whole-genome and whole-exome sequencing.

Wenming Xiao, The Somatic Mutation Working Group of the SEQC-IIConsortium. _Center for Devices and Radiological Health, Silver Spring, MD_.

Clinical applications of precision oncology require accurate tests that can distinguish tumor-specific mutations from errors introduced at each step of next generation sequencing (NGS). For NGS to successfully improve patient lives, discriminating between true mutations and artifacts is crucial. We systematically interrogated somatic mutations in paired tumor normal cell lines to identify factors affecting detection reproducibility and accuracy. Different types of samples with varying input amount and tumor purity were processed using multiple library construction protocols. Whole-genome and whole-exome sequencing were carried out at six sequencing centers followed by processing with nine bioinformatics pipelines to evaluate their reproducibility. We identified artifacts due to sample and library processing and evaluated the capabilities and limitations of bioinformatics tools for artifact detection and removal. By examining the interaction and effect of various wet lab and computational parameters concomitantly, here we recommend actionable best practices for mutation detection in clinical applications using NGS technologies.

#3526

Incorporation of unique molecular identifiers (UMIs) into unique dual sample indexing (UDI) improves the accuracy of quantitative next generation sequencing.

Pingfang Liu, Keerthana Krishnan, Camille X. Devoe, Bradley W. Langhorst, Eileen Dimalanta, Theodore B. Davis. _New England Biolabs, Inc., Ipswich, MA_.

Accurate analysis of quantitative NGS data is critical for low frequency variant detection, identification of differentially expressed transcripts, and correct diagnosis and patient care in a clinical NGS setting. Two major factors affecting sequencing accuracy are 1) PCR duplication arising from amplification of library molecules; and 2) errors introduced during library preparation and actual sequencing on the flow cell. Standard practice for identification and removal of PCR duplicates relies on aligning reads to the same genomic coordinates. However, this approach can not differentiate between PCR duplicates and reads originating from unique molecules with identical ends. The most effective method for error correction is Duplex Sequencing, which utilizes UMIs to tag both strands of each individual DNA duplex followed by building consensus sequences. Unfortunately, this approach is labor intensive and cost-prohibitive for complex genomes and large target panels. Therefore, a simple and reliable approach for duplicate removal and error correction would greatly facilitate the wider adoption of NGS technology for diagnostics and clinical applications.

In this study, we incorporate UMIs into UDI systems and assess the effect on the accuracy of quantitative sequencing assays. We first study the effectiveness of various computational methods to account for UMIs and remove base-calling errors introduced during sequencing. We then analyze the utility of UMIs for 1) unique and low abundance transcript identification and accurate transcript quantification; and 2) error correction in low frequency variant detection in genomic sequencing from both high quality cell line DNA and low quality FFPE DNA. In addition, we demonstrate that combining unique dual sample indexing with UMI molecular barcoding further improves data analysis accuracy, especially on patterned flow cells. Our approach involves a simple new UMI-containing UDI adaptor design that can also be applied to other sequencing methods and platforms.

#3527

Combined high-throughput DNA genotyping and protein quantification in single cancer cells.

Benjamin Demaree,1 Cyrille Delley,1 David Ruff,2 Sebastian Treusch,2 Dennis Eastburn,2 Keith Jones,2 Adam Abate1. 1 _University of California, San Francisco, San Francisco, CA;_ 2 _Mission Bio, Inc., San Francisco, CA_.

Clonal genetic diversity is a hallmark of tumor biology. In many cancers, clonal evolution has been implicated as a driver of tumor initiation, progression, and relapse, and can be a prognostic indicator of disease outcome. Cancer cells are also phenotypically heterogeneous, constituting features which can enable intravasation and metastasis, or which can impart immunoevasive capabilities. Clinically, a combination of single-cell genotype and phenotype data from cancer biopsies could provide valuable insights into oncological disease states, such as metastatic risk or drug response. Here we present a multiomic technology for the simultaneous, high-throughput capture of DNA genotype and protein information from single cells, demonstrated through the analysis of acute myeloid leukemia (AML) derived cell lines. The experimental workflow leverages the high-throughput single-cell microfluidic droplet technology on the Mission Bio Tapestri platform, a system optimized to conduct multiplex targeted genomic DNA amplification from >50 AML-associated loci. In our modified workflow, cells are stained with a pool of monoclonal antibodies conjugated with barcoded oligonucleotides, enabling a sequencing-based readout of single-cell protein signatures. The sequencing data is analyzed with a bioinformatic pipeline that separates antibody signal from targeted genotyping data on a cell-by-cell basis. We find that on a mixed population of myelogenous leukemia cells and B-cells, single-cell genotype information effectively distinguishes between cell lines, while the protein data independently recapitulates the biological signal from conventional flow cytometric methods. In conclusion, this technology opens up a new era for discovery using multiomic analysis of the complex relationships between genotype and phenotype in cancer, paving the way for future studies analyzing primary tissue samples.

#3528

Novel enzymatic DNA fragmentation integrated into library construction.

Vaishnavi Panchapakesa, Lynne Apone, Karen Duggan, Bradley Langhorst, Chen Song, Pingfang Liu, Timur Shtatland, Christine Rozzi, Christine Sumner, Fiona Stewart, Eileen Dimalanta, Theodore Davis. _New England Biolabs, Ipswich, MA_.

Next Generation Sequencing (NGS) is rapidly becoming a powerful tool, by providing a high resolution and global view of the cancer genome mainly through whole genome, whole exome and transcriptome sequencing. This knowledge is important to support drug development targeted towards precision medicine. Obtaining high quality sequencing data begins with optimal sample preparation. DNA fragmentation is one of the critical first steps in the construction of high quality libraries for NGS, but current fragmentation methods create a bottleneck on library preparation throughput. We have developed an efficient, one tube fragmentation system that integrates enzyme-based DNA fragmentation with end-repair and dA-tailing in a single step, followed by adaptor ligation in the same tube. This streamlined method is compatible with a wide range of DNA samples and input amounts, resulting in reproducible and reliable fragment sizes.

Genomic DNA (100pg to 500ng) with various GC content, was used in the construction of libraries, that were sequenced on an Illumina platform. For low input samples, PCR amplification was performed prior to sequencing. Libraries constructed using intact genomic DNA and this novel library preparation method produced substantially higher yields than standard methods and mechanically-fragmented DNA. Enzyme-based shearing increases library yield by reducing DNA damage and sample loss. High library yields also enable PCR-free workflows from as little as 50ng starting material. This method can be used for DNA extraction and library preparation from whole blood and dried blood samples.Sequencing quality of libraries generated with inputs ranging from 100pg to 500ng DNA show similar coverage uniformity and fragment size distribution, with minimal GC bias.

We have developed an enzymatic DNA fragmentation kit that enables the construction of high quality libraries from a range of sample types and inputs with minimal sequence bias, high yields and low duplication rates. This method eliminates the need for expensive equipment to fragment DNA as well as numerous cleanup and liquid transfer steps thereby reducing the time, cost and errors associated with library construction. These advances in NGS technologies, supported by powerful bioinformatics tools, promises to revolutionize cancer research, diagnosis and therapy.

#3529

Sample quality control of cell-free DNA.

Eva Graf. _Agilent Technologies, Waldbronn, Germany_.

Sequencing of cell-free DNA (cfDNA) is possible due to the establishment of low input library protocols for next-generation sequencing workflows. Accurate quantification of cfDNA samples is essential to determine suitable input amounts for cfDNA library preparation prior to sequencing. The main component of cfDNA samples is the mononucleosome with a size around 170 bp, sometimes with additional species representing nucleosome multimers. Further, cfDNA samples may contain larger DNA fragments dependent on pre-analytical sample treatment or extraction method. High molecular weight material can negatively influence library preparation and subsequently result in lower sequencing depth. Therefore, reliable quantification of cfDNA requires a method that separates DNA fragments by size, such as electrophoresis. This poster shows the use of an automated electrophoresis platform performing cfDNA quantification with region analysis. Moreover, the results include a score to qualify cfDNA samples according to their contamination level with high molecular weight material. This allows defining a threshold for objective sample qualification prior to library preparation. The analysis features are described with examples of typical sample patterns.

#3530

Gene expression alterations associated with histologic aggressiveness in stage I lung adenocarcinomas.

Jiarui Zhang,1 Eric Burks,2 Jennifer Beane,1 Travis Sullivan,1 Gang Liu,1 Steven Dubinett,3 Marc Lenburg,1 Avrum Spira1. 1 _Boston University School of Medicine, Boston, MA;_ 2 _Lahey Hospital & Medical Center, Boston, MA; _3 _David Geffen School of Medicine at UCLA, Los Angelos, CA_.

RATIONALE:

The National Lung Screening and Nelson Trials demonstrated a 20% and 26% (for men) reduction, respectively in lung cancer mortality for patients screened using low-dose CT. However, lung cancer screening has the potential for over-diagnosis of indolent tumors. Therefore, we sought to identify molecular features that could distinguish indolent from aggressive early stage lung tumors based on histologic aggressiveness profiling, with the ultimate goal being to translate these findings into biomarkers that could inform post-biopsy/surgery management.

METHODS:

63 FFPE samples of stage I lung adenocarcinomas were included for pathologic annotation and Whole Exome sequencing. An average of 48.2±15.6 million total reads per sample were generated with 78.4%±4.8% reads uniquely mapped. Tumors were categorized into 3 groups based on histologic features previously associated with tumor aggressiveness: Minimally-Aggressive (n=18): containing zero aggressive components (e.g. zero solid/cribriform); Medium-Aggressive (n=19): not yet dominated by aggressive components; Highly-Aggressive (n=26): solid/cribriform predominant. Tumor histology was characterized for percentage of lepidic, acinar, papillary, micro-papillary, solid and cribriform components. Negative binomial models were used to identify genes whose expression was associated with tumor histology. Estimate Algorithm was used to estimate immune cell type infiltration of these tumors.

RESULTS:

430 genes (FDR q<0.05) were differentially expressed between the three histologic groups. Genes with elevated expression in the Medium- and Highly-Aggressive subgroups were enriched for genes with roles in cell-cycle regulation and the EMT transition. The genes elevated in the Minimally-Aggressive subgroup were enriched for genes with roles in inflammatory pathways and cytokine production. This gene signature was also associated with tumor invasiveness and mitotic grades (p<0.05). Using the Estimate Algorithm, we compared whether the three histologic groups had differential immune cell infiltrations using hallmark gene sets of lymphocytes and myeloid cells. We identified that Minimally-Aggressive subgroups had significantly higher Th-1, Tfh, Th17 and NK cell infiltration than Medium- and Highly-Aggressive subgroups (adjusted p value<0.05).

CONCLUSION:

We identified gene expression alterations among stage I adenocarcinomas associated with histologic features of tumor aggressiveness. This gene signature may help identify more indolent tumors and potentially impact their post biopsy/surgery clinical management.

#3531

Modular RNA expression analysis using whole transcriptome and targeted analysis in single cells on BD Rhapsody.

Margaret Nakamoto, Gretchen Lam, Christina Chang, Eleen Shum. _BD Biosciences, Menlo Park, CA_.

Advances in genomics have allowed high-throughput single-cell whole transcriptome analysis (WTA) to enable the discovery of biomarkers and cellular pathways that may be important in diseases. As a complementary tool to WTA, targeted analysis can provide high sensitivity of single cell gene expression data with lower sequencing cost. Here we demonstrate using the BD Rhapsody platform in a modular fashion to generate both WTA and targeted sequencing libraries from the same sample. Utilizing the archivability of the BD Rhapsody beads, we first subsampled a portion of the beads to generate WTA data, from which we identified genes that are important for T cell exhaustion. We then designed a targeted panel to profile the rest of the beads from the same experiment. The study demonstrates the versatility of the BD Rhapsody single cell platform and the ability to conduct different workflows on the same sample. This approach offers flexibility and choice for users in experimental design and allows users to easily transition from novel target discovery to robust data validation using targeted panels.

#3532

Tumor mutational burden (TMB) assessment and variant detection using an ultra-high multiplexed 20,000 amplicons NGS panel via a rapid 4 hour workflow.

Kathryn E. Pendleton, Yang L. Liu, Lifeng Lin, Lucie S. Lee, Jeffery Liu, Guoying Liu, Zhitong Liu. _Paragon Genomics, Hayward, CA_.

Tumor mutational burden (TMB) is currently of high interest in the field of immuno-oncology due to its correlation with patient response to checkpoint inhibitor chemotherapy. Traditionally, TMB is calculated using whole exome sequencing; however, targeted sequencing approaches provide better coverage of the genetic regions of interest at lower costs. While hybrid-capture based target enrichment methods are well-established, the 2-5 day workflows are time consuming and require specialized equipment and highly trained operators. Here we present an ultrafast, 4-hour method for preparing target enriched NGS libraries for assessing TMB that would streamline and lower the cost of immuno-oncology studies. This multiplex-PCR-based technology provides a highly efficient, accurate and robust method for unbiased enrichment of tens of thousands of target regions while minimizing non-specific primer-primer interactions and GC bias and maximizing coverage uniformity. We demonstrate this using a highly-multiplexed prototype NGS panel that contains ~20,000 amplicons and covers 355 genes for assessment of TMB.

Sequencing-ready NGS libraries were prepared using the Paragon Genomics CleanPlex® target enrichment technology. The 3-step workflow combines target enrichment and NGS library preparation. The protocol includes an ultra-high multiplex PCR step to amplify regions of interest with target-specific primers, a background cleaning step to remove non-specific PCR products, and a final PCR to add Illumina sequencing adapter and sample indexes. Libraries were made using 20ng of genomic DNA and sequenced on an Illumina NextSeq® platform. Sequencing metrics such as on-target rates were calculated, and variants were identified using Paragon Genomics' variant calling algorithm.

Using the CleanPlex technology, this prototype TMB panel exhibits >95% uniformity at 0.2X mean, limited GC bias, and >94% detection rate for mutants with 5% allele frequencies. The CleanPlex background cleaning step was essential for removing the undesirable byproducts of the multiplexed reaction step. CleanPlex technology demonstrates that it is capable of creating high quality amplicon libraries with high uniformity, low GC bias, and sensitive variant calling even in ultra-multiplexed libraries with ~20,000 amplicons with a workflow under 4-hours.

#3533

Low-frequency variant detection in cell-free DNA by integrating double-stranded unique molecular identifiers with ultrafast amplicon-based library preparations.

Lucie S. Lee, Yang Lily Liu, Kathryn Pendleton, Jeffrey Liu, Lifeng Phil Lin, Guoying Liu, Zhitong Liu. _Paragon Genomics, Hayward, CA_.

The use of liquid biopsy has seen tremendous growth in recent years as cell-free DNA (cfDNA) is a noninvasive and easily obtainable sample type with many diagnostics and prognostic values. While liquid biopsy can potentially enable new applications, such as early cancer detection, treatment monitoring, and drug resistance screening, it also presents challenges to accurate variant detection due to the low fraction of mutant DNA present in theses samples, which can be easily buried by the artifacts and background noise from systematic PCR and sequencing errors. While some success has been made using adapters with unique molecular identifiers (UMIs) in hybrid capture-based methods and performing deep sequencing, such approaches suffer from long, tricky, and tedious workflows and disappointingly low on-target rates. Additionally, the large amount of cfDNA required for hybrid capture workflows is not realistic to obtain from patients.

We have developed the CleanPlex® UMI technology to provide a fast, simple, and reliable NGS solution for low-frequency variant detection. The technology features three simple steps to generate molecular-barcoded and target-enriched NGS libraries in under 4 hours. This amplicon-based method consists of a multiplex PCR reaction for molecular barcoding, followed by a biochemical reaction for removal of redundant PCR products, and finally a second round of PCR to add Illumina adapter sequences and sample indexes. To validate this new method, we designed an NGS panel targeting frequently mutated hotspots in 23 genes associated with lung cancer. NGS libraries were prepared with commercial reference cfDNA at 0.1% to 0.5% minor allele frequencies (mAF) and sequenced on an Illumina NextSeq®.

Overall, we obtain high detection sensitivity for low-frequency alleles even at low DNA inputs. When including UMI in the analysis, we observe significant reductions in false positive calls. Nearly all known mutations covered by the CleanPlex UMI panel (12 out of 13) are detected at 0.1% mAF using 50ng of the reference cfDNA. At 0.25% mAF, all known mutants are detected with 100% PPV using 50ng of DNA. With only 20 ng of cfDNA, the method can achieve the same 100% detection at 0.5% mAF. The CleanPlex® UMI technology demonstrates high sensitivity with low false positive rate, for the detection of low-frequency alleles.

#3534

Analytical performance of a comprehensive genomic profiling system to detect actionable genetic alterations in NSCLC.

Kelly Gerding, Laurel Keefer, Christine McCord, Amy Greer, Shantanu Shewale, Nicole Barkley, Eileen Sagini, Dorhyun Johng, Kenneth Valkenburg, Caitlin Gilley, Colby Ganey, Alvis Hu, Diandra Denier, Lorenzo Jones, Christina Oliveras, Gregory Joseph, Kartikeya Joshi, James Hernandez, Christopher Gault, Eniko Papp, Peibing Qin, Sonya Parpart-Li, James White, Mark Sausen, Siân Jones. _Personal Genome Diagnostics, Baltimore, MD_.

Background Comprehensive genomic profiling in NSCLC has become increasingly important for clinical management of advanced stage patients. Alteration status of EGFR, ALK, BRAF, and ROS1 are validated biomarkers linked to approved drugs, with many clinical trials enrolling on the basis of additional biomarkers, including Tumor Mutation Burden (TMB). It is therefore important to develop accurate and sensitive tools for tumor profiling, particularly in NSCLC where biopsy materials are limited. Here, we present results from ongoing analytical performance studies with the PGDx elio tissue complete - IUO assay, verifying our capabilities to accurately identify a range of genomic alteration types, comprising SNVs, indels, translocations, and TMB.

Methods >300 specimens in NSCLC, comprising FFPE tissue and characterized cell lines were analyzed on our >500-gene targeted panel. Accuracy of the results were compared to orthogonal methods, such as whole exome sequencing (WES), IHC, FISH, and on-market IVD assays. Results were analyzed for the positive percent agreement (PPA) and negative percent agreement (NPA) for all variants assessed. Analytical studies were performed to assess the limit of blank, limit of detection, and repeatability for detection of these variants.

Results Clinical FFPE and characterized cell line specimens (representing EGFR and BRAF SNVs, EGFR exon 19 deletions, and ALK and ROS1 translocations) were evaluated and demonstrated high concordance between PGDx elio tissue complete - IUO and orthogonal methods, with high PPA and NPA for SNV, indel, and translocation alterations analyzed. Repeatability studies demonstrated 100% PPA and 100% NPA for all variants assessed across operators, instruments, and days. The accuracy for panel-wide sequence mutations were assessed by comparing the results of >50 clinical samples (representing >500 SNVs and ~40 indels) run on PGDx elio tissue complete IUO and a validated NGS method, resulting in high PPA and NPA across all alterations analyzed. Reported alterations between PGDx elio tissue complete IUO and the independent NGS method displayed high sensitivity for analyzed SNVs (≥4% MAF) and indels (≥6% MAF). Finally, comparison of TMB results to WES data demonstrated TMB can be accurately and consistently reported from this panel, across a range of DNA inputs (50-200 ng) and tumor purities (10-30%).

Conclusions The PGDx elio tissue complete - IUO >500-gene assay system, including our proprietary bioinformatics, provides accurate and reproducible results for the detection of clinically relevant genetic alterations in this NSCLC study. Further verification and validation studies of this gene panel are ongoing. The PGDx elio tissue complete assay will employ a decentralized, kitted model, increasing clinical accessibility to NGS and allow for delivery of highly accurate and timely results.

#3535

Identification of multiple hepatocellular carcinoma using genomic approach.

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

Multiple hepatocellular carcinoma (HCC) is categorized into two types; multicentric hepatocarcinogenesis (MC) and intrahepatic metastasis (IM). Although clinical discrimination of the types of multiple HCC has been proposed, it is quite difficult to determine it correctly even after histological diagnosis. In order to diagnose multiple HCC as MC or IM, we compared pairs of multiple HCC samples from 60 patients with multiple HCC and 2 patients with pairs of primary HCC and extrahepatic metastasis such as adrenal grand and lung metastasis with regard to molecular aberrations. Exome or capture sequences showed that more than 70% of somatic mutations were common in the pairs of IM, which were consistent with the result of a pair of primary HCC and extrahepatic metastasis, while no common somatic mutations was detected in genomic MC pairs. Notably, more than 60% of patients who were clinically diagnosed metachronous MC were genetically IM due to the concordance of mutation and shows significantly frequent tumor thrombus, suggesting that genomic IM pairs developed from the common ancestor. Epigenetically, methylation profiles were similar in a pair of IM, while those of MC were different each other. Consequently the accuracy of clinical diagnosis of multiple HCC was only 53.3%. In addition, cell-free DNA was analyzed using exome sequence in five patients with recurrent HCC, and found that somatic mutations of cell free DNA were similar to the primary HCC only in genomic IM but not in genomic MC cases. Intriguingly, overall survivals of patients with clinical IM was significantly shorter than those with clinical MC, while overall survivals of patients with genomic IM and genomic MC were almost similar. Taken together, comparison of mutations in a pair of HCCs makes it possible to classify multiple HCC into MC and IM, which is difficult to be correctly determined in clinical practice. In addition our data showed that it is not clinically important to determine the types of multiple HCC. Furthermore liquid biopsy for mutation analysis is available to make treatment plans for multiple HCC patients.

#3536

Advantages of ssDNA over dsDNA library preparation for capturing cell-free tumor DNA in plasma.

Jing Zhu,1 Jingyong Huang,2 Peng Zhang,2 Manish Kohli,3 Chiang-Ching Huang,4 Liang Wang2. 1 _Harbin Medical University, Harbin, Heilongjiang, China;_ 2 _Medical College of Wisconsin, Milwaukee, WI;_ 3 _Mayo Clinic, Rochester, MN;_ 4 _University of Wisconsin, Milwaukee, WI_.

Single-stranded DNA (ssDNA) sequencing library method has shown to enrich shorter and degraded DNA fragments than double-stranded DNA (dsDNA) library. The goal of this study is to determine whether ssDNA libraries enriches more circulating tumor DNA (ctDNA) in plasma cell-free DNA (cfDNA). We used 2ng cfDNA to prepare dsDNA libraries and ssDNA libraries. We also prepare pure-ssDNA libraries by skipping the DNA denaturation step to capture the pre-existing ssDNA only. To calculate ctDNA content, we mapped fastq files to human genome and binned the mapped reads into 1 Mb genomic windows. We normalized the read counts using two unrelated healthy controls. The resulting ratios were transformed with log2 and adjusted for GC content. The fully normalized log2 ratios were subjected to segmentation using the copy number analysis method. After the segmentation, we selected the most significantly deleted genomic region with segment size >20 Mb in each library pair and calculated ctDNA content using formula: 100 × (1-2log2 ratio). We also calculated the plasma genomic abnormality (PGA) score (a composite score algorithm to reflect overall ctDNA burden in peripheral blood) to estimate ctDNA burden. Overall, we prepared and sequenced a total of 27 libraries including ten dsDNA libraries, ten ssDNA libraries and seven pure-ssDNA libraries, using ten cfDNAs from cancer patients. We received an average of 24264998 raw read sequences and 19495014 mappable reads per sequencing library. Duplicate rate in the ssDNA libraries (mean=0.057%, range=0.046-0.083%) and pure-ssDNA libraries (mean=0.06%, range= 0.054-0.065%) was significantly lower than in dsDNA libraries (mean=0.2%, range=0.14-0.249%) (p<0.001 and p<0.01, respectively), indicating that ssDNA-based library preparation method may better preserve the diversity and complexity of ctDNAs. Library insert size analysis showed that ssDNA libraries were on average 14bp shorter (range from 6 bp to 21 bp) than their matched dsDNA libraries. We also found the Ampure XP beads ratio influenced the library size. We observed consistently higher ctDNA content in ssDNA libraries than in matched dsDNA libraries (p<0.0005). Comparing ssDNA libraries (mean ctDNA content=34%) to their matched dsDNA libraries (mean ctDNA content=30.7%), the increased ctDNA content ranged from 1.3% to 6.4% (mean=3.3%). ctDNA content was also significantly higher in pure-ssDNA libraries than in their matched dsDNA libraries (p<0.005). The increased ctDNA content ranged from 1.0% to 6.9% (mean=3.6%). The PGA scores were always higher in the ssDNA libraries (p<0.0001) or pure-ssDNA libraries (p<0.005) compared to the matched dsDNA method. Our results demonstrate that ssDNA libraries enrich more ctDNA than dsDNA libraries do. Pre-existing ssDNA in plasma is abundant and provides sufficient resource for genomic analysis of ctDNA.

#3537

Targeted deep sequencing of driver mutations in airway epithelial cells from smokers.

Daniel J. Craig, Thomas M. Blomquist, Erin L. Crawford, James C. Willey. _Univ. of Toledo Health Science Campus, Toledo, OH_.

Background: Inter-individual variation in risk for lung cancer is based on variation in both exposure to environmental factors and genetic predisposition. As such, it is reasonable to hypothesize that prevalence of mutations in the airway epithelium will vary based on the combined effects of these lung cancer risk factors. Genes most commonly mutated in non-small cell lung cancer were recently reported from The Cancer Genome Atlas (TCGA) project. The purpose of this study was to use targeted deep sequencing to assess the prevalence of these mutations in bronchial epithelial cells (BEC) of individuals at high demographic risk for lung cancer.

Methods: We enrolled 19 individuals with varying demographic risk for lung cancer, who were undergoing standard-of-care bronchoscopy into an IRB-approved research study in which we collected 1-5 million BEC via bronchoscopic brush biopsy and extracted genomic DNA. 10 subjects were smokers with lung cancer, 6 subjects were smokers without lung cancer, and 3 subjects were non-smokers without lung cancer. For targeted deep sequencing, we prepared synthetic spike-in competitive internal standards for each of 12 gene loci commonly mutated in lung cancer based on TCGA results and combined them into an IS mixture (ISM). DNA aliquots from each patient were quantified and mixed with equivalent genome equivalents of ISM prior to library preparation to control for technical artifacts associated with library preparation and NGS platform-specific error. The DNA input varied depending on sample quantity; there were 5 samples with 50,000 genome copies loaded, 12 samples with 100,000 genome copies loaded, and 2 samples with 1 million genome copies loaded. This approach enabled reliable measurement of 95% confidence limits of variant allele frequency (VAF), controlled for technical sequencing error, and allowed confident call of variants at a VAF of 0.1% and lower. Libraries for specimens from each of the 19 subjects were first sequenced using the Illumina MiSeq platform to ensure equal coverage across targets and samples. Deeper sequencing was then obtained using the Illumina NextSeq platform.

Results: Preliminary results showed that smokers harbored mutations up to 1.6% in gene loci associated with non-small cell lung cancer according to TCGA, while none were identified in non-smoking controls.

Conclusion: Initial results suggest that airway epithelial cells in smokers acquire somatic mutations that gradually lead to malignant conversion. Deep sequencing replicate experiments are underway to expand these results.

#3538

Analysis of error profiles in deep next-generation sequencing data.

Xiaotu Ma, Jinghui Zhang. _St Jude Children's Research Hospital, Memphis, TN_.

Background

Sequencing errors are a key confounding factor for detecting low frequency genetic variants for cancer screening, testing, treatment and surveillance through deep next-generation sequencing (NGS). However, there is a lack of comprehensive understanding of errors introduced at various steps of a conventional NGS workflow, such as sample handling, library preparation, enrichment PCR, and sequencing. In this study we systematically investigated the above question by using the current NGS technology.

Results

We discovered that the substitution error rate can be computationally suppressed to 10-5~10-4, which is 10~100-fold lower than current reports. We then quantified substitution errors attributable to sample handling, library preparation, enrichment PCR, and sequencing using multiple deep sequencing datasets from multiple sequencing centers. We show that 1) error rate differs by nucleotide substitution types, ranging from 10-5 for A>C/T>G, C>A/G>T, and C>G/G>C changes to 10-4 for A>G/T>C changes; 2) C>T/G>A errors exhibit strong sequence context dependency; 3) sample-specific effects dominate elevated C>A/G>T errors; 4) target enrichment PCR lead to ~6-fold increase of overall error rate; 5) more than 70% of hotspot variants, such as BRAF V600E, can be detected at 0.1%~0.01% frequency with the current NGS technology by applying in-silico error suppression.

Conclusions

We present the first comprehensive analysis of error sources in conventional NGS workflows. The error profiles revealed by our study highlight new directions for further improving NGS accuracy both experimentally and computationally which will lead to enhanced precision for deep sequencing.

#3539

Impact of formalin time on targeted NGS performance in FFPE tissue.

Thomas M. Blomquist,1 Erin Crawford,1 James Willey,1 Joshua Xu,2 Onco-panel Working Group of the Sequencing Quality Control Phase 2 (SEQC2) Consortium. 1 _University of Toledo, Toledo, OH;_ 2 _Food and Drug Administration, Jefferson, AR_.

Targeted next generation sequencing (NGS) panel testing is now a standard of care for management of advanced stage cancer. At present, most targeted NGS oncology testing occurs with formalin fixed paraffin embedded (FFPE) tissue. This is due to the established utility and prevalence of FFPE in routine histologic classification, grading, staging, and ancillary testing. In addition, FFPE materials are an optimally suited medium for determining adequacy of the specimen prior to downstream NGS targeted panel testing. For these reasons, as well as a growing trend of minimally invasive procedures, clinical specimens are increasingly prioritized for FFPE to meet the bulk of standard oncology care. Currently, formalin fixation time guidelines do exist for specific immunohistochemical and in-situ hybridization oncology tests. However, specific FFPE processing guidelines have yet to be established that would optimize targeted NGS panel testing performance.

In this study, our aim was to develop contrived FFPE materials using the FDA-led Sequencing Quality Control Phase II (SEQC2) consortium working group 2 (WG2) cell line materials. The overarching goal was to use these FFPE materials to identify an upper limit of formalin time associated with minimal impact on somatic variant calling performance in targeted NGS oncology panels. The SEQC2 - WG2 normal lymphoblast cell line was selected as a well-characterized test material to undergo systematic alteration in formalin fixation time (1, 2, 6 and 24 hours) prior to "routine" tissue processing and embedding (FFPE). These FFPE cell-block materials, derived from a single sample source (with both extracted FFPE DNA as well as shaved sections), were distributed to multiple labs running various targeted NGS amplicon and hybridization oncology panels.

The expected outcome of these studies is to provide general guidance to the community on how formalin time in FFPE specimens impacts targeted NGS oncology panel testing performance. In addition, we hope to identify genomic regions (internal control regions) that serve as markers for the full range of formalin time FFPE-effect. This information will assist in improving the lower limit of detection for variant calling (<5% variant allele frequency).

#3540

A complete solution for high throughput single cell targeted multiomic DNA and RNA sequencing for cancer research.

Dalia Dhingra, Kaustubh Gokhale, Nianzhen Li, Pedro Mendez, Shu Wang, Manimozhi Manivannan, Adam Sciambi, Keith Jones, Charlie Silver, Dennis Eastburn, David Ruff. _Mission Bio, South San Francisco, CA_.

Recent advancements in single cell analysis technologies are now able to provide insights into genomic DNA content, RNA expression and protein surface markers. In bulk assays, the effect of genetic variation on gene expression would be masked by the heterogeneity inherent in tumor cells. Additionally, for cancer immunotherapy studies that rely on gene editing, single cell resolution is necessary to minimize possible off target effects. However, the ability to simultaneously interrogate multiple intracellular analytes, such as genomic DNA and RNA, have proved difficult to implement in a high throughput single cell workflow. We report the development of a complete solution that enables both targeted genomic DNA and RNA sequencing from individual cells.

This workflow relies on the Tapestri microfluidic droplet platform, where up to 20,000 cells can be sequenced in each run. Leveraging proprietary cell barcoding, novel primer design strategies and enzymatic manipulation of cellular contents, DNA and RNA multiplex targeted sequencing panels provide for independent barcoded sequence information from both overlapping mRNA and corresponding genomic DNA regions. In addition, amplification primers can be designed to target separate gDNA and non-overlapping RNA transcript. Sequencing is followed by an integrated analysis solution that assigns the reads from both the gDNA and RNA to each cell.

Feasibility of the targeted nucleic acid workflow has been shown with inputs from mixed cancer cell lines. A targeted sequencing panel with overlapping mRNA and gDNA regions was designed covering oncogenes, tumor suppressor genes, and known fusions. Expected SNVs and indels were detected and gene expression measured for thousands of cells per run with high cell recovery. This complete solution for single cell multiomics on the Tapestri platform has the power to quantitatively and unambiguously link genotypic and phenotypic data, giving insight into cancer progression.

#3541

Characterization of olfactory receptor upregulation in invasive breast cancer.

Shirin Masjedi, Laurence J. Zwiebel, Todd D. Giorgio. _Vanderbilt University, Nashville, TN_.

Despite some progress in early detection and new therapies, breast cancer remains the second most common cause of cancer-related deaths among women. Majority of deaths occur due to invasion and metastasis among triple-negative breast cancer (TNBC) patients. Gene profiling of mammary tumors has enabled identification of novel genes with potential roles in cancer invasion using next generation sequencing technologies. This study is the first to examine upregulation of olfactory receptors (ORs) among a large invasive breast carcinoma population and begins to establish a correlation of OR genes in breast cancer invasion. Invasive breast carcinoma cases from the TCGA database were used. Among data from 1100 unique patients, 960 cases of invasive breast carcinoma had RNAseq of their primary tumors in addition to exome sequencing data. The abundance of all 408 human coding OR genes among the 960 cases were analyzed using a three-factor stratification algorithm, and 30 OR genes were identified as highly abundant among 198 breast invasive carcinoma patients. Supervised clustering of the 198 patients based on the similarity of OR upregulation levels identified five subpopulations among the invasive carcinoma patients in which only one OR gene (OR2A7, OR2B6, OR2C1, OR2W3) was significantly upregulated. Abundance of OR2W3 was significantly elevated in two subpopulations with 30- and 19-fold increase compared to their level of abundance in normal mammary tissue. These two subpopulations were composed of 74% and 70% TNBC tumor subtypes and included all the stage iv cancer patients in the study. Transcript abundance of 21 genes included in the Oncotype DX gene expression diagnostic test was measured for each patient subpopulation. The subpopulation with abundance in OR2W3 was correlated with significant upregulation of invasion genes (CTSV, MMP-11) and the subpopulation with abundance of OR2B6 was correlated with significant upregulation of proliferation genes (MKI67, AURKA, BIRC67, CCNB1, MYBL2). Interestingly, only the cases with significant upregulation of OR2W3 were correlated with significantly reduced survival probability compared to invasive breast carcinoma patients not expressing OR2W3. Furthermore, analysis of OR transcript abundance levels among 56 human breast cancer cell lines showed that 3 ORs (OR2A7, OR2B6, OR2W3) were exclusively upregulated in one cell group with 2, 1.3 and 2.1-fold increase compared to their upregulation levels in a human mammary epithelial cell line (MCF10A). The cell group with significant upregulation of OR2W3 was composed of 83% TNBC cell lines. These results show a consistent elevated upregulation of the OR2W3 gene in all the invasive breast tumors and cell lines. Our ongoing discovery research suggests that OR2W3 may be an indicator of breast cancer invasion. It is also possible that OR2W3 has signaling or other mechanistic roles in breast cancer progression, metastasis and reduced survival.

#3542

Turbolase: A radically streamlined high throughput sample prep to sequencing workflow.

Dustin Masser, Robert Stedtfeld, Justin Lenhart, Vladimir Makarov, Laurie Kurihara. _Swift Biosciences, Ann Arbor, MI_.

Normalase is a novel enzymatic library normalization method that eliminates library quantification and manual concentration adjustment of each sample prior to library pooling, and the uniform sample processing eliminates numerous error-prone pipetting steps. When combined with Swift 2S Turbo rapid library kits, a highly streamlined 'Turbolase' workflow is created that is readily automated on the Hamilton Star and other platforms, where simple bulk processing improves throughput and reduces cost for NGS laboratories. Swift 2S Turbo kits comprise two enzymatic steps and a single purification that completes DNA fragmentation, end repair and adapter ligation. This is followed by standard library amplification using Normalase PCR primers to condition the libraries and produce a required minimum yield in excess of the 4 nM final concentration. This is followed by a single purification and two 15-minute Normalase incubation steps that; 1) enzymatically select 4 nM of each library, and 2) enzymatically normalize each library to 4 nM within a single pool. The pools can then be directly sequenced without further purification. Normalase allows for greater than 10-fold variation in input quantity, while generating less than 10% variation in sample representation within a pool resulting in optimal cluster density and sample balance for Illumina sequencing. Because of the unique workflow, Normalase also eliminates index hopping for libraries sequenced on patterned flow cells. The 'Turbolase' workflow is compatible with full-length indexed adapters that have been added by ligation as well as workflows that require indexing PCR primers to complete library construction during library amplification. Normalase is also compatible with library preparation kits available from other vendors. Normalase for pooling of libraries for pre-hybridization capture is currently under development, which will make multiplexed hybridization capture protocols streamlined and robust from library generation to sequencing.

#3543

Clinical whole genome sequencing at scale.

Alyssa MacBeth, Tera Bowers, Betty Woolf, Maegan Harden, Niall Lennon, Stacey Gabriel. _Broad Institute of MIT and Harvard, Cambridge, MA_.

Whole genome sequencing (WGS) offers the greatest potential to comprehensively and accurately identify all forms of human genetic variation. The accessibility of WGS data for diagnostic use has increased due to cost reductions in recent years, driving the need for a high quality, clinically validated offering. Our platform has completed an extensive benchmarking study of the Illumina PCR-free whole genome pipeline to establish clinical validity for the end-to-end laboratory, analytical, and computational processes. Our benchmarking study is comprehensive in nature, including measuring the precision, robustness, limits of detection for variant classes, and contamination estimates. A cohort of well-characterized reference controls and clinical samples with previously identified pathogenic variants were used to establish the performance characteristics of our clinical WGS test, which at 30X coverage has >99% analytical sensitivity for SNVs and >98% analytical sensitivity for small insertions and deletions. Our platform is capable of operating at a scale that supports applications from individual clinics (cancer and medical genetics related applications) as well as large scale ambitious collaborative projects such as the All Of Us program which aims to generate clinical grade whole genome variant calls for 1 million healthy research participants.

#3544

High performance multiplexed targeted enrichment sequencing from FFPE tissues.

Mark Consugar,1 Leonardo Arbiza,1 Kristin Butcher,1 Siyuan Chen,1 Hutson Chilton,1 Richard Gantt,1 Yehudit Hasin-Brumshtein,1 Jim Laugharn,2 Jayne Simon,2 Ulrich Thomann,2 Christina Thompson,1 Ramsey Zeitoun1. 1 _Twist Bioscience, San Francisco, CA;_ 2 _Covaris, Inc., Woburn, MA_.

Library construction for Next-Generation Sequencing (NGS) using formalin-fixed paraffin-embedded (FFPE) samples offers unique challenges in acquiring high-quality sequencing data due to wide distribution of sample quality. Specifically, differences in formalin fixation methods lead to crosslinked and/or degraded nucleic acid and inconsistent extraction yields. Hence, FFPE extraction and library construction methods must be carefully considered for target enrichment applications. In collaboration, Covaris and Twist Bioscience demonstrate a complete library preparation and target enrichment solution that generates ready-to-sequence multiplexed libraries directly from FFPE tissue.This workflow leverages the Covaris truXTRAC FFPE total Nucleic Acid Plus Kit and oneTUBE-10 shearing with the world-class performance of Twist Bioscience's Target Enrichment Solutions. Covaris, the Gold Standard for DNA shearing in NGS, also offers pre-analytical products that leverage Adaptive Focused Acoustics® (AFA)® technology. In this FFPE-specific application, the Covaris truXTRAC FFPE total Nucleic Acid Plus Kit and oneTUBE-10 shearing enable full emulsification of paraffin and disaggregation of tissue for highly efficient nucleic acid extraction and generation of size specific DNA libraries. With the Twist Bioscience Human Core Exome kit, the resulting libraries are indexed, pooled, and target enriched with uniquely optimized DNA probes to generate ready-to-sequence high quality multiplexed libraries.Using the aforementioned workflow, results from processing numerous FFPE tissue types are presented. Sequencing results demonstrate large improvements in general Picard metrics that include uniformity (Fold_80 < 1.8), sequencing depth (20X coverage >85% with 100X downsampling), and duplication rates (<6%) when compared to similar published studies. These results demonstrate a validated solution for library preparation and targeted exome sequencing of FFPE samples that can be integrated into automated workflows. The truXTRAC kit and AFA® technology from Covaris generate size specific DNA libraries from FFPE samples which, when paired with Twist Bioscience's superior target enrichment workflow, deliver multiplexed libraries for high performance targeted sequencing.

### miRNAs as Tumor Suppressors/Oncogenes

#3545

High expression of miR-30a is associated with favorable breast cancer survival.

Yuki Obata,1 Dionyssios Katsaros,2 Nicoletta Biglia,3 Yi Shen,4 Yuanyuan Fu,4 Zhanwei Wang,4 Herbert Yu4. 1 _Kinjo Gakuin University, Nagoya, Japan;_ 2 _University of Torino School of Medicine, Turin, Italy;_ 3 _University of Torino School of Medicine, Mauriziano Hospital, Turin, Italy;_ 4 _University of Hawaii Cancer Center, Honolulu, HI_.

Introduction: MicroRNAs (miRNAs) are single-stranded, small non-coding RNAs, consisting of about 20 nucleotides and studies have shown that miRNAs may play important roles in the development and progression of cancer. Members of the miR-30 family, including miR-30a, are reported to play different roles as oncogenes or tumor suppressor genes depending on the type of cancer in several studies. We studied miR-30a expression in breast cancer in relation to disease features and patient survival.

Methods: We measured miR-30a expression in tumor samples of 509 breast cancer patients by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Using the study-specific tertile distribution as cut-off, miR-30a expression data were grouped into low, medium, and high three categories. Hazards ratios (HRs) and 95% confidence intervals (CIs) were calculated to assess the association between miR-30a expression and breast cancer survival using the Cox proportional hazards regression model, and the analysis was adjusted for age at surgery, tumor grade, disease stage, and hormone receptor status. Overall survival (OS) was defined as the time interval from the date of surgery to the date of death or last follow-up. Disease-free survival (DFS) was the time interval from surgery to recurrence or last follow-up. We also retrieved breast cancer provisional data in The Cancer Genome Atlas (TCGA) using the web-based tool cBioPortal (http://www.cbioportal.org/index.do) and analyzed the association between miR-30a and breast cancer survival.

Results: Patients with estrogen receptor (ER)-positive tumors had higher expression of miR-30a compared to those with ER-negative tumors (p = 0.0080). Patients with progesterone receptor (PR)-positive tumors also had higher miR-30a expression than those with PR-negative tumors (p = 0.0038). Survival analysis showed that patients with high expression of miR-30a had 58% reduction in risk of relapse (HR = 0.42, 95% CI = 0.21-0.83, p = 0.013) compared to those with low expression. This association remained significant (HR = 0.43, 95% CI = 0.21-0.89, p = 0.023) after adjustment for age at surgery, disease stage, tumor grade, and hormone receptor status. A significant association between miR-30a and overall survival was also observed in the TCGA data (HR = 0.60, 95% CI = 0.41-0.88, p = 0.0088).

Conclusions: Our results suggest that high expression of miR-30a may reduce the risk of breast cancer relapse and miR-30a may act as a tumor suppressor in breast cancer.

#3546

miRNA expression of spontaneous non-small cell lung cancers in p53 mutant transgenic mice following exposure to PRIMA-1.

Javier A. Feito,1 Abeer Almiman,1 Li Gao,1 Shirley Tang,2 Kathleen Dotts,2 Miguel Villalona-Calero,3 Wenrui Duan1. 1 _Herbert Wertheim College of Medicine, Florida International University, Miami, FL;_ 2 _Comprehensive Cancer Center, Ohio State University, Columbus, OH;_ 3 _Miami Cancer Institute, Miami, FL_.

The tumor suppressor protein p53 is a key regulator of cell cycle control, apoptosis, and genomic stability in response to various cellular stresses. Loss of function of p53 causes an inability by the cell to regulate these important processes and results in uncontrolled cell proliferation, which leads to tumor growth. Mutations in the TP53 gene are commonly associated with cancers, and for this reason TP53/p53 is one of the most important genes in cancer research. PRIMA-1 targets mutated p53 protein and has been shown to restore its wild type function and to induce apoptosis in human tumor cells, but the underlying mechanisms behind it are still not well understood. Micro RNAs (miRNAs) are small, noncoding RNA molecules that act as gene expression regulators. The involvement of miRNA in restoring tumor suppressor function of mutant p53 by Prima-1 remains unclear. The aim of this research is to investigate the associations between expression of miRNA and the PRIMA-1 anticancer treatment.

We have developed a lung tumor model in transgenic mice in which the human mutant p53(273H) is expressed in a lung specific manner, under the control of the surfactant protein C promoter. These mice developed lung adenocarcinomas at an age range of 12-15 months. A micro-CT scanner was used on the mice to screen for lung tumors. To evaluate feasibility and preliminary activity of PRIMA-1 on the murine spontaneous lung tumor, we treated 4 lung tumor bearing mice via intraperitoneal (i.p.) injection with PRIMA-1 at a dose of 100 mg/kg in 0.2 ml PBS every other day for two weeks. A control group containing 3 tumor bearing mice was treated with PBS only. After two weeks of treatment, all mice were then sacrificed for tissue collection. Total RNA was isolated with Trizol method from the frozen lung tumor tissues and matching lung tissues. miRNA array was carried out with a NanoString counter. miRNA expression level was compared between the treated and control groups.

After comparing 599 miRNA expressions in lung tumor tissue between PRIMA-1 treated and non-treated mice, 26 miRNAs were changed at least 3-fold. From these 26, 23 of were decreased and only 3 were increased. Of particular interest was that 9 miRNAs changed at least 6 folds post Prima-1 treatment as comparing to control group. These were miR-194, miR-1937a, 1937b, miR-34c, miR-192, miR-1949, miR-2135, miR-3472, miR-712, and miR-1931. Because PRIMA-1 restores the tumor suppression function of mutant p53 and to induce cell cycle arrest or apoptosis in tumor cells containing mutant p53, PRIMA-1 may act as an anti-cancer drug through regulating miRNAs. Because the great majority of the miRNA expression levels among the miRNA with bigger expression changes in PRIMA-1 treated group comparing to the non-treated controls are reduced, it implies that these miRNAs may promote tumor development. Mir-194, for example, has been reported to have tumor suppressor function in lung tissue.

#3547

MicroRNA-496 inhibits triple negative breast cancer cell proliferation by targeting DEL-1.

Dong Won Baek, Jae-Hwan Jeong, Soo Jung Lee, Jiyeon Lee, Yee Soo Chae, Wan WooK Kim, Jieun Kang, Ho Yong Park, Jin Hyang Jung, Ji Yun Jeong, Ji Young Park, Keon Uk Park. _Kyungpook National University Hospital, Daegu, Republic of Korea_.

Background and Objectives: Del-1 is linked to the pathogenesis of various cancers including breast cancer; however, the regulation of Del-1 expression remains unclear. The current study investigated how microRNA-496 (miR-496) regulates Del-1 expression in triple negative breast cancer (TNBC).

Methods: Del-1 mRNA and miR-496 were measured by quantitative PCR in breast cancer cells (MDA-MB-231, MCF7, SK-BR3, and T-47D) and tissues from 30 patients with TNBC. The effects of miR-496 on cell proliferation, migration, and invasion were determined in MTT, wound healing, and Matrigel Transwell assays, respectively.

Results: In MDA-MB-231, miR-496 levels were remarkably low and Del-1 mRNA was higher compared to other breast cancer cell lines. Luciferase reporter assays revealed that miR-496 binds the 3′-UTR of Del-1 and that Del-1 expression is downregulated by miR-496 mimics. Furthermore, miR-496 inhibited the proliferation, migration, and invasion of MDA-MB-231 cells. The effects of miR-496 on cell proliferation were additive with those of miR-137, another miRNA that regulates Del-1 expression. Moreover, in the 30 TNBC specimens, miR-496 was downregulated (P < 0.005) and the levels of Del-1 in the plasma was significantly elevated as compared to normal controls (P = 0.0142). TCGA data showed the correlation of miR-496 expression with better overall survival in patients with early TNBC.

Conclusions: In in silico and in vitro analyses, we showed that Del-1 is a target of miR-496 in TNBC and thereby affects cancer progression. Our findings suggest that miR-496 and Del-1 might act as modulating factors in TNBC and are new biomarkers for patients with TNBC.

#3548

Differential miRNA expression levels and risk of lung adenocarcinoma in transgenic mice.

Myia Aiges,1 Abeer Almiman,1 Li Gao,1 Kathleen Dotts,2 Miguel A. Villalona-Calero,3 Wenrui Duan1. 1 _Florida International University, Miami, FL;_ 2 _The Ohio State University Cancer Center, Miami, FL;_ 3 _Miami Cancer Institute, Miami, FL_.

In 2018, 154,100 lung cancer deaths are projected to occur in the United States. Currently, there are very few predictors to identify lung cancer in its early stages. Recent studies have demonstrated a link between microRNAs (miRNAs) and specific cancers. Dysregulation of miRNAs in cancer can lead to upregulate oncogenes and downregulate tumor suppressors. Previously, we developed a line of transgenic mice expressing the human mutant p53 (273H) gene. The transgenic mice were observed to have accelerated risk of spontaneous adenocarcinomas at the age of 13-14 months. However the mechanisms involved in lung tumorigenesis at this age cohort is still unclear. Herein, we report our analysis on miRNA expression in lung tissue between a low risk cohort and high risk cohort. To investigate the role of mutant p53 in lung tumorigenesis, a p53(273H) transgenic mouse model was developed. Murine lung tissue were harvested from age groups of 7 months (low risk group with risk of 5% of lung cancer), and the 13.5 months (high risk group with risk of 27% of lung cancer). RNA was then extracted with trizol, and miRNAs were quantified using a Nanostring counter miRNA array. The miRNA expressions were normalized and averaged. The normalized miRNAs from high risk group (n=3) were compared with the miRNAs obtained from the low risk group (n=3). We screened 600 different miRNA expressions from these age cohorts. Total of 19 miRNAs that had an up-regulation of at least 5-fold in the high risk cohort as compared with the low risk cohort including some important miRNAs in human lung cancer (e.g mmu-miR-2133, mmu-miR-2140, mmu-miR-142-5p, mmu-miR-2138, mmu-miR-2134, mmu-miR-2135, mmu-miR-452, mmu-miR-703, mmu-miR-690, mmu-miR-206, mmu-miR-706, mmu-miR-691, mmu-miR-208a, mmu-miR-574-5p, mmu-miR-1). Four miRNAs had a down regulation at least 5-fold in the high risk cohort including mmu-miR-720, mmu-miR-1937c, and mmu-miR-1937a+mmu-miR-1937b.We found a group of microRNAs to be expressed abnormally high and another group microRNA to be expressed abnormally low in the high risk group as comparing to the low risk group. We compared the data to our murine lung cancer microRNA profile and found a group of miRNA expression potentially correlated to risk of lung cancer development in mice. Most human lung cancers are at an advanced stage when they are first found. These cancers are very hard to cure. However, if a lung cancer is found at an earlier stage when it is small and before it has spread, the early stage lung cancer can be successfully treated. Further studies are necessary to employ these miRNAs as early diagnostic biomarkers in prediction of human lung cancers.

#3549

Elevated miR-141-3p inhibits renal cell carcinoma aggressiveness by targeting epithelial-to-mesenchymal transition pathway.

Pritha Dasgupta,1 Priyanka Kulkarni,1 Shahana Majid,1 Varahram Shahryari,1 Nadeem S. Bhat,2 Yutaka Hashimoto,1 Marisa Shiina,1 Guoren Deng,1 Sharanjot Saini,1 Soichiro Yamamura,1 Yuichiro Tanaka,1 Rajvir Dahiya1. 1 _University of California San Francisco/VA Medical Center, San Francisco, CA;_ 2 _University of Miami, Miami, FL_.

Objective: Clear cell renal cell carcinoma (ccRCC), a common histological subtype of renal cell carcinoma (RCC) are categorized by their aggressive nature and comprise 90% of metastatic RCCs. Despite recent advances, management of the disease in the advanced metastatic phase is a significant challenge. Expression of miR-141-3p (miR-141) is low and function as tumor suppressor in various cancers. However, its association with long non-coding RNAs (lncRNAs) in RCC is not well understood. This study shows that miR-141 interacts with lncRNAs, and plays vital role in the regulation of stemness and epithelial-to-mesenchymal transition (EMT) in RCC.

Experimental Design: Human renal cancer cell lines (ACHN and Caki-1), normal renal epithelial cells, RPTEC and tumor tissues were used for this study. We analyzed the expression of miR-141 in tissue samples and cell lines, and studied the function of miR-141 in kidney cancer progression using in vitro and in vivo models. Statistical analysis was performed to determine the clinical significance of miR-141 in kidney cancer patients.

Results: Reduced expression of miR-141 was observed in ccRCC clinical specimens and cell lines. To elucidate the epigenetic role of miR-141 silencing in RCC, methylation status of CpG islands in its putative promoter region was analyzed. Result showed significant increase in miR-141 expression after 5-Aza-CdR treatment indicating that promoter hypermethylation is responsible for its inactivation. Ectopic expression of miR-141 reduced cell proliferation, clonogenicity, migration, invasion and induced apoptosis and cell cycle arrest compared to controls. An increase in cleaved PARP, cleaved caspase-3 and epithelial marker (CLDN1) was observed with a concomitant decrease in stemness (KLF4, Nanog) and EMT markers (FN1, VIM). We further investigated the biological significance of miR-141 in RCC. Loss of miR-141 function in RPTEC cells induced pro-cancerous characteristics. In addition, we examined lncRNAs (CDKN2B-As1, PCAT1 and PVT1) that bind to miR-141 and are overexpressed in RCC clinical samples compared to controls. Reduced expression of these lncRNAs was observed in RCC cells with overexpression of miR-141, supporting the notion that miR-141 interacts with these lncRNAs in RCC. Finally, in vivo experiment in nude mice revealed that intra-tumoral administration of miR-141 in the established tumors significantly suppressed tumor growth compared to controls. Furthermore, statistical analysis of patient samples showed that miR-141 may serve as a RCC diagnostic biomarker.

Conclusion: Our results demonstrate that miR-141 overexpression inhibits RCC progression and inhibits epithelial-to-mesenchymal transition. These studies also show that miR-141 may be a useful RCC biomarker for early detection and monitoring RCC progression.

#3550

**microRNA-211 promotes aggressive melanoma growth** in **** vivo **by epigenetic modification, and contributes to BRAFV600E inhibitor resistance via ERK5 signaling.**

Bongyong Lee,1 Anupama Sahoo,2 Junko Sawada,1 Dimitrios G. Zisoulis,3 John Marchica,1 Sanjay Sahoo,2 Fabiana I Alves De Lima Layng,4 Darren Finlay,4 Joseph Mazar,5 Piyush Joshi,1 Masanobu Komatsu,1 Kristiina Vuori,4 Garth Powis,4 Petrus R. de Jong,4 Animesh Ray,6 Ranjan J. Perera1. 1 _Johns Hopkins School of Medicine, St. Petersburg, FL;_ 2 _Sanford Burnham Prebys Medical Discovery Institute, Orlando, FL;_ 3 _Regulus Therapeutics Inc., San Diego, CA;_ 4 _Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA;_ 5 _Nemours Children's Hospital, Orlando, FL;_ 6 _Keck Graduate Institute, Claremont, CA_.

The microRNA miR-211 is an established participant in melanomagenesis, but controversy exists as to whether it acts as a bone fide tumor suppressor or oncogene. Here we ectopically expressed miR-211 in the BRAF v600E-mutant A375 melanoma cell line and examined its effect in xenografts in vivo. The miR-211 ectopic expression promoted aggressive tumor xenograft growth with extensive cell proliferation, and angiogenesis. ChIP-seq and single cell sequencing analysis of xenograft tissues demonstrated that aggressive tumor formation is partly associated with H3K27me3 and H3K4me3, and migration of cells from mouse tissues to tumor locus. Interrogation of xenograft transcriptomics data revealed activation of the ERK5 pathway, itself negatively regulated by miR-211 target genes, BIRC2 and DUSP6, further confirmed as direct miR-211 target genes by RNA immunopurification with RNA-seq (RIP-seq) and site-directed mutagenesis. miR-211 conferred resistance to the BRAF inhibitor vemurafenib, and MEK inhibitor cobimetinib with corresponding increases in ERK5 phosphorylation. The miR-211-ERK5 axis may represent a novel therapeutic target, but however, miR-211 is exquisitely pleiotropic in the complex in vivo tumor environment and its context must be considered carefully in diagnostic and therapeutic development.

#3551

Dysregulation in microprocessor processing of a primary miRNA, mir-21, leads to downregulation of a tumor suppressor, GHR, progressing hepatocellular carcinoma.

Seokju Park,1 Hee Doo Yang,2 Jwawon Seo,1 Seokwoo Nam,2 Jin-Wu Nam1. 1 _Hanyang University, Seoul, Republic of Korea;_ 2 _The Catholic University of Korea, Seoul, Republic of Korea_.

Improper miRNA biogenesis produces the 5' variants of miRNA with a shifted seed region (also called isomiRs). Although the presence of stably abundant isomiRs were repeatedly reported, it is still unclear whether the generation of isomiRs results either from the mistake of microprocessor or from the regulation by other intrinsic or extrinsic factors, and whether the isomiRs participate in critical cellular processes, particularly in cancer progress. Here, we performed both high-throughput RNA sequencing (RNA-seq) and small RNA sequencing (sRNA-seq) from 75 hepatocellular carcinoma (HCC) and normal samples (fresh frozen) of Korean HCC cohort. Combined analysis with other publicly available sequencing data, showed that although the expression signatures of isomiRs largely follow those of canonical ones, the relative abundance of some isomiRs are dynamically changed over cancer progression, forming new isomiR-target interactions. Particularly, the biogenesis of highly abundant miR-21 is dysregulated by interaction with HNRNPC to primary mir-21 during cancer progress, producing relatively more isomiR-21-5p in HCC. Introducing isomiR-21-5p mimic or antagomir against isomiR-21-5p, and the dysregulation of HNRNPC expression perturbed the relative abundance of isomiR-21-5p, leading to dysregulation of miRNA targeting on a tumor suppressor gene, GHR, and affecting cancer progress. Our study not only describes that isomiR-21-5p is an independent prognostic marker and a potential therapeutic target of HCC but also presents that the production of isomiRs is tightly regulated by other extrinsic factors in cells.

#3552

MiRNA-150 targets SUFU and promotes EMT by activating the Wnt/β-catenin signaling pathway in human gastric cancer.

Yin Peng, Xiaojing Zhang, Xianling Feng, Xinmin Fan, Zhe Jin. _Shenzhen University, Shenzhen, China_.

Mounting evidence has shown that miRNA-150 is aberrantly upregulated in gastric cancer (GC) and associated with gastric carcigonenesis; however, the underlying oncogenic mechanism is elusive. Here, we have discovered that overexpressed miRNA-150 targets tumor suppressor SUFU to promote gastric cell proliferation, migration and EMT via Wnt signaling activation. MiRNA-150 is highly expressed in GC tissues and cell lines in which SUFU is downregulated. Furthermore, the expression level of miRNA-150 is negatively related to SUFU expression level. MiRNA-150 activates GC cell proliferation and migration. MiRNA-150 inhibitors promote cytoplasmic localization of β-catenin, leading to repression of Wnt signaling. In addition, overexpression of the miRNA-150 activates EMT while inhibition of miRNA-150 suppresses EMT. Anti-SUFU siRNAs rescue the inhibitory effects of miRNA-150 antagonists on Wnt activation and EMT. Moreover, inhibition of cell proliferation, migration and colony formation caused by miRNA-150 antagonist is alleviated by anti-SUFU siRNA. Taken together, these findings suggest that miRNA-150 is oncogenic and promotes GC cell proliferation, migration and EMT by activating Wnt signaling, at least in part, via suppression of SUFU.

#3553

**Targeting miR-361-3p suppresses the growth of human oral squamous cell carcinoma cells** in vitro ** & **in vivo **.**

Norihiko Tokuzen, Koh-ichi Nakashiro, Himiko Ogawa, Hiroyuki Goda. _Ehime Univ. Graduate School of Medicine, Toon, Ehime, Japan_.

MicroRNAs (miRNAs) are small non-coding RNAs with size of 20-25 nucleotides, and inhibit protein translation by binding the 3'-untranslated region (3'-UTR) of target mRNA. Each miRNA can regulate multiple mRNAs and each mRNA can be targeted by a number of miRNAs. In cancer, miRNAs can act as not only tumor suppressor genes but also oncogenes. Addiction to oncogenic miRNAs (OncomiRs) may provide therapeutic opportunities in human cancers such as oncogene addiction. In this study, we have attempted to identify OncomiRs in human oral squamous cell carcinoma (OSCC) cells and considered whether targeting miRNAs can be possible for cancer therapy. Functional screening for OncomiRs in human OSCC cells was performed with the use of miRCURY LNATM microRNA Knockdown Library-Human v12.0 (EXIQON). We transfected 918 locked nucleic acid (LNA)/DNA antisense oligonucleotides for specific human mature miRNAs into human OSCC cells (GFP-SAS). After 80 hours, each cell growth was evaluated by WST-8 assay. We identified LNA/DNA antisense oligonucleotide against miR-361-3p (LNA-miR-361-3p) which showed remarkable growth inhibition in GFP-SAS cells. Subsequently, we confirmed the target specificity of LNA-miR-361-3p by quantitative RT-PCR (qRT-PCR). LNA-miR-361-3p significantly reduced the expression of miR-361-3p. Co-transfection of synthetic mature miR-361-3p abrogated the growth inhibitory effect of LNA-miR-361-3p in GFP-SAS cells. Furthermore, transfection of synthetic mature miR-361-3p resulted in 20% increase of cell growth in GFP-SAS cells. Next, we identified odd-skipped related 2 (OSR2) as a target gene of miR-361-3p by the use of microarray, GeneSpring GX, and Ingenuity Pathway Analysis (IPA). The expression of OSR2 after transfection with LNA-miR-361-3p was significantly induced, and co-transfection of the OSR2 3'-UTR luciferase reporter plasmid and LNA-miR-361-3p into GFP-SAS cells produced higher luciferase activity than cells in co-transfected with LNA-non target (LNA-NT). Finally, we assessed the effect of LNA-miR-361-3p on the in vivo growth of GFP-SAS cells. We administered LNA-miR-361-3p into the xenograft tumors every 3 days. We found that LNA-miR-361-3p significantly reduced the size of subcutaneously xenografted GFP-SAS tumors, compared to the control group treated with LNA-NT. The expression of miR-361-3p in excised tumors was examined by qRT-PCR. LNA-miR-361-3p suppressed the expression levels of miR-361-3p compared with the control tumors. These results suggest that miR-361-3p support the growth of human OSCC cells and that targeting miR-361-3p may be a useful therapeutic approach for patients with OSCC.

#3554

miR-139 is associated with improved prognosis in patients with localized prostate cancer.

Tania Benatar,1 Robert K. Nam,2 Elizabeth Kobylecky,1 Yutaka Amemiya,3 Arun K. Seth4. 1 _Sunnybrook Research Institute, Sunnybrook Health Sciences Centre, Toronto, Ontario, Canada;_ 2 _Division of Urology, Toronto, Ontario, Canada;_ 3 _Sunnybrook Research Institute Genomics Facility, Toronto, Ontario, Canada;_ 4 _Univ. of Toronto, Department of Laboratory Medicine and Pathobiology, Toronto, Ontario, Canada_.

Background: We previously identified a panel of five miRNAs associated with biochemical recurrence and metastasis following prostatectomy based on NGS-based whole miRNome discovery and qPCR-based validation analysis. In this analysis, we examine the effect of miR-139-5p, one of the down-regulated miRNAs identified in the panel, in greater detail.

Methods: Using a cohort of 585 patients treated with radical prostatectomy, we examined the prognostic significance of miR-139 (dichotomized around the median) using the Kaplan Meier method and Cox proportional hazard models. We validated these results using The Cancer Genome Atlas (TCGA) data. We created cell lines that over-expressed miR-139 or transiently transfected cells using miR-139 mimics for functional assays. Finally, we examined pathways through which miR-139 may function using prediction algorithms and confirmed targets by Western blotting and reporter assays.

Results: MiR-139 down-regulation was significantly associated with a variety of accepted prognostic factors in prostate cancer, including Gleason score, pathologic stage, margin positivity and lymph node status. MiR-139 was associated with prognosis: the cumulative incidence of biochemical recurrence and metastasis were significantly lower among patients with high miR-139 expression (p=0.0004 and 0.038, respectively). After adjusting for known prognostic factors, patients with high miR-139 expression had significantly lower risk of recurrence (HR 0.77, 95% 0.58-1.04). Validation in the TCGA dataset showed a significant association between dichotomized miR-139 expression and biochemical recurrence (OR 0.52, 95% CI 0.33-0.82). Over-expression of miR-139 in prostate cancer cells led to a significant reduction in cell proliferation and migration compared to control cells, with cells arrested in G2 of cell cycle. IGF1R, RUNX1 and AXL were identified as potential gene targets of miR-139 based on their association with prostate cancer growth pathways and multiple miRNA binding site prediction tools. The reporter assays using luciferase gene constructs containing the predicted miRNA targeting sequence from IGFR1 and RUNX1 verified them as direct targets of miR-139. Furthermore, Western blotting of prostate cancer cells demonstrated RUNX1 and AXL expression were inhibited by miR-139 treatment, which was reversed by addition of miR-139 antagomir. Examination of the molecular mechanism of growth inhibition by miR-139 revealed the downregulation of activated Akt and cyclin D1, with upregulation of the CDK inhibitor p21.

Conclusions: miR-139 is associated with improved prognosis in patients with localized prostate cancer, which may be mediated through inhibition of IGF1R, RUNX1 and/or AXL and their associated growth signaling pathways.

#3555

miR-374a-5p induces tumor progression in triple negative breast cancer.

Yesol Kim, Dasom Son, Do Yeon Kim, Jong Hoon Park. _Sookmyung Women's University, Seoul, Republic of Korea_.

Triple negative breast cancer (TNBC) has higher aggressiveness and poorer outcomes compared with other subtypes of breast cancer. However, the genomic and molecular aberrations of TNBC are largely unknown, with cytotoxic chemotherapy remaining the major treatment for TNBC patients. In this study, miR-374a-5p was discovered as a novel TNBC-specific miRNA and its functions and the molecular mechanisms involved were investigated. Combined gene expression profiling of miRNA-microarray and human transcriptome dataset analysis revealed that miR-374a-5p is specifically upregulated in TNBC patients. Functional studies using in vitro and in vivo TNBC models, as well as survival analysis of TNBC patients, indicated that upregulated miR-374a-5p promotes tumor progression in TNBC. miR-374a-5p was also found to directly target arrestin beta 1 (ARRB1) that is specifically downregulated in TNBC patients in several human genomic datasets. Overexpressed ARRB1 reduced TNBC cell growth and migration, and the ARRB1 expression level is inversely correlated with the histological grade of the breast cancer and positively associated with TNBC patient survival, suggestive of a tumor-suppressive function of ARRB1 in breast cancer. Interestingly, increased ARRB1 activates AMPK in TNBC cells, which is associated with the expression of miR-374a-5p. Taken together, the findings suggest that miR-374a-5p is a novel prognostic marker and therapeutic target in the treatment of TNBC.

#3556

MiR-506 suppresses tumor progression by reprogramming macrophage polarization in pancreatic ductal adenocarcinoma.

Longhao Sun,1 Qianqian Song,2 Weijun Tian,1 Zhixiang Zhang1. 1 _Tianjin Medical University General Hospital, Tianjin, China;_ 2 _Wake Forest Baptist Medical Center, Winston-Salem, NC_.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancy owing to its aggressive nature and tumor-associated macrophages (TAMs) significantly promote disease progression by representing an immunosuppressive phenotype in PDAC. Macrophages could be functionally reprogrammed to opposite phenotype owning to different microenvironment and STAT3 (signal transducer and activator of transcription 3) activation may play an essential role in TAM polarization, directly contributing to local immunosuppression. Our recent study demonstrated that MiR-506 functioned as a tumor suppressor in many cancer types through the regulation of multiple pathways and directly targeted STAT3 in PDAC cells. In this study, we hypothesized that MiR-506 could affect tumor microenvironment and exert a tumor suppressive function in PDAC by reprograming TAM polarization through targeting STAT3 in TAM. Our results provided evidence that MiR-506 downregulation was associated with less immunosuppressive TAMs accumulation and better prognosis in human PDAC. MiR-506 could reduce the infiltration of immunosuppressive TAMs and inhibit the PDAC progression in mouse model. MiR-506 reprogramed macrophage polarization from an immunosuppressive phenotype to immune-activated phenotype through direct targeting the STAT3 axis. Silencing and inhibiting STAT3 recapitulated the effects of MiR-506. These findings expand the known mechanisms of MiR-506-mediated tumor suppression to reprogramming TAM polarization and suggest a strategy for using MiR-506 as an anti-STAT3 approach to PDAC immunotherapy.

#3557

miR-181a is a key driver of genomic instability and ovarian cancer tumorigenesis through the regulation of RB1.

Matthew Knarr,1 Lily Kwiatkowski,1 Anil Nagaraj,1 Ronny Drapkin,2 Analisa DiFeo3. 1 _Case Western Reserve University, Cleveland, OH;_ 2 _University of Pennsylvania, Philadelphia, PA;_ 3 _University of Michigan, Ann Arbor, MI_.

Epithelial ovarian cancer (EOC) is one of the deadliest gynecological cancers currently diagnosed in women with approximately 20,000 new cases and 15,000 deaths per year. Ovarian cancer can be stratified into distinct subtypes of which high-grade serous ovarian cancer (HGSOC) comprises 90% of all cases and has the highest mortality rate. Reducing the high mortality rate associated with HGSOC currently has two primary barriers: 1) most HGSOCs are diagnosed at a post-metastatic stage and 2) up to 70% of patients recur within 5 years. Both of these limitations are due to the fact that the drivers of tumor initiation are poorly understood. In order to change the status quo of the last 30 years, a new approach that focuses on understanding the origins of HGSOC is required. Our lab has recently discovered that miR-181a promotes key hallmarks of HGSOC progression in fallopian tube secretory epithelial cells (FTSECs) which are precursor cells of HGSOC. We show that enhanced expression of miR-181a in FTSECs results in increased proliferation, survival, and anchorage independent growth in vitro. Furthermore, FTSECs with miR-181a overexpression are able to form tumors in vivo whereas FTSECs that overexpress a control scramble miRNA cannot. Using cell cycle analysis, dynamic live-cell imaging, and SNP array analysis, we also found that miR-181a overexpression increases aneuploidy and genomic instability in FTSECs. We show that miR-181a promotes defects in nuclear structure, ruptures in the nuclear envelope, as well as mitotic and cytokinetic defects affecting proper chromosome segregation. A combination of global mRNA expression profiling and target prediction software identified the tumor suppressor retinoblastoma (RB1) as a target for miR-181a mediated FTSEC tumorigenesis. Using an RB1 3'UTR luciferase assay, along with stable expression of an shRNA against RB1 in FTSECs, we are able to show that miR-181a directly targets RB1 and that knockdown of RB1 phenocopies miR-181a mediated tumorigenesis. Taken together, our data shed light on a novel mechanism that promotes genomic instability and tumorigenesis in the early phases of HGSOC development. Our results suggest miR-181a as a promising early detection biomarker as well as a prognostic tool to guide patient treatment.

#3558

Interrogation of onco-microRNA 125b reveals androgen receptor as a key upstream transcriptional factor that is targetable in female gastric cancer.

Ben Liu,1 Meng Zhou,1 Da Yang,2 Qinghua Wang,1 Qiang Zhang,1 Xiangchun Li,1 Meng Yang,1 Qiong Wang,1 Lian Li,1 Luyang Liu,1 Hongji Dai,1 Fengju Song,1 Hong Zheng,1 Wei Zhang,3 Kexin Chen1. 1 _Tianjin Medical Univ. Cancer Inst. & Hosp., Tianjin, China; _2 _University of Pittsburgh, PA;_ 3 _Wake Forest Baptist Comprehensive Cancer Center, Wake Forest Baptist Medical Center, NC_.

Background & Aims: Our prior study revealed a miRNA-regulatory network by an integrated analysis of microRNA and mRNA profiling of gastric cancer (GC). It defined a GC microRNA subtype that was associated with poor survival in GC cases. In this study, we further demonstrate that onco-microRNA miR-125b, a key node in this microRNA regulatory network, is upregulated in GC and associated with poor overall survival by interfering in the apoptosis pathway and this pathway can be regulated by androgen receptor (AR). Our study also clarifies the potential clinical utility of the AR antagonist Bicalutamide on GC treatment.

Methods: To further investigate the role of AR-miR-125b axis in GC, we conducted both in vitro experiment and clinical study with a cohort of 373 GC samples. The expression of miR-125b and AR was analyzed and compared with patient survival, and its implications were evaluated between genders of GC cases. We explored the tumor suppressive effect of bicalutamide using in vitro assays and in vivo subcutaneous xenotransplanted tumor model of human GC cell lines in nude mice.

Results: The markedly up regulated expression of AR-miR-125b axis was validated in 373 GC samples. Univariate and multivariate analyses find that the miR-125b and/or AR were associated with poor prognosis and this trend was more pronounced among female than male cases. Pathway analysis shows that the predicted targets of miR-125b are highly involved in apoptosis/program death pathway. The robust apoptosis genes, BIK and CASP6 are validated as the directed targets of miR-125b. miR-125b can suppress apoptosis and increase cell growth, migration and invasion in GC cell lines. Furthermore, low expression of BIK and CASP6 in GC were associated with poor disease-free survival. There was a notable positive correlation between miR-125b and AR level in GC samples. Chip, EMSA and luciferase-binding assay confirmed that AR can direct regulated miR-125b expression as a transcriptional factor. We examined AR antagonist bicalutamide to inhibit proliferation and increased apoptosis in AR positive gastric cancer cells. The effect of bicalutamide is more apparent in female than in male nude mice.

Conclusions: Our findings suggest that miR-125b regulated apoptosis pathway is important to GC progression and that the AR/miR-125 may be an important clinical biomarker for GC survival and possible therapeutic target for GC treatment.

#3559

A crowd-control approach for castration-resistant prostate cancer therapeutics.

Girish C. Shukla. _Cleveland State Univ., Cleveland, OH_.

Castration-resistant prostate cancer (CRPC) is defined by affecting intrinsic cellular mechanisms including dysregulated androgen signaling, aerobic glycolysis (Warburg effect), epithelial-mesenchymal transition (EMT) and dysregulation of transcription factors including androgen receptor (AR) and c-Myc. Development of CRPC post Androgen-deprivation therapy is indicated by tumor microenvironment heterogeneity, compensating cellular pathways and recurrence of AR expression leading to the failure of androgen-signaling inhibitors including Enzalutamide and Abiraterone Acetate. We have discovered that miR-644a potentiates Enzalutamide efficacy both, in vitro and in vivo prostate cancer models. In depth analyses revealed that miR-644a downregulated expression of diverse tumor microenvironment regulators including c-Myc, AR, AR co-regulators and the anti-apoptosis factors Bcl-xl and Bcl-2, EMT factors ZEB1, cdk6, and Snail. Enhanced miR-644a expression also suppressed the Warburg effect by direct down-regulating c-Myc, Akt, IGF-1R and GAPDH expression. Taken together, our data demonstrate that the adjunctive miRNA expression down-regulated several oncogenesis promoting cellular pathways and functions cooperatively with Enzalutamide. We propose that a "crowd-control" approach to pacifying aggressive tumorigenesis and metastasis supporting cellular pathways by a miRNA with a wide range of gene targets is likely to potentiate the efficacy of cancer treating drugs and potentially preventing the drug-resistance.

#3560

MicroRNA-665 promotes metastasis of breast cancer by targeting nuclear receptor subfamily 4 group A member 3.

Xinge Zhao. _Sun Yat-sen University Cancer Center, Gaungzhou, China_.

Background: It is the result of metastasis to the lymph nodes or other organs that leads breast cancer (BC) patients to death. Then the early detection of metastasis in BC is helpful to regulate progression of BC and then predict prognosis of BC patients. Since the abnormal level of miRNAs are bound up with BC and metastasis, of which may act as biomarkers to predict prognosis of BC, we find that expression level of miR-665 is ectopic up-regulated in BC patients and it may serve as an estimable biomarker of prognosis of BC patients. Methods: The expressions of miRNA were detected by our current microarray system and verified by quantitative real-time reverse transcription-PCR (qRT-PCR). In Student's t-test and Kaplan-Meier analysis, the levels of miR-665 were positively correlated with poor survival in BC patients. Multivariate survival analysis identified either miR-665 expression or TNM stage as an independent prognostic factor of BC. Diverse roles of miR-665 played in various cellular functions were examined by loss-of-function and gain-of-function effects in vitro and in vivo. Dual-luciferase reporter gene experiment was then used to confirm the direct complementation between miR-665 and NR4A3, accompanied by activating the epithelial-mesenchymal transition (EMT). Furthermore, siRNAs targeting NR4A3 were used to rescue cell migration and invasion in miR-665 knockdown cells. Results: Expression of miR-665 was upregulated which predicted poor prognosis in BC patients. Since miR-665 may regulate key processes of tumorigenesis, targeting of NR4A3 was proved to promote cell migration and invasion by activating the EMT. Accordingly, either silencing of miR-665 or over-expressing of NR4A3 rescued the migration and invasion phenotype in vitro. Conclusions: Our study suggests that miR-665 plays an important part in progression of BC and promotes cell migration as well as invasion by down-regulation of NR4A3. The data collectively indicate that miR-665 is an onco-miRNA and may represents a potential predictor for diagnosis and prognosis of BC.

#3561

MicroRNA-214 inhibits prostate cancer cell proliferation, migration, invasion and increases drug sensitivity by targeting PTK6.

Patrice Cagle, Suryakant Niture, Anvesha Srivastava, Malathi Ramalinga, Leslimar Rios-Colon, Uchechukwu Chimeh, Deepak Kumar. _North Carolina Central University, Durham, NC_.

MicroRNAs (miRNAs) are involved in numerous physiological and pathological processes and play a critical role in cancer progression and the role miRNA-214 (miR-214) in prostate cancer remains elusive. In the present study, we investigated the effect of expression of miR-214 on cell survival/migration/invasion, cell cycle regulation, and apoptosis. Differential expression of miR-214 was found between Caucasian, African American prostate cancer cells compared with normal prostate epithelial cells. Expression of miR-214 in prostate cancer cells inhibited cell proliferation and colony forming capability, and induced apoptosis. miR-214 also inhibited cell migration and 3D spheroid invasion in prostate cancer cells. Interestingly, miR-214 targets protein tyrosine kinase 6 (PTK6) and reduced PTK6 expression. The restoration of PTK6 expression significantly blocked the inhibitory effect of miR-214 on cell proliferation. Moreover, inactivation of PKT6 by ibrutinib and expression of miR-214 significantly enhanced the cell death in prostate cancer cells. Collectively the data suggest that miR-214 functions as a tumor suppressor in prostate cancer, and may become a potential therapeutic target in prostate cancer.

#3562

**Exosomes secreted by AC133** + **/CD34** + **cells harbor invasion potentiating miRNAs.**

Ghada Ben Rahoma, Rachana Maniyar, Sanjukta Chakraborty, Sarnath Singh, Anitha Srinivasan, Abraham Mittelman, Jan Geliebter, Raj K. Tiwari. _New York Medical College, Valhalla, NY_.

Despite the exciting progresses in the treatment of breast cancer, the effectiveness of the current therapeutic modalities is still restricted by drug toxicity, resistance, and lack of predictive and prognostic biomarkers. Breast cancer continues to be the second leading cause of cancer death among women in the U.S. Therefore, the development of new therapeutic targets and further understanding of the tumor microenvironment is extremely critical for accelerating the progress against breast cancer. Human AC133+/CD34\+ stem cells are a highly promising and novel therapeutic option for targeting tumor angiogenesis. We and others have described the incorporation of bone marrow derived AC133+/CD34+/KDR+ cells in the neovasculature around implanted tumors supporting their growth and metastasis. Many mediators have been involved in the cross talk between AC133+/CD34\+ cells, endothelial cells, and the tumor cells, but most of these have insufficient clinical benefits as reported by several trials. In this study, we evaluated the secretome of the AC133+/CD34\+ stem cells that were isolated by positive selection from human umbilical cord blood and their role in breast cancer progression. Using flow cytometry, we show that the high proliferative AC133+/CD34\+ stem cells maintain their capacity to differentiate in to AC133+/CD34+/KDR\+ endothelial progenitor cells even after long period of in vitro expansion. In order to evaluate the effect of AC133+/CD34\+ stem cells on breast cancer cells, a proliferation (XTT) assay was performed using conditioned medium (CM) from AC133+/CD34\+ stem cells and examined on MCF-7 and MDA-MB-231 proliferation. As anticipated, CM significantly induced breast cancer cells proliferation. This effect was in part due to the high expression of a large range of proinflammatory and proangiogenic cytokines in the CM of the AC133+/CD34+ cells. In particular, angiogenin, GRO, IL-8, MCP, and TIMP2. Next, we examined if exosomes, a component of paracrine secretion are involved in the paracrine effect of the AC133+/CD34\+ stem cells. Surprisingly, exosomes from AC133+/CD34\+ stem cells significantly increased MCF-7 and MDA-MB-231 proliferation at a comparable level as the CM. Further analysis of the exosomes using miRNA array screen reveals that exosomes of AC133+/CD34 cells are highly enriched with oncogenic miRNAs including miR-21-5p, miR-142-3p, and miR-223-3p. These miRNAs are up-regulated in breast cancer. Several studies have confirmed their role in mediating breast cancer cells invasiveness. However, miR-142-3p and miR-223-3p are exclusively expressed in hematopoietic cells. Therefore, we propose that shuttling of the exosomes between AC133+/CD34+cells and breast cancer cells induces breast cancer invasiveness. The analysis of the paracrine interactive mediators between breast cancer cells and AC133+/CD34\+ cells is likely to yield viable novel clinically translatable therapeutic targets.

#3563

Exosomal transfer of tumor-associated macrophage derived miR-6068 promote ovarian cancer progression.

Seyda Baydogan,1 Jianting Sheng,2 Nermin Kahraman,1 Pinar Kanlikilicer,1 Hamada Ahmed Mokhlis,1 Sayra Dilmac,1 Stephen T. C. Wong,2 Bulent Ozpolat1. 1 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Houston Methodist Research Institute, Houston, TX_.

Ovarian cancer, especially high-grade serous ovarian cancer (HGSOC), is the deadliest gynecological cancer with 50-70% 5-year mortality rates. Every year more than 22,000 new cases of ovarian cancer and 15,000 deaths are anticipated within the United States only. Recent clinical and experimental evidence indicates that tumor-associated macrophages (TAMs), the most abundant cells in the tumor microenvironment, play a significant role in tumor growth and progression by contributing to angiogenesis, invasion, metastasis, and drug resistance, leading to poor clinical outcomes and significantly shorter patient survival in HGSOC. More than 50% of cells in the peritoneal tumor microenvironment and malign ascites consist of TAMs in ovarian cancer (OC) patients. Especially, M2 macrophages have been shown to support tumor proliferation and promote tumor progression, angiogenesis, and drug resistance. But the mechanisms of these oncogenic effects are still not clear. The goal of our study to investigate the role of TAM-derived exosomes, which are 30-100nm microvesicles released from cells and are key factors in communication between cancer cells and the tumor microenvironment. To this end, we evaluated differentially expressed miRNAs in high-grade ovarian cancer cells (OVCAR3, OVCAR 432 and OVCAR5) after treatment with exosomes-derived from TAMs (M2 phenotype) using the Affymetrix Gene Chip miRNA 4.0 microarrays. We identified several miRNAs, including miR-6068 that we validated by qPCR and found it to be significantly upregulated in both HGSOC cells and their exosomes. We demonstrated that transfection of HGSOC cells with miR-6068 significantly increased proliferation, migration and invasion capacities of the cells in vitro, suggesting that this miR-6068 act as an oncogenic miR (oncomiR). Using in silico prediction algorithms we found that miR-6068 has binding sites on the 3'-untranslated region (3'-UTR) of PTPN4 gene encoding a phosphatase and demonstrated that it miR-6068 suppresses PTPN4 expression by Western blot and qPCR. Inhibition of PTPN4 by siRNA significantly induced cell proliferation in OC cells, suggesting that PTPN4 acts as a tumor suppressor. In conclusion, our results suggest that miR-6068 has an oncogenic role in ovarian cancer progression by targeting PTPN4 and may be a novel effective therapeutic target for ovarian cancer.

#3564

MicroRNA-145 gene therapy for pancreatic cancer using exosome as carrier source derived from cancer cells.

Kamalika Samanta, Sheema Khan, Saini Setua, Sonam Kumari, Nirnoy Dan, Murali Mohan Yallapu, Meena Jaggi, Subhash Chandra Chauhan. _University of Tennessee Health Science Center, Memphis, TN_.

Background-Pancreatic cancer (PanCa) is the third deadliest cancer in United States with a poor survival rate. Despite extensive research efforts, there is no substantial progress in cancer therapeutics due to barrier against intracellular drug delivery. Our group has previously identified that microRNA-145 (miR-145) is downregulated in PanCa, the restoration of which inhibits tumor growth and enhances gemcitabine sensitivity. However, lack of an effective tumor-specific delivery system remains an unmet clinical challenge for successful translation of microRNAs. Herein, we have utilized exosomes (extra cellular vesicles) isolated from pancreatic cancer cells as a vehicle for delivering miR-145 in PanCa cells. Exosomes, unlike other vectors for gene delivery, are non-immunogenic in nature and can protect the RNA/gene of interest from digestion making them a more efficient vehicle for gene delivery.

Method- Exosomes were isolated from pancreatic cancer cells HPAF-II, AsPC-1 by the principle of precipitation using exosome isolation reagents. Physico-chemical characterization (DLS, TEM), presence of exosomal marker (CD63; immunoblotting), protein concentration (Bradford assay) and cellular internalization (confocal microscopy) of the exosomes were performed. miR-145 transfection was performed in pancreatic cancer cells using exofectamine. miR-145 restitution and anti-cancer efficacy was investigated using in vitro functional assays for cell viability (MTT), migration (Boyden chambers), invasion (Matrigel), colonogenicity and tumor spheroid formation. The effect of exosome mediated miR-145 restitution was investigated using Western blotting and PCR analysis.

Results- Exosomes show effective size and zeta potential (AsPC-1:164 nm -9.4mV respectively; HPAF-II: 157 nm -9.2 mV), which is ideal for drug delivery purposes. The purification of exosomes was confirmed by analyzing the expression of exosomal markers, such as CD63. Immunofluorescence for CD63 expression confirmed the efficient delivery of exosomes in PanCa cells. miR-145 loaded exosome treatment efficiently delivered miR-145 into the cancer cells and inhibited the tumorigenic features such as, proliferation, migration, invasion and colonogenicity of PanCa cells. The restoration of lost miR-145 levels using exosome mediated delivery was confirmed in PanCa cells using PCR analysis. MicroRNA-145 fold expression was found to be high at both pH, 7.4 and 6.8, which further indicates relevance for their utilization in the delivery of therapeutic modalities.

Conclusion- Our observations offer importance of the utilization of exosomes for delivery of therapeutic modalities and developing personalized medicine in PanCa.

#3565

Role of novel microRNA 4287 at a frequently deleted chromosome 8p region in prostate cancer.

Divya Bhagirath, Thao Yang, Laura Tabatabai, Shahana Majid, Soichiro Yamamura, Rajvir Dahiya, Yuichiro Tanaka, Sharanjot Saini. _Veterans Affair Medical Center San Francisco and University of California San Francisco, San Francisco, CA_.

Deletion of chromosome (chr)8p21-22 region that has been traditionally associated with the loss of homeodomain protein, NKX3.1 and with tumor initiation, is frequently observed in prostate cancer (PCa). More significantly, loss of this region is associated with aggressiveness and poor prognosis of PCa, thereby emphasizing a key role of this region in advanced prostate cancer. In a paradigm shifting hypothesis, we proposed that this frequently deleted locus is associated with a cluster of miRNA genes - miR-3622a/b and miR-383- that are lost in PCa and play an important mechanistic role in PCa progression and metastasis by regulating Epithelial-mesenchymal-transition (EMT) and stemness. Extending our hypothesis, in this study we evaluated the role of miR-4287, a miRNA gene located within this region in PCa. We profiled the expression of miR-4287 in laser capture microdissected (LCM) PCa tissues and matched adjacent normal regions by real time PCR. miR-4287 expression was down regulated in ~79% of tissue samples. High miR-4287 expression was observed in 20% of cases. Analyses of miR-4287 expression in prostate cell lines showed that its expression is specifically attenuated in PCa cell lines compared to normal or immortalized prostate epithelial cells. Further, we evaluated the functional role of miR-4287 in prostate cancer by overexpressing miR-4287 precursor in PCa cell lines PC3 and LNCaP. miR-4287 overexpression in PCa cell lines led to decreased cellular viabilities as compared to control cells. Moreover, PCa cell lines overexpressing miR-4287 showed an increase in apoptosis and G0/G1 arrest. Further, we found that miR-4287 inhibits epithelial to mesenchymal transition (EMT) in PCa as its overexpression in PCa cell lines led to increased E-cadherin and decreased vimentin expression concomitant with morphological changes consistent with mesenchymal to epithelial transition (MET). We identified SRC, SFPR4 and CD44 as potential miR-4287 targets. Taken together, our data suggests that miR-4287 plays a significant role as tumor suppressor in PCa pathogenesis. 

### Noncoding RNAs 2

#3566

SLCO4A1-AS1 **promotes tumorigenesis of colorectal cancer by stabilizing** SLCO4A1 **.**

Yuri Choi,1 Chae Hwa Kwon,2 Seon Jin Lee,2 Daye Jeon,1 Do Youn Park,1 Sojeong Lee,1 Ahrong Kim1. 1 _Pusan National University, Busan, Republic of Korea;_ 2 _Pusan National University Hospital, Busan, Republic of Korea_.

Long noncoding RNAs (lncRNAs) constitute an important component of tumor biology, but the molecular mechanism through lncRNAs modulating colorectal cancer (CRC) development and progression remains unclear. Herein we analyzed RNA sequencing data of CRC patients (147 tumor and 47 matched normal tissues) to identify significant lncRNAs involved in CRC. We found 6032 lncRNAs (15%) and selected three up-expressed (ELFN1-AS1, SLCO4A1-AS1, and LINC02418) and six down-expressed (MIR22HG, BCYRN1, SATB2-AS1, LINC01752, CMAHP, and LINC01082) lncRNAs according to fold change and p-value. In Kaplan-Meier survival analysis, CRC patients with high expression of SLCO4A1-AS1 had poorer prognosis than the patients with low expression of SLCO4A1-AS1, and the other lncRNA expressions were not associated with prognosis. To concentrate on the biological function of SLCO4A1-AS1, we investigated the relationship between SLCO4A1-AS1 and SLCO4A1. They were validated in tumor and normal tissues by qRT-PCR and SLCO4A1 expression was also confirmed by immunohistochemistry, indicating their positive correlation. After knocking down of SLCO4A1-AS1, SLCO4A1 protein level was decreased and cell proliferation was suppressed in CRC cell lines. However, SLCO4A1-AS1 level was not changed by knock-down of SLCO4A1. It demonstrated that SLCO4A1-AS1 plays oncogenic role through influencing on SLCO4A1. Thus, SLCO4A1-AS1 might be useful biomarkers for CRC diagnosis and prognosis.

#3567

A novel E2F1-regulated lncRNA, XLOC_000190, is required for S phase progression and cell proliferation.

Doron Ginsberg, Tali Nizri-Megnaji, Esther Baruch. _Bar Ilan University, Ramat Gan, Israel_.

Long non-coding RNAs (lncRNAs) are major regulators of many cellular processes including cell cycle progression and cell proliferation. The pivotal transcription factor E2F1 induces both proliferation and cell death and is a critical downstream target of the tumor suppressor RB. The RB/E2F pathway is often inactivated in human tumors resulting in deregulated E2F activity. Here, we report that the human lncRNA XLOC_000190 is an E2F1- regulated lncRNA that plays a role in S phase progression. XLOC_000190 levels are elevated upon activation of E2F1. Furthermore, knockdown of E2F1 and E2F3 reduce XLOC_000190 levels and endogenous E2F1 binds the XLOC_000190 promoter. Moreover, expression of XLOC_000190 is cell cycle regulated and peaks near G1/S transition and in early S. Inhibition of XLOC_000190 expression increases percentage of S phase cells, suggesting that XLOC_000190 plays a role in S phase progression. Also, silencing of XLOC_000190 in cells that are synchronized at the G1/S delays progression of cells through S phase. In agreement with its suggested role in S phase, prolonged silencing of XLOC_000190 inhibits proliferation of human cancer cells in culture. XLOC_000190 is a nuclear lncRNA and its gene is localized downstream to the protein-coding gene CDKN2C that encodes the p18ink4c CDK inhibitor. In search for XLOC_000190 mode of action we do not detect any changes in the expression of this neighbor upon XLOC_000190 silencing. However, XLOC_000190 silencing leads to a significant decrease in the level of Ribonucleotide Reductase Regulatory Subunit M2 (RRM2), which is a critical player in S phase progression. Additional data indicate that RRM2 mediates, at least in part, the effect of XLOC_000190 on S phase progression. Importantly, low levels of XLOC_000190 are associated with increased survival of kidney cancer patients.

In conclusions, our data identify XLOC_000190 as a novel E2F-regulated lncRNA that has a potential role in human cancer and regulates cell-cycle progression and cell proliferation, at least in part, via regulation of RRM2.

#3568

**Metastasis-promoting role of** H19 **long non-coding RNA in pancreatic cancer cells.**

Norihiko Sasaki,1 Masashi Toyoda,1 Hisashi Yoshimura,2 Yoko Matsuda,3 Tomio Arai,3 Yoko Itakura,1 Fujiya Gomi,1 Junko Aida,1 Toshiyuki Ishiwata1. 1 _Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan;_ 2 _Nippon Veterinary and Life Science University, Tokyo, Japan;_ 3 _Tokyo Metropolitan Hospital and Institute of Gerontology, Tokyo, Japan_.

The H19 long non-coding RNA is highly expressed and carries out various functions in different types of cancers. Recently, we reported that H19 contributes to the metastasis of pancreatic ductal adenocarcinoma (PDAC) cells and its inhibition reduces metastasis in vivo. However, the molecular mechanisms underlying the metastasis-promoting role of H19 in PDAC cells remain unclear. With a focus on cancer stem cells (CSCs), we elucidated the mechanisms by which H19 regulates PDAC metastasis through the overexpression and knockdown of H19 in PDAC cells. To determine whether H19 is expressed heterogeneously or homogeneously in human PDAC cells, we examined its expression in PANC-1 cells using a highly sensitive in situ hybridization technique. Under 2D-culture conditions, PANC-1 cells showed heterogeneous H19 expression and the presence of small populations of H19-expressing cells. In contrast, numerous H19-expressing PANC-1 cells were detected in 3D-cultured spheres. These results suggest that H19 is expressed in CSC-like cells among PANC-1 cells. To investigate the involvement of H19 in the development of CSC characteristics, we examined self-renewal ability, anti-cancer drug resistance, and CSC-marker expression. Sphere formation of PDAC cells depended on H19 expression. However, other CSC characteristics of the cells, including CSC-marker expression and anticancer-drug resistance were unaffected by H19 levels. In addition to its role in the development of CSC characteristics, we investigated the involvement of H19 in stromal invasion, which is a key step in the metastatic cascade. Although the invasion ability of PDAC cells was dependent on H19 expression, metalloproteinase activity, a key mediator of invasion, was independent of H19 expression. During the process of invasion, a critical event is the adhesion of cancer cells to the extracellular matrix. Therefore, we investigated whether H19 contributes to this cell-to-matrix adhesion step. We found that H19 promoted cell adhesion by regulating the expression of integrins and CD24. Notably, the increased adhesion of H19-overexpressing cells was blocked by an anti-β1-integrin antibody, which resulted in the inhibition of sphere formation and invasion. Taken together, H19 plays critical roles in CSC self-renewal and cell adhesion of PDAC cells that lead to invasion and metastasis. Our findings suggest that H19 represents a novel therapeutic target for the metastasis of pancreatic cancer.

#3569

Long non-coding RNA HAR1B is a novel player in parathyroid adenoma tumorigenesis.

Annamaria Morotti,1 Chiara Verdelli,2 Vito Guarnieri,3 LuciaAnna Muscarella,3 Rosa Maria Silipigni,4 Silvana Guerneri,4 Leonardo Vicentini,5 Sabrina Corbetta,6 Valentina Vaira1. 1 _University of Study of Milan; IRCCS Ca' Granda Foundation, Milan, Italy;_ 2 _IRCCS Istituto Ortopedico Galeazzi, Milan, Italy;_ 3 _IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo (Foggia), Italy;_ 4 _IRCCS Ca' Granda Foundation, Milan, Italy;_ 5 _IRCCS Istituto Auxologico Italiano, Milan, Italy;_ 6 _IRCCS Istituto Ortopedico Galeazzi; University of Milan, Milan, Italy_.

Background. Long non-coding RNAs (lncRNAs) have been implicated in the regulation of several physiological processes such as cell growth, differentiation and proliferation. Although lncRNAs functions in human diseases have not been completely disclosed, their role in endocrine cancer pathogenesis is emerging. We previously identified a long non-coding RNAs signature clearly distinguishing parathyroid adenomas (PAds) and carcinomas from normal glands. In particular, HAR1B was upregulated in PAds harbouring the common chromosome 11q loss of heterozygosity (Chr11q-LOH) compared with wildtype PAds, suggesting a potential new mechanism involving lncRNAs in 11q LOH-related parathyroid tumorigenesis.Here, we get preliminary insights into HAR1B ncRNA deregulation and function in human parathyroid adenomas.

Methods. Genomic aberrations of parathyroid adenomas (n=24, PAds) were analyzed by array Comparative Genomic Hybridization (aCGH). HEK293T cells were used for HAR1B and MEN1 transient silencing and Tazemetostat (EPZ-6438) treatment and analyzed for cancer pathways. Human PAds-derived parathyroid cells were transiently silenced for MEN1 expression and analyzed for HAR1B expression. Cancer pathways potentially involved in parathyroid adenomas were investigated by qPCR using the Human Molecular Mechanisms of Cancer- TaqManTM Array.

Results. PAds harbouring Chr11q-LOH (n=10) showed HAR1B upregulation compared to PAds with normal haplotype (n=12; p<0.0001). In vitro PAds-derived parathyroid cells silenced for MEN1 expression showed an increase in HAR1B expression levels. We tested the hypothesis that the inhibitory complex PRC2 is involved in HAR1B regulation, since its promoter is predicted to be regulated by EZH2. We found that the selective EZH2 inhibition by Tazemetostat increased HAR1B levels, in HEK293T cells. Next, we analysed expression levels of EZH2 target genes AXIN2, CCND1 and CDKN1A. All transcripts were significantly different between Chr11q-LOH and normal haplotype PAds. Moreover, H3K27me3 protein levels were higher in PAds with normal haplotype compared to Chr11q-LOH PAds, suggesting a higher EZH2 activity in PAds with normal haplotype. Lastly, HAR1B silencing in HEK293T cells induced upregulation of LEF1 and WNT1 mRNA levels and a reduction in CDKN1A protein levels.

Conclusions. We identified HAR1B as a new candidate gene in parathyroid tumorigenesis associated with Chr11q-LOH. Indeed, our data suggest that HAR1B could undergo epigenetic silencing and that MEN1 can contribute to its regulation. Moreover, we preliminary document that HAR1B may be involved in the Wnt signalling pathway in parathyroid tumors.

#3570

Investigating coding and non-coding RNA in papillary thyroid cancer.

Sina Dadafarin,1 Anvita Gupta,2 Katharine Dermigny,1 Leyla Cavdar,1 Brandon Pecchia,1 Melanie Jones,3 Timmy O'Connell,4 JK Rasamny,1 Nina Suslina,5 Codrin Iacob,5 Monica Schwarcz,1 Ameet Kamat,1 Cameron Budenz,1 Craig Berzofsky,1 Deya Jourdy,1 Tali Lando,1 Stimson Schantz,5 Sarnath Singh,1 Edward Shin,5 Augustine Moscatello,1 Raj Tiwari,1 Jan Geliebter1. 1 _New York Medical College, Valhalla, NY;_ 2 _Guggenheim Partners, NY;_ 3 _United States Military Academy Preparatory School West Point, NY;_ 4 _Sema4 Genomics, CT;_ 5 _New York Eye and Ear Infirmary, NY_.

Thyroid cancer is among the most common endocrine malignancies, with papillary thyroid cancer (PTC) accounting for approximately 80% of new thyroid cancer cases in 2017 (ACS). Despite the high sensitivity (95%) of ultrasound-guided fine needle aspiration biopsies (FNAB), approximately 20% of FNA biopsies are indeterminate, which require resection, despite the fact that about 50% are benign. The outcome is that many patients undergo surgical resection of benign disease resulting in avoidable iatrogenic morbidity, and about $700 million in health care costs. Thus, identifying diagnostic/prognostic molecular signatures of PTC would greatly reduce the number of costly, unnecessary resections following indeterminate biopsies. Following consenting of NYEEI patients, surgery, and diagnosis by the pathologist, RNA was prepared from PTC and matched-normal tissue samples, rRNA eliminated and RNA-Seq performed (100bp, paired-end). STARv2.5.2b/ htseq-countv0.6.1 and DESeq2 were used to align raw sequences, and measure transcript abundance. Preliminary bioinformatics analysis was performed with Advaita's iPathway Software. Over 1500 protein-coding transcripts, and 386 lincRNAs achieved 1.5 fold level differential expression (p= 0.05). Gene Ontology enrichment analysis indicated that locomotion, cell motility, signaling, cell differentiation and cell communication were among the most statistically significantly biological processes altered between PTC and matched-normal tissue. Cytokine, signal transducer, ion channel activity, receptor and growth factor activities were among the most statistically significantly molecular functions altered between PTC and matched, normal tissue. Additionally, Pathway Analysis indicated that cell adhesion molecules, cytokine-cytokine receptor, ECM-receptor, cancer, proteoglycans and Jak-Stat signaling were significantly altered. Thyroid differentiation scores (TDS) were calculated for each patients and correlations between differentially expressed lincRNA's and the TDS were identified. We expect followup experiments modulating the expression of lincRNAs most strongly correlated to the TDS will augment the expression of iodine-handling genes and differentiation in PTC cell lines. We anticipate that the detailed bioinformatics analysis of our coding and noncoding databases in addition to evidence from in vitro studies will yield new diagnostic/prognostic biomarkers, and therapeutic targets.

#3571

Identification of a novel imatinib upregulated lncRNA family that is required for efficient cellular transformation by Abl oncogene.

Jilong Chen, Xuefei Wang. _Fujian Agriculture and Forestry University, Fuzhou, China_.

Bcr-Abl oncogene is generated by a reciprocal translocation between chromosome 9 and 22 in human genome, giving Bcr-Abl protein with constitutive tyrosine kinase activity that causes chronic myeloid leukemia (CML) . Few acute lymphoblastic leukemia (ALL) patients also contain Bcr-Abl oncogene. Owing to the development of imatinib, over 90% of CML patients can be cured in recent years. However, the functional relevance of lncRNAs in Bcr-Abl-mediated leukemia remains obscure. Here, we identified a conserved, imatinib-upregulated lncRNA (IUR) family, named lncRNA-IUR37. Upregulation of lncRNA-IUR37 has been detected in both human and mouse Abl-transformed cell lines after imatinib treatment. Interestingly, lncRNA-IUR37 expression levels were significantly lower in leukemic cells derived from Bcr-Abl-positive ALL patients than those in normal control group. Furthermore, altering lncRNA-IUR37 expression remarkably affected survival of Abl-transformed leukemic cells, and tumorigenesis induced by these leukemic cells in xenograft mouse model. Knockdown of lncRNA-IUR37 in transgenic mice significantly promoted Bcr-Abl-mediated primary bone marrow transformation, and leukemia development in leukemia mouse model. These results indicate that lncRNA-IUR37 functions as a suppressor gene in Bcr-Abl-induced tumorigenesis. In addition, we demonstrated that lncRNA-IUR37 directly bound and affected the phosphorylation of STAT5, thereby impacting expression of transferrin receptor protein (TfR or CD71) to regulate leukemic cell survival. Together, our observations suggest that lncRNA-IUR37 is critically involved in Bcr-Abl-mediated leukemogenesis, and provide novel insights into complicated mechanisms underlying cellular transformation by Bcr-Abl oncogene.

#3572

SNPs in lncRNA genes are associated with non small cell lung cancer in a Chinese population .

Ruoyang Wang, Biyun Qian. _Shanghai Jiao Tong University School of Medicine, Shanghai, China_.

Background: Recent genetic studies indicate that single nucleotide polymorphisms (SNPs) in the genes encoding non-coding RNAs are associated with lung cancer susceptibility. But the comprehensive analysis of SNPs in long non-coding RNA (lncRNA) genes has not been elucidated. Methods: Based on the previous expression microarray of lncRNAs, we genotyped 17 SNPs located in 13 selected lncRNA genes by the TaqMan method in 1294 non-small cell lung cancer (NSCLC) cases and 1729 controls. Then we followed up NSCLC patients to get the survival data, and conduct logistic regression, Kaplan-Meier method and Cox hazards regression analysis to explore the associations of SNPs with NSCLC risk and overall survival (OS). Results: We identified 4 SNPs significantly associated with NSCLC risk. For rs498238, compared with CC genotype, TT genotype could decrease the risk of NSCLC (adjusted OR=0.32, 95%CI=0.11-0.92, p=0.034). As for rs16901995, TT genotype showed significantly protective effect compared with CC genotype (adjusted OR=0.76, 95%CI=0.59-0.99, p=0.040). For rs219741 dominant model, increased risk of NSCLC was evident in younger patients (age <60) compared those with wild type (adjusted OR=1.47, 95%CI=1.03-2.10, p=0.033). Rs3113503 demonstrated a controversial effect on NSCLC risk. No SNPs showed effects on overall survival of NSCL patients. Conclusion: Our findings indicated that genetic variants within lncRNA genes may predict NSCLC risk in Chinese population.

#3573

Identification and functional characterization of prognostic long non coding RNA LADDER in lung cancer.

Sathiya Pandi Narayanan,1 Sudhanshu Shukla,1 Jean Tien,1 Palak Shah,1 Sunita Shankar,1 Sethuramasundaram Pitchaiya,1 Srihari Srinivasan,1 Yuping Zhang,1 Xiaoming Wang,1 Lanbo Xiao,1 Xuhong Cao,1 Susan Freier,2 Andrew Watt,3 Shuling Guo,3 Felix Feng,4 David Beer,1 Saravana M. Dhanasekaran,1 Arul M. Chinnaiyan1. 1 _University of Michigan, Ann Arbor, MI;_ 2 _Ionis Pharamaceuticals, CA;_ 3 _Ionis Pharmaceuticals, CA;_ 4 _University of California, San Francisco, CA_.

Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related deaths in the world. Disease subtypes include lung adenocarcinoma (LUAD), which accounts for nearly 50% of lung malignancies, and lung squamous carcinoma (LUSC). Disease diagnosis at late stages and limited therapeutic options are the major causes for the poor prognosis of lung cancer patients. Additionally, lack of discriminatory diagnostic and prognostic biomarkers have hampered therapeutic management and, consequently, NSCLC patients are treated as one entity. In this modern era, the development of next generation based RNA sequencing (RNA-Seq) methodologies enable us to accurately quantify the transcriptional abundance in cells in a highly reproducible and reliable manner. By utilizing this technology, we sequenced a large cohort of lung cancer samples and leveraged several publically available RNA-Seq datasets for analysis to facilitate a deeper understanding of the disease. Furthermore, from the large RNA-Seq datasets, we also discovered long noncoding RNAs (lncRNA). These transcripts, which are longer than 200 nucleotides that are not translated into protein, have tissue-, lineage-, and cancer-specific expression patterns. In our previous study, we developed a highly significant prognostic marker gene signature by analyzing 255 LUAD patients from The Cancer Genome Atlas (TCGA) dataset. Using a training cohort, 96 genes including 5 lncRNAs had significant prognostic associations with lung cancer. Further, a stepwise regression model identified a four-gene signature, including one lncRNA (LADDER). The four-gene signature was then validated in two independent patient cohorts from the TCGA and in-house data. Functional studies showed that inhibition of LADDER lncRNA using both siRNA and ASOs decreased cell proliferation in lung cancer cells. In addition, knockdown of LADDER lncRNA using ASOs decreased lung cancer cell growth in vivo. Taken together, we have identified the lncRNA LADDER, a member of a four-gene prognostic signature, to play a functional role in lung cancer progression as well as serve as a robust prognostic marker.

#3574

Identification of long non-coding RNAs involved in chromosomal instability in a cellular model of prostate cancer.

Rogelio Montiel Manriquez. _Unidad de Investigación Biomédica en Cáncer - IIB - UNAM - Instituto Nacional de Cancerología, Mexico City, Mexico_.

Most of the human genome is transcribed into RNA, but only 2% encodes proteins, leading to a large group of non-coding RNA which, without constitutive RNA, is divided into 2 groups: small (sncRNA) and long (lncRNAs) non-coding RNAs. In this study we used RNA-seq data to identify new lncRNAs which could be involved in chromosomal instability (CIN) in prostate cancer (PCa). Our aim is to study their molecular mechanism and potential role as prognostic biomarkers in this disease.

Using RNA-seq data from two prostate cell lines, neoplastic (LNCaP) and non-neoplastic (PrEC), we have identified an unannotated long lncRNA adjacent to CEP55 (centrosomal protein 55), a gene associated with CIN. We have named this 1.6 kb transcript as lncRNA- CEP55. qPCR experiments showed it is downregulated in the neoplastic cell line, in contrast to the adjacent coding gene, CEP55, which is upregulated in the same cell line and downregulated in PrEC. Our data suggest this new lncRNA might be a possible repressor for CEP55. Changes in expression of CEP55 have been associated to CIN in other cancer cell lines.

LINC02043 is an annotated lncRNA. We identified a differential expression between PrEC and LNCaP using RNA-seq data. LINC02043 is adjacent to the protein-coding gene RFC4; this gene is associated with CIN in different human cancers. We performed qPCR experiments and found LINC02043 to be downregulated in LNCaP, whereas the adjacent coding gene RFC4 is upregulated; the opposite is found in PrEC cell line. Our findings suggest LINC02043 could have a possible repressor role in RFC4. This protein-coding gene is related to mismatch repair and DNA double-strand break repair, abnormalities in this coding gene have been associated with CIN in other cancer cell lines. LINC02043 could play an important role in the cis-regulation of RFC4.

lncRNA-CEP55 and LINC02043 are important targets of study, these lncRNAs could play an essential role in regulating the expression of adjacent genes related to CIN, an enabling characteristic that facilitates the development of the hallmarks of cancer. Further analysis is needed to validate their mechanisms of action and their potential use as prognostic biomarkers in PCa.

Particularly in PCa, CIN has lately been related to cancer progression. The role of lncRNAs as drivers of CIN in PCa is a promising field in order to understand PCa progression and may play a potential role as biomarkers in this disease.

#3575

Dysregulated expression of lncRNAs in glioblastoma multiforme and their association with overall survival.

Marek Vecera,1 Jan Oppelt,1 Lenka Radova,1 Radim Lipina,2 Stefan Reguli,2 Martin Smrcka,3 Radim Jancalek,4 Michal Filip,5 Marketa Hermanova,6 Leos Kren,7 Jiri Sana,1 Alena Kopkova,1 Julia Kovacova,1 Ondrej Slaby1. 1 _CEITEC - Central European Institute of Technology, Masaryk University, Brno, Czech Republic;_ 2 _Department of Neurosurgery, University Hospital Ostrava, Ostrava, Czech Republic;_ 3 _Department of Neurosurgery, University Hospital Brno, Faculty of Medicine, Masaryk University, Brno, Czech Republic;_ 4 _Department of Neurosurgery, St. Anne's University Hospital, Brno, Czech Republic;_ 5 _Department of Neurosurgery, Tomas Bata Regional Hospital, Zlin, Czech Republic;_ 6 _First Department of Pathological Anatomy, St. Anne's University Hospital, Brno, Czech Republic;_ 7 _Department of Pathology, University Hospital Brno, Faculty of Medicine, Masaryk University, Brno, Czech Republic_.

Introduction: Glioblastoma multiforme (GBM) is the most frequent primary brain malignancy of astrocytic origin. The prognosis remains very poor with the median overall survival (OS) being between 12 and 16 months from diagnosis despite early use of conventional medical therapy. Identifying new therapeutic targets, as well as prognostic and predictive biomarkers for accurate stratification of patients is therefore of utmost importance. Long non-coding RNAs (lncRNAs) are regulators of gene expression having critical impact on both physiological processes and the molecular pathology of GBM, indicating their potential as biomarkers and therapeutic targets.

Material and Methods: Our study included 219 GBM patients and 29 patients with non-dominant anterior temporal cortexes resected during surgery for intractable epilepsy. Informed consent approved by the local Ethical Commission was obtained from each patient. RNA (RIN > 8) from 77 specimens was used for next-generation RNA sequencing (RNAseq). rRNA depletion and cDNA library preparation were done with RiboCop rRNA Depletion Kit V1.2 (Lexogen) and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB), respectively. RNAseq was performed using NextSeq 500 High Output Kit and NextSeq 500 instrument (both Illumina). 8,414 lncRNAs and their sequential variants with non-zero RPKM at least in one sample were statistically evaluated. The alignment and target counts were performed with CLC genomic workbench. Selected significantly dysregulated lncRNAs between GBM and non-tumor controls were analyzed in a larger cohort of 188 specimens by qRT-PCR and the expression data normalized to PPIA was then evaluated by Mann-Whitney U test.

Results: Statistical analysis revealed 538 (P < 0.001) dysregulated lncRNAs in GBMs compared to non-tumor brain tissue samples. The expression of top 10 downregulated lncRNAs (SNAI3-AS1, LINC00882, RFPL1S, MIR137HG, TTLL7-IT1, PWAR6, LINC00634, LINC00632, DGCR5, LINC00982; logFC ≤ -2; P < 0.001) and 1 upregulated lncRNA (BTN2A3P; logFC ≥ 2; P < 0.001) in GBM and non-tumor controls was successfully validated by qRT-PCR (P < 0.0001). Moreover, the statistical analysis revealed 22 lncRNAs significantly dysregulated between patients with OS less than 12 months and those with OS equal or more than 12 months (P < 0.01).

Conclusion: We observed significant dysregulation of lncRNAs in GBM tissues compared to non-tumor controls based on the results of both RNASeq and qRT-PCR. We also found 22 lncRNAs to be dysregulated in relation to overall survival. Our study indicates that lncRNAs could serve as promising diagnostic and prognostic biomarkers in GBM. This work was supported by Ministry of Health of the Czech Republic grant nr. NV18-03-00398, grant of Czech Grant Agency nr. 17-17636S, and by the Ministry of Education, Youth and Sports of the Czech Republic under the project CEITEC 2020 (LQ1601).

#3576

MALAT1 is essential for A-NHEJ pathway choice during B cell immunoglobulin class switch recombination.

Jianjun Zhao, Yi Hu, Jianhong Lin, Hua Fang. _Cleveland Clinic Lerner Research Institute, Cleveland, OH_.

Metastasis-associated lung adenocarcinoma transcript 1(MALAT1) has been found high expressed in multiple cancer type including B cell lymphoma and multiple myeloma. In human cancer murine models, deficiency of MALAT1 reduce the tumor burden and malignancy phynotype. We have identified MALAT1 in alternative-non-homozygous end joining(A-NHEJ) pathway by binding to PARP1 and LIG3 in multiple myeloma cells, two key components of the A-NHEJ protein complex. To investigate requirments for MALAT1 in A-NHEJ pathway, by using MALAT1 deficient mice, we found that the contribution of MALAT1 in the context of AID induced DNA breaks in CSR of B cells in mice. MALAT1 is required for A-NHEJ DNA repair pathway, but not the NHEJ pathway, which is the major DNA repair pathway for CSR. To further prove the hypothesis that MALAT1 promote A-NHEJ pathway in B cells, we have engineered mice that conditionally overexpress MALAT1 in B lymphocytes confers a strong A-NHEJ activity in CSR. More importantly, we also found IgH/c-myc translocations are promoted in MALAT1 B cell specific transgenic mice. Thus, we identified MALAT1 as a critical noncoding RNA in regulating A-NHEJ pathway during CSR.

#3577

A p53-responsive long noncoding RNA GUARDIN is essential for genome integrity.

Lei Jin,1 Wang Lai Hu,2 An Xu,2 Yu Fang Wang,3 Rick F. Thorne,1 Xu Dong Zhang,1 Mian Wu2. 1 _Univ. of Newcastle, Callaghan, Australia;_ 2 _University of Science and Technology of China, China;_ 3 _Sichuan University, China_.

The list of long non-coding RNAs (lncRNAs) involved in the p53 pathway of the DNA damage response is rapidly expanding, but whether lncRNAs have a role in maintaining the de novo structure of DNA is unknown. Here, we demonstrate that the p53-responsive lncRNA GUARDIN is important for maintaining genomic integrity under steady-state conditions and after exposure to exogenous genotoxic stress. GUARDIN is necessary for preventing chromosome end-to-end fusion through maintaining the expression of telomeric repeat-binding factor 2 (TRF2) by sequestering microRNA-23a. Moreover, GUARDIN also sustains breast cancer 1 (BRCA1) stability by acting as an RNA scaffold to facilitate the heterodimerization of BRCA1 and BRCA1-associated RING domain protein 1 (BARD1). As such, GUARDIN silencing triggered apoptosis and senescence, enhanced cytotoxicity of additional genotoxic stress and inhibited cancer xenograft growth. Thus, GUARDIN may constitute a target for cancer treatment.

#3578

Comprehensive pharmacogenomic analysis establishes lncRNAs as protein-coding independent biomarker of drug response in human cancers.

Aritro Nath,1 Eunice Y. Lau,2 Adam M. Lee,1 Paul Geeleher,3 William C. Cho,2 R. Stephanie Huang1. 1 _University of Minnesota, Minneapolis, MN;_ 2 _Queen Elizabeth Hospital, Hong Kong, Hong Kong;_ 3 _University of Chicago, Chicago, IL_.

Somatic alterations in the cancer genome influence a patient's response to anti-cancer therapeutics. Thus, identifying context-specific alterations associated with differential drug sensitivity can improve patient response prediction and therapeutic selection. The discovery of pervasive dysregulation of long non-coding RNAs (lncRNA) in human cancers and their perceived role in gene regulation has led to the speculation that lncRNAs may also play a role in determining cancer drug response. However, it is unclear whether lncRNAs can augment the existing, predominantly protein coding, repertoire of pharmacogenomic biomarkers of anti-cancer agents.

We performed comprehensive analysis of whole genome and transcriptome data from over 1000 cancer cell lines and drug screening data for over 500 anti-cancer agents to systematically analyze over 5 million lncRNA-drug associations. We developed statistical and machine-learning frameworks to study these associations while controlling for the effects of confounding PCGs. Sparse regression and network-based methods demonstrated the predictive ability and biological relevance of literature-supported and novel lncRNAs biomarkers. Using regression analysis of lncRNA expression controlling for neighboring PCGs against drug response, we established that a large proportion of the lncRNAs candidates are PCG-independent biomarkers and potent predictors of cancer drug response. We further identified response-associated somatic alterations specifically in lncRNA genome that do not overlap PCGs. In addition, we demonstrated that collinear lncRNAs might be biologically relevant determinants of drug response. As an example, we identified EGFR-AS1 and MIR205HG as novel predictors of anti-EGFR therapy, which explain a significantly larger proportion of variability in erlotinib and gefitinib response as compared to EGFR mutations and copy-number alterations in drug screens and in patient data. We validated our findings in 16 non-small cell lung cancer and erlotinib-resistant cell lines. Our knockdown experiments revealed mechanisms of erlotinib sensitivity mediated by the two lncRNAs without influencing EGFR expression.

In conclusion, our comprehensive analysis generated unprecedented insights into the role of lncRNAs in cancer drug response that reaches beyond PCGs. This study will serve as a foundation for future cancer pharmacogenomic studies and will be an invaluable resource for investigators seeking insights into mechanisms of drug response.

#3579

Long non-coding RNA ADAMTS9-AS2 inhibits ovarian tumor growth via Annexin A1.

Cristian Rodriguez-Aguayo, Emine Bayraktar, Lingegowda S. Mangala, Cristina Ivan, Gabriel Lopez-Berestein, Anil K. Sood. _UT MD Anderson Cancer Ctr., Houston, TX_.

Non-coding RNAs (ncRNA), which account for more than 75% genome transcripts, have unique functions in regulating cancer pathogenesis. Long non-coding RNAs (lncRNAs) are key regulators of gene expression and are involved in cancer progression and metastasis. However, the role of long non-coding RNAs (lncRNAs) in high-grade serous ovarian cancer (HGSC) is not well understood. We used the NCode™ non-coding RNA array to screen downregulated lncRNAs in HGSCs. After analyzing the array data of lncRNAs in HGSCs (n=29) and normal ovarian (n=11) samples, the probes mapping to the top 25 most down-regulated non-coding RNAs were analyzed. Two lncRNAs (ADAMTS9-AS1 and ADAMTS9-AS2) were identified as the antisense of gene ADAMTS9, and we validate by in vitro translation assay, q-RT-PCR, in-situ hybridization and the biological functions of ADAMTS9-AS2 in ovarian cancer cells. Moreover, using A2780-CP20 orthotopic mouse models of ovarian cancer, we further demonstrated restoring ADAMTS9-AS2 resulted in profound reduction of tumor growth and metastasis. Here, we demonstrate that ADAMTS9-AS2 is highly down-regulated (5.01x10-13 and 1.6x10-19) in ovarian cancers. Overexpression of ADAMTS9-AS2 leads to significantly increased apoptosis (7.5% control and 19.5% AS2OE) and decreased proliferation (49.5% control and 37.0% AS2OE) compared with controls. Consistently, increased expression of ADAMTS9-AS2 led to significantly reduced tumor burden in A2780-CP20 orthotopic ovarian cancer mouse models. We identified the promoter region of Annexin A1 as the major target of ADAMTS9-AS2. The binding of lncRNA ADAMTS9-AS2 to the promoter region can facilitate the expression of tumor suppressor Annexin A1 thereby decrease the proliferation and increase apoptosis of cancer cells. Collectively, our data point to an important role for ADAMTS9-AS2 as a tumor suppressor in ovarian cancer.

#3580

Long noncoding RNA LINC00P8 functions as ceRNA to regulate RAC1 function by sponging miR-4715-5p in lung cancer.

Juze Yang,1 Jiani Yi,1 Xinyi Qian,1 Mengqian Yu,1 Qiongzi Qiu,2 Xufan Li,1 Jia Li,1 Yiling Jiao,1 Bingjian Lu,2 Enguo Chen,1 Yan Lu,2 Pengyuan Liu1. 1 _Sir Run Run Shaw Hospital and Institute of Translational Medicine, School of Medicine, Hangzhou, Zhejiang, China;_ 2 _Center for Uterine Cancer Diagnosis & Therapy Research of Zhejiang Province, Women's Reproductive Health Key Laboratory of Zhejiang Province, Department of Gynecologic Oncology, Women's Hospital and Institute of Translational Medicine, School of Medicine, Hangzhou, Zhejiang, China_.

Backgrounds: Long noncoding RNAs (lncRNA) are emerging as key players in the development and progression of cancers. However, the overall biological roles and clinical significance of most lncRNAs in lung carcinogenesis are unclear. Here, we identified a novel LINC00P8 and explored its functional role in lung cancer.

Experimental design: First, we analyzed LINC00P8 expression in RNA-sequencing data of lung cancerous and noncancerous tissues from The Cancer Genome Atlas. Then, the functional role and mechanism of LINC00P8 were further investigated by loss-of-function and gain-of-function assays in vivo and in vitro.

Results: LINC00P8 as an oncogene is significantly upregulated in lung cancer tissues and associated with poor prognosis. Knockdown of LINC00P8 induce growth arrest and metastasis in vitro, and inhibit tumorigenesis in mouse xenografts. Mechanistically, LINC00P8 functions as a ceRNA for miR-4715-5p, thereby leading to the depression of its endogenous target Rac family small GTPase 1 (RAC1).

Conclusions: As a novel regulator, LINC00P8 is upregulated in lung cancer, and LINC00P8-miR-4715-5p-RAC1/PAK1 axis plays a critical role in lung cancer carcinogenesis and progression. Our findings may provide bona fide targets for anti-cancer therapies in lung cancer.

#3581

LncRNA THAP9-AS1 promotes pancreatic cancer growth via sponging miR-484 and interacting with YAP to enhance YAP activity.

Nan Li,1 Guohua Yang,1 Zhimin He,1 Wanqing Liu,2 Haiying Liu,1 Guopei Zheng1. 1 _Affiliated Cancer Hospital & Institute of Guangzhou Medical University, Guangzhou, China; _2 _Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences; Department of Pharmacology, School of Medicine; Integrative Biosciences Center, Wayne State University, Detroit, MI_.

This study was supported by grants from the National Natural Science Foundation of China (No. 81872197, 81672616, 81401989 and 81602016); Guangdong Natural Science Funds for Distinguished Young Scholars (No.2016A030306003); Guangdong Special Support Program (No.2017TQ04R809); Science and Technology Program of Guangzhou (No.201710010100 and 201804010001).

Abnormal expression of long non-coding RNAs (lncRNAs) has been observed in various cancer types, providing probability to exploit new diagnostic markers, prognostic factors and therapeutic targets in cancer treatment. Our analysis showed lncRNA THAP9-AS1 was highly overexpressed in pancreatic cancer. Here, we aimed to investigate the roles and mechanisms of THAP9-AS1 in pancreatic cancer. We confirmed the overexpression of lncRNA THAP9-AS1 in pancreatic cancer and validated the no protein-coding potential. THAP9-AS1 promotes pancreatic cancer cells growth in vitro and in vivo. Mechanically, THAP9-AS1 exerts its effects via enhancing YAP signaling. Ectopic YAP expression disrupts the effects of THAP9-AS1 knockdown. Inversely, YAP knockdown overcomes the effects of THAP9-AS1 overexpression. THAP9-AS1acts as a competing endogenous RNA for miR-484, leading to YAP upregulation. Moreover, THAP9-AS1 binds YAP protein and inhibits the phosphorylation mediated inactivation of YAP by LATS1. Reciprocally, YAP/TEAD1 complex promotes THAP9-AS1 transcription to form a feed-forward circuit. Importantly, THAP9-AS1 level positively correlates with YAP expression in pancreatic cancer tissues. THAP9-AS1 and YAP overexpression predicts a poor outcome of pancreatic cancer patients. Our findings indicate that THAP9-AS1 plays an important role in pancreatic cancer growth via enhancing YAP signaling at two aspects. THAP9-AS1/YAP axis may serve as a potential biomarker and therapeutic target for pancreatic cancer treatment.

#3582

Circular RNA circPLEHM3 acts as the sponge of microRNA-9 to suppress ovarian carcinoma progression.

Lei Zhang, Pengyuan Liu, Bingjian Lu, Weiguo Lu, Yan Lu. _Zhejiang Univ., Hangzhou, China_.

Circular RNAs(circRNAs) are a class of stable and widespread endogenous noncoding RNA. CircRNAs have been reported to play essential roles in cancer biology, and have potential as biomarkers and therapeutic targets for cancer intervention. Here, we screened the expression profiles of human circRNAs in 27 ovarian cancer and 26 normal ovary tissues, and found circPLEKHM3 (hsa_circ_0001095) was significant down-regulated in ovarian cancer tissues. Patients with lower circPLEKHM3 expression tended to have poorer prognosis. Functionally, circPLEKHM3 overexpression inhibited cell growth, migration and epithelial-mesenchymal transition (EMT) in vitro and in vivo, but its knockdown reversed these effects. Further analyses showed that circPLEKHM3 sponges miR-9 to regulate the expression of BRCA1, DNAJB6 and KLF4, and inactivates the AKT1 expression. In addition, AKT inhibitor MK-2206 blocked the tumor-promoting effect of circPLEKHM3 knockdown, and potentiated taxol induced growth inhibition of ovarian cancer cells. Taken together, our findings demonstrated that circPLEKHM3 functions as a tumor suppressor in ovarian cancer cells by targeting the miR- 9/BRCA1/DNAJB6 /KLF4/AKT1 axis and may provide a prognostic biomarker and therapeutic target in ovarian cancer.

#3583

Differential expression of noncoding RNAs in black compared with white laryngeal cancers.

Kristianna Fredenburg, Jinmai Jiang, Tom Schmittgen. _University of Florida, Gainesville, FL_.

Background: Compared with other races, Blacks have maintained a higher incidence, prevalence, and poorer survival for laryngeal squamous cell carcinoma (LSCC). To date, a molecular basis remains undefined. Noncoding RNAs (ncRNAs), in particular microRNAs, have been found to be dysregulated in cancers with disparate outcomes. Ultraconserved RNAs (UCR), a highly conserved, new class of ncRNA, are regulated by miRNAs and differentially expressed in cancer. No studies to date have explored the dysregulation of these elements in Black compared with White LSCC. We present, here, our miRNA and UCR data from Black LSCC, White LSCC, and normal mucosal tissues.

Methods: RNA was isolated from 32 fresh frozen tumors (16 Black and 16 White) and normal mucosal tissues (10 White and 1 Black). For miRNA analysis, small RNA library construction and sequencing were performed. Differentially expressed miRNAs identified through sequencing were considered for qPCR validation using the criteria of FDR <0.05, FC >1.5. The expression of 481 transcribed-UCRs (T-UCR) was profiled using primers designed to each T-UCRs. A T-UCR was considered expressed in a particular sample if the mean CT ≤ 35. miRNAs and T-UCR were considered differentially expressed if the fold change between the comparative groups was greater than 1.5-fold and p < 0.05 (FDR).

Results: Our data show that there are significant differences in the somatic expression of two miRNAs, miR-9-5p and miR-191-5p, where both miRNAs were decreased in Black LSCC compared with White. However, when compared to normal mucosa, miR-9-5p was significantly overexpressed only in White LSCCs not in Black. In contrast, miR-191-5p was significantly decreased in Black LSCC compared to normal mucosa but not in White. This trend was seen also in our T-UCR data, where compared to normal mucosa, uc.424 was significantly overexpressed only in Black LSCC whereas uc.401 and uc.012 were significantly overexpressed only in White LSCC. Overall comparison of tumor to normal mucosa revealed 4 T-UCRs, uc.401, uc.389, uc.382, uc.218 were overexpressed in tumor compared to normal. No differences in T-UCR expression was identified in Black LSCC compared to White LSCC.

Conclusions: In summary, our data show that there are somatic expression differences and baseline expression differences in ncRNAs between Black and White LSCC. Our data suggest that gene regulatory mechanisms may differ in Black and White LSCC. Additionally, we are the first to identify T-UCR expression differences in laryngeal cancer. Through their interplay with miRNAs, these elements may contribute to LSCC tumorigenesis. Future studies will focus on uncovering the functional consequences of our differentially expressed ncRNAs and determining the manner in which they impact disparate outcomes seen in LSCC.

#3584

Role of long noncoding RNA LINC00461 in EGFR-TKIs resistant non-small cell lung cancer.

Ji Yun Lee, Thi-Thu-Trang Luu, Duc-Hiep Bach, Ruoci Hu, Donghwa Kim, Sang Kook Lee. _Seoul National University, Seoul, Republic of Korea_.

Chemo-resistance is one of the major factors limiting the therapeutic efficacy and prognosis of non-small cell lung cancer (NSCLC) patients, especially with patients who harbor epidermal growth factor receptor (EGFR)-activating mutations. Accumulating evidence suggests that aberrant expression of long noncoding RNAs (lncRNAs) is in part associated with carcinogenesis. Therefore, we tried to identify lncRNAs and their potential target genes in drug-resistant NSCLC. We evaluated lncRNAs with drug-resistant NSCLC using microarray data and in vitro assays. W found that lots of lncRNAs were differentially expressed between the drug-resistant NSCLC cells (HCC827-gef) and its parental (HCC827) cells. Among them, LINC00461 was found to be up-regulated lncRNAs, and highly correlated with the drug resistance. Taken together, the present study provides the expression profiles of chemo-resistant lncRNAs and identifies LINC00461 as a potential biomarker associated with EGFR-TKI resistance in non-small cell lung cancer cells.

This work was supported by a National Research Foundation of Korea (NRF) Grant funded by the Korean Government (MEST) (NRF-2016M3A9B6903499).

### Targeting Metabolism for Cancer Therapy

#3585

Amino acid transporter SLC6A14: A novel drug target for colorectal cancer.

Mohd Omar Faruk Sikder, Sathish Sivaprakasam, Vadivel Ganapathy. _TTUHSC, Lubbock, TX_.

SLC6A14 is a Na+/Cl− -dependent amino acid transporter capable of transporting 18 of the 20 proteinogenic amino acids, including arginine, leucine (mTOR activators) and glutamine (necessary for nucleotide biosynthesis). This transporter is expressed at basal levels in normal colon but significantly upregulated in colorectal cancer (CRC). However, the relevance of this upregulation to disease progression remains unknown. We postulated that deletion of SLC6A14 or pharmacological blockade of its function would suppress CRC by depleting amino acids and interfering with mTOR signaling selectively in tumor cells. To test this postulate, we first used Slc6a14-null mice. With two different models of spontaneous CRC (Apcmin/- and DSS/AOM), we found the tumor incidence and tumor growth were much lower in mice with Slc6a14-null background than in mice with Slc6a14. To evaluate the impact of pharmacologic blockade of the transporter on tumor growth we used a syngeneic tumor mouse model with MC-38 cells (a mouse CRC cell line); blockade of Slc6a14 with alpha-methyl tryptophan (α-MT) markedly reduced tumor growth. We then determined the transcriptome profiles of colonic epithelial cells and colonic non-epithelial cells from wild type mice and Slc6a14-null mice by RNAseq. There were hundreds of genes that were either upregulated or downregulated in the null mice. Ingenuity pathway analysis (IPA) of these data revealed predictive activation of canonical AMPK signaling pathway in colonic epithelial of Slc6a14-null mice. AMPK has anticancer activities by modulating multiple pathways including negatively regulating mTOR signaling. Moreover, upstream analysis showed predictive inactivation of two important tumor growth and survival signaling pathways ERK and EGF in the colon of the null mice. Likewise, significant upregulation of APC downregulated 1 (APCDD1) in epithelial cells in null mice implies predictive suppression of canonical Wnt signaling pathway. We also found marked differences in fecal microbiota as a result of Slc6a14 deletion. There was an increase in the relative abundance of beneficial microbiota including those capable of generating short chain fatty acids and lactic acid in null mice. We conclude that deletion of Slc6a14 or its pharmacological blockade protects against CRC by inducing amino acid starvation in tumor cells, thereby causing changes in multiple signaling pathways and in colonic microbiome. These studies identify SLC6A14 as a novel drug target for the treatment of colorectal cancer.

#3586

**Silencing of** GLSiso2 **(GAC) decreases cell proliferation and induces cell death in glioblastoma cell line.**

Yollanda Moreira Franco, Roseli Silva Soares, Sueli M. Oba-Shinjo, Suely Kazue Nagahashi Marie. _University of Sao Paulo, Sao Paulo, Brazil_.

The metabolic reprogramming is currently recognized as one of the hallmarks of cancer. Tumor cells have unusual metabolic activity in comparison to normal cells, and ability to reprogram their metabolic machinery to meet their biosynthetic and bioenergetic needs. Factors such as hypoxia, mutation in oncogenes and changes in signaling pathways may induce an upregulation of anabolic processes and suppression of catabolic pathways that leads to the maintenance of bioenergetics, redox status, cell signaling, and biosynthesis, which support rapid cell proliferation. Tumor cells can use a variety of alternative energy production pathways depending on the availability of nutrients. Glutaminolysis, the process where glutamine is transported into the cells and converted into α-ketoglutarate (α-KG) to enter into the tricarboxylic acid (TCA) cycle, is regulated positively in cancer. Glutaminase (GLS) is the main regulator of this pathway and presents two forms in humans: kidney-type glutaminase (KGA or GAC) and liver-type GLS2 (LGA or GAB), both with distinct tissue distribution, regulation and functions. The GLS presents two isoforms: GLSiso1 and GLSiso2, and their regulation are not yet well understood. The aim of this study was to analyze the expression of GLS in astrocytoma of different malignant grades. Additionally, in vitro assays were performed in a glioblastoma cell line (U87MG) to access GLSiso2 functions in astrocytoma. Differential expression levels of both isoforms were detected in 173 astrocytomas of our group cohort. A gradual increase of both isoforms expression levels was observed in parallel to the increase of the tumor malignancy. However, high expression level of GLSiso1 was detected in normal brain samples, while very low expression of GLSiso2. Hence, the isoform 2 is more eligible and relevant as a therapeutic target. To analyze the role of GLSiso2 in gliomagenesis, the isoform-specific transcript expression was inhibited by the interfering RNA method in U87MG cells. The efficiency of silencing was confirmed at gene and protein levels by quantitative real time PCR and western blotting, respectively. Additionally, GLSiso2 silenced cells presented significant decrease of cell proliferation, a temozolamide-senzitizing effect and an increase of cell death, when compared to control cells. Total GLS protein distribution was analyzed in different grades of astrocytoma through immunohistochemistry. The higher the malignant grade of astrocytomas, the higher the expression of GLS observed in tumor cell cytoplasm. Interestingly GLS was also expressed in endothelium, with higher expression in GBM cases compared to lower grades of astrocytoma and non-neoplastic brain tissues. Further analysis of mechanistic role of GLS isoforms in the endothelium cell compartment might better clarify a possible complementary effect in the metabolic reprogramming over the tumor growth.

#3587

Targeting cancer metabolism: A novel approach for improved radiotherapy.

Suchet Taori, Sarwat Naz, Janet Gamson, Askale Mathias, John Cook, James B. Mitchell. _National Institutes of Health, Bethesda, MD_.

One key distinguishing hallmark of cancer cells includes a deregulated cellular metabolism that can be reprogrammed to preferentially exhibit dependence on glycolysis over oxidative phosphorylation (OXPHOS) even in the presence of oxygen (commonly known as "Aerobic Glycolysis" or the Warburg Effect). The bi-directional conversion of glucose to lactate in the presence of oxygen is mainly driven by the activity of the lactate dehydrogenase enzyme (LDH), of which, its A isoform is highly overexpressed in a variety of tumors. In the present study we characterized two novel LDH inhibitors (NCI-006 and NCI-737) in combination with ionizing radiation (IR) for their anti-cancer and radio-sensitization effects across tumor types. This study primarily examined effects of this novel combination therapy in cell lines of Pancreatic (MiaPaca, Hs766T, Panc-1), Lung (H460, A549), Head and Neck (FaDU, UMSCC-1), and Prostate (DU145, PC3) origin. Our preliminary results indicated that targeting LDH in conjunction with IR can enhance radiosensitivity under both hypoxic and normoxic conditions across glycolytic tumor cell lines while not affecting non-glycolytic/normal cells (1522, skin fibroblast) in vitro. Further we established that this enhanced radiosensitivity could be attributed to reduced DNA repair as seen by enhanced expression of y-H2AX and reduced ATP generation. Our results also indicated that increased expression of lactate and glucose transporters, combined with cellular bioenergetics changes, leads to a reprogramming of cells to OXPHOS. Lastly, we found that inhibition of OXPHOS and NAD+ in conjunction with these novel LDH inhibitors promotes metabolic synthetic lethality in vitro. As cancer patients continue to receive radiation for local tumor control, we hope that future studies targeting cancer metabolic vulnerabilities in combination with IR in vivo will enhance the therapeutic ratio of radiotherapy.

*The LDH inhibitors used in this study were provided by the NCI Experimental Therapeutics (NExT) Program

#3588

Identification of a potent, orally bioavailable and selective MCT4 Inhibitor for the treatment of solid Warburg tumors.

Nina Hambruch, Barbara Herkert, Christian Gege, Aurelie Mallinger, Johannes Fabian, Olaf Kinzel, Yansong Wang, Gero Fink, Michael Albers, Anette Funk, Annika Bittman-Waetzig, Tilly Fleckenstein, Floriane Braun, Claus Kremoser. _Phenex Pharmaceuticals AG, Heidelberg, Germany_.

A key metabolic alteration in tumor cells is an elevated glycolysis rate, resulting in the production of increased amounts of protons and lactate leading to lactic acidosis within the tumor microenvironment. The tumor-promoting effects of lactic acidosis are versatile, ranging from stimulation of proliferation, angiogenesis and metastasis to the reshaping of the tumor immune environment to a tumor-permissive phenotype. In tumors lactate is transported across the plasma membrane by the monocarboxylate transporters 1 and 4 (MCT 1/4) in symport with a proton. While MCT1 is expressed almost ubiquitously at low levels, MCT4 expression is regulated by hypoxia-inducible factor 1α (Hif1α), the central player in the cellular response to hypoxia. Thus, MCT4 is present in hypoxic, highly glycolytic cells. Given their central role in prevention of intracellular acidification and lactate transport, MCTs appear to be indispensable for glycolytic tumor growth. Since MCT4 has the strongest implication on the development and aggressiveness of diverse cancer types it has emerged as a new target for the treatment of solid tumors.

We have identified MCT4 specific inhibitors structurally based on an internal compound library using cell-based lactate export assays. A subsequent extensive medicinal chemistry program was able to significantly improve activity and selectivity of the primary structures. Cellular target engagement was verified by cell-based assays like cellular thermal shift assay (CETSA). These efforts resulted in the identification of highly potent, orally bioavailable and selective MCT4 inhibitors (MCT4 IC50 < 20nM; MCT1 IC50 > 100µM) with good physicochemical properties and beneficial pharmacokinetics in mouse.

In vivo efficacy and tumor lactate modulation were evaluated in NCI-H358 xenograft and MC38 syngeneic mouse models. Thereby we explore the potential of our MCT4 inhibitor to act as a single anti-cancer agent or to synergize with other established therapies like angiogenesis inhibitors or cancer immunotherapeutics.

#3589

Targeting metabolic reprogramming and OXPHOS as a viable anti-melanoma strategy.

Gang Cheng,1 Jacek Zielonka,1 Micael Hardy,2 Michael B. Dwinell,1 Balaraman Kalyanaraman1. 1 _Medical College of Wisconsin, Milwaukee, WI;_ 2 _Aix Marseille University, Marseille, France_.

Invasive melanoma is an aggressive form of skin cancer with poor clinical outcomes. Nearly 50% of melanoma patients exhibit oncogenic BRAFV600E mutations. Existing BRAF kinase inhibitor drugs provide a short-term benefit prior to the rapid onset of drug resistance. Emerging results suggest that BRAF and other kinase inhibitors induce metabolic reprogramming from glycolytic energy production to oxidative phosphorylation (OXPHOS) in a subset of malignant melanomas. Metabolic reprogramming to OXPHOS is emerging as an important mechanism of resistance to BRAF kinase inhibitors and is associated with worse prognosis. Consequently, there is a significant opportunity for multimodal therapeutic approaches that selectively target the metabolic reprogramming associated with resistance to BRAF kinase inhibitors and promote immune-targeted therapy. We have previously developed mitochondria-targeted drugs by exploiting the enhanced negative mitochondrial membrane potential of cancer cells to selectively target and disrupt tumor growth. We hypothesized that metabolic reprogramming from glycolysis to OXPHOS in BRAF inhibitor-resistant melanoma cells would make them susceptible to OXPHOS inhibitors. In this study, we have modified the structure of a naturally occurring polyphenol, magnolol into a mitochondria-targeted-magnolol (Mito-MGN). We tested the effects of Mito-MGN on mitochondrial respiration and bioenergetic function, cellular redox status, and mitophagy in wild type (WT) and BRAF-resistant (metabolically reprogrammed) melanoma cells. Our data show that Mito-MGN inhibits mitochondrial respiration, melanoma cell proliferation and invasion at concentrations more than 200-fold lower than that of magnolol. Furthermore, Mito-MGN increases ROS formation and oxidation of mitochondrial peroxiredoxin. Preliminary results also indicate that BRAF inhibitor resistant melanoma cells exhibit enhanced OXPHOS as a consequence of metabolic reprogramming and that Mito-MGN effectively inhibits proliferation and mitochondrial respiration in the resistant cells. Mito-MGN inhibited mitochondrial complex I activity in both WT and resistant cells. The IC50 values to inhibit complex I-mediated respiration was consistent with the values calculated for inhibition of cell respiration. We conclude that inhibition of complex I is a key initial event that is responsible for the anti-proliferative effects observed in the presence of Mito-MGN against wild-type as well as drug-resistant melanoma cells.

#3590

Mechanisms and inhibition RIOK2 for obesity-driven prostate cancer.

Everardo Macias,1 Jiyoon Cho,1 Drew Rosowicz,1 Zahra Heidari,1 Sungyong You,2 Erick J. Maravilla,1 Jen-Tsan Chi,1 Stephen J. Freedland2. 1 _Duke University Medical Center, Durham, NC;_ 2 _Cedars-Sinai Medical Center, Los Angeles, CA_.

Obesity is associated with greater risk of high-grade prostate cancer (PC), recurrence after therapy, metastases, and PC specific mortality. The fact that obesity is a PC risk factor creates an opportunity to identify and target the mechanisms underlying obesity-enhanced lethal PC. We exploited this link by performing an shRNA in vivo screen in obese mice targeting the entire kinome, which found Right Open Reading Frame Kinase 2 (RIOK2) as essential for PCs in obese mice. We found RIOK2 gene signature is correlated with a 56% increased risk of metastatic PC. Moreover, when we queried only men with a BMI ≥ 30 kg/m2 we found that high levels of RIOK2 gene signature increased the risk of metastasis by an additional 50%. RIOK2, a serine/threonine kinase, is critical for de novo ribosome maturation and assembly, which is essential to maintain the high proliferation rates in cancer cells. Indeed, we found that RIOK2 loss reduced de novo protein synthesis in PC cells and decreased cell viability. Conversely, RIOK2 overexpression enhanced PC cell growth. As our goal is to find actionable PC targets to reduce obesity effects on PC, we conducted a protein structure based compound screen in silico and identified a lead kinase inhibitor of RIOK2 (kiRIOK2). Our lead kiRIOK2 inhibits cell proliferation, protein synthesis, anchorage-independent growth and invasion of multiple PC lines in the µM range. SAR optimized kiRIOK2 have increased efficacy and bioavailability. Our studies are providing probes and mechanistic insights to further investigate the role of RIOK2 in PC biology. In the long-term, this may help mitigate the added risk of obesity on PC metastasis and PC-specific mortality.

#3591

**Genetic and pharmacological inhibition of fatty acid synthase (FASN) attenuates prostate cancer driven by** Pten **loss.**

Caroline F. Ribeiro,1 Débora C. Bastos,2 Hubert Pakula,1 Thomas Ahearn,3 Jéssica Nascimento,1 John S. Clohessy,4 Lorelei Mucci,3 Silvio M. Zanata,5 Giorgia Zadra,1 Massimo Loda1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _University of Campinas, Piracicaba, Brazil;_ 3 _Harvard T. H. Chan School of Public Health, Boston, MA;_ 4 _Beth Israel Deaconess Medical Center, Boston, MA;_ 5 _Universidade Federal do Paraná, Curitiba, Brazil_.

Inactivation of the tumor suppressor gene phosphatase and tensin homologue (PTEN) is a common alteration in prostate cancer, occurring in the majority of metastatic castration-resistant prostate cancer (mCRPC) cases, and is associated with a more aggressive phenotype and worse prognosis. Another hallmark of prostate cancer is the dysregulation of lipid metabolism with overexpression of fatty acid synthase (FASN), the key enzyme in the de novo synthesis of fatty acids. FASN activity is essential for the generation of new cellular membrane lipids and signaling molecules, conferring growth and survival advantages for cells that overexpress it. Increased FASN expression enhances the aggressive phenotype associated with PTEN loss. To study how inhibition of FASN affects prostate carcinogenesis, we developed a mouse model combining prostate-specific homozygous knockout (KO) of Pten and Fasn genes. Deletion of Fasn, Pten or Fasn/Pten was achieved by expressing Cre recombinase under the control of a Probasin promoter, generating different genotypes: PtenloxP/loxPFasnwt/wtCrepos (P-KO), Ptenwt/wtFasnloxP/loxPCrepos (F-KO) and PtenloxP/loxPFasnloxP/loxPCrepos (F/P-dKO). Inhibition of FASN decreased the weight and volume of the ventral and anterior lobes of murine prostate when compared with Pten single KO mice. Histopathological analysis of the tissues also revealed a reduction in reactive stroma in the ventral and anterior lobes of F/P-dKO in comparison to the P-KO group. This phenotype is suggestive of inhibition of microinvasion. To assess whether FASN inhibition impairs cancer aggressiveness, we tested a novel FASN inhibitor compound, IPI-9119 (Infinity Pharmaceuticals), in epithelial cells from prostate of mice heterozygous for Pten loss. Pharmacological inhibition of de novo lipogenesis lead to decreased cell proliferation and reduction in invasion and motility. Finally, combined loss of PTEN with FASN overexpression assessed in 660 prostate cancer patients with 14.2 years of median follow up, was associated with lethality. Taken together, these data suggest that endogenous lipogenesis supports invasion in a PTEN null background. Since de novo lipogenesis contributes to the aggressive phenotype induced by PTEN loss in murine prostate, targeting lipid metabolism may represent an attractive therapeutic strategy in the context of prostate cancer driven by PTEN loss.

#3592

Evaluation of OT-82, a nicotinamide phosphoribosyltransferase inhibitor (NAMPTi), as a potential therapeutic option for Ewing sarcoma.

Anna Gibson, Choh Yeung, Arnulfo Mendoza, Sameer Isaaq, Christine Heske. _National Cancer Institute, Bethesda, MD_.

Targeting NAMPT, the rate-limited enzyme in the NAD salvage pathway, is a potentially attractive anticancer strategy, since cancer cells preferentially rely on this pathway to produce NAD. We explored the use of NAMPTi in Ewing sarcoma (ES), an aggressive pediatric malignancy for which novel therapeutic options are critically needed. We previously showed that ES cells are exceptionally sensitive to NAMPTi compared to other cancer types and have single agent activity in vitro and in vivo. We also demonstrated that combining NAMPTi with PARP inhibitors results in enhanced efficacy in vitro and in vivo. In this study, we further characterized the mechanisms of action of OT-82, a NAMPTi expected to enter early phase clinical trials, in ES cells and evaluated various dosing schedules for OT-82 combinations with cytotoxic therapy. On-target activity of OT-82 was assessed using NAD quantification assays and rescue experiments with multiple NAD intermediates. Characterization of NAD salvage pathway enzyme expression was performed by western blot. Mechanistic studies of cell cycle analysis, DNA damage, and cell death were performed using flow cytometry, western blotting, and comet assays. Glycolytic profiles of ES cell lines were analyzed using the Agilent Extracellular Flux Analyzer. Orthotopic ES cell line and PDX models in immunodeficient mice were used to evaluate in vivo combinations. Tumor measurements were performed with calipers twice weekly and toxicity was assessed with weekly body weights and general observations. Tumors were harvested at intermediate time points and at endpoints for biology and pharmacodynamic studies. Treatment with OT-82 revealed dose-dependent reduction of NAD both in vitro and in vivo. Addition of NMN, the product of NAMPT, rescued ES cell viability, indicating on-target activity of OT-82. While delayed NMN administration up to 48 hours after treatment with OT-82 rescued viability, a 72-hour delay rendered the effects irreversible in all cell lines. Treatment with OT-82 resulted in DNA damage, G2/M arrest and induction of apoptosis at 72 hours. Extracellular flux analysis indicated that OT-82 treatments resulted in a decrease in both oxidative and glycolytic activity. While NAMPT expression did not correlate with sensitivity to OT-82, low expression of NAPRT, an enzyme in a parallel NAD salvage pathway, appeared to be predictive of greater sensitivity to OT-82. Further, ES cells that expressed NAPRT could be fully rescued with addition of NA, the substrate of NAPRT, whereas low expressers could not. In vivo studies of the combination of OT-82 and irinotecan revealed enhanced antitumor activity. Studies of dosing schedule and tumor biology are ongoing and will be reported. OT-82 is an on-target NAMPTi that may be a novel targeted approach for the treatment of ES, especially in rational combinations.

#3593

PDK1 is a preferential metabolic target in glioblastoma.

Maheedhara R. Guda, Swapna Asuthkar, Andrew J. Tsung, Kiran K. Velpula. _Univ. of Illinois College of Medicine, Peoria, IL_.

Glioblastoma (GBM), the most common adult primary malignant brain tumor, carries a high morbidity and mortality despite therapeutic advances. GBM's progression is associated with a transition from oxidative phosphorylation to aerobic glycolysis as its main source of energy. Our laboratory recently confirmed that overexpression of pyruvate dehydrogenase kinase isoform 1 (PDK1) positively contributes to GBM progression. Here we report a novel role of mitofusin 1 (MFN1), a mitochondrial outer membrane GTPase that is a key regulator of mitochondrial fusion associated with glycolysis. We have shown that both PDK1 and MFN1 are highly co-expressed in glioblastoma patient specimens. Data obtained from The Cancer Genome Atlas demonstrated a positive correlation between PDK1 and MFN1 mRNA levels and the survival of GBM patients. The data obtained from the subtyped glioma stem cells corroborated with the datamining studies. To directly explore the potential MFN1-PDK1 interplay, we used CRISPR/Cas9 to suppress MFN1 expression in proneural, classical and mesenchymal subtypes of GBM stem cells (CSC). In another experiment, using ChIP assay, we showed that both PDK1 and MFN1 are under the transcriptional control of Myc oncogene. We present evidence that silencing MFN1 regulates altered metabolism in GBM CSC by increasing ROS levels, altering mitochondrial membrane potential, reduced lactate metabolites via NMR spectroscopy. Taken together, our results indicate that combined targeting of PDK1 and MFN1 shift Warburg effect towards oxidative phosphorylation, and may have increased therapeutic potential for glioblastoma patients.

#3594

GLUT1 as a metabolic target in glioblastoma therapy.

Collin M. Labak, Maheedhara R. Guda, Neha Jain, Chase P. Smith, Charles P. Cain, Andrew J. Tsung, Kiran K. Velpula. _University of Illinois College of Medicine-Peoria, Peoria, IL_.

Glioblastoma (GBM) is an aggressive diffuse glioma with poor prognosis due to lack of sound therapeutic options. As in many aggressive tumors, GBMs create a hypoxic microenvironment and switch to glycolytic metabolism from oxidative phosphorylation in what is known as the Warburg Effect. Here, we hypothesize that strategically inhibiting influx of glucose via targeting GLUT1 may reverse the Warburg Effect and selectively slow tumor progression. Nanostring data suggests an aberrant metabolic clincial profile in our cohort of GBM patients. Datamining studies and immunoblot analysis were conducted on these same human GBM specimens to demonstrate upregulated GLUT1. Mass spectrometric analysis was performed on GLUT1 precipitated from mesenchymal subtype patient-derived xenograft (PDX). Zonula Occludens adhesion studies were carried out to determine GLUT1 inhibition's effects on cell-cell adhesion structures. Finally, ATP, lactate release, and glucose uptake assays were performed on PDX and GLUT1-silenced PDX to demonstrate a normalized metabolic phenotype when GLUT1 is silenced. Our work suggests that GLUT1 overexpression plays a major role in the metabolic profile of mesenchymal subtype GBM, and that targeting its overexpression could prove effective in reversing the Warburg Effect in GBM cells and ultimately improving patient outcomes.

#3595

DSP-0692, a selective SCD inhibitor, enhances the anti-tumor activity of anti-PD1 antibody.

Eiji Sugaru, Yudai Furuta, Yoshikazu Nagagaki, Satoshi Ikeda, Yuichi Fukunaga, Hiroki Umehara, Tsuguteru Otsubo, Manabu Watanabe, Shingo Tojo, Miki Hashizume, Yasushi Matsuki, Hitoshi Ban. _Sumitomo Dainippon Pharma., Co., Ltd., Osaka, Japan_.

SCD (Stearoyl-CoA Desaturase), a key enzyme for lipid metabolism, is overexpressed in various types of cancer and plays an important role in cancer cell proliferation. We have previously shown that the identification and the functional characterization of DSP-0692, a novel and selective SCD inhibitor. SCD expression was induced in tumor cells cultured in sphere conditions, which were resistant to conventional agents within therapeutic concentration. DSP-0692 showed strong growth inhibition when tumor cells were cultured in sphere conditions, but had little impact on cells cultured under adherent conditions. In addition, DSP-0692 decreased the expression of stemness-related genes in tumor cells as well as in tumor tissues. Treatment with DSP-0692 suppressed tumor growth in multiple models both in single agent or in combination with conventional chemotherapies. DSP-0692 also inhibited WNT/beta-catenin signaling pathway through two different mechanisms (inhibition of Wnt3A secretion and nuclear translocation of beta-catenin). DSP-0692 exhibited lower IC50 value to sphere cells carrying mutations related to WNT/beta-catenin signaling pathway than those without these mutations. Blockade of the coinhibitory checkpoint molecule PD-1 has emerged as an effective treatment for many cancers. However, despite successes in the clinical setting, only a limited subpopulation respond to PD-1 blockade. We hypothesized that the deregulated lipid metabolism of tumor cells and microenvironment function as a barrier to antitumor immunity, and therefore that normalization of lipid metabolism with DSP-0692 might restore response to immunotherapy. Combination of DSP-0692 with anti-PD-1 antibody in the syngeneic CT-26 colon model, a resistant model to anti-PD-1 antibody, augmented tumor regressions relative to each single agent alone. DSP-0692 induced PD-L1 and PD-L2 expression in CT-26 cells, indicating that it is one of a mechanism of restoration of the effect of anti-PD-1 antibody. Our data suggest that remodeling the lipogenic tumor microenvironment has potential to convert patients resistant to immunotherapy into those who may receive clinical benefit of immunotherapy.

#3596

The combinatory effect of bevacizumab and metabolic inhibitors on angiogenesis and autophagy in gastrointestinal cancer cells.

Nadine Mahfouz,1 Rita Ammoury,1 Mayssam Moussa,1 Charbel Khalil,1 Joe Aoun,2 George Hilal1. 1 _Saint Joseph Univ., Beirut, Lebanon;_ 2 _St. Elizabeth's Medical Center, Boston, MA_.

Background: Bevacizumab is anti-VEGF drug that has shown therapeutic. However, treatment with bevacizumab has been associated to the development of resistance in several types of cancer. Glycolysis has been considered as a potential target for anti-cancer therapies and several molecules that target this pathway have been developed, including 2-Deoxy-D-glucose (2-DG) and 3-Bromopyruvate (3-BP). Autophagy is a process that degrades cellular organelles and proteins and maintains cellular biosynthesis during nutrient deprivation or metabolic stress. Since anti-angiogenic therapy and metabolic inhibitors both lead to nutrient deprivation in cancers, we investigated how a combination therapy would affect angiogenesis and autophagy in gastrointestinal cancer cells.

Methods: AGS and Caco-2 cells were treated with glycolysis inhibitors 2-DG (4 mM) and 3-BP (20 µM), in the presence or in the absence of bevacizumab at 100 µg/ml. Cell proliferation was assessed using tetrazolium salt. VEGF protein levels were quantified by ELISA. Autophagy and apoptosis markers' expression was assessed by quantitative real time PCR. Tube formation by human umbilical vein endothelial cells (HUVECs) was assessed using ECMatrix™.

Results: Our results showed that combining bevacizumab to 2-DG decreased AGS and Caco-2 cells' proliferation rates compared to each of these compounds alone. Interestingly, VEGF quantification showed that 2-DG increased VEGF secretion, in contrary to 3-BP. Moreover, our results showed that 3-BP reduced tube formation by HUVECs and amplified bevacizumab's anti-angiogenic effect when combined together. In autophagy markers' assessment, our results showed that 2-DG, 3-BP, and bevacizumab treatment increased FOXO1, FOXO3, LC3II, and HIF-1α expression. However, Beclin-1 expression, a protein implicated in the autophagic programmed cell death, decreased following bevacizumab treatment while it was upregulated by 2-DG and 3-BP. Hence, it was important to evaluate apoptotic markers' expression, caspase-3 and BAX. Our results showed that bevacizumab decreased caspase-3 and BAX expression while their expression was upregulated by 2-DG and that its combination to bevacizumab reestablished their levels.

Conclusions: Taken together, our results suggest that 2-DG and 3-BP, both improve cancer cells response to bevacizumab treatment by increasing its antiangiogenic outcome and reversing its anti-apoptotic effect. Moreover, it was interesting to find that bevacizumab treatment decreased Beclin-1, caspase-3, and BAX expression, suggesting a possible implication of autophagy in the development of resistance to bevacizumab. Hence, further studies are required in order to uncover the means by which bevacizumab modulates cancer cells autophagy and metabolism, and the effect of these changes on cancer cells survival.

#3597

BAY 2402234: Preclinical evaluation of a novel, selective dihydroorotate dehydrogenase (DHODH) inhibitor for the treatment of diffuse large B-cell lymphoma (DLBCL).

Ashley Eheim,1 Sven Christian,1 Hanna Meyer,1 Detlef Stoeckigt,1 Claudia Merz,1 Katja Zimmermann,1 Marcus Bauser,1 Andrea Haegebarth,1 Steven Ferrara,1 David B. Sykes,2 David T. Scadden,2 Stefan Gradl,1 Andreas Janzer1. 1 _Bayer AG, Berlin, Germany;_ 2 _Massachusetts General Hospital, Boston, MA_.

DLBCL is the most common type of non-Hodgkin lymphoma. It is an aggressive and fast growing tumor with two major molecular subtypes: germinal center (GCB) and activated B-cell like (ABC). While the majority of patients are 60 years or older, DLBCL can occur at any age.

Despite a cure rate of around 50% the need for novel therapies remains high, especially for relapsed/refractory DLBCL patients not eligible for stem cell transplant.

DHODH is a key enzyme in the de novo pyrimidine synthesis converting dihydroorotate to orotate. We recently discovered its role in AML differentiation (Sykes et al 2016, Cell) and we are investigating the novel DHODH inhibitor BAY 2402234 in an ongoing phase I study in myeloid malignancies (NCT03404726). Further screening of non-leukemia cancer types identified DLBCL as highly responsive to DHODH inhibition in preclinical studies.

Here, we disclose for the first time the functional preclinical characterization of BAY 2402234 in DLBCL. BAY 2402234 is a selective low-nanomolar inhibitor of human DHODH enzymatic activity. In vitro it potently inhibits proliferation of DLBCL cell lines in the sub-nanomolar to low-nanomolar range. The anti-proliferative effects can be rescued by uridine supplementation which bypasses DHODH via the salvage pathway and demonstrates the on-target specificity of the inhibitor. In vivo, BAY 2402234 exhibits strong in vivo anti-tumor efficacy in monotherapy in subcutaneous models derived from patient-derived xenograft (PDX) and cell lines representing various DLBCL subtypes, including GCB and ABC. Dose dependent target engagement and drug exposure of BAY 2402234 could be observed by increases in plasma dihydroorotate levels and unbound plasma drug levels after treatment with the inhibitor.

Based on preclinical data presented herein we plan to start clinical investigations of BAY 2402234 in patients with DLBCL in early 2019.

#3598

Phosphoglycerate mutase 1 inhibitor causes metabolic disadvantages in glioblastoma by rescue of FBP1 repression.

Sungmin Lee, Hyunwoo Kim, Hyunkoo Kang, BuHyun Youn. _Pusan National University, Busan, Republic of Korea_.

Radiotherapy is a primary treatment for patients with glioblastoma (GBM). Despite high therapeutic efficacy, the tumor cells that survive after radiotherapy may cause side effects. In this study, we found that fructose 1,6-bisphosphatase 1 (FBP1) which is a rate-limiting enzyme in gluconeogenesis was downregulated upon treatment with ionizing radiation (IR). IR-induced infiltrating GBM has overexpressed Ets1 which was suggested to be a transcriptional repressor of FBP1. Besides, glucose uptake and extracellular acidification rates were increased upon FBP1 downregulation, which indicated an elevated glycolysis level. We found that emodin, an inhibitor of phosphoglycerate mutase 1 derived from natural substances, significantly suppressed the glycolysis rate and the IR-induced GBM aggressiveness in the in vivo orthotopic xenograft mouse models. We propose that the downregulation of FBP1 level reprogrammed the metabolic state of GBM, and thus, FBP1 is a promising therapeutic target regulating GBM metabolism following radiotherapy.

#3599

BAY 2402234: Preclinical evaluation of a novel, selective dihydroorotate dehydrogenase (DHODH) inhibitor for the treatment of colorectal carcinomas.

Claudia Merz,1 Sven Christian,1 Ashley Eheim,1 Henrik Seidel,1 Ralf Lesche,1 Merlin Luetke- Eversloh,1 Hanna Meyer,1 Steven Ferrara,2 Marcus Bauser,1 Andrea Haegebarth,1 Stefan Gradl,1 Andreas Janzer1. 1 _Bayer AG, Berlin, Germany;_ 2 _Broad Institute, Cambridge, MA_.

Colorectal carcinoma (CRC) is the third most common malignancy in the US, being responsible for approximately 50,000 deaths per year. The overall 5-year survival rates are high for localized disease (~90%), but low for metastatic disease (~14%), thereby underlining the high unmet medical need for novel therapeutics for CRC patients.

DHODH is a key enzyme in the de novo pyrimidine synthesis, converting dihydroorotate to orotate. We recently discovered its role in AML differentiation (Sykes et al 2016, Cell) and we are investigating BAY 2402234 in an ongoing phase I study in myeloid malignancies (NCT03404726). Since then, several publications have also described a role of DHODH in solid tumor indications.

Herein we disclose the functional preclinical characterization of the novel DHODH inhibitor BAY 2402234 in CRC. BAY 2402234 is a selective low-nanomolar inhibitor of human DHODH enzymatic activity. In vitro it potently inhibits proliferation of CRC cell lines in the sub-nanomolar to low-nanomolar range. The anti-proliferative effects can be rescued by uridine supplementation which is known to bypass DHODH via the salvage pathway and demonstrates the on-target specificity of the inhibitor. Interestingly, not all CRC cell lines are sensitive to the inhibitor. Similar to the in vitro observations, BAY 2402234 exhibits strong in vivo anti-tumor efficacy in monotherapy in some, but not all, subcutaneous CRC xenograft models. A large CRC PDX in vivo screen revealed tumor growth inhibition in over 60% of the models in response to BAY 2402234 treatment. To elucidate the mode of action of BAY 2402234 in CRC we analyzed transcriptomic changes in CRC PDX models in vivo after single treatment with BAY 2402234, and identified several hundred differentially regulated genes at early time points. These in vivo PDX models were also used to explore potential effects of BAY 2402234 treatment on a large panel of metabolites.

These preclinical results support the clinical evaluation of BAY 2402234 in patients with CRC, and such a clinical study is planned for early in 2019.

#3600

Antibiotics suppress growth of breast cancer cells and synergize cytotoxicity of 2-Deoxy-D-glucose: Treating cancer like an infection.

Xianpeng Jiang, Amanda Benoit, Toby Jiang, Robert L. Elliott. _Sallie A. Burdine Breast Foundation, Baton Rouge, LA_.

Mitochondria evolved from free-living bacteria via endocytosis within a eukaryotic host cell millions of year ago. We hypothesized that antibiotics might damage mitochondria of human cancer cells and lead to oxidative damage of cancer cells. We examined three classes of antibiotics, azithromycin, doxycycline and ciprofloxacin in the human breast cancer cell lines MCF-7 and MDA-MB-231. The combination of antibiotics and the anti-glycolytic agent, 2-Deoxy-D-glucose (2-DG) was also tested in the cells. Cell growth was measured by MTT [3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide] assay. Mitochondrial membrane potential and reactive oxygen species were tested by fluorescent microscopy. Gene expression and DNA damage were measured by real time polymerase chain reaction (qPCR) and ELISA. All antibiotics suppressed mitochondrial membrane potential and growth of MCF-7 and MDA-MB-231 cells in a dose dependent pattern. The IC50 of all three antibiotics were less than clinical patient's maximum serum concentration (Cmax) or concentrations in tissues. The IC50 of ciprofloxacin in MCF-7 (1.25-2.5µM) and MDA-MB-231 (2.5-3.5µM) are much lower than the Cmax 7.7µM while patients take 500mg every 12 hours orally in clinical practice. The DNA oxidative damage metabolite, 8-OHdG, in the media was significantly increased after cancer cells were cultured in the media containing antibiotics for 24 hours. Three antibiotics upregulated gene expression of glycolytic enzymes including hexokinase 2(HK2), phosphofructokinase 1 (PFKM) and pyruvate kinase muscle isozyme M2 (PKM2), lactate dehydrogenase A (LDHA), and glucose transporters 1 (SLC2A1) and 3 (SLC2A3). The antibiotics increased uptake of 3[H]-2-DG of MCF-7 and MDA-MB-231. These results suggest that cancer cells increase aerobic glycolysis to produce ATP after antibiotic treatment. Thus, we combined 2-DG and antibiotics to treat the breast cancer cell lines. The antibiotics and 2-DG synergistically inhibited growth of the breast cancer cells. MDA-MB-231, a chemo-drug resistant cell line, has similar sensitivity to antibiotics and 2-DG as the MCF-7, a drug sensitive cell line. In summary, azithromycin, doxycycline and ciprofloxacin suppress the growth of breast cancer cells and augment the cytotoxicity of 2-DG to breast cancer cells. These findings suggest we can treat cancer with antibiotics similar to treating an infection.

#3601

Dietary phosphatidylcholine depletion reduces tumor development independent of autotaxin inhibition in hyperlipidemic mouse models of breast cancer.

Fredrick O. Onono, Ebubechi Adindu, Baoxiang Yan, Sony Soman, Courtney Hammill, Andrew J. Morris. _University of Kentucky, Lexington, KY_.

Lysophosphatidic acid (LPA) is a pleiotropic growth-factor lysophospholipid that promotes cancer cell survival, growth, migration, invasion, metastasis, and resistance to chemotherapy and radiotherapy. The main precursor for LPA, lysophosphatidylcholine (LPC) is derived majorly from the highly abundant phosphatidylcholine (PC). Hydrolysis of LPC to the circulating LPA is catalyzed by the enzyme autotaxin (ATX), a secreted lysophospholipase D. We investigated the effects of altering dietary intake of PC and pharmacological inhibition of ATX on breast cancer development in mice. To formulate diets with defined PC levels we used tandem mass spectrometry to measure the concentration of PC in various components of fat used to manufacture rodent diets. We determined that casein is the main source of PC when present in rodent diets. High fat diets (60% kcal fat) were formulated with casein (high PC) or amino acids in place of casein (low PC). To study the effects of these diets on breast cancer development, six-week old female C57Bl6 low density lipoprotein receptor knockout mice (LDLr-/-) were fed high fat diets for 12 weeks. Subsequently, E0771 syngeneic mammary cells were injected into the inguinal mammary fat pads of mice fed continuously on high fat diets. At the time of tumor cell inoculation, the animals were divided into groups to either receive daily dosage of a novel potent ATX inhibitor or vehicle control. Primary tumor growth was monitored using orthogonal caliper measurements and tumor volumes estimated from width2 × length/2. The number of metastatic nodules in the lungs were counted after staining with India ink. In high fat diet mouse models of breast cancer we observed that feeding mice with diets in which casein is replaced with amino acids decreases primary tumor growth. Contrary to our expectation, treatment with a potent ATX inhibitor resulted in no significant effect on primary tumor growth and an increased number of metastatic nodules compared with vehicle-treated mice. Moreover, treatment with the ATX inhibitor reversed the beneficial effects of casein replacement and instead resulted in enhanced tumor growth and metastasis. These studies suggest that dietary casein promotes primary tumor growth in the hyperlipidemic E0771 models of cancer and that targeting ATX inhibition is not efficacious in these models. Our results show that under certain hyperlipidemic conditions consumption of dietary casein can promote primary tumor growth. We also provide evidence emphasizing the need for further investigation in ascertaining the impact and utility of ATX inhibitors in breast cancer treatment.

#3602

JHU395, a nervous tissue penetrant glutamine antagonist, restricts growth of malignant peripheral nerve sheath tumor with inhibition of nucleotide synthesis.

Kathryn M. Lemberg,1 Liang Zhao,1 Ying Wu,1 Jesse Alt,1 Joanna Marie H. Aguilar,1 Jenny Lam,1 Alexandra J. Gadiano,1 Rana Rais,1 Pavel Majer,2 Jaishri Blakeley,1 Barbara S. Slusher1. 1 _Johns Hopkins, Baltimore, MD;_ 2 _Institute of Organic Chemistry and Biochemistry, Czech Acadmy of Sciences, Prague, Czech Republic_.

Malignant peripheral nerve sheath tumor (MPNST) is a sarcoma that occurs in ~10% of patients with neurofibromatosis type I (NF1). Incomplete surgical resection at diagnosis leads to a 4-year event-free survival rate of <30%. Thus, improved treatments are needed. Recent evidence suggests that perturbing glutamine utilization warrants further exploration as a potential therapeutic strategy for MPNST. Competitive glutamine antagonists (GA) have been studied in several clinical trials for sarcoma previously, but clinical development was hampered by gastrointestinal (GI) toxicity. JHU395 is a broadly active nervous tissue penetrant GA. JHU395 delivers active GA preferentially to nervous tissue which may result in less GI toxicity than was observed in past trials. The primary goals of this study were to evaluate JHU395 in preclinical models of MPNST and to investigate tumor metabolic changes in response to JHU395. We investigated glutamine antagonism on growth of MPNST cells in culture and murine flank MPNST. JHU395 was administered orally for 14 days to mice bearing inoculated tumors derived from the NPcis (NF1+/-;p53+/-) genetically engineered model of MPNST. Tumors were measured every other day and tumor volume was calculated as the primary endpoint. Concurrent evaluations of GI and neurotoxicity were performed as secondary endpoints. GA levels in tumor versus plasma were compared using a previously validated bioanalytical method. In vivo stable isotope labeled glutamine (15N2\- or 13C5-labeled) flux analysis and targeted liquid chromatography-mass spectrometry (LC-MS) metabolomics were performed on murine tumors. Compared to immortalized Schwann cells, growth of MPNST was preferentially inhibited by GA (IC50=8 micromolar versus >30 micromolar). JHU395 delivered GA to MPNST cells with >4-fold higher cell-to-plasma ratio compared to native GA and maintained ~2.5-fold higher tumor-to-plasma GA levels in vivo. Mice treated with JHU395 had >40% smaller mean tumor volume compared to controls with no overt toxicity. Quantitiative bioanalysis revealed that JHU395 treated tumors had >60% higher glutamine levels compared to controls 30 minutes after oral dosing. In vivo flux analysis demonstrated that JHU395 preferentially affects tumor glutamine utilization for nucleotide synthesis, while glutamine-derived substrates for the citric acid cycle appear unaffected. In summary, JHU395 inhibits growth of MPNST with inhibition of glutamine utilization in nucleotide synthesis and is well-tolerated. Nervous tissue penetrant glutamine antagonism is a feasible and effective therapeutic approach in preclinical models of MPNST. Future studies will investigate JHU395 in combination with nucleotide synthesis inhibitors in MPNST and investigate JHU395 efficacy in additional sarcoma models including patient-derived samples.

#3603

Sphingomyelin synthase inhibition in sarcomas results in ceramide accumulation and apoptosis.

Jared J. Barrott, Trevor Meldrum, Farjana Afrin, Srinath Pashikanti. _Idaho State University, Pocatello, ID_.

Sphingolipid metabolism is critical to cellular homeostasis especially as cells undergo mitosis. It is, therefore, unsurprising that the sphingolipid pathway is upregulated in a number of a cancers. Sphingoid bases such as sphingosine and ceramide are rapidly synthesized to provide the building blocks of other sphingolipids. Inhibition of downstream enzymes that convert sphingosine and ceramide into sphingolipids can lead to an increase of ceramide, which results in cellular toxicity and initiation of apoptosis. Using the marine natural product Jaspine B and chemically synthesized analogues, we investigated the potency of inhibiting sphingomyelin synthase in sarcoma cell lines. Sphingomyelin synthase is a downstream enzyme that converts ceramide into sphingomyelin by adding the polar head group phosphorylcholine to ceramide. Jaspine B and its analogues exhibited submicromolar potency against several human sarcoma cell lines. It was determined that the inhibition of sphingomyelin synthase resulted in an increase in ceramide and sphingosine levels and that sphingomyelinase enzymatic activity increased. While the mitochondria mass remained the same, the membrane potential of the mitochondria was significantly affected by the presence of the inhibitors. In conclusion, inhibitors of sphingolipid metabolism are viable therapeutic options with high untapped potential to treat cancer.

#3604

Synthetic lethality therapy for lung cancers with compromised glutathione homeostasis.

Xiang Yan,1 Bingliang Fang2. 1 _The Chinese PLA General Hospital, Beijing, China;_ 2 _MD Anderson Cancer Center, Houston, TX_.

Glutathione (GSH)/GSH reductase (GSR) and thioredoxin/thioredoxin reductase (TXNRD) are two major compensating thiol-dependent antioxidant pathways that maintain protein dithiol/disulfide balance. We hypothesized that functional deficiency in one of these systems will render cells dependent on compensation by the other system for survival, providing a mechanism-based synthetic lethality approach for treatment of cancers. The human GSR gene is located on chromosome 8p12, a region frequently lost in human cancers. GSR deletion was detected in about 6% of lung adenocarcinomas in The Cancer Genome Atlas database. To test whether loss of GSR sensitizes cancer cells to TXNRD inhibition, we knocked out or knocked down the GSR gene in human lung cancer cells and evaluated their response to the TXNRD inhibitor auranofin. The results showed that GSR deficiency sensitized lung cancer cells to this agent. A correlation analysis of auranofin sensitivity and gene expression levels in a panel of 129 NSCLC cell lines revealed that auranofin sensitivity correlated with the expression levels of the GSR, glutamate-cysteine ligase catalytic subunit (GCLC), and NAD(P)H quinone dehydrogenase 1 (NQO1) genes. In NSCLC patient-derived xenografts with reduced expression of GSR and/or GCLC, growth was significantly suppressed by treatment with auranofin. Together, these results provide proof-of-concept evidence that cancers with compromised expression of enzymes required for GSH homeostasis or with chromosome 8p deletions that include the GSR gene may be targeted by a synthetic lethality strategy with TXNRD inhibitors.

#3605

O-linked N-acetylglucosamine transferase as a potential therapeutic target for metastatic gastric cancer.

Joon Young Hur,1 Jeeyun Lee,1 Kyoung-Mee Kim,1 Sun-Ju Byeon,1 Inkyoung Lee,2 Won Ki Kang1. 1 _Samsung Medical Center, Seoul, Republic of Korea;_ 2 _Biomedical Research Institute, Samsung Medical Center, Seoul, Republic of Korea_.

Introduction

Many cancer types display elevated O-GlcNAcylation and aberrant expression of O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Here, we describe metabolic reprogramming linked to aberrant increased O-GlcNAcylation, and then inhibition of O-GlcNAcylation as a potential therapeutic target for gastric cancer (GC) treatment.

Methods

We utilized 17 established GC cell lines including 6 primary cell lines (AGS, OCUM-2M, MKN45, SNU1, SNU484, and SNU719), 4 metastatic cell lines (MKN1, MKN28, MKN74, and SNU216), 6 ascite cell lines (SNU5, SNU19, SNU601, SNU620, SNU638, and SNU668), and 1 pleural effusion cell line (KATO III). We collected 21 matched pairs of primary GC and normal gastric tissues and Patient-derived cells (PDCs) (N = 10) at the Samsung Medical Center. PDCs and all cells were grown in RPMI-1640 medium supplemented with 10% FBS. For immunoprecipitation, cells were transfected with pFlag-OGT or siOGT, washed with cold PBS, and lysed in a buffer. Protein extracts (300-500 μg) were incubated with protein G-agarose beads (sc-2002; Santa Cruz)

Results

Cell lines of primary, metastasis and ascites showed expression of O-GlcNAc and OGT. OGT expression is increased in tumor cell and ascites and western blot results showed that OGT expression is more increased in ascites than tumor cell. OGT knockdown inhibited MKN1 cell growth and colony formation. To determine the effect of OGT knockdown on tumor growth in vivo, BALB/c nude mice were injected subcutaneously in the bilateral flank with siC and siOGT transfected MKN1 cells (1 x 107 cells). OGT overexpression enhances O-GlcNAcylation of NFκB p65, c-Myc, and K-Ras. NFκB p65, c-Myc, and K-Ras immunoprecipitates obtained from the cellular extracts were analyzed by immunoblotting for O-GlcNAc. OGT knockdown inhibited PDC (collected from malignant ascites) cell growth. OGT knockdown inhibited GC PDC (1 x 104 cells) cell invasion. Targeting OGT with siRNA in xenograft models decreases the growth of tumors.

Conclusion

Depletion of OGT inhibits cancer cell proliferation and OGT knockdown significantly delays tumor growth in xenograft model. Future work will take advantage of approaches to explain the impact of O-GlcNAc modification on GC.

#3606

Blockade of the PPARα metabolic checkpoint with TPST-1120 suppresses tumor growth and stimulates anti-tumor immunity.

Chan C. Whiting,1 Nick Stock,2 Davorka Messmer,2 Traci Olafson,2 Derek Metzger,1 Amanda Enstrom,1 Jennifer McDevitt,1 David Spaner,3 Peppi Prasit,2 Dipak Panigrahy,4 Ginna Laport1. 1 _Tempest Therapeutics, San Francisco, CA;_ 2 _Inception Sciences, San Diego, CA;_ 3 _Sunnybrook Research Institute, Toronto, Ontario, Canada;_ 4 _Beth Israel Deaconess Medical Center, Boston, MA_.

Tumors evolve to modulate metabolism to promote their own survival and to suppress tumor-specific immunity. Hypoxic conditions in the tumor microenvironment (TME) induce fatty acid oxidation (FAO), and diverse malignancies are reliant on this metabolic pathway. Additionally, suppressive immune cell populations including M2 macrophages, myeloid-derived suppressor cells and regulatory T cells preferentially utilize FAO. Peroxisome proliferator-activated receptor alpha (PPARα) is the principal transcription factor that regulates the expression of FAO genes, and this metabolic checkpoint is critical for tumor proliferation. TPST1120 is a first-in-class selective competitive antagonist of the human PPARα. To test the hypothesis that blocking FAO with TPST-1120 confers anti-tumor efficacy, we assessed TPST-1120 in multiple syngeneic and xenograft mouse models. Blockade of PPARα with TPST-1120 mediated potent anti-tumor immune responses and significant tumor regression in syngeneic models of breast, lung, colon, pancreatic and melanoma in addition to xenograft models of CLL, AML, pancreatic and melanoma cancers as a monotherapy or in combination with chemotherapy. In pancreatic and breast cancer models, TPST-1120 augmented regression of tumor growth in combination with chemotherapy. In combination with anti-PD1, TPST-1120 treatment resulted in significant reduction of tumor growth in ovarian orthotopic (ID8) and colon (MC38) models; cured mice were completely protected against autologous tumor challenge, strongly suggesting immunological T cell memory against the primary tumor. Studies in genetic knock-out mice indicated that macrophages and antigen cross-presenting dendritic cells are required for TPST-1120 activity, mediated through thrombospondin-1(TSP-1) and stimulator of interferon genes (STING). Consistent with prior reports, inhibition of PPARα with TPST-1120 skewed macrophages in vivo toward an M1 effector phenotype. These results provide the rationale for evaluating TPST-1120 in patients with advanced malignancies. A Phase 1/1b open-label, dose-escalation and dose-expansion study of TPST-1120 as a single agent or in combination with systemic anti-cancer therapies is planned in early 2019.

#3607

**Inhibition of mutant IDH enzymes reduces production of 2-HG but does not restore wild type IDH activity** in vitro **or** in vivo **.**

Katie Sellers, Taryn Sleger, Sebastien Ronseaux, Rohini Narayanaswamy, Thomas Roddy, Brandon Nicolay. _Agios Pharmaceuticals, Inc., Cambridge, MA_.

Mutations in isocitrate dehydrogenase (IDH) 1 and 2 occur in variety of malignancies, and notably >70% of low grade gliomas. These mutations lead to neomorphic enzymatic activity that results in the production of the oncometabolite (D)-2-hydroxyglutarate (2-HG) through the consumption of NADPH. IDH mutant cells and tumors have been shown to have heightened sensitivity to reactive oxygen species (ROS) and radiation, though it is unclear if this sensitivity is due to a loss of IDH wild type enzymatic NADPH production, the neoenzymatic activity of the mutant enzyme, or a combination of these activities. In support of the hypothesis that sensitivity to ROS and radiation is due to a reduced IDH wild type activity in IDH mutant cells, we previously demonstrated that combined treatment of mutant IDH mouse xenografts with AG-881, a potent orally available mutant IDH inhibitor, and radiation resulted in superior tumor growth inhibition than either modality alone. To extend this investigation, we have now developed a novel assay to read out the wild type activity of IDH1/2 enzymes in both cell-based assays and mouse xenograft models. Using an isotopic labeling approach that allows us to fully differentiate the activity of wild type IDH1/2 from the mutant enzymatic activity in the same cell or tumor, we observed that mutant IDH cells and tumors have a significantly reduced level of wild type IDH1/2 activity compared to normal tissue or IDH wild type tumors. Importantly, pharmacologic inhibition of mutant IDH1/2 that resulted in >90% reduction in 2-HG production in mutant cells and tumors did not restore the IDH1/2 wild type activity. These data support our previous observations and suggest that therapeutic strategies targeting the reduced activity of wild type IDH enzymes, such as modalities that induce ROS or treatment with radiation, would be effective when combined with mutant IDH inhibitors.

#3608

BPM31510 exploits differential redox vulnerabilities between normal and glioblastoma cells to mediate its anti-cancer effect.

Jiaxin Sun,1 Seema Nagpal,1 Chirag Patel,1 Milton Merchant,1 Tiachang Jang,1 Anne R. Diers,2 Shiva Kazerounian,2 Stephane Gesta,2 Niven R. Narain,2 Rangaprasad Sarangarajan,2 Lawrence Recht1. 1 _Stanford Medicine, Palo Alto, CA;_ 2 _BERG LLC, Framingham, MA_.

Glioblastoma is an aggressive cancer, the proliferative capacity of which is correlated with glycolytic metabolism. BPM31510 is a novel formulation for delivery of supraphysiological levels of ubidecarenone to the mitochondria, enabling cancer specific metabolic switches. It is being studied in Phase I clinical trials versus a number of tumors, including glioma. Here, the effects of ubidecarenone on viability and redox homeostasis of glioma and non-tumorigenic cells was assessed using in vitro monoculture and coculture systems and an in vivo preclinical model. BPM31510 administration (50 mg/kg bid i.p., beginning 4-8 days post-inoculation) resulted in over a 20% long term survival rate in C6 tumor-bearing rats. We next compared BPM31510 effects in vitro between glioma lines (rat C6, human U251) and murine NIH3T3 fibroblasts, as a stromal control. In monocultures, decreased growth was observed in glioma lines and NIH3T3 with increasing BPM31510 doses; however, glioma lines were 2-fold more sensitive to BPM31510 compared to NIH3T3 cells (IC50 glioma lines: 230 µM vs IC50 NIH3T3: >460 µM). To investigate the differential sensitivity to BPM31510, a coculture system was developed by coincubating 2 x 105 C6-GFP labeled cells and NIH3T3 cells. After 6 days of coculture, the percentage of C6 relative to NIH3T3 cells was lowest at doses of BPM31510 between 115 µM and 230 µM, evidence of greater sensitivity to BPM31510-induced cytotoxicity in the C6 glioma cells than the non-tumorigenic component. At higher doses, differential effects on cell viability were less apparent. The level of superoxide, a central reactive oxygen species important in redox homeostasis, was also assessed using Mitosox in cocultures. At a BPM31510 dose which resulted in maximal differential viability between C6 and NIH3T3 cells (230 μM), the maximal differential superoxide level was likewise greatest. The basal differential in Mitosox signal was 9-fold between C6 and NIH3T3 cells, and it increased to over 50-fold upon treatment with BPM31510 (230 μM), implying that BPM31510 exploits differential redox vulnerabilities between C6 and NIH3T3 to mediate its anti-cancer activity. At high doses of BPM31510, differential effects on superoxide levels were less apparent. In summary, BPM31510 has marked anti-cancer activity in rats implanted with C6 glioma, and its differential effects on the viability of normal and transformed cells are associated with maximal differences in BPM31510-induced superoxide production. Together, these data suggest that differential redox vulnerabilities between tumorigenic and non-tumorigenic cells may underpin the anti-cancer activity of BPM31510, and identification of in vivo correlates of redox indices may represent an avenue to improved measurement of anti-cancer efficacy as well as define patient populations responsive to BPM31510.

#3609

Metformin treatment outcomes in relation to obesity and cation transporter expression in mouse models of endometrial cancer.

Aruljothi Muralidharan, Hui Guo, Jianjun Han, Lu Zhang, Wenchuan Sun, Yajie Yin, Chunxiao Zhou, Ruth S. Everett, Dhiren R. Thakker, Victoria L. Bae-Jump. _University of North Carolina, Chapelhill, NC_.

Purpose: Obesity is associated with increased risk and worse outcomes for endometrial cancer (EC). Metformin, a frontline therapy for type 2 diabetes, is thought to have both indirect and direct anti-cancer effects via decreasing circulating insulin/glucose and activating AMPK/inhibiting mTOR signaling, respectively. Due to its positive charge (pKa 12.4) and hydrophilicity (logD -6.13 at pH 7.4), metformin requires cation transporters for cellular uptake where it can then activate AMPK; and thus, variation in transporter expression may be critical for response to metformin for cancer treatment. Thus, we explored the inter-relationship of obesity, cation transporter expression and the anti-tumorigenic efficacy of metformin in a genetically engineered endometrioid EC mouse model.

Methods: LKB1fl/fl p53fl/fl mice were fed a low-fat diet (10% calories from fat) vs. a high-fat diet (60% calories from fat) to mimic diet-induced obesity, starting at 3 weeks of age (n=10 mice/group). AdCre was injected at 6 weeks of age to induce EC. Mice were treated for four weeks with placebo or metformin (200 mg/kg/day, oral gavage) following tumor onset. Cell proliferation/apoptotic markers and downstream targets of AMPK/mTOR signaling were evaluated in the ECs by immunohistochemistry. Cation transporter expression was assessed in the ECs by Western blotting and RNA sequencing.

Results: Obesity led to a doubling of endometrial tumor size in obese vs. lean mice (1.469g vs. 0.75g). Metformin was more potent in inhibiting tumor growth in obese vs. lean mice (78% vs. 60%) compared to the respective controls (p<0.05). Metformin (i) decreased expression of Ki-67 and cyclin D1 (cell proliferation markers) by 65% and 15%, respectively, in tumors from obese mice and by 18% and 5%, respectively, in tumors from lean mice vs. their respective controls (p<0.05); (ii) increased cleaved caspase-3 (marker of apoptosis) in tumors from obese and lean mice (2.3 fold and 2.1 fold) vs. their respective controls (p<0.05); (iii) increased phosphorylated AMPK expression and decreased expression of phosphorylated S6 in ECs from obese and lean mice vs. their respective controls (p<0.05); (iv) increased cation transporter OCT3 and PMAT gene expression in only tumors from obese mice, with a trend towards higher protein expression of OCT1 and 3 and MATE1 and 2 with metformin treatment in ECs from both lean and obese mice.

Conclusion: Our data suggest that metformin has greater anti-tumorigenic efficacy in obese vs. lean mice with EC. In addition, cation transporters were expressed in the endometrial tumors and were affected by metformin treatment. Studies are underway in EC patients to understand the impact of obesity and transporter expression on response rates to metformin in the clinic.

#3610

Cellular fatty acid uptake is enhanced in tamoxifen-resistant breast cancer cells.

Sewon Hwang, Mi-Ock Lee. _Seoul National Univ. College of Pharmacy, Seoul, Republic of Korea_.

The most accepted therapeutic regimen for treating ER-alpha breast cancer is Tamoxifen. Yet over 30% of patients gain resistance. Recently, lipid metabolism has been rising as a new mode of cancer drug resistance. In order for a rapid growth, cancer cells show a strong avidity for lipid which they obtain by de novo lipogenesis or increasing the uptake of exogenous lipids. Lipid metabolism may confer drug resistance, but little is known about its association with endocrine resistance. Thus, we aimed to investigate the association of lipid metabolism in the context of tamoxifen resistance. First, fatty acid translocase and membrane-associated fatty acid binding protein, both of which are responsible for fatty acid uptake, were overexpressed in MCF7/TAMR-8 and T47D/TR-1, TR-2 cell lines. Also the mRNA level of fatty acid activating enzyme, long-chain fatty-acid-coenzyme A, were elevated as well. Next, through Oil-red-O staining and Nile red staining followed by FACS analysis, we examined whether tamoxifen-resistant cells actually increase fatty acid uptake. When supplemented with free fatty acids, tamoxifen-resistant cell lines showed higher efficiency in fatty acid uptake compared to the control cell lines. Subsequently when the amount of triglyceride accumulation was measured, more TG were present in resistant cells at the basal level and after free fatty acid treatment. Taken together, these results suggest lipid uptake by cancer cells may play a role in driving tamoxifen-resistance in breast cancer.

#3611

Erythropoietin can cancelchemo-resistances viadown regulation of ABC transporters.

Hirofumi Matsui, Hiromi Kurokawa. _University of Tsukuba, Tsukuba, Japan_.

Introduction: Erythropoietin (EPO) is a glycoprotein cytokine which stimulates red blood cell production (erythropoiesis) in the bone marrow. The receptor of it (EPOR) was reported to exist on the cancer cellular membrane: more than 85% cancer cell expresses EPOR. Several reports suggested to play an important role for cancer cellular growth, however, the role of EPOR in cancer cells is not confirmed. The authors have studied the cancer cellular porphyrin accumulation, metabolism and excretion to clear the pathogenetic mechanism which involved the high concentration of porphyrins. The authors found that reactive oxygen species (ROS) accelerate both the cancer cellular porphyrin accumulation and the effect of photodynamic therapy. However, we cannot confirm which substrate is a key of signal transduction. EPO has been known used to be up-regulated at the metabolic acidosis via the stabilization of HIF-1α, a nuclear factor. Since HIF-1α can be stabilized by not only acidosis but also the high concentration of nitric oxide (NO), NO rich cancer cells may involve EPOR. Moreover, the purpose of EPO-EPOR system is likely to survive the hypoxic condition, it may keep intracellular heme/ porphyrins. The authors' hypothesis is EPO-EPOR system in cancer cells may down-regulate ABCG2 which excretes heme/ porphyrins, and several anti-cancer drugs. Moreover, if so, the excretion of several anti-cancer chemo reagents should be inhibited to involve more effective chemotherapy.

Methods: To prove these possibilities, the authors carry out following experiments with gastric and colorectal cancer cells. The existence of EPOR, the amount of ABC transporters (B1, C5, G2) after the recombinant EPO (rEPO) exposure (dose dependency and time dependency) were analyzed by western blotting. Cytotoxicity of anti-cancer drugs with or without rEPO was measured with the Cell Counting Kit-8.

Result: (1) EPOR existed in each cell line used in this study. (2) rEPO treatment involved down regulation of ABC transporters (B1, C5, G2) in dose dependently. (3) ABCG2 expression decreased until 24 hours after rEPO treatment. However, its expression re-increased 48 hours after treatment. (4) The 24 hours pretreatment of EPO significantly increased the effect of doxorubicin, paclitaxel, irrinotecan, cisplatin, 5-FU and oxaliplatin even in chemo-resistant cells.

Conclusions: rEPO accelerates the effect of anti-cancer drugs via the down regulation of ABC transporters.

#3612

**Decreased vitamin C uptake mediated by** SLC2A3 **promotes leukemia progression and impedes Tet2 restoration.**

Jun Liu,1 Junshik Hong,2 Kwangsung Ahn,3 Sungsoo Yoon2. 1 _Cancer Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea;_ 2 _Department of Internal Medicine, Seoul National University Hospital, Seoul, Republic of Korea;_ 3 _PDxen Biosystems Inc., Seoul, Republic of Korea_.

Background: Two new studies in Nature and Cell, from Agathocleous et al. (2017) and Cimmino et al. (2017), respectively, showed that vitamin C regulates HSC function and suppresses leukemogenesis by modulating Tet2 activity. However, little is known about the relationship between vitamin C transporter and hematological malignancies.

Purpose: In this study, we investigated the role of SLC2A3, a gene that encode GLUT3 which mainly transport glucose and oxidized form vitamin C, in hematological malignancies. Through this research we identified a set of biomarkers which can predict the effect of vitamin C treatment and develop stagey to improve this putative innovative treatment.

Results: We analyzed gene expression patterns of the major GLUT family genes in primary AML blast cells using previously published microarray datasets (GSE37307) and (GSE9476) and determined that SLC2A3 gene expression was significantly decreased in blast cells compared with normal hematopoietic cells. By contrast, expression of SLC2A1, SLC2A2 and SLC23A2 was not significant different in between primary AML cells and normal hematopoietic cells. Next, we analyzed the relevance of SLC2A3 expression to the survival of AML patients from TCGA and TARGET database and found that below-median SLC2A3 expression was associated with inferior overall survival. Subsequently, we detected the SLC2A3 mRNA expression level on 4 AML cell lines and 3 DLBCL cell lines, there were very low expression level on OCI-AML-3 and OCI-LY1. To check whether decreased expression of SLC2A3 has an impact on the Vitamin C effect, we treated the 7 cell lines with dose dependent vitamin C, up to 500 μM, 48 hours. The cell viability assay showed that OCI-AML-3 and OCI-LY1 have no response to Vitamin C, however the proliferation of NB4, HEL, HL60, OCI-LY19 and Toledo was suppressed by 250 μM Vitamin C.

Conclusions: SLC2A3 expression was decreased in AML blast cell and down regulation of SLC2A3 was closely associated with the poor outcomes of AML patients. In vitro study showed that SLC2A3 was essential for the effect of Vitamin C. Therefore, SLC2A3 could be a potential biomarker to predict the effect of Vitamin C treatment. In addition, through targeting SLC2A3, efficacy of Vitamin C treatment could be improved. 

## CANCER CHEMISTRY

### Drug Delivery and Nanoparticles

#3613

Mononuclear phagocytic system occupancy to increase nanomedicines based treatment efficacy.

matthieu germain, Laurence Poul, Julie Devalliere, Marion Paolini, Audrey Darmon, Maxime Bergere, Oceane Jibault, Francis Mpambani. _Nanobiotix, Paris, France_.

Introduction: To efficiently deliver treatment, nanomedicines must exhibit sufficient blood bioavailability for further accumulation at the target site. So far, a large part of the administered dose remains useless due to the high rate of clearance by the mononuclear phagocytic system (mainly by Kupffer cells). Here we propose a new approach to redefine nanomedicines bioavailability by priming the body before receiving the treatment as a sequential administration of a nanoprimer before the nanomedicine. The nanoprimer is a nanoparticle designed to transiently occupy the main pathway responsible for the limited bioavailability of nanomedicines. As such, the nanoprimer allows to redefine the bioavailability of different nanomedicines and improve treatment's outcomes for a large panel of therapeutic agents (e. g. small molecules or nucleic acids).

Methods: Optimization of nanomedicines bioavailability was performed using a liposomal nanoprimer with specific physico-chemical properties. First we evaluated the nanoprimer's biodistribution by in vivo imaging system, and its accumulation within the different hepatic cell populations by flow cytometry. Then we evaluated the impact of this nanoprimer on the bioavailability of different nanomedicines: for this, blood bioavailability and biodistribution of irinotecan loaded liposomes or mRNA loaded nanoparticles (lipidic and polymeric) were evaluated by HPLC and fluorescence respectively. We compared distribution of each nanomedicine administered intravenously alone or 10min after a single intravenous (IV) injection of nanoprimer on HT29 (colorectal adenocarcinoma) xenografted mice. Finally, the impact of nanoprimer on efficacy was evaluated by a tumor growth delay experiment for irinotecan loaded liposomes by IV administration of nanoprimer 10min before irinotecan loaded liposomes in HT29 xenografted mice. Treatment cycle was repeated one week later. For nucleic acid loaded nanoparticles, the impact of the nanoprimer was evaluated by measuring transfection efficiency on HT29 xenografted mice 24h after IV administration of the nanoprimer 10min before nanomedicine.

Results: liposomal nanoprimers present a rapid accumulation in the liver with a preferential localization in Kupffer cells and liver sinusoidal endothelial cells. This accumulation leads to transient cells saturation and decreased hepatic trapping of the nanomedicines. Increased bioavailability resulted in a higher accumulation of irinotecan loaded liposomes within the HT29 tumor and in an increased efficacy. Such a bioavailability increase is also related with the transfection profile obtained for the nucleic acid loaded nanoparticles.

Here we showed that a same nanoprimer could be used to prime the body to receive different types of nanomedicines and improve treatment's outcomes. Such approach may decrease compromises between bioavailability, efficacy and toxicity.

#3614

Targeted 3DNA-siHuR nanocarrier therapy for pancreatic ductal adenocarcinoma.

Grace McCarthy,1 Christopher Schultz,1 Aditi Jain,1 Teena Dhir,1 Charles Yeo,1 Jessica Bowers,2 Lou Casta,2 Kelly Rhodes,2 Lori Getts,2 Robert Getts,2 Trevor Baybutt,1 Adam Snook,1 Jonathan Brody1. 1 _Thomas Jefferson University, Philadelphia, PA;_ 2 _Genisphere, Hatfield, PA_.

The poor overall survival rate of pancreatic ductal adenocarcinoma (PDAC), less than 10% over 5 years, is largely due to the majority of patients presenting with overt metastatic burden or post-surgical recurrence of micrometastases. Due to these factors, new treatment methods are needed to effectively target both primary and metastatic tumors.

We have previously established that the mRNA binding protein Human antigen R (HuR), is an important molecule for PDAC progression. In a tumor microarray of matched normal and malignant patient tissues, we found active HuR staining in 79% of tumor samples, which was not seen in normal pancreatic tissue (n=70). We have shown that homozygous CRISPR knock-out of HuR results in xenograft lethality, as well as sensitization of PDAC cell lines to standard-of-care chemotherapy. Therefore, in the current experiments we have utilized a targeted 3DNA nanocarrier for the delivery of an RNA silencer against HuR in order to enhance tumor susceptibility to chemotherapy. Treatment of PDAC cell lines with this 3DNA nanocarrier was highly effective in knocking down both HuR and established HuR targets (DCK, IDH1, PIM1).

To increase the specificity of the 3DNA nanocarrier, we sought to discover targeting moieties specifically upregulated in PDAC. We found increased folic acid receptor (FAR) expression in HS 766T, MIA PaCa-2, and PANC-1 PDAC cell lines with an average fold change of 2.6, in comparison to normal pancreatic cells. Additionally, patient tissue samples showed elevated FAR levels in 100% of tumors (n=80). Thus, by attaching folic acid to the 3DNA nanocarrier (FA-3DNA), we were able to target the nanocarrier specifically to tumors, both primary and metastatic. This was detected by tagging FA-3DNA with Alexa750 fluorophore and monitoring its movement in mice bearing luciferase expressing-MIA PaCa-2 tumors. FA-3DNA had a longer half-life and increased delivery to tumors, as compared to non-targeted 3DNA.

We also demonstrated that treatment of mice with FA-3DNA conjugated with an RNA silencer for HuR (FA-3DNA-siHuR) led to a significant decrease in HuR mRNA and protein, in comparison to mice treated with 3DNA containing a control RNA silencer (FA-3DNA-siControl). Subsequent in vivo experiments showed prolong survival of mice treated with FA-3DNA-siHuR, as compared to mice treated with FA-3DNA-siControl (p=0.04).

Our previous work has established HuR as a promising therapeutic target in PDAC. In both in vitro and in vivo models, we have shown FA-3DNA-siHuR is capable of targeting tumors directly for the inhibition of HuR. These results suggest treatment with FA-3DNA-siHuR will sensitize tumors to standard of care chemotherapy, which can be introduced systemically or incorporated into the 3DNA nanocarrier for direct delivery. The latter could potentially increase the efficiency of the chemotherapy, and also of critical importance, reduce the toxic adverse effects of systemic chemotherapy.

#3615

Design for a flexible localized drug delivery system.

Marzenna Wiranowska,1 Ryan Toomey,2 Rana Falahat,2 Norma Alcantar2. 1 _Univ South Florida Morsani College of Medicine, Tampa, FL;_ 2 _Univ South Florida, Tampa, FL_.

This project focuses on studying the optimal design of a drug delivery system based on niosomes (non-ionic surfactants vesicles) embedded in a hydrogel network (chitosan) to ensure full control of the release time and dosage of cancer treatment drugs for localized delivery. Here we present data on the molecular interactions facilitating specific binding at tumor cell surfaces to aid in the treatment of ovarian, brain and lung cancers to name a few. We have done three types of characterizations to determine the basic design parameters for a personalized cancer treatment. First, we determined how fundamental surface interactions can be optimized to target and attack tumor integrity and boundaries. We used confocal microscopy and Xenogen imaging to determine how data from cells and in-vivo models compare. Second, we determined the parameters of chemotherapeutic agents that can act individually or in a cocktail configuration in both in-vitro and in-vivo studies. We used light scattering and transmission electron microscopy to determine the topography for guidance on the design of the drug delivery process. Third, we found that specific targeting can be accomplished by optimizing the binding between drug molecules and mucin 1 (MUC1) found on the surface of epithelial-derived tumors. We used attenuated total internal reflection Fourier transform infrared (ATR-FTIR) spectroscopy to elucidate the interactions that enhance the selectivity of our drug delivery system with respect to model cancer systems. In addition, we used chlorotoxin, a carrier-type polypeptide known to bind to a broad number of tumors. By identifying and optimizing these two methodologies, we have found that tumor and normal cells can be selectively targeted. The significant advantage of applying a localized treatment is the avoidance of systemic exposure to chemotherapy drugs thereby limiting their side effects.

#3616

Towards intracellular targeting: Cytosolic delivery using TAT-trimers.

Ole Tietz, Rod Chalk, Katherine A. Vallis. _University of Oxford, Oxford, United Kingdom_.

Large therapeutic biomolecules, particularly antibodies, are generally directed against cancer cell surface antigens to facilitate antibody-antigen interaction. This limitation excludes a rich array of potential intracellular cancer targets, which has prompted research to solve the problem of transport of large molecules across the cell membrane and their release into the cytoplasm. However, current efforts suffer from endosomal entrapment and lack of efficacy due to low intracellular concentration ranges. Using the archetypal cell penetrating peptide (CPP) TAT, we report the design, synthesis and evaluation of two novel trimeric TAT clusters with enhanced efficiency in the low micromolar range. In addition, we demonstrate that geometry and conformation of TAT clusters affects internalization properties and mechanism. TAT-trimers were synthesized using copper catalyzed 2+3 cycloaddition between tetrakis core structures and azide / alkyne modified TAT (49-57) peptide. The trimeric clusters were furnished with AlexaFluor488 (AF488) for in vitro detection. Tri-TAT A is designed around a smaller, more strained core structure than tri-TAT B, which is designed around a larger core structure and more flexible. Both trimers, as well as monomeric AF488 labelled TAT (mono-TAT), were evaluated for their cell penetrating properties using live-cell fluorescence confocal microscopy in HeLa and CHO cells. The uptake of tri-TAT A and tri-TAT B into HeLa and CHO was found to be significantly higher than that of mono-TAT (1 μM; 60 min); in addition, the uptake of the trimers (1 μM) was found to be significantly higher than that of 10 μM mono-TAT, indicating that clustering TAT peptides improves their efficacy. Interestingly, we found that tri-TAT A shows homogenous cytosolic uptake and nucleolar staining at 1 μM, while tri-TAT B shows no evidence of cytosolic uptake or nucleolar staining in either HeLa or CHO cells. The fluorescence signal in cells treated with tri-TAT B was focal in nature, which is typical of endosomal entrapment. These results suggest that the geometry of the core structure and the conformational flexibility of TAT trimers is of decisive importance to the efficacy of cytosolic uptake. Time course experiments with tri-TAT A indicate that the homogenous cytosolic signal spreads evenly from the membrane into the cytosol, suggesting that the trimer translocates directly across the membrane, rather than or in addition to entry into the cytosol via endosomal escape. TAT-trimers were successfully conjugated to antibodies and antibody fragments (Fab). These constructs showed favourable internalisation properties in preliminary cell uptake studies in comparison single TAT constructs. Trimeric arrangement of CPPs on a rigid scaffold represents a promising approach to promoting cell uptake of anticancer drugs directed against intracellular targets and a significant improvement over recently reported CPP approaches.

#3617

Trastuzumab-conjugated gold nanoparticles as novel HER2-targeted therapeutics against trastuzumab-resistant gastric cancer.

Kento Kumon, Tetsushi Kubota, Shinji Kuroda, Nobuhiko Kanaya, Yoshihiko Kakiuchi, Tomoko Tsumura, Satoru Kikuchi, Shunsuke Kagawa, Hiroshi Tazawa, Toshiyoshi Fujiwara. _Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama city, Japan_.

While trastuzumab (Tmab), a monoclonal antibody that targets the human epidermal growth factor receptor 2 (HER2), has greatly contributed to improve survival outcomes in metastatic gastric cancer, an issue of concern is that no current HER2-targeted therapeutic agent is effective against Tmab-resistant gastric cancer. Nanotechnology, which has progressed rapidly in recent years, greatly contributes to progress in medical fields including cancer therapy, and especially, gold nanoparticles (AuNPs) are attention-grabbing nanomaterials that are promising drug carriers with unique properties of high in vivo stability and a large surface area available for attachment of materials such as antibodies. Methods: We created HER2-targeted AuNPs by conjugating Tmab onto their surface (T-AuNPs) and examined their therapeutic efficacy and cytotoxic mechanisms using HER2-postive Tmab-resistant (MKN7) or Tmab-sensitive (NCI-N87) gastric cancer cell lines in vitro and in vivo. We also examined T-AuNPs on HER2-negative MKN74 gastric cancer cells with adenoviral vector-mediated overexpression of the HER2 extracellular domain (HER2-ECD). Results: The size of T-AuNPs was 85.39 ± 0.68 nm and the surface was negatively charged with 39.43 ± 0.85 mV. In in vitro assays, T-AuNPs showed 6-fold higher cytotoxic activity than Tmab against NCI-N87 cells. Then, T-AuNPs showed significantly stronger cytotoxic effects than controls against both MKN7 and NCI-N87 cells although Tmab had no effect on MKN7 cells. Autophagy was proven to play an important role in T-AuNPs cytotoxic mechanisms. This autophagy was considered to be induced by HER2-dependent internalization of T-AuNPs, some of which were interestingly located in the cytoplasm independent of lysosomal encapsulation. Although T-AuNPs were not cytotoxic towards MKN74 cells, they became cytotoxic following HER2-ECD overexpression. Finally, Intratumoral injection of T-AuNPs showed potent antitumor effects, which were mediated by autophagy, against NCI-N87 and MKN7 subcutaneous tumors in vivo mouse models. Conclusion: HER2-targeted AuNPs with conjugated Tmab can be a promising strategy for the development of novel therapeutic agents to overcome Tmab resistance in gastric cancer.

#3618

The mechanism of functional lipoprotein-like nanoparticle (FLip-NP) induced lymphoma cell death.

Jonathan S. Rink, Shuo Yang, Adam Lin, Reem Karmali, Colby S. Thaxton, Leo I. Gordon. _Northwestern University, Chicago, IL_.

Introduction: Diffuse large B-cell lymphoma (DLBCL) cells have an increased demand for cholesterol and cholesteryl esters to maintain membrane anchored, pro-survival signaling pathways, such as B-cell receptor (BCR) signaling. We have previously reported that synthetic, biomimetic functional lipoprotein gold nanoparticles (FLip-NPs) induce lymphoma cell death in vitro and in vivo through targeted, receptor-mediated cholesterol depletion (Yang et al PNAS 2013) and that cellular cholesterol levels influence response to FLip-NPs (Rink et al Molecular Pharmaceutics 2017). Given the critical role of cholesterol in maintaining proper membrane organization to enable intracellular signaling, we hypothesized that FLip-NP-induced cholesterol depletion reorganizes the plasma membrane, leading to reduced membrane anchored signaling. Additionally, we hypothesized that FLip-NPs will synergize with small molecule inhibitors of BCR-related kinases, leading to enhanced lymphoma cell death.

Methods: SUDHL4 [germinal center (GC) DLBCL] and Ramos [Burkitt's lymphoma] cells were used for experiments. Confocal fluorescent microscopy was used to visualize changes in membrane organization following FLip-NP treatment. Changes in the localization and phosphorylation of signaling kinases (e.g. AKT, ERK1/2) following FLip-NP treatment were measured using immunoprecipitation, phospho-kinase arrays, and western blot assays. The Amplex Red Cholesterol Assay was used to measure total cellular cholesterol, while cell viability was quantified using the MTS assay.

Results: In SUDHL4 and Ramos cells, FLip-NPs induced a reorganization of the plasma membrane, clustering of the HDL receptor, scavenger receptor type B1. This reorganization correlated with decreased phosphorylation of the signaling kinases AKT, ERK1/2, LCK and LYN, as well as a reduction in total cellular cholesterol. Given the decreases in pro-survival kinase phosphorylation, we investigated whether small molecule inhibitors of these kinases enhanced FLip-NP efficacy. FLip-NPs synergized with inhibitors of PI3K (idelalisib, duvelisib), SYK (R406), and AKT (GDC-0068), increasing lymphoma cell death. Finally, addition of a PTEN inhibitor, to prevent AKT de-phosphorylation, rescued Ramos and SUDHL4 from FLip-NP induced cell death.

Conclusion: These data demonstrate that FLip-NPs reduce B-cell lymphoma cell cholesterol, inducing changes in the organization of the cell membrane and, subsequently, a series of changes to membrane-anchored pro-survival and pro-proliferative signaling pathways. Enhancing cholesterol starvation strategies, such as the combination of the targeted cholesterol depletion agent FLip-NPs with small molecule inhibitors of signaling kinases, represents a novel therapeutic strategy for DLBCL and may be applicable to other cholesterol dependent malignancies.

#3619

Nanoparticle formulation of VERU 111 for pancreatic cancer treatment.

Vivek K. Kashyap, Bilal B. Hafeez, Qinghui Wang, Neeraj Chauhan, Prashanth K. Nagesh, Murali M. Yallapu, Duane D. Miller, Wei Li, Meena Jaggi, Subhash C. Chauhan. _University of Tennessee Health Science Center, Memphis, TN_.

Background: Pancreatic cancer (PanCa) is one of the leading causes of cancer-related mortality in the United States due to very limited therapeutic options. Thus, developing novel therapeutic strategies will help for the management of this disease. We recently identified VERU-111, a novel synthetic molecule which showed potent anti-cancer effect against PanCa via targeting clinically important βIII and βIV tubulin isoforms. In this study, we synthesized and characterized its novel nanoformulation (MNP-VERU) and evaluated its therapeutic effects in vitro and xenograft mouse model.

Methods: MNPs were prepared by chemical precipitation method and loaded with VERU-111 using diffusion method. This formulation was characterized for particle size, chemical composition, and drug loading efficiency, using various physico-chemical methods (TEM, FT-IR, DSC, TGA, and HPLC). The internalization of MNP-VERU was achieved after 6 hours incubation with MNP-VERU in PanCa cells. To determine therapeutic efficacy of MNP-VERU, we performed various in vitro (MTS, wound healing, boyden chamber real-time xCELLigence, and apoptosis assays) and in vivo (mouse tumor xenograft) studies using PanCa. Effect of MNP-VERU on various key oncogenic signaling pathways, and miRNAs was evaluated by Western blot, immunohistochemistry (IHC), confocal microscopy, qRT-PCR and in situ hybridization (ISH) analyses respectively.

Result: Our novel MNP-VERU formulation provided average size of 110 nm in dynamic light scattering (DLS) and exhibited -8.23 to -11.65 mV zeta potential with an outstanding loading efficiency (94%). Cellular uptake and internalization studies demonstrate that MNP-VERU escape lysosomal degradation, providing efficient endosomal release to cytosol. MNP-VERU showed remarkable anti-cancer potential in various PanCa cells (Panc-1, AsPC-1, HPAF-II, BxPC-3, MiaPaca) and more effectively repressed βIII and βIV tubulin isoforms via restoring the expression of miR-200c. MNP-VERU more effectively suppressed AsPC-1 cells derived xenograft tumors in athymic nude mice.

Conclusions: Taken together, our results suggest that MNP-VERU has more anti-cancer potential than free VERU-111 against PanCa. MNP-VERU may reduce the toxicity and improve the bioavailability of free VERU-111 and could be used for the management of PanCa.

#3620

**Tumor targeted delivery of glutamine antagonist: Use of CES1** -/- **mice.**

Rana Rais,1 Jesse Alt,1 Ranjeet Dash,1 Lukáš Tenora,2 Pavel Majer,2 Barbara S. Slusher1. 1 _Johns Hopkins University, Baltimore, MD;_ 2 _Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague, Czech Republic_.

6-diazo-5-oxo-L-norleucine (DON), a potent glutamine antagonist, broadly blocks glutamine utilizing reactions critical for the synthesis of nucleic acids, amino acids, proteins and the generation of alpha-ketoglutarate for energy metabolism. DON has shown robust efficacy in multiple preclinical cancer models and exploratory clinical studies. Although promising, development of DON was halted due to its dose-limiting gastrointestinal (GI)-toxicities, as the GI system is highly dependent on glutamine utilization. Given DON's promising efficacy, we developed novel tumor cell-targeted glutamine antagonists intended to circulate intact as inert prodrug/s in plasma and be preferentially biotransformed to DON in tumor cells. Our prodrug strategy of utilizing elevated protease activities in cancer tissues (e.g. Histone deacetylase, Cathepsin-L) led to the discovery of a highly innovative prodrug pharmacophore. Using a well-defined screening paradigm, we discovered compound 6, (isopropyl 2-(6-acetamido-2-(adamantane-1-carboxamido) hexanamido)-6-diazo-5-oxohexanoate), that showed stability in plasma, liver and intestinal homogenates, yet was readily cleaved to DON in P493B human tumor cells. When directly compared to DON, compound 6, exhibited a 55-fold enhanced P493B cell-to-plasma ratio. In a time-dependent study, compound 6 showed sustained DON delivery to P493B cells while maintaining minimal release in human plasma. Moreover, in a cell proliferation assay, compound 6 showed dose-dependent inhibition of P493B cell growth. One challenging aspect of prodrug development is the selection of appropriate animal model, as it can be confounded by interspecies variation in metabolism, specifically in the varying levels of the carboxylesterase enzyme, CES1, which is highly abundant in rodent plasma but not present in human plasma. We hypothesized that CES1-/- mice would recapitulate human metabolism and serve as a suitable model for our prodrug containing ester promoiety on its carboxylate. Using plasma from CES1-/- mice, wild-type mice and human, we confirmed that compound 6 exhibited similar stability in CES1-/- mice and human plasma but not in wild-type mice plasma. We then performed pharmacokinetic evaluation in C57BL/6 CES1-/- mice bearing flank murine EL4 tumors. Following subcutaneous dosing (1mg/kg DON equivalent), compound 6 exhibited excellent pharmacokinetics with a ~5-fold higher DON tumor exposures (AUC= 5.1 nmol/g*h) versus plasma (1.1 nmol/ml*h) and a 11-fold higher tumor exposures versus GI-tissues (toxicity site; AUC = 0.45 nmol/ml*h). These studies describe discovery of a tumor targeted glutamine antagonist. In addition, we introduce a murine model, that recapitulates human metabolism and can be broadly utilized in prodrug development. Future studies will investigate the dose dependent efficacy and safety of compound 6 in tumor bearing C57BL/6 CES1-/- mice.

#3621

Targeted hyaluronic acid nanoparticles improve treatment response in pancreatic cancer.

Mohammad Raheel Jajja, Lei Zhu, Dazhi Wang, Charles A. Staley, Bassel El-Rayes, David A. Kooby, Lily Yang. _Emory University, Atlanta, GA_.

Pancreatic cancer has poor response to chemotherapy with desmoplastic stroma identified as a delivery barrier. We have developed a nanoparticle (NP) drug system using an engineered ligand of receptor binding region of urokinase plasminogen activator (ATF) and the catalytic domain of metalloprotease (MMP14) carrying SN38 (CPT-11 analog). Hyaluronic acid (HA), a naturally occurring protein was chosen as the backbone to produce a biocompatible NP. HA can also bind to CD44, highly expressed in epithelial cancers. Dual uPAR, CD44 targeting allows for targeted delivery and facilitates receptor mediated endocytosis of NP-drug complex. MMP14 activity degrades extracellular matrix to deplete the stromal barrier. SN38 was encapsulated and recombinant ATFmmp14 conjugated to surface of self-assembled hyaluronic acid spheres (200nm) to form the complete particle (HANP). Cytotoxicity assays were conducted to determine IC50 in cell lines. Patient derived xenograft (PDX) model of a drug resistant pancreatic cancer was used for efficacy studies. Orthotopically implanted tumors were treated with HANP (10mg/kg SN38 dosage) weekly for 6 weeks via tail vein and overall survival compared to conventional therapy. HANP in-vitro cytotoxicity was greater than conventional Irinotecan (>80x) and liposomal irinotecan (>900x) in a PDX derived cell line (Table 1). In-vivo efficacy study demonstrated a significant improvement in survival of PDX bearing mice with HANP (n=9) (median survival 50 days), compared to FOLFIRINOX (n=9) and Gemcitabine-nab-Paclitaxel (n=9) (median survival 37 days each) and no treatment (n=13) (median survival 22 days). Combination of HANP with a modified-FOLFIRINOX regimen (n=9) led to median survival >72 days (p<0.001). HANP alone or in combination with a modified FOLFIRINOX led to significantly improved survival in a pancreatic cancer PDX model. Further studies are underway to evaluate preclinical PD/PK for eventual translation as targeted therapy for pancreatic cancer.

In-vitro cytoxicity assay for IC50 determination

---

Drug | IC50 (uM)

HANP | 0.01

SN-38 | 0.012

Irinotecan | 0.8

Liposomal Irinotecan | 9

#3622

Differential effect of liposomal C6-ceramide/doxorubicin targeted to nucleolin and conventional combinations against triple negative breast cancer stem cells.

Ana F. Cruz,1 Nuno A. Fonseca,2 Nélio Gonçalves,3 Vera Moura,4 Sérgio Simões,1 João Nuno Moreira1. 1 _Center for Neurosciences and Cell Biology; Fac Pharmacy; Univ. of Coimbra, Coimbra, Portugal;_ 2 _Center for Neurosciences and Cell Biology, Univ. of Coimbra; TREAT U, Coimbra, Portugal;_ 3 _Center for Neurosciences and Cell Biology, Univ. of Coimbra, Coimbra, Portugal;_ 4 _TREAT U and Center for Neurosciences and Cell Biology, Univ Coimbra, Coimbra, Portugal_.

The interconversion between cancer stem cells (CSCs) and non-stem cancer cells (non-SCCs) in response to environmental stimulus, has urged the need to identify markers common to both cell sub-populations. This is the case of cell surface nucleolin, a target for drug delivery, offering the opportunity to target both cell populations as previously demonstrated (Fonseca, Biomaterials 2015). Herein, it is hypothesized that the intracellular delivery of the developed liposomal synergistic combination of doxorubicin (DXR) and the sphingolipid C6-ceramide (C6), functionalized with a nucleolin-binding peptide, [F3]L-DC6, will imbalance the CSC niche of triple-negative breast cancer (TNBC), relative to a clinically used combination of DXR and cisplatin (Cis). Cytotoxicity against (bulk) TNBC cell lines (MDA-MB-231, MDA-MB-468 and HS-578T) was assessed by the rezasurin assay. The impact over the putative CSCs population (ALDHhigh, as determined by the Aldefluor assay) was evaluated by flow cytometry. Moreover, assessment of the effect of each drug combination on the in vitro stemness properties of TNBC cells (the mRNA levels of pluripotency transcripts OCT4 and NANOG and mammosphere-forming capacity) was also performed. The intracellular delivery of the DXR:C6 (1:1 molar ratio) combination provided by the formulation targeted to nucleolin ([F3]L-DC6) enabled a significant increase in bulk cell death, compared with the counterpart single drug formulation. In fact, only the targeted liposomal DXR:C6 combination enabled a 90% death in the case of MDA-MB-231 cells, for an exposure as short as 4 h. This was in contrast with the lack cytotoxic benefit of the (non-liposomal) DXR:Cis combination over each drug alone, against each one of the TNBC cell lines used. Despite increasing concentrations of the DXR:Cis combination, enabled higher (bulk) cell death, the percentage of ALDHhigh viable cells increased by 1.5-fold relative to the untreated control, in the case of MDA-MB-468 cells. Interestingly, the opposite effect was observed for the DXR:C6 combination targeted to nucleolin, where increasing concentrations enabled higher (bulk) cell death, along with a 5-fold decrease of the percentage of viable ALDHhigh putative CSCs (relative to untreated control). These observations were concomitant with alterations of stemness properties favoring the latter. Overall, the enhanced efficacy of the synergistic DXR:C6 combination targeted to nucleolin, enables a significant decrease of the percentage of chemoresistant triple negative breast CSC, besides non-SCCs, in contrast to observations with clinically used drug combination. Thus, this novel strategy offers the potential to address tumor recurrence and drug resistance associated with CSC, in an unmet medical need as TNBC.

#3623

Mesenchymal stem cells engineered with TAT peptide functionalized nanoparticles improve therapeutic efficacy of paclitaxel in an orthotopic lung tumor model.

Swayam Prabha, Gopikrishna Moku, Buddhadev Layek, Jayanth Panyam. _University of Minnesota, Minneapolis, MN_.

Tumors are characterized by uneven vascular perfusion with near normal blood flow in the outer most regions, while the inner regions can be avascular. In addition, elevated interstitial fluid pressure and rigid extracellular matrix compromise intra-tumoral solute transport and hence limited drug efficacy. Mesenchymal stem cells (MSCs) have the unique advantage of being able to migrate specifically to both primary tumors and metastases following systemic administration. However, poor payload capacity of MSCs limits their use as drug delivery carriers. To address this issue, we investigated polymeric nanoparticles that were functionalized with transactivator of transcription (TAT) peptide. Paclitaxel loaded PLGA nanoparticles (15-16 % w/w paclitaxel; diameter of 225 ± 7 nm; and zeta potential of −15 ± 4 mV) were prepared by emulsion-solvent evaporation method, followed by TAT-conjugation to the surface of nanoparticles via maleimide-thiol chemistry. Nanoengineered MSCs were generated by incubating MSCs in suspension with 100 µg/mL paclitaxel loaded nanoparticles for 4 hours at 37°C. Our studies demonstrated that TAT functionalization improved the paclitaxel loading in MSCs. In addition, engineering MSCs with TAT functionalized nanoparticles did not affect the differentiation and migration properties of MSCs. Further, MSCs engineered with TAT peptide functionalized nanoparticles resulted in significant inhibition of tumor growth and overall improved survival in a mouse orthotopic model of lung cancer compared to that with free drug treatment. In summary, our results demonstrated that MSCs nanoengineered using TAT functionalized nanoparticles can serve as an efficient carrier for tumor specific delivery of anticancer drugs, resulting in greatly improved therapeutic efficacy.

#3624

Improved intratumor drug delivery and therapeutic efficacy in pancreatic cancer by albumin-Cisplatin complex.

Chao Kong, Junxiao Ye, Zhengsheng Liu, Weijian Kong, Mengnan Sun, Huiqin Liu, Fang Yuan, Feng Qian. _Tsinghua University, Beijing, China_.

Background: The tumor microenvironment in pancreatic cancer is widely known for its abundant stroma and lacking of tumor blood vessels. Many effective therapeutic agents evaluated in vitro failed to show any benefits in vivo due to the inefficient drug delivery into the tumors. Albumin was reported to naturally accumulate in several types of solid tumors including sarcomas, lung cancer, etc. In this study, we systematically investigated the tumor accumulation and penetration of albumin in pancreatic cancer, and evaluated whether albumin could improve the intratumor drug delivery and therapeutic efficacy by albumin-drug complex.

Methods: We prepared a red fluorescence dye (6-TMR)-labeled albumin and studied the tumor accumulation and penetration of albumin in both orthotopic xenograft and the genetically engineered KPC mouse model by in vivo fluorescence imaging and confocal microscope. To further investigate the drug delivery capacity of albumin in pancreatic cancer, we used Cisplatin as a model drug and prepared albumin-Cisplatin complex. Organ and intratumor drug distribution of Cisplatin were measured by ICP-MS and LA-ICP-MS imaging, respectively.

Results: Albumin-6-TMR showed tumor-selective accumulation in both orthotopic xenograft and KPC tumors, with about 50-fold higher radiant efficiency than that of free 6-TMR in KPC tumors at 12 h. Intriguingly, the confocal results indicated that albumin-6-TMR could penetrate into the interior of tumor tissues and stay for at least 72 h while no obvious fluorescence was seen in free 6-TMR group. The mechanism of albumin accumulation and penetration in pancreatic tumors is still under investigation. Moreover, administration of albumin-Cisplatin complex significantly reduced Vd by nearly 25 times and increased AUC of Cisplatin in plasma by 33 times, and improved the MRT and AUC of Cisplatin in orthotopic xenograft tumors by 10 and 29 times, respectively. The LA-ICP-MS imaging was being used to confirm intratumor penetration and distribution of Cisplatin in KPC tumors. Finally, albumin-Cisplatin complex exhibited reduced systemic toxicity (the maximum tolerated dose was more than 60 mg/kg of Cisplatin, i.v.) and enhanced inhibition of tumor growth in subcutaneous PANC-1 xenograft mouse models. Importantly, albumin-Cisplatin complex prolonged the survival of orthotopic MIA PaCa-2 xenograft mouse models compared with free Cisplatin.

Conclusion: In summary, we have shown for the first time that albumin as a drug carrier could improve intratumor drug delivery in both orthotopic xenograft and KPC tumors. And albumin-Cisplatin complex could significantly reduce systemic toxicity and improve therapeutic efficacy of Cisplatin in preclinical pancreatic cancer models. Together, albumin can be a promising drug carrier and provides a new insight into anti-cancer drug delivery in pancreatic cancer.

#3625

Nanodelivery platform for targeting mutant-KRAS and improving response to gemcitabine therapy.

Nirnoy Dan, Sheema Khan, Saini Setua, Sonam Kumari, Pallabita Chowdhury, Kamalika Samanta, Meena Jaggi, Murali Yallappu, Subhash Chauhan. _UTHSC, Memphis, TN_.

Background: Although, surgical resection and chemotherapy are the gold standard for treating pancreatic cancer (PanCa), poor patient survival remains the problem. Despite being one of the most common oncogenes in human cancer, to date, no success has been achieved to inhibit KRAS. Targeting KRAS has been shown to synergize anti-cancer activity of gemcitabine in pancreatic cancer. Herein, we have developed a supermagnetic iron oxide (SP) nanoparticles for the sustained delivery of KRAS siRNA to the tumor and simultaneous sensitization of gemcitabine in PanCa.

Methods: PanCa cells, AsPC1 and Panc-1 were used in the study. A precipitation approach was employed to develop the SP formulation. These particles were further conjugated with siKRAS (12D; most common KRAS mutation) and investigated for its anticancer efficacy alone and in combination with gemcitabine. Particles were investigated for size, physico-chemical characterization (Dynamic light scattering), hemocompatiblity (hemolysis assay) and the complexation of siKRAS (gel retardation assay). Cellular internalization and uptake of the particles were investigated using FAM labelled siRNA and Prussian blue assay. kRAS silencing was confirmed at both mRNA and protein levels using quantitative reverse-transcription PCR and Western blotting, respectively. Anti-cancer efficacy of SP-siKRAS particles alone or in combination with gemcitabine treatment was determined using in vitro functional assays for cell viability (MTT), migration (Boyden chambers), invasion (Matrigel), colonogenicity and tumor spheroid formation.

Results: Our results demonstrate optimal particle size (190.137 nm) and zeta potential (18.73mV) of SP-siKRAS formulation. SP-siKRAS efficiently internalized in PanCa cells and suppressed KRAS G12D expression as well as its downstream targets, YAP and PDL-1. SP-siKRAS improved gemcitabine response as observed through enhanced inhibition of cell proliferation, clonogenicity, migration, and invasion of pancreatic cancer cells. Additionally, SP-siKRAS together with gemcitabine resulted in the activation of death related mechanisms in PanCa cells, such as Bax, bcl-2, PARP cleavage. Interestingly, SP-siKRAS inhibited the secondary tumorsphere formation in combined PanCa and cancer associated fibroblast (CAFs) cells, alone as well as in combination with gemcitabine. At day 14, analysis of secondary tumorspheres, treated with SP-siKRAS revealed diminished levels of KRAS G12D through PCR. This further provides a clinical validation demonstrating potential of SP-siKRAS particles to efficiently silence KRAS expression. SP-siKRAS also exhibited haemocompatibility, suggesting its potential of silencing KRAS without being toxic to the body.

Conclusion: Therefore, SP-siKRAS provide a highly efficient and safe platform for silencing KRAS and improving gemcitabine therapy in pancreatic cancer.

#3626

Develop novel nanoparticle formulations of disulfiram copper for cancer therapy.

Wu Chen, Wen Yang, Landon F. Stewart, David T. Coombs, Jianzhong Shen, Pengyu Chen, Feng LI. _Auburn University, Auburn, AL_.

Despite remarkable progress in cancer treatment, drug resistance remains a significant issue for prostate cancer, breast cancer, and others. Disulfiram (DSF), an alcohol-aversion drug, has been repurposed for cancer treatment and overcome drug resistance. DSF and copper ions form a copper diethyldithiocarbamate (Cu-DSF) complex which has a potent anticancer activity. However, the poor aqueous solubility of Cu-DSF creates a significant formulation challenge, and there is no formulation available for clinical use. We developed a Stabilized Metal Ion Ligand Nanocomplex (SMILE) technology to prepare Cu-DSF nanoparticle (NP) formulations where in situ formed DSF-Cu NPs were stabilized by an optimal amount of stabilizers (e.g. poly(ethylene glycol)-poly(lactide)). The SMILE technology involves a novel formulation design and an innovative preparation process using a 3D-printed microfluidic device. After optimizing the protocol, we can prepare Cu-DSF NPs with size in the sub-100 nm range which are suitable for intravenous injection and can target solid tumors through enhanced permeability and retention (EPR) effects. Cu-DSF NPs prepared with SMILE method showed high drug loading efficiency (above 90%) and high drug concentration (at least 2 mg/mL). The drug concentration of Cu-DSF NPs developed in our study was much higher than those in micelle NP formulations prepared with the classical film-dispersion method. Since we used generally recognized as safe (GRAS) excipients approved by the US Food and Drug Administration (FDA) or other excipients with well-recognized safety profiles, the developed NP formulations will have less regulatory hurdle for FDA approval. Because of the novel preparation process and unique formulation design, the SMILE technology can produce Cu-DSF NPs on a large scale and thus paved the way for its mass production and commercialization. We also determined the anticancer effects of Cu-DSF NPs with multiple assays including MTT assay, colony-forming assay, calcein-AM/propidium iodide staining, and others. Cu-DSF NPs showed excellent anticancer activity against various prostate cancer and breast cancer cells as well as drug-resistant cancer cells. In summary, we developed a novel SMILE method to prepare Cu-DSF NP formulations which could address drug delivery and formulation challenges of DSF-based chemotherapy and facilitate the clinical translation.

#3627

Development of CLENs for future treatment & diagnostic detection using cell models of drug-resistant ovarian cancer.

Negar Amini, Joshua Emerson, Jenel Clement. _MCPHS University, Worcester, MA_.

Current guidelines suggest the use of a platinum agent and a taxane as first line therapy for ovarian cancer where total surgical resection is limited. Liposomal preparations of these drugs have demonstrated therapeutic value by increasing drug uptake and reducing toxicity. The following research aims to improve target specificity by employing a multi-strategic approach to drug targeting and treatment in vitro. Cellular membrane lipid-extracted nanoliposomes (CLENs) were constructed in part from lipid extracts (LE) derived from the membrane of multidrug resistant ovarian cancer (SKOV-3-MDR) cell lines, and more conventional components of liposomes. Formulations of greatest interest were selected after comparing cellular uptake studies of conventional liposomes to various types of CLENs composed of mixed ratios of DOPC, cholesterol, and LE. Cellular uptake was measured by rhodamine fluorescence (RF) following 24 hours incubation in 48 well plates, where RF was used as an indicator of cellular uptake. To determine effectiveness of LE, formulations with and without synthetic cationic lipid DOTAP were compared. Flow cytometry was used to evaluate liposome binding. Additional studies include drug loading efficiency and SRB cytotoxicity studies involving cisplatin, and the effect of additional inclusion of MAG-C (magnetic material) in the SKOV-3 lipid extract-containing liposomes. CLENs containing 15% LE demonstrated greater uptake compared to CLENs prepared with 0% and 5% LE content. Minimal uptake was observed in non-target cancer cell lines (prostate cancer-CRL-1740 and gliobastoma-U87MG), as well as with normal pancreas, non-malignant (hTERT) cell line. The fluorescence intensity values reported for SKOV-3-MDR, CRL-1740, U87-MG and hTERT, were 437 ± 153, 295 ± 67, 57 ± 6, and 29 ± 6, respectively. The additional inclusion of DOTAP showed significant cellular uptake with and without LE. Flow cytometry showed greater initial binding of CLENs to target cells when LE was included in preparations. Incorporation efficiency of cisplatin into SKOV-3 CLENS was 67%. Chemotherapy drug-loaded CLENs is an appealing vehicle as it addresses many parameters. Early findings suggest that CLENS is a suitable vehicle for targeting and treatment using a drug-resistant ovarian cancer cell line. CLENS allows for faster initial nanoparticle binding, cell-type specific uptake, and enhances cytotoxicity. Our encouraging results corroborate this theorem, showing greater targeting with reduced uptake by non-target cells. Additional studies involving MAG-C are currently underway.

#3628

Comparison of CD44 expression in pancreatic cancer, pancreatic stem and normal pancreatic cells: Development of CLENs for tumor targeting and therapy using cell models.

Dotun D. Adegunle, Eugene Boakye Ansah, Danny Nguyen, Kanchi Sanghvi, Drew Goodrich, Robert Campbell. _MCPHS University, Worcester, MA_.

Purpose: The incidence of drug resistance in pancreatic cancer correlates with poor prognosis, increased disease progression and unfavorable treatment outcomes. CD44, a surface membrane proteoglycan has been identified to be directly involved in pancreatic cancer drug resistance and metastasis. As such, the aim of this research was to screen various pancreatic cells for their expression of CD44, and to begin the early design and development of cell membrane lipid-extracted nanoliposmes (CLENs) for enhanced targeting and therapy.

Methods: Three pancreatic cancer cells- Panc-1, MiaPaCa 2, MS1 VEGF and a normal cell line- hTERT- were employed for this study. Stem cell, Mia PaCa-2, served as a positive control for screening purposes. Four liposome compositions were prepared using various ratios of DOPC, Chol, DPPE-PEG5000, and cellular membrane lipid-extracted (LE) was derived from target cell, Panc 1. To evaluate CD44 glycoprotein expression, cells were seeded in 48-well plates and incubated overnight. Fluorescence measurements were determined at excitation wavelength of 485/25 and emission wavelength at 528/20 nm. G44-26 monoclonal antibody was used for CLENs antibody conjugation studies. A Sephadex G-25 column was used for separation of bound and unbound antibody material, and fluorescence detection was used for cell culture analysis. Fluorescence detection was performed at excitation and emission wavelengths of 530/25 and 590/25, respectively.

Results: The relative level of CD44 expression for each of the cell lines was as follows. Panc1>Mia PaCa-2 = hTERT>MS1-VEGF. There was a statistically significant difference between Panc-1 and Mia Paca 2 (P<0.001), between hTERT and MS1 VEGF (P<0.05), and between Panc-1 and hTERT (P<0.0001). The inclusion of Panc1-LE in combination with additional liposome components did not increase cellular uptake significantly. The conjugation of G44-26 monoclonal antibody to the liposomes increased average size of all 4 preparations. Average size for CLENs and G44-26 monoclonal antibody -CLENs increased from 140.75 ± 3.22 nm to 250.23 ± 15.31 nm.

Conclusion: Future studies are required to demonstrate the effect of G44-26 monoclonal antibody liposome conjugates to further exploit the optimal expression of CD44 for pancreatic drug targeting.

#3629

Development of Combrestatin loaded vascular cell modified CLENS for targeting cellular models of tumor vascular endothelia in vitro.

Kanchi S. Sanghvi, Robert Campbell, Guang Yan. _MCPHS University, Worcester, MA_.

Purpose: The American Cancer Society predicts that about 0.6 million people would die of cancer and about 1.7 million new cases will be diagnosed in the year 2018. One important goal of cancer treatment is to minimize uptake of the cytotoxic drug agent by normal tissues. CLENs (cell membrane lipid-extracted nanoliposomes) have been developed to enhance targeted delivery of drugs to cancer cells located in the tumor interstitial matrix environment. The purpose of this study was to evaluate the use of CLENs for tumor vascular targeting.

Methods: We expanded tumor endothelial cells derived from the pancreas followed by membrane extraction and the development of MS1-VEGF CLENs for vascular targeting. We evaluated the benefits of the additional inclusion of conventional liposome components (DOPC/Chol/DPPE-PEG5000) in various ratios, including the synthetic cationic lipid DOTAP (50 mol%), known for its vascular targeting characteristics when employed with conventional liposome components. We performed cellular uptake studies to determine relative level of selectivity in connection with the inclusion of LE (lipid extract) material. We determined also the effect of the additional inclusion of DOTAP in CLENs for targeting. We separated free drug from drug incorporated in CLENs using dialysis cassettes with MWCO of 10 kD. We evaluated the efficiency of Combrestatin A4 Phosphate (a microtubule-destabilizing agent) loading in CLENs by HPLC.

Results: The addition of (50 mol%) cationic lipid DOTAP content in CLENs increased the overall zeta potential of the preparation from -14.26 ± 3.65 mV to 10.24 ± 4.23 mV. The average particle size for MS1-VEGF CLENs was approximately 137 ± 7 nm. The cellular uptake studies revealed that CLENs consisting of 70 mol% LE demonstrated the greatest selectivity towards MS1 VEGF cells, when compared to minimal uptake observed for non-target human glioblastoma (U87-MG) cell control. However, the data suggest also an overwhelming cationic lipid effect when DOTAP was included in the CLENs. Where overall cellular uptake increased, the selectivity afforded by LE decreased. Preliminary drug loading studies (between 20 and 45%) suggested that LE inclusion in nanoliposomes influenced the capacity for drug loading.

Conclusion: The inclusion of MS1-VEGF lipid extract material in nanoliposomes improved vascular selectivity. The additional inclusion of DOTAP enhanced targeting, but the preference for target versus non-target cells had diminished. Additional studies are currently underway.

#3630

RNA nanoparticles as a carrier for targeted drug delivery into cancer cells.

Piotr G. Rychahou,1 Sijin Guo,2 Eun Y. Lee,1 Nicole Rychagov,1 Jon Thorson,1 Peixuan Guo,2 B. Mark Evers1. 1 _University of Kentucky, Lexington, KY;_ 2 _The Ohio State University, Columbus, OH_.

Colorectal cancer (CRC) is the second leading cause of cancer deaths in the US; the inefficiency of chemotherapeutic treatments has severely limited the treatment options in patients with CRC metastasis. Polyvalent RNA nanoparticles have demonstrated metastatic tumor homing without accumulation in normal organ tissues surrounding metastatic tumors; the flexibility in constructing trimers also enables the assembly of polyvalent nanoparticles to carry drug molecules for therapeutic purposes. The purpose of our study was to: (i) demonstrate PI3K/mTOR inhibitor conjugation to RNA nanoparticles, and (ii) pH-sensitive intracellular drug delivery by folate receptor targeting (FA)-3WJ-PI-103 nanoparticles in CRC cells.

Methods. (1) PI-103-3WJ-Folate nanoparticle binding, internalization and drug release was evaluated in HT-29 (human CRC), SK-OV-3 (human ovarian cancer), JAR (human choriocarcinoma), HCT116 (human CRC), and Caco-2 (human CRC) cell lines. (2) Confocal microscopy was used to examine RNA nanoparticle binding and internalization by endocytosis. (3) Western blot was used to confirm drug delivery with PI-103-3WJ-Folate nanoparticle.

Results. (1) Drug release from fully assembled RNA nanoparticles was confirmed after 3WJ-PI-103 nanoparticles (5 µM) incubation in PBS pH 2.5, 3, 3.5 for 4h at 37°C and analysis of pAkt (Ser473) expression, a marker of PI3K inhibition; 1.5 mM 3WJ-PI-103 was minimum dose required to inhibit PI3K/Akt signaling in cancer cell lines. (2) Rapid folate-receptor mediated RNA nanoparticle internalization was observed within 2-4 h in HT29, HCT116, JAR and SK-OV-3 cell lines by confocal microscopy. (3) Cancer cells were treated with FA-3WJ-PI-103 nanoparticles directly to evaluate all steps of targeted receptor-mediated drug delivery-receptor binding, endocytosis, pH-triggered drug release and endosomal drug escape into the cytoplasm. HCT116 and JAR cells were treated with FA-3WJ-PI-103 in 0% and 1% folate-free media for 4h, media was replaced and evaluated 3h later for PI3K pathway activation. PI3K/Akt signaling was inhibited after direct treatment with FA-3WJ-PI-103 nanoparticles at 5 mM drug concentration; 3WJ-PI-103 particles were used as a control.

Conclusions. A critical finding of our current study was the ability to conjugate PI3K/mTOR inhibitor PI-103, via a pH-selective linker, to RNA nanoparticles and the demonstration of intracellular drug delivery into cancer cells and drug release in a pH-dependent fashion. Our results demonstrate the potential of receptor-selective drug delivery to cancer cells with high FRα expression and represents a promising approach for treatment of CRC metastases.

#3631

Development of targeted nanoformulation of talazoparib for combined chemoradiation therapy in lung cancer.

Bijay Singh, Mostafa Abdelhalim, Stephanee Warrington, Srinivas Sridhar. _Northeastern University, Boston, MA_.

Poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi) therapy exploits a synthetic lethality strategy in cancers with inherent damage in DNA repair or transcription pathways. Talazoparib is a potent PARPi that is currently indicated for oral inhibitor therapy in several cancer clinical trials. Oral administration of these inhibitors typically results in poor bioavailability and tumor accumulation. In contrast, nanoparticle formulation provides a safe vehicle for parenteral administration of therapeutic drugs with sustainable release reducing systemic toxicity and also protect from surveillance of immune cells, thereby increasing the bioavailability of the drugs in vivo. Moreover, targeting strategy for the nanoparticles will improve the accumulation of drugs in the tumors. Here, we developed a targeted formulation of Talazoparib (NanoTLZ) which was decorated with anti-EGFR antibody as a ligand to ameliorate the cellular uptake of NanoTLZ via EGFR-mediated endocytosis. In vitro tests showed that NanoTLZ is more effective in cell growth inhibition than free Talazoparib. When combined with radiation with different doses from 2-10Gy, NanoTLZ showed a strong radiosensitization effect as evidenced with almost no colonies formation at 6 Gy of radiation dose. The high therapeutic efficacy of combined chemoradiation therapy in Calu 6 cell line can be attributed to the higher accumulation of NanoTLZ in the cancer cells as compared to the free Talazoparib which is also marred with efflux of the drug from the cells. The slow and sustained release of Talazoparib from nanoparticle formulation inside the cells lead enhanced inhibition of DNA repair pathways. These studies provide very encouraging results to evaluate the efficacy of these nanoparticles in lung cancer animal models.

#3632

Enhanced efficacy of optimized phenethyl isothiocyanate and cisplatin co-encapsulated liposome nanoparticle for treatment of non-small cell lung cancer.

Mengwei Sun, Yi Shi, Anthony Di Pasqua. _Binghamton University, Binghamton, NY_.

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, which is the leading cause of cancer-related death in the United States. Accounting for about 85% of all lung cancers, NSCLC is difficult to treat and its survival rates are low. After decades of basic and clinical research, the most effective treatments still involve the first-generation anticancer agent cisplatin (CDDP) in combination with other drugs. Naturally-occurring compound, phenethyl isothiocyanate (PEITC), can be used to sensitize NSCLC cells to CDDP. We have previously proved that co-encapsulation of PEITC and CDDP in liposomes enhances the cytoxicity toward NSCLC cells, compared to free PEITC or CDDP. In this study, liposomal-PEITC-CDDP is optimized and the release of PEITC and CDDP from the nanoparticle is characterized. In vitro studies show that liposomal-PEITC-CDDP has a higher cytotoxicity toward both A549 and H596 human NSCLC cell lines than toward WI-38 and BEAS-2B human normal lung cell lines. Therfore, we have prepared an efficacious therapy with a potential for fewer side-effects due to low off-target toxicity.

#3633

Evauation of doxorubicin-thanine co-loaded nanoparticles for cancer chemotherapy.

Ikumi Sugiyama, Yasuyuki Sadzuka. _Iwate medical University, Iwate, Japan_.

[Purpose] Novel nanoparticles have the potential to increase the efficacy of cancer chemotherapy. Recently, the use of two chemotherapeutic drugs load to liposomes has been studied. In particular, cytarabine-daunorubicin (1:5) co-loaded liposome (CPX-351) has high therapeutic efficacy against acute myelocytic leukemia. We previously reported that a combination of theanine (a specific amino acid of green tea) with doxorubicin (DOX) had a stronger antitumor effect than DOX alone. The effect is caused by the competitive inhibition of glutamate transport by theanine as a glutamate derivative via glutamate transporters in tumor cells. Subsequently, decreased glutamate levels in tumor cells result in lower glutathione, which makes efflux of DOX from cells difficult. Thus, it was speculated that specific accumulation of DOX, caused by theanine, increases the therapeutic index. In the present study, we examined the availability of a DOX-theanine co-loaded liposome as drug carrier for cancer chemotherapy.

[Methods] DOX-theanine co-loaded liposome (DT-lip) was prepared using the thin-lipid method. DOX and theanine were loaded at the lipid membrane layer and internal water layer, respectively. P388 leukemia cells were used to study the influence of DT-lip on DOX influx and efflux. The cell suspension was incubated with DT-lip at 37°C, and the DOX concentration in tumor cells was determined. In the antitumor effect study in vivo, tumor-bearing mice were intravenously administered DT-lip (DOX 2.5 mg/kg) at 17th, 20th, and 23rd days after tumor cell implant. Tumors and normal tissue were removed 48 h after last administration. Tumor weight and DOX concentration in tissue were measured.

[Results and Discussion] DT-lip was prepared with three ratios of DOX to theanine (1:0.5, 1:1, and 1:4, w/w). In the influx study, the DOX concentration in tumor cells was the same for the different ratios. DOX influx into tumor cells in the DT-lip group was enhanced compared to liposomes loaded with DOX only (D-lip). At 30 min, the intracellular DOX concentration in the DT-lip was 1.4 fold higher than that in the D-lip group. Conversely, there was no practical difference in DOX efflux from tumor cells between the D-lip and DT-lip group. In other words, DT-lip had an influence on influx system, and this phenomenon differed from the previous results for theanine′s role in the DOX efflux system. This findings suggest that liposomalization of theanine + DOX changed the theanine connected integrated mechanism in tumor cells. In vivo, the antitumor effect of DT-lip was superior to combined administration of both solutions. The adverse effects of DT-lip treatment on normal tissue did not increase because DOX concentration in tissue was comparable across group. In conclusion, the DT-lip, a liposome co-loaded DOX and theanine, is a novel antitumor formulation for cancer chemotherapy.

#3634

Tannic acid-docetaxel scaffold nanoparticles for improved prostate cancer therapy.

Prashanth Kumar Bhusetty Nagesh, Pallabita Chowdhury, Elham Hatami, Sonam Kumari, Vivek Kumar Kashyap, Bilal Hafeez, Sheema Khan, Manish Kumar Tripathi, Meena Jaggi, Subhash C. Chauhan, Murali M. Yallapu. _Univ. of Tennessee Health Science Ctr., Memphis, TN_.

Purpose: Prostate cancer (PrCa) is one of the leading causes of cancer-related deaths among men in the United States. Chemotherapy with docetaxel holds great promise and important therapeutic option for PrCa treatment. However, chemotherapy is often associated with incomplete cell death and leads to persistent senescent phenotype. Such phenotypic cancer cells survive and promotes tumor development, recurrence, and drug resistance. Altogether, targeting these cells can enhance chemotherapy outcomes. In the current study, we established a tannic acid-docetaxel scaffold nanoparticles (TDS NP) platform that allows for facile targeting and inhibition of drug induced senescence in prostate cancer.

Methods: TDS NP were prepared by solvent evaporation and extrusion process using a series of tannic acid and docetaxel weight ratios. The scaffold self-assembly formation was determined using a steady-state fluorescence quenching, FT-IR, XRD, DSC, and TGA. The surface and morphological properties of TDS NPs were determined by dynamic light scattering and transmission electron microscopy. The biocompatibility assessment was characterized by hemolysis assay. C4-2 and PC-3 cell lines were used as PrCa model systems for in vitro and in vivo studies. The cellular internalization (in vitro) and accumulation (in vivo) was examined using dye-tagged TDS NPs. The superior in vitro anti-cancer and metastatic potential of TDS NPs were evaluated using proliferation, colony formation, migration, invasion, and immunoblotting assays. The anti-senescence ability of TDS NPs was examined through β-Galactosidase Staining Kit. A PC-3 xenograft mouse model was used to examine its superior therapeutic activity over free docetaxel treatment.

Results: A series of physico-chemical analyses confirmed the integrity of TDS NP. An optimized formulation exhibited a spherical shape particle formulation with 124.13 ± 2.16 nm with a negative zeta potential of -14.0 mV. TDS NPs formulation exhibited superior internalization capacity in PrCa cells in a dose- and time-dependent manner. In vitro functional studies confirm superior therapeutic activity of TDS NPs over free docetaxel treatment. Enhanced pro-apoptotic while downregulating anti-apoptotic effects with treatment of TDS NPs in comparison to native drug. A profound inhibition of β-galactosidase activity was noticed with TDS NPs treatment. In vivo biodistribution studies confirm efficient accumulation of TDS NPs in mice. In addition, TDS NPs showed robust antitumor activity against PC-3 xenografts. Such improved activity was correlated with increased inhibition of senescence and induced apoptosis.

Conclusion: In summary, we developed a novel tannic acid-docetaxel scaffold nanoparticle formulation that is capable of inhibiting senescence, minimizing drug resistance, and inducing apoptosis in prostate cancer.

#3635

RNA-based nanostructures for therapeutic siRNA delivery.

Yonggang Ke, DongMoon Shin, Georgia Chen. _Emory University, Atlanta, GA_.

Short interfering RNA (siRNA) has emerged as a promising molecular therapeutic tool for targeted cancer treatment. However, systemically administered siRNA has demonstrated only limited success, due to limited delivery to cancer cells. Therefore, the lack of a robust and versatile siRNA delivery system is a critical issue in translating this therapeutic tool for cancer treatment. Recent developments in DNA nanotechnology have made programmable DNA nanoparticles (DNPs) a potent drug delivery platform. This study focuses on the development of a novel RNA-based siRNA delivery system to knockdown Bcl2 gene, as a targeted cancer therapeutic. By eliminating the use of DNA, the RNA-based nanodelivery can potentially create more robust siRNA delivery vehicles for targeted cancer therapeutics.

#3636

Generation of polymeric nanoformulation of ormeloxifene for cervical cancer treatment.

Neeraj Chauhan,1 Murali M. Yallapu,1 Diane M. Maher,2 Bilal B. Hafeez,1 Mohammed Sikander,1 Meena Jaggi,1 Subhash Chauhan1. 1 _UTHSC, Memphis, TN;_ 2 _Sanford Research, Sioux Falls, SD_.

Background: Cervical cancer (CxCa) is one of the most common death related cancers among women around the world and associated with poor 5-year survival rate. Therefore, there is an urgent need to develop newer treatment modalities. Ormeloxifene (ORM) is a non-steroidal, Selective Estrogen Receptor Modulator (SERM) that is used as an oral contraceptive in humans. Recent investigations suggest that ORM exhibits potent anti-cancer activity against various types of cancers. Nanoparticulates offer targeted delivery of anti-cancer drugs with minimal toxicity and promise newer approaches for cancer treatment. Therefore, nanotherapy approach outstands over traditional chemotherapy which is not site specific and often associated with various side effects. Thus, pursuing this novel nanotherapy approach, we have developed ORM nanoformulation using PLGA (poly [lactic-co-glycolic acid]); an FDA approved biodegradable polymer.

Methods: We generated ORM loaded PLGA nanoformulation (PLGA-ORM) employing nanoprecipitation method. PLGA-ORM was characterized for its physicochemical properties such as particle size, FT-IR, DSC and drug loading. We next performed cellular uptake studies in Caski and SiHa cell lines at different concentrations (5, 10, 20 and 25 µM) and temperatures (4° and 37° C) utilizing fluorescent microscope, flow cytometer and TEM. To evaluate the cellular uptake mechanism of PLGA-ORM, we did another flow cytometer experiment using various endocytosis pathways' inhibitors. Next, we determined anti-proliferative activities of PLGA-ORM by performing MTS and colony formation assays. Furthermore, we investigated anti-tumoral functions of PLGA-ORM in an orthotopic mice model of cervical cancer.

Results: Our optimized PLGA-ORM showed particle size around 250 nm measured by DLS. ORM was completely miscible in this formulation as indicated by FT-IR and DSC spectra, which resulted in excellent drug loading of about 80% done by HPLC. PLGA-ORM nanoformulation exhibited improved internalization in dose, time and energy dependent manner through endocytosis mediated pathways in both Caski and SiHa cell lines. Additionally, we employed MTS and colony forming assays to determine the short- and long-term effects of PLGA-ORM on these cells, results showed that this formulation had an improved inhibition of cellular proliferation and clonogenic potential compared to free ORM. Furthermore, PLGA-ORM nanoformulation exhibited superior anti-tumorous activities in an orthotropic cervical cancer mouse model compared to free ORM.

Conclusion: Our findings suggest that, this PLGA-ORM nanoformulation has great potential for repurposing the drug and becoming a novel modality for cervical cancer management which needs to be developed as a lead therapy approach with appropriate clinical investigations.

#3637

Development of a polyethylenimine conjugated liner multiblock polymer to deliver VEGF siRNA for triple negative breast cancer.

Yuanke Li, Zhen Zhao, Kun Cheng. _University of Missouri-Kansas City, Kansas City, MO_.

Vascular endothelial growth factor (VEGF) plays important roles in the angiogenesis process and is correlated with high proliferation and metastasis of breast cancer, particularly in triple negative breast cancer (TNBC). Downregulation of VEGF expression with siRNA in cancer cells is therefore a potential strategy for TNBC treatment. A polyethylene glycol based bio-degradable, linear multiblock polymer was synthesized and conjugated with branched low-molecular-weight poly(ethyleneimine) (PEI) to form nanocomplexes with a VEGF siRNA. The nanocomplex can protect siRNA from serum degradation and deliver the VEGF siRNA into TNBC cells with 72% silencing activity and low cytotoxicity. In vitro activity studies showed that the siRNA nanocomplexes significantly inhibit 64% migration and 67% invasion of TNBC cells. Moreover, the nanocomplex exhibited efficient tumor penetration in a 3D tumor spheroid model, suggesting a good penetration capability of the nanocomplex in tumor microenvironment in vivo. More importantly, the VEGF siRNA nanocomplex efficiently inhibit tumor growth in vivo and successfully downregulate VEGF expression in the tumor. These results suggested that VEGF siRNA is a promising anti-tumor agent for TNBC therapy, and the PEI 1800 conjugated bio-degradable multiblock polymer is a promising system to deliver siRNAs to TNBC cells.

#3638

Combination of metformin and metronomic liposomal doxorubicin exerts a robust anticancer effect in triple negative breast cancer by inhibiting breast cancer stem cells & the Wnt/beta-catenin pathway.

Indranil Banerjee,1 Subhayan Das,1 Chandan Kanta Das,1 Bikash Ch. Jena,1 Deblina Bharadwaj,1 Anjan K. Pradhan,2 Swadesh K. Das,2 Paul B. Fisher,2 Mahitosh Mandal1. 1 _Indian Institute of Technology Kharagpur, Kharagpur, India;_ 2 _Virginia Commonwealth University, Richmond, VA_.

Introduction: The high death rate due to breast cancer is often attributed to aggressive triple negative breast cancer (TNBC). We used the combination of metronomic pegylated liposomal doxorubicin (PLD), and metformin {selectively inhibits cancer stem cells (CSCs)} for effective management of TNBC xenografts generated in mice. Down-regulation of β-catenin is responsible for the efficacy of the combination of metronomic PLD and metformin in treating aggressive TNBC.

Methodology: PLD was prepared using the ammonium sulfate gradient method. Morphology, particle size, zeta potential measurement, drug entrapment efficiency, and drug release studies were performed to characterize the prepared liposomal formulation. Cell culture studies were performed on MDA-MB-231 and MDA-MB-468 cell lines (i.e., TNBC cells). MTT assays, mammosphere assays, apoptosis assays, quantitative real-time polymerase chain reactions (qPCR), western blotting experiments were performed. In vivo anticancer efficacy of the combination therapy {antidiabetic dosing of metformin: 150 mg/kg body weight every day, and metronomic dosing of PLD: 1 mg/kg body weight with respect to doxorubicin (Dox) every other day for 3 weeks} was verified in an MDA-MB-231 xenograft model.

Results: Monodisperse PLD having dimensions less than 100 nm was successfully prepared (confirmed by AFM, TEM, and DLS). Zeta potential measurement showed that PLD was negatively charged (- 12.1 ± 2.3 mV, n = 3). Encapsulation efficiency and drug release were found to be satisfactory. IC50 (i.e., 50 % inhibitory concentration) of PLD was found to be in nanomolar range with respect to Dox in TNBC cell lines. However, a combination of metformin (500 µM) and PLD resulted in a leftward shift of the concentration-response curve such that the IC50 value of PLD was further reduced in the nanomolar range with respect to Dox. A similar trend was seen in the apoptosis assay. Metformin works together with PLD to reduce non-stem cancer cells, CSCs, and stem cell markers as indicated by the reduction in the number of mammospheres, qPCR results, and western blotting experiments. We also found attenuation of β-catenin in TNBC cells by the combination treatment regimen, as suggested by qPCR and western blotting. Some of the in vitro observations were also confirmed in the MDA-MB-231 xenograft model.

Conclusion: Our experiments demonstrate that down-regulation of β-catenin is responsible for the strong anticancer effects of the combination of metformin and PLD in vitro and in vivo. Further studies are warranted with this combination regimen in different cancer types as metformin is an approved anti-diabetic drug with an acceptable safety profile, and metronomic chemotherapy is gaining popularity in treating different cancer types.

#3639

EGCG-gold nanoparticles exhibit greater anti-tumor activity over conventional gold nanoparticles or EGCG due to potential synergistic interactions.

Suhash Reddy Chavva, Sachin Kumar Deshmukh, Rajashekhar Kanchanapally, Nikhil Tyagi, Jason W. Coym, Ajay P. Singh, Seema Singh. _University of South Alabama, Mobile, AL_.

Epigallocatechin gallate (EGCG) possesses significant anti-tumor activity and binds to laminin receptor over-expressed on cancer cells with high affinity. However, the use of EGCG in therapeutics is limited due to its poor bioavailability and limited stability. Gold nanoparticles (GNPs) serve as excellent drug carriers and protect the conjugated drug from enzymatic metabolization. Here, we investigated if EGCG-formulated GNPs (E-GNPs) would demonstrate superior anti-cancer activity compared to EGCG or conventionally-synthesized citrate-GNPs (C-GNPs) due to their synergistic interactions. Cell viability studies showed greater growth inhibition by E-GNPs, compared to EGCG, or C-GNPs. Cellular uptake studies revealed that, unlike C-GNPs, E-GNPs were taken-up more efficiently by cancerous cells than noncancerous cells. Furthermore, data showed that E-GNPs induced more apoptosis in cancer cells, compared to EGCG, and C-GNPs. From the mechanistic standpoint, we observed that E-GNPs inhibited the nuclear translocation and transcriptional activity of NF-κB with greater potency than EGCG, whereas C-GNPs were only minimally effective. Altogether, our data suggest that E-GNPs can serve as effective anti-cancer agents.

#3640

Cancer cell membrane coated biomimetic nanoparticles as decoys for disrupting cancer cell-stromal cell interactions.

Jiefu Jin, James Barnett, Flonne Wildes, Sridhar Nimaggada, Zaver Bhujwalla. _Johns Hopkins Univ. School of Medicine, Baltimore, MD_.

Introduction: Biomimetic nanoparticles (NPs) combining synthetic and biological materials have flexibility and functionality for disruptive strategies.1 Stromal cells such as cancer associated fibroblasts (CAFs) mediate many of the aggressive characteristics of cancer and play a crucial role in proliferation, invasiveness, metastasis, and angiogenesis of cancer.2 NPs coated with cancer cell membrane fractions (CCMFs) inherit the repertoire of surface proteins from cancer cells, making them potentially useful as decoys to disrupt cancer cell-stromal cell interactions. Here, we coated cancer cell membranes onto poly(lactic-co-glycolic acid) (PLGA) NPs to form CCMF-PLGA NPs, and characterized their protein profile, size, purity, cellular internalization, and integrity. We investigated, for the first time, the ability of these "artificial cancer cells" to disrupt fibroblast-mediated migration and lung metastasis.

Method: CCMFs were isolated upon cell homogenization, and sucrose density gradient centrifugation. PLGA NPs were prepared by nanoprecipitation. CCMFs and PLGA NPs were mixed and physically extruded through a porous membrane to obtain CCMF-PLGA NPs. The protein profile of CCMFs was analyzed by western blot with antibodies against cell fraction markers.. The experimental lung metastasis model was established by intravenously injecting MDA-MD-231 cells constitutively expressing luciferase (231-luc) through the tail vein of nude mice. The treated group was inoculated with the same number of 231-luc cells mixed with CCMFs-PLGA NPs and injected weekly with CCMF-PLGA NPs in the following weeks. Lung metastasis was monitored in vivo for three consecutive weeks by bioluminescence imaging (BLI). At the end of treatment, lungs were isolated and inflated. Metastatic nodules were imaged with BLI and examined by histology.

Results: Plasma membrane purity was confirmed from western blot analysis that showed the significant enrichment of Na+/K+-ATPase, negligible amount of GPR78 or GAPDH in CCMFs. Confocal fluorescence microscopy and flow cytometry confirmed a "right-side" out orientation of CCMF-PLGA NPs and the integrity of membrane-associated proteins after membrane isolation and PLGA coating. When CCMFs or CCMF-PLGA NPs were added to HMFs in the transwell assay, fewer cancer cells migrated towards HMFs, identifying the unique ability of CCMF-PLGA NPs to disrupt HMF-cancer cell interactions. In the lung metastasis study, mice treated with CCMF-PLGA NPs had significantly less incidence and metastatic burden confirmed by BLI and histology. CCMF-PLGA NPs hold promise as decoys to disrupt cancer cell-stromal cell interactions.

References: 1. Fang, R. et al. Small 2015; 2. Shiga, K. et al. Cancers 2015. Supported by NIH R21 CA198243, R35 CA209960, and a grant from the Emerson Collective.

#3641

Biodegradable polysaccharides based paclitaxel formation for ovarian cancer therapy.

Jian Bao, Hui Zhang, Aili Rong, Bajin Han, Nazar Filonov, Jun Li. _ZY Therapeutics, Inc, Research Triangle Park, NC_.

Over 90% of pipeline therapeutic molecules have low solubility and lack of suitable delivery vehicle, which results the treatment potential could be limited. To address this challenge, ZY Therapeutics developed a tunable biodegradable polysaccharide-based nanoparticle drug delivery platform. The first candidate ZY-010-PNP is a nanoformulation using folate-modified polysaccharide encapsulating paclitaxel (PTX). ZY-010-PNP is supplied as a lyophilized powder that is readily reconstituted in saline to form a transparent suspension. The reconstituted formulation has an average particle size < 150 nm and remains stable for 24 hours at room.

Drug release profile of ZY-010-PNP in human plasma was evaluated from 20 min to 4 hrs at different concentrations, using the stable isotope tracer ultrafiltration assay developed at NCI. The ZY-010-PNP formulation released 20-40% of the encapsulated PTX in human plasma within 20 min, and 60-100% was released over the subsequent 4-hour period. The data clearly showed that the drug release profile of ZY-010-PNP was time / concentrations dependent in serum, in contrast with burst release of all other marketed Taxane formulations.

The in vitro efficacy was evaluated in human ovarian cancer SKOV3 cells. ZY-010-PNP induced dose- and time-dependent cell death and showed a similar potency as the benchmark drug in terms of cytotoxicity. However, the vehicle showed very low toxicity on SKOV3 and THLE-2 epithelial cells.

The PK properties of ZY-010-PNP were also studied. ZY-010-PNP administrated on rats yielded the highest total plasma PTX level (Cmax), followed by a rapid declining and relatively longer elimination phase (t1/2) in comparison with the benchmark drug.

The in vivo efficacy of ZY-010-PNP was demonstrated in two xenograft models, Triple Negative Breast Cancer (4T1) and Ovarian Cancer (SKOV3). In the SKOV3 study, multiple doses (10, 30 and 45 mg/kg) of ZY-010-PNP were tested in comparison to the benchmark drug. ZY-010-PNP at doses of over 30 mg/kg demonstrated complete tumor growth inhibition. Body weight (group means up to 15%) dropped during 30 and 45 mg/kg dosing period, but it was regained quickly post administration.

In summary, a lyophilized dosage form of PTX formulation was developed with time and concentration dependent drug release profile. The in vitro and in vivo efficacy studies showed a similar tumor growth inhibition effect compared to the benchmark formulation. The PK profile demonstrated that ZY formulation yielded the highest total plasma Taxane level and relatively longer elimination phase. ZY's vitamin-modified-polysacchride drug delivery platform showed advantages on improved stability, easy reconstitution, no foaming and no immunogenicity in compare to other benchmark delivery vehicle. The proprietary tunable platform can be applied to other high potency with low solubility molecules to increase the tolerance and reduce the toxicity of these drugs.

#3642

Combination nanotherapy using the PARP inhibitor talazoparib and cyclin dependent kinase inhibitor dinaciclib.

Paige Baldwin, Adrienne Orriols, Srinivas Sridhar. _Northeastern University, Boston, MA_.

Introduction: PARP inhibitors exploit defects in DNA repair pathways to selectively target cancerous cells. As such, Talazoparib (TLZ), a potent PARP inhibitor, offers a way to target the biology of a number of cancers with DNA repair defects until these tumors develop resistance. PARP inhibitors must be used in combination with other inhibitors or chemotherapeutics to reverse resistance and sensitize non-responsive tumors. Dinaciclib, a potent cyclin dependent kinase (CDK) inhibitor, has been shown to sensitize both BRCA wild-type tumors and PARP inhibitor resistant tumors to PARP inhibition through disruption of homologous recombination. In clinical trials, Talazoparib and Dinaciclib have both demonstrated hematologic toxicities, suggesting a combination of these drugs would result in compounded toxicity, leading to dose reduction and an ineffective combination. Nanoparticle delivery systems offer a means to modify the toxicity profiles of these drugs and enhance the therapeutic window, therefore allowing for effective combination treatment.

Methods: Separate nanoformulations of Talazoparib (NanoTLZ) and Dinaciclib (NanoDCB) were optimized, and pharmacokinetics and pharmacodynamics assessed. Nanoformulations were tested alone and in combination in vitro to ensure NanoDCB could sensitize a model with no known DNA repair defects to NanoTLZ. The combination of the two nanoformulations was then assessed for efficacy and toxicity in orthotopic MDA-MB-231 xenografts.

Results: Robust formulations of NanoTLZ and NanoDCB were developed. Each nanoformulation extended the half-life of the drug it encapsulates. A constant low dose of Dinaciclib sensitized MDA-MB-231 cells to Talazoparib, significantly lowering the IC50 value. As a single agent NanoDCB was more effective in vitro than free Dinaciclib. In vivo, the combination of the two nanoformulations was more effective than either single nanoformulation or the combination of the two free drugs. Assessments of hematologic toxicities are underway, but thus far, there were no signs of gross toxicity in the combination therapy group.

Conclusions: The combination of NanoDCB and NanoTLZ has provided an effective method for sensitizing tumors to PARP inhibition that are otherwise nonresponsive to this therapy. The development of two separate nanoformulations has allowed for tailored dosing. These long-circulating nanoformulations have proven more effective than the free drugs in stabilizing tumor growth and were well tolerated. This work was supported by ARMY/W81XWH-16-1-0731.

## TUMOR BIOLOGY

### Biology and Signaling in Pediatric Cancer

#3643

**Genetic models reveal that the novel** VGLL2-NCOA2 **fusion oncogene leverages embryonic programs for sarcomagenesis.**

Genevieve C. Kendall,1 Sarah Watson,2 Lin Xu,1 Collette LaVigne,1 Whitney Murchison,1 Dinesh Rakheja,1 Franck Tirode,3 Olivier Delattre,2 James Amatruda1. 1 _UT Southwestern Medical Center, Dallas, TX;_ 2 _Institut Curie Research Center, Paris, France;_ 3 _Centre Léon Bérard, Lyon, France_.

Rhabdomyosarcoma (RMS) is an aggressive pediatric cancer characterized by a misregulation of skeletal muscle developmental pathways. To date, identified oncogenic drivers predominantly include RAS mutations or chromosomal translocations and gene fusions between PAX3 or PAX7 and FOXO1. RNAseq analysis of sarcomas with non-canonical gene fusions has identified new potential genetic drivers of tumorigenesis that have not been rigorously functionally validated for their transformation capacity and biological activity. One new fusion is a chromosomal translocation and inversion between chromosomes 6 and 8, which acts to juxtapose two transcriptional co-activators, VGLL2 and NCOA2. This VGLL2-NCOA2 fusion was identified in congenital rhabdomyosarcoma clinical cohorts by us and others, and characterizes aggressive RMS that express MYOD and MYOG histological markers. However, evidence of VGLL2-NCOA2 transformation capacity has not been verified, hindering insights into its functional contributions to tumorigenesis. Here, we interrogate the function ofVGLL2-NCOA2 using complementary genomic patient data and zebrafish model systems. We utilized the Tol2 transposon system to express mosaic human VGLL2-NCOA2 during early development, and found that VGLL2-NCOA2 is sufficient for tumorigenesis, and results in aggressive tumors with high penetrance by 75 days of age in zebrafish. Further, the histology of zebrafish tumors resembles the human disease, and tumors express markers indicative of RMS such as myog and desma. A cross-species RNAseq of patient and zebrafish VGLL2-NCOA2 RMS tumors highlights a significant enrichment and overlap between gene expression signatures. Finally, mapping the gene expression signatures of VGLL2-NCOA2 zebrafish RMS tumors along the spectrum of zebrafish embryogenesis indicates a clustering with developmental stages corresponding to early somitogenesis, highlighting their arrested developmental nature. Hence, we have generated the first animal model of human VGLL2-NCOA2 tumorigenesis, and have applied this model to understand the biology and identify potential therapeutic targets for this newly identified disease.

#3644

Novel NOTCH1-ROS1 gene fusion drives distinct molecular mechanisms in a rare pediatric angiosarcoma.

Payal Jain,1 Sudarshan Iyer,2 Joshua Straka,3 Lea Surrey,3 Jennifer Pogoriler,3 Tiffany Smith,3 Christine Busch,3 Marilyn Li,3 Elizabeth Fox,3 Adam C. Resnick,1 Monika Davare,2 Angela J. Waanders3. 1 _Children's Hospital of Philadelphia/University of Pennsylvania, Philadelphia, PA;_ 2 _Oregon Health and Sciences University, Portland, OR;_ 3 _Children's Hospital of Philadelphia, Philadelphia, PA_.

Angiosarcomas are extremely rare, malignant childhood tumors, which often arise in deep soft tissue and liver as well as exhibit a wide anatomical distribution. Due to the rarity of this diagnosis in pediatrics, the development of novel targeted therapeutic options are limited. Here, we diagnosed a case of pediatric angiosarcoma (pAS), and to identify putative driver genomic aberrations, we performed genomic sequencing via a Comprehensive Next Generation Sequencing Solid Tumor Panel, including RNA-sequencing by anchored multiplex PCR (ArcherDx) for 106 fusion partner genes. We identified a novel NOTCH1-ROS1 gene fusion generated from a rearrangement of chromosomes 9 and 6. Subsequent reverse transcription and Sanger sequencing validated the expression of NOTCH1-ROS1 in the pAs. While both NOTCH1 and ROS1 are established proto-oncogenes, and have been separately involved in gene fusions across diverse cancer types, this is a previously unreported fusion in this rare pediatric cancer. We also identified a germline TP53 p.T123Wfs*12 pathogenic variant on follow-up sequencing, thus revealing Li Fraumeni syndrome in this angiosarcoma patient.Notch1-ROS1 is a novel, uncharacterized fusion protein and its functional oncogenic potential or tumorigenic sufficiency is unknown. We cloned NOTCH1-ROS1 cDNA and generated stably expressing heterologous cell lines to test its oncogenic potential using cellular models of transformation and tumorigenicity. Our analysis revealed that Notch1-ROS1 is a potent oncogene capable of inducing neoplastic transformation in cells, and demonstrated that its expression alone is sufficient for tumor formation in a murine allograft model. Notch1-ROS1 oncogenesis is driven via activation of several downstream pathways through a collaboration between the Notch1 and Ros1 domains. We also tested the efficacy of ROS1-targeted tyrosine kinase inhibitors (TKI) in these model systems; our data show dose-dependent suppression of Notch1-Ros1 driven cellular activity and concurrent inhibition of tumor growth as well as prolonged survival after oral monotherapy.Overall, this study reveals the first known NOTCH1-ROS1 alteration in a case of pAs. Given the extreme rarity of this malignant disease, it is challenging to expand to a larger cohort in a short duration. However, our findings on the mechanistic underpinnings of Notch1-ROS1 provide a deeper understanding of how this novel fusion drives tumor growth. Furthermore, drug-targeting studies provide requisite preclinical evidence for the possibility of utilizing ROS1-TKI in pA patients harboring this or other ROS1-fusions. Overall, these data suggest that ongoing genomic profiling of these rare tumors may reveal actionable drivers and holds the promise to improve patient outcomes.

#3645

Cooperation between canonical Wnt and TGF-beta pathways promotes sarcoma angiogenesis.

Allegra G. Hawkins, Elisabeth A. Pedersen, Wei Jiang, Sydney Treichel, Colin Sperring, Jay Read, Brian Magnuson, Rajiv M. Patel, Dafydd Thomas, Rashmi Chugh, Elizabeth R. Lawlor. _University of Michigan, Ann Arbor, MI_.

Local and metastatic progression of solid tumors depends on crosstalk between tumor cells and the tumor microenvironment (TME), including both stromal cells and the extracellular matrix (ECM). We recently showed that high Wnt/beta-catenin activity in Ewing sarcoma correlates with diminished patient survival and that canonical Wnt signaling alters the tumor secretome, influencing ECM protein composition. In light of this, we investigated the hypothesis that Wnt/beta-catenin supports tumor progression by modulating tumor: TME crosstalk. Our results reveal that, in discrete tumor cell sub-populations, beta-catenin activation sensitizes cells to TGF-beta ligands through derepression of the TGF-beta receptor, TGFBR2, resulting in canonical Wnt-induced, TGF-beta-dependent upregulation of TGF-beta targets. Significantly, these Wnt/TGF-beta responsive targets include multiple AngioMatrix genes that are known to alter the TME to promote angiogenesis, including tenascin-C and collagens. Studies of Ewing sarcoma models, in vitro and in vivo, as well as in two independent patient cohorts, confirm that a direct relationship exists between beta-catenin and TGF-beta activation in tumor cells and angiogenesis in the local TME. Mechanistically, this is due, in part, to tenascin C-mediated promotion of endothelial cell proliferation. Thus, functional cooperation between canonical Wnt and TGF-beta signaling in Ewing cells induces secretion of pro-angiogenic factors. This study reveals a novel link between Wnt and TGF-beta signaling in Ewing sarcoma and illustrates the critical contribution of tumor cell heterogeneity and tumor: TME crosstalk to sarcoma progression.

#3646

**(Epi-)genomic homogeneity in radiation-induced glioblastoma with recurrent** PDGFRA **amplification and loss of** CDKN2A/B **following primary acute lymphatic leukemia and medulloblastoma.**

Maximilian Y. Deng,1 Dominik Sturm,1 Elke Pfaff,1 Gnana P. Balasubramanian,1 Jens Schittenhelm,2 Martin Ebinger,2 Martin U. Schuhmann,2 Andrey Korshunov,1 Andreas von Deimling,1 Olaf Witt,1 Stefan M. Pfister,1 David T. Jones1. 1 _German Cancer Research Center, Heidelberg, Germany;_ 2 _University Hospital of Tübingen, Tübingen, Germany_.

As an essential pillar of today's cancer treatment, radiation therapy has led to improved survival rates of patients with childhood malignancies including leukemia and central nervous system (CNS) tumors. However, long-term complications such as radiation-induced malignancies occur in a subset of patients following radiation therapy, especially observed in pediatric patients due to their long follow-up period in case of survival. Radiation-induced glioblastomas (RIGs) have been reported in patients after treatment with cranial irradiation for various primary malignancies such as acute lymphoblastic leukemia (ALL) and medulloblastoma (MB). Histopathologically, most RIGs are best described as high-grade gliomas resembling de novo glioblastoma, and histopathological features to distinguish RIGs from their sporadic counterparts are lacking. Recent large-scale genomic and epigenomic analyses have revealed key genetic alterations in various types of CNS tumors. Here, we performed comprehensive (epi-)genomic and gene expression profiling of RIGs following radiation therapy for primary MB (n=24) and ALL (n=8). DNA methylation profiling demonstrates a high similarity of global DNA methylation patterns among RIGs, regardless of the primary malignancy. Known genetic alterations in high-grade gliomas such as PDGFRA amplification (53%, 17/32) and loss of CDKN2A/B (63%, 20/32) occur in RIGs. None of the RIGs harbored somatic hotspot mutations in genes encoding histone variants H3.3 and H3.1/H3.2 or IDH1/2, which are frequently observed in high-grade glioma subtypes of children and young adults. Germline alterations causing cancer predisposition syndromes were not found more frequently in RIG patients than in patients suffering from high-grade gliomas without previous irradiation treatment. The genetic homogeneity of RIGs with the absence of histone 3 and IDH1/2 mutations suggests that RIGs share a common cell of origin, which might be particularly vulnerable to radiation. We performed in vitro drug screens on patient-derived RIG tumor spheres, exhibiting amplified and overexpressed PDGFRA, alongside with non-PDGFRA-amplified GBM cell lines to assess the potential efficacy of RTK inhibitors. The median latency time between cranial irradiation and RIG occurrence was 4.5 years (range: 2.5-15). Patients treated for ALL were diagnosed with RIG at age 9-14 years, exposing a particularly vulnerable but narrow time frame for RIG occurrence in ALL patients, in contrast to RIG patients treated for MB (range: 3-39 years). In summary, our study uncovers diagnostic biomarkers and potential targetable alterations in RIG, which could become relevant for the stratification into future clinical trials with e.g. specific RTK inhibitors, with the objective of improving the outcome of survivors of childhood cancer.

#3647

Single cell RNA sequencing reveals mitogenic and progenitor gene programs inBRAF-rearranged pilocytic astrocytomas.

Zachary J. Reitman,1 Brenton Paolella,1 Guillaume Bergthold,1 Kristine Pelton,1 Robert Jones,1 Sarah Becker,1 Claire E. Sinai,1 Haley Malkin,1 Ying Huang,1 Leslie Grimmett,1 Zachary T. Herbert,1 Yu Sun,1 Jessica Weatherbee,1 John Alberta,1 John Daley,1 Orit Rozenblatt-Rosen,2 Rosalind Segal,1 Daphne Haas-Kogan,1 Mariella G. Filbin,1 Mario L. Suva,3 Aviv Regev,2 Charles Stiles,1 Mark W. Kieran,4 Liliana Goumnerova,5 Keith L. Ligon,1 Alex K. Shalek,2 Pratiti Bandopadhayay,1 Rameen Beroukhim1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Broad Institute of Harvard and MIT, Cambridge, MA;_ 3 _Massachusetts General Hospital, Boston, MA;_ 4 _Bristol Myers Squibb, Cambridge, MA;_ 5 _Boston Children's Hospital, Boston, MA_.

Single-cell RNA sequencing (scRNAseq) has been performed across a range of intermediate to high-grade gliomas that each harbor multiple driver mutations. Pilocytic astrocytoma (PA), the most common childhood brain tumor, is a low-grade glioma. PAs frequently harbor oncogenic KIAA1549-BRAF fusions, and exhibit low rates of other driver mutations. While PAs exhibit a favorable prognosis compared to the higher-grade gliomas, treatment morbidity and tumor recurrence can represent major challenges for some PA patients. We performed scRNAseq of both tumor and non-tumor cells in newly-diagnosed PAs that contained KIAA1549-BRAF rearrangements. To confidently distinguish tumor cells from nontumor cells, we sorted cells by glial progenitor marker A2B5 status and profiled KIAA1549-BRAF fusion status of cells using a sensitive quantitative PCR-based assay. Results were validated using RNA in situ hybridization. When compared to higher-grade gliomas, a higher proportion of the PA tumor cells exhibited a differentiated, astrocyte-like phenotype. A smaller proportion of cells exhibited a progenitor-like phenotype with evidence of proliferation. These progenitor-like tumor cells expressed a mitogen-activated protein kinase (MAPK) program that was absent from higher-grade gliomas. Similar patterns of expression of genes associated with the astrocyte-like and MAPK gene programs were also seen in formalin fixed, paraffin embedded PA tissues using RNA in situ hybridization. Immune cells, especially microglia, comprised almost half of all cells in the PAs and accounted for differences in bulk expression profiles between tumor locations and subtypes. These single cell transcriptional data reveal a transcriptional developmental hierarchy in a pediatric low grade glioma that is skewed towards more mature brain cells compared to higher-grade gliomas. The results indicate that future analyses of bulk PA tissues should attempt to account for considerable infiltration by nontumor cells. Finally, the finding that a MAPK gene program is not uniformly expressed in PA tumor cells has implications for ongoing clinical investigations of therapies directed at the MAPK pathway for the treatment of PA.

#3648

Protein phosphatase 1 regulatory subunit 1A regulates cell cycle progression in Ewing sarcoma.

Wen Luo, Marcela Laukova, Sarah Phillips, Janet Ayello, Mitchell S. Cairo. _New York Medical College, Valhalla, NY_.

Ewing sarcoma (ES) is a highly malignant pediatric bone and soft tissue tumor characterized by the expression of chimeric fusions of EWS and ETS family transcription factors, mostly EWS/FLI, as a consequence of chromosomal translocation. The dominant role of EWS/FLI in ES indicates that insights into the biology of this aberrant transcription factor and its downstream targets in ES initiation and progression could lead to the discovery of novel therapeutic strategies. We recently identified protein phosphatase 1 regulatory subunit 1A (PPP1R1A), a potent protein phosphatase 1 (PP1) inhibitor, as one of the core EWS/FLI targets and a potential specific therapeutic target for primary and metastatic ES. Small molecule compound inhibition of PPP1R1A and the associated PKA/PPP1R1A/PP1 signaling pathway impaired tumor growth and metastasis in ES xenograft mouse model. In the current study, we seek to identify the underlying mechanisms of PPP1R1A mediated tumorigenesis and metastasis and define additional role of PPP1R1A in ES pathogenesis to facilitate discovery of additional therapeutic targets. We performed shRNA induced knockdown (iR1A) and CRISPR knockout (R1A-KO) of PPP1R1A in ES cells and found that iR1A and R1A-KO cells proliferated much slower than the control cells. Cell cycle analysis showed that PPP1R1A depleted cells were arrested in G1 to S phase transition which could be rescued by re-expression of 35D, a constitutively active PPP1R1A. We found that Rb, whose phosphorylation releases cell cycle arrest in G1 phase, was hypo-phosphorylated upon PPP1R1A knockdown and hyper-phosphorylated after 35D rescue. Furthermore, depletion of PPP1R1A increased expression levels of cell cycle inhibitors p21 and p27, but not other G1 phase associated proteins. Results from RNA-seq and qRT-PCR analysis showed that PPP1R1A knockdown decreased the level of total mRNA but increased that of poly-adenylated mRNA of a subset of replication-dependent histone genes, suggesting a compensation mechanism of the loss of normal pre-mRNA processing of the histone genes in G1-arrested PPP1R1A knockdown cells. Our findings demonstrate that PPP1R1A promotes cell cycle progression through G1 to S phase by down-regulating cell cycle inhibitors p21 and p27 which leads to Rb hyper-phosphorylation and de-repression of cell cycle. Since PPP1R1A is specifically highly expressed in ES but not its putative cell of origin, mesenchymal stem cells (MSCs), our results indicate that PPP1R1A serves as an ES specific cell cycle modulator. Recently, IGF-1R inhibitors have shown synergistic effect with cell cycle modulators such as CDK4/6 inhibitors. Thus, combination of PPP1R1A inhibition with IGF-1R inhibitor may be explored for synergistic specific and effective tumor control in ES. Further studies for identification of the molecular mechanisms by which PPP1R1A regulates p21 and p27 may reveal more therapeutic targets for ES treatment.

#3649

**Allelic imbalance in** KMT2A **-rearranged infant acute lymphoblastic leukemia.**

Byunggil Yoo,1 Midhat S. Farooqi,1 Rumen Kostadinov,2 Warren Cheung,1 Emily Farrow,1 Shannon Kelley,2 Neil Miller,1 Bing Ge,3 Margaret Gibson,1 Patrick Brown,2 Erin M. Guest,1 Tomi Pastinen1. 1 _Children's Mercy Hospital and Clinics, Kansas City, MO;_ 2 _Johns Hopkins University, Baltimore, MD;_ 3 _McGill University, Montreal, Quebec, Canada_.

Background

Infant acute lymphoblastic leukemia (ALL) is a malignant disorder with poor clinical outcome. It is well-known that infant ALL cases with rearrangement of the KMT2A gene (KMT2A-r) have an even poorer prognosis than non-KMT2A-r cases. Interestingly, KMT2A-r infant ALL cases have remarkably few other genetic alterations. We hypothesized that non-coding events in cancer genomes (e.g. loss of expression or methylation) may play a role in this disease. Such events can be captured using genomic analyses in haploid genomes using analytical approaches for allelic imbalance quantification. Here, we examine whether allelic imbalance is a feature of infant ALL.

Methods

We performed whole genome sequencing (WGS) and RNA sequencing on peripheral blood or bone marrow specimens from 29 KMT2A-r cases and 14 non-KMT2A-r cases at diagnosis (DX), remission (MD), and relapse (RL) as applicable. WGS data from MD samples was phased using a 1000 Genomes reference panel. Biallelic expression was measured on the phased genome for transcripts with at least 2 SNPs and at least 15 aligned reads. Lesser allele fraction (LAF; allele fraction for the less prevalent allele) for DX/RL versus MD samples was compared for transcripts that had LAFs available in at least 3 cases using t-test. Transcript-level p-values were aggregated at the gene level using the Sidak method.

Results

Allelic imbalance in expression (LAF <= 0.2) was observed in an average of about 600 genes per sample in infant ALL regardless of timepoint. Disease-specific allelic imbalance (skewed in DX samples but not in paired MD samples) was detected in 431 genes for the KMT2A-r cohort and 77 genes for the non-KMT2A-r cohort. A total of 38 genes with allelic imbalance were shared between the two cohorts. Notably, KMT2A was observed to be imbalanced in KMT2A-r samples. Genes of known significance that were found to be skewed included HOXA9 and PARP8.

Discussion

Our study suggests that allelic imbalance quantification may help uncover novel molecular mechanisms in infant ALL, especially KMT2A-r cases. However, similar to gene expression, the patterns of allelic imbalance in KMT2A-r cases at DX do not allow efficient prediction of which patients go on to relapse.

#3650

Integrative mass spectrometry and RNA-sequencing identifies DLK1 as a candidate immunotherapeutic target in neuroblastoma.

Amber K. Weiner,1 Alexander B. Radaoui,2 Nathan M. Kendsersky,1 Jo Lynne Harenza-Rokita,2 Simone Sidoli,1 Karina L. Conkrite,2 Zalman Vaksman,2 Komal Rathi,2 Pichai Raman,2 Daniel Martinez,2 Tricia Bhatti,2 Matthew Tsang,2 Bruce Pawel,2 Benjamin A. Garcia,1 John M. Maris,2 Sharon J. Diskin2. 1 _Univ. of Pennsylvania School of Medicine, Philadelphia, PA;_ 2 _Children's Hospital of Philadelphia, Philadelphia, PA_.

BACKGROUND. Neuroblastoma (NB) is an embryonal tumor of the sympathetic nervous system that accounts for 12% of childhood cancer deaths. Despite multimodal therapy, survival probability for high-risk NB patients remains below 50% and relapsed NB is largely incurable. To date, the cell surface landscape (surfaceome) of NB remains poorly defined. An unbiased survey of these proteins will facilitate the identification of candidate immunotherapeutic targets for preclinical validation.

METHODS. To identify proteins on the cell surface of NB cells, we performed plasma membrane protein extraction utilizing a sucrose density gradient methodology followed by nano-liquid chromatography coupled to mass spectrometry (nLC-MS/MS) in NB cell lines (n=12) and patient derived xenografts (PDX; n=10). We next integrated MS data with RNA-sequencing (NB=153; Normal=7859) data to evaluate proteins with an annotated extracellular domain differentially expressed in NB compared to normal tissues. Candidate immunotherapeutic targets were validated by immunohistochemistry on NB tumor and normal tissue microarray (TMA) and in-vitro functional studies were performed following genetic manipulation of candidate targets to assess cell proliferation, differentiation and viability.

RESULTS. We yielded on average 66% (range:60-68%) membrane protein enrichment with high reproducibility between biological replicates (80%; range:78-84%) and identified 4826 unique membrane proteins. Our approach confirmed known cell surface proteins in development as immunotherapeutic targets in NB (ALK, GPC2, NCAM1, DLL3 and CD276). We prioritized DLK1 for further evaluation due to it being the only candidate with expression directly associated with a super enhancer element (P=6.09X10-5). Genetic depletion of DLK1 resulted in neurite outgrowth suggesting induction of terminal differentiation (P=7.26X10-5). Full proteome analysis of DLK1 knockdown and control using MS showed regulation of proteins that promote outgrowth of neurites (P=3.37X10-3) and development of neurons (P=3.76X10-3).

CONCLUSION. We have developed the first MS-based surfaceome of NB. DLK1 is an epigenetically regulated oncoprotein in a large subset of high-risk NBs. We are currently developing antibody drug conjugate therapeutics designed for NBs with proven overexpression of DLK1.

#3651

**Increased prevalence of germline monoallelic** RECQL4 **mutations in children with cancer.**

Jamie L. Maciaszek, Gang Wu, Kayla Hamilton, Rose B. McGee, Zhaoming Wang, Regina Nuccio, Stacy Hines-Dowell, Lynn Harrison, Elsie L. Gerhardt, Annastasia Ouma, Scott Newman, Aman Patel, Joy Nakitandwe, Elizabeth Azzato, Alberto S. Pappo, Sheila A. Shurtleff, David W. Ellison, James R. Downing, Melissa M. Hudson, Leslie L. Robison, Victor Santana, Jinghui Zhang, Kim E. Nichols, Chimene A. Kesserwan. _St. Jude Children's Research Hospital, Memphis, TN_.

RECQL4 encodes an essential helicase that repairs DNA damage and maintains genomic stability. Biallelic pathogenic germline mutations in RECQL4 cause the autosomal recessive (AR) Rothmund-Thomson, Baller-Gerold, and RAPADILINO syndromes. Predisposition to cancer has been observed in all three syndromes, with osteosarcoma (OS) representing the greatest risk.

Monoallelic pathogenic variants in DNA damage response genes (e.g., ATM, NBN) are associated with a moderate increase in cancer risk. Building upon this notion, we sought to determine whether monoallelic RECQL4 loss of function (LOF) variants contribute to childhood cancer, particularly OS. Here, LOF variants were defined as nonsense, frameshift, or canonical splice altering variants that are classified as pathogenic or likely pathogenic based on the 2015 ACMG Guidelines. Among 4,436 pediatric cancer patients at St. Jude and 1,127 in the National Cancer Institute TARGET database (total: 5,563 patients), we identified 20 individuals (0.36%; tumor types in Table) harboring germline monoallelic RECQL4 LOF variants. Compared to reference controls in the Genome Aggregation Database (gnomAD), we observed an enrichment of RECQL4 LOF variants in pediatric cancer patients (P = 0.046, prevalence ratio [PR] = 1.51). We next assessed for enrichment of RECQL4 LOF variants across tumor types (Table). This examination revealed a significant association between monoallelic RECQL4 mutations and leukemia (P = 0.032, PR = 1.91) and more notably, with OS (P = 0.0028, PR = 7.03) where 1.7% of pediatric OS patients carried monoallelic RECQL4 LOF variants. No evidence of association was observed for the other tumor types examined.

Our data provide the first evidence of an association linking germline monoallelic RECQL4 LOF variants to childhood cancer, especially OS. Examination of larger cohorts are warranted to elucidate the extent to which these mutations increase the risk for leukemia and OS and the mechanisms by which they promote tumor formation. | |  | |  | |

|

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

|

Pediatric Cancer Patients | gnomAD

Controls | Cancer Risk

(Fisher's Exact Test)

|

Average age at cancer diagnosis (range) | Carriers,

RECQL4mut/wt | Total Patients,

RECQL4wt/wt | Carriers, RECQL4mut/wt | Total Controls, RECQL4wt/wt | Prevalence Ratio (95% CI) | P Value

All cancers | 7 (0.3 - 18) | 20 | 5563 | 281 | 120659 | 1.51 (0.99, 2.30) | 0.046

OS | 10 (6 - 16) | 4 | 243 | 281 | 120659 | 7.03 (2.65, 18.69) | 0.0028

CNS tumor | 5 | 1 | 579 | 281 | 120659 | 0.74 (0.10, 5.26) | 0.74

GCT | 0.3 | 1 | 75 | 281 | 120659 | 5.74 (0.81, 40.88) | 0.16

Leukemia | 4 (3 - 11) | 11 | 2431 | 281 | 120659 | 1.91 (1.07, 3.41) | 0.032

Lymphoma | 10 (7 - 18) | 3 | 586 | 281 | 120659 | 2.19 (0.71, 6.76) | 0.16

OS = osteosarcoma; CNS tumor = central nervous system tumor; GCT = germ cell tumor

#3652

USP7 heterozygous loss-of-function affects T-cell differentiation in pediatric T-ALL.

Timothy I. Shaw,1 Li Dong,1 Anthony High,1 Yu Liu,1 Bensheng Ju,1 Kanisha Kavdia,1 Vishwajeeth Pagala,1 Bridget Shaner,1 John Easton,1 Chenxi Qian,1 Jiyang Yu,1 Janke Janke,1 John Kim Choi,1 Junmin Peng,1 Wei Gu,2 James R. Downing,1 Jinghui Zhang1. 1 _St Jude Children's Research Hospital, Memphis, TN;_ 2 _Columbia University, New York City, NY_.

Ubiquitin-specific-processing protease 7 (USP7), a protein deubiquitinase, is one of the most frequently mutated genes (33%) in the TAL1 subtype of pediatric T-lineage acute lymphoblastic leukemia (T-ALL). However, the functional effect of USP7 haploinsufficiency on T-ALL pathogenesis remains elusive. To understand USP7 haploinsufficiency's impact on T-ALL, we performed gene expression analysis on 42 non-early T-cell precursor T-ALL RNAseq samples downloaded from phs000218. The gene expression analysis suggested that USP7 haploinsufficiency (USP7-mut N = 12; USP7-wt N = 30) down-regulated the expression of T-cell maturation markers (e.g. RAG1, RAG2, and CD1B), which were negatively regulated by TAL1 [1]. RNAseq on a T-ALL cell line with USP7 knocked down by shRNA also found the same TAL1 negatively regulated gene set, further supporting an increase of TAL1 activity in the USP7 mutated T-ALLs. To examine whether the T-cell maturation was affected by USP7 heterozygous knockout, we generated a conditional knockout (cKO) mouse model by cross-breeding the transgenic vav1-cre mice with the USP7fl/fl mice to obtain heterozygous USP7fl/wt-vav1-cre. Thymocytes isolated from cKO mice were co-cultured with the OP9-Δ1 cells in the medium supplied with cytokines. The cell surface differentiation markers, CD4 and CD8 were stained for flow cytometry detection, and we observed an increase of double-negative cells and a decrease of double-positive cells in USP7-het-KO mice versus control (N = 4 in each group; p-value < 0.05), consolidating the disrupted T-cell development ex vivo. To further understand USP7's mechanism to regulate TAL1, we analyzed proteins that interacted with USP7 by affinity purification-mass spectrometry (AP-MS) and immune-precipitated followed by western-blotting (IP/WB). AP-MS with the anti-USP7 antibody revealed that USP7 directly interacts with TAL1 in Jurkat cells; AP-MS with the anti-TAL1 antibody also reciprocated the USP7-TAL1 interactions. Whole proteome analysis, through tandem-mass-tag and two-dimensional liquid chromatography-tandem mass spectrometry, revealed that USP7 knockdown down-regulated TRIM27, a deubiquitin target of USP7. IP/WB further confirmed the interaction between USP7, TRIM27, and TAL1, suggesting a possible synergistic relationship between USP7 and TRIM27 to regulate TAL1. In conclusion, our finding demonstrates heterozygous loss of function of USP7 dysregulates T-cell maturation by enhancing TAL1 activity. Future work on TRIM27 could further shed light on the mechanism underlying USP7's ability to regulate TAL1. Reference: [1] Sanda et al. Cancer Cell 22, 209 (2012).

#3653

Immunogenomic landscape of pediatric solid malignancies.

Jun S. Wei,1 Andrew S. Brohl,2 Young K. Song,1 Sushma Najaraj,1 Vineela Gangalapudi,1 Ashley Walton,1 Xinyu Wen,1 Marc Ladanyi,3 Javed Khan1. 1 _National Cancer Inst., Bethesda, MD;_ 2 _H. Lee Moffitt Cancer Center, Tampa, FL;_ 3 _Memorial Sloan Kettering Cancer Center, New York, NY_.

Malignancy remains the leading cause of disease-related death in children. To identify potential tumor-driving molecular targets and immunogenomic profiles in pediatric cancers, we performed RNA-seq analysis on a cohort of 792 pediatric solid malignant tumors across 14 different diagnoses in conjunction with additional 147 normal tissues for comparison. Sequencing data was analyzed for expressed mutations, fusion events, and expressional patterns, providing therapeutic targets and rich cancer biology for these childhood cancers. Furthermore, we describe immunogenomic features of the tumors including immune cell infiltrate, neoantigen expression, expression of immunomodulatory molecules, and T cell receptor repertoire. Across the cohort, we observed a striking correlation between the expressed neoantigen burden in tumors and enrichment of the effector immune signatures. Histology-specific immunogenomic patterns were also apparent. Several of the pediatric cancers such as alveolar soft part sarcoma and osteosarcoma exhibit rich immune cell infiltration and evidence for activated T cell activities, whereas others such as Ewing's sarcoma and yolk sac tumors generally have a very low T cell infiltration. We demonstrate that RNA-seq is a powerful tool to identify clinically relevant and histology-specific recurrent mutations, novel oncogenic fusions, and translationally relevant immunogenomic patterns for pediatric cancers. This study also represents one of the largest of its type to date and provides a framework for future translational efforts in pediatric cancer.

#3654

Recurrent deletions of 3q13.31 in human osteosarcoma commonly affect TUSC7 and LINC00901.

Baptiste Ameline,1 Michal Kovac,1 Karim H. Saba,2 Maxim Barenboim,3 Michaela Nathrath,4 Karolin H. Nord,2 Daniel Baumhoer1. 1 _Bone Tumour Reference Center at the Institute of Pathology, University Hospital Basel, Basel, Switzerland;_ 2 _Department of Laboratory Medicine, Division of Clinical Genetics, Lund University, Lund, Sweden;_ 3 _Pediatric Oncology Center at the Department of Pediatrics, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany;_ 4 _Pediatric Oncology Center, Department of Pediatrics, Technische Universität München, Munich, Germany and Pediatric Hematology and Oncology, Klinikum Kassel, Kassel, Germany_.

Background: Osteosarcoma, the most common primary tumor of bone, generally harbors complex structural rearrangements. In this study, we identified frequent mono- and bi-allelic deletions of the two long non-coding RNAs TUSC7 and LINC00901, both located on the long arm of chromosome 3 (3q13.31). Their precise biological function remain largely unknown, although it was suggested that TUSC7 may participate in micro-RNA trapping and thereby could be involved in tumor suppressor gene regulation. Moreover, the presence of a TP53 responsive element upstream of their coding region suggests an involvement in the convoluted TP53 signaling pathway. Methods: Whole genome sequencing data (n=104) and Affymetrix Cytoscan arrays (n=52) were used to derive copy-number profiles of each tumor and interrogated for LINC00901 and TUSC7 deletions. These findings were validated in an independent set of 102 formalin-fixed and paraffin-embedded tissue samples using RNA in situ hybridization techniques. Results: Mono- and bi-allelic deletions were detected in 68 (43.5%) sequenced and array-profiled osteosarcomas. Of these, 34 tumors (50%) acquired deletions in both genes whilst additional focal deletions of TUSC7 and LINC00901 were acquired in 11 (16%) and 19 (28%), respectively. The four remaining cases (6%) revealed copy number losses within the intergenic region between TUSC7 and LINC00901. In addition, reduced amounts or complete losses of hybridisation signals were detected in similar proportions in the independent set of FFPE samples. Conclusions: We identified recurrent 3q13.31 deletions in 68/156 (43,5%) human osteosarcomas which seems remarkable in a tumor well known for its high amount of genomic complexity and intertumoral heterogeneity. TUSC7 and LINC00901 might act as downstream effectors of the TP53 pathway and could be functionally equivalent to TP53 inactivation in case of copy number loss. To further elucidate the consequences of 3q13.31 aberrations we aim to correlate our findings with transcriptome data which is currently ongoing.

#3655

Mapping the osteogenic lineage and identification of the cell of origin of osteosarcoma through high-dimensional analysis.

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

Osteosarcoma (OS) is the most common primary malignant bone tumor in children and young adults. Neo-adjuvant chemotherapy combined with wide resection increased the 5-year survival rate to 60%-70%. However, treatment and outcome of OS have not changed in several decades. OS is histologically defined by the presence of malignant osteoid, suggesting it could be derived from anywhere in the osteogenic lineage between mesenchymal stem cells (MSC) and mature osteoblasts, but the cell of origin of OS is still undefined. Tracing the cells from which most cancers formed are characterized by its multipotent and/or unipotent stem cell nature. Studies on the cellular origin of tumors from phenotypic lineage-tracking and its derived progeny can reveal tissue-specific tumor-initiating cells. Investigating cell of origin of OS and osteogenic differentiation using developmentally relevant model using MSC to OB differentiation is promising. Here we developed a high-dimensional analytic pipeline using single-cell mass cytometry (CyTOF) and RNA sequencing to discover the unrecognized progenitor-osteoblast populations in the osteogenic lineage. For the data analysis, the population clustering using Flowsom and ConsensusClusterPlus on arcsinh transformed FCS data combined with t-SNE plot visualization shows that combination of CD49f, CD95, CD56, CD117 expression enables the identification and future isolation of the progenitor populations. Also, when comparing OS with the populations from MSC to OB, there are progenitor -osteoblast / mature osteoblast-like populations in tumor cells, but no MSC-like component is found. The expression level of CD56 and CD117 are significantly higher in OS compare to MSC and OB, which warrants future investigation of new therapeutic strategy. Isolation of the progenitor-osteoblast cells, gene expression comparison between OS and these progenitors, validation of their differentiation potential and tumor-initiating capacity is still underway. In this study, we developed a CyTOF based pipeline to discover the progenitor populations in the osteogenic lineage. By comparison of the OS and these progenitor cells, we may be able to unveil the cell of origin and new targeted treatment strategy of OS.

#3656

**The mTOR-activating GATOR2 complex is essential in** PAX3-FOXO1- **positive rhabdomyosarcoma.**

Amit J. Sabnis, David V. Allegakoen, Trever G. Bivona. _UCSF, San Francisco, CA_.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood, and can be genomically classified into fusion-positive and fusion-negative subsets. The former is driven by PAX3-FOXO1 or related chimeric transcription factors and has few other genetic alterations; the latter harbors recurrent mutations within the PI3K-RAS pathway. Despite these fundamental differences in cancer initiating events, current therapeutic approaches are identical between the two groups aside from chemotherapy intensification for some fusion-positive patients given their poorer outcomes.

Current pharmacologic strategies do not permit direct targeting of the PAX3-FOXO1 oncoprotein, so we undertook a CRISPRi genetic screen to identify genes whose loss was toxic to PAX3-FOXO1+ RMS cells, but tolerated in cells with knockdown of the fusion protein. We hypothesized that this would identify targetable dependencies created by PAX3-FOXO1 expression that might permit precision therapy for fusion positive RMS. Through this screen, we identified two genes within the mTOR-activating GATOR2 complex, MIOS and WDR24, as essential for the growth of PAX3-FOXO1 positive RMS cells.

We find that loss of MIOS is toxic to fusion-positive RMS cells due to a loss of mTORC1 signaling that normally initiates from signals of amino acid sufficiency. Restoration of mTORC1 activation by amino acids could be achieved by genetic suppression of either DEPDC5, a component of the RAG GAP GATOR1 complex that is normally inhibited by GATOR2, or of TSC1, which suppresses mTORC1 activity in the absence of PI3K signaling. Either genetic alteration completely rescued fusion positive RMS cells from the deleterious effects of MIOS loss. Correspondingly, we found that a fusion-negative RMS cell line with an activating NRAS mutation was insensitive to MIOS loss, suggesting that in this genetic context, amino acid signaling through GATOR2 is no longer necessary to activate mTORC1 and promote RMS survival.

In contrast, loss of WDR24 was toxic to both fusion positive and fusion negative RMS cells, and could not be rescued by genetic reactivation of mTORC1. Preliminary data demonstrate alterations in lysosome size and quantity after WDR24 suppression, adding to existing data in Drosophila that this gene plays a critical, mTOR-independent role in lysosome biogenesis, and showing that this role supports RMS survival.

As mTOR inhibitors are being tested in frontline phase 3 studies in North America, our work lends timely molecular insight into how the genetic basis of distinct RMS subtypes can influence the signals that promote mTORC1 function to support cancer cell survival. Based on our finding that amino acid activation of mTORC1 is a fusion-positive dependency, we are now testing combined mTOR inhibition with amino acid depletion as a precision therapeutic strategy in preclinical models of this aggressive childhood cancer.

#3657

Defining the role of the RNA-binding protein MSI2 in neuroblastoma.

Adeiye Pilgrim, Selma Cuya, Dongdong Chen, Robert Schnepp. _Emory University School of Medicine, Atlanta, GA_.

Neuroblastoma (NB) is the most common extracranial solid tumor of childhood with a 5-year survival rate approaching 50% in high-risk patients and like other pediatric cancers, lacks many targetable mutations. Thus, novel therapeutic approaches are needed. Previous research has shown that the RNA-binding protein (RBP), LIN28B, enhances MYCN expression and induces NB formation. Additionally, we previously demonstrated that LIN28B-RAN-AURKA signaling drives NB oncogenesis. These results highlight the role of RBPs in modulating NB tumorigenesis.

The Musashi family includes RBPs that have been previously shown to be associated with worse prognosis in adult malignancies, including colon cancer and pancreatic cancer, among others. Thus, we speculated that the Musashi family might influence aggressive phenotypes in pediatric tumors, such as neuroblastoma. MSI2 is robustly expressed in neuroblastoma tumors, and, indeed, increased levels of Musashi-2 (MSI2) correlate with worse NB prognosis. In order to begin to elucidate its role in NB tumorigenesis, we knocked-down MSI2 in human NB cell lines using multiple independent short hairpin RNAs (shRNA) and small interfering RNAs (siRNA) and examined the impact on proliferation, survival, and downstream target genes relative to scrambled control RNA-treated cells. From these experiments, we observed decreased colony formation in clonogenic assays, increased apoptosis and decreased proliferation. To gain an initial understanding of the downstream targets that MSI2 influences, we performed Nanostring analysis in 2 human NB cell lines, Kelly and SK-N-DZ and found that knocking down MSI2 led to a downregulation of many cancer-related genes including IDH2 and other metabolic targets. IDH2 is a mitochondrial enzyme and its mutant is known to be involved in gliomagenesis. Changes in IDH2 expression have also been linked to epigenetic alterations. Finally, wild-type IDH2is upregulated in lung cancer and contributes to tumor growth. Therefore, we are currently examining the functional relationship between MSI2 and IDH2 in NB.

#3658

Characterization of the role of L3MBTL3 in medulloblastoma tumorigenesis.

Honglai Zhang, Ester Calvo Fernandez, Claire Peabody, Rork Kuick, Sung-Soo Park, Thomas Saunders, Sandra Camelo-Piragua, Jean-Francois M. Rual. _Univ. of Michigan, Ann Arbor, MI_.

Medulloblastoma is the most common malignant brain tumor of childhood. Therapeutic approaches to medulloblastoma have led to significant improvements but are achieved at a high cost to quality of life. The Notch pathway governs cell proliferation in many biological contexts, including medulloblastoma tumorigenesis. Using our proteomic platform, we discovered an interaction between RBPJ, a key co-factor of Notch for the mediation of Notch signals, and L3MBTL3, a methyllysine reader for which deletions are observed in medulloblastoma. We demonstrated that L3MBTL3 is part of a molecular mechanism linking the KDM1A demethylase to Notch signal modulation. We hypothesize that malfunction of this molecular mechanism may contribute to the previously suggested tumor suppressor role of L3MBTL3 in medulloblastoma. In a survival analysis using our L3mbtl3 KO mouse in combination with a genetically engineered mouse model of medulloblastoma, we validated our hypothesis that L3mbtl3 is a tumor suppressor in this disease context. Our discovery provides insights into the role of the L3MBTL3 in medulloblastoma that could be harnessed in the future for the therapeutic benefit of medulloblastoma patients.

#3659

Alyref is a novel binding partner and co-factor for MYCN-driven oncogenesis in neuroblastoma.

Zsuzsanna Nagy,1 Maxwell Kanikevich,1 Jessica Koach,1 Chelsea Mayoh,1 Daniel Carter,1 Tao Liu,1 Yanhua Du,2 Cizhong Jiang,2 Michelle Haber,1 Murray Norris,1 Belamy Cheung,1 Glenn Marshall1. 1 _Children's Cancer Institute, Kensington, Australia;_ 2 _School of Life Sciences and Technology, Tongji University, Shanghai, China_.

MYCN amplification is a poor prognostic indicator in neuroblastoma associated with high-risk disease. Therapies that directly repress the MYCN oncogenic signal in neuroblastoma are limited. We and others have shown that MYCN requires multiple cofactors to increase its protein stability in neuroblastoma cells, so that the very high MYCN levels required to drive tumourigenesis can be achieved. Here, we have identified ALYREF, a nuclear molecular chaperone protein, as a novel regulator of MYCN function in neuroblastoma. High expression of ALYREF predicted poor neuroblastoma patient survival and substantially correlated with MYCN levels in a large dataset (n=649) of human neuroblastoma tumour samples. ALYREF mRNA expression was also significantly increased in ganglia cells from the homozygous TH-MYCN neuroblastoma mouse in comparison to ganglia from wild-type littermates. Using co-immunoprecipitation and mass spectrometry, we identified ALYREF, as a direct binding partner of nuclear MYCN protein. Chromatin immunoprecipitation showed that MYCN bound the ALYREF gene promoter, and knockdown MYCN by MYCN siRNAs decreased ALYREF expression. A set of overexpression and knockdown experiments in MYCN-amplified neuroblastoma cells revealed that MYCN and ALYREF form a positive forward feedback expression loop. Overexpression of ALYREF further increased MYCN expression and protein stability in MYCN-amplified neuroblastoma cells. We found that ALYREF had a critical function in regulating the turnover of MYCN protein through transcriptional repression of the E3 protein ubiquitin ligase, NEDD4. The stabilized N-Myc oncoprotein enhanced ALYREF expression and stimulated cell growth of neuroblastoma. Additionally, we demonstrated that ALYREF plays a significant role in maintaining cell viability and proliferation of MYCN-amplified neuroblastoma cells. Taken together, our findings demonstrate a crucial role for ALYREF in regulating MYCN function and suggest that ALYREF-mediated stabilization of MYCN protein contributes to the development and progression of the disease. Thus inhibition of ALYREF activity using small molecules is a potential therapy for MYCN-amplified tumours.

#3660

A link between miRNAs and mRNA translation elongation: The let7-eEF2K axis in MYC-driven pediatric tumors adaptation to nutrient deprivation.

Alberto Delaidelli,1 Gian Luca Negri,1 Brian Cho,1 Simran Sidhu,1 Stefan Pfister,2 Michael Taylor,3 Gabriel Leprivier,4 Marcel Kool,2 Poul Soresnsen1. 1 _BC Cancer Research Centre, Vancouver, British Columbia, Canada;_ 2 _German Cancer Consortium (DKTK), Heidelberg, Germany;_ 3 _Arthur and Sonia Labatt Brain Tumor Research Centre, Toronto, Ontario, Canada;_ 4 _University Hospital Düsseldorf, Düsseldorf, Germany_.

BACKGROUND/OBJECTIVES: MYC family proteins are implicated in many human cancers, but their therapeutic targeting has proven challenging. MYCN and MYC amplification in childhood neuroblastoma (NB) and medulloblastoma (MB) are associated with aggressive disease and high mortality, underscoring a dire need for novel therapies. Let-7 microRNAs (miRNAs) inhibit tumor progression and regulate metabolism by degrading several mRNAs, including MYC. Let-7 miRNAs are therefore frequently repressed in cancer, including MYC-driven NB and MB. We previously reported that the mRNA translation elongation regulator eukaryotic Elongation Factor-2 Kinase (eEF2K) is a pivotal mediator of cancer cells adaptation to nutrient deprivation (ND). Publicly available transcriptomic database analyses indicate that eEF2K expression significantly correlate with MYCN and MYC expression in multiple tumor cohorts. Our preliminary data also indicate that the eEF2K 3' untranslated region (UTR) harbors a potential binding site for let-7 miRNAs. In addition, eEF2K mRNA and let-7 miRNA expression negatively correlates in NB and MB, suggesting a potential regulation of the former by the latter. We therefore hypothesized that let-7 down-regulation induces eEF2K expression, thereby supporting MYC-driven NB and MB adaptation to ND and tumor progression.

METHODS: Immunohistochemistry for eEF2K substrate (p-eEF2) was performed on NB and MB tissue microarrays to link results with MYC expression and outcome. Effects of eEF2K pharmacological and genetic inhibition on NB and MB cell survival were evaluated in vitro by MTT assay and PI staining. The ability of let-7 to degrade eEF2K mRNA was assessed by let-7 miRNAs transfection into MB cells, followed by RT-PCR and Western Blotting for eEF2K. Binding of let-7 to the eEF2K 3'UTR was validated by luciferase reporter assay. Finally, NB xenograft mouse models were used to confirm in vitro observations.

RESULTS: High eEF2K activity is linked to MYC over-expression and reduced survival in NB and MB (p<0.05). Pharmacological and genetic inhibition of eEF2K significantly reduces survival of MYC/MYCN-amplified NB and MB cell lines under ND. Let-7 miRNAs transfection decreases eEF2K mRNA and protein levels (by ~40-50%), and down-regulation of luciferase activity by let-7 miRNAs is impaired upon mutation of the let-7 binding site on the eEF2K 3'UTR. Knockdown of eEF2K determines a twofold growth decrease of MYCN-amplified NB xenografts when mice are kept under caloric restriction diet.

CONCLUSIONS: Let-7 miRNAs degrade eEF2K mRNA by binding to its 3'UTR, indicating that let-7 repression in MYC-driven NB and MB is partially responsible for eEF2K increased levels and activity. Moreover, the let-7-eEF2K axis represents a critical mechanism for MYC-driven NB and MB adaptation to ND, constituting a promising therapeutic target.

#3661

GLI1 contributes to proliferation, survival and drug-resistance of Ewing sarcoma (EWS) cells.

Joon Won Yoon, Marilyn Lamm, Philip Iannaccone, David Walterhouse. _Ann & Robert H. Lurie Children's Hosp. of Chicago Research Ctr., Chicago, IL_.

Ewing sarcoma (EWS) is characterized by TET-ETS fusions, most commonly the EWSR1-FLI1 fusion. GLI1, a transcription factor in the Hedgehog (HH) signal transduction pathway and target of canonical HH signaling, is a direct transcriptional target of EWSR1-FLI1. A role for the HH signaling pathway and GLI1 in acquisition of a multidrug resistance phenotype has been previously observed in several human cancers. The role of the HH signal transduction pathway and GLI1 in the biology of EWS remains incompletely understood. We hypothesize that the majority of EWS cell lines express GLI1 based on non-canonical up-regulation by EWSR1-FLI1 and that further up-regulation of GLI1 may be associated with the development of drug resistance. Here, we show that EWS cell lines (CHLA-9, CHLA-10, CHLA-258, TC-32 and TC-71) express canonical HH pathway components (PTCH1, SMO, GLI1 and GLI3) and that cells lines established at the time of recurrence have higher GLI1 expression (CHLA-258 and TC-71) than those established at the time of diagnosis (CHLA-9, CHLA-10 and TC-32). Exposure to Sonic HH ligand in order to activate canonical HH signaling did not affect cell viability (MTT assay), proliferation (BrdU assay), or apoptosis (caspase 3/7 assay). However, inhibition of GLI1 activity with GANT61 reduced GLI1 expression (qRT PCR), cell viability (MTT assay) and cell proliferation (BrdU assay) but increased apoptosis (caspase 3/7 assay) and sensitivity of the cells to vincristine (VCR) (MTT assay), suggesting GLI1 plays roles in each of these processes. We then established VCR-resistant EWS cell lines (TC-71 [45-fold increase in the IC-50] and TC-32 [27-fold increase in the IC-50]) by exposing cells to serially increasing concentrations of VCR. We used an 86-gene cancer drug resistance PCR array (Qiagen) to characterize gene expression differences between untreated vs. recurrent EWS cell lines and between parental vs. VCR-resistant EWS cell lines. Among the changes in gene expression, we showed higher GLI1 expression in the recurrent EWS cell lines (CHLA-258 cells [4.8-fold] and TC-71 [3.4-fold]) vs. untreated CHLA-9 cells, and in VCR-resistant TC-71 cells (2.2-fold) vs. parental TC-71 cells. Down-regulation of GLI1 by GANT61 or GLI1 siRNA enhanced sensitivity of the cells to VCR. Our results suggest that GLI1 expression contributes to proliferation, survival and drug-resistance of EWS cell lines. Strategies to inhibit GLI1 may have therapeutic benefit in EWS.

#3662

Plasticity of transcriptional and epigenetic cellular states in neuroblastoma is driven by core lineage transcription factors.

Johan van Nes, Tim van Groningen, Linda Valentijn, Danny Zwijnenburg, Ellen M. Westerhout, Mohamed Hamdi, Jan Koster, Rogier Versteeg. _Academic Medical Center, Amsterdam, Netherlands_.

Background: Cellular identity in development and disease is driven by Core Regulatory Circuitries (CRCs) of lineage transcription factors that associate with super-enhancers. We showed that neuroblastoma includes two types of tumor cells with divergent gene expression profiles. Undifferentiated mesenchymal (MES) cells and lineage-committed adrenergic (ADRN) tumor cells have divergent phenotypes, super-enhancer (SE) landscapes and Core Regulatory Circuitries (van Groningen et al., Nature Genetics, 2017).

Results: We study five pairs of MES- and ADRN-type cell lines, each of which are derived from the tumor of individual patients. These isogenic cell lines can show spontaneous bidirectional transdifferentiation. As the mechanisms of reprogramming in cancer are poorly understood, we studied the mechanism of MES and ADRN transdifferentiation. We identified a MES-specific Core Regulatory Circuitry consisting of 20 super enhancer-associated transcription factors. Amongst them were NOTCH and MAML transcription factors. Indeed MES cells were found to have an active NOTCH signaling. Inducible expression of NOTCH3-IC in ADRN cells induced a step-wise reprogramming of the ADRN transcriptome towards a dedifferentiated MES state. This transition induced genome-wide remodeling of the H3K27ac landscape and a switch from ADRN SEs to MES SEs. The NOTCH3-IC transgene activated a transcriptional feed-forward cascade including NOTCH ligands, -receptors and -cofactors to amplify the NOTCH signaling levels. Blocking of this endogenous feed-forward loop with a γ-secretase inhibitor showed that this cascade was essential to achieve MES reprogramming. The endogenous NOTCH feed-forward cascade maintained the induced MES state, also after abrogating expression of the NOTCH3-IC transgene. The induced MES cells and stable MES cell lines were resistant to chemotherapy, highlighting their clinical importance. Accordingly, we found that MES cells are strongly enriched in post-treatment samples, suggesting that MES cells play a role in resistance and relapse development. Since neuroblastoma is presumed to originate from the sympathetic nervous system, we analyzed normal sympathetic lineage development at single-cell resolution. We found that MES tumor cells resembled non-malignant precursor cells of the sympatho-adrenal (SA)-lineage, while ADRN cells expressed SA-lineage differentiation genes.

Conclusions: Our results demonstrate that the divergent transcriptional states of cancer cells resemble stages of normal lineage development. Lineage TFs induce transdifferentiation via remodeling of the epigenetic and transcriptional landscapes, mimicking spontaneous interconversion. Plasticity of CRCs and lineage identity may have profound implications for treatment strategies in neuroblastoma.

#3663

Neuropeptide Y stimulates neuroblastoma cell migration via Y5R/RhoA pathway.

Nouran Abualsaud,1 Lindsay Caprio,1 Abrar Bakr,1 Lamia Alamri,2 Ewa Krawczyk,1 Susana Galli,1 Sung Hyeok Hong,1 Joanna Kitlinska1. 1 _Georgetown University, Washington, DC;_ 2 _George Washington University, Washington, DC_.

Neuroblastoma (NB) is a pediatric malignancy that originates from precursors of the sympathetic neurons and expresses neuronal proteins, such as neuropeptide Y (NPY). NPY is a neurotransmitter released from sympathetic nerves, acting via its Y1-Y5 receptors (Y1R-Y5R). NB cells constitutively express Y2R, while Y5R is induced in pro-apoptotic conditions. Our previous data indicated that Y2R is involved in NB cell proliferation and angiogenesis, while Y5R acts as a pro-survival factor. In NB patients, elevated serum NPY levels are associated with poor prognosis and metastasis. Interestingly, in NB tissues, tumor cells with angioinvasive phenotype have a strong expression of Y5R on the leading edge of the penetrating cell. Thus, the goal of our study was to determine the role of NPY and its receptors in NB dissemination. To this end, we assessed the effect of NPY system perturbations on NB cell motility (IncuCyte Live Cell Analysis system), cytoskeleton organization (phalloidin staining) and activity of a cytoskeleton regulator, RhoA (ICC, pull-down assay). The role of particular NPY receptors in these processes was validated by their overexpression in CHO-K1 cells. Treating NB cells with NPY stimulated NB cell migration in a dose-dependent manner, with a peak of activity at 10-8M and lower effects at higher and lower peptide concentrations. This effect was inhibited by Y5R antagonist, while in some cell lines further reduction was observed when combining Y5R and Y2R antagonists. The stimulatory effect of NPY on cell migration was associated with cytoskeleton remodeling, which included a higher number of filopodia positive for Y5R and co-localization of Y5R with an active form of RhoA at both leading and trailing edges of a migrating cell. In line with this role for Y5R in NB motility, expression of this receptor was up-regulated by hypoxia, a known pro-metastatic factor. Consistent with the results on native NB cells, overexpression of Y5R in CHO-K1 cells significantly increased their motility, while transfection with Y1R or Y2R had no such effect. Stimulation of CHO-K1/Y5R cells with NPY increased migration in a dose-dependent manner, while blocking Y5R inhibited cell motility. Moreover, NPY exerted a chemotactic effect for CHO-K1/Y5R cells. Like in NB cells, in CHO-K1 transfectants, Y5R co-localized with active RhoA in both leading and trailing edges of migrating cells. In line with this, CHO-K1/Y5R cells had elevated basal RhoA activity, while NPY stimulation further increased RhoA-GTP levels. Altogether, our data implicate Y5R as the main NPY receptor mediating its stimulatory effect on cell motility. In NB cells, Y2R may further enhance this effect. The Y5R-induced cell motility is associated with cytoskeleton remodeling driven by RhoA activation. The concentration-dependent activity of NPY and its chemotactic effect indicates that the peptide may spatially regulate NB cell migration within tumor tissue.

#3664

Hypoxia-induced phenotypic and metabolic changes in Ewing sarcoma cells trigger bone metastasis.

Shiya Zhu, Akanksha Mahajan, Sung-Hyeok Hong, Susana Galli, Congyi Lu, You-Shin Chen, Sara Misiukiewicz, Stacey Chung, Jason Tilan, Joanna B. Kitlinska. _Georgetown Univ., Washington, DC_.

Ewing Sarcoma (ES) is an aggressive malignancy that arises in children and young adults. Although the survival for patients with localized tumors is relatively high, the metastatic form carries a dismal prognosis, particularly when bone metastases are present. The frequency of metastases and osseous dissemination increases in patients with necrotic ES tumors. In line with this, hypoxia, the major cause of tumor necrosis, has been shown to increase metastatic potential of ES cells. Previous data from our laboratory demonstrated that in an ES orthotopic xenograft model, primary tumor hypoxia specifically promotes bone metastasis, which is associated with accumulation of cells with enlarged nuclei and frequent chromosome gains in bone invasion areas. We have also shown that the progeny of hypoxia-induced polyploid ES cells (ES≈4n cells) preferentially metastasized to bone. Thus, the goal of our study was to determine the mechanisms enabling osseous dissemination of ES≈4n cells. To this end, we used FUCCI Cell Cycle Sensor followed by DNA staining with Hoechst 33342 and cell sorting to isolate tetraploid cells (4n) from hypoxia-exposed SK-ES1 ES cells. Subsequently, we compared the metastatic properties of their progeny (H-SK-ES1≈4n) with a diploid cell population selected from normoxic SK-ES1 cells (N-SK-ES1-2n cells). We have found that H-SK-ES1≈4n cells have increased motility and invasiveness, as compared to the N-SK-ES1-2n cells. H-SK-ES1≈4n cells had also an increased ability to grow in hypoxia in 2D culture and in soft agar. In line with this, H-SK-ES1≈4n cells were highly sensitive to a glycolysis inhibitor, 2D-glucose, but not an inhibitor of oxidative phosphorylation, metformin. Moreover, H-SK-ES1≈4n cells were more sensitive than controls to a growth-inhibitory effect of AICAR, a blocker of anabolic metabolism. In contrary, the tetraploid cell progeny were highly resistant to doxorubicin, particularly under hypoxic conditions, which in contrast sensitized N-SK-ES1-2n and wild type SK-ES1 cells to chemotherapy. Altogether, our data implicate the following mechanisms underlying osseous dissemination of the hypoxia-induced poplyploid cell progeny: 1) increased motility and invasiveness facilitating escape from the primary tumor; 2) ability to survive under low oxygen tension characteristic for bone tissue. Moreover, we have shown that the cells initiating bone dissemination are highly resistant to conventional chemotherapy, while strategies targeting their metabolic dependencies may be more successful in treatment of patients with bone metastases.

#3665

Integrative analysis of whole-genome and RNA sequencing in high-risk pediatric malignancies.

Marcus R. Breese,1 Avanthi T. Shah,1 Alex G. Lee,1 Bogdan Tanasa,1 Stanley G. Leung,1 Aviv Spillinger,1 Heng-Yi Liu,1 Inge Behroozfard,1 Phuong Dinh,1 Florette K. Hazard,2 Arun Rangaswami,2 Sheri L. Spunt,2 Norman J. Lacayo,2 Tabitha Cooney,3 Jennifer G. Michlitsch,3 Anurag K. Agrawal,3 E. Alejandro Sweet-Cordero1. 1 _University of California San Francisco, San Francisco, CA;_ 2 _Stanford University, Palo Alto, CA;_ 3 _UCSF Benioff Children's Hospital Oakland, Oakland, CA_.

Clinical use of gene-panel based sequencing has become increasingly common in the management of pediatric cancer patients. For many patients, gene-panel tests have identified clinically actionable findings. However, highly targeted approaches will miss unanticipated (but potentially clinically meaningful) or novel alterations. In diseases with unknown or complex etiologies, including many pediatric high-risk and solid tumors, an unbiased approach may yield more actionable findings. To accomplish this, we examined the feasibility and utility of whole genome sequencing (WGS) and RNA sequencing (RNAseq) in the management of high-risk pediatric oncology patients.

Here we describe our experience with an expanded cohort of over 100 high-risk pediatric oncology patients, with a combination of solid tumors, brain tumors, and leukemia/lymphomas represented. The majority of patients were deemed high-risk due to relapsed/refractory disease. An additional group of patients were defined as high-risk at time of initial diagnosis due to metastatic disease, a rare tumor, prior history of another cancer type, an undifferentiated tumor, or less than 50% estimated overall survival. WGS (tumor/germline) and RNAseq were used to characterize available samples and compared to results from panel testing for each patient (performed as part of their clinical evaluation). When possible, multiple samples from an individual patient were collected (i.e. specimens obtained at biopsy, resection, relapse, and/or from metastatic sites). Somatic DNA samples were sequenced to an average depth of at least 60X and germline samples to at least 30X. RNAseq was performed to a depth of at least 20 million paired-end reads for each sample. WGS samples were analyzed for single nucleotide variants (SNVs), structural rearrangements (SV), and copy-number alterations (CNA). RNAseq samples were analyzed to identify known and novel gene-fusions, to measure allele specific expression of SNVs, and to perform gene-expression outlier analysis. Expression of variants (SNV/SV) identified using WGS were confirmed using RNAseq. For gene expression outliers detected using RNAseq, the WGS data was used to predict possible mechanisms for the aberrant expression (such as CNA, gene fusions, or promoter hijacking).

This analysis suggests that WGS and RNAseq analysis is feasible in a clinical setting and can reliably identify variants reported on gene panel tests. However, the use of WGS/RNAseq resulted in additional clinically informative findings while also enabling novel research to further advance our understanding of these rare and highly aggressive pediatric malignancies.

#3666

**The genomic landscape and clonal evolution of tumours arising in** TP53 **mutation carriers.**

Nicholas Light,1 Matthew Zatzman,1 Nathaniel Anderson,1 Vallijah Subasri,1 Mehdi Layeghifard,1 Ana Novokmet,1 James Tran,1 Richard de Borja,1 Fabio Fuligni,1 Joshua Schiffman,2 David Malkin,1 Adam Shlien1. 1 _The Hospital for Sick Children, Toronto, Ontario, Canada;_ 2 _Huntsman Cancer Institute, Salt Lake City, UT_.

Li-Fraumeni syndrome (LFS) is a familial cancer predisposition syndrome (CPS) caused by germline mutations in TP53, associated with a high frequency of sarcomas, breast cancers, adrenocortical carcinomas and CNS tumours. Recently, our group and others have identified distinct mutational signatures, defined by the spectrum and context of somatic mutations, in tumours arising in individuals with another CPS, constitutional mismatch repair deficiency (CMMRD). CMMRD tumours frequently harbor somatic mutations disrupting the proofreading function of the DNA polymerases POLE and POLD1, which consequently leads to an ultra-hypermutant cancer genome, with early MMR mutational signatures and late POLE/POLD1 mutational signatures. This ultrahypermutant tumor phenotype is essentially diagnostic for the syndrome and provides a rational for immune checkpoint inhibitors which have shown success in this context. Based on our results in CMMRD tumors we hypothesize that LFS tumours might likewise harbor distinct mutational events and/or evolutionary dynamics from sporadic tumours of the same histiotype. Although several cancer genomics landscape studies have included a handful of tumours from LFS patients, to our knowledge no study to date has attempted to comprehensively characterize the cancer genomes of LFS patients. To investigate the somatic mutational events driving tumourigenesis in LFS we performed whole-genome sequencing (WGS) analysis of 22 tumours derived from patients with pathogenic germline TP53 mutations. Tumours from germline TP53 wildtype patients were analyzed by the same methods to serve as a control data set. For each tumour sample, where possible, we performed WGS on multiple spatially distinct micro-dissected tumour regions in order to reconstruct the evolutionary history of each cancer. Somatic variant calling was performed at high sensitivity using in silico reconstructed high-depth bulk WGS (80-120X coverage), with somatic mutations, structural variants, copy number alterations, mutational signatures and subclones identified using MuTect2, delly, battenberg, SigProfiler and phyloWGS respectively. High confidence variants were identified using in-house designed filtering pipelines. The resulting analyses reveals the life history of LFS cancers is marked by a high frequency of early catastrophic genomic rearrangement events, a diverse range of somatic driver events and in at least some cases marked intratumoural spatial heterogeneity of CNVs and SNVs.

#3667

The Pediatric Brain Tumor Atlas - Transforming the landscape of research.

Yuankun Zhu,1 Yiran Guo,1 Allison P. Heath,1 Pichai Raman,1 Elizabeth Appert,1 Jennifer Mason,1 Bo Zhang,1 Karthik Kalletla,1 Miguel A. Brown,1 Natasha Singh,1 Bailey K. Farrow,1 Parimala Killada,1 Meen Chul Kim,1 Alex Felmeister,1 Mateusz P. Koptyra,1 Sabine Mueller,2 Michael Prados,3 Jena V. Lilly,1 Rishi Lulla,4 Adam C. Resnick,1 Javad Nazarian,5 Phillip B. Storm,1 Angela J. Waanders1. 1 _Children's Hospital of Philadelphia, Philadelphia, PA;_ 2 _UCSF, San Francisco, CA;_ 3 _UCSF Benioff Children's Hospital, San Francisco, CA;_ 4 _The Warren Alpert Medical School of Brown University, Providence, RI;_ 5 _George Washington University School of Medicine and Health Sciences, Washington, DC_.

The Pediatric Brain Tumor Atlas (PBTA) is a cloud-based, cross-platform, and data-rich collaborative effort to accelerate discoveries for therapeutic intervention in children diagnosed with brain tumors. Pediatric brain tumors are the leading cause of disease-related death in children and despite advances in therapy, morbidity and mortality rates remain poor. There have been large scale efforts to genomically profile these cancers in the past, however public access to the resulting datasets are limited at best. In contrast, the PBTA, created as a multi-center effort by Children's Brain Tumor Tissue Consortium (CBTTC) and Pacific Pediatric Neuro-Oncology Consortium (PNOC), has a goal of characterizing over 1,600 pediatric brain tumor samples and making the data publicly available to cancer researchers world-wide. PBTA comprises comprehensive clinical data in addition to whole exome sequencing, whole genome sequencing (WGS), RNA sequencing (RNASeq), miRNA sequencing, and proteomics. To address the need to inform novel discovery and clinical implementation of genomic approaches for diagnostic/therapeutic purposes, PBTA utilizes a cloud-based scientific environment, the CAVATICA portal. CAVATICA provides near real-time integration, dissemination, processing, and sharing of associated PetaByte-sized data by leveraging Amazon Web Services and powerful genomic workflows. It also enables dbGaP approved users to access TCGA and other datasets hosted by NCI's Cancer Genomics' Cloud, allowing for cross-disease studies. While CAVATICA manages raw sequencing data, processed annotations and biospecimen querying is enabled for PBTA via PedcBioPortal, a data visualization/analysis application further integrating additional public and deposited datasets. PBTA data can further be accessed via the KidsFirst platform, which integrates both CAVATICA and PedcBioPortal, and allows for querying of cancer and non-cancer genomic data for subsequent analytics, visualization, and discovery. The 1st dataset release of PBTA occurred on September 10th, 2018. In this release there are over 30 different types of pediatric brain tumors representing over 1,000 subjects. This data is available on CAVATICA, KidsFirst, and PedcBioPortal and users can seamlessly move between applications. Data types include those for matched tumor/normal samples, such as WGS, RNASeq, proteomics, longitudinal clinical data, imaging data (MRIs and radiology reports), histology slide images, and pathology reports. CBTTC/PNOC promote real time data release with no embargo period, allowing PBTA to have is up-to-date data releases with no embargo. The combination of cloud-based analytic platforms such as KidsFirst/CAVATICA/PedcBioPortal with data from both genomics and clinical practice serves to define a new paradigm for pediatric cancer research and collaborative discovery.

#3668

A LIN28B-PBK Axis promotes neuroblastoma dissemination and aggression.

Dongdong Chen,1 Julie Cox,2 Jayabhargav Annam,1 Melanie Weingart,2 Grace Essien,1 Komal Rathi,2 Priya Khurana,2 Selma M. Cuya,1 Robert W. Schnepp1. 1 _Aflac Cancer and Blood Disorders Center, Department of Pediatrics, Division of Pediatric Hematology, Oncology, and Bone Marrow Transplant, Emory University School of Medicine, Atlanta, GA;_ 2 _Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Children's Hospital of Philadelphia, Philadelphia, PA_.

LIN28B is an RNA binding protein that plays key roles in normal development and, when deregulated, oncogenesis; mechanistically, it blocks the processing of the let-7 family of tumor suppressors and binds mRNAs directly. We previously demonstrated that LIN28B induces neuroblastoma proliferation, in part by regulating the expression of RAN GTPase and Aurora kinase A (AURKA). However, given the widespread metastases seen within neuroblastoma, we speculated that LIN28B might also influence neuroblastoma dissemination. We used gain and loss of function approaches to genetically manipulate transcripts of interest in neuroblastoma cells and measured effects on self-renewal, invasion, and downstream signaling. To examine the impact of LIN28B on dissemination, we generated GFP-luciferase expressing neuroblastoma cell line models in which LIN28B levels were manipulated, injected these lines into the tail veins of NSG mice, and tracked dissemination using an IVIS Spectrum system. Results show that depletion of LIN28B significantly delayed the onset of tumor metastasis, reduced tumor burden, and extended mouse survival (104 days versus 50 days, p<0.0001) compared to control cells. While LIN28B did not impact anoikis resistance, it did increase both tumorsphere number and size, linking self-renewal to metastatic dissemination. Additionally, LIN28B promoted cellular invasion. These effects were largely opposed by let-7. We next sought to understand how LIN28B promotes aggression and metastasis, specifically focusing on novel networks that are currently therapeutically targetable. Given our discovery of AURKA as a novel LIN28B target, we speculated that LIN28B might promote the expression of additional oncogenic kinases, perhaps revealing novel therapeutic possibilities to target the LIN28B network. We evaluated the TARGET dataset of neuroblastoma tumors and, focusing on the top 10 kinases most significantly and positively correlated with high LIN28B expression, nominated PBK for further study (4/10 of top correlated kinases). PBK (PDZ-binding kinase) is a Ser/Thr protein kinase expressed in normal embryonic tissues and various tumor types that plays a role in both mitosis and metastasis. Depletion of PBK mimicked the effects of LIN28B depletion, with respect to self-renewal and invasion. Depletion of LIN28B and overexpression of let-7 both reduced PBK protein expression, suggesting that PBK is a direct or indirect let-7 target. Taken together, our findings suggest that LIN28B/let-7 shapes neuroblastoma aggression, in part through influencing PBK, a kinase not previously implicated in the pathogenesis of neuroblastoma or other aggressive pediatric solid tumors. Current studies are further dissecting the functional and molecular relationships among LIN28B, let-7, and PBK in neuroblastoma.

#3669

Defining an ELAVL1-Musashi 2 signaling cascade in neuroblastoma tumorigenesis.

Selma M. Cuya,1 Adeiye Pilgrim,1 Komal Rathi,2 DongDong Chen,1 Robert Schnepp1. 1 _Emory University, Atlanta, GA;_ 2 _University of Pennsylvania, Philadelphia, PA_.

Background: Patients with high-risk neuroblastoma endure an extremely intense, multidrug treatment regimen and yet, approximately half of them die, thus mandating a better approach to this cancer. Our laboratory is investigating the overarching hypothesis that RNA binding proteins (RBPs), which play key roles in balancing self-renewal, differentiation, and cell proliferation, are deregulated in neuroblastoma, and drive oncogenic signaling networks. Therefore, RBPs may constitute novel targets for therapeutic development. In this work, we focus on Musashi 2 (MSI2), an RBP that has been shown to play a critical role in the maintenance of stem cell populations and in the formation of aggressive tumors, notably in acute myeloid leukemia and in colon cancer. The role of MSI2 in neuroblastoma, however, has not been assessed and is the focus of this investigation.

Methods: To test this hypothesis, we used in silico analysis of neuroblastoma gene expression datasets annotated with clinical parameters. Additionally, we used shRNAs to manipulate transcripts of interest in neuroblastoma cells and measured effects on cell proliferation, cell cycle status, and apoptosis.

Results: Gene expression profiling revealed that MSI2 is robustly expressed in neuroblastoma cell lines, tumors, and patient derived xenografts, within both the MYCN and non-MYCN amplified context. Immunoblotting in a representative panel of lines confirmed the expression of MSI2 at the level of protein. High MSI2 expression was associated with more advanced stages of neuroblastoma across multiple independent datasets and was correlated with worse survival. Depletion of MSI2 using four independent shRNAs led to 2 to 3-fold decreased proliferation across four cell lines that was due in part to increased apoptosis. Another RBP, ELAVL1, is also expressed within neural lineages, and has been shown to regulate MSI1 expression. We hypothesized that ELAVL1 might similarly promote MSI2 expression and we demonstrated that, ELAVL1 depletion led to decreased MSI2 expression.

Conclusions: MSI2 is robustly expressed in multiple models of neuroblastoma and appears to drive increased proliferation and survival. Currently, we are investigating the functional interplay between ELAVL1 and MSI2 providing a foundation for designing strategies to target the ELAVL1-MSI2 interaction for therapeutic effect.

#3670

Transcriptional co-activators TAZ/YAP are novel regulators of PAX3-FOXO1 transcriptional programing and fusion-positive rhabdomyosarcoma cancer cell stemness.

Breanne A. Burgess, Katherine K. Slemmons, Corinne M. Linardic, Michael D. Deel. _Duke University, Durham, NC_.

Introduction: Fusion Positive Rhabdomyosarcoma (FP-RMS), a soft tissue sarcoma of adolescents and young adults, is driven by the oncogenic transcription factor PAX3-FOXO1 (P3F). Although most patients with FP-RMS initially respond to therapy, tumors frequently become refractory or relapse, underscoring a need to target resistance mechanisms and cancer stem cells. Because P3F is inherently disordered with no viable drug binding sites, it is currently not amendable to direct inhibition. Our prior work demonstrated that transcriptional co-activators TAZ and YAP are highly abundant in human FP-RMS and mediate several cancer phenotypes including apoptosis, differentiation, proliferation, and xenograft tumor growth. TAZ/YAP are essential co-activators for TEADs and AP1, which are among the top enriched transcription factor motifs at P3F binding sites. Here, we show that TAZ/YAP complex with P3F to positively regulate P3F transcriptional activity and that TAZ/YAP mediate FP-RMS stemness.

Methods: Functional interactions of TAZ/YAP and P3F were investigated using P3F reporters, immunoblots, and co-immunoprecipitation (co-IP); while co-IP-coupled mass spectrometry (IP-MS) to identify the TAZ/YAP/P3F interactome is currently underway. For reporter assays, gain- and loss-of-function vectors expressing control, wild-type TAZ/YAP (WT), constitutively active TAZ/YAP (S89A/S127A), or shRNA knockdown (KD) were used. Epitope-tagged TAZ, YAP, and P3F were used for co-IPs and IP-MS. To investigate the role of TAZ/YAP in stemness, we evaluated the expression of stem cell genes in FP-RMS 3D-spheres expressing vector, WT, S89A/S127A, or KD. We are now performing RNA-Seq as an unbiased approach to examine genes and pathways regulating FP-RMS stemness.

Results: TAZ/YAP functionally augment P3F transcriptional activity in reporter assays and also regulate expression of P3F targets. Immunoblotting of 293T cells co-expressing epitope-tagged proteins demonstrate an interaction between TAZ/YAP and P3F. A TAZ/YAP/P3F complex is also seen in co-IPs of endogenous proteins in FP-RMS cells. Serial passaging of spheres increases TAZ/YAP expression, suggesting these may be important for stem cell enrichment. Indeed, expression of SOX2, OCT4, and NANOG are TAZ/YAP-dependent, whereby their expression decreases with KD and increases with S89A/S127A. Functionally, TAZ/YAP are required for FP-RMS sphere formation in limiting dilution assays.

Conclusions: These studies expand on prior work showing TAZ/YAP are potent oncoproteins in FP-RMS. We identify a novel complex between TAZ/YAP and P3F, show TAZ/YAP are positive regulators of P3F transcriptional activity, and demonstrate that TAZ/YAP regulate FP-RMS cancer cell stemness. Thus, targeting TAZ/YAP could be a vulnerability of P3F and for inhibiting the cancer stem cell population.

#3671

Visualize 10,000 whole-genomes from pediatric cancer patients on St. Jude Cloud.

Xin Zhou, Clay Mcleod, Scott Newman, Zhaoming Wang, Michael Rusch, Kirby Birch, Michael Macias, Jobin Sunny, Gang Wu, Jian Wang, Edgar Sioson, Shaohua Lei, Robert J. Michael, Aman Patel, Michael N. Edmonson, Stephen V. Rice, Andrew Frantz, Ed Suh, Keith Perry, Carmen Wilson, Leslie L. Robinson, Yutaka Yasui, Kim E. Nichols, Gregory T. Armstrong, James R. Downing, Jinghui Zhang. _St. Jude Children's Research Hospital, Memphis, TN_.

Whole-genome sequencing (WGS) is invaluable for investigating genetic abnormalities contributing to the initiation, progression and long-term clinical outcome of pediatric cancer. St. Jude Cloud (https://www.stjude.cloud/) hosts 10,000 (10K) harmonized WGS samples generated from: 1) St. Jude/Washington University Pediatric Cancer Genome Project, 2) the Genomes for Kids Clinical Trial, 3) the St. Jude Lifetime Cohort Study, and 4) the Childhood Cancer Survivor Study. To enable on-the-cloud discovery and eliminate the need for data download, we developed GenomePaint, an interactive genomics browser, to explore the somatic and germline variants of the 10K genomes with rich annotation.

Germline variants in cancer predisposition genes were annotated for pathogenicity. Using GenomePaint, users can compare pathogenic variants from a locus of interest across multiple cancers or test for association of a germline variant with a specific cancer type on the fly. By matching germline variants to somatic mutation hotspots from www.cancerhotspots.org, we annotated potential germline mosaic mutations including IDH1 R132H, FBXW7 R465C, and KRAS A146T. For noncoding variants, we investigated overlap with ATAC and DNase peaks in 50 cancer cell lines along with transcription factor motif change predictions. These features will enable exploration of the functional impact of genetic variations with potential clinical status such as genetic risk for a specific cancer type, genetic association with age of onset, or development of subsequent malignancies for pediatric cancer survivors.

GenomePaint also provides an integrated view of somatic SNV/indel, copy number variation, loss-of-heterozygosity, structural variation, and gene fusion. These are shown together with tumor gene expression at the single tumor level. GenomePaint also presents allele-specific expression (ASE) and outlier expression as an indicator for assessing dysfunction of regulatory regions caused by genomic variants. Cloud-based on-the-fly ASE analysis is also available for user's samples with paired DNA and RNA sequencing results. Such gene expression integration will drive novel insights about the functional aspects of somatic coding and noncoding mutations in pediatric cancer.

The innovative visualization of whole-genome sequencing data generated from 10K pediatric cancer patients on the St. Jude Cloud enables genomic discovery by scientists and clinicians through exploration of this unprecedented resource.

#3672

Insulin-like growth factor 2 (IGF-2) mRNA binding protein 3-mediated regulation of CD164-CXCR4 axis impacts aggressiveness of Ewing sarcoma.

Caterina Mancarella,1 Maria Cristina Manara,1 Arthur M. Mercurio,2 Katia Scotlandi1. 1 _IRCCS Istituto Ortopedico Rizzoli, Bologna, Italy;_ 2 _University of Massachusetts Medical School, Worcester, MA_.

INTRODUCTION: The genomic stability of primary and metastatic Ewing sarcoma (EWS), the second most common primary bone tumor in pediatric age, evokes epigenetic or mRNA translational reprogramming as mechanisms involved in tumor progression. The oncofetal RNA-binding protein (RBP) insulin-like growth factor 2 (IGF-2) mRNA binding protein 3 (IGF2BP3) controls stability and translation of transcript targets and represents a major determinant of EWS prognosis and cell migration. Reported IGF2BP3 mRNA targets involved in cancer cell motility include CD164 (endolyn), a cell surface receptor for sialomucin which induces migration interacting with chemokine receptor CXCR4. Here we aim to dissect the role of IGF2BP3 as a critical regulator of CD164-CXCR4 axis in relation to EWS malignancy.

MATERIAL AND METHODS: Ribo-immunoprecipitation (RIP) assay and qRT-PCR were employed to test IGF2BP3-mRNAs interactions. CD164 and CXCR4 expression was assessed by qRT-PCR and western blot in stable IGF2BP3 knockdown EWS cellular models compared to controls. Soft-agar growth, migration and CXCR4 expression were investigated after treatment with anti-CD164 siRNA or scramble control. Cell growth and migration in hypoxic compared to normal conditions was assessed by Trypan Bleu and transwell migration assays after exposure of cells with differential expression of IGF2BP3 to hypoxia mimetic agent CoCl2 (100 µM). Samples of 79 paraffin-embedded EWS primary tumors were included in a tissue microarray (TMA) and processed for immunohistochemical evaluation of IGF2BP3, CD164 and CXCR4 to establish correlations (Chi-square test).

RESULTS: Within the panel of reported IGF2BP3 transcript targets involved in cancer invasiveness (CD44, MMP9, CD164), CD164 was the most abundantly enriched transcript after immunoprecipitation with anti-IGF2BP3 antibody in EWS cells. IGF2BP3 knockdown, beyond decreasing the invasive potential of EWS cells, down-regulated CD164 and, interestingly, its partner CXCR4, a major mediator of stress-response including hypoxia. Functional studies confirmed that CD164 silencing impaired soft-agar growth and migration of EWS cells and decreased the expression of CXCR4. Accordingly, presence of IGF2BP3/CD164/CXCR4 axis favored cell growth and migration in hypoxic conditions. In EWS patients, a direct correlation between IGF2BP3 and CD164 (p-value minor than 0.001) or CXCR4 (p-value equal to 0.035) was found, confirming the soundness of the in vitro results at clinical setting.

CONCLUSIONS: This study describes CD164 as a novel mediator of EWS aggressiveness and it highlights a connection between IGF2BP3 and the CD164/CXCR4 axis that may further explain the oncogenic role of IGF2BP3 in this tumor. (Grants: AIRC IG2016_18451 and RF Ministry of Health PRWEB 7153 to KS)

### Epigenetic Regulation, Plasticity, and DNA Repair of Cancer Stem Cells

#3673

The BMI1/Sp1/IGF1R pathway mediates pemetrexed resistance in non-small cell lung cancer cells.

Wen-Ling Wang,1 Peng-Ju Chien,1 Wen-Wei Chang,1 Bing-Yen Wang2. 1 _Chung Shan Medical University, Taichung, Taiwan;_ 2 _Changhua Christian Hospital, Changhua, Taiwan_.

Background: Lung cancer is the leading cause of cancer death in Taiwan and worldwide and non-small cell lung cancers (NSCLCs) account for more than 85% of lung cancers. Pemetrexed is approved for several steps of nonsquamous NSCLC therapy but drug resistance often occurs in long-term treatment. Cancer stem cells (CSCs) are considered as the rare subpopulation of cancer cells with the features of tumor initiation, drug resistance, and metastatic capability. The signaling pathway and potential targets contribute to the maintenance of pemetrexed-resistant cancer stem cell remain unknown.

Methods: To investigate the mechanism of pemetrexed resistance in lung cancers, the pemetrexed-resistant A549 cells were established (A400). The sphere-formation assay, western blot analysis and real-time qPCR were employed to identify cancer stem cells and study their properties.

Result: We first discovered that the pemetrexed-resistant A549 cells (called A400) contained more CSC activity than the parental A549 cells including the increased number of tumorspheres and the upregulation of BMI1, c-Myc, Sox2 and p-IGF1Rtyr1165/1166 expression. The treatment of PTC-209, a BMI1 inhibitor, displayed a greater inhibitory effect in cell proliferation in A400 than A549 cells and it suppressed tumorsphere formation capability of A400 cells. In addition, PTC-209 treatment downregulated the expression of IGF1R, p-IGF1Rtyr1165/1166, and Sp1, a positive regulatory transcriptional factor in IGF1R expression, in A400 cells. We also found that Sp1 was increased in A400 when compared to parental A549 cells. With the treatment of Sp1 inhibitor in A400 cells, the expression of IGF1R was suppressed without changing BMI1 level. Finally, we examined the therapeutic effect of BMI1 inhibition in vivo and resulted found that intraperitoneal administration of PTC-209 enhanced the pemetrexed efficacy in NOD/SCID mice harboring A400 xenograft tumors.

Conclusion: Our data reveal that BMI1/Sp1/IGF1R pathway is upregulated in pemetrexed resistant NSCLCs and targeting BMI1 could be a therapeutic strategy in NSCLCs when pemetrexed resistance occurs.

#3674

Protein levels of Piwi-like 1 and -2 protein are prognostic factors for muscle invasive urothelial bladder cancer patients.

Markus Eckstein, Rudolf Jung, Katrin Weigelt, Danijel Sikic, Robert Stöhr, Carol Geppert, Abbas Agaimy, Verena Lieb, Arndt Hartmann, Bernd Wullich, Sven Wach, Helge W. Taubert. _Univ. of Erlangen, Erlangen, Germany_.

The Piwi-like proteins are essential for stem-cell maintenance and self-renewal in all multicellular organisms. We analyzed the expression of Piwi-like 1 and Piwi-like 2 by immunohistochemistry (IHC) in 95 muscle invasive bladder cancer (MIBC) samples using tissue microarray. Application of an immunoreactive score (IRS) revealed 37 and 45 patients who were Piwi-like 1 and -2 positive (IRS>2). IHC results were correlated with clinico-pathological and survival data. The expression of both proteins was positively correlated with each other, lymph node metastasis and expression of CK20 and GATA 3. A negative correlation for both proteins was detected for disease-specific survival (DSS), recurrence, Ki67/MIB1 proliferation index, and CK5 expression. Detection of Piwi-like 1 protein positivity was associated with poor DSS (P=0.019; log rank test, Kaplan-Meier analysis), and in multivariate Cox's analysis (adjusted to tumor stage and tumor grade), it was an independent prognostic factor for DSS (RR=2.16; P=0.011). Piwi-like 2 positivity was associated with DSS (P=0.008) and relapse-free survival (RFS; P=0.040), and in multivariate Cox's analysis, Piwi-like 2 positivity was an independent prognostic factor for DSS (RR=2.46; P=0.004) and RFS (RR=3.0; P=0.003). Most interestingly, in the basal type patient subgroup (CK5+/CK20−), Piwi-like 2 positivity was associated with poorer DSS, OS and RFS (P<0.001, P<0.001 and P=0.006; log rank test). In multivariate analysis, Piwi-like 2 positivity was an independent prognostic factor for DSS (RR=10.49; P=0.001), OS (RR=5.71; P=0.001) and RFS (RR=6.35; P=0.016). In summary, Piwi-like 1 and -2 positivity are associated with clinico-pathological factors and survival. Therefore, both Piwi-like proteins are suggested as biomarkers for MIBC patients.

#3675

Elucidating the role and function of APOBEC3 DNA deaminases in myeloproliferative neoplasms.

Jane Isquith,1 Qingfei Jiang,1 Raymond Diep,1 Jessica Pham,1 Frida Holm,2 Catriona Jamieson1. 1 _UCSD, San Diego, CA;_ 2 _Karolinska Institutet, Stockholm, Sweden_.

The prevalence of upregulated apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3 (APOBEC3) transcripts as well as a distinct mutational signature has been identified and presented extensively in numerous cancer types. Previously, this family of cytidine deaminases has been studied in innate immunity, as they are active in the conversion of cytosine to uracil to restrict retroviral replication. Recent studies employing whole exome sequencing of multiple cancer types have shown a significant increase in both APOBEC3 transcript level as well as an increase in mutations indicated as a result of APOBEC3 driven mutagenesis. However, the expression and role of these enzymes in cancer initiation and progression as well as the mechanisms by which the APOBEC3 enzymes elicit a response in the cancer microenvironment remains unknown, especially in hematopoietic malignancies. Here, we elucidate the APOBEC3 mutation phenotype in Myeloproliferative Neoplasms (MPNs) as well as the naïve cell populations of CD34\+ cord blood and normal aged samples. Through RNA sequencing we have found a cell type and context specific nature of these enzymes, notably the upregulation of APOBEC3G (A3G) in the Myelofibrosis (MF) stem and progenitor cell population as compared to normal aged counterparts. There is also significant differential expression of APOBEC3C (A3C), APOBEC3D (A3D), and APOBEC3F (A3F) in MF disease states compared to normal controls. By cloning the APOBEC3 enzymes into lentiviral expression vectors, we can now study the physiological effects of changes in APOBEC3 transcript level in relation to the known changes in expression seen in many cancers, focusing on the upregulation of A3G through lentiviral overexpression. Using these techniques, we will also elucidate the mutation signature of APOBEC3G in CD34\+ cord blood as compared to the known mutagenesis pattern derived from the editing signature of APOBEC3B. Upon APOBEC3G overexpression we can connect the prevalence and proposed activity of the enzymes to the physiological deregulation present in hematopoietic malignancies. We have found that A3G induces a proliferative burst in normal CD34\+ cord blood, which leads us to study the effects of APOBEC3 upregulation in regards to proliferation, differentiation and self-renewal in normal and malignant cells as well as their potential function in disease initiation and progression in MPNs.

#3676

Novel tumor suppressive role of Peptidylarginine deiminase IV involving cancer stem cell regulation in breast cancer models.

Humberto J. Ochoa, Nellie Moshkovich, Binwu Tang, Howard H. Yang, Maxwell P. Lee, Lalage M. Wakefield. _National Cancer Institute, Rockville, MD_.

Breast cancer accounts for >500,000 deaths annually worldwide. Despite therapeutic advances, treated patients often relapse with metastases. Emerging research indicates that relapse may be driven by a subpopulation of tumor cells termed cancer stem cells (CSCs), that are uniquely capable of self-renewal and have enhanced drug resistance. Understanding the properties of these CSCs is critical for development of more effective therapies. Peptidyl arginine deiminase 4 (PADI4) is an enzyme that catalyzes deimination of arginine to citrulline. Citrullination of histones modulates expression of key stem cell transcription factors in embryonic stem cells (Christophorou, et al., 2014, Nature 507:104), so we hypothesized that PADI4 could be involved in CSC regulation. We showed that PADI4 mRNA and protein are expressed at varying levels across a panel of breast cancer cell lines, and we knocked down PADI4 in two high expressing lines, MCF10Ca1h and MDAMB231-LM2. PADI4 knockdown did not alter tumor cell proliferation in vitro, but it enhanced migration and invasion and increased cell survival. Importantly, PADI4 knockdown increased clonogenicity and enhanced tumorsphere formation, suggesting that it specifically increases the CSC population. In vivo studies showed higher tumor initiation efficiency following PADI4 knockdown, and increased lung metastasis. PADI4 is predicted to have multiple isoforms with unknown biological activity that complicate PADI4 expression-based analysis in patient data sets. We circumvented this issue by pharmacologically inhibiting PADI4 in MCF10Ca1h to generate a transcriptomic signature reflecting PADI4 gene activity. This PADI4 gene activity signature was shown to correlate with lower tumor grade and better disease outcome in transcriptomic datasets from human ER+ breast cancers. In conclusion, our findings suggest that PADI4 functions as a tumor suppressor in breast cancer, in part through effects on the CSC.

#3677

Downregulation of FOXO3a by DNMT1 promotes breast cancer stem cell properties and tumorigenesis.

Zhimin He, Hao Liu, Ying Song, Guopei Zheng. _Affiliated Cancer Hospital & Institute of Guangzhou Medical University, Guangzhou, China_.

Breast cancer stem cells (BCSCs) are tumor initiating cells that can self-renew and are highly tumorigenic and chemoresistant. Recent evidence indicated that FOXO3a plays an important role in regulating CSCs properties. However, the biologic function and detailed molecular mechanism of FOXO3a in BCSCs remains largely elusive. Here, we demonstrated that FOXO3a was hypermethylated and downregulated in breast cancer cells and tissues. Overexpression of FOXO3a potently attenuated the BCSCs properties in vitro and inhibited tumorigenesis in vivo, whereas knockdown of FOXO3a increased these properties. Further mechanistic investigation revealed that FOXO3a was functionally linked to the inhibition of the FOXM1/SOX2 pathway. Moreover, we found that SOX2 directly transactivated DNMT1 gene expression and then feedback inhibited FOXO3a expression. Clinically, we observed a significant inverse correlation between FOXO3a and FOXM1 or SOX2 expression levels. Loss of FOXO3a expression also predicted poor prognosis in breast cancer. In addition, we demonstrated that inhibition of DNMT activity suppressed tumor growth via regulating of FOXO3a/FOXM1/SOX2 signaling in breast cancer. Our findings suggest an important role of DNMT1/FOXO3a/FOXM1/SOX2 pathway in regulating BCSCs properties, identifying potential therapeutic targets for breast cancer.

Keywords: breast cancer, cancer stem cell, FOXO3a, DNMT

#3678

Ovarian cancer stemness is driven by a novel L1/Stat3/Src axis.

Marco Giordano,1 Alessandra Villa,2 Giovanni Bertalot,3 Fabrizio Bianchi,4 Stefano Freddi,3 Ugo Cavallaro1. 1 _Unit of Gynecological Oncology Research - European Institute of Oncology, Milano, Italy;_ 2 _Philochem AG, Zurich, Switzerland;_ 3 _European Institute of Oncology, Milano, Italy;_ 4 _ISBReMIT - Institute for Stem-cell Biology, Regenerative Medicine and Innovative Therapies, San Giovanni rotondo, Italy_.

Introduction

Ovarian cancer (OC) is the most lethal gynaecological malignancy due to the lack of peculiar symptoms in its early phase (Bowtell, 2010). OC often relapses as a chemoresistant disease within 3 years after surgical debulking (Lengyel, 2010; Sun et al, 2007).

These pathological hallmarks raised the hypothesis that OC is a cancer-stem cell (CSC)-driven disease. Indeed, a "stem-like" chemoresistant subset of OC cells is more tumorigenic than the bulk tumor cell population and is able to form spheroids in vitro (Bapat et al, 2005; Pan et al 2012). This pointed to ovarian CSC (OCSC) as an attractive target for OC-eradicating therapies.

The cell adhesion molecule L1 has been implicated in OC progression (Zecchini et al, 2008). While a few reports implicated L1 in stemness, its role in OCSC has not been investigated. Based on these observations, we aimed to investigate the functional role of L1 in the physiopathology of OCSC.

Experimental procedures

OVCAR3 and Ov90, two OC cell lines with high and low endogenous L1 levels, respectively, were employed for loss- and gain-of-function studies. Sphere-forming efficiency (SFE) was determined as the fraction cells within the bulk population able to overcome anoikis and proliferate in suspension cultures, and used as an in vitro proxy for CSC frequency. Tumorigenesis in NSG mice was employed as in vivo assays for cancer stemness. BBI608 and SU6656 were used as STAT3 and SRC activity inhibitor, respectively.

Results

L1 was both required and sufficient for self-renewal in OC. Indeed, L1 silencing prevented tumor formation while its forced expression in OCSC-enriched sphere cultures promoted tumor initiation in vivo.

Mechanistically, L1 increased dramatically the expression and the activation of STAT3 in OCSC and promoted its nuclear localization. L1 per se was sufficient for OC sphere formation even under very stringent culture conditions in which, once again, STAT3 was activated in L1-expressing OCSC only. Moreover, STAT3 activity was required for L1-induced clonogenic advantage, which occurred in a JAK-independent manner. Alternatively, L1-induced SFE was mediated by SRC-dependent STAT3 activation. SRC inhibition, in turn, reduced FAK activation in L1-expressing OCSC. Thus, a novel L1/STAT3/SRC axis appears to sustain OCSC pathophysiology.

L1 is also an optimal target for OC eradication since it confers chemoresistance to OCSC while its targeting reduces OCSC frequency. On these premises, L1 targeting should be explored in combined treatments with chemotherapeutic agents.

Conclusion

L1 can be considered as a new player and potential target in the context of OCSC, and the L1/STAT3/SRC interplay emerges as a novel mechanism in OC initiation and progression. Therefore, this work might pave the way to novel therapeutic strategies for the eradication of such a devastating disease.

#3679

A novel mechanism for pancreatic cancer stem cell maintenance: PAF1C-independent role of PAF1.

Saswati Karmakar,1 Sanchita Rauth,1 Rama Krishna Nimmakayala,1 Srikanth Barkeer,1 Mohd W. Nasser,1 Satyanarayana Rachagani,1 Dario Ghersi,2 Moorthy P. Ponnusamy,1 Surinder K. Batra1. 1 _University of Nebraska Medical Center, Omaha, NE;_ 2 _University of Nebraska, Omaha, Omaha, NE_.

Background: Cancer stem cells (CSCs), a minor subset of cancer cells, mediate aggressiveness, metastasis, and drug resistance of many cancers including the deadly pancreatic cancer (PC). Molecular governance of pancreatic CSCs remains elusive, mandating the need for their better molecular characterization to improve management of PC. PD2 (Pancreatic Differentiation 2) or RNA Polymerase II-Associated Factor 1 (Paf1) is a core component of human PAF1 complex (PAF1C), which regulates transcription elongation and mRNA processing. Human PAF1C consists of 5 members: PAF1, LEO1, CDC73, CTR9 and SKI8. Recently, PAF1 has emerged as a novel pancreatic CSC marker that enhances tumorigenic and metastatic potential of PC. Paf1 also maintains the self-renewal of mouse embryonic stem cells and ovarian CSCs via its interaction with OCT3/4, a major regulator of pluripotency. However, the mechanistic role of PAF1 in CSC maintenance and CSC-mediated PC pathogenesis is poorly understood.

Hypothesis: We hypothesize that "PAF1 forms a sub-complex exclusive of PAF1C, wherein PAF1 functions as the master-regulator for maintaining pancreatic CSCs by regulating stem cell gene signature".

Experimental Design: PAF1 knockdown (KD) was used to identify its targets and role in tumorigenesis. PAF1 sub-complex in CSCs was identified using immunoprecipitation (IP) and mass spectrometry (MS). ChIP-Seq was performed to confirm binding of PAF1 on its target genes.

Results: Inducible KD of PAF1 led to a significant reduction in tumor burden from orthotopically implanted human PC cells in athymic nude mice, indicating its role in pancreatic tumorigenesis. CSCs from different PC cell lines demonstrated higher expression of PAF1 along with CSC markers and PAF1 KD caused a significant decrease in CSC and self-renewal markers analyzed through Western blotting and immunofluorescence. Reciprocal co-IP and MS revealed that PAF1 interacted with Phf5a, DDX3, and hnRNP-K in CSCs. PAF1 KD in CSCs showed significant down-regulation of tumorigenic and stemness maintenance genes by RNA-Seq and PCR array analysis. PAF1 along with its binding partners occupied Nanog promoter in pancreatic CSCs. Further, KD of PAF1 in CSCs did not affect the expression of other PAF1C components, whereas individual KD of single PAF1C components decreased other remaining PAF1C members including PAF1. Moreover, other PAF1C components did not interact with Phf5a, a PHD-fold harboring nuclear protein in CSCs, suggesting that PAF1 forms a sub-complex with Phf5a, independent of PAF1C that functions in CSC maintenance. Depletion of Paf1 from mouse pancreas since birth using a CRISPR/Cas9-based conditional Paf1 knockout mouse model severely affected exocrine pancreas development with peri-ductal sclerosis and inflammation.

Conclusion: Altogether, PAF1 functions as the master-regulator for CSC maintenance by forming a sub-complex, which regulates CSC-network genes in PC.

#3680

SRSF2-dependent MBD2v2 expression is induced by obesity and promotes tumor-initiating triple negative breast cancer stem cells.

Emily A. Teslow,1 Cristina Mitrea,2 Bin Bao,1 Ramzi M. Mohammad,1 Lisa A. Polin,1 Gregory Dyson,1 Kristen S. Purrington,1 Aliccia Bollig-Fischer1. 1 _Karmanos Cancer Institute at Wayne State University SOM, Detroit, MI;_ 2 _Wayne State University, Detroit, MI_.

Obesity is a risk factor for triple negative breast cancer (TNBC) incidence and poor outcomes, the underlying molecular biology of which remains unknown. We previously identified in TNBC cell cultures that expression of epigenetic reader methyl-CpG-binding domain protein 2 (MBD2), specifically the alternative mRNA splicing variant MBD2v2, is dependent on reactive oxygen species (ROS) and is crucial for maintenance and expansion of cancer stem cell-like cells (CSCs). The relevance of CSCs is that they are a subpopulation of cancer cells recognized as the source of malignant tumor initiation, and give rise to drug resistance and metastatic recurrence. Because obesity is coupled with inflammation and ROS, we hypothesized that obesity could fuel an increase in MBD2v2 expression to promote the tumor-initiating CSC phenotype in TNBC cells in vivo. In this study we sought to characterize the role of obesity in regulating MBD2v2 expression in TNBC tumors, and better understand the mechanism regulating MBD2v2 expression in TNBC cells. Analysis of TNBC patient datasets revealed associations between high tumor MBD2v2 expression and high relapse rates and body mass index (BMI). Stable gene knockdown/overexpression methods were applied to TNBC cell lines to elucidate that MBD2v2 expression is governed by ROS-dependent expression of the serine and arginine-rich splicing factor 2 (SRSF2). Analysis of TNBC patient datasets also revealed an association between high tumor SRSF2 expression and high relapse rates and BMI. We employed a diet-induced obesity (DIO) mouse model to investigate if obesity influenced MBD2v2 expression and increased tumor initiation capacity of inoculated TNBC cell lines. MBD2v2 and SRSF2 levels were increased in TNBC cell line-derived tumors, which formed more frequently in DIO mice, relative to tumors in lean control mice. Stable MBD2v2 overexpression increased the CSC fraction in culture and increased TNBC cell line tumor initiation capacity in vivo. SRSF2 knockdown resulted in decreased MBD2v2 expression, decreased CSCs in TNBC cell cultures and hindered tumor formation DIO mice. The data provide concurring evidence that SRSF2-regulated MBD2v2 expression is induced by obesity and drives TNBC cell tumorigenicity, and thus provides molecular insights in support of the epidemiological evidence that obesity is a risk factor for TNBC. The majority of TNBC patients are obese and rising obesity rates threaten to further increase the burden of obesity-linked cancers, which reinforces the relevance of this study.

#3681

RAD51AP1 is a novel therapeutic target for cancer prevention and treatment.

Allison E. Bridges, Pragya Rajpurohit, Parth Patel, Nagendra Singh, Putter D. Prasad, Muthusamy Thangaraju. _Augusta University, Augusta, GA_.

Although much progress has been made in recent years in its treatment and prevention, cancer is still the second leading cause of death in the United States. Surgical removal of the tumor is not possible in all cancer types; therefore, chemotherapy and radiation therapy have become the standard course of treatment and are often the only option for more late stage and metastatic tumors. Unfortunately, chemotherapy and radiation therapy resistance are the greatest challenge for physicians trying to eradicate disease, prevent tumor recurrence, and inhibit distant metastasis. This resistance is derived from a heterogeneous population of cells within the tumor known as cancer stem cells (CSCs). CSCs are able to maintain a higher capacity for self-renewal due to a more efficient DNA repair system. CSCs are actively cycling, therefore, the repair mechanism of homologous recombination (HR) plays a significant role in repairing double-stranded DNA breaks that inevitably accumulate. Several studies have examined the link between efficient DNA repair and CSC self-renewal and found that proteins involved in HR repair are elevated in many human cancers. RAD51-associated protein 1 (RAD51AP1), which is responsible for the successful resolution of HR during DNA repair, is overexpressed in wide variety of human cancers. The present study sought to determine the functional role of RAD51AP1 in CSC self-renewal and its relevance to cancer growth and progression and also drug resistance. Our studies provide evidence that RAD51AP1 plays a critical role in CSC self-renewal and maintenance in breast, lung, and colon cancers. To determine the functional role of RAD51AP1 in cancer growth and progression, we generated genetically engineered mouse (GEM) models in breast and lung in WT (Rad51ap1+/+) and Rad51ap1-knockout (KO, Rad51ap1-/-) background and found that Rad51ap1 deletion significantly delayed the time of tumor formation and distant metastasis, which in turn increase the overall survival. Rad51ap1 inactivation in human breast and lung cancer cell lines, significantly reduced tumor growth in xenograft mouse model. This findings are also recapitulated in mouse mammary tumor cell lines (AT3 and 4T1). We also investigated the functional role of RAD51AP1 on colon cancer growth and progression using AOM/DSS and Apcmin/+ models of colon cancer and found that smaller tumor burden in KO mice compared to WT suggesting that Rad51ap1 may have a significant role in tumor growth. Taken together, these data demonstrate that RAD51AP1 plays a critical role in CSC growth and self-renewal in many human cancers and RAD51AP1 could be a novel therapeutic target for cancer prevention and treatment.

#3682

Therapeutic targeting of stem cell self-renewal in childhood medulloblastoma: Strategies for blocking recurrence.

David Bakhshinyan, Michelle Kameda-Smith, Branavan Manoranjan, Ashley Adile, Chitra Venugopal, Sheila Kumari Singh. _McMaster Univ. Medical Ctr., Hamilton, Ontario, Canada_.

Introduction: Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Group 3 MB patients face the highest incidence of metastasis and poor overall patient survival. The early onset and highly aggressive nature of MB suggest a stem cell origin, where a highly self-renewing transformed cell of the postnatal cerebellum drives MB tumorigenesis. In this work, we explore how WNT signaling and other essential drivers of self-renewal, BMI1 and MSI1, promote MB progression. We subsequently generate new strategies to therapeutically target mechanisms of MB stem cell self-renewal that drive treatment resistance and relapse in Group 3 MB.

Experimental procedures: We apply stem cell assays, patient-derived human-mouse xenograft (PDX) models, and genomic and bioinformatic profiling of recurrent patient-derived MB. Our established brain tumor initiating cell (BTIC) model provides an excellent tool for the examination of developmental pathways implicated in MB.

New Unpublished Data: A small molecule Bmi1 inhibitor, PTC-028, induced a remarkable decrease in self-renewal as well as reduction of local and spinal metastatic disease in recurrent MB, which is striking as no prior drug has shown efficacy against recurrent Group 3 MB. Although mouse and human neural stem cells (NSCs) express Bmi1 and are mildly sensitive to Bmi1 inhibitors, no significant toxicity was observed in either mouse or human NSCs upon PTC-028 treatment, at doses that induced efficacious killing of MB cells. Another novel therapeutic paradigm includes activating Wnt signaling in otherwise non-Wnt MB, which abrogates self-renewal and tumorigenicity of these highly aggressive tumors. For safe and non-toxic activation of Wnt in preclinical models, we identified L807mts, a novel inhibitor that functions through a substrate-to-inhibitor conversion mechanism within the catalytic site of GSK. A final therapeutic strategy to target self-renewal lies in the discovery of the targetable MB-specific interactome of the RNA binding protein (RBP) Musashi1, another key regulator of stem cell self-renewal. Msi1 is overexpressed in Group 3 MB compared to normal cerebellum, and is associated with poor patient prognosis. shRNA knockdown of Msi1 decreased the self-renewal capacity of MB stem cells and significantly decreased tumor burden and increased survival in our PDX model. Finally, comparative eCLIP (enhanced cross-linking and immunoprecipitation) of MB stem cells and normal NSCs, combined with mass spectrometry and RNA-sequencing of shMsi1 MB cells, has elucidated novel therapeutic targets in the RBP interactome of Msi1

Conclusion: Characterization and therapeutic targeting of self-renewal mechanisms unique to MB BTICs may provide an opportunity to limit treatment-resistant stem cell populations from driving patient relapse in recurrent Group 3 MB, a disease currently lacking any targeted therapies.

#3683

IL32 **expression is epigenetically regulated in EpCAM-/Cd49f- basal-like breast cancers and can be suppressed by the bromodomain inhibitor JQ1.**

Emma V. Gray,1 Caroline E. Dyar,1 Maria Ouzounova,2 Max S. Wicha,3 Hasan Korkaya,4 Austin Y. Shull1. 1 _Presbyterian College, Clinton, SC;_ 2 _Cancer Research Centre of Lyon, Lyon, France;_ 3 _University of Michigan Rogel Cancer Center, Ann Arbor, MI;_ 4 _Georgia Cancer Center, Augusta, GA_.

Metastatic basal-like breast cancers are believed to correspond with EpCAM-/Cd49f- cancer stem cell (CSC) enrichment. As well, basal-like breast cancers typically correspond with tumor inflammation and immunoediting phenotypes. However, the exact interplay between CSCs and the inflammatory signature of basal-like breast cancers is not well understood. To provide insight regarding the clinical overlap between breast cancer stem cells and tumor inflammation, we compared the 450K DNA methylation profile of EpCAM-/CD49f- CSCs from the isogenic MCF10A p53-/PTEN- breast cell line against the corresponding EpCAM+/CD49f+ and EpCAM-/CD49f+ subpopulations to determine whether differential DNA methylation occurred within the promoters of immune-related genes in CSCs. In addition, we also overlapped the 450K DNA methylation profile from 16 established breast cancer cell lines of varying EpCAM-/CD49f- concentrations to compare against the isolated CSCs. Based on our results, we identified 1432 differentially methylated promoter regions overall (ANOVA FDR p-value <0.001) and found IL32 to be differentially hypomethylated in the EpCAM-/CD49f- enriched cell lines. This hypomethylation of IL32 corresponded with increased expression of the beta isoform of IL32. Results from the cell lines were mirrored in The Cancer Genome Atlas (TCGA) breast cancer datasets, which revealed decreased promoter DNA methylation and increased gene expression of IL32 in basal-like patients. Further analysis of TCGA data using Gene Set Enrichment Analysis (GSEA) revealed that transcripts that tightly correlate with IL32 expression were preferentially involved in NF-kappaB mediated inflammation, with specific examples including REL, CCL5, PIK3CD, and IDO1. Furthermore, publicly available H3K27Ac and BRD4 ChIPseq data revealed that the IL32 promoter in the basal-like breast cancer cell line SUM159PT contains a high presence of H3K27 acetylation and BRD4 recruitment, with the latter event being disrupted by JQ1 treatment. These results complemented qRT-PCR results showing the IL32-beta isoform being quickly suppressed by 1uM JQ1 in SUM159PT as well as chick chorioallanotoic membrane (CAM) xenograft assays demonstrating suppressed metastasis and neovascularization of SUM159PT treated with JQ1. Collectively, these findings highlight the potential impact of IL32 promoter hypomethylation in basal-like breast cancer stem cells and how the overall epigenetic signature may predispose CSCs towards an immunomodulatory phenotype.

#3684

**The TGFβ2-Snail1-miRNA** TGFβ2 **circuitry is critical for the development of aggressive functions in breast cancer.**

Zhimin He,1 Guopei Zheng,1 Zhijie Zhang,1 Hao Liu,1 Binhua Peter Zhou2. 1 _Affiliated Cancer Hospital & Institute of Guangzhou Medical University, Guangzhou, China; _2 _Department of Molecular and Cellular Biochemistry, Markey Cancer Center, College of Medicine, University of Kentucky, KY_.

There have been contradictory reports on the biological role of transforming growth factor-βs (TGFβs) in breast cancer, especially with regard to their ability to promote epithelial-mesenchymal transition. Here, we show that TGFβ2 is preferentially expressed in mesenchymal-like breast cancers and maintains the epithelial-mesenchymal transition phenotype, correlating with cancer stem cell -like characteristic, growth, metastasis and chemo-resistance and predicting worse clinical outcome. However, this is only true in ERα- breast cancer. In ERα+ luminal-type breast cancer, estrogen receptor interacts with p-Smads to block TGFβ signaling. Furthermore, we also identify a microRNAs (miRNAs) signature (miRNAsTGFβ2) that is weakened in TGFβ2-overexpressing breast cancer cells. We discover that TGFβ2-Snail1 recruits EZH2 and BMI1 to convert miRNAsTGFβ2 promoters from an active to repressive chromatin configuration and then repress miRNAsTGFβ2 transcription, forming a negative feedback loop. On the other hand, miRNAsTGFβ2 overexpression reverses the mesenchymal-like traits in agreement with inhibition of TGFβ2-Snail1 signaling in breast cancer cells. These findings clarify the roles of TGFβ2 in breast cancer and suggest novel therapeutic strategies based on the TGFβ2-Snail1-miRNATGFβ2 loop for a subset type of human breast cancers.

Keywords: TGFβ, EMT, miRNA, Snail1, feedback loop This study was supported by grants from the National Natural Science Foundation of China (No.81672616, 81872197, 81402196, 81272450 and 81772825); Guangdong Natural Science Funds for Distinguished Young Scholars (2016A030306003); Guangdong Special Support Program (2017TQ04R809).

#3685

HOTAIR regulation and functionality in ovarian cancer stem cells.

Weini Wang, Fang Fang, Ali Ozes, Kenneth P. Nephew. _Indiana University School of Medicine, Bloomington, IN_.

Ovarian cancer (OC) is the fifth leading cause of cancer-related death among American women. Persistence of OC stem cells (OCSCs) is believed to contribute to resistance to platinum-based chemotherapy and disease relapse. We have previously shown that epigenetic changes in OCSCs play a role in post-therapy OCSC persistence and demonstrated that is it possible to target OCSC using epigenetic therapies. HOXC transcript antisense RNA (HOTAIR) has been shown to be associated with chemoresistance and to be overexpressed in many types of cancers, including high-grade serous OC (HGSOC). HOTAIR interacts with Polycomb Repressive Complex 2 (PRC2) and due to its histone methyltransferases activity plays a key role in chromatin remodeling. Because HOTAIR is a known epigenetic regulator of differentiation and developmental genes in OC and other cancers, we hypothesize that HOTAIR is a key epigenetic regulator in OCSCs and therapeutically targeting HOTAIR in OCSCs will prevent tumor relapse. The goal of this study is to understand the role of HOTAIR in regulating OCSCs and further dissect the mechanism of action of HOTAIR in OCSCs. Aldehyde dehydrogenase (ALDH) activity and FACS was used to separate OCSCs from non-OCSCs in a panel of HGSOC cell lines (OVCAR3, CAOV3, OVCAR5, Kuramochi, COV362). Quantitative RT-PCR analysis revealed that HOTAIR was overexpressed in OCSC cells compared to non-OCSC cells. Knockout of HOTAIR using CRISPR-Cas9 system significantly decreased OCSC population and stemness-related phenotypes, including spheroid formation and colony formation ability. Overexpression of HOTAIR in HGSOC cells significantly increased these stem-like characteristics. Furthermore, targeting HOTAIR using peptide nucleic acid (PNA), which blocks interaction with Enhancer of Zeste Homologue 2 (EZH2), significantly decreased the OCSC population, indicating HOTAIR functions through EZH2 in regulating OCSCs. Combining the HOTAIR-targeting PNA with other epigenetic inhibitors, including the hypomethylating agent guadecitabine and the EZH2 inhibitor GSK503, significantly decreased proliferation and colony formation ability of HGSOC cells. We suggest that a better understanding of HOTAIR will facilitate identifying epigenomic alterations and chromatin landscape that contributes to OCSC phenotypes. Targeting HOTAIR in combination with epigenetic therapies may represent a therapeutic strategy to prevent tumor relapse.

#3686

Regulatory role cancer stem cells (CSCs)-derived exosomal miRNAs in ovarian cancer growth and progression.

Fahriye Duzagac,1 Gabriel Lopez-Berestein,1 Pinar Kanlikilicer,1 Cristian Rodriguez-Aguoya,1 Roberto Cardenas-Zuniga,1 Ummu Guven,1 Gulperi Oktem,2 Bulent Ozpolat1. 1 _University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Ege Universitesi Medical Faculty, Izmir, Turkey_.

Ovarian cancer (OC) is the most lethal cancer in all of the gynecological cancers.Despite the recent advancements in the treatment of OC, it remains the seventh most common cancer in women worldwide. Cancer stem cells (CSCs) are a unique cell population described by their ability to indefinitely self-renew, proliferate, initiate, and propagate. Evidence suggest that the interaction between cancer stem cells with their microenvironment play a critical role for cancer progression. Exosomes which are the key players of microenviroment are nano-sized (50-180nm) endosomal pathway-derived vesicles that present in almost all biological fluids.Transporting information via exosomes and exosomal miRNAs is deemed to be the crucial way of intercellular communication that is as essential as the cell-to-cell contact-dependent signaling. As a key regulator of cellular signaling, both normal, and cancer stem cells secrete exosomes to orchestrate diverse autocrine and paracrine functions which alter tumor micro‐environment, growth, progression and putative immunological function. There is still not much known about how ovarian cancer stem cells (OCSCs) interact with their tumor microenvironment to promote oncogenic phenotype. Thus, in this study we investigate whether the OCSCs-derived exosomal miRNAs mirror and orchestrate that of the tumor microenvironment and thus could be used diagnostically in ovarian cancer patients.To this end,we evaluated significantly differentially expressed miRNAs between OCSCs exosomes and their cells of origin using the Affymetrix Gene Chip miRNA 4.0 microarrays. Several miRNAs were found to be significantly upregulated in both OCSCs and their exosomes. In addition we found many common up/down regulated miRNAs (miR-103a-3p, miR-20a-5p, miR-125b-5p, miR-17-5p, miR-27a-3p, miR-8075, miR-4487) with the microarray data in which tumor-associated macrophage (TAM) exosomes and their cells of origin were compared. We also found that OCSCs secrete exosomes enriched in miR-103a-3p, which has been shown to increases cancer migration, invasion and angiogenesis and induces polarization of M0 type macrophages toward immunosuppressive M2-type macrophages. In addition TAMs promote cancer stem cell-like properties by enhancing their epithelial–mesenchymal transition capacity. We are currently validating the identified miRs and investigating the role of these miRs in OC cells and CSCs proliferation, migration, invasion and tumor growth. Our findings also indicate that the interaction between TAMs and OCSCs are bidirectional. Overall, our data suggest that OCSCs and OCSCs secreted exosomes may contribute tumor microenvironment especially by regulating TAMs by transferring oncogenic miRs and using them as a progression and invasion tools within tumor microenvironment.

#3687

Colorectal cancer stem cell expressing POU5F1 promotes liver metastasis.

Norikatsu Miyoshi,1 Shiki Fujino,1 Masaru Sasaki,1 Kazuhiro Saso,1 Hidekazu Takahashi,1 Naotsugu Haraguchi,1 Taishi Hata,1 Chu Matsuda,1 Tsunekazu Mizushima,1 Masaki Mori,2 Yuichiro Doki1. 1 _Osaka University, Osaka, Japan;_ 2 _Kyusyu University, Fukuoka, Japan_.

Background: The gene POU5F1, encoding the POU domain, class 5, is a transcription factor and the expression is found in embryonic stem cells. POU5F1 gene controls self-renewal and differentiation, and regulates pluripotency and proliferation in embryonic stem cells. However, the role of POU5F1 in metastatic CRC (mCRC) is not well known. We focused on the relationship between POU5F1 gene expression and liver metastasis of CRC and clarify the role and abilities of POU5F1-positive CRC cells.

Methods: The study included 158 patients with CRC who underwent surgery from 2009 to 2011. The correlations between the POU5F1 gene expression and the clinical parameters were assessed, and liver-metastasis-free survival (LMFS) was evaluated in these patients. POU5F1-EGFP-positive cells were established by the construct with lentiviral vector, and we examine their sub-population and ability. The capacity to form liver metastasis in vivo was examined using CRC cell lines and primary cultured CRC cells.

Results: In the survival analysis, LMFS was significantly poor in the high POU5F1 expression group compared to the low-expression group (P=0.008). Multivariate analyses showed that POU5F1 expression (P=0.015) and TNM stage (p<0.001) significantly correlated with LMFS. POU5F1-EGFP-positive cells highly expressed stem-cell-associated markers and they had self-renewal and differentiation abilities. POU5F1-high cells actively formed liver metastasis.

Conclusion: The POU5F1expression correlated with liver metastasis in CRC patients. POU5F1-positive cells have the self-renewal and differentiation abilities as cancer stem cells. POU5F1 contributes to the ability forming liver metastasis in CRC.

#3688

iPSC-derived cancer organoids recapitulate genomic and phenotypic alterations of c-Met-mutated hereditary kidney cancer.

Jin Wook Hwang,1 Christophe Desterke,1 Olivier Feraud,1 Stephane Richard,1 Sophie Ferlicot,2 Virginie Verkarre,3 Jean Jacques Patard,4 Julien Loisel-Duwattez,2 Adlen Foudi,1 Frank Griscelli,5 Annelise Bennaceur Griscelli,1 Ali G. Turhan1. 1 _Paris Sud University, Villejuif, France;_ 2 _Paris Sud University, Bicetre, France;_ 3 _Université Paris Descartes, Paris, France;_ 4 _Centre Hospitalier de Mont de Marsan, Mont de Marsan, France;_ 5 _Université Paris Descartes, Villejuif, France_.

Patient-specific, tumor-derived organoids represent a powerful novel technology to model cancer, but the requirement of collection of fresh tumor cells and low efficiency of organoid generation represents significant bottlenecks. The induced pluripotent stem cell (iPSC) technology offers the possibility to generate an unlimited and reproducible source of cancer organoids from patient-derived iPSC bearing an oncogenic mutation. We report here for the first time, the generation of patient-specific iPSC-derived kidney organoids in type 1 papillary renal cell carcinoma (PRCC) with hereditary c-met-mutation. To this end we first reprogrammed hematopoietic cells of a patient with type 1 PRCC using Oct4, Sox2, Klf4 and c-Myc. The iPSC cell line generated teratoma in NSG mice and expressed pluripotency markers Tra-1-60 and SSEA-4. As compared control iPSC lines, the c-Met mutated iPSC expressed high levels of phosphorylated c-Met protein. To determine the feasibility of generation of kidney organoids from the iPS cells we have used 3D culture in low attachment cultures giving rise to organoids expressing markers of kidney progenitors (PODXL, LTL) with presence of tight junctions and brush borders in tubular structures at transmission electron microscopy. Importantly, the c-met-mutated kidney organoids expressed PRCC markers in vitro with presence of cells expressing cytokeratin 7 and TFE3. When injected under the kidney capsule of NSG mice, c-met-mutated kidney organoids generated teratomas in which we have found structures with glomerular or tubular origin of such structures with the expression of Nephrin and Cytokeratin 7. Gene expression profiling of c-met-mutated iPSC-derived organoid structures showed striking molecular similarities with signatures found in a large cohort of PRCC patient samples and identified the expression of 11 common genes shared with c-Met-mutated iPSC-derive organoids and primary cancers. Among these, BHLHE40 and KDM4C, well-known factors involved in PRCC pathogenesis, were also expressed in tumors obtained in vivo after transplantation of c-met-mutated kidney organoids in immunodeficient mice. To determine the expression of these two markers discovered by the iPSC-derived organoid technology, we have analyzed tumor biopsies from 5 primary PRCC wit and without c-MET mutation. This analysis showed overexpression of the

BHLHE40 and KDM4C only in the c-met-mutated PRCC tumors, as predicted by c-met-mutated organoid transcriptome. These data represent therefore the first proof of concept of the generation of "renal carcinoma in a dish" model using c-met-mutated iPSC-derived organoids, opening new perspectives for discovery of novel disease markers and novel drugs for future precision medicine strategies.

#3689

Tumor-specific gain-of-function tGLI1 transcription factor is a novel mediator of breast cancer stem cells and a novel transcriptional activator of cancer stemness genes.

Sherona R. Sirkisoon,1 Richard L. Carpenter,2 Tadas Rimkus,1 Daniel Doheny,1 Dongqin Zhu,1 Noah R. Aguayo,1 Marlyn Anguelov,1 Austin Arrigo,1 Fei Xing,1 Michael Chan,1 Jimmy Ruiz,1 Linda J. Metheny-Barlow,1 Roy Strowd,1 Jiayuh Lin,3 Boris C. Pasche,1 Waldemar Debinski,1 Kounosuke Watabe,1 Hui-Wen Lo1. 1 _Wake Forest Univ. School of Medicine, Winston Salem, NC;_ 2 _Indiana University, Bloomington, IN;_ 3 _Univ. of Maryland School of Medicine, Baltimore, MD_.

Breast cancer is the second leading cause of cancer-related mortality in women; metastasis to distant organs results in 90% of deaths for these patients. Cancer stem cells (CSCs) are considered the drivers of metastasis. Despite our current knowledge of breast CSCs, there still remains a significant challenge in managing patients with the metastatic breast cancer, underscoring the need for identifying novel regulators of breast CSCs. The hedgehog pathway is an important mediator of stem cells; however, the effect of truncated glioma-associated oncogene homolog 1 (tGLI1), a nuclear effector of the hedgehog pathway and a gain-of-function GLI1 transcription factor, on breast CSCs has never been investigated. Herein, we investigated whether tGLI1 is implicated in breast CSCs by evaluating tGLI1 expression levels in cells grown as monolayer versus mammospheres, as a representation of the stem cell population, and found tGLI1 to be induced in mammosphere culture. Overexpression of tGLI1 promoted mammosphere-forming ability of breast cancer cells, as well as, increased the breast CSC population defined by CD44high/CD24low expression. Further, tGLI1 overexpression transformed normal mammary epithelial cells resulting in increased mammosphere formation and enhanced anchorage-independent growth of immortalized human mammary epithelial HMLE cells. Functional and biochemical assays further showed that tGLI1 promotes breast CSC self-renewal by transcriptional activation of stemness genes including a novel tGLI1 target gene, OCT4, a recently reported tGLI1 target gene (CD44), and known GLI1 target genes (Nanog and SOX2). Bioinformatic analysis of breast cancer patient datasets revealed that activated tGLI1 is associated with shortened time to develop metastasis to the lung, bone, and brain. Furthermore, tGLI1 activation is enriched in HER2-enriched and triple-negative breast cancers, the subtypes with the highest propensity to metastasize, compared to luminal subtypes. Gene Set Enrichment Analysis showed that high tGLI1 activation is enriched in breast cancer with high gene signatures of breast CSCs, radioresistance, and metastasis. We further validated these results by immunohistochemical staining of paired primary breast tumors with lymph node metastases and found that that expression of tGLI1, but not GLI1, was increased in lymph node metastases and that tGLI1 was expressed at higher levels (84-91%) of lymph node-positive metastatic HER2-enriched and triple-negative breast tumors. Lastly, tGLI1 knockdown resulted in decreased mammosphere formation of breast cancer cells and decreased expression of stemness genes, OCT4, CD44, and Nanog. Taken together, these findings establish a novel role that tGLI1 plays in mediating breast CSCs and implicate tGLI1 in facilitating breast cancer metastasis.

#3690

SOX9-driven lineage plasticity controls basal-like breast cancer progression.

Wenjun Guo, John Christin, Chunhui Wang, Jihong Cui, Maja Oktay. _Albert Einstein College of Medicine, Bronx, NY_.

Accumulating evidence suggests cell plasticity plays a key role in tumor heterogeneity and therapeutic resistance. Basal-like breast cancer (BLBC), an aggressive breast cancer subtype, is a prime example of the involvement of cell plasticity in tumor development. Although originated from estrogen receptor (ER)-negative luminal cells in the mammary gland, BLBC acquires substantial basal cell features and contain a collection of basal-like, luminal-like and bipotent cancer cells. However, what role luminal-to-basal reprogramming plays in BLBC initiation and progression is unclear. Neither is the mechanism driving such lineage plasticity.

We recently discovered that ER-negative luminal cells are maintained by lineage-restricted stem cells in addition to multipotent stem cells. The ER-negative luminal stem cells specifically express the transcription factor SOX9. In this current study, we demonstrated that SOX9 is required for the activity of ER-negative luminal stem cells using Sox9 conditional knockout mice. Interestingly, SOX9 is overexpressed in human BLBC and correlated with poor prognosis. Using C3(1)/Tag BLBC mouse models carrying a Sox9-GFP reporter, we found that Sox9 is greatly upregulated in in a subset of cells in DCIS lesions during BLBC development. These Sox9-high cells are enriched in K14+/K8+ multipotent cells, which shows multipotent differentiation capability in in vivo transplantation and in vitro organoid culture. Importantly, conditional knockout of SOX9 in C3(1)/Tag mice suppressed reprogramming of luminal cells to multipotent cells and blocked progression of DCIS to invasive carcinoma. Mechanistically, Sox9 is required for activation of canonical and non-canonical NF-kB pathways, which are both required for ER-negative stem cell activity. Together, our results suggest that hyper-activation of the ER-negative stem cell program induces lineage plasticity to promote DCIS progression to invasive BLBC.

#3691

XPC inhibits lung cancer cell dedifferentiation by suppressing Snail expression.

Shurui Cai, Dayong Wu, Ananya Banerjee, Lu Liu, Chunhua Han, Tiantian Cui, Qi-En Wang. _Ohio State Univ., Columbus, OH_.

Lung cancer remains a leading cause of cancer-related deaths in the United States with unfavorable prognosis mainly due to tumor relapse and metastasis, which are recently believed to be caused by a specific population of cancer cells within tumor termed "cancer stem cells (CSCs)". These cells share the common characteristic of self-renewal and differentiation as normal stem cell, but also show resistance to chemotherapy and radiation therapy. Thus, targeting CSC populations in lung tumors is critical to the prevention of tumor metastasis. Xeroderma pigmentosum group C (XPC) was first recognized as a DNA repair protein. As a DNA repair factor, XPC plays an important role in preventing carcinogenesis. However, it has also been reported that XPC insufficiency is associated with poor treatment outcomes for a variety of cancers, and low expression of XPC is correlated with poor prognosis of lung cancer patients, suggesting that XPC may suppress lung cancer progression. Here, we show that downregulation of XPC expanded lung CSCs characterized by CD133+, while overexpression of XPC limited this cell population, as well as reduced the tumorigenic potential of the lung cancer cell line. Furthermore, we found that XPC knockdown is able to promote the CD133--to-CD133+ cell conversion in A549 cells, indicating that XPC can inhibit lung cancer cell dedifferentiation. Mechanistic investigation demonstrated that XPC can suppress Snail expression by directly binding to the promoter region of the SNAI1 gene, leading to the enrichment of histone H3 trimethylation at serine 27 (H3K27me3) and loss of histone H3 acetylation at serine 27 (H3K27Ac) in this region. Given that Snail plays a critical role in the induction of epithelial-mesenchymal transition (EMT), which is a major mechanism for the acquisition of stem cell-like properties, we believe that XPC can limit the CSC population by inhibiting cancer cell dedifferentiation. In summary, we conclude that low expression of XPC in lung tumors can de-repress the expression of Snail, promote EMT, and increase the de novo production of CSCs, eventually facilitating lung tumor progression.

#3692

The effect of endothelin 1 on cancer stem cell development.

Akimasa Seno,1 Masaharu Seno2. 1 _Okayama University, Detroit, MI;_ 2 _Okayama University, Okayama, Japan_.

Although we have been demonstrating the induction of cancer stem cells from ESCs or human/mouse iPSCs reprogrammed from normal fibroblasts using cancer cell conditioned media, the exact inducing factor is still unknown. Previously, we compared the gene expression profiles among hiPS/ESC, cancer stem like cells induced from hiPSC with cancer cell conditioned media, cancer stem cells obtained from cancer tissue or cell culture, and cancer cells. In this study, the expression of endothelin 1 was found commonly upregulated in different cancer stem cell groups. To investigate the roles of endothelin 1 in the development of cancer stem cells, the significance of endothelin 1 signaling including endothelin 1 in the cancer cell conditioned media and the expression of endothelin 1 receptors in iPSCs as well as in the developed cancer stem cells were quantitatively and qualitatively evaluated.

#3693

GLIS1 can replace MYC to generate induced pluripotent stem cells from ovarian cancer cells.

S Bindhya,1 C Sidhanth,1 S Krishnapriya,1 R P. Nagare,1 M Garg,2 T S. Ganesan1. 1 _Cancer Institute (WIA), Chennai, India;_ 2 _Amity Institute of Molecular Medicine & Stem Cell Research (AIMMSCR), New Delhi, India_.

Ovarian cancer is the most common cause of mortality among gynecological cancers, despite advances in treatment. Recurrence is common and is due to development of drug resistance. One of the reasons for drug resistance is the persistence of cancer stem cells. To understand the role of CSCs, it is essential to capture and propagate cells continuously in culture. Reprogramming cancer cells to induced pluripotent stem cells (iPSCs) is an approach to achieve this. An ovarian cancer cell line, PEO4 (high grade serous adenocarcinoma), was initially reprogrammed into iPSCs using the classical four factors OCT4, SOX2, KLF4 and MYC (OSKM) using STEMCCA by lentivirus transduction. Embryonic stem cell (ESC)-like bodies appeared between 8 to 15 days post-transduction. Morphology of the colonies resembled that of ESC colonies with defined border and tightly-packed cells. Two individual clones were further characterized. The reprogrammed PEO4-OSKM-iPSCs expressed alkaline phosphatase and pluripotency markers, NANOG, OCT4, SSEA4, TRA-1-60 and TRA-1-81 by immunofluorescence. Further, reverse-transcriptase-polymerase chain reaction (RT-PCR) analysis showed expression of the pluripotency markers NANOG, SOX2, OCT4, TERT, NESTIN, DMNT, DPPA4 in PEO4-OSKM-iPSC which were absent in the parental PEO4 cells. PEO4-OSKM-iPSC cells could be differentiated in vitro with appropriate growth factors into ectodermal, mesodermal and endodermal lineages. MYC was replaced with GLIS1 in the lentiviral cassette and PEO4 cells were able to be transformed to iPSCs. The transfection efficiency was two fold better with OCT4-SOX2-KLF4-GLIS1 with larger colonies. Individual iPSC colonies expressed all pluripotency markers and were able to differentiate into all 3 lineages. Characterization of iPSC cells for expression of cell surface markers specific for serous adenocarcinoma, showed that CD133, EPHA1, CD44 and LGR5 were expressed. Cell viability assays demonstrated that IC50 of cisplatin in parental PEO4 cells (15uM) was less as compared to iPSC cells (32uM) (p<0.03) and similarly, IC50 of paclitaxel was less in parental PEO4 (17 uM) as compared to iPSC cells (27 uM) (p<0.02). These results demonstrate for the first time that an ovarian cancer cell line derived from a patient with high grade serous adenocarcinoma can be reprogrammed. Further, GLIS1 can successfully replace MYC as a transcription factor to generate induced pluripotent stem cells.

#3694

TonEBP promotes chemoresistance and recurrence of hepatocellular carcinoma via DNA repair of cancer stem cells.

Jun Ho Lee,1 Jae Hee Suh,2 Soo Youn Choi,1 Hyun Je Kang,1 Orlando D. Schärer,1 Neung Hwa Park,2 Hyug Moo Kwon1. 1 _Ulsan National Inst. of Science & Technology, Ulsan, Republic of Korea; _2 _Ulsan University Hospital, Ulsan, Republic of Korea_.

Hepatocellular carcinoma (HCC) is common cancer with a high rate of recurrence and mortality. In addition to diverse etiological agents and wide heterogeneity in individual tumors, inherent resistance to chemotherapeutic agents and a very high rate of recurrence are the major contributing factors to HCC-related death. Cancer stem cells (CSCs) are believed to play a major role in these pathological properties due to its highly efficient DNA damage responses. However, mechanistic understanding of liver CSCs is still lacking, unlike other cancers. Tonicity-responsive enhancer binding protein (TonEBP), also known as NFAT5, is a central component of the pro-inflammatory enhanceosome in which TonEBP bridges activated transcription factors to histone acetyltransferase p300 on gene promoters. Although inflammation is intimately associated with the pathogenesis of HCC, the role of TonEBP is unknown. We aimed to identify a function of TonEBP in HCC and liver CSCs. We previously reported that TonEBP promotes hepatocellular carcinogenesis and expression of TonEBP predicts recurrence, metastasis, and death of patients with HCC. Three common sites of TonEBP action in response to diverse etiological agents leading to tumorigenesis and tumor growth were found: cell injury and inflammation, induction by oxidative stress, and stimulation of the COX-2 promoter. The goal of the following study was to understand how TonEBP mediates recurrence and metastasis of HCC. In HCC cells, TonEBP was required for self-renewal and tumorigenic potential of liver CSCs. In addition, TonEBP knockdown reduced the maintenance of liver CSCs. TonEBP mediated DNA repair of the CSCs caused by cisplatin, UV, and mitomycin C - both inter- and intra-strand crosslinks. Chemoresistance by highly activated DNA repair is contributed by TonEBP. We found that TonEBP-mediated DNA repair was carried out by ERCC1/XPF dimer. TonEBP interacted with ERCC1/XPF dimer through rel-homology domain (RHD) and was required for DNA recruitment of the dimer. RHD is required for TonEBP-mediated self-renewal of liver CSCs. TonEBP-ERCC1/XPF complex activated ATM serine/threonine kinase in response to DNA damage leading to activation of NF-κB, STAT3, and expression of pro-inflammatory cytokines, which stimulate the self-renewal of CSCs. Of note, in a cohort of 296 patients with HCC, expression of ERCC1-XPF predicted recurrence, metastasis, and death with high significance in multivariate analyses in TonEBP dependent manner. We conclude that TonEBP-ERCC1/XPF plays a pivotal role in DNA repair-associated chemoresistance and recurrence via liver CSCs. As such, the TonEBP-ERCC1/XPF complex is an attractive target for the prevention of recurrence and effective chemotherapy in HCC. 

### Novel Model Organisms and Approaches

#3695

A protein tyrosine phosphatase 4A3 (PRL-3)/Wnt signaling axis as a novel therapeutic target in acute lymphoblastic leukemia (ALL) relapse.

Meghan G. Haney, Kristin O'Leary, Jessica S. Blackburn. _University of Kentucky, Lexington, KY_.

Acute Lymphoblastic Leukemia (ALL) is the most common pediatric malignancy and 15-20% of patients experience disease relapse, which is frequently more aggressive and treatment resistant than the primary disease, and often has unfavorable outcome. Relapse occurs because conventional chemotherapies are unable to reliably and completely eliminate leukemia stem cells (LSCs), which are the only cells within the leukemia with the ability to self-renew and remake or replenish a leukemia from a single cell. Eliminating LSCs can greatly improve patient outcome; for example, differentiation therapy, which blocks LSC self-renewal, has improved the survival of Acute Pro-myelocytic Leukemia to >95%.

We have previously completed a >8,000 animal screen in a zebrafish Myc-induced ALL model and identified a panel of zebrafish ALL, in which the leukemia stem cell frequency is ~1 in every 10 cells, and identified a unique leukemia stem cell signature. The Wnt pathway was highly expressed by LSCs, and has also emerged in mouse studies as having an important role in LSC self-renewal in T-ALL. Current Wnt inhibitors have unacceptably toxicity in the clinic. I have found that Protein Tyrosine Phosphatase 4A3 (PTP4A3 or PRL3) is highly expressed by ALL cells that also express Wnt pathways genes, and is not expressed by normal cell types. In a zebrafish Myc-induce ALL model, PRL3 expression significantly enhanced leukemia stem cell frequency, while inhibition of PRL3 reduced LSC numbers in vivo. In human cells, I found that PRL3 regulates the expression of downstream Wnt pathway target genes, and we are currently testing the effects of small molecule inhibition of PRL3 on the phosphorylation status of proteins involved in Wnt signaling, to define the mechanism of PRL3 action in this pathway.

Apart from identifying novel interacting partners in the Wnt pathway, I have also developed a novel zebrafish reporter line that can be used to identify other small molecules that block Wnt signaling. In these fish, Wnt expressing leukemia stem cells are fluorescently tagged. These leukemias will be used for in vivo drug screens to identify FDA-approved compounds that block Wnt signaling and/or target leukemia stem cells without adverse effects on developing zebrafish larvae.

In total, my research will define a novel role for the phosphatase PRL3 in self-renewal of leukemia stem cells via activation of Wnt signaling. These data will demonstrate that "untargetable" oncogenes like Wnt can be targeted by inhibiting easily druggable critical interacting proteins, such as phosphatases like PRL3. This work is also likely to have a positive translational impact by identifying FDA-approved drugs that block LSC self-renewal in ALL and other types of Wnt and/or PRL3 dependent cancers.

#3696

PHF6 **loss drives IL7R oncogene addiction in TLX1 driven T-ALL.**

Siebe Loontiens,1 Kaat Durinck,1 Suzanne Vanhauwaert,1 Lisa Depestel,1 Mariana L. Oliveira,1 Givani Dewyn,1 Charles De Bock,2 João T. Barata,3 David Langenau,4 Jan Cools,2 Tom Taghon,1 Pieter Van Vlierberghe,1 Frank Speleman1. 1 _Ghent University, Ghent, Belgium;_ 2 _KU Leuven, Leuven, Belgium;_ 3 _Universidade de Lisboa, Lisboa, Portugal;_ 4 _Harvard Medical School, Boston, MA_.

T-cell acute lymphoblastic leukemia (T-ALL) is a genetically heterogeneous disease. The PHF6 gene is frequently targeted by loss-of-function mutations or deletions, with the highest prevalence in TLX1 or TLX3 rearranged T-ALLs. To gain insights into the putative function of PHF6 as a tumor suppressor in the T-cell lineage, we investigated the effects of PHF6 knock down during normal and malignant thymocytes. Notably, we observed broad effects on the investigated transcriptomes suggesting an important role for PHF6 in gene regulation. Furthermore, IL7R was identified as a common transcriptional target that was significantly upregulated upon PHF6 knockdown in both normal and malignant T cells. IL7R encodes a cytokine receptor critically involved in normal thymic development and which also acts as a bona fide oncogene in subset of primary T-ALLs. Thus, loss of PHF6 might further boost oncogenic addiction of leukemic T-cell lymphoblast to IL7-induced JAK-STAT signaling.

To further explore the role of PHF6 inactivation in TLX1 driven leukemogenesis in vivo, we performed zebrafish modeling. For this, we generated a stable tg(rag2:TLX1, rag2:GFP) overexpressing as well as a phf6 knock out zebrafish line. These lines were crossed and offspring was monitored for T-ALL formation. Interestingly, three fish out of a cohort of 80 animals developed leukemia between 10 to 18 months of age. These leukemias originated from the thymus, spreaded throughout the whole body and were transplantable. Thus far, no leukemia was detected in PHF6 mutated or TLX1 overexpressing only zebrafish. Leukemic cells obtained from tumors that developed in the PHF6null TLX1rag2-TLX1/GFP animals were subjected to RNA-, ATAC- and H3K27ac ChIP-sequencing to assess the epigenetic status of the IL7R locus. In addition, exome-, and CNV-sequencing was performed to identify somatic lesions that cooperated loss of Phf6 during TLX1 driven T-cell transformation in zebrafish. Furthermore, additional injections of TLX1 in combination with an activating IL7R mutant into phf6 mutant zebrafish are currently ongoing to monitor additional effects on accelerated tumor formation. In conclusion, our data suggest that loss of PHF6 drives TLX1 mediated leukemogenesis, at least in part, by increasing surface IL7R expression. Therefore, we believe that increased addiction to oncogenic JAK-STAT signaling may render PHF6 mutant leukemic cells more sensitive to JAK inhibitors, a notion that we are currently investigating in our TLX1/PHF6 and TLX1/PHF6/IL7R zebrafish models.

#3697

Optimization of human cancer cell xenografts into zebrafish larvae for high-throughput drug screening.

Stephen R. Dockins, Jessica S. Blackburn, Meghan G. Haney, Bradley Wilson. _University of Kentucky, Lexington, KY_.

The use of zebrafish in cancer xenograft models has grown rapidly in recent years. This is primarily due to the fact that this model takes advantage of the ease of in vivo imaging and the high-throughput screening capabilities that zebrafish have to offer. However, researchers have yet to come to a consensus on standardized procedure to utilize zebrafish for xenograft of human cells. This study aims to optimize a zebrafish xenografting protocol for various human cancers with the intention of performing high-throughput drug screening.

We tested the survival of 3 different zebrafish strains when incubated at 28°C, 34°C and 37°C. There was no difference in survival between the AB, SAT, and Casper strains of zebrafish at any temperature. Human leukemia, breast, lung, colon, and brain cancer cell lines were fluorescently labelled with Vybrant DiI cell staining dye and injected at a concentration of 100, 250, 500, or 1000 cells per 2nL droplet volume into dechorionated 2-day-old zebrafish embryos. The injection sites used were the brain, caudal vein, duct of Cuvier (DC), eye, pericardium, perivitelline space (PVS), and yolk sac. As expected, injections became more difficult and time consuming when using higher cell counts as the injector needle clogged more often. Similarly, injection sites that required more precision like the caudal vein or eye took the longest to inject successfully and had lower engraftment rates than sites like the yolk or DC. We determined that using 500 cells per injection was optimal as engraftment level was comparable to 1,000 cell injections but had improved survival and reduced injection time. Optimal injection site varied between cell lines, but overall, injections into the duct of Cuvier resulted in consistently high survival and engraftment rates with one of the lowest injection times.

We are in the process of determining the extent to which the injection site affects chemotherapy response on human cells implanted into zebrafish. We are also preforming a high-throughput drug screen on human leukemia cells implanted into zebrafish to provide proof-of-principle that these methods are useful in identifying novel anti-cancer compounds. In total, this work will establish standard operating procedures for use of xenograft in zebrafish, providing new opportunities in personalized medicine and drug discovery.

#3698

Conditionally reprogrammed cells from patient-derived xenograft to model neuroendocrine prostate cancer development.

Xinpei Ci,1 Jun Hao,1 Xin Dong,2 Hui Xue,2 Rebecca Wu,2 Anne M. Haegert,1 Colin C. Collins,1 Dong Lin,1 Yuzhuo Wang1. 1 _University of British Columbia, Vancouver, British Columbia, Canada;_ 2 _BC Cancer, Vancouver, British Columbia, Canada_.

Treatment-emergent neuroendocrine prostate cancer (t-NEPC) is a lethal subtype of advanced prostate cancer that occurs via NE transdifferentiation of prostate adenocarcinomas in response to androgen receptor (AR)-inhibition therapy. Study of t-NEPC has been hampered by a lack of clinically relevant models. We previously established a unique and first-in-field patient-derived xenograft (PDX) model of adenocarcinoma (LTL331)-to-NEPC (LTL331R) transdifferentiation. In this study, we established conditionally reprogrammed (CR) cells from the adenocarcinoma PDX tumor line LTL331. These LTL331-derived CR (LTL331-CR) cells retained the same genomic mutations of the parental tumor and, can be genetically manipulated and continuously propagated in vitro. Further androgen deprivation treatment on LTL331-CR cells showed no effect on cell proliferation. Transcriptomic analyses of the LTL331-CR cells revealed profound downregulation of androgen response pathway, and enrichment of stem/progenitor-like marker genes, compared with the parental tumor LTL331. Notably, when grafted back into the subrenal capsule of male NOD/SCID mice, these LTL331-CR cells gave rise to NEPC tumors directly as manifested by histological expression of NE markers. Transcriptomic analyses of the newly developed NEPC tumors also demonstrated marked enrichment of NEPC signature genes and loss of AR signaling genes. This study provides a novel strategy to investigate the mechanisms underlying t-NEPC development with a unique PDX by enabling gene manipulation ex vivo and subsequent functional evaluation in vivo.

#3699

Tumor budding: A predictor for metastasis in the CAM xenograft model.

Julienne K. Muenzner,1 Raphela A. Ranjan,1 Markus Eckstein,1 Ramona Erber,1 Philipp Kunze,1 Carol I. Geppert,1 Matthias Ruebner,1 Tobias Baeuerle,2 Arndt Hartmann,1 Regine Schneider-Stock1. 1 _University Hospital Erlangen, Friedrich-Alexander University Erlangen-Nuremberg, Erlangen, Germany;_ 2 _University Hospital Erlangen-Nuremberg, Erlangen, Germany_.

Background: Nowadays, breast cancer represents both the most frequently diagnosed type of cancer as well as the leading cause of cancer associated death in women. Clinically, breast cancer tumors can be stratified into four major subgroups based on the expression of estrogen receptors (ER) and progesterone receptors (PR), the proliferation marker Ki67 and the expression/amplification of HER2/neu. Hormone receptor (HR) positive tumors generally have a slightly more favorable prognosis than the other two subgroups and can be treated with agents interfering with hormone signaling. A targeted therapy approach is also applied for HER2/neu amplified tumors by using agents that inhibit HER2/neu. In contrast, patients with triple negative breast cancers (TNBCs), i.e. tumors lacking HR expression as well as HER2/neu amplification, have a fairly poor outcome due to the high aggressiveness of this subgroup, especially if not responding to neoadjuvant therapy.

Methods: In our study, we investigated phenotypical and functional characteristics of the two breast cancer cell lines MCF-7 and MDA-MB-231 that resemble the HR+ and the TNBC subgroup, respectively. In vitro, we were specifically interested in the invasiveness of the cells, thus determining their aggressiveness in a 3D spheroid invasion assay. In addition, we applied the in vivo chorioallantoic membrane (CAM) xenograft assay to histologically analyze the tumor growth patterns and evaluate metastasis formation using a human-specific Alu-PCR method and an in vivo imaging system (IVIS).

Results: As anticipated, the highly aggressive TNBC cell line MDA-MB-231 generated CAM micro-tumors much larger than those derived of the HR+ MCF-7 cell line. Moreover, we were able to verify different characteristic histological features of TNBC and HR+ tumors in the CAM xenograft model. In this regard, the MDA-MB-231 cells showed the distinct growth pattern of a poorly differentiated adenocarcinoma and a highly infiltrative growth into the CAM with strong tumor budding (clusters of ≤5 cells) at the invasive front. The high tumor budding rate could be correlated to an extremely invasive potential of the TNBC cells in vitro as well as to a strong metastasis formation in the in vivo CAM assay.

Conclusion: Our findings suggest that the CAM xenograft assay represents a suitable model to mimic the clinical situation of breast cancer patients. For the first time, we could show that tumor budding is correlated with metastasis formation in this alternative xenograft system.

#3700

Investigating the therapeutic potential of parthenolide for hematopoietic neoplasms in dogs.

Lisa J. Schlein, Barbara Rose, Aubree Peterson, Douglas Thamm. _Colorado State University, Fort Collins, CO_.

The purpose of this research is to explore the therapeutic potential of parthenolide (PTL) to treat various hematopoietic neoplasms in dogs; additionally, some dog breeds are enriched for development of mast cell neoplasia and histiocytic sarcoma, providing translational study populations for rare and deadly human diseases. Growth inhibition assays were performed using a panel of canine mast cell, histiocytic sarcoma, lymphoma, and leukemia cell lines, with PTL alone or in combination with redox-perturbing standard-of-care therapeutics. Cell death was assessed using flow cytometry. Immunofluorescence and immunoblotting were used to assess NFκB localization and phosphorylation, respectively. All immortalized canine cell lines evaluated are sensitive to PTL therapy and undergo dose-dependent apoptosis following exposure to drug. PTL exposure leads to inhibition of NFκB, as evidenced by immunofluorescent nuclear exclusion and decreased p65 phosphorylation. Preliminary studies indicate that some standard-of-care therapeutics synergize with PTL. These initial studies show that PTL is a promising therapeutic for hematopoietic neoplasms in dogs. Murine modeling and investigation of redox perturbance are underway and will further investigate PTL's potential in the clinical setting.

#3701

Fn14 overexpression in RCAS/tv-a rat gliomas promotes tumor progression, increases invasion and decreases survival.

Nina Connolly,1 Heather Ames,1 Nhan L. Tran,2 Anthony Kim,1 Jeff Winkles,1 Graeme Woodworth1. 1 _Univ. of Maryland, Baltimore, Baltimore, MD;_ 2 _Mayo Clinic, Phoenix, AZ_.

Fibroblast growth factor-inducible 14 (Fn14) is the cell surface receptor for the tumor necrosis factor (TNF) family member TNF-like weak inducer of apoptosis (TWEAK). The Fn14 gene is not normally expressed in healthy tissues, but expression is significantly increased in many primary solid tumor types, including glioblastoma (GBM). High Fn14 expression in tumors is often associated with the stimulation of cancer cell invasion leading to diffuse, aggressive tumors and metastasis. To investigate the effects of increased Fn14 expression in brain tumors, two different tumor types were developed using the RCAS/tv-a transgenic rat model: a low-Fn14, "proneural-like" (PDGFA/shP53 derived) tumor and a high-Fn14 (PDGFA/shP53/Fn14-derived) tumor. Interestingly, rats with high-Fn14 brain tumors had a significantly shorter median survival compared to those with low-Fn14 with 42 days and 70 days median survival, respectively. Using MR proton spectroscopy, both tumor types exhibited the markers of a malignant glioma such as increases in choline to creatinine ratio (Cho/Cr) and decreases in neuronal activity (NAA); however, high-Fn14 tumors also displayed an increase in lactate during tumor progression which is often a sign of high grade, necrotic tumors and poor prognosis. Morphologically, the two tumor subtypes appear quite different. H&E staining of the high-Fn14 tumors showed a highly malignant tumor with several areas of pseudopalisading necrosis within the tumor core. This is different than the somewhat more homogeneous morphology of the low-Fn14 proneural-like tumor. The pattern of tumor cell invasion also differs between the two brain cancer subtypes. High-Fn14 tumors show a more human GBM-like pattern by invading along the white matter tracts whereas low-Fn14 tumors appear to invade more locally with minimal white matter invasion. High-Fn14 tumors were positive for Fn14 throughout the tumor including the invasive rim of the tumor, which is also seen in human GBM, whereas the low-Fn14 tumor revealed very little staining even at the invasive rim. These findings indicate that increased Fn14 expression creates a more malignant brain tumors that develops and behaves more aggressively then low-Fn14 tumors.

#3702

**Establishment and characterization of a novel** MYC/BCL2 **double-hit high-grade B-cell lymphoma cell line HLA-3.**

Hui Guo, Hui Zhang, Krystle Nomie, Liang Zhang, Kelley P. Murfin, Michael Wang. _University of Texas MD Anderson Cancer Center, Houston, TX_.

The majority of MYC/BCL2 double hit lymphoma (DHL) morphologically resembles diffuse large B-cell lymphoma (DLBCL) or unclassified B-cell lymphoma, with intermediate features of DLBCL and Burkitt's lymphoma. DHL also typically results in shorter survival time than other lymphomas. To further understand DHL's biology and identify potential effective agents against DHL, we established a unique MYC/BCL2 high-grade B-cell lymphoma cell line with morphological DLBCL features. The HLA-3 DHL cell line was derived from a MYC/BCL2 high-grade B-cell lymphoma patient-derived xenograft (PDX) model. We performed flow cytometry immunophenotypic analysis and established the HLA-3 cell line after 7 months of continuous culture. We utilized molecular and cellular techniques, including flow cytometry and polymerase chain reaction (PCR) analysis, to characterize the HLA-3 cell line. We performed reverse phase protein array (RPPA) analysis to determine its protein signaling profile. To identify agents with anti-DHL activity, we screened the effects of a panel of agents alone and in combination with ibrutinib on the cell viability of this newly established cell line. The HLA-3 cell line was optimally maintained at a density between 1 and 2 × 106 cells/mL and successfully proliferated in a single-cell suspension without cellular clump formation. The HLA-3 cells were EBV-negative and possessed a germinal center B cell immunophenotype. Moreover, the immunophenotypes of the primary patient tumor cells and HLA-3 cells were virtually identical. The HLA-3 cells were positive for CD19, CD45, CD35, CD10 and HLA-ABC, negative for CD3, CD5, CD40, CD34 and TdT, and were dim for CD22, CD56, CD8, CD4 and CD23. Representative flow cytometry histograms showed 99% of the HLA-3 cells were positive for CD19 and CD45, with no detectable surface kappa and lambda light chains. The HLA-3 cells expressed a high level of CD38, an indicator of MYC rearrangement. Similar to the original lymphoma cells, the HLA-3 cells showed dim/low CD20 expression. Based on RPPA data, we constructed a diagram showing activation of the ATM/Chk2 DNA repair and the IGF1R/PI3K/mTOR signaling pathways in the HLA-3 cells. In support of this upregulated activity, we found that 5/7 tested commercial PI3K/mTOR inhibitors significantly reduced the viability of the HLA-3 cells in a dose-dependent manner, with AZD8055 the most sensitive of the tested inhibitors against the cell line. Further, DNA repair pathway inhibitors BMN673 and RG7112 were effective against the HLA-3 cell line. The newly characterized high-grade B-cell lymphoma cell line will provide useful in vitro and in vivo models for biological studies related to human DHL, which is refractory to current therapies and urgently needs novel therapeutic approaches. This work suggests the PI3K/mTOR and DNA repair pathways may be therapeutic targets for MYC/BCL2 double-hit high-grade B-cell lymphoma.

#3703

Development of oncopig urothelial carcinoma cell lines.

Aisha Qazi,1 Faith M. Thomas,1 Sulalita Chaki,1 Noah Robertson,2 Shovik Patel,1 Kyle M. Schachtschneider,3 Lawrence B. Schook1. 1 _University of Illinois, Urbana-Champaign, Urbana, IL;_ 2 _Albion College, Albion, IL;_ 3 _University of Illinois, Chicago, Chicago, IL_.

Urothelial carcinoma (UC), more commonly known as bladder cancer, affects approximately 81,000 people a year in the United States and is the fourth most common type of cancer among men. Age also plays a tremendous factor, as 90% of people affected by bladder cancer are older than 55, and if the cancer has metastasized the 5-year survival rate is 35%. This requires the advancement of early detection and treatment methods for bladder cancer. The development of clinically relevant systems to serve as a bridge between preclinical murine studies and human clinical practice is of vital importance. The Oncopig Cancer Model (OCM) is a novel transgenic swine platform that recapitulates human cancer through development of site/cell specific tumors after Cre recombinase induced expression of heterozygous KRASG12D and TP53R167H transgene. In this study, we standardized a protocol for the development and growth of Oncopig UC cell lines that faithfully recapitulates in vitro and in vivo features of human bladder cancer.

The focus of this preliminary research was to successfully create OCM UC cell lines that mimic features of human UC. OCM urothelial cell lines (n=7) were isolated from whole bladders collected from male Oncopigs at euthanasia. OCM urothelial cell lines were exposed to Cre recombinase 48 hours post isolation to induce expression of KRASG12D and TP53R167H transgenes, resulting in development of OCM UC cell lines. Differences in migration rates were observed between OCM urothelial and UC cell lines, confirming cellular invasiveness. RT-PCR was performed after several passages to confirm KRASG12D and TP53R167H gene expression, which was observed in OCM UC but not urothelial cell lines. Uroplakin II and pan-Cytokeratin (PCK-26) are markers for UC and urothelial cells, respectively, and are used to diagnose UC clinically. OCM UC cells stained positively for Uroplakin II and PCK-26, confirming their identity as malignant UC cells. Future work is needed to confirm OCM and human UC display similar histological and molecular features, and to develop strategies for in vivo OCM UC tumor development.

#3704

**Crisper screening in a xenograft model for** in vivo **drug moa analysis.**

Michele Melton,1 Athena Bast,1 Alastair Leeson-Payne,2 Maximilian Blanck,2 Steffen Lawo,2 Deeds Stacy,1 Benedict C.S. Cross2. 1 _Horizon Discovery, Saint Louis, MO;_ 2 _Horizon Discovery, Waterbeach, United Kingdom_.

Functional genomic screening with CRISPR has provided a powerful and precise new way to interrogate the phenotypic consequences of gene manipulation in high-throughput, unbiased analyses. Rapid development of pooled lentivirus and deep-sequencing-led approaches have allowed us and others to exploit this technology in target ID, target validation, drug MOA analysis and patient stratification.Until now, these exquisitely powerful screening technologies have been deployed predominantly in simple in vitro analyses. Whilst these in vitro approaches allow for high quality hit discovery and at whole-genome level scale, they are not able to adequately capture the impact of tumour microenvironment and heterogeneity on the genetic perturbations under study. To address this, we present here the development and validation of a platform for pooled CRISPR knock-out screening in xenograft models of tumour growth.

A critical step in appropriate study design is the evaluation of the cell uptake following implantation. Since this directly contributes to the tumour growth and cell number, understanding cell survival dynamics post-implantation is necessary to determine the appropriate experimental scope. We used a barcoding approach coupled to next generation sequencing to determine the response and contribution of individual A375 melanoma cells to final tumour composition and used this to calibrate the experimental design for robust pooled-based CRISPR screening analysis.

Following the determination of experimental conditions, a proof-of-concept drug mechanism of action analysis was conducted using a custom designed cancer genome CRISPR knock-out library. A375 cells were first infected with the CRISPR library and selected ex vivo ahead of implantation and treatment of established tumour-bearing nude mice with a defined therapeutic regime of the BRAFV600E selective inhibitor, Vemurafenib. Tumours were harvested and the identification of genes which selectively modulate the tumour response to drug were identified by next generation sequencing using our optimised screening analysis platform.

Our results provide a robust experimental validation and define a novel platform which will allow the rapid and definitive in vivo evaluation of both drug and gene interactions using powerful CRISPR-based screening tools.

#3705

Identification of frequent HER2 activating mutations in canine primary pulmonary adenocarcinoma.

Gwendolen Lorch,1 Karthigayini Sivaprakasam,2 Victoria Zissman,2 Nieves Perdigones,2 Tania Contente-Cuomo,2 Alexandra Nazareno,2 Salvatore Facista,2 ShukMei Wong,2 Winnie Liang,2 Joseph M. Amann,3 Sara L. Sinicropi-Yao,3 Michael J. Koenig,3 Krista La Perle,3 Timothy G. Whitsett,4 Muhammed Murtaza,2 Jeffrey Trent,2 David P. Carbone,3 William P. Hendricks2. 1 _Ohio State Univ. College of Veterinary Med., Columbus, OH;_ 2 _Translational Genomics Research Institute, Phoenix, AZ;_ 3 _Ohio State University, Columbus, OH;_ 4 _Dignity Health, Phoenix, AZ_.

Spontaneous primary canine lung cancers are aggressive malignancies that are increasingly common in dogs. They share clinicopathologic features with human lung cancers in never-smokers, but their genetic underpinnings are unknown. As never-smoker lung cancer incidence increases in humans, a need exists for improved biologic understanding and development of new models to fuel translational research. Here, we describe for the first time the detailed clinical and genetic features of primary canine lung cancers through case review and multi-platform sequencing of 90 primary canine lung tumors and cell lines. We performed multi-platform genomic profiling including whole exome sequencing (WES), amplicon panel-based sequencing of 250 regions of 51 cancer genes, and single nucleotide polymorphism (SNP) array-based analysis of tumors and matched constitutional DNA. We profiled 76 cPAC, 11 canine pulmonary adenosquamous carcinomas (cPASC) and three canine pulmonary squamous cell carcinomas (cPSCC). The median age at the time of diagnosis was 11 year old. The most common affected pure breed was the Labrador retriever (19%). Five cPACs assessed by WES displayed low mutation burden with a median of 64 somatic single nucleotide variants (SNVs), 19 somatic copy number variants (CNVs), and 1 structural variant (SV), and frequent C>T substitutions (80%) at NpCpG trinucleotides, a common mutation signature seen across human and canine cancers. Based on WES and amplicon sequencing, we discovered somatic, coding HER2 (ERRB2) point mutations in 37% of cPAC, but none in cPASC or cPSCC. Additional recurrently mutated genes in cPAC included CDKN2A/B (~40%), TP53 (~12%), K/HRAS (~7%), SMAD4 (~5%), and PTEN (~5%). In cPASC, KRAS and TP53 were the most frequently mutated genes (18% each). In cPSCC, no recurrently mutated genes were identified, but individual somatic coding mutations were found in BRAF and PTPN11. The majority (93%) of HER2 mutations were hotspot V659E, located on the transmembrane domain (TMD), and comparable to activating mutations at this same site in human cancer. We were able to detect this mutation not only in biopsied tumors, but also in the plasma of 33% (2/6) of dogs with localized V659E-positive tumors. Other somatic HER2 mutations identified were similarly located in the HER2 juxtamembrane domain and TMD including A664T and K676E. HER2 point mutations occurred in the absence of significant focal amplification (assessed by WES and SNP array) or overexpression (assessed by qRT-PCR and IHC). We also showed that HER2 mutation correlated with constitutive phosphorylation of Akt in cPAC cell lines. Furthermore, HER2 V659E-mutant lines displayed hypersensitivity to the HER2 kinase inhibitors neratinib (IC50, 23nM) and lapatinib (IC50, 168nM) relative to HER2 wild-type cell lines (IC50, >2500nM). These data bear implications for comparative understanding of HER2-mutant lung cancer across species.

#3706

The CRISPR-Cas9 minipig: A transgenic toolbox pig to produce specific genome editing in designated tissues.

Martin F. Berthelsen,1 Maria Riedel,1 Mikkel H. Vendelbo,2 Aage K. Alstrup,2 Frederik Dagnæs-Hansen,2 Yonglun Luo,2 Simone S. Møller,2 Henrik Callesen,3 Jannik E. Jakobsen,1 Martin K. Thomsen1. 1 _Aarhus University, Aarhus N, Denmark;_ 2 _Aarhus University, Aarhus C, Denmark;_ 3 _Aarhus University, Tjele, Denmark_.

The aim of the project is to generate a toolbox pig model. This is being done because the pig probably is the best non-primate model in translational medicine. Unfortunately, pig models have proven difficult to produce. The two main complications have been the cloning of viable pigs and that just a few genes are altered, which often is not sufficient to induce the desired disease. Especially, models based on multiple genetic modifications of the pig genome, e.g. cancer, are challenging to generate. This was changed with the development of the CRISPR-Cas9 technology, which made it possible to perform multiple in vivo gene editing.

We have cloned an inducible Cas9 expressing minipig and have obtained healthy F1 offspring. Activation of the Cas9 expression is attained by transduction with adeno-associated virus that delivers both the recombinase and the guide RNA to the designated tissue. This design provides spatial and temporal control of the activation. The Cas9 expression is mediated by a ubiquitous promotor, which also co-expresses fluorescent proteins for expression analysis. Transgenic Cas9 expression is observed in all major organs with strong expression in the epithelia, muscles and hepatocytes. Successful in vitro gene editing has been conducted in primary keratinocytes and fibroblast obtained from the transgenic pigs, which confirms the genetic design.

By delivery of our construct by adeno-associated virus to the skin of the pigs, In vivo activation of the transgenic construct has been achieved. To validate the model, lung cancer has been induced in nine pigs by alteration a panel of cancer related genes including STK11, TP53, PTEN, and KRAS, all key drivers of human lung cancer. To obtain the constitutive active KRAS-G12D isoform of the KRAS oncogene a repair template was supplied. The pigs are being followed by PET/CT scanning in combination with 18F-FDG as a tracer. Preliminary data indicates abnormalities/cancer initiation in the pigs eight months after induction.

In conclusion, an inducible Cas9 expressing minipig allows rapid gene editing to study different scenarios of human cancer in a pig model. The development of the Cas9 pig will extend the variety of pig disease models, both to generate a model of lung cancer as proposed but also models for other cancers.

#3707

2D organoid (2DO) as human tumor model for predicting therapeutic effect of anti-cancer drugs.

Shiki Fujino,1 Norikatsu Miyoshi,1 Kazuhiro Saso,1 Masaru Sasaki,1 Masayoshi Yasui,2 Masayuki Ohue,2 HIdekazu Takahashi,1 Naotsugu Haraguchi,1 Chu Matsuda,1 Taishi Hata,1 Tsunekazu Mizushima,1 Masaki Mori,3 Yuichiro Doki1. 1 _Osaka Univ. Graduate School of Medicine, Osaka, Japan;_ 2 _Osaka International Cancer Institute, Osaka, Japan;_ 3 _Kyusyu Univ. Graduate School of Medicine, Kyusyu, Japan_.

Primary cultured cells derived from an individual patient's tumor have phenotypic heterogeneity unlike established cancer cell lines. We established organoid in two-dimensional (2D) culture with the reproducibility of clinical phenotypic heterogeneity as a human tumor model, named isolated tumor-derived cancer cells (iCCs). The success rate of iCC growth in vitro was 100%, passage was 90%, and establishment of a xenograft model from iCC was 80%. Established 2DO can proliferate well, and are easier to use in several assays compared with reported organoid in 3D culture. However, established 2DO also can be easily cultured in a 3D culture system. iCC populations were analyzed by flow cytometry. Most iCCs expressed epithelial cell adhesion molecule (EpCAM), and the high- and low-expressing CD44 groups were identified. Seral markers were examined, and we reviled that TRA1-81-positive cells in the high-expressing CD44 group were necessary for replicating clinical tumor heterogeneity. Moreover, basic fibroblast growth factor (bFGF) was necessary to keep TRA1-81-positive cells. Next, we did anticancer drug assay using iCCs from 10 patients and predicted the clinical efficacy of drugs. Six patients received chemotherapy after their surgery, and four patients of them had distant metastases. The in vitro sensitivity was compared to clinical outcomes (RECIST criteria) in four patients that all had distant metastases. The concentration of anti-cancer drugs was set as estimated concentration in human tissue. In the examination using 5-FU and oxaliplatin, the survival rates of iCCs were more than 85% in one progressive disease (PD) patient. The survival rates of iCCs were less than 83% in three stable disease (SD) patients (median, 70%). The survival rates were also less than 83% in two patients who underwent adjuvant chemotherapy with no recurrence (median 70%). After 3 months chemotherapy, PD patient's cell survival rates were more than 85%, and SD patient's cell survival rates were less than 83%. After 6 months chemotherapy, PD patient's cell survival rates were more than 81%, and SD patient's cell survival rates were less than 75%. Therefore, the cut-off value was considered to be 83-85% for 3 months to keep SD, and 75-80% for 6 months. However, more examination is need to develop a prediction model we can use as a clinical application. In conclusion, our primary culture model may be a novel tool as a human tumor model with tumor heterogeneity, and it will be used to predict therapeutic effect of anti-cancer drugs in the clinical field.

#3708

Multiomic validation of canine simple carcinoma as a model for human basal-like breast cancer.

Joshua L. Watson, Yuan Feng, Shaying Zhao. _University of Georgia, Athens, GA_.

Roughly 20% of all breast cancer (BC) cases in humans are classified as basal-like breast cancer (BLBC), the deadliest subtype with a five-year survival prognosis lower than any other subtype. BLBC usually lacks the clinically targetable receptors estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). One of the main challenges in BLBC research is the lack of a spontaneous cancer model. To address this deficiency, we performed a comprehensive dog-human multiomic comparison. Briefly, we characterized the genomic and transcriptomic alterations of >100 spontaneous canine mammary cancer cases, including simple carcinoma (SC), a common subtype in pet dogs, from our own data and published studies by others. We then evaluated the dog-human molecular homology by comparing our canine findings to the studies of BLBCs from The Cancer Genome Atlas (TCGA).

Our study shows a strong dog-human molecular homology. First, cross-species PAM50 classification indicates significant grouping of canine SC with human BLBC, but not other BC subtypes. This is supported by unsupervised clustering of a panel of recently published ~1800 cancer genes that characterize human BLBC. Second, gene set enrichment analysis with 44 BC molecular signatures, 20 of which are BLBC-specific, indicates that canine SCs have the same enrichment patterns as human BLBCs. Third, canine SCs show the same overall copy number alteration (CNA) patterns as human BLBCs. Specifically, canine SCs have significantly more amplified/deleted genes and a much greater percentage of the genome with CNA than other mammary cancer subtypes of the dog, which is also true for human BLBC. Furthermore, canine SC shares significantly more amplified/deleted genes with BLBC than any other BC subtypes. Importantly, these shared genes are strongly enriched in known BLBC signatures, including the 8q23-24 amplicon (containing the oncogene MYC) and chromosomal instability regulation. Finally, as with the PAM50 analysis, unsupervised cross-species clustering with amplified/deleted genomic regions and genes indicates a strong homology between canine SCs and human BLBCs.

In conclusion, canine SC faithfully recapitulates the molecular features of human BLBC, and can effectively serve as a much-needed spontaneous cancer model for BLBC research. Canine SC will accelerate basic research, e.g., providing a novel dog-human comparison strategy for cancer driver-passenger discrimination. Canine SC will also accelerate the bench-to-bedside translation by bridging the gap between preclinical studies in mice and human clinical trials.

#3709

The interplay between Aldehyde dehydrogenase 1 and TGF-β in therapeutic resistant glioblastoma.

Chan-Chuan Liu,1 Meng-Xuan Lin,2 I-Chun Yeh,3 Chun-I Sze1. 1 _The Institution of Basic Medical Science, Department of Cell Biology and Anatomy, College of Medicine, National Cheng Kung University, Tainan City, Taiwan;_ 2 _Department of Cell Biology and Anatomy, College of Medicine, National Cheng Kung University, Tainan City, Taiwan;_ 3 _Department of Radiation Oncology, Kuo General Hospital, Tainan City, Taiwan., Tainan City, Taiwan_.

Glioblastoma (GBM) is the most common and lethal primary brain tumor. In spite of aggressive treatment, easily developing resistance results in GBM recurrence and poor prognosis. TGF-β is a well-known molecule which induces epithelial-to-mesenchymal transition and participates in cancer resistance. Aldehyde dehydrogenase 1 (ALDH1) is a new cancer stem cell marker. How ALDH1 contributes to GBM resistance remains poorly understood. Therefore, we hypothesized that the interplay between ALDH1 and TGF-β may play a role in GBM resistance. We mimicked clinical standard GBM treatment procedure, which utilizes consecutive radiation and temozolomide (TMZ), and selected the median effective dose of radiation and the TMZ dose of growth inhibition of 50% to develop three therapeutic-resistant models by using U87MG and 1306MG cell lines. It included resistance against radiation, TMZ, and radiation plus TMZ. To develop radiation resistance, U87MG required 2 Gy irradiation for four times while 1306MG required 3.5 Gy irradiation for six times. On the other hand, to establish TMZ resistance, U87MG required TMZ treatment for three times while 1306MG required TMZ treatment twice. U87MG is a low-ALDH1 expression cell line, in contrast, 1306MG is high-ALDH1 expression cell line. They showed differential responses to radiation and TMZ. Interestingly, while GBM developed TMZ resistance, U87MG increased ALDH1 expression, however, 1306MG reduced ALDH1 expression. These cell lines elevated TGF-β secretion as they developed resistance against radiation (995.4 pg/ml vs. 1234.1 pg/ml in U87MG; 25.7 pg/ml vs. 1607.7 pg/ml in 1306MG) and radiation plus TMZ (995.4 pg/ml vs. 1058.1 pg/ml in U87MG; 25.7 pg/ml vs. 1207.0 pg/ml in 1306MG). Also, therapeutic-resistant GBM accelerated cell proliferation, enhanced cell motility, and displayed TGF-β-induced mesenchymal differentiation by up-regulating N-cadherin and fibronectin. Developing resistance increased ALDH1 expression. Disulfiram (DSF) is a drug which is able to inhibit ALDH activity. We found that inhibiting ALDH1 by DSF sensitized GBM to TMZ in both parental and TMZ-resistant GBM. Moreover, DSF or ALDH1 activity specific inhibitor NCT-501 reduced cell mobility, reversed mesenchymal differentiation via down-regulating N-cadherin and fibronectin, and decreased CD133 expression. Combined DSF and TGF-β receptor inhibitor exaggerated cytotoxicity and enhanced cell mobility inhibition in radiation resistant 1306MG and radiation plus TMZ resistant U87MG. Therefore, the differential expression of ALDH1 may influence the sensitivity of treatment and response to TGF-β inhibitor. Accordingly, ALDH1 may contribute to GBM resistance via modulating TGF-β signaling. Targeting ALDH1 and TGF-β receptor may improve the efficacy of GBM therapy.

#3710

**Establishment of a patient-derived acute myeloid leukemia (AML)** ex vivo **platform for evaluation of novel therapeutic agents.**

Bhavna Verma, Swetha Tati, Bruce Ruggeri, Amy Wesa. _Champions Oncology, Inc., Rockville, MD_.

Acute myelogenous leukemia (AML) is the most common acute leukemia in adults. A hematologic cancer, the disease is highly heterogeneous, with multiple subtypes. Despite advances in treatment, the long-term survival for AML remains poor and the development of novel treatments is an unmet need. Due to the highly divergent subtypes and mutation profiles in AML, the use of patient-derived models may improve drug discovery and development. To address this, we have established a short-term culture system that supports the growth of primary AML cells ex vivo to permit the evaluation and/or screening of candidate agents. Our AML bank is comprised of patient-derived specimens across a range of subtypes (which includes M1, M2, M4, M5, and others), and includes models with common mutations in FLT3 (ITD), IDH1/IDH2 and NPM. Primary AML specimens were characterized for common mutations by TruSight sequencing and for surface marker expression by flow cytometry. The ex vivo assay system was evaluated for the ability to support the survival and expansion of 22 primary AML specimens. Among these, 15 had evidence of proliferation, 5 had no net expansion, and 2 failed to survive. Extension of the culture period for up to 14 days was feasible, with most models having equal or increased cell numbers by the end of the culture period (8 of 10 evaluated). All models stably expressed CD33 throughout the assay. To verify the applicability of this system for drug testing, a standard of care agent cytarabine (ara-C) was assessed for each of the AML models. Cell growth/viability was assessed using Celltiter-Glo assay. Concentration-dependent responses to ara-C were observed across multiple models (IC50 10 nM to 150 nM), indicating a range of relatively sensitive to resistant AML models. A cohort of models were evaluated in vivo for sensitivity to ara-C. Engraftment of the systemic patient-derived xenograft AML models (into NOG or NOG-EXL mice) was evaluated in circulation by flow cytometry. When engrafted, AML-bearing mice were randomized into ara-C or vehicle control groups. Two weeks later, mice were evaluated for the presence of human CD45+CD33+ AML cells. Models that were relatively sensitive to Ara C ex vivo (IC50 < 30 nM) showed a greater in vivo response as evidenced by a 60-80% reduction in the mean circulating AML cells versus models with >100 nM IC50 values, that had little response. In conclusion, Champions offers over 30 well characterized models of AML for study in ex vivo culture and in vivo systemic xenograft models. The diversity in these AML models is reflective of patient diversity, enhancing their utility in the evaluation of novel therapeutic candidates. These data indicate the feasibility of utilization of these primary models, ex vivo as well as in vivo, for drug discovery for AML, from screening to preclinical efficacy modeling.

#3711

Development, maintenance, and characterization of porcine hepatocellular carcinoma cell lines.

Shovik S. Patel,1 Sulalita Chaki,1 Faith M. Thomas,1 Aisha Qazi,1 Kyle M. Schachtschneider,2 Matthew Stewart,1 Elizabeth Pollack,1 Ron C. Gaba,3 Lawrence B. Schook1. 1 _University of Illinois at Urbana-Champaign, Champaign, IL;_ 2 _University of Illinois at Chicago, Chicago, IL;_ 3 _University of Illinois at Chicago, Champaign, IL_.

Biomedical research of liver cancer requires effective model cell lines and animals in order to translate diagnostic and treatment strategies into clinical practice. An ideal translational model to study hepatocellular carcinoma (HCC) would be the use of a genetic pig model of HCC due to the many similarities between pigs and humans which include anatomy, physiology, metabolism and genetics. This study utilized the Oncopig Cancer Model (OCM), a transgenic pig model that develops site and cell specific tumors through Cre recombinase induced expression of KRASG12D and TP53R167H transgenes. Our objective was to develop an in vitro HCC model using primary hepatocytes isolated from Oncopig liver tissue. In order to develop Oncopig HCC cell lines, Oncopigs (n=36) underwent liver resection to remove a portion (5 to 20 grams) of their liver for hepatocyte isolation. Post isolation mean cell yield was 1.8 million cells per gram of tissue ranging from 2.2 million cells to 6.5 million cells. After incubation (24 hours post hepatocyte isolation), primary hepatocytes were transfected with adenoviral vector encoding Cre recombinase (AdCre), resulting in KRASG12D and TP53R167H expression and transformation of hepatocytes into HCC cell lines. Also, AdCre transfected cells will have GFP expression. Successful transfection rates (77% to 99% with a mean of 87.9%) were confirmed by fluorescent microscopy. Phenotypic characterization of OCM HCC cells was then performed. Cell migration assays were performed to characterize enhanced cell migration of transformed cells (mean t1/2 gap = 4 hours) and polymerase chain reaction (PCR) assays were done after several passages to confirm KRASG12D and TP53R167H gene expressions. HCC lines were injected subcutaneously in the abdomen of SCID or NSG mice (n=34). Each mouse was given 2 injections with each injection site receiving 5 million cells. Mouse injections resulted in 59 tumors (86.8% tumor growth success). Mean volume for the tumors was 285.3 mm3 ranging from 18.84 mm3 to 1766.3 mm3. Following confirmation of tumor development in SCID or NSG mice, HCC cell lines were autologously injected into Oncopigs (n=29). Each Oncopig was given 6 injections subcutaneously in the abdomen with each injection site having 10 million cells Autologous injections to Oncopigs were capable of growing tumors. These results indicate that a porcine HCC model can be created and used for further tumor biology research.

#3712

Proliferative and invasive colorectal tumors in pet dogs provide unique insights into human colorectal cancer.

Jin Wang,1 Tianfang Wang,1 Yanfang Sun,1 Yuan Feng,1 William C. Kisseberth,2 Carolyn J. Henry,3 Irene Mok,4 Susan E. Lana,4 Kevin Dobbin,1 Nicole Northrup,1 Elizabeth W. Howerth,1 Shaying Zhao,1 Houjian Cai1. 1 _University of Georgia, Athens, GA;_ 2 _Ohio State University, Columbus, OH;_ 3 _University of Missouri, Columbia, MO;_ 4 _Colorado State University, Fort Collins, CO_.

Spontaneous tumors in pet dogs represent a valuable but undercharacterized cancer model. To better use this resource, we performed an initial global comparison between proliferative and invasive colorectal tumors from 20 canine cases and evaluated their molecular homology to human colorectal cancer (CRC). We sequenced 15 canine intestinal samples for WGS and 26 for RNA-seq. We investigated alterations in the genome and transcriptome, using state-of-the-art analysis tools. We aslso investigated their microbiome, by mapping WGS and RNA-seq read pairs that could not be placed onto the canine genome to two microbial genome databases: The Human Microbiome Project database and all bacterial genomic sequences. We performed the same analysis with TCGA (The Cancer Genome Atlas) colon cancer data. Based on our analysis, proliferative canine tumors harbor overactivated WNT/β-catenin pathways and recurrent CTNNB1 (β-catenin) mutations S45F/P, D32Y and G34E. Invasive canine tumors harbor prominent fibroblast proliferation and overactivated stroma. Both groups have recurrent TP53 mutations. We observed three invasion patterns in canine tumors: collective, crypt-like and epithelial-mesenchymal transition (EMT). We detected enriched Helicobacter bilis and Alistipes finegoldii in proliferative and crypt-like tumors, but depleted mucosa-microbes in the EMT tumor. Additionally, guided by our canine findings, we classified 79% of 478 human colon cancers from TCGA into four subtypes: primarily proliferative, or with collective, crypt-like or EMT invasion features. Their molecular characteristics match those of canine tumors. We showed that consensus molecular subtype 4 (mesenchymal) of human CRC should be further divided into EMT and crypt-like subtypes, which differ in TGF-β activation and mucosa-microbe content. Our canine tumors share the same pathogenic pathway as human CRCs. Dog-human integration identifies three CRC invasion patterns and improves CRC subtyping.

#3713

Strong expression of EZH2 and H3K27me3 is associated with poor survival time in small cell lung cancer.

Ana Paula Fernandes,1 Rita deCassia S Alves,2 Guilherme Watte,3 Philipp Kunze,4 Adriana Vial Roehe,1 Regine Schneider-Stock4. 1 _Federal University of Health Sciences Porto Alegre, Porto Alegre, Brazil;_ 2 _Federal University of Health Sciences Porto Alegre, Porto Alegre, Germany;_ 3 _Pontifical Catholic University of Porto Alegre, Porto Alegre, Brazil;_ 4 _University of Erlangen-Nürnberg, Erlangen, Germany_.

BACKGROUND: Small-cell lung cancer (SCLC) is a highly aggressive neoplasm, characterized by early development of metastasis and very poor clinical outcome. The therapeutic possibilities are limited and poorly changed over the past three decades. Enhancer of zeste homolog 2 (EZH2) is a member of the polycomb repressive complex 2. It is involved in epigenetic gene silencing through the trimethylation of H3K27 and it has been identified as a biomarker of aggressive and highly proliferating tumors, emerging as a potential target for cancer therapy.

OBJECTIVE: We aimed to correlate the expression profile of the EZH2 and H3K27me3 proteins in tumor tissues of non-treated SCLC patients with patient´s outcome.

METHODS: EZH2 and H3K27me3 expression were analyzed by immunohistochemistry in 48 SCLC tumor biopsies. The expression of the markers was quantified in a semi quantitative way and correlated with clinic pathological factors and patients overall survival. To mimic an EZH2 loss, H209 SCLC cells were treated with an EZH2 inhibitor for 48h and the in vivo growth pattern was investigated in the chorioallantoic membrane (CAM) assay.

RESULTS: All patients died within one year after diagnosis. There was a heterogeneous expression pattern for EZH2 and H3K27me3. Strong expression of EZH2 and H3K27me3 was observed in appr. 50% of the patients. The Kaplan Meier curve showed a significant association of high expression pattern of EZH2 and shorter overall patient survival time after 12 and 25 weeks of follow up, respectively (p=0.030; p=0.014) Different markers of tumor aggressiveness after EZH2 inhibition were investigated in the CAM model.

CONCLUSION: Strong EZH2 and H3K27me3 expression in non-treated SCLC patients was related to worse prognosis. EZH2 inhibitors should be considered in future trials for SCLC.

#3714

Efficient derivation and expansion of tumor cell lines from primary and xenotransplanted pancreatic, ovarian and renal tumors.

Olaf Hardt,1 David Agorku,1 Anne Langhammer,1 Franziska Zickgraf,2 Felix Geist,2 Elisa M. Noll,2 Christian Eisen,2 Andreas Bosio,1 Martin R. Sprick,2 Andreas Trumpp2. 1 _Miltenyi Biotec GmbH, Bergisch Gladbach, Germany;_ 2 _Heidelberg Institute for Stem Cell Technology and Experimental Medicine, Heidelberg, Germany_.

Cancer cell lines are widely used as in vitro models to study tumor biology and for efficacy testing of novel anti-cancer therapeutics. In the past, most of this work has been done using established cell lines that have been cultured for decades. Extensive in vitro propagation has been shown to lead to the acquisition of multiple genetic and epigenetic alterations in cell lines, leading to decreased heterogeneity and the lack of tumor initiating as well as multi-lineage differentiation capacity. Consequently, established cell lines only insufficiently resemble the characteristics of tumors and thus have limited applicability as in vitro models for example. To overcome this hurdles, cell lines can be derived from primary cancer biopsies. However, this process is very inefficient for most tumor entities. In addition, most of the media used include largely undefined serum, such as FBS, which has been shown to drive primary tumor cell cultures to a more differentiated state when used over multiple passages. We have developed advanced, serum-free media for derivation and expansion of tumor cell lines from pancreatic, ovarian or renal tumors. Our media have been optimized concerning formulation, stability, and usability and allow for efficient generation of primary cell lines from both, patient and xenotransplanted tumors. Primary cell lines derived with our media retained their tumorigenic potential and could therefore be xenotransplanted and propagated in immunodeficient mice. Notably, the resulting tumors closely resembled the initial patient tumor as shown on the histomorphological as well as functional level. Moreover, the primary cell lines closely resembled essential characteristics of the parental tumor in vitro, including expression of subtype-specific markers, cellular heterogeneity, as well as genetic and epigenetic signatures. Taken together, we have developed serum-free medium for efficient derivation and expansion of tumor cell lines from primary and xenotransplanted pancreatic, ovarian and renal tumors, allowing for the establishment of easily accessible in vitro as well as corresponding xenografting vivo models. This facilitates the translation of in vitro findings directly into in vivo settings, allowing for more reliable pre-clinical modelling.

#3715

Cancer as a consequence of breaking through evolutionary constraints on longevity.

Jaime F. Modiano, Aaron L. Sarver. _Univ. of Minnesota, Minneapolis, MN_.

It is well known that the risk of cancer increases with age, but when viewed through a comparative lens, this risk is species specific and exists at a relatively low background in the animal kingdom. The exceptions, where cancer is a major cause of mortality, are humans and companion dogs and cats. Indeed, it is estimated that more than 50% of pet dogs that reach the age of 10-years will die from cancer. Colloquially, there is a perception that there is a "cancer epidemic" in dogs caused by exposures to real or perceived mutagens, but this hypothesis is based on presumptions for which supporting evidence is weak or absent. Here, we propose an alternative hypothesis, that replacing natural selection with artificial selection for form and function, and superimposing longevity that breaks the evolutionary constraints for that species inevitably creates cancer-prone phenotypes. Modern pet dogs have achieved a social status equivalent to human family members. With this have come better health care, reduced early mortality, and greater longevity. Although dogs were first domesticated15,000 to 20,000 years ago and breeds were derived over the last 200 to 400 years, significant gains in longevity and the recognition of cancer as a major cause of mortality in pet dogs have taken place over the past 40 to 60 years, coinciding with the transition of dogs from working animals to pets. Breaking through the evolutionary constraint on longevity comes at a price: long-lived species such as elephants, whales, mole rats, and others have acquired a diversity of cancer-protective mechanisms, achieving a balance between longevity and their reproductive and energetic investments over tens of millions of years. But no such cancer-protective mechanisms have been identified in dogs, or for that matter in humans. In both species, risk increases with age, but in dogs, risk is magnified by artificial selection. Osteosarcoma provides an example that can be generalized. The underlying causal factor is selective breeding for large size, but this is only able to manifest itself because modern pet dogs exceed their evolutionarily expected lifespan by at least 2 or 3 times. We conclude that appreciating the role of longevity as a major cause of cancer in pet dogs, as well as in other model organisms will improve our ability to design and interpret experiments that will show greater fidelity when translated to humans, where the same risk factors are operative, although they are partly masked by the influence of behavioral or occupational exposures to strong mutagens.

#3716

Conditional reprogramming technology for Next Generation Living Biobanks (NGLB).

Xuefeng Liu. _Georgetown Univ. School of Medicine, Washington, DC_.

As one of the next generation biobanks, living biobanks (iPSC, organoids, CRC, PDX, etc.) and related functional analyses are rapidly growing in basic research, clinics, and industry (especially immune-oncology and targeting therapies) as well. Most recently NCI launched PDMR (patient-derived model repository) using PDX, CRC and organoid cultures, NCI/ATCC/Broad/Sanger launched HCMI (Human Cancer Models Initiative) using Organoids and CRC cultures. In this study, we established a conditional reprogramming (CR) protocol which allows to generate cell cultures from cryopreserve fresh tissue specimens from surgery, core biopsies, needle biopsies, and brushed cells.It has been extremely difficult to generate primary cultures from tumor specimens, it has not been possible to expand and indefinitely propagate cells derived from adult tissues while retaining lineage-commitment, normal growth control and differentiation potential. The CR method rapidly expands both normal and malignant epithelial cells from diverse anatomic sites and mammalian species and does not require transfection with exogenous viral or cellular genes. Establishment of cell cultures from both normal and tumor tissue (many types) is highly efficient. The robust nature of the technique is exemplified by the ability to produce 2 x 106cells in 5 days from a core biopsy of breast tumor. Normal cultures retain a normal karyotype and differentiation potential and cell lines derived from tumors retain their tumorigenic phenotype. We were also able to generate their corresponding fibroblast cultures from the same specimens. These fibroblasts can be immortalized by exogenously expressed hTERT. This approach allows to establish in vitro "epithelial-stroma interactions" to study normal cell differentiation and tumor microenvironment. The ability to produce inexhaustible cell populations from small biopsies and cryopreserved specimens has the potential to transform biobanking repositories (termed as NGLB - next generation living biobanks) and current pathology practice by enabling genetic, biochemical, metabolomic, proteomic, and biological assays, including chemosensitivity testing as a functional diagnostics tool (or a living biomarker) for precision cancer medicine. CR cells can be also an inexhaustible resource to generate CR cells derived iPSC, organoids, and xenografts (CDXs).

#3717

Animal model of cirrhosis with hepatocellular carcinoma: A reliable tool for testing new therapies.

Keerthi Kurma,1 Zuzana Macek Jilkova,1 Patrice N. Marche,1 Nathalie Sturm,2 Hervé Lerat,1 Thomas Decaens3. 1 _Institute for Advanced Biosciences, La Tronche, France;_ 2 _CHU-Grenoble Département d'Anatomie et de Cytologie Pathologiques, La Tronche, France;_ 3 _Clinique Universitaire d'Hépato-gastroentérologie, Pôle Digidune, La Tronche, France_.

Background and aim: Liver cancer is now the second most common cause of cancer related death worldwide, with hepatocellular carcinoma (HCC) accounting for the majority of these cases . 90% of HCC are associated with liver fibrosis or cirrhosis developed from chronic liver injuries.Small animal models represent essential tools in cancer research. As fibrosis/cirrhosis modifies liver vascularization, extracellular matrix composition, and drugs metabolism, it is essential to use a cirrhotic animal model to test HCC drugs for their efficiency against tumour initiation and/or progression. Current mouse model failed to reproduce all fibrosis stages, especially cirrhosis. One of the rodent models that most faithfully reproduce human cirrhosis is diethyl nitrosamine-injured rats (DEN rats).

The aim of our project is to deeply characterize DEN-induced HCC rat model during cirrhosis progression and HCC development with special focus on liver inflammatory micro-environment.

Method: 6-weeks-old Fischer 344 male rats were treated weekly with intra-peritoneal injections of 50 mg/kg DEN. 9 rats are sacrificed before starting DEN-injections at 0 week (control group), after 8 weeks of injections (8 weeks), after 14 weeks of injections (14 weeks) and at 20 weeks after start of DEN-injections (20 weeks). Histopathological analysis, immunohistochemical and FACS analysis were performed. Results were analysed in a double blind manner.

Results: Chronic DEN treatment induces tumour development that starts at 14 weeks. Tumour size significantly increases between 14 and 20 weeks but not tumour number. DEN injection was associated with a significant increase of hepatocyte proliferation at 8 weeks, 14 weeks, and 20 weeks when compared to 0 weeks. Similarly, CD34 positive cells were increased, suggesting enhanced angiogenesis at 14 & 20 weeks when compared to 0 weeks. DEN-induced liver fibrosis was significantly and gradually enhanced as suggested by upregulation of fibrosis deposition at 8 weeks, 14 weeks, 20 weeks compared to 0 weeks. We also observed significant decrease in CD4+ and increase in CD8+ T lymphocytes in the blood and non-tumour liver tissue at each time points. In parallel, CD8+ T lymphocytes were significantly decreasing in hepatic tumour tissue. CD152 expression was significantly increasing in blood samples and non- tumour liver tissues at 8, 14 and 20 weeks compared to 0 weeks.

Conclusion: DEN-induced HCC rat model displays tumour initiation, development and different stages of liver fibrosis, up to cirrhosis and also helps to understand related modulation of immune micro environment. Indeed we demonstrated CTLA-4 expression is upregulated in this model. In this context, DEN-induced cirrhotic HCC rat model might be a relevant tool as pre-clinical models to evaluate new HCC treatment efficacy and tolerance in liver cirrhotic background.

#3718

ID8-Luc syngeneic ovarian cancer model for preclinical evaluation of immunomodulatory molecules.

Sumithra Urs, Sheri Barnes, Stacey Roys, Maryland R. Franklin. _MI Bioresearch, Ann Arbor, MI_.

Ovarian cancer (OC) is a gynecological malignancy with a high mortality rate of over 14,000 deaths annually in the US. Although ~ 80% of OC patients initially go into remission after 1st line treatment (surgery and chemotherapy), more than 60% relapse within 16-18 months. Low neoantigen burden and the immunologically "cold" nature of OC makes it a challenging malignancy. Therefore, novel methods such as immunotherapy and/or combined therapies are essential to improve clinical outcome of patients. This work evaluated the ID8-Luc murine ovarian carcinoma model to determine sensitivity to immunomodulatory agents. C57BL/6 albino mice implanted intraperitoneally with ID8-Luc cells were evaluated for growth and sensitivity to checkpoint inhibitors anti-PD-1, anti-PD-L1, anti-CTLA-4 and the chemotherapy agent paclitaxel. Disease progression was monitored weekly by in vivo bioluminescence imaging (BLI). Baseline immune profile of the model was determined by collecting ascites from untreated mice and analyzed with panels for myeloid and lymphoid cell populations by flow cytometry. ID8-Luc cells successfully established tumors with 7-8 days doubling time, as determined by BLI, and a median overall survival time of ~ 40-50 days. Formation of ascites resulted in weight gain and extended abdomen at advanced disease state. Necropsy showed solid tumor nodules within the peritoneal cavity including involvement of pancreas, liver, spleen and abdominal wall. Baseline immune profiling of ascites indicated presence of B cells (23%), granulocytic myeloid derived suppressor cells (G-MDSCs; 15%), TAMs (25%) and a low percentage of T cells (3-4%). This was suggestive of a possibly "cold" tumor model. Paclitaxel is a front-line chemotherapy option in OC. We find that the ID8-Luc model has 60% tumor free survivors (TFS) following treatment. To determine how this model responds to immune checkpoint inhibition we tested response to anti-PD-1, anti-PD-L1 and anti-CTLA-4 antibodies. Treatment was initiated at either 7 (early) or 14 (late) days post tumor cell implant and we found that the overall response varied depending on the timing of treatment initiation. Early treatment with either anti-PD-1 or anti-PD-L1 resulted in complete regression of tumors (100% TFS) while the model was refractory to anti-CTLA-4 treatment. Late treatment initiation of anti-PD-1 or anti-PD-L1 resulted in an "all or nothing" response (40% complete response and 60% non-response). In addition, reducing the antibody dose from 10mg/kg to 5mg/kg did not make a material impact on the anti-tumor activity of these agents in the late treatment regimen. New models to further the preclinical testing of agents for OC is vital. This work characterizes the baseline immune profile of the murine ID8-Luc model along with response to checkpoint inhibitors to enable the rational design of combination strategies in the preclinical setting.

#3719

Systemic and subcutaneous modeling of A20 murine B cell lymphoma in mice: A comparative assessment.

Sheri R. Barnes, Sumithra Urs, David Draper, Scott Wise, Maryland Rosenfeld Franklin. _MI Bioresearch, Ann Arbor, MI_.

To advance immuno-oncology drug development, there is a need for well-characterized syngeneic tumor models. In addition to subcutaneous modeling, orthotopic models are equally important for preclinical development as they are posited to have improved clinical translatability compared to subcutaneous implantation. To this end, we have generated both subcutaneous and systemic A20 murine B cell lymphoma models.

Specific to orthotopic modeling, a luciferase-enabled A20 model (A20-luc) was created to monitor tumor progression by bioluminescence imaging. Systemic parental A20 or A20-luc models show slight differences in median survival (34.5 days and 39 days, respectively) but similar take rates and clinical manifestations, including abdominal distension and metastatic foci in the liver. These data suggest that parental A20 and A20-luc behave similarly in systemic disease.

To understand how checkpoint blockade and costimulatory agonists act on A20, anti-mPD-1, anti-mPD-L1, anti-mCD137, and anti-mGITR were evaluated for efficacy against both established subcutaneous and systemic A20 models. Our data indicate that neither anti-mPD-1 nor anti-mGITR demonstrate antitumor activity in either model. Anti-mPD-L1 is inactive in systemic disease but shows some efficacy on A20 subcutaneous tumors with delayed growth observed in 40% of mice. Contrastingly, the costimulatory agonist anti-mCD137 elicits a stronger response, with 50% of established subcutaneous A20 tumors having delayed growth including two complete responses and one tumor free survivor. Similarly, in systemic A20 disease, 50% of animals treated with anti-mCD137 exhibited a profound decrease in luciferase signal. While modestly responsive, both A20 and A20-luc show promise for use in combination strategies.

To determine whether this modest activity is due to an infiltration of immunosuppressive cells into the tumor, the composition of immune cells in established subcutaneous A20 tumors was evaluated. Despite modest activity, 50% of total CD45+ infiltrate were T cells and NK cells, 7% were MDSCs and macrophages, and the remaining were dendritic in nature. As high T cell infiltrate is more common in tumors responsive to immunotherapy, these infiltrated T cells are hypothesized to lack signals required for activation or are reactive to non-tumor-associated antigen. Similar assays with metastatic foci resulting from systemic disease are planned.

Both subcutaneous and orthotopic A20 models exhibit favorable characteristics for evaluation of investigational agents. Response to immunotherapy is minimal to moderate in both models but are generally aligned. The high infiltrate of T cells in the subcutaneous model have potential for exploitation of this model in development of agents targeting T cells. Taken together, our results indicate that A20 is a favorable syngeneic tumor model for both subcutaneous and orthotopic applications.

#3720

Blockade of the short-form of prolactin receptor induces FOXO3a/EIF-4EBP1-mediated cell death in uterine cancer.

Yun-Fei Wen, Ying Wang, Shaolin Ma, Anca Chelari Raicu, Keith A. Baggerly, Anil K. Sood. _UT MD Anderson Cancer Ctr., Houston, TX_.

Purpose: Extra-pituitary prolactin (PRL) is involved in development and progression of uterine carcinoma. However, the underlying mechanisms of PRL/PRLR axis in uterine cancer are not well understood, and current means of targeting this axis are limited. Here, we used a potent PRL antagonist (G129R) to interfere with oncogenic activities of uterine cancer cells and further inhibit tumor growth.

Experimental Design: We performed integrated analyses by comparing expression of PRLR isoforms in a broad panel of tumors and normal countparts using TCGA and GTex databases. Therapeutic effects of G129R treatment in uterine cancers were assessed in both orthotopic PTENwt and PTENmut models. Additionally, we carried out reverse phase protein assay (RPPA) at in vivo condition from the resulting tumors and at in vitro conditions to interrogate downstream targets and mechanism of action for G129R. Further functional analyses including cell viability, cell cycle and nascent protein synthesis were accessed in two types of uterine cancer cells under serum-depleted condition.

Results: We found that PRLR_S1a, a short form of PRLR that is generated from alternative splicing during transcription, is expressed in human uterine cancers to a greater extent than other isoforms. Blocking activities of the PRL/PRLR_S1a axis with the antagonist G129R significantly reduced growth and progression of both orthotopic PTENwt and PTENmut uterine tumors. Data from reverse-phase protein arrays (RPPA) by in the resulting tumors showed that pathways involved in epithelial-mesenchymal transition such as PI3K/mTOR activities were significantly downregulated by G129R treatment. In addition, based on the RPPA data from the in vitro uterine cancer cells treated with G129R as monotherapy or in combination with PRL, we discovered a novel function of G129R in initiating cell death mediated by nuclear FOXO3a and inducing the G0/G1 arrest in both PTENwt and PTENmut uterine cancer cells. Furthermore, G129R decreased the nascent protein synthesis in these uterine cancer cells mediated by transcriptional suppressor EIF-4EBP1. Collectively, our findings reveal a new role of the PRLR_S1a isoform in uterine cancer and identify G129R as a potential therapeutic in uterine cancer.

#3721

**Improved fitting of HRMAS NMR spectra for** ex vivo **metabolomic analysis of glioma tissue.**

Selin Ekici, Ren Geryak, Stewart G. Neill, Hui-Kuo Shu, Candace C. Fleischer. _Emory University School of Medicine, Atlanta, GA_.

Gliomas are aggressive brain tumors with high rates of treatment resistance and low survival rates. High-resolution magic angle spinning (HRMAS) nuclear magnetic resonance (NMR) spectroscopy can quantify metabolite concentrations in glioma tissue, but most analysis techniques require manual peak selection and integration that prevents accurate and reliable quantitation of overlapping peaks and large macromolecule baselines. Our goal was to compare the effectiveness of operator-independent LCModel analysis of glioma spectra acquired from free induction decay (FID) and Carr-Purcell-Meiboom-Gill (CPMG) pulse sequences.

HRMAS NMR spectra were acquired using FID and CPMG sequences from 14 histologically-confirmed glioma tissue samples (WHO grade II (n=5), III (n=5), and IV (n=4)) collected during surgical resection from human brain tumor patients. Metabolite concentration ratios and Cramer-Rao lower bounds (CRLBs) were estimated using LCModel. Metabolite CRLBs were compared using a paired two sample t-test. Differences in concentrations as a function of WHO grade were determined with ANOVA and Tukey-Kramer post-hoc tests. Significance was determined by p<.05.

Metabolite CRLBs for lactate and myo-inositol were significantly lower for CPMG compared to FID spectra (p<.05). Most metabolites could be quantified with LCModel from spectra acquired with CPMG where many metabolites acquired with the FID sequence were not detected. For example, lactate was quantifiable from 3 of the spectra acquired with FID compared to 12 spectra with CPMG. The use of the CPMG sequence reduces quantification errors by eliminating confounding baseline signals. LCModel facilitates improved separation of overlapping resonances compared to manual peak integration, supporting the use of operator-independent methods for metabolic spectral analysis.

Comparison of metabolite concentrations as a function of WHO grade revealed significant differences in lactate and glutamine plus glutamate normalized to creatine (p<.05). 2-hydroxyglutarate (2-HG) was also detected in 9 out of 13 isocitrate dehydrogenase (IDH)-mutated samples using both sequences. IDH-mutated tissue should produce 2-HG; however, these results are promising as 2-HG can be difficult to quantify in 1D NMR due to spectral overlap.

In conclusion, LCModel can reliably quantify HRMAS spectra acquired with the CPMG sequence but is less reliable with the FID sequence. Increases in lactate and glutamine plus glutamate concentrations as a function of tumor grade were consistent with previous results using HRMAS for glioma metabolic analysis, and 2-HG was detected in 1D HRMAS spectra acquired with both sequences. We expect that improved spectral fitting will contribute to future NMR-based metabolomics studies in glioma.

#3722

**Novel Optical Coherence Tomography (OCT) technology for no-invasive 3D** ex vivo **drug activity profiling.**

Sumeer Dhar,1 Akihiro Ueda,2 Takemitsu Muira,2 Yasushi Kuromi,2 Yuki Mori,2 Hiroki Fujimoto2. 1 _SCREEN LifeScience, Amstelveen, Netherlands;_ 2 _SCREEN Holdings, Kyoto, Japan_.

Drug discovery and early to late drug development process have relied on 2D monolayer based in vitro assay platforms and small animal models. The limitations posed by 2D in vitro assays for evaluation of targeted and immunomodulatory agents have prompted researchers to develop novel 3D in vitro and ex vivo platforms utilizing the tumor microenvironment like conditions for better understanding of mechanisms of novel targeted and immunomodulatory drug/compound entities. Therefore, rapid development of robust 3D ex vivo cell culture as drug efficacy testing system for drug discovery, development and profiling of standard of care and Immunotherapeutic molecules have led to understanding the relevance of these platforms within the scientific and clinical community. 3D ex vivo platforms are being diligently evaluated as better predictive drug efficacy testing tools in preclinical as well as clinical space in the quest for profiling of novel anticancer entities (as single or two drug combinations). Within this context, there has been growing need in improved imaging and analysis of complex 3D structures (Multicellular tumor spheroids/Organoids) containing multiple cellular components such as, Tumor cells, stromal cells, various immune cells to monitor signaling pathways and assess immune events including T cell infiltration as drug efficacy measurement. At SCREEN LifeScience, we have developed a novel OCT based technology for imaging and analysis of complex 3D structures, such as, spheroid /organoid for growth & morphological profiling, quantification of internal cavities, drug sensitivity testing to capture the events leading to tumor cell death and assessing mechanism of drug action. Additionally, this technology allows us perform large tissue imaging, non-invasive monitoring of macro and sprouted neo-vasculature without the need for fluorescent staining for providing quantitative information about the vascular morphological changes, thereby allowing for the evaluation of anti-angiogenic drugs in real time. In summary, the OCT technology shown to be robust and versatile technology useful for studying multiple disease models, including cancer. Its utility in regeneration medicine is being further evaluated as a versatile non-invasive platform.

#3723

Fatty acid synthase: A new therapeutic target for pancreatic cancer.

Travis Vander Steen,1 Li Ding,1 Yu Zhang,1 George Kemble,2 Daniel Billadeau,1 Ruth Lupu1. 1 _Mayo Clinic, Rochester, MN;_ 2 _3-V Biosciences, Menlo Park, CA_.

Pancreatic cancer is the fourth leading cause of cancer related deaths in the United States and continues to be hindered with limited treatment options. One of the current therapeutic regimens for pancreatic cancer often include the nucleoside analog gemcitabine (Gem), while it has been used for many years, the overall survival rate lingers around 6%. New therapies are needed to treat this disease. Fatty acid synthase (FASN) has received increased attention as a potential target for cancer therapy in the last decade. FASN is the key enzyme in the de novo synthesis of palmitate from malonyl-CoA. Palmitate is the precursor other long-chain fatty acids (FA). However, the precise mechanism by which pharmacological interference with endogenous FA biogenesis might facilitate apoptosis in tumor cells remains unresolved. We herein explored the molecular relationship between FASN activity, oxidative stress/redox balance, and the intrinsic apoptotic pathway in FASN overexpressing-dependent pancreatic cancer cells. About 68% of Pancreatic Ductal Carcinomas (PDA) expresses high levels of Fatty acid synthase (FASN), and its expression is correlated with disease free survival. A newly developed FASN inhibitor with excellent pharmaceutical properties was used for our studies TVB-3166 (in vitro) and TVB-3664 (in vivo) and ten different PDX derived cells were used for the studies. Pharmacological blockade of FASN activity significantly increased the NADPH/NADP+ ratio, promoted the production of reactive oxygen species (ROS). Accordingly, the inhibition of endogenous FA synthesis promoted mitochondrial membrane permeabilization, cytochrome c release, and apoptotic cell death. Each step of the FASN inhibition/BH3-only protein axis could be reversed with a ROS scavenger or the FASN end-product palmitate, thus confirming that the delineated pro-apoptotic cascade likely reflects on-target effects of FASN inhibition. Most importantly, the in vivo studies using PaCa-PDX reveal a significant decrease in tumor growth and furthermore a synergistic effect of between TVB-3664 and Gem. In conclusion, our findings uncover a novel FASN-dependent mitochondrial priming that links de novo FA biosynthesis with the intrinsic apoptotic threshold in cancer cells. The discovery that FASN-inhibited cancer cells exist in an apoptosis-prone state highly sensitive to BH3 mimetics warrants clinical exploration in FASN-overexpressing pancreatic cancer patients. 

### Radiation Tissue Tolerance, Immunity, and in Vivo Effects of Radiation

#3724

Molecular mechanisms of TTField action determined by measurements and modelling of electro-conductive properties of microtubules.

Aarat P. Kalra,1 Sahil Patel,1 Asadullah Bhuiyan,1 Jordane Preto,1 Vahid Rezania,2 John D. Lewis,1 Karthik Shankar,1 Jack Tuszynski1. 1 _University of Alberta, Edmonton, Alberta, Canada;_ 2 _Grant MacEwan University, Edmonton, Alberta, Canada_.

Biological effects of AC electric fields at frequencies between 100-300 kHz discovered a decade ago are being applied to cancer cells as a therapeutic modality in the treatment of glioblastoma multiforme (GBM). They are called Tumor Treating Fields (TTFields) as they disrupt cell division. Based on our electro-conductive measurements and modeling, we provide an assessment of possible molecular-level mechanisms. Computer simulations and experimental measurements carried out for microtubules and actin filaments are presented. Charge and dipole values for monomers and dimers as well as polymerized forms of these proteins are summarized. Continuum approximations for cable equations describing actin filaments and microtubules compare favorably to measurements in buffer solutions showing soliton waves and transistor-like amplification of ionic signals, respectively. AC Conductivity and capacitance of tubulin and microtubules have been measured and modeled in the range of frequencies between 1 Hz and 1 MHz. A dramatic change in conductivity occurs when tubulin forms microtubules. In living cells, this signals a conductive phase transition coinciding with mitosis in dividing cells. This process is allowed by TTField penetration into the cleavage furrow in dividing cells and provides the most significant mechanistic explanation of the observed effects. We provide estimates of the forces, energies and power involved in the action of TTFields on microtubules and kinesin motors. These calculations are compared and contrasted with typical values experienced at a cell level and provide strong arguments for real physical effects of TTFields in dividing cells. We also show results of DLS and TEM measurements on microtubules and tubulin oligomers in solution, which allow us to quantify these processes under controlled conditions. In conclusion, the most likely candidates to provide a quantitative explanation of these effects are ionic condensation waves around microtubules as well as dielectrophoretic effects on the dipole moments of microtubules.

#3725

Numerical simulation of tumor treating fields effects on cell structures: Mechanism and signaling pathway candidates.

Kristen W. Carlson,1 Nirmal Paudel,2 Jack A. Tuszynski,3 Zeev Bomzon4. 1 _BIDMC/Harvard Medical School, Cambridge, MA;_ 2 _IEEE, Greenville, NC;_ 3 _University of Calgary, Edmonton, Alberta, Canada;_ 4 _Novocure Ltd, Haifa, Israel_.

Tumor Treating Fields (TTFields) have become a fourth modality for cancer treatment. Mild electric fields (~1-4 V/cm) produce few side effects and significantly extend overall survival of glioblastoma patients, and TTFields are in clinical trials for a variety of tumor cell types. Our goal is to uncover TTFields' mechanism and cell signaling pathways by numerically modeling their effects on sub-cellular structures, such as microtubules (MTs) and their interactions with motor proteins. METHODS: We have built finite element models in COMSOL Multiphysics (tm) of the MT and its micro-environment to test hypotheses on TTFields' mechanism of action by predicting effects on sub-cellular structures. RESULTS: One model prediction is that current density induced in the MT counter-ion layer by TTFields essentially shunts electric current within them. The strongest current flows through the counter-ion layer surrounding the MT's C-termini and energy density in this layer likely exceeds the level to disrupt motor protein 'walk' along the MT. The energy density is predicted at 10-20 Joules when both the field and the MTs are aligned with the cell axis. A second mechanism examined by our model is disruption of the 'foot' of kinesin, released from its C-terminus contact by ATP (10-19 Joules). The final phase of the walk is driven by thermal buffeting of the forward foot randomly positioning it near enough to the C-terminus for electrostatic forces to bind it. A stall force ~10-19 - 10-16 N from TTFields would prevent diffusion and disrupt the kinesin walk. A recent clinical study segregating patient cohorts treated vs. not treated with dexamethasone found overall survival indefinitely for the non-dexamethosone cohort, leading us to hypothesize that TTFields activate the intrinsic Bcl2-mediated apoptotic signaling pathway. Future modeling will seek to tie disruption of motor protein transport along MTs to activating intrinsic apoptosis, e.g. via failure to silence the G2 cell cycle checkpoint. CONCLUSION: Our modeling predicts that TTFields in cytosol induce electric currents along MTs that are strong enough to disrupt key cellular functions such as the kinesin walk and C-termini transitions, both of which are crucial for motor protein transport. Hence, TTFields disrupt the most delicate mechanisms involved in the carefully-orchestrated succession of steps in mitosis.

#3726

Preclinical activity of PSMA-TTC (BAY 2315497) in combination with androgen receptor antagonists in prostate cancer models.

Stefanie Hammer,1 Urs B. Hagemann,1 Sabine Zitzmann-Kolbe,1 Aasmund Larsen,2 Chrstine Ellingsen,2 Oliver von Ahsen,1 Jenny Karlsson,2 Roger M. Bjerke,2 Olav B. Ryan,2 Pascale Lejeune,1 Hartwig Hennekes,1 Alan Cuthbertson,2 Dominik Mumberg1. 1 _Bayer AG, Berlin, Germany;_ 2 _Bayer AS, Oslo, Norway_.

Targeted alpha therapy (TAT) agents are able to deliver high linear energy transfer alpha-radiation selectively to tumors. The PSMA targeted thorium-227 conjugate PSMA-TTC (BAY 2315497) is a TAT approach for mCRPC consisting of a human anti-PSMA antibody covalently linked to the chelator moiety (3,2 HOPO) radiolabeled with the alpha emitter thorium-227. PSMA-TTC has shown strong anti-tumor activity in PSMA-positive prostate cancer models (Hammer et al. AACR 2017/2018). Androgen receptor (AR) antagonists like enzalutamide have been effective in improving overall survival in CRPC patients, however, not all patients respond to these therapies and those responding develop resistance and progression of disease.

Herein we evaluate in prostate cancer models combination treatments of PSMA-TTC with enzalutamide and the novel AR antagonist darolutamide, which has recently completed a clinical phase 3 trial.

AR antagonists induced PSMA levels in LNCaP and C4-2 prostate cancer cells in vitro resulting in increased sensitivity to growth inhibition by PSMA-TTC. In vivo, the combination of PSMA-TTC with enzalutamide was tested in the hormone- and enzalutamide-sensitive patient-derived prostate cancer model ST1273 (South Texas Accelerated Research Therapeutics, San Antonio, Texas). A single i.v. injection of PSMA-TTC at 250 kBq/kg (0.14 mg/kg) resulted in stable disease in 50% and partial response in 38% of the animals 40 days after dosing. Daily treatment with enzalutamide (30 mg/kg po for 28d) resulted in stable disease in 86% and partial response in 14% of the animals. Combining PSMA-TTC and enzalutamide at the mentioned concentrations achieved tumor reduction in all animals, with partial response in 67% and complete response in 33% of the animals 40 days after start of treatment. No significant adverse effects on body weight were detected compared to vehicle treated animals in any of the groups. Additionally, the combination of PSMA-TTC with darolutamide was tested in the enzalutamide resistant patient-derived prostate cancer model KUCaP-1 (provided by Prof. O. Ogawa, University of Kyoto, Japan). PSMA-TTC monotherapy at 150 kBq/kg (0.43 mg/kg) injected twice at an interim of two weeks resulted in 79% tumor growth inhibition and darolutamide treatment (200 mg daily) achieved 60% tumor growth inhibition compared to vehicle treated animals. The combination of PSMA-TTC with darolutamide increased tumor growth inhibition compared to vehicle to 85% and, importantly, 77% of the animals showed stable disease or partial response until 57 days after start of treatment.

In summary, combining PSMA-TTC with AR antagonists shows promising preclinical data in patient-derived prostate cancer models with increased response rates even in an enzalutamide-resistant model. Clinical investigation of this targeted alpha pharmaceutical investigational agent alone and in combination is warranted.

#3727

An orthotopic xenograft model for studying reirradiation and glioblastoma evolution.

Joseph H. McAbee,1 Barbara H. Rath,1 Xiaolin Wu,2 Uma Shankavaram,1 Kevin Camphausen,1 Philip J. Tofilon1. 1 _National Cancer Institute, National Institutes of Health, Bethesda, MD;_ 2 _Frederick National Laboratory for Cancer Research, Frederick, MD_.

Glioblastoma (GBM), the most common and malignant primary adult brain cancer, has a median survival of 15 months despite multimodal treatment involving surgical resection and chemo-radiotherapy. Ongoing clinical trials seek to examine the safety and benefit of retreatment with radiation therapy for recurrent glioblastoma. By adding a reirradiation protocol to a previously described glioma stem-like cell (GSC) initiated orthotopic xenograft model, this study seeks to better understand the impact of radiotherapy on both primary and recurrent GBM evolution and to establish an in vivo model for studying reirradiation. Therefore, we intracranially implanted CD133+ NSC11 cells, and other GSC lines, into nude mice. After 21 days, bioluminescence imaging was performed to confirm the presence of tumor prior to randomization into control and radiation therapy groups (3x5Gy). After treatment tumors were imaged weekly to track changes in BLI ratios. Once the average BLI ratio for the treated mice was found to be between 1 and 10, the mice were rerandomized into control (3x5Gy-Control) and radiation therapy groups (3x5Gy-3x5Gy). Following treatment, brain samples were collected at various time points out to morbidity to investigate changes in tumor morphology and histology. Further, tumors from morbid mice were collected for viral integration site analysis (VISA), whole-exome sequencing (WES), and NanoString gene expression analysis. Survival analysis demonstrated a significant survival advantage for mice undergoing radiation therapy (+34.2 days) compared to controls. A further survival advantage was found for mice undergoing reirradiation (+30.0 days) compared to mice receiving only one course of radiation. On gross examination of morphology and H&E/SOX2 staining, brains bearing irradiated tumors and reirradiated tumors contained tumor tissue that was more likely to efface olfactory bulb(s) and less infiltrative than control tumors. These histological changes were followed up with VISA which revealed that control tumors harbor fewer clones than in vitro lines and that irradiated tumors harbor the fewest clones of all. Gene expression and IPA analyses showed that pathways involved in cell movement, survival, and proliferation are differentially regulated between irradiated and control tumors. WES was performed to compare gene mutation patterns between reirradiated, irradiated, and control samples. Our results demonstrate that radiation, a central component of glioblastoma treatment, can have wide-ranging effects on the evolution of this dynamic tumor after initial presentation and recurrence. We have demonstrated for the first time the utility of a GSC-initiated orthotopic xenograft model for studying recurrent GBM biology and evolution. This reirradiation model may provide the opportunity to design and test more effective recurrent GBM treatment strategies that are centered around recurrent biology.

#3728

**Inverse effect of** 28 **Si and** 56 **Fe radiation on intestinal tumorigenesis vs. carcinogenesis in APC** 1638N/+ **mice.**

Santosh Kumar, Shubhankar Suman, Bhaskar V.s. Kallakury, Bo-Hyun Moon, Albert J. Fornace, Kamal Datta. _Georgetown University, Lombardi Cancer Center, Washington, DC_.

Space radiation is a major risk factor for gastrointestinal (GI) cancer in astronauts and is a matter of concern. Earlier we have reported higher relative effects for intestinal tumorigenesis in APC1638N/+ mice after exposure to different heavy ion radiation spanning a range of LET values. The purpose of the current study was to histologically grade intestinal tumors into adenoma vs. adenocarcinoma and assess relative effects of different heavy ion radiation on tumorigenesis vs carcinogenesis. Tumors from male APC1638N/+ mice 150 d after exposure to whole-body sham, γ (0.3 keV/μm), 12C (13 keV/μm), 28Si (70 keV/μm), or 56Fe (148 keV/μm) radiation were harvested, fixed, paraffin embedded, and sectioned. Radiation doses were 0.1 Gy, 0.5 Gy and a equitoxic dose of 2.0 Gy γ-rays. H&E stained tumor sections from at least 50 tumors per study group were used for histological grading into adenoma and adenocarcinoma. Hotspot mutations for p53 and Kras genes were assessed using targeted PCR followed by amplicon sequencing in 20 tumors per study group. Our previous published data showed that among the three LETs tested relative intestinal tumorigenic effects (relative to γ-rays) peaked at a LET of 70 keV/μm (28Si) with 13 keV/μm (12C) showing the least effect and 148 keV/μm (56Fe) showing an intermediate effect. In contrast, the current study demonstrates that the frequency of adenocarcinoma peaked after 56Fe with the least effect after 12C and an intermediate effect after 28Si. Relative to γ-rays, the carcinoma frequencies were 42.4+0.9, 13.2+0.61 and 8.0+1.14 higher after 56Fe at doses of 0.1, 0.5 and 2.0 Gy respectively. Whereas the relative carcinoma frequencies of 34.2+1.35, 14.0+0.95 and 9.0+2.25 were observed after 28Si at doses of 0.1, 0.5 and 2.0 Gy respectively. While Kras mutation analysis of tumors did not show any difference among radiation types, p53 mutation analysis demonstrate a higher number of mutations in exon 5 in 56Fe relative to 28Si samples. Collectively, our data show that while LET-dependent tumorigenic effects followed a parabolic shape, carcinogenic effects followed a more linear-like pattern with highest adenocarcinoma percentage and frequency at 148 keV/μm (56Fe). Although inverse effects of LET on tumorigenesis vs. carcinogenesis could in part be attributed to the beams' differential physical properties, our mutation data on p53 supports differential effects of the beams on tumor suppressor mutation.

#3729

Heavy-ion space radiation exposure is a potential risk factor for gastrointestinal tumorigenesis even at extremely low doses.

Shubhankar Suman, Santosh Kumar, Bo-Hyun Moon, Jerry Angdisen, Bhaskar VS Kallakury, Albert J. Fornace, Kamal Datta. _Georgetown Univ., Washington DC, DC_.

Heavy-ion radiation (HZE)-induced carcinogenesis is a major concern in astronauts planning to undertake long-term deep space exploration, such as a mission to the Mars. Due to high-LET (linear energy transfer) characteristics of HZE ions present in deep space environment, the Mars mission is expected to substantially enhance gastrointestinal cancer risk in astronauts, relative to low-LET radiation. Previously, using three different mouse models of human colorectal cancer (APCmin/+, APC1638N/+ and IL10-/- mouse) we have unequivocally demonstrated a significantly higher risk of intestinal and colonic tumorigenesis after exposure to energetic heavy ions. This study aims to obtain quantitative GI-tumorigenesis data at space relevant doses (5-50 cGy) of heavy ions covering a broad-spectrum of linear energy transfer (2-148 keV/μm), relative to γ-radiation.

The APC1638N/+ mouse was used due to its best signal-to-noise ratio among all previously studied mouse models of human gastrointestinal cancer. Both female and male APC1638N/+ mice (6-8 weeks, n=20) were whole-body exposed to sham-radiation, γ-rays, 4He (2 keV/μm), 12C (13 keV/μm), 16O (22 keV/μm), 28Si (69 keV/μm), and 56Fe (148 keV/μm) -ion radiation at 5, 10, and 50 cGy dose at the NASA Space Radiation Laboratory (NSRL) in Brookhaven National Laboratory. Mice were euthanized at 150 d after radiation exposure and intestinal and colon tumor frequency and size were scored and analyzed as a function of dose, LET, and gender.

The highest increase in tumor frequency was observed after 28Si followed by 56Fe, 16O, 12C, and 4He radiation, and male preponderance for tumorigenesis was evident for each radiation type and dose. At 50 cGy dose, no significant difference in tumorigenesis was observed between γ and 4He. However, at lower doses (5 and 10 cGy) significantly higher tumorigenesis was noted after 4He exposure. Furthermore, calculation of relative biological effectiveness (RBE) for tumorigenesis showed the highest value with 28Si and lower doses showed greater RBE relative to higher doses. No statistical difference in tumorigenesis pattern was evident between 16O and 12C at all studied doses. In addition no significant change in tumorigenesis pattern was evident between 28Si and 56Fe in female mice. Analysis showed greater tumorigenesis per unit of radiation (per cGy) at lower doses suggesting radiation-induced tumorigenesis reaching a saturation point at higher doses.

The lack of understanding on radiation quality effects is one of the major uncertainties in space radiation exposure-associated cancer risk prediction. Using a broad-spectrum of HZE-ions we have demonstrated the dependence of gastrointestinal tumorigenesis on radiation quality even at very low doses, which has implications in cancer risk prediction for astronauts and also for accessing secondary cancer risks after particle radiotherapy.

#3730

1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol mitigates the hematopoietic syndrome of lethal acute radiation syndrome in mice.

Yong-Jae Kim,1 Jinseon Jeong,2 Ki-Young Sohn,1 Do Young Lee,1 Sun Young Yoon,2 Jae Wha Kim2. 1 _Enzychem Lifesciences, Daejeon, Republic of Korea;_ 2 _Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea_.

The acute radiation syndrome (ARS) is a broad term used to describe a range of signs and symptoms that reflect severe damage to specific organ systems and that can lead to death within hours or several months after exposure. In this study, we investigated the efficacy of EC-18 for the development of a medical countermeasure for ARS by analyzing IR-induced mortality and morbidity. First, we established a murine model of the ARS by exposing eleven week old male and female BALB/c mice to 6.0-6.5 Gy doses of total body irradiation (TBI; γ-ray, 60Co, 1553 R/min ), and assessed for 30 day survival, mean survival time and lethality dose (LD). The LD70/30 with confidence interval (CI) was 6.11Gy (5.98-6.22Gy). To determine the efficacy of EC-18 in IR-induced mortality, we exposed BALB/c mice to a 6.11Gy dose (LD70/30) of TBI and orally administered 10-250 mg/kg/day of EC-18, starting one day after irradiation. As a result, 6.11Gy of γ-radiation caused the death of 80% of the animals of positive control group within 23 days, with an average life span (ALS) of 17.9 days. The percentages of survival of the irradiated mice with EC-18 10, 50, and 250 mg/kg were 20%, 40%, and 80% with ALS of 19.3, 22.3, and 28.2 days, respectively. Moreover, the LD70/30 dose of γ-ray irradiation caused a substantial decrease in the body weight of the mice. The administration of EC-18 effectively prevented severe weight loss induced by irradiation. Next, we investigated the efficacy of EC-18 for hematopoietic ARS (H-ARS) by analyzing the kinetics of white blood cells (WBC), red blood cells (RBC), and platelets. A single whole body exposure of γ-radiation (6.11Gy) rapidly exhausted all kinds of WBC counts, and the administration of EC-18 significantly attenuated γ-radiation-induced depletion of WBCs in the irradiated mice. Especially, the administration of EC-18 substantially reduced γ-radiation-induced reduction of the ANC. The mean first day of neutropenia (ANC<500cells/μL) of control and EC-18-treated cohorts was 1.8±1.09 and 2.2±1.09 days, respectively. Although EC-18 did not protect the irradiated mice from experiencing severe neutropenia, it effectively reduced the duration of severe neutropenia from 13.0 days to 7.2±1.79 days. In addition, EC-18 significantly increased the mean nadir of ANC after γ-ray irradiation from 4.0±5.48 cells/μL to 20.0±10.00 cells/μL. In addition, the administration of EC-18 in the irradiated mice remarkably attenuated the rapid reduction of RBCs and hemoglobin. When exposed to a supra-lethal dose (8Gy) of γ-radiation, the two of five mice in the control cohort experienced severe skin discoloration and edema formation on the front right feet and hemorrhagic telangiectasia on the tales on day 10. EC-18 remarkably improved γ-radiation-induced skin damage in the irradiated mice. Based on the observations in this study, we concluded that EC-18 has potential as a medial countermeasure for ARS.

#3731

A novel heat shock protein 90-targeted photosensitizer (HS-201) enables enhanced tumor-specific photodynamic therapy of inflammatory breast cancers.

Kensuke Kaneko, Takuya Osada, Philip F. Hughes, Timothy A. Haystead, Michael A. Morse, Herbert K. Lyerly. _Duke University, Durham, NC_.

Background: Photodynamic therapy (PDT) is an effective local anti-cancer modality applied for the treatment of early stage disease and palliation of advanced disease, and could be used in managing inflammatory breast cancer (IBC). Although PDT can be focused by limiting the area of near infrared (nIR) light exposure, normal tissue injury due to the photosensitizer (PS) uptake by non-tumor tissues remains a barrier to broader applicability. We reported that selective delivery of a compound to malignant cells by exploiting their heat shock protein 90 (Hsp90) activity could be used for imaging breast cancers. This work was extended by the development of an Hsp90 targeted PS (HS201), in which a well characterized PS, verteporfin (VP), has been chemically tethered to a small molecule Hsp90 inhibitor and assessed the tumor-specificity and antitumor efficacy of HS201-based PDT (HS201-PDT).

Methods & Results: HS201 was taken up by human breast cancer cell lines, including IBC in vitro more readily than VP, resulting in increased tumor killing in vitro after exposure to a laser with a 690 nm wavelength. The in vivo selectively of HS201 uptake in tumor tissue was confirmed in both human breast cancer xenograft models and spontaneous tumors from HER2-transgenic mice. HS201 accumulation in the tumor was dramatically enhanced after the first laser exposure due to the feed forward increase in Hsp90 expression in the treated tumor cells, and thus a repeated laser exposure was conducted to enhance antitumor effects. This strategy enhanced antitumor effects, as MDA-MB-231 tumors implanted into SCID-beige mice responded to HS201-PDT but not to VP-PDT. Human xenograft breast cancer cell lines, including the IBC cell lines SUM149 and KPL-4, as well as non-IBC cell line BT474M1 and the patient-derived breast cancer xenograft HCI-013, were also tested in SCID-beige mice and demonstrated significantly suppressed tumor growth by HS201-PDT.

Conclusions: HS201 showed selective uptake and longer retention by tumor cells, including IBC, especially when the tumors were exposed to local laser treatment. Due to the advantage of this feed-forward control of HS201 distribution, HS201-PDT demonstrated superior anti-tumor effects compared to VP-PDT against various human breast cancer xenografts in mice. These results suggest that HS201-PDT may be used in managing IBC, and may have an important role in anti-cancer therapy.

#3732

Targeted photodynamic therapy enhances the therapeutic efficacy of combination therapy (PDT and chemotherapy) on chemoresistant melanoma cells.

Lester M. Davids,1 Fleury Nsole Biteghe,2 Eden Padayachee,2 Stefan Barth2. 1 _University of Pretoria, Pretoria, South Africa;_ 2 _University of Cape Town, Cape Town, South Africa_.

Cutaneous melanoma remains refractory to current therapies such as chemotherapy. Failure of treatment has been attributed to enhanced self-renewal capacity and the over-expression of ABC transporters by therapy resistant populations. A need therefore exists to identify and evaluate new approaches. Alternative combinatorial approaches comprising chemotherapeutics, photodynamic therapy (PDT) and targeted-PDT have been proposed to overcome resistance.

Objective

This study set to investigate the efficacy of novel (targeted-PDT-IR700) and standard combinatorial effect of PDT and chemotherapy (Hypericin-PDT and DTIC) as an adjunctive combination therapy treatment to decrease cellular chemoresistance in melanoma. This was assessed in an in vitro model, to show differences in killing of melanoma cells.

Methodology Single chain variable fragments (scFv) of panitumumab were cloned into a transient eukaryotic SNAP expression vector co-expressing cytosolic proteins and transfected into HEK293T cells. Purified recombinant protein was confirmed by SDS-PAGE and Western blot analyses. Benzylguanine (BG) modified IR700 was irreversibly conjugated in a 1:1 stochiometric ratio to the 1711(scFv)-SNAP using the self-labelling property of the suicide enzyme SNAP-tag. Binding activity and cytotoxicity was documented on EGFR expressing melanoma cells using flow cytometry, confocal microscopy and XTT assays. Cytotoxic activity, self-renewal capacity and ABC transporters expression (ABCB5 and ABCG2) after the combination therapy (HYP-PDT and DTIC) was determined using XTT, clonogenic assays and flow cytometry.

Results Binding and internalization of purified 1711(scFv)-SNAP conjugated to BG-modified Alexa 488 or IR700 was successfully confirmed by confocal microscopy and flow cytometry. Cell viability showed cell specific cytotoxicity of the photosensitizer labeled fusion proteins upon light induction, at nanomolar concentrations which were lower than those used for the non-targeted combinatorial approach (HYP-PDT and DTIC). Moreover, DTIC resistant melanoma cells were less sensitive to therapeutic treatment than their non-resistant counterpart. This correlated with an increased self-renewal capacity and ABC transporter expression in DTIC resistant melanoma in comparison to their non-resistant counterpart. This study describes a proof of concept of selective targeting and elimination of EGFR expressing melanoma cells by targeted photoimmunotherapy using a panitumumab 1711-scFv fused to SNAP tag and conjugated to near infrared photosensitizer IR700 which improve therapeutic efficacy with lower concentration and reduced sides effect. This work highlights the efficacy of targeted PDT and suggest that it is highly applicable to improve the current clinical arsenal of therapies.

#3733

Development of photoimmunotherapy for colon cancer in nude mice models.

Hannah Hollandsworth,1 Siamak Amirfakhri,1 Filemoni Filemoni,1 Sahar Razemjooie,1 Paul Yazaki,2 Robert Hoffman,3 Michael Bouvet1. 1 _UCSD, San Diego, CA;_ 2 _City of Hope, CA;_ 3 _AntiCancer, San Diego, CA_.

Introduction: Photoimmunotherapy (PIT) uses tumor specific monoclonal antibodies conjugated to photosensitizer phthalocyanine dye IR700 to produce cytotoxicity. To date, there is no published data on the use of PIT in colorectal cancers, which could be useful as an adjunct to surgery to prevent local recurrence, or as a primary therapy.

Methods: Humanized anti-CEA antibody (m5A) was conjugated to IR700CW dye (LI-COR). In vitro PIT was performed using a human colon cancer cell line LS174T. Cancer cells (2x103) were seeded on a 96-well plate and incubated for 24 hours. m5A-IR700CW containing media was added to the cells. Cells received either 2 minutes of PIT or no treatment. PIT was administered at 4 J/cm2, 8 J/cm2 or 16 J/cm2. Cell viability was measured on a microplate reader with CellTiter 96 Aqueous One Solution (Promega, Madison, WI). Subcutaneous implantation of LS174T was performed on the bilateral flanks of four male nude mice. Tail vein injection of 25 micrograms of m5A-IR700CW reconstituted in 100 microliters PBS was performed for each mouse. Animal imaging was performed with a LI-COR Pearl Trilogy imaging system. Right flank tumors of three mice were treated with a 690 nm laser at 150 mW/cm2 for 30 minutes, delivering a total of 270 J/cm2. A non-treated mouse served as the control. Images were obtained prior to PIT, immediately after PIT and 10 minutes, 20 minutes, 30 minutes and 24 hours after treatment. Maximum fluorescence intensity was measured at each time point. Tumor size was measured prior to treatment, 24 hours, 48 hours, 5 days and 10 days after treatment.

Results: In vitro PIT was effective to kill the cancer cells in a dose response manner. In vivo PIT decreased tumor fluorescence intensity in a time-dependent manner. PIT arrested tumor growth in all mice, compared to the control, which demonstrated continuous tumor growth.

Conclusion: PIT affects cancer cell death in vitro and arrests tumor growth in colon cancer models.

#3734

Cancer cell-targeted photoimmunotherapy elicits immunogenic cell death and activates the innate and adaptive immune response in the tumor microenvironment.

Michelle A. Hsu, Stephanie M. Okamura, Daniele M. Bergeron, Deepak Yadav, Jerry J. Fong, Roger Heim, Miguel Garcia-Guzman. _Rakuten Aspyrian, San Diego, CA_.

INTRODUCTION: Cancer cell-targeted photoimmunotherapy (PIT) is a platform technology under development for the treatment of various cancers. PIT is a drug + device combination that utilizes monoclonal antibodies conjugated to a dye (IRDye 700DX) that are activated with nonthermal red light illumination to induce rapid cell death by necrosis. Binding of the antibody-dye conjugate to cancer cells followed by photoactivation with nonthermal red light elicits rapid necrosis of the cancer cells bound to the antibody conjugate, providing very high cancer cell specificity. Given the rapid cell necrosis induced by PIT treatment, we hypothesized that PIT also induces immunogenic cell death (ICD) as a step to activate immune cells in the tumor microenvironment. The objective of this study was to evaluate, through in vitro and in vivo experiments, whether PIT results in ICD of targeted cancer cells and activation of the innate and adaptive immune response.

METHODS: Human cancer cells (A431 and FaDu cells) were targeted by PIT, and evaluated for ICD markers in vitro. Human dendritic cells exposed to supernatants of PIT-killed cancer cells were evaluated for activation markers and cytokine production. An immunocompetent mouse model for PIT was also developed to determine intratumoral immune activation after treatment with PIT.

RESULTS: After photoactivation, PIT-targeted human cancer cells upregulated cell surface ICD markers Hsp70, Hsp90, and calreticulin, as well as release of intracellular HMGB1. Human dendritic cells exposed to PIT-killed cell supernatants exhibited markers of immune activation (CD86 and MHCII), and secreted proinflammatory cytokines including TNF, IP-10, IL-1β, MIP-1a, MIP-1b, and IL-8. In an immunocompetent mouse model, tumors treated by PIT displayed increased percentage of intratumoral CD11c+ dendritic cells with activation markers MHCIIhigh, CD80, and PD-L1. In addition, intratumoral natural killer cells from PIT treated tumors displayed increased cytotoxic activity (CD3-DX5+CD69+ and CD3-DX5+CD107a+), as well as an increased population of total CD3+ CD8+ T cells, compared to non-PIT treated tumors.

CONCLUSION: Cancer cells killed by PIT undergo ICD, which results in the activation of intratumoral innate and adaptive immune response in a preclinical mouse model. Combination studies with PIT and immune modulators are warranted to explore potential synergistic anticancer effects.

#3735

An anti-tumor immune response is evoked by partial-volume single dose radiation.

Adriana Haimovitz-Friedman, Johnn Humm, Joseph O. Deasy, Jedd Wolchok, Taha Merghoub, James Russell, Hongyan Li, Robert Samstein, Sadna Budhu, Simon Powell, Chloe Bodden, Ela Markovsky, Qing Chen. _Mem. Sloan Kettering Cancer Ctr., New York, NY_.

Purpose: To study tumor growth delay resulting from partial irradiation in preclinical mouse models.

Methods and Materials: We investigated 67NR murine orthotopic breast tumors in both immunocompetent and nude mice. Treatment was delivered to 50% or 100% of the tumor using a 2x2 cm collimator on a micro-irradiator. Radiation response was modulated by treating with anti-CD8 and anti-ICAM antibodies. Similar experiments were performed using the less immunogenic Lewis Lung Carcinoma (LLC) mouse model. Tumor growth delay and γH2AX phosphorylation were measured and immune response was assessed by immunofluorescence and flow cytometry at 1 and 7 days post-radiotherapy (RT). Tumor expression of cellular adhesion molecules was also measured at different times post-RT.

Results: Partial irradiation led to tumor responses similar to fully exposed tumors in immunocompetent mice, but not in nude mice. After a single dose of 10Gy, infiltration of CD8+ T cells was observed, along with increased expression of ICAM. The response to 10Gy in hemi-irradiated tumors was abrogated by treatment with either anti-CD8 or anti-ICAM antibodies. Similar responses were obtained in the less immunogenic LLC mouse model delivering 15Gy to half the tumor volume. Treatment with FTY720, a compound that inhibits T cell egress from lymph nodes, did not affect tumor response at the time of CD8+ T cells infiltration in the non-irradiated area of the tumor, indicating that the most likely source of these cells is the irradiated portion of the hemi-irradiated tumors. In addition, a significant abscopal effect was observed after partial irradiation with a single dose of 10Gy in the 67NR model.

Conclusions: In these models, radiation controls tumor growth both directly through cell killing and indirectly through immune activation. This raises the possibility that this effect could be induced in the clinic.

#3736

Space radiation-induced decline in gut autophagy and expansion of both mitotic and senescent population denotes an aging phenotype with enhanced cancer risk.

Kamal Datta, Shubhankar Suman, Bo-Hyun Moon, Albert J. Fornace. _Georgetown Univ., Washington, DC_.

Exposure to space radiation is known to alter a myriad of cellular processes that could potentially affect astronauts' health during and after long duration's space missions. Due to high-LET (linear energy transfer) characteristics of energetic heavy ions (HZE) present in space, astronauts are predicted to be at higher risk for gastrointestinal cancer development. Proliferative epithelial cells in gastrointestinal (GI) tissue are highly radiosensitive and in our previous studies we have demonstrated a persistent stress phenotype in crypt cells after heavy ion space radiation exposure. However, uncertainty remains in our understanding of how heavy ions induce chronic stress and inflammation is linked to higher cancer risk. The purpose of the current study was to assess late effects of heavy ion (56-Fe) on the autophagy process that is required to recycle damaged cellular components and its role in HZE-induced gastrointestinal carcinogenesis. C57BL6/J mice were exposed to 1.6 Gy of 56-Fe radiation at NASA Space Radiation Laboratory (NSRL). Mice were euthanized 2-month after radiation and intestinal tissues were analyzed for oxidative stress and associated DNA damage, and alterations in antioxidant response and autophagy signaling. Further, cells underdoing mitosis, autophagy and stress-induced senescence were also scored and alterations in cell signaling pathways with established role in carcinogenesis were evaluated. Energetic heavy ions exposure resulted in a long-term increase in intracellular reactive oxygen species (ROS), accompanied with decreased expression of key antioxidant genes (Gpx, SOD and catalase). Increase in both mitochondrial and cytosolic ROS was evident as both loss of mitochondrial membrane potential (MMP) and increase in prooxidant Nox1 expression was found. Paradoxical increases in phospho-histone-3 positive (mitotic) and p16 positive (senescent) cells were found, however decreases in autophagy positive (LC3B) cells was noted in HZE-exposed mouse intestine. Upregulation of upstream mTOR-PI3K-AKT signaling and p62 were noticed in long-term suppression of autophagy (Atg12, LC3B and Beclin-1) after HZE-exposure. Here, we show that space radiation downregulated autophagy processes, which are an important cellular mechanism involved in suppression of carcinogenesis. When considered along with upregulation of proliferative pathways and increased oxidative stress, our data suggest increased risk of chronic GI diseases including cancer due to late persistence of damaged organelles in cells after energetic heavy ion radiation exposures. Collectively, this study demonstrates a unique phenomenon of heavy ion-induced suppression of autophagy concurrent with increases in both mitotic and senescent cells indicating a premature aging phenotype with enhanced cancer risk.

#3737

Targeting the YKL40 pathway inhibits monocyte-mediated cell migration of prostate cancer cells following radiotherapy treatment.

Chris W. Armstrong,1 Melissa LaBonte Wilson,1 Kelly M. Redmond,1 Suneil Jain,1 David J. Waugh2. 1 _Queen's University Belfast, Belfast, United Kingdom;_ 2 _Queensland University of Technology, Brisbane, Australia_.

Introduction: Loss of the tumor suppressor PTEN is associated with relapse following radiotherapy (RT) in men with prostate cancer (PCa). We have previously shown that PTEN-deficient tumors display increased monocyte and macrophage infiltration. Co-culture models have confirmed that monocyte-derived paracrine signaling factors can significantly alter the radiation response of PCa cells. The aim of this study is to identify monocyte-derived cytokines that can be therapeutically targeted in combination with RT to reduce the occurrence of relapse in men with PTEN-deficient PCa.

Methods: Techniques employed included: cytokine arrays to detect radiation-induced proteins secreted from monocytes; IF and IHC on FFPE tumor sections; in vitro biological assays extending to RT-PCR, ELISA, immunoblotting and transwell migration assays; and analysis of relevant genes in the FASTMAN radiotherapy patient cohort.

Results: Cytokine array analysis of monocytes with and without prior ionizing radiation exposure identified several upregulated secreted proteins - the most abundant of which was YKL40 (CHI3L1). This was further potentiated in co-culture with PCa cell lines. Secretion of YKL40 was confirmed as being monocyte-derived by ELISA and this was further supported by IF co-staining of YKL40 with a monocyte marker (F4/80) in PTEN-deficient tumour sections. Exposure of PCa cell lines to conditioned media (CM) from irradiated monocytes increased cell migration - an effect that was also observed by direct addition of rhYKL40. Previous studies have reported the role of syndecan-integrin complexes in mediating YKL40 signal transduction. Immunoblotting confirmed expression of these receptors (Sdc1 and αvβ3) on PCa cell lines. Using siRNA to knockdown Sdc1 expression inhibited YKL40-induced activation of the FAK pathway and subsequently attenuated YKL40-induced cell migration. In addition, YKL40 was shown to increase expression of genes involved in both actin cytoskeleton rearrangement and epithelial-mesenchymal transition. Use of a FAK inhibitor (PF-573228) successfully prevented the induction of these genes and attenuated the capacity of YKL40 to drive cell migration. The clinical relevance of our observations is supported by high expression of YKL40-receptor and signaling mediators in prostate cancer biopsy tissue and its correlation to increased biochemical recurrence following RT treatment.

Conclusion: Use of a YKL40-pathway-targeted agent alongside radiotherapy may reduce disease recurrence in men with high-risk clinically-localized PTEN-deficient PCa by preventing radiation-induced cell migration and metastatic escape. Current and future experiments will aim to confirm this via a series of relevant in vivo studies.

#3738

A STING independent type-1 interferon response induced by fractionated radiotherapy coincides with altered tumor growth and clonogenicity.

Ruben S. Goedegebuure,1 Esther A. Kleibeuker,1 Kitty C. Castricum,1 Jaap van den Berg,1 Sarah Derks,1 Henk M. Verheul,1 Ben J. Slotman,1 Adrian Harris,2 Victor L. Thijssen1. 1 _Amsterdam UMC, Vrije Universiteit Amsterdam, Cancer Center Amsterdam, Amsterdam, Netherlands;_ 2 _Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom_.

Introduction To improve radiotherapy efficacy, insight in the dynamic biological responses that occur during clinical radiation schedules is vital. In the current study we explored gene expression changes during the course of fractionated radiotherapy (FrRT) that might be targeted with combination treatment strategies.

Methods Human cell lines HT29, SW480, HCT116, COLO320, RKO (colorectal carcinoma), D384 (glioblastoma) and OE19 (esophageal adenocarcinoma) as well as HT29 xenograft tumors in nude mice were irradiated up to 3 to 5 weeks with daily fractions of 2 Gy for 5 days per week. In vivo tumor growth was monitored daily and in vitro clonogenic survival was determined every other day. At the end of each treatment week tumors and cells were harvested for RNA isolation. Subsequently, gene expression analysis was performed by RNA sequencing and micro-array analysis. Altered gene expression levels were validated by qPCR. Protein expression was determined by ELISA and Western blot.

Results Tumor growth of the HT29 xenografts declined after 2 weeks of FrRT compared to non-irradiated tumors. In irradiated cell lines, clonogenic survival analyses revealed a log-linear decline in survival in the first 2 weeks, after which a steady-state-like phase was reached up to 5 treatment weeks. Gene expression analysis of both xenograft tumors as well as irradiated cell lines at this 2 week time point identified a type-1 interferon (IFN) mediated signaling pathway as the most significantly enriched biological process. The induction of a selection of IFN-stimulated genes (ISGs) was confirmed by qPCR. Comparable responses were observed in both STING positive (HT29, SW480, HCT116, D384) and negative (COLO320, RKO, OE19) cell lines. To get more insight in the dynamics of the IFN response we analyzed ISGs expression at the end of each treatment week. This revealed a peak in ISGs expression after 10 to 15 fractions, both in vivo and in vitro. The ISGs expression levels decreased with continuation of treatment but always remained above the basal level of non-irradiated cells. Single dose irradiation up to 10 Gy also induced dose-dependent ISGs expression, albeit typically 5 to 10 fold lower as compared to FrRT. Subsequent protein and mRNA expression analyses consistently revealed induction of IFN beta expression, with peak levels measured after 10 to 15 fractions. In addition, occasional induction of IFN lambda 2/3 protein and mRNA expression was observed.

Conclusion FrRT induces an intrinsic type-1 IFN response which occurs independent of STING. This response is associated with induction of IFN beta expression, which peaks after 2 to 3 weeks of treatment and coincides with a shift in in vivo tumor growth and in vitro clonogenic survival. Targeting this response during FrRT might be exploited as a novel combination treatment.

#3739

Characterization of the effects of DIM (3,3'-Diindolylmethane) on irradiated tumors and anti-tumor immunity.

Renxiang Chen, Lijun Li, Lorreta Yun-Tien Lin, Albert J. Fornace, Heng-Hong Li. _Georgetown Lombardi Comp. Cancer Ctr., Washington, DC_.

The overall goal of this project is to determine the efficacy of DIM (3,3'-Diindolylmethane) to protect normal tissues during radiotherapy but not enhance the survival of irradiated tumors. We tested expression change of p53-downstream genes in the absence and presence of DIM after radiation exposure using hTERT-immortalized human mammary epithelial cells. Radiation-induced gene inductions for both p21 and Wip1 were found attenuated in cells pretreated with various concentrations of DIM compared to the vehicle control. To reveal DIM's effects on the global transcriptome of irradiated cells, we performed whole genome gene expression microarray analysis. Pathway analysis results showed that the differentially expressed genes are involved in cell cycle, p53 network, DDR, and related pathways. Gene regulation pattern at the later post-radiation time point in the presence of DIM shows similarity with that of early time point with no DIM, which suggests that DIM treatment delays responses of cell-cycle-related genes. We monitored tumor growth after localized radiation in tumor xenograft models of MDA-MB-231 and HCC1937 human breast carcinoma cell lines using immunocompromised NSG mice. The fractionated regimen consists of a total dose of 24 Gy in four fractions (6 Gy per fraction given every other day) with administration of DIM or vehicle at one hour prior to each fraction. DIM showed no significant effect on the growth of irradiated tumors, i.e. DIM did not affect the treatment efficacy of radiation on tumors. To investigate effects of DIM on tumor microenvironment, we treated tumors with fractioned radiation in an E0771 syngeneic tumor model. DIM treatment enhanced tumor infiltration of lymphocytes population. These studies support the goal of developing DIM as a clinical radioprotector in order to improve the therapeutic index by allowing higher doses of radiation to improve local tumor control by reducing late dose-limiting normal tissue toxicity by radiation.

#3740

Ionizing radiation-induced transcriptomic changes in triple-negative breast cancer cells reveal putative mechanisms of treatment resistance.

Victor T. Lin, Tshering D. Lama-Sherpa, Rajeev S. Samant, Lalita A. Shevde. _University of Alabama at Birmingham, Birmingham, AL_.

Breast cancer is the most common malignancy and the second leading cause of cancer-associated deaths in women. Of these, triple-negative breast cancers (TNBCs) remain the most difficult to treat. Unlike hormone-driven breast cancers, TNBCs that remain in remission past the five-year mark rarely relapse and are considered functionally cured. Thus, investigating mechanisms of treatment resistance in order to improve remission rates is of paramount importance. In this study, we use RNA-Seq to analyze global transcriptomic changes in TNBC cells in response to DNA damage induced by ionizing radiation (IR). Applying gene set enrichment analysis (GSEA), we have identified a number of expression signatures that represent putative mechanisms of resistance to treatment. In prior work, we have demonstrated the role of the transcription factor Gli1, the terminal effector of the Hedgehog (Hh) signaling pathway, in regulating the expression of DNA repair proteins in response to genotoxic damage with cisplatin. We have previously shown that Hh inhibition blunts the expected upregulation of specific proteins involved in single-strand DNA repair in ovarian cancer cells. Here, we expand on our earlier studies by examining TNBC cells with high levels of Hh activity and querying how global transcriptomic changes in response to IR are altered by Hh inhibition. Our data suggest that rational manipulation of these pathways may sensitize TNBCs to treatment and improve the functional cure rate of these tumors.

#3741

Identification of RNA binding proteins influenced by ionizing radiation through RNA interactome capture.

Stacey L. Lehman, Theresa Wechsler, Gaelyn C. Lyons, Lisa M. Jenkins, Kevin Camphausen, Philip J. Tofilon. _National Cancer Institute, Bethesda, MD_.

Cells respond to ionizing radiation (IR) through the activities of constitutively expressed proteins and through changes in gene expression. Constitutively expressed proteins play well-defined roles in major components of the cellular radioresponse, such as DNA repair and cell cycle. However, the contribution of changes in gene expression to the radioresponse is less understood. We have previously demonstrated through polysome profiling and microarray analysis that translational control is a key component of radiation-induced changes in gene expression. In response to IR, specific transcripts are recruited to or away from polysomes without affecting the global polysome profile of the cells. One mechanistic explanation for this observation is that IR influences the activity of RNA binding proteins (RBPs), which regulate the inclusion or exclusion of transcripts from polysomes. Here, we utilized RNA interactome capture (RIC) to identify RBPs bound to transcripts in control and irradiated human cancer cells. Cells were either mock irradiated or treated with 2Gy. One hour post-IR, RNA and protein complexes were crosslinked with 254 nm UV light. mRNA was isolated from cell lysates with oligo(dT) magnetic beads followed by several rounds of highly stringent washes to remove non-specific interactors. RNA was removed from crosslinked proteins by RNase digestion. The proteins were separated on SDS-PAGE gels and subjected to in-gel trypsin digestion, and the subsequent peptides were analyzed by label-free quantification mass spectrometry using an Orbitrap Fusion mass spectrometer. Proteome Discoverer 2.2 was used to search the data against human proteins from the UniProt database using SequestHT with a 1% false discovery rate. We found that we were able to efficiently pull down RNA:protein complexes with a crosslinking dose of 150 mJ/cm2. As determined by silver staining, the protein pattern of crosslinked samples differed greatly from protein isolated from whole cell lysates. By western blot, this method highly enriched for known RBPs, such as PTBP1 and CUGBP1, while contamination from non-RBPs, such as actin and tubulin, was not detected. By mass spectrometry, we identified hundreds of RBPs in each replicate above the no crosslinking background control for both untreated and irradiated cells. However, more RBPs were consistently detected in the control cells. Examples of RBPs preferentially binding in control cells include SSB, a protein involved in a variety of RNA metabolic processes, and U2AF2, a protein involved in RNA splicing. Overall, these data demonstrate that RIC is a sensitive and rigorous method to identify RBPs and that RBPs may play a role in radiation-induced post-transcriptional gene regulation. Preferential binders identified by this method may provide further insight into pathways regulating tumor cell radiosensitivity.

#3742

Exosome-mediated EGR1 transportation controls the radiation-induced abscopal effect in head and neck cancer.

Mohammady Akbor, Liang-Ting Lin. _The Hong Kong Polytechnic University, Kowloon, Hong Kong_.

Head and neck cancer (HNC) are one of the vital types of metastatic cancer, and radiotherapy (RT) is the convincing treatment regarding its location. RT can regulate cancer growth in not only directly irradiated cells but also non-irradiated remote cells by abscopal or bystander effects. It is evident that radiation-induced Early Growth Response 1 (EGR1) is involved in the control of such type cancer progression. Also, exosomes are established carriers for biological factors basically in cell communication. In this study, we would like to identify the EGR1 as a functional molecule that carried by the radiation-induced exosome to control the HNC growth to disclose the molecular mechanism of the abscopal effect. In this case, we used FaDu cell, a hypopharyngeal cancer, for in vitro experiments. Irradiation was given to the cells in a dose-dependent manner from 0 to 12 Gy followed by exosome isolation using miRCURY Exosome Cell Kit. Characterization of the isolated exosomes was performed by Nanoparticle Tracking Analysis (NTA) for vesicle sizes and concentration, and by transmembrane markers CD63 and CD9 through western blot. We treated unirradiated FaDu cells with isolated exosomes to address the phenotypic changes. MTT assay and colony-forming assay observed the cell viability and survival. Moreover, the expression of EGR1 in both radiation-induced exosome and the exosome-treated cells were determined by western blot. To validate the causal effect of exosomal transportation of EGR1, we assessed the viability changes in EGR1-silenced FaDu cells with exosome treatment. Our results indicated that the number of exosomes was increased while the amounts of protein in each isolated exosome were also increased in a radiation dose-dependent manner. CD63 and CD9 had been identified through western blotting. Moreover, the enhanced expression of EGR1 was observed in both radiation-induced exosomes and the cells treated with isolated exosomes. The possible abscopal effect was evident by the significant decrease of the viability of unirradiated FaDu cells treated with radiation-induced exosomes. Besides, elevated expression of EGR1 was noticed in the radiation-induced exosomes comparing to non-irradiated cohorts. Notably, EGR1-containing exosomes can suppress the proliferation of the unirradiated FaDu cells. Alternatively, radiation-induced EGR1-containing exosome failed to inhibit the proliferation of EGR1 knockdown FaDu cells. In conclusion, radiation-induced EGR1 via exosome transportation contributes to the mechanism of the abscopal effect for metastatic head and neck cancer.

#3743

Neutrophil extracellular traps and their implication with radioresistance in muscle invasive bladder cancer.

Surashri Shinde-Jadhav, Jose Joao Mansure, Roni Rayes, Mina Ayoub, Jonathan Spicer, Wassim Kassouf. _McGill University Health Centre, Montreal, Quebec, Canada_.

PURPOSE: Radiotherapy modifies diverse components of the tumor microenvironment and inflammation plays a pivotal role in modulating radiation responsiveness of tumors. Neutrophils are one of the first-line responders during the acute phase of inflammation and are increasingly being recognized as drivers of tumor progression. One mechanism by which neutrophils play a role in tumor progression is through the formation of neutrophil extracellular traps (NETs). NETs are web-like structures expelled by the neutrophil composed of DNA studded with various proteins. Initially, this was described as a mechanism of antimicrobial defense but lately NETs have been associated with a variety of adverse effects, such as pathogenesis of autoimmune diseases, surgical stress, tumor progression, and metastasis. Recent studies show that the protein High Mobility Group Box-1 (HMGB1), a key player in radioresistance can in fact promote NETs. Importantly, the impact of NETs has not yet been explored in the context of radiation, so we sought to explore this further.

METHODS: In vitro: a) Human neutrophils isolated from healthy donors were stimulated with 50ng of rHMGB1 for 4 hours. NETs were quantified through Sytox green fluorescence. b) Neutrophils were co-cultured with irradiated or non-irradiated conditioned media from UM-UC3 human bladder cancer cell line in combination with glycyrrhizin (GLZ), a natural inhibitor of HMGB1. In vivo: Murine bladder cancer cell line (MB49) was subcutaneously implanted into flanks of C57BL/6 and NETosis deficient PAD4-/- mice. Tumors were irradiated (2x5Gy) using the XRAD Smart Irradiator. Intraperitoneal injections of GLZ were used to modulate HMGB1 and intramuscular injections of DNAse were used to deplete NETs. Tumor volumes were measured using a digital caliper till endpoint.

RESULTS: Our in vitro results demonstrate incubation of neutrophils with 50ng rHMGB1 significantly induced NETs formation compared to controls (p<0.0001) and this was reversed through addition of GLZ (p<0.0001). Similarly, co-culture of neutrophils with irradiated MB49 conditioned media induced NETs formation (p=0.01) and this effect was reversed with GLZ (p=0.009). Our in vivo results demonstrate that NETosis deficient mice treated with an HMGB1 inhibitor significantly improves response to radiation therapy. PAD4-/- mice treated with GLZ showed delayed tumor growth kinetics (p=0.023) and increased overall survival post radiation (p=0.0231) compared to all other irradiated arms: C57BL/6, PAD4-/-, C57BL/6 + DNAse and C57BL/6 + GLZ. Similarly, C57BL/6 mice treated with DNAse and GLZ also showed a delay in tumor growth kinetics post radiation (p<0.0001).

CONCLUSION: NETs may induce radioresistance through interactions with HMGB1. Highlighting the role of HMGB1 in NET formation will provide valuable information on the responses that occur in the tumor microenvironment post radiation therapy.

#3744

Repurposing the neuroprotective agent dimethyl fumarate against white matter damage and cognitive decline after radiotherapy.

Alexandra Taraboletti, Albert J. Fornace. _Georgetown University, Washington, DC_.

Radiation therapy (RT) is a critical tool for the treatment of metastatic brain tumors, most commonly glioblastoma multiforme (GBM), however, patients can have significant cognitive impairment due to the exposure to ionizing radiation. Up to 50%-90% of adult patients that survive these brain tumors will develop radiation-induced cognitive dysfunction within 3-6 months. Brain injury from IR is characterized by white matter damage from the loss of myelin-producing oligodendrocyte cells, subsequent demyelination, and vascular inflammation. While neuro-oncology outcomes are often concerned with survival, strategies to ameliorate and understand radiation-induced demyelination after IR treatment are needed to preserve and improve patient quality of life (QOL). Here we investigate the ability of dimethyl fumarate (DMF), an established neuroprotective agent, to amend damage and demyelination caused by RT in oligodendrocyte cells versus glioma cells. Our in vitro study confirms that DMF acts opposingly in each cell type at lower doses (10 nM), but proves toxic in both the tumor and normal model at high concentrations (100 µM). DMF also increased survival rates in oligodendrocytes after radiation. Using metabolomics, we also noted that oligodendrocyte cells upregulated TCA cycle intermediates in response to DMF treatment with RT. This work provides a basis for in vivo dosing and timing strategies using DMF to cause cessation of ROS-driven oligodendrocyte loss and inflammation after RT. Ultimately, DMF is a promising therapy that could be used to ameliorate radiation-induced demyelination, promoting patient QOL.

#3745

Mechanisms of HIF2-mediated small intestine radioprotection.

Carolina J. Garcia Garcia,1 Suman Govindaraju,2 Marimar de la Cruz Bonilla,1 Cullen M. Taniguchi1. 1 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Moffitt Cancer Center, Tampa, FL_.

Pancreatic cancer is the fourth-leading cause of cancer-related deaths in the United States, and it is projected to become the second by 2030. The 5-year overall survival rate for locally advanced pancreatic cancer (LAPC) remains < 5%. Chemotherapy regimens often fail due to poor drug delivery and penetration into the tumor. Because surgery is not feasible by definition in LAPC, radiation therapy (RT) is the only option to achieve local control. However, irradiating the pancreas is challenging because potentially curative doses are limited by the neighboring small intestine, which can only tolerate a maximum of 50Gy. A selective radioprotector of the intestinal tract could allow pancreatic cancer patients to receive a higher, and potentially definitive, dose of radiation by increasing the duodenum's tolerance to survive radiation injury. The EGLN prolyl hydroxylases are cellular oxygen sensors that regulate cell survival and metabolism through the degradation of hypoxia-inducible factors (HIFs) in the presence of oxygen. Our group has shown that HIF2 stabilization through genetic or pharmacologic inhibition of the EGLN family prevents death from radiation-induced gastrointestinal toxicity in mice without protecting the primary tumor. To understand the mechanism by which HIF2 mediates this protection, we generated intestinal organoids from mice with a Cre-inducible allele that expresses a non-degradable form of human HIF1 or HIF2. Whole transcriptomic analysis revealed HIF1 and HIF2 have distinct gene responses in the small intestinal crypt. HIF2 induced the expression of known radiation modulators and genes involved in oxidative damage response, tissue healing, and intestinal homeostasis such as the non-canonical Wnt5a. We validated HIF2 drives Wnt5a expression in multiple organoid model systems. We then generated knockouts (KO) of Wnt5a or Ror2, its cognate receptor, using the Cre/lox system to test if Wnt5a is necessary and sufficient for radioprotection. By using the spheroid formation assay, a modified clonogenic assay, we found Wnt5a KO organoids had decreased crypt regeneration following RT. Our preliminary data suggest Wnt5a protects the duodenum from RT by increasing intestinal stem cell survival.

#3746

Fasting in mice enables abdominal radiation dose escalation in the setting of pancreatic cancer by mitigating small intestinal toxicity.

Marimar de la Cruz Bonilla, Kristina M. Stemler, Sabrina Jeter-Jones, Tara N. Fujimoto, Jessica Molkentine, Gabriela M. Asencio Torres, Cullen M. Taniguchi, Helen Piwnica-Worms. _MD Anderson Cancer Center, Houston, TX_.

Surgical resection is the only potentially curative treatment for pancreatic cancer, but only 15-20% of patients have resectable tumors. In unresectable cases, stereotactic body radiotherapy (SBRT) may be used to give tumor-directed radiotherapy (RT). Unfortunately, this can cause severe gastrointestinal (GI) toxicity due to proximity of the pancreatic head to the duodenum. Protecting the intestine from the toxic side-effects of radiation may enable dose-escalation that could achieve more effective local control of disease. We and others have previously shown that a fast of 24 hours protects mice from lethal doses of the DNA-damaging agent etoposide. In this study, we demonstrate that a 24 hour fast also protects mice from lethal doses of total-abdominal radiation. Histologic analyses using the Withers-Elkind microcolony assay show that fasting protected small intestinal (SI) stem cells from radiation damage and promoted early regeneration. To show a proof-of-principle for the use of this radioporotective maneuver in cancer therapy, we used an orthotopic model of pancreatic cancer using KPC tumor cells syngeneic to C57BL/6. Here, we show that fasting-mediated intestinal protection enabled dose escalated SBRT for treatment of these orthotopic tumors. RT with fasting-mediated radioprotection delayed tumor growth and improved survival compared to controls. Given this robust phenotype, we developed a 3D culture ex vivo assay using intestinal stem cell-enriched epithelial spheroid cultures. We modified these intestinal spheroids with a bioluminescent reporter and used these cells to develop a modified clonogenic assay for 3D culture that can be used to identify novel radioprotectors, such as a fasting mimetic. Taken together, these results suggest that fasting protects small intestinal stem cells, allowing animals to receive potentially curative doses of abdominal radiation that would otherwise be lethal. Future work will aim to identifying the mechanisms by which fasting confers intestinal protection and drug candidates that can be used to mimic this fasting-mediated protection.

#3747

Radium 223 inhibits prostate cancer in bone via zonal cytotoxicity.

Eleonora Dondossola,1 Stefano Casarin,2 Claudia Paindelli,1 Elena De-Juan-Pardo,3 Dietmar Hutmacher,3 Christopher Logothetis,1 Peter Friedl1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _Houston Methodist Research Institute, Houston, TX;_ 3 _Queensland University, Australia_.

Bone metastases are the initial site of progression and account for many complications experienced by men with metastatic prostate cancer (PCa), with limited therapeutic options. Targeting the bone environment has recently resulted in the approval of Radium-223 (Rad-223), a new life-prolonging therapy for patients with metastatic castrate-resistant prostate cancer. Rad-223 is a rare earth metal radioisotope that displays chemical properties similar to calcium, becomes enriched in bone after in vivo administration and emits alpha particles with locally high energy but limited penetrance in tissues (<100 µm). Confoundingly, the clinical response to Radium-223 is often followed by detrimental relapse and progression, and whether Radium-223 causes tumor-cell directed cytotoxicity in vivo remains unclear. We hypothesized that limited radiation penetrance in situ defines outcome and addressed the principles discriminating Radium-223 efficacy from failure by combining 3D intravital microscopy, in silico modeling and end-point analysis in preclinical PCa models in bone. Radium-223 induced profound but zonally confined cancer cell lethality along the bone interface (200-300 µm), while the more distant tumor core remained unperturbed. As consequence, macro-lesions persisted and grew, whereas micro-tumors in the bone niche showed severe growth delay or eradication. The relative inefficacy in controlling large tumors points to application of Radium-223 in secondary prevention of early bone-metastatic disease and regimens co-targeting the tumor core or broadening the zonal toxicity.

### Tumor Dormancy, Metastasis, and the Metastatic Niche

#3748

Implantable tumor attracting niche models to study disseminated tumor cell biology.

Ryan Carpenter, Jungwoo Lee. _University of Massachusetts Amherst, Amherst, MA_.

Metastasis is the leading cause of death in cancer but remains the most poorly understood aspect of tumor biology. This can be attributed to the lack of relevant experimental models that can recapitulate the complex and lengthy progression of metastatic relapse. Mouse models have been widely used to study metastasis, however they are limited for prolonged investigation of the post-dissemination phase of cancer cell biology due to the rapid onset of actively growing primary and secondary tumors. This limitation has left a critical gap in understanding the long-term bi-directional crosstalk between DTCs and their local microenvironment due to the relatively short experimental timeframe. Here we introduce an implantable tissue engineered metastasis model that can overcome the fundamental restrictions of existing mouse models and substantiate the role of the tumor microenvironment in the reactivation of dormant DTCs with molecular and cellular detail.

Subcutaneous implantation of porous collagen-coated acrylamide scaffolds, fabricated as previously described (1-3), modulate the foreign body response to induce the formation of a robust vascularized tissue. During tissue formation and local inflammation, the scaffold generates a pre-metastatic niche, characterized by the recruitment of circulating tumor cells and given time, the formation of overt metastases. However, mice became moribund before meaningful investigation of dormant tumor cell biology could occur due to increasing tumor burden. We addressed this limitation via intact transplantation of the DTC microenvironment to tumor-free mice. Scaffolds have been maintained in secondary mice for up to 10 weeks. Compared to transplanted DTC-bearing lung tissue, scaffolds maintained an inactive phenotype for longer periods. At the end of this period, transplanted scaffolds captured three distinct DTC phenotypes: dormant, early colonization, and overt metastasis. Multiplex immunohistostaining was used to dissect the local microenvironment during the transition from single DTCs to overt metastasis. The local vasculature and Ly6G+ cells were observed to have differential roles during metastatic development. These experiments have been demonstrated in humanized NSG mouse models capable of generating scaffold niches containing human stromal, tumor (PC-3), and immune cells, as well as a spontaneous tumor model (MMTV-PyMT) with an intact immune system.

Our results demonstrate the potential of a tissue engineering approach to generate pre-metastatic niches and investigate the transient activity of the DTC niche during tumor cell awakening and subsequent metastasis. We envision that implantable microenvironments will be an enabling tool to study DTC biology and aid in the development of anti-metastatic therapies.

References: Carpenter R. Nat. Biomed. Eng, 2018. (2) Lee J. PNAS, 109(48), 19638-43, 2012. (3) Bersani F. Cancer Research, 74(24), 7229-38, 2014.

#3749

**Metabolic reprogramming by** GCN1 **mediates metastatic reactivation and outgrowth at liver metastatic site in response to glutamine deprivation in pancreatic cancer.**

Surajit Sinha, Jonathan Hernandez, Reed Ayabe, Michael Wach, Samantha Ruff, Alok Ranjan, Kirsten Remmert, Imani Alexander. _National Institutes of Health, Bethesda, MD_.

Introduction: Disseminated tumor cells in the circulation upon extravasation into a secondary organ like the liver encounter hostile conditions like nutrient deprivation and oxidative stress and may undergo dormancy or growth arrest. The molecular drivers and mechanisms that enable disseminated tumor cells to overcome such adverse growth conditions and revert to proliferative stage are unknown.

Method: To identify these molecular drivers, a retroviral cDNA library was generated from a highly metastatic pancreatic cell line (M-4964Liv) derived from a KPC mouse which forms macroscopic liver lesions upon splenic injection in mice. The cDNA library was then transduced into a dormant pancreatic KPC cells (D-4964Liv) cells which does not give liver lesions upon splenic injection in mice. This methodology will allow all the cDNA's from the metastatic cells to express in the dormant cells but only those cDNA's which can trigger the dormant cells to outgrow will give rise to a metastatic liver lesion.

Results: The screening strategy led to the identification of general control of amino-acid synthesis 1-like 1 (GCN1L1). The human homologue GCN1 has been shown to regulate the activation of mammalian amino acid sensor general control non-derepressible 2 (GCN2) under amino acid starvation in budding yeast but how GCN1 regulates metastatic reactivation is not known. We show that GCN1 is activated under physiologic glutamine levels in the liver which is around 0.5 mM compared to standard tissue culture levels of 2-4 mM. Mechanistic interrogation revealed that GCN1 activates both the mammalian amino acid sensors: the general control nonderepressible 2 (GCN2) and the mechanistic target of rapamycin complex 1 (mTORC1) leading to metastatic outgrowth in vivo. Contrary, knockdown of GCN1 dramatically impairs the activation of GCN2 and mTORC1 and abrogates liver metastatic outgrowth upon splenic injection in immunocompetent mice.

Conclusions: These results suggest that under physiologic glutamine levels, induction of GCN1 activates two different pathways: One pathway impinges on ATF4 and the other on mTORC1. While ATF4 activation promotes metabolic homeostasis by inducing de novo amino acid synthesis, the activation of mTOR pathway ensures re-entry in to the cell cycle leading to metastatic reactivation and outgrowth.

#3750

Conversion surgery in unresectable advanced gastric cancer and cancer dormancy as a prognostic marker.

Hun Jee Choe,1 Jin Won Kim,1 Song-Hee Han,1 Kui-Jin Kim,1 Jeong-Min Kim,2 Koung Jin Suh,1 Ji-Won Kim,1 Keun-Wook Lee,1 Hye Seung Lee1. 1 _Seoul National University Bundang Hospital, Seongnam-si, Gyeonggi-do, Republic of Korea;_ 2 _Chung-Ang University Hospital, Seoul, Republic of Korea_.

Background: While additional gastrectomy has not shown superiority compared to conventional systemic therapy in unresectable gastric cancer in REGATTA trial, there have been several subsequent attempts to find a role for conversion surgery with a curative intent in selected patients. The aim of this study was to analyze the conversion surgery data and to see if there was a role of cancer dormancy markers to select patients for conversion surgery.

Patients and Methods: From the pathology database at Seoul National University Bundang Hospital, we identified 49 patients treated with chemotherapy followed by gastrectomy in initially unresectable gastric cancer from January 2006 to August 2016. Of these, 26 patients were analyzed to explore the role of conversion surgery with a curative intent. As cancer dormancy markers, NR2F1, nanog, mig6, and pERK were evaluated using immunohistochemistry staining.

Results: Twenty-six (53%) from a total of forty-nine patients received conversion surgery. The median age was 58 years (range, 39-78) and the duration of prior chemotherapy before conversion surgery was 5.1 months (range, 3.1-13.9). Category 2 with para-aortic lymph node involvement was the most prevalent disease status (57.5%). At the time of conversion surgery, complete response, partial response, and stable disease status had been established with chemotherapy in 2, 15, and 3 patients, respectively. Of these, R0 resection was accomplished in twenty-two (85%) patients. Median overall survival (OS) in all patients that underwent conversion surgery, defined as the time from initiation of chemotherapy to death by any cause, was 36.1 months (95% CI, 29.6-51.4). In patients that underwent R0 resection, disease free survival (DFS) from conversion surgery and OS from initial chemotherapy was 15.1 months (95% CI, 13.9-43.8) and 37.8 months (95% CI, 31.7-57.1), respectively. Patients with a shorter duration of prior chemotherapy were associated with both longer OS and DFS from conversion surgery (p < 0.001, both). Less advanced pathologic stage at the time of conversion surgery was also associated with longer OS from conversion surgery (p=0.045). Regarding cancer dormancy markers from initial biopsy specimens, stronger expression (moderate to strong) of NR2F1, nanog, and mig6 showed a tendency for longer DFS after conversion surgery (p=0.018, p=0.834, p=0.344, respectively) and also longer OS after conversion surgery (p=0.027, p=0.225, p=0.359, respectively).

Conclusion: When advanced gastric cancer showed a favorable response after chemotherapy, a survival of over 3 years was observed in patients who received a curative conversion surgery, despite the initial unresectability. We may expect survival benefits from conversion surgery in certain subgroups, with better initial response to palliative chemotherapy and higher levels of specific cancer dormancy proteins in the tumor.

#3751

A mode of treatment resistance of metastatic tumor cells: Propagation exceeds elimination in number.

Chie Kudo-Saito,1 Yukinori Ozaki,2 Keiichi Kinowaki,2 Hidetaka Kawabata,2 Yamato Ogiwara1. 1 _National Cancer Center Research Institute, Tokyo, Japan;_ 2 _Toranomon Hospital, Tokyo, Japan_.

Purpose: Distant recurrence following metastasis is a major cause of cancer-associated death. Interfering spread and re-growth of the disseminated tumor cells, which could be cancer stem-like cells (CSCs) via EMT, must be greatly helpful for improving prognosis of cancer patients. However, little is known about the molecular mechanisms underlying the refractoriness. In this study, we attempted to elucidate the mechanism how the dormant CSCs would wake up from the sleeping at the metastatic lesions.

Results: C57BL/6 mice were subcutaneously and intravenously implanted with murine melanoma B16-F10 cells or B16-F10 cells transduced with snail gene as an EMT control, and 3 weeks later, the metastasized tumor cells (F10-BM or F10-BM-snail+) were harvested from the bone marrow, a niche for CSCs, or from the subcutaneous tumors as a control (F10-primary). We comprehensively analyzed these clones for EMT-related molecular expressions and biological functions in vitro and in vivo. The F10-primary cells were round and adhesive having high expression of epithelial markers such as E-cadherin, but not mesenchymal markers such as Snail, and the F10-BM-snail+ cells were small and spindle having high expression of mesenchymal markers, but not epithelial markers. In contrast, the F10-BM cells were giant and very adhesive with high expression of epithelial markers, but still keeping high Snail expression in the cytoplasm. Interestingly, these cells were polyploidy having a number of nuclei in a cell, and proliferated significantly slower than the F10-primary cells. However, once receiving stimulation with a low concentration of chemotherapeutics, the F10-BM cells aggressively proliferated through generation of many progeny cells, looking resistant to the treatment. In the in vivo settings, the F10-BM tumor growth was significantly slower after implantation in mice, and the mice survived for a longer term than the F10-primary-implanted mice. As seen in the in vitro culture, the F10-BM tumors quite rapidly grew in response to treatments such as chemotherapeutics and immune checkpoint inhibitors, and the treated mice died significantly earlier than the nontreated F10-BM-implanted or the F10-primary-implanted mice, suggesting hyperprogression that tumor cell propagation might exceed elimination in the mice.

Conclusions: These data suggest that dormant and polyploidy giant cells could be a risk factor of hyperprogression of the metastatic tumors after treatments. We are now identifying a specific molecule regulating this mechanism utilizing cDNA microarray analysis.

#3752

**Fabrication and validation of an** in vitro **bone marrow microenvironment for the study of prostate cancer progression.**

Molly Kozminsky, Lydia Sohn. _UC Berkeley, Berkeley, CA_.

Metastasis is responsible for the vast majority of cancer deaths, motivating the investigation of how the disease progresses in the face of each obstacle put forth by the body. While disseminated tumor cells (DTCs) are detected in the bone marrow of many prostate cancer patients, only 1% of DTCs yield macrometastases. There is pressing need for an in vitro system to study the characteristics of the interactions with the microenvironment that influence prostate cancer progression. High-throughput DNA-directed single-cell patterning was used to create an in vitro bone-marrow microenvironment that provides replicates of these miniature systems within an easy-to-images conventional chamber slide. This system enables the study of the contributions of several cell types present in the bone marrow microenvironment. Cell types in the in vitro system to be represented by cell lines include bone cells (osteoblasts and osteoclasts), cells of hematopoietic lineage (macrophages, osteoclasts), and endothelial cells. Optimization strategies for cell culture conditions, pattern design, and fabrication parameters will be described as well the incorporation of prostate cancer cells to study cell-cell interactions. Applying high-throughput DNA-directed cell-patterning system yielded a highly complex cellular system that can be used to answer open questions about prostate cancer metastasis.

#3753

Patient derived xenograft colorectal cancer in a micro-vascularized tumor on a microfluidic chip.

Benjamin Anbiah,1 Iman Hassani,1 Bulbul Ahmed,1 Nicole Habbit,1 Michael Greene,1 Balabhaskar Prabhakarpandian,2 Elizabeth Lipke1. 1 _Auburn University, Auburn, AL;_ 2 _CFD Research Corporation, Huntsville, AL_.

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in the United States. Cellular and biochemical cues from the tumor microenvironment (TME) modulate tumor aggressiveness including metastasis and treatment failure with cancer therapeutics. Conventional tumor models exhibit several shortcomings including lack of cell heterogeneity which limits their potential to examine more complex phenomena associated with drug testing applications. Patient-derived xenografts (PDX) represent an alternative model which maintains tumor heterogeneity. Physiological correlation of current preclinical models is limited by the degree of endothelial vascularization of tumor and incorporation of the tumor cell heterogeneity with an appropriate extracellular matrix to form a three-dimensional (3D) tumor. Herein, we have developed a micro-vascularized tumor on a chip model to examine the dynamics of tumor cell metastasis using CRC PDX lines.

PDX tumors established from stage II, III-B, IV adenocarcinomas were propagated subcutaneously in SCID-NOD mice. Two days prior to tumor dissociation, the tumor chip containing an intricate vascular network for the growth of endothelial cells was primed and coated with 200 µg/ml fibronectin. EA.hy926 endothelial cells were seeded in the vascular channel and were allowed to form a lumen over 48 hours. The excised PDX tumor was dissociated and the cells (20 x 106 cells/ml) were mixed with a polymer precursor containing Poly(ethylene glycol)-fibrinogen and the photoinitiator eosin Y, then seeded into the primary tumor chamber and crosslinked under visible light. Serum containing media was perfused through the vascular channel at a constant flow rate of 1µl/minute. Metastasis was observed by maintaining the chip long-term.

Significant differences (p = 0.034) in tumor cell migration in the chip (intravasation, tumor cell circulation through the vascular channel, invasion and extravasation) over time were observed between the PDX CRC stages. Stage II tumor cells intravasated to the vascular channel by day 8 followed by circulation and adhered to the endothelium as single circular cells or clusters by day 15. Stage III-B tumor cells were observed to intravasate, circulate, and adhere to the vascular channel by day 8, and the endothelium was overtaken by the cancer cells by day 8. Stage IV tumor cells began intravasation from the primary chamber to the vascular channel and circulated through the endothelium by day 8, while invasion to the secondary chamber adjacent to the primary chamber was observed by day 15. The Stage IV tumor cells also formed clusters in the vascular channel at a distant site and extravasated to the secondary chamber which is consistent with the staging of the tumor. Finally, this chip can be used for screening anti-cancer drugs for CRC patients.

#3754

**Interaction of engineered patient-derived mesenchymal stem cells encapsulated in fibrin glue with the glioblastoma microenvironment:** Ex vivo **validation of novel cellular therapeutic paradigms for brain cancer.**

Cesar A. Garcia,1 Rawan Alkharboosh,2 Adip G. Bhargav,3 Natanael Zarco,1 Hugo Guerrero Cazares,1 Alfredo Quinones-Hinojosa1. 1 _Mayo Clinic, Jacksonville, FL;_ 2 _Mayo Clinic Graduate School, Rochester, MN;_ 3 _Mayo Clinic School of Medicine, Rochester, MN_.

Purpose: The aim of the present study was to use an Ex Vivo model to assess the feasibility of applying an intraoperative local administration of cellular therapy to treat Glioblastoma (GBM). The proposed therapy employs human Mesenchymal Stem Cells (hMSC's) engineered with non-viral transfection to express a protein of interest. The engineered hMSC's are then encapsulated in an FDA approved Fibrin glue that can be embedded into a resection cavity post-surgical resection.

Methods: hMSC's were isolated from patient fat samples with enzymatic digestion, transfected with polymeric Nanoparticles to express mCherry, encapsulated in fibrin glue, and seeded onto murine organotypic brain slice cultures containing a human derived GBM tumor that was grown for 10 weeks after orthotopic injection. Tumors were grown with Brain Tumor initiating cells (BTICs) that were isolated from patient samples and transduced to express GFP for Ex vivo imaging. Cellular interactions on the ex vivo model were observed for three weeks with 3D time-lapse imaging and confocal microscopy. PI staining was also conducted to assess hMSC viability after Fibrin glue encapsulation.

Results: During the first 73 hours after initial seeding of the encapsulated hMSC's (red) onto the brain slice, BTICs (green) migrated from the primary tumor bulk, proliferated at the border of the Fibrin gel where it met the tissue, and eventually invaded the gel where they co-localized with hMSC's (yellow). Confocal images taken at 13- and 20-day time points show that hMSC's retained mCherry expression, escaped from the Fibrin glue, proliferated, and migrated throughout the tumor microenvironment. PI staining also indicated high viability (>95%) after incubation in Thrombin (Fibrin glue component).

Discussion: Isolated hMSC's were successfully transfected and retained expression of mCherry for up to three weeks within the tissue, showing that engineered cells can possess prolonged protein expression. Future non-viral modifications can include anti-glioma proteins or other therapeutic agents. Cell-cell interactions indicate that encapsulated hMSC's attract and promote migration of BTICs into the gel. This could mean that when applied in clinic or in vivo, migrating tumor cells can be recruited into the resection cavity where they can be better targeted with adjuvant chemo- and radiotherapies. Overall, hMSC's were viable in Fibrin glue components and were able to migrate throughout the tumor micro-environment showing that these cells hold the capacity to travel throughout brain tissue over time and deliver anti-glioma cargo within that environment. Future directions will involve testing the validated treatment paradigm with therapeutic anti-glioma cargos and further in vivo studies.

#3755

Generation of a sandwich based 3D hydrogel to support human mammary fibroblast viability and proliferation.

Anna Abbott,1 Jana Byrd,2 Abby Hielscher2. 1 _Liberty University, Lynchburg, VA;_ 2 _GA-PCOM, Suwanee, GA_.

Myofibroblasts are activated fibroblasts which play a role in breast tumor growth, metastasis and therapy resistance. One factor which has been shown to facilitate the transition of fibroblasts to myofibroblasts is increased matrix stiffness, a phenomenon which has been reported in 2D, but not widely investigated in 3D. One of the limitations associated with 3D cultures is that cells are unable to remodel an environment which is too rigid and fail to remain viable as a result. To this end, we sought to develop a mechanically tunable 3D hydrogel to investigate whether this platform was supportive for growth of human mammary fibroblasts (HMFs). To accomplish this, we generated a sandwich-based gelatin hydrogel in which we cultured HMFs between two mechanically tuned hydrogels. The bottom hydrogel consisted of gelatin cross-linked with microbial transglutaminase (mTG) to generate compliant (30µg), moderate (100µg) and stiff (200µg) mTG hydrogels. HMFs were seeded on these hydrogels and then a compliant hydrogel was plated atop the HMFs, generating the 3D hydrogel sandwich. As a control, HMFs were cultured in 2D atop compliant, moderate and stiff hydrogels. After a period of 2 and 4 days, HMFs cultured in the 2D and 3D hydrogels were investigated for changes in viability and growth using EtBr/Calcein and WST, respectively. At days 2 and 4, results showed significantly more live cells than dead in all 3D hydrogels, indicating that HMFs remained viable despite increases in mechanical stiffness of the bottom hydrogel. Differences in viability between HMFs grown in 2D vs 3D were negligible with the exception for HMFs in 2D compliant hydrogels which had a significantly greater proportion of live cells in comparison to HMFs in 3D compliant hydrogels. Analyzing changes in proliferation, we found that proliferation increased over the 4-day culture period for HMFs grown in all 3D hydrogels, but when compared to one another, HMFs were most proliferative in the compliant 3D hydrogels than in the moderate and stiff 3D hydrogels. Analyzing differences in 2D and 3D cultures, it was found that HMFs were more proliferative in 2D as opposed to 3D. To investigate changes in morphology, a property which is influenced in part by changes in the mechanical properties of the substrate, HMFs in 2D and 3D hydrogels were analyzed for changes in circularity using Image J. HMFs in the compliant 3D hydrogel were more circular at days 1 and 2 in comparison to HMFs in moderate and stiff hydrogels. At days 3 and 4, HMFs in all 3D hydrogels exhibited similar elongated morphologies. Comparing 2D and 3D hydrogels, HMFs were more elongated overall in 2D than in 3D in each of the hydrogels. Overall, the results indicate that HMFs remain viable and proliferate over the culture period in the mechanically tuned 3D hydrogels. This work supports the use of this platform to investigate the role of mechanical stiffness on fibroblast acquisition of a myofibroblast phenotype.

#3756

Highly multiplexed proteomic assessment of the human acute myeloid leukemia bone marrow microenvironment.

Bogdan Popescu,1 Katherine Lindblad,1 Giovanna Fantoni,2 Gege Gui,1 Janet Valdez,1 Meghali Goswami,1 Christin DeStefano,1 Catherine Lai,1 Angélique Biancotto,2 Julián Candia,2 Foo Cheung,2 Julie Thompson,1 Laura W. Dillon,1 Christopher S. Hourigan1. 1 _National Institutes of Health, National Heart, Lung and Blood Institute, Bethesda, MD;_ 2 _National Institutes of Health, National Institute of Allergy and Infectious Diseases, Bethesda, MD_.

Acute myeloid leukemia (AML) is a genetically heterogenous and often fatal cancer of the hematopoietic system. Even after achieving an initial complete remission, more than half of such AML patients experience clinical relapse due to the persistence of "minimal" residual disease (MRD). Ex-vivo studies have hypothesized that the interactions between residual leukemic cells and the local microenvironment in the bone marrow may play a key role in their survival and chemoresistance.

Therefore, we performed for the first time a global examination of the proteomic profile of the bone marrow microenvironment in AML patients using a highly multiplexed method based on the ability of slow off-rate modified aptamers (modified small single-stranded oligonucleotides) to bind target proteins with high specificity and affinity at slow dissociation rates. Ten relapsed/refractory adult AML patients and age-matched healthy subject controls were recruited, under an IRB-approved protocol, for research bone marrow aspirate (BMA) and blood serum collection. The supernatant resulting from centrifugation of BMA and serum samples were processed and analyzed on a SOMAscan™ hybridization microarray platform for the detection and quantification of 1,305 target proteins (Somalogic, CO). Data was corrected using Hybridization Control and Median Signal Normalization methods. Significant differences were found between AML and healthy donor bone marrow, such that 133 analytes were differentially expressed in the AML group (Wilcoxon rank sum test FDR p<0.05, fold change >1.5); 85 over-expressed and 48 under-expressed. In addition to proteins already known to be elevated in AML patients (eg: Erythropoietin, Thrombopoietin, Hepcidin and Ferritin) and dysregulation of pathways previously identified as disease relevant (eg: Arginase), we also discovered multiple statistically significant differences in levels of soluble proteins that are not currently known to be associated with AML pathogenesis or treatment. Comparative analysis between blood serum and bone marrow determined that 82 of the 133 candidates have differential expression that was specifically restricted to the bone marrow. The STRING database was queried for pathway analysis of enriched protein sets in the AML group and clustered analytes with molecular functions including cytokine activity (GO:0005125, n=11, p=1.93e-08), cytokine receptor binding (GO:0005126, n=12, p=3.17e-08) and signal transducer activity (GO:0004871, n=19, p=9.23e-05).

Using a high-throughput proteomic technology we have identified an AML bone marrow microenvironment-specific profile comprised of both proteins with known implications in leukemic pathogenesis and also several novel candidates from biologically plausible pathways that, once validated, may provide mechanistic insight and opportunity for therapeutic targeting.

#3757

Probing glioblastoma and its microenvironment at single cell resolution.

Sen Peng,1 Sanhita Rath,1 Saumya Bollam,1 Jenny Eschbacher,2 Shwetal Mehta,2 Nader Sanai,2 Michael Berens,1 Seungchan Kim,3 Harshil Dhruv1. 1 _Translational Genomics Research Institute (TGen), Phoenix, AZ;_ 2 _Barrow Neurological Institute, Phoenix, AZ;_ 3 _Prairie View A &M University, Phoenix, AZ_.

Single-cell sequencing (scSeq) is a powerful tool to investigate cancer genomics at single cell resolution. Multiple studies have recently illuminated intratumoral heterogeneity in glioblastoma, however, the majority focused on molecular complexity of tumor cells, without taking into account unexplored host cell types that contribute to the microenvironment around GBM tumor. To address the glioblastoma microenvironment composition and potential tumor-host interactions, we performed deep coverage (176k average reads per cell) scSeq of freshly resected primary GBM patient tissue without implementing any tumor cell enrichment strategies. scSeq libraries for 902 cells were prepared using 10X Gemcode platform and sequenced on Illumina NextSeq 500. This run was of high quality with 2,663 median genes per cell and low mitochondrial gene percentage (median < 5%). We used Cell Ranger analysis pipelines and Seurat packages to classify individual cells into 10 clusters and visualize them using t-SNE two-dimensional projections. We then identified the signature gene set for each cluster, relative to all other cells. Pathway analysis of each cluster signature along with known GBM microenvironment cell signatures revealed glioma tumor population along with surrounding microglia/marcophages, astrocytes, pericytes, oligodendrocytes, T cells and endothelial cells. Cell type markers identified by single cell transcriptomics were validated by IHC analysis. Microenvironmental composition and single cell signature will be confirmed through single nuclei sequencing of preserved (Frozen) tumor sample. Our results demonstrate the cellular diversity of brain tumor microenvironment and lay a foundation to further investigate the individual tumor and host cell transcriptomes that are influenced not only by their cell identity but also by their interaction with surrounding microenvironment.

#3758

Targeting ovarian cancer induced peritoneal carcinomatosis by inhibition of signaling axis between Interleukin-6 (IL-6) and serine protease inhibitor Kazal type 1 (SPINK1).

Christine Mehner,1 Erin Miller,2 Mathew A. Coban,2 Alexandra Hockla,2 Derek C. Radisky,2 Evette S. Radisky2. 1 _Mayo Clinic Graduate School of Biomedical Sciences, Jacksonville, FL;_ 2 _Mayo Clinic Cancer Center Jacksonville, Jacksonville, FL_.

Ovarian cancer remains a challenging disease, with an average 5 year survival rate of 46%. At time of diagnosis, over 70% of patients have advanced metastatic disease, for which there are no effective treatment options. Of patients with metastatic disease, more than 70% of patients develop peritoneal carcinomatosis, making the peritoneum one of the main areas of dissemination.

We have previously found that expression of the serine protease inhibitor Kazal type 1 (SPINK1) by ovarian cancer cells drives anoikis resistance, allowing the tumor cells to circumvent apoptosis protocols and facilitating tumor cell metastasis. For this new study we focused on ovarian clear cell carcinoma (OCCC), a histotype of ovarian cancer that has particularly poor prognosis if diagnosed in late stage. Using OCCC cell lines, we found that knockdown of SPINK1 reduces cell survival and stimulates apoptotic pathways in OCCC cells, when cultured under attachment-free conditions to mimic metastatic disease. We then identified Interleukin-6 (IL-6) as an upstream regulator of SPINK1 expression and a potential therapeutic target for metastatic OCCC: we showed that IL-6 silencing reduced SPINK1 mRNA expression and cell survival, while treatment with recombinant IL-6 increased SPINK1 expression and attachment-free survival. We developed a new OCCC

mouse model and showed that knockdown of SPINK1 results in significant reduction of metastatic lesions, highlighting the impact of SPINK1 on OCCC metastasis. In ongoing studies we are testing intraperitoneal injections of FDA approved IL-6 inhibitors in a preclinical model to assess impact on abdominal dissemination of tumor cells.

By targeting the IL-6 – SPINK1 signaling axis, we aim to develop therapeutic approaches to reduce the metastatic burden and peritoneal carcinomatosis of late stage disease, resulting in better prognosis and survival of patients with ovarian clear cell carcinoma.

#3759

Host and neoplastic cell secretions of proteolytic enzymes potentiate metastasis.

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

The presence of macrophages within breast tumors is associated with metastatic potential. Tumor-associated macrophages are often stimulated by Interleukin-4, resulting in a pro-tumorigenic (M2-like) phenotype and the secretion of growth factors and proteases, including the lysosomal protease cathepsin L (CTSL). CTSL is often secreted by breast cancer cells and stromal cells and contributes to tumor invasion, metastasis, and angiogenesis. We hypothesized that secretion of the proteolytic enzyme cathepsin L by both tumor-associated macrophages and neoplastic cells facilitate tumor invasion and dissemination. Initial studies found that inhibiting CTSL using the novel cathepsin L/K inhibitors KGP94 and KGP207 prevented in vitro M2 macrophage invasion and reduced macrophage-stimulated invasion of 4T1 murine breast cancer cells. Interestingly, KGP94 and KGP207 also reduced the expression of several M2-associated markers, suggesting that CTSL activity may be important for Interleukin-4-driven M0 to M2 differentiation. Furthermore, CTSL shRNA knockdowns revealed that CTSL supplied from both the tumor cell and the macrophage population is important for tumor cell invasion. These data suggest that tumor cells and macrophages may both contribute to the CTSL-driven metastatic phenotype of breast cancer. Taken together, these studies highlight the importance of CTSL in macrophage functions and suggest that cathepsin inhibition strategies may be therapeutically beneficial by impairing the progression of tumors with high infiltration of M2 macrophages.

#3760

Identifying site-specific disparities in the tumor microenvironment of primary and metastatic disease.

Krysten E. Vance, Micheal A. Hollingsworth, Paul Grandgenett. _UNMC, NE_.

The tumor microenvironment (TME), or all cellular and molecular components in and around the tumor, has a well-documented impact on cancer cell proliferation, chemoresistance, and overall patient survival. However much of the research into the TME has been limited to the primary site. How the TME of metastases compares to the primary lesions remains poorly characterized, even though metastasis is responsible for 90% of all cancer deaths. Understanding these differences is critical to treating advanced cancer patients. To study this our lab examined the TME in 22 matched sets primary pancreatic tumors and liver metastasis obtained from end-stage pancreatic cancer patients via our rapid autopsy program. We used a multiplex-immunofluorescent technique that allows staining of a single slide with up to 35 antigens and characterized the presence, position and functional statues of cells that comprise the pancreatic TME. These included: tumor cells, vasculature, fibroblasts, macrophages, mast cells, basophils, eosinophils, B cells, T cells, and NK cells, along with multiple subtypes of these. Based on our preliminary data from this, we hypothesize that the organ determines the features of the TME. Analyzing primary tumors and metastases revealed that both the composition and spatial distribution of different cell types are distinct between the primary tumor and liver metastases. PAM clustering of the cellular featured grouped primary tumor with primary and metastasis with metastatis 87% of the time. As the TME has been shown to impact chemotherapeutic response, we further examined chemotherapeutic effect between sites. Comparing gemcitabine (standard of care) and untreated patients, we observed distinct site-based responses. In the primary tumors with gemcitabine treatment there was a significant increase in inflammatory cells (neutrophils and macrophages) in comparison to untreated patients, while in the liver gemcitabine induced an increase in desmoplasia (SMA). Combined this data indicates that the distinct TME's exist within the same patient in an organ specific manner which can impact chemotherapeutic response. Understand these differences is therapeutically critical to patients whose therapeutic strategies involve targeting the TME to improve drug delivery, modulate immune suppression or otherwise modulate the microenvironment.

#3761

Regulation of metastasis by CD8 T lymphocytes.

Robiya Joseph,1 Rama Soundararajan,1 Suhas Vasaikar,1 Fei Yang,1 Sevinj Isgandarova,2 Lin Tian,3 Monika Haemmerle,1 Barbara Mino,1 Tieling Zhou,1 Geraldine Vidhya Raja,1 Esmeralda Ramirez Pena,1 Petra Den Hollander,1 Neeraja Bhangre,1 Crystal Shin,3 Melisa Martinez,4 Jaime Rodriguez Canales,1 Jeffrey Chang,5 Anil Sood,1 Ignacio Ivan Wistuba,1 Don L. Gibbons,1 Jeffrey M. Rosen,3 Ghanashyam Acharya,3 Navin Varadarajan,4 Xiang H. Zhang,3 Sendurai A. Mani1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _Texas A &M Health Science Center, Houston, TX; _3 _Baylor College of Medicine, Houston, TX;_ 4 _University of Houston, Houston, TX;_ 5 _UT Health Sciences Center at Houston, Houston, TX_.

Metastatic breast cancer is the most dreadful malignant disease that accounts for the majority of cancer-related deaths worldwide among women. A number of studies have shown that the tumor microenvironment (TME) plays a crucial role in regulating metastasis. It is therefore imperative to understand the dynamic interactions between cancer cells and their microenvironment to examine the molecular interaction and to effectively target cancer cells. TME comprises a variety of cells including immune cells which can influence tumor survival, growth and metastasis. Tumor-infiltrating lymphocytes (TILs), in particular, the CD8 T lymphocytes, has emerged as a promising prognostic marker for immunotherapy in a variety of cancers. However, the key molecular factors that regulate the cross-talk between tumor cells and CD8 T lymphocytes and its impact on metastatic traits in breast cancer is still inconclusive. Platelets are crucial components of the tumor microenvironment that are known to modulate tumor promotion and metastasis. The contribution of platelets and platelet secreted molecules are also carefully examined in metastasis of various cancers. The primary objective of this study is to investigate the role of CD8 T lymphocytes and platelets in breast tumor progression using isogenic tumor lines that form identical primary tumors but differ in their ability to develop metastasis.

#3762

Single-cell analysis of cancer-associated fibroblast heterogeneity in non-small cell lung cancer: Mapping molecular phenotypes in tumors.

Sara Waise, Christopher J. Hanley, Rachel Parker, Christian H. Ottensmeier, Matthew Rose-Zerilli, Gareth J. Thomas. _University of Southampton, Southampton, United Kingdom_.

This work aims to characterise the heterogeneity and spatial relationships of the cancer-associated fibroblast (CAF) population in non-small cell lung cancer.

Fresh human lung tissue was dissociated for sixty minutes to extract the maximum possible proportion of fibroblasts. Single-cell RNA sequencing was performed using a droplet-barcoded platform (Drop-seq). Quality control was performed on the raw sequencing data and resulting gene expression matrix. Bioinformatic analysis was performed using multiple packages in R. Spatial relationships between cell types were assessed using a multi-immunohistochemical (IHC) staining technique.

We developed a workflow for efficient processing of raw Drop-seq data including quality control, normalisation and visualisation. Low-quality events were identified by integrating previously-described and novel quality-control metrics into a machine learning (random forest) model, and demonstrated that this approach improves clustering quality. Applying this method to samples from twelve non-small cell lung cancer (NSCLC) patients, we identified 5 distinct fibroblast subtypes; 3 predominantly derived from normal tissue and 2 largely from tumor samples. Of the normal subtypes, one showed gene expression consistent with the previously-described "inflammatory" fibroblast phenotype. Trajectory analysis identified a branched differentiation process from normal to CAF phenotypes, suggesting that these cells share a common initial activation before differentiation to either a "matrix remodelling" or "hypoxic" subtype. The prevalence and impact of these sub-populations appears to differ between NSCLC subtypes. The "matrix remodelling" subtype is present in both adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC), but confers a negative prognostic effect in LUAD only; the "hypoxic" phenotype appears relatively LUSC-specific. Multiplexed IHC using identified cluster markers demonstrated that these subtypes have different spatial distributions and relationships to other cell types.

CAF remain a poorly-characterised population, despite their abundance in most solid cancers. No single molecular marker identifies all CAF, and there has been a scarcity of evidence regarding the existence of distinct subtypes and whether such subgroups have different functions. Our analysis has revealed five distinct CAF subtypes in NSCLC. In addition to divergent differentiation pathways, these subtypes have differential gene set enrichment, indicative of functional differences. In keeping with this, the phenotypes show distinct prognostic impact across NSCLC subtypes. Characterisation of CAF subgroups associated with aggressive tumor progression may facilitate identification of novel stromal targeting strategies.

#3763

Effect of cancer-associated fibroblasts on lymphatic vessel formation in malignant melanoma.

Shusaku Maeda, Masakazu Yashiro, Hisashi Motomura, Takaharu Hatano, Heishiro Fujikawa. _Osaka City University Graduate School of Medicine, Osaka, Japan_.

Introduction: It has been reported that cancer-associated fibroblasts (CAFs) in tumor microenvironment may play an important role for the progression and metastasis of cancer cells. Some reports suggested that CAFs may promote tumor lymphangiogenesis. In malignant melanoma, lymph node metastasis is one of important prognostic factors. Tumor lymphangiogenesis may be controlled not only by tumor cells but also tumor microenvironment such as CAFs, however no study about the interaction between CAFs and human lymphatic endothelial cells (HLECs) was found. Then, we evaluated the effect of CAFs on lymphatic vessel formation in malignant melanoma.

Materials and methods: We used CAFs, normal tissue associated fibroblasts (NAFs) established from a patient of malignant melanoma, and HLECs. Serum-free conditioned medium was obtained from NAFs (NAF-CM) or CAFs (CAFs-CM). CAFs and NAFs were cultured with or without supernatant from malignant melanoma cells. HLECs were cultured by EBM with 50% of NAF-CM or CAFs-CM. Control group was cultured with 50% EBM and 50% of serum-free medium. The effect of each CM on the proliferation of HLECs was examined by CCK assay and tube formation assay. Tube formation was assessed at 12 hours of incubation by fluorescence microscope.

Result: CAFs-CM significantly decreased the proliferation of HLECs in comparison with NAFs-CM after 48, 72 and 96 hours of incubation by CCK assay. In tube formation assay, tube length and number of tubes and junctions were also significantly shorter and smaller in CAFs group, in compared with the control and NAFs group.

Conclusion: CAFs might have a suppressive effect on HLECs, resulting in inhibition of lymphangiogenesis.

#3764

TLR3 facilitate breast cancer metastasis to lymph node.

Odalys J. Torres-Luquis, Sulma Mohammed. _Purdue University, West Lafayette, IN_.

Tumor cells can only enter the lymphatic circulation at the interface between the invasive edge of the tumor and the adjacent host stroma that contains the lymphatic circulation. The existence of this distance barrier means that it would be unlikely for tumor cells to passively enter the lymph drainage. Instead, Toll-like receptors (TLRs) are important mediators for active migration and chemotaxis for tumor cells entry into the lymphatic drainage from the primary tumor. Here we use a microsurgical technique to collect lymph circulating tumor cells (LCTC) prior to its entry into sentinel lymph node in rat models and determine if the TLR expression induces the migration and tumor progression. In our results, we observed high levels of TLR3 expression in LCTC when cells were treated with Poly (I:C), a synthetic ligand for TLR3 induction. NFkB activation was also observed in LCTCs, after TLR3 activation this pathway can express genes involved in cancer progression. In addition, when looking at the expression of cytokines and chemokines factors present in the lymph or secreted by the tumor cells, CXCL10 had a high expression on induced TLR3 of LCTC and its ligand CXCR3 was also expressed in the lymph. CXCL10 is involved in chemotaxis, induction of apoptosis, and regulation of cell growth. The CXCL10/CXCR3 signaling mediates paracrine interactions between tumor and stromal cells that govern leukocyte trafficking and angiogenesis. Suppression of CXCR3 or receptors signaling limits metastasis suggesting CXCR3 contributes to the metastatic process. TLR3 Knockdown prevented tumor growth and metastasis in rat. Our data suggest that for breast cancer metastasis, TLR3 might play a role in active migration of tumor cells and tumor progression. Activation of TLRs in tumor cells can dampen the anti-tumor functions of infiltrating immune cells, altering the inflammatory response and promoting cancer progression.

#3765

Somatic JAK-STAT mutations in subtypes of aggressive B-cell lymphomas with DLBCL morphology.

Elena Vigano,1 Gerben Duns,1 Daisuke Ennishi,1 Clementine Sarkozy,1 Bruce Woolcock,1 Faith Cheung,1 Elizabeth Chavez,1 Stacy S. Hung,1 Katsuyoshi Takata,1 Anja Mottok,2 Randy Gascoyne,1 Kerry J. Savage,1 Ryan Morin,3 David W. Scott,1 Christian Steidl1. 1 _BC Cancer, Vancouver, British Columbia, Canada;_ 2 _Institute of Human Genetics, University Hospital Ulm, Ulm, Germany;_ 3 _Simon Fraser University, Burnaby, British Columbia, Canada_.

Aberrant activation of the JAK-STAT pathway is a hallmark of a variety of lymphomas and can alter the lymphoma cells secretome and the composition of the tumor microenvironment (TME). The up-regulation of the immune regulatory chemokine CCL17 has been shown to be mediated by STATs-dependent mechanism in primary mediastinal B cell lymphoma (PMBCL). Here, we assembled a cohort of 340 R-CHOP-treated aggressive lymphomas with diffuse large B cell lymphoma (DLBCL) morphology to investigate JAK-STAT mutations (targeted gene sequencing), copy number (CN) alterations (SNP arrays), gene expression (RNAseq) and TME composition (IHC). The cohort was evaluated using FISH for BCL2, BCL6 and MYC rearrangements and the molecular classification assay, Lymph2Cx, to distinguish ABC vs GBC vs double-hit lymphomas with DLBCL morphology (DH-DLBCL). Among the 317 evaluable cases, 26 were DH-DLBCLs, 101 ABC, 155 GCB and 35 unclassified. In addition, we used the recently published Lymph3Cx assay (Mottok A. et al., Blood 2018) to identify cases with a molecular PMBCL (mPMBCL) expression signature within the GCB-DLBCL group (6.5%, 10/155 cases). Twenty-five of 155 GCB cases (16.1%) were classified as 'uncertain PMBCL/DLBCL'. Mutational analysis of all cases (n=340) revealed the presence of JAK-STAT pathway mutations at the following frequencies: SOCS1 21.6%, STAT6 4.8%, IL4R 5.3% and 9p24 amplification 11.3%. These mutations were significantly enriched in the mPMBCL (n=10) group compared to the bona fide GCB (n=118) and intermediate phenotype group (uncertain PMBCL/DLBCL) (n=25) (p<0.05). The relative frequency of any JAK-STAT mutations was 100% (10/10) in mPMBCL, 29.2% (35/118) in bona fide GCB and 56% (14/25) in uncertain PMBCL/DLBCL. Among the GCB samples with follow-up data (n=107, incl. bona fide GCB and mPMBCL), cases carrying mutations in IL4R (n=11, 10.3%) had an inferior disease-specific survival (p=0.011) and time to progression (p=0.0048) after R-CHOP therapy. Interestingly, IL4R mutations were also detected in the DH-DLBCL group with an incidence of 11.5% (3/26 cases). Mutations in the extracellular and transmembrane domains of IL4R resulted in gain-of-function mutations leading to constitutive activation of the JAK-STAT pathway in vitro. IL4R gain-of-function increased CCL17 expression through a STAT6-dependent mechanism in vitro. Similarly, gene expression analysis of primary patient samples carrying activating IL4R mutations displayed increased CCL17 expression (p<0.05), which positively correlated with the level of the T-regulatory marker FOXP3 (p<0.05) by IHC. In summary, aberrant JAK-STAT activation driven by alterations, such as activating IL4R mutations, plays a significant role in altering chemokine expression profiles and TME changes. Our data strongly suggest the biological and clinical relevance of Lymph3Cx to define a subgroup of aggressive lymphoma harboring a molecular PMBCL signature.

#3766

**Embryonal metastasis assay, an** in vivo **test of circulating tumorspheres to detect metastatic predisposition.**

Peter Geck,1 James Kubilus,1 Andrew Makarovskiy,2 Viktoria Denes3. 1 _Tufts Univ. School of Medicine, Boston, MA;_ 2 _Tufts Medical Center, Boston, MA;_ 3 _University of Pecs, Pecs, Hungary_.

INTRODUCTION One of the unsolved problems of cancer is its progression into systemic, metastatic disease. Malignant cells are released into circulation very early (circulating tumor cells or CTCs), but distant metastases are disseminated only by cancer stem cells (CSCs) that survive circulation. We investigated a unique feature of CSCs, formation of cell clusters (spheroids or tumorspheres) in vitro. We found that it also takes place in vivo, cancer tumorspheres circulate in blood and we showed that they carry cancer stem cells and strongly correlate with metastatic disease. In this project we developed a filtration method to collect live circulating cancer stem cell in tumorspheres and searched for a method to establish a prognostic assay to predict their metastatic potential.

METHODS The conventional approaches use knockout mice to assay metastasis (NOD-SCID mice, etc.), but he high cost, the complex procedure and the lengthy assay (5 weeks) ruled them out as a routine clinical assay. Our goal was to establish an in vivo translational assay to detect metastatic predisposition early, with a chance to prevent systemic progression. We found that the rapidly progressing environment in the chicken embryo offers critical advantages to test the metastatic potential of a patient's cancer. Easy access to chorioallantois circulation and to the embryo, the lack of immune response, the rapid turnaround time (5 days) and the technical simplicity rendered the new embryonal metastatic assay a promising and cost effective translational method. We used tumorspheres of both highly metastatic and non-metastatic cell lines. For image analysis a highly metastatic cell line (MDA-MB-436) was labeled by Green Fluorescent Protein. To model the technique of labeling real life patient spheroids (DiI membrane dye and protein tracing by CFDA-SA), we used the highly metastatic MDA-MB-231 and the non metastatic MCF7 and T47D cell lines. For quantitative analysis of embryonal metastasis we used methods applied in microchimerism detection. Embryonal DNA/RNA was extracted 5-9 days after cancer spheroid implantation and used qPCR to detect human sequences.

RESULTS In the embryonal metastatic assays, fluorescent and confocal imaging detected metastatic cells in the embryo. DiI and CFDA-SA labeling of native tumorspheres indicated a level of toxicity and needed to be optimized. Molecular detection of human microchimerism in the embryos was extremely sensitive and generated quantitative metastatic data.

CONCLUSIONS The results indicate that the embryonal metastasis assay using molecular microchimerism detection has the potential for a sensitive and quantitative method to assess the metastatic predisposition of a cancer patient. The method needs only an incubator, DNA extraction and a thermocycler, easily available in most hospitals and offers cost effective, relevant in vivo prognostic data within days.

#3767

Characterization of the protumorigenic role of MSLN in peritoneal metastases in pancreatic cancer.

Leela Rani Avula, Michael Rudloff, Salma El-Behaedi, Danielle Arons, Xianyu Zhang, Christine Alewine. _National Institutes of Health, Bethesda, MD_.

Peritoneal metastases develop in 70-80% of nonresectable patients with pancreatic cancer, and the onset of clinically significant ascites is associated with rapid demise. Mesothelin (MSLN) is a GPI-linked cell-surface glycoprotein expressed in >90% of pancreatic ductal adenocarcinomas (PDAC) but not in healthy pancreas nor in the parenchyma of other vital organs. Due to this differential expression, multiple therapeutics targeting MSLN have already reached clinic. Overexpression of MSLN has been implicated in increased aggressiveness of PDAC, both in laboratory models and in patients. Here, we have used CRISPR-Cas9 gene editing to delete MSLN from the KLM-1 pancreatic cancer cell line (MSLN KO) and have examined the growth of these cells in culture and in nude mice compared to mock deleted cells (WT). The cells were also transduced to express GFP/Luciferase(Luc) in order to allow for monitoring of tumor by bioluminescence imaging (BLI) in the mouse intraperitoneal (IP) cavity. MSLN KO and WT cells grew at the same rate in culture and as subcutaneous xenografts in nude mice. But when cells were inoculated IP, KO cells grew poorly compared to WT cells or did not grow at all. When MSLN KO cells grew, visible tumors were located primarily at the inoculation site. In contrast, WT cells grew extensive metastases throughout the IP cavity. GFP/Luc+ tumor cells adhered to the mesothelial lining of the peritoneum could be detected by BLI and histology as early as 24h post IP injections. By 72h, the MSLN KO tumors had significantly decreased microvascular density as determined by CD31 staining, lower metastatic dissemination and growth as detected by BLI and H&E staining, and decreased proliferation as determined by Ki67 quantification. These differences were maintained even at timepoints out to 2 weeks. Our results suggest that MSLN might enhance peritoneal carcinomatosis of PDAC by positively regulating vascular remodeling/angiogenesis and tumor cell proliferation within the IP cavity. The mechanism of MSLN action has been previously ascribed to activation of MAPK or NF-kB pathways or signaling through MUC16, the only known binding partner of MSLN. However, we found no changes in phosphorylated p38 or ERK, nor in total levels of the NF-kB target OCT-2 upon KO of MSLN expression. Since we also observed protumorigenic effect of MSLN in a MUC16 deficient pancreatic cancer cell line, it is unlikely that MUC16 is responsible for the protumorigenic activity of MSLN. We are currently performing comprehensive molecular studies to uncover key upstream and downstream signaling factors responsible for the protumorigenic effects of MSLN in peritoneal dissemination.

### Tumor Evolution and Heterogeneity 2

#3768

Single-cell characterization and lineage tracking of recurrent pediatric rhabdomyosarcoma.

Anand G. Patel, Elizabeth Stewart, Michael Clay, Xiang Chen, Michael Dyer. _St. Jude Children's Research Hospital, Memphis, TN_.

A critical challenge for the effective treatment of pediatric solid tumors is tumor recurrence. In pediatric rhabdomyosarcoma (RMS), 70% of patients with metastatic disease will experience relapse despite treatment with chemotherapy, radiation and/or surgery. Those patients with relapse have particularly dismal outcomes with less than 20% survival over 5 years. These poor outcomes underscore the need for a better understanding of RMS, particularly the biologic processes responsible for recurrence and progression. Currently, a major obstacle towards improving RMS treatment has been that we, as a field, do not know which cells within RMS tumors evade treatment toxicity, and how these cells are selected for in the presence of chemotherapy.

We have previously developed a panel of orthotopic patient-derived xenografts (O-PDXs) that encompass the clinical and molecular diversity of RMS. In this study, we have leveraged single-cell RNA-sequencing (scRNA-seq) of our O-PDX system to characterize the intratumoral heterogeneity within this panel. Analysis of single-cell transcriptomic data demonstrate that fusion-negative RMS O-PDXs bear two distinct subpopulations: (1) a major subpopulation encompassing approximately 99% of the tumor cells, and (2) a minor population that are non-cycling and express genes from a variety of mesenchymal lineages. This two-population intratumoral structure is stable within O-PDXs during passaging of xenografts.

To extend these findings, we have developed a workflow using lentiviral transduction of O-PDX cells with a high complexity barcode library. This library integrates a unique 18-mer oligonucleotide barcode into each transduced cell within the 3'-untranslated region of blue fluorescent protein. Thus, all cells within an O-PDX with the same barcode are progeny of the same ancestral cell. We have validated longitudinal tracking of barcode diversity using genomic DNA and within the libraries generated during scRNA-seq. Mice bearing barcoded O-PDXs have undergone chemotherapy dosed to pharmacokinetically mimic pediatric therapy regimens; O-PDXs regress during treatment and ultimately recur following completion of therapy, akin to the clinical experience with RMS. Recurrent O-PDXs bear divergent barcodes, indicating that chemotherapy selects for tumor cells independent of the originating lineage of those cells. Further studies to evaluate the changes that occur in recurrent O-PDXs cells are underway.

#3769

Detection of allele specific loss of heterozygosity in 70,000 patients with ctDNA.

Jing Zhao, Stephen R. Fairclough, Darya Chudova, Richard Lanman, AmirAli Talasaz. _Guardant Health, Redwood City, CA_.

Background: Loss of Heterozygosity (LoH) is a common feature of tumor biology. LoH frequently arises from defects in Homologous Recombination Repair (HRR) and results in uni-parental deletions that manifest as (LoH). In the absence of a driving force, the likelihood of allelic loss is equal, so in a population, the rate of retention and loss of a given allele is equal, but allele specific loss (or retention) is known to occur.

Methods: Using a novel analysis method incorporating observed allele frequency and copy number variation, we investigated the Guardant Health database of variants detected from 70,000+ samples from whole blood samples from patients with advanced solid tumors that were tested using a 73-gene cell-free DNA next-generation sequencing panel (Guardant360, Guardant Health, Redwood City, CA) to identify allele specific loss.

Results: Analysis of the Guardant Health database revealed that LoH frequently manifests as allele imbalance with the observed mutation allele frequency (MAF) of the retained allele exceeding an observed allele frequency of 50% and the lost allele having observed mutation allele frequencies below 50% in an individual sample. This imbalance occurs because allele frequency is a relative measurement and loss of one allele causes the relative abundance of the remaining allele to increase in a proportional amount. Population analysis reveals that the majority of of alleles are lost without preference, but certain alleles may be more prone to retention or loss.

Conclusions: The detection of allele specific loss from ctDNA provides insight into the underlying tumor biology and the selective pressures shaping tumor evolution over the course of treatment.

#3770

Unexpectedly high subclonal mutational diversity in human colorectal cancer and its significance.

Robert A. Beckman,1 Brendan Kohrn,2 Kaitlyn Loubet-Senear,2 Jasmin Dunn,2 Jacintha OSullivan,3 Mary Bronner,4 Lawrence A. Loeb2. 1 _Lombardi Cancer Center, and Innovation Center for Biomedical Informatics Georgetown University Medical Center, Washington, DC;_ 2 _University of Washington, Seattle, WA;_ 3 _University College Dublin, Dublin, Ireland;_ 4 _University of Utah, Salt Lake City, UT_.

Human colorectal cancers (CRC) contain numerous positively selected clonal somatic mutations in 10% or more of the cells. In addition, subclonal mutations in a fraction of cells contribute to phenotypic heterogeneity. Several groups have shown either by analysis of the number of unique subclones as a function of sequencing depth, or by the evaluation of synonymous to nonsynonymous mutation ratios, that most subclonal mutations are not selected and evolve neutrally. Because of the branching nature of tumor evolution, the clonal mutations arise in the founder cell, or very early thereafter. Subclonal mutations appear next, the rare ones on progressively smaller branches of the evolutionary tree. Here, we apply Duplex Sequencing (DS) methodology to quantify subclonal mutations in 5 fresh human MSI-low CRC diagnostic samples with unprecedented depth (104) and accuracy (10-7), and confirm neutral evolution further forward in time than previously known. We find that CRCs without known DNA repair deficits harbor unexpectedly many subclonal mutations; indicating that the mutational diversity of CRCs has been greatly underestimated. The "effective mutation frequency", or mutation frequency per new cell added to the tumor (taking cell death into account) is also unexpectedly high: 6 X 10-7 per base. Given a genome length of 3 X 109, a new daughter cell (taking turnover into account) would have ca 2000 new private mutations compared to its parent. Further, the smallest clinically diagnosable tumor has ca 109 cells, leading to violation of the common modeling assumption that a given mutation arises uniquely in only one cell ("infinite sites assumption"), since at an effective mutation frequency of 6 X 10-7, a cell generation leading to the formation of 109 new cells would produce the same mutation in 600 of those cells. We have developed a new theoretical approach to model neutral evolution independent of the infinite sites assumption, and find that intratumoral heterogeneity in clinical tumors is also underestimated by previous theoretical approaches. Our novel experimental and theoretical methods show that every possible somatic point mutation is present when a tumor is clinically detectable, leading to pre-existing resistant cells to any therapy. We can more accurately predict emergence of cells simultaneously mutationally resistant to multiple non-cross resistant therapies with increasing cell number; targeting genetic instability itself may prevent this.

#3771

Oncogenes and tumour-suppressors drive differential retinoblastoma evolution.

Adriana Salcedo,1 John D. Watson,1 Hilary Racher,2 Diane Rushlow,2 Shadrielle Melijah G. Espiritu,1 Doroto H. Sendorek,1 Stephenie D. Prokopec,1 Donco Matveski,2 Fouad Yousif,1 Julie Livingstone,1 Brenda L. Gallie,3 Paul C. Boutros1. 1 _Ontario Institute for Cancer Research, Toronto, Ontario, Canada;_ 2 _Impact Genetics, Bowmanville, Ontario, Canada;_ 3 _Hospital for Sick Children, Toronto, Ontario, Canada_.

Unlike any other tumour type, retinoblastomas can be driven by only two tumour-initiating events: bi-allelic loss of the tumour suppressor RB1 or amplification of the MYCN oncogene. These mutations drive morphologically indistinguishable tumours that arise at different ages, providing a unique model system to evaluate the influence of initiating mutation on tumour evolution. We performed high-resolution copy number analysis of 101 retinoblastoma and whole genome sequencing of 23. These data reveal that different initiating mutations cause probabilistic changes in somatic point and copy number mutations, but large changes in genomic rearrangements and mitochondrial number. Independent of the initiating mutation, retinoblastomas harbour multiple subclonal populations that preferentially accrue point mutations after subclonal diversification. These subclonal structures suggest caution when choosing chemotherapeutic strategies for primary treatment. Overall, initiating mutations influence evolutionary trajectory more than specific driver mutations: RB1- and MYCN-driven tumours harbour distinct mutational processes, sequences of mutation acquisition and patterns of subclonal diversification. These data show how tumour-initiating mutations drive clinical behaviour by subtly biasing multiple evolutionary processes. Validation in other tumour types is underway.

#3772

Inter- and intra-tumoral variations in ASCL1, NEUROD1, and POU2F3 transcriptional programs underlie three distinct molecular subtypes of small cell lung cancers.

Carl M. Gay, Lixia Diao, C. Allison Stewart, Yuanxin Xi, Robert J. Cardnell, Stephen G. Swisher, Jack A. Roth, Bonnie S. Glisson, Jing Wang, John V. Heymach, Lauren A. Byers. _UT MD Anderson Cancer Ctr., Houston, TX_.

Accounting for 15% of all lung cancer diagnoses, small cell lung cancer (SCLC) is an aggressive malignancy with dismal clinical outcomes, due in part to failure to define SCLC molecular subsets and identify the unique, targetable vulnerabilities therein. Recent data has begun to delineate these subsets by uncovering inter-tumoral heterogeneity in features such as DNA damage response, EMT, and neuroendocrine (NE) status. An integrated analysis of clinical samples to determine the implications of this heterogeneity broadly on SCLC classification has not yet been performed. Using RNAseq data from 81 SCLC tumor samples, we applied non-negative matrix factorization (NMF) which identified three clusters, each enriched for unique transcriptional programs driven by ASCL1 (30/81), NEUROD1 (24/81), or POU2F3 (27/81). These three genes encode transcription factors which define mutually exclusive NE-high, NE-low, and novel tuft cell variants of SCLC. Bulk RNAseq analyses of SCLC CTC-derived xenograft (CDX) models validated the clustering analysis in vivo. However, single-cell RNAseq from these same models reveals subtle evidence of intra-tumoral and intra-cellular heterogeneity in transcriptional programs that appear mutually exclusive in bulk analyses, suggesting plasticity among these variants. Guided by our NMF results, we performed RNAseq-based supervised clustering to classify each of 60 SCLC cell lines into ASCL1-driven, NEUROD1-driven, and POU2F3-driven clusters. Then, using reverse phase protein array data for these lines, we derived proteomic signatures for each cluster as follows: ASCL1-driven: E-cadherinhigh/TTF-1high/cMYClow, NEUROD1-driven: E-cadherinlow/TTF-1low/cMYChigh, and POU2F3-driven: E-cadherinhigh/TTF-1low/cMYChigh (ANOVA p<0.001 for each). Other targetable proteins vary significantly among these clusters including BCL-2 (lower in NEUROD1-driven; p<0.001) and PD-L1 (higher in ASCL1-driven; p=0.04). Correlating these clusters with IC50 values for over 500 drugs we find unique vulnerabilities. For example, POU2F3-driven lines were more sensitive to PARP inhibitors (PARPi), despite lacking biomarkers of PARPi sensitivity in SCLC (e.g. high SLFN11, low ATM). ASCL1-driven lines were more resistant to aurora kinase inhibitors (AURKi), as predicted given their lower expression of cMYC, a predictor of AURKi sensitivity in SCLC. Our data suggest that SCLC is subdivided on the basis of three unique transcriptional programs and that each subtype is characterized by diverse protein expression and drug responses. Single-cell analyses suggest, however, that multiple transcriptional programs may coexist within a single tumor or, even, a single cell, thus providing a potential novel mechanism for lineage switching and therapeutic resistance.

#3773

Investigating the tumor-host evolutionary arms race as a possible strategy for cancer therapy.

Arig A. Ibrahim Hashim,1 Dominique Abrahams,1 Kim Luddy,1 Robert Gillies,1 Christina L. Richards,2 Joel Brown,3 Robert Gatenby3. 1 _Moffitt Cancer Center, Tampa, FL;_ 2 _University of South Florida, Tampa, FL;_ 3 _Moffitt cancer Center, Tampa, FL_.

As first proposed by Cairns and Nowell more than 60 years ago, cancer is a Darwinian process so that cancer progression and resistance to therapy are governed by natural selection and eco-evolutionary dynamics. This creates an "evolutionary arms race" between the cancer cells and the tumor suppressor mechanisms of the host. To mechanistically investigate these dynamics we have conducted an experiment in which we selected for resistance to growth of implanted Lewis lung (mouse) cancer tumor (LL/2-Luc-M38) cells in immuno-deficient (SCID) mice and immuno-competent (C57BL/6). At each round a fixed numbers of LL/2-Luc-M38 cells were implanted in 10 male SCID and 10 male C57BL/6 mice. Tumor growth was measured and the two animals that exhibited the slowest tumor growth were bred and their male progeny were used as host in the subsequent round. After 12 generations, the tumors grew at approximately 1/10th the rate compared their initial generation. We observed that the hosts evolved cancer suppression through two general strategies. The evolved SCID mice suppressed tumor growth via mechanical restriction with increased in collagen deposition in and around the tumor. In contrast BL/6 mice enhance their immunologic response. We investigated the molecular properties of tumor cells growing in the different cohorts suing microarray. We identified genes significantly different in their expression of cancer cells harvested at different days following injection in Wild Type and Evolved SCID and BL/6 mice using Principle component analysis (PCA) method. The genes were functionally classified based on Gene Ontology and the majority was found to be related to integrin binding, cell adhesion molecular binding, and extra cellular matrix (ECM) binding in SCID mice with collagen, type XII, alpha (Col12a1) being the significantly decreased (results validated with RT-PCR (**p=0.009)). However, in BL/6 mice Molecular studies, contrary to expectation, did not show significant changes in expression of immune-related genes. Rather we found a decrease in expression of genes associated with the angiogenesis and ATP production as well as increased expression of genes regulating EMT. Thus, our results highly suggest that the counter adaptive strategies deployed by the cancer cells in response to the host suppressive mechanisms is host dependent and can be a possible target for therapy.

#3774

Elevated cancer evolution dynamics: Emergence of polyploid cancer cells in response to multimodal therapy as an adaptive response on both individual and collective levels.

Gonzalo Torga,1 Ke-Chih Lin,2 Kayla Myers,1 Kimberly Smith,1 Robert H. Austin,2 Kenneth J. Pienta1. 1 _Johns Hopkins University, Baltimore, MD;_ 2 _Princeton University, Princeton, NJ_.

Polyploidy has been often associated with poor prognosis and has been described to some extent in histological samples for most types of solid tumors. In cancer biology, it has been proposed to induce the gain of a stem-like phenotype and drive tumor progression by increasing the potential for cellular transformation. Experiments with chemotherapy gradients in our engineered microfluidic device with prostate cancer cell lines exhibited a stiff polyploidy distribution pattern of heterogeneous cell populations across the gradient. This demonstrates the generation of polyploid giant cancer cells (PGCCs) is an emergent response to high doses across the microfluidic drug stress landscape. Furthermore, in these experiments we observed polyploidization events occurring concurrently with the development of drug resistance and delayed relapse by non-polypoidal cell budding off from PGCCs leading to a complete repopulation, even in the high-dose areas of the array. Recent publications suggest polyploidy might play a crucial role in resistance to chemo-, hormone- and radio-therapy across most solid tumors investigated, thus independent of the therapy's mechanism of action. To confirm these findings, we conducted experiments in conventional cell culture using different chemotherapeutic agents and ionizing radiation in several cell lines and confirmed the development of polyploidy was a common feature among them. Although these data supporting the association of polyploidy with resistance are extensive, the effects of these therapies on tumor heterogeneity and the cancer stem niche remains unknown. Different molecular mechanisms, such as mitotic slippage, aborted cytokinesis, endo-replication or cell fusion can lead to polyploidy. We hypothesize a subset of polyploid cells with stem-like features may be the reservoir of therapeutic resistance in cancer. In this work we focused on the characterization of this subset of cells within the tumor leading to the polyploid-stem phenotype, particularly the subpopulation with potential to reverse polyploidy and repopulate tumor heterogeneity after therapy. In order to establish causality and chronology of these phenomena, as well as quantify the complex population dynamics involved in this process, we engineered several cell lines from various common types of solid tumors with two fluorescence biosensors enabling simultaneous monitoring of SOX2/OCT4 activity and cell cycle status at the single-cell level using high-throughput confocal videomicroscopy. The emergence of polyploid cancer cells in response to multimodal therapy as an adaptive response on both individual and collective levels may be a hallmark of elevated cancer evolution dynamics and constitutes a promising potential target to prevent relapse.

#3775

Characterization of intratumoral heterogeneity in drug sensitivity and modeling of drug combination effects using subclonal cell populations derived from a single breast cancer cell line.

Hendrik J. Kuiken,1 Chandler M. Friend,1 Steven M. Corsello,2 Christopher C. Mader,3 Joan S. Brugge1. 1 _Harvard Medical School, Boston, MA;_ 2 _Dana-Farber Cancer Institute, Boston, MA;_ 3 _Broad Institute of MIT and Harvard, Cambridge, MA_.

Intratumoral heterogeneity can have profound effects on tumor progression and drug resistance. While tumor cell heterogeneity has been described at many levels, there is a poor understanding of the extent of heterogeneity in drug sensitivity within a single tumor, the dynamics of tumor cell subpopulations during treatment with anti-cancer agents and non-cell autonomous mechanisms of drug resistance. The overall objective of this study is to characterize heterogeneity in drug sensitivity within a model for human breast cancer and to model and evaluate the effect of drug combinations on the dynamics of subpopulations within heterogeneous mixtures.

We have characterized 31 subclonal cell populations (SCPs) that were generated through single cell cloning of the triple-negative breast cancer cell line MDA-MB-468. These SCPs display considerable heterogeneity with respect to morphology, proliferation rate, colony formation in soft agar and tumorigenicity in mice. To examine the extent of heterogeneity in drug sensitivity, we designed and screened a 200-small molecule compound library in 24 SCPs and the parental cell line. We observed considerable heterogeneity in drug sensitivity and selected 36 compounds for validation, including GPX4 inhibitor ML-210 and gemcitabine, to which the SCPs displayed 12-fold and 150-fold differences in the EC50, respectively. The results of these validation experiments were used to identify transcriptional programs and individual genes which expression correlates with drug sensitivity. We have generated and will evaluate predictive models for drug combination using the SCP drug sensitivity profiles. To test the effect of individual drugs and combinations, as well as different treatment schedules on heterogeneous populations, we will examine the dynamics of individual SCPs within mixed cultures. To this end, we have transduced 22 SCPs with CloneTracer lentiviruses, each encoding for a unique DNA barcode. The distribution of these unique barcodes can be determined by next generation sequencing. Previously, we have found that propagation of a 22 SCP mixture results in reproducible changes in the composition of mixed monolayer cultures with approximately half of the SCPs having a relative abundance of 1% each in the mixed population following propagation for four months.

The results of this study show that there is considerable heterogeneity in drug sensitivity among clonal subpopulations derived from a single cancer cell line. Ongoing experiments will provide insight into the effect of drug combinations and unique treatment schedules on distinct tumor cell subpopulations within heterogenous mixtures. Moreover, using this panel of SCPs, we will be able to test for and investigate non-cell autonomous modes of drug resistance, which may guide the development of new drug combinations.

#3776

Spatially resolved immunogenomic analyses reveal diverse sub tumoral microenvironments in the context of melanoma immunotherapy.

Akash Mitra,1 Miles C. Andrews,1 Whijae Roh,2 Mariana P. de Macedo,3 Alexandre Reuben,1 Fernando Carapeto,1 Feng Wang,1 Sangeetha M. Reddy,1 Khalida Wani,1 Christine Spencer,1 John Miller,1 Aislyn Schalck,1 Latasha D. Little,1 Donald A. Sakellariou-Thompson,1 Curtis Gumbs,1 Wen-Jen Hwu,1 Chantale Bernatchez,1 Jianhua Zhang,1 Patrick Hwu,1 Nicholas Navin,1 Padmanee Sharma,1 James P. Allison,1 Jennifer Wargo,1 Alexander J. Lazar,1 Philip A. Futreal1. 1 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _The Broad Institute, Boston, MA;_ 3 _Hospital A. C. Camargo, Sao Paolo, Brazil_.

Sustained periods of apparent clinical benefit despite lack of objective response are well known in a subpopulation of advanced melanoma patients. Inter-individual heterogeneity in response of separate tumors is common, characterizing complex overall response patterns. The molecular and cellular dynamics facilitating such long-term survival and heterogeneous response is poorly understood, particularly in the era of exposure to multiple potentially active therapies. We studied an exceptional case of long-term survival in a patient with non-responding metastatic melanoma in order to characterize the clonal and microenvironmental factors active across 3 time points.

We performed immunogenomic analyses of 3 metachronous tumors, including a systemic therapy-naïve mass, 67 intratumor sub-regions of a non-responding mass during PD-1 inhibitor therapy, and a post-PD-1 inhibitor mass. We profiled samples using whole exome sequencing, RNA-sequencing (RNA-seq), immunohistochemistry (IHC), and T cell receptor sequencing. Longitudinal, spatial, and cross-modal analyses were performed.

Longitudinal analyses identified mutations in several genes known to be associated with targeted or immune therapy resistance affecting distinct metastases. Genomic intratumoral heterogeneity (ITH) was primarily driven by subclonal copy number alterations that showed evidence of spatially-distinct evolution which may be in response to selective pressures at the tumor margin. RNA-seq revealed an unexpectedly high degree of ITH characterized by limited group-level gene or pathway associations with physical or immune characteristics of each site. Spatially-distinct pockets of immune activation and suppression were observed throughout the PD-1 inhibitor resistant metastasis despite a largely immune-excluded phenotype seen on IHC. A specific T cell Vβ CDR3 rearrangement was identified as dominant and recurrent not only across multiple spatial points within a single tumor mass, but also across metachronous tumors spanning the patient's disease course. Immunophenotyping of the T cell population with single-cell RNA-seq suggested repeated T-cell priming events leading to the persistence of both activated and exhausted T cells bearing the same TCR-β at any given time.

Our findings highlight an unexpected level of genomic and immune heterogeneity in metastatic melanoma tumors of a long-term surviving patient. The observed degree of ITH across local tumor microenvironments reiterates the inherent limitations to identifying robust and reproducible predictive biomarkers of therapy response based on limited physical sampling of tumors. Further spatiotemporal analysis of metastatic lesions in the context of immune checkpoint blockade will be required to determine how the mechanisms driving convergent microenvironmental phenotypes may be harnessed for therapeutic gain.

#3777

Exploring phenotypic mosaicism in a mouse model of glioma towards understanding intratumoral heterogeneity.

Toshiro Hara. _Salk Institute, La Jolla, CA_.

The lethality of glioblastoma multiforme (GBM) and the failure of treatment is largely attributable to the heterogeneous properties of this cancer. We aim specifically to characterize the driving event for GBMs to acquire a heterogeneous phenotype during progression. Central to achieving this goal is the ability to interrogate possible sources of heterogeneity that guide cellular states that are similar to those identified in human patients. We therefore employ a mouse model of glioma that recapitulates the pathophysiology and gene expression signatures of human GBM. While initiated with identical oncogenic drivers, this mouse model unexpectedly displays heterogeneous phenotypes between animals. Our preliminary data using time-series single-cell transcriptomics approach shows that transformed cells, even without acquiring additional genetic alterations, switch cellular states at the beginning of tumor formation and one population commit themselves to expand to establish gliomas that are mainly comprised of one cell type. We further observe the strong association of microenvironmental cues with glioma cell populations and their roles in the state transitioning of glioma cells. Through the characterization of mechanisms that drive phenotypic mosaicism, we hope to uncover general principles that govern tumor progression and heterogeneity, and then ultimately provide novel therapeutic strategies to cure GBM.

#3778

Uncovering the role of glioma stem cells in glioblastoma chemo-resistance and recurrence.

Gabriele Riva,1 Charles Couturier,1 Phuong Uyen Le,1 Xiaohua Yan,1 Yu Chang Wang,2 Marie Christine Guiot,1 Ioannis Ragoussis,2 Kevin Petrecca1. 1 _McGill University, Montreal Neurological Institute, Montréal, Quebec, Canada;_ 2 _McGill University, Genome Quebec, Montréal, Quebec, Canada_.

Glioblastoma (GBM) is the most common malignant brain tumor. Despite multimodal aggressive therapies, the median survival after diagnosis is 14 months. The failure of current therapies is due to the presence within the tumor of a subpopulation of cancer cells with stem-like properties, called glioma stem cells (GSCs), which are responsible for chemo-resistance and recurrence. The molecular mechanisms involved in chemo-resistance of this cell subpopulation and its role in recurrence are still largely unknown. In this work, we isolated GSCs from seven recurrent GBM patients and we performed single-cell RNA-sequencing (scRNA-seq) on them. The analysis showed the persistence of a conserved neurodevelopmental hierarchy in recurrent GBM with a glial-like progenitor population at the top. Furthermore, we extended this analysis by sequencing the tumor bulk of seven recurrent GBMs in order to investigate deeply cancer heterogeneity in recurrences. Our data displayed the presence of many different cell populations that recapitulate the multiple steps of the physiological neural differentiation, suggesting that this capability is not affected by standard therapies. Afterwards, we confirmed the stem-like properties of our recurrent GSCs, verifying by immunofluorescence the expression of several stem-ness markers, such as PROM1, MASH1, OLIG2 and SOX2, and assessing the tumorigenicity in vivo of these cells. Standard therapies are able to eliminate only differentiated cancer cells, but they do not eradicate the cancer stem cell subpopulation. In order to defeat GBM and avoid recurrence, GSCs have to become the goal of future therapies. Considering chemo-resistance as an intrinsic and natural property of these cells, we performed a pathways analysis comparing the scRNA-seq datasets of recurrent GBMs with scRNA-seq datasets of primary GBMs and fetal nervous tissues to identify shared molecular pathways. Intriguingly, several pathways involved in DNA repair, cell cycle control and stem cell maintenance, such as BRCA1, ATM, E2F4 and FOXM1 pathways, were uniformly overexpressed by the progenitor cells of the three groups, representing new molecular targets for therapeutic purposes. Afterwards, we tested in vitro an E2F4 inhibitor on GSCs derived from de novo and recurrent GBMs, obtaining stunning results in terms of cell growth inhibition and cell death induction. Furthermore, we pretreated recurrent GCSs with this molecule and we injected them in mice: data showed the ability of the drug to reduce tumor growth sharply and prolong the survival. Currently, we are evaluating the efficacy in vivo of this compound on a recurrent GBM mouse model, administering the drug with intracerebroventricular pumps. This project could seriously help to decipher GSCs chemo-resistance, as well as their role in GBM relapse, also providing the necessary knowledge for the development of new targeted therapies.

#3779

Clonal tracing reveals different mechanisms of resistance to immune checkpoint blockade.

Shengqing Gu,1 Xihao Hu,1 Xiaoqing Wang,1 Peng Jiang,1 Ziyi Li,2 Nicole Traugh,1 Xia Bu,1 Xiaofang Xing,3 Gordon J. Freeman,1 Myles Brown,1 Xiaole S. Liu1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Tongji University, China;_ 3 _Peking University Cancer Hospital, Beijing, China_.

Immune checkpoint blockade (ICB) therapy has improved patient survival in a variety of cancers, but its resistance presents a major clinical challenge. It remains unclear whether therapeutic resistance is driven predominantly by refractory clones of cancer cells or by immunosuppressive microenvironment. To address this, we utilized syngeneic transplantation model and performed clonal tracing of cancer cells to monitor the response to ICB. Different cancer clones displayed variable sensitivity to ICB treatment, reflecting intratumoral heterogeneity in therapy response. Furthermore, different recipients manifested distinct resistance mechanisms. ICB-responders developed resistance through selection and expansion of pre-existing ICB-resistant cancer clones, whereas non-responders presented with irresponsive microenvironment rather than dominance of resistant cancer clones. Extrapolation of the tumor-infiltrating immune repertoire revealed that higher IgG and lower IgA strongly correlated with better ICB response. This biomarker was further validated in multiple clinical cohorts. Our approach discriminates different sources of resistance to ICB, and identifies humoral immunity as biomarker of ICB response.

#3780

Spatially resolved transcriptional patterns of PARP inhibitor-resistant high grade serous ovarian cancer PDX tissue.

Benjamin King, Elke Van Oudenhove, Selim Misirlioglu, Ernesto Arostegui Fernandez, Douglas Levine, Timothee Lionnet. _NYU School of Medicine, New York, NY_.

Ovarian cancer is the leading cause of mortality among gynecological malignancies; 70% of patients who initially respond to therapy eventually relapse and die. Within highly heterogeneous tumors, resistance originates from a small number of cells that express beneficial genes and receive favorable cues from their microenvironment. Understanding the mechanisms of drug resistance therefore requires the knowledge of which cell types compose the tumor, which genes they express, and where they are located respective to other cells and tissue landmarks. To this end, we have explored cell type and transcriptional heterogeneity in homologous recombination deficient patient-derived xenograft (PDX) models of high grade serous ovarian cancer (HGSOC) treated with the PARP inhibitor talazoparib. We are using single-cell RNA sequencing (scRNA-seq) to identify the discrete cell types present in drug naïve and drug resistant PDX tissue and to characterize the transcriptional changes driving the resistant phenotype. We are using single-molecule hybridization chain reaction (smHCR) to fluorescently label mRNAs and diSPIM light sheet microscopy to visualize fluorescently labeled mRNAs within large volumes of PDX tissue. We are currently developing computational strategies to deconvolve the 3D spatial organization of PDX cell types and to understand where and when resistance genes are expressed. This integrated scRNA seq and smHCR microscopy framework has allowed us to identify rare cell populations within HGSOC PDXs and to visualize their 3D localization relative to other cell types and tissue features, and it has the potential to elucidate how transcription of key genes supports transition to a resistant state. The insights gained from single cell transcriptional and 3D spatial profiles has the potential to greatly advance our understanding of how the 3D tumor tissue microenvironment allows and encourages rare cells with pre-resistant transcriptional programs to escape PARP inhibitor treatment. Further, this research has the potential to be predictive of disease progression and to suggest additional therapeutic targets that could be exploited as part of novel and/or personalized combination therapy regimens. An additional advantage is that this approach could be generalizable to many types of cancer to characterize the interplay between tissue organization and diverse processes such as tumor evolution, metastasis, and drug resistance.

#3781

Cancer archetypes co-opt and adapt the transcriptional programs of existing cellular states.

Itai Yanai,1 Maayan Baron,1 Isabella S. Kim,2 Reuben Moncada,1 Yun Yan,1 Nathaniel R. Campbell,3 Richard M. White2. 1 _NYU School of Medicine, New York, NY;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 3 _Weill Cornell/Rockefeller/Sloan-Kettering Tri-Institutional MD-PhD Program, Memorial Sloan Kettering Cancer Center, New York, NY_.

Tumors evolve as independent systems comprising complex survival-ensuring functions; however, the nature of these distinct processes and their recurrence across cancers is not clear. Here, we provide evidence that melanoma cancer cells can be classified to three 'archetypes' that co-opt the neural crest, mature melanocytes, and stress gene expression programs, respectively, have a unique subclonal structure as well as tumor locations, and are conserved between zebrafish and human melanomas. Studying the natural history of a zebrafish melanoma tumor at the single-cell level, we found that one archetype exclusively exhibits the signature of the Warburg effect, suggesting that a shifting balance in energy production occurs differentially in the tumor. Deconvolving bulk human melanomas, we found that patients with a dominant fraction of the neural crest archetype show worse survival rates, indicating a clinical relevance for the composition of archetypes. Finally, we provide evidence that extending our approach to other cancer types can reveal universal and cancer-specific archetypes

#3782

Tracking the evolution of nasopharyngeal carcinoma metastasis.

Ming Yuan Chen, Zhi-Xiang Zuo, Rui You, Xiao-Long Zhang, Mei Lin. _Sun Yat-sen University Cancer Center, Guangzhou, China_.

Understanding the evolutionary history of distant metastasis in Nasopharyngeal carcinoma (NPC) is important for curing NPC and has not been systematically investigated to date. Here, we profiled whole exomes/genomes of 142 bulk samples, and transcriptomes of 60 bulk samples from 42 NPC patients with matched primary tumor, and regional lymph node/distant metastasis tumor tissues. Among them, 55755 single cells from 2 patients was also sequenced. In addition, we analyzed radiomic data from 600 NPC patients. We observed that NPC primary, regional lymph node metastasis and distant metastasis tumors had concordant mutation profiling and intratumor heterogeneity. Furthermore, we found the genetic events selected during metastasis were enriched in pathways related to metastasis, such angiogenesis, Notch and Wnt signaling. Some critical genetic events were identified, such as mutations in FAT1, RPLP1, TET2, NFKBIA, NOTCH1/3 and ARID1A, copy number variations in NOTCH2 and ARID1A. According to the mutation based phylogenetic tree and single cell RNA-Seq data, two distinct dissemination routes of distant metastases were observed, including lymphatic dissemination from regional lymph node metastases and hematogenous dissemination from primary tumors. We observed that the two different metastatic dissemination routes had different genetic mechanisms revealed by profound genomic and transcriptomic evidence. The hematogenous and lymphatic route had distinct mutation signatures, where hematogenous route was significantly enriched with higher DNA mismatch repair related mutation signature compared to lymphatic route. The bulk and single cell RNA-Seq data showed that the lymphatic route had higher activity of Epithelial-mesenchymal transition (EMT) signaling, while the hematogenous route was characterized by higher expression of INF-γ response genes and higher fraction of exhausted CD8+ T cells. Moreover, the two dissemination routes exhibited evidently different clinical outcomes. The radio image data showed that the hematogenous and lymphatic routes significantly differed in the size of primary lesion, and the range of involved lymph nodes. The radiomic data further indicated that the hematogenous and lymphatic routes could be clearly separated according to radiomic features. By applying the radiomic model to a larger cohort, we found hematogenous route had poorer survival outcome compared to lymphatic route. Finally, at cellular resolution, we observed the primary tumor cells could be separated into two distinct clones, including non-metastatic clone and metastatic-like clone. The metastatic-like clone was characterized by a significant reduction of a ribosomol protein, RPL22. We experimentally confirmed that the reduction of RPL22 could activate NFKB pathway, thereby promoting NPC cell metastasis.

#3783

**Effective MEK inhibitor combinations to target tumor heterogeneity in** EGFR **mutant transformed SCLC.**

Atsuko Ogino,1 Xinmeng Jasmine Mu,2 Mika Lin,1 Antonio Calles,1 Stephen Wang,3 Man Xu,3 Lynette M. Scholl,4 Geoffrey R. Oxnard,1 Paul Kirschmeier,3 Pasi A. Jänne1. 1 _Dana-Farber Cancer Insitute, Boston, MA;_ 2 _Broad Institute of MIT and Harvard, Cambridge, MA;_ 3 _Belfer Center for Applied Cancer Sciences, Boston, MA;_ 4 _Brigham and Women's Hospital, Boston, MA_.

Background: Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are effective for the treatment of EGFR-mutant non-small lung cancer (NSCLC). In almost all cases, however, the response duration is limited and resistance ultimately emerges. Transformation of NSCLC to small cell lung cancer (SCLC) occurs as one of the resistance mechanisms to EGFR-TKIs. To date, the mechanism associated with this transformation is largely unknown, necessitating the need to establish an effective treatment strategy for patients with transformed SCLC (tSCLC).

Methods: We isolated cancer cells that grow as floating aggregates (F) and adherent monolayers (Ad) from the pleural effusion of three patients with tSCLC to establish patient derived cell lines (DFCI112F, DFI112ad, DFCI190F, DFCI190ad, DFCI283F, DFCI283ad). In parallel, patient derived xenograft (PDX) models from these patients were successfully established by subcutaneous implantation of pleural effusions in NSG mice. Transcriptomic and genomic characterization of the cell lines were performed using whole-exome sequencing (WES) and RNA sequencing. These cell lines were further treated with a panel of agents, to screen for effective combination therapies.

Results: Biological characterization of the created cell lines revealed that the floating aggregates have neuroendocrine (NE) features, while the adherent cell population presents non-NE/mesenchymal characteristics. The histology of the PDX tumors was confirmed as SCLC. According to WES, all tSCLC cell lines retained the original activating EGFR mutations. RB1 and TP53 were universally inactivated. Co-culturing the floating-NE cells with the adherent non-NE cells promoted the proliferation of the floating-NE cells in vitro. NE cells (DFCI112F) and non-NE cells (DFCI112ad) were injected into nude mice either as single or admixed populations. Tumor growth was observed only in admixed cell populations. In an effort to target both NE and non-NE populations, we performed a drug screen and demonstrated that MEK inhibitors are selectively efficacious against non-NE populations. We further evaluated the MEK inhibitor combination therapies with agents targeting NE populations (cisplatin, ABT-263 and JQ1), which effectively eliminated both NE and non-NE components in vitro. Our further study demonstrated that ABT-263 in combination with MEK inhibitors presented a synergistic effect, which could be a promising therapeutic combination for tSCLC patients.

Conclusion: We demonstrate that the cancer cell lines established from tSCLC patients consist of heterogeneous population of cells with NE and non-NE/mesenchymal characteristics. The crosstalk between these two cell populations is observed and our findings underscore the importance of targeting tumor heterogeneity as a promising treatment strategy for patients with tSCLC. 

### Viral and Microbiome-associated Carcinogenesis

#3784

Role of HPV-human fusion transcripts in oropharyngeal cancer development.

Lisa Pinatti, Heather Walline, Thomas Carey. _University of Michigan, Ann Arbor, MI_.

Human papillomavirus (HPV) genomic integration is frequently seen in oropharyngeal squamous cell carcinoma (OPSCC). Integration into cellular genes can cause the generation of HPV-host fusion transcripts, which have been associated with worse survival in OPSCC patients, but their functional consequences are unknown, as HPV alone is capable of transformation. The forced expression of fusion transcripts versus HPV transcripts in spontaneously immortalized human oral keratinocytes (NOKSI) could demonstrate their effects on cell behavior. Our research focuses on characterizing HPV integration sites and fusion transcripts in cell lines and tumors, followed by in vitro analysis of fusion transcript function. By Detection of Integrated Papillomavirus Sequences (DIPS-PCR) and RT-PCR, we have identified integration events and fusion transcripts in a subset of samples in both intergenic and genic regions of the genome. We began our analysis with a fusion transcript reading from HPV16 E6-E7-E1 into the tumor suppressor gene TP63 from UM-SCC-47. The full fusion transcript and the HPV portion only were cloned into lentiviral vectors, and stable NOKSI populations expressing these transcripts were selected. These cultures, as well as control cultures, were subjected to proliferation, migration and invasion assays. Subsequent in-depth molecular characterization and in vivo studies will help clarify their effects on cancer formation and metastasis. These studies will advance our understanding of how fusion transcripts lead to worse patient survival and help develop new therapies for these patients.

#3785

**Identification of potential cellular targets for Epstein-Barr virus encoded microRNAs miR-BART7 and miR-BART9 by** in silico **analysis.**

Brunno F. Caetano, Bárbara G. Müller-Coan, Rafael L. Coan, Bruno E. Fantinatti, Deilson E. de Oliveira. _UNESP - São Paulo State Univ., Botucatu, São Paulo., Brazil_.

The Epstein-Barr virus (EBV), formally designated human herpesvirus 4 (HHV-4), is an ubiquitous virus that causes lifelong latent infection in over 90% of the world's human adult population. EBV infection is regarded as carcinogenic to humans and associated with a variety of human cancers, especially endemic Burkitt lymphoma and nasopharyngeal carcinomas. EBV was the first human virus described to encode viral microRNAs (miRs), but the activity of EBV-encoded miRs in carcinogenesis remains to be fully elucidated. In this study we sought to identify novel potential targets for EBV-encoded miR-BART7 and miR-BART9 using in silico analysis. Mature sequences for EBV-miR-BARTs 7-3p, 7-5p, 9-3p, and 9-5p were retrieved from miRBase (http://www.mirbase.org, version 22) and 3'-UTRs from Homo sapiens were obtained from Ensembl (https://www.ensembl.org, version 92). RNAhybrid, miRanda, and PITA softwares were used for target predictions of miR-binding sites within 3'-UTRs of human mRNAs. Overlapped targets were collected using a custom Python script in such a way that only genes predicted by all the three algorithms were selected and used for gene-enrichment pathway analysis using the Cytoscape with the Reactome FI database plug-in (version 3.6.0). Each gene in a given pathway was counted as one hit using another custom Phyton script, and results were considered significant when P-value <0.05. As result, high-scoring predicted targets for EBV-miR-BARTs 7 and 9 were found to affect pathways involved in immune responses, gene expression, metabolism of proteins, and signal transduction. By targeting key tyrosine kinases pathways, such as epidermal growth factor receptor (EGFR), mitogen-activated protein kinase (MAPK), and vascular endothelial growth factor (VEGF), EBV-miR-BART7-3p was predicted to play a significant role in cell proliferation, cell cycle progression, and tumorigenesis. On the other hand, EBV- miR-BART7-5p was found to regulate adaptive immune system antigen presentation via MHC class II, cytokine signaling, and growth hormone receptor signaling. EBV-miR-BART9-3p was found as a relevant regulator for post-translational modification of proteins, WNT/β-catenin signaling, and pre-mRNA processing, while EBV-miR-BART9-5p could exert a role on signaling mediated by Rho GTPases, G protein- coupled receptors (GPCRs), gene silencing by small RNAs and RNA polymerase II transcription pathways. In conclusion, the results indicate pathways and genes that may be substantially targeted by EBV-miR- BARTs 7 and 9 and deserve further experimental investigation to disclose possible roles of EBV miRs on pathogenesis of cancers associated with viral infection.

This work was supported by Sao Paulo Research Foundation (FAPESP) under grants 17/20352-0, 17/22312-5 and 17/23393-9.

#3786

A study of HPV induced anal cancer progression in a raft tissue culture system.

Irina V. Getun,1 Abidemi Ajidahun,1 Janice Milici,2 Sreejata Chatterjee,2 Abul Elahi,1 Leah E. Hendrick,1 Evan S. Glazer,1 Louisa Balazs,1 Craig Meyers,2 David Shibata1. 1 _University of Tennessee Health Science Center, Memphis, TN;_ 2 _Penn State College of Medicine, Hershey, PA_.

Introduction: Anal cancer is one of few malignancies for which the incidence continues to rise in the US. It is highly associated with high risk (HR) HPV infection with HPV 16 and 18 accounting for the vast majority of cases. Understanding the molecular mechanisms underlying anal cancer progression would be valuable for the purposes of developing novel clinical management strategies. However, there remains a paucity of experimental models allowing for the study of HPV-associated anal cancer development. Herein, we describe an in vitro raft tissue culture system model to study anal squamous neoplastic progression via anal intraepithelial neoplasa (AIN) induced by HPV infection.

Methods: Primary human anal keratinocyte (HAK) cells were transfected with either HPV16 or 18 DNA and grown in a raft tissue culture system. The progression of dysplastic changes was sequentially examined at ~5 passage intervals by histologic review including hematoxylin-eosin (H&E) staining, immunohistochemistry (p16, Ki67, cytokeratins CK5/6, CK8/18 and CK10) and in situ hybridization (ISH) with an HPV16/18 RNA probe cocktail. The degree of dysplasia was determined by the degree of disorganization of the epithelial architecture, presence of mitotic/dysplastic cells, p16 positivity, diffuse Ki67 staining, increased number of basal-parabasal type cells (CK5/6 and CK10 markers) and lack of epithelial differentiation.

Results: Advanced dysplastic changes (corresponding to AIN2-3 lesions) were already apparent at the earliest of initial passages of transfected HAK cells (passage 8 for HPV16 and 7 for HPV 18). Later passages suggested development of AIN3/squamous cell carcinoma in situ in both lines with HPV16 HAK cells showing progressively increased labeling of CK8/18 and HPV18 HAK cells demonstrating increasing cell keratinization and epithelial disintegration. In both cell lines, HPV was present in both episomal and integrated forms throughout all stages of progression as suggested by ISH signal patterns.

Conclusions: A raft tissue culture model system has been developed to study HPV-induced dysplastic progression in human anal keratinocytes. Importantly, this system is of great interest as an in vitro tool to characterize the sequential molecular alterations associated with the development of anal cancer which in turn, may have implications for novel screening, prevention and treatment strategies.

#3787

Reduced expression of INPP4B by promoter hypermethylation in Epstein-Barr virus-associated gastric carcinoma.

Yiwen Ma,1 Jichao Tan,2 Weihong Dai,1 Yu Zhang,1 Yu Zhang,1 Rongjin Lin,1 Yan Xu1. 1 _First Hospital of China Medical University, Shenyang, China;_ 2 _Baoji Center Hospital, Baoji, China_.

Background: Epstein-Barr virus-associated gastric carcinoma (EBVaGC) is attracting increasing attention due to distinct molecular and clinical characteristics. However, its carcinogenesis still remains unclear. Previous studies demonstrated that aberrant DNA methylation is one of the pathogenic mechanisms of EBVaGC. Inositol polyphosphate 4-phosphatase type II (INPP4B), as a component of PI3K/AKT pathway, is implicated in the regulation of crucial cellular processes and tumorigenesis. This study aims to reveal the correlation between INPP4B expression and EBV infection, as well as investigate the inactivation mechanism of INPP4B in EBVaGC.

Methods: EBV status and INPP4B expression were examined in gastric carcinoma (GC) tissue microarray (301 cases) using EBV RNA probe and immunohistochemistry, respectively. The correlation of EBV status and INPP4B expression was analyzed in GC by Chi-Squared Test. The INPP4B promoter methylation was investigated in GC cell lines: SNU-719 (EBV positive), AGS (EBV negative) and SGC-7901 (EBV negative) with methylation-specific polymerase chain reaction (MSPCR). After treatment with 5-Aza-2′-deoxycytidine, the INPP4B promoter methylation and expression were detected using MSPCR, western blot, and qPCR.

Results: INPP4B expression is decreased in 146 of 301 GC cases (48.5%). In 272 evaluable GC cases, EBV was present in 40 cases (14.71%). In addition, Lower expression of INPP4B is significantly correlated with EBV infection (p<0.05). There was a significantly decreased expression of INPP4B in EBV positive cell line (SNU-719) compared to EBV negative cell line (AGS and SGC-7901). Moreover, aberrant CpG island hypermethylation of INPP4B was found in SNU-719 rather than AGS and SGC-7901. After treatment with 5-Aza-2′-deoxycytidine, INPP4B protein and mRNA are dramatically up-regulated, while unmethylated DNA became detectable in SNU-719.

Conclusions: Our results suggest that the reduction of INPP4B expression is commonly found in GC, particularly in EBVaGC. Promoter hypermethylation may be one of the mechanisms of INPP4B inactivation.

#3788

Sensitive detection of oncoviruses integrated into a comprehensive tumor immuno-genomics platform.

Gabor Bartha, Robin Li, Shujun Luo, John West, Richard Chen. _Personalis, Inc, Menlo Park, CA_.

Introduction

HPV, HBV, HCV and EBV viruses are causally linked to over 11% of cancers worldwide while KSHV, HTLV and MCV are linked to an additional 1%. As use of immunotherapy expands to a broader variety of cancers, it is important to understand how these oncoviruses may be impacting the overall immune response in patients as part of the tumor and tumor microenvironment. However, typical cancer diagnostic and genomic biomarker assays do not include oncovirus detection.

Methods

To enable detection of these oncoviruses, we have developed ImmunoID NeXT, a novel augmented exome and transcriptome based platform that comprehensively characterizes tumor and tumor microenvironment from a single sample. As part of the platform assay design we developed targeting for the following oncoviruses: HPV, HBV, HCV, EBV, KSHV and HTLV. Furthermore we developed analytics for detecting these viruses from both the DNA and RNA data. To test the ability of the platform to detect these oncoviruses, we identified a set of 11 EBV cell lines from Coriell in which EBV was used as a transformant. We obtained another 23 cell lines from ATCC containing HPV16, HPV18, HPV45, HPV68, HBV, EBV, KSHV, HTLV1 and HTLV2 in which the oncoviruses were known to be in the tumors from which the cell lines were created. We have also started running a series a FFPE tumor samples of unknown status. We extracted DNA and RNA from all these samples, made libraries and sequenced them on a NovaSeq to 30G. High quality read counts were computed for all targeted oncoviruses.

Results

We detected EBV in all the Coriell cell lines in both DNA and RNA indicating strong sensitivity of the platform. Wide dynamic range suggests quantification may be possible as well. In the DNA there was no signal from any other oncovirus indicating high specificity. In the ATCC samples we detected 23 out of 23 oncoviruses expected in both the DNA and RNA. We detected all the different types of oncoviruses that we targeted except for HCV because it wasn't in any sample. In all but one case the signals were strong. False positive detections appear to be rare. Much less than 1% of the overall exome/transcriptome reads map to oncoviruses in all cases.

Conclusions

Oncovirus detection integrated into an augmented exome and transcriptome assay is sensitive and specific. Oncoviral characterization of the tumor as part of a comprehensive tumor immunogenomics platform can serve and an important additional biomarker for understanding immunotherapy response.

#3789

Genomic instability changes acquired by HPV16 E6/E7 targeting multipolar mitoses aneuploidy: Cellular sequelae.

Eva McGhee,1 Mengtao Li,2 Yi-Ling Lin,2 Khadijah Lang,1 Meidrah Tyler,1 Judith Okoro,3 Mai Do,1 Naomi Long,1 Jenna Cormier,1 Adin Handler,1 Julian Handler,1 Sameeran Das,1 Benjamin Liu,1 Anthonia Duru,4 Billy Ballard,5 Roland Pattillo,6 Jay Vadgama7. 1 _Charles R. Drew University. of Medicine and Science, Los Angeles, CA;_ 2 _University of California Los Angeles, Los Angeles, CA;_ 3 _University of California Berkeley, Berkeley, CA;_ 4 _John Hopkins University, Baltimore, MD;_ 5 _Meharry Medical College, Nashville, TN;_ 6 _Morehouse School of Medicine, Atlanta, GA;_ 7 _Charles R. Drew Univ. of Medicine & Science, Los Angeles, CA_.

Malignant progression of high-risk Papillomavirus (HPV), associated lesions is a slow and inefficient process. HPV a non-enveloped epitheliotropic double stranded DNA virus, is an etiological agent of oral cancer. Oral cancer is related to the persistent infection by high-risk HPV type 16, E6/E7 oncoproteins. The E6/E7 complex significantly contribute to the carcinogenic genetic instability effect of high-risk HPV through the degradation of two gatekeeper proteins, p53 and pRB, respectively. This process is associated with viral genome integration into a host cellular chromosome and accumulation of numerical and structural chromosome aberrations. Viral genome integration results in consistent, sustained and dysregulated expression of the E6 and E7 oncogenes, and continued E6/E7 expression is necessary for maintenance of the transformed phenotype of HPV positive cancer cell. Induction of genomic instability by high-risk HPV E6/E7 demonstrates that HPV also drive malignant progression through targeting the nuclear and mitotic apparatus protein-1 (NuMA), and through subversion of DNA double strand break repair by HPV E6/E7 induced centrosome abnormalities. This in turn may cause abnormal localization of the microtubule protein dynein. The NuMA provides a cohesive force that guarantees the integrity of the mitotic spindle poles. However, there are no studies addressing the relationship between HPV16 infection-oral cancer, NuMA expression, and genomic instability. The aim of this study determined if HPV16 E6/E7 subverts centrosome coalescence in oral cancer caused by HPV oncogenes targeting the NuMA protein. In our experimental procedures, we proposed a novel approach to study HPV-induced chromosomal changes with reconstructed human oral epithelium in SCID mouse. We used HPV16, which is the most common HPV type found in oral cancer. Two plasmids were used to produce HPV-16 E6/E7. The first plasmid p16sheLL expresses the two viral capsid proteins, L1 and L2. The second plasmid pBR322HPV16 contains the full length HPV-16 genome. This approach closely mimics the architecture of normal human oral epithelium. In our study, we first focused on analyzing selected cellular processes involving multipolar mitoses. We examined Dynein localization in mitotic cells by immunofluorescence. We also analyzed progressive morphological and cytopathic changes in HPV-infected oral epithelia by using molecular cytogenetic analysis. In summary these data indicate that NuMA expression is alternated, and HPV16 E6/E7 oncoproteins can promote increased upregulation of genomic instability by induction of centrosome abnormalities and inhibition of DNA double strand break repair. We also found High-risk HPV16 E6/E7 oncoproteins induced mitotic defects and genetic aneuploidies through induction of supernumerary anaphase bridges, supernumerary centrosomes, and DNA double strand breaks.

#3790

Molecular analysis of HPV genotype in head and neck cancers in a tertiary hospital in Ghana.

Babatunde M. Duduyemi, Francis A. Yeboah, Evans Aboagye. _Kwame Nkrumah University of Science and Technology, Kumasi, Ghana_.

Introduction: Human papillomavirus (HPV) has been implicated in head and neck cancers (HNC), and patients now present at a much younger age without a strong alcohol or tobacco history. This work aims to establish the prevalence of HPV in head and neck squamous cell carcinoma (HNSCC) in our center as very minimal work has been done in Ghana in this area.

Method: The study was to detect HPV DNA and genotypes from histologically confirmed HNSCC. Formalin-fixed paraffin embedded (FFPE) tissue blocks and slides of HNSCC cases were retrieved consecutively. Genomic DNA was then prepared from 10μm FFPE tissue section using the Quick-DNATM FFPE kit. A nested multiplex PCR assay that combines degenerate E6/E7 consensus primers and type-specific primer was utilized for the detection of HPV genotypes (16, 18, 31, 33, 35, 45, 51, 52, 56, 58, 59, 6/11, 42, 43 and 44) in three cocktails.

Results: A total of 301 HNSCC cases were extracted from the surgical day book, with male preponderance of 72.1% and a mean age of 54.97 years. The most common site of occurrence was the oral cavity (28.9%), followed by larynx (26.2%). Majority of the samples of patients aged <30 years were poorly differentiated (65.7%). HPV E6/E7 oncogenic DNA was detected in 18% (n=100), with 17 being HPV-16 and 1 both HPV-16 and HPV-18. A higher prevalence of HPV-positive DNA was found in males (83.3%), and there was a trend towards patients older than 56 years likely to have HPV tumours (OR=1.378). Larynx was the commonest site for HPV-positive HNSCC (38.9%), followed by oropharyngeal site (33.3%).

Conclusion: The overall prevalence of HPV DNA (genotypes 16 and 18) in our study was 18%, more common in younger people and mainly male. This suggests that males should be considered in screening and vaccination against HPV to prevent HNSCC.

#3791

Understanding the function of LMP1 CTAR3 in EBV-associated cancers.

Analise McGreal,1 Aimee Minko,1 Melissa A. Visalli,1 Robert J. Visalli,1 Gretchen L. Bentz2. 1 _Mercer University School of Medicine, Savannah, GA;_ 2 _Mercer University School of Medicine, Macon, GA_.

Background: Over 90% of the human population is infected with Epstein-Barr virus (EBV), which has been linked to cancers such as Hodgkin's lymphoma and nasopharyngeal carcinoma. The principal oncoprotein responsible for cellular proliferation, increased cell invasion, evasion of apoptosis, and metastasis observed in EBV-associated cancers is latent membrane protein-1 (LMP-1). LMP-1 constitutively activates multiple signal transduction pathways via its cytoplasmic C-terminal activating regions (CTARs). Although, the functions of CTAR1 and CTAR2 regions have been extensively defined, the functions of a third regulatory region, CTAR3, are less understood. CTAR3 participates in protein sumoylation, which regulates evasion of immune responses and oncogenesis. The CTAR3 region is composed of 11-amino acid repeats and JAK binding motifs that contribute to the sumoylating activity of LMP-1. Variation in the number of repetitive elements has been noted in clinical isolates, but the exact function of these elements in oncogenesis is unknown. Therefore, the present study aims to generate EBV LMP-1 CTAR3 mutations within the repetitive elements to study their role in oncogenesis.

Methods: Recombineering using an EBV-containing bacterial artificial chromosomes (BACs) was utilized to prepare mutant CTAR3 constructs with a N-terminal hemagglutinin (HA) epitope tag. Mutant BACs were transfected into HEK 293 cells and clones selected using hygromycin. Expression of mutant CTAR3 proteins was detected by immunoblot using HA antibody.

Results: Preliminary results indicate the successful generation of infectious EBV clones containing the following LMP-1 CTAR3 mutations: (1) HA - Wild Type, (2) HA - JAK Only, (3) HA - No JAK, and (4) HA - ΔCTAR3. Clones for each mutant were selected after transfection into HEK 293 cells. Expression of mutant CTAR3 proteins was confirmed via immunoblot. The ability of the mutants to produce infectious virus was determined after induction of HEK CTAR3 clones and infection of B cells (RAJI) with culture supernatant. All CTAR3 mutants produced infectious virus and expressed CTAR3 mutant proteins of the expected molecular weight.

Conclusions: An EBV-BAC was recombineered to create EBV strains with targeted mutations in CTAR3. The novel BAC mutants will be used to assess the function of CTAR3 domains in EBV replication and oncogenesis. The role that CTAR3 plays in envelopment and egress during lytic replication will be determined. Additionally, the role of CTAR3 during EBV-mediated immortalization of naïve B-cells will be examined. Ultimately, mutant EBV strains will be studied in a SCID mouse model to determine the role of CTAR3 sumoylation in cell migration and metastasis in vivo. Overall, further appreciation of the role of LMP-1 in tumorigenesis will identify potential targets for therapeutic intervention in patients with EBV-related cancers.

*Analise McGreal and Aimee Minko contributed equally to this work.

#3792

Restoring DREAM assembly in HPV-positive cancer cell lines promotes growth suppression.

Fatmata Sesay,1 Siddharth Saini,1 Claire James,2 Jessica Felthousen,1 Iain Morgan,2 Larisa Litovchick1. 1 _VCU Massey Cancer Center, Richmond, VA;_ 2 _VCU School of Dentistry, Richmond, VA_.

Human Papillomaviruses (HPV) 16 and 18 are recognized as causative factors in 4-5% of all human cancers, including the majority of cervical and oropharyngeal carcinomas. The HPV vaccine is expected to reduce the incidence of HPV-driven cancers in the future. However, millions of people in the US already infected with high-risk HPV are still at risk. Therefore, it remains a priority to improve our understanding of the mechanism of progression of the initial HPV infections to cancer. Furthermore, there are currently no therapies that specifically target HPV-positive cancers, despite evidence that viral genes continue to contribute to growth and survival of established cancer cell lines. In high risk HPV (those that cause cancer), viral protein E7 binds to pRb via a LxCxE motif to relieve repression of E2F1-mediated transcription of S-phase genes, whereas the E6 protein interacts with the tumor suppressor p53 protein and targets it for degradation. Therefore, the consequence of E6 and E7 expression in the host cell is an aberrant induction of cellular proliferation and suppression of apoptosis, which contribute to oncogenic transformation. Recently, it has also been demonstrated that E7 interaction with the pRb-like protein p130 disrupts the transcriptional repressor DREAM (dimerization partner, RB-like, E2F and MuvB) complex. In G0/G1, p130 serves as a scaffold to recruit E2F4/5-DP1/2 transcription factors, and the MuvB core complex of five proteins, to the promoters of approximately 800 cell-cycle regulated genes. In S-phase, DREAM dissociates and the MuvB core binds to B-Myb to promote expression of mitotic genes. The LIN52 subunit of the MuvB core serves as an adaptor that mediates the binding between p130 and MuvB core proteins. Our data show that LIN52 binds to the LxCxE-binding cleft in p130, and that the E7 protein can compete for p130 binding to LIN52 therefore disrupting the DREAM complex. Indeed, we found that the DREAM assembly was impaired in human cancer cell lines HeLa and SiHa containing integrated viral genomes of HPV18 and HPV16, respectively. Using our recently solved structure of the p130-LIN52 complex, we identified a LIN52 mutant (LIN52 S20C) that can compete for HPV E7 binding to p130. Stable expression of S20C-LIN52 in HeLa and SiHa cells resulted in significantly increased DREAM complex assembly as well as suppression of proliferation and colony formation in 2D and 3D cultures. Our findings identify the DREAM complex as an important tumor suppressor disrupted by high risk HPV, and support the rationale for targeting the interaction between the oncogenic HPV proteins and host cell factors as a therapeutic approach for treatment of HPV-positive cancers and pre-cancerous lesions.

#3793

Understanding the function of LMP1 CTAR3 in EBV-associated lymphomas.

Tabithia Ross, Gretchen L. Bentz. _Mercer University School of Medicine, Macon, GA_.

Background: Our present study aims to determine the function of the repetitive elements in the biology of Epstein-Barr virus (EBV) Latent Membrane Protein-1 (LMP1). Latent EBV infection is associated with distinct lymphoid and epithelial malignancies, and LMP1 is the principal oncoprotein of the virus. LMP1 has three C-terminal activating regions (CTARs) that induce multiple signal transduction pathways resulting in sustained cellular proliferation, increased cellular survival, and its oncogenic nature. CTAR1 and CTAR2 have been extensively characterized, but CTAR3 is much less studied. CTAR3 contains the LMP1 repetitive elements, which consist of 11-amino acid repeats and PxxPxP/Jak-interacting motifs. Analysis of over 200 clinical isolates revealed that there is significant variability in the number of 11-aaR (1.5-7.5 repeats) and PxxPxP-motives (1-2 repeats), but the exact function of these elements in oncogenesis is unknown. The findings of the study will greatly add to the understanding of the oncogenic nature of LMP1 and help to elucidate a specific purpose for the repetitive elements found within CTAR3.

Methods: Using EBV B95.8 as our wild type strain, we constructed GFP-tagged LMP1 expression constructs that contain (1) wild type CTAR3; (2) PxxPxP only (no 11-aaR); (3) 11-aaR only (no PxxPxP); and (4) ∆CTAR3 (no 11-aaR or PxxPxP). These expression constructs were transfected into HEK 293 cells to assess the effect of the repeats on the trafficking and oncogenic potential of LMP1.

Results: Findings suggest that the 11-aaR are necessary for correct intracellular and extracellular trafficking of LMP1. Loss of the 11-aaRs results in decreased plasma membrane-expressed LMP1 and the accumulation of LMP1 in intracellular membranes. Further, loss of the 11-aaR inhibits LMP1-induced cell migration, which suggests that the 11-aaR contribute to the oncogenic nature of LMP1.

Conclusions: The number of 11-aaR found in LMP1 CTAR3 is variable in nature. Here we show that loss of the 11-aaR inhibits the normal biology of the oncoprotein, in that LMP1 trafficking is altered and the ability of the oncoprotein in promote cell migration is abrogated. Therefore, we propose that the CTAR3 11-aaR are essential to the oncogenic nature of LMP1. The role of these repeats during the transformation process and during tumorigenesis will be determined. Ultimately, further appreciation of the role of LMP1 CTAR3 in the biology of the virus may help identify potential targets for therapeutic interventions for EBV-associated malignancies.

#3794

**Understanding** H. pylori **-related gastric MALToma using multi-omics data.**

Hyojin Song,1 Eun Ae Kang,2 Hosim Soh,2 Sungyoung Lee,2 Hongseok Yun,2 Hyunsoo Chung,2 Jaeyoung Chun,2 Sung-Soo Yoon,2 Youngil Koh2. 1 _Seoul National University College of Medicine, Seoul, Republic of Korea;_ 2 _Seoul National University Hospital, Seoul, Republic of Korea_.

Mucosa-associated lymphoid tissue lymphoma (MALToma) is an extranodal B cell lymphoma, predominantly associated with bacterial infection of the stomach by Helicobacter pylori (H. pylori). Infection of H. pylori can be divided into four steps: 1) entrance into host and survival; 2) motility and chemotaxis; 3) Interaction between adhesion-receptors and colonization, and 4) release of toxins and damage to host. To date, pathogenesis of bacteria related lymphoma and individual functions of H. pylori genes are not yet unearthed. Hence, we aim to characterize the carcinogenic mechanism of H. pylori-related MALToma throughout this study.We obtained fresh gastric tissues of both tumor and adjacent normal region from MALToma patients. Using the tissue and saliva samples, we generated 12 sets of whole exome and transcriptome sequencing data. To identify the gene expression pattern, we aligned the raw data using STAR algorithm and quantified expression of genes in both H. pylori and human genome. Next, we estimated the relative abundance of immune cells in each tissue, employing a linear support vector regression (SVR) based analytical algorithm, CIBERSORT. Besides, we investigated somatic mutations in the patients using our internal pipeline.Gene expression profiling of H. pylori enabled us to identify frequently expressed genes in MALToma, and among them, aliphatic amidase (aimE) was expressed in 9 of 21 lesion tissues. The role of aimE is to contribute to ammonia production, which is analogous to that urease. Then, we explored gene expression pattern of immune checkpoint modulators. We determined that major groups of high CD112, CD155, and VISTA expression, and minor groups of high CD86 and PD-L1 expression, signifying cell adhesion and regulation of T cell activation.From CIBERSORT data, we found that increment of resting CD4 memory T cells and decrease of plasma cells in pair-wise MALToma donors. Since CD4+ T cells are abundantly distributed in the gastric cellular infiltrates, this immune cell composition implies that CD4 memory T cells might have been re-activated by exposure to bacterial antigens. In addition, majority of B cells including plasma cells die after they fight against infected cells, and this supports the decreased plasma cells in the tumor tissues.Using exome sequencing data, we identified two recurrent missense mutations (K169R and N175S) of a cell cycle gene, CDC27, in ~20% of MALToma patients. across the donors. Previous studies mentioned that impaired functions of CDC27 facilitate cell growth as it fails to regulate the cell growth inhibition.To understand carcinogenesis of H. pylori-related MALToma, we focused on determining gene expression patterns, immune cell composition, and recurrent somatic mutations. In conclusion, we demonstrated expression profiling of both H. pylori genes and human checkpoint modulators, estimation of immune cell abundance, and verification of recurrent DNA mutations, in MALToma.

#3795

Antibiotics induce mitochondrial dysfunction, DNA damage and aerobic glycolysis of human mammary epithelia and fibroblast.

Robert L. Elliott,1 Xianpeng Jiang2. 1 _Mastology Center, Baton Rouge, LA;_ 2 _Sallie A. Burdine Breast Foundation, Baton Rouge, LA_.

Mitochondria evolved from free-living bacteria after being endocytosed by eukaryotic host cells millions of year ago. We hypothesized that antibiotics could cause mammalian mitochondrial damage while causing bacterial lethality. Mitochondrial toxicity of three antibiotics, azithromycin, doxycycline and ciprofloxacin, were tested in human mammary epithelia MCF-12A and fibroblast by fluorescent and transmission electron microscopy. Gene expression and DNA damage were tested by real time polymerase chain reaction (qPCR) and ELISA. We found antibiotics suppressed the mitochondrial membrane potential gradient of MCF-12A and fibroblast. Ultrastructural studies showed that antibiotics caused vacuolated swollen mitochondria with disrupted cristae in MCF-12A and fibroblast, compared to the ultrastructure of mitochondria in the cells without antibiotic treatment. Fluorescent microscopy revealed antibiotics induce mitochondrial reactive oxygen species (ROS), superoxide, after 3 hours of culture. The DNA oxidative damage product, 8-OHdG, was significantly increased in the media after MCF-12A and fibroblast were cultured in antibiotic media for 24 hours. Antibiotics upregulated gene expression of hypoxia inducible factor 1 alpha (HIF1a), glycolytic enzymes including hexokinase 2(HK2), phosphofructokinase 1 (PFKM) and pyruvate kinase muscle isozyme M2 (PKM2), lactate dehydrogenase A (LDHA), and glucose transporters 1 (SLC2A1) and 3 (SLC2A3) in MCF-12A and fibroblast. Lactate production was also increased in the culture media of healthy cells MCF-12A and fibroblast cells, confirming aerobic glycolysis, or "the Warburg effect", the method to generate energy after cells are treated with antibiotics. In summary, antibiotics caused mitochondrial toxicity, ROS overproduction, DNA oxidant damage, upregulation of HIF1a gene and aerobic glycolysis in healthy mammalian cells. Over-usage of antibiotics could contribute to tumorigenesis and neurodegeneration and aggravate existing mitochondria-associated diseases.

## EXPERIMENTAL AND MOLECULAR THERAPEUTICS

### Drug Resistance 5

#3796

Selonsertib, an ASK1 inhibitor, antagonizes ABCB1- and ABCG2-mediated chemotherapeutic drug resistance.

Ning Ji,1 Yuqi Yang,1 Chao-Yun Cai,1 Zi-Ning Lei,1 Jing-Quan Wang,1 Pranav Gupta,1 Suneet Shukla,2 Suresh V. Ambudkar,2 Dexin Kong,3 Zhe-Sheng Chen1. 1 _St. John's University, Jamaica, NY;_ 2 _National Cancer Institute, NIH, Bethesda, MD;_ 3 _Tianjin Medical University, Tianjin, China_.

The overexpression of ATP-binding cassette (ABC) transporters has known to be one of the most important mechanisms responsible for the development of multidrug resistance (MDR). Selonsertib, an apoptosis signal-regulating kinase 1 (ASK1) inhibitor, is in phase III clinical trial for the treatment of non-alcoholic steatohepatitis (NASH). In this study, we investigated whether selonsertib could antagonize MDR mediated by ABC transporters, involving in ABCB1, ABCG2, ABCC1 and ABCC10. The results showed that selonsertib significantly reversed ABCB1- and ABCG2-mediated MDR, but not MDR-mediated by ABCC1 or ABCC10. Mechanistically, our studies indicated that the reversal effect of selonsertib was related to the attenuation of the efflux function of ABCB1 and ABCG2 transporters, consequently enhancing intracellular accumulation of substrate drugs. Meanwhile, selonsertib, at reversal concentration, affected neither the expression level of ABCB1 and ABCG2 nor the localization of corresponding proteins in subcellular level. Selonsertib stimulated the ATPase activity of both ABCB1 and ABCG2 in a concentration-dependent manner. Our in silico docking study showed that selonsertib could interact with the substrate-binding sites of both ABCB1 and ABCG2. This study provides a clue into a novel treatment strategy, which includes a combination of selonsertib with antineoplastic drugs to circumvent ABCB1- or ABCG2- mediated MDR.

#3797

AsiDNA abrogates acquired resistance to PARP inhibitors.

Wael Jdey, Pauline Lascaux, Françoise Bono. _Onxeo, Paris, France_.

Purpose: PARP inhibitors (PARPi) are approved for the treatment of homologous recombination (HR)-deficient cancers. Despite the success of this approach, drug resistance remain a clinical hurdle, and several attempts failed to decrease this resistance. In the current study, we propose a novel therapeutic strategy, based on drug combination to abolish the onset of resistance to PARPi.

Experimental design: We performed a set of experiments to induce resistance to PARPi and assess the impact of AsiDNA addition to decrease this resistance occurrence. AsiDNA is a double stranded DNA molecule (decoy oligo-nucleotide) that mimics double stranded DNA breaks (DSBs) to interfere with DNA repair, by over-activating a "false" signaling of DNA damage through DNA-PK and PARP enzymes. We characterized the effects of long term treatment with the PARPi olaparib and talazoparib on resistance selection in two different cell lines, BC227 and NCI-H446, and we further analyzed the effect of low doses of AsiDNA on the occurrence of acquired resistance to these PARPi. Finally, we addressed the mechanisms underlying resistance emergence and abrogation.

Results: Long term exposure of cancer cells to PARPi induces the emergence of resistance in all the tested independent populations (11 BC227 independent populations and 3 NCI-H446 for talazoparib, 5 BC227 independent populations for olaparib), raising the question of the clinical benefit of long-term maintenance monotherapy with PARPi. Interestingly, double-treated populations with AsiDNA (2.5µM - low non cytotoxic dose) and talazoparib or olaparib showed a significant lower probability of resistance occurrence. In fact, 9/11 (or 5/5) BC227 populations treated with AsiDNA and talazoparib (or AsiDNA and olaparib) remained sensitive up to 5 cycles of treatment, and 3/3 NCI-H446 populations treated with AsiDNA and talazoparib. Interestingly, AsiDNA is also able to partially revert talazoparib resistance in BC227 and NCI-H446 resistant populations. As described, PARP1, EZH2 and/or 53BP1 protein expression loss or reduction are important mechanisms leading to PARPi resistance. We confirmed in these experiments that treatments with PARPi significantly modify the expression and activity of these proteins and we demonstrated that co-treatment with AsiDNA inhibits these changes. These results indicate that AsiDNA may abrogate and reverse PARPi acquired resistance by the normalization of involved protein expression and activity.

Conclusion: Our results highlight the therapeutic interest of combining AsiDNA and PARPi to recapitulate drug-driven PARPi resistance abrogation. As PARPi have obtained FDA approval, and AsiDNA is being currently tested in a clinical trial, a potential exists for a rapid clinical confirmation of AsiDNA-induced PARPi resistance abrogation and reversion.

#3798

Acetate supplementation eliminates hypoxia mediated resistance to differentiation therapy in neuroblastoma cells.

Yang Li,1 Yiren Zhou,1 John M. Maris,2 Amato J. Giaccia,1 Jiangbin Ye1. 1 _Stanford University, Stanford, CA;_ 2 _The Children's Hospital of Philadelphia, Philadelphia, PA_.

Neuroblastoma is the most common extracranial solid tumor in childhood. Retinoic acid (RA) leads to cell cycle arrest and induces cell differentiation in neuroblastoma cells, thus it has been used to treat neuroblastoma as differentiating agent for decades. However, therapy resistance often occurs and the overall survival remains low. Hypoxia, a common metabolic stress existing in tumor microenvironment, increases tumor resistance to RA by promoting dedifferentiation of neuroblastoma cells. The aim of this study was to elucidate the mechanisms underlying hypoxia-mediated therapy resistance and develop therapeutic approach to improve the response to differentiation therapy. By RNA-Seq analysis, we identified a group of neuron-specific differentiation markers that induced by RA treatment, while the induction was diminished under hypoxia. Associated with the silencing of differentiation markers expression, a significant loss of histone acetylation of H3K9, H3K27 and total H3 was observed under hypoxia. Metabolomics analysis indicated hypoxia inhibited mitochondrial oxidative phosphorylation to decrease intracellular acetyl-CoA and citrate, which provides acetyl-CoA for histone acetyltransferase. Importantly, acetate supplementation restored cellular acetyl-CoA level, histone acetylation, and the expression of differentiation markers under hypoxia. Differentiation morphology was also restored by acetate supplementation under hypoxia. In conclusion, our finding suggested hypoxia contributed to the resistance to RA-induced differentiation by reprogramming cellular metabolism and inducing histone hypoacetylation. Acetate supplementation is an efficacious therapeutic approach to improve the sensitivity of hypoxic tumor to RA-based differentiation therapy.

#3799

Large-scale pharmacogenomics based drug discovery for ITGB3 dependent chemoresistance in mesenchymal lung cancer.

Haeseung Lee,1 Ok-Seon Kwon,2 Wankyu Kim,1 Hyuk-Jin Cha2. 1 _Ewha Womans University, Seoul, Republic of Korea;_ 2 _Seoul National University, Seoul, Republic of Korea_.

Even when targets responsible for chemoresistance are identified, drug development is often hampered due to the poor druggability of these proteins. We systematically analyzed therapy-resistance with a large-scale cancer cell transcriptome and drug-response datasets and predicted the candidate drugs based on the gene expression profile. Our results implicated the epithelial-mesenchymal transition as a common mechanism underlying resistance to chemotherapeutic drugs. Notably, we identified ITGB3, whose expression was abundant in both drug resistance and mesenchymal status, as a promising target to overcome chemoresistance. We also confirmed that depletion of ITGB3 sensitized cancer cells to conventional chemotherapeutic drugs by modulating the NF-κB signaling pathway. Considering the poor druggability of ITGB3 and the lack of feasible drugs to directly inhibit this protein, we took an in silico screening for drugs mimicking the transcriptome-level changes caused by knockdown of ITGB3. This approach successfully identified atorvastatin as a novel candidate for drug repurposing, paving an alternative path to drug screening that is applicable to undruggable targets.

#3800

HDAC8 regulates plasticity and escape from therapy in BRAF mutant melanoma.

Michael Emmons,1 Fernanda Flores,1 John Koomen,1 Edward Seto,2 Jane Messina,1 Eric Lau,1 Jonathan Licht,3 Keiran Smalley1. 1 _H. Lee Moffitt Cancer Ctr. & Res. Inst., Tampa, FL; _2 _George Washington University, Washington DC, DC;_ 3 _University of Florida, Gainesville, FL_.

Melanoma cells are highly plastic and have the ability to switch to a dedifferentiated, invasive phenotype in response to multiple stimuli. We here show that exposure to melanoma cell lines and patient specimens to multiple stresses including BRAF-MEK inhibitor therapy, hypoxia and UV-irradiation leads to an increase in HDAC8 expression/activity, and in turn, the adoption of a drug-resistant, invasive phenotype. Systems level analyses using mass spectrometry-based phosphoproteomics and RNA-Seq demonstrated HDAC8 to be involved in pathways that regulated cell cycle entry, ribosome function, RNA binding, regulation of the cytoskeleton and MAPK pathway signaling. Introduction of HDAC8 into drug-naïve melanoma cells conveyed resistance in vitro and in in vivo xenograft models. The HDAC8-mediated BRAF inhibitor resistance was mediated through upstream receptor tyrosine kinase (RTK) activation, Ras/CRAF/MEK/ERK signaling and the suppression of apoptosis through increased stabilization of Mcl-1 and the inhibition of pro-apoptotic BIM. Among the multiple RTKs increased by HDAC8, EGFR emerged as a direct transcriptional target, and it was found that EGFR inhibition could overcome HDAC8-mediated tolerance to BRAF inhibition. Although it is known that HDACs primarily function at the histone level, they can also regulate signaling through the modulation of cytoplasmic protein acteylation. In line with this, we observed that HDAC8 introduction decreased the acetylation of c-JUN, leading to an increase in its transcriptional activity and the enrichment for an AP-1 gene signature. Mutation of putative acetylation sites in c-JUN (K268R, K271R, K273R) reduced the transcriptional activation of c-JUN in melanoma cells and conveyed resistance to BRAF inhibition through increased MAPK pathway activity. In vivo xenograft studies confirmed the key role of HDAC8 in therapeutic adaptation, with either a pan-HDAC inhibitor (panobinostat) or an HDAC8 inhibitor (PCI-34051) enhancing the durability of response to BRAF inhibitor therapy. We have thus identified HDAC8 as a key driver of phenotype switching in melanoma that regulates the responses to multiple cellular stresses and conveys resistance to BRAF inhibition. Our studies demonstrate that isoform-specific HDAC8 inhibitors could be an excellent strategy to limit the adaptation of melanoma cells to multiple stresses and therapeutic interventions, including the BRAF-MEK inhibitor combination.

#3801

Reversing chemoresistance in high grade serous ovarian cancer.

Jayeta Saxena, Francesco Nicolini, Michelle Lockley. _Barts Cancer Institute, Queen Mary University of London, London, United Kingdom_.

Background: High grade serous ovarian cancer (HGSOC) is the most common ovarian cancer subtype. Although it is initially responsive to platinum-containing chemotherapy, the emergence of chemo-resistant cells is inevitable. Most women with HGSOC die of platinum-resistant cancer, and the five-year overall survival for ovarian cancer has changed little in recent decades. The aim of this project is to identify and validate existing, licensed drugs that selectively target resistant cancer cells in combination with cisplatin or carboplatin.

Methods: Established chemo-sensitive HGSOC cell lines Ovcar4, COV318 and OVSAHO were passaged in vitro with increasing doses of either Cisplatin or Carboplatin to achieve a 5-10 fold difference in IC50 in resistant compared to sensitive parental cells, reflecting the chemo-resistance commonly found in patient tumor samples. We screened a library of 1170 FDA-approved drugs in Ovcar4 and carboplatin-resistant Ovcar4 (Ov4Carbo) cell lines and viability was analyzed using Cell Titre Glow (CTG). Ovcar4 or Ov4Carbo cells were injected and intraperitoneal tumor growth was assessed in female CD1 nude mice as in vivo model to assess the effects of drug combinations.

Results: Using a high throughput drug screen, we identified Chloroxine as one of the top hits that re-sensitized resistant Ov4Carbo cells to Carboplatin. We have further validated Chloroxine in the platinum sensitive/resistant cell pairs in our novel HGSOC cell line panel (Ovcar4/Ov4Cis; Ov4CarboRFP; Ov4Carbo single cell clone 7 and COV318/Cov318Cis).

Previous literature has identified kappa-type opioid receptor, OPRK1 as a potential binding target for Chloroxine. We found that the OPRK1 agonist, U50488 reproduced the synergy with Carboplatin in resistant Ov4Carbo cells, as observed with Chloroxine. Additionally, OPRK1 antagonist, nor- Binaltorphimine (nor-BNI) reduced the combined effects of Chloroxine and Carboplatin in Ov4Carbo cells suggesting that Chloroxine acts via OPRK1 receptor.

We have tested Ovcar4, Ov4Carbo and Ov4Cis in vivo and found that all formed tumors in the peritoneum of nude mice and sequential bioluminescent readings correlated with survival. We are currently testing the effects of Chloroxine or OPRK1 agonist, U50488 in combination with Carboplatin in our in-vivo models.

Conclusion: We have identified Chloroxine, an FDA-approved clinical drug that combines synergistically with carboplatin or cisplatin to induce cell death in chemo-resistant cell lines. We expect that understanding the mechanism of this synergy will provide novel insights into the pathophysiology of chemotherapy resistance in high grade serous ovarian cancer. In addition, as the pharmacokinetics and toxicity profiles of these FDA-approved drugs have already been established, this 'repurposing' of commonly used drugs will allow for a faster bench-to-bedside translation.

#3802

Interleukin-4 receptor-targeted delivery of Bcl-xL siRNA sensitizes tumors to chemotherapy and inhibits tumor growth.

Soo-Woong Lee,1 Jae-Won Yoon Yoon,1 Guruprasath Padmanaban,1 Rang- Woon Park,1 Moon-Chang Baek,1 Dong-Kyu Kim,2 Won-Jong Kim,3 Byungheon Lee1. 1 _Kyungpook National University, Daegu, Republic of Korea;_ 2 _Daegu Gyeongbuk Medical Innovation foundation, Daegu, Republic of Korea;_ 3 _Pohang University of Science and Technology, Daegu, Republic of Korea_.

IL-4 receptor (IL-4R) is commonly up-regulated on tumor cells, and interactions between the receptor and Interleukin-4 (IL-4) can induce the expression of anti-apoptotic proteins, including Bcl-xL. This contributes to tumor cell survival and their resistance to chemotherapy. In this study, we exploited IL-4R-targeted delivery of Bcl-xL siRNA to IL-4R-expressing tumor cells in order to sensitize them to chemotherapy. To target IL-4R, an IL-4R-binding peptide, IL4RPep-1, was attached to branched polyethyleneimine-superparamagnetic iron oxide nanoparticles (BPEI-SPION). These nanoparticles were then complexed with Bcl-xL-targeting siRNA. IL-4R-targeted BPEI-SPION/Bcl-xL siRNA more efficiently reduced Bcl-xL gene expression and enhanced cytotoxicity of doxorubicin in MDA-MB231 breast tumor cells compared to untargeted BPEI-SPION/Bcl-xL siRNA. The siRNA was released from the complexes after 15 h of incubation at pH 5.5 and was stable in the complexes up to 72 h in the serum. The IL-4R-targeted BPEI-SPION/siRNA was internalized by cells through IL-4R, successfully escaped the endosomes, and was dispersed into the cytoplasm. Near-infrared fluorescence and magnetic resonance imaging demonstrated that in vivo tumor homing and accumulation of IL-4R-targeted BPEI-SPION/siRNA were both higher than untargeted BPEI-SPION/siRNA. The IL-4R-targeted BPEI-SPION/Bcl-xL siRNA, in combination with doxorubicin, significantly inhibited tumor growth in mice compared to untargeted BPEI-SPION/Bcl-xL siRNA. These results suggest that the IL-4R-targeted delivery of Bcl-xL siRNA to IL-4R-expressing tumors can sensitize tumors to chemotherapy and enhance the efficacy of anti-tumor therapeutics. Keywords: Bcl-xL, BPEI-SPION, chemotherapy, IL-4R, siRNA

#3803

Targeting 17q23 amplicon to overcome the resistance to anti-HER2 therapy in HER2+ breast cancer.

Yunhua Liu,1 Xiaoming He,2 Xiongbin Lu,1 Xinna Zhang1. 1 _Indiana University, Indianapolis, IN;_ 2 _University of Maryland, College Park, MD_.

Amplification of chromosome 17q23 is a frequent genomic event that occurs in ~ 11% of human breast cancers. The 17q23 amplification is enriched in HER2+ breast cancers, which is significantly correlated with poor clinical outcomes. Previous studies have identified the oncogenic phosphatase WIP1 gene in the amplicon, which functions as a master inhibitor in DNA damage response. While the possibility of any other protein-coding oncogenes in the WIP1-containing 17q23 amplicon was ruled out, our analysis of human breast cancer genomics uncovered an oncogenic microRNA gene, MIR21, in a majority of the WIP1-containing amplicons. Accordingly, the 17q23 amplification results in aberrant expression of WIP1 and miR-21, which not only promotes breast tumorigenesis, but also leads to resistance to anti-HER2 therapies. Inhibiting WIP1 and miR-21 using small molecular inhibitor against WIP1 (GSK2830371) and anti-miR-21 oligonucleotides selectively inhibits the proliferation, survival and tumorigenic potential of HER2+ breast cancer cells harboring 17q23 amplification. However, the in vivo bioavailability of the two agents in their free form is poor. To overcome the resistance of trastuzumab-based therapies in vivo, we developed pH-sensitive nanoparticles for specific co-delivery of the two agents into breast tumors. The nanoparticles can improve the encapsulation efficiency of anti-miR-21 oligonucleotides but also effectively capture carbon dioxide (CO2) into the nanoparticle to achieve the 'nano-bomb' effect for triggered drug release under the reduced pH in tumors. The two agents (inhibitors of miR-21 and WIP1)-laden nanoparticles can be used to efficiently kill trastuzumab-resistant HER2+ breast cancer cells, leading to a profound reduction of the tumor growth in vivo. These results demonstrate the great potential of the combined treatment of WIP1 and miR-21 inhibitors for the HER2+ breast cancers resistant to anti-HER2-based therapies.

#3804

The potent AXL kinase inhibitor, TP-0903, is active in pre-clinical models of EGFR positive non-small cell lung cancer.

Ryan Mangelson, Ethika Tyagi, Peter Peterson, Adam Siddiqui-Jain, Clifford J. Whatcott, David J. Bearss, Steven L. Warner. _Tolero Pharmaceuticals, Inc., Lehi, UT_.

Lung cancer is the leading cause of cancer related death in the US (2017). Non-small cell lung cancer (NSCLC) accounts for more than 80% of all lung cancer cases. While EGFR inhibitors (EGFRi) have shown activity in NSCLC, single agent response rates often do not exceed 10%. EGFR mutation is a common oncogenic driver in NSCLC, appearing in between 13% and 47% of all NSCLC patients depending on ethnicity. Third generation EGFRis, such as osimertinib, were approved upon demonstrating improved progression-free survival (PFS) rates versus standard therapy. However, EGFR inhibitor resistance, including resistance to osimertinib, has been reported to result from the epithelial-to-mesenchymal transition (EMT) that is coincident with increased AXL kinase expression. The AXL RTK drives EMT and constitutes a bypass survival pathway for tumor cells under EGFRi treatment pressure. We have shown that treatment with TP-0903, our potent AXL inhibitor, leads to a reversal of the mesenchymal phenotype in multiple cancer models. We therefore hypothesized that TP-0903 treatment may potentiate EGFRi treatment in cancer, and in particular EGFR mutant NSCLC. To interrogate our hypothesis, we treated cells with TP-0903 and assessed changes in cell viability with the celltiter-glo assay, changes in mRNA expression using RT-qPCR, and protein expression changes, using standard immunoblotting. In cell viability assays in the H1650 NSCLC cell line, TP-0903 showed an EC50 of 39 nM, while osimertinib showed an EC50 of 2.2 µM. In mRNA and protein assays, we observed changes consistent with a reversal of the mesenchymal phenotype. Following treatment, Slug mRNA expression was inhibited as much as 3.8-fold. However, E-cadherin expression was increased by 1.6-fold. To assess the combination in vivo, we utilized the H1650 xenograft model for NSCLC. In pharmacodynamic assessment of EMT markers in vivo, Snail protein expression was reduced as much as 56% following a single dose of TP-0903 (40 mpk, at 24hrs). In assessment of treatment efficacy in vivo, and with TP-0903 treatment (40 mpk, qd), we observed 60% tumor growth inhibition (%TGI) over the course of a 21-day treatment regimen. With osimertinib treatment (20 mpk, qd), we observed 121 %TGI. However, with the combination, we observed 140 %TGI. Due to its ability to reverse the aggressive mesenchymal phenotype of cancer cells, TP-0903 is a promising agent with the potential to have single agent activity and combined synergy with targeted anti-cancer agents. A Phase I trial with this investigational agent is ongoing, which includes patients with EGFR positive non-small cell lung cancer (clincaltrials.gov, NCT02729298). Taken together, the current study supports continued development of AXL inhibitors in NSCLC, especially in combination with EGFRis.

#3805

Artesunate reduces tumor growth in sunitinb-resistant renal cell carcinoma cells.

Eva Juengel,1 Sascha Markowitsch,1 Holger H. Erb,1 Thomas Efferth,2 Axel Haferkamp1. 1 _University Medical Center Mainz, Mainz, Germany;_ 2 _Johannes Gutenberg University Mainz, Mainz, Germany_.

Introduction: Renal cell carcinoma (RCC) is the most common cancer of the kidney. Due to therapy resistance effect of the approved compounds, like the tyrosine kinase inhibitor (TKI) sunitinib, is limited. Thus, new treatment approaches are urgently needed. Artesunate (ART) is derived from Traditional Chinese Medicine (TCM), showing anti-tumor activity, also in urologic tumors. However, little is known about the efficiency of ART on therapy-resistant RCC. Therefore, we investigated the effect of ART on sunitinib-resistant RCC cell lines.

Material and Methods: Parental (=sensitive) and sunitinib-resistant (1 µM) RCC cell lines, KTC-26, A498, and Caki-1, were exposed to ART (1-100 µM) for 24, 48 and 72 hours. ART untreated cells served as controls. Tumor cell growth, proliferation, cell cycling and the expression of cell cycle regulating proteins were then evaluated. Aside, the appearance of apoptotic and necrotic effects was determined.

Results: Exposure to different concentrations of ART significantly reduced the tumor cell growth and proliferation of parental and sunitinib-resistant cells in a time- and dose-dependent manner, compared to the controls. At the same time, exposure to ART resulted in a cell cycle arrest in the G0/G1 phase. Notably, this effect was more prominent in the sunitinib-resistant cells. G0/G1 phase elevation was associated with a decrease in the percentage of S and G2/M phase cells. The observed changes in cell growth and cell cycle phases were accompanied by distinct modulations of the cell cycle regulating proteins, indicating for an inhibitory regulation of ART through cell cycle arrest. Indeed, neither necrotic nor apoptotic effects events were detectable after ART treatment, corroborating this assumption.

Conclusion: Due to our data we conclude that ART has anti-tumor effects not only in parental but also in sunitinib-resistant RCC cells. Thus, adding ART to the standard therapies might be a promising treatment strategy for patients with advanced RCC. Further investigations are necessary to verify our findings.

#3806

Overcoming drug-tolerant cancer cell subpopulations showing AXL activation and epithelial-mesenchymal transition is critical in conquering ALK-positive lung cancer.

Shinji Nakamichi, Seike Masahiro, Akihiko Miyanaga, Akiko Takahashi, Rintaro Noro, Kaoru Kubota, Akihiko Gemma. _Nippon Medical School, Tokyo, Japan_.

Anaplastic lymphoma kinase tyrosine kinase inhibitors (ALK-TKIs) induce a dramatic response in non-small cell lung cancer (NSCLC) patients with the ALK fusion gene. However, acquired resistance to ALK-TKIs remains an inevitable problem. In this study, we aimed to discover novel therapeutic targets to conquer ALK-positive lung cancer. We established three types of ALK-TKI (crizotinib, alectinib and ceritinib) -resistant H2228 NSCLC cell lines by high exposure and stepwise methods. We found these cells showed a loss of ALK signaling, overexpressed AXL with epithelial-mesenchymal transition (EMT), and had cancer stem cell-like (CSC) properties, suggesting drug-tolerant cancer cell subpopulations. Similarly, we demonstrated that TGF-β1 treated H2228 cells also showed AXL overexpression with EMT features and ALK-TKI resistance. The AXL inhibitor, R428, or HSP90 inhibitor, ganetespib, were effective in reversing ALK-TKI resistance and EMT changes in both ALK-TKI-resistant and TGF-β1-exposed H2228 cells. Tumor volumes of xenograft mice implanted with established H2228-ceritinib-resistant (H2228-CER) cells were significantly reduced after treatment with ganetespib, or ganetespib in combination with ceritinib. Some ALK-positive NSCLC patients with AXL overexpression showed a poorer response to crizotinib therapy than patients with a low expression of AXL. ALK signaling-independent AXL overexpressed in drug-tolerant cancer cell subpopulations with EMT and CSC features may be commonly involved commonly involved in intrinsic and acquired resistance to ALK-TKIs. This suggests AXL and HSP90 inhibitors may be promising therapeutic drugs to overcome drug-tolerant cancer cell subpopulations in ALK-positive NSCLC patients for the reason that ALK-positive NSCLC cells do not live through ALK-TKI therapy.

#3808

225 **Ac-CD33 Radioimmunotherapy potently increases the sensitivity of resistant acute myeloid leukemia lines to the Bcl-2 inhibitor venetoclax by mediating a reduction in cellular Mcl-1 levels.**

Ravendra Garg,1 Eileen Geoghegan,2 Kevin J. Allen,1 Wojciech Dawicki,1 Ekaterina Dadachova,1 Dale L. Ludwig2. 1 _University of Saskatchewan, Saskatoon, Saskatchewan, Canada;_ 2 _Actinium Pharmaceuticals, New York, NY_.

Acute myeloid leukemia (AML) is a complex hematological disease often occurring in older patients. While a number of new targeted therapies have been recently approved, patient outcomes remain poor. In the US, about 19,520 new cases AML occur annually and approximately 10,670 will die from the disease. Venetoclax is a promising new targeted therapy that is under regulatory review in the US for use in combination with a hypomethylating agent (HMA) or with low-dose cytarabine (LDAC) for the treatment of newly diagnosed patients with acute myeloid leukemia (AML) who are ineligible for intensive chemotherapy. Recently, several studies have demonstrated that increased expression of Mcl-1 is a mediator of resistance to venetoclax in AML and other hematologic malignancies. Indeed, significant up-regulation of Mcl-1 has been reported in patients with relapsed/refractory AML and venetoclax treatment itself has been shown to increase Mcl-1 levels tumor cell lines. 225Ac-lintuzumab is a clinical stage radioimmunotherapy targeting CD33 that has shown evidence of single agent activity in relapsed/refractory AML. 225Ac-lintuzumab enables delivery of the potent alpha emitting warhead 225-actinium (T1/2 = 10 days; high linear energy transfer = 6.83 MeV; and short path length = 40-100 μm or about 3-5 cell diameters) directly to tumor cells. This elicits DNA cluster breaks and double strand breaks to kill within a short radius, thereby limiting damage to normal tissue. In this study, we demonstrate that 225Ac-lintuzumab is capable of dramatically enhancing the potency of venetoclax when used in combination in both venetoclax sensitive and resistant AML cell lines. AML lines U937 and OCI-AML3 are Mcl-1 positive AML lines which are highly resistant to venetoclax (IC50 > 1 μM). While treatment of these lines with single agent venetoclax at 500 nM was ineffective at suppressing tumor cell growth in either model, the combination of 40 nCi 225Ac-lintuzumab plus 500 nM venetoclax induced a statistically significant increase tumor cell killing in vitro. While 225Ac-lintuzumab can directly effect tumor cell killing by multiple mechanisms, we investigated the potential for 225Ac-lintuzumab-directed DNA damage to suppress Mcl-1 levels as a potential mechanism of enhancing the potency of venetoclax in these models. Cell lines were incubated with titrations of 225Ac-lintuzumab prior to analysis of Mcl-1 protein by ELISA. In both cell lines, treatment with 225Ac-lintuzumab lead to a significant depletion of Mcl-1 levels in cells. This supports a mechanistic basis of Mcl-1 suppression when 225Ac-lintuzumab is used in combination with venetoclax to increase the potency of resistant AML cells to the Bcl-2 inhibitor. Results of in vivo tumor xenograft studies will also be presented.

#3809

Modulation of MRP1 activity reverses chemotherapy resistance in adult cancer cells.

Kimberley M. Hanssen,1 Christine C. Gana,1 Madeleine S. Wheatley,1 Denise M. Yu,1 Claudia L. Flemming,1 Richard Young,2 Gavin M. Wright,2 Gwenaelle Conseil,3 Catherine J. Kennedy,4 Anna deFazio,4 Ben Solomon,2 Susan P. Cole,3 Murray D. Norris,1 Michelle Haber,1 Jamie I. Fletcher1. 1 _Children's Cancer Institute Australia, Randwick, Australia;_ 2 _Peter MacCallum Cancer Centre, Melbourne, Australia;_ 3 _Queen's University Cancer Research Institute, Kingston, Ontario, Canada;_ 4 _The Westmead Institute for Medical Research, Westmead, Australia_.

Multidrug resistance protein 1 (MRP1) is an ATP-dependent efflux pump that extrudes chemotherapeutic drugs from cancer cells, preventing their intracellular accumulation and thus their efficacy. MRP1 also regulates intracellular levels of glutathione (GSH), an antioxidant that when elevated in cancer provides resistance against chemotherapy- and radiotherapy-induced oxidative damage. The different binding sites for drugs and GSH on MRP1 has allowed the development of small molecule MRP1 modulators that block chemotherapy efflux whilst simultaneously enhancing GSH efflux to deplete GSH. As no therapeutic options are available to combat MRP1-mediated multidrug resistance, we have been investigating MRP1 modulators for their efficacy in sensitizing MRP1-expressing cancers to treatment. MRP1 immunohistochemistry staining in ovarian and non-small cell lung cancer (NSCLC) patient samples was assessed to determine the frequency of high MRP1 expression. High MRP1 staining was frequently observed in the subcellular compartment (37%, 47%) and, to a lesser extent, plasma membrane (7%, 16%) in ovarian and NSCLC patient tumour samples respectively. In NSCLC, subcellular MRP1 was associated with poorer overall survival in late stage disease (p=0.0095) and membrane MRP1 with poorer disease-free survival in Stage 4 cancer (p=0.01449). Analysis for ovarian cancer is ongoing. As MRP1 is therefore an attractive target for inhibition, we used membrane vesicle uptake assays to compare the ability of modulators to inhibit [3H]-labelled MRP1 substrate transport. 7-(difluoromethyl)-N-4-morpholinyl-5-phenylpyrazolo[1,5-a]pyrimidine-3-carboxamide (7914321) was identified as an analogue of the MRP1 inhibitor Reversan with improved potency. Interestingly, this inhibition was GSH dependent. Exploring further this relationship with GSH, the high MRP1-expressing A549 lung and SKOV3 ovarian cancer cell lines were treated with 7914321 alone or in combination with the GSH synthesis inhibitor buthionine sulfoximine (BSO), then GSH levels were determined by glutathione recycling assay. 7914321 stimulated GSH efflux in both cell lines in an MRP1-dependent manner and strongly synergised with BSO to cause near complete GSH depletion. To determine the potential of this synergistic GSH depletion to chemosensitize cells, the viability of A549 and SKOV3 cells treated with chemotherapy, 7914321, and BSO was assessed by clonogenic and resazurin-based cytotoxicity assays. 7914321/BSO significantly diminished clonogenicity in high but not low MRP1-expressing cells, and further sensitized cancer cell lines to MRP1-substrate chemotherapeutics compared to the single agents. Together, these findings provide preliminary evidence that 7914321 is a potent MRP1 modulator whose dual effects on MRP1 place it in a unique position to potentially enhance the therapeutic window to treat chemoresistant MRP1-overexpressing cancers.

#3810

Establishment of an in vitro assay that phenocopies tumor response to PARP inhibitors in vivo.

Olivier Déas, Romain Rousseau, Sophie Banis, Kathleen Flosseau, Enora Le Ven, Jean-Gabriel Judde, stefano cairo. _XenTech, Evry, France_.

Poly (adenosine diphosphate-ribose) polymerase inhibitors (PARPi) have recently emerged as therapeutic options for patients with homologous recombination-deficient (HRD) breast or ovarian cancer, two heterogeneous diseases associated with high mortality rates. As shown in several clinical studies, patient response to PARPi is invariably followed by eventual mid-long term resistance and progression under treatment. The molecular processes contributing to PARPi-resistance are at present under-explored. Therefore, a huge effort is being made to better understand how to overcome resistance and to identify ad-hoc combinations with other targeted therapies to improve tumor response and extend progression-free survival. We have previously published the PARPi-response profile of a panel of 40 breast cancer (BC) patient-derived xenografts (PDXs). The models tested showed heterogeneous response to PARPi, and only partial association with the genomic status of BRCA genes, the only currently acknowledged clinical marker to select patients that can benefit from PARPi administration. Although these models are ideal preclinical tools for the evaluation of drug combinations to improve tumor response to PARPi as single agent, the use of these models for early preclinical evaluation of combination efficacy is not straightforward in reason of the limited throughput and of the ethical issue with respect of the 3Rs. The use of cellular models is still considered as a standard early preclinical test to evaluate drug response before moving to in vivo assays. However, the main concern when using in vitro models is the representativeness of the results obtained when transposed to in vivo models. In this study, we have setup an in vitro assay that recapitulates the response to PARPi observed in vivo in our BC PDXs, with particular focus on the models that show resistance to PARPi. To this aim we generated 9 cellular models from 9 BC PDX models: HBCx-2, HBCx-3, HBCx-6, HBCx-8, HBCx-9, HBCx-17, HBCx-19, HBCx-39 and T174. Two cellular models (HBCx-3, and HBCx-19) were established from ER+ BC PDX and 7 from ER- BC PDXs and two models, HBCx-8 and HBCx-17, harbor BRCA1 and BRCA2 mutation, respectively. Seven out of 9 models are resistant to PARPi in vivo, with HBCx-6 PDX showing partial tumor regression and HBCx-17 PDX showing tumor stabilization upon treatment. Several different 2D-culture experimental conditions, namely different cell growth conditions, drug concentrations, duration of cell exposure to drugs, time points and readouts, have been tested to evaluate response to olaparib. The results showed that a 2D colony assay is the best experimental strategy to faithfully evaluate tumor cell sensitivity, minimizing the false positive results when compared to the in vivo data.. This cell panel will be used to identify combination of PARPi with a library of FDA-approved targeted therapy to identify the treatments to be moved forward in vivo.

#3811

**Overcoming** in vivo **chemotherapy resistance in triple negative breast cancer** via **targeting lysyl oxidase (LOX).**

Ozge Saatci,1 Umar Raza,2 Pelin Ersan,2 Carolyn Banister,1 Suhail Ansari,2 Madeleine Arseneault,3 Phillip Buckhaults,1 Yasser Riazalhosseini,3 Ozgur Sahin1. 1 _University of South Carolina, Columbia, SC;_ 2 _Bilkent University, Ankara, Turkey;_ 3 _McGill University, Montreal, Quebec, Canada_.

Triple negative breast cancer (TNBC) is the most aggressive subtype, and treatment relies solely on chemotherapy due to lack of effective targeted therapies. Chemoresistance is common among TNBC patients, leading to poor survival rates. To identify novel targets that could re-sensitize TNBC to standard chemotherapy, we developed chemoresistant TNBC tumors in vivo, then performed whole transcriptome sequencing and pathway analysis on the tumor cells. This identified a novel intersection between integrin-extracellular matrix (ECM) interaction and hypoxia in acquired chemoresistance of TNBC, which was mainly driven by overexpression of a highly central ECM re-modulator, lysyl oxidase (LOX). Inhibition of LOX overcomes doxorubicin resistance in ex vivo, in vitro, and in vivo models of aggressive TNBC, and potentiates doxorubicin response as a first-line therapy in treatment naïve samples in vivo and in a 3D organoid model. Mechanistically, upregulation of LOX in hypoxic tumors treated with chemotherapy activates Integrin Subunit Alpha 5 (ITGA5) and its associated Focal Adhesion Kinase/Src complex FAK/Src, leading to chemoresistance. Hypoxia also downregulates the expression of miR-142-3p, which targets HIF1A, LOX and ITGA5, leading to further activation of the HIF1A/LOX/ITGA5 axis and downstream FAK/Src signaling. Importantly, higher expression of LOX or ITGA5, or lower expression of miR-142-3p was associated with shorter relapse-free survival, but only in chemotherapy-treated TNBC patients. Taken together, these results provide pre-clinical rationale for development and testing of LOX inhibitors to potentiate chemotherapy response in either drug resistant or treatment-naïve TNBC patients.

#3812

Overcoming de novo and acquired modes of cetuximab resistance by broad-spectrum receptor tyrosine kinase inhibitors.

Bhuminder Singh, Ramona Graves-Deal, Galina Bogatcheva, Robert J. Coffey. _Vanderbilt University Medical Center, Nashville, TN_.

MEK inhibition has been shown to delay emergence of cetuximab resistance in colorectal cancer (CRC) (Nat Commun 22;6:8305, 2015). By exploiting a novel 3D culture system, we herein show that three broad-spectrum tyrosine kinase inhibitors overcome de novo and acquired resistance to cetuximab. By culturing a CRC cell line, HCA-7 in 3D in type I collagen, we previously identified two novel modes of cetuximab resistance. In 3D culture, HCA-7-derived CC are sensitive to cetuximab, while SC exhibit de novo cetuximab resistance (PNAS 114:E2852-E2861, 2017). Continuous exposure of CC to cetuximab resulted in colonies that acquired resistance to cetuximab, designated CC-CR, which was dependent in part on increased levels of lncRNA MIR100HG that leads to increased WNT signaling (Nat Med 23:1331-1341, 2017). We previously showed increased MET and RON tyrosine phosphorylation in SC cells and that MET/RON inhibitor, crizotinib overcomes SC cetuximab resistance. We now show that crizotinib also overcomes acquired cetuximab resistance in CC-CR. A phospho-RTK array showed increased phosphorylation of several RTKs, including MET and RON, in SC and CC-CR cells compared to cetuximab-sensitive CC counterparts. Furthermore, other multi-RTK inhibitors cabozantinib and BMS-777607 helped overcome cetuximab resistance, as measured by 3D colony growth and activation state of key signaling molecules. Conversely, addition of RTK ligands HGF and NRG1 induced cetuximab resistance in CC cells, which could be blocked by addition of crizotinib and this was associated with disorganized spindle positioning during cell division, leading to multi-layering of CC cultures. By FACS analysis, we observed increased cell cycle arrest in G1 phase in SC and CC-CR cultures treated with cetuximab and crizotinib. Furthermore, we show that crizotinib overcomes cetuximab resistance in vivo in SC nude mice xenografts. Finally, in a direct comparison between vertical suppression (EGFR/MEK inhibition) and parallel inhibition (EGFR/multi-RTK inhibition), we observed a higher degree of synergy with the latter strategy. The non-overlapping toxicities of cetuximab and crizotinib warrants consideration for its clinical deployment.

#3813

PHB2 inactivation by AKAP-BIG3 is required for progression of HER2-overexpressing breast cancer.

Tetsuro Yoshimaru,1 Yosuke Matsushita,1 Mitsunori Sasa,2 Yasuo Miyoshi,3 Toyomasa Katagiri1. 1 _Tokushima University, Tokushima, Japan;_ 2 _Tokushima Breast Care Clinic, Tokushima, Japan;_ 3 _Hyogo College of Medicine, Nishinomiya, Japan_.

Overexpression of the human epidermal growth factor receptor 2 (HER2) in breast cancer is linked to aggressive tumor behavior and a poorer prognosis. Anti-HER2 monoclonal antibody, trastuzumab extends the overall survival of patients with HER2-overexpressing breast cancer, but resistance to trastuzumab and/or other HER2-targeted therapies remains a serious clinical problem. Our previous studies demonstrated that Brefeldin A-Inhibited Guanine nucleotide-exchange protein 3 (BIG3), which is exclusively overexpressed in the majority of breast cancers, functions as a cancer specific A-kinase anchoring protein that binds protein kinase A (PKA) and protein phosphatase 1 (PP1Cα), thereby inactivating prohibitin2 (PHB2) suppressive activity through its dephosphorylation on Ser39. We further developed dominant-negative stapling peptide (stERAP), which is designed to specifically disrupt binding of BIG3-PHB2 complex and improve duration of their anti-tumor effects. Here we newly report that stERAP has significant suppressive effects on the HER2 signaling network by activating the tumor suppressive ability of PHB2. stERAP resulted in intrinsic PHB2 release from BIG3, followed by rapid phosphorylation on Ser39, Thr42 and Thr169 via PKCα, TTK and MK5, respectively. Importantly, PHB2 released from BIG3 by stERAP treatment translocated into nucleus, followed by acting as a transcriptional co-repressor by recruiting HDAC1 and NcoR through its Ser39-phosphorylation. More importantly, we observed that serine/threonine phosphorylations of PHB2 are necessary for disruption of HER2-HER3 hetero-dimerization and NF-κB signalling activation which are associated with acquired resistance to trastuzumab. We demonstrated that weekly treatment of stERAP completely suppressed the proliferation of trastuzumab-resistant HER2-positive breast cancer cells in vitro and in vivo. We are currently examining the association of both BIG3 overexpression and PHB2 dephosphorylation with poor prognosis of patients with HER2-positive breast cancers by immunohistochemical staining, and will report these results at this meeting. We demonstrated for the first time that PHB2 inactivation by the AKAP-BIG3 (BIG3/PKA/PP1Cα tri-complex) is required for oncogenic HER2 signalling activation in breast cancer cells, and stERAP may be a promising anti-cancer drug for trastuzumab-resistant breast cancer.

#3814

Overcoming multidrug resistance (MDR) in colorectal cancer by modulating exosome-mediated transfer of MDR transporter regulatory machineries.

Kenneth KW To, Christy WS Tong, Mia MX Wu, Wei Yan. _Chinese Univ. of Hong Kong, Hong Kong, Hong Kong_.

Aim: The study aims to investigate the circumvention of multidrug resistance (MDR) in colorectal cancer by modulating exosome-mediated transfer of MDR transporter regulatory machineries from resistant to sensitive cells. Background: Colorectal cancer is a leading cause of cancer related death worldwide. MDR is a major unresolved obstacle to successful cancer chemotherapy in colorectal cancer. It is most commonly associated with an increased efflux of anticancer drugs by MDR transporters including ABCG2. Exosomes are a unique form of extracellular lipid vesicles, which are released upon the fusion of multivesicular bodies with plasma membranes on cell surface. Tumor-derived exosomes are emerging as important cell-to-cell couriers of oncogenic materials through the horizontal transfer of mRNAs, microRNAs or proteins during tumorigenesis. Recently, tumor-derived exosomes have been reported to mediate the transfer of the drug resistance phenotype. We hypothesize that regulatory machineries of MDR transporters may be transferred via exosomes from drug resistant donor cells to sensitive recipient cells, thereby conferring spread of MDR. Method: Exosome-mediated transfer of selected regulatory mediators from ABCG2-overexpressing resistant S1M180 cells to the parental S1 colorectal cancer cell line was evaluated. Exosomes were isolated from S1M180 cells-conditioned medium by the standard differential centrifugation method. The secretion of ABCG2-regulatory microRNAs into exosomes by S1M180 was studied by microRNA microarray. The uptake of fluorescence-labeled exosome from S1M180 cells by S1 cells was visualized by fluorescence microscopy. The alteration of a newly identified miR-203-DNMT3b-ABCG2 regulatory pathway in the recipient sensitive cells after incubation with exosomes derived from the resistant cells was examined by qPCR and Western blot analysis. Result: Exosomes were successfully isolated from ABCG2-overexpressing S1M180 cells and confirmed by the expression of exosomal markers CD63 and HSP70. S1M180-derived exosomes increased the viability of the parental S1 cells upon treatment with SN38 or 5-fluorouracil. After incubation with S1M180-derived exosomes, remarkably higher expression of miR-203 but lower expression of DNMT3b was found in the parental S1 cells, presumably leading to the induction of ABCG2 to mediate the transferred resistance phenotype. Inhibition of exosome uptake by a putative inhibitor dynasore was found to prevent ABCG2 induction and re-sensitize the exosome-treated parental cells to SN38 treatment. Conclusion: MDR cells-derived exosomes may spread the drug resistance phenotype by transferring the miR-203-DNMT3b-ABCG2 regulatory machinery to sensitive cells. Inhibition of this exosomal transfer of regulatory materials may prevent the spread of MDR and sensitize cancer cells to chemotherapy.

#3815

Overcoming acquired resistance of Lapatinib in breast cancer with Palbociclib.

Da Eun Jeong,1 Ahrum Min,1 Seongyeong Kim,1 So Hyeon Kim,1 Yoonjung Park,1 Yaewon Yang,2 Kyung-Hun Lee,3 Seock-Ah Im3. 1 _Cancer Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea;_ 2 _Department of Internal Medicine, Chungbuk National University Hospital, Seoul, Republic of Korea;_ 3 _Biomedical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea_.

Background : Lapatinib is a dual tyrosine kinase inhibitor that interrupts the ErbB family pathways in many solid tumors. However, the vast majority of patients who initially respond to the treatment will eventually relapse due to acquired resistance. The defects in cell cycle regulation via increased levels of cyclin D-CDK4/6 complex is well known mechanism of the acquired resistance to lapatinib. Thus, CDK4/6 inhibition can be considered as an attractive strategy to overcome lapatinib resistance. In this study, we investigated whether palbociclib, a palbociclib can overcome the resistance to lapatinib by modulating cell cycle regulation.

Methods : Acquired resistant SK-BR-3-cells was established by treating lapatinib consistently. The features of lapatinib resistance (LR) cell lines were evaluated by MTT, FACS, Western blotting, qPCR, methylation assay and WES. The antitumor effects of palbociclib in LR cell lines were evaluated through changes in cell cycle progression, proliferative ability and signaling transduction.

Results : In LR cell lines, the change of the molecules that were related to G1/S transition of cell cycle was prominent. It was confirmed that cell cycle progression and proliferation were significantly faster in LR cells compared to parental cells. Upregulation of cyclin D1, CDK4, CDK6 was shown in LR cells but cyclin-dependent kinase inhibitor, p16 was downregulated. The increase of Cyclin D1, CDK4, and CDK6 occur at the transcription level, and the epigenetic alteration of CDKN2A was observed. We identified that the palbociclib inhibited the cell cycle progression and reduced the cell viability significantly in LR cells by cell cycle arrest via blocking hyperphosphorylation of Rb. It was also confirmed that these changes resulted in apoptosis.

Conclusion : The changes of cell cycle molecules that were related to G1/S transition was observed in LR cells. Upregulation of cyclin D1, CDK4, CDK6, and deficiency in CDKN2A led to the increasing cell cycle progression and prolfieration in LR cells. Therefore, inhibition of these molecules by palbociclib could be an effective strategies for overcoming lapaitnib resistance.

#3816

Corilagin could help to overcome PARP inhibitor resistance by inhibiting ERK signaling pathways.

Baoxin Luan,1 Hongbo Zhao,1 Robert C. Bast, Jr.,2 Zhen Lu,2 Yinhua Yu1. 1 _OB/GYN Hospital of Fudan Univ., Shanghai, China;_ 2 _The University of Texas, M.D. Anderson Cancer Center, Houston, TX_.

We have identified that Corilagin is a major anti-tumor active component extracted from a well-known hepatoprotective and antiviral medicinal herb, Phyllanthus niruri L. Our previous work found that Corilagin inhibited the growth of ovarian cancer cells via TGF-β/AKT/ERK signaling pathways. Corilagin also sensitizes Paclitaxel and Carboplatin by primarily inhibiting Snail-glycolysis pathways. Recently, we have investigated if Corilagin could give a help to overcome poly ADP ribose polymerase (PARP) inhibitors (PARPi) resistance in ovarian cancer cells. As we knew, PARP inhibitors are DNA repair drugs, have been approved for ovarian cancer therapy, but drug resistance is a faced problem, combination therapy may give helps. In this study, two pairs of PARPi sensitive/resistant cells: A2780CP/A2780CP_R and UMB1.289/UMB1.289_R (Sci Transl Med. 2017; 9:392) were used. Cells were cloned again and confirmed their sensitivities to PARPi. The IC50 of PARPi-BMN673 is 0.02μM to A2780CP, > 10μM to A2780CP_R, 0.12μM to UMB1.289 and 60μM to UMB1.289_R. The combination assays showed Corilagin had almost equally cytotoxic activities to these two pairs and had synergistic effects with BMN673 to A2780CP_R and UMB1.289_R . Using CulcuSyn, the combination indexes (CI) presented at IC50 are 0.17365 and 0.62787 in A2780_R and UMB1.289_R, respectively. Signaling analysis found that PARPi did down-regulate the expression levels of PARP and up-regulate of pH2AX, PARPi only inhibited pERK activity in sensitive cells, but not in resistant cells. In contrast, Corilagin did not have DNA repair function, but it inhibited pERK activity in both sensitive and resistant cells. In summary, we demonstrate that combined treatment with Corilagin and PARPi evokes synergistic cytotoxic effects in two pairs of ovarian cancer cell models. Corilagin is an herb medicine with lower toxic effects to normal cells, especially hepatoprotective, which could be an ideal complimentary medicine when combinating with PARPi in ovarian cancer therapy.

#3817

Systems biology identifies that Gleevec reverses taxane resistance in solid tumors by selective inhibition of a novel +Tip microtubule-binding variant.

Prashant V. Thakkar,1 Katsuhiro Kita,1 Giuseppe Galletti,1 Neel S. Madhukar,1 Kyle Cleveland,1 Isabel Barasoain,2 Jose Fernando Diaz,2 Olivier Elemento,1 Manish A. Shah,1 Paraskevi Giannakakou1. 1 _Weill Cornell Medical College, New York, NY;_ 2 _Centro de Investigaciones Biológicas, Madrid, Spain_.

The microtubule (MT) cytoskeleton is a validated therapeutic target in oncology, evidenced by the wide use of taxanes in solid tumors including gastric cancer (GC). Post-hoc analysis of the clinical trial that led to docetaxel approval in GC, revealed that patients with diffuse histological subtype were intrinsically resistant to taxane chemotherapy. Using a panel of GC cell lines intrinsically sensitive or resistant to taxanes, we showed lack of drug-target engagement in the resistant lines, despite unimpaired intracellular drug accumulation and absence of tubulin mutations. We discovered a novel, truncated variant of the MT +TIP binding protein CLIP1, hereafter CLIP1S, which was significantly enriched in the resistant cells. Mass-spec proteomics and 5'RACE showed that CLIP1S lacked the first 150 amino acids, thus, missing the Cap-Gly domain required for MT +TIP localization. Confocal microscopy of endogenous or exogenous tagged proteins revealed that CLIP1S was indeed mislocalized from the +TIP to the MT lattice in contrast to +TIP localization of canonical CLIP1. Stable CLIP1S-Knock Down (KD) entirely reversed taxane-resistance (~300 fold), establishing causation between CLIP1S and taxane resistance. Quantitation of drug-binding kinetics using live-cell imaging of Flutax-2 (fluorescently-labeled taxane) in native cytoskeletons, showed that CLIP1S caused Flutax-2 to have significantly reduced affinity and increased dissociation rates from MTs, as compared with cells expressing only the canonical CLIP1. CLIP1S-KD, fully restored Flutax-2 binding, implicating CLIP1S in impeding taxane-MT interaction. Co-administration of chemical probes specific for the low affinity taxane binding site on MT surface further implicated CLIP1S in partially obstructing the MT pore thereby restricting taxane access in the MT lumen where the high affinity taxane binding site is located. Computational analyses of RNA-seq data from untreated or taxane-treated sensitive and resistant GC cells using a novel bayesian drug-target identification algorithm predicted Imatinib (Gleevec™) as a drug that could overcome CLIP1S mediated taxane resistance. Indeed, imatinib completely reversed taxane resistance, phenocopying the sensitization observed with the CLIP1S-KD. Most importantly, we showed that imatinib reversed taxane resistance by specific inhibition of CLIP1S in a dose-dependent manner as early as 3 h post-treatment. Taken together, these data identify an entirely novel mechanism of taxane resistance that involves obstruction of the MT pore in the presence of a previously unknown +TIP variant. Through systems biology we identified imatinib as the first specific CLIP1S inhibitor, thereby repurposing imatinib as a novel therapeutic to overcome clinical taxane resistance in GC and beyond.

#3818

BGJ398, a pan-FGFR inhibitor, overcomes paclitaxel resistance in urothelial carcinoma with FGFR1 overexpression.

Haram Ryu,1 Se Hyun Kim,1 Chan-Young Ock,1 Koung Jin Suh,1 Ji Yun Lee,1 Ji-Won Kim,1 Jin Won Kim,1 Jeong-Ok Lee,1 Yu Jung Kim,1 Keun-Wook Lee,1 Soo-Mee Bang,1 Jee Hyun Kim,1 Jong Seok Lee,1 Joong Bae Ahn,2 Kui-Jin Kim,1 Sun Young Rha2. 1 _Seoul National Univ. Bundang Hospital, Seongnam, Republic of Korea;_ 2 _Yonsei University College of Medicine, Seoul, Republic of Korea_.

Introduction: Paclitaxel (PTX) is commonly used to treat urothelial carcinoma (UC) after platinum-based chemotherapy has failed. However, single-agent taxane therapy is not sufficient to inhibit tumor progression and drug resistance in advanced UC. Epithelial-to-mesenchymal transition (EMT) induced by fibroblast growth factor receptor (FGFR)1 signaling has been proposed as a mechanism of PTX resistance, but it is unclear whether this can be overcome by FGFR1 inhibition. The present study investigated whether FGFR1 overexpression contributes to PTX resistance and whether FGFR inhibition can enhance PTX efficacy in UC.

Materials and Methods: UC cell lines: UMUC-14, RT4, T24, J82, HTB5 and HTB9 cells were used. Cells were treated with various concentration of PTX (0 to 20 nM) and BGJ398 (0 to 6 μM). The half-maximal inhibitory concentrations (IC50) were measured by the CellTiter-Glo assay. Cell cycle was analyzed after propidium iodide (PI) staining via flow cytometry. Changes in cell cycle, apoptosis and EMT-associated protein levels were assessed by Western Blot analysis. The migratory and colony formative property were analyzed by wound healing assay and colony forming assay, respectively. RNA interference was performed to assess the dependence of PTX sensitivity on pro-apoptotic proteins.

Results: We showed that mesenchymal-type T24 and J82 cell lines were highly resistant to PTX (0 to 20 nM) or BGJ398 (0 to 6 μM) monotherapy compared to FGFR3-positive epithelial-type (i.e., RT4 and UMUC-14) UC cell lines. We further found that PTX (10 nM) combined with BGJ398 (6 μM) caused synergistic effects on sub G1 accumulation and apoptosis compared to PTX or BGJ398 monotherapy in T24 and J82 cells, which were accompanied by downregulation of cyclin D1 protein and upregulation of gamma-histone 2A family member X, cleaved caspase-9 and cleaved poly (ADP-ribose) polymerase (PARP). Additionally, 10 nM of PTX combined with 6 μM of BGJ398 synergistically suppressed mesenchymal-type UC cell migration and colony formation via downregulation of EMT-associated factors including snail, slug and ZEB1. FGFR1 knockdown enhanced the antitumor effect of PTX (10 nM) by upregulation of apoptosis-associated markers, cleaved caspase-9 and PARP.

Conclusions: We investigated the mechanism of resistance to PTX in UC mediated by FGFR1 and found that combined treatment with BGJ398 can enhance the efficacy of PTX in UC cell lines with mesenchymal features. Our results demonstrate that combination with FGFR1-targeted therapy improves the antitumor efficacy of standard cytotoxic chemotherapy, which is a strategy that warrants further investigation in clinical trials in selected UC patients with FGFR1 overexpression.

#3819

Amphiregulin in damaged tumor microenvironment potentiates therapeutic resistance and enables PD-L1-mediated immunosuppression.

Qixia Xu,1 Jean-Philippe Coppé,2 Judith Campisi,3 Eric Lam,4 Yu Sun5. 1 _Shanghai Jiao Tong University School of Medicine, Shanghai, China;_ 2 _University of California San Francisco, San Francisco, CA;_ 3 _Buck Institute for Research On Aging, Novato, CA;_ 4 _Imperial College London, London, United Kingdom;_ 5 _Chinese Academy of Sciences, Shanghai, China_.

Therapeutic resistance is the major barrier to conquer human cancer, the most lethal pathology that claims numerous lives per year. Although identification of resistance mechanisms is the key to develop optimal strategies, progress has been limited due to long term failure to reveal the most critical targets. Cancer cells exhibit high plasticity even in clinical settings, a phenomenon that contributes to disease refraction by developing intrinsic or acquired resistance. Here we highlight the multifaceted and genetically conserved senescence-associated secretory phenotype (SASP), which encodes a large array of soluble factors including amphiregulin (AREG). Expression of AREG is markedly inducible by DNA damage to stromal cells in the tumor microenvironment (TME), a process mediated predominantly by the NF-κB complex and C/EBP family. Stromal AREG remarkably enhances the malignancy of cancer cells including acquired resistance, mainly by activating the EGFR pathway and causing a distinct epithelial to endothelial transition (EET). Paracrine AREG induces PD-L1 expression in recipient cancer cells, thereby creating an immunosuppressive TME via immune checkpoint activation. Therapeutic targeting AREG by a monoclonal antibody not only remarkably deprived cancer cells of chemoresistance, but achieved prominent efficiency when combined with classical chemotherapy in humanized animals with restored immunocompetency. Stromal AREG enters circulation and is readily detectable in the peripheral blood of post-treatment patients, representing a novel liquid biopsy biomarker for therapeutic evaluation and disease prognosis. Our study substantiates the potential of in vivo SASP in driving the TME-mediated drug resistance and shaping an immunosuppressive niche, and provides the proof-of-principle of targeting major SASP factors to effectively improve therapeutic index with reduced mortality.

#3820

Discovery of adaptive resistance pathways and anti-resistance combination therapies from phosphoproteomic data using graphical models.

Augustin Luna,1 Heping Wang,2 Ozgun Babur,3 Chris Sander,1 Anil Korkut2. 1 _Harvard Medical School, Boston, MA;_ 2 _UT MD Anderson Cancer Center, TX;_ 3 _Oregon Health & Science University, Portland, OR_.

Introduction. Resistance to targeted therapies, either intrinsic (pre-existing at the time of treatment) or acquired (emerges as the tumor adapts to therapy), is a major challenge in oncology. Blocking multiple escape routes using drug combinations is the best solution to drug resistance. However, the discovery of effective combinations remains a challenging task due to complexity of the underlying biological processes and inter-tumor heterogeneity. Here, we developed a statistical pathway analysis method that (i) reveals pathways involved in drug activity and adaptive resistance and (ii) nominates combination therapies to down-regulate the resistance pathways. The method is based on the rationale that (i) targeting a specific genomic aberration may lead to activation of compensatory pathways (e.g. via feedback loops in the short term) and subsequent resistance, and (ii) collective changes in pathway activities are better predictors of resistance and mitigation strategies than abundances of individual molecules.

Method. We construct a network model of signaling interactions using an adjusted graphical LASSO (GLASSO) algorithm from RPPA data. This is supplemented with prior pathway information from multiple signaling databases (using Pathway Commons) to estimate sparse directed graphical models. Next, we combine the network with the cell- type-specific drug response data to calculate a target score (TS) for each protein. The TS quantifies the adaptive pathway responses to a perturbation by integrating the change in the level of a (phospho)protein and its pathway neighborhood in response to a perturbation. A high TS corresponds to involvement in adaptive response (e.g. RTK upregulation by MEK inhibitor via a feedback loop) and a low TS corresponds to the activity of the drug (e.g. ERK phosphorylation inhibition by MEK inhibitor). Finally, by identifying the subnetworks with enriched, high TS values, we determine the adaptive resistance pathways. We validate the resulting predictions (combinations of the original drug perturbation with drugs targeting the resistance pathways) experimentally.

Applications. The method is amenable to calculations for hundreds of samples treated with individual (or combinations of) drugs in multiple doses and/or time points and interrogated for thousands of molecular entities (mRNA or proteomic). With longitudinal data, we will be able to explain the evolution of drug resistance and potentially the optimum time points for intervention. The protocol is defined with a focus on RPPA data, but can be adapted to other kinds of molecular data associated with adaptive responses. We applied our method to BET-BRD inhibition in ovarian and breast cancers to compare resistance/response pathways in cells with varying sensitivity. The analysis nominated cell-type-specific anti-resistance combinations involving BET inhibitors.

#3821

Taxane resistance in breast cancer controlled by the APC tumor suppressor.

Emily Astarita,1 Megan Dieringer,2 Jenifer R. Prosperi3. 1 _University of Notre Dame, South Bend, IN;_ 2 _Indiana University South Bend, South Bend, IN;_ 3 _Indiana Univ. School of Medicine-South Bend, South Bend, IN_.

Adenomatous Polyposis Coli (APC) is a multi-domain tumor suppressor protein that binds to proteins including β-catenin, axin, and microtubules (MTs). APC is lost in many epithelial cancers and up to 70% of sporadic breast cancers, with a tendency towards triple negative breast cancers (TNBCs). In a mouse breast cancer model of APC loss, MMTV-PyMT;ApcMin/+, our laboratory previously demonstrated that APC loss resulted in metaplastic-like tumors, a subtype of TNBCs that often develops resistance to chemotherapy. Using the human breast cancer cell line, MDA-MB-157, we created APC knockdown cells (APCKD) using lentiviral mediated shRNA knockdown of APC, which demonstrated resistance to the Taxane family chemotherapeutic agent, Paclitaxel (PTX). Given that PTX and APC both alter MT dynamics, we sought to understand the molecular mechanisms of APC-mediated resistance. Based on the mechanism of action of PTX, we hypothesized that genes involved in the G2/M transition would be responsible for PTX resistance. We have also performed an unbiased analysis of transcriptomic changes downstream of APC loss to identify potential therapeutic targets to overcome PTX resistance. Cell cycle analysis demonstrated that the cell cycle profile of APCKD cells was not drastically changed after PTX treatment. However, PTX induction of p21 specifically in the APCKD cells suggests that APCKD cells may become senescent to avoid apoptosis. We next analyzed the expression of the G2/M checkpoint proteins, CDK1 and cyclin B1, by western blot. CDK1 expression was significantly increased in APCKD cells compared to control with no changes in CDK1 phosphorylation (Thr14, Thr161, Tyr15). Interestingly, there was no effect of PTX treatment in either cell line. Based on these findings, we performed combination studies using a CDK1 inhibitor (RO-3306) with PTX treatment. PARP (total and cleaved) was used to measure apoptosis, and showed enhanced cleavage in the APCKD cells specifically after combination treatment. In addition to the cell cycle analysis, we performed RNA-sequencing on parent and APCKD cells to understand the transcriptomic changes downstream of APC loss. Upon validation of results by qRT-PCR and western blot, we identified that APCKD cells had altered expression of ZNF366, ADAMTSL1, SELENBP1, and CYB5R2. These 4 genes are known to be involved in the development of breast cancer and response to chemotherapy. Ongoing studies in the laboratory will investigate the effect of manipulating expression of these genes. Overall, our studies strive to understand the molecular nature of PTX resistance in APCKD cells, and to identify a therapeutic target to work as a targeted treatment for some TNBC types.

#3822

ORIC-101 reverses a GR-driven EMT-like phenotype and sensitizes TNBC cells to chemotherapy.

Haiying Zhou, Jessica Sun, Wayne Kong, Yosup Rew, Xiaohui Du, John Eksterowicz, Daqing Sun, Qiuping Ye, Omar Kabbarah, Valeria R. Fantin. _ORIC Pharmaceuticals, South San Francisco, CA_.

The Glucocorticoid Receptor (GR) is a member of the superfamily of nuclear hormone receptors that is activated by human cortisol and synthetic glucocorticoids such as dexamethasone (Dex). Upon ligand binding, GR translocates to the nucleus and regulates the expression of a wide spectrum of genes involved in diverse biological processes, including inflammation, immunity, metabolism, cell cycle, and differentiation. Dysregulated cortisol levels are associated with poor prognosis, drug resistance, and increased cancer recurrence. Multiple studies have shown that GR inhibition reverses resistance to chemotherapy in cancers of epithelial origin including prostate, bladder, renal, ovarian, pancreatic, and triple negative breast cancer (TNBC).

We have recently reported the discovery of ORIC-101, a potent GR antagonist with a unique cytochrome P450 inhibition profile that makes this compound particularly suitable for combination with taxanes such as paclitaxel (Rew Y et al, 2018). Consistent with previous reports, our data showed that activation of GR promoted growth of TNBC cells in 3D culture conditions and protected TNBC cells from paclitaxel. Treatment with ORIC-101 fully reversed these effects. To understand the molecular basis of the observed GR-mediated chemotherapy resistance, we set out to isolate the pool of TNBC cells that escaped from paclitaxel treatment in the presence of Dex. Molecular profiling of these "chemotherapy escapees" pointed to a number of glucocorticoid-regulated biological pathways, including basal stem cell lineage genes and mesenchymal markers, suggesting the acquisition of an EMT-like phenotype in chemo-resistant cells. In support of this finding, we found using ChIP-seq analysis that GR directly bound within the promoter/enhancer regions of well-established EMT genes such as SNAI2 and FN1, and regulated their expression in response to Dex treatment. Functionally, RNAi-mediated knockdown of SNAI2 partially restored sensitivity to paclitaxel, suggesting that the GR-driven EMT phenotype contributes to paclitaxel resistance in TNBC cells. Consistent with the in vitro observations, immunohistochemical analysis showed that GR activation upregulated the levels of both basal stem cell and mesenchymal markers in "chemotherapy escapees" from paclitaxel-treated TNBC xenografts. Importantly oral administration of ORIC-101 fully blocked these effects.

Altogether, we found that activation of GR drove an EMT-like phenotype in TNBC cells in vitro and in vivo. ORIC-101 reversed these effects and sensitized TNBC cells to chemotherapy. Our findings thus provide mechanistic insights into the role of GR as a mediator of therapy resistance in TNBC. Clinical evaluation is being planned to assess the therapeutic potential of ORIC-101 in combination with standard-of-care chemotherapeutic agents.

#3823

Inhibition of homologous recombination, PARP inhibitor, or dianhydrodulcitol overcomes temozolomide-resistance in glioma cells.

SHIGEO OHBA, Yuichi Hirose. _Fujita Health University, Toyoake, Japan_.

Glioblastoma is one of the most aggressive tumors in the central nervous system tumors. The standard therapy for glioblastomas is maximal safe resection, followed by radiation therapy and chemotherapy with temozolomide (TMZ). The acquisition of resistance to TMZ is big problem. TMZ is a DNA-methylating agent, delivering a methyl group to DNA (O6-guanine, N7-guanine and N3-adenine). The primary cytotoxic lesion, O6-methylguanine, mispairs with thymine, leading to futile DNA mismatch repair (MMR), formation of double strand breaks and eventual cell death, when O6-methylguanine DNA methyltransferase (MGMT) is absent. N7-methylguanine and N3-methyladenine are repaired by base excision repair. The object of the study was to reveal the mechanisms of resistance to TMZ and to find the way to overcome the resistance in glioma cells. Several clones of TMZ-resistant U251 were obtained and analyzed. #3 clone showed G2 arrest after TMZ exposure and this arrest was abrogated sooner compared to parental U251. TMZ did not induce G2 arrest in #8 clone. The expression of MGMT was not found in U251 parental cells, #3 cells nor #8 cells. The ability of homologous recombination (HR) was increased in #3 clone, and suppression of HR resensitized #3 clone to TMZ. The protein levels of MSH6, which was one of the main proteins associated with MMR, was reduced in #8 clone. Inhibition of base excision repair by PARP inhibitor resensitized #8 clone to TMZ. Dianhydrodulcitol, alkylating agent, suppressed the proliferation in U251 and even U251-derived resistant clones. These effects were shown in U87MG-dereived resistant clones and MGMT-proficient cell lines. To select the way according to the mechanisms of resistance to TMZ could overcome the resistance in glioma cells.

#3824

**Increased production of endogenous hydrogen sulfide (H** 2 **S) induced resistance to 5-FU and Oxaliplatin in colon cancer cells.**

Shanwen Chen, Shuai Zuo. _Peking University First Hospital, Beijing, China_.

The combination of 5-FU and Oxaliplatin has been widely adopted as one of the first line chemotherapeutic regimens for colon cancer patients. However, the development of chemoresistance can lead to early recurrence and metastasis to distant organs, thus remaining a leading cause of death from colon cancer. In the present study, the increased production of hydrogen sulfide (H2S) in colon cancer tissues was validated and the effect of inhibition of production of endogenous H2S on the responses of colon cancer cells to 5-FU and Oxaliplatin was further analyzed. The results suggested that inhibition of the enzymatic activity of cystathionine-β-synthase (CBS), a major producer of H2S in colon cancer cells, significantly decreased the IC50s of both 5-FU and Oxaliplatin. Inhibition of CBS leads to decreased PI3K-Akt signaling and consequently decreased expression of thymidylate synthetase (TYMS), which is the target of 5-FU. Inhibition of CBS utilizing aminooxyacetic acid (AOAA) leads to increased production of reactive oxygen species (ROS) and exaggerated mitochondrial damages in colon cancer cells. Analysis performed on CompuSyn confirmed the synergetic effect of AOAA with both 5-FU and Oxaliplatin. In vivo imaging of tumorigenesis in nude mice further confirmed the effect of inhibition of CBS on the therapeutic effect of both 5-FU and Oxaliplatin. Together, these results suggested that inhibition of production of endogenous H2S might be a promising target for chemoresistance in colon cancer.

#3825

Targeting activated PI3K/mTOR signaling overcomes resistance to CDK4/6-based therapies in preclinical ER+ breast cancer models.

Neil A. O'Brien,1 Martina SJ McDermott,1 Dylan F. Conklin,1 Alex Gaither,2 Tong Luo,1 Raul Ayala,1 Suruchi Salgar,1 Emmanuelle DiTomaso,2 Naveen Babbar,2 Faye Su,2 Sara A. Hurvitz,1 Ronald Linnartz,2 Kristine Rose,2 Samit Hirawat,2 Dennis J. Slamon1. 1 _UCLA, Los Angeles, CA;_ 2 _Novartis Institutes for BioMedical Research, Cambridge, MA_.

Addition of CDK4/6 inhibitors such as palbociclib, ribociclib or abemaciclib to endocrine-based therapies significantly improves progression-free and in some subgroups, overall survival in patients with advanced estrogen receptor-positive (ER+) breast cancer. However, acquired resistance to CDK4/6 inhibitors remains a significant unmet clinical need. It is critical to ascertain the mechanisms by which tumor cells evade CDK4/6 based therapy. In this study we screen multiple in vitro and in vivo models of acquired resistance to CDK4/6 inhibitors to identify potential resistance/escape pathways and targets for pharmaceutical intervention to overcome resistance. ER+ breast cancer cell lines of diverse molecular backgrounds were conditioned to acquire resistance to CDK4/6 inhibitors through either long-term culture in the presence of clinically relevant concentrations of inhibitors or as cell line xenografts treated through progression on a CDK4/6 inhibitor plus fulvestrant. Baseline and pharmacodynamic changes in cell signaling were measured using reverse phase protein array (RPPA) and RNAseq analysis. Acquired resistance to CDK4/6 inhibitors was associated with decreases in phosphorylated-Rb (pRb) and ER-alpha protein and increases in pAKT and pS6 relative to isogenic controls. The p110α-selective PI3K-inhibitor, alpelisib, in combination with fulvestrant or ribociclib/fulvestrant blocked pAKT signaling in xenografts progressing on palbociclib/fulvestrant and induced significant tumor regressions. Apelisib plus fulvestrant also produced robust anti-tumor responses in xenografts progressing on ribociclib/fulvestrant. Triple combination treatment with ribociclib/alpelisib/fulvestrant induced significant regressions in resistant tumors and in treatment naïve models, where complete tumor regressions occurred regardless of PIK3CA mutation status. Regressions were maintained for >9 weeks post withdrawal of treatment, indicating that therapeutic resistance may be prevented by this triple combination approach. RPPA analysis of responding tumors identified sustained inhibition of both PI3K- and CDK4/6:Rb-pathway signaling accompanied by activation of pro-apoptotic proteins. These data support clinical investigation of targeting PI3K with alpelisib in breast cancers progressing on CDK4/6 based therapies and investigation of upfront triple combination therapy prior to acquisition of resistance to CDK4/6.

### Epigenetic Targets

#3826

**CCS1477, a potent and selective p300/CBP bromodomain inhibitor, is targeted & differentiated from BET inhibitors in prostate cancer cell lines **in vitro **.**

Nigel Brooks,1 Amy Prosser,2 Barbara Young,2 Luke Gaughan,3 Paul Elvin,4 Neil Pegg1. 1 _CellCentric Ltd, Cambridge, United Kingdom;_ 2 _Sygnature Discovery, Nottingham, United Kingdom;_ 3 _Northern Institute for Cancer Research, Newcastle, United Kingdom;_ 4 _Oncognition, Cambridge, United Kingdom_.

Background: CCS1477 is a potent and selective p300/CBP bromodomain inhibitor, currently in a Ph1 trial for patients with metastatic castration resistant prostate cancer (mCRPC). CCS1477 works by inhibiting the expression and function of the androgen receptor (AR), as well as inhibiting c-Myc. Bromodomain and extraterminal domain (BET) protein inhibitors are also being developed in mCRPC. We have established a BET inhibitor (BETi) resistant 22Rv1 prostate cancer cell line and used this, alongside parental 22Rv1 cells, to characterise the differential effects of p300/CBP vs BET bromodomain inhibition.

Methods: 22Rv1 cells which express both the wild-type and splice variant forms of AR were cultured in the presence of increasing concentrations (30-500nM) of JQ1 for several months. A parallel set of 22Rv1 cells were cultured in the presence of vehicle (0.1% DMSO). The anti-proliferative effects of JQ1, iBet762, OTX-015 and CCS1477 (10 nm-10 µM dose range) was determined in resistant and parental 22Rv1 cells in a 5d CellTitre Glo assay. The effects of combining CCS1477 with JQ1 was measured in parental 22Rv1 cells. Protein biomarker (AR, AR-splice variant, c-Myc) responses were measured by Western blot and qPCR was used to determine changes in the expression of selected genes (AR, AR-V7, c-Myc, KLK3, TMPRSS2). Gene expression microarrays (Clariom D) were used to assess global gene expression changes in cells treated for 24h with 500nM CCS1477 or JQ1.

Results: JQ1 resistant 22Rv1 cells were significantly less sensitive to JQ1 compared with parental cells. (IC50; Res, 7.3 µM vs parental, 0.06 µM). There was also cross-resistance to other chemically distinct BET inhibitors, iBET762 and OTX-015. JQ1 potently inhibited c-Myc protein and gene expression in parental cells, a response that was abrogated in the JQ1 resistant line. The inhibitory effects of JQ1 on AR gene and protein expression were reduced in the resistant line. In contrast potent anti-proliferative effects of CCS1477 were retained in JQ1 resistant cells, as was the inhibitory effect on c-Myc and AR. Combination of CCS1477 & JQ1 resulted in a highly synergistic inhibitory effect on proliferation in normal 22Rv1 cells. Global gene expression analysis revealed significantly fewer altered genes after CCS1477 (27 up, 119 down) compared to JQ1 (196 up, 655 down).

Conclusions: These studies provide three lines of evidence for a differentiated mode of action of CCS1477 vs BETi. First, CCS1477 continues to inhibit proliferation and relevant response biomarkers in a cell line that is resistant to BETi. Second, there is a synergistic, rather than additive effect of combining CCS1477 with JQ1. Third, there are significantly fewer genes and a distinct pattern of gene change after CCS1477 vs. JQ1. Collectively, these data point to a differentiated and more selective profile after p300/CBP inhibition with CCS1477.

#3827

Antitumor activity of ODM-207, a novel BET bromodomain inhibitor, in nonclinical models of ER+ breast cancer as single agent and as a combination treatment.

Julia Lindqvist,1 Mari Björkman,1 Reetta Riikonen,1 Daniel Nicorici,1 Elina Mattila,1 Mahaboobi Jaleel,2 Chandrasekhar Abbineni,2 Anu-Maarit Moilanen1. 1 _Orion Corporation Orion Pharma, Turku, Finland;_ 2 _Aurigene Discovery Technologies Limited, Bangalore, India_.

Introduction: The bromodomain and extraterminal (BET) family of proteins are chromatin readers that promote the transcription of several important cell identity genes. BET proteins also control expression of many genes that play an essential role in the pathogenesis of human cancer, including cell-cycle and proliferation-regulating genes. The small-molecule BET inhibitors block BET binding to chromatin and have shown antitumor activity in a variety of pre-clinical cancer models. In this study, we evaluated the anticancer activity of the novel BET inhibitor, ODM-207, in ER+ breast cancer models as a single agent and in combination with other cancer drugs.

Methods: ER+ breast cancer cell lines were studied for sensitivity to ODM-207 and the in vivo efficacy was assessed using the ER+ Ma3366 patient-derived xenograft model. For gene expression analyses, breast cancer cells were treated with ODM-207 or reference BET inhibitor JQ1 and differentially expressed genes were analyzed by RNA-sequencing. The ability of ODM-207 to regulate anticancer signaling pathways was validated by western blotting. Synergistic drug interactions were profiled using five-concentration dose response matrices.

Results: ODM-207 is a novel BET inhibitor structurally distinct from JQ1 that shows antiproliferative activity in a broad panel of cancer cell lines. The strongest antitumor activity could be observed in hormone-dependent prostate and breast cancer models. In this study, we show that ODM-207 effectively inhibits the proliferation of ER+ breast cancer cell lines by inducing cell cycle arrest in G0/G1-phase. Additionally, ODM-207 suppresses the growth of ER+ patient-derived breast cancer xenograft tumors. ODM-207 as well as JQ1 targeted several pathways important for cancer progression such as MYC, estrogen response and cell cycle gene signatures. The inhibition of key cell cycle regulators, such as CDK4 and Cyclin D1, were further verified. The cyclin D1:CDK4/6 axis plays a significant role in the development, and currently, treatment of ER+ breast cancer together with endocrine therapy. Interestingly, ODM-207 was shown to synergize with palbociclib in vitro in ER+ breast cancer cell lines: the combination of ODM-207 and CDK4/6 inhibitor palbociclib achieved greater cell proliferation inhibition than either drug alone at sub IC50 concentrations. Notably, the ODM-207 and palbociclib combination did not cause the induction of an obvious senescent-like phenotype as compared to palbociclib alone, but rather affected cell survival cellular assays.

Conclusions: In summary, ODM-207, which is currently in Phase I clinical trials for treating solid tumors, causes significant growth inhibition in pre-clinical models of ER+ breast cancer and enhances antiproliferative activity of palbociclib, providing a rationale for development of a combination therapy.

#3828

Differential transcriptional response of MYC to bromodomain inhibition in HPV positive head and neck squamous cell carcinoma.

Hassler Rengifo, Albert R. Wang, Mohammad Titi, Kwang Nickel, Randy Kimple, Gopal Iyer. _University of Wisconsin-Madison, Madison, WI_.

Differential transcriptional response of MYC to bromodomain inhibition in HPV positive head and neck squamous cell carcinoma

Targeting of bromodomain (BRD) and extra-terminal domain (BET) family protein activity has the potential to reduce proliferation in many cancers. We hypothesize that targeting BRDs using its inhibitors will lead to reduced proliferation in human papilloma virus (HPV) infected head and neck cancer squamous cell carcinoma (HNSCC). HPV infected HNSCC is predicted to surpass the incidence of cervical cancer by 2020. Amongst various HPV serotypes, HPV16 is classified as oncogenic for a various number of cancer sites including head and neck squamous cell carcinoma (HNSCC). Integration of HPV into host genomes is not part of its life cycle; however, the exception is HNSCC wherein E6 and E7 viral proteins predominantly drive its tumor progression. HPV integrated HNSCC cells will be utilized to unravel the fundamental question of epigenetic regulation of viral-host interaction using the HPV-positive HNSCC model. Results from our lab indicate that despite high nanomolar IC50 in cell proliferation assays, downregulation of MYC expression was not observed using BRD inhibitor in all MYC expressing HNSCC cell lines. Further interrogation revealed differential phosphorylation of tumor suppressors - p53 and pRb might influence the sensitivity to BRD inhibition, which in turn could explain the contrasting regulation of MYC RNA levels. Intriguingly, combining radiation with bromodomain inhibitors led to increased radio-sensitization suggesting that BRDs have a role to play in DNA damage repair pathway and altered cell cycle regulation. Taken together, we will present MYC dependent and independent mechanism following BRD inhibition in HPV positive HNSCC.

#3829

NEO1132 and NEO2734, novel dual bromodomain inhibitors of both BET and CREBBP/EP300, compared to single BET or CREBB/EP300 inhibitors in diffuse large B cell lymphoma.

Filippo Spriano,1 Eugenio Gaudio,1 Chiara Tarantelli,1 Gaetanina Golino,1 Luciano Cascione,1 Emanuele Zucca,2 Anastasios Stathis,2 Francis Giles,3 Francesco Bertoni1. 1 _Università della Svizzera Italiana, Institute of Oncology Research, Bellinzona, Switzerland;_ 2 _Oncology Institute of Southern Switzerland, Bellinzona, Switzerland;_ 3 _Epigene Therapeutics Inc, Montreal, Quebec, Canada_.

Lymphoma cells have frequent deregulation of their epigenome. The Bromodomain (BRD) and Extra-Terminal domain (BET) proteins are key regulators of the transcription process. The acetyltransferases cyclic AMP response element binding protein (CREB)-binding protein (CBP) and the E1A interacting protein of 300 kDa (EP300) are highly homologous BRD-containing transcriptional co-activators and are often mutated in diffuse large B cell lymphoma (DLBCL). Targeting the individual classes of proteins is a new therapeutic approach, with BET inhibitors having both preclinical and early clinical anti-lymphoma activity. NEO2734 and NEO1132 are novel chemically distinct dual inhibitors of both BET and CREBBP/EP300 proteins with different affinity for their targets. Here, we present initial data exploring their anti-tumor activity in DLBCL models. Lymphoma cell lines were exposed to increasing doses of compounds for 72h followed by MTT assay. Twenty-seven DLBCL cell lines were exposed to NEO2734 and NEO1132. NEO2734 showed anti-tumor activity with a median IC50 of 157 nM (95% C.I., 135-214). While, NEO1132 showed a lower activity compared to NEO2734, with a median IC50 of 400nM (95% C.I., 316-622). For both compounds, cell lines derived from activated B-cell-like (ABC) DLBCL (n.=8) were more sensitive than those derived from germinal center B-cell (GCB) DLBCL (n.=19) (P=0.009, P=0.03 respectively). No differences were observed based on double hit MYC/BCL2 (yes, n.=6; no, n.=14), MYC (translocation: yes, n=8; no, n.=13), BCL2 (translocation: yes, n=12; no, n.=6), TP53 (inactive: yes, n=14; no, n.=6), CREBBP (mutated, n.=10; wild type, n=16), or EP300 (mutated, n.=5; wild type, n.=20) gene status. All the cell lines were also exposed to a BET inhibitor (birabresib, OTX015) (Boi et al, Clinical Cancer Res 2015) and to a CREBBP/EP300 inhibitor (CBP30) (Hammitzsch et al, PNAS 2015). The median IC50 values of the two molecules were 237 nM (95% C.I., 171-344) and 5.5 μM (95% C.I., 4.2-8.3 μM) respectively. The four compounds presented a similar pattern of anti-proliferative activity across all the cell lines (NEO2734 vs NEO1132 R2=0.98, P < 0.001; NEO2734 vs birabresib: R2=0.84, P < 0.001; NEO2734 vs CBP30, R2 = 0.73, P < 0.001; birabresib vs CBP30, R2 = 0.73, P < 0.001) but with different degrees of IC50. NEO2734 was significantly more potent than birabresib (P=0.0182), CBP30 (P<0.001) and NEO1132 (P<0.001). The higher activity of NEO2734 compared to NEO1132 may be attributable to its superior potency in binding CREBBP and EP300, confirming the importance of the BET/CREBBP/EP300 simultaneous inhibition. The novel dual BET and CREBBP/EP300 inhibitors NEO2734 and NEO1132 showed potent in vitro anti-tumor activity across a large panel of DLBCL cell lines and their activity appear to be proportional to the binding affinity for both BET and CREBBP/EP300 proteins.

#3830

Novel bromodomains and extra-terminal motif (BET) inhibitors developed against estrogen receptor positive, fulvestrant- and CDK4/6-resistant breast cancer models.

Rui Xiong, Yangfeng Li, Jiong Zhao, Debra Tonetti, Gregory Thatcher. _Univ. of Illinois at Chicago, Chicago, IL_.

Approximately 70% of breast cancer patients have estrogen receptor positive (ER+) tumors. Fulvestrant (FUL), a selective estrogen receptor degrader (SERD) able to ablate ER, was first introduced to clinical practice in 2002 as the second and third line treatment for ER+ metastatic breast cancer. In August 2017, FUL was added to first-line therapy in postmenopausal women by demonstrating superiority compared to first-line AI therapy, anastrozole, with improved progression-free-survival (PFS) and a 20% reduction in the risk of disease progression or death. However, acquisition of FUL-resistance in both first-line setting or as combination therapy with CDK4/6 inhibitor has been observed clinically, leaving patients and oncologists with chemotherapy as the only backup therapy. Little is known about resistance mechanisms to FUL alone or in combination with CDK4/6 inhibitors. Here, we showed that targeting epigenetic vulnerabilities in breast cancer using novel bromodomain and extra-terminal (BET) inhibitors can effectively inhibit growth of tamoxifen-, FUL- and CDK4/6- resistant breast cancer cells. We designed and developed a novel series of pyridinone-based BET inhibitors, optimized by growth inhibition in tamoxifen- and FUL- resistant breast cancer cell lines (MCF7:CFR) in both 2D and 3D cultures. Optimized compounds showed superior in vitro activity to BET inhibitors in clinical trials. Selected compounds were further evaluated for pharmacokinetics and shown efficacy in endocrine-resistant xenograft models.

#3831

Selective targeting BET family BDII bromodomain with ABBV-744 and BCL-2 with venetoclax (ABT-199) is synergistic in primary acute myeloid leukemia models.

Tianyu Cai,1 Vinitha Kuruvilla,1 Xiaoyu Lin,2 Tamar Uziel,2 Xin Lu,2 Lu Zhang,2 Qi Zhang,1 Lina Han,1 Antonio Cavazos,1 Yu Shen,2 Marina Konopleva1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _AbbVie Inc., North Chicago, IL_.

Despite advances in understanding of the biology of acute myeloid leukemia (AML), cure remains elusive for the majority of patients. ABT-199 (Venetoclax) is a small-molecule BH3 mimetic that selectively inhibits BCL-2 causing cell death. ABBV-744 is a highly selective inhibitor for the BDII of BET family proteins, exhibiting greater than 300-fold more potent binding affinity to the BDII bromodomain of BRD4 relative to BDI (Warren Kati AACR 2018; Xiaoyu Lin AACR 2018). In this study, we evaluated the anti-leukemia efficacy of the concomitant BCL-2 blockade by venetoclax and of BDII inhibition with ABBV-744 in primary AML samples.

First, anti-leukemia activity of venetoclax and ABBV-744 was examined in 12 primary AML samples with diverse genomic alterations. The combination significantly enhanced cell death (61.4% ± 8.7%), as compared to the single agent treatment (51.4% ± 9.3% in venetoclax 10 nM group and 22.2% ± 3.4% in ABBV-744 50 nM group, p<0.001). ABBV-744 inhibited cell proliferation in the majority of AML cases (31.7% ± 8.2 %), and the cell growth suppression was more profound in the combination group (82.9% ± 6.9%, p<0.001). Most importantly, three of 12 patients were resistant to venetoclax, but two of these were sensitive to ABBV-744 or ABBV-744/venetoclax combination. We next performed the whole genome transcriptome analysis of pre-treatment AML cells by RNA-sequencing (RNA-seq). The samples which were sensitive to venetoclax and to the combination with ABBV-744 were characterized by high levels of BCL2 and mid-low level of MCL1 expression. In addition, low mRNA expression of AR, IL1R1 expression and high CCND1 expression correlated with response of primary AML cells to the combination of venetoclax and ABBV-744.

To test the efficacy of this regimen in vivo, we established a patient-derived xenograft (PDX) from an AML patient in NSG mice. After 21 days of therapy, flow cytometry data demonstrated significantly reduced leukemia burden in venetoclax treated group (9.5% ± 1.7%) but not in ABBV-744 group (22.3% ± 5.8%) compared to controls (30.8% ± 3.9%), with lowest tumor burden in the combination group (5.0% ± 0.8%, p<0.01). No significant impact on mice' weight was noted, and no clinical signs of toxicity recorded over the course of therapy. The experiment is ongoing, and the survival analysis will be reported.

Next, the anti-leukemia efficacy of ABBV-744 was tested in 7 additional AML PDX models. In all of the 7 models, Combination of ABBV-744 and venetoclax treatment delayed AML progression compared to untreated mice (survival days: 141 vs 105, 275 vs 153, 62 vs 46, 136 vs 119, 138 vs 77, 129 vs 116, 94 vs 86).

In summary, combinatorial blockade of BDII bromodomain and of BCL-2 anti-apoptotic pathway facilitates apoptotic cell death and suppresses proliferation in the majority of primary AML cells and produces anti-AML activity in AML PDX models in vivo.

#3832

Synergistic anticancer effects of epigenetic drugs JQ1 and SAHA in adenoid cystic carcinoma of the lacrimal gland.

Ravi Doddapaneni, Wensi Tao, David Tse, Daniel Pelaez. _University of Miami Miller School of Medicine, Miami, FL_.

Background: Adenoid cystic carcinoma is the most common epithelial malignancy of the lacrimal gland and has poor prognosis despite current treatment strategies. The aims of this study were to evaluate the anticancer efficacy of the dual inhibition of bromodomain proteins (by JQ1) and HDAC proteins (by SAHA or Vorinostat) in lacrimal gland adenoid cystic carcinoma (LGACC) and to elucidate the underlying mechanism of action.

Methods: LGACC tissue samples were collected from patients and tissue digestion was done by a collagenase digestion method. Molecular characterization of LGACC primary cell cultures was done by immunocytochemistry. LGACC cells were treated with JQ1 and SAHA individually or in combination. The synergistic effect of JQ1 and SAHA combination was determined by the CellTiter-Glo luminescent cell viability assay using Combenefit software. Migration capability of LGACC cells after drug treatment was examined by a wound healing assays. The expression levels of genes associated with LGACC malignancy were investigated after treatment with JQ1 and SAHA for 48hrs by real time PCR. The expression of c-MYB protein was detected by western blot analysis.

Results: LGACC primary cultures were successfully established in-vitro. Immunofluorescence of these cells revealed positive antigenicity for epithelial, proliferative and tumor markers such as FGFR1, MYB and NICD; slightly positive antigenicity for CK-5 and CK-7; and negative antigenicity for E-cadherin.. We found that synergism between JQ1 and SAHA was highly significant as shown by synergy matrix plot and 3D surface plot. Interestingly, migration capacity of LGACC cells was significantly (p<0.002) inhibited by the combination treatment compared to either drug alone.. Our gene expression analysis revealed that the expression levels of Notch1 (p<0.04), Hes1 (p<0.05), Hey1 (p<0.008), Maml1 (p<0.013) and CDKNB (p<0.04) were downregulated significantly in LGACC cells treated with the combination compared to single drug treatment. Furthermore, western blot analysis revealed that combination treatment significantly (p<0.05) downregulated MYB expression (0.9 fold). Currently, investigations are being conducted to evaluate the synergistic anticancer effect of JQ1 and SAHA in LGACC tumor xenografts in nude mice.

Conclusion: In conclusion, we successfully generated LGACC cell lines in-vitro. Our results demonstrated that the combination of bromodomain inhibitor JQ1 and histone deacetylase inhibitor SAHA behaves in a synergistic manner and has superior anticancer activity against LGACC cancer cells via targeting the Notch pathway. Taken together, these findings indicate that LGACC is strikingly sensitive to epigenetic inhibitors, which may be quickly implemented for patient use.

#3833

Pharmacology Team Leader.

InHwan Bae, WonJeoun Kim, JiSook Kim, JiYoung Song, JungSoo Nam, Moonsub Lee, Jooyun Byun, Hyeongki Kim, TaeHun Song, Seokhyun Hong, KyuHang Lee, YoungGil Ahn, YoungHoon Kim, Kwee Hyun Suh. _Hanmi Pharmaceutical, Hwaseung, Republic of Korea_.

Introduction: Global changes in the epigenetic landscape are known as the hallmark in cancer and the histone demethylase Lysine-specific demethylase 1 (LSD1) is one of the novel targets for the therapy of various malignant diseases. Also LSD1 overexpression is associated with poor prognosis in various cancers. LSD1-mediated epigenetic modification is known to play a key role in the regulation of gene expression by removing the methyl groups from methylated lysine 4 and lysine 9 of histone H3. Alterations in histone methylation lead to aberrant silencing of expression of multiple genes involved in tumor survival and in cell cycle. Moreover, epigenetic dysregulation plays a critical role in pathogenesis of various types of cancers such as acute myeloid leukemia (AML) or small cell lung cancer (SCLC). In this study, we have characterized a novel LSD1 inhibitor and assessed its potential as a novel therapy for AML and SCLC patients.

Materials and Methods: Novel LSD1 inhibitor, HM97211 and its derivatives were designed and synthesized as the active biologic inhibitory compound against the AML and SCLC cells. Biochemical enzyme assay was performed to identify enzyme selectivity of HM97211 derivatives. Standard proliferation assay, immunoblotting, and apoptosis analysis were carried out to validate the potency of HM97211 derivatives in AML and SCLC cell lines. In vivo anti-tumor activity was evaluated in AML and SCLC cell xenograft mice models. Tumor sizes were measured and tumor samples were analyzed to define the mechanisms of action.

Results: HM97211 derivatives showed higher selectivity toward LSD1 compared to other FAD-utilizing enzyme as well as other epigenetic enzymes. HM97211 derivatives alter expression of various genes and induce differentiation and apoptosis, resulting in growth suppression in a panel of AML and SCLC cells. Moreover, HM97211 derivatives significantly induced programmed cell death in AML and SCLC cells. Oral administration of HM97211 derivatives inhibited tumor growth of human AML and SCLC xenograft models as a single agent at doses exhibiting significant. Efficacy of HM97211 derivatives was further evaluated in combination treatment with standard chemotherapies in human AML and SCLC xenograft model.

Conclusion: Collectively, results of this study suggested that novel reversible LSD1 inhibitor, HM97211 derivatives, can be a strong therapeutic agent for AML and SCLC patients. Hanmi presents a new insight to epigenetics application in anticancer therapy.

#3834

Epigenetic modification of oncogenes or tumor suppressor genes by thymoquinone in triple negative breast cancer.

Md. Asaduzzaman Khan, Meiling Zheng, Junjiang Fu. _Southwest Medical University, Luzhou, China_.

Background and Objective: Triple negative breast cancer (TNBC) is a molecular subtype, and most aggressive and deadly among breast cancers. Different molecular subtypes of breast cancer have distinct molecular mechanisms, and in case of TNBC, the mechanisms are complex, and still obscure. Development of therapeutic strategies are thus also difficult against TNBC. Thymoquinone (TQ) is a natural compound, which has been found effective against TNBC cells, but the mechanism of TQ action in TNBC is not clear. In this study, we have focused on TQ's genome-wide epigenetic effect on TNBC cell line BT549. Methods: The BT549 cells were treated with 1-10 µM of TQ for cellular growth analysis, and isolated DNA for dot blot analysis with anti-5-methyl cytosine (anti-5MC) antibody to observe whole genome methylation status affected by TQ. Also the chromatin immunoprecipitation (ChIP) was performed with anti-methyl binding domain protein (anti-MBD1) to identify DNA sections, of which methylation status is changed by TQ.

Results: Cell viability assay showed that TQ dose-dependently reduced BT549 cell growth. DNA dot blot analysis results indicated no obvious changes in total methylation status change in genomic DNA. However, ChIP-seq confirmed that methylation level was affected (either increased or decreased) in certain genomic locations by TQ treatment. Some of the locations are proximal or close to some genes, indicating that this epigenetic modification might have influence on these genes' expression pattern.

Conclusion: Thus, here in this preliminary research study, we indicate some genes, which might be epigenetically targeted by TQ in TNBC cells. Funding support: National Natural Science Foundation of China (Grant No. 81850410555, 81672887, 81172049)

#3835

In vivo evaluation of the Menin inhibitor VTP-50469 against Ewing sarcoma preclinical models: A report from the Pediatric Preclinical Testing Consortium (PPTC).

Peter J. Houghton,1 Raushan Kurmasheva,1 Gerard M. McGeehan,2 Steve W. Erickson,3 Beverly Teicher,4 Malcolm Smith4. 1 _Greehey Children's Cancer Research Institute, San Antonio, TX;_ 2 _Syndax Pharmaceuticals, Waltham, MA;_ 3 _RTI International, Research Triangle Park, NC;_ 4 _National Cancer Institute, Bethesda, MD_.

Background: Menin and MLL-containing trithorax complexes play important roles in developmental transcription programs by promoting gene expression through depositing H3K4me3 marks on gene promoters. Menin and MLL1, (lysine methyltransferase 2A, KMT2A) are reported to be overexpressed in Ewing sarcoma. Further, inhibition of the menin-MLL interaction by MI-503 resulted in loss of menin and MLL1, resulting in inhibition of proliferation and tumorigenicity of Ewing sarcoma cells (Svoboda et al., Oncotarget, 2017). VTP-50469 is a small molecule that disrupts menin-MLL1 interactions and is highly active against MLL-rearranged leukemia xenograft models. To extend these results, the PPTC evaluated VTP-50469 in vivo against a set of Ewing sarcoma xenografts.

Methods: VTP-50469 was administered twice daily (BID) at a dose of 120 mg/kg or 100 mg/kg orally (PO) for 28 days, a dose and schedule that is highly effective in leukemia MLL-rearranged models. Standard PPTC methods for assessing time to event (EFS T/C = ratio of median time to event for treated versus control animals). Objective response measures similar to clinical measures were applied (Houghton, Pediatr Blood Cancer 2007;49:928-940).

Results: VTP-50469 was generally well tolerated during the efficacy testing phase. Among 70 animals treated with VTP-50469 in efficacy testing, 3 (4.3%) experienced toxic death. As a single agent, VTP-50469 statistically significantly prolonged time to event in 4 of 7 Ewing sarcoma models. In models for which the time to event was significantly prolonged, the extent of prolongation was modest, with EFS T-C values ranging from 3.6 to 7.8 days and with EFS T/C values ranging from 1.24 to 1.74. None of the models tested showed objective responses, but rather showed progressive disease as their best response. The minimum relative tumor volumes were significantly smaller compared to control for 2 of 7 Ewing sarcoma models. However, the mean minimum relative tumor volumes were all greater than 1.0, indicating the absence of tumor regression.

Conclusions: VTP-50469 was adequately tolerated in the Ewing sarcoma xenograft treatment cohorts that were studied. While a number of the models studied showed statistically significant effects on tumor growth rate, the effect size was generally small and tumor regression was not observed. As this dose and schedule of VTP-50469 is highly effective against MLL-rearranged leukemia xenograft models, these results suggest that the menin-MLL1 interaction may play a less critical role in maintenance of Ewing sarcoma. (Supported by CA199297 and CA199222)

#3836

Efficacy of CDK9 inhibitor-based combinations as therapy for post-myeloproliferative neoplasm (MPN) secondary (s) AML.

Dyana T. Saenz, Warren C. Fiskus, Taghi Manshouri, Christopher P. Mill, Joseph D. Khoury, Srdan Verstovsek, Kapil N. Bhalla. _MD Anderson Cancer Center, Houston, TX_.

Standard AML chemotherapy and JAK inhibitor (ruxolitinib) treatments are ineffective against post-myeloproliferative neoplasm (MPN) sAML. Mutations in JAK2, MPL and calreticulin, and co-epimutations, result in a dysregulated transcriptome, which is responsible for transformation of MPN to sAML and for its therapy-refractoriness. CDK9 is the catalytic subunit of pTEFb that phosphorylates serine 2 in the heptad-repeats of the C-terminal domain of RNA pol II (RNAP2). CDK9 also phosphorylates and inactivates negative transcript elongation factors (NELF and SPT5), which induces promoter-proximal pause-release of RNAP2 to enable productive mRNA transcript elongation. The bromodomain extra-terminal (BET) protein BRD4 recruits pTEFb to the chromatin to promote RNAP2-mediated elongation of transcripts, including those of c-Myc, Bcl-xL, PIM1 and MCL1, important for cell growth and survival of post-MPN sAML cells. Here, we determined the effects of inhibiting BRD4-CDK9-RNAP2 axis in post-MPN sAML cells. Treatment with the CDK9 inhibitor (CDK9i) NVP2 or BAY1143572 (B) dose-dependently induced apoptosis of ruxolitinib-sensitive (Rux-S) sAML cultured cells lines HEL92.1.7 (HEL) and SET2, as well as of patient-derived sAML blast progenitor cells (BPCs), while sparing normal CD34+ progenitor cells (HPCs). CDK9i-induced lethality in sAML cells was associated with attenuation of protein levels of S2-phosphorylated RNAP2, as well as depletion of mRNA and protein levels of c-Myc, PIM1, MCL1, Bcl-xL and XIAP. ATAC-Seq analyses demonstrated that treatment with NVP2 or B caused marked alterations in chromatin accessibility, including large peak losses on the chromatin with binding sites of TFs such as ERG, PU.1, STAT5, c-Myc, and RELA. This was associated with attenuation of mRNA expression of cMyb, c-Myc, RUNX1, LMO2, RELB, NFKB2, c-FLIP, MCL1, CDK6 and Bcl-xL. Following engraftment of luciferase transduced HEL cells into NSG mice, daily treatment with B at 10 mg/kg for 3 wks significantly reduced sAML burden and improved survival of the NSG mice (p < 0.002). Co-treatment of NVP2 or B with ruxolitinib or BCL2/Bcl-xL inhibitor ABT263 synergistically induced apoptosis of ruxolitinib-sensitive sAML cells (CI values of < 1.0). Treatment with NVP2 or B, or with BETi OTX015, also induced apoptosis of ruxolitinib persister/resistant (Rux-P) sAML cells (which demonstrate > 10-fold higher ruxolitinib IC50 values than Rux-S cells). This was associated with attenuated protein levels of p-RNAP2, c-Myc, PIM1, Bcl-xL and XIAP. Co-treatment with OTX015 and NVP2 or B synergistically induced apoptosis of not only HEL and SET2 but also HEL-RuxP and SET-RuxP cells, as well as of patient-derived sAML BPCs (CI values < 1.0). These findings confirm that targeted inhibition of BRD4-CDK9-RNAP2 leads to high level of pre-clinical efficacy against ruxolitinib-sensitive and ruxolitinib-refractory sAML cells.

#3837

JQ1 induces cell death and disrupts BET protein binding to the MYC locus in cholangiocarcinoma cells.

Samuel C. Fehling, Aubrey L. Miller, Karina J. Yoon. _University of Alabama at Birmingham, Birmingham, AL_.

Cholangiocarcinoma (CCA) is an aggressive bile duct neoplasm which is typically not diagnosed until late stage. The current standard of care, resection followed by gemcitabine with or without cisplatin, is relatively ineffective. Additionally, most patients are ineligible for surgical resection, and postoperative chemotherapy rarely prolongs overall survival. No previously identified mutations (KRAS, SMAD4 and p53) have been shown to drive disease progression. We and others have shown that the proto-oncogene c-Myc is expressed at relatively high levels in CCA cells compared to uninvolved normal tissue, suggesting this oncogene as a prospective therapeutic target in CCA. To evaluate whether c-Myc inhibition affected the proliferation of CCA cells, we evaluated the efficacy of the bromodomain and extraterminal domain (BET) inhibitor JQ1 on c-Myc expression and cell proliferation of CCA cells. JQ1 functions as an acetyl lysine (K-Ac) mimetic which binds to the K-Ac binding pocket of BET protein family members (BRD2, BRD3, BRD4 and BRDT). This binding competitively inhibits the association of BET proteins with K-Ac residues of nuclear proteins and inhibits c-Myc expression. Using a patient-derived xenograft (PDX) model of CCA, we showed previously that JQ1 downregulated c-Myc expression and inhibited tumor growth. Therefore, we hypothesized that JQ1 induces CCA cell death through disruption of BET protein binding to the promoter region of the MYC locus, inducing its downregulation. Our data show that JQ1 decreased c-Myc RNA and protein expression as well as the expression c-Myc transcriptional targets CHK1 and BRCA2. JQ1 also decreased CCA cell viability, clonogenic potential and induced apoptosis of KKU-055 CCA cells in vitro. The likely mechanism of JQ1-mediated c-Myc reduction has been postulated to involve competitive inhibition of the K-Ac binding function of the BET protein BRD4. Our current chromatin immunoprecipitation (ChIP) assays indicate that BRD2 binds to the MYC transcriptional start sites (TSS) P1 and P2, while BRD4 primarily binds MYC TSS P1 in KKU-055 cells. JQ1 precludes these BET proteins from binding their respective sites. When exposed to the IC50 of JQ1, ChIP pulldown with an anti-BRD2 antibody displays a near 100% reduction in binding of BRD2 to the MYC TSS while an anti-BRD4 antibody displays a 70% reduction in binding. In addition, stable BRD2 knockdown models of KKU-055 display reduced c-Myc protein expression by 60 to 80%, while BRD4 knockdown models showed no such reduction. The data demonstrate that in addition to BRD4, BRD2 contributes to regulation of expression of c-Myc in CCA cells. Research funded by NIH/NCI R21 CA205501

#3838

Targeting epigenetic regulator BMI-1 in alveolar rhabdomyosarcoma.

Cara E. Shields,1 Selma M. Cuya,1 Sarah K. Chappell,1 Komal Rathi,2 Shiv Patel,1 Sindhu Potlapalli,1 Robert W. Schnepp1. 1 _Emory University, Atlanta, GA;_ 2 _University of Pennsylvania, Philadelphia, PA_.

Background: Rhabdomyosarcoma (RMS) is an extremely aggressive soft tissue sarcoma which affects mainly children. There are two subtypes: alveolar rhabdomyosarcoma (ARMS) and embryonal rhabdomyosarcoma (ERMS). ARMS is characterized by PAX-FOXO1 fusion proteins, whereas subsets of ERMS harbor alterations within RAS and TP53 pathways. Currently, the outcomes for ARMS (especially when metastatic) remain dismal, thus underscoring the urgent need to identify novel targets for this cancer. The genomic landscape of many pediatric cancers, including ARMS, is relatively sparse. This led us to ask whether key epigenetic factors are driving tumor aggressiveness and could constitute novel approaches for treating ARMS. Notably, the epigenetic complexes PRC1 and PRC2 are overexpressed in a variety of sarcomas and are associated with worse overall survival. We took a hypothesis-based approach and focused on PRC1. We discovered that B lymphoma Mo-MLV insertion region 1 (BMI-1), a protein member of PRC1, is overexpressed in ARMS cells. BMI-1 is a known oncogene in other cancers, but its potential oncogenic role in ARMS and other pediatric malignancies has not yet been interrogated; thus we aim to study it within this context.

Methods: To analyze the function of BMI-1 in ARMS, we depleted the protein in ARMS cell line models by both shRNA/siRNA knockdown and measured expression, cell proliferation and apoptosis. We utilized two small molecule inhibitors, PTC-209 and PTC-028, to obtain IC50s in these cell lines, then determined effects on cell proliferation and apoptosis.

Results: We examined RNA-Seq tumor datasets and determined that BMI1 is robustly expressed in ARMS tumors. Additionally, we confirmed that BMI-1 is also overexpressed in ARMS cell lines at the levels of RNA and protein. Next, we depleted BMI-1 using multiple shRNAs and siRNAS and found that this led to striking (~70%) decreases in cell growth. We also observed increased levels of apoptosis within knockdown cells. Given these results, we asked whether small molecule inhibitors of BMI-1 mediated similar phenotypes, and so we used the inhibitors PTC-209 and PTC-028. PTC-209 is a first-generation BMI-1 inhibitor, while PTC-028 is a second-generation orally available inhibitor with higher potency. Both compounds inhibited BMI-1 function and greatly reduced cell proliferation in ARMS cell lines within the nanomolar range; however, as expected, PTC-028 showed a more pronounced effect compared to PTC-209.

Conclusions: BMI1 supports proliferation and survival in cell line models of ARMS. Both chemical and pharmacologic inhibition of BMI1 led to striking decreases in cell proliferation. Currently, we are further investigating the molecular impact of BMI1 inhibition, with plans to investigate its effectiveness within an in vivo ARMS model. Targeting BMI-1 pharmacologically could provide a novel therapeutic option for patients with ARMS and may apply more broadly to other sarcomas.

#3839

Comparison of thio-deoxy-cytidine (TdCyd) and aza-thio-deoxy-cytidine (Aza-TdCyd) in solid and liquid tumor cell lines and PPTC pediatric xenografts.

Beverly A. Teicher,1 Richard B. Lock,2 Kathryn Evans,2 Peter J. Houghton,3 Raushan T. Kurmasheva,3 Richard Gorlicki,4 Steve Erickson,5 Donn Wishka,1 Joel Morris,1 Michael Difilippantonio,1 Jerry E. Collins,1 Malcolm A. Smith,1 James H. Doroshow1. 1 _National Cancer Inst., Bethesda, MD;_ 2 _Children's Cancer Institute, Sydney, Australia;_ 3 _UT Health Science Center, San Antonio, TX;_ 4 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 5 _RTI International, Research Triangle Park, NC_.

Cytidine analogs target DNA methyltransferases as at least one of their mechanisms of action. The mechanism(s) of action of these compounds are incompletely elucidated and vary. TdCyd and Aza-TdCyd are thio-nucleoside analogs of cytidine which differ by a single nitrogen atom in the cytidine ring. Here the activity in solid and liquid tumor cell lines, and pediatric tumor xenografts is compared.

Methods: Cytotoxicity was examined after 2, 3 and 7-day exposure in 10 solid tumor lines and 6 liquid tumor lines. TdCyd was administered (1mg/kg) ip, daily for 5 days for 4 weeks (total 20 doses). Aza-TdCyd was administered (1.5, 1.0 or 0.5 mg/kg) ip, daily for 5 days for 4 weeks (total 20 doses). For solid tumors (sc), events were defined as 4X increase in tumor volume from day 0, for acute lymphoblastic leukemias (ALL) as %hCD45 cells exceeding 25%. The Kaplan-Meier method was used to compare time-to-event across treated and control groups. The objective response categories are progressive disease (PD), stable disease (SD), partial response (PR), complete response (CR), and maintained complete response (MCR) [Pediatr Blood Cancer 2007;49:928-40].

Results: Aza-TdCyd was, in general, a more active antitumor agent than TdCyd. In cell culture, after 7-days exposure Aza-TdCyd was 3-to 4-fold more potent than TdCyd in 10 solid tumor lines (IC50 1.2 vs 3.7 uM) and similarly more potent in 6 liquid tumor lines (0.03 vs 0.11 uM). TdCyd and Aza-TdCyd were tested in Ewing sarcoma, rhabdoid tumor, rhabdomyosarcoma, osteosarcoma, and pediatric acute lymphoblastic leukemia (ALL) patient-derived xenografts. The sarcomas were implanted subcutaneously, and the leukemias were implanted intravenously. At tolerable doses, the anticancer activity of both TdCyd and Aza-TdCyd were modest in the Ewing sarcoma (4) and rhabdomyosarcoma (4) models. The activity of both compounds was also modest in 5 out of 6 osteosarcoma models. However, the median survival of mice bearing the OS-9 osteosarcoma doubled (42 days) upon treatment with TdCyd and increased nearly 4-fold (84 days) in mice treated with Aza-TdCyd. While TdCyd had modest ALL activity, Aza-TdCyd increased median survival from 9.1(2-16) days in controls to >70 in 7/9 ALL models. Aza-TdCyd antileukemic activity was maintained at lower doses of the agent with median survivals of 75.4 days at 1.5mg/kg, 72.7 days at 1.0 mg/kg and 62.0 days at 0.5 mg/kg. Both TdCyd (NCT02423057) and Aza-TdCyd (NCT03366116) are in phase 1 clinical testing. Supported by NCI Grants: CA199222, CA199221, CA199297, CA199288, CA199000 and NCI Contract No. HHSN261200800001E.

#3840

ATF3 drives synergy between protein disulfide isomerase (PDI) inhibitors and epigenetic cancer therapy.

Ravyn M. Duncan,1 Leticia Reyes,1 Katelyn Moats,1 Reeder M. Robinson,1 Holly A. Stessman,2 Nathan G. Dolloff1. 1 _Medical University of South Carolina, Charleston, SC;_ 2 _Creighton University Medical School, Omaha, NE_.

The clinical potential of epigenetic cancer therapy has not been fully realized. Histone deacetylase inhibitors (HDACi), for example, only benefit a minor fraction of all cancer patients despite thousands of preclinical studies demonstrating their antitumor effects. In this study we show that a new structural class of protein disulfide isomerase (PDI) that was discovered by our lab dramatically enhances the antitumor effects of HDACi and select other epigenetic modifiers. We set out to identify synergistic interactions between our lead PDI inhibitor, E64FC26, and FDA-approved drugs in the NCI Approved Oncology Drug Set VIII of ~130 marketed drugs. We identified robust and previously uncharacterized synergy with all four HDACi in the set (i.e., panobinostat, romidepsin, vorinostat, and belinostat) in a broad range of solid and heme-malignancies. For example, E64FC26 potentiated panobinostat-induced cytotoxicity by 240-fold in PANC-1 pancreatic cancer cells and by 20-fold in T98G glioblastoma cells. Synergy with HDACi involved an apoptotic response measured by the activation of caspase 3, 8, 9 and the cleavage of the caspase 3 substrate PARP. PDI mediates proper protein folding, and western blotting and confocal microscopy confirmed the accumulation of misfolded poly-ubiquitinated protein aggregates in treated cells, which we showed was a critical component underlying the synergistic effects of the combination. To further understand the mechanism, we profiled the transcriptomes of combination treated cells using comparative RNA-Seq. These experiments revealed a convergence on ATF3 and DDIT3, two candidates in the ER stress pathway. RNA-Seq results were confirmed by qPCR and western blotting analysis in a range of tumor types. ATF3 played an apparent dominant role in driving the synergy between PDI and HDAC inhibitors, as knockdown of ATF3 completely ameliorated synergy between the two drug classes. We further clarified this molecular mechanism to show that PDI inhibition induces an ER stress response that leads to the induction of ATF3, which is further potentiated in the presence of HDACi, leading to the synergistic effects of the drug combination. These effects were not limited to HDACi, as similar results were observed in combinations with other epigenetic targeted therapies. We went on to characterize the efficacy and tolerability of PDI and HDAC inhibitor combinations in multiple mouse models, including pancreatic and glioblastoma xenograft models as well as multiple myeloma xenotransplant models. In summary, this study presents the potent antitumor combination of PDI and HDAC inhibitors and demonstrates a key mechanistic role of the ATF3 transcription factor. The combination of our developmental PDI inhibitor and HDACi offers a new dual therapeutic strategy and the opportunity to amplify and rescue previously unrealized activity of epigenetic therapy in oncology.

#3841

**Therapeutic targeting of** FLT3 **mutations in AML via menin-MLL1 and FLT3 inhibition.**

Margarita M. Dzama,1 Martha C. Taubert,1 Kerstin Kunz,1 Johanna Rausch,1 Chun-Wei Chen,2 Annalisa Mupo,3 Matthias Theobald,1 Thomas Kindler,1 Richard P. Koche,4 George S. Vassiliou,5 Scott A. Armstrong,6 Michael W. Kühn1. 1 _University Medical Center, Mainz, Germany;_ 2 _Beckman Research Institute, City of Hope, CA;_ 3 _University of Cambridge, Cambridge, United Kingdom;_ 4 _Memorial Sloan Kettering Cancer Center, New York City, NY;_ 5 _Wellcome Trust Sanger Institute, Cambridge, United Kingdom;_ 6 _Dana-Farber Cancer Institute, Boston, MA_.

Acute myeloid leukemias (AML) that are driven by MLL1 (KMT2A)-fusion proteins (MLL-f) or NPM1 mutations (NPM1mut) are both associated with aberrant expression of HOX and MEIS1 transcription factors and commonly harbor mutations in the gene encoding the receptor tyrosine kinase FLT3. Inhibition of the menin-MLL1 interaction has been shown to be a therapeutic opportunity in MLL-f driven leukemias and we recently demonstrated that this interaction is a dependency in NPM1mut AML. MI503 a specific small molecule menin-MLL1 inhibitor reduces dramatically cell growth and reverses leukemogenic gene expression including MEIS1 and FLT3. To determine global transcriptional changes associated with menin inhibition we performed RNA sequencing upon MI503 treatment in NPM1mut OCI-AML3 and MLL-f driven MV411 cells. MEIS1 and its putative target gene FLT3 were found to be among the most significantly downregulated genes. MEIS1 and FLT3 were consistently downregulated in various human and murine leukemia cell lines driven by MLL-f or NPM1mut. Allele specific qPCR confirmed profound downregulation of the mutant FLT3 allele in the MLL-f driven MV411 and MOLM13 cells as well as murine Npm1mut/+Flt3ITD/+ cells that all harbor a FLT3-ITD mutation. FLT3 surface expression was also substantially reduced upon MI503 treatment as assessed by FACS. Next, we assessed combinatorial menin- and FLT3 tyrosine kinase inhibition using MI503 and the 2nd generation FLT3 inhibitor AC220. The two drugs worked in a synergistic way to promote growth inhibition and enhanced apoptosis compared to single drug treatment or vehicle alone in MOLM13 and MV411 cells. HL60 and NB4 AML cells lacking NPM1mut, MLL-f, or FLT3-ITD showed no response. Drug synergism was also observed in the murine NPM1mut/+FLT3ITD/+ AML cells when combining MI503 with ponatinib a tyrosine kinase inhibitor with activity against the FLT3-ITD F692L resistance mutation that has been described in these cells. Of interest, ectopic expression of Hoxa9-Meis1 resulted in upregulation of Flt3 and rescued the antiproliferative effect of combined menin- and FLT3-inhibition. Combined menin- and FLT3 inhibition reduced FLT3 phosphorylation more than AC220 or MI503 alone most likely reflecting the joint effect of AC220 mediated inhibition of FLT3 phosphorylation and transcriptional FLT3 suppression via MI503. Transcriptional profiling revealed substantial silencing of FLT3 downstream signature genes including MYC upon combinatorial treatment. In vivo treatment of leukemic MV411 xenografts with combined MI503 and AC220 resulted in significantly enhanced reduction of leukemia burden that was observed with single drug treatment compared to vehicle controls. Altogether, our data show that simultaneous menin- and FLT3 inhibition has a synergistic effect against leukemogenic FLT3 signaling and may represent a novel therapeutic concept for MLL-f and NPM1mut driven AML with concomitant FLT3-ITD.

#3842

Co-targeting BET and MEK as salvage therapy for MAPKi and checkpoint inhibitor resistant melanoma.

Jessie Villanueva,1 Ileabett M. Echevarria-Vargas,1 Patricia I. Reyes-Uribe,1 Adam Guterres,1 Amruta Ronghe,1 Delaine M. Zayas-Bazan Burgos,1 Qin Liu,1 Xiaowei Xu,2 David W. Speicher1. 1 _The Wistar Institute, Philadelphia, PA;_ 2 _University of Pennsylvania, Philadelphia, PA_.

Despite novel therapies for melanoma, drug resistance remains a significant hurdle to achieving optimal responses. NRAS mutant melanoma is an archetype of therapeutic challenges in the field, which we used to test drug combinations to avert drug resistance. We show that the BET family member BRD4 is expressed at high levels in NRAS-mutant melanoma, and that high levels of BRD4 are associated with poor patient survival. Combining BET and MEK inhibitors synergistically curbed the growth of NRAS mutant melanoma and prolonged the survival of mice bearing tumors refractory to MAPK inhibitors and immunotherapy. Transcriptomic analysis revealed that combining BET and MEK inhibitors mitigated a MAPK- and checkpoint-inhibitor resistance transcriptional signature, downregulated the transcription factor TCF19, and induced apoptosis. Notably, TCF19 levels were downregulated in post-treatment biopsies from patients that responded to targeted or immunotherapies. In contrast, TCF19 levels were upregulated in tumor samples from non-responder patients. Furthermore, global proteomic analysis of PDX derived from patients resistant to immune checkpoint inhibitor indicate that the major downstream effects of concurrent BET and MEK inhibition is lipid metabolism. We are investigating the impact and mechanism whereby lipids modulate therapy response. Together our studies demonstrate that co-targeting MEK and BET can offset therapy resistance, offering a potential salvage strategy for melanomas with no other therapeutic options, and possibly other treatment-resistant tumor types.

#3843

LSD1 modulation by allosteric ligands.

Wei B. Emond,1 Matthis Geitmann,1 Malin Jarvius,2 Konrad Koehler,1 Per Källblad,1 Mia Niklasson,3 Vendela Parrow,2 Rajiv Sawant,1 Maria Sjöberg,1 Johan Winquist,1 Anna Segerman,3 Ulf Bremberg1. 1 _Beactica AB, Uppsala, Sweden;_ 2 _SciLifeLab Drug Discovery and Development, Uppsala, Sweden;_ 3 _Uppsala University, Uppsala, Sweden_.

LSD1 has emerged as a potential therapeutic target for a number of cancer types (e.g. AML, SCLC, colorectal, breast, liver, prostate, glioblastoma, Ewing sarcoma), as well as sickle cell anaemia and Alzheimer's disease. Irreversible LSD1 catalytic inhibitors have shown limited clinical efficacy in AML and SCLC, while solid tumours are largely unaddressed. This is in contrast to LSD1 knockdown with impact on a wide range of cancers, indicating that LSD1 functions other than enzymatic should be targeted.

We have developed novel small molecules that modulate LSD1 via an allosteric site - without inhibiting its enzymatic function - inducing a 60% reduction of nuclear LSD1 levels. The sensitivity profile in a cancer cell line panel is unique and dissimilar to >300 diverse reference compounds. In vitro efficacy is observed in glioma-initiating clones that are highly resistant to standard-of-care temozolimide as well as catalytic LSD1 inhibitors. Efficacy in the sub-µM range is observed with other solid tumour models, e.g. prostate cancer. The compounds exhibit synergy (Bliss independence >40%) with HDAC inhibitors as evaluated by viability in cellular cancer models, including lung, liver and glioblastoma.

Pharmacokinetic studies show good blood-brain-barrier penetration and oral availability of the allosteric LSD1 modulator BEA-17. A repeat dose of 25 mg/kg was well tolerated by NOD SCID mice, leading to µM level accumulation in the brain. Results from orthotopic glioblastoma PDX models, and in vivo hollow-fiber models of other solid tumours will be presented, as well as mechanistic insights from biophysical assays and gene expression analysis.

#3844

Anti-tumor efficacy of SMARCA degraders in pre-clinical models of cancer.

Leena Khare Satyam,1 Sanjita Sasmal,2 Manoj K. Pothuganti,1 Sreevidya M.R.,1 Ashokk Ettam,2 Sireesha Nunna,2 Marla Roshaiah,2 Shankaraiah Chithaluru,2 Rangarao Pallepati,2 Amitkumar A. Pawar,2 Leela P. Narukulla,2 Raghunadh A. Sripathi,2 Suraj Tgore,1 Rakesh P. Nankar,1 Charamanna KB,1 Nagesh Gowda S,1 Kiran Aithal,1 Samiulla DS,1 Subhendu Mukherjee,1 Shekar Chelur,1 Kavitha Nellore,1 Girish Daginakatte,1 Murali Ramachandra,1 Susanta Samajdar1. 1 _Aurigene Discovery Technologies Ltd., Bangalore, India;_ 2 _Aurigene Discovery Technologies Ltd., Hyderabad, India_.

SMARCA2/BRM and SMARCA4/BRG1 are the mutually exclusive DNA-dependent ATPases within the SWI/SNF complexes, which function in mobilizing nucleosomes to regulate transcription, DNA replication and repair, and higher-order chromosome dynamics. SMARCA4 is mutated in a number of cancers, which generally lack targetable oncogenes. Genetic silencing studies have established a requirement of SMARCA2 for survival of tumor cells lacking SMARCA4. SMARCA4-deficient patient population represents 10%-20% of NSCLC cases, ∼5% pancreatic cancer patients and ∼10% ovarian cancer patients where SMARCA2 is overexpressed. Interestingly, SMARCA4 is highly expressed without mutation in certain tumor types, where overexpression contributes to increased proliferation and survival. SMARCA4 knockdown in these tumors leads to inhibition of proliferation and also increase sensitivity to known chemotherapeutic agents, supporting the validity of targeting SMARCA4. Although genetic silencing of SMARCA2 leads to potent anti-proliferative activity in SMARCA4-deficient cancer cell lines, pharmacological studies with a probe capable of binding to SMARCA2 and SMARCA4 bromodomain have failed to show such an anti-proliferative phenotype. These findings triggered us to evaluate chemical degradation as an alternate approach to target SMARCA2/4 altered cancers. Optimization of bifunctional molecules with binding moieties for SMARCA2/4 and E3 ligase to induce proteasome-mediated degradation resulted in the identification of selective SMARCA2 and SMARCA4 degraders. These degraders showed selectivity against other bromodomain containing proteins such as BRD4, CBP and p300 in Western blot analysis. Functional analysis of a preferential SMARCA2 degrader in a panel of cell lines indicated a potent anti-proliferative activity in the context of SMARCA4 mutation. Additionally, these compounds displayed acceptable drug-like properties including solubility, metabolic stability and pharmacokinetics in mice. Dose-dependent tumor growth inhibition was observed in a SMARCA4-deficient lung cancer xenograft model and a syngeneic model of lymphoma at well-tolerated doses. Observed efficacy was correlated with the target degradation in the tumor supporting the potential to further develop them for cancer therapy. Based on the reported vulnerability of SMARCA4-deficient cell lines of diverse tumor origin to agents targeting PARP, PI3K/AKT and EZH2, combination effects with SMARC2 degrader are being interrogated.

#3845

Anti-tumor potential of Hydralazine, a DNMT1 inhibitor, on breast cancer growth and progression.

Parth B. Patel. _Medical College of Georgia - Augusta University, Augusta, GA_.

Breast cancer (BC) is the second largest diagnosed cancer among women, with approximately 266,120 new cases of BC in the United States. Thus far BC is understood as a disease driven by genetic abnormalities. Specifically, recent studies provide evidence that epigenetic modifications, such as methylation profiles, play a critical role in BC development regarding tumor suppressor genes. Despite there being many DNMT inhibitors, such as 5- azacytidine (Vidaza), they are ineffective versus solid tumors and they elicit a cytotoxic response. Thus, it is vital that a drug that is able to halt BC growth and progression is investigated. This project hopes to test the anti-tumor potential and therapeutic potential of Hydralazine (HDZ) because of its ability to act as a DNA methylation inhibitor, specifically DNMT1 and DNMT3A. In addition, despite HDZ being currently tested in clinical cancer trials for its antihypertensive effect, its mechanism of antitumor action remains undefined.We analyzed the anti-cancer potential of HDZ on two human BC cell lines and a mouse mammary tumor cell line (AT3). Cells were treated with different doses of HDZ (0, 1, 10, 25, and 50 M) for different time points (1, 2, 3, 7, and 14 days) and cell viability was measured by MTT assay. From these experiments, we optimized the time and concentration of HDZ in BC cells. Finally, we treated the cells with the appropriate concentration and time and then prepared RNA and protein for gene expression analysis, measured DNMT1 activity, and analyzed stem cells status using cell surface markers (CD24/CD44). We also tested the anti-tumor potential of HDZ in a mouse model using AT3 cells. AT3 cells were implanted in the mammary fat pads and grown to the tumor size of 250 mm3. Then, we treated the mice with HDZ and monitored the tumor growth. Tumor tissues harvested from these mice will be used for gene expression analysis, measure DNMT1 activity, histological analysis and analyze the stem cell status such as Oct4, Nanog, Gata4, Slug, and Sox2.All 4 BC cell lines demonstrated a slightly lower cell viability from the MTT when treated for 3 days and used at the highest dosage. In addition, through the use of RT-PCR, it has been shown that the levels of DNMT1 and CD24 expressions have been slightly downregulated. Western Blot analysis showed, the protein levels of DNMT1 and CD24 also decreased slightly. We also found that HDZ treatment significantly reduced tumor growth and volume in the mouse model. These findings suggest that the drug HDZ has a slight anti-tumor property on breast cancer growth and progression in the cell and mouse models. Although not as significant as we originally thought, the future implications of this project would be to complement HDZ with another drug such as a stem cell inhibitor. We believe that this dual therapy would synergistically work to halt BC progression and growth much more efficiently than any monotherapy for the treatment of triple-negative breast cancer.

#3846

Targeting epigenetic dysregulation in isocitrate dehydrogenase mutant glioblastoma.

Thomas K. Sears, Sandipan Datta, Gino Cortopassi, James Angelastro, Kevin Woolard. _University of California, Davis, Davis, CA_.

Glioblastoma (GBM) is an incurable form of brain cancer with a median survival time (MdST) of ~15 months after treatment with chemoradiotherapy. Identification of a CpG Island Methylator Phenotype (CIMP) subtype of GBM (G-CIMP), represents a significant clinical discovery, as these patients have an enhanced survival, with a MdST of 3 years. G-CIMP is characterized by a mutation in isocitrate dehydrogenase 1 or 2 (IDH1/2), which results in production of the oncometabolite 2-hydroxglutarate, an inhibitor of α-ketoglutarate-dependent demethylases. This results in aberrant DNA methylation, transcriptional repression, and may drive tumorigenesis. Here, we present data showing that G-CIMP is significantly more sensitive in vitro to inhibitors of the two arms of epigenetic regulation of transcription: the transcriptional activation arm and the transcriptional repression arm. Drug targets include the Bromodomain and Extraterminal (BET) and Histone Deacetylase (HDAC) proteins, involved in epigenetic transcriptional activation of oncogenes and repression of tumor suppressors, respectively. G-CIMP cells show a profound loss of cell viability at submicromolar concentrations using the small molecule inhibitors JQ1 and Panobinostat, which target BET and HDAC proteins, respectively. Western blot analysis indicates that BET inhibition elicits preferential loss of histone phosphorylation and acetylation in G-CIMP, notably serine 10 of Histone 3 and lysine 27 of Histone 3 (H3K27Ac), respectively. Both epigenetic marks are associated with positively regulating transcription. H3K27Ac is a superenhancer with known functions in activating oncogene transcription. These data ultimately suggest that G-CIMP tumor cells are reliant on established epigenetic transcriptional regulation to maintain cell viability. The global methylation status of these tumors may make them particularly sensitive to pharmacologic disruption of these epigenetic pathways. These studies provide a strong foundation for future preclinical work evaluating the prospective treatment of G-CIMP with combinatorial therapy using BET and/or HDAC inhibitors.

#3847

Knockout of DNMT1 using CRISPR gene-editing technology confers resistance to DNMT inhibitors in human breast cancer cells.

Angelo B. Laranjeira, Erich Huang, Larry Rubinstein, Dat Nguyen, Sherry X. Yang. _Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, MD_.

DNA methyltransferase (DNMT) 1 plays an important role for DNA replication and cell proliferation, and it is implicated in the maintenance and propagation of DNA methylation patterns. Targeting DNMT represents a promising approach for epigenetic therapy in cancer. To better elucidate the function of DNMT1 on the treatment effect of DNMT inhibitors in cancer cells derived from human solid tumors, we used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system (Cas9) to edit DNMT1 gene in MCF-7 breast carcinoma cells. The co-transfection of the DNMT1 target sequence that directs to exon 1 of the gene in pCas-guide vector and a donor DNA vector effectively knocked down DNMT1 protein in MCF-7 cells as detected by Western blot. After 96h of drug exposure, IC50s of azacitidine (5-azacytidine) and decitabine (5-aza-2'-deoxycytidine) were all higher than 10 µM in DNMT1 knockout (DNMT1-/-) cells assessed by MTT; in contrast, IC50s were 1.44 µM and 10 µM in parental (DNMT1+/+) cells. As for the cellular survival in the presence of drugs, a remarkable increase in the colony formation was observed in DNMT1-/- cells compared to DNMT1+/+ cells. The annexin-V/propidium iodide test by flow cytometry provided evidence of lower percentages of necroptosis in the DNMT1-/- cells than DNMT1+/+ cells when the cells were exposed to these inhibitors for 48 hours at 5 µM (7.12 ± 5.24 vs. 39.0 ± 2.65 by azacitidine, p = 4.11 × 10-5, and 5.77 ± 4.19 vs. 20.0 ± 6.66 by decitabine, p = 4.11 × 10-5); in addition, there were large fractions of viable cells in the DNMT1-/- cells versus DNMT1+/+ cells (85.86 ± 6.39 vs. 56.86 ± 4.00 for azacitidine treatment, p = 4.11 × 10-5 and 87.4 ± 5.56 vs. 76.2 ± 9.36 for decitabine, p = 5.60 × 10-3). The findings demonstrate that knockout of DNMT1 using CRISPR/Cas9 method leads to an increased resistance to azacitidine and decitabine, in corroboration with our previous data in DNMT1 knockout HCT116 colorectal cancer cells. Therefore, DNMT1 is critical to antitumor activity of DNMT inhibitors in human solid tumor cell lines.

#3848

In vivo **evaluation of the LSD1 inhibitor SP-2577 against Ewing sarcoma and rhabdomyosarcoma preclinincal models: A report from the Pediatric Preclinical Testing Consortium (PPTC).**

Peter J. Houghton,1 Steve W. Erickson,2 Beverly Teicher,3 Malcolm A. Smith,3 Ruolan Han,4 Raushan Kurmasheva1. 1 _Greehey Children's Cancer Research Institute, San Antonio, TX;_ 2 _RTI International, Research Triangle Park, NC;_ 3 _National Cancer Institute, Bethesda, MD;_ 4 _Salarius Pharmaceuticals, Houston, TX_.

Background: Lysine-specific demethylase 1 (LSD1/KDM1A) acts as a transcriptional co-regulator and utilizes flavinadenine dinucleotide (FAD) as a cofactor to remove mono- and di-methyl groups from histone H3 lysine-4 (H3K4) and lysine-9 (H3K9). SP-2577, in contrast to other LSD1 inhibitors in clinical evaluation, is a reversible inhibitor of LSD1. SP-2509 (a tool compound for SP-2577) reversed the EWSR1-FLI1 mediated transcription profile and oncogenic phenotype. To follow-up on these results, the PPTC evaluated SP-2577 against a set of Ewing sarcoma and rhabdomyosarcoma xenografts. Methods: SP-2577 was administered at a dose of 100 mg/kg intraperitoneally (IP) for 28 days and was tested against 7 Ewing sarcoma and 4 alveolar and 1 embryonal rhabdomyosarcoma xenografts. Standard PPTC methods for assessing time to event (EFS T/C = ratio of median time to event for treated versus control animals). Objective response measures similar to clinical measures were applied (Houghton, Pediatr Blood Cancer 2007;49:928-940). Results: SP-2577 was generally well tolerated during the efficacy testing phase. Among 120 animals treated with SP-2577 in efficacy testing, 9 (7.5%) experienced toxic death, and the median maximum weight loss was 1.8% (range 0.0% to 13.7%). As a single agent, SP-2577 induced a statistically significant prolongation in time to event in 3 of 7 Ewing sarcoma models and 4 of 5 rhabdomyosarcoma models. The extent of prolongation of time to event was small for most of the models for which there was a statistically significant prolongation in time to event, with EFS T-C values ranging from 1.9 to 14 days and with EFS T/C values ranging from 1.15 to 2.79. None of the models tested showed objective responses, but rather showed progressive disease as their best response. The minimum relative tumor volumes were significantly smaller compared to control for 4 of 7 Ewing sarcoma models and for 2 of 5 rhabdomyosarcoma models. However, the mean minimum relative tumor volumes were all greater than 1.0, indicating the lack of tumor regression. Pharmacodynamic effects of SP-2577 that were studied included c-Myc, N-Myc, HMOX1, histone H3, and histone H3(K4) dimethyl levels. There were no consistent changes in global histone H3(K4) dimethyl levels, and there was no obvious decrease in either c-Myc or N-Myc, whereas HMOX1 was slightly increased by treatment in two Ewing sarcoma models. Conclusions: SP-2577 was adequately tolerated in the Ewing sarcoma and rhabdomyosarcoma xenografts treatment cohorts that were studied. While a number of the models studied showed statistically significant effects on tumor growth rate, the effect size was generally small and tumor regression was not observed. (Supported by CA199297 and CA199222)

#3849

Structure-based PROTAC design demonstrates BAF complex ATPase vulnerabilities in cancer.

Manfred Koegl,1 William Farnaby,2 Claire Whitworth,2 Michael Roy,2 Emelyne Diers,2 Nicole Trainor,2 Steffen Steurer,1 Carina Riedmueller,1 Teresa Gmaschitz,1 Johannes Wachter,1 Christian Dank,1 Michael Gallant,1 Bernadette Sharps,1 Klaus Rumpel,1 Elisabeth Traxler,1 Romario Lorenz,1 Oliver Petermann,1 Peter Greb,1 Harald Weinstabl,1 Gerd Bader,1 Andreas Zoephel,1 Alexander Weiss-Puxbaum,1 Joerg Rinnenthal,1 Heribert Arnnhof,1 Nicola Wiechens,2 Meng-Ying Wu,2 Tom Owen-Hughes,2 Peter Ettmayer,1 Mark Pearson,1 Darryl B. McConnell,1 Alessio Ciulli1. 1 _Boehringer-Ingelheim, Vienna, Austria;_ 2 _University of Dundee, Dundee, United Kingdom_.

Targeting subunits of BAF/PBAF complexes has been proposed as an approach to exploit cancer vulnerabilities, yet small molecules developed to date have remained largely ineffectual. Here we develop PROTAC degraders of the BAF ATPase subunits SMARCA2 and SMARCA4 using a bromodomain ligand and recruitment of the E3 ubiquitin ligase VHL. High-resolution ternary complex crystal structures and biophysical investigation guided the rational optimization of ACBI1, a potent cooperative SMARCA2/4 degrader. ACBI1 induced antiproliferative effects and cell death caused by SMARCA2 depletion in SMARCA4 mutant cancer cells, and in acute myeloid leukemia cells dependent on SMARCA4 ATPase activity. These findings exemplify a successful biophysics and structure driven campaign to degrade targets previously considered undruggable, and pave the way towards new therapeutics for the treatment of tumors sensitive to the loss of BAF complex ATPases.

#3850

The potent catalytic p300HAT inhibitor A-485 induces rapid decay and reduction in breast cancer ERBB2 mRNA expression.

Gary K. Scott, Jacob Rose, Lei Wei, Birgit Schilling, Christopher Benz. _Buck Inst. for Research on Aging, Novato, CA_.

We have recently demonstrated that siRNA knockdown of p300 promotes the selective loss of oncogenic breast cancer transcripts such as ERBB2 while housekeeping transcripts like GAPDH are largely unaffected. Utilizing the potent p300 HAT inhibitor A-485 to treat the ERBB2 overexpressing SKBR3 breast cancer cells, ERBB2 transcripts experienced accelerated decay relative to decay induced by actinomycin D with cells entering growth arrest by 24 h, a situation similar to the growth arrest observed following p300 siRNA knockdown. Interestingly, previous studies from our lab identified a 3'UTR dependent mechanism promoting accelerated decay of ERBB2 transcripts following inhibition of class-1 histone deacetylases (e.g. HDAC1/2) using the approved therapeutic Romidepsin (FK228). In this situation mass spectrometry (MS) determined that within 2 h of FK228 treatment p300 becomes hyperacetylated at multiple lysine sites (K970, K1542, K1546, K1549, K1550, K1551, K1554, K1555, K1558, K1560, K1674, K1760) which include those lysines thought to regulate the activity of its HAT domain. The rapid reduction in ERBB2 mRNA levels induced by either 2-4 h of FK228 or A-485 treatment raises the interesting question of how either gain or loss of p300HAT enzymatic function induces accelerated ERBB2 mRNA decay? In regard to FK228 treatment, p300 siRNA experiments identified a 28 kDa species, presumed to be monoubiquitinated H2B, dependent upon p300 for FK228 induced acetylation and where knockdown of RNF20, an E3 ligase promoting H2B monoubiquitination, blunted the FK228 induced decay of ERBB2 mRNA. By immunoprecipitating p300 from cells treated with either vehicle or FK228 (2 h) and then assaying immunoprecipitates for histone acetyl transferase activity, we observed that FK228 induced hyperacetylation appears to increase p300HAT enzymatic activity. Additionally, it was observed that pretreating cells for 1 h with A-485 (2 μM) followed by 5 h of FK228 treatment (0.5 uM) produced complete loss of FK228 induced p300 hyperacetylation together with greatly reduced ERBB2 mRNA levels. Altogether, these findings point to a novel 3'UTR-dependent transcript regulatory mechanism wherein active but not hyperacetylated or hypoacetylated p300 stabilizes both new and mature ERBB2 transcripts, whose stability is then selectively and rapidly lost when p300 is either hyperacetylated by FK228 or functionally crippled by either siRNA knockdown or A-485 catalytic inhibition. While this novel ERBB2 mRNA stability mechanism is still in need of further structural definition, it can be effectively targeted by mechanistically different approaches and small molecule anticancer agents including the clinically approved class-I HDAC inhibitor, Romidepsin (FK228), and the investigational catalytic p300HAT inhibitor, A-485. 

### Novel Antitumor Agents 2

#3851

An Aurora kinase inhibitor BPR6K471 inhibits tumor growth and reduces the cancer stem cell-like properties of small cell lung cancer.

Chun-Ping Chang, Yi-Yu Ke, Wen-Hsing Lin, Dai-Hui Jhuo, Wan-Ping Wang, Chia-Hua Tsai, Yen-Ting Chen, Yu-Jie Su, Ming-Chun Hung, Zhong-Wei Wu, Po-Chu Kuo, Teng-Kuang Yeh, Ching-Ping Chen, Jen-Shin Song, Chiun-Tong Chen, Chuan Shih, Ya-Hui Chi. _National Health Research Institutes, Miaoli County, Taiwan_.

Small cell lung cancer (SCLC) is one the most aggressive tumors with poor survival rate. SCLC patients often present with metastasis at time of diagnosis, excluding surgery as a treatment option. While patients show high response rate to standard chemotherapy such as cisplatin/etoposide, they soon develop drug resistance and disease progression. Therefore, new therapeutic strategies are urgently needed for SCLC. Several characteristics of SCLC such as aggressiveness and drug resistance could attribute to the existence of a cancer stem cell (CSC) subpopulation in SCLC. The SCLC cell line NCI-H446 has been shown to present high degree of stemness and express stem cell markers including CD133, OCT4, MYC and Nestin. Here we develop a pyrimidine-based small molecule BPR6K471 which potently inhibits Aurora kinase activities in enzymatic- and cell- based assays. BPR6K471 efficiently (IC50 = 66 nM) inhibits proliferation of NCI-H446, and reduces the expression of stem-cell markers. In addition, intravenous injection of BPR6K471 inhibits >90 % progression of NCI-H446 in a mouse xenograft model. These results suggest that targeting Aurora kinases may be a potential therapeutic strategy to combat the CSC subpopulation in SCLC.

#3852

Development of1,2-bis(hydroxymethyl)benzo[g]pyrrolo[2,1-a]phthalazine hybrids as potent anticancer agents to small cell lung cancer.

Tai-Lin Chen, Vicky Jain, Yi-Wen Lin, Tsann-Long Su, Te-Chang Lee. _Academia Sinica - Inst. of Biomedical Sci., Taipei, Taiwan_.

Angiogenesis is a hallmark of cancer development, especially in lung cancer and colorectal cancer. Unfortunately, the rapid development of resistance to anti-angiogenesis agents has limited the clinical benefits for the treatment. To improve the efficacy of anti-angiogenesis agents, we designed and synthesized a series of new hybrid molecules that are composed of benzophthalazine (anti-angiogenesis moiety) and bis(hydroxymethyl)pyrrole (DNA interstrand crosslinking moiety). We demonstrate that these compounds are the dual function inducing DNA crosslinking and anti-angiogenesis. 1,2-Bis(hydroxymethyl)benzopyrrolo[2,1-a]phthalazine derivatives displayed potent cytotoxicity to leukemia and a batch of solid tumor cell lines. Among them, small cell lung cancer (SCLC) cells were the most susceptible cells with the IC50 values ranged from dozens of nanomolar to submicromolar. Significantly, we observed total tumor remission by treatment of SCLC H526 (TP53 WT) xenografts with formulated compound 8 at the dose of 20mg/kg, 5 times/week for 2 weeks. In addition, compound 8 was also more potent than cisplatin in suppression of growth of SCLC H211 (TP53 mutant) xenografts. Combination treatment of H211 cells with compound 8 and cisplatin displayed the synergistically cell killing. Our preliminary results further showed that co-treatment of compound 8 and low dose cisplatin significantly suppressed tumor growth and prolonged the survival rate of mice bearing H211 xenografts. In contract to cisplatin, treatment of mice with compound 8 did not cause body weight lost, indicating low systematic toxicity of compound 8. In summary, our results indicate that bis(hydroxymethyl)benzopyrrolo[2,1-a]phthalazine hybrids, either alone or in combination with other therapeutic drugs, are potent anticancer agents against SCLC and deserve for future development.

#3853

CD70 antibody-drug conjugate: A novel therapeutic agent for platinum-resistant ovarian carcinoma.

Mayu Shiomi,1 Shinya Matsuzaki,1 Ruriko Nakae,1 Satoshi Nakagawa,1 Akiko Okazawa,1 Eiji Kobayashi,1 Yutaka Ueda,1 Satoshi Serada,2 Tetsuji Naka,2 Tadashi Kimura1. 1 _Osaka University Graduate School of Medicine, Suita City, Japan;_ 2 _Kochi University Medical Hospital, Kochi City, Japan_.

Objective: The development of therapeutic agents for platinum-resistant ovarian carcinoma remains a significant issue. Currently, the use of antibody-drug conjugates (ADCs) represents an innovative therapeutic approach for various cancers. In this study, we examined the antitumor effect of a CD70-ADC on platinum-resistant serous ovarian carcinoma (SOC).

Methods: The CD70 expression was examined in A2780 cells (an SOC cell line) by western blotting and fluorescence activated cell sorting analysis. Cisplatin resistant A2780 cell lines (A2780 CisR cells) were generated, and the IC50 values for the A2780 parent and A2780 CisR cells were determined using modified MTT assays. The A2780 CisR cells were transfected with siRNA to suppress the CD70 expression. The suppression was confirmed by western blotting. Next, the CD70 monoclonal antibodies were generated to investigate their antitumor effect on the A2780 parent and A2780 CisR cells. ADCs, which consisted of CD70 monoclonal antibody linked to monomethyl auristatin F (MMAF, the tubulin polymerization inhibitor), were generated, and their antitumor effect against the A2780 CisR cells was investigated in vitro and in vivo. After the A2780 CisR-xenograft tumors were treated with CD70-ADC, immunohistochemistry (IHC) analysis was performed using antibodies against the mitotic marker, anti-phospho-histone H3.

Results: The expression of CD70 was observed in the A2780 CisR cells but not in the A2780 parent cells. CD70 silencing in A2780 CisR cells did not significantly reduce the cisplatin IC50 value nor did it inhibit cell proliferation. The CD70 monoclonal antibodies did not inhibit the proliferation of the A2780 parent and CisR cells. These results suggest that inhibiting the expression of CD70 does not affect the sensitivity of cisplatin and the growth of the tumor. Next, we generated the CD70-ADC. The CD70-ADC did not reach IC50 in the A2780 cells; however, the IC50 value was 0.104 nM for the A2780 CisR cells. CD70-ADC successfully exhibited antitumor effects in the CD70-ADC-treated mice in comparison with the control, i.e., the ADC-treated mice (162.4 vs. 534.2 mm3; p<0.001). IHC staining of the A2780 CisR-xenograft tumors with phosphorylated-histone H3 was performed. The percentage of tumor cells undergoing mitosis was markedly decreased after treatment with CD70-ADC, but not with the control IgG-ADC. These results suggest that MMAF was effectively delivered to the CD70-expressing tumor cells by the anti-CD70 mAb, causing mitotic arrest.

Conclusion: Our results revealed that CD70-ADC could be a novel therapeutic agent for platinum-resistant SOC.

#3854

Preclinical characterization of a novel non-cyclic dinucleotide small molecule STING agonist with potent antitumor activity in mice.

Zezhou Wang, Peter Dove, David Rosa, Bolette Bossen, Simone Helke, Marilyse Charbonneau, Laura Brinen, Karen Dodge, Gloria H. Lin, Carole Galligan, Natasja N. Viller, Mark Wong, Vivian Lee, Tina Catalano, Robert A. Uger, Malik Slassi, Jeff Winston. _Trillium Therapeutics Inc., Mississauga, Ontario, Canada_.

The cGAS-STING pathway plays a pivotal role in sensing aberrant cytoplasmic DNA fragments derived from pathogens or host tumor cells and initiating an innate immune response. STING activation induces type I IFNs and a cascade of other pro-inflammatory cytokines that activate the innate immune system and subsequently recruit adaptive immune cells. STING functions as a key mediator bridging the innate and adaptive immune responses by stimulating anti-tumor antigen presentation and promoting effective T cell priming, thereby making STING activation an attractive target for cancer immunotherapy. Currently, all known STING agonists in clinical trials are based on a cyclic dinucleotide (CDN) scaffold. These molecules mimic the endogenous STING ligand cGAMP which displays limited potency and cell permeability. Here we present the preclinical characterization of a novel non-CDN small molecule STING agonist, TTI-10001.

TTI-10001 exhibited potent and broad activity in a panel of HEK293T-STING reporter cell lines expressing each of the five human STING (hSTING) alleles or murine STING (mSTING). Activation of the STING pathway was confirmed by an increase of both phospho-STING and phospho-IRF3 after TTI-10001 treatment, and TTI-10001-dependent induction of reporter activity was ablated in the presence of an inhibitor of the downstream STING signaling kinase TBK1. TTI-10001 showed a favorable safety profile with no activity on hERG or major CYP450 isoforms and was well tolerated in mice with no weight loss or overt morbidity after repeat intratumoral administration. Mice dosed with TTI-10001 displayed increased levels of phospho-STING and phospho-IRF3 in tumors as well as elevated expression of various pro-inflammatory cytokines including IFNβ, TNFα, and IL-6. This in vivo activation of STING signaling correlated with significant anti-tumor activity in multiple syngeneic mouse tumor models.

In summary, we have generated a novel non-CDN small molecule STING agonist with potent activity on the five hSTING alleles and mSTING. Our compound exhibits strong in vitro and in vivo induction of the STING pathway as well as potent anti-tumor activity. Taken together, these results highlight the potential of TTI-10001 and support further evaluation and development of this molecule as a novel cancer immunotherapy agent.

#3855

Preclinical investigation of novel ALDH1A1 inhibitors in pancreatic cancer.

Betelehem W. Yacob, John J. Arcaroli, Maraake Taddese, Todd M. Pitts, Stacey M. Bagby, Sarah J. Hartman, S.Lindsey Davis, Christopher H. Lieu, Alexis D. Leal, Wells A. Messersmith. _University of Colorado Denver-AMC, Aurora, CO_.

Introduction: Pancreatic adenocarcinoma remains a devastating disease, with a predicted 5-year survival at the time of diagnosis of less than 10%, and likely the number 2 cause of U.S. cancer deaths in the next decade. The lack of effective therapies and resistance to cytotoxic agents contribute to the poor outcomes in this patient population. Aldehyde dehydrogenases (ALDHs) are a family of enzymes that play important biological and metabolic roles in the human body. ALDH activity has been identified in many human malignancies as a marker of tumor initiating cells. In pancreatic cancer, ALDH1A1 has been shown to be upregulated in cancer stem cells (CSCs) and has been attributed to tumorigenesis and chemotherapeutic resistance.

Methods: Six novel ALDH1A1 inhibitor drugs from the National Center for Advancing Translational Sciences (NCATS) were screened on human pancreatic cancer cell lines using the Cell Titer-Glo Assay. Proliferation assays were also performed with the three best performing inhibitors on a total of eight human pancreatic cancer cell lines. The best performing ALDH1A1 inhibitor drug were chosen and tested in combination with gemcitabine and paclitaxel on patient-derived tumor organoids (PDTOs). The combination effects were assessed over a 7-day period using the IncuCyte ZOOM live cell imager and CellTiter-Glo 3D Assay.

Results: We assessed the anti-proliferative effects of 6 novel ALDH1A1 inhibitors on 4 pancreatic cancer cell lines. Two out of 4 pancreatic cell lines showed moderate treatment effects to 3 ALDH1A1 inhibitors. Further evaluation of one of the ALDH1A1 inhibitor on 8 additional pancreatic cancer cell lines revealed similar results. While several pancreatic cell lines showed some moderate activity at higher levels of drug exposure, most of the pancreatic cancer cell lines treated were more resistant. We next investigated the treatment effects of ALDH1A1 inhibitor in combination with gemcitabine or paclitaxel on PDTO 269 and 272. A combination effect with the ALDH1A1 inhibitor and paclitaxel was observed after 72 hours of drug exposure in both PDTO 269 and 272. In contrast, treatment with the ALDH1A1 inhibitor with gemcitabine did not further decrease growth when compared to single agent ALDH1A1 and gemcitabine.

Conclusions: Although single agent activity of novel ALDH1A1 inhibitors is limited at significantly reducing cellular proliferation, inhibition of ALDH1A1 in combination with paclitaxel appears to have potent anti-tumor activity in PDTOs. These findings support further investigation of this combinational therapy for the treatment of pancreatic cancer. Additional studies are currently underway to evaluate this combinational activity in more preclinical pancreatic cancer models and to elucidate the underlying mechanisms whereby the inhibition of ALDH1A1 enhances the anti-tumor potential of paclitaxel.

#3856

MTLCEBPA, a drug candidate for hepatocellular-carcinoma enhances efficacy of Sorafenib.

Nagy A. Habib,1 Kai-Wen Huang,2 Vikash Reebye,1 Mikael Sodergren,1 John Rossi3. 1 _Imperial College London, London, United Kingdom;_ 2 _National Taiwan University, Taipei, Taiwan;_ 3 _Beckman Research Institute, City of Hope, Duarte California, CA_.

MTLCEBPA, a non-toxic therapeutic oligonucleotide formulated inside SMARTICLES (liposomal nanoparticles) is currently under a Phase I study for patients with advanced hepatocellular carcinoma (HCC). We have demonstrated in multiple in vivo liver disease models that MTLCEBPA can improve liver function and inhibit HCC tumor growth. Here we show that upregulation of hepatic CEBPA expression by MTLCEBPA enhances the effects of Sorafenib in a (Diethylnitrosamine) DEN induced cirrhotic HCC rat model by significantly reducing liver tumor volume after only two weeks of combination treatment when compared to untreated control or single agent treatment only.

In this study, we established cirrhosis and HCC by exposing male Wistar rats to DEN for 6 weeks. Animals were randomized into 5 groups comprising of:

1. PBS control;

2. MTL-CEBPA treatment (TIW at 3mg/kg) for one week;

3. MTL-CEBPA treatment (TIW at 3mg/kg) for two weeks;

4. Sorafenib TIW at 10mg/kg for two weeks; and

5. MTLCEBPA at 3mg/kg at week one combined with Sorafenib 10mg/kg at week 2 group.

The results here indicate that Sorafenib combined with MTLCEBPA significantly inhibits tumor growth when compared to single agent treatment. Tumor sizes averaged at 644.7mm3 (± 88.57) in group 1 compared to 326mm3 (± 53.05) in group 2; 199.7mm3 (± 54.73) in group 3 and 299.5mm3 (± 77.1) in group 4. In combination treatment (group 5) the tumor

sizes averaged at 101.3mm3 (± 37.48). Serum AFP change (posttreatment values - pre-treatment) was more prominent in the MTLCEBPA/Sorafenib combination treatment (group 5) where there was a 4-fold reduction in values after treatment when compared to control groups 1-4. Serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and total bilirubin remained unchanged across all the treatment groups suggesting no deterioration in liver function during the study period.

These results suggest that MTLCEBPA improved the efficacy of Sorafenib in this DEN induced HCC model, indicating a promising therapeutic option for advanced HCC patients who can no longer be treated with potentially more effective local therapies.

#3857

Discovery and structure-activity relationship studies of TP-2846: a novel tetracycline antileukemia agent.

Cuixiang Sun,1 Peng Zhao,1 Diana Hunt,1 Kathryn Kerstein,1 Joseph Newman,1 Sara McKellip,2 Robert Bostwick,2 Jacques Dumas,1 Xiao-Yi Xiao1. 1 _Tetraphase Pharmaceuticals, Watertown, MA;_ 2 _Southern Research Institute, Birmingham, AL_.

Tetracyclines are broad-spectrum antibacterial agents. They inhibit bacterial protein synthesis by binding to the 30S subunit of the bacterial ribosome. Besides tetracyclines' potent antibacterial activity, there is accumulating in vitro and in vivo evidence that tetracycline derivatives, including COL-3, doxycycline, minocycline, and tigecycline (Škrtić, Cancer Cell (2011) 20(5): 10.015), also possess anticancer properties against both liquid and solid tumors. Several tetracyclines have been investigated in various cancer clinical trials. However, most of these human trials, including the recent phase 1 study of tigecycline in patients with acute myeloid leukemia (AML) (Reed, Cancer Med (2016) 5(11): cam4.845), have yielded disappointing results. We believe that the failure of tigecycline in the AML trial is due to its inadequate in vitro potency (single-to-double digits µM range). To identify more potent anticancer tetracyclines, we explored the anticancer, particularly antileukemia activities of novel tetracycline analogs created by the proprietary tetracycline total synthesis platform during our antibacterial drug discovery efforts.

A collection of 2368 novel, structurally diverse tetracycline analogs was screened against the leukemia cell line THP-1 in an HTS format (386-well). From the 68 hits (2.9% hit rate) with sub-µM antiproliferation activity, five lead compounds derived from two sub-series were selected for further profiling, including TP-2846, which displayed one of the lowest GI50 values at 0.75 µM. As a comparison, tigecycline had a GI50 of 29 µM in the same assay. The five lead compounds were subsequently evaluated for antiproliferation activity in other liquid and solid tumor lines, including MV4-11 (0.020 µM), MOLT-4 (0.09 µM), K-562 (0.43 µM), HL-60 (0.37 µM), CCRF-CEM (0.067 µM), KG-1 (0.27 µM), Kasumi-1 (0.16 µM), U-87 (0.09 µM), HepG2 (1.6 µM), CCL-247 (0.26 µM), CCL-222 (0.48 µM), HTB-77 (0.26 µM), and CRL-1923 (6.2 µM) (values in parentheses are GI50 of TP-2846). TP-2846 and another lead compound were also screened against the NCI60 panels. TP-2846 demonstrated the highest overall antiproliferation potency against most cell lines tested and is 10-50 folds more potent than tigecycline.

Using MV4-11 as the primary screening cell line, we subsequently evaluated and studied the structure-activity relationships of more than 170 analogs of TP-2846 with systematic variations at the C4, C7, and C8 positions of the tetracycline core. Although a number of new analogs displayed comparable antiproliferation activity in MV4-11, TP-2846 remains the most potent analog overall in vitro and was selected to be further profiled in vitro and in vivo (see accompanying abstracts "In vitro characterization of TP-2846: a novel tetracycline antileukemia agent" and "In vivo activities of TP-2846: a novel tetracycline antileukemia agent").

#3858

Inflammation and oncogene in hepatocellular carcinoma: Clinical relevance and experimental targeted therapy.

Wei Wang,1 Jianwen Cheng,2 Jiang-Jiang Qin,1 Bo Hu,2 Bhavitavya Nijampatnam,3 Sadanandan Velu,3 Xin-Rong Yang,2 Jia Fan,2 Ruiwen Zhang1. 1 _University of Houston, Houston, TX;_ 2 _Fudan University, China;_ 3 _University of Alabama at Birmingham, Birmingham, AL_.

Hepatocellular carcinoma (HCC) poses a major public health problem worldwide and novel, effective therapeutic approaches for this devastating disease are urgently needed. The overexpression or activation of the MDM2 oncogene frequently occurs in HCC and is linked to cancer growth, poor survival, metastasis, and resistance to treatment. We have hypothesized that MDM2 is a promising therapeutic target for HCC therapy. We have recently discovered that the transcription factor NFAT1 upregulates MDM2 expression, promoting cancer cell growth, migration, invasion, and angiogenesis. The present study was designed to demonstrate the role of the NFAT1-MDM2 pathway in HCC progression and its value in developing HCC targeted therapy. We systemically investigated the expression and association of MDM2 and NFAT1 in 254 pairs of human HCC and matched non-cancerous tissue samples. High-throughput virtual and cell-based screenings were carried out for search for lead compounds targeting both NFAT1 and MDM2. The in vitro and in vivo anti-HCC activities and underlying mechanisms of action were further evaluated in HCC cell lines with various p53 backgrounds. Our results demonstrated that, compared with the non-malignant peritumoral tissues MDM2 and NFAT1 were overexpressed in HCC tissues, which were correlated with poor prognosis and increased metastasis. Based on molecular modeling study, Biacore assay, and pull-down assays, one of the lead compounds, named MA242, was identified as a potent and selective MDM2 and NFAT1 dual inhibitor. Mechanistically, M242 directly bound to MDM2 and NFAT1 proteins with high affinity and induced their protein degradation. MA242 also inhibited NFAT1-mediated MDM2 transcription. At cellular level, MA242 decreased cell proliferation, and induced apoptosis and G2/M phase arrest in various HCC cell lines, regardless of the p53 status. Furthermore, M242 inhibited the tumor growth and metastasis in orthotopic and patient-derived xenograft (PDX) models of HCC, without any obvious host toxicity. In conclusion, MDM2 and NFAT1 expression can serve as predictors of survival in patients with HCC. Dual MDM2 and NFAT1 inhibitors may represent a first-in-class clinical candidates for the treatment of HCC, including tumors lacking functional p53. This project is supported by NIH R01 CA186662 and R01 CA214019 and ACS RSG-15-009-01-CDD.

#3859

Targeting CDK9 and MCL-1 by a New CDK9/p-TEFB Inhibitor with and without 5-fluorouracil in esophageal adenocarcinoma.

zhimin Tong,1 Alicia Mejia,1 Omkara Veeranki,1 Anuj Verma,1 Arlene Correa,1 Viren Patel,2 Rashmi Dokey,1 Luisa Solis,1 Barbara Mino,1 Riham Kathkuda,1 Jaime Canales,1 Steven Lin,1 Sunil Krishnan,1 Scott Kopetz,1 Mariela Blum,1 Jaffer Ajani,1 Wayne Hofstetter,1 Dipen M Maru1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _Duke University School of Medicine, NC_.

Background: CDK9 inhibitors are anti-tumorigenic against several solid tumors including esophageal adenocarcinoma (EAC). However, efficacy of CDK9 inhibitor as adjuvant to chemotherapy and target proteins shared between these two drugs are not known.

Methods: Efficacy of a new CDK9 inhibitor, BAY1143572 with and without 5-fluorouracil (5-FU) on tumor growth was tested in vitro and xenograft models of EAC. We tested effects of these agents on MCL-1 in vitro. We also analyzed a possible role of MCL-1 as a predictor of survival in patients with EAC.

Results: BAY1143572 had a dose-dependent anti-proliferative and pro-apoptotic effects in vitro. BAY1143572 and 5-FU had strong synergy in vitro. Median tumor volume of FLO1 or ESO26 xenografts in cohort treated with combination of two drugs was 82% or 55% smaller than the cohort treated with 5-FU alone, and 46 % or 33% smaller than the cohort treated with BAY114352 alone (p<0.05). BAY1143572 transcriptionally downregulated MCL-1 by inhibiting HIF-1α binding to the MCL-1 promoter. 5-FU enhanced BAY1143572 induced MCL-1 downregulation in vitro. Stable overexpression of MCL-1 reduced apoptosis by BAY1143572 and 5-FU. Higher tumor cell MCL-1 expression correlated with shorter overall survival in patients with EAC treated with chemoradiation and surgery.

Conclusion: CDK9 inhibitor and 5-FU are synergistic and MCL-1 is a target of CDK9 inhibitor in EAC.

#3860

Discovery and characterization of novel anti-cancer small molecule inhibitors of Sec61.

Eric Lowe,1 Janet L. Anderl,1 Andrea R. Fan,1 Ying Fang,1 Jing Jiang,1 Henry W. Johnson,1 Christopher J. Kirk,1 Dustin McMinn,1 Tony Muchamuel,1 Meera Rao,1 Phillip P. Sharp,2 Jack Taunton,2 Jinhai Wang,1 Jennifer A. Whang1. 1 _Kezar Life Sciences, South San Francisco, CA;_ 2 _University of California, San Francisco, San Francisco, CA_.

Secreted and membrane proteins play key roles in malignant transformation and tumor growth, including autocrine growth factor expression, receptor oncogene signal transduction pathways, metastasis, and immune system evasion. The biogenesis of most secreted and transmembrane proteins involves cotranslational translocation of nascent polypeptides into the endoplasmic reticulum through the Sec61 translocon. Broad inhibition of protein secretion can be mediated by natural product-derived Sec61 inhibitors that also possess anti-tumor activity, suggesting that Sec61 represents an untapped therapeutic target for cancer treatment. Here we describe a process for discovering and evaluating novel Sec61 inhibitors with varying degrees of selectivity for therapeutically relevant targets in oncology.

We used HEK293 reporter cell lines stably transfected to express secreted and transmembrane proteins of interest fused to luciferase to enable high-throughput screening of potential inhibitors of Sec61-dependent protein secretion. As expected, target selectivity was largely dependent on respective signal sequences. However, full-length protein constructs were found to be 5-10 fold less sensitive to inhibition compared to reporters containing only the signal sequence plus 10 amino acids, suggesting that additional interactions beyond the signal sequence are involved in promoting cotranslational translocation. To verify that novel compounds specifically targeted Sec61, we found that compounds were unable to inhibit the activity of luciferase localized to the cytosol and had reduced potency in reporter cells expressing a Sec61 mutant known to confer resistance to published inhibitors such as cotransin, mycolactone, and apratoxin. We assessed compound selectivity using a library of 37 transiently expressed Sec61-client proteins of therapeutic relevance, revealing 4 distinct chemical scaffolds differentiated by target selectivity profile. Consistent with data from published compounds, broad inhibitors of protein secretion had anti-tumor activity. Of particular interest were compounds able to block expression of multiple immune checkpoints including PD-1, PD-L1, CTLA-4, and CD96. Activity in primary cells, such as receptor expression on activated T-cells and cytokine release from stimulated PBMC, was assessed to verify specificity. Compounds with adequate in vitro metabolic stability were tested in mice for pharmacokinetics as well as inhibition of target protein secretion and global inhibition of Sec61 activity.

Taken together, our data suggest that Sec61 is a novel therapeutic target which represents a unique opportunity to both selectively block expression of multiple proteins involved in tumor growth and globally modulate protein homeostasis, resulting in tumor cell death.

#3861

Proxalutamide (GT0918), a novel androgen receptor (AR) antagonist, targeting resistance mechanisms to AR signaling-directed castration-resistant prostate cancer (CRPC) therapies.

Liandong Ma,1 Qianxiang Zhou,1 Yang He,1 Jie Chen,1 Karl Zhou,1 Qingqing Zhou,1 Chunyun Chen,1 Jason Shaoyun Xiang,1 Xiaobing Deng,2 Luhua Lai,2 Youzi Tong1. 1 _Suzhou Kintor Pharmaceuticals, Inc., Suzhou, China;_ 2 _Peking University, Beijing, China_.

Reactivation of androgen receptor (AR) plays a key role in prostate cancer growth, especially in castration-resistant prostate cancer (CRPC) progression. Accumulated data have demonstrated that the AR reactivation is resulted from AR amplification, AR overexpression and mutations in AR ligand binding domain (LBD) as well as AR splicing variants (AR-V7). This AR reactivation also drives the resistance to AR targeted therapies, including 1st and 2nd generations of AR antagonists, such as Bicalutamide (Bica) and Enzalutamide (Enza, MDV3100). Therefore, the development of novel and effective AR antagonists is in urgent need for overcoming AR reactivation for patients with metastatic castration-resistant prostate cancer (mCRPC).

Here we report a novel AR binding pose of Proxalutamide (GT0918), an AR antagonist, in silico of drug's mode of action. We also reveal the effects of GT0918 on the transactivation of the wild type AR (wtAR) and AR mutants with comparisons to 1st and 2nd generations of AR antagonists (Bicalutamide and Enzalutamide). GT0918 was reported to have higher binding affinity than Bica (11.4X) and Enza (3.5X) in AR ligand binding assays previously. To gain detail insights into the mode of action of GT0918 and Enza in AR LBD, structure-based computer modeling was performed. The mode of drug action shows that GT0918 binds to AR LBD pocket. This compound not only fits in the AR LBD pocket very well, but also its structure pushes AR Helix 12 away, resulting in the decreased interaction between AR Helix 12 and LBD pocket, which creates a unique and significant difference from other AR blockers. GT0918 was further assessed in luciferase-based AR transactivation assays. The results showed GT0918 significantly inhibited the androgen-induced transactivation of the wtAR. Importantly, it also blocked the transcriptional activity of tested mutant ARs arising to targeted AR therapies, including the AR mutant F877L that converts the Enzalutamide and Apalutamide (ARN-509) from antagonist to agonist activity. In clinical trial setting, some mCRPC patients progressed on Enzalutamide and bicalutamide showed stable disease and durable responses after received GT0918 from various doses. In conclusion, our results support the clinical development of GT0918 in prostate cancer patients who progressed on Enzalutamide or/and AR blockers.

#3862

AB474, a potent orally bioavailable inhibitor of arginase, for the treatment of cancer.

Sterling C. Eckard, Yu Chen, Dana Piovesan, Nell Narasappa, Timothy W. Park, Jarek Kalisiak, Eric T. Newcomb, Manmohan R. Leleti, Divyank Soni, Elaine Ginn, Jie Chen, Dan DiRenzo, Kristen Zhang, Lixia Jin, Stephen W. Young, Matthew J. Walters, Ulrike Schindler, Jay P. Powers. _Arcus Biosciences, Hayward, CA_.

Introduction: It has been demonstrated that myeloid derived suppressor cells (MDSCs) have a direct role in tumor immune evasion, and increased MDSCs are associated with reduced overall survival in several types of cancer. Elevated levels of circulating MDSCs have been shown to correlate with a blunted response to checkpoint blockade. MDSCs secrete arginase, which depletes arginine, leading to decreased T cell activity and a suppressed anti-tumor response. Here we will present the characterization of AB474, a highly potent small molecule inhibitor of arginase.

Methods: Arginase inhibition was measured using a coupled enzyme assay in the presence and absence of both mouse and human serum. IC50 was determined in buffer, human plasma, and serum by LC-MS/MS quantification of ornithine, while plasma protein binding was determined by equilibrium dialysis. Tumor transcript analysis was performed on homogenized whole murine tumors using Taqman probes. Arginase inhibition by AB474 was tested on human CD4 and CD8 T cells, with proliferation and effector functions being determined in the presence of recombinant and endogenous human ARG1. Assays were performed using standard CD2/CD3/CD28 activation conditions: proliferation was measured by flow cytometry, and IFNγ secretion by ELISA.

Results: AB474 inhibits arginase with low-nanomolar potency. It effectively inhibits both recombinant and endogenous arginase, while displaying low plasma protein binding across species. Pharmacokinetic characterization of AB474 demonstrated oral bioavailability in both mice and rats, displaying qualities compatible with human dosing. Measuring transcript of Arg1 in murine tumor models revealed expression in both tumor and immune compartments. Flow cytometry analysis shows ARG1+ MDSCs exist in both the tumor and periphery, with higher expression levels seen in Ly6c+ mMDSC populations. Consistent with the elevation of arginase in tumor-bearing mice, levels of arginine are decreased when compared to naïve littermates. Addition of arginase to activated CD4 or CD8 T-cells results in a significant suppression of proliferation, CD3 expression, and IFNγ secretion. Addition of AB474 results in a significant restoration of normal T cell effector functions (P < 0.001).

Conclusions: AB474 is a potent orally bioavailable inhibitor of arginase. Inhibition of arginase will block one of the primary immune-suppressive functions of MDSCs resulting in restored T cell activation and anti-tumor responses.

#3863

Specifically targeting PDIA1 with indene inhibitors leads to bortezomib-potentiation in multiple myeloma.

Reeder M. Robinson, Leticia Reyes, Ravyn Duncan, Nathan Dolloff. _Medical University of South Carolina, Charleston, SC_.

Protein disulfide isomerase (PDI) is an emerging therapeutic target in oncology. PDI inhibitors have demonstrated a unique propensity to overcome resistance to existing cancer therapies, although drug candidates have not yet progressed to the stage of clinical development. We recently reported the discovery of lead indene compound E64FC26 as a potent pan-PDI inhibitor that enhances the cytotoxic effects of proteasome inhibitors (PIs) in panels of Multiple Myeloma (MM) cells and MM mouse models [1]. An extensive medicinal chemistry program has led to the generation of a diverse library of indene-containing molecules with varying degrees of PI-potentiating activity. The results show detailed structure activity relationships (SAR) with a high degree of correlation between potency of PDIA1 inhibition and bortezomib (Btz) potentiation in MM cells. Inhibition of PDIA1 in MM leads to ER and oxidative stress characterized by the accumulation of misfolded poly-ubiquitinated proteins and the induction of UPR biomarkers ATF4, CHOP, and Nrf2. Furthermore, an advanced derivative, E64FC65, was identified as a PDIA1 selective inhibitor as it was significantly weaker against PDIA3, PDIA4, TXNDC5, PDIA6, and thioredoxin when compared to E64FC26 (Table 1). We purified the redox-active a and a' thioredoxin-like domains of PDI and found that E64FC65 only inhibited the a domain while E64FC26 inhibited both a and a' domains. The PDI isoform and domain selectivity of E64FC65 imparted increased anti-cancer activity while reducing toxicity against primary activated human T-cells. Additionally, E64FC65 exhibited improved pharmaceutical properties in ADME assays. This work shows the generation of a PDIA1 a domain specific indene inhibitor that potentiates Btz cytotoxicity in MM cells while abrogating toxicity in normal cells. E64FC65 is thus a favorable PDI inhibitor candidate for further development.

#3864

A novel PDE10/β-catenin pathway inhibitor, MCI-030, for the treatment of colorectal cancer.

Antonio B. Ward,1 Xi Chen,1 Jacob Valiyaveettil,1 Kevin Lee,1 Wen-Chi L. Chang,2 Yulia Maxuitenko,1 Veronica Ramirez-Alcantara,1 Kristy Berry,1 Luciana Madeira da Silva,1 Bing Zhu,1 Tyler Mattox,1 Michael R. Boyd,3 Adam B. Keeton,1 Margie L. Clapper,2 Harry S. Cooper,2 Gary A. Piazza1. 1 _Mitchell Cancer Institute, Mobile, AL;_ 2 _Fox Chase Cancer Center, Philadelphia, PA;_ 3 _ADT Pharmaceuticals, Orange Beach, AL_.

Over 90% of colorectal cancers harbor mutations in β-catenin or pathway components (e.g. APC) that stabilize β-catenin, causing nuclear translocation and constitutive Tcf-mediated transcription of genes encoding proteins essential for the proliferation and survival of tumor cells. We recently reported that the cyclic nucleotide degrading phosphodiesterase (PDE) isozyme PDE10 is overexpressed in colorectal cancers relative to normal tissue. Its expression and enzymatic activity are essential for colon tumor cell growth, as evidenced by knockdown of PDE10 expression using siRNA or inhibition of enzyme activity using known inhibitors such as PF-2545920. PDE10 inhibition in tumor cells expressing high levels of PDE10 causes increased intracellular cGMP levels to activate PKG and phosphorylate β-catenin, which induces ubiquitination and proteasomal degradation to suppress nuclear translocation and Tcf transcriptional activity. Conversely, ectopic expression of PDE10 in normal colonocytes or precancerous adenoma cells causes increased levels of β-catenin and the expression of proteins (e.g. cyclin D and survivin) essential for the proliferation and survival of tumor cells. To identify novel antitumor PDE10 inhibitors, we screened a chemically diverse library of indenes for PDE10 and tumor cell growth inhibitory activity. Following extensive chemical optimization, MCI-030 emerged as a potent and selective inhibitor of tumor cell growth. Similar to PF-2545920, but with appreciably greater potency and tumor cell selectivity, MCI-030 inhibited colon tumor cell growth by activating cGMP/PKG signaling to phosphorylate and induce β-catenin degradation. MCI-030 also inhibited colon tumor cell spheroid formation and reduced spheroid size and growth at concentrations that inhibit PDE10. Oral administration of MCI-030 significantly inhibited colon tumor formation in the Apc+/min-FCCC mouse model without discernable toxicity. Importantly, unlike PDE10 inhibitors developed to cross the blood-brain barrier for the treatment of CNS disorders, MCI-030 lacks the sedation side effects. Together, these findings support preclinical development of MCI-030 for the treatment of colorectal cancer as a novel PDE10 inhibitor capable of selectively inhibiting the growth of tumors harboring β-catenin or APC mutations. Funding provided by NCI grants R01CA131378, R01CA148817, R01CA197147, and R01CA155638.

#3865

Discovery of novel DHODH and OXPHOS inhibitors.

Matthias Versele,1 Philippe Selhorst,1 Kristine Metzger,1 Marnik Nijs,1 Hugo Klaassen,1 Amuri Kilonda,1 Damien Marchand,1 Philippe Arzel,1 Dominique Lambin,1 Jean-Christophe Vanherck,1 Arnaud Marchand,1 Patrick Chaltin,1 Cyril Corbet,2 Olivier Feron2. 1 _Centre for Drug Design and Discovery (CD3), KU Leuven, Leuven, Belgium;_ 2 _Université Catholique de Louvain (UCL), Brussels, Belgium_.

Inhibition of mitochondrial metabolism for treatment-resistant tumors has attracted renewed attention. Pharmacologic inhibition of mitochondrial oxidative phosphorylation (OXPHOS) is efficacious in preclinical models of chemo-resistant AML, glycolysis-deficient glioma, and Swi/Snf mutant lung cancer, and is associated with an apparent robust safety index (Molina et al., 2018, Nat Med 24: 1036-1046; Lissanu Deribe et al., 2018, Nat Med 24: 1047-1057). In addition, immune-suppressive cell types in the tumor micro-environment depend on OXPHOS metabolism, including CD4+ regulatory T-cells (Tregs), whereas tumor-infiltrating effector T-cells instead rely on glycolytic metabolism (Angelin et al., 2017, Cell Metab. 25: 1282-1293).

Here, we have used a phenotypic drug discovery approach to identify selective inhibitors of mitochondrial -but not glycolytic- tumor cell metabolism. Based on initial hits derived from a high-throughput screening campaign, a chemical series was optimized to achieve single digit nM potency (best IC50= 2 nM) in targeting OXPHOS-dependent cancer cells (grown on lactate as sole carbon source) but not glycolytic cells (grown on glucose; IC50 > 10 microM). Target deconvolution within this chemical series revealed 2 distinct mechanisms. One chemical subseries are direct inhibitors of the mitochondrial enzyme, dihydroorotate dehydrogenase (DHODH). Consistent with previous reports on DHODH inhibitors, these compounds potently impair AML cell proliferation (best IC50= 10nM) through induction of myeloid cell differentiation -a trait that can be rescued by providing exogenous uridine to the cell cultures (IC50 > 10 microM). A second chemically distinct subset of compounds inhibit mitochondrial OXPHOS, but does not inhibit DHODH. As expected, these compounds selectively target OXPHOS-dependent cancer cell lines, and display a robust selectivity window (determined in glycolysis-dependent cell lines). Moreover, these inhibitors do not affect a mixed-lymphocyte reaction (MLR) assay. In-depth metabolomic profiling in cancer cells fueled by glucose, lactate or glutamine, the precise molecular target of these compounds, and further in vivo characterization of these compounds will be presented. Finally, the unique opportunity to simultaneously inhibit both DHODH and OXPHOS using dual inhibitors will be evaluated in chemo-resistant AML models.

#3866

Decursin inhibits tumor growth, migration and invasion through the regulation of CXCR7 expression in gastric cancer.

Solbi Kim,1 Nayoung Kim,1 Mina Joo,1 HueungJin Jeon,1 Hyewon Ryu,2 Hyo Jin Lee3. 1 _Chungnam National University, Department of Medical Science, Daejeon, Republic of Korea;_ 2 _Chungnam National University, Daejeon, Republic of Korea;_ 3 _Chungnam National University, Department of Internal Medicine and Cancer Research Institute, Daejeon, Republic of Korea_.

Background: CXCR7 is overexpressed in a variety of cancer and is known to promote cell migration, invasion and metastasis. Therefore, inhibition of CXCR7 can prevent tumor progression. In this study, we investigated the role of decursin on the CXCR7 expression and its antitumor effects in gastric cancer.

Methods: CXCR7-overexpressing gastric cell lines were established, and the expression of membrane CXCR7 was confirmed by flow cytometry and immunocytochemistry. Cell viability and anoikis assay were performed using CCK-8 reagent. The effect of decursin on cell migration and invasion was tested using transwell system, and the cleavage of PARP and caspase-3 was performed in western blotting and was confirmed by flow cytometry using apoptosis detection kit. The inhibition of tumor growth by decursin was also demonstrated in vivo by measuring tumor growth using subcutaneous injection mouse model.

Results: CXCR7 overexpression significantly increased the invasion and migration, and tumor growth in gastric cancer cells. Administration of decursin decreased the membrane expression of CXCR7 in a concentration dependent manner and significantly inhibited tumor progression by suppressing cell migration, invasion and proliferation. In addition, decursin treatment impeded in vivo tumor growth in BALB/c nude mice. CXCR7-induced STAT3 and c-Myc activation were inhibited by decursin, whereas pro-apoptotic pathway was activated.

Conclusion: Decursin inhibited the cell growth, migration and invasion via downregulating the membrane expression of CXCR7 in gastric cancer in vitro and suppressed tumor growth in vivo suggesting that decursin might be used as a therapeutic agent alone or in combination with other anticancer agents in gastric cancer.

#3867

Microsomal prostaglandin E synthase-1: A therapeutic target in bladder cancer.

Benjamin Leeland Woolbright, Jordan Barney, Van Schloegel Schloegel, Erika Abbott, John Arthur Taylor. _Univ. of Kansas Medical Ctr., Kansas City, KS_.

Introduction and Objective: Prostaglandin E2 (PGE2) is a lipid-derived signaling agent that promotes tumorigenesis via increasing stemness, proliferation, and angiogenesis. PGE2 levels are elevated in multiple cancers, including bladder (BCa) where they may play a pathological role. PGE2 is generated in part by cyclooxygenase enzymes (COX-1/COX-2). Clinical trials using the COX-2 specific inhibitor celecoxib to prevent progression and recurrence of non-muscle invasive BCa report hazard ratio reductions; however, celecoxib fails to significantly outperform standard of care. These studies are dose-limited by cardiotoxicity present with COX inhibition, but have suggested higher doses of celecoxib may yield significant results. Microsomal PGE2 synthase-1 (mPGES-1) is one of two terminal enzymes (downstream of COX-1/2) associated with PGE2 production. Inhibition of mPGES-1 does not affect synthesis of the prostaglandins responsible for cardiotoxicity but still reduces PGE2 levels. Using a model of carcinogen induced BCa, we have previously established mPGES1-/- have significantly smaller bladder tumors by weight, reduced pathological stage, reduced PGE-2 metabolites, and reductions in percent tumor involvement in the bladder compared to WT mice. The purpose of this study was to further understand mPGES mediated tumorigenesis using in vitro cellular models. We hypothesized pharmacological inhibition with the mPGES-1 specific inhibitor MF63 would reduce PGE2 levels leading to reduced cellular proliferation and reduced PGE2 release in vitro.

Methods: mPGES1 protein and RNA levels were measured in benign (UROtsa) or malignant BCa cell lines using western blotting and qPCR respectively. PGE2 levels were assessed by ELISA. Cell proliferation was assessed using the hexosaminidase assay and colony formation assays. Cell death was assessed using lactate dehydrogenase (LDH) release.

Results: In vitro, BCa cell lines HT-1376, RT-4 and HTB-9 significantly upregulate mPGES1 as compared to a benign urothelial cell line (UROtsa). To assess the capacity of MF63 to effect PGE2 production, we treated HT-1376 and HTB-9 cells with MF63 at increasing concentrations and assessed PGE2 levels. MF63 significantly reduced PGE2 production after 24h in both cell lines. Furthermore, pharmacological inhibition of mPGES1 with MF63 at concentrations of 10µM and above significantly reduced cellular proliferation after 48 or 72 hours using both colony formation and the hexosaminidase assay to assess proliferation. Finally, concentrations of 50µM MF63 robustly induced cell death as measured by LDH release.

Conclusions: These data show that inhibition of mPGES1 in vitro with the inhibitor MF63 results in decreases in PGE2 levels and reduced cellular proliferation, likely due in part to cell death induction. mPGES1 inhibitors are expected to have improved safety profiles and given our results, may have therapeutic potential in BCa.

#3868

FND 4b decreases proliferation and increases apoptosis of triple negative breast cancer through AMPK activation.

Jeremy Johnson, Piotr Rychahou, Vitaliy M. Sviripa, Heidi L. Weiss, David Watt, B. Mark Evers. _University of Kentucky, Lexington, KY_.

Introduction. Triple negative breast cancer (TNBC), which comprises 15-20% of all breast cancers, is the deadliest and most aggressive subtype. TNBC affects a younger patient population and is associated with an increased risk of distant recurrence, higher rates of metastases, earlier time to recurrence, and worse prognosis after recurrence. AMP-activated protein kinase (AMPK) is a major cellular energy regulator that has recently been implicated in cancer progression. AMPK can act as a tumor suppressor by decreasing anabolism and inducing cell cycle arrest. FND 4b is a novel AMPK activator that has been shown to inhibit growth and induce apoptosis in colon cancer, but its effects have not been studied in breast cancer. The purpose of this project is to test the effects of FND 4b on AMPK activation, cellular proliferation, and apoptosis in several types of breast cancer—with a particular emphasis on TNBC.

Methods. (i) To assess signaling pathways, estrogen-receptor positive (ER+) breast cancer cells (MCF-7 and T-47D), TNBC cells (MDA-MB-231 and HCC-1806), and breast cancer stem cells were treated with FND 4b (0, 1, 2.5, 5, 10, and 20 µM) for 24 h. Immunoblot analysis assessed phosphorylated and total forms of AMPK, acetyl CoA carboxylase (ACC), and ribosomal protein S6 as well as cyclin D1 and cleaved PARP. (ii) Growth assays with sulforhodamine B (SRB) were performed by treating ER+ breast cancer cells and TNBC cells with FND 4b (0, 2.5, 5, 10, and 20 µM) for 72 h and then quantifying cellular protein content. Proliferation was also assessed by treating all cell lines with FND 4b (0 or 5 µM) for 72 h and then counting viable cells. (iii) Cell death was assessed by treating ER+ breast cancer cells and TNBC cells with FND 4b (0, 2.5, 5, and 10 µM) for 72 h and then quantifying fragmented DNA in the cytoplasm by ELISA.

Results. (i) FND 4b treatment for 24 h increased AMPK activation with concomitant decreases in ACC activity, phosphorylated S6, and cyclin D1 in ER+ breast cancer, TNBC, and breast cancer stem cells. (ii) FND 4b treatment for 72 h (5 µM) decreased proliferation in all breast cancer cells, while dose-dependent decreases in growth were also observed in ER+ breast cancer and TNBC cells. (iii) Increases in apoptosis were found in ER+ breast cancer and one TNBC cell line with FND 4b treatment for 72 h.

Conclusions. Our findings indicate that FND 4b can decrease proliferation for a variety of breast cancers by activating AMPK—with notable effects on TNBC. The reductions in growth were mediated through decreases in fatty acid synthesis (ACC), mTOR signaling (S6), and cell cycle flux (cyclin D1). ER+ breast cancer cells were more susceptible to FND 4b-induced apoptosis, but MDA-MB-231 cells still underwent apoptosis with higher dose FND 4b treatment. Further development of FND compounds—with extensions to animal studies—could result in a novel therapeutic agent in the treatment of TNBC.

#3869

The reversible LSD1 inhibitor SP-2509 promotes anti-tumor immunity in small cell carcinoma of the ovary-hypercalcemic type (SCCOHT).

Raffaella Soldi, Alexis Weston, Trason Thode, Rhonda Lewis, Mohan Kaadige, Hariprasad Vankayalapati, William Hendricks, Sunil Sharma. _TGEN, Phoenix, AZ_.

Purpose: Small cell carcinoma of the ovary-hypercalcemic type (SCCOHT) is a rare ovarian cancer of young women characterized by SWI/SNF ATPase SMARCA4 (BRG1) genetic inactivation alongside epigenetic silencing of the homolog ATPase SMARCA2 (BRM). Because the SWI/SNF complex associates with histone modifying complexes (HMCs) containing targetable epigenetic enzymes, the lack of SWI/SNF ATPase activity is hypothesized to interrupt normal function of HMCs that result in altered epigenetic landscape. Lysine-specific histone demethylase 1 (LSD1 or KDM1) regulates the chromatin landscape and gene expression by removing specific methyl groups from methylated proteins including histone H3. It associates with a number of transcriptional regulatory complexes including SWI/SNF and acts in a context-dependent manner. We and others have found that LSD1 is among the most highly expressed histone modifiers in SCCOHT. SCCOHT cell lines are very sensitive to the reversible LSD1 inhibitor SP-2509 compared to other epigenetic agents like EZH2 and bromodomain inhibitors, suggesting that LSD1 dependence is a defining feature of these SWI/SNF-deficient ovarian cancers. Recently it has been shown that LSD1 inhibition stimulates an INF-dependent anti-tumor immunity and overcomes resistance to checkpoint blockade through induction of Endogenous Retroviruses (ERVs) in breast cancer. Interestingly, SCCOHT have been shown to exhibit an immune-active tumor microenvironment with PD-L1 expression in both tumor and stromal cells that strongly correlated with T-cell infiltration (TIL). In this study we investigate the ability of SP-2509 to promote anti-tumor immunity and T-cell infiltration in SCCOHT.

Experimental Design: SCCOHT cell line BIN67, COV434 and SCCOHT1 were subjected to RT-PCR to evaluate ERVs induction and INF expression in response to SP-2509 treatment. SCCOHT cells were also co-cultured with peripheral blood mononuclear cells (PBMCs) in a 3D platform to evaluate T-cell infiltration following SP-2509 treatment.

Results: SP-2509 promotes ERV-mediated immune response in SCCOHT cell lines. Additionally, SP-2509 stimulates T-cell infiltration in SCCOHT in a spheroid platform.

Conclusion: Our data suggest that the reversible LSD1 inhibitor SP-2509 stimulates an INF-dependent anti-tumor immunity in SCCOHT cells in vitro and in 3D platform. Given that LSD-1 is a potential therapeutic target in SWI/SNF mutated SCCOHT, the combination of SP-2509 and PD-1 inhibitor represents a plausible combination to induce or augment immunogenic responses in these tumors.

#3870

The addition of CB-839 to temozolomide significantly reduces glioma aspartate and glutamate in an IDH mutated patient derived glioma xenograft model.

Sani Haider Kizilbash,1 Danielle M. Burgenske,1 Samuel McBrayer,2 Sandhya Devarajan,1 Shiv K. Gupta,1 Taro Hitosugi,1 Lihong He,1 Mark A. Schroeder,1 Brett L. Carlson,1 Marina Gelman,3 Charles A. Kunos,4 Joel M. Reid,1 Alex A. Adjei,1 Jann N. Sarkaria1. 1 _Mayo Clinic, Rochester, MN;_ 2 _Dana-Farber Cancer Institute, Boston, MA;_ 3 _Calithera Biosciences, South San Francisco, CA;_ 4 _National Cancer Institute, Bethesda, MD_.

Background: IDH mutated gliomas are critically dependent on glutaminase for glutamate biosynthesis. CB-839 is a novel glutaminase-1 inhibitor which has demonstrated efficacy in both genetically engineered and patient derived IDH mutated glioma cells, especially in combination with radiation. These preclinical studies evaluate the pharmacodynamic and pharmacokinetic impact of combining CB-839 with TMZ in patient derived xenograft (PDX) glioma models.

Methods: GBM164 is a PDX model derived from an IDH1 mutant, 1p/19q non-codeleted glioma. D- and L-2HG levels in untreated GBM164 tumors were compared to GBM6 tumors (IDH wildtype glioma PDX) by GC/MS. Athymic nude mice bearing GBM164 flank tumors were treated with CB-839 (200 mg/kg PO BID x 9 doses) and/or temozolomide (50 mg/kg PO daily x 5 doses) and sacrificed four hours after the last dose to harvest plasma, normal brain and flank tumor. Tumor metabolomics were assessed by GC/MS. DNA damage signaling in tumors was assessed by Western blotting for KAP1 / Chk1 / Chk2 phosphorylation and H2AX / Rad51. Tumor, plasma and brain pharmacokinetics were assessed by LC/MS/MS.

Results: Total 2HG levels in GBM164 were 18-fold higher than GBM6. CB-839 monotherapy reduced tumor glutamate (18% reduction, p = 0.15) and aspartate (30% reduction, p = 0.06) when compared to vehicle, however these changes did not reach statistical significance. The combination of CB-839 and TMZ more significantly reduced both tumor glutamate (30% reduction, p = 0.03) and aspartate (34% reduction, p < 0.001) when compared to TMZ monotherapy. CB-839 did not significantly increase DNA damage compared to vehicle, and the combination of CB-839 and TMZ did not significantly increase DNA damage compared to TMZ monotherapy. The mean tumor: plasma ratios of CB-839 concentrations were 0.68 and 0.58 in mice treated with CB-839 monotherapy and CB-839/TMZ, respectively. The mean brain: plasma ratios of CB-839 concentrations were 0.07 and 0.12 in mice treated with CB-839 monotherapy and CB-839/TMZ, respectively.

Conclusion: The addition of CB-839 to TMZ significantly reduces glioma aspartate and glutamate in an IDH1 mutant PDX glioma model, without any impact on DNA damage. Survival studies are in progress to assess the efficacy of CB-839 when used in combination with RT and/or TMZ.

#3871

A novel TACC3 inhibitor as an anti-cancer agent in breast cancer.

Ozge Akbulut,1 Deniz Lengerli,2 Ozge Saatci,3 Elif Duman,4 Urartu Seker,4 Burcu Caliskan,2 Erden Banoglu,2 Ozgur Sahin3. 1 _Bilkent University, Ankara, Turkey;_ 2 _Gazi University, Ankara, Turkey;_ 3 _University of South Carolina, Columbia, SC;_ 4 _Institute of Materials Science and Nanotechnology, Ankara, Turkey_.

Transforming Acidic Coiled-Coil Containing Protein 3 (TACC3) is a microtubule-associated protein that is localized on mitotic spindles and ensures proper chromosomal segregation and microtubule stability. TACC3 is frequently amplified or mutated, and was demonstrated to be a prognostic factor in a broad spectrum of cancers which makes it a highly attractive therapeutic target. TACC3 knockdown was demonstrated to cause mitotic spindle defects and prevent proper mitotic progression causing apoptotic cell death. Inhibition of TACC3 with the currently available small molecule inhibitors, KHS101 and SPL-B, has been shown to reduce the growth of glioblastoma xenografts, and suppress tumor growth in ovarian cancer xenografts, respectively. However, none of these TACC3 inhibitors is being tested in clinics potentially due to low systemic stability or high IC50 (inhibitory concentration 50) values. Here, by combining rational drug design and screening approaches, we aimed to identify a novel and more potent TACC3 inhibitor that is effective in in vitro and in vivo systems and that can be used as a mitotic blocker in breast cancer (BC). A library of test compounds was generated by replacing certain functional groups of already available TACC3 inhibitors with their isosteric equivalents to improve potency and drug-like properties. Then, the generated compounds were tested in vitro in terms of growth inhibition in a panel of breast cancer cell lines of different breast cancer subtypes. The compounds were also compared based on their effect on the cellular processes that TACC3 is involved in, and were further compared with genomic TACC3 inhibition by siRNAs. Finally, the most promising agent was tested in vivo in terms of its anti-tumorigenic effects. BO-264 was identified as the most potent TACC3 inhibitor that effectively kills breast cancer cells of different subtypes when administered at nanomolar dose. Moreover, it demonstrated superior inhibitory effects on mitotic progression, DNA repair, and cellular viability as compared to currently available inhibitors. Oral administration of BO-264 suppressed tumor growth in breast cancer xenografts at a dose that caused no apparent toxicity. Specificity of BO-264 to TACC3 protein was tested by kinome profiling and further validated by state-of-art binding assays. Currently, we are testing the pharmacokinetics and pharmacodynamics properties and organ toxicity of our inhibitor and performing the detailed molecular characterization of BO-264-mediated anti-tumor effects. Overall, our preclinical findings suggest that our novel compound (BO-264) is potentially a specific TACC3 inhibitor, causing growth inhibition in breast cancer cell lines and xenograft models by inducing mitotic arrest, DNA damage and apoptosis. Considering the critical importance of TACC3 as a cancer biomarker and driver of disease progression, BO-264 has great potential to be used as a novel therapeutic strategy in BC.

#3872

**Targeting multi-functional ERK-protein complexes** in vivo **.**

Tamer S. Kaoud,1 William H. Johnson,1 Nancy D. Ebelt,1 Andrea Piserchio,2 Diana Zamora-Olivares,1 Sabrina V. Ravenstein,1 Jacey R. Pridgen,1 Ramakrishna Edupuganti,1 Rachel Sammons,1 Micael Cano,1 Mangalika Warthaka,1 Pengyu Ren,1 Eric V. Anslyn,1 Kenneth Y. Tsai,3 Ranajeet Ghose,2 Kevin N. Dalby1. 1 _The University of Texas at Austin, Austin, TX;_ 2 _The City College of New York, New York, NY;_ 3 _Moffitt Cancer Center, Tampa, FL_.

Mutations in pathways that enhance the activity of ERK1 and ERK2 are frequently present in human cancers, reflecting their important roles in tumor initiation, and progression. This notion is reinforced by observations in BRAF V600E mutant melanoma where the majority of the mechanisms of resistance to FDA-approved combination therapies targeting BRAF and MEK involve reactivation of ERK1 and ERK2. Recently, the direct targeting of the ERK enzymes using ATP-competitive inhibitors has emerged as an attractive strategy to overcome acquired resistance to current combination therapies. The ERK enzymes employ unique mechanisms of molecular recognition to engage protein components of the MAPK pathway. Here we report the potent targeting of an ERK-protein docking interaction by a small molecule thiotriazole, which abrogates ERK signaling in vivo. The thiotriazole binds covalently to a highly conserved cysteine residue within the D-recruitment site of ERK1/2 with more than a 100-fold discrimination over other MAPKs (e.g. JNK1/2, p38MAPKs and ERK5). Treatment of various BRAF-inhibitor naive or inhibitor-resistant melanoma cell lines expressing BRAF V600E with the thiotriazole for 2 hours induces dose-dependent inhibition of ERK activation and downstream signaling. Inhibition is maintained for up to 5 hours following thiotriazole washout and induces apoptosis and growth inhibition. Treatment of mice carrying a BRAFV600E A375 melanoma xenograft with the thiotriazole blocked tumor growth. Transient expression of a mutant form of ERK2, which is resistant to the thiotriazole, promotes survival of A375 and HEK 293 cells treated with thiotriazole. This study lays the foundation for developing a new modality for the treatment of solid tumors driven by excessive ERK signaling.

#3873

CHOP10/TRB3/Akt signaling regulates ER stress apoptosis in colon cancer cells treated with prostamide-J.

Hussam Albassam, Daniel A. Ladin, Rukiyah Van Dross. _East Carolina University, Greenville, NC_.

The endoplasmic reticulum (ER) is a cellular organelle that is primarily responsible for oxidative protein folding. When the protein folding load in cells exceeds the protein folding capacity, ER stress occurs. The ER stress pathway is a prominent regulator of cancer cell death and as a result, ER stress inducers are being exploited pharmacologically. Cytotoxic ER stress is typically regulated by the transcription factor, C/EBP homologous protein 10 (CHOP10). Transcriptional products of CHOP10 include pro-apoptotic genes, such as tribbles-related protein 3 (TRB3), death receptor-5 (DR5), and ER oxidoreductase 1α (ERO1α). Our previous data showed that apoptosis mediated by 15deoxy, Δ12,14 prostamide J2 (15dPMJ2) occurred in an ER stress-dependent manner. However, the signaling pathway that regulates 15dPMJ2 mediated ER stress apoptosis has not been identified. Therefore, the goal of this study is to determine the role of CHOP10 and its transcriptional targets in ER stress apoptosis that is induced by 15dPMJ2 in colon cancer cells. According to our data, 15dPMJ2 was 3-fold more effective in inducing apoptosis in the human colon cancer cell line, HCT116, compared to the non-tumorigenic colon cell line, FHC. The induction of apoptosis in HCT116 cells occurred coincident with a significant increase in CHOP10 protein expression. In addition, blockade of the ER stress pathway with 4-phenylbuterate (PBA) or salubrinal prevented apoptosis indicating that cell death is reliant upon ER stress. To determine the role of CHOP10 in this process, CRISPR/Cas9 CHOP10 knockout HCT116 cells (CHOP10-KO-HCT116) were generated. As anticipated, 15dPMJ2 increased CHOP10 expression in wt-HCT116 cells but not in CHOP10-KO-HCT116 cells. Furthermore, 15dPMJ2 increased the expression of ERO1α, DR5, TRB3, and it induced apoptosis in wt-HCT116 cells, but not in CHOP10-KO cells. Interestingly, the induction of TRB3, but not DR5 or ERO1α, was completely inhibited in cells devoid of CHOP10 expression. Therefore, the activity of protein kinase B (PKB)/Akt, a known TRB3 target, was investigated. CHOP10 stimulation by 15dPMJ2 inactivated Akt in wt-HCT116 cells, but not in CHOP10-KO cells. These findings suggest the importance of CHOP10, TRB3, and Akt in the control of the ER stress-mediated apoptosis in response to 15dPMJ2 treatment. Thus, 15dPMJ2 is a potential chemotherapeutic agent that inhibits PKB/Akt survival signaling by upregulating the CHOP10/TRB3 pathway to prevent colon cancer growth.

#3874

Efficacy of the Wnt/Beta-Catenin pathway inhibitor RXC004 in genetically-defined models of cancer.

Simon Woodcock, Inder Bhamra, Cliff Jones, Alicia Edmenson Cook, Catherine Eagle, Caroline Phillips. _Redx Pharma, Cheshire, United Kingdom_.

Background: RXC004, a potent and selective inhibitor of the Wnt/β-Catenin pathway regulator porcupine, is being investigated in a safety and tolerability study in cancer patients with solid tumours (NCT03447470). Wnt pathway alterations, including loss-of-function RNF43 mutations and RSPO gene fusions, result in higher levels of the Wnt receptor Fzd on the cell surface, increasing Wnt-ligand dependent signalling. These alterations are implicated in colorectal, gastric, pancreatic and biliary cancer. We present pre-clinical data on the direct tumour-targeting effects of RXC004 in genetically-defined models of cancer.

Methods: RXC004 in vitro effects on sensitive (RNF43 mutant or RSPO fusion) or insensitive (APC/β-Catenin mutant) colorectal and pancreatic tumour cells lines were assessed against a panel of potential Wnt target genes measured using qPCR. To test if these effects translated in vivo, RXC004 was evaluated in several RNF43 mutant or RSPO fusion tumour line efficacy, PK and PD studies. A range of oral doses and schedules of RXC004 were tested for either 7 or 26 days, for PK/PD or efficacy measurements respectively. Efficacy was measured by tumour volume/weight, RXC004 PK was measured in plasma and tumour, and PD effects were measured by tumour qPCR and histological methods.

Results: In sensitive cell lines, RXC004 inhibited expression of the Wnt pathway negative feedback genes Axin2 and RNF43. Decreases in c-Myc correlated with the anti-proliferative effects of RXC004. Additionally, MMP7 and CD44 were downregulated, whilst Mucin gene expression increased. In contrast, RXC004 had no anti-proliferative effects on APC mutant colorectal cancer cells in vitro, and no modulation of these Wnt target genes. In vivo, RXC004 demonstrated significant efficacy and PD effects in multiple RNF43 mutant and RSPO fusion xenograft models. Focusing on an RSPO fusion model, RXC004 reduced tumour volume in a dose-dependent manner at 1.5 and 5 mg/kg QD, and 1.5mg/kg BID. In addition, 1.5mg/kg BID of RXC004 scheduled 5 days on, 2 days off also gave significant efficacy. PK analysis was consistent with the predicted dose response, showing excellent absorption and tumour distribution. PD analysis confirmed the RXC004-induced Wnt target gene changes identified in vitro translated in vivo, whilst histology showed a clear reduction in the proliferation marker Ki67 and concomitant increase in the staining of Mucins, indicative of mucinous differentiation of the tumour cells.

Conclusion: Taken together, these data demonstrate that RXC004 monotherapy has the potential to benefit patients with tumours bearing RNF43 mutations or RSPO fusions, supporting a genetically-defined patient selection strategy for ongoing RXC004 clinical studies.

#3875

A novel STAT3 decoy inhibitor prevents lung cancer development.

Christian Njatcha, Jiayi Wang, Huiyu Li, Mariya Farooqui, Jill M. Siegfried. _University of Minnesota, Minneapolis, MN_.

Exposure to tobacco carcinogens such as NNK leads to mutations in genes like K-RAS in premalignant lesions, which progress to adenocarcinoma. Early events in carcinogenesis are also characterized by inflammation. Central to these processes, STAT3 activation has pleotropic pro-tumor effects on proliferation, angiogenesis, and immune evasion. We previously showed that a "decoy" cyclic STAT3 oligonucleotide (CS3D) produced robust antitumor effects in human lung adenocarcinoma models. CS3D mimics the STAT3 DNA response element and acts as a "decoy" to which STAT3 binds, blocking its transcriptional activity. Effects of CS3D were compared to an inactive mutant construct unable to bind to STAT3 (CS3M). Therapeutic effects in human lung cancer xenografts suggested that CS3D might also impair lung cancer development. To test this, mice were exposed to NNK (3mg/kg) for 4 weeks (wks) followed by a 1-wk washout phase to model ex-smokers. Mice were then treated 3 times per wk with CS3D or CS3M (5mg/kg, IV) for 8 wks. Tumor burden and size were then assessed at 8 and 20 wks after the end of treatment. To evaluate effect of CS3D on the development of tumors with K-RAS activating mutations (since STAT3 knockout has been reported to promote KRAS mutant lung cancer), we isolated DNA from tumors by laser capture microdissection and assessed the incidence of G12D activating mutations. CS3D caused a reduction in preneoplasias (P<0.001), tumor size (P<.05), and tumor number (P<.05) by 30%, 50%, and 40% respectively, compared to CS3M. Immunohistochemistry (IHC) showed a tumor microenvironment (TME) that is less primed for tumor progression. Markers of angiogenesis (VEGF) and cell cycle progression (MYC) were downregulated by CS3D (P<.05). Strikingly, IHC analysis at 20 wks after the final treatment revealed a substantial decrease in STAT3 and NF-kB activation (P<.05) suggesting a long-lasting effect of CS3D. However, we detected higher levels of IL-6 at 8 wks and 20 wks after the end of treatment which could indicate a rescuing feedback loop. IHC also showed a 2.6 fold increase in macrophage infiltration at 8 wks. Sequencing of DNA from residual tumors collected at 20 wks (n = 10) showed no significant difference in tumors with K-RAS mutation between CS3D (33.3%) and CS3M (27.3%), showing that pharmacological inhibition of STAT3 with CS3D doesn't select for K-RAS mutant tumors. To examine immunomodulation, mice were exposed to 4 wks of NNK with a 1-wk washout phase and 1 wk of CS3D or CS3M treatment. Lungs were collected for flow cytometry analysis to define the immune cell profile. CS3D favored an antitumor immune response over an immunosuppressive TME by increasing the number of lung M1 macrophages while decreasing M2 macrophages and MDSCs. Ratio of M1 to M2 was 4:1 with CS3D and 1:3 with CS3M. This is the first evidence that CS3D has chemopreventive properties in lung cancer without promoting KRAS-driven tumors, and modulating the TME is involved in the preventive effect.

#3876

Thymoquinone induces the expression of clock genes in breast cancer cells.

Aliaa A. Alamoudi,1 Hanan A. Bashmail,1 Ghada M. Ajabnoor,1 Hani Choudhry,1 Mohammed A. Hassan,1 Alia M. Aldahlawi,1 Ahmed A. Al-abd2. 1 _Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia;_ 2 _Medical Division, National Research Centre, Giza, Egypt, Giza, Egypt_.

Thymoquinone is naturally occurring bioactive compound which showed cumulative preclinical evidence of anticancer effects. However, its exact anti-cancer activity remains to be elusive and is currently being studied. In this study, we assessed the role of thymoquinone as a potential therapy in human breast cancer cell lines in addition to its role in altering molecular clock genes expression, which in turn has been associated with tumorgenesis. Sulfa-Rhodamine-B assay (SRB) assay was used to evaluate the cytotoxic effect of thymoquinone in breast cancer cell lines (MCF-7 and T47D). Following 72 h of exposure, thymoquinone showed cytotoxic effects against MCF-7 and T47D with IC50's of 44.4±3 µM and 152±11 µM respectively. Further investigation showed that thymoquinone showed some affect on the cell cycle distribution in both cell lines. It significantly increased the cell population in S-phase in MCF-7cells (P<0.05), while causing a significant G1 phase arrest in T47D cells (p<0.01). Interestingly, treatment of both cell lines with non-cytotoxic concentrations of thymoquinone resulted in a significant increased expression of clock genes, measured by reverse transcription–quantitative PCR (RT-qPCR). Thymoquinone appeared to increase the expression of BMAL-1 (1.4±0.1 fold), CLOCK (3.4±0.9 fold), PER-1(5.6±0.5 fold), CRY-1 (4.4±0.4 fold), CRY-2(2.2±0.6 fold) in MCF-7 cells. Similarly, thymoquinone increased the expression of BMAL-1 (1.4±0.3 fold), CLOCK (1.4±0.3 fold), PER-1(1.4±0.3 fold), CRY-1 (1.5±0.4 fold), and CRY-2 (1.8±0.1 fold) in T47D cells . In conclusion, thymoquinone possesses a potential in combating breast cancer alone. Despite the promising anti-proliferative activity of thymoquinone against breast cancer cells, the better understanding of these effects is needed to provide a novel approach for the treatment of breast cancer as a disease. Here, we postulate that the drug could be modulating clock genes expression, which ultimately can affect key molecules in cell cycle and cell proliferation. Further studies are needed to confirm this and to elucidate the exact underlying mechanism.

#3877

IND-enabling characterization of DRD2/3 imipridone antagonist ONC206 for oncology.

Varun V. Prabhu,1 Abed Rahman Kawakibi,1 Neel Madhukar,2 Mathew J. Garnett,3 Ultan McDermott,3 Cyril H. Benes,4 Lakshmi Anantharaman,5 Neil Charter,5 Sean Deacon,5 Alexander VanEngelenburg,6 Joseph B. Rucker,7 Benjamin J. Doranz,7 Jessica Rusert,8 Robert Wechsler-Reya,8 Olivier Elemento,2 Martin Stogniew,1 Wolfgang Oster,1 Sharon DeMorrow,9 R. Benjamin Free,10 David R. Sibley,10 Joshua E. Allen1. 1 _Oncoceutics, Inc, Philadelphia, PA;_ 2 _Weill Cornell Medicine, New York, NY;_ 3 _Wellcome Trust Sanger Institute, United Kingdom;_ 4 _Massachusetts General Hospital, Boston, MA;_ 5 _Eurofins DiscoverX, Fremont, CA;_ 6 _HistoTox Labs, Inc, Boulder, CO;_ 7 _Integral Molecular, Inc, Philadelphia, PA;_ 8 _Sanford Burnham-Prebys Medical Discovery Institute, San Diego, CA;_ 9 _Texas A &M College of Medicine, Temple, TX; _10 _NINDS/NIH, Rockville, MD_.

Dopamine receptor D2 (DRD2) is a G protein-coupled receptor that is overexpressed and critical for survival in several cancers. ONC201, an imipridone small molecule, is a DRD2/3 antagonist in Phase II advanced cancer clinical trials with a compelling safety and efficacy profile. We evaluated the binding target, anti-tumor activity, biodistribution and safety of ONC206, a chemical derivative of ONC201 with the same imipridone core structure. GPCR profiling with β-Arrestin recruitment revealed that ONC206 selectively antagonizes dopamine receptors DRD2 and DRD3. ONC206 exhibited a Ki of ~320nM for DRD2 with complete specificity across human GPCRs and complete DRD2 antagonism. Schild analyses of ONC206 in cAMP and β-Arrestin recruitment assays revealed hallmarks of non-competitive DRD2 antagonism, unlike antipsychotics but similar to ONC201. Shotgun mutagenesis across DRD2 identified 7 residues critical for ONC206-mediated antagonism at orthosteric and allosteric sites. While 6 mutated residues were also critical for ONC201-mediated antagonism, the impact and magnitude of different mutants varied between the two compounds and one of the allosteric residues was unique to ONC206. In vitro profiling of ONC206 in >1000 GDSC cancer cell lines demonstrated broad nanomolar efficacy (GI50 <78-889nM). TCGA and tissue microarrays analyses revealed that malignant DRD2 expression was highest in pheochromocytoma, high grade gliomas, neuroblastoma, medulloblastoma, Ewing's sarcoma and cholangiocarcinoma. Accordingly, ONC206 demonstrated nanomolar in vitro sensitivity in these tumor types. Similar to ONC201, a DRD2+/DRD5- RNA expression signature in the GDSC panel predicted significantly enhanced ONC206 sensitivity. ONC206 reduced the viability of normal human fibroblasts at micromolar doses (GI50 > 5µM), suggesting a wide therapeutic window. Robust inhibition of tumor growth without body weight loss was observed in HuCCT1 cholangiocarcinoma and MHH-ES-1 Ewing's sarcoma subcutaneous xenografts with 50-100 mg/kg oral ONC206 weekly or every 2 weeks. Biodistribution studies in Sprague-Dawley rats revealed a ~12 µM plasma Cmax with a half-life of ~6 hours upon a single oral dose of 50 mg/kg. Additionally, 5-10 fold higher ONC206 concentrations were observed in adrenal gland, bile duct, brain and bone marrow relative to plasma concentrations. GLP toxicology studies with weekly oral ONC206 in Sprague-Dawley rats and beagle dogs at doses above or equivalent to efficacious doses revealed no dose-limiting toxicities. In both species, observations at the highest dose were mild and reversible. The no observed adverse event level (NOAEL) was ≥ 16.7 mg/kg in dogs and ≥ 50 mg/kg in rats, which both correspond to a human dose of approximately 500 mg assuming standard allometric scaling. These results provide rationale for a 50 mg starting ONC206 dose in dose escalation clinical trials in patients with DRD2-dysregulated tumors.

#3878

**Newly identified p53-pathway restoring small molecule, CB002 analog #4 induces apoptosis and appears non-toxic** in vivo **.**

Liz J. Hernandez Borrero,1 Avital Lev,2 Lanlan Zhou,2 David T. Dicker,2 Wafik S. El-Deiry2. 1 _The Pennsylvania State University, Hershey, PA;_ 2 _Fox Chase Cancer Center, Philadelphia, PA_.

The transcription factor encoding TP53 tumor suppressor gene is mutated in more than half of human cancers. TP53 accounts for therapy resistance and poor patient survival in colorectal and other cancers. Upon cellular stress, p53 protein determines cell fate through the regulation of cell senescence, cell cycle arrest, and apoptosis pathways. Distinct from other tumor-suppressors, mutated p53 can acquire a gain-of-function activity, leading to chemotherapy resistance and a more aggressive tumor phenotype. Direct therapeutic targeting of transcription factors is challenging especially when reactivation is needed; thus, to date, there are no FDA approved drugs for p53 functional restoration in tumors harboring p53 mutations. To overcome this, we have screened for small molecules capable of restoring wild-type p53 pathway signaling rather than direct physical restoration of its function. We identified a small molecule, CB002 analog #4 capable of restoring the p53 pathway in HCT116, SW480 and DLD-1 colorectal cancer cell lines as indicated by the increased expression of p53-target genes NOXA, p21 and DR5, and apoptotic markers upon treatment. We found that analog #4 enhances ethidium homodimer staining of dead cells and decreases the number of positive Ki67 staining cells in colorectal cancer patient organoids. Sub-G1 analysis demonstrated that analog #4 does not induce apoptosis in normal human fibroblasts and preliminary in vivo studies indicate that it can be safely administered to mice. We are investigating the in vivo efficacy of analog #4 in addition to its mechanism of action through micro-array experiments. Our data supports the concept that small molecules can restore the p53-pathway as a therapeutic strategy that can be translated into a clinical setting.

#3879

Inhibition of CXCR4 driven colorectal cancer progression by Nef-M1 peptide.

Venkat R. Katkoori,1 Zeynoire Anderson,1 Upender Manne,2 Harvey Bumpers1. 1 _Michigan State Univ., East Lansing, MI;_ 2 _University of Alabama at Birmingham, Birmingham, AL_.

Introduction: The Nef-M1 peptide (Nef-M1) competes effectively with the natural ligand of CXCR4, SDF-1α, and exhibits anti-tumorigenic activity in human colorectal cancer (CRC). However, the molecular mechanisms of action of Nef-M1 on CRC remain unclear. In this study, we evaluated the inhibition of CXCR4 driven molecular signaling mechanisms that is involved in CRC progression.

Experimental Design: Cell line derived xenografts (CDX), patient derived explant (PDE) and in vitro cell based models were used to examine the mechanisms of action of Nef-M1. The severe combined immunodeficient (SCID) mice with tumor were treated intraperitoneally with either Nef-M1 or scrambled amino acids sequence Nef-M1 (sNef-M1) that is the control. Preparation of CRC tissue, explant complete media, and cultures and treatment for 48 hours were performed for the PDE study. Sections from tumors were evaluated by immunostaining (IHC) for signaling proteins thus, implicating CRC progression. Western blot (WB) analyses were also performed on lysates of both cell lines and tumors to assess the effect of Nef-M1 on the signaling pathways that promote CRC progression.

Results: The present study revealed that PDE recapitulate multiple biological features of the disease and these were found to be very similar to the corresponding original CRC. CXCR4 overexpressing CRC cells displayed activation of CXCR4/CREB signaling as demonstrated by an increased activation of CREB, and expression of B-Myb and APOBEC3B (A3B). IHC analysis for Ub-H2B, a stem cell signaling protein that correlates with advanced disease and metastasis indicated that Nef-M1 treated CDX or PDE tumors had low expression of Ub-H2B. However, sNef-M1 treated tumors had high expression of Ub-H2B. WB analyses indicated that Nef-M1 not only suppressed the expression of Ub-H2B, but also significantly suppressed the expression of A3B. Cells expressing CXCR4 became susceptible to Nef-M1-induced inhibition of Akt, mTOR activation and Ub-H2B expression.

Conclusions: These results indicate that Nef-M1 suppresses CXCR4 driven activation of CXCR4/CREB signaling and the expression of Ub-H2B. Therefore, Nef-M1 inhibition of these signaling pathways may be a promising therapeutic strategy for CRC. This work was supported by NIH/NCI Workforce Diversity Grant R21-CA171251.

#3880

In vivo **activities of TP-2846: a novel tetracycline antileukemia agent.**

Xiao-Yi Xiao,1 Cuixiang Sun,1 Jian Zhou,1 Noriaki Tatsuta,1 Joseph Newman,1 Douglas White,2 Barbara Hibner,2 Arijit Chakravarty,2 Jacques Dumas1. 1 _Tetraphase Pharmaceuticals, Watertown, MA;_ 2 _Fractal Therapeutics, Inc., Cambridge, MA_.

The pharmacokinetics (PK) of TP-2846 was studied in mice, rats, and monkeys via IP, PO, and IV administration. In a mouse PK study with TP-2846 dosed IP at 10 mg/kg, Cmax, T1/2, and AUC0-inf values of 5340 ng/mL, 6.81 h, and 5944 ng∙h/mL were observed, respectively. In an IV PK study in mice, TP-2846 was dosed at 1, 3, and 6 mg/kg, and the various PK parameters at each respective dose are: Cmax = 1142, 5881, 8518 ng/mL; T1/2 = 6.15, 5.15, 6.92 h; AUC0-inf = 704, 2174, 4205 ng∙h/mL. In a rat PK study, TP-2846 was dosed at 5 mg/kg IV and PO. The various PK parameters by each dosing route are: Cmax = 7180, 4.39 ng/mL; T1/2 = 9.64, 9.41 h; AUC0-inf = 9390, 21.0 ng∙h/mL. In a PK study in monkeys, TP-2846 was dosed at 1, 2, 4, and 6 mg/kg IV. Key PK parameters at each respective dose are: Cmax = 734, 1071, 1835, 2715 ng/mL; T1/2 = 9.97, 12.5, 15.7, 22.4 h; AUC0-inf = 4128, 8252, 19179, 30871 ng∙h/mL. TP-2846 demonstrated good half-lives and exposures across all three species. Low oral bioavailability was observed when TP-2846 was dosed in rats. Dose linearity of plasma exposure was shown when varying levels of TP-2846 was dosed in mice and monkeys.

The in vivo efficacy of TP-2846 was evaluated in a number of leukemia mouse xenograft models including MV4-11, MOLT-4, CCRF-CEM, HL-60, KG-1, Kasumi-1, and K-562. TP-2846 demonstrated potent in vivo efficacy in MV4-11 with a Tumor Growth Inhibition (TGI) value of 111.4% (p = 0.001) when dosed IP at 12.5 mg/kg, QD, 4 days on/6 days off, for two cycles, while cytarabine and tigecycline had a TGI value of 23.5% and -49.2%, respectively, at their reported optimal dosing regimens. TP-2846 was also highly efficacious in HL-60 xenograft models with a TGI value of 109.1% (p < 0.001) when dosed IP at 20 mg/kg, QDx3, for two weeks. In an IV study in HL-60, TP-2846 demonstrated dose-responding antitumor efficacy with TGI values of 5.38% (p = 1.000), 34.73% (p = 0.643), 103.50% (p = 0.015), and 104.87% (p = 0.013), respectively, when dosed at 1, 2, 4, and 6 mg/kg, QDx3, for two weeks. To understand the PK parameters that drive the efficacy of TP-2846, studies with varying dosing levels and dosing schedules were conducted in the HL-60 model. Subsequent PK/PD modeling using the resulting data sets suggested that AUC is the primary efficacy driver for TP-2846 in the xenograft model.

In summary, TP-2846 displayed favorable PK profiles across multiple species and demonstrated potent, dose-responding, AUC-driven efficacy in several mouse leukemia xenograft models. These data sets, combined with other desirable in vitro, in vivo, and chemical-physical properties, support the continued pre-clinical development of TP-2846 as a new antileukemia agent.

### Pharmacokinetics and Pharmacodynamics / Preclinical Toxicology

#3881

Relating tumor drug concentrations to target effect with semi physiologic PK-PD modeling in drug development: An application using a novel dCK inhibitor.

Soumya Poddar,1 Mina Nikanjam,2 Edmund Capparelli, Thuc Le, Liu Wei, Caius Radu. 1 _UCLA, Los Angeles, CA;_ 2 _UCSD, San Diego, CA_.

Introduction: Deoxycytidine kinase (dCK) is essential for DNA synthesis through salvage pathways and DI-87 is a novel dCK inhibitor in preclinical development. This study used PET imaging to measure dCK inhibition and pharmacokinetic-pharmacodynamic (PK-PD) modeling to relate tumor drug levels to tissue with dCK activity and tumor shrinkage.

Methods: Oral DI-87 was administered to NSG mice with plasma and tumor PK assessed over 24 hrs by mass spectrometry. NSG mice with CEM tumors were administered varying doses of DI-87 followed by the [18F]CFA PET probe and PET imaging. NSG mice with CEM tumors were administered thymidine and DI-87 concurrently and tumor growth monitored. PK-PD modeling was conducted with NONMEM (v. 7.3).

Results: DI-87 plasma concentrations peaked at 3 hr; tumor peak concentrations occurred later. Tumor concentrations were less than one third of plasma concentrations. Full dCK inhibition by PET imaging occurred between 10 and 25 mg/kg and decreasing doses led to faster recovery of activity. Full recovery of enzyme activity occurred by 36 hrs with full inhibition being maintained at the 12 hr time point at the 25 mg/kg dose. Maximal growth inhibition occurred with full dCK inhibition over the dosing range (25 mg/kg BID) when DI-87 was given in combination therapy with thymidine; thus increased doses led to more persistent dCK inhibition with predictable increased growth inhibition.

Conclusions: Combining PET probes with PK-PD modeling led to an optimized dosing schedule for combination therapy with DI-87.

#3882

Exposure-toxicity analysis of unbound regorafenib and its active metabolites by dose escalation strategy with low starting dose in patients with colorectal cancer.

Hironaga Satake,1 Takeshi Suzuki,2 Chiyo K. Imamura,2 Yasutaka Sukawa,2 Toshiki Masuishi,3 Yosuke Kumekawa,4 Shinsuke Funakoshi,5 Masahito Kotaka,6 Yoshiki Horie,7 Sadayuki Kawai,8 Hiroyuki Okuda,9 Tetsuji Terazawa,10 Chihiro Kondoh,11 Ken Kato,12 Kenichi Yoshimura,13 Hideki Ishikawa,14 Yasuo Hamamoto,2 Narikazu Boku,12 Hiromasa Takaishi,2 Takanori Kanai2. 1 _Kobe City Medical Center General Hospital, Japan;_ 2 _Keio University School of Medicine, Japan;_ 3 _Aichi Cancer Center Hospital, Japan;_ 4 _Saitama Cancer Center, Japan;_ 5 _Tokyo Saiseikai Central Hospital, Japan;_ 6 _Sano Hospital, Japan;_ 7 _St. Marianna University School of Medicine, Japan;_ 8 _Shizuoka Cancer Center, Japan;_ 9 _Keiyukai Sapporo Hospital, Japan;_ 10 _Osaka Medical College, Japan;_ 11 _Toranomon Hospital, Japan;_ 12 _National Cancer Center Hospital, Japan;_ 13 _Kanazawa University, Japan;_ 14 _Kyoto Prefectural University of Medicine, Japan_.

Background: As regorafenib (REG) and its active metabolites are extensively bound to serum proteins, their unbound exposure are considered to be more relevant to pharmacological and toxicological responses than total (unbound plus bound) exposure. We thus investigated the relationships between toxicity and serum unbound concentrations of REG and active metabolites M-2 and M-5 in patients with colorectal cancer.

Patients and Methods: REG was administered orally once daily for the first 21 days of each 28-day cycle. The dose was started at 120 mg/day, and escalated to standard dose of 160 mg/day on day 15 of the first cycle in patients who experienced neither hand-foot skin reaction nor grade ≥2 REG-related adverse events (AEs) without dose reduction or interruption until day 14. Serum concentrations of REG, M-2 and M-5 were evaluated on days 8, 15, and 22 of the first cycle in patients without interruption of REG treatment until each sampling point. Unbound fraction was obtained by equilibrium dialysis, and concentrations were determined by the UPLC-MS/MS method. AEs were graded according to CTCAE ver. 4.0.

Results: Among all 68 enrolled patients, 57 patients on day 8, 42 patients on day 15, and 23 patients on day 22 were assessable. On day 8, the median total concentrations of REG, M-2 and M-5 in serum were 6801 nM (range, 2487-17621), 2596 nM (321-12107), and 834 nM (49-12315), respectively. Unbound fraction of REG, M-2 and M-5 varied from 0.019-0.441 %, 0.000-0.477 % and 0.041-2.381 %, respectively, showing no association with levels of serum albumin (28-50 mg/mL) which is a major binding protein. Serum concentrations of total REG or sum of total REG, M-2 and M-5 (total SUM) on day 8 were not related with maximum grade (grade ≤2 vs. grade ≥3) of AEs (excluding laboratory abnormalities) in the first cycle. On the other hand, higher serum concentrations of unbound REG on day 8 tended to associate with severity of maximum grade of AEs in the first cycle (P=0.0711), and concentrations of sum of unbound REG, M-2 and M-5 (unbound SUM) on day 8 were significantly correlated with maximum grade of AEs in the first cycle (P=0.0345). In addition, concentrations of total REG and unbound REG on day 8 in six patients whose dose were escalated to 160 mg/day on day 15 were significantly lower than those in 50 patients whose dose were not escalated (total, 3978 vs. 7005 nM in median, P=0.0270; unbound, 2834 vs. 7244 pM in median, P=0.0455). There were also significant association between concentrations of unbound REG or unbound SUM on day 15 and severity of REG-related AEs (grade 1 vs. grade ≥2) on day 15 (P=0.0495, P=0.0126, respectively).

Conclusion: Unbound exposure was well correlated with toxicity in patients treated with REG. Exposure of sum of REG, M-2 and M-5 was associated with toxicity more than exposure of REG alone. Serum albumin levels didn't affect unbound fraction of REG, M-2 and M-5.

#3883

High unbound plasma concentration of M-2, an active metabolite, is associated with shorter survival in patients with metastatic colorectal cancer who received regorafenib.

Yutaro Kubota,1 Ken-ichi Fujita,1 Takehiro Takahashi,2 Yu Sunakawa,3 Hiroo Ishida,1 Kazuyuki Hamada,1 Wataru Ichikawa,2 Takuya Tsunoda,1 Yusuke Masuo,4 Yukio Kato,4 Yasutsuna Sasaki1. 1 _Showa University School of Medicine, Tokyo, Japan;_ 2 _Showa University Fujigaoka Hospital, Kanagawa, Japan;_ 3 _St. Marianna University School of Medicine, Kanagawa, Japan;_ 4 _Kanazawa University, Ishikawa, Japan_.

Background and Aims: Regorafenib is an oral multikinase inhibitor which showed survival advantage in the later-line chemotherapy in patients with metastatic colorectal cancer (mCRC). This anticancer drug is sequentially metabolized to pharmacologically active metabolites M-2 and M-5 by the hepatic CYP3A. A randomized phase II trial has recently revealed that initial dose of 80 mg/day followed by dose escalations (40 mg/week) was superior to the standard dosing strategy (160 mg/day) with respect to overall survival (OS), resulting in the NCCN guideline recommendation for this dose escalation strategy, although scientific index for rational dose estimation is still limited. The aim of this prospective study was to clarify the association of pharmacokinetics (PK) with clinical outcome of regorafenib in patients with mCRC.

Methods: Consecutive patients with mCRC administered regorafenib at Showa University Hospital were prospectively enrolled in this study. Patients received regorafenib 160 mg once daily for the first 3 weeks of each 4 weeks cycle. Blood samples for PK analysis were obtained on day1 (0-48 h) skipping the second dose on day2, and if possible day15 of treatment (0-24 h). Plasma concentrations of the regorafenib, M-2, and M-5 were analyzed by HPLC or LC-MS/MS. Unbound fraction of these compounds were measured by equilibrium dialysis method.

Results: A total of 36 patients were enrolled between October 2013 and June 2017. The median progression free survival (PFS) was 1.9 months, and the median OS was 6.4 months. Area under the total (protein bound plus unbound) plasma concentration-time curve (AUC) on day1 was the highest in regorafenib (88.5 ± 53.8 µM·h), followed by M-2 and M-5 (37.0 ± 32.9 and 5.9 ± 6.5 µM·h, respectively), whereas AUC calculated based on unbound plasma concentration (uAUC) of M-2 was the highest (9.8 ± 12.0 nM·h), followed by M-5 and regorafenib (2.5 ± 4.3 and 1.8 ± 1.7 nM·h, respectively), reflecting ~10-fold higher unbound fraction in M-2 and M-5 than regorafenib. uAUC of M-2 measured on day15 was also higher than those of M-5 and regorafenib. Patients with the M-2 uAUC of 9.8 nM·h or higher had significantly shorter PFS than those with the uAUC of lower than 9.8 nM·h (30 vs. 74 days, p=0.0092). The highest uAUC value of M-2 may be compatible with its association with PFS, implying that unbound form of M-2 could be primarily important for clinical response.

Conclusion: We thus found that uAUC of active M-2 was the highest as compared to those of regorafenib and M-5, which was associated with shorter PFS.

#3884

Advancing Glioblastoma drug regimen development to support combination therapy through integrated PKPD modeling and simulation-based predictions.

Saugat Adhikari,1 Harlan E. Shannon,2 Karen E. Pollok,2 Robert E. Stratford2. 1 _Purdue University, Indianapolis, IN;_ 2 _Indiana University, Indianapolis, IN_.

Despite advancements in therapies, such as surgery, irradiation (IR) and chemotherapy, outcome for patients suffering from glioblastoma remains fatal; the median survival rate is only about 15 months. Even with novel therapeutic targets, networks and signaling pathways being discovered, monotherapy with such agents targeting such pathways has been disappointing in clinical trials. Development of tumor resistance, particularly to temozolomide (TMZ), creates a substantial clinical challenge.

The primary focus of our work is to rationally develop novel combination therapies and dose regimens that mitigate resistance development. Specifically, our aim is to combine TMZ with small molecule inhibitors that are either currently in clinical trials or are approved drugs for other cancer types, and which target the disease at various resistance signaling pathways that are induced in response to TMZ monotherapy. To accomplish this objective, an integrated PKPD modeling approach is used. The approach is largely based on the work of Cardilin, et al, 2018. A PK model for each drug is first defined. This is subsequently linked to a PD model description of tumor growth dynamics in the presence of a single drug or combinations of drugs. A key outcome of these combined PKPD models are tumor static concentration (TSC) curves of dual or triple combination drug regimens that identify combination drug exposures predicted to arrest tumor growth. This approach has been applied to TMZ in combination with abemaciclib (a dual CDK4/6 small molecule inhibitor) based on data from a published study evaluating abemaciclib efficacy in combination with TMZ in a glioblastoma xenograft model (Raub, et al, 2015).

A PKPD model was developed to predict tumor growth kinetics for TMZ and abemaciclib monotherapy, as well as combination therapy. Population PK models in immune deficient NSG mice for temozolomide and abemaciclib were developed based on data obtained from original and published studies. Subsequently, the PK model was linked to tumor volume data obtained from U87-MG GBM subcutaneous xenografts, again using both original data as well as data from the Raub, et al, 2015 study. Model parameters quantifying tumor volume dynamics were precisely estimated (coefficient of variation < 30%). The developed PKPD model was used to calculate plasma concentrations of TMZ and abemaciclib that would arrest tumor growth, as well as combinations of concentrations of the two drugs that would accomplish the same endpoint. This so-called TSC curve for the TMZ and abemaciclib combination pair evidenced an additive effect of the two agents when administered together. These results will be presented. In addition, results from on-going PKPD studies of TMZ in combination with two other small molecule inhibitors, RG7388, an MDM2 inhibitor, and GDC0068, an AKT inhibitor, will also be presented.

#3885

Evaluation of pharmacokinetics of proxalutamide, a novel androgen receptor antagonist, in treatment for mCRPC patients via CTC enumeration and AR biomarker analysis.

Lincy Chu,1 Phoebe Zhang,2 Amanda Anderson,1 Mark Landers,1 Yipeng Wang,1 Karl Zhou2. 1 _Epic Sciences, San Diego, CA;_ 2 _Suzhou Kintor Pharmaceuticals, Suzhou, China_.

Introduction: Proxalutamide (code name: GT-0918) is a novel androgen receptor (AR) blocker and has shown efficacy in the treatment of castration resistant prostate cancer. Proxalutamide binds to AR and inhibits androgen-induced receptor activation. A Phase 1/2 clinical trial (ClinicalTrials.gov Identifier: NCT02826772) was conducted to identify dose-limiting toxicities (DLTs) and to assess safety, tolerability, and PK of GT0918 in pts with mCRPC who have progressed on standard of care and experimental therapies in the United States. To better understand patient responses and pharmacokinetics of GT0918, we sought to examine CTC enumeration, AR-N term expression, and AR-V7 nuclear expression using Epic CTC platform for patients treated with GT0918.

Methods: A total of 18 metastatic castrate resistant prostate cancer (mCRPC) patients were recruited under either dose escalation or expansion studies and blood samples were collected at 3 time points: C1D1, C3D1, and C6D1/EOS. Patients with histologically confirmed mCRPC who had progressed after both hormonal therapy (abiraterone or enzalutamide) and chemotherapy (e.g., docetaxel) were eligible. Dosages of 300, 400, and 500 mg were evaluated in this study. CTCs were detected using the Epic Sciences platform and assessed for full length AR status using an AR N-terminus specific antibody and for nuclear localized AR-V7 truncation. AR-V7 nuclear localization was confirmed by trained technician.

Results: 18/18 (100%) of patients had detectable CTCs, with 12/18 (67%) patients having AR-N term+ CTCs. 7/12 (58%) of AR-N term+ patients were AR-V7 positive. Reduction of CTC/mL, AR+ CTC/mL and % of AR+ CTC in follow up draws were observed in the whole cohort, with 500mg dose group showing more reduction than others. Sample accrual and patient follow up is ongoing and the correlation of CTC, AR, and AR-V7 status with the clinical outcomes will be investigated once the data becomes mature.

Conclusions: In this clinical trial, we evaluated the pharmokinetic effects of GT0918 on CTC enumeration and Androgen Receptor CTC status. GT0918 might serve as a potential alternative for late stage mCRPC patients who have developed resistance to both hormone and chemotherapy treatments. The role of CTC as PK marker or patient selection device is ongoing.

#3886

Impact of nanosuspension of CZ48, a lactone stabilized camptothecin, on the biliary excretion and enterohepatic recycling of its active moiety, camptothecin.

Diana S-L Chow,1 Yu Jin Kim,2 Dong Dong,3 Xiaohui Li4. 1 _Univ. of Houston, Houston, TX;_ 2 _University of Arizona, Tucson, AZ;_ 3 _Jinan University Medical School, Guangzhou, China;_ 4 _Food and Drug Admnistration, Rockville, MD_.

The biliary excretions of CZ48, a lactone-stabilized camptothecin (CPT), and CPT were evaluated in bile duct-cannulated SD rats after a single oral administration of CZ48 in two different types of formulations, co-solvent solution (CoS, control, in the mixture of DMSO, PEG 400, and EtOH (2:2:1, v/v/v) at 10 mg/ml) and nanosuspension (NS with stabilizers of TW 80 and F-108, sustained release of CZ48). Enterohepatic recycling was further characterized by collecting bile intermittently or continuously to modulate the recycling system in the rats. The results demonstrated that CZ48 and CPT were secreted into the bile as their parent forms with the dominant secretion of CPT as compared to CZ48. The AUCs0-12h of CPT in bile were 213.45 ± 232.09 and 250.40 ± 134.83 ng/ml*h/[mg/kg] in CoS and NS groups, respectively, which were 18.39 and 34.16 times higher than those of CZ48 (11.61 ± 7.99 ng/ml*h/[mg/kg] in the CoS group and 7.33 ± 2.22 ng/ml*h/[mg/kg] in the NS group). On the contrary, the AUCs0-12h of CZ48 in plasma were 6.10 and 6.80 times higher than those of CPT in CoS and NS groups, respectively. There was no significant impact of NS on the plasma AUCs0-12h and biliary excretions of CZ48 and CPT. Their concentrations in plasma and biliary excretions were sustained for 8 or 12 h post oral dose. Moreover, a pharmacokinetic co-model for CZ48 and CPT in plasma and bile was developed to characterize the biliary secretion and saturable process of enterohepatic recycling of CZ48 and CPT in rats.

#3887

Population pharmacokinetic/pharmacodynamic evaluation of the relationship between glasdegib exposure and safety endpoints in cancer patients.

Ana Ruiz-Garcia,1 Naveed Shaik,1 Catriona Jamieson,2 Michael Heuser,3 Geoffrey Chan4. 1 _Pfizer Clinical Pharmacology, San Diego, CA;_ 2 _Moores Cancer Center, University of California, La Jolla, CA;_ 3 _Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany;_ 4 _Pfizer Oncology, Collegeville, PA_.

Glasdegib inhibits Hedgehog signaling through Smoothened, and is in clinical development for treating acute myeloid leukemia. This exposure-response (ER) analysis estimated the association between glasdegib exposure and incidence of the cluster terms DYSGUESIA, MUSCLE SPASMS, RENAL TOXICITY, and QT INTERVAL PROLONGED as safety endpoints (D, MS, RT, and QT) in patients receiving glasdegib as monotherapy or in combination with chemotherapy and evaluated the effect of moderators in the defined ER relationship for the different cluster terms. Ordinal logistic regression was used to model the incidence of adverse events (AEs). AEs were graded from 0-4 using the National Cancer Institute Common Terminology Criteria for Adverse Events version 4.03. Base models were developed for each safety endpoint, selecting the most significant glasdegib exposure metric. Covariates in Table 1 were evaluated for inclusion in the model, with categorical covariates included if the incidence per category was >10%. Patients received single-agent glasdegib (N=70; 5-640 mg once daily [QD]); or glasdegib (N=202, 100-200 mg QD) with low-dose cytarabine (LDAC), decitabine, or daunorubicin and cytarabine. Estimated pharmacokinetic (PK) parameters were used to derive glasdegib exposure metrics and added to the safety data analysis. Glasdegib exposure appeared to have a statistically significant association with cluster term safety endpoints D, MS, RT, and QT. The variables age on MS; and BWT and BCCL on RT help explain the AE Grade distribution. At the 100 mg QD clinical dose, the predicted probabilities of the highest AE Grade (≥3 for MS, RT and QT, and 2 for D) were 11.3%, 6.7%, 7.7%, and 2.5% for D, MS, RT, and QT respectively. Overall, the predicted probability of developing an AE of any severity for these safety endpoints was low. Therefore, no starting dose adjustments may be required based on the observed safety profile.

Table 1. Potential Covariates Evaluated:

---

Category | Variable

Treatment | Glasdegib monotherapy, glasdegib+LDAC, glasdegib+decitabine, and glasdegib+DNR/Ara-C

Demographics | Sex (Male versus Female), Race, Age (years), BWT (kg)

Safety laboratory information at baseline: | BBIL (mg/dL), BAST (IU/L), BALT (IU/L), BALB (g/dL), BSCR (mg/dL), BCCL (mL/min), BHGB (g/dL), and BWBC (109 cells/L)

Baseline Disease Status: | BECOG PS

Ara-C=cytarabine; BALB=baseline albumin; BALT=baseline alanine transaminase; BAST=baseline aspartate transaminase; BBIL=baseline total bilirubin; BCCL=baseline creatinine clearance; BECOG=baseline Eastern Cooperative Oncology Group; BHGB=baseline hemoglobin; BSCR=baseline serum creatinine; BWBC=baseline white blood cells; BWT=baseline body weight; dL=deciliter; DNR=daunorubicin; g=gram; IU=international unit; kg=kilogram; L=liter; LDAC=low-dose cytarabine; min=minute; mg=milligram; mL=milliliter; PS=performance status.

#3888

**Intracranial evaluation of the in vivo pharmacokinetics, brain distribution, and efficacy of rucaparib in** BRCA **-mutant, triple-negative breast cancer.**

Minh Nguyen,1 Liliane Robillard,1 Thomas C. Harding,1 Jim J. Xiao,1 Andrew D. Simmons,1 Hartmut Kristeleit,2 Michelle (Mingxiang) Liao1. 1 _Clovis Oncology, Inc., Boulder, CO;_ 2 _Guy's and St Thomas' NHS Foundation Trust, London, United Kingdom_.

Background: The poly(ADP-ribose) polymerase inhibitor rucaparib is approved for use in recurrent ovarian cancer; however, there are limited data on the activity of rucaparib in patients with central nervous system (CNS) involvement. The goals of these studies were to evaluate the pharmacokinetics (PK) and pharmacodynamics (PD) of rucaparib and to correlate these results with the efficacy observed in a BRCA1-mutant xenograft model of triple-negative breast cancer (TNBC). In addition, a case report where rucaparib demonstrated clinical activity in the CNS of a patient with BRCA2-mutant breast cancer is presented.

Methods and Results: In a PK study in CD-1 mice, rucaparib demonstrated limited brain penetration after a single oral dose of 150 mg/kg, with a mean free brain-to-plasma AUC ratio (Kpuu, brain) of 0.09. The free brain Cmax of rucaparib was 8.59 ng/mL, which was comparable to its free cerebrospinal fluid Cmax (6.69 ng/mL) but significantly lower than the total Cmax measured in the brain (118 ng/mL). The antitumor efficacy of rucaparib was evaluated in orthotopic and intracranial tumor models using the BRCA1-mutant MDA-MB-436 TNBC cell line. In the orthotopic setting, rucaparib ≥50 mg/kg BID resulted in >100% tumor growth inhibition (TGI) after 28 days of dosing. Higher levels of rucaparib were observed in the tumor relative to plasma at all doses evaluated. A PK-PD analysis showed an inverse correlation between poly(ADP-ribose) and rucaparib levels in the plasma and tumor, with decreased poly(ADP-ribose) levels correlating with greater TGI. To evaluate intracranial efficacy, a luciferase labelled MDA-MB-436 cell line was employed and tumor burden was evaluated by weekly bioluminescence measurements. Rucaparib 150 mg/kg BID demonstrated efficient suppression of tumor growth after 43 days of dosing, with >100% TGI. In support of these preclinical findings, a patient with germline BRCA2-mutant metastatic breast cancer who had progressive CNS disease following whole brain radiation therapy was treated with rucaparib 600 mg PO BID for 9 months under a compassionate use program. A reduction of multiple metastatic brain lesions was observed based on MRI scans taken before and after rucaparib treatment. The patient also experienced complete resolution of neurological symptoms.

Conclusions: In vivo PK studies confirmed the limited brain penetration of rucaparib in a murine model with an intact blood-brain barrier. Nevertheless, antitumor efficacy was observed in a BRCA1-mutant intracranial TNBC mouse model, and clinical activity has been observed in a patient with germline BRCA2-mutant breast cancer and CNS involvement who was treated with rucaparib. Further studies are warranted to elucidate the activity of rucaparib in the brain.

#3889

Population pharmacokinetic/pharmacodynamic evaluation of the effect of glasdegib exposure on cardiac repolarization (QT interval) in cancer patients.

Luke Fostvedt,1 Naveed Shaik,1 Giovanni Martinelli,2 Andrew Wagner,3 Ana Ruiz-Garcia1. 1 _Pfizer Clinical Pharmacology, San Diego, CA;_ 2 _Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori, IRST IRCCS, Meldola, Italy;_ 3 _Dana-Farber Cancer Institute, Boston, MA_.

Glasdegib inhibits Hedgehog signaling through Smoothened. Glasdegib (100 mg once daily [QD]) is in clinical development as a first-line treatment for adults with acute myeloid leukemia (AML). In two dose-escalation studies, involving cancer patients with either hematologic malignancies or advanced solid tumors, instances of QT interval prolongation occurred at high dose levels. The current analysis characterizes the exposure-response (E-R) relationship between the mean heart rate corrected QT interval (QTc) and glasdegib plasma concentration using data from these two studies. The glasdegib single-agent, dose-escalation studies collected pharmacokinetic (PK) and triplicate electrocardiogram (ECG) pair measurements in patients (n=70) treated over a wide dose range (5-640 mg QD). Fridericia, Bazetts, and population-specific correction factors were examined to determine which correction factor would generate QTc values independent of the underlying heart rate. Linear mixed effects modeling was used to characterize the exposure-response (E-R) relationship between glasdegib plasma concentration and the QTc interval. Potential covariates that may be sources of variability in this E-R relationship (ie, gender, age, and study) were evaluated using a stepwise covariate modeling approach. A parametric bootstrap was performed to estimate the mean QTc change from baseline at the expected mean maximum steady-state plasma concentration (Cmax) at the 100 mg QD therapeutic dose, as well as at the supratherapeutic Cmax (a mean 40% increase observed in the presence of a strong CYP3A inhibitor with the most extreme observed individual increase of 100%). The magnitude of the QTcF change was characterized using a 95% confidence interval at the Cmax concentrations of interest. Glasdegib did not directly affect heart rate. The Fridericia and population-specific correction factors were adequate in removing the heart rate dependence of the QT interval. Based on the model, a positive relationship was established between glasdegib plasma concentration and the QTcF interval. None of the demographic characteristics explored as potential covariates had a statistically significant impact on the E-R relationship. The predicted ΔQTcF mean (upper bound of 95% CI) from baseline at the therapeutic Cmax was 5.30 (6.54) msec. At supratherapeutic concentrations (40% increase and 100% increases over therapeutic Cmax) the change from baseline was 7.42 (8.74) msec and 12.09 (14.25) msec, respectively. The potential for glasdegib exposure to affect the QTc interval was well characterized in this PK/PD analysis. The upper bound (95% CI) of the model predicted ΔQTc from baseline at the therapeutic and supratherapeutic exposures fell below the 20 msec threshold of clinical concern for oncology drugs. A subsequent Thorough QT study confirmed the findings of this analysis.

#3890

Pharmacodynamic and proteomic analysis of combined gemcitabine/nab-paclitaxel in patient-derived pancreatic cancer xenograft models.

Jin Niu,1 Xue Wang,2 Shichen Shen,3 Ju Qu,1 Donald Mager,1 Robert M. Straubinger1. 1 _University at Buffalo, Buffalo, NY;_ 2 _Roswell Park Cancer Institute, Buffalo, NY;_ 3 _New York State Center of Excellence in Bioinformatics and Life Sciences, Buffalo, NY_.

Gemcitabine (GEM) combined with albumin-bound paclitaxel nanoparticles (nab-PTX) is the first-line therapy for patients with metastatic pancreatic adenocarcinoma (PDAC). However, the in vivo mechanisms of drug action and interaction have not been investigated quantitatively for this combination. We evaluated the effects of GEM (100 mg/kg iv weekly) and nab-PTX (equivalent to 30 mg/kg PTX iv weekly), alone or combined, on tumor volume progression in 3 patient-derived xenograft (PDX) PDAC models in SCID mice. The MS1-based quantitative proteomic analysis (IonStar), which offers highly reproducible and accurate quantification with extremely low missing data (<0.5%), was conducted on Days 1, 4, and 8 after dosing to evaluate tumor pharmacodynamic responses in protein expression. Proteins were quantified based on species-specific peptides in order to identify responses of the PDAC cells (human) and tumor-associated stromal cells (murine). A population pharmacokinetic/pharmacodynamic model was developed to quantify tumor growth inhibition by the individual and combined drugs. Tumor growth kinetics were described by a composite function of exponential growth transitioning to linear growth. Treatment effects were represented by a 2nd-order, concentration-dependent parameter k that leads to cell death following delays arising from signal transduction. All 3 PDX models responded to the combination therapy, but with different sensitivities. PDX#14312 showed the least inhibition by the drugs alone (k[GEM]=13.6, k[PTX]=37.0 /day/mM), compared to the other PDXs (#18254: k[GEM]=68.1, k[PTX]=125; 18269: k[GEM]=112, k[PTX]=51.2 /day/mM). Compared to the more potent single-drug treatment for each PDX, the combination resulted in 1.33-fold greater cell death in #14312, 1.39 fold in #18269 and 1.19 fold in #18254. Model simulations showed distinct temporal profiles of cytotoxic signals in the 3 PDXs. Pathway analysis showed that the top changes mediated by the drug combination in PDX#14312 PDAC cells included extracellular matrix organization (FDR-corrected p-value = 7E-5), synthesis of nucleotide di- and triphosphates (p = 0.012), and interferon signaling (p = 0.04), whereas top changes in stroma included increased platelet activation (p = 3.8E-4) and extracellular matrix organization (p = 0.046). These processes represent potential contributors to the resistance of PDX#14312 to the drug combination. Top changes in #18269 and #18254, which were sensitive to the combination, included increased glycolysis (p = 0.038), decreased fatty acid beta-oxidation (p = 3E-5) and DNA replication (p = 0.0058) in PDAC cells, and increased complement activation (p < 1E-5) in stroma. Overall, inter-patient variability in PDAC responsiveness to GEM/nab-PTX therapy may be associated with tumor stress responses partially resembling wound healing processes.

#3891

A phase 1 study to evaluate the pharmacokinetics (PK) and safety of niraparib in Chinese patients with epithelial ovarian cancer (OC).

Jian Zhang,1 Yu Nong Gao,2 Ge Lou,3 Ru Tie Yin,4 Jian Mei Hou,5 James Yan,5 Zhi Yi Zhang,6 Xiao Hua Wu1. 1 _Shanghai Cancer Center, Fudan University, Shanghai, China;_ 2 _Beijing Cancer Hospital, Beijing University, Beijing, China;_ 3 _Harbin Medical University Cancer Hospital, Harbin, China;_ 4 _Department of Gynecology and Obstetrics, West China Second University Hospital, Sichuan University, Chengdu, China;_ 5 _Clincal Science, Zai Lab, Shanghai, China;_ 6 _Tesaro, Inc., MA_.

Purposes: To characterize the PK and safety of niraparib as a maintenance therapy in Chinese OC patients.

Methods: Eligible patients were randomized 1:1:1 to 100, 200, or 300 mg once daily cohort. Plasma samples were collected following single and multiple dosing and analyzed using a validated LC/MS/MS method. PK parameters were analyzed by standard non-compartmental approach with WinNonlin. A population PK (Pop PK) model was derived from pooled PK data of current study and two previous PK studies in Caucasian: the dose escalation and expansion study (PN001), and the ENGOT-OV16/NOVA sub-study. Non-linear mixed effect modeling was done with NONEM®.

Results: 36 patients were randomized and included in the PK and safety analysis set. Niraparib was rapidly absorbed after dosing with tmax~3h. The exposure of niraparib was dose proportional, while other PK parameters such as t1/2, accumulation ratio were dose independent (Table 1). The POP-PK analysis model was established using PK data from 144 Caucasians and 39 Chinese. Chinese patients had a slightly higher Cmax than Caucasian patients but similar total exposure (AUC0-∞) after a single dose. Simulation by final Pop PK model indicated comparable total exposure (AUC0-∞) and Cmax between Chinese and Caucasian patients at the steady-state following multiple daily doses. Treatment emergent AEs (TEAEs) occurred in 97.2% of all patients treated with niraparib. The most frequent (>20%) TEAEs were consistent with the known safety profile of PARP inhibitors. TEAEs of Grade 3/4 reported in ≥ 5% patients included decreased platelet count (13.9%), decreased neutrophil count (11.1%), anemia (8.3%) and increased gamma-glutamyl transferase (5.6%).

Conclusions: The PK profile of niraparib in Chinese was consistent with its known profile in Caucasian. Niraparib in Chinese OC patients has a manageable safety profile, similar to that observed in global studies. (NCT03551171)

#3892

Population pharmacokinetics of siltuximab: An analysis across tumor types.

Mina Nikanjam, Jin Yang, Edmund V. Capparelli. _UCSD, La Jolla, CA_.

Introduction: Siltuximab is an IL-6 inhibiting monoclonal antibody which has been studied in solid tumors and hematologic malignancies, but no comprehensive pharmacokinetic (PK) analysis has been conducted to determine the impact of disease on siltuximab disposition.

Methods: Siltuximab pharmacokinetic data from 7 Phase I and II clinical trials included healthy subjects, Castleman's disease, ovarian cancer, non-Hodgkin's lymphoma, multiple myeloma, smoldering multiple myeloma, chronic lymphocytic leukemia, renal cell carcinoma, and solid tumors with KRAS mutations. Siltuximab was most commonly dosed at 11 or 15 mg/kg (range 1 - 20 mg/kg) with 70% of patients receiving multiple doses. Plasma samples were obtained 0.33 h - 27 weeks after dosing with siltuximab concentrations determined using a validated ELISA assay. The PK analysis was performed using nonlinear mixed effects modeling (NONMEM 7.3) and included data from 465 subjects (7,761 samples).

Results: Siltuximab concentration data was best fit with a two-compartment model. Healthy volunteer status (HV), Castleman's disease (CD), albumin (ALB), and ALT were independent predictors of clearance (CL), while ALB, HV, serum creatinine (SCR), weight (WT), and smoldering multiple myeloma predicted volume of distribution (Vdss). There was no impact of dose level on CL or Vdss. In the final PK model CL was described as:

CL (mL/hr) = 8.93 x (ALB/4.1)-0.843 x (ALT/17)-0.0964 x 1.24 (if HV) x 0.765 (if CV);

between subject variability was 41.0%. The final model was used to simulate a dose of 11 mg/kg for 1000 subjects with the median (IQR) exposure (AUC) and CL as follows: | |

|

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

|

CL (mL/hr) | AUC (mcg*day/mL) | Half-lifeβ (days)

Healthy volunteers | 6.61 (4.99 – 8.63) | 4,855.8 (3,717.4 - 6,426.3) | 24.5 (19.2 – 33. 6)

Castleman's Disease | 11.11 (8.48 - 14.36) | 2,884.3 (2,233.7 - 3,782.5) | 19.1 (14.6 - 25.0)

Other tumor types | 9.13 (6.93 - 11.52) | 3,514.5 (2,786.2 - 4,627.0) | 22.2 (17.5 - 30.5)

Conclusions: This combined population PK analysis demonstrates that clearance can differ with disease type consistent with previously published variations in IL-6 activity. These population differences along with significant between subject variability suggest that clinical and disease factors influence siltuximab disposition and may need to be considered in dosing requirements.

#3893

Melatonin-tamoxifen drug conjugates: Mechanism against breast cancer cells, and pharmacokinetic assessment in female mice.

Mahmud Hasan,1 Thomas D. Wright,1 Saugat Adhikari,2 Mohamed A. Marzouk,3 Alaina Pericoloso,1 Kelsey Murgas,1 Miranda Burgman,1 Benton P. Miller,1 Ulrike Holzgrabe,3 Darius P. Zlotos,4 Robert E. Stratford,5 Paula A. Witt-Enderby1. 1 _Duquesne Univerity, Pittsburgh, PA;_ 2 _Purdue University, Indianapolis, IN;_ 3 _University of Wuerzburg, Germany;_ 4 _The German University in Cairo, Cairo, Egypt;_ 5 _Indiana University School of Medicine, Indianapolis, IN_.

Background: Melatonin-tamoxifen drug conjugate linked with a 5-carbon chain (C5) exhibited uterus protection in female mice, which did not occur when the same doses of unlinked melatonin and tamoxifen were co-administered. Anti-cancer actions of C5 and 4 others, with varying carbon lengths (C2, C4, C9, C15), were further tested in phenotypically diverse breast cancer (BC) cell lines—MCF-7(ER+/PR+), MMC(HER2+) and triple negatives(MDA-MB-231 and BT-549) in terms of cell viability and migration. In all BC lines, C4 and C5 exhibited superior potency and efficacy compared to C2, C9, C15 and unlinked melatonin and tamoxifen or 4-OH-tamoxifen controls. In this study, C4 and C5 were further tested for their anti-cancer effects in tamoxifen-resistant MCF-7 cells(TamR-MCF-7); their pharmacokinetic parameters were assessed in vitro and in vivo; and the mechanisms underlying C4- and C5-mediated anti-cancer actions were also determined by use of inhibitors against MEK1/2, MEK5 or PI3 kinase and western blot analysis.

Results: C4 and C5 exhibited similar potency (TamR-MCF-7 IC50 = 4.22µM and 7.21µM; MCF-7 IC50 = 3.62nM and 440mM) and efficacy (TamR-MCF-7 = 83% and 81% inhibition; MCF-7= 71% and 90% inhibition) to inhibit TamR-MCF-7 cell viability as they did in MCF-7 cells. Regarding metabolism, C4 and C5 displayed similar CYP-mediated pharmacokinetic profiles as tamoxifen in both mouse and human liver microsomes where 40% loss of tamoxifen, C4 or C5 occurred by 10 min in mouse vs. 20 min in human. In vivo, the Tmax for C5 (oral) was 24h compared to 0.5h for C4 (oral) resulting in lower Cmax (8.9 ng/mL) and AUC0-24 (1.6 hr.ng/mL) for C5 compared to C4 (Cmax =102.6 ng/mL and AUC0-24 =143.7 hr.ng/mL) and lower oral bioavailability (0.11%) for C5 compared to C4 (4.52%). Regarding mechanisms, the effect of C4- or C5-mediated protein modulation was "context-specific" dependent upon BC phenotype (i.e., ER+, HER2+ or ER-/PR-/HER2-); the number of carbons linking melatonin to tamoxifen (i.e., 4 carbons for C4 or 5 carbons for C5); and the concentration (i.e., 1nM or 10µM) of C4 or C5 tested. For MCF-7, MMC and MDA-

MB-231 cells, inhibition of MEK1/2, MEK5 and PI3K enhanced but did not block C4's or C5's anti-cancer actions; while for BT-549 cells, Bix02189 blocked C5's effects. Also, in MCF-7 cells, pERK1/2, NF-κB and Runx2 were significantly modulated by C4 or C5; for MMCs, C4 and C5 modulated NF-κB and β1-integrin expression; for MDA-MB-231, MEK1/2 and MEK5 played a more central role possibly through co-modulation of NF-κB; and for BT-549, C5-mediated effects on pERK5 or PI3K-dependent regulation of pERK1/2 may underlie its anti-cancer actions.

Conclusions: Melatonin-tamoxifen drug conjugates (C4 and C5) may be superior to tamoxifen therapy alone in a variety of cancers, including TamR BC.

#3894

**Determination of the efficacious Entrectinib exposures required for pathway inhibition and anti-tumor activity in a subcutaneous and intracranial** TPM3-NTRK1 **mutant tumor model.**

Cecile C. de la Cruz,1 Thomas Hunsaker,2 Faye Vazvaei,3 Dragomir Draganov,3 Li Yu,3 Mark Merchant2. 1 _Genentech, South San Franciso, CA;_ 2 _Genentech, South San Francisco, CA;_ 3 _Roche, New York, NY_.

Entrectinib is a selective and brain penetrant inhibitor with potency against TRKA, TRKB, TRKC, ROS1 and ALK currently in clinical development for the treatment of tumors harboring NTRK or ROS1 gene fusions. In multiple non-clinical models harboring NTRK or ROS1 gene fusions, entrectinib demonstrates potent tumor growth inhibition leading to tumor regressions. Entrectinib achieves exposure in the brain across multiple species with brain-to-plasma concentration ratios of 0.4 in mice, 0.6-1.5 in rats, and 1.4-2.2 in dogs after multiple oral administration. Previously Cook, et al. demonstrated that entrectinib demonstrates anti-tumor efficacy in a BCAN-NTRK1 glioma model, however only testing higher doses. Here we sought to better understand the effective exposures of entrectinib required for pathway inhibition and anti-tumor efficacy through dose-ranging efficacy, pharmacokinetics (PK) and pharmacodynamics (PD) studies using the TPM3-NTRK1 KM12-Luciferase (KM12-Luc) tumor model inoculated subcutaneously or intracranially. In the subcutaneous setting, entrectinib treatment results in significant dose-dependent inhibition of TRKA pathway signaling for up to 12 hours and anti-tumor activity with doses of 5 mg/kg (dosed orally (PO), every day (QD)) and above resulting in strong pathway suppression and tumor regressions. PK analysis demonstrated that an exposure of AUC24h of ≥20 µM.hr was associated with potent TRKA pathway inhibition and tumor growth inhibition. In the intracranial setting, entrectinib similarly demonstrated a dose-dependent effect on inhibition of tumor bioluminescence and enhanced animal survival. Pathway inhibition was observed at all doses of entrectinib with significant activity observed at doses of 5 mg/kg, PO, QD and above. In contrast to tumors inoculated subcutaneously, entrectinib doses of 15 mg/kg, PO, twice daily (BID) or 30 mg/kg, PO, QD were required to strongly inhibit anti-tumor efficacy with the highest dose of 60 mg/kg, PO, BID achieving full suppression of intracranial tumor growth. These data help to establish a model connecting entrectinib drug exposure in circulation and in the brain to pathway suppression and anti-tumor efficacy in an NTRK gene fusion tumor model, which support clinical use of entrectinib in patients with brain NTRK or ROS1 gene fusion tumors.

#3895

A phase I-II pharmacokinetic drug-drug interaction evaluation of oral palbociclib in combination with vemurafenib in patients suffering metastatic melanoma with BRAF V600 mutated and CDKN2A loss & expression of Rb.

Samuel Huguet,1 Thierry Lesimple,2 Arthur Geraud,1 Mouna Amini-Adle,3 Caroline Dutriaux,4 Laetitia Da Meda,5 Florence Capelle,5 Zineb Ghrieb,5 Samia Mourah,6 Olivier Madar,1 Didier Bouton,7 Mathieu Resche-Rigon,8 Celest Lebbe,5 Keyvan Rezai1. 1 _Inst. Curie, Saint Cloud, France;_ 2 _Centre Eugene Marquis, Rennes, France;_ 3 _Institut de cancerologie des Hospices Civils de Lyon, Lyon, France;_ 4 _Hopital Saint Andre CHU Bordeaux, Bordeaux, France;_ 5 _Hopital Saint Louis, Paris, France;_ 6 _GH Saint Louis Lariboisière, FW, Paris, France;_ 7 _APHP DRCI, Paris, France;_ 8 _Université Paris-Diderot, Paris, France_.

Background: A novel combination of vemurafenib (VM), a selectif inhibitor of BRAF V600 mutated protein + oral palbociclib (Palbo) a highly selective reversible oral inhibitor of cyclin-dependent kinases (CDK) 4 and 6, could provide a synergistic antitumor activity in patients with metastatic melanoma harbouring BRAF V600 mutation and CDKN2A loss. VM has been shown to be an inductor of CYP3A4 which is also mainly involved in Palbo metabolism.

In this study we investigated the potential pharmacokinetic (PK) drug-drug interactions (DDI) related to the association of VM and Palbo.

Methods: metastatic melanoma harbouring BRAF V600 mutation and CDKN2A loss patients were treated with a 14 days on followed by 7 days off dosing schedule of Palbo + continuous bid dosing of VM. Dose levels [DL, Palbo (mg/day)/VM (mg/bid)] ranged from 25/720 to 200/960. For PK analysis, 7 time point samples were collected on D1C1, D21C1, and D7C2, for Palbo and VM assays. Plasma concentrations of Palbo and VM, were measured using ultra high performance liquid chromatography (UHPLC) coupled with tandem mass spectrometry validated methods.

Population PK (PPK) was modeled using a non linear mixed effect model program (Monolix version 2018r) by computing the maximum likelihood estimator of the parameters without any approximation of the model (no linearization). The following parameters were calculated absorption constant (Ka); apparent distribution volume (V/F); apparent clearance (CL/F)

Results: A total of 18 patients were treated by the combination of Palbo + VM. VM and palbo plasma concentrations were measured in 236 and 275 blood samples respectively. A one-compartment open model with linear elimination adequately described Palbo concentration-time courses. The inter-individual variabilities (ISV) could be well estimated for all stuctural parameters. The PPK parameters obtained for the structural model were: Ka = 0.791 h-1, CL/F=76.0L/h, V1/F=2830 L. Albuminemia had a significant impact on palbo CL. A one-compartment model adequately fitted the VM plasma concentration-time data. The PPK parameters were CL/F=0.133 L/h, V/F=83.6 L and Ka has been fixed at 0.2 h-1. Body weight (BW) was the best size descriptor when VM CL and V terms were normalized to a mean BW of 70 Kg according to an allometric scaling rule.

Conclusions: The PPK modeling satisfactorily described the plasma Plabo and VM time-concentration curves in patients. The main covariate effect was related to BW and albuminemia. There is no significant evidence for drug-drug PK interaction between Palbo and VM. PK-PD modeling will be performed.

#3896

**Distinct pharmacokinetics, tissue distribution and CNS penetration of ABI-009 (** nab **-Sirolimus).**

Shihe Hou,1 Anita Schmid,2 Neil Desai2. 1 _Aadi Bioscience, Inc., Pacific Palisades, CA;_ 2 _Aadi Bioscience, Inc., Millington, NJ_.

Background: Activation of the mTOR signaling pathway is frequently observed in multiple types of cancer. The poor solubility and low oral bioavailability of the mTOR inhibitor sirolimus limit its development as an anticancer therapy. ABI-009 (nab-sirolimus) is an injectable nanoparticle form of human albumin-bound sirolimus developed with a proprietary nanoparticle albumin-bound (nab®) technology. Currently, numerous phase 1 and 2 clinical studies are ongoing with ABI-009, including a registrational phase 2 study for the treatment of malignant perivascular epithelioid cell carcinoma (PEComa), and various oncology (bladder cancer, soft-tissue sarcomas, neuroendocrine tumors, colorectal cancer, glioblastoma, and various childhood cancers) as well as in non-oncology indications (pulmonary arterial hypertension (PAH), pediatric epilepsy, and mitochondrial diseases). Several nonclinical studies were conducted to evaluate pharmacokinetics, tissue distribution and in particular, CNS penetration of ABI-009.

Methods: Rats were intravenously (IV) administered with a single dose of ABI-009 at 1.7, 9.5, and 17 mg/kg. Blood and organs were collected at 2, 8, 24, 72, and 120 hrs to analyze for sirolimus concentrations.

Results: There is a generally linear dose response with sirolimus concentrations in blood, heart, lung, liver, and pancreas following a single dose of IV ABI-009. Sirolimus concentrations in blood and well-perfused organs had high peaks at earlier time points but dropped quickly with time, with the highest concentrations observed in lung. In contrast, sirolimus concentrations in brain rose slowly at first, but maintained at a steady level over time. After 5 days, sirolimus level in the brain was similar to other organs, suggesting substantial and prolonged drug distribution to the brain with ABI-009. Organ/blood ratios of most organs increased with time, with the brain/blood ratios increasing more significantly over time. Higher initial dose of ABI-009 resulted in increased brain/blood ratios.

Conclusions: ABI-009 administered IV demonstrated a distinct PK profile compared with oral mTOR inhibitors. The PK of ABI-009 is characterized by higher Cmax and AUC, and rapid tissue distribution. Combined with efficient tumor penetration observed in a xenograft study, results of this study support the potential use of ABI-009 for the treatment of cancers in different organ sites, including lung, brain, liver, and pancreas. These findings also warrant further clinical studies in non-oncology indications including PAH and disorders involving the CNS.

#3897

Stability and safety evaluation of STRO-002, a site-specific anti-folate receptor alpha antibody-drug conjugate for the potential treatment of ovarian and endometrial cancers.

Willy Solis, Venita De Almeida, Cristina Abrahams, Xiaofan Li, Tyler Heibeck, Maureen Bruhns, Adam Galan, Heidi Hoffman, Robert Kiss, Trevor Hallam, Mark Lupher. _Sutro Biopharma, South San Francisco, CA_.

Antibody-drug conjugates (ADCs) constitute an expanding class of therapeutic molecules in preclinical and clinical development for multiple oncology indications. Folate receptor alpha (FolRα) is a glycosylphosphatidylinositol-linked cell-surface glycoprotein that is overexpressed in ovarian, endometrial, lung, and triple negative breast cancers. In contrast, FolRα is minimally expressed on normal tissues, thus, making it an amenable ADC target. STRO-002, a site-specific ADC, is comprised of 3-aminophenyl hemiasterlin cytotoxic drug (SC209) conjugated to an aglycosylated anti-FolRα IgG1 antibody through a protease-cleavable linker at two engineered non-natural amino acids in each heavy chain (DAR ~4).

In vitro and in vivo stability studies, showed that STRO-002 is highly stable in circulation, with the warhead SC209 predominantly accumulating in tumor tissue. Toxicology and toxicokinetic studies were conducted to determine the safety profiles for STRO-002 and its metabolite SC209 in cynomolgous monkeys and rats, respectively. In single-dose toxicology studies in rats, SC209 was tolerated up to 0.6 mg/kg with the main toxicity findings predominantly found in hematopoietic/lymphoid tissue, consistent with tubulin targeting chemotherapeutic drugs. In a tissue cross-reactivity study with normal human tissues, STRO-002-specific membranous staining was detected in Fallopian tubes, kidney (tubules), and placenta (trophoblasts). In a definitive safety study in cynomolgus monkeys, pharmacologically relevant model for toxicity testing, STRO-002 was tolerated at up to 9 mg/kg when intravenously administered on days 1 and 22 followed by a 4-week observation period. The most prominent STRO-002-related antigen-independent toxicity finding was dose-related, minimal to marked, and cyclical neutropenia. There were no antigen-dependent toxicity findings and no evidence of ocular toxicity. Toxicokinetic analysis confirmed dose proportional STRO-002 exposures (total antibody, ADC, and SC209 catabolite) at all doses tested. In summary, STRO-002 ADC has a favorable nonclinical safety and pharmacokinetic profiles and a Phase I study of STRO-002 in patients with ovarian and endometrial cancer has been initiated.

#3898

Preclinical evaluation of DM21, a next-generation maytansinoid payload with a stable peptide linker.

Wayne Deats, Wayne Widdison, Juliet Costoplus, Bahar Matin, Nicole McBrine, Laura Bartle, Olga Ab, Richard Gregory, Jan Pinkas. _ImmunoGen, Inc., Waltham, MA_.

Antibody drug conjugates (ADCs) are designed to target surface antigen(s) expressed at higher levels on cancer cells compared to normal cells. ADCs are internalized and the antibody component is subsequently degraded in catabolic vesicles to release cytotoxic metabolites that can kill the cell. Membrane permeable metabolites can also diffuse into and kill neighboring cells (also called bystander cells) via a mechanism known as bystander killing, resulting in greater tumor cell killing. Non-antigen-mediated mechanisms of antibody and ADC uptake are also known to occur, and the maximum tolerated dose for most ADCs is driven by target independent delivery.

DM21 is a peptide-cleavable immolative maytansinoid payload that was designed to allow ADCs to efficiently release hydrophobic metabolites better than conjugates utilizing the disulfide linked maytansinoid DM4. DM21 ADCs typically have similar direct in vitro cytotoxicity as DM4 ADCs against antigen positive cells, but have much greater bystander killing activity in assays where antigen positive cells are mixed with antigen negative cells.

To evaluate the toxicity of DM21 as an ADC, it was conjugated to the non-targeting, chimeric anti-soybean trypsin inhibitor antibody (chKTI), and administered to cynomolgus monkeys. Two groups of 5 male cynomolgus monkeys received a single intravenous dose of chKTI-DM21 at dose levels of 11 and 22 mg/kg (204 and 408 µg/kg DM21), while a concurrent group of 5 male monkeys was administered the formulation buffer as a control group. Three monkeys/group were sacrificed on Day 5 (terminal necropsy) to assess acute toxicity, and the remaining two monkeys/group were sacrificed on Day 29 (recovery necropsy) to assess the recovery, persistence, or progression of any effects. Toxicity was determined based upon clinical observations, body weights, ophthalmic examinations, and clinical and anatomic pathology. Plasma and serum samples were also collected to evaluate the toxicokinetic (TK) profile.

chKTI-DM21 was well tolerated at both doses. There was no effect on body weight gain, and clinical observations were limited to reddened/darkened skin, scabbing, and soft/liquid feces. Effects noted on clinical pathology parameters included alterations in erythroid and leukocyte parameters, increased platelet counts and fibrinogen, and transient increases in ALT and AST without histopathologic correlates. The target organ noted at the terminal necropsy was the large intestine (cecum, colon, and rectum), but all findings were resolved by the recovery necropsy indicating reversibility. Toxicokinetic analysis of the samples showed that chKTI-DM21 has dose proportional exposure and apparent stability of the peptide linkage in cynomolgus plasma.

In conclusion, DM21 is a promising maytansinoid payload with a high-degree of bystander activity and a favorable toxicity profile.

#3899

Nanoparticle reduces hepatotoxicity of cancer treatment by controlled release and Kupffer cell uptake.

Yu Mi, Feifei Yang, Andrew Z. Wang. _UNC Chapel Hill, Chapel Hill, NC_.

Background: One of the major concerns in cancer treatment and clinical translation of many anti-cancer compounds has been their potential toxicities, especially hepatotoxicity. Nanoparticle (NP) holds great potential to solve the problem, as many clinical studies have not shown increased hepatotoxicity for nanotherapeutics with higher accumulation in the liver. However, there are few studies investigating how nanoparticle assists in reducing hepatotoxicity. Herein, we demonstrated that nanoformulation of known hepatotoxic anti-cancer compounds showed lower hepatotoxicity than their small molecule counterparts. Importantly, we demonstrated that slower drug release is associated with reduced hepatotoxicity. We also showed that that Kupffer cell uptake in the liver reduce nanotherapeutics' hepatotoxicity.

Methods: Two different antineoplastic drugs with high hepatotoxicity, SN-38 and wortmannin (Wtmn), were encapsulated into lipid shell-PLGA core nanoparticles separately. NP release kinetics were controlled by adjusting lipids/polymer ratio for fast (Fast-NPs), medium (Medium-NPs) and slow drug release profile (Slow-NPs), respectively. Hepatotoxicity of NPs or free drugs was analyzed by assessing serum ALT and AST levels post intravenous injection at ½ MTD in CD-1 mice. IHC staining of HO-1 and Mn-SOD was also used to show liver damage. In vitro hepatotoxicity of primary hepatocytes after Kupffer cell uptake was assessed by MTS assay and LDH assay. In vivo macrophage depletion was achieved using clodrosome.

Results: Nanoformulations of SN-38 and Wtmn showed lower ALT and AST levels than their small molecule counterparts 12 h and 24 h post treatment. The liver damage was further confirmed with IHC staining of Mn-SOD and HO-1. To address the effect of drug release kinetics on hepatotoxicity, we showed that over 90% of drugs were released within 24 h in Fast-NP. While the release rate of Slow-NPs was 66.8% for SN-38 and 67.5% for Wtmn. We showed that Slow-NPs of Wtmn resulted in minimal increase of ALT and AST levels. Compared to the baseline in untreated mice, the ALT level was 2.3-fold for Slow-NPs, 4.4-fold for Medium-NPs and 6-fold for fast-NPs; meanwhile, the AST level was 1.6-fold, 2.5-fold and 3.6-fold, respectively. To assess the effect of Kupffer cell uptake in hepatotoxicity, we found that the toxicity of the nanotherapeutics for primary hepatocytes significantly reduced after Kupffer cell uptake in vitro. We further confirmed it in vivo by depleting Kupffer cells in CD-1 mice. We demonstrated that ALT and AST levels of nanotherapeutics significantly increased to the levels comparable to free drugs after Kupffer cell depletion.

Conclusions: We demonstrate that nanoparticle reduces hepatotoxicity of cancer treatment by controlled release and Kupffer cell uptake. Our work bridges an important knowledge in nanoparticle drug delivery and clinical translation of nanomedicine.

#3900

The Safety and efficacy profile of a PD-L1 directed, Engineered Toxin Body, as a novel targeted direct-cell kill approach for the treatment of PD-L1 expressing cancers.

Hilario J. Ramos, Asis K. Sarkar, Sara Le Mar, Brigitte Brieschke, Joseph D. Dekker, Veronica R. Partridge, Pablo A. Maceda, Michaela M. Sousares, Garrett L. Robinson, Aimee Iberg, Shaoyou Chu, Jensing Liu, Jack P. Higgins, Erin K. Willert. _Molecular Templates, Austin, TX_.

Engineered Toxin Bodies (ETBs) are comprised of a deimmunized Shiga-like toxin subunit A (SLTA) genetically fused to an antibody-like targeting domain. The antibody targeting domain allows for specific targeting of cancer cells while the SLTA component promotes self-internalization of ETBs, an activity that allows for the delivery of an enzymatic and permanent ribosomal destruction against targeted cells even in the context of non-or-poorly internalizing receptor targets. Molecular Templates has developed PD-L1-targeting ETBs as an approach to directly target tumor cells and overcome resistance mechanisms against PD-1 and PD-L1 antibodies. The cytotoxicity delivered by PD-L1-specific ETBs is engineered to be independent of a requirement for tumor infiltrating lymphocytes (TILs), high tumor mutational burden, or modulatory effects of the tumor microenvironment. Further, the activity is not dependent on blockade of the PD-1/PD-L1 checkpoint axis. Thus, PD-L1 targeting ETBs represent a distinct class of therapeutics with direct cell-kill mechanism of action and ability for activity in patients who have progressed on current standard of care or checkpoint therapy. In this study, we highlight the efficacy and safety profile of MT-6020, a human and cynomolgus cross-reactive, PD-L1 targeted, ETB. MT-6020 retains potent catalytic activity and mediates enzymatic destruction of ribosomes at comparable levels to wild-type SLTA in a cell free model. In addition, MT-6020 binds to human NSCLC, Melanoma, and TNBC tumor cell lines with nM affinity and mediates cellular cytotoxicity via ribosomal destruction at low nM to sub-nM potency. MT-6020 binds to cell lines expressing non-human primate (NHP)-PD-L1 and elicits cytotoxic responses comparable to those observed on human tumor target cells. MT-6020 demonstrated pharmacodynamic and pharmacokinetic effects and displayed a favorable tolerability profile in a repeat dose NHP study at doses that are above the presumed therapeutically active concentration. Further our lead PD-L1 ETB, MT-6035, is built upon the MT-6020 scaffold and can deliver a viral peptide for cell surface presentation to and targeting by a specific antiviral CTL population (antigen seeding technology (AST)) for a second and complementary mechanism for tumor cell destruction. MT-6020 and MT-6035 represent a novel approach to targeting and destroying tumors expressing PD-L1 that is unlikely to be inhibited by resistance mechanisms to current checkpoint inhibitors, is well tolerated in relevant toxicity models, and has the capacity for activity in indications where standard of care has failed. Molecular Templates is poised to initiate clinical development of the PD-L1 targeted-ETB (AST), MT-6035, in 2H - 2019.

#3901

Nonclinical safety evaluation of CA102N, a hyaluronic acid (HA) and nimesulide (Nim) conjugate, for colorectal cancer treatment.

Eskouhie Tchaparian, Louis Lin, Elin Hsu, Ivy Chen, Albert Wu. _Holy Stone Healthcare Co., Ltd., Taipei, Taiwan_.

CA102N is a conjugate of the biological polymer hyaluronic acid (HA) and Nimesulide (Nim), a cyclooxygenase-2 (COX-2) inhibitor that represents a promising new approach to addressing cancer treatment. Similar to an antibody-drug conjugate or a pro-drug, CA102N enhances the efficacy of Nim through targeting and binding of HA to CD44 receptors highly expressed in solid tumors. Pharmacological (in vitro and in vivo) studies indicated that CA102N exhibits apoptotic and tumor growth inhibitory activities against colorectal cancer. The MOA of CA102N was demonstrated to be related to apoptosis and disruption of the PI3K/Akt/mTOR pathway in addition to inhibition of the COX-2/PGE2 related pathways.

Safety of CA102N was assessed in acute dose range finding and repeat-dose intravenous toxicity studies in rats and beagle dogs. Three dose levels including the Maximum Tolerated Dose (MTD) from DRF study, a middle dose and a low dose equivalent to the proposed clinical dose were administered twice weekly (BID). A 28-day recovery group was also included in the rat study.

Overall CA102N was well tolerated in both species. CA102N did not induce any significant effects on the CNS, the cardiovascular and respiratory systems in animal models. Some changes in hematological parameters were observed only in rats at the highest dose (Std 10: ≥ 600 mg/kg/day ), however, most of these observations were reversible. No hERG inhibition (in vitro) was observed suggesting the risk of QT prolongation with CA102N is low. The Highest Non-Severely Toxic Dose (HNSTD) of CA102N in the dog was determined to be 280 mg/kg/day (7.24 mg/kg in Nim equivalents ) and the rat NOAEL was 400 mg/kg/day (10.16 mg/kg in Nim equivalents ). These doses are 2.3 times (rat) and 5.6 times (dog) of nimesulide dose to be evaluated (up to 0.72 mg/kg of nimesulide) in the proposed phase 1 study.

On the basis of the mechanism of action and the nonclinical safety profiles, toxicity associated risks to patients appear to be low at the proposed clinical dose levels. A Phase I study of CA102N in patients with locally advanced or metastatic colorectal cancer is ongoing.

#3902

Increased sensitivity for detecting cytokine release syndrome with cancer immunotherapy using a PBMC humanized NSG-SGM3 mouse model.

Chunting Ye,1 Michael Brehm,2 Mingshan Cheng,1 Leonard Shultz,1 Dale Greiner,2 James Keck1. 1 _The Jackson Laboratory, Sacramento, CA;_ 2 _University of Massachusetts Medical School, Worcester,, MA_.

We have described a novel humanized NSG (NOD-scid IL2Rgammanull, NOD-scidIL2Rgnull; JAX stock number 005557) mouse model to measure cytokine release syndrome (CRS) after treatment with immunotherapeutic monoclonal antibodies (mAb). This humanized mouse model is based on PBMC engraftment of NSG mice and provides a rapid, robust and sensitive platform to assess CRS. Exposure of PBMC-engrafted NSG mice to mAb therapeutics was characterized by clinical readouts of cytokine production and decreases in body temperature. Moreover this model enabled the evaluation of differential responses between individual donors and allowed a more comprehensive evaluation of CRS than in vitro assays. However, human immune system engraftment in NSG mice injected with PBMC is dominated by human CD3+ T and NK cell in early time point. To expand the application of our model to additional human immune cell types, we explored the NSG-SGM3 (NOD-scid IL2Rgnull-3/GM/SF; JAX stock number 013062) strain as a model to assess CRS. The NSG-SGM3 mouse expresses human KITLG, CSF2 and IL-3 as transgenes and supports increased development of human myeloid and Treg populations following engraftment with umbilical cord CD34+ hematopoietic stem cells as compared to NSG mice. We hypothesized that PBMC-engraftment of NSG-SGM3 mice would augment human myeloid and T cell functionality in vivo. We compared NSG and NSG-SGM3 that were humanized with PBMC from identical donors and evaluated both the human immune cell engraftment profile by flow cytometry and CRS induction with single mAb agent and combination therapy. For PBMC engraftment, six weeks old female NSG and SGM3 mice were irradiated and then injected with human PBMC. Five days after PBMC injection, levels of total human cells (CD45+), and human immune cell subsets including CD3+ T cells, CD33+ myeloid cells, CD14+ monocytes, CD19+ B cells, and CD56+ NK cells, were measured by flow cytometry. Overall no significant differences were observed in the levels of human immune cells between PBMC-injected NSG and NSG-SGM3 mice. Six days after PBMC injection, mice were treated with mAb therapeutics including anti-CD28, pembrolizumab, Anti-thymocyte globulin and Lenalidomide as single agents or in combination. While rapid production of human cytokines (IFN-γ, IL-6, IL-10, IL-2, IL-4 and TNF) was detected in both PBMC-injected NSG and NSG-SGM3 mice, the overall cytokine levels were significantly higher in NSG-SGM3 mouse. Moreover, a lower PBMC concentration was compared in NSG and SGM3 mice for measuring CRS. Overall our results indicate that NSG-SGM3 mice humanized with PBMC are a precise and sensitive model to measure human CRS with cancer immune-therapies.

#3903

**Acute toxicity studies of ethanolic fruit-skin extract of** Annona muricata **.**

Wisdom O. Iyanda-Joel, Sandra O. Israel-Ovirih, Shalom N. Chinedu, Emeka EJ Iweala. _Covenant University, Ota, Nigeria_.

Recent reports have shown that the currently high demand for Annona muricata fruits and plant products is most likely due to their selective cytotoxic activity, where extracts and subsequent pure compounds exhibited more toxicity to cancer cell lines than standard drugs and even increased the viability of normal cells at certain concentrations. The current study explored the phytochemical screening and acute toxicity profile of ethanolic fruit-skin extract of Annona muricata (ESA) in female Wistar rats. Phytochemical screening were carried out following stipulated protocols while twenty-five female Wistar albino rats were treated with 2000 mg kgbwt-1and 5000 mg kgbwt-1 ESA for 24 - 72 hours and after 14 days to evaluate their toxicity against biochemical (ALT, AST, ALP, urea, creatinine, triglycerides, cholesterol, albumin and total protein), hematological (RBC, WBC, PCV, platelet count, MCV, MCH and MCHC, neutrophil, lymphocyte and monocyte) and histopathological parameters. The phytochemical screening of extract collectively revealed the presence of alkaloids, flavonoids, phenols, quinones, saponins, steroids, tannins and terpenoids. No sign of toxicity was observed in animals treated with ESA in the first critical four hours of dosage neither any mortality recorded. Toxicological assessment of ESA showed significant difference in values of extract-treated animals mostly at 5000 mg kgbwt-1 compared to normal control on ALT, ALP, WBC, M, N, PCV, MCH, MCV and platelet count within 72 hours of treatment compared to normal control but mostly later reversed after 14 days. Histopathology results also showed significant differences in morphology of certain organs compared to control with reversal of some lesions after 14 days. Ultimately, ethanolic fruit-skin extract of Annona muricata is non-toxic at moderate concentrations and may serve as a huge potential for nature-friendly antiproliferative and antitumour agents.

#3904

Pharmacokinetic modeling of serum platinum reveals extent of long-term exposure and associated comorbidities after cisplatin treatment.

Matthew R. Trendowski,1 Omar El-Charif,1 Zepeng Mu,1 Mark J. Ratain,1 Heather Wheeler,2 Paul C. Dinh,3 Darren R. Feldman,4 Shirin Ardeshir-Rouhani-Fard,3 Robert J. Hamilton,5 David J. Vaughn,6 Chunkit Fung,7 Taisei Mushiroda,8 Lawrence H. Einhorn,3 Lois B. Travis,3 M. Eileen Dolan1. 1 _University of Chicago, Chicago, IL;_ 2 _Loyola University Chicago, Chicago, IL;_ 3 _Indiana University, Indianapolis, IN;_ 4 _Memorial Sloan-Kettering Cancer Center, New York, NY;_ 5 _Princess Margaret Cancer Centre, Toronto, Ontario, Canada;_ 6 _University of Pennsylvania, Philadelphia, PA;_ 7 _J.P. Wilmot Cancer Institute, University of Rochester Medical Center, Rochester, NY;_ 8 _RIKEN Center for Integrative Medical Science, Yokohama, Japan_.

Platinum is detectable in the plasma years after completion of cisplatin treatment. Although it has been hypothesized that circulating platinum contributes to the severity and persistence of cisplatin's adverse effects, results have been inconsistent due to the lack of an effective pharmacokinetic model that takes into account time since therapy and the use of relatively small patient cohorts. Here, we report the largest pharmacokinetic study to date of 1,013 testicular cancer survivors (TCS) assessed 1-35 years after completion of cisplatin-based chemotherapy, and perform a genome-wide association study (GWAS) to assess genetic contributions to our pharmacokinetic phenotype computed from serum platinum levels. Eligible TCS given 300 or 400 (± 15 mg/m2) cisplatin underwent extensive audiometric testing and clinical examination, as well as completed questionnaires at the time of follow-up. We then built an empirical pharmacokinetic model by considering time since treatment and cumulative cisplatin dose received, fitting the data to a power (log-log linear) model as a log-log transformation linearized relationship with time. This power model was used to generate a residual platinum value (RPtV) that likely correlates with the area under concentration-time curve. Using linear regression, we found positive associations between RPtV and several clinically relevant phenotypes including the cumulative burden of morbidity for cisplatin-induced toxicities (β = 0.08, p = 8.42x10-3), peripheral sensory neuropathy (β = 0.14, p = 5.45x10-3), Raynaud's phenomenon (β = 0.09, p = 0.04), hypercholesterolemia (β = 0.22, p = 0.02), LDL-cholesterol (β = 2.67x10-3, p = 0.01), luteinizing hormone (β = 0.02, p = 0.01), and age at clinical examination (β = 0.02, p = 2.04x10-7). Multinomial regression indicated that these associations are much more prominent in patients designated with high RPtV (> 1 standard deviation above the mean). In addition, we found a strong negative association with creatinine clearance (β = -9.04x10-3, p = 1.03 x10-10). GWAS implicated two SNPs that met genome-wide significance: rs78225733 (p = 6.9 x 10-9) in a gene desert on chromosome 12 and rs45623132 (p = 1.1 x 10-8), which is intronic to meningioma 1 (MN1). Taken together, our novel pharmacokinetic model indicates serum platinum levels are associated with several cisplatin-induced toxicities and comorbidities, and that finding methods by which to reduce circulating platinum once patients have been cured of their disease could be an effective strategy to improve their overall quality of life.

#3905

Translational efficacy and safety modeling and simulation to support the clinical development of JNJ-64619178, a PRMT5 inhibitor.

Yue Guo,1 Nahor Haddish-Berhane,1 Hillary J. Millar,1 Tinne Verhulst,2 Tony Greway,1 Junguo Zhou,1 Loeckie DeZwart,2 Dana Gaffney,1 Joseph Portale,1 Dirk Brehmer,2 An Boeckx,2 Erika Van Heerde,2 Daniele Ouellet1. 1 _Janssen R &D, PA; _2 _Janssen R &D, Belgium_.

Protein arginine methyltransferase 5 (PRMT5) is an epigenetic enzyme with oncogenic properties. JNJ-64619178 (JNJ178) is a potent, selective, structurally unique PRMT5 inhibitor with good preclinical efficacy in inhibiting the growth of hematologic and solid tumor cell lines. Toxicology studies showed that JNJ178 decreased reticulocytes and neutrophils in rats and dogs. The objectives of translational modeling and simulation were to understand the exposure-response relationship of both safety and efficacy and provide guidance to the first-in-human clinical development of JNJ178.

Experimental data for the PK/PD (Pharmacokinetics/Pharmacodynamics) modeling included: plasma concentration after single dose of JNJ178 in non-tumor bearing mice, plasma concentration and PD markers of dimethylation of arginine (%SDMA in plasma and %SMD1/3-Me2 in tumor, respectively) after multiple doses (1 to 10 mg/kg) QD (once daily) of JNJ178 in H1048 (human small cell lung carcinoma) xenografts, and tumor volume in four xenograft mouse models (A427, human lung carcinoma; H441, human lung adenocarcinoma; H520, human squamous cell lung carcinoma; and H1048). Plasma PK were first described by a standard two-compartment model and used as a driver of PD and tumor volume (efficacy). Plasma and tumor PD were modeled using an indirect response model. A hybrid tumor growth coupled with transit compartment mediated tumor killing model was used to fit the tumor volume data. To predict the safety profile of JNJ178, lifespan based indirect response model for erythropoiesis and Friberg myelosuppression model were used to simulate hemoglobin and neutrophil kinetics in human.

The PK/PD model described the data well and validated the hypothesis that PD is driven by trough concentration. Based on the exposure-response relationship from the four xenograft models, the trough concentration needed to achieve tumor stasis for mouse was determined. In addition, the level of inhibition in tumor and plasma PD marker that was associated with tumor stasis was identified. Together with human PK parameters scaled using allometry, the dose range needed to achieve target therapeutic exposure for a typical human subject was predicted. Simulation results from erythropoiesis and Friberg myelosuppression models informed the optimal doing schedules for certain dose levels that would allow hematological toxicity to be manageable with <40% reduction in hemoglobin and >1.0 x 109/L neutrophil counts at all times. Overall, a translational modeling and simulation approach that considers safety and efficacy has been instrumental in the design of the first-in-human clinical development of PRMT5 inhibitor JNJ178 regarding selection of dose and schedule.

#3906

PK-PD modelling of an anti-CTLA-4 antibody on immune checkpoint humanized mice.

Madeline Lee,1 Jie Xiang,1 Tian Gan,2 Yuelei Shen,2 Chaoshe Guo2. 1 _Biocytogen, Wakefield, MA;_ 2 _Biocytogen, Beijing, China_.

Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4; also known as CD152) is expressed on the surface of T cells, where it primarily suppresses the early stages of T cell activation. In preclinical studies, blockade of CTLA-4 led to an increase in T-cell proliferation. Anti-CTLA-4 monoclonal antibodies (mAbs) have been shown to be an effective approach for tumor immunotherapy. Preclinical pharmacokinetic (PK) and pharmacodynamic (PD) characterization of these antibodies is essential for designing first-in human trials.

PK prediction is especially challenging for monoclonal antibodies attributed to target-mediated drug disposition (TMDD). We generated a humanized B-hCTLA-4 mouse model, in which exon 2 of the murine CTLA-4 gene was replaced with its human counterpart. In homozygous mice, only human protein expression can be detected, while mouse CTLA-4 was knocked out. Using B-hCTLA-4 mice, we developed a mAb PK/PD model to characterize the anti-tumor effects of hCTLA-4 antibodies.

MC38 cancer cells were inoculated into both C57BL/6 wild type and humanized CTLA-4 animals. After administering different antibody concentrations (ranging from 3.0, 1.0, 0.3, 0.1mg/kg), serum samples from each animal were taken at specific time points for PK/PD analysis. An NCA approach was used to determine serum concentrations of mAb in mice. The levels of 13 cytokines (IL-23, IL-1α, IFN-r, TNF-a, IL-6 etc.) were quantified using LEGENDplex bead-based immunoassays. The PK half-life was 50~100h and the cytokine concentration ranged from 0-2000pg/ml depending on the dose used. The Tumor Growth Inhibition (TGI) value was also evaluated as a PD effect in the MC38/B-hCTLA-4 model. The TGI value reached nearly 100% when the dose was above 0.3mg/kg. The PK/PD assays showed that the anti hCTLA-4 mAb tested has a dose-dependent tumor inhibition effect in hCTLA-4 mice. Results from TMDD model data showed that the humanized mice reached QuasiSteady State faster than wild type mice, indicating that the humanized mice are an appropriate TMDD model. In conclusion, humanized B-hCTLA-4 mice are a promising PK/PD model for CTLA-4 mAb preclinical trials.

### PI3K/AKT Inhibitors

#3907

Synergistic effect of targeting both CDK4/6 and mTORin sarcoma cell lines.

Xiaochun Wang, David Goldstein, Philip Crowe, Jia-Lin Yang. _University of New South Wales, Sydney, Australia_.

Background: The phosphatidylinositide-3-kinase/Protein kinase B (AKT)/ mammalian target of rapamycin (mTOR) signaling is central for cancer growth, survival and motility. One of the key restraints on cell growth downstream is retinoblastoma (Rb) tumour suppressor, which is negatively regulated by the cyclin-dependent protein kinases (CDK) 4/6 protein. We hypothesize that blocking both the CDK4/6 and the mTOR allows the double brakes to tumour growth.

Aim: Our aim is to investigate whether combination therapy using mTOR and CDK4/6 inhibitors (palbociclib and ridaforolimus) would have a synergistic growth inhibitory effect in sarcoma.

Methods: The effect and mechanism of palbociclib and ridaforolimus was investigated in a panel of twelve sarcoma cell lines by Crystal-violet colorimetric and Western blot assays. HT1080 fibrosarcoma metastatic mouse model was investigated for in vivo therapeutic study.

Results: Palbociclib showed anti-proliferation in all cell lines (IC50s: 0.1-1.1µM), while ridaforolimus had growth inhibition in 10/12 cell lines (IC50s: 0.4-26nM). The palbociclib-ridaforolimus combination achieved synergistic effect (CIs: 0.1-0.9) in 9/12 cell lines, including applying the drugs in different sequence (together or pre-treatment with one drug for 24-48 hours) and ratio (1:1, 1:2, 1:4, 2:1 or 4:1). The Western blot demonstrated that the 2-drug combination further blocked both CDK/Rb/E2F and AKT/mTOR pathways, via promoting apoptosis. In the experimental lung metastatic HT1080 mouse model, using palbocilib (10 and 30mg/kg, every other day) and ridaforolimus (0.1 and 0.3mg/kg, daily for 5 days per week) for 4 weeks synergistically (CI = 0.7) inhibited the development of lung metastases (mean number of colonies: 132 in low combination dose and 38 for high combination dose) compared to monotherapy [243 (palbociclib) and 255 (ridaforolimus) in low dose, 113 and 97 for high dose) and vehicle control (286). In the orthotopic fibrosarcoma HT1080 mouse model, the palbociclib-ridaforolimus combination therapies achieved significant (p<0.001) and synergistic (CI=0.00375) growth inhibitory effect compared to monotherapies and control groups.

Conclusions: This study demonstrated that palbociclib-ridaforolimus combination is active in a variety of sarcoma subtypes and worthy of further development towards a clinical trial.

#3908

The combination of NVP-BEZ235 & endostatin exerts synergistic anticancer activity against triple-negative breast cancer in vitro and in vivo.

Xiaoshan Wang, Xiao Zhang, Fanghua Li. _Academy of Medical Sciences & Sichuan Provincial People's Hospital, University of Electronic Science and Technology of China, Chengdu, China_.

Background: Triple-negative breast cancer(TNBC) take up 15% of invasive breast cancer. TNBC has no effective molecular targets & unfavourable prognosis. We investigated the effects of NVP-BEZ235, a novel dual PI3K/mTOR inhibitor, a combine with or not an angiogenesis inhibitor Endostatin in the orthotopic TNBC model & explored its anti-tumor & anti-angiogenesis mechanisms.

Material & Methods: Assess sensitivity of the cells to the drug by MTT assays.Orthotopic xenografts were performed for tumor growth and survival studies. Additionally, Immunohistochemistry for VEGF and MVD(CD34) was performed using the EnVision/HRP technique,Serum VEGF was detected by ELISA method.

Results: BEZ235 effectively inhibited cell proliferation in vitro & provided additive effects when combine with Endostatin.Treatment with BEZ235 & Endostatin resulted in inhibition of tumor growth & prolongation of survival time of tumor-bearing mice in vivo.Immunohistochemical analysis revealed that intratumoral proliferation & angiogenesis was significantly suppressed. Furthermore, Changing of the number of Serum VEGF support the finding of angiogenesis inhibition too.

Conclusion: Our findings suggest that BEZ235 exerts antitumor effects against TNBC & enhances effects of Endostatin through inhibition of cell proliferation & tumor angiogenesis. This approach may represent a promising combination targeted therapy for TNBC treatment.

#3909

Potential antitumor effect of a pan-PI3K inhibitor ZSTK474 on human sarcoma cell lines.

Shingo Dan, Naomi Tamaki, Nachi Namatame, Yuya Yoshizawa, Mutsumi Okamura, Yumiko Nishimura, Kanami Yamazaki, Shin-ichi Yaguchi. _Japanese Fdn. for Cancer Research, Tokyo, Japan_.

Phosphatidylinositol-3 kinase (PI3K) is a key signaling molecule for tumor growth and survival, thus the PI3K pathway has been thought to be a promising target for cancer therapy. We previously identified a novel phosphatidylinositol-3 kinase (PI3K) inhibitor, ZSTK474, by its similarity of antiproliferative profile across the JFCR39 cancer cell line panel to a known PI3K inhibitor, LY294002. Since then, ZSTK474 has been evaluated in preclinical models as well as in clinical trials in the US and Japan against a range of advanced solid tumors. The US trial revealed that three of four sarcoma patients exhibited stable disease, suggesting possible clinical benefit in this disease. Treatment of patients with advanced sarcoma remains challenging due to lack of effective medicine. Therefore the results of clinical trial prompted us to examine antitumor activity of this compound in various subtypes of sarcomas. To this end, we developed a sarcoma panel comprising of 17 human sarcoma cell lines from a variety of subtypes and investigated the effect of ZSTK474 across the panel and compare it with those of other molecularly targeted agents and clinically used chemotherapeutic agents for the treatment of sarcoma, such as doxorubicin, paclitaxel and gemcitabine. As a result, ZSTK474 exhibited a unique antiproliferative profile that was similar to other PI3K inhibitor but clearly different from conventional chemotherapeutic drugs examined, as we previously reported using JFCR39 carcinoma cell line panel. Indeed, ZSTK474 inhibited PI3K-downstream pathways, in parallel to growth inhibition, in all the sarcoma cell lines examined, showing proof-of-concept of PI3K inhibition. Of note, ZSTK474 exerted a potent antiproliferative effect on SW684, a fibrosarcoma cell line and MES-SA/Dx5, and a uterine sarcoma cell line that acquired resistance to doxorubicin via overexpression of P-glycoprotein, while both exhibited resistance to most of the chemotherapeutic agents examined. Moreover, ZSTK474 induced extensive apoptosis selectively in Ewing's sarcoma and alveolar rhabdomyosarcoma cell lines, both of which harbor chromosomal translocations and resulting fusion genes, EWSR1-FLI1 and PAX3-FOXO1, respectively, whereas remaining sarcoma cell lines as well as carcinoma cell lines examined so far hardly underwent apoptosis. Additional cell lines from synovial sarcoma harboring SS18-SSX1/2 fusion gene also underwent apoptosis, suggesting preferential induction of apoptosis in oncogenic chromosomal translocation positive sarcoma cells. Finally, animal experiments using nude mice bearing human sarcoma xenograft confirmed the antitumor activity of ZSTK474 in vivo, with superior efficacy observed in translocation-positive cells. These results suggest that ZSTK474 could be a promising drug candidate for sarcomas, especially those harboring oncogenic chromosomal translocation.

#3910

Tetrandrine inhibits signal transduction through PI3K/Akt/mTOR and PERK/eIF2á pathways to target aberrantly activated cap-dependent translation in cancer cells.

Sergey Slepenkov, Praveen Jaiswal, Sweaty Koul, Hari K. Koul. _LSU Health Sciences Ctr. - Shreveport, Shreveport, LA_.

The PI3K/Akt/mTOR pathway plays a key role in the control of cap-dependent mRNA translation and is also an important driver of tumor progression. Owing to mutations in PTEN across pan cancer, this pathway is activated in several solid tumors. mTOR signaling enhances tumor growth by stimulating both the global translation rates and the translation of a selected subset of sensitive transcripts, including mRNAs, encoding oncogenes, growth factors, anti-apoptotic factors - c-Myc, ornithine decarboxylase, Cyclin D1, VEGF, IGF, BCL-2, and survivin in several solid tumors. The foregoing data demonstrate that abnormal translational control in cancer, implemented by oncogenic signaling via mTOR and other pathways, may represent an attractive therapeutic target. Tetrandrine (Tet) a bis-benzylisoquinoline alkaloid with broad anticancer activity attracts increasing attention lately. However, the underlying molecular mechanisms were not studied systematically. In the present studies, we evaluated the effects of Tet on aberrantly activated cap-dependent translation and on its principal regulator - Akt / mTOR signaling pathway in cancer cells for the first time in any system. The anti-proliferative effects of Tet was evaluated in several cancer cell lines (LNCaP, 22Rv1, BXPC3, MiaPaCa2, A498, UOK262 HCC827, etc) representing many solid tumors. Using capped mRNA reporter construct, we evaluated the effect of Tet on translation in 22Rv1 and LNCaP PC cell cultures. We found that Tet was not able to inhibit translation of mRNA on the ribosomes per se in the in vitro translation system. However, Tet was operational in LNCaP and 22Rv1 cells, inhibiting cap-dependent translation rapidly and efficiently during the first few hours. A subsequent study of the polysomal distribution in LNCaP and 22Rv1 cells showed that Tet mainly inhibited the initiation phase of translation. Importantly, the translation-inhibiting effect of Tet correlated with its growth-suppressive activity in concentration and time-dependent manner. Our study of the PI3K/Akt signaling showed that Tet inhibited signaling to mTOR and its downstream effectors in PC cells. This explains the inhibition of cap-dependent mRNA translation and the suppression of cancer growth. Collectively, presented results show a new, previously unknown "translation side" of the anticancer activity of Tet. This novel insight may help with potential therapeutic applications of Tet in the treatment of cancer in general and prostate cancer in particular.

#3911

Akt inhibitor SC66 promotes cell sensitivity to cisplatin in chemoresistant ovarian cancer cells through inhibition of COL11A1 expression.

Yi-Hui Wu, Cheng-Yang Chou. _National Cheng Kung University, Tainan, Taiwan_.

Background and aims: Akt plays a central role in regulating cell growth and cell cycle progression and is regarded as a promising therapeutic target. We examined whether Akt inhibition by SC66 is therapeutically efficacious in the treatment of ovarian cancer as a single agent and in combination with chemotherapy drugs.

Methods: The expressions of phospho-Akt in ovarian cancer patients were evaluated by immunohistochemistry. Using eight human ovarian cancer cell lines, we determined the effect of SC66 by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, western blot, and apoptosis assays. We evaluated the association between phospho-Akt/mTOR, COL11A1 and SC66 sensitivity. We also determined the effect of SC66 on tumor growth using mice inoculated with human ovarian cancer cells.

Results: Elevated phospho-Akt levels in cancerous tissue were associated with short progression-free survival and overall survival. Cell sensitivity to SC66 was inversely correlated with phospho-Akt and COL11A1 expression levels as well as resistance to cisplatin or paclitaxel. SC66 inhibited phosphorylation of Akt and its downstream effectors 4E-BP1 and p70S6 kinase. SC66 also attenuated expression of TWIST1 and Mcl-1, factors important in cell invasiveness and anti-apoptosis, respectively. SC66 sensitized chemoresistant cells to cisplatin and paclitaxel treatment and promoted apoptosis. In addition, SC66 inhibited COL11A1 expression via decreased binding of CCAAT/enhancer-binding protein beta (c/EBPβ), reducing chemoresistance and decreasing binding of nuclear transcription factor Y (NF-YA) to COL11A1. A mouse xenograft experiment demonstrated that SC66 treatment caused a reduction in tumor formation and enhanced the therapeutic efficacy of cisplatin.

Conclusions: This study demonstrates the role of Akt in ovarian tumor progression and chemoresistance and supports the application of SC66 as a therapy for ovarian cancer.

#3912

The PI3K/mTOR dual inhibitor PF-04691502 shows antitumor activity in Sezary cells and in a xenograft mouse model.

Antonella Bresin,1 Cristina Cristofoletti,1 Francesca Monzo,1 Elisabetta Caprini,1 Mauro Helmer Citterich,1 Alessandra Frezzolini,1 Alessandro Monopoli,1 Roberto Benucci,1 Maria Cantonetti,2 Enrico Scala,1 Giandomenico Russo,1 Maria Grazia Narducci1. 1 _IDI-IRCCS, Rome, Italy;_ 2 _University of Tor Vergata, Rome, Italy_.

Introduction: Cutaneous T-cell Lymphomas (CTCLs) are extranodal non-Hodgkin's lymphomas deriving from neoplastic transformation of T lymphocytes that home to the skin. Mycosis fungoides (MF) is the most diffuse form, usually having an indolent course but presenting in some cases as an aggressive disease with a poor prognosis. Sézary syndrome (SS) is a rare and aggressive variant, with erythroderma, leukemia, and lymph node involvement. Despite recent advances, CTCLs are still incurable cancers and the clinical symptoms are highly disabling. Among the landscape of genomic alterations identified in CTCL, we and others have described alterations of the PI3K pathway members (loss of PTEN, LKB1 and PDCD4, and gain of p70S6K). As a consequence, several SS patients show hyper-activation of PI3K/AKT/mTOR pathway. Hence, inhibition of this pathway represents a potential therapeutic choice against CTCL.

Objective: In this work, we investigated the therapeutic potential of the dual-PI3K/mTOR inhibitor PF-04691502 (hereafter PF-502) against CTCL.

Materials and Methods: PF-502 therapeutic efficacy was evaluated in four CTCL cell lines and in patient-derived SS cells, for its ability to inhibit cell growth, to induce apoptosis and to inhibit chemotaxis toward the chemokine SDF-1. Moreover, to evaluate PF-502 therapeutic effects in vivo we used a xenograft mouse model, consisting of nude mice transplanted with the CTCL-derived HH cell line.

Results: PF-502 inhibited the cell growth of all the CTCL cell lines (0.07-0.32 µM, IC50 range), and of tumor cells derived from six SS patients (2.3 µM, mean IC50). The mechanism of action of PF-502 in CTCL cell lines was a combination of apoptosis induction and G1 block, while in SS patients it was mostly based on a strong apoptosis induction. Of note, PF-502 showed an only modest effect in T-lymphocytes from 3 healthy donors. PF-502 inhibited also cell migration toward the chemokine SDF-1, in both the systems. This result might have an important clinical implication because we have recently demonstrated that malignant lymphocytes are activated by the skin microenvironment. Thus, blocking the homing to the skin could affect the disease course. Finally, PF-502 has a robust antitumor activity in the xenograft mouse model, by reducing tumor volume (400 mm3 in treated mice vs 936 mm3 in controls) as well as tumor weight (0.2 g in treated mice vs 0.56 g in controls). Moreover, the Kaplan-Meier curve, in these mice, revealed that PF-502 treatment prolongs survival (P<0.005).

Conclusions: Our results strongly support the therapeutic potential of targeting PI3K/AKT/mTOR pathway in CTCL.

#3913

**PDK1 drives susceptibility of** NOTCH1 **mutant head and neck squamous carcinoma to PI3K/mTOR pathway inhibition.**

Vaishnavi Sambandam,1 Li Shen,1 Shaohua Peng,1 Tuhina Mazumdar,1 Curtis Pickering,1 Jeffrey Myers,1 Jing Wang,1 Mitchell Frederick,2 Faye Johnson1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _Baylor College of Medicine, Houston, TX_.

Head and neck squamous cell carcinoma (HNSCC) is primarily driven by the loss of tumor suppressor genes. Although approaches to target driver oncogenes have been clinically successful, targeting tumor suppressors has been challenging. To address this translational gap, we previously demonstrated that HNSCC cell lines with loss of function (LOF) NOTCH1 mutations (NOTCH1MUT n=14) were more sensitive to 6 PI3K pathway inhibitors (Omipalisib, Alpelisib, Bimiralisib, Dactolisib, Copanlisib, Apitolisib) than NOTCH1WT cells (n=45). In contrast to PIK3CAMUT cell lines, NOTCH1MUT lines underwent significant apoptosis after PI3K/mTOR pathway inhibition. NOTCH1MUT lines also showed significantly reduced clonogenic growth and significant tumor growth inhibition in both oral orthotopic and subcutaneous xenograft models. We employed CRISPR-Cas9 gene editing technology to knock out NOTCH1 gene in two NOTCH1WT lines, PJ34 and UMSCC49. After Omipalisib treatment, NOTCH1-KO lines showed decreased cell viability compared to parental lines. The NOTCH1 knock out lines showed significantly increased apoptosis after Omipalisib treatment compared to parental lines (PJ34 KO: 1.62-fld, P<0.003; UM49 KO: 1.37-fld, P=0.002). Both NOTCH1 KO lines also showed increased cleaved PARP and cleaved caspase 3 by western blot analysis. With NOTCH1 knock out lines, treatment significantly decreased number of colonies (PJ34 KO: 1.35-fld, P<0.005; UM49 KO: 1.62-fld, P<0.0001). As no canonical pathways account for the underlying mechanism of sensitivity, we measured 301 proteins by reverse phase protein array (RPPA) in NOTCH1MUT and NOTCH1WT lines after Omipalisib treatment. Several proteins were differentially regulated in NOTCH1MUT cells compared with NOTCH1WT lines including PDK1, FoxM1 and p-ERK. We then investigated PDK1 because it is activated by PI3K, and can regulate FOXM1 and ERK which were also inhibited more robustly in NOTCH1MUT HNSCC. To test the role of PDK1 in PI3K/mTOR inhibition, we over expressed PDK1 in NOTCH1MUT cells and it successfully led to decreased apoptosis after Omipalisib treatment as compared to the parental lines. The combination of AKT and PDK1 inhibition led to decreased cell viability in all four NOTCH1WT lines tested as compared to single agents. The combination also led to significantly increased apoptosis in all NOTCH1WT cell lines tested as demonstrated by cleaved PARP and Caspase 3 levels by western blot analysis. Our research is the first to establish a therapeutic vulnerability of NOTCH1MUT HNSCC to any class of drugs and may inform the development of the first biomarker-driven targeted therapy for HNSCC. Also, we may uncover combination therapies and define a heretofore-unknown mechanism of PDK1 regulation. Because NOTCH1 LOF mutations are common in other squamous carcinomas, including skin, esophagus and lung, our findings will likely have implications beyond HNSCC.

#3914

mTOR pathway components dictate cell response to Src inhibitors in prostate cancer cells.

Yao Dai, Dietmar W. Siemann. _Univ. of Florida College of Medicine, Gainesville, FL_.

Src inhibitors have been actively evaluated in the clinic for the treatment of advanced prostate cancer however the effects are modest. One interpretation is that oncogenic signaling pathways are redundant and blocking one pathway is not sufficient to yield considerable efficacies. mTOR pathway is commonly hyperactivated in prostate cancer. However, the functional role of mTOR in Src inhibitor-mediated response remains largely unknown. In the current study, we combined mTOR inhibitor rapamycin and Src inhibitor dasatinib to detect functional behaviors in prostate cancer cells. Cell proliferation and clonogenic survival were detected by MTT and colony formation assay, respectively. Wound-healing" scratch assay was conducted to monitor cell migration, and Matrigel-based transwell assay was applied to assess cell invasion. Protein expression was detected by Western blotting. Gene silencing was detected by siRNA transfection. We found that two agents combined together showed greater inhibition on proliferation and clonogenic survival than either agent alone. Such combination effects were additive or synergistic depending on the cell types, as reflected by combination index calculation. In addition, cell migration and invasion were inhibited in a greater extent by rapamycin plus dasatinib, compared to either treatment alone. At the molecular level, co-targeting mTOR and Src lead to clear inhibition of Src and its downstream pathway focal adhesion kinase (FAK), Akt and mTOR downstream components S6RP and 4E-BP1. Interestingly, knockdown 4EBP1, but not S6RP, attenuated dasatinib-mediated functional inhibition. These data indicate that adding mTOR inhibitors to Src-based intervention may achieve better treatment outcomes. Such combination could be evaluated as a novel treatment strategy for prostate cancer patients who are less responsive to Src inhibitors. In addition, 4EBP1 may be used as a candidate predictive biomarker for monitoring clinical effectiveness of Src inhibitors in prostate cancer.

#3915

YH25248, a selective PI3K delta inhibitor, shows synergistic effect with an anti-PD-L1 antibody.

Jinhwi Park, Ho-Woong Kang, Hyun-Mo Koo, Jongsuk Park, Tae-Wang Kim, Se-Woong Oh, Soongyu Choi. _Yuhan Research Institute, Seoul, Republic of Korea_.

Phosphatidylinositol 3-kinase (PI3K) plays critical roles in numerous intracellular processes. Of the four known subtypes, PI3Kδ subtype is primarily expressed in immune cells and has been reported to promote B cell differentiation and regulatory T (Treg) cell activation. Treg cell-mediated immunosuppression is thought to be a major resistance mechanism for immune checkpoint inhibitors. We therefore hypothesized that an effective PI3Kδ inhibitor could enhance anti-cancer immunity when combined with immune checkpoint inhibitors. Through recent efforts, we have discovered YH25248, an oral PI3Kδ-specific inhibitor. YH25248 effectively inhibits PI3Kδ (IC50 = 10nM) showing more than 100-fold selectivity over α, β, and γ subtypes, and exhibited favorable kinase selectivity in 463 kinase panels. In cellular experiments, phospho-AKT was effectively blocked (IC50 = 7 nM), with basophil activation, a known PI3Kδ specific signal, was also inhibited. YH25248 reduced the activity of Treg cells (CD4+/CD25+) isolated from the splenocytes of CT26 tumor-bearing mice, while sparing conventional T cells (CD4+/CD25-). When YH25248 was orally administered (10 mg/kg) to mice, drug exposure in plasma was reasonable, with a half-life of 4.3 hours, while once-daily oral administration of YH25248 (10 ~ 60 mg/kg) significantly inhibited tumor growth of CT26 colon carcinomas in mouse. When the immune cells of tumors were analyzed by flow cytometry, the number of Treg cells was reduced, CD8 + T cells increased. In this model, YH25248 (30 mg/kg, QD) showed synergistic efficacy when combined with an anti-PD-L1 antibody (200 μg/mice, 10F.9G2 clone). In other syngeneic models using 4T1 or MC38 cells, YH25248 (30 mg/kg, QD) also exhibited similar synergistic efficacy in combination with the anti-PD-L1 antibody. In conclusion, YH25248 is a potent, selective, oral inhibitor of PI3Kδ that reduces tumor growth in syngeneic mouse tumor models by increasing the ratio of CD8+ to Treg cells. The combination of YH25248 and anti-PD-L1 antibody resulted in substantially greater tumor growth inhibition in several syngeneic models. These data support the promising potential of YH25248 for combination therapy with immune checkpoint inhibitors to increase therapeutic response rates.

#3916

Unbiased phospho-proteomic profiling of mouse breast cancer models with DIA mass spectrometry refines CanPath prototype, a platform for predictive cancer pathway modeling.

Magdalena Bober,1 Monika Banko-Bielecka,1 Daniel Heinzmann,1 Oliver Rinner,1 Nicholas Dupuis,1 Christoph Wierling,2 Huaibiao Li,3 Thomas Kessler,2 Artur Muradyan,2 Louisa Krützfeldt,2 Moritz Schütte,2 Felix Dreher,2 Aspasia Ploubidou,3 Bodo Lange2. 1 _Biognosys AG, Schlieren, Switzerland;_ 2 _Alacris Theranostics GmbH, Berlin, Germany;_ 3 _Fritz Lipmann Institute, Jena, Germany_.

Background CanPathPro is designed to build and validate a combined experimental and systems biology platform, which will be used in testing cancer signaling hypotheses. It combines highly defined mouse and organotypic experimental systems, high-dimensional data including next generation sequencing and quantitative proteomics, and computational models for data integration, visualization and modelling. Mouse cancer models are characterized by quantitative transcriptome, quantitative mass spectrometry including phospho-proteome, histopathology and histochemistry. Phospho-proteomic data has been obtained using data independent acquisition (DIA) and used to refine signaling models in the mouse cell lines. Currently, the model integrates modules of the signal transduction pathways Egfr/Erbb2, Fgfr, insulin, Akt, Mtor, Myc, Ras, Hippo, Met, Tgfbr, Il6, integrin, Wnt, apoptosis and cell cycle regulation integrating in total about 380 different genes as well as related proteins and phospho-proteins Methods Two mouse mammary gland model cell lines, (Cdh1-fl/AKT1[E17K] and Cdh1-fl + PTEN-fl) were each treated with DMSO, a Pik3ca inhibitor (Wortmannin), or an Akt inhibitor (MK-2206). Cells were lysed and proteins were denatured, followed by reduction, alkylation and digestion with trypsin. The resulting peptides were desalted and enriched for phosphopeptides with TiO2 beads and cleaned up for mass spectrometry. A phosphopeptide library was generated from pooled phosphoenriched samples using LC-MS/MS shotgun measurements and included 22,893 phosphosites from 3,549 protein groups. DIA data was acquired on a Q Exactive HF mass spectrometer with a gradient length of 60 - 120 minutes on a C18 Easy LC 1200 nano-liquid chromatography system. The DIA data was extracted and processed with Spectronaut 11 (Biognosys) for analysis. Results In the AKT1[E17K] samples, 13,396 peptides (21,862 phospho-peptides) were quantified in the DIA runs and 12,297 peptides (19,928 phospho-peptides) were quantified in PTEN-fl samples. Under treatment with MK2206 and Wortmanin, 859 phosphopeptides from 548 protein groups were significantly changed across all comparisons in the AKT1[E17K] samples and with the same treatments 2,276 phosphopeptides from 976 proteins were significantly changed in the PTEN-fl cells. Based on this data 11 functionally relevant new phosphosites have been added to the model including ones on: EIF4B, FOX03, MAP2K4, PAK1, RAF1, and ULK1. Conclusions Phospho-proteomic profiling of cell lines using DIA mass spectrometry enables deep characterization of the phospho-signaling cascades modulated through small molecule inhibitors.

#3917

**Double** PIK3CA **mutations in** cis **enhance oncogene activation and sensitivity to PI3K alpha inhibitors in breast cancer.**

Neil Vasan,1 Pedram Razavi,1 Jared Johnson,2 Hong Shao,1 Hardik Shah,3 Alesia Antoine,3 Erik Ladewig,1 Alexander Gorelick,1 Eneda Toska,1 Guotai Xu,1 Abiha Kazmi,1 Matthew T. Chang,4 Barry S. Taylor,1 Maura N. Dickler,5 Komal Jhaveri,1 Raul Rabadan,6 Ed Reznik,1 Melissa L. Smith,3 Robert Sebra,3 Lewis C. Cantley,2 Maurizio Scaltriti,1 Jose Baselga7. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _Weill Cornell Medical College, New York, NY;_ 3 _Mount Sinai School of Medicine, New York, NY;_ 4 _Genentech, South San Francisco, CA;_ 5 _Eli Lilly, Indianapolis, IN;_ 6 _Columbia University, New York, NY;_ 7 _Vall d'Hebron Institute of Oncology, Barcelona, Spain_.

Activating mutations in PIK3CA, the gene coding for the catalytic subunit (p110α) of phosphoinositide 3-kinase (PI3K), are the most frequent oncogenic alterations in all cancers, including estrogen receptor-positive (ER+) breast cancer. There are many distinct oncogenic PIK3CA mutations including major hotspot mutations (e.g. E542K, E545K, H1047R) which are predictive of response to PI3Kα inhibitors. While responses to PI3Kα inhibitors can be variable, some patients with single hotspot mutant tumors derive a deep and prolonged clinical benefit. In a comprehensive analysis of the genomic data available in breast cancer, we observed double PIK3CA mutations and investigated their potential biological relevance and potential correlation with sensitivity to PI3Kα inhibitors. We detected double PIK3CA mutations in 10-15% of all PIK3CA-mutant cancers including breast cancer. Double PIK3CA mutations in breast cancer are defined by a pattern of co-expression of a major hotspot mutation associated with a recurrent second-site PIK3CA mutation ('minor mutation'). We have found that these double PIK3CA mutations are compound, that is in cis on the same allele, using single molecule real time sequencing (SMRT-seq) of fresh breast tumor samples. Double compound PIK3CA mutations result in increased PI3K activity and downstream signaling compared to single hotspot mutants in nontransformed cells and in ER+ breast cancer cells. Double compound mutations also increase cell proliferation and xenograft growth compared to single hotspot mutants. Biochemical experiments using recombinant PI3K compound mutant protein complexes reveal a combination mechanism of action, with simultaneous PI3K destabilization and increased membrane binding compared to single hotspot mutants, leading to increased kinase activity. Importantly, these compound mutations predict for increased sensitivity to PI3Kα inhibitors compared to single hotspot mutants in both preclinical models and also in patients with breast cancer. Together, our data support that double compound PIK3CA mutations enhance the oncogenicity of single hotspot PIK3CA mutations and this increased dependency results in increased sensitivity to PI3Kα inhibitors compared to single hotspot mutations. We propose that double compound activating mutations are an additional mechanism of oncogenic transformation in addition to other known mechanisms such as gene amplification, point mutation, or gene translocation. We speculate that double compound mutant PIK3CA may function as a clinical biomarker of increased sensitivity to PI3K-directed targeted therapies and may improve the therapeutic window of PI3K inhibitors in ER+ breast cancer and other PIK3CA-mutant tumor histologies.

#3918

Νovel small molecule modulators of the hotspot PIK3CA mutants identified by computational and experimental approaches.

Zoe Cournia,1 Paraskevi Gkeka,1 Hari Leontiadou,1 Ioannis Galdadas,1 Christina Athanasiou,1 Antriana Papadimitropoulou,1 Vasiliki Lazani,2 Maria Pavlaki,3 Bogos Agianian,3 Savvas Christoforidis,4 Argiris Efstratiadis1. 1 _Academy of Athens Biomed. Res. Fndn., Athens, Greece;_ 2 _Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas & University of Ioannina, Ioannina, Greece; _3 _Democritus University of Thrace, Alexandroupoli, Greece;_ 4 _Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas & University of Ioannina, Athens, Greece_.

Kinases are intensely pursued as drug targets for cancer treatment. Usually, kinase inhibitors target the active center (ATP binding site). However, the similarity of this site across many kinases often results in non-selective inhibition. Accordingly, an alternative approach is being pursued for the identification of allosteric inhibitors targeting relatively less conserved binding sites, thus higher selectivity can be attained. ΡΙ3Κα is the most frequently mutated kinase in human cancers, with 80% of the mutations occurring in two hotspots of the PIK3CA gene, which encodes the catalytic subunit of ΡΙ3Κα: 1) the H1047R mutation, which is found at the kinase (membrane binding) domain and 2) the E545K mutation, which is found at the helical domain (amino acid replacement with charge reversal). To assess the activation mechanisms of PIK3CA mutants, extensive microsecond Molecular Dynamics (MD) simulations were performed to examine conformational differences between the wild type (WT) and mutant proteins. The MD results were used to predict alterations in the allosteric networks of the WT upon each of the mutation. Binding site analysis of allosteric action in the kinase domain identified cavities in the vicinity of the H1047R mutation and the membrane-binding region. Virtual screening and subsequent biochemical assays were used to identify several PI3Kα inhibitors acting at a micromolar concentration. The effect of the inhibitors on WT and H1047R mutant ΡΙ3Κα-membrane associations was subsequently studied by SPR experiments, which showed that these small molecules alter the protein-membrane affinity, unlike other known ΡΙ3Κα inhibitors such as wortmannin. It was also observed that the inhibitory concentration remained unchanged even under conditions of increased ATP concentration, indicating that these inhibitors are non-competitive. The most promising small molecules were further tested in vivo using xenografts and genetically modified mouse models. Micro PET/CT imaging of the tumors showed reduction in volume after treatment. Immunohistochemical analysis of the xenografts showed reduction of proliferation and increase in apoptosis after treatment, in comparison to the controls. Details about the results with the E545K will be presented at the meeting.

#3919

GSK458, a novel oral dual PI3K/mTOR Inhibitor, is effective in preclinical model of T-cell lymphoma alone and in combination therapies.

Juan J. Gu,1 Lianjuan yang,2 Jing He,3 Weina Shen,4 Cory Mavis,1 Francisco Hernandez-Ilizaliturri1. 1 _Roswell Park Comprehensive Cancer Center, Buffalo, NY;_ 2 _Shanghai Dermatology Hospital, Shanghai, China;_ 3 _Peking University People's Hospital, Beijing, China;_ 4 _Fudan University Shanghai Cancer Center, Shanghai, China_.

Purpose: Despite T cell lymphoma (TCL) responses well at the initial chemotherapy, the outcome of TCL remains poor when it becomes systematic disease with refractory or relapse (r/r) after several cycle of treatment. Constitutively activated PI3K-mTOR pathway was found variety of cancers including TCL, and dual inhibition of PI3k-mTOR has been shown preclinically and clinically successful in B-cell lymphoma. However, the activity of dual inhibition of PI3k-mTOR in T-cell malignancies remain utterly unknown. GSK458 is a potent oral dual inhibitor of pan PI3K (α, β, γ and δ) and mTOR (mTOR1 and mTOR2). Preclinical study showed GSK458 had broad antitumor activity in vitro and in vivo. In 2016, phase I clinical trial of GSK458 was competed and the dosage was durable in multiple tumor types. Here we extensively characterized the activity and the mechanism of action of GSK458 in preclinical model of T-cell lymphoma alone and in combination therapies.

Methods: This study included a panel of T cell lymphoma lines as well as primary samples derived from lymphoma patient, including J45 and SupT-1 are PTCL as T-cell lymphoblastic lymphoma; whereas MJ, HH and H9 are CTCL represented Mycosis fungoides (MF), aggressive leukemic MF and the most aggressive form of CTCL Sezary syndrome (SS) respectively. In vitro viability assay was performed as single agent of GSK458 and in combination with other drugs. Differences in cellular metabolic difference were examined by ATP levels, low mitochondria potential and glucose update utilizing Cell-Titer Glo assays, DiOC6 and 2-NDG flow analysis, respectively. Cellular apoptosis and cell cycle distribution were evaluated by Annexin V and propidium iodide staining followed by flow cytometry. PI3K and mTOR downstream pathway phosphorylation status were detected by flow cytometry. Apoptosis proteins were detected by western blot. The regulative T cells were evaluated by flow cytometry.

Results: GSK458 had potent anti-tumor activity as single agent and potentiated the activity of chemotherapy or small inhibitors, such as doxorubicin, carboplatin, venetoclax, brentuximab, romdespsin and proteasome inhibitors in TCL pre-clinical models. GSK458 sufficiently blocked the downstream targets of PI3K-mTOR, such as phosphorylation status of Akt serine473 and threonine308; phosphorylation of mTOR and GSK3β. At metabolic level, GSK458 reduced ATP production, decrease glucose uptake, lowered mitochondrial membrane potential and triggered apoptosis in TCL. Moreover, GSK458 arrested the cell cycle at G1. In our study, we found after exposure to GSK458, the percentage of regulatory T cell among the total CD4+ T cells were decreased.

Conclusion: Based on these preclinical results, GSK458 appeared as a novel and promising drug that is possible to develop clinical trial in T-cell lymphoma. (Supported by Roswell Park Cancer Institute Alliance Foundation Grant) 

### Preclinical Radiotherapeutics

#3920

Differential effects of Jumonji family demethylase and EZH2 H3K27 methyltransferase inhibition on radiation sensitivity of H3K27M mutant DIPG.

Anatoly Y. Nikolaev, John B. Fiveash, Eddy S. Yang. _University of Alabama at Birmingham, Birmingham, AL_.

Diffuse intrinsic pontine glioma (DIPG) is an aggressive pediatric brainstem tumor with a 5-year survival of <1% (1). Up to 80% of DIPG tumors contain a specific K27M mutation in one of two genes encoding histone H3 (H3K27M). 60-75% of H3K27M mutations occur in gene encoding histone variant H3.3, which has been linked to reduced overall survival and poor response to first line radiation therapy (1). Recent evidence indicates that H3K27M inhibits the enzymatic activity of Polycomb Repressive Complex 2 through interaction with the EZH2 histone methyl transferase (2). A specific inhibitor of Jumonji family histone demethylase GSK-J4 was recently reported to restore H3K27 tri-methylation patterns in human DIPG cells and improve survival of H3K27M mutant orthotopic xenograft brainstem tumor models (3). Furthermore, a genetic deletion of EZH2 had also been shown to improve survival of H3K27M mouse model of DIPG, suggesting that EZH2 may be a potential therapeutic target (4). Given these data, we investigated the effects of Jumonji demethylase inhibitor GSK-J4 and EZH2 inhibitor tazemetostat on radiosensitization of human H3K27M DIPG cells. We found that GSK-J4 inhibitor had a growth inhibitory effect on H3K27M mutant DIPG cells as a single agent (96% growth inhibition at 1.5 micro-molar concentration, P<0.0001). Strikingly, GSK-J4 inhibitor also displayed radiosensitizing effects when combined with ionizing radiation at sub-micromolar concentrations of the drug (34.4% growth inhibition at 0.37 micro-molar concentration when combined with 4 Gy XRT vs 23.3% with XRT alone, P<0.0001 for both; no significant growth inhibition as single agent). Contrary to previously reported results with EZH2 inhibition in H3K27M neural stem cell model of DIPG, EZH2 inhibitor tazemetostat had no effect on human H3K27M DIPG cells as a single agent or when combined with ionizing radiation. This result is, however, not unexpected, given the proposed inhibitory effect of H3K27M mutation on EZH2 activity in DIPG disease pathogenesis. In conclusion, Jumonji family methyltransferase inhibitor GSK-4J sensitizes H3K27M DIPG cells to ionizing radiation, while EZH2 methyltransferase inhibitor has no effect. This result appears to be consistent with the recently proposed epigenetic mechanism of DIPG pathogenesis (1, 2). 1. Johung TB, et al. Diffuse intrinsic pontine glioma: new pathophysiological insights and emerging therapeutic targets. Curr Neuropharmacol. 2017;15:88-97. 2. Lewis PW, et al. Inhibition of PRC2 activity by a gain-of-function H3 mutation found in pediatric glioblastoma. Science. 2013;340:857-61. 3. Hashizume R, et al. Pharmacologic inhibition of histone demethylation as a therapy for pediatric brainstem glioma. Nat Med. 2014;20:1394-6. 4. Mohammad F, et al. EZH2 is a potential therapeutic target for H3K27M-mutant pediatric gliomas. Nat Med. 2017;23:483-492.

#3921

Poly(ADP-ribose) polymerase 1 (PARP-1) targeting radionuclides are highly cytotoxic across a panel of 20 neuroblastoma cell lines.

Hwan Lee,1 Adam Mansfield,1 Minu Samanta,2 Sean W. Reilly,1 Kuiying Xu,1 Sean D. Carlin,1 John M. Maris,2 Robert H. Mach,1 Mehran Makvandi,1 Daniel A. Pryma1. 1 _University of Pennsylvania, Philadelphia, PA;_ 2 _Children's Hospital of Philadelphia, Philadelphia, PA_.

Introduction: Neuroblastoma is the most common extracranial solid malignancy in childhood, with only up to 50% 5-year survival in high-risk cases. It overexpresses the nuclear enzyme PARP-1, which can be targeted for specific delivery of radionuclides to DNA. Two such novel radiolabeled small molecule therapeutics are [211At]MM4, containing an alpha particle-emitting astatine-211, and [125I]KX1, containing an Auger electron-emitting iodine-125.

Methods: Twenty human neuroblastoma cell lines were screened in vitro for sensitivity to [211At]MM4 and [125I]KX1, with non-targeted radionuclides and non-radioactive PARP-1 inhibitors as controls. Next, one sensitive and one resistant cell line was chosen for dosimetry based on radiopharmacology and cellular geometry properties measured specific to each therapeutic and cell line. Monte Carlo simulation was then performed to calculate the radiation dose to the cell nucleus at 50% cell kill. Relative biological effectiveness (RBE) of alpha and Auger particles against gamma irradiation was determined. Finally, the mechanism responsible for differential radiosensitivity across the twenty cell lines was examined by correlation with PARP-1 mRNA level.

Results: [211At]MM4 and [125I]KX1 showed closely correlated (R2=0.93) cytotoxicity across the twenty neuroblastoma cell lines. [211At]MM4 demonstrated 102-103 times and 108-109 times greater cytotoxic potency compared to unbound 211At and its non-radioactive PARP-1 inhibitor analog respectively. More sensitive cell lines demonstrated greater sensitivity to [211At]MM4 even when normalized against their sensitivity to alpha radiation (R2=0.40, p<0.01). [125I]KX1 also showed 104-106 times greater potency than its non-radioactive analog. No cell kill by biochemical inhibition of PARP-1 was seen at therapeutic doses of [211At]MM4 and [125I]KX1.The sensitive and resistant cell lines had similar PARP-1 binding of [211At]MM4 and [125I]KX1, but only the sensitive cell line had significantly higher RBE of alpha (10.8) and Auger (3.1) radiation compared to external gamma irradiation. RBE of alpha against Auger therapy was similar in both sensitive (3.5) and insensitive (3.6) cell lines. Cytotoxic sensitivity to PARP-1 targeted radiopharmaceutical was not correlated with PARP-1 mRNA level (R2=0.10, p=0.17).

Conclusion: Delivery of alpha and Auger emitters directly to neuroblastoma nuclear DNA by radiolabeled PARP-1 inhibitors results in enhanced cytotoxicity without requiring enzymatic PARP-1 inhibition. Sensitivity to radiolabeled PARP-1 inhibitors is characterized by increased RBE against gamma irradiation and only partially explained by inherent radiosensitivity, warranting further investigation of underlying biological factors. This promising efficacy in vitro can be examined in vivo and in other cancer types for broader application.

#3922

Biodistribution of Ra-224 and its daughter Pb-212 after intraperitoneal infusion of Ra-224 labeled microparticles in rats.

Jesper Fonslet,1 Carsten H. Nielsen,1 Lotte K. Kristensen,1 Ida S. Jorstad,2 Kim Lindland,2 Roy H. Larsen,2 Øyvind S. Bruland,3 Andreas Kjaer,4 Tina B. Bønsdorff2. 1 _Minerva Imaging, Copenhagen, Denmark;_ 2 _Oncoinvent, Oslo, Norway;_ 3 _Oslo University Hospital, Oslo, Norway;_ 4 _Department of Clinical Physiology, Nuclear Medicine & PET and Cluster for Molecular Imaging, Department of Biomedical Sciences, Rigshospitalet and University of Copenhagen, Copenhagen, Denmark_.

Introduction: Radium-224 (Ra-224) labeled calcium carbonate (CaCO3) microparticles were developed with the intent to treat micrometastases located in the abdominal cavity. The slowly degradable microparticles act as carriers for the α-emitter Ra-224 and ensure high intraperitoneal (IP) retention of the radioactivity without cellular targeting. This novel α-therapy has a short action range in tissue and is designed to confine the radiation exposure to the IP cavity, treating both linings of the peritoneal surfaces and liquid volumes. The distribution of the Ra-224 labeled microparticles was examined by planar gamma imaging, SPECT and CT. The ex vivo biodistribution of Ra-224 and its progeny Pb-212 was also determined.

Experimental procedures: Fifteen female Wistar rats were infused IP with Ra-224-labeled microparticles (200-400 kBq, 100 mg CaCO3). The injections were performed by use of a perforated catheter (pigtail catheter), followed by a Plasmalyte flush to mimic the planned clinical administration. The Ra-224 labeled microparticles were imaged using both planar gamma imaging, SPECT and CT to evaluate the distribution over time in the abdominal region. Ex vivo biodistribution was performed 2, 24, 96, 168 and 336 hours after particle infusion and organs were harvested for activity measurements. The activity was determined at 2 hours and 48 hours after harvesting for all samples. This allowed differentiation between the biodistribution of Ra-224 and the biodistribution of its progeny Pb-212. Furthermore, 4 rats were injected IP with free Ra-224 and 2 rats were injected intravenously with free Ra-224 to achieve a baseline for the biodistribution. These control animals were euthanized at 24 and 168 hours, and 168 hours, respectively.

Results: Based on both CT and SPECT images, the particles distributed to the entire peritoneum, albeit with local areas of high microparticle concentration. Over the course of the experiment there was, as expected, a modest but evident systemic leakage of Ra-224 from the particles in the peritoneal cavity. The amount was estimated by the bone uptake of radioisotopes Pb-212 and Ra-224, using IP administered free Ra-224 as reference. The retention of Ra-224 in the peritoneal cavity was found to be > 87% at 24 hours and > 77% at 168 hours. Redistribution of the progeny Pb-212 was observed as modest uptake in the kidneys.

Conclusions: High peritoneal retention of both Ra-224 and its progeny Pb-212 after IP injection of Ra-224 labeled CaCO3 microparticles, was found in rats with high translational value to the clinical setting. SPECT imaging supported distribution of the Ra-224 labeled microparticles to the entire peritoneal lumen in the animals. SPECT and CT revealed some clusters of labeled microparticles. Due to the short range of the therapeutic relevant α-particles, the clusters are not expected to have an impact on therapeutic or safety aspects.

#3923

Rationally designed combination therapies against metastatic castration-resistant (mCR) prostate cancer (PC).

Katharina Lueckerath, Andreea D. Stuparu, Soumya Poddar, Joseph R. Capri, Thuc M. Le, Caius G. Radu, Johannes Czernin. _University of California Los Angeles, Los Angeles, CA_.

Radionuclide therapy (RNT) is an emerging treatment modality for mCRPC that utilizes the radio-labeled peptidomimetic 177Lu-PSMA617 to target prostate-specific membrane antigen (PSMA) which is overexpressed on PC. Despite RNT being effective, only 50% of patients with PSMA\+ tumors respond to RNT, and relapses occur uniformly in this group. Poor understanding of the underlying resistance mechanisms to RNT represents a key barrier to development of more effective PSMA targeted RNT against mCRPC. Here, we provide insight into signaling adaptations triggered by PSMA-RNT in PC, and establish new, clinically applicable combination therapies against major resistance mechanisms to RNT.

RNT-induced (phospho)proteomic alterations in the C4-2 PC mouse model were profiled using mass spectrometry. The relevance (i) of thus identified, significantly altered kinases for mediating resistance to RNT, and (ii) of pharmacological targeting of these kinases in combination with RNT was validated across a panel of PSMA+ PC cells (immunoblot, flow cytometry, tumor cell growth inhibition), and in vivo using the C4-2 PC mouse model (treatment efficacy as determined by computed tomography volumetry).

Kinase substrate enrichment analysis of C4-2 tumors revealed a crucial role of DNA damage response (DDR) signaling in mitigating the effects of RNT. Ataxia telangiectasia and Rad3-related (ATR) was one of the most activated kinases following RNT. Treating a panel of PC cell lines with an ATR inhibitor (ATRi) in combination with ionizing radiation (IR) sensitized cells to IR, by increasing DNA damage (pH2A.X) and cell death (Annexin V/PI), and impairing DNA damage signaling (pCHEK1/2) and colony forming capacity. In mice with C4-2 tumors, combining with 177Lu-PSMA617 RNT with the ATRi resulted in synergistic tumor growth inhibition. However, tumors were not eradicated and started to re-grow ~80 days post-RNT. These findings suggested that PC can evade the effects of ATRi/RNT and targeting these escape mechanisms may result in improved tumor control. Based on our phosphoproteomic dataset we hypothesized that adding inhibitors of ataxia-telangiectasia mutated (ATM) or poly ADP ribose polymerase (PARP) to the ATRi/RNT combination might improve therapeutic efficacy. Both triple combination treatments (ATMi/ATRi/IR PARPi/ATRi/IR) resulted in significantly enhanced DNA damage, cell death, and cell growth inhibition compared to ATRi/RNT alone in all cell models tested. In ongoing studies, we investigate the efficacy of the triple combinations in vivo and will report on resulting data.

Our analysis identified DDR signaling as a major cause for limited RNT efficacy in mCRPC. Using clinically viable inhibitors of key-effectors in DDR pathways, we established rationally-designed, synergistically acting combination therapies that significantly improve PSMA-RNT in mCRPC.

#3924

PARP inhibition as a radiosensitizing strategy to improve locoregional control in inflammatory breast cancer.

Anna Michmerhuizen, Andrea Pesch, Leah Moubadder, Meleah Cameron, Amanda Zhang, Nicole Hirsh, Meilan Liu, Kari Wilder-Romans, Lori J. Pierce, Reshma Jagsi, Corey Speers. _University of Michigan, Ann Arbor, MI_.

Purpose: Sustained locoregional control of disease is a significant issue in patients with inflammatory breast cancer (IBC) with local control rates <70% at 5 years in these patients. Given the unsatisfactory outcomes, there is a clear need for intensification of local therapy, including radiation, for these patients. Inhibition of poly(adenosine diphosphate-ribose) polymerase 1 (PARP1), a DNA repair protein, represents a novel and promising strategy for radiosensitizing aggressive tumors. The purpose of this study was to investigate radiosensitization by PARP1 inhibition in IBC and to determine the mechanism of radiosensitization.

Methods: Pharmacologic PARP1 inhibitors, olaparib and veliparib, were used at various concentrations (10-5 to 10-12 M). Proliferation assays were used to determine the IC50s of these inhibitors in three independent IBC cell lines (SUM 149, SUM 190, MDA-IBC-3). Clonogenic survival assays were used to determine the radiosensitization in cell lines after pharmacologic PARP inhibition (PARPi). DNA damage was quantified using γH2AX staining in IBC cell lines. Transcriptomic, protein and signaling pathway changes were measured using RNA-Seq and RPPA, respectively. In vivo tumor growth was also measured in CB17-SCID mice with varying control and treatment groups (16-20 tumors/group) that included radiation (RT) alone, olaparib alone, and RT+olaparib.

Results: PARPi via olaparib and veliparib had limited single agent efficacy in all three independent IBC models with IC50 values >10 µM. Despite limited single agent efficacy, sub micromolar concentrations of PARPi with RT led to significant radiosensitization with radiation enhancement ratios of 1.20-1.35 in the same three models (500 nM in SUM 190, 500 nM in MDA-IBC-3, 2 nM in SUM 149). This effect was partially dependent on BRCA 1/2 mutational status. Radiosensitization was due, at least in part, to delayed resolution of double strand DNA (dsDNA) breaks at 12, 16, and 24 hours in all models. Additionally, this effect is mediated, at least in part, by a non-homologous end joining (NHEJ) mechanism and was not dependent on homologous recombination (HR). RNA-Seq and RPPA protein pathway data identify key pathways that regulate the radiosensitization in these IBC models. Additionally, xenograft studies show significant differences in tumor doubling and tripling times between treated and untreated tumors.

Conclusion: Our study demonstrates that the PARP1 inhibition improves the therapeutic index of radiotherapy in IBC cell lines. Combined PARPi with RT leads to unresolved dsDNA breaks and decreased clonogen survival. These studies provide the rationale for a phase II randomized trial of RT +/- PARPi (olaparib) in women with IBC that has recently opened for accrual (SWOG 1706 - NCT03598257). This work was supported by a grant from Komen for the Cure and NIH T-32-GM007315.

#3925

A screen for epigenetic radiosensitizers in small cell lung cancer.

Mansi K. Aparnathi,1 Lifang Song,1 Ratheesh Subramaniam,2 Richard Marcellus,2 Rima Al-awar,2 Benjamin H. Lok1. 1 _Princess Margaret Cancer Center, University Health Network, Toronto, Ontario, Canada;_ 2 _Drug Discovery Program, Ontario Institute of Cancer Research, Toronto, Ontario, Canada_.

Objective: Small cell lung cancer (SCLC) is a highly aggressive neuroendocrine malignancy. Post-platinum-based chemotherapy, rapid relapse of chemoresistant tumor is commonly seen. Unidentifiable causative mutations and from reversible nature of acquired chemoresistance, involvement of epigenetic switch regulating SCLC progression is evident. Aim of this screen is to identify epigenetic modifiers that could sensitize SCLC cells to ionizing radiation (IR).

Methods: To recapitulate molecular diversity of patients, a range of SCLC cell lines (n=10) from classic or variant and ASCL1 or NeuroD1 sub-groups, driven by varying MYC family amplifications were picked. Short term viability experiment was setup in 3 groups: (a) Epigenetic monotherapy: cells were screened with 10 epigenetic modifiers (0 to 10 µM). (b) IR monotherapy: cells were irradiated from 0 to 8 gy. (c) Combination therapy: cells were treated with different doses of epigenetic probes and IR. Radiosensitization was measured as dose modifying factor at SF63 for each epigenetic modulator.

Results: (a) Epigenetic monotherapy: All 10 cell lines were responsive to GSK-J4 (KDMi, IC50 3.1-0.9 µM), SAHA (HDACi, IC50 2.9-0.79 µM) and JIB04 (Jumonji KDMi, IC50 74-2.4 nM). Other interesting epi-probe to which 9/10 cell lines responded was JQ1 (BET-BRDi, IC50 5.7 to 0.15 µM, except SBC-5). Compounds with lesser universal potency were MS023 (PRMTi), UNC0642 (G9a/GLPi) and UNC1999 (EZH2i). All cell lines were irresponsive to PFI3 (SMARCA2/4 BRDi), BAY598 (SMYD2 MTi) and OICR-9429 (WDR5i). (b) IR monotherapy: All 10 cell lines demonstrated different sensitivities to 4 days of IR. They're ranked in descending order of radiosensitivity as H446, H82, SHP77, LX22, H889, H1092, H196, H69, SBC5, H526. (c) Epi-probes + IR combination therapy: Epi-probes ranked in descending order of number of cell lines they radiosensitized are JQ1, JIB-04, GSK-J4, SAHA, UNC0642, BAY598, MS023, UNC1999, OICR9429 and PFI3. Interestingly, drugs that demonstrated potency as a single agent did not necessarily radiosensitize the cell lines. On the contrary, certain epi-probes that were ineffective as monotherapy radiosensitized a few of the cell lines. H82 is a sensitive cell line which was radiosensitized by all epi-probes used.

Conclusions: This screen shows that treating cells with IR in conjunction with epigenetic modifiers may potentially improve therapeutic efficacy of radiotherapy in SCLC. Further validation is being done with long term colonogenic assays followed by in vivo studies for the potent radiosensitizing candidate drugs. It would be therapeutically relevant to correlate the probability of radiosensitization by an epi-probe with the molecular profile of the patients for more predictive targeted therapeutics.

#3926

**MSLN-TTC (BAY 2287411) induces immunogenic cell death and secretion of pro-inflammatory cytokines** in vitro **and triggers an immune memory effect against a mouse tumor model.**

Urs B. Hagemann,1 Pascale LeJeune,1 Jenny Karlsson,2 Christoph A. Schatz,1 Alan S. Cuthbertson,2 Hartwig Hennekes,1 Karl Ziegelbauer,1 Dominik Mumberg1. 1 _Bayer Ag, Berlin, Germany;_ 2 _Bayer AS, Oslo, Norway_.

Mesothelin (MSLN)-targeted thorium conjugate (MSLN-TTC; BAY 2287411) is the first targeted alpha therapy in clinical development for the treatment of patients suffering from MSLN-positive mesothelioma and ovarian cancer (NCT03507452). It consists of the MSLN-targeting antibody anetumab, covalently attached to a 3,2-HOPO chelator complexing the alpha-emitter thorium-227. The main mode of action of MSLN-TTC is the induction of clustered DNA double-strand breaks upon alpha decay of thorium-227, resulting in cell death. The preclinical efficacy of MSLN-TTC has been demonstrated previously. The tumor control achieved by external beam radiation is partly driven by immune-stimulatory effects (Vatner et al; Frontiers in Oncology 2014). The essential mechanisms can be attributed to different pathways, including (a) induction of immunogenic cell death and (b) activation of the STING-pathway resulting in the secretion of pro-inflammatory cytokines (Vanpouille-Box C et al; NatComm 2017). We therefore investigated whether the targeted alpha therapy MSLN-TTC is also able to induce both of these pathways in vitro. The human ovarian cancer MSLN-expressing cell line OVCAR-3 was exposed to MSLN-TTC resulting in a dose dependent upregulation of markers of immunogenic cell death, e.g. calreticulin and HMGB-1. Further, exposure of OVCAR-3 cells to MSLN-TTC resulted in the secretion of pro-inflammatory cytokines, including IFN-β, IL-6 and IP-10. The efficacy of MSLN-TTC was evaluated in a syngeneic subcutaneous tumor model. As MSLN-TTC is not cross-reactive to murine MSLN, an MC38 cell line stably transfected with human MSLN was established (MC38-hMSLN). In vitro, MSLN-TTC induced specific reduction of MC38-hMSLN cell viability. Following single dose administration to MC38-hMSLN tumor-bearing mice, MSLN-TTC induced dose dependent antitumor activity with complete tumor eradication in 10 out of 36 treated animals. Interestingly, when tumor-free animals were re-inoculated with MC38-hMSLN cells 121 days post treatment, no tumor growth was observed. In contrast, tumors grew in animals inoculated with B16F10 cells, suggesting the development of an immune memory response against MC38-hMSLN cells. In summary, the data presented demonstrate that MSLN-TTC is able to induce immunogenic cell death and secretion of pro-inflammatory cytokines in vitro. Further, MSLN-TTC monotherapy evokes an immune-stimulatory effect in vivo.

#3927

**Radium-223 α-particle radiation: Characterization of the** in vitro **effects on cancer cells in monotherapy and in combination with DNA repair inhibitors.**

Kristina Bannik, Sabine Zitzmann-Kolbe, Arne Scholz, Sabrina Jarke, Marco Jarzombek, Andreas Sutter, Gerhard Siemeister, Dominik Mumberg. _Bayer AG, Berlin, Germany_.

Purpose: Targeted alpha therapy (TAT) represents an emerging treatment approach attempting to deliver systemic radiation selectively to cancer cells and bypassing mechanisms of acquired resistance while minimizing systemic toxic effects. Radium-223 dichloride (Ra-223) is a first-in-class TAT shown to prolong survival in patients with metastatic castration-resistant prostate cancer and bone metastases. Yet, the understanding of the biological effects of α-particle irradiation is still limited. Here, we used a novel in vitro assay designed to assess alpha irradiation specific effects to investigate the biological effects of Ra-223 in various cancer cell lines. Furthermore, the combination of α-particle irradiation with DNA repair inhibitors was tested.

Methods: The following cancer cell lines were used: lung (H460), ovarian (OVCAR-3, COV362, COV644, ES2) and prostate (22Rv1, LNCaP). The effects of different radiation doses and exposure times of Ra-223 on various biological parameters were analyzed using a Transwell® system where cells grow on a 10 µm membrane located on top of an underlying Ra-223 coating.

Results: After 4 hours of exposure to α-particle irradiation (5, 10 and 20 kBq/3.8 cm2), a dose-dependent induction of DNA double strand breaks (DSB) as assessed by 53BP1 positivity was observed. Exposure to α-particle irradiation (10 and 20 kBq/3.8 cm2) for 1 hour dose-dependently induced DNA damage in a comet assay. Exposure to α-particle irradiation (5 kBq/3.8 cm2) for 1, 4 and 8 hours time-dependently reduced the surviving fraction in the subsequent colony formation assay. The frequency of micronuclei formation was increased in an activity- and time-dependent manner after α-particle exposure at 5, 10, 20, 40 kBq/3.8 cm2 for 1, 4 and 8 hours. In the same assay, the number of dead cells was significantly increased after an 8-hour exposure in all dose groups tested. Pre-incubation with the ATR inhibitor BAY 1895344 (5 nM for 2 hours prior to radiation) synergistically enhanced micronuclei formation (< 5% vs 12% vs 42% in ATR inhibitor only vs α-particle irradiation only vs ATR inhibitor + α-particle irradiation, respectively).

Conclusion: Using several cancer cell lines, we demonstrated that short term exposure to α-particle irradiation in the range of 1 to 8 hours is sufficient to induce DNA damage including DNA DSB which leads to cellular damage and cell death. Synergistic effects were observed when Ra-223 was combined with the ATR inhibitor BAY 1895344 .

#3928

N-cadherin(CDH2) expression in mesenchymal glioblastoma modulates radiation sensitivity.

Christopher P. Cifarelli, J. Austin Vargo, Ian MacFawn, Steven M. Frisch. _West Virginia University, Morgantown, WV_.

Background: Radiation is a mainstay of treatment in patients diagnosed with glioblastoma. Despite maximal surgical and chemo-radiotherapeutic treatment, the disease remains uniformly fatal, with local disease recurrence within the previously radiated field comprising the major pattern of progression. According to the molecular subtyping of GBM, there is a predominance of mesenchymal GBM cells within the recurrent tumor mass, with characteristic increased expression of CD44, N-cadherin, L1CAM, TWIST1 and phosphorylated STAT3. Moreover, these populations of cells are known to be resistant to additional salvage radiation. The current study aims to evaluate the role of N-cadherin in the relative radiation resistance of mesenchymal subtype GBM.

Methods: Overall survival data from the TCGA was analyzed using Cox regression hazard ratios based on N-cadherin expression across multiple molecular subtypes of GBM along with age and MGMT covariates. Using the U3035 and U87MG cells, the analyses of N-cadherin expression or exogenous exposure in the setting of external beam radiation were performed via stable shRNA knockdowns or the use of recombinant human N-cadherin ectodomain-Fc fusion proteins in clonogenic survival assays. Orthotopic implantations were performed in NSG mice with the endpoints of overall survival and tumor size, as determined by luciferase imaging. Subcellular localization of RAD51 was accomplished by confocal microscopy. Statistical analyses were performed using SPSS and GraphPad Prism with significance at p<0.05.

Results: N-cadherin expression above the median among the mesenchymal subtype of GBM in the TCGA dataset correlated with a significant decrease in overall survival (HR 1.924, p=0.029). The elimination of N-cadherin expression in normally high expressing U3035 GBM cells led to a significant increase in the ability of radiation to induce cell death in clonogenic assay (17.2% U3035 vs 2% U3035 Δ N-cadherin at 8Gy; 12.7% U3035 vs 1.75% U3035 Δ N-cadherin at 20Gy) and an increase in the presence of double stranded DNA breaks (DSBs) on TUNEL assay. N-cadherin ectodomain-Fc fusion protein exposure in U87 cells resulted in a decrease in NSG mouse mean OS (17.6±0.25 days) compared to control radiated mice (22.3±1.1 days). Conversely, N-cadherin knockdown in U3035 cells resulted in further decrease in tumor bioluminescence beyond that seen in 4Gy x 4 radiation treatment of native U3035 xenografts (21% vs 4% of baseline).

Conclusions: The reduction of N-cadherin expression in mesenchymal U3035 GBM cells increases the efficacy of radiation-induced cell death. This finding is accompanied by a decrease in the nuclear localization of RAD51 following DSBs induced by ionizing radiation, indicating a potential crosstalk between DNA damage repair mechanisms and calcium dependent cell-cell adhesion.

#3929

Hypothesis-driven biomarkers for radiotherapy stratification from multi-omic profiling in rectal cancer biopsies.

Enric Domingo,1 Andrew Blake,1 Susan Richman,2 Keara Redmon,3 Matthew Alderdice,3 Aikaterini Chatzipli,4 Claire Hardy,4 Celina Whalley,5 Chieh-Hsi Wu,1 Andrew Beggs,5 Ultan McDermott,4 Phillip Dunne,3 Ian Tomlinson,5 Graeme I. Murray,6 Leslie M. Samuel,7 Tim Maughan1. 1 _University of Oxford, Oxford, United Kingdom;_ 2 _Leeds Institute of Cancer and Pathology, Leeds, United Kingdom;_ 3 _Queens University, Belfast, United Kingdom;_ 4 _Wellcome Trust Sanger Institute, Cambridge, United Kingdom;_ 5 _University of Birmingham, Birmingham, United Kingdom;_ 6 _University of Aberdeen, Aberdeen, United Kingdom;_ 7 _Aberdeen Royal Infirmary, Aberdeen, United Kingdom_.

Background: Neoadjuvant radiotherapy is commonly used to treat rectal cancer but patients have different levels of response and/or toxic effects. Although a range of molecular biomarkers potentially associated with radiotherapy stratification have been described, they have never been formally tested.

Methods: As part of the Stratification in COloRecTal cancer (S:CORT) programme, we collected a cohort of 135 rectal biopsies from patients subsequently treated with neoadjuvant radiotherapy and capecitabine as part of their standard of care. We attempted multi-omic profiling and tested by linear regression different pre-defined, hypothesis-driven biomarkers for radiotherapy response: Hypoxia signature, DNA Damage Response Deficiency (DDRD), Consensus Molecular Subtypes (CMS), ColoRectal Instrinc Subtypes (CRIS), RadioSensitivity Index (RSI), TGF-β fibroblast signature, mutations in APC, TP53, KRAS and ATM/ATR, Mutation burden, Chromosomal instability, 8 different Immunophenotypes, stromal content and CpG Island Methylator Phenotype (CIMP).

Results: Profiling of these small biopsies was successful for RNA arrays, targeted DNA sequencing and DNA methylation arrays in 131 (97%), 112 (83%) and 70 (52%) paraffin blocks respectively. After adjustment by the known clinical confounders T and N stage, seven candidates were found to be statistically associated with complete pathological response: DDRD scores, Cytotoxic lymphocytes, B lineage and CMS1 with radiosensitivity and TGF-β fibroblasts, RSI and CMS4 with radioresistance. Multivariable stepwise regression models showed that B lineage, CMS1 and TGF-β fibroblasts were independently associated with complete response (OR 36.61 [3.64-367.81], p=0.002; OR 43.48 [5.97-316.53], p=0.0002; OR 0.11 [0.01-0.81], p=0.03 respectively). The model showed good predictive value in a ROC curve with AUC=0.84. A statistically optimal cut-off resulted in 86% specificity and 71% sensitivity with 82% patients correctly classified as complete or non-complete responders.

Conclusions: Multi-omic profiling from small biopsies is feasible. The tumor microenvironment is a key player for prediction of complete response to radiotherapy in rectal cancer which has potential to be informative for patient stratification and modulated to improve outcome.

#3930

Oligosaccharyltransferase inhibition reduces glioma cell invasion and migration.

Marta Baro,1 Jann N. Sarkaria,2 Joseph N. Contessa1. 1 _Yale Univ., New Haven, CT;_ 2 _Mayo Clinic, Rochester, MN_.

Purpose: Glioblastoma (GBM) is the most common and aggressive primary brain neoplasm. Cell migration, invasion, and resistance to therapy lead to a high rate of recurrence in GBM. Glycoprotein receptor overexpression or activation caused by mutation is critical for the development and progression of many tumors including GBM. N-linked glycosylation is an endoplasmic reticulum co- and post-translational modification common to RTKs and other glycoproteins that is crucial for receptor activation. We hypothesized that altering N-linked glycosylation with an oligosaccharyltransferase (OST) inhibitor (NGI-1), could effectively disrupt GBM cell proliferation, migration, and invasion.

Experimental Procedures: The effects of OST inhibition on migration and invasion were assessed in vitro by wound healing assay, Boyden chamber assay and 3D Matrigel assay in glioma cell lines (D54, SKMG3, U251, T98G and 42-MG) and in patient-derived cell lines (GBM6, GBM26 and GBM76) in the presence or the absence of NGI-1. Cell proliferation was determined by the MTT assay over 5 days of drug treatment. Radiation survival was evaluated by clonogenic assay. Cell adhesion molecules were measured by phospho-blot assay.

Results: OST inhibition reduced cell proliferation in a dose-response manner with a significant reduction of 50% or greater over a time course of 5 days. These responses were observed in 6 of the 8 glioma and patient-derived cell lines tested while only small effects were found in the less sensitive U251 and GBM6 cell lines. A 70 to 80% reduction of tumor cell invasion was observed after NGI-1 treatment in T98G, SKMG3 and GBM76 cells with a 40 to 50% reduction in U251, D54 and GBM6 cells. Interestingly, neither 42-MG or GBM26 cells showed a decrement in invasion with the treatment of NGI-1, suggesting this effect is mediated by discrete cell signaling pathways. These results also correspond to a significant reduction of radiation clonogenic survival caused by OST inhibition in SKMG3 and D54 cells. In matrigel assays significant changes in 3D tumor growth and morphology were also observed over a time course of 72h following NGI-1 treatment. Together this work shows that cell lines with ErbB-driven signaling (D54 and SKMG3) and patient-derived cell lines with the A289T EGFR activating mutation (GBM26 and GBM76) are sensitive to OST inhibtion with NGI-1 treatment. Changes in glycosylation of cell adhesion molecules, such as integrins and NCAMs, were also observed and studies to define the glycoprotein targets that regulate glioma migration and invasion are underway.

Conclusion: This study suggests that the inhibition of N-glycosylation with a small molecule OST inhibitor suppresses migration and invasion of glioma cell lines and GBM patient-derived cell lines and supports further preclinical investigation in malignant glioma.

#3931

The irradiated tissue microenvironment and its role in breast cancer recurrence: Enhanced macrophage infiltration promotes tumor cell recruitment.

Benjamin C. Hacker,1 Steven M. Alves,1 Dadi Jiang,2 Albert C. Koong,2 Amato J. Giaccia,3 Edward E. Graves,3 Marjan Rafat1. 1 _Vanderbilt University, Nashville, TN;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 3 _Stanford University, Stanford, CA_.

Triple negative breast cancer (TNBC) recurrence rates remain high despite aggressive therapeutic intervention, including surgery, chemotherapy, and radiotherapy (RT). Recent studies suggest that circulating tumor cell re-seeding of primary tumors may facilitate recurrence rather than persistent tumor cells in the irradiated surgical bed. However, the role of the microenvironment in recurrence is not well understood. An emerging risk factor for breast cancer patients is lymphopenia or abnormally low systemic lymphocyte counts following therapy. In this study, we examined whether lymphopenia following therapy correlates to local recurrence. To investigate the relationship between prolonged low lymphocyte count and recurrence, we evaluated radiation effects on tumor and immune cell recruitment to normal tissues using an orthotopic breast cancer model of lymphopenia. We tested the hypothesis that local recurrence in some instances is due to the attraction of circulating tumor cells to irradiated tissues. Local cytokine secretion as well as protein expression levels were analyzed to evaluate how tumor-stromal interactions modulate tumor cell recruitment to sites of damage. Recurrence free survival at 5 years for TNBC patients with sustained low ALC 1-5 months after RT (n=37) was 62.5% compared with 97% for patients with normal ALC (n=46; p<0.0001). Ex vivo BLI analysis revealed that normal tissue irradiation attracted tumor cells to the mammary fat pad (MFP) and surrounding tissues in BALB/c mice with depleted CD8+ T cells. Macrophage infiltration was greatly enhanced in BALB/c mice with depleted CD8+ T cells (p<0.0001) and necessary for tumor cell recruitment. Luminex analysis of MFPs in mice lacking CD8+ T cells after RT showed enhanced cytokine secretion of factors that regulate the inflammatory microenvironment and influence tumor cell proliferation and invasion. RPPA analysis of irradiated MFPs in the absence of CD8+ T cells revealed factors that promote epithelial-mesenchymal transition. Taken together, these results demonstrate that the response of the tissue microenvironment to therapy is dependent on the immune milieu and may encourage local recurrence. Our study establishes the importance of considering tumor subtype and immune function when assessing failure and outcome risks in breast cancer. We show that normal tissue radiation response may play a role in modulating tumor and immune cell migration. These results suggest that the stroma facilitates local recurrence in breast cancer through influencing immune and tumor cell behavior following RT.

#3932

Enhancing the effectiveness of radiation therapy with arabinoxylan rice bran (MGN-3/ Biobran) in mouse bearing solid tumor.

Nariman K. Badr El-Din,1 Sayed K. Areida,1 Kavan O. Ahmed,2 Mamdooh Ghoneum3. 1 _Mansoura Univ., Mansoura, Egypt;_ 2 _Al Qalam University College, Kirkuk, Iraq;_ 3 _Charles R. Drew University of Medicine and Science, Los Angeles, CA_.

Purpose: Enhancing tumor radio-responsiveness via natural radiosensitizer agents is a promising approach for improving the efficacy of radiation therapy. In the present work, we examine the radiosensitizing effect of arabinoxylan rice bran (MGN-3/Biobran), a potent biological response modifier, on fractionated X-ray irradiation of Ehrlich solid tumor-bearing mice and elucidate the molecular mechanism underlying its effect.

Materials and Methods: Swiss albino mice were inoculated in the thigh with Ehrlich ascites carcinoma cells. Mice bearing tumors were treated with Biobran (40mg/kg/day, i.p.), 5 days/week, beginning on day 11 post inoculation until day 30. Mice received X-ray ionizing radiation (IR) with or without Biobran at a dose level 6 gray (Gy) divided into three fractionated doses (2 Gy) each with a dose rate of 0.85 Gy/min on days 12, 14 or 16 post inoculation. Tumor growth was measured; cell cycle analysis, apoptosis, and apoptotic regulator expression were analyzed by flow cytometry and RT-PCR; DNA fragmentation was detected by gel electrophoresis; and safety parameters were evaluated.

Results: IR-induced tumor regression was enhanced in mice treated with Biobran. Tumor weight was reduced relative to control by 31% for IR alone, by 46% for Biobran alone, and by 57% for co-treatment. Relative to control, Biobran alone and IR alone arrested the hypodiploid cells in the sub-G1-phase by 102% and 85%, respectively, signifying apoptosis, while co-treatment enhanced IR induced apoptosis by 123% and maximized the apoptosis/proliferation ratio. Co-treatment also potentiated early apoptosis in cancer cells as detected by Annexin V/PI and augmented DNA laddering pattern. In addition, co-treatment profoundly upregulated protein and mRNA expression of p53, Bax, and caspase-3 and downregulated Bcl-2 expression. Bax/Bcl-2 ratio was increased relative to control by 3.4 fold for IR alone and maximized at 9.5 fold for co-treatment. With regard to safety parameters, co-treatment markedly modulated the decrease in body weight and the increase of liver and spleen weight of mice treated with IR alone, and it decreased IR-induced elevation of serum enzyme levels (AST, ALT, and GGT).

Conclusion: This study concludes that MGN-3/Biobran potently enhances radiation therapy-induced tumor regression by potentiating apoptosis and minimizing toxicities related to radiation therapy. MGN-3/Biobran may have utility in human cancer patients undergoing radiotherapy and could warrant clinical trials. MGN-3/Biobran was supplied by Daiwa Pharmaceutical Co., Ltd., Japan.

#3933

NADPH oxidases 4 inhibition using a hyaluronic acid nanoparticle drug delivery system sensitized therapeutic response to chemo and radiotherapy in drug resistant breast cancer.

Lei Zhu,1 Yi Zhao,1 Wei Ping Qian,1 Dazhi Wang,1 Bing-Hua Jiang,2 Lily Yang1. 1 _Emory University, Atlanta, GA;_ 2 _University of Iowa, Iowa City, IA_.

Objectives: Resistance to therapy is the major unmet challenge in clinical management of breast cancer. NADPH oxidases 4 (NOX4) is a critical NADPH oxidase subunit because it constitutively reacts with hydrogen peroxide (H2O2), generating ROS that is associated with tumorigenesis, metastasis, and invasion. A high level of NOX4 activity has been associated with drug resistance in human cancers. To improve the therapeutic efficacy, we have developed a hyaluronic acid nanoparticle (HANP) carrying a NOX4 inhibitor and examined the anti-tumor effect in combination with radiotherapy (RT) or chemotherapy.

Methods: Biodegradable polymeric HANP was synthesized and encapsulated with GKT831 (HANP/GKT831), a novel inhibitor of NOX4. Targeted delivery of GKT831 with HANP was first studied by optical imaging in an orthotopic human breast cancer patient tissue derived xenograft (PDX) model. Subsequently, the ability of HANP/GKT831 in inhibition of tumor cells and sensitization of tumor cells to chemotherapy and RT was evaluated in breast PDX models following intravenous administration of HANP/GKT831 (containing 5 mg/kg equivalent dose of GKT831) in combination with Doxorubicin (DOX, 5 mg/kg) and RT (2 Gy) for 5 times, once per week. Furthermore, mechanism of NOX4 inhibition on improving therapeutic response in breast cancer was investigated.

Results: We have developed HANP/GKT831 with drug loading efficiency of 15% (w/w) determined by high performance liquid chromatography (HPLC). The selective accumulation of HANP/GKT831 in breast PDX tumors following systemic delivery was demonstrated by optical imaging. Following five treatments, nude mice bearing breast PDX tumors that received HANP/GKT831 in combination with DOX or RT had significant stronger tumor growth inhibition compared with either DOX or RT alone (73.71±13.87% and 79.33±19%, respectively) as well as the non-treatment control group (86.33±16.25% and 78.15±14.27%, respectively). Examination of tumor tissues revealed that the level of poly (ADP-ribose) polymerase (PARP) expression was reduced in tumors treated with HANP/GKT831 in combination of RT or DOX, which induces cell death through DNA damage.

Conclusions: We have developed a polymeric HANP carrying a NOX4 inhibitor (HANP/GKT831). We found that HANP/GKT831 could be efficiently delivered into tumors following systemic administration and significantly enhanced tumor response to Dox treatment or radiotherapy in two multi-drug resistant breast PDX tumor models. Therefore, HANP/GKT831 is a promising therapy agent for targeted therapy of breast cancer. Currently, mechanism of the effect on chemo- and radiotherapy sensitization is under investigation. One of the possible mechanisms is the downregulation of DNA-damage repairing functions, such as downregulation of DNA damage repair enzyme PARP.

#3934

Bcl2-expressing quiescent type B neural stem cells in the SVZ are resistant to concurrent temozolomide/X-irradiation.

Brent D. Cameron, Geri Traver, Joseph T. Roland, Daniel Dean, Levi Johnson, Kelli L. Boyd, Rebecca A. Ihrie, Jialiang Wang, Michael L. Freeman. _Vanderbilt University School of Medicine, Nashville, TN_.

The subventricular zone (SVZ) is the largest source of neural stem cells (NSCs) in the adult brain. Emerging research indicates that NSCs within the SVZ may be cells of origin for WHO grade IV astrocytoma (glioblastoma, GBM)1. GBM consists of multiple fractions of proliferative and/or quiescent stem-like cells that are thought to be lineally related. GBM located adjacent to the SVZ are very resistant to standard of care concurrent temozolomide (TMZ)/X-irradiation (XRT) therapy, a consequence, it is hypothesized, of their NSC origin2. An important, unanswered question is the origin of this resistance. While a significant effort has been undertaken to study proliferating cells, the origins of quiescent cell resistance are not well understood. Normal NSCs adjacent to the SVZ are mainly quiescent. We rationalized that a fundamental understanding of the response of quiescent NSCs to TMZ/XRT would be informative and aid in our understanding of GBM resistance. For 5 consecutive days cohorts of C57BL/6 mice were administered TMZ (0 or 50 mg.kg i.p.). One hr later 0 or 2 Gy was administered to the brain. Transcardial perfusion was performed on day 6 for half the mice. The remaining mice received adjuvant TMZ (100 mg/kg) or vehicle on days 19-22 and transcardial perfusion was performed on day 82. 10 µm coronal brain sections were obtained and immunostained for well characterized markers of type B NSCs (GFAP and Sox2) and type A neuroblasts (Dcx). Immunofluorescence was imaged using a Leica Aperio Versa 200 slide scanning microscope. Cell Profiler software was used to quantify type B and A cells in the SVZ in all cohorts. Proliferating type A cells were exquisitely sensitive to 5 days of concurrent TMZ/XRT treatment whereas quiescent NSCs located within 30 µm of a dorsal or dorsolateral ventricle were very resistant. NSCs in mice exposed to concurrent and adjuvant therapy were also resistant and importantly, able to repopulate type A cells to sham/control levels. 53BP1 foci formation, a surrogate for DNA DSBs, was quantified in Sox2- and Dcx-expressing cells using confocal microscopy following a single TMZ/XRT exposure. Foci formation, measured 6 min to 24 hrs after TMZ/XRT, was not statistically different between cell types (P>0.05). Because TMZ/XRT induced an apoptotic response in A but not in B cells, as marked by cleaved Caspase-3 staining, we investigated expression of Bax and Bcl2 on a per cell basis. Bax expression was not significantly different for type A or B cells (P>0.05). In contrast, type B NSCs expressed 5-fold more Bcl2 than type A neuroblasts (P< 0.001). In conclusion, we demonstrate that type A neuroblasts are sensitive to TMZ/XRT but can be repopulated by inherently resistant type B NSCs given sufficient time. The resistance of quiescent NSCs to TMZ/XRT is associated with high basal expression of anti-apoptotic proteins. 1Lee et al Nature 2018, 560:243-47; 2Smith et al J Neurooncol 2016, 128:207-16

#3935

PLK4 inhibition as a strategy for non-small cell lung cancer radiosensitization.

Thomas J. Hayman,1 Mark R. Bray,2 Jacqueline M. Mason,2 Tak W. Mak,2 Joseph N. Contessa1. 1 _Yale University, New Haven, CT;_ 2 _University Health Network, Toronto, Ontario, Canada_.

Background/Purpose: Lung cancer remains the leading cause of cancer-related death. Concurrent chemoradiation is the standard of care for locally advanced non-small cell lung cancer (NSCLC). Despite advancements in treatment, local control remains an issue and survival is suboptimal, highlighting the need for novel therapeutic approaches. Polo-like kinase 4 (PLK4) is a serine/threonine kinase that controls centriole duplication. PLK4 is highly expressed in multiple malignancies including lung cancer. CFI-400945 is a specific PLK4 inhibitor currently undergoing clinical trial evaluation. As radiation causes cell death primarily via a mitotic death, we hypothesized that disruption of the mitotic machinery by inhibition of PLK4 would enhance the effects of radiation.

Methods: The effects of CFI-400945 on cell survival and radiation sensitivity were examined using the clonogenic survival assay in two KRAS-mutant lung cancer cell lines (H460 and A549). Confocal microscopy was utilized to determine the effects of PLK4 inhibition on radiation-induced mitotic catastrophe (defined as the presence of 2 or more distinct nuclear lobes within a single cell). Cell cycle distribution by flow cytometry was determined after treatment with CFI-400945 and radiation.

Results: Exposure of H460 and A549 lung cancer cell lines to 10nM CFI-400945 for 48h reduced clonogenic survival to 63.9% and 83.1% respectively. CFI-400945 treatment of H460 and A549 cells resulted in radiosensitization of both cell lines with dose enhancement factors (DEF) of 1.60 and 1.31. To understand the mechanism of enhanced radiosensitivity with CFI-400945 treatment the effects on radiation-induced mitotic catastrophe were determined. In both H460 and A549 cells the combination of radiation and PLK4 inhibition enhanced the proportion of cells undergoing mitotic catastrophe. The combination of CFI-400945 and radiation resulted in increased G2/M arrest and appearance of >4N populations by flow cytometric analysis when compared to drug or radiation treatment alone.

Conclusion: PLK4 inhibition with CFI-400945 enhances the radiosensitivity of lung cancer cell lines via mitotic catastrophe and G2/M arrest and these data support additional pre-clinical testing.

#3936

Efficacy of single agent radium-223 in the syngeneic MBT-2 bladder cancer bone growth model in mice.

Tiina E. Kähkönen,1 Mari I. Suominen,1 Jenni H. Mäki-Jouppila,1 Birgitta Sjöholm,2 Ilmari Ahonen,3 Dominik Mumberg,4 Karl Ziegelbauer,4 Jussi M. Halleen,1 Sanna-Maria Käkönen,2 Arne Scholz4. 1 _Pharmatest Services Ltd., Turku, Finland;_ 2 _Aurexel Life Sciences Ltd., Askainen, Finland;_ 3 _Vincit Oy, Turku, Finland;_ 4 _Bayer AG, Berlin, Germany_.

Radium-223 dichloride (Ra-223, Xofigo®) is a targeted alpha therapy which prolongs the survival of castration-resistant prostate cancer (CRPC) patients with symptomatic bone metastases. As a calcium-mimetic, Ra-223 selectively binds to hydroxyapatite and targets areas of high bone turnover such as bone metastases. Here, we report the effects of Ra-223 on development and progression of osteolytic bone lesions and on survival in the syngeneic intratibial MBT-2 murine bladder cancer model in immunocompetent mice.

Female 5-7-week-old C3H/HeNHsd mice were injected intratibially with 5x105 MBT-2 cells on day 0. The mice were stratified based on body weight (n=40 per group) on day 5 and Ra-223 (300 kBq/kg, i.v.) or vehicle control was administered on days 5, 33, and 61. The tumor-induced osteolytic lesion area was imaged using radiography on days 10, 18, 25, 76, and at sacrifice. The mice were sacrificed when they met the pre-defined sacrifice criteria (> 20% body weight loss, palpable tumor outgrowth from the bone or general deterioration of health status). The study was terminated on day 158.

In this study, the first mice met the sacrifice criteria on study day 24 and the survival curves plateaued at 52% after 84 days in the control group and at 68% after 112 days in the Ra-223 treatment group. Survival on day 40 was significantly improved in the Ra-223 treatment group compared to the vehicle control (p< 0.001), but the effect was not statistically significant at the end of the study. Based on X-ray imaging 25 days after cancer cell inoculation, Ra-223 treatment also significantly decreased the bone lesion area compared to the vehicle treatment (p=0.0041). After initially sacrificing the mice with extensive bone lesions, tumor growth in bone was stabilized around day 25. The main reason for sacrifice at a later stage was breathing difficulties due to lung metastases, and in the Ra-223-treated mice only one mouse was sacrificed due to bone metastases. Ra-223 treatment was well-tolerated; no body weight loss was observed.

In summary, radium-223 dichloride (Xofigo®) demonstrates moderate single agent in vivo efficacy in the intratibial MBT-2 murine bladder cancer model of osteolytic bone metastasis. Retaining a functional immune system in syngeneic mouse models is particularly relevant for the characterization of potential immune-stimulatory effects of Ra-223. The promising results obtained in this model warrant further investigation of Ra-223 in combination with immuno-oncological treatments like checkpoint inhibitors.

#3937

MSLN-TTC (BAY 2287411) demonstrates increased activity in comparison to standard of care chemotherapy in models of acquired drug resistance.

Urs B. Hagemann,1 Sabine Zitzmann-Kolbe,1 Alexander Kristian,2 Carolyn Sperl,1 Christoph A. Schatz,1 Roger M. Bjerke,2 Alan S. Cuthbertson,2 Hartwig Hennekes,1 Karl Ziegelbauer,1 Dominik Mumberg1. 1 _Bayer Ag, Berlin, Germany;_ 2 _Bayer AS, Oslo, Norway_.

Mesothelin (MSLN)-targeted thorium conjugate (MSLN-TTC; BAY 2287411) is a targeted alpha therapy consisting of the MSLN-targeting antibody anetumab, covalently attached to a 3,2-HOPO chelator complexing the alpha-emitter thorium-227. We previously demonstrated the potent in vivo activity of MSLN-TTC in cell line and patient-derived xenograft models, and further, demonstrated its combination potential with DNA damage repair inhibitors. MSLN-TTC is currently in clinical development for the treatment of patients with MSLN-positive mesothelioma and ovarian cancer (NCT03507452). Development of cellular resistance mechanisms are often co-evolutionary to the treatment regimen given to the respective individual. As such, it is well described that ovarian cancer patients have a decreased overall survival when tested positive for markers of acquired drug resistance (ADR), e.g. phospho-glycoprotein (P-gp) (Herzog TJ et al; Oncotarget 2016). However, MSLN-TTC should also be effective independent of P-gp expression, as the major mode of action of MSLN-TTC is the induction of irreparable DNA double-strand breaks upon alpha decay by thorium-227, resulting in apoptotic cell death. To confirm this hypothesis, we first determined the involvement of P-gp in cellular resistance mechanisms in ovarian cancer. To this end, immunohistochemical staining of ovarian cancer patient samples for P-gp was performed. Expression of P-gp was observed in 15% (7/48) primary and relapsing specimens. We then evaluated the potency of MSLN-TTC in MSLN-expressing cell line models in vitro using the isogenic cell line pair OVCAR-8 (P-gp negative) and NCI-RES-ADR (P-gp positive) in comparison to the standard of care (SOC) therapies cisplatin, paclitaxel, doxorubicin and vinorelbine. Whereas paclitaxel and doxorubicin showed a 100- and 10-fold decrease in in vitro potency on the P-gp positive cell line NCI-RES-ADR vs the P-gp negative cell line OVCAR-8, the potency of MSLN-TTC on the two isogenic cell lines remained unaffected.

To further confirm and explore these observations, in vivo xenograft models were conducted. Similar to the in vitro results, the in vivo efficacy of MSLN-TTC was not affected by the P-gp status in the OVCAR-8 vs NCI-RES-ADR xenograft model. In contrast, limited efficacy of paclitaxel and doxorubicin was observed in the P-gp-positive MSLN-expressing NCI-RES-ADR and Hela-MATU-ADR models. Similar, vinorelbine showed no efficacy in the Hela-MATU-ADR xenograft. In summary, these data demonstrate that the efficacy of MSLN-TTC is independent of the P-gp status. Further, these data suggest that MSLN-TTC may be active in patients relapsing after standard therapy.

#3938

Acoustic immune priming of radiation therapy eradicates primary tumor and reduces distant metastases in triple-negative murine breast cancer.

Saurabh Singh, Claudia Gutierrez-Chavez, Karin Skalina, Wade Koba, Indranil Basu, Stephen Barry, Chandan Guha. _Albert Einstein College of Medicine, New York, NY_.

Introduction: Recently, we have shown that low energy focused ultrasound (FUS) treatment reversed tumor-induced T cell tolerance in the B16F10 melanoma model. FUS also induced protein unfolding and translocation of cellular chaperones on the cell surface, thereby providing danger signals for immune activation. To further study immune priming using FUS, we designed a novel immune priming ablation therapy, whereby non-ablative low energy FUS was delivered before ablative radiation therapy in murine triple-negative breast cancer for generating an in situ vaccine.

Methods: Orthotopic, palpable (>5mm) 4T1 tumors, a poorly immunogenic, aggressive, triple-negative murine metastatic mammary carcinoma in Balb/c mice, were treated with FUS (15W (100 W/cm2) and 75W (500W/cm2); 100% duty cycle for 3 seconds using custom transducer in multi-frequency mode with or without RT (20Gy) for three consecutive days. Tumor growth, survival, and metastases were monitored. The tumor immune-infiltrate population was analyzed at day 7 post-treatment using flow cytometry. 18F-FDG PET was used to image distant metastases. For the abscopal study, mice were implanted with a primary (treated) and secondary (non-treated) contralateral tumor and monitored for tumor growth on both sites.

Results: FUS treatment (75W) in combination with RT eradicated 86% (6/7) of tumors. Tumor growth was also considerably reduced in RT alone and 15W+RT groups compared to non-treated, with a relapse of 43% (3 of 7) in both the groups. There was significant reduction in regional metastatic spread in draining lymph nodes (DLN) (RT, 50% versus FUS (75W)+RT, 22%) and pulmonary metastases (RT, 60% versus FUS (75W)+RT, 22%). Tumors relapsed in DLN and lungs after FUS+RT treatment in only 2 out of 9 mice. At 7 weeks, FDG-PET scans showed no uptake in FUS+RT treated groups, compared to metastatic uptakes in lungs, lymph nodes, and liver in 3 out of 7 mice, treated with RT alone. There was a significant increase in tumor-infiltrating CD3+ lymphocytes, IFNg+CD8 T cells, Granzyme B+ CD8+ T cells and NKT cells, upon treatment with FUS+RT compared to the RT alone group. In contrast to RT alone, FUS+RT promoted suppression of untreated abscopal tumors in contralateral flank.

Summary: In this study, we found that local treatment with FUS+RT resulted in primary tumor control, a significant reduction in distant metastasis with improved long-term survival and suppression of unirradiated, abscopal tumors, indicating that focal therapy with a combination of acoustic immune priming with ablative RT induced systemic immunity. Our acoustic immune priming ablation therapy provides a foundation for using physical energy-based focal therapies in combination with immunotherapies for in situ tumor vaccines.

#3939

Exploiting tumor treating fields induced downregulation of BRCA1 pathway for novel combination therapies.

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

A new physical cancer treatment modality called Tumor Treating Fields (TTFields) has demonstrated effectiveness in the treatment of solid tumors in vitro and in vivo. TTFields therapy is a non-invasive cancer treatment modality that delivers low intensity (1-3 V/cm), intermediate frequency (100-300 kHz) alternating electric fields to the tumor. The TTFields delivery device called Optune (NovoCure), has been approved for recurrent and newly diagnosed glioblastoma and clinical trials are ongoing for other cancers. The primary mechanism of TTFields action is thought to be interference with mitosis. We monitored temporal gene expression changes in 5 non-small cell lung cancer (NSCLC) cell lines whose response to TTFields is variable and found that the expression of the BRCA1 DNA damage repair pathway, as well as other DNA repair/checkpoint pathways were significantly downregulated (P<0.05) upon TTFields exposure. We monitored the dynamics of DNA double strand break repair to study functional relevance of BRCA1 pathway downregulation and found that TTFields treatment slowed the repair of ionizing radiation (IR)-induced DNA damage and interestingly, TTFields alone increased the number of γH2AX foci and the incidence of chromatid aberrations. Furthermore, as a function of TTFields exposure time, decreased replication fork speed and increased R-loop formation was identified, suggesting that TTFields induce replication stress. We carried out DNA/RNA immunoprecipitation sequencing (DRIP-Seq) analysis and are currently mapping R-loop formation within the genome. Based upon these newly identified mechanisms of TTFields action, i.e., enhancing DNA damage, reducing DNA repair capacity and increasing replication stress, led us to hypothesize that by applying TTFields first, a conditional vulnerability environment would develop rendering cells more susceptible to novel combination therapies. For example, cell killing by ionizing radiation exposure is enhanced when NSCLCs are exposed to TTFields after radiation. However, TTFields exposure prior to IR treatment is more effective compared to IR treatment prior to TTFields treatment. Furthermore, the combination of cisplatin together with TTFields also suggests synergistic effects in NSCLC cells. TTFields exposure concomitant with the PARP inhibitor Olaparib followed by radiation was synergistic compared to radiation or Olaparib alone or in combination, although the degree of sensitization and synergy varied across the different NSCLC cell lines. Taken together our results suggest that TTFields may enhance the efficacy of radiation if used as a neoadjuvant therapy or as concomitant therapy with different chemotherapy agents to improve patient outcome in clinic.

#3940

Acute total body ionizing radiation induces long-term adverse effects and immediate changes in cardiac protein oxidative carbonylation in the rat.

Elliot T. Rosen,1 Dmitry Kryndushkin,1 Baikuntha Aryal,1 Yanira Gonzalez,1 Leena Chehab,1 Jennifer Dickey,1 Steven Mog,2 V Ashutosh Rao1. 1 _FDA-CDER, Silver Spring, MD;_ 2 _FDA-CFSAN, College Park, MD_.

Radiation-induced heart disease presents a significant challenge in the event of an accidental radiation exposure as well as to cancer patients who receive acute doses of irradiation as part of radiation therapy. We utilized the Wistar-Kyoto and spontaneously hypertensive rat model, previously shown to demonstrate drug-induced cardiomyopathy, to evaluate the acute and long-term effects of sub-lethal total body irradiation at two, four, and fifty-two weeks. We further examined irreversible oxidative protein carbonylation in the heart immediately following irradiation. Both males and females sustained reduced growth and anemic conditions over a one-year period as reflected by reduced body weight and low red blood cell count. Increased inflammation was detected by elevated IL-6 serum levels in males at four weeks. Serum cardiac troponin T and I analyses revealed indications of cardiomyopathy at earlier time points while more variability in the troponin levels was observed at one year. Echocardiography at two weeks following 5.0Gy treatment revealed hypertrophy and decrease in cardiac output in the short term, but significant differences were not observed at the one-year timepoint. Cardiac output was decreased in both males and females after two weeks, and statistically significant in females. Following irradiation, the heart tissue showed an increase in total protein carbonylation accompanied by DNA damage indicated by an increase in γ-H2AX. We present novel findings of several proteins that showed a marked change in carbonylation profile including those of mitochondrial origin. Overall, we present findings of acute oxidative protein, DNA damage, and long-term cardiomyopathy in the irradiated animals.

#3941

Novel cancer therapy approach based on Auger effect mediated by monoenergetic x-ray.

Erh-Hsuan Lin,1 Wen-Wong Kuo,2 Wan-Ting Tseng,2 Chi-Chieh Cheng,2 Shih-Feng Tseng,2 Wei-Li Chen,2 Chia-Gee Wang,2 Cheng-Wen Wu1. 1 _National Yang-Ming University, Taipei, Taiwan;_ 2 _Nanoray Biotech Co., Ltd., Taiwan, Taipei, Taiwan_.

The Auger effect is a physical phenomenon in which the filling of an inner-shell vacancy of an atom is accompanied by the emission of an electron from the same atom. An Auger electron is estimated to have a travel distance around 2-100 μm, within which an energy up to 106 gray is released. Therefore, if Auger effect can be induced in cancer cell DNA, it could mediate immense damage concentrated in cancer genome, providing a potential approach for cancer therapy. Auger effect can be induced by radioactive isotope itself, like 125I, or monoenergetic X-Ray in combination with specific heavy atom. Conventionally, monoenergetic X-Ray can be produced only in a synchrotron radiation accelerator, which hinders its application in medical use.

Here, our study showed that a novel design of X-Ray tube allowing electron beams to pass through the target metal can generate monoenergetic X-ray efficiently. This design substantially minimizes the size of X-Ray generator to be portable, and can generate 3-fold higher X-Ray production than traditional X-ray tube under the same voltage and current-exposure time. The monoenergetic X-Ray generated by the novel design (denominated as NanoRay hereafter) contains >20% of K-characteristic photon, up to 29-fold higher than traditional X-Ray in the same energy range.

For anti-cancer analysis, 14 and 33 keV NanoRays were combined with Thymidine analogues BrdU and IdU, respectively, in cancer cell treatment. The results showed that while single treatments showed low cytotoxicity, the combination of Nanorays with BrdU or IdU induced significant cancer cell death synergistically in vitro. In contrast, combined treatments of conventional radiation with BrdU or IdU did not show any synergistic effect. In vivo tumor treatment combining 33 keV NanoRay and IdU showed a tumor reduction rate up to 80% as compared to Nanoray alone. Likewise, combination of conventional radiation with IdU showed no synergistic anti-tumor effect. DNA damage analysis found that NanoRay produced 2-fold more double-strand breaks (DSBs) in cancer cells incorporating BrdU or IdU than control cells, validating the Auger effect induced and applied in cancer genome damage.

In summary, the current study presents that NanoRay, a new generation of medical X-Ray with highly specific monoenergetic spectrum, can be generated from a transmission tube within a portable apparatus. When NanoRay is applied to cancer DNA incorporating specific heavy atoms, such as BrdU or IdU, it induced significantly more pronounced DNA DSBs and cell death through Auger effect, thus lowering the dose necessary for corresponding anti-tumor effect induced by conventional radiation.

NanoRay serves as a novel medical X-Ray source with low dose, low cost, and high efficiency, holding great promise in future cancer therapy.

#3942

Animal studies evaluating the safety of Tumor Treating Fields (TTFields) in the torso.

Shiri Davidi, Mijal Munster, Shay Cahal, Moshe Giladi, Eilon D. Kirson, Uri Weinberg, Yoram Palti. _Novocure, Haifa, Israel_.

TTFields therapy is an anti-neoplastic treatment modality targeting dividing cells by disrupting microtubules and septin filaments localization. TTFields therapy received FDA approval for the treatment of glioblastoma based on phase 3 studies demonstrating efficacy and high safety profile. TTFields are currently being tested as a treatment option for solid tumors in the torso. The aim of this work is to study whether TTFields' safety profile is maintained in the torso, and specifically in tissues with high cellular proliferation rates. TTFields (2-3 V/cm) were applied for 2 weeks using the Novo-TTF 100 system to the rat torso at 150 and 200 kHz, intensities and frequencies known to be effective for the treatment of NSCLC, mesothelioma, pancreatic cancer, ovarian cancer and hepatocellular carcinoma. Throughout the treatment course, all animals underwent daily clinical examination by a certified experienced veterinarian. At the end of treatment, animal were euthanized and an experienced independent pathologist performed histological comparative evaluation of all major internal organs. No changes in the following parameters were observed: activity level, food intake, drinking, stools, motor neurological status and respiration. Further, no changes in weight were observed between the treated and control groups. No significant changes were observed in complete blood count and differential between the different groups. Histological analysis did not reveal any increase in pathological findings in the TTFields treated animals group. Taken together, these results demonstrate that safety of TTFields application at frequencies of 150-200 kHz to the rat torso and specifically to tissues with high cellular proliferation rates. Additional work is now underway to better understand the specificity of TTFields' effects on rapidly dividing tumor cells.

#3943

Inhibition of ribosomal protein RPS6KB1 as a strategy to improve conventional therapy for prostate cancer.

Alison D. Clark,1 Suleman S. Hussain,2 Shih-Bo Huang,1 Roble G. Bedolla,1 Mohammad A. Obeidat,1 Niko Papanikolaou,1 Chun Lin Lin,1 Tim H. Huang,1 Yogesh Gupta,1 Yidong Chen,1 Hiroshi Miyamoto,3 Rita Ghosh,1 Addanki P. Kumar1. 1 _University of Texas Health Science Center San Antonio, San Antonio, TX;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 3 _University of Rochester Medical Center, Rochester, NY_.

Radiation therapy (RT) is a standard treatment for organ confined prostate cancer (PCa). Although dose escalation increases local control, further escalation hampers effectiveness due to toxicity. Additionally, intrinsic and acquired resistance to radiation causes progression to advanced metastatic PCa. Therefore, there is an urgency for addition of adjuvant therapies that synergize with radiation to improve therapeutic efficacy. We have identified a novel combination regimen (Nexrutine, Nx, a natural compound plus radiation) that demonstrated strong synergistic tumor growth inhibition in vitro (in multiple prostate cancer cell lines) and in transgenic adenocarcinoma of mouse prostate (TRAMP) model in part through downregulation of ribosomal protein S6K (RPS6KB1). To understand the precise molecular mechanism of synergy between Nx and radiation, we conducted transcriptome profiling of prostate cancer cells treated with single and combination modalities and followed up with pathway analysis, clinical validation, and in vitro gain and loss of function studies. Analyses of these data identified significant alterations in expression of genes across all experimental groups and genes that were unique to each group. Functional annotation using DAVID analysis of the top 200 genes in the combination group revealed 'cell cycle regulation' as the top pathway altered in the combination treatment. Remarkably, 22 genes enriched in the cell cycle pathway (including RPS6KB1) showed significant upregulation in high Gleason human prostate tumors. Interestingly cBioportal data indicated that alterations of RPS6KB1 in human prostate tumors is mutually exclusive with expression changes of AKT or NFĸB. Computational molecular modeling studies showed that Berberine (Ber), one of the active constituents of Nx, interacts with RPS6KB1 in a novel mode. Additional biochemical and molecular investigations using RPS6KB1 KO cells divulged that KO cells are relatively (i) resistant to Ber and (ii) more sensitive to NFĸB inhibitor-induced proliferation inhibition compared to wild type LNCaP cells. Collectively, our findings (i) identified RPS6KB1 as a direct target of Ber; and (ii) demonstrate that RPS6KB1 is a clinically relevant target of prostate cancer with possible role in radiosensitization. This work is supported in part by SABER*IRACDA grant K12GM11726 (ADC) and VA-Merit Award I01 BX 003876 (APK).

#3944

MKI-1 is a small molecule inhibitor of MASTL with antitumor and radiosensitizer activity in breast cancer cells.

Ah-Young Kim, Jae-Sung Kim. _Korea Institute of Radiological and Medical Sciences, Seoul, Republic of Korea_.

Although microtubule-associated serine threonine kinase like (MASTL) is an emerging target for selective anticancer treatment, a small-molecule inhibitor of MASTL with antitumor activity had not been reported yet. This study showed the new MASTL inhibitor, MKI 1 (MASTL Kinase Inhibitor 1), with antitumor activity and radiosensitizing effect in breast cancer in vitro and in vivo. MKI 1 was identified and validated its inhibitory activity to MASTL by in silico and in vitro analysis, respectively. MKI 1 inhibited phosphorylated ENSA, a direct substrate of MASTL, but not the other AGC kinases such as Akt signaling in breast cancer cells. In addition, MKI 1 reduced various oncogenic properties of breast cancer cells rather than normal breast cells. Furthermore, MKI 1 increased the radiosensitivity of breast cancer cells and breast cancer stem cells in response to irradiation. We also confirm the in vivo antitumor activity and radiosensitizing effects of MKI 1 in breast tumor xenograft model. Therefore, our results provide the new line of a small-molecule inhibitor of MASTL, MKI 1, with the antitumor and radiosensitizer activity in breast cancer in vitro and in vivo.

## CLINICAL RESEARCH

### Immunomodulation by Chemotherapy and Targeted Agents

#3945

The folate pathway inhibitor pemetrexed pleiotropically enhances effects of cancer immunotherapy via immunogenic tumor cell death and T cell-intrinsic mechanisms.

David A. Schaer, Nelusha Amaladas, Zhao Hai Lu, Erik Rasmussen, Andreas Sonyi, Darin Chin, Andrew Capen, Yanxia Li, Catalina Meyer, Bonita Jones, Xiaodong Hong, Shuang Luo, Carmine Carpenito, Kenneth Roth, Alexander Nikolayev, Bo Tan, Manisha Brahmachary, Krishna Chodavarapu, Frank Charles Dorsey, Jason Manro, Thompson Doman, Greg Donoho, David Surguladze, Gerald Hall, Sandaruwan Geeganage, Michael Kalos, Ruslan Novosiadly. _Eli Lilly, New York, NY_.

Combination strategies leveraging chemotherapy and immunotherapy have held the promise as a method to improve benefit to cancer patients. However, most chemotherapies have detrimental effects on immune homeostasis and do not induce immunogenic cell death. The positive phase III trial of pemetrexed/carboplatin with the PD-1 antibody pembrolizumab in NSCLC (Keynote-189) lead to the first chemotherapy/immunotherapy combination ever approved. While this suggests a positive interaction between pemetrexed-based chemotherapy and PD-1 therapy, the underlying mechanism is unknown. Therefore, it is important to understand the role of pemetrexed in modulating antitumor immune response to assure application of this therapy to the appropriate patients. To characterize the effects of pemetrexed on intra-tumor immune response, murine tumor models which were sensitive to pemetrexed and known to be sensitive to PD-L1, were treated with pemetrexed with or without carboplatin, or anti-mouse PD-L1. In MC38 tumors, pemetrexed monotherapy demonstrated a trend towards an increased frequency of intra-tumor leukocytes that was accompanied by immune-related gene expression changes indicative of enhanced T cell infiltration and/or activation. Gene expression induced by pemetrexed was largely unaffected by carboplatin. Treatment of both MC38 and Colon26 tumor cells in vitro with pemetrexed induced release of HMGB1, indicative of immunogenic cell death. Although proliferation of primary human T cells was slightly reduced by pemetrexed, at clinically relevant concentrations, treatment lead to an enhanced T cell activation phenotype, including upregulation of multiple interferon gamma-induced genes, and increased mitochondrial respiration. This correlated with improved antigen specific in vitro cytotoxic activity of OT-1 TCR transgenic CD8 T cells when treated with pemetrexed during priming with OVA peptide. Treatment with pemetrexed and PD-L1 demonstrated a combination benefit compared to either monotherapy in both tumor models. Pathway Analysis of gene expression data revealed that improved antigen presentation, enhanced T cell and cytokine signaling and an engagement of CD4+ T cell-mediated immunity during the combination. This correlated with upregulation of MHC-I & II on monocytes, macrophages and tumor cells, suggesting increased immune priming. Accordingly, treatment with S1P1R antagonist (FTY720, preventing T cell LN egress) after initiation of therapy resulted in a loss of combination efficacy. This data suggests that pemetrexed promotes intra-tumor T cell-mediated immune response through immunogenic tumor cell death and increased activation and metabolic fitness of T cells. The combination of these effects results in enhanced T cell function leading to an improved anti-tumor efficacy in combination with PD-L1 antibody

#3946

Tumor checkpoint inhibitor profiling for an optimal clinical response.

Rachana R. Maniyar, Sanjukta Chakraborty, Ghada Ben Rahoma, John J. Degliuomini, Marc Wallack, Jan Geliebter, Raj K. Tiwari. _New York Medical College, Valhalla, NY_.

The last three decades have seen a steady rise in melanoma, the most aggressive form of skin cancer. The discovery of checkpoint inhibitors has revolutionized the treatment landscape for melanoma; anti-CTLA4 and anti-PD1 showing great successes in clinic. While checkpoint molecules and co-stimulatory molecules are traditionally present on cells involved in immune activation, mounting evidence suggests that tumor cells also express these molecules. Our laboratory has characterized five patient-derived melanoma cell lines, MEL-2, MEL-V, 3MM, KFM, and GLM-2 and screened them for the expression of a comprehensive list of 29 co-stimulatory and co-inhibitory molecules compared to normal adult melanocytes. We see a differential mRNA expression of many of these immune-regulatory molecules, including BTLA, HVEM, CD160, CD226 and TIM1. Western blots and immunofluorescence confirmed the presence of these molecules at the protein level. A flow cytometry analysis demonstrated that BTLA, HVEM, CD160, TIM1 and CD226 are present on the membrane of these patient derived melanoma cells; implying that they are capable of engaging their respective ligands and exerting a functional role in immunomodulation. These findings were additionally validated in melanoma tissues, obtained from patients, by immunohistochemistry. Interestingly, treatment of MEL-2, MEL-V, KFM and GLM-2, our BRAFV600E containing patient derived cells, with BRAFV600E inhibitor PLX4032 led to the upregulation of these molecules. This upregulation was coupled with an increase in transcription factor MITF, a binding site for which is present in the promoter region of all the upregulated molecules. Co-culture experiments with immune cells, demonstrate a modulatory role played by the HVEM/BTLA/CD160 axis molecules that are present on tumor cells. The presence of these additional immune-regulatory molecules signify the existence of a robust tolerance mechanism induced by these checkpoint inhibitors. Our studies underscore the importance of characterizing tumor profiles to enable the selection of an optimal treatment regimen combining targeted small molecule inhibitors, and checkpoint inhibitors to maximize T cell killing, and tumor clearance.

#3947

Mechanisms of action of a neoantigen-targeting antibody NEO-201.

Philip Martin Arlen, Massimo Fantini, Justin David, Christina M. Annunziata, Kwong Y. Tsang. _Precision Biologics, Inc., Bethesda, MD_.

NEO-201 is a humanized IgG1 monoclonal antibody (mAb) that reacts to tumor-associated antigens (TAA) derived from pooled allogeneic colon tumor tissue extracts. NEO-201 recognizes tumor-associated variants of CEACAM family members. This mAb is remarkably tumor-specific in its staining profile and reacts to a wide range of human carcinoma cell lines by flow cytometry and tumor tissues by immunohistochemistry. NEO-201 exhibited both ADCC and complement-dependent cellular cytotoxicity (CDC) activity against human carcinoma cell in vitro and counteracted the growth of human pancreatic xenograft tumors in vivo. We investigated an additional mechanism of action of NEO-201 and to further confirm the rationale for clinical testing. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a cell-surface protein expressed by immune cells and tumor cells, and it can inhibit T cell function similar to PD-1 and CTLA-4. CEACAM1 is also a potent inhibitor of natural killer (NK) cell function. Binding between CEACAM1 on NK cells and CEACAM1 or CEACAM5 on tumor cells inhibits activation signaling by NKG2D, which prevents NK cell cytolysis and permits tumor cells to evade NK killing. This study was designed determine whether NEO-201 blocks the CEACAM1 inhibitory pathway to restore antitumor functionality to NK cells. In vitro assays using human tumor cell lines were conducted to identify CEACAM family members bound by NEO-201. Functional assays were conducted to assess the ability of NEO-201 to potentiate the in vitro killing of tumor cells by the NK cell line NK-92, which expresses CEACAM1 and lacks CD16 and the ability to mediate ADCC. NEO-201 was found to react with distinct variants of CEACAM5 and CEACAM6, but not with CEACAM1 or CEACAM8. Expression profiling revealed that various NEO-201+ cell lines expressed differing levels of the native forms of CEACAM5/6 vs. the NEO-201-reactive variant forms of these molecules. Functionally, NEO-201 treatment augmented the cytolytic activity of NK-92 cells against NEO-201+ tumor cells in proportion to their level of CEACAM5 expression (average increase of 2-fold), but not against NEO-201+ cells that only expressed CEACAM6. This study demonstrates that NEO-201 is capable of an additional function. This antibody can block the interaction between tumor cell CEACAM5 and NK cell CEACAM1 to reverse CEACAM1-dependent inhibition of NK cytotoxicity. Experiments are in progress to determine the involvement of NK cell CEACAM1 and/or other checkpoint pathways in this mechanism of action. These results suggest that NEO-201 may potentially reverse CEACAM1-dependent immunosuppression of NK cells in patients whose tumors express the NEO-201-reactive variant of CEACAM5.

#3948

The sequential administration of vinca alkaloids and intermittent cyclophosphamide primes antigen-presenting and NK cells and significantly improves in vivo efficacy of anti-PD-L1 in triple-negative breast cancer models.

Stefania Orecchioni, Loredana Vecchi, Paolo Falvo, Lucilla Luzi, Patrizia Mancuso, Chiara Camisaschi, Francesco Bertolini. _European Institute of Oncology, Milan, Italy_.

Checkpoint inhibitors (CIs) have shown an unprecedented clinical activity in a large variety of cancer types, but only in a fraction of patients. To increase the number of responders, preclinical models are studied to define effective combinatorial regimens and the best window of therapeutic opportunities. We recently described a synergy between anti-PD-1 and -PD-L1 CIs and several types and dosages of chemotherapy in immunocompetent models of triple negative breast cancer (TNBC), likely due to unique effects of chemotherapeutics over circulating and tumor-infiltrating, suppressive, antigen-presenting and effector immune cell populations (Orecchioni et al, Br J Cancer 2018). To find a high-order drug combination, we have further studied by multiparametric flow cytometry and single-cell transcriptomic a panel of different chemotherapy drugs (including microtubular poisons, alkylating agents, topoisomerase inhibitors and antimetabolites) used in vivo at different dosages and schedule. Our aim was to define a combination, dose and schedule able to promote a) dendritic cell maturation, b) release and c) uptake of cancer-associated antigens from targeted neoplastic cells, d) cross-priming between antigen-presenting cells (APCs) and T cells, e) NK cell activation, and f) inhibition of suppressor immune cell populations. Vinca alkaloids vinblastine and vinorelbine (V), at low-medium dosages, were the most effective in a), b), c) and d). After V administration, Tregs were reduced, and circulating and tumor-infiltrating APCs showed a "maturation to activation" program with increased expression of several antigens including CD40, CD80, and CD86. The alkylating agent cyclophosphamide (CTX) targeted Tregs, mobilized APC progenitors, activated and increased the number of circulating and intratumoral NK cells. These effects were significantly increased when CTX was administered in an intermittent fashion (every 6 days) at low-medium doses. As the APC-NK cell axis has been recently found to be pivotal in CI-mediated, anti-tumor cell immunity, our data suggested a preclinical trial in immunocompetent orthotopic models of metastatic (post-mastectomy) BALB mice injected with 4T1 or EMT-6 TNBC cells. Among a large panel of different combinations and dosages, the sequential administration of V, intermittent CTX and anti-PD-L1 was the only able to completely abrogate TNBC local and metastatic growth, and this effect was not observed in T and NK cell-depleted mice. Taken together with recent data from randomized, combinatorial studies in TNBC patients (Schmid et al, NEJM 2018), our findings suggest that CIs might be included in future combinatorial regimens aimed at improving the percentage of patients receiving a clinical benefit and prolonging the duration of this benefit.

#3949

Characterization of MP1115; a new natural killer cell activator.

Pitchaimani Kandasamy,1 Jason E. Duex,1 Greg Miknis,1 Julie Lang,2 Ana Chauca-Diaz,1 Chris Johnson,1 Colleen Hudson,1 Jakub Staszak-Jirkovsky,1 Neal C. Goodwin1. 1 _Machavert Pharmaceuticals, Aurora, CO;_ 2 _University of Colorado Denver School of Medicine, Aurora, CO_.

MP1115 is in development as a new immuno-oncology therapeutic lead comprised of bioactive phospholipid containing nanoparticles with activities unique to phospholipid active pharmaceutical ingredients. MP1115 demonstrated potent activation of NK cells in vitro and in vivo and had a secondary anti-tumor cell proliferation mechanism of action. Methods: MP1115 was formulated as ~100 nM diameter nanoparticles formulated in buffered sucrose and stably stored frozen. MP1115 nanoparticles were also loaded with a CDK8 inhibitor test compound at a 30 percent loading efficiency. MP1115 was tested for anti-tumor cell proliferation in 2D and 3D in vitro assays, NK cell activation in ex vivo splenocyte and NK cell activation assays, and anti-tumor efficacy and NK cell activation in vivo in immunocompetent and immunodeficient mice. MP1115-CDK8 inhibitor loaded nanoparticles were tested in vitro for increased anti-tumor cell proliferation compared to non-loaded MP1115 nanoparticles and non-loaded CDK8 inhibitor controls. Results: MP1115 demonstrated increased NK cell activation compared to a CDK8 test compound as measured by IFN-γ release in an ex vivo mouse splenocytes-Yac-1 mouse lymphoma assay (p<0.0001). This activity was maintained with splenocytes from immunocompetent BALB/c mice, immunodeficient BALB/c scid mice, and with purified NK cells from each strain background. MP1115 demonstrated increased NK cell activation of mouse splenocytes in vivo in immunocompetent BALB/c mice compared to controls (p<0.0001), as seen by increased CD69 and CD107a signal, as well as the significant killing of target Yac-1 cells ex vivo (p<0.001). The levels of activation and killing observed were similar to that achieved by a CDK8 inhibitor test compound (p>0.9999). MP1115 also successfully demonstrated the ability to deliver a CDK8 test compound payload to human non-Hodgkin's lymphoma cells in vitro with increased anti-tumor cell proliferation compared to controls (p<0.0001). MP1115 achieved increased anti-tumor cell proliferation efficacy in acute myeloid leukemia, multiple myeloma, non-Hodgkin's lymphoma, non-small cell lung cancer, KRAS mutant colorectal cancer models in vitro, and in pancreatic cancer tumor models in vivo. MP1115 demonstrated high tolerability in rodent models when dosed at over 200 mg/kg daily for multiple doses. MP1115 is currently being validated in additional in vivo models including human immune system mice with human xenografts. Conclusions: MP1115 is being advanced to IND-enabling studies based on demonstrated low toxicity, high systemic delivery, potent NK cell activation, and anti-tumor cell proliferation activities.

#3950

Rovalpituzumab tesirine enhances the anti-tumor efficacy of PD-1 blockade in a murine model of small cell lung cancer with endogenous Dll3 expression.

Philip M. Vitorno,1 Chen-Hua Chuang,1 Christine Moore,1 Tolga Turan,2 Ronald Ferrando,1 Aaron Nichols,1 Shravanthi Madhavan,1 Laura R. Saunders1. 1 _AbbVie, South San Francisco, CA;_ 2 _AbbVie, Redwood City, CA_.

Rovalpituzumab tesirine (Rova-T) is an antibody drug conjugate (ADC) comprised of a DLL3-targeted antibody conjugated to a potent cytotoxic pyrrolobenzodiazepine (PBD) toxin that is currently being investigated clinically in phase 3 trials for small cell lung cancer (SCLC). Cytotoxic DNA damaging agents including PBDs may elicit immunologic cell death (ICD), a form of apoptosis that enhances immune activation (Rios-Doria J 2017). Blockade of PD-1, an immune checkpoint receptor, using Nivolumab in 3L+ SCLC patients, showed an objective response rate (ORR) of 11.9%, with ORR higher in patients with high tumor mutation burden (Hellmann MD 2018). Here we report that sub-efficacious doses of Rova-T in combination with anti-PD-1 mAb resulted in enhanced anti-tumor activity in KP1, a murine SCLC syngeneic tumor model (Park KS 2011) that endogenously expresses Dll3, compared to Rova-T alone or anti-PD-1 mAb alone. In contrast, a single cycle of cisplatin + etoposide (C/E) standard of care chemotherapy alone showed some tumor burden reduction, but no additive activity was seen for combination C/E + anti-PD1 mAb. Rova-T alone, anti-PD1 alone, and the combination increased CD4+ and CD8+ T-cell infiltration in the tumor, as measured by gene expression analysis, flow cytometry and immunostaining of tumors after treatment. A multiplex immunofluorescence panel showed that the increased lymphocytes are restricted to the surrounding stroma in tumors treated with Rova-T or anti-PD1 alone. Unique to the Rova-T + anti-PD1 treated tumors, lymphocytes were found throughout the tumor, and they expressed granzyme B, a marker of activation. CD8 but not CD4 T-cell depletion before and during in vivo treatment with Rova-T + anti-PD1 reduced the anti-tumor efficacy, supporting a role for cytotoxic CD8 T cells in the combination efficacy. In addition to enhancing T-cell infiltration, Rova-T alone and Rova-T + anti-PD1 increased expression of PD-L1, the immune suppressive ligand of PD-1, on tumor cells. Mice treated with a single efficacious dose of Rova-T alone or Rova-T + anti-PD1 showed durable tumor suppression (>50 days) and did not develop tumors when re-injected with 1 million tumor cells on the opposite flank, indicating that treatment conferred immunologic memory and sustained anti-tumor immunity. Together, these data provide pre-clinical rationale for the ongoing combination SCLC clinical trials of Rova-T and Nivolumab (NCT03026166) and Rova-T and ABBV-181 (NCT03000257).

#3951

Apalutamide reworks the immune composition of prostate tumors.

Marco A. De Velasco,1 Yurie Kura,1 Naomi Ando,1 Noriko Sato,1 Masahiro Nozawa,1 Kazuhiro Yoshimura,1 Kazuko Sakai,1 Kazuhiro Yoshikawa,2 Kazuto Nishio,1 Hirotsugu Uemura1. 1 _Kindai University Faculty of Medicine, Osaka-Sayama, Japan;_ 2 _Aichi Medical University, Japan_.

Prostate cancers depend on androgen receptor (AR) signaling for survival making it a major therapeutic target. Apalutamide is an oral nonsteroidal AR antagonist that is currently indicated for the treatment of patients with non-metastatic castration-resistant prostate cancer. Apalutamide hinders AR-mediated transcriptional activity by impeding AR nuclear translocation and binding to DNA in cancer cells. AR is also expressed by stromal and immune cells and can modulate innate and adaptive immune responses, moreover, androgen ablation can both hinder and enhance antitumor immunity. Thus far, success from immune checkpoint blockade monotherapy has been lacking in prostate cancer patients and novel strategies using of other modalities are being investigated. Here, we characterize the effect of apalutamide therapy on antitumor immunity in a preclinical mouse model of prostate cancer to assess its value as potential partner for combination with immunotherapy. Conditional prostate-specific Pten-knockout mice were treated with apalutamide (30 mg/kg/d, 5 days on/2 days off) or vehicle for a period of 4 or 8 weeks to coincide with androgen sensitivity and the shift towards castration resistance. Tumor reductions after 4 or 8 weeks of treatment with apalutamide were 38.2% (P<0.01) and 46.2% (P<0.001), respectively. Flow cytometric analysis of tumor tissues revealed 1.3 (P=0.033) and 1.9 (P<0.001) -fold increases of leukocyte (CD45+) infiltration in mice treated with apalutamide for 4 or 8 weeks, respectively. CD8+ T cell infiltration was 2.7-fold higher at 8 weeks, however, tumor reactive PD1+/CD8 T cells were 2-3-fold greater for apalutamide-treated mice at weeks 4 and 8. Quantitative immunohistochemical analysis confirmed increased intraepithelial CD8+ T and granzyme B-positive cells. Apalutamide treatment was also associated with increased peritumoral T regulatory cells and intraepithelial neutrophils. Immuno-profiling using a qRT-PCR-based panel of immune-relevant genes associated apalutamide therapy with an increased IFN-γ inflammatory signature, antigen presentation/dendritic cell, natural killer cell, T regulatory cell, tumor associated macrophage, and myeloid-derived suppressor cells (MDSC). These data also revealed a trend towards decreased CD8 T cell activation at week 8 accompanied by increased expression of the immunological checkpoints Ctla4, Tim3, and 2b4. In conclusion, our findings suggests that apalutamide-induced tumor cell death attracted phagocytes (macrophages and dendritic cells) that led to a innate and adaptive immune cell responses. While, cytotoxic T cell activity was improved, it was limited by the development of an immunosuppressive tumor microenvironment replete with MDSCs and T regulatory cells. Nevertheless, treatment with apalutamide turned immunologically "cold" tumors into more immunologically reactive tumors that may become more susceptible to targeted immunotherapy.

#3953

FF-10832 combined with immune checkpoint blockade shows favorable anti-tumor activity.

Tadaaki Ioroi, Takashi Komori, Takeshi Matsumoto, Susumu Shimoyama, Takayuki Kobayashi, Hidetoshi Murao, Shinichi Watanabe, Shinji Hagiwara, Takefumi Hara. _FujiFilm Corporation, Kanagawa, Japan_.

Introduction: We previously reported that FF-10832, a liposomal gemcitabine, exerted anti-tumor activity superior to that of gemcitabine with high stability, long plasma half-life, and preferential tumor accumulation in preclinical tumor models. Beneficial gemcitabine immune-modulating effects in tumor microenvironments have also been reported. We investigated the immune-modulating effects of FF-10832 by evaluating its anti-tumor activities combined with immune checkpoint blockade.

Methods: Murine syngeneic tumor models were created by subcutaneous implantation of MBT-2 (bladder) or EMT6 (breast) tumor cells and tumor volume evaluated during and after treatments. Treatments included 2 or 4 mg/kg of FF-10832 or 240 mg/kg of gemcitabine intravenously once a week, and 10 mg/kg of an immune checkpoint inhibitor intraperitoneally twice a week for 3 weeks.

Results: In the MBT-2 tumor model, although tumor growth was slightly inhibited by anti-PD-L1 mAb (clone: 10F.9G2) alone, only one out of nine mice showed complete response. Although FF-10832 decreased tumor growth during treatment (3 weeks), remission was incomplete, and tumor growth continued after treatment was discontinued. In contrast, in combination, tumor growth was inhibited even after treatment withdrawal; three out of nine mice achieved complete remission, and median survival improved (vehicle control and anti-PD-L1 mAb: 25 days; FF-10832: 28 days; combination: >49 days). This combination effect was comparable to that of gemcitabine combination. Previously, FF-10832 has also shown anti-tumor effects in a gemcitabine-insensitive BxPC-3 xenograft tumor model, so the potential benefit in combination with checkpoint inhibitors was evaluated in a gemcitabine-insensitive EMT6 tumor model. FF-10832 significantly inhibited tumor growth relative to that of gemcitabine in this model, but all mice showed tumor re-growth after treatment withdrawal. Anti-CTLA-4 mAb (clone: 9H10) alone showed complete response in only one out of eight mice. Strikingly, FF-10832 in combination with anti-CTLA-4 mAb exerted superior anti-tumor activity compared to that with gemcitabine, as shown by tumor regression in all mice and complete regression in seven out of eight mice. Further analysis to clarify the mode of action underlying the combination effect is ongoing now. These results indicate that the effect of immune checkpoint inhibitors is enhanced by FF-10832 regardless of tumor sensitivity to gemcitabine.

Conclusion: Although immune checkpoint blockade is thought to be a breakthrough for cancer treatment, only a limited patient population has achieved favorable outcomes. Our results suggested that FF-10832 enhances the anti-tumor activity of immune checkpoint blockade, which could significantly improve treatment outcomes.

#3954

Evaluating the compatibility of tumor treating electric fields with key anti-tumoral immune functions.

Gil Diamant, Hadar Simchony, Tamar Shiloach, Anat Globerson-levin, Lital Gasri Plotnitsky, Zelig Eshhar, Niv Pencovich, Rachel Grossman, Zvi Ram, Ilan Volovitz. _Tel Aviv Sourasky Medical Center, Tel Aviv, Israel_.

Background: Combining Tumor Treating electrical Fields (TTFields) with immunotherapy is a rational approach due to their different mechanisms of action (MOA) and to TTFields' ability to induce immunogenic cell death (ICD). Conversely, TTFields may interfere with immune functions critical for effective T cell responses.

Methods: T cells from healthy donors' peripheral blood or from viably dissociated glioblastoma samples were cultured under normal or TTFields conditions, with or without superantigen-stimulation. Eight-color flow cytometry was used to assess T cell responses by monitoring select pivotal antitumoral functions: proliferation (CFSE), IFNγ secretion, cytotoxic degranulation (CD107a), activation/exhaustion (PD1) and viability. Direct cytotoxicity was evaluated using chimeric antigen receptor (CAR) T cells.

Results: The viability of stimulated T cells that attempted to proliferate decreased under TTFields, in line with TTFields' MOA. Small or no reductions in viability were found in activated T cells that did not attempt to proliferate and in unstimulated T cells.

The functionality of stimulated peripheral-blood T cells and tumor-infiltrating T cells (TILs) under TTFields was unhindered: T cells exhibited comparable PD1 upregulation, IFNγ secretion and CD107a expression as controls. T cell polyfunctionality, associated with effective antitumoral responses, was retained under TTFields conditions. PD1-expressing TILs, a subset containing most of the tumor antigen-specific TILs, exhibited unaltered viability and functionality under TTFields. CAR T-cells, which utilize the same killing machinery as unmodified T cells, exhibited unaltered cytotoxic capability under TTFields.

Immunohistochemical evaluation of GBM samples before TTFields treatment and after recurrence showed that some patients had accommodated large increases in their CD8 and CD4 counts. RNA-Seq performed on GBM samples from 6 standardly-treated and 6 TTFields-treated patients before treatment and after recurrence. The data shows differential increases in TTFields-treated patients to controls, in the expression of immune genes associated with favorable prognosis (e.g. t-bet, NKG2D, ICOS-L, CD70) and concurrent decreases in genes associated with poor prognosis (e.g. IL4, TSLP, various complement genes).

Conclusions: The preclinical data showed that all antitumoral T cell functions examined, but proliferation, were unhindered by TTFields. The clinical data showed that TTFields may shift treated tumors to a state more conducive of anti-tumoral immune responses. Our findings support the further preclinical and clinical investigation into combining TTFields with immunotherapy.

#3955

Tumor macrophage-mediated antibody dependent cell phagocytosis (ADCP) is theprimary mechanism mediating HER2 mAb (Trastuzumab) anti-tumor responses which can be synergistically enhanced by CD47 innate immune checkpoint blockade.

Li-Chung Tsao, Jun-Ping Wei, Gang-jun Lei, Tao Wang, Xiao-Yi Yang, Cong-Xiao Liu, H. Kim Lyerly, Zachary C. Hartman. _Duke Univ. Medical Ctr., Durham, NC_.

Approximately 20% of breast cancers (BC) are defined by HER2 overexpression and treated using HER2-specific monoclonal antibodies (mAb), such as Trastuzumab. Previous studies have demonstrated Trastuzumab can limit signaling, elicit antibody dependent cell cytotoxicity (ADCC), antibody dependent phagocytosis (ADCP), and adaptive immune responses to HER2, however its primary therapeutic mechanism of action (MOA) remains unclear. As HER2 mAb efficacy is subverted in advanced cancers, understanding and boosting their MOA is of critical clinical interest and scientific significance for HER2+ BC as well as for other targeted mAbs.

Using a series of fully murine Trastuzumab mAbs of different isotypes, we found that the IgG2A (high A/I) isotype was essential for tumor growth suppression, suggesting an innate immune MOA. In contrast to previous reports, our studies revealed that NK cells, neutrophils, T-cell, or B-cell populations were unnecessary for this anti-tumor MOA. However, we found that a murine IgG2A Trastuzumab (mTras) altered the immune microenvironment in vivo through neutrophil suppression (~4 fold, p<.001) but stimulated a significant expansion (~7 fold, p<.001) and activation of resident tumor associated-macrophages (TAMs). Using DiD dye-labelled tumors, we confirmed that mTras elicited striking HER2-specific ADCP in ~50% of resident TAMs (versus ~16% in control), while in vitro studies confirmed that mTras-mediated macrophage anti-tumor efficacy was dependent upon ADCP, but not ADCC, through activation of FCGR4. Critically, we also found BC CD47 expression suppressed ADCP and resulted in suboptimal mTras anti-tumor efficacy. To explore this innate resistance pathway, we utilized CD47 KO BC lines and CD47 mAbs and found that both significantly enhanced mTras-mediated ADCP in vitro (>4 fold, p<.01) without altering ADCC activity. Moreover, we also found that this combination significantly enhanced TAM expansion and activation in vivo, which significantly enhanced human HER2+ BC anti-tumor efficacy (88% complete regression vs. 0% for single agent) and prolonged survival in multiple murine HER2+ BC models, including an endogenous treatment-resistant HER2+ BC mouse model.

Thus, our study demonstrates the primary MOA of a clinically relevant HER2 mAb requires engagement with TAMs to stimulate their expansion and elicit ADCP, which could be significantly enhanced by CD47-SIRPa blockade. This suggests that the strategic use of CD47-SIRPa innate blockade may allow for significant enhancement of anti-tumor immunity in combination with other cancer antigen targeting mAb therapies (targeting EGFR, CD20, etc) as a more effective strategy to stimulate anti-tumor immune responses in advanced immunosuppressive cancers.

#3956

A novel compound screen for enhancing T-cell based immunotherapy identifies aurora kinases as a targetable mechanism for tumor immune escape in pancreatic ductal adenocarcinoma preclinical models.

Emily L. Ashkin, Deborah Silverman, Simone Punt, Soraya Zorro Manrique, Leila Williams, Yunfei Wang, Patrick Hwu. _MD Anderson Cancer Center, Houston, TX_.

Harnessing a patient's immune system by attenuating endogenous immune checkpoints on T-cells has led to dramatic, durable tumor rejection for multiple tumor types. Nonetheless, many cancers, including pancreatic ductal adenocarcinoma (PDAC), still do not respond to immunotherapy. PDAC remains largely resistant to all therapies, leading to its notoriety as one of the most lethal malignancies, with a 6% 5-year survival rate. We aimed to find novel, targetable mechanisms by which tumors escape the adaptive immune system and resist immunotherapy. We conducted a high throughput screen of 1280 compounds producing a synergistic effect with T-cell killing to assess differences and similarities among distinct pathways inclusive between PDAC and melanoma. These screens identified several aurora kinase inhibitors as synergistic with autologous T-cell killing of tumor target. Here we present results underlying the mechanism by which aurora kinase inhibition enhances autologous T-cell mediated killing and may improve T-cell-mediated cancer immunotherapy. These findings may contribute to the design of new therapeutic combination strategies for enhancing immunotherapy treatment of melanoma, PDAC, and other "immunologically cold" tumors.

#3957

**MIV-818 stimulates an anti-tumor immune response** in **** vitro **and enhances the effects of pembrolizumab.**

Mark Albertella, Ana Slipicevic, Richard Bethell. _Medivir AB, Huddinge, Sweden_.

Background: MIV-818, a nucleotide prodrug of troxacitabine monophosphate, designed to target the active metabolite to the liver while minimizing systemic exposure, has recently entered phase 1 clinical development for the treatment of liver cancers. Preclinical studies have demonstrated that some DNA damaging agents may further enhance antitumor immune responses in addition to direct cytotoxic effects. We therefore investigated whether MIV-818 induces similar immunomodulating effects.

Methods: Immune-mediated tumor cell killing was determined by co-culturing labelled SKOV3 cancer cells and PBMC and quantified over time using the IncuCyte ZOOM system. Effects on inflamed host tumor microenvironments (TME) were investigated by cytokine profiling co-cultures of activated PBMCs, primary fibroblasts or endothelial cells and HT29 colon adenocarcinoma cells. Effects on SEB-stimulated PBMC were determined by quantitating thymidine incorporation and cytokine expression in supernatants.

Results: MIV-818 caused a dose-dependent inhibition of proliferation of SKOV-3 cells, with no significant induction of cell death (IC50 7.5nM; ≤5% apoptosis). In contrast, co-incubation of MIV-818 with PBMCs induced profound apoptosis of the cancer cells (74%+/-25 compared to 37%+/-23 from PBMCs alone). 40nM MIV-818 gives equivalent induction of apoptosis compared to 20ug/ml pembrolizumab. In a more complex model of the immune TME, MIV-818 treatment impacted inflammatory and angiogenesis-related responses in both the stromal and vascular co-culture systems, increasing IFNγ, IL-10, IL-17A, and TNFα in a manner comparable to that of pembrolizumab alone while also inhibiting IL-6. Combining MIV-818 with pembrolizumab further enhanced IL-10, IL-17A and IFNy, while also modulating immune-related (GranB, IL-2, IL-6), and the inflammation-related cytokines (VCAM-1, IP-10, TNFα). MIV-818 demonstrated dose-dependent inhibitory effect on proliferation of SEB-stimulated PBMCs. Despite this growth inhibitory effect, it led to a strong increase in IL-2 levels both as a single agent and which was further enhanced in combination with pembrolizumab.

Conclusions: MIV-818 exerts immunomodulating effects in vitro by increasing immune cytokine levels and PBMC-mediated cancer cell killing, which are further enhanced in combination with pembrolizumab. The effects of MIV-818 on tumor immune activation are being assessed in an ongoing phase 1/2 clinical trial, and these data could support future combination of MIV-818 with PD-(L)1 inhibitors in patients with HCC and other liver cancers.

#3958

**FAK inhibition reduces tumor infiltration of Tregs via restricting CCL5 release and boosts the present therapeutic regimen for BRAF** V600E **-mutated colorectal carcinoma.**

Luping Yuan,1 Wentao Pan,2 Suna Zhou,1 Qiuyun Luo,1 Xianglei Yan,1 Yuxin Zhang,1 Mengxian Pan,2 Lin Zhang,1 Miaozhen Qiu,1 Dajun Yang1. 1 _State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-Sen University Cancer Center, Guangzhou, China;_ 2 _Suzhou Ascentage Pharma Inc., Jiangsu, China_.

Background: BRAFV600E-mutated colorectal carcinoma (CRC) is a refractory disease with poor prognosis and limited therapeutic approaches. Recently, researches have indicated that the recruitment of Tregs in tumor tissue promoted by BRAFV600E is responsible for the weakened antitumor immunity. Whereas, as the main targeting therapy for this intractable carcinoma, BRAFV600E inhibitor has little response. In addition, FAK, being the novel therapeutic target in CRC, is considered to have an impact on the release of chemokines. In this study, we sought to explore the capacity of FAK inhibitor restricting Treg infiltration in tumor and its mechanism, as well as the synergistic antitumor effect with the present therapeutic strategy for BRAFV600E-mutated CRC.

Methods: Cell proliferation was determined by CCK-8 assay and colony formation assay. Migration ability was detected by transwell assay. Cell cycle and cell group analysis were assessed by flow cytometry. Western blot, Elisa and RT-qPCR analysis were conducted for mechanism exploration. Immunohistochemistry and immunofluorescence analysis were performed to confirm the link between BRAFV600E, FAK and Tregs. CRC xenograft models were established to detect the Treg recruitment and evaluate the synergistic antitumor effect in vivo.

Results: BRAFV600E-mutated CRC was of higher expression of FAK, and hence more sensitive to FAK inhibitor APG-2449. Besides, BRAFV600E-mutated in CRC was linked with more Treg infiltration in tumor site, therefore contributed to the attenuated antitumor immunity. What's more, despite directly inhibiting tumor proliferation and migration, one of the important mechanisms of FAK blockade restricting the growth of BRAFV600E-mutated CRC, was by downregulating the release of Treg chemokine CCL5 from tumor cells through FAK-IL33 axis. With the recruitment of Tregs decreasing induced by FAK inhibitor APG-2449, the number of CD8+T cells relatively elevated in tumor tissue. In BRAFV600E-mutated CRC xenotransplantation models, we revealed that the combination of FAK inhibitor APG-2449 and Vemurafenib was of no significant difference with the combination of Vemurafenib and cetuximab. While adding APG-2449 to the current combination strategy achieved greater tumor suppressing effect than not adding.

Conclusions: BRAFV600E-mutated CRC is associated with a higher expression level of FAK, as well as the tumor infiltration of Tregs. FAK inhibitor APG-2449 can promote antitumor immunity by restricting Treg number in tumor tissue, and shows a synergistic effect with the present therapeutic strategy for BRAFV600E-mutated CRC treatment.

#3959

Ibrutinib immunomodulation via CTLA4 downregulation in CLL.

Max Yano, Priscilla Do, Xiaokui Mo, Natarajan Muthusamy, John C. Byrd. _The Ohio State University, Columbus, OH_.

Chronic lymphocytic leukemia (CLL) is the most common lymphoid malignancy in the United States. It causes significant morbidity and mortality, largely through its suppressive effects on the immune system. Our group has previously demonstrated that CLL cells express CTLA4, an immune checkpoint molecule normally found on activated T cells. CTLA4 is brought to the leukemic cell surface after interaction with a CLL-reactive activated T cell and functions to decrease CD80 expression on neighboring cells, thereby decreasing T cell activation. Previous data indicated a decrease in CTLA4 expression on T cells in patients treated with ibrutinib or acalabrutinib, two irreversible Bruton's Tyrosine Kinase (BTK) inhibitors. Here, we expand our understanding of CTLA4 regulation in CLL and demonstrate that ibrutinib treatment directly modulates CTLA4 expression on CLL cells while indirectly modulating T cell CTLA4 expression.

In CLL cells, we demonstrate that in vitro treatment with ibrutinib suppresses CTLA4 at each stage of its expression - mRNA, total protein, and surface trafficking (mean reductions: mRNA 53%, p>.0001, n=18; total protein 36%, p=.044, n=10; surface protein 35%, p=.0006, n=10). In contrast, treatment with acalabrutinib (a BTK-specific inhibitor) decreases mRNA (mean reduction 44%, p=.006, n=14) while sparing protein expression and trafficking. This means that CTLA4 surface protein (and therefore CTLA4 function) is regulated by non-BTK "off-target" effects of ibrutinib, while transcription is at least partly BTK-dependent. Comparing patient samples obtained before treatment versus after five months on ibrutinib, we demonstrate that CTLA4 suppression does occur with in vivo treatment (mean reduction: 54% total, p=.047; 40% surface, p=.034; n=5). In contrast to its in vivo effects and direct suppression on CLL cells, in vitro ibrutinib treatment failed to suppress T cell expression of CTLA4. This indicates that the T cell CTLA4 modulation seen in treated patients is due to indirect effects and points to different regulatory mechanisms in T versus CLL cells. Previous published data indicates that IFNγ levels in CLL patients decrease with ibrutinib treatment – here, we show that IFNγ can stimulate CTLA4 expression on CLL cells (qPCR mean increase 80%, p=.012, n=10). This indicates a second mechanism of CTLA4 reduction in patients receiving this treatment, via loss of IFNγ stimulation.

Overall, we demonstrate a new mechanism of immunomodulation by ibrutinib – decreased CTLA4 on CLL cells. We demonstrate a different regulatory mechanism driving CTLA4 expression in CLL cells vs. T cells, and show that the decrease in T cell CTLA4 expression during ibrutinib treatment occurs indirectly. While recent efforts have developed more selective BTK inhibitors in an effort to decrease medication side effects, these results indicate that off-target immunomodulatory effects may be an underappreciated benefit from ibrutinib treatment.

#3960

Low doses of Docetaxel in combination with anti-prostate cancer immunotherapy lead to complete tumor-free outcome through Galectin-3 silencing.

Daniel Compagno,1 Carolina Tiraboschi,2 Lucas Gentilini,2 Enrique Corapi,2 Felipe M. Jaworski,2 Anne Chauchereau,3 Diego J. Laderach4. 1 _IQUIBICEN-UBA, NA, Argentina;_ 2 _IQUIBICEN-UBA, Cuidad Autonoma de Buenos Aires, Argentina;_ 3 _INSERM U981, France;_ 4 _INSERM U981, Cuidad Autonoma de Buenos Aires, Argentina_.

Based on animal model results and the analysis of clinical data from metastatic and castration resistant prostate cancer (mCRPC) patients, we were able to demonstrate the advantages and the mechanisms by which Docetaxel(DTX)-based chemotherapy induces the correct pre-conditioning of the tumour microenvironment to allow high cytotoxic T cell activity to avoid prostate tumour growth and recurrence. First, analysis of the gene expression of human PCa cells showed that treatment with DTX decreases the expression of Galectin-3 (Gal-3) in either chemo-sensitive or -resistant prostate cancer (PCa) cell lines. In addition, using a murine PCa model (derived from TRAMP mice), treatment of TRAMP-C1 (TC1) cells or tumors with a low dose of DTX lowered the expression of Gal-3 but not Gal-1 (among others) in this cell line and tumors as well (reversible silencing of about 98% (in vitro) and 55% (in vivo)). More importantly, Gal-3 downregulation was confirmed in clinical samples of mCRPC patients treated by taxane-based chemotherapy. To evaluate whether this DTX-dependent Gal-3 downregulation in PCa tumors could be one of the principal molecular factors responsible for chemotherapy enhancement of immunotherapy, we decide to inoculate mice with an autologous BM-DC vaccine loaded with lysates from TC1 cells pretreated by DTX in vitro or expressing an anti-Gal-3 shRNA. This protocol completely protects the animals to the growth of Gal-3 deficient-PCa tumors. We then decided to go further in the understanding of the mechanisms that promote this effective anti-PCa immune response, and analysed the proliferation ability of lymphocytes in the presence or absence of TC1 tumor cells, expressing or not Gal-3. The results were encouraging as we demonstrated that Gal-3 down-regulation in tumors (through the use of TC1-shGal-3 cells or TC1-treated with low doses of DTX) was sufficient to restore the capability of CD8+ T cells to be activated and to proliferate as well as in the absence of tumor cells. Interestingly, we showed by ex vivo cytotoxicity assays that these activated and proliferating CD8+ T lymphocytes after vaccination of mice with TC1-lysate loaded BM-DC are capable of killing Gal-3 downregulated TC1 cells and protect prostate cancer recurrence and metastasis development in mice after primary tumor-resection. In conclusion, our results strongly suggest that Gal-3 is one of the principal immunosuppressor in prostate cancer that limits the success of immunotherapy. We then show that DTX-based chemotherapy would promote a decrease of Gal-3 expression by the tumors to create a permissive tumor microenvironment for successful immunotherapy. Finally, we propose a rational protocol combining no toxic/low doses of Docetaxel with simplified immunotherapy to completely control tumor recurrence for all PCa patients and chemoresistant as well.

#3961

Alternating electric fields (TTFields) induce immunogenic cell death resulting in enhanced antitumor efficacy when combined with anti-PD-1 therapy.

Tali Voloshin, Noa Kaynan, Shiri Davidi, Yaara Porat, Anna Shteingauz, Mijal Munster, Rosa S. Schneiderman, Catherine Tempel Brami, Einav Zeevi, Karnit Gotlib, Shay Cahal, Aviran Itzhaki, Moshe Giladi, Eilon D. Kirson, Uri Weinberg, Adrian Kinzel, Yoram Palti. _Novocure, Haifa, Israel_.

Tumor Treating Fields (TTFields) are a clinically applied anti-neoplastic treatment modality delivered via noninvasive application of low intensity, intermediate frequency, alternating electric fields. In this study we evaluated whether TTFields-induced cell death is immunogenic. For evaluation of immunogenic cell death (ICD), cultured murine cells were treated with TTFields using the inovitro system. ICD was characterized by the pre-apoptotic exposure of calreticulin (CRT) on the cell surface, secretion of adenosine triphosphate (ATP), and release of the chromatin-binding protein high mobility group B1 (HMGB1). For detection of ER stress, phosphorylation of the translation initiation factor eIF2α was assessed. TTFields effect on autophagy was evaluated using electron microscopy and immunoblot and immunofluorescence evaluation of LC3. For evaluation of the effect of TTFields on dendritic cells (DCs), bone marrow derived dendritic cells were co-incubated with TTFields treated LLC-1 cells and phagocytosis by DCs and DCs maturation were evaluated using flow cytometry. For in-vivo studies, mice orthotopically implanted with LLC cells were treated with TTFields, the immune checkpoint inhibitor anti-PD-1 or a combination of the two modalities. Tumor volume was monitored and flow cytometry analysis was performed for phenotypic characterization of infiltrating immune cells. We demonstrate that cancer cells that die under TTFields application exhibit release of HMGB1, ATP depletion from cells , and ER stress leading to CRT translocation to the cell surface. Moreover, we show that TTFields treated cells promote phagocytosis by DCs, DC maturation in vitro, and initiate inflammation in vivo. We also show that the combined treatment of lung tumor-bearing mice with TTFields plus the immune checkpoint inhibitor anti-PD-1 led to a significant decrease in tumor volume compared to anti-PD-1 alone or to the control group. Significant increases in CD45+ tumor infiltrating cells were observed in the TTFields plus anti-PD-1 group. These infiltrating cells, specifically macrophages and DCs, demonstrated upregulation of surface PD-L1 expression. Correspondingly, cytotoxic T-cells isolated from these tumors have shown higher levels of IFN-γ production relative to untreated mice. Our results demonstrate the potential of TTFields therapy to induce ICD. We also demonstrate robust efficacy of concurrent application of TTFields and anti PD-1 therapy in a mouse model of lung cancer. These data suggest that combining TTFields with anti-PD-1 might achieve tumor control by further enhancing antitumor immunity.

#3962

Chemotherapy induces alteration of programmed death ligand-1 expression in gastric cancer.

Kohei Yamashita, Masaaki Iwatsuki, Shiro Iwagami, Kojiro Eto, Yuki Koga, Yuki Kiyozumi, Yoshifumi Baba, Naoya Yoshida, Hideo Baba. _Kumamoto University, Japan, Kumamoto, Japan_.

Background Cancer immunotherapy targeting the programmed cell death 1 (PD-1) / programmed death ligand 1 (PD-L1) pathway have been demonstrated favorable therapeutic effect in gastric cancer (GC). However, it remains unclear that PD-L1 expression in GC tissue can be a biomarker to predict the therapeutic effect of immune checkpoint inhibitors. We hypothesize that prior therapy, including chemotherapy and radiation therapy can lead to the alteration of PD-L1 status, resulting in imprecise evaluation of PD-L1 expression. The aim of this study is to clarify the effect of cytotoxic agents on PD-L1 expression in GC cells in time and dose depending manner. Methods PD-L1 expression of GC cell lines were confirmed by quantitative PCR and western blotting. Two gastric cancer cell lines, which have low PD-L1 expression, are selected and used in subsequent in vitro experiment. We evaluated the expression of PD-L1 in these cell lines by quantitative PCR and western blotting after exposure to cytotoxic agents at various concentrations and exposure times. Furthermore, we examined whether the expression of PD-L1 induced by cytotoxic agents was maintained after ceasing the 5-FU exposure. The gene pathway regulating PD-L1 induction was also examined. Results The PD-L1 expression of the selected GC cell lines were upregulated , depending on the concentration and exposure time of cytotoxic agents. Furthermore, the PD-L1 expression induced by cytotoxic agents was maintained even after cessation of exposure. We clarify that the upregulation of PD-L1 expression was induced via p38 MAPK activation. Conclusion We demonstrated that cytotoxic agent can alter the expression of PD-L1 in GC cells through the activation of p38 MAPK pathway in dose and time depending manner. Our findings suggested that the modified administration of immune checkpoint inhibitors can improve the antitumor response, leading to promising new treatment strategy against GC.

#3963

Deep serial analysis of the TCR/BCR CDR3 adaptome repertoire in peripheral blood of pancreatic cancer patients in a randomized study of neoadjuvant chemotherapy with or without autophagy inhibition.

Yue Wang, Michael T. Lotze. _University of Pittsburgh, Pittsburgh, PA_.

Pancreatic cancer has a paucity of dendritic cells and almost no T lymphocytes. Here, we used Next Generation Sequencing following dimer avoidance multiplexed PCR (DAM-PCR) immune repertoire to study the characteristics and diversity of the TCR repertoire among PDAC (Pancreatic Ductal Adenocarcinoma) patients who underwent chemotherapy with or without Hydroxycholoroquine followed by surgical extirpation. We sequenced the TCR α,β,γ, and δ chains and the B-cell receptor (BCR) heavy chain (IgH) complementary-determining region 3 (CDR3) repertoire in peripheral blood mononuclear cells (PBMCs) from 10 PDAC patients before (Pre-chemo) and after chemotherapy (Post-chemo) and following operation (Postop). The distribution of CDR3, VD indel, and DJ indel lengths were similar among the Prechemo, Postchemo and Postop groups. The TCR repertoire diversity of PDAC patients was much lower compared to healthy donors (HD). The diversity index (D50) of TCR α and β's in PDAC patients who received HCQ during chemotherapy was less than in patients without HCQ (p<.05). TRBV expression increased and some public sequences diminished at individual time points following chemotherapy and postop. Full analysis of an ongoing study will be presented.

#3964

Chemotherapeutic drug-induced tumor neoantigen discovery.

Julie M. Rumble,1 James L. Mobley,1 Richard Jones,2 Michael Ford,2 Michael Pisano1. 1 _Cayman Chemical, Ann Arbor, MI;_ 2 _MS Bioworks LLC, Ann Arbor, MI_.

Checkpoint inhibitors have shown significant efficacy against a variety of cancers, changing the way many cancers are treated. However, only a subset of patients can benefit from these therapies currently, and finding ways to increase the number of patients who can is a primary interest. Tumor mutational burden is one biomarker for checkpoint inhibitor efficacy, which may point to effective strategies for increasing tumor response to therapy. Combination of checkpoint inhibitors with existing chemotherapy is being pursued in several clinical trials, and has been shown to have synergistic effects in a mouse tumor model. Chemotherapeutic drugs work in many ways to kill tumor cells, notably by attacking DNA and its replication machinery to prevent proliferation. These DNA-targeting drugs can cause mutations in cells that are not killed outright. We hypothesize that some chemotherapeutics can cause DNA mutations that are translated into proteins and peptides that make their way into the HLA class I peptide presentation pathway. If these neoantigens are seen by CD8+ T cells as foreign, T cells will attack these tumor cells. This could provide one mechanism of action for an increase in therapeutic efficacy in tumors treated with combinations of chemotherapeutics and checkpoint inhibitors. We test the ability of two drugs, SN-38 (an active metabolite of irinotecan) and oxaliplatin to induce neoantigens by sequencing peptides from class I molecules in a colon cancer cell line.

#3965

Circulating levels of IFN gammaand neutrophil counts in breast cancer patients who received balixafortide and eribulin combination therapy.

Johann Zimmermann, Garry Douglas, Barbara Romagnoli, Debra Barker, Daniel Obrecht. _Polyphor Ltd., Allschwil, Switzerland_.

Background: Balixafortide (B) is a highly selective antagonist of the chemokine receptor CXCR4. Clinical proof-of-concept in combination with eribulin (E) was achieved in a recent Phase 1 single arm dose-escalation trial in patients with metastatic HER2-negative breast cancer (NCT01837095). Safety and tolerability of B in combination with E was similar to that of B or E monotherapy.

Anti-cancer effects of CXCR4 antagonists include sensitization of tumor cells to chemotherapy, suppression of metastatic spread, inhibition of angiogenesis, and activation of immune cells.

Methods: Plasma samples from patients who received initial E monotherapy (1.4 mg/m2, run-in cycle) or combination of B and E (escalating doses of B intravenous up to 5.5 mg/kg plus E 1.4 mg/m2 in 21 days cycles) were immediately processed and stored at -80°C. Circulating levels of IFN-γ, an important marker of anti-tumor immune response, were determined by a high-sensitive S-Plex assay (Meso Scale Diagnostics). Absolute neutrophil counts (ANC) were measured by automated hematology instruments.

Results: E monotherapy did not increase IFN-γ levels in the first 8 hours post dosing, but led to an increase after 24 hours. Combination with B modulated IFN-γ levels and led to a sustained ANC increase 5 days post dosing. The increase pre vs post treatment can be up to 100-fold and receded prior to the next treatment cycle. Conclusions: E treatment led to a strong, transient increase of plasma IFN-γ levels in breast cancer patients modulated by B. The ANC increase by B may balance possible neutropenic actions of E.

#3966

Combined Rho-kinase inhibition and immunogenic cell death triggers and propagates immunity against cancer.

In-San Kim, Gi Beom Kim, Gi-Hoon Nam, Yeonsun Hong. _Biomedical Research Institute, KIST, Seoul, Republic of Korea_.

Activation of T-cell immune response is critical for the therapeutic efficacy of cancer immunotherapy. Current immunotherapies have shown remarkable clinical success against several cancers; however, significant responses remain restricted to a minority of patients. Here, we show a therapeutic strategy that combines enhancing the phagocytic activity of antigen-presenting cells with immunogenic cell death to trigger efficient antitumour immunity. Rho-kinase (ROCK) blockade increases cancer cell phagocytosis and induces antitumour immunity through enhancement of T-cell priming by dendritic cells (DCs), leading to suppression of tumour growth in syngeneic tumour models. Combining ROCK blockade with immunogenic chemotherapy leads to increased DC maturation and synergistic CD8+ cytotoxic T-cell priming and infiltration into tumours. This therapeutic strategy effectively suppresses tumour growth and improves overall survival in a genetic MMTV/Neu tumour model. Collectively, these results suggest that boosting intrinsic cancer immunity using immunogenic killing and enhanced phagocytosis is a promising therapeutic strategy for cancer immunotherapy.

#3967

Comparison of neoadjuvant FOLFIRINOX alone vs FOLFIRINOX + stereotactic body radiation as immune-modulators of the pancreatic adenocarcinoma microenvironment.

Matthew R. Farren, Walid Shaib, Mohammad Zaidi, Layal Sayegh, David Kooby, Shishir Maithel, Alyssa Krasinskas, Olatunji Alese, Juan Sarmiento, Bassel El-Rayes, Gregory B. Lesinski. _Winship Cancer Institute of Emory University, Atlanta, GA_.

Pancreatic ductal adenocarcinoma (PDAC) has dismal five-year survival (<9%). We examined the impact of neoadjuvant FOLFIRINOX alone or in combination with stereotactic body radiation (SBRT) on biomarkers in the PDAC tumor microenvironment (TME). We hypothesize conventional therapies may unmask novel targets in the TME that can be leveraged to enhance the efficacy of immunotherapy in PDAC. We conducted a pilot study using FFPE-tumor sections from PDAC patients who underwent upfront surgical resection (resection-only) or who received neoadjuvant FOLFIRINOX or FOLFIRINOX + SBRT (FOX or FOX+S, respectively) prior to resection (n=6/group). Average time (weeks) from end of neoadjuvant therapy - surgery was 6.6 ± 2.9 (FOX) or 7.0 ± 1.2 (FOX+S). Using the NanoString GeoMX platform to conduct multiplexed analysis of protein targets (~30 analytes) in spatially distinct regions of tumor tissue, we analyzed individual regions in each tumor that were enriched for malignant cells (Pan-Cytokeratin+), stroma (SMA+) or immune cells (CD45+). Across all tumor regions, FOX treatment was associated with increased CD14, CD56, PD-L1, phosphorylated (p-)STAT3, p-AKT, and PTEN compared to resection-only. In contrast, in FOX+S treated patients none of these were elevated in comparison to resection-only, and PTEN was significantly lower. Additionally, CD3, CD4, and CD8 expression—which did not differ between resection-only and FOX, were all significantly lower in FOX+S treated patients. Similar effects were observed in CD45-enriched regions. Whereas FOX treatment was associated with increased CD14, PD-L1, p-STAT3, p-AKT, PTEN, B7-H3, GZMB and β-catenin compared to resection-only, none of these were increased in FOX+S, while PTEN was again significantly decreased. Moreover, CD3 and CD4 expression were also significantly decreased in FOX+S. In tumor-rich regions, we found increased CD3, CD44, CD45RO, and CD56 in FOX-treated tumors as compared to resection-only. However, there was no difference in these markers when comparing resection-only and FOX+S. In addition, CD4 expression was lower in FOX+S samples while FOXP3 expression was unchanged, suggesting a relative enrichment of regulatory T cells. Finally, in αSMA-rich regions (stromal), FOX treatment was associated with increased p-STAT3 and significantly decreased CD4 and VISTA expression. FOX+S exhibited no such increase in p-STAT3 and further significant decreases in CD4 and VISTA expression, while also being associated with significantly lower CD3 and CD8 expression. This data provide insight into the immunological effects of standard of care neoadjuvant therapy for resectable/borderline-resectable PDAC. This work provides data to guide strategic new combination therapies for pancreatic cancer.

#3968

OT-101/Chemotherapy - A novel mechanism of action (MOA) in pancreatic cancer immunization therapy.

David Nam, Larn Hwang, Vuong Trieu. _Oncotelic Inc., Agoura Hills, CA_.

Background: OT-101 is a phosphorothioate antisense oligodeoxynucleotide targeting the human TGF-β2 mRNA. OT-101 breaks down immune tolerance and activates immunity against the tumor. The Subsequent Chemotherapy (SC) releases tumor antigens and boosts the immunity response along with further epitope expansion. The phase 1/2 clinical data for OT-101/SC is presented here along with supporting preclinical MOA studies.

Methods: Total of 37 pts, 2nd line and beyond received OT-101 with option to go on SC (OT-101/SC) or Best Supportive Care (BSC) (OT-101/BSC). Overall survival (OS) was compared using log-rank statistics. Stratification by treatment line, schedule, metastasis location, disease control (DC), and baseline CA19-9, was performed. For cytokine analysis, plasma levels of 31 cyto-/chemokine were measured over 8 time points during 3 cycles of intravenous OT-101 therapy and a subset of 12 patients.

Results: In vitro cell kill assay and in vivo xenograft study were performed with human PBMC and OT-101. OT-101 reduced TGF-β2 secretion and increased LAK cell-activity against all tumor lines by 400% and 364% in comparison to the untreated control and the Lipofectin control, respectively. Addition of active rh-TGF-β2 protein suppressed the cytotoxic activity of the immune cells in a dose dependent manner. Preclinical- LS174T xenograft was treated with PBMC or PBMC + OT-101. OT-101 significantly enhanced the activity of PBMC against the xenograft. Median OS (mOS) of the 18 pts receiving OT-101/SC was 9.4mos vs. the 19 pts on OT-101/BSC (2.8mos, p=0.0004). Pts with only liver mets had a mOS of 9.5mos while those with liver mets and others only had a mOS of 4.7mos (p=0.0077). Among the former, one patient had complete response beyond 77.3mos and another had stable disease with OS of 40.3mos. OS was higher for liver mets. only group with OT-101/SC - 12.4mos versus 1.9mos, p=0.0006. There were 16 of 37 pts with DC, with mOS of 9.7mos vs. 3.0mos (p<0.0001). OS was higher for DC group with OT-101/SC - 11.8mos vs. 5.0mos, p=0.0021. Patients exhibited spike in IL-8 level which returned to basal level at treatment stop. R2 relating the IL-8 spike and OS were 0.8522 and 0.9895 and p values were 0.0011 and 0.0053 for pts treated subsequently with SC and BSC, respectively. The two lines intersect at the origin with higher response for those with OT-101/SC, suggesting exaggerated response expected for antigen boost during the SC phase.

Conclusions: Escalating Intratumoral heterogeneity (IH) resulting in xenogenization, which is countered by overexpression of TGF-β. Here we report on the use of OT-101/SC to break immune tolerance to pancreatic cancer (PC) for the cure. The MOA for OT-101/SC is consistent with the reactivation of immunity during TGF-β suppression and subsequent boosting/expansion of immunity during SC. Contrary to traditional tumor vaccine- this is universally applicable to all patients.

#3969

TP-0903, a potent AXL receptor tyrosine kinase inhibitor, enhances the activity of anti-PD-1 therapy in a metastatic preclinical syngeneic model of breast cancer.

Yuka Kumagai,1 Jun Oishi,1 Megumi Nakamura,1 Jason M. Foulks,2 Clifford J. Whatcott,2 Steven L. Warner,2 David J. Bearss,2 Masashi Goto1. 1 _Sumitomo Dainippon Pharma Co., Ltd., Osaka, Japan;_ 2 _Tolero Pharmaceuticals, Inc., Lehi, UT_.

The high rate of non-responders to immune checkpoint inhibitors (ICI) represents significant challenges in the field of immune-oncology. Breast cancer has emerged as a cancer type that responds poorly to ICI. Therefore, there is an urgent need to better understand how cancer cells escape immune surveillance and resist immune attacks. Cancer cell clones with mesenchymal features seem to be less susceptible to attacks by immune cells. Hence, epithelial-to-mesenchymal transition (EMT) targeting agents such as TP-0903 are emerging as potential combination candidates to enhance immune checkpoint inhibition. AXL is overexpressed in a variety of cancers, often driving the pathogenesis and progression of disease. AXL overexpression induces cancer growth, invasion, metastasis, drug resistance, and mesenchymal characteristics. Aside from cancer cells, AXL is also expressed on several types of immune cells including macrophages and dendritic cells. Recently, it has been reported that AXL plays an important role as a negative regulator of immune response, contributing to an anti-tumor immune suppression. We have previously identified TP-0903 to be a potent small molecule inhibitor of AXL (IC50=14 nM) capable of reversing EMT. In this study, we evaluated whether TP-0903 could enhance the ICB effect in an ICB-resistant triple negative, metastatic breast cancer mouse model (4T1). In the 4T1 syngeneic model, anti-PD-1 monotherapy failed to inhibit tumor growth indicating that this tumor is resistant to anti-PD-1 treatment. The combination of TP-0903 and anti-PD-1 resulted in statistically significant tumor growth inhibition versus TP-0903 monotherapy (p<0.05). We hypothesized that the combination effect was influenced by potential CD8+ T cell depletion, and further investigated the effects of TP-0903 on immune cells in spleen and tumors. Tumor growth inhibition was found to be associated with a decrease in myeloid-derived suppressor cells (MDSC) in the spleen and an increase in infiltration and activation of dendritic cells (DCs) in the tumor. Gene expression analysis revealed that TP-0903 treatment decreased multiple immunosuppressive cytokines and chemokines including IL-6 and G-CSF in vivo. These results suggest that TP-0903 modulates the immune-suppressive tumor microenvironment (TME) to reinvigorate T cell immunity in anti-PD-1 resistant 4T1 tumors. In conclusion, AXL inhibition with TP-0903 modulates TME and enhances the anti-tumor effects of ICBs in an anti-PD-1 resistant, metastatic breast cancer mouse model. These findings support additional clinical testing of TP-0903 in combination with ICBs, which is currently being investigated in patients with advanced solid tumors in an ongoing clinical study (NCT02729298).

#3970

PARP inhibitors increase expression of Type I interferon regulated genes mainly through trapping.

Monica E. Wielgos, Petar Jelinic, Douglas A. Levine. _NYU Langone Health, New York, NY_.

Background: Currently, there are multiple ongoing clinical trials testing the efficacy of various poly (ADP-ribose) polymerase (PARP) inhibitors in combination with immune checkpoint blockade across many cancer types. However, there is no rationale as to which PARP inhibitor is best to use in these studies. Two PARPis (rucaparib and talazoparib) have been reported to activate IFN signaling genes through the stimulator of interferon genes (STING) pathway. The objective of this study is to characterize the effect of all clinically active PARPis on Type I interferon (IFN) regulated genes. We also aim to identify whether this effect is dependent on CHK1, which we have previously reported to be activated by PARP trappers (rucaparib, olaparib, niraparib, and talazoparib) and not by the mainly catalytic inhibitor (veliparib).

Methods: CCL5 and CXCL10 gene expression levels were measured using qRT-PCR in 293T after two days of treatment with the PARP inhibitors (velipairb, rucaparib, olaparib, niraparib, and talazoparib). CXCL10 mRNA levels were also measured in HGSOC BRCA wild-type cell lines (OVCAR3 and CAOV3) after two days of treatment with veliparib and talazoparib. We also measured CCL5 and CXCL10 expression after veliparib and niraparib treatment in 293T cells with stable CHK1 overexpression.

Results: The four PARP trappers consistently increased CCL5 and CXCL10 expression in 293T cells. Although, veliparib had little to no effect on the expression of these two genes as compared to the other four PARPis. CXCL10 mRNA levels were significantly upregulated after talazoparib treatment (p<0.01) but not affected after veliparib treatment in CAOV3 cells. Similarly, an increase in CXCL10 gene expression was observed in OVCAR3 cells (p<0.05). Although, veliparib also had a modest but not significant effect on CXCL10 levels. CCL5 and CXCL10 gene expression consistently increased when treated with niraparib in 293T cells with stable CHK1 overexpression. This same effect was not observed after veliparib treatment in 293T cells that stably overexpressed CHK1.

Conclusions: Our results suggest that all PARP trappers promote expression of IFN Type I regulated genes which may be dependent on CHK1 expression. These findings may better define the most ideal PARP and immune checkpoint inhibitor combination.

#3971

MEK inhibitors limit inflammatory cytokine production and modulate the immune response in the setting of Cholangiocarcinoma..

Amanda N. Ruggieri,1 Matthew R. Farren,1 Mark Yarchoan,2 Nilofer S. Azad,2 Lauren Dennison,2 Bassel El-Reyes,1 Gregory B. Lesinski1. 1 _Emory University, Atlanta, GA;_ 2 _Johns Hopkins University, Balitmore, MD_.

Cholangiocarcinoma (CC) is a rare but aggressive cancer of the bile ducts with a five-year survival rate of only 2% in patients with metastatic disease. MAP kinase signaling is important in the pathogenesis of CC and serves as a key regulator of cell proliferation and survival. Our data indicate this pathway is constitutively active in several CC cell lines and accompanied by production of immunomodulatory cytokines including IL-6, MCP-1 and GM-CSF. Cobimetinib is a potent and selective inhibitor of MEK that has been utilized clinically in patients with advanced CC. Although MEK inhibitors (MEKi) have been studied in a variety of solid tumors, the mechanisms by which they act upon CC cells are not completely defined. These agents have modest single agent activity in CC, and their impact in combination with immunotherapy is under active investigation. We hypothesized that MEKi can modulate cytokine and chemokine expression from CC tumors, thereby complementing immunotherapy in this aggressive disease. Using a novel panel of human CC cell lines, we demonstrated a variable impact of cobimetinib on in vitro cell proliferation, with a reduction in viability no greater than 35%. However, MEKi reproducibly limited cytokine (GM-CSF and IL-1β) and chemokine (MIP-1α) production from human CC cell lines in vitro. The ability of cobimetinib to limit MAPK signal transduction in these cells was confirmed via immunoblot. These data complement prior published observations by Ebert et al.in 2016 indicating that single agent MEKi is sufficient to elicit T cell infiltration into tumors. We have completed enrollment of a Phase II clinical trial (NCT03201458) evaluating the efficacy of cobimetinib in combination with the PD-L1 inhibitor, atezolizumab. An extensive series of correlative studies are underway in peripheral blood and tumor biopsies to examine the impact of cobimetinib on modulating cytokine and chemokine signatures in the context of PD-L1 targeted therapy. These data will provide new insight into the impact of MEK inhibitors as a complement to immunotherapy in a disease that is refractory to most conventional therapies.

#3972

Investigation of tumor intrinsic PD1/PD-L1 in canine urothelial carcinomas as a spontaneous translational model for human invasive bladder cancer.

Yu Jia Wang,1 Katarzyna Pietrzak,2 Mikolaj Kocikowski,2 David Argyle,1 Ted Hupp,3 Maciej Parys1. 1 _University of Edinburgh, Roslin, United Kingdom;_ 2 _University of Gdansk, Gdansk, Poland;_ 3 _University of Edinburgh, Edinburgh, United Kingdom_.

Urothelial carcinoma (UC) is the fourth most common malignancy in human and the most common form of urinary bladder cancer in dogs. This neoplasia tends to be invasive at advanced stages hence novel therapeutic approaches are urgently needed. As urothelial carcinomas share many similarities between the dog and human in the immunological responses, dogs are a perfect model for studying immunotherapeutic approaches. Recently immuno-checkpoint blockade using anti-PD1/PD-L1 antibodies have shown a promising effect and durable responses in the treatment of UC. Although until recently the PD1/PD-L1 axis was considered to be mostly affecting immune cells, recent reports identified, that PD-1/PD-L1 interaction is also capable of pro-tumorigenic signaling in melanoma and other cancers. Yet, little is known regarding this phenomenon in UC. In our study, we aim at identification of the expression level of PD-1 in canine UC and subsequently characterize the effect of overexpression and CRISPR-based knock-out of PD-1 and PD-L1. Using flow cytometry and immunocytochemistry, we identified that PD-1 is expressed on the surface of canine UC cell line. Interestingly, incubation with high concentration of PD-1 antibody decreased the proliferation rate of the tumor. After transfection with a PD-1 overexpression vector, we notice a significant morphological change and decrease in proliferation and colony formation. Our result provides the first report on PD-1 intrinsic signaling play as an important role in tumor growth of canine UC and thus may also be responsive to immune checkpoint blockade. The effect identified further strengthens the dog as a potential translational model for UC immunotherapy.

#3973

Nivolumab-based ELISA revealed increased serum PD-1 levels in advanced RCC patients.

Kosuke Mizutani,1 Kengo Horie,1 Taku Kato,1 Keita Nakane,1 Kyojiro Kawakami,2 Yasunori Fujita,2 Masafumi Ito2. 1 _Gifu Univ. Graduate School of Medicine, Gifu, Japan;_ 2 _Tokyo Metropolitan Institute of Gerontology, Japan_.

Introduction: Antibody therapy has been successfully used for several caners, but the efficacy is still limited. To select patients that respond well to the therapy, development of companion diagnostics is essential. Although PD-L1 expression determined by immunohistochemistry has been shown to be a marker to select patients with non-small-cell lung cancer who will benefit from antibody therapy, its potential for Nivolumab treatment in renal cell carcinoma (RCC) patients is unclear. Detection of the soluble form of PD-1 in serum has been reported, but there are few studies demonstrating its clinical role in RCC. Antibody-based assays may rely on characteristics and quality of antibody used. We hypothesized that the use of Nivolumab as a capture antibody in ELISA would give more clinically relevant results to evaluate the serum PD-1 levels in RCC patients.

Materials and Methods: After approval of the study by the Medical Review Board of Gifu University, a total of 71 participants were recruited (22; healthy volunteers, 29; localized RCC and 20; RCC patients with metastasis or recurrence). Serum PD-1 levels were determined using PD-1 Human ELISA Kit (Thermo Fisher Scientific Inc., MA, USA). For Nivolumab-based ELISA, a capture antibody included in the kit was changed to Nivolumab. Nivolumab was coated to 96-well microplates using a commercially available kit.

Results: Medians of the serum PD-1 levels measured by conventional ELISA were 31.8, 29.8 and 34.3 pg/ml in healthy volunteers, localized RCC patients and those with metastasis or recurrence, respectively, which showed no statistically significant difference. When the same set of patients were analyzed by Nivolumab-based ELISA, medians were 24.6, 23.7 and 46.4 pg/ml and the serum PD-1 levels were significantly elevated in patients with metastasis or recurrence (P = 0.013) compared to controls and localized patients. Of the 29 localized patients, 3 patients with relatively higher serum PD-1 levels at initial diagnosis relapsed later. Among 6 patients with metastasis who were treated with Nivolumab, well responders showed relatively lower serum PD-1 levels before treatment.

Conclusion: The serum PD-1 levels determined using Nivolumab were increased in advanced RCC patients. Our results suggest that the detection of target molecules using a therapeutic antibody may yield more clinically relevant information, thus providing a novel approach to develop companion diagnostics.

#3974

In vivo CRISPR-Cas9 library screen for liver cancer therapeutic targets essential for T cell killing.

Haixin Zhao,1 Chenlu Geng,2 Wenrong Zhou,1 Zhengang Peng,1 Qunsheng Ji,1 Yong Cang1. 1 _WuXi AppTec, Shanghai, China;_ 2 _Zhejiang University, Hangzhou, China_.

T-cell checkpoint inhibition by antibodies blocking programmed cell death protein 1 (PD1) has been a major breakthrough in cancer therapeutic development. However, anti-PD1 drugs approved by FDA are ineffective in treating all cancer types or in all patients in a responsive cancer type. Therefore, it is important to stratify responsive patient population and identify synergistically effective combination therapies. Primary hepatocellular carcinoma (HCC) is a human malignancy with a high incidence, mortality and recurrence rate worldwide. We established a syngeneic mouse HCC model using a murine hepatoma cell line, Hepa1-6, which, interestingly, shrunk in volume shortly after reaching up to 400 mm3. The tumor failed to resume growth unless treated with anti-CD4/CD8 antibodies to deplete endogenous T cells, suggesting that an adaptive T cell immunity suppresses the late-stage tumor growth. To identify the genetic regulators of the cancer elimination, we employed CRISPR-Cas9 based genome-wide knockout screening strategy to identify the potential targets essential for cancer cell sensitivity to the T cell immunity. Mouse xenograft models derived from hepa1-6 cells infected with the library were treated with anti-CD4/CD8 antibodies or anti-mPD1 antibody. Deep sequencing of the tumors revealed T cell-dependent enrichment of guide RNAs targeting 241 genes with variable functions. We have performed a second round of in vivo screen with a CRISPR library focused on these candidate genes. We will discuss the technical challenges of the in vivo screen as well as the broad application of the technology for discovery of immune oncology targets.

### Novel Strategies for Biomarker Identification and Use in Cancer 2

#3975

Tumor mutation burden and microsatellite instability in colorectal cancer.

Francesca Fenizia,1 Raffaella Pasquale,1 Cristin Roma,1 Francesca Bergantino,1 Anna Maria Rachiglio,1 Nicoletta Chicchinelli,1 Paolo Graziano,2 Gerardo Botti,1 Fabiana Tatangelo,1 Giosuè Scognamiglio,1 Christopher Allen,3 Matilde Lambiase,1 Alessia Iannaccone,1 Nicola Normanno1. 1 _INT Fondazione Pascale, Napoli, Italy;_ 2 _IRCCS "Casa Sollievo della Sofferenza", San Giovanni Rotondo, Italy;_ 3 _Thermo Fisher Scientific, Houston, TX_.

Introduction In colorectal cancer (CRC), the use of immunotherapy is currently based on the evaluation of microsatellite instability (MSI) for the identification of patients who may benefit from this treatment. Recently, tumour mutation burden (TMB) has been evaluated as a useful biomarker to predict response to immune checkpoint (IO) therapy. TMB can be measured by means of targeted next generation sequencing with panels covering approximately 1 Mb of the human genome. We performed TMB analysis on tumor cell lines and CRC samples and compared the results with MSI status to explore the relationship between these two biomarkers.

Materials and methods MSI status was evaluated by means of routine immunohistochemistry (IHC), the Idylla MSI assay (Biocartis) and with fluorescence capillary electrophoresis-based DNA fragment size analysis of a T17 mononucleotide sequences within intron 8 of heat shock protein 110 kDa HSP110 (17 polythymine, T17). TMB evaluation was performed with the Oncomine Tumor Mutation Load Assay (Thermofisher) on the Ion S5XL platform. Data analysis was carried out using Ion Reporter Software v5.6. TMB was calculated as the total number of somatic single nucleotide variants (SNVs) divided by number of bases with sufficient coverage.

Results We first evaluated the TMB in 13 tumor cell lines and correlated the results with massively parallel sequencing data from >1600 genes for the same cell lines available on cBioPortal, demonstrating an high correlation between the Oncomine Tumor Mutation Load Assay and the TMB value obtained by wide genetic profiling (R² = 0.976). We next analyzed a cohort of 23 MMR deficient (MMR-D) and 32 MMR-proficient (MMR-P) formalin fixed paraffin embedded (FFPE) CRC, as classified by IHC. The MMR data were compared to TMB and revealed TMB values not in line with the expected results, since some samples in the MMR-D group had low TMB values (TMB range: 5.5-134.4). We re-evaluated the 23 MMR-D samples by means of the Idylla System. The Idylla MSI assay re-classified 7 samples as microsatellite stable (MSS) and categorized the remaining 16 samples as MSI-High (MSI-H). The results were confirmed by the T17 analysis. In the MSS group (n. 39), the mean and median TMB value were 11.48 and 10.39, with TMB ranging from 4.88 to 33.29. The mean and median TMB for the MSI-H group (n. 16) were 43.89 and 38.63, and the TMB values ranged from 19.63 to 134.4. While the medians of the two groups were significantly different (Mann-Whitney test P<0.0001), these data confirm a high heterogeneity of TMB within MSI-H CRC. One MSS case displayed a TMB close to the median TMB of the MSI-H group (TMB: 33.29). The MSS status of this sample was confirmed by the Idylla System.

Conclusions These data suggest that routine IHC methods for MSI evaluation can lead to a wrong classification of CRC. On the other hand, TMB assessment might better identify patients who may benefit from IOs within both the MSI-H and the MSS subgroups.

#3976

"Fit-for-purpose" PIK3CA assays for patient stratification in breast cancer clinical trials.

Hua C. Gong, Sharmila Manjeshwar, Violet Abraham, Shawn Rivera. _Navigate BioPhama, a Novartis Subsidiary, San Diego, CA_.

Background: Prospective clinical trials stratifying by PIK3CA genotype is necessary to determine if the mutation predicts for increased sensitivity to agents targeting the phosphoinositide 3-kinase (PI3K) pathway. It is crucial to select appropriate assays to optimize operational, scientific and timeline requirements for patient enrollment in a real world setting. Here we report application of three different PIK3CA mutation test methods during the course of a PI3K TKI development.

Methods: We customized Sanger sequencing, cobas® assay and a proprietary investigational use only (IUO) assay to detect point mutations in PIK3CA in order to accommodate the development needs at the different stages of clinical development of PIK3CA TKIs. We performed method comparison studies to determine the most cost effective and "fit-for-purpose" assay for clinical trial testing. Specimen requirements, assay performance characteristics, turnaround-time (TAT) and valid result rates were assessed to select the best test method for patient stratification to meet the requirements of each stage of drug development.

Results: PIK3CA test results can be obtained from a single, 5μm FFPE section with ≥0.1% and ≥10% tumor in the IUO and cobas® tests respectively. Sanger Sequencing requires longer processing time and ≥30% tumor content. The cobas® assays were validated in FFPE samples and can reliably detect PIK3CA mutations in exons 9 and 20 with ≥5% mutation level. The method comparison study showed that the concordance rate between Sanger sequencing and cobas® PIK3CA assay is 86% among the 360 samples tested while it is 96% between the cobas® assay and the IUO assay among 395 samples tested. The IUO assay is slightly more sensitive, and calls out the exact mutation while the cobas® assay may not. The Sanger sequencing demonstrated the lowest sensitivity and required more DNA input, which resulted in more samples failing to yield a valid result. The cobas® assay is a more user-friendly assay allowing better laboratory efficiency and TAT. Patients selected using the IUO assay showed a median progression-free survival nearly twice as long among those receiving a PI3K TKI compared to placebo.

Conclusions: The most important factor in selecting a testing platform for clinical trial patient stratification is the assay performance characteristics. We employed the Sanger sequencing assay in the initial trial testing, then switched to the cobas® assay, and finally to the IUO assay to meet the requirements as the drug progressed through different stages of clinical development. The IUO assay has the performance characteristics that meet the regulatory requirements and a validated clinical utility in selecting patients for PI3K TKI treatment, thus on its way to a companion diagnostic submission.

#3977

Clinical validation of cell-free circulating tumor DNA to detect therapy resistance and disease progression in metastatic colorectal cancer patients.

Iris van 't Erve,1 Alessandro Leal,2 Karen Bolhuis,3 Jillian Phallen,2 Nicole van Grieken,3 Veerle Coupé,3 Annegien Broeks,1 Daan van den Broek,1 Victor Velculescu,2 Cornelis Punt,3 Gerrit Meijer,1 Remond Fijneman1. 1 _Netherlands Cancer Institute, Amsterdam, Netherlands;_ 2 _Sidney Kimmel Comprehensive Cancer Center, Baltimore, MD;_ 3 _Amsterdam Academic Medical Centers, Amsterdam, Netherlands_.

Introduction: DNA mutation analysis in individual patients paves the road towards personalized medicine, in which prognosis and therapy prediction are determined based on gene mutations. For example, it is known that RAS mutations are a negative predictor biomarker for response to therapeutic monoclonal antibodies directed against the epithelial growth factor receptor (EGFR) in metastasized colorectal cancer (mCRC) patients. Anti-EGFR treatment is expensive and 80-90% of the patients who do not benefit from anti-EGFR therapy suffer from the negative side-effects. Therefore, better prediction and monitoring of anti-EGFR treatment response is necessary. Cell-free circulating tumor DNA (ctDNA) derived from blood plasma is expected to improve stratification by early detection of therapy resistance and disease progression.

Aim: The general aim of this study is to advance towards clinical implementation of ctDNA-based tests as molecular biomarkers to improve disease management of mCRC patients. In particular, we investigate the added value of liquid biopsy mutation analysis for (1) assessment of primary and acquired resistance to anti-EGFR treatment using a gene panel, compared to the conventional tissue analyses, and (2) for detection of disease progression, compared to conventional computed tomography (CT) imaging.

Methods: CAIRO5 is a multicenter, randomized, phase 3 clinical trial of the Dutch Colorectal Cancer Group and includes patients with initially unresectable, liver-only mCRC, as confirmed by a central panel of liver surgeons/radiologists. Liquid biopsies are longitudinally collected in cell-save Streck tubes at baseline and during follow-up, in parallel with the CT imaging (every 2 to 3 months). In ctDNA, RAS/RAF hotspot mutations are analyzed by droplet digital PCR (ddPCR), while a panel of 33 genes will be analyzed using ultrasensitive next generation sequencing approaches.

Results: The nation-wide multicenter logistics for longitudinal blood sample collection and plasma processing has been established, with participation of more than 55 Dutch hospitals. At present (November 2018), over 350 patients have been included from whom more than 850 blood samples were obtained. Based on tissue analyses, 59% of these patients harbors a RAS/RAF mutation (48% KRAS, 5% NRAS, 6% BRAF). Concordance between tissue biopsy and liquid biopsy was determined in a subset of patients and showed a >90% concordance for KRAS at codons 12, 13 or 61. In addition, radiologic evaluations of the CT images are collected and the lead time for recurrence will be compared with ctDNA analysis. Results of a first selection of patients will be presented.

Conclusion: The liquid biopsy ctDNA and clinical data obtained in this study will provide the input for a health technology assessment yielding recommendations for clinical implementation of ctDNA applications.

#3978

Direct analysis of exosome-bound proteins and nucleic acids using the ExoVerita™ Flex platform.

Jean M. Lewis,1 David Searson,1 Alfred Kinana,1 Heath Balcer,1 Debrah Thompson,2 David Bodkin,3 Juan P. Hinestrosa,1 Rajaram Krishnan1. 1 _Biological Dynamics, Inc., San Diego, CA;_ 2 _HTG Molecular Diagnostics, Tucson, AZ;_ 3 _Cancer Center Oncology Medical Group, La Mesa, CA, San Diego, CA_.

Exosomes carry signals between cells and may influence cancer development, metastasis, and drug resistance. Unfortunately, current tools for exosome isolation and analysis have been limited to examining a specific class of markers, such as DNA, RNA, and proteins. We have developed a microelectrode array chip that uses a novel Alternating Current Electrokinetic method to perform rapid, affinity-free isolation of exosomes. The chip runs on our proprietary ExoVeritaTM Flex platform. Isolated exosomes can then be analyzed in situ or can be lysed to obtain DNA and RNA for use in downstream assays, such as sequencing.

In this study, we used the ExoVerita Flex to isolate exosomes directly from plasma specimens obtained from cancer subjects with lung, colorectal, or pancreatic cancers as well as healthy donors. Isolated exosomes were analyzed in situ for exosome-bound proteins and nucleic acids, and then purified following isolation to examine their nucleic acid expression profiles. Using the ExoVerita Flex with plasma specimens and antibodies targeting the exosome-bound proteins CEA, PD-L1, GPC-1 and CD63, we observed differences in protein expression levels among the different cancer types (N = 35) and healthy donors (N = 20), such as increased PD-L1 expression in non-small cell lung cancer subject samples and elevated levels of CEA in colorectal cancer subject samples (N = 10). Furthermore, we co-stained samples with antibodies and DNA- or RNA-selective dyes to simultaneously measure exosome-bound proteins and circulating nucleic acid levels in the same sample at the same time.

RNA sequencing analysis was performed by extracting RNA from the isolated exosomes using a proprietary buffer, and then performing RT-ddPCR and miRNA-Sequencing. RT-ddPCR analysis of the PGK-1 housekeeping gene RNA transcript confirmed the presence of viable RNA following extraction. miRNA profiles from isolated exosomes were generated using a targeted RNA sequencing assay and used for repeatability and differential expression studies.

The ExoVerita Flex platform offers a novel, unbiased method for isolation and characterization of exosome-bound proteins and circulating nucleic acids while being compatible with standard downstream analysis. The ability to perform two-dimensional protein & nucleic acid analysis in the same sample run, at the same time, is both unique and unprecedented and will enable promising future clinical studies in cancer looking at diverse classes of biomarkers directly from blood.

#3979

Allele frequencies of TP53 in liquid biopsy of castration-resistant prostate cancer patients were correlated with clinical response : A pilot study.

Ju Won Kim, Hye Sun Lee, Yeul Hong Kim, Yoon Ji Choi, Won Jin Jang, Jeong Yoon Choi, Kyong Hwa Park. _Korea University Anam Hospital, Seoul, Republic of Korea_.

Circulating cell-free DNA in the blood of castration-resistant prostate cancer patients harbors tumor-specific genomic information. Here, we investigated common genomic variants and whether these data correlate with treatment response.

Minimum 20ng of cfDNA were extracted from plasma of CRPC patients. Hybrid-capture based genomic profiling (Axen Cancer Panel 1, Macrogen) was performed to sequence 88 genes in each samples. The data was presented with mutated gene, DNA change, allele frequency(AF). The final reports provided each variants' clinical effect based on Clinvar reference. We considered 'pathologic', 'likely pathologic', and 'unknown significance' as meaningful variants.

Total 22 CRPC patients who were receiving chemotherapy and/or androgen receptor antagonists participated in our study. Patients' median age was 73.5 years old, and almost all the patients had bone metastasis (21/22, 95.5%). Follow up samples were available in 17 patients at the time of response evaluation. Thus, 39 samples were analyzed with NGS platform. Median 6 mutations were detected per each analysis, and 3 of them were clinically pathogenic or had unknown significance. Most common meaningful variant was TP53 (21/39, 53.8%), followed by ALK (16/39, 41.0%) and PTEN (15/39, 38.5%). In TP53 gene, c.734G>A, c.659A>G, and c.844C>T missense variants were detected. We also compared number of mutations in 17 pairing samples according to patients' clinical response based on Recist Criteria v1.1. 6 out of 8 patients in PR group had pathogenic TP53 mutation initially; 3 of them disappeared, and 3 of them showed decreased AF in f/u data. In PD group of another 8 patients, 1 out of 3 initially mutated TP53 showed increased AF and 2 new TP53 mutations appeared in f/u analysis. Median delta between the number of meaningful mutated gene was -1 in PR groups (range -4 ~ +6), and +0.5 in PD groups (range -2 ~ +4). Each group had one outstanding value against the tendency, which showed +6 in PR and -2 in PD. Without these outliers, median delta was -2 in PR and +1 in PD. The outlier in PR group with a significant increase (Δ+6) in the number of mutation showed favorable results in PSA and bone scan. However, soon after that, his next follow up imaging study was presented with highly aggravated bone metastasis. With this case, we suspected that cfDNA could function as an early surrogate marker of upcoming treatment failure.

We examined the common variants in cfDNA of CRPC patients and consequences of treatment responses. Pathologic TP53 mutation was detected more than half of the analysis, and its AF tended to change as treatment went on. The total amount of variants was also correlated with clinical response. If cfDNA sequencing data and clinical aspect are conflicting, the increasing number of mutation could be a sign of treatment failure. We will present more data from 30 patients at the time of conference.

#3980

Molecular profiling of cell-free DNA and RNA in the blood of patients with non-small cell lung cancer.

Jordan Reese, Leisa Jackson, Hestia S. Mellert, Gary Pestano. _Biodesix, Inc., Boulder, CO_.

Lung cancer is the leading cause of cancer-related deaths in the United States with Non-Small Cell Lung Cancer (NSCLC) being the most commonly diagnosed subtype. However, up to 30% of advanced NSCLC patients are not eligible for tissue biopsy. As a result, liquid biopsies are becoming increasingly utilized in clinical testing as they are non-invasive and have an overall decreased risk to patients. This approach also addresses other challenges associated with tissue-based profiling, including tumor heterogeneity and low yield of quality nucleic acid for the identification of actionable targets of treatment. In this study, cell-free total nucleic acid (cfTNA) was isolated from donor patient plasma samples collected and shipped at ambient temperatures in blood collection tubes to the Biodesix CAP/CLIA laboratory in Boulder, CO. cfTNA was profiled with a targeted cancer NGS (next generation sequencing) panel, using the Ion Torrent GeneStudio S5 Plus System. Variant analyses were conducted using a threshold of ≥0.3% percent variant allele frequency to assess concordance in the patient donor specimens. A high level of concordance (R2=0.99) was observed between inter-laboratory/inter-instrument NGS runs using 6 clinical samples. In the 0.1% single nucleotide variant (SNV) positive control sample which encompasses 23 hotspots, there was slightly lower concordance (R2=0.90) due to low variant allele frequency (0.06-0.3%). Validation studies are in progress and include RNA fusions and additional variants present in blood from patients diagnosed with NSCLC.

#3981

Exosomal microRNA profiles in peritoneal fluids as a therapeutic biomarker for peritoneal metastasis of gastric cancer.

Hideyuki Ohzawa, Akira Saito, Yuko Kumagai, Hironori Yamaguchi, Yoshinori Hosoya, Naohiro Sata, Joji Kitayama. _Jichi Medical University, Shimotsuke, Tochigi, Japan_.

Objectives: Peritoneal metastasis (PM) frequently occurs in patients with gastric cancer and has poor prognosis even with systemic chemotherapy. Intraperitoneal chemotherapy (IPC) using paclitaxel (PTX) combined with systemic chemotherapy has demonstrated a remarkable clinical efficacy for gastric cancer with PM. However, there is no reliable biomarker to reflect the response of peritoneal lesions as well as to predict the outcome of the patients receiving IPC. Although peritoneal fluids are considered to contain many factors derived from disseminated tumor cells in peritoneal cavity, little information is available so far. In this study, we examined exosomal miRNA profiles derived from peritoneal fluids in patients with gastric cancer and explored possible biomarkers useful for IPC of the patients with PM.

Methods: Peritoneal fluids were collected from 2 groups; with or without PM. Peritoneal fluids were obtained either laparoscopically, during open surgery or from subcutaneous infusion port connected to an intraperitoneal catheter. Exosomal fractions were isolated using ultracentrifuge method (150,000 xg, 70 min, 4°C) and the presence of exosome was confirmed with nanotracking analysis. Total RNA including small RNA was extracted from exosomal fractions and expression analyses of miRNAs were performed using RT-PCR.

Results: At first, we comprehensively analyzed the expression of miRNAs between 11 samples with PM and 14 samples without PM and identified 11 miRNAs which showed particularly different expression pattern between the 2 groups. Among them, 4 miRNAs (miR-21-5p,miR-223-3p, miR-342-3p and miR-92a-3p) were selected and further analysed using 51 samples derived from the peritoneal fluids of gastric cancer. Expression of these 4 miRNAs were significantly up-regulated in samples with PM which was consistent with formeranalysis. Moreover, expression of miR-21-5p was significantly upregulated in in samples fromT4 tumor (invaded serosa or adjacent structure) as compared with those from T1 to T3 tumor.

Conclusion: We identified several dysregulated exsosomal miRNAs derived from peritoneal fluids of the patients with PM from gastric cancer, which were supposed to reflect the tumor burden on peritoneum. Exosomal miRNA profiles might be useful biomarkers to determine the presence of PM as well as the response to IPC.

#3982

**Isolation, culture & characterization ofthe circulating tumor cells from breast cancer patients' peripheral blood **in vitro **.**

Pan Zhao, Huirong Zhang, Chang Liu, Jianhong Wang, Wenbin Zhou, Chang Zou. _Shenzhen People's Hospital, Shenzhen, China_.

Background: Circulating tumor cells (CTCs) are rare tumor cells in peripheral blood. As a few studies showed CTCs would be a kind of intriguing drug targets and more beneficial for cancer patients if there genomic and proteomic information could be deciphered dynamically. However, the rarity, challenging isolation as well as the primary culture procedures limited the analysis of CTCs insightfully.

Methods: The method of density gradient centrifugation was used to obtain PBMCs. Cells adherent and suspension culture for collecting the CTCs.

Results: There was a cell cluster which named CTC-3 came out in the plate. CTC-3 steadily formed clones, displayed epithelial phenotype and showed stem-cell like properties. Immunofluorescence identification of CTC-3 were DAPI-positive, Cytokeratin-positive, and CD45-negative. The karyotyping results indicated that chromosomes were aneuploidthe, the chromosome number of CTC-3 varied from 56 to 60. CTC-3 had larger nuclear/cytoplasmic ratio and limitless replicative potential. Comparing with the MCF-7, CTC-3 grew more faster than MCF-7 from the fifth day. Furthermore, the sphere formation ability of CTC-3 was significantly higher than MCF-7 in vitro. Besides, the tumorigenesis of CTC-3 cells was almost the same as MCF-7 in vivo by the analysis of xenografting in immunodeficient mice. For drug resistance, CTC-3 cells were more resistant to anti-tumor drugs after 3D culture compared with 2D, survive rates of 3D CTC-3 cells after treatment were more than 80%. Finally, the RT-PCR results shows the expression of Vimentin, E-cadherin and CD44 were significantly higher in CTC-3.

Conclusion: We have successfully isolated and expanted a new breast cancer cell line from the breast cancer patient, which could provide more drug sensitivity information of the patient and open up novel avenues towards the understanding of breast cancer.

Keywords: Circulating tumor cell; Breast cancer

#3983

An efficient new detection method of colorectal cancers from blood samples.

Joon Kim,1 Seon Hahn Kim,1 Kyung-Hwan Cho,1 Yeongkeun Kwon,1 Tae-Sung Kim,1 YouJin Jung,2 Hag Dong Kim2. 1 _Korea University, Seoul, Republic of Korea;_ 2 _HAEL Lab, Seoul, Republic of Korea_.

Colorectal cancer incidence in Korea is known to be one of the highest in the world. However, the detection methods from patients' blood samples show relatively inefficient. Ribosomal protein S3 (rpS3), a multifunctional protein with a DNA repair endonuclease activity, has been known to be overexpressed and secreted from cancer cells but not from normal cells. Western blotting using antibodies against rpS3 protein revealed that this protein is overexpressed from cancer tissues of colorectal cancer patients comparing to normal tissues from the same patients. However, other proteins including other ribosomal proteins did not show this phenomenon. We also tested the secretion level of the protein using newly developed S3-1 antibody in the serum samples from 50 colorectal cancer patients and 50 healthy people to test the possibility as a cancer marker. All the diagnosed blood samples which were randomly selected among subjects in National Biobank of Korea were collected and serum was acquired by centrifugation. Samples were analyzed by dot blotting and ELISA methods. The detection level of S3-1 from the serum of colorectal cancer patients showed significantly higher than that from healthy control group. Also more than 60% from colorectal cancer patients' serum showed sensitivity signals with 90% specificity which are significantly better than those of CEA or CA19-9 showing a possibility as a novel colorectal cancer biomarker.

#3984

Detection of CTCs from whole blood of lung cancer patients using the automated Liquid Scan.

Rolf Muller,1 Sangeetha Purushotham,1 Paul Diaz,1 Alena Bartakova,1 Judy Muller-Cohn,1 Jerry Lu,1 Veronica Cheung,1 Roksolana Melnychuk,1 Jennifer Barber-Singh,1 Elizabeth Fabio,1 Hatim Husain,2 Malgorzata Witek,3 Maryam Zomorrodi,1 Mateusz Hupert,1 Steven Soper,3 Matt Jackson,2 Catherine Chen2. 1 _BioFluidica, San Diego, CA;_ 2 _University of California San Diego, San Diego, CA;_ 3 _University of Kansas, Lawrence, KS_.

Blood contains a wealth of diverse tumor biomarkers, including circulating tumor cells (CTCs), extracellular vesicles (EVs), and cell free DNA (cfDNA) allowing us to advance development of technologies to aid in detection and management of cancer and other related diseases. Biomarkers released from tumor cells harbor important signatures of disease including mutations, copy number variations, methylation changes, and/or chromosomal rearrangements that can be used as biomarkers to monitor disease progression or to tailor individualized treatment options in this era of personalized medicine. We describe the use of the Liquid Scan, an automated liquid biopsy platform to interrogate lung cancer patient blood for CTCs, cfDNA and EVs.For CTC diagnostics, whole blood from patient sample is run through Biofluidica's customized chips coated with cancer specific antibodies. CTCs bound to the chips were eluted live and used for standard screening tests and molecular characterization of the tumor. We were able to capture CTCs from all stages (I, II, III, IV) of the lung cancer patient blood samples using EpCAM antibodies coated on the chip surface. CTCs detected from Stage I patients using minimal blood samples (1 ml) suggests Biofluidica's microfluidic chip technology can detect pre-symptomatic disease and early detection of residual disease or relapse.

The automated Liquid Scan is also used for capture and isolation of cfDNA and EVs with different processing protocols, microfluidic chips and reagents. Whole blood or plasma is applied to the chip that contains 1.4 million diamond shaped posts. The specific biomarker is captured and released from the chip for analysis. Here we demonstrate the ability of the platform to efficiently capture cfDNA and EVs from cell lines and plasma from patient samples with minimal background resulting in the ability to further analyze these biomarkers for genetic mutations. As a measure of tumor burden a comparison is made on the relative abundance of circulating tumor DNA (ctDNA) compared to background level of cfDNA originating from normal cells. Biofluidica's Liquid Scan automated platform will help to provide excellent patient care that delivers high quality data suitable to assist in clinical decisions as per cancer stage, response to treatment and early detection. Biofluidica's Liquid Scan can reduce expensive and painful biopsy procedures generally used in the current health care system.

#3985

Dynamic changes of tumor-derived extracellular vesicles and related RNAs in blood samples of NSCLC patients.

Rita Lampignano,1 Martin Neumann,2 Vera Kloten,1 Nina Kessler,1 Anna Babayan,2 Laura Keller,3 Ines Stevic,3 Klaus Pantel,3 Thomas Krahn,1 Thomas Schlange,1 for the IMI CANCER-ID consortium. 1 _Bayer AG, Wuppertal, Germany;_ 2 _Qiagen GmbH, Hilden, Germany;_ 3 _University Cancer Center Hamburg/Eppendorf, Hamburg, Germany_.

Liquid biopsy is defined as molecular analysis of rare cells or cell-free nucleic acids circulating in blood or in other biofluids. The concept of liquid biopsy aims at closely monitoring the status of a disease or treatment efficacy in a simple, fast, cost efficient way and at any point in time with minimal risk and burden for the patient. It has been suggested that the most reproducible approach to investigate "liquid" mRNAs would be to focus on cell-free extracellular vesicle (EV)-transcripts rather than on circulating cell-free mRNAs, as the former are well protected from degradation and therefore more stable. However, the volumes of available blood samples can be limiting for molecular analysis and rare cells as well as cell-free nucleic acids are normally present in low abundance. Therefore, detection of rare events or quantification of limited material requires robust, highly sensitive technologies like droplet digital PCR (ddPCR).

In this pilot study, we investigated the absolute expression of 10 EV-transcripts typically involved in cancer development and chemotherapy resistance in longitudinal serum samples of 13 non-small cell lung cancer (NSCLC) patients (age range 37-76 years) treated with cisplatin at the Department of Inner Medicine II, University of Ulm, between May 2011 and August 2012.

Blood samples were collected at baseline, after 3-6 months and after 9-12 months of chemotherapy from patients showing objective clinical response. Then, EV-RNAs were isolated by sequentially processing obtained serum samples with miRCURY® exosome kit and miRNeasy kit (both Qiagen, Hilden, Germany). Afterwards, the status of selected EV-transcripts was investigated via ddPCR (Biorad, Milan, Italy).

After start of chemotherapy, we observed a downregulation of at least 2-fold of the following potentially cancer-related EV-transcripts : PTEN, ERBB2, FOSL1, IL-8, MET, RPS27A, SF3B1 and of the following housekeeping EV-mRNAs: ACTB, HIST1H3H and HSPA1A, in all patients. Interestingly, in none of patients´ samples, the expression of EV-AKT1 and of EV-PD-L1 transcripts could be detected.

Our preliminary results demonstrates significant dynamic changes of cancer-related EV-mRNA expression in serum samples of NSCLC patients during cisplatin treatment, thus suggesting a potential overall decrease of EVs in response to chemotherapy. Moreover, despite the small amounts of mRNA in extracellular vesicles from patient blood, using ddPCR differential mRNA levels could be determined in patient serum and expand the scope of biomarker analysis of EVs.

This work is supported by IMI JU & EFPIA (grand no. 115749, CANCER-ID). Samples from patients and healthy volunteers, respectively, were collected under signed informed consent.

#3986

**Digital droplet PCR measurement for plasma** HER2 **amplification in patients with AGC.**

Kyoungmin Lee,1 Kazuko Sakai,2 Min-Hee Ryu,1 Jae-Joon Kim,1 Young Soo Park,1 Young-Soon Na,1 Jungeun Ma,1 Hana Na,1 Kazuto Nishio,2 Yoon-Koo Kang1. 1 _Asan Medical Center, Seoul, Republic of Korea;_ 2 _Kindai University Faculty of Medicine, Osaka, Japan_.

Introduction: With the recent introduction of human epidermal growth factor receptor 2 (HER2) targeted therapies for patients with advanced gastric cancer (AGC), determining HER2 status is essential to select the patients who may benefit from this treatment. The current standard method assessing HER2 positivity is immunohistochemistry (IHC) or in situ hybridization assays, but this way of HER2 assessment has weaknesses especially when considering tumor heterogeneity. Using digital droplet polymerase chain reaction (ddPCR) technique, we evaluated HER2 amplification in both tissue and plasma samples collected from AGC patients and analyzed clinical implication of HER2 amplification in circulating tumor DNA.

Method: Biopsied tissue and plasma samples were collected from patients with metastatic or recurrent AGC who were enrolled in AGC biomarker study conducted at Asan Medical Center. Samples were obtained before the start of anti-cancer treatment. HER2 amplification in DNA from tissue and cell-free DNA from plasma were determined by ddPCR, performed in Kindai University.

Results: Samples of 63 patients with HER2 positive GC and 37 patients with HER2 negative GC enrolled between April 2014 and October 2017 were included in the analysis. As expected, the median HER2 copy number (CN) was higher in patients diagnosed as HER2 positive than negative, both for tissue (4.54 vs 0.95, P < 0.001) and plasma (1.49 vs 1.07, P < 0.001). ROC curve analysis showed 83.8% specificity and 69.8% sensitivity for tissue HER2 positivity at 1.17 CN of plasma HER2. In patients receiving anti-HER2 therapy (n=57), there was a trend for worse progression-free survival (PFS) outcome in patients with higher plasma HER2 CN (>1.5 vs ≤1.5), although no statistical significance was seen (median PFS 6.67 vs 8.19 months, P = 0.172). In multivariate Cox regression analysis including the age (>60 vs ≤60), HER2 IHC intensities (2+ vs 3+), and risk groups determined by Koo et al.'s prognostic model (Good vs Moderate vs Poor), higher plasma HER2 CN (>1.5 vs ≤1.5) was significantly associated with poor PFS (HR 2.22, 95% CI 1.14-4.32, P = 0.019). However, when tumor burden (sum of largest diameter of all measurable lesions) was combined as another factor in patients with measurable disease (n=39), plasma HER2 CN was no longer an independent factor (HR 1.38, 95% CI 0.58-3.27, P = 0.466) and larger tumor burden (>6.5cm vs ≤6.5cm) was found to be an independent prognostic factor for poor PFS (HR 2.63, 95% CI 1.12-6.21, P = 0.027), instead. Of note, plasma HER2 CN showed a positive relationship with tumor burden multiplied by tissue HER2 CN in linear regression (β=0.32, P = 0.002).

Conclusion: Using the ddPCR technique, we found that plasma HER2 CN was significantly increased in HER2 positive GC patients compared to negative patients. However, plasma HER2 CN did not provide more information on predicting therapeutic effects than the tumor burden in patients receiving anti-HER2 therapy.

#3987

Predicting MCL1 inhibitor sensitivity in large cell line panels using a gene expression signature.

Andreas Wernitznig,1 Dorothea Rudolph,1 Matthias Samwer,1 Norbert Schweifer,1 Francesca Trapani,1 Tobias Wunberg,1 Heribert Arnhof,1 Teakyu Lee,2 John L. Sensintaffar,2 Edward T. Olejniczak,2 Cyril H. Benes,3 Stephen W. Fesik,2 Norbert Kraut1. 1 _Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria; _2 _Vanderbilt University, Nashville, TN;_ 3 _Massachusetts General Hospital, Boston, MA_.

MCL1, an anti-apoptotic member of the BCL-2 family of proteins, is a key regulator of cancer cell survival and a known resistance factor to anti-cancer drugs, making it a highly desirable target for therapeutic intervention. Recently several MCL1 inhibitors have entered Phase I clinical development. Data derived from large cancer cell line panels suggest, that cell lines of hematopoietic origin are more broadly sensitive to MCL1 inhibition, than cell lines derived from solid tumor types. In particular, for the therapy of solid tumor patients with an MCL1 inhibitor, a patient selection biomarker would be highly desirable.

Published in vitro data using various MCL1 inhibitors, siRNA or CRISPR/Cas9 technology show that tumor cell lines with low BCL-XL gene expression are mostly sensitive to MCL1 inhibition, down-regulation or inactivation. Here we show that by adding the gene expression data of additional six genes (including the MCL1 binding partner BAK1) to BCL-XL, a supervised learning predictor was applied and could reach a performance of almost 80% correctly classified solid tumor cell lines. This new predictor has been applied to either tumor samples, adjacent normal tissues or normal tissue samples from TCGA and GTEx. Briefly, most normal tissue samples are categorized as being sensitive. Moreover, solid tumor samples in contrast to solid tumor cell lines are predicted to be broadly sensitive to MCL1 inhibition with a predicted anti-tumor effect rate of 60% to 95%. In summary, our work describes the translational challenges using cell line-derived predictors on ex vivo tumor samples.

#3988

Identifying prognostic biomarkers of hepatocellular carcinoma by weighted gene coexpression network analysis.

Yi Bai,1 Junyu Long,1 Jianzhen Lin,1 Xu Yang,1 Dongxu Wang,1 Xiaobo Yang,1 Xinting Sang,1 Jiahui Liu,2 Haitao Zhao1. 1 _Department of Liver Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China; _2 _YuceBio, Shenzhen, China_.

Purpose: Exploring the molecular mechanisms of prognosis in patients with hepatocellular carcinoma (HCC) could assist in identifying novel biomarkers for effective therapy against this deadly disease. Hence, this study was designed to determine the prognostic biomarkers of HCC and to investigate potential mechanisms. Experimental Design: The RNA-Seq data as well as clinical data were downloaded from The Cancer Genome Atlas (TCGA) database, aiming at identifying differentially expressed genes (DEGs) between nontumorous tissues and HCC. Then, weighted gene coexpression network analysis (WGCNA) was performed for determining significant modules associated with overall survival (OS). Results: The mRNA expression profiles from 374 HCC patients in TCGA were included to determine 3270 DEGs. Then, WGCNA was carried out on the 3270 DEGs, and 10 coexpressed gene modules were determined. Pearson's correlation analysis revealed that the turquoise module significantly positively correlated with OS and the blue module significantly negatively correlated with OS, followed by seven hub genes with high connectivity were identified in these two modules. Based on the Kaplan-Meier (K-M) survival curves and receiver operating characteristic (ROC) curves, the seven hub genes could predict prognosis for patients with HCC well, and the results were validated by GSE54236 dataset contained 78 HCC patients. Additionally, the univariate and multivariate Cox regression analyses showed that the seven hub genes were independent prognostic factors for patients with HCC. Conclusions: In conclusion, the current results provide novel insights into the prognostic biomarkers of HCC, which may contribute to the development of molecular targeted therapy for HCC.

#3989

Identification of biomarkers for UM-164 in triple-negative breast cancer.

Nathan M. Merrill, Nathalie M. Vandecan, John P. Lloyd, Eric J. Lachacz, Peter J. Ulintz, Sofia D. Merajver, Matthew B. Soellner. _University of Michigan, Ann Arbor, MI_.

Triple negative breast cancer (TNBC) is one of the deadliest forms of breast cancer due to limited treatment options beyond conventional chemotherapy. While making up approximately 10-20% of total breast cancer cases in the US, TNBC carries a serious burden of disease as it tends to be more aggressive, higher grade, and have a poorer prognosis than other forms of breast cancer. Though new therapies, such as PARP inhibitors, have shown promise in clinical trials in very select patients (e.g. BRCA1 mutation carriers), there is a general lack of predictive markers of therapeutic efficacy to identify subsets of patients most likely to positively respond to a given targeted or conventional therapy. Thus, there is a critical need for biomarkers of drug efficacy in TNBC to help design precision medicine clinical trials based on systematically derived predictive biomarkers. To fill this gap, we devised a strategy to identify biomarkers of drug efficacy in TNBC. As a paradigm of our approach, we treated a panel of 23 TNBC cell lines with our potent c-Src/p38 inhibitor, UM-164, and determined a drug sensitivity score (DSS) in each line. We then calculated correlations of DSS with a variety of molecular readouts, including RNA sequencing, reverse-phase protein array, DNA sequencing array, and Nanostring RNA and miRNA arrays. From these correlations, we have identified expression DJ1, MAPK11, and PCM1 as strong predictors of UM-164 efficacy in TNBC. Building on this, we used our results to identify companion drugs that lead to synergy or test others that could instead result in antagonism when used in combination. To verify our results, independently targeting the DJ1 pathway, we show that AKT inhibition results in synergy when combined with UM-164. The outcome of this research yields a robust and generalizable methodology for the identification of biomarkers in cancer to design Phase I-III trials and for generating the mechanistic evidence of effective rational combinations.

#3990

Circulating tumor DNA assessed through amplicon-based next-generation sequencing and its clinical application.

Hitoshi Zembutsu, Hiroki Osumi, Eiji Shinozaki, Kensei Yamaguchi, Masato Ozaka, Takashi Sasaki, Marie Muramatsu, Naoki Sasahira. _Cancer Institute, JAPAN, Tokyo, Japan_.

Circulating tumor DNA (ctDNA) studies focusing on only one or a few genes to monitor the disease progress or treatment response are unlikely to find its clinical significance. Hence, the development of cell-free DNA (cfDNA) panel covering hundreds of mutation hotspots is important for the establishment of clinically practical ctDNA detection system. We enrolled 101 patients with metastatic colorectal cancer (mCRC) who received chemotherapy. Amplicon-based genomic profiling of 14 genes, which are commonly mutated in CRC, in plasma by next-generation sequencing (NGS) was performed to evaluate the feasibility of this assay, and was compared with their clinical parameters and RAS status in matched tissue samples. Somatic mutations of the 14 genes in plasma cfDNA were detected in 88 patients (87.1%) with mCRC. Mutations in TP53, KRAS and APC genes were detected in 70 (69.3%), 39 (38.6%) and 24 (23.7%) patients, respectively. Mutant allele frequencies in cfDNA were significantly associated with metastasis (liver, P=0.00004, lymph node, P=0.008, number of metastatic organs, P=0.0006), tumor markers (CEA, P=0.000007, CA19-9, P=0.006, LDH, P=0.00001), and tumor diameter (maximum, P=0.00002, sum of diameter, P=0.00009). The overall concordance rate of RAS status between ctDNA and matched tissue was 77.2% (78/101). Furthermore, we enrolled 76 patients with stage lV pancreatic cancer, and their genomic profiling of 14 genes in plasma were performed to evaluate the feasibility of the cfDNA panel. We observed that patients with lower ctDNA level showed significantly longer overall survival (P=0.00000026). Our data confirmed that mutant allele in cfDNA can be sensitively detected by amplicon-based NGS system. These results suggest that ctDNA could be a novel diagnostic biomarker to monitor changes in mutational status and tumor burden in patients with mCRC, and a predictive marker for survival in patients with advanced pancreatic cancer.

#3991

ONCOTARGET (ONCOALVO): A custom NGS panel for therapeutic decision in solid tumors refractory to conventional therapy.

Andre M. Murad,1 Jose Claudio Casali-da-Rocha,1 Juliana G. Carneiro,1 Ana L. Godard2. 1 _Laboratorio Personal Diagnostica, Belo Horizonte, Brazil;_ 2 _Universidade Federal de Minas Gerais, Belo Horizonte, Brazil_.

Next-generation sequencing (NGS) of tumor biopsies of both solid tumors, as well as hematological malignancies, using commercially available platforms has broadly entered routine clinical practice in medical oncology. This molecular diagnostic approach is now used mainly to test for predictive biomarkers for patients with no available further standard therapies, as diagnostics rely on genomic testing of molecular alterations to enable effective cancer treatment. However, the high cost of large multigenic panels and especially the sequencing of the entire tumor exome is a concern, especially in a developing country such as Brazil. Here we report the clinical application of a panel of 57 genes that we call ONCOTARGET (ONCOALVO) which is based on the Thermo Fisher Oncomine Focus Assay, an integrated, commercially available NGS assay for the rapid and simultaneous detection of single nucleotide variants, short insertions and deletions, copy number variations, and gene rearrangements in 52 cancer genes with therapeutic relevance, but with 5 additional genes: the homologous recombination repair (HRR) genes BRCA1, BRCA2, ATM and CHEK2 that determine tumor sensitivity to inhibitors of the PARP enzyme and also KDR, which determines sensitivity to regorafenib. Twenty-five diagnostic samples of formalin-fixed, paraffin-embedded material submitted for molecular testing over a 8-month time period were analyzed so far. All patients had advanced solid tumors already refractory to conventional systemic therapy. Libraries were prepared from isolated nucleic acids and sequenced on the Ion Torrent S5 sequencer. Sequencing datasets were analyzed using the Ion Reporter software. From the samples of the 39 patients tested, we found 30 potential therapy target pathogenic gene aberrations in 22 (56.4%) patients: KRAS - 7 (23.3%), BRCA1 - 6 (20%), BRCA2 - 5 (16.6%), PIK3CA - 3 (10%), MAP2K1 - 2 (6.6%), BRAF-V600E - 2 (6.6%) mutations; and also one of each: ERBB2 (3.3%), mTOR (3.3%), KIT (3.3%), ALK (3.3%) mutations and one (3,3%) CCND1 amplification. In 3 (7,69%) patients there were double mutations: BRCA1 and BRCA2 in 2 cases and BRCA2 and KRAS and one case. The Oncotarget workflow enabled a turnaround of 18 frac12 days. Taken together, ONCOTARGET was found to be a convenient tool for fast, reliable, broadly applicable and cost-effective targeted NGS of tumor samples in routine diagnostics and therapeutic decisions with a potential to become an important assay for precision oncology in Brazil.

#3992

Comparison of AKR1B10, 2SC, and FH as biomarkers for HLRCC detection.

Terhi Ahvenainen,1 Outi Uimari,2 Anne Ahtikoski,2 Kati Kämpjärvi,1 Ralf Bützow,3 Pia Vahteristo1. 1 _University of Helsinki and Research Programs Unit, Helsinki, Finland;_ 2 _Oulu University Hospital, University of Oulu, and Medical Research Center Oulu, Oulu, Finland;_ 3 _The Laboratory of Helsinki University Hospital (HUSLAB), Helsinki University Hospital, and University of Helsinki, Helsinki, Finland_.

HLRCC is an autosomal dominant tumor predisposition syndrome characterized by cutaneous and uterine leiomyomas (ULs), and increased risk for aggressive type 2 papillary renal cell carcinoma (RCC). The syndrome is caused by germline mutations in fumarate hydratase (FH). Loss of FH causes dysfunction of the Crebs cycle and an increase of succinated proteins, which can be detected using anti-2-succinocysteine (2SC) antibody. Recently, we have observed that aldo-keto reductase family 1, member B10 (AKR1B10) is overexpressed in RNA-level in ULs with biallelic loss of FH. Here, we compared anti-AKR1B10, anti-2SC, and anti-FH antibodies in detection of FH loss in protein-level in HLRCC-associated ULs.

The study material consisted of 142 formalin-fixed paraffin-embedded (FFPE) UL samples collected at the University hospitals in Finland. The sample series include 77 FH-deficient UL samples from 25 HLRCC patients and 65 sporadic conventional ULs. HLRCC background was verified by detected germline mutation of FH. Four 0.8 mm cores of representative FFPE tumor tissue were used to construct tissue microarrays. Immunohistochemistry (IHC) was executed using an anti-AKR1B10 (1:300, H00057016-M01, Abnova), anti-2SC (1:2000, provided by Dr. Norma Frizzell), and anti-FH (1:1000, sc-100743, Santa Cruz Biotechnology, Inc) antibodies. Expression was detected by BrightVision (Immunologic) and DAB Quanto (Thermo Fisher Scientific) systems. Pathologist specialized in gynecological pathology evaluated the IHC staining from the smooth muscle tumor cells.

AKR1B10 and 2SC detected all 77 HLRCC samples. AKR1B10 displayed either mild (12/77, 16%) or strong (65/77, 84%) expression and succination level was strong in all cases. Loss of FH expression was displayed in 68/77 (88%) of the FH-deficient ULs. All conventional ULs displayed negative AKR1B10 and 2SC staining and expressed FH. FH was expressed either mildly (15/65, 23%) or strongly (50/65, 77%) in the samples.

Reliable methods for FH-deficient UL detection in the clinics are currently lacking. Here, comparison of AKR1B10, 2SC, and FH showed that all three biomarkers identified HLRCC-ULs with 100% specificity. The sensitivity was 100% with AKR1B10 and 2SC, and 88% with FH. It seems that even with known mutation in FH, in some cases the likely inactive protein is retained in tumor cells and is thus detectable by anti-FH.

To conclude, we show that anti-AKR1B10 and anti-2SC detect FH-deficient ULs with 100% specificity and sensitivity. Anti-FH is lacking sensitivity in 12% of HLRCC cases. Reliable detection of FH-deficient ULs enables the patients and their families to be directed to genetic counseling, family planning, and regular renal monitoring.

#3993

Programmed cell death ligand-1 (PD-L1) and CD8 expression profiling identifies an immunologic subtype of pancreatic ductal adenocarcinomas with favorable survival.

Won Jin Ho, Ludmila Danilova, Qingfeng Zhu, Teena Vithayathil, Ana De Jesus-Acosta, Nilo Azad, Daniel Laheru, Elana Fertig, Robert Anders, Elizabeth Jaffee, Mark Yarchoan. _Johns Hopkins University, Baltimore, MD_.

Unselected cases of pancreatic adenocarcinoma (PDAC) have not shown clinical benefit from single agent immune checkpoint therapy, but a subset of PDAC are known to upregulate pathways involved in acquired immune suppression. Further delineation of immunologic subtypes of PDAC is key to smarter trial designs and progress toward improved immunotherapy strategies. To accomplish this, we first employed clinical survival and RNA expression data from 155 patients in The Cancer Genome Atlas (TCGA) to investigate the relationship between immune modulating checkpoints and immune subset marker, CD8A, and their impact on survival in PDAC patients. Among the markers explored, overexpression of PD-L1 (HR=2.55, p=0.001) and IDO1 (HR=2.24, p<0.01) were individually associated with poor survival. PD-L1 expression was not significantly associated with stage or tumor mutational burden of the PDACs. While CD8A expression alone was not correlated with survival, stratifying the analysis based on PD-L1 and CD8 expression identified a subtype characterized by low PD-L1 and high CD8A with favorable survival (p=0.004). Similarly, the combination of low IDO1 and high CD8A expression was associated with favorable survival (p=0.038). We further extended these observations using an independent PDAC cohort of 33 patients from our institution via immunohistochemistry, again observing that low PD-L1 / high CD8 subtype associates with positive prognosis (p=0.021). Although PDAC is regarded as a poorly immunogenic cancer type, these findings infer that baseline T cell infiltration into PDAC is a feature of long term survival and highlights the importance of developing future immunotherapeutic strategies based on data-supported biomarkers to refine patient selection. 

### Special Populations / Biostatistics in Clinical Trials

#3994

Genomic characterization of melanoma in the elderly reveals molecular drivers of progression and prognosis associated with immunotherapy response.

Stephen P. Smith,1 Caroline Gaudy-Marqueste,2 Rajive Kumar,3 Richard Marais,4 Eduardo Nagore,5 Amaya Viros4. 1 _University of Cambridge, Cambridge, United Kingdom;_ 2 _Aix-Marseille University, Marseille, France;_ 3 _Deutsches Krebsforschungszentrum, Heidelberg, Germany;_ 4 _University of Manchester, Manchester, United Kingdom;_ 5 _Instituto Valenciano de Oncologia, Valencia, Spain_.

Melanoma incidence and mortality particularly affects elderly patients. 85% of melanoma deaths occur in patients older than 60, and age is a powerful independent predictor of outcome. The genomic characteristics driving melanoma in the aged population have not been previously investigated.

We have undertaken a comprehensive genomic analysis of age-specific molecular characteristics in metastatic cutaneous melanoma and discovered a core set of 4 driver genes that when mutated in older patients is a powerful predictor of poor outcome. We validate these findings in international independent primary melanoma replication cohorts. This signature stratifies patient prognosis more accurately than the current staging guidelines from early to late stages, making it relevant to immediate clinical practice.

Checkpoint inhibitors are showing promising results in the adjuvant setting to treat early stage melanoma patients, and the cost burden to health care will limit access to these drugs. Importantly, elderly patients benefit from immune checkpoint inhibitor therapies, and there are currently no accepted predictors of response used to prioritize patients for these costly therapies in the adjuvant or advanced disease stages. Previous studies have shown a higher mutation burden and neo-antigen load are indicators of response.

Critically, we show elderly melanoma patients are the population subset most likely to present with a higher mutation burden, and our signature strongly correlates with melanomas of higher mutation burden and neo-antigen load. We validate these findings across multiple primary and metastatic melanoma cohorts. Finally, stratifying old patients by our 4-gene outcome signature is also associated with immunotherapy response in the metastatic stage. Our study demonstrates that stratifying patients by age will allow improved discrimination of prognosis and association with response to treatment.

Current selection of patients is based on AJCC stage alone, which means more patients have to be enrolled and more time is required to discriminate the effect of novel adjuvant immunotherapy strategies. Our work, validated across multiple cohorts, directly proposes enrolling patients with high genetic risk of poor outcome and improved rate of immunotherapy response using a simple 4-gene DNA signature that can be easily translated into clinical practice.

#3995

BACH2 **and** PRDM1 **gene** **expression changes in T and B cells according to age of healthy individuals and significance in chronic lymphocytic leukemia patients.**

Pushpamali De Silva, Vu Luan Dang Chi, Basile Stamatopoulos, Soizic Garaud, Vincent Thibaud, Hugues Duvillier, Jean-Nicolas Lodewyckx, Catherine Sibille, Karen Willard-Gallo, Dominique Bron. _Institut Jules Bordet, ULB, Brussels, Belgium_.

Background: Aging is a risk factor for developing malignant hemopathies including chronic lymphocytic leukemia (CLL). We evaluated the relevance of BACH2 and PRDM1 gene expression in the aging of immune cells and the immunodeficiency associated with CLL.

Methods: Peripheral blood mononuclear cells were isolated from untreated CLL patients (n=41) and age-matched healthy donors (HD; n=60). T and B cells were purified (95%-99%) by magnetic isolation. BACH2, PRDM1, PD1, PDL1 and CDKN2A (p16INK4A) transcripts were quantified using RT-qPCR. ß-galactosidase activity was measured by flow cytometry. Cell apoptosis was analyzed after intracellular oxidative stress induced by etoposide treatment. ChIP-qPCR was done to find BACH2-apoptotic target genes in purified CD4+ T cells from HDs with/without etoposide treatment. Prognostic value of BACH2 and PRDM1 expression in purified CD19+ B cells was analyzed retrospectively in a cohort of CLL patients (n= 270), and correlated with clinical parameters.

Results: BACH2 expression in HDs is significantly downregulated while PRDM1 expression increased in CD4+, CD8+ T and CD19+ B cells according to age groups. BACH2 expression is further reduced in T and B cells from CLL patients compared to age-matched HDs. Also, PRDM1 is significantly upregulated in T cells from CLL patients but not in leukemic B cells. PD1 expression is significantly upregulated in T cells in the older vs younger HDs and also in CLL patients compared with age-matched HDs. High PDL1 expression is also strongly correlated with increased age in HD B cells with a further increase detected in leukemic B cells. A strong inverse correlation was observed between BACH2 and PD1 in T cells; and between BACH2 and PDL1 in B cells from HDs. We also analyzed the link between BACH2 expression and senescence markers; CDKN2A and ß-galactosidase. CDKN2A expression inversely correlated with BACH2 in CD4+, CD8+ T and CD19+ B cells. ß-galactosidase activity showed an increase compared to BACH2 deficient lymphocytes in older compared to young HDs, suggesting that BACH2 deficiency leads to the accumulation of senescent cells. A strong correlation was observed between age-related BACH2 downregulation and a decrease in CD4+ T and CD19+ B cell apoptosis after etoposide treatment. ChIP-qPCR assay confirmed that BACH2 binds to genes involved in apoptosis and after etoposide treatment, BACH2 binding was repressed. In our retrospective cohort, increased BACH2 expression was correlated with improved treatment-free survival (p=0.0352).

Conclusion: Decrease of BACH2 and increase of PRDM1, PD1 and PDL1 in T and B cells from CLL patients and aged-matched HDs, are significantly correlated with aging and could be part of immunosenescence. Involvement of BACH2 in resistance to drug-induced apoptosis of HD lymphocytes was documented and is currently investigated in the CLL cohort.

#3996

Search for prognosis prediction factors in treatment selection for elderly patients with EGFR negative advanced stage non-small cell lung cancer patients.

Yoshiko Kaneko, Takako Mouri, Nobuyo Tamiya, Tadaaki Yamada, Junji Uchino, Koichi Takayama. _Kyoto Prefectural University of Medicine, Kyoto, Japan_.

Introduction: Since 40% of Japanese lung cancer patients are older than 75 years old, it is necessary to consider a specific strategy to treat such elderly lung cancer patients. From that aspect, it is very important to define frail or vulnerable elder populations who have lots of heterogenic health background in treatment of lung cancer. In this study, we have tried to identify prediction factors which significantly correlates to overall survival (OS) of such elderly advanced lung cancer patients by using of patient status and laboratory data before treatment.

Objects and methods: Patients more than 75 years old who enrolled in this retrospective study were diagnosed as an advanced and EGFR negative non-small cell lung cancer during 2013-2017, and they were not available for any molecular targeted therapeutics. OS of best supportive care (BSC) cohort and chemotherapy with Docetaxel monotherapy and more cohort from those elderly patients were analyzed by Kaplan-Meier analysis.

Results: Clinical Question 1; What is a prediction factor for OS of BSC patients?

Answer 1; From analysis of 29 BSC patients (age 15-94 years old, median 82), it is revealed that low performance status (PS =0,1) stage at diagnosis (p=0.03) are significantly correlated to longer OS , and also hypoalbuminemia is significantly correlated to worse OS (11 wks vs 35 wks). Age at diagnosis did not correlate to duration of OS. ( more vs less than 80 years old:15 wks vs 37 wks, P=0.13)Clinical Question 2; Does chemotherapy significantly prolong OS of elderly patients?

Answer 2; Kaplan-Meier analysis of elderly patients (PS 0-2) less than 80 years old who were treated by chemotherapies at least with DOC monotherapy showed longer OS than that of 29 BSC patients ( 84 wk vs 15 wk, p=0.006). However, no significant difference to OS (p=0.378) has been observed to BSC patients in advanced stage with better PS (0-2). Multivariate analysis for OS indicated significant correlation of hypoalbuminemia (p=0.002), but no correlation of chemotherapy (p=0.202) has been observed.

Conclusion: 1, EGFR mutation-negative advanced NSCLC patients elder than 80 years old who were treated by chemotherapies did not showed significantly longer OS than that of BSC patients. 2, PS and hypoalbuminemia before treatment may be prediction factors for longer OS of elder lung cancer patients.

#3997

Outcomes of geriatric sarcoma patients enrolled in phase 1 clinical trials.

Shiraj Sen,1 Roberto C. Pestana,2 Kenneth Hess,2 David S. Hong,2 Ishwaria M. Subbiah,2 Anthony Conley,2 Gerald S. Falchook,1 Robert S. Benjamin,2 Shreyas Patel,2 Funda Meric-Bernstam,2 Vivek Subbiah2. 1 _Sarah Cannon Research Institute, Denver, CO;_ 2 _MD Anderson Cancer Center, Houston, TX_.

Background: Despite the rising incidence of sarcoma in the geriatric population, there is a paucity of older adults enrolled on clinical trials. Whether older sarcoma patients treated on early phase clinical trials have any meaningful differences in clinical benefit or survival outcomes compared to younger patients remains unknown.

Methods: We analyzed clinical and next generation sequencing data from sarcoma patients treated on phase 1 trials at MD Anderson Cancer Center (MDACC) and performed logistic and Cox proportional hazards regression analyses to compare response rate (RR), median time to progression (mTTP), clinical benefit rate (CBR; defined as CR, PR, or SD > 6 months), and median overall survival (mOS) between patients younger and older than 65 years of age.

Results: Among the 406 patients with advanced sarcomas (321 soft tissue sarcoma, 85 bone sarcomas) treated on phase 1 trials at MDACC from May 2006 to May 2018, median age was 53 (range 11-84), 48% were female, and patients had a median 3 prior lines of therapy (range 0-9). The most commonly treated soft tissue sarcoma subtypes included leiomyosarcoma (n=66; 16%), liposarcoma (n=52; 13%), GIST (n=44; 11%), UPS (n=14; 3%), and synovial sarcoma (n=11; 3%) and most commonly treated bone sarcomas included osteosarcoma (n=34; 8%), chondrosarcoma (n=28; 7%), and Ewing's sarcoma (n=25; 6%). 25% (n=102) of sarcoma patients treated on phase 1 protocols were age 65 or older. RR in patients younger than 65 years was 7% compared to 8% in those 65 or older, odds ratio 1.21 (95% CI 0.52, 2.83), p=0.66. mTTP for patients younger than 65 was 2.7 months compared to 3.7 months in those 65 or older, hazard ratio 0.90 (95% CI 0.72, 1.14), p=0.39. CBR in patients younger than 65 years was 22% compared to 31% in those 65 or older, odds ratio 1.65 (95% CI 1.00, 2.72), p=0.05. mOS in patients younger than 65 years was 16.1 months compared to 20.0 months in those 65 years or older, hazard ratio 1.08 (95% CI 0.80, 1.46), p=0.61.

Conclusion: Heavily pretreated, metastatic sarcoma patients aged 65 or older enrolled on phase 1 studies at MDACC had no significant differences in response rate, time to progression, and overall survival compared to their younger counterparts. In fact, older sarcoma patients demonstrated an improved clinical benefit rate compared to younger patients. Older patients should be encouraged to enroll on early phase clinical trials when clinically appropriate and broad eligibility criteria for such studies are needed to ensure older sarcoma patients are not excluded from these trials.

#3998

Phase II trial of afatinib in elderly patients over 75 years of age withEGFR mutation positive non-small cell lung cancer.

Kageaki Watanabe,1 Satoshi Oizumi,2 Hidenori Mizugaki,3 Yuka Fujita,4 Toshiyuki Harada,5 Taichi Takashina,6 Satoshi Igawa,7 Ryo Ko,8 Takamasa Hotta,9 Hiroyuki Minemura,10 Sho Saeki,11 Shigehiro Yagishita,12 Akinobu Hamada12. 1 _Tokyo Metropolitan Cancer and Infectious Diseases Center Komagome Hospital, Tokyo, Japan;_ 2 _National Hospital Organization Hokkaido Cancer Center, Hokkaido, Japan;_ 3 _Hokkaido University Hospital, Hokkaido, Japan;_ 4 _National Hospital Organization Asahikawa Medical Center, Hokkaido, Japan;_ 5 _JCHO Hokkaido Hospital, Hokkaido, Japan;_ 6 _Iwamizawa Municipal General Hospital, Hokkaido, Japan;_ 7 _Kitasato University, Tokyo, Japan;_ 8 _Juntendo University Graduate School of Medicine, Tokyo, Japan;_ 9 _Shimane University, Shimane, Japan;_ 10 _Fukushima Medical University, Fukushima, Japan;_ 11 _Kumamoto University, Kurokami, Japan;_ 12 _Division of Molecular Pharmacology, National Cancer Center Research Institute, Tokyo, Japan_.

Background: Although reports on the use of gefitinib and erlotinib in elderly patients are occasionally found, reports on afatinib are rare. According to the analysis of 54 Japanese patients in the LUX-Lung3 study, an afatinib dose reduction from 40 mg/day, was necessary for 76.0%. However, the prolonged administration was possible after a dose reduction to 20 mg/day, and antitumor effects were maintained with the reduced dose.

Material and methods: The efficacy and safety of afatinib at 30 mg/day in PS0-1 patients who were over 75 years of age with EGFR mutation positive non-small cell lung cancer were studied. The primary endpoint was the response rate (RR), and the planned number of registered cases was set at 35, with a threshold RR of 50%, an expected RR of 75%, α of 0.05, and β of 0.1. The secondary endpoints were progression-free survival (PFS), overall survival (OS), the incidence rate of adverse events (AE), QOL survey (FACT-L), and trough plasma concentration of afatinib at steady state (PK, collected between 8th to 15th day of oral administration).

Results: The data of 35 patients were collected from May 2015 to August 2017. Patient background was, median age of 79 years (75-92), male/female: 8/27, PS 0/1: 8/27, adenocarcinoma/NSCLC: 30/5, IIIA/IIIB/IV/postoperative recurrence (TNM 7th edition): 2/2/22/9, and exon19del/exon21L858R/exon19del+exon21L858R: 15/19/1. The best overall efficacy was PR/SD/PD/NE: 28/4/1/2, and the RR was 80.0% (95% CI, 63.1-91.6). The median PFS and OS were 16.3 months (95% CI, 11.8-27.0) and not reached. The main AEs were rash 69%, diarrhea 60%, and paronychia 51%. While the initial afatinib dose was 30 mg, nine (26%) patients continued with 30 mg, 23 (66%) were reduced to 20 mg, and three (8%) discontinued due to AEs (2 ILD and 1 stomatitis). Treatment-related death was not observed. There is no significant change of QOL at baseline, after 4, 8, and 12 weeks. PK analyses showed steady state plasma concentration as 22.8 ng/mL which was comparable to reported plasma concentration of 40 mg afatinib in LUX-LUNG3 and 6 trials (24.3 ng/mL). No obvious PK differences were found according to dose reduction, adverse event, and response.

Conclusions: Afatinib at 30 mg/day could be an effective treatment option for elderly patients, over 75 years of age, with good PS. (UMIN 0000177050)

#3999

Advanced lung adenocarcinoma cell bank (ALACB) : A comprehensive preclinical platform.

Hyeong-Seok Joo,1 Dong Hwi Kim,1 Seok-Young Kim,1 Ji-Yeon Lee,2 Mi-Ran Yun,1 Han-Na Kang,1 Byoung Chul Cho,2 Hye Ryun Kim2. 1 _JEUK Institute for Cancer Research, Seoul, Republic of Korea;_ 2 _Yonsei Cancer Center, Seoul, Republic of Korea_.

Purpose: Patients with advanced lung adenocarcinoma often lack large clinical specimens required for molecular testing which limits next therapeutic options. Patient-derived cells (PDC) from malignant effusions can predict patient drug responses and provide a valuable tool for studying drug resistance mechanisms. In this study, we created an Advanced Lung Adenocarcinoma Cell Bank (ALACB) consisting of 28 unique PDCs established from patients who progressed on various tyrosine kinase inhibitor TKIs including 3rd generation EGFR TKI osimertinib.

Experimental design: Patient malignant effusion samples were tested for malignancy, processed, observed by light microscopy, and cultured with appropriate media and supplements. The criteria for established PDCs include the following: sharing the same driver mutation as the patient; free of stromal fibroblasts confirmed by FACS staining; recapitulating patient's drug response; and can be cryopreserved and re-grown. Established PDCs were further authenticated by STR profiling and microplasma testing to maintain cell quality. We were able to perform various in vitro assays and next generation sequencing for detailed analysis of individual PDC.

Results: We were able to successfully establish 28 PDCs incorporating unique patient characteristics. Among the total 146 samples, we observed a success rate of 30% only accounting 95 malignancy positive samples. Established PDCs consists of 20 EGFR mutation positive cell lines, 3 with ALK rearrangements, 6 with ROS1 rearrangements, 1 with BRAF K601E mutation and 1 with KRAS G12D mutation. Seven PDCs were established from patients who progressed on osimertinib, two from olmutinib (HM61713), and others from 1st and 2nd generation EGFR TKI such as gefitininb and erlotinib. We were able to newly generate 3 PDCs harboring a T790M mutation with an activating EGFR mutation. Moreover, we are one of the few labs to successfully establish an EGFR del19/T790M/C797S triple mutant PDC showing resistance to osimertinib treatment in vitro. Other established PDCs harbored rare EGFR mutations including exon 20 insertion, L861Q and G719X/S768I. PDC with EGFR G719X/S768I compound mutation showed high sensitivity to afatinib but was resistant to gefitininb and osimertinib treatment. Three ALK-positive PDCs were derived from crizotinib-resistant, alectinib-resistant and ceritinib-resistant tumors. Furthermore, of 6 ROS1 fusion positive PDCs, 3 were derived from TKI-naïve tumors, and 3 from crizotinib-resistant tumors.

Conclusions: Successfully established PDCs reflecting patient genomic profiles and drug response is a valuable resource for biological assays, generating in vitro drug-resistant models, and evaluating novel drugs and therapeutic targets. Further advances in drug development combined with a library of cell bank will facilitate lung cancer translational research.

#4000

Development of a patient navigation training program for the Caribbean context.

Kimlin T. Ashing,1 KImberly Badal2. 1 _City of Hope, Duarte, CA;_ 2 _Caribbean Cancer Research Institute, Trinidad and Tobago_.

Introduction

For the Caribbean context, a patient navigator (PN) is one who is responsible for coordinating and streamlining care in a complex cancer care continuum by providing support in physical, mental and emotional challenges; educating and empowering patients in their journey; tracking patient medical records electronically and addressing barriers to care. As a first step to introducing this role to the Caribbean, a comprehensive training program for new navigators was developed and conducted.

Methods

A multi-disciplinary steering committee consisting of a behavioral scientist, two oncology registered nurses and educators, a clinical psychologist, two cancer survivors, a cancer researcher, a physician and an experienced oncology nurse navigator was formed in Trinidad and Tobago. The steering committee had these objectives: (i) develop a comprehensive list of PN core competencies; (ii) determine PN skill requirements; (iii) develop and execute a training curriculum that met the PN core competencies.

Results

Two categories of navigators and requisite skill requirements were developed for each (Table 1). Three skill domains were defined as patient navigator skills, research skills and professional skills. Each domain was then divided into specific competencies which translated into 11 curriculum topics. Candidates who met the skill requirements filled a form-based application, provide a curriculum vitae and completed 0.5hr scenario-based interview. A 5-day, 30 contact hour patient navigation training program was successfully conducted with candidates from 4 Caribbean territories. Results of the pre- and post- test assessment as well as the training evaluation will be reported in the presentation.

Conclusion

A productive international collaboration between City of Hope an NCI designated Comprehensive Cancer Center and CCRI a civil society cancer organization successfully developed and conducted a comprehensive program to train patient navigators for the Caribbean context.

Table 1: Skill requirements for oncology nurse navigators and lay navigators

---

|

Oncology Nurse Navigator  | Lay Navigator

Primary Skill Requirements | Oncology-trained Registered Nurse. Registered Nurse with 2 years clinical experience in Oncology | Cancer survivor. Care giver

Alternative Skill Requirements Skills | Retired medical personnel. Any Registered Nurse | Any person with interest, holding a minimum Bachelor degree in a related area of expertise such as counselling pharmacy, psychology, social work etc.

#4001

Unraveling the molecular mechanism of racial disparity in hepatocellular carcinoma (HCC): A link between type I interferon (IFN-I) signaling and HCC disparity.

Saranya Chidambaranathan-Reghupaty,1 Mikhail Dozmorov,1 Rachel Mendoza,1 Zhao Lai,2 Paul B. Fisher,1 Trevor Reichman,1 Devanand Sarkar1. 1 _Virginia Commonwealth University, Richmond, VA;_ 2 _University of Texas Health Science Center, San Antonio, TX_.

Among HCC patients, there is a statistically significant increase in incidence and mortality as well as a decrease in 5-year survival rates in African-American (hereafter referred to as black) patients compared to non-Hispanic white (hereafter referred to as white) patients. Currently, there is a gap in our understanding of the molecular mechanism underlying this racial disparity. The purpose of this study is to unravel this molecular mechanism.

Gene expression profiles were analyzed by RNA-sequencing (RNA-Seq). Canonical pathway analysis of the differentially expressed genes was performed by Ingenuity Pathway Analysis (IPA). Differential expression of the genes was validated by qRT-PCR and immunohistochemistry. The role of the identified genes in regulating proliferation was determined by transient knockdown by siRNA in a patient-derived xenograft (PDX) cell line established from a black HCC patient followed by MTT assay.

In TCGA database, RNA-Seq data from 377 HCC patients were available, of which 17 were black and 184 were white. 279 genes were identified, with a p-value<0.01, that are differentially regulated between black and white patients. IPA analysis identified Type I Interferon (IFN-I) signaling as the only pathway that showed statistically significant activation in black patients. RNA-Seq on HCC samples from 14 white and 18 black patients from the tissue bank at VCU Medical Center identified 283 differentially regulated genes and IPA analysis revealed that the only pathway that showed statistically significant activation in black patients is the IFN-I signaling pathway. Activation of the IFN-I signaling pathway in black patients was identified even after correction of the differential gene expression data for HCV status, indicating that the observed findings are independent of HCV infection. Four interferon stimulated genes (ISGs), ISG15, IFI6, MX1 and OAS1, showing significantly increased expression in black HCC patients in both the TCGA database and our own analysis, were chosen for further analysis by qRT-PCR in HCC patient-derived xenografts (PDX) samples from black and white patients, both HCV positive and negative. The levels of the four ISGs were very low in two HCV-negative white HCC PDX lines. In three HCV-negative black HCC PDX lines, their expression levels were significantly and robustly higher. In a HCV-positive black HCC PDX line, their expression was further higher. The results for OAS1, ISG15 and MX1 were validated at the protein level using immunohistochemistry. Transient knock down of IFI6 and ISG15 significantly decreased proliferation of a PDX line established from a black HCC patient.

Persistent activation of the IFN-I signaling pathway might be a key determinant of racial disparity in black HCC patients. This pathway might be exploited to develop targeted treatment that work best for black HCC patients.

#4002

Uptake of cervical cancer screening among HIV positive women at a tertiary healthcare center in Nigeria.

Chibuike F. Chukwunyere, David O. Awonuga. _Federal Medical Centre Abeokuta, Abeokuta, Nigeria_.

BACKGROUND: Cervical cancer currently ranks the commonest gynecological cancer in Nigeria. This could be attributed to poor performance of screening strategies due to economic and patient factors. HIV (human immunodeficiency virus) infection is associated with increased risk of cervical cancer and there is lack of sufficient data on cervical cancer screening among HIV positive patients in our setting. Inefficient cervical cancer screening is related to late diagnosis and increased mortality associated with cervical cancer in our setting.

AIM: This study was to assess the uptake and attitude of HIV positive women in relation to cervical cancer screening.

METHODOLOGY: This was a cross-sectional study of women diagnosed with HIV that presented for care at the department of obstetrics and gynecology of Federal Medical Centre Abeokuta. A total of 52 women were recruited from July, 2017 to June, 2018. Knowledge of Pap smear, HPV DNA test and colposcopy were tested among the participants. A pretested questionnaire was given to respondents to assess factors associated with uptake of cervical cancer screening and SPSS version 23 was used for cross tabulation and to perform logistic regression of the factors associated with cervical screening among the subjects.

RESULTS: The mean age of the subjects was 36.5±6.8 years. Out a total of 52 respondents, only 15 (29%) were aware of at least one method of cervical cancer screening. Women with tertiary level of educational status comprised 11(78%) of the subjects that are aware of cervical cancer screening. Only 6 (12%) of the HIV positive subjects were aware of the increased risk of cervical cancer associated with HIV infection. Among the 15(29%) of subjects that are aware of cervical cancer screening, 9(17%) were screened because of symptoms and signs of lower genital tract condition. Women between 40 and 50 years of age (OR= 2.41), presence of gynecological symptoms (OR= 2.97), educational attainment (OR=2.54), duration of HIV diagnosis >5yrs (OR=2.99), were strong predictors of uptake of cervical cancer screening in our setting.

CONCLUSION: The knowledge and uptake of cervical cancer screening was very poor among HIV positive patients in our setting despite the increased risk associated with HIV infection, incorporation of cervical cancer screening among health education and intervention to improve its uptake by the agencies involved in HIV care will lead to improved uptake and ultimately decrease mortality associated with cervical cancer

REFERENCES

1. Zayyan MS, Akpa M, Dawotola DA, Oguntayo AO, Kolawole AO. Quality of life in patients with advanced cervical cancer in Nigeria. Sahel Med J 2018;21:61-9

#4003

Colon sessile serrated polyps associate with endometrial polyps.

Hassan Brim,1 Taraneh Tarjoman,1 Shirin Ganjali,1 Saman Azam,1 Negin Farsi,1 Edward Lee,1 Babak Shokrani,1 Farshad Aduli,1 Carla Williams,1 Adeyinka Laiyemo,1 Akbar Soleimani,1 Zaki Sherif,1 Mehdi syed Nouraie,2 Hassan Ashktorab1. 1 _Howard University Hospital, Washington, DC;_ 2 _University of Pittsburgh, Pittsburg, PA_.

Background: Colon and endometrial polyps are current health issues and their incidences are on the rise. Due to conflicting reports of higher colon polyps in patients with endometrial polyps, we aimed to evaluate if there is such an association in African Americans, a population at high risk for colorectal cancer.

Method: We retrospectively reviewed all female patients who had endometrial (EN) presentations (n=3,600) and colonoscopy (n=14,888) at Howard University Hospital from January 2004 to December 2015. We selected cases with endometrial poly and control without this diagnosis who underwent colonoscopy. Clinical [Body Mass Index (BMI), diabetes (DM), hypertension (HTN), Tamoxifen intake, age] and pathological (histology, location, type of lesions) features of Cases and Controls were collected. Association between EN and colon polyp was tested using multivariable logistic regression analysis. We excluded the patients with endometrial cancer.

Results: We recruited 664 cases and 118 controls. Cases were statistically significant older (median age of 60 vs. 57 in controls) and had higher rates of smoking and DM than controls. However, the overall colon polyp prevalence in the two groups was not statistically different (54% vs 52% in controls and cases, respectively). In subgroups of subject with colon polyp, sigmoid and rectal polyps were more prevalent in controls than cases. Sessile serrated polyps/adenomas (SSPs) were more frequent in cases (8% vs. 2%, p=0.03) while benign mucosa was more so in controls. Whether considering the overall study population or just those with colon polyps, SSPs lesions associated with an Odds Ratio of 4.6 (p=0.02) for EN polyp occurrence, after adjusting for confounder.

Conclusion: Our study shows that the overall colon polyp's prevalence was similar in endometrial polyp patients compared to endometrial-polyp free controls. However, a significant association between SSP with endometrial polyps was noted. Females with colon lesions of the SSP type might benefit from a screening for endometrial polyp in an age-independent manner.

#4004

Unique patient signatures underlie health disparities in ethnically diverse patients with pancreatic cancer.

Miles E. Cameron,1 Patrick W. Underwood,1 Jinping Lai,1 Jennifer B. Permuth,2 Andrew R. Judge,1 Jose G. Trevino1. 1 _University of Florida Health Science Center, Gainesville, FL;_ 2 _Moffitt Cancer Center and Research Institute, Tampa, FL_.

Introduction: Health disparities in pancreatic cancer (PC) exist but how the underlying biology contributes to this is poorly described. Black Americans have a higher prevalence, present with more advanced disease, and suffer from higher mortality rates than other ethnic groups. Cancer cachexia, or cancer-associated muscle wasting, affects more than 80% of patients with PC. Unique muscular characteristics exist in different ethnic groups which might be contributing to pancreatic cancer health disparities. We hypothesize that Black patients with PC have a distinct muscular profile and are more affected by cancer cachexia than Whites and that unique genomic variations might contribute to this phenomenon in PC tumors between Black and White patients.

Methods: Black (including African Americans and Afro-Caribbeans) and non-Hispanic White surgically resected patients with PC between 2010 and 2017 were case matched by age, gender, and tumor grade. Muscular indices were measured from CT scans by dividing the area of the psoas muscles by the L3 vertebral body area to normalize for patient size. Whole-exome sequencing was performed on DNA from tumor and adjacent benign parenchyma.

Results: Healthy Blacks have a greater psoas index than Whites (0.8995 vs. 0.6988, p=0.004). When comparing patients with PC, Blacks and Whites were found to have similar psoas indices. Black patients had a greater percent decrease in psoas index than White patients (-25.29% vs. -12.37%, p=0.04). Tumor size inversely correlated to psoas index in Blacks (r=0.4330, p=0.007), but not in Whites (r=0.0025, p=0.9). Similar to tumor size, the positive lymph node ratio (LNR) inversely correlated to psoas index in Black patients (r=0.3930, p=0.01), but not in White patients (r=0.0867, p=0.2). LNR was significantly greater in Whites than Blacks (0.0627 vs. 0.2253, p=0.002), but overall survival was similar in both groups (15.8 vs. 14.3, p=0.7). In whole-exome sequencing, there were 22 mutations, of which several harbored implications in chemoresistance (ABCF1, ANAPC1), metastasis (CYP2A7, CYP21A2), and pathways of tumorigenesis (PHACTR4, PPP1R12B), and had 100% penetrance in Blacks but not in Whites. Seven mutations were present only in tumors of Whites but not Blacks.

Conclusion: Black Americans with PC suffer more muscle loss in the early stages of disease than Whites. A decreased psoas index in Black patients is associated with worse prognostic factors. In our genetic analysis, we identified 27 novel mutations not before reported in the literature. We conclude that recognizing biological variance in patients may underlie health disparities and allow investigations toward defining specific targets in a diverse group of patients with PC.

#4005

Glycemic microenvironment and ovarian cancer risk in underserved minority populations.

Desmond Henry,1 Crystal Washington,2 Marley Rashad,1 Guihong Fan,2 Veena Rao,1 Monica Frazier2. 1 _Morehouse School of Medicine, Atlanta, GA;_ 2 _Columbus State University, Columbus, GA_.

Studies have shown that ovarian cancer risk is associated with diabetes mellitus (DM). Moreover, the notion that cancer growth and progression is linked to obesity is also widely accepted. However, the impact of the glycemic and adipose microenvironment is often overlooked. Epidemiologic studies of these microenvironments in medically underserved populations are also overlooked. The aim of this study was to determine the relationship between DM and ovarian cancer incidence and progression in underserved minority populations.

A retrospective study of ovarian cancer patients diagnosed over at 10 year period (2008 – 2018) at one location was performed. Data abstracted for this study included demographic, pathologic, glycemic, DM and BMI data of 142 women with ovarian cancer. Pearson chi-square (p<0.05) was used to compare variables.

26 (18%) of 141 patients had a DM diagnosis and 128 (91%) of the 141 were from an underserved minority group. There was an inverse relationship between tumor size and stage. Patients in later stages had significantly smaller tumors than those in earlier stages of ovarian cancer. There was also a direct correlation between hemoglobin a1c (HbA1c) levels and tumor staging in patients with more advanced disease having slightly higher HbA1c levels (5.2, 5.5, 5.3 and 5.8 HbA1c levels Stages 1 - 4, respectively) And although there continues to be discussion on the best glycemic indicator for determining risk, we did find a similar correlation between staging and blood glucose levels. Lastly, when investigating the relationship between BMI and ovarian cancer staging, it appears that stage 1 occurrence was irrespective of BMI measurements (range 16.3 – 63.2). However, in stages 2 (range 17 – 22.8), 3 (range 21.5 – 33.9) and 4 (range 20.9 – 41.2), there was a positive correlation between BMI and staging. This result suggests that ovarian cancer progression is associated with BMI.

Overall, when using cancer stage as an indicator of ovarian cancer progression, this data suggests that while tumor size is inversely related to progression, the glycemic microenvironment along with the increased presence of adipose tissue is positively correlated with ovarian cancer progression.

#4006

Effect of Prior Cancer on Trial Eligibility and Treatment Outcomes in Nasopharyngeal Carcinoma: Implications for Clinical Trial Accrual.

Ya-Qin Wang,1 Jia-Wei Lv,1 Ling-Long Tang,1 Xiao-Jing Du,1 Lei Chen,1 Wen-Fei Li,1 Xu Liu,1 Ying Guo,2 Ai-Hua Lin,3 Yan-Ping Mao,1 Ying Sun,1 Yu-Pei Chen,1 Jun Ma1. 1 _Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative, Guangzhou, China;_ 2 _Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou, China;_ 3 _School of Public Health, Sun Yat-sen University, Guangzhou, China_.

Background: In cancer trials, prior cancer is a common exclusion criterion. We evaluated the characteristics of prior cancer exclusion criteria in nasopharyngeal carcinoma (NPC) trials and determined its prognostic effect on patients with NPC.

Methods: We reviewed NPC trials for prior cancer exclusion criteria. Then we estimated the effect of prior cancer among NPC patients using the Surveillance, Epidemiology, and End Results database. Propensity score-matching was used to compensate for differences in baseline characteristics between patients with and without prior cancer.

Results: There were 109 clinical trials involving 10,437 patients; 49 trials (45%) excluded patients with prior cancer. Prior cancer exclusion was more common in recent or phase III trials. We identified 10,195 NPC patients; 6.2% had prior cancer. More than 70% of these cancers were in situ/localized/regional and diagnosed relatively close to the NPC diagnosis (median 3.3 years). Patients with certain prior cancer type (prostate, breast, gynecological, hematological), time of diagnosis (>5 years ago), or stage (in situ/localized) did not have inferior survival compared with patients with no prior cancer. We tested one form of prior cancer exclusion criteria in an NPC cohort resembling a modern trial population: it did not adversely affect overall and NPC-specific survival.

Conclusions: Many NPC trials excluded patients with prior cancer, which impacts trial accrual and generalizability. Our findings suggest that broader inclusion in trials of patients with NPC with prior cancer might not affect trial outcomes. More research is needed to understand the appropriateness of this exclusion policy across cancer types and trials.

#4007

Decipher the myth between progression-free survival and overall survival in HER-2 positive metastatic nreast cancer: Anti-HER2 antibody as the constant winner---A hypothesis-driven meta-regression analysis.

I-Chun Chen,1 Fu-Chiang Hu,2 Ching-Hung Lin,1 Yen-Shen Lu1. 1 _Dept. of Oncology, National Taiwan Univ. Hospital, Taipei, Taiwan;_ 2 _National Taiwan University College of Medicine, Taipei, Taiwan_.

Purpose: Traditionally, the predictive value of progression free survival (PFS) hazard ratio (HR) on overall survival (OS) HR in metastatic breast cancer (MBC) was low. We observed that when trials were designed as adding on an anti-HER2 antibody, either trastuzumab or pertuzumab, to the control treatment, the OS increment is usually greater than the PFS increment. We aim to investigate the interaction of increment of median PFS and median OS in HER-2 positive MBC stratified by drug difference patterns between treatment arms using trial-level information.

Methods: All randomized phase II or III trials of systematic therapy specifically for HER-2 positive MBC with available OS information were included after a systematic literature review. Treatment arms with shorter PFS were defined as control arm in the analysis. The correlation and interaction of HR for PFS and OS as well as the absolute increment for median PFS and OS were evaluated by fixed-effect or random-effect meta-regression meta-analysis. Drug difference between two treatment arms were specified as a categorical variable in the analysis.

Results: A total of 28 trials with 10928 patients published between January 1999 and December 2017 were included in the final analysis. The correlation of median PFS HR (0.73, 95% C.I.= 0.68-078) and median OS HR (0.82, 95% C.I.= 0.77-0.87) was modest as reported by other investigators. However, when the drug difference between treatment arms was anti-HER2 antibody, the absolute increment in median OS is 2 fold of the absolute increment in median PFS (p = 0.0005), showing a linear correlation. This linear correlation was not observed when the drug difference between treatments was non-anti-HER2 antibody. Anti-HER2 antibody as drug difference would translate the absolute increment of median PFS into absolute increment of median OS, suggesting a good predictive value of median PFS on median OS.

Conclusion: Anti-HER2 antibody as the drug difference between treatment arms provides consistently longer OS improvement than PFS improvement in HER-2 positive MBCs.

### Tumor Markers to Assess the Biology and Clinical Course of Cancer 2

#4008

Association of lung cavitation development with apatinib treatment outcomes in advanced gastric and NSCLC patients with multichemotherapy failure.

Man Jiang, Chuantao Zhang, Dong Liu, Na Zhou, Yongjie Wang, Helei Hou, Tianjun Li, Hongying Lv, Jingjuan Zhu, Chuanyu Zhang, Xiaochun Zhang. _The Affiliated Hospital of Qingdao University, Qingdao, China_.

Purpose:

Patients who receive antiangiogenic agents commonly develop cavitation in their lung lesions. This symptom has also been found in apatinib-treated patients with lung tumors. The present study evaluated the frequency and clinical outcomes of patients who developed tumor cavitation following apatinib treatment.

Experimental Design:

This was a retrospective study of 189 patients with lung tumors who were treated with 250 mg per day of apatinib between February 1, 2015, and May 19, 2017. Clinical data were retrieved from their medical records, and chest imaging findings were documented. Survival data were analyzed with Kaplan-Meier estimates and were compared with a log-rank test. OR values were analyzed for the effect factor of cavitation development.

Results:

Cavitation development showed a benefit for patients on apatinib therapy regardless of whether they had primary or metastatic lung cancer. For patients without cavitation, metastatic lung cancer had greater 6-month LRC, PFS and OS rates than did primary lung cancer. No significant association was found between histological type and survival analysis in apatinib-treated NSCLC patients. Increased CEA levels promoted PFS in patients on apatinib who developed cavitation. However, changes in CEA values did not predict LRC, OS or PFS in patients who developed no cavitation. OR analysis showed that increased CEA levels were a risk factor for cavitation development.

Interpretation:

This was a retrospective real-world study approved by the ethics committee of the Affiliated Hospital of Qingdao University. Patients with advanced or metastatic gastric cancer and NSCLC who had progressed or relapsed after undergoing at least two lines of systemic therapy in accordance with NCCN guidelines and had never received antiangiogenic therapy were included.

Conclusions:

Lung cavitation development is common with apatinib therapy and is a potential prognostic biomarker. CEA is not a prognostic biomarker but is a prospective biomarker for lung cavitation in apatinib-treated patients.

#4009

GLUT3 variant is associated withprognosis of patients with non-small cell lung cancer after surgical resection.

Shin Yup Lee,1 Sook Kyung Do,1 Sun Ha Choi,1 Jin Eun Choi,1 Hyo Gyoung Kang,1 Mi Jeong Hong,1 Dong Won Baek,1 Seung Soo Yoo,1 Ji Woong Son,2 Jae Yong Park1. 1 _Kyungpook National Univ. School of Med., Daegu, Republic of Korea;_ 2 _Konyang University Hospital, Daegu, Republic of Korea_.

This study was conducted to explore whether polymorphisms of glucose transporter 3 (GLUT3) gene affect the prognosis of patients with non-small cell lung cancer (NSCLC) after surgical resection. Four single nucleotide polymorphisms (SNPs) in GLUT3 were investigated in a total of 782 patients with NSCLC who underwent curative surgery. The association of the SNPs with overall survival (OS) and disease free survival (DFS) was analyzed. Among the four SNPs investigated, GLUT3 rs7309332C>T was significantly associated with OS and DFS in multivariate analyses. The SNP was associated with significantly worse OS (adjusted hazard ratio [aHR] = 1.62, 95% confidence interval [CI] = 1.04-2.53, P = 0.03, under recessive model), and worse DFS (aHR = 1.64, 95% CI = 1.18-2.29, P = 0.003, under recessive model). When stratified by tumor histology, the association between the GLUT3 rs7309332C>T and OS/DFS was not limited to either squamous cell carcinoma (SCC) or adenocarcinoma (AC), although the significant association remained only in AC for OS (P = 0.40 for SCC and P = 0.04 for OS) and only in SCC for DFS (P = 0.03 for SCC and P = 0.08 for OS). When AC patients were stratified according to EGFR mutation status, the SNP was significantly associated with DFS in patients with EGFR mutant tumors (aHR = 2.47, 95% CI = 1.15-5.30, P = 0.02, under recessive model), but not in those with EGFR wild-type tumors. This study suggests that genetic variation in GLUT3 may be useful in predicting survival of patients with early stage NSCLC.

#4010

Adjuvant chemotherapy based on the intratumoral expressions of class III β tubulin and thymidylate synthase improved disease-free survival in patients with resected locally advanced non-small cell lung cancer.

Ryota Sumitomo, Tatsuya Hirai, Hiyoaki Murakami, Yosuke Otake, Cheng-long Huang. _Kitano Hospital, OSAKA, Japan_.

Objectives: Although the molecular targeted therapy and immune-check point inhibitors improved survival of some populations of patients with non-small cell lung cancer (NSCLC), most of patients still remain to need conventional chemotherapy. Adjuvant chemotherapy is recommended for patients with resected locally advanced NSCLC. However, selection of the optimal adjuvant chemotherapy regimen is difficult. Regarding the predictive biomarkers for chemotherapy, the intratumoral expressions of class III β tubulin (TUBB3) has been reported to be a biomarker for taxanes, and thymidylate synthase (TS) for 5-fluorouracil (5-FU) derivatives and pemetrexed. Therefore, we conducted a prospective clinical study to evaluate the effectiveness of tailor-made adjuvant chemotherapy based on TUBB3 and TS.

Methods: Between November 2011 and December 2016, 54 patients with pathological stage IIB to IIIB NSCLC who underwent complete resection were studied. The expressions of TUBB3 and TS were estimated by immunohistochemistry. When less than 50% of the tumor cells in a given specimen were positively stained for TUBB3, the tumor was classified as TUBB3-low. When less than 50% of the tumor cells in a given specimen were positively stained for TS, the tumor was classified as TS-low. Concerning to adjuvant chemotherapy, patients with TUBB3-low tumors were preferably treated with Taxane-based chemotherapy, and patients with TS-low tumors were preferably treated with chemotherapy including 5-FU derivatives or pemetrexed. Patients who could receive tailor-made adjuvant chemotherapy based on evaluations of TUBB3 and TS were assigned to tailor-made group. Patients who could not receive tailor-made adjuvant chemotherapy were assigned to non-tailor-made group, due to several reasons, such as both TUBB3-high and TS-high tumors and renal dysfunction.

Results: Among 54 tumors, 39 tumors were adenocarcinomas, 14 were squamous cell carcinomas, and 1 was adenosquamous cell carcinoma. Regarding pathological stage, 19 tumors were stage IIB, 32 were stage IIIA, and 3 were stage IIIB. Concerning to survival, the disease-free survival (DFS) rate was significantly higher in tailor-made group than in non-tailor-made group (5-year DFS rate: 58.6% vs 12.0%; P < 0.001). In particular, the DFS rate was significantly higher in tailor-made group than in non-tailor-made group, both in stage IIB (5-year DFS rate: 83.3% vs 28.6%; P = 0.013) and stage IIIA (5-year DFS rate: 43.8% vs 8.3%; P =0.034). The overall survival of the tailor-made group tended to be longer than that of non-tailor-made group (5-year overall survival rate: 76.6% vs 65.8%; P = 0.160).

Conclusion: Adjuvant chemotherapy based on the intratumoral expressions of TUBB3 and TS is considered to be clinically useful for patients with resected locally advanced NSCLC.

#4011

Notch 1/2/3 mutation as a potential biomarker for immunotherapy in smokers with non-small cell lung cancer via increasing tumor mutational burden.

Xiaohua Hong,1 Aijun Shen,2 Yu Xu,3 Chengcheng Li,3 Guoqiang Wang,3 Li Liu1. 1 _Cancer Center, Union Hospital, Medical college,Hua-zhong University of Science and Technology, Wuhan, China;_ 2 _Division of Anti-tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, Shanghai, China;_ 3 _The medical department, 3D Medicines Inc., Shanghai, China_.

Background: Smoking status have been discovered to be associated with the response of anti-PD-1/PD-L1 treatment in advanced non-small cell lung cancer (NSCLC), on account of the smoking-induced higher tumor mutational burden (TMB). However, merely partial smokers with NSCLC exhibit high TMB and respond to anti-PD-1/PD-L1 therapy. The underlying signalling pathway that involved in the accumulation of mutational burden and response to immunotherapy in smokers with NSCLC has not been clarified.

Methods: Data from Rizvi study (n = 240, nivolumab, tissue-sample NGS) and OAK study (n = 642, atezolizumab vs. docetaxel, blood-sample NGS) was retrieved and analysed as training cohorts, followed with validation in our independent NSCLC cohort (n = 44, anti-PD-1/PD-L1, blood-sample NGS). In survival analysis, Kaplan-Meier curves were compared by log-rank test, and the hazard ratio (HR) was determined through a multivariable Cox regression model.

Results: In Rizvi cohort, mutation of Notch 1/2/3 was associated with higher TMB in smokers with NSCLC (P < 0.001) not in non-smokers with NSCLC (P = 0.908). Notch 1/2/3mut was associated with prolonged progression-free survival (PFS) compared with Notch 1/2/3wt in smokers with NSCLC (P = 0.015; HR, 0.51, 95% CI, 0.30-0.88), while not in non-smokers with NSCLC (P = 0.73; HR, 0.83, 95% CI, 0.30-2.35). Consistent results were observed in OAK study that Notch 1/2/3mut was associated with higher TMB (P < 0.001) and longer PFS exclusively in current smokers with NSCLC treated

with immunotherapy (P = 0.018; HR, 0.28, 95% CI, 0.10-0.81). No association of Notch 1/2/3 status and PFS was observed in smokers with NSCLC treated with chemotherapy (P = 0.80; HR, 0.92, 95% CI, 0.47-1.78). We further validated these results in our independent cohort. Similar upregulation of TMB was observed in the Notch1/2/3-mutated smokers with NSCLC, and Notch1/2/3mut improved DCR (100% vs. 41.2%, P = 0.065) and significantly prolonged PFS (P = 0.046; adjusted HR, 0.12, 95% CI, 0.01-0.96).

Conclusions: Notch1/2/3 mutation may serve as a novel predictor of response to anti-PD-1/PD-L1 treatment in smokers with NSCLC via upregulating TMB.

#4012

Small RNA-sequencing identifies a microRNA signature predictive of response to FOLFOX-based adjuvant therapy in stage II and III colorectal cancer.

Raju Kandimalla,1 Uthra Balaji,1 Jinghua Gu,1 Marta Mendiola,2 Francesc Balaguer,3 Luis Bujanda,4 Joan Maurel,5 Jaime Feliu,2 Ajay Goel1. 1 _Baylor Scott &White Research Institute and Charles A. Sammons Cancer Center, Dallas, TX; _2 _La Paz University Hospital (IdiPAZ), CIBERONC, cátedra UAM‐AMGEN, Madrid, Spain;_ 3 _Hospital Clinic, CIBERehd, IDIBAPS, University of Barcelona, Barcelona, Spain;_ 4 _Instituto Biodonostia, Universidad del País Vasco (UPV/EHU), Centro de Investigación Biomédica en Red de Enfermedades Hepaticas y Digestivas (CIBERehd), San Sebastián, Spain;_ 5 _Hospital Clinic of Barcelona, IDIBAPS, Barcelona, Spain_.

Purpose: Current NCCN guidelines for identifying high-risk stage II/III colorectal cancer (CRC) who may benefit from FOLFOX-based adjuvant chemotherapy remain inadequate. While stage III CRC patients often receive six months of oxaliplatin-based therapy following radical surgery, such treatments are frequently toxic and do not benefit all patients. The use of adjuvant chemotherapy in stage II CRC patients is even more controversial. Hence, identification of high-risk CRC patients with the highest likelihood to benefit from such treatments is of paramount clinical importance. Since microRNAs (miRNAs) have emerged as key functional players and important disease biomarkers, using genomewide miRNA expression profiling, we explored their potential as predictive biomarkers of therapeutic response in CRC.

Experimental design: Small RNA-sequencing was performed in a cohort of stage II/III CRC patients who received, at least 6 months of FOLFOX-based therapy (n=71; 30 with recurrence, and 41 without recurrence). Identification of differentially expressed miRNAs was done using DESeq2, and biomarker prioritization using LASSO-Cox's proportional hazards model and AUC (area under the curve) analysis, using recurrence-free survival (RFS) as an outcome. The miRNA-signature was validated by using miRCURY LNA miRNA assays in three, independent patient cohorts of tissue (fresh frozen and FFPE) and serum specimens obtained from stage II/III CRC patients (n=91, 77 and 82, respectively). Univariate and multivariate Cox proportional hazard models were used to evaluate the performance of the miRNA classifier, in conjunction with known clinicopathological risk factors and the microsatellite instability (MSI) status.

Results: The small RNA-expression profiling led to the identification of a 13-gene miRNA classifier that significantly predicted recurrence free survival (HR: 2.72, 95% CI: 1.96-3.76, p<0.0001) with an AUC of 0.90 (95% CI: 0.83-0.97). This classifier was successfully validated in two tissue cohorts, with impressive AUCs of 0.78 (95% CI: 0.64-0.90, fresh frozen cohort) and 0.86 (95% CI: 0.69-1.00, FFPE cohort), respectively. Importantly, our miRNA signature yielded an impressive AUC of 0.73 (95% CI: 0.60-0.88), even in the preoperative serum specimens from CRC patients. Multivariate analyses revealed that our miRNA classifier was the only independent predictor of response to FOLFOX-based treatment in stage II and III CRC patients.

Conclusions: We report a novel miRNA-based classifier, which robustly predicts response to FOLFOX-based adjuvant chemotherapy in stage II/III CRC patients. Validation of this classifier in pre-operative serum provides an attractive opportunity to explore its potential for disease monitoring during the longitudinal follow-up in CRC patients.

#4013

**CoA Synthase (** COASY **): A novel predictive marker and functional protein that mediates radiation resistance via PI3K signaling in rectal cancer.**

Sylvain Ferrandon, Jennifer DeVecchio, Leonardo Duares, Hanuman Chouhan, Jacqueline Davenport, Georgios Karagkounis, Matthew Orloff, David Liska, Matthew F. Kalady. _Cleveland Clinic, Cleveland, OH_.

Purpose: Rectal cancer response to neoadjuvant chemoradiation is prognostic for disease-free and overall survival, with better responders enjoying better outcomes. Understanding the biology underlying better responses to therapy could lead to more personalized treatment approaches. The purpose of this study was to evaluate a newly identified biomarker and explore its mechanism of action.

Experimental Design: Pretreatment human rectal cancer samples underwent DNA microarray analysis and profiles were compared to post-chemoradiation histologic regression scores. A candidate gene, COASY (which encodes for Coenzyme A Synthase), was stably knocked down by shRNA in colorectal cell lines, and the effects on response to radiation were tested in a xenograft model. The biological function of COASY was first assessed by Gene Set Enrichment Assay (GSEA). Its effects on cell death, cell survival, and DNA repair capacity in colorectal cell lines was analyzed. Immunoprecipitation of COASY followed by LC-MS/MS analysis was done to identify COASY partners.

Results: COASY gene expression was overexpressed in rectal cancer compared to normal rectum and was significantly correlated to the tumor regression score. Tumors from cell lines knocked down for COASY had a strong delayed tumor growth, less proliferation, and more apoptosis after irradiation than the control cell line using in vivo xenograft models. In addition, a higher cell death rate after irradiation was observed in vitro in two colorectal cancer cell lines knocked

down for COASY. Mechanistically, a physical interaction between COASY and the PI3K regulatory subunit PI3K-P85α was identified; which led to a modulation of AKT and mTOR phosphorylation after irradiation. Furthermore, there were more residual double DNA strand breaks after irradiation in cell lines knocked down for COASY accompanied by a decrease in DNA-PK and MRE11 protein levels.

Conclusion: COASY serves as a predictive biomarker for rectal cancer response to neoadjuvant chemoradiation and its increased levels correlate with poor response. In addition to its role as a predictive biomarker, COASY has biological relevance leading to increased radiation resistance via the PI3K pathway-dependent increase in DNA repair capacity.

#4014

**Clinical validation of Lantern Pharma's Response Algorithm for Drug Positioning and Rescue (RADR** TM **).**

Umesh Kathad, Yuvanesh Vedaraju, Aditya Kulkarni, Barry Henderson, Gregory Tobin, Panna Sharma, Arun Asaithambi. _Lantern Pharma Inc, Dallas, TX_.

Lantern Pharma has developed a technology platform termed RADRTM that can be used to predict true responders before conducting a clinical trial in order to achieve higher success rates. RADRTM is an Artificial Intelligence (Al)-based machine learning approach for complex biomarker identification and patient stratification. RADRTM is a combination of three automated modules working sequentially to generate drug- and tumor-specific gene signatures predictive of response. RADRTM integrates biological knowledge, data-driven feature selection, and robust Al algorithms to facilitate hypothesis-free drug- and cancer-specific biomarker development. We present retrospective analyses performed as part of RADRTM validation using at least 9 independent datasets of patients from selected cancer types treated with approved drugs including chemotherapy, targeted therapy and immune-oncology agents. Pre-treatment patient gene expression profiles along with corresponding treatment outcomes were used as algorithm inputs. Model training was typically performed using an initial set of genes derived from cancer cell line data when available, and further applied to a subset of patient data for model tuning and final gene signature development. Model testing and performance computation were carried out on patient records held out as blinded datasets. The response prediction accuracy, true positive rate (TPR), true negative rate (TNR) false discovery rate, positive predictive value and Matthew's Correlation Coefficient were among the model performance metrics calculated. On average, RADRTM achieved a response prediction accuracy of 80% during clinical validation. For instance, in an analysis of 92 breast cancer patients, RADRTM generated a signature of 18 genes whose expression level was predictive of Paclitaxel treatment response at an overall accuracy of 78% and 81% TPR/ 76% TNR. The above results imply that the application of the RADRTM program to this Paclitaxel trial in breast cancer patients could have potentially reduced the number of patients in the treatment arm from 92 unselected patients to 24 biomarker-selected patients to produce the same number of responders. Moreover, we cite published evidence correlating genes from this 18-gene signature with increased Paclitaxel sensitivity in breast cancer. The value of the platform architecture is derived from its validation through the analysis of about 6 million oncology-specific clinical data points, more than 120 drug-cancer interactions, and over 600 patient records. Thus, by implementing unique biological, statistical and machine learning workflows, Lantern Pharma's RADRTM technology is capable of deriving robust biomarker panels for pre-selecting true responders for recruitment into clinical trials which may improve the success rate of oncology drug approvals.

#4015

Synthetic lethality-based predictive biomarker identification of splicing modulators in lung cancer.

Claire E. Repellin, Puja Patel, Yihui Shi, Helena Gong, Thomas R. Webb, Lidia Sambucetti, Subarna Sinha. _SRI International, Menlo Park, CA_.

Identification of predictive biomarkers for targeted drugs is an active research area in precision oncology. Usually, a genetic alteration in the drug target is used to identify responders, which can limit the use of the drug in other responsive cancers. Alternatively, cell line-based drug sensitivity data are used to build machine-learning models to identify predictive biomarkers. These methods require large amounts of data and the black-box models developed are often hard to interpret.

Synthetic lethality provides an alternate approach for predictive biomarker identification of targeted drugs. In synthetic lethal (SL) interactions, a defect in one gene leads to dependency on a second gene. Neither defect by itself is essential for survival, but together they lead to cell death. Thus, the inhibition of the drug target in the presence of a cancer-specific genetic alteration would result in cancer cell death. The genetic alterations can be used as biomarkers for the drug. We developed a novel computational pipeline for predictive biomarker identification based on our tool, Mining Synthetic Lethals (MiSL), that mines SL interactions from pan-cancer primary tumor data (such as TCGA). Since MiSL utilizes primary human tumor data, it enables the identification of SL interactions in the native context of human tumors and is more likely to find relationships relevant to in vivo tumor biology than shRNA/CRISPR screens. To discover biomarkers of a targeted drug, we used MiSL to identify genetic alterations that are SL with targets of the drug in a specific cancer type.

As proof of principle, we applied our method to identify biomarkers of sudemycin-D6 (SD6), a potent splicing modulator, in non-small cell lung cancer (NSCLC). First, we identified putative drug targets of SD6. Since SD6 is known to inhibit SF3B1, any gene belonging to the same protein complex as SF3B1 was termed a SD6 drug target. Using these SD6 drug targets, our MiSL-based pipeline identified KRAS mutations, present in ~30% of NSCLC patients, as a SD6 biomarker. To confirm our predictions, we treated a panel of KRAS-mutant-and-dependent NSCLC lines (H358, H23 and H441) and a KRAS-WT NSCLC line (H2228) with SD6 and measured cell viability. All KRAS-mutant lines tested were significantly more sensitive to SD6 (> 5-30X lower IC50) than the KRAS-WT line. Furthermore, the KRAS-mutant lines were also more sensitive (5-20X lower IC50), compared to the KRAS-WT line, to a splicing modulator of a different class – a kinase inhibitor that phosphorylates components of the spliceosome. In vivo studies are underway to further validate our in vitro findings.

This study identifies KRAS mutation as a novel predictive biomarker for splicing modulators in NSCLC, which could lead to new therapeutic options for KRAS-mutated NSCLC. Furthermore, our work demonstrates the feasibility of a synthetic lethality-based pipeline to predict biomarkers for targeted drugs.

#4016

PIK3IP1 regulates prostate cancer cell migration and proliferation and is a noninvasive biomarker of the disease.

Andrej Jedinak,1 Katherine Kaplan,1 Kevin Loughlin,2 Marsha Moses1. 1 _Boston Children's Hospital, Boston, MA;_ 2 _Brigham and Women's Hospital, Boston, MA_.

Prostate cancer (PCa) is the second most common epithelial cancer in men after lung cancer. PCa shares similar clinical symptoms with benign prostate hyperplasia (BPH) making it very challenging for physicians to make an accurate diagnosis. Prostate specific antigen (PSA) has been used for several decades to facilitate clinical decision making but unfortunately lacks sensitivity and specificity and cannot effectively distinguish between these two prostate diseases. Therefore, there is a need for new biomarkers that can discriminate between these two diseases with high accuracy. Using a global proteomics approach supported by extensive validation, we have found significantly (P<0.002) elevated levels of Phosphoinositide-3-Kinase Interacting Protein1 (PIK3IP1) in the urine of PCa patients with early disease compared to patients with BPH. These data motivated us to examine the potential role of this protein in prostate cancer development and progression. We began by transiently transfecting multiple prostate cancer cell lines including DU145, PC-3 and LNCaP with a PIK3IP1 plasmid DNA and used these cells to conduct proliferation and migration studies in vitro. We found that there was a significant inhibition of PCa cell proliferation (P<0.05) and migration (P<0.007) in all cell lines tested. We validated these finding with DU145 stable clones established by lentiviral transduction with a PIK3IP1 vector. Stable clones overexpressing PIK3IP1 significantly suppressed proliferation and especially migration and invasion of DU145 cells. We next transiently transfected DU145, PC-3 and LNCaP cells with PIK3IP1 plasmid DNA and performed phospho-protein array experiments to identify signaling molecules that might be modulated by PIK3IP1 to mediate these activities. These data revealed altered levels of molecules involved in several pathways including MAPK. We are currently conducting further in vitro and in vivo studies to assess the potential effects of PIK3IP1 on the modulation of prostate cancer progression and metastasis. In summary, we have identified and validated the urinary detection of PIK3IP1 as a novel liquid biopsy biomarker for localized PCa and have demonstrated that it can modulate both the migration and proliferation of several PCa cells including DU145, PC-3 and LNCaP. (Supported by The Ellison Foundation and the Advanced Medical Research Foundation)

#4017

Clinical-genomic models of node-positive breast cancer: training, testing, and validation.

Tzu-Ting Huang,1 Tzu-Pin Lu,2 Lei Lei,3 Skye H. Cheng1. 1 _Koo Foundation Sun Yat-Sen Cancer Center, Taipei, Taiwan;_ 2 _National Taiwan University, Taipei, Taiwan;_ 3 _Cancer Institute, University of Mississippi Medical Center, Jackson, MS_.

Purpose: We developed and validated clinical-genomic models for stratifying node-positive breast cancer patients into low- and high-risk groups.

Experimental design: The four datasets were (1) training group (n = 112); (2) testing group (n = 46); (3) validation group (n = 388); and (4) external validation group (n = 655). Patients who had undergone mastectomy or breast-conserving surgery and mRNA microarray analysis of their primary tumor tissue with a pathological stage of N0-N2 were enrolled. The validation groups included node-positive patients. Using pre-set cutoffs obtained from the training group, the models were tested and validated in the three other independent groups.

Results: Two clinical-genomic models were developed to predict any recurrence (recurrence index [RI]-local recurrence [LR]) and distant recurrence (RI-DR). In the validation dataset, the RI-LR distinguished between low- and high-risk groups according to 10-year LR-free interval (LRFI; 100% vs. 93.3%, P = 0.014), relapse-free interval (RFI; 88.4% vs. 79.0%, P = 0.017), and relapse-free survival (RFS; 82.2% vs. 73.3%, P = 0.029). The RI-DR distinguished the low- from the high-risk group according to 10-year RFI (90.2% vs. 79.3%, P = 0.007) and RFS (81.0% vs. 74.2%, P = 0.031). RI-DR was associated with regional node irradiation in N1-N2 patients (hazard ratio: 3.7, 95% confidence interval 1.2-12.0) by multivariate analysis. The RI-DR- and RI-LR-gene panels performed similarly in the external validation dataset with hazard ratios of 1.27 (0.93-1.74, P= 0.127) and 1.54 (1.11-2.13, P= 0.0094), respectively, in high-risk patients.

Discussion: The RI-DR shows better performance in partitioning N1-N2 patients into low- and high-risk groups than the RI-LR for any recurrence, although the latter is superior for predicting LRR.

#4018

Pan-cancer predictions of drug sensitivity based on protein-coding and non-coding RNA biomarkers.

John P. Lloyd, Nathan Merrill, Eric Lachacz, Jun Z. Li, Sofia Merajver, Matthew Soellner. _University of Michigan, Ann Arbor, MI_.

Molecular characteristics that accurately forecast tumor response to drug therapies represent an important target for precision cancer treatment. However, it is not known whether tumor drug response can be predicted in a tissue-agnostic manner or if predictions must be established within historically-defined cancer types and subtypes. To address this question, we utilized public datasets to test whether multi-omics biomarkers in 346 tumor cell lines representing 17 cancer types can predict response to five small molecule therapies (2 MEK, 2 PI3K, and 1 FGFR inhibitors). The pool of potential biomarkers tested included 51,268 total RNA expression levels and DNA variants. We identify a panel of 45 gene expression levels capable of accurately predicting response to MEK inhibitors (AUC-ROC >0.90), indicating that tumor response to small molecule drugs can be predicted regardless of cancer type. We found that RNA expression levels, notably among non-coding sequences, were most important for predictions. Experimental validation of the 45-gene panel is demonstrated based on predicted and observed MEK inhibitor sensitivity in 12 cell lines from 5 cancer types without previously known drug sensitivities. Successful pan-cancer predictions of drug response in cancer cell lines represents a critical step toward truly personalized treatment in real time, based on the characteristics of individual tumors.

#4019

High TERT expression in NSCLC is associated with poor prognosis.

Tianhong Li,1 Weijie Ma,1 Qianping Li,1 Sixi Wei,1 Tao Wang,1 Jingyi Xiang,2 Cheng Liu2. 1 _UC Davis Comp. Cancer Ctr., Sacramento, CA;_ 2 _Eureka Therapeutics, Inc., Emeryville, CA_.

Background: Activation of human telomerase reverse transcriptase (TERT) has been linked to tumorigenesis and aggressive tumor behavior. This study was undertaken to evaluate if TERT expression was correlated with clinicopathological parameters and survival in patients with non-small cell lung cancer (NSCLC).

Methods: TMA slides made from 254 NSCLC patients (cohort 1 and cohort 2), including 127 adenocarcinoma (LUAD), 78 squamous carcinomas (LUSC) and 46 other types, were included. TMA Formalin-fixed and paraffin-embedded (FFPE) tissue sections were stained using the immunohistochemistry stain (IHC). The percentage of positive nuclei was determined for each case and the cut-off value was set as 5%, 20% and 50% positive tumor cells. TERT expression levels were analyzed for associations with patient survival rates and clinicopathological parameters (such as age, gender, tumor grade, tumor stage, metastasis). Survival analysis was performed using the Kaplan-Meier method.

Results: TERT was positive in 199/254 (78%) of the samples. High TERT expression was seen in tumor specimens compared with normal adjunct tissues (p < 0.001). Kaplan-Meier curves indicated that high TERT expression was significantly associated with poor overall survival in NSCLC patients (p < 0.001 and p < 0.0001, 20% and 50% cut-off, respectively). Multivariate analysis of survival demonstrated that TERT expression at 50% cutoff was an independent prognostic factor for NSCLC patients (HR = 0.1461, 95% CI: 0.0862-0.2475, p < 0.0001). High TERT expression was also associated with short survival of LUSC and LUAD patients at 50% cutoff (p < 0.0001). No significant association with OS was observed at 5% cutoff. There was also significant correlation between TERT expression with metastasis but no correlation with clinical stage, histological type and tumor differentiation.

Conclusion: Our study showed that high TERT expression is present in 78% of NSCLC tumors and is associated with poor prognosis. TERT may serve as a prognostic biomarker and novel drug target for NSCLC.

#4020

Correlation between the expression of glutathione metabolism-related genes and prognosis in ovarian clear cell carcinoma.

Hiroshi Asano,1 Ryosuke Matsuoka,2 Kanako C. Hatanaka,2 Yutaka Hatanaka,2 Tatsuya Kato,2 Yosuke Konno,2 Takashi Mitamura,2 Hirotoshi Akita,1 Yoshihiro Matsuno,2 Hidemichi Watari1. 1 _Faculty of Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan;_ 2 _Hokkaido University Hospital, Sapporo, Japan_.

Background: Ovarian clear cell carcinoma (OCCC) is more prevalent in Japan than western countries, exhibits chemoresistant phenotype and poor survival. Although the expression of the genes related to glutathione (GSH) metabolism had been reported as one of the possible chemoresistant mechanisms, correlation between the expressions of these genes and prognosis in OCCC patients remains unclear. In this study, we aim to investigate 1) enrichment pathway analysis in OCCC in comparison with high-grade serous carcinoma (HGSC), and 2) correlation between the expression of gamma-glutamyl transpeptidase 1 (GGT1), one of the enzymes in GSH metabolism and prognosis.

Methods: We consecutively collected surgically resected tissue specimens from 33 ovarian cancer patients; 15 patients with OCCC and 18 patients with HGSC. The tissue specimens were collected in the vials containing RNA later® within 10 min. after resection of tumors. Total RNAs were extracted from the tumor tissues, and comprehensive gene expression analysis was performed using Ion AmpliSeq™ Transcriptome Human Gene Expression Kit (Thermo Fisher Scientific). For prognostic analysis, we used a tissue microarray (TMA) from 56 consecutive patients pathologically diagnosed with OCCC in our hospital, and assessed by immunohistochemistry (IHC) using H-score ranging from 0 to 300. Overall survival (OS) was estimated by Kaplan-Meier method, and analyzed by Cox-regression hazard model in multivariate analysis.

Results: Gene expression profiles revealed that TCF1/2 target genes were up-regulated in OCCC samples in comparison with HGSC. In addition, genes related with amino-acid transporters, such as SLC3A1, a cystine transporter, and genes related to GSH metabolism, including GGT1, CSE, and GPX3 were also up-regulated. In the OCCC patients with high expression level of GSH-related genes, FOXP3 was down-regulated but the T-cell inflamed genes were not varied compared to the OCCC patients with low expression level. The expression level of GGT1 in gene expression analysis was strongly correlated with H-score of GGT1 in IHC (r = 0.7850, p = 0.0023). In prognostic analysis, there were no significant differences during follow up period (57.5 months vs. 53 months), FIGO stage (I: 50% vs 64%), optimal surgery (92% vs. 98%), or recurrent rate (42% vs. 30%) between GGT1 positive (n = 44) and negative (n = 12) OCCC in IHC. However, platinum-drug resistant recurrent rate was significantly higher in GGT1 negative OCCC than GGT1 positive OCCC (42% vs 14%, p = 0.027). OS in GGT1 negative OCCC was significantly worse than that of GGT1 positive OCCC (5-year OS was 42% vs 72%, p = 0.0226), and negative expression of GGT1 was one of the independent poor prognostic factors in OCCC.

Conclusion: Negative expression of GGT1 in OCCC might be a poor prognostic maker via recruitment of regulatory T cells in tumor microenvironments.

#4021

A seven-lncRNA signature has a good value of prognosis in esophageal squamous cell carcinoma.

Nuoqing Weng. _Sun Yat-Sen University Cancer Center, Guangzhou, China_.

Background: Long non-coding RNAs (lncRNAs) were used as prognostic biomarkers in many types of cancer. We aimed to recognize a signature that can forecast the prognosis in patients with esophageal squamous cell carcinoma (ESCC).

Method: Using a 2520 probe microarray, we retrospectively analysed lncRNAs expression profiles in 141 samples of ESCC and 81 paired non-cancer specimens that were served as the training set from SUN YAT-SEN University Cancer Center (Guangzhou, China) to evaluate the association between the signature and clinical outcomes. We randomly selected 40 paired tumor tissues and adjacent normal tissues from the above samples for the quantitative RT-PCR to ensure the credibility of microarray data. Then we conducted the quantitative RT-PCR in another 103 samples of ESCC to identify the signature. We applied Kaplan-Meier method and log-rank tests to assess the prognostic power of signature with overall survival (OS) and disease-free survival (DFS).

Result: Being differently expression between esophageal squamous cell carcinoma tissues and no-cancer specimens, 338 lncRNAs were selected after filtration. A further analysis were conducted that identified a seven-lncRNA signature in the training set. We calculated the risk score according to the signature and classified ESCC patients as high-risk or low-risk group. Patients in high-risk group had shorter overall survival (HR = 3.555, 95%CI 2.195-5.757, p < 0.0001) and disease-free survival (HR 2.537, 95%CI 1.646-3.909, p < 0.0001) when compared with low-risk group in the training set, and we found the same conclusion in the independent set for OS (HR = 2.662, 95%CI 1.588-4.464, p < 0.0001) and DFS (HR 2.389, 95%CI 1.447-3.946, p < 0.0001). The multivariate Cox proportional hazards regression analysis indicated that the signature is an independent factor for predicting survival. Combing the signature and TNM stage was more powerful than TNM stage alone in prognosing outcomes both in the training set (AUC: 0.778 vs 0.681, p = 0.0127) and in the independent set ((AUC: 0.796 vs 0.713, p = 0.0539).

Conclusion: The seven-lncRNA signature might win a place as a valuable prognostic factor in patients survival with ESCC and guide a more individual treatment when cooperating with the TNM stage system.

#4022

Novel evidence for m6A methylation regulators as prognostic biomarkers and potential therapeutic targets in gastric cancer.

Tadanobu Shimura,1 Raju Kandimalla,1 Yuji Toiyama,2 Yoshinaga Okugawa,2 Masato Kusunoki,2 Ajay Goel1. 1 _Center for Gastrointestinal Research; Center from Translational Genomics and Oncology, Baylor Scott & White Research Institute and Charles A. Sammons Cancer Center, Baylor University Medical Center, Dallas, TX; _2 _Department of Gastrointestinal and Pediatric Surgery, Division of Reparative Medicine, Institute of Life Sciences, Mie University Graduate School of Medicine, Mie, Japan_.

Purpose: N6-methyladenosine (m6A) post-transcriptional RNA modification represents the most abundant epigenetic alteration in eukaryotic cells. These m6A modifications are regulated by a cascade of enzymes and cofactors, categorized as 'writers' [methyltransferase-like 3 (METTL3), METTL14, and Wilms' tumor 1-associating protein (WTAP1)], 'erasers' [fat mass and obesity-associated protein (FTO) and alkylated DNA repair protein AlkB homolog 5 (ALKBH5)] and 'readers' [YTHDF1, and YTHDF2 (YTH N6-Methyladenosine RNA Binding Protein)]. While emerging evidence indicates that these enzymes play a central role in RNA metabolism (e.g. RNA stability, translation, splicing, transport and localization), their clinical significance, if any, remains unclear. Herein, we for the first time, systematically unraveled the functional role, as well as the clinical significance of m6a regulators in gastric cancer (GC).

Experimental design: First, we investigated the expression of the seven m6A regulator genes (FTO, METTL3, METTL14, WTAP, ALKBH5, YTHDF1, and YTHDF2) in a large cohort of 171 GC patients by qRT-PCR assays. Subsequently, a multivariate Cox-regression model was developed using the 7-gene panel to evaluate its predictive potential, followed by Kaplan Meier analyses for survival outcomes. In addition, we undertook a series of functional studies to investigate the oncogenic role of FTO in GC cells, and subsequent validation of our findings in an animal model.

Results: Gastric cancer patients with low-expression of METTL3, METTL14, ALKBH5, WTAP and YTHDF1 demonstrated significantly poor OS (p=0.02, 0.001, 0.01, 0.02 and 0.02, respectively), while patients with high FTO expression exhibited markedly worse OS (p<0.0001). Furthermore, the cumulative risk-score derived from these gene panel also significantly associated with poor OS, with a corresponding hazard ratio of 5.47 (95% CI: 3.18-9.41, p<0.0001). We observed that FTO expression was frequently up-regulated in GC cell lines, particularly in those with epithelial-mesenchymal-transition (EMT) features. Functional studies with FTO knockdown in HGC27 and AGS cells inhibited cell proliferation (p=0.005, and <0.0001, respectively) and migratory potential (p<0.0001, respectively); while its overexpression in MKN28 cells resulted in enhanced proliferation and migration (p<0.0001, and 0.007, respectively). Finally, confirming our in-vitro findings, FTO suppression led to significant tumor growth inhibition in a xenograft animal model (p<0.0001).

Conclusions: We provide first evidence that m6A regulators may serve as important prognostic biomarkers in GC patients. Our functional studies reveal that FTO is an important oncogene, and may be a novel therapeutic target in patients with gastric cancer.

#4023

Co-occurring mutations in recurrent/persistent head and neck squamous cell carcinoma (HNSCC) patients.

Alok R. Khandelwal,1 Kelsey Poorman,2 Tara Moore-Medlin,1 Xiaohui Ma,1 Abhijit Gundale,1 Ronald Horswell,3 San Chu,3 Michelle Winerip,2 Cherie-Ann O. Nathan1. 1 _LSU Health-SHV, Shreveport, LA;_ 2 _Caris Life Sciences, Phoenix, AZ;_ 3 _Pennington Biomedical Research Center, Baton Rogue, LA_.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer and lacks effective targeted therapies. HPV(-) patients have a 50-60% recurrence rate and could benefit from adjuvant therapy. Although HPV(+) patients have a significantly better survival, 20% have persistent/recurrent disease. Therefore, biomarkers could potentially help identify patients that would benefit from adjuvant targeted agents. Our objective was to evaluate if the mutational and biomarker analysis of tumor samples from OPSCC patients predict recurrence and/ or persistence in patients undergoing definitive therapy with curative intent. 44 advanced stage OPSCC patients that underwent comprehensive genomic profiling by Caris Life Sciences were included in this retrospective study. Next Generation Sequencing (NGS) on genomic DNA from FFPE tumors was performed using the Illumina Nextseq (592-gene, n=17)/MiSeq (44-gene, n=23) platform. Tumors were analyzed for total mutational load (TML), CNV's and microsatellite instability status. IHC for tumor protein expression of ERCC1, PD-L1, RRM1, TrkA/B/C, TS and TUBB3 was performed using automated platforms. The ASCO/CAP scoring criteria and the cutoff points from published evidence was used in IHC evaluation. Of the 44 patients, 27 were HPV(+) tumors and 11 had HPV(-) disease. Patients with lack of progression free survival data were excluded. Only 4 patients in the entire cohort harbored a pathogenic PIK3CA mutation. Among HPV(+) patients, 22 patients were TP53 WT and 2 patients were found to be TP53 mutant. Although there was no significant change in the TML in smokers compared to nonsmokers (mean 8.77 vs 6.5, respectively; p=0.253), TML was higher in HPV(-) smokers compared to HPV(+) nonsmokers (mean 10.33 vs 6.5, p=0.0661). Our study presented a higher incidence of recurrent/persistent disease in the Caucasians (60%) in comparison to African-Americans (21%). Considering the sample size in the current study, the racial difference in outcomes appears likely to be present regardless of HPV status or disease state. The co-occurrence of multiple deleterious mutations was observed in 70% of the non-responders. Particularly, mutations in CHEK2, PTCH, MUTYH were noted in 50% of the recurrent/persistent patients. CNV mutations in SMAD2, MALT1, NFKBIA in 50% of the recurrent/persistent patients were found. The co-occurrence of multiple mutations were absent in responders in spite of an appreciable sample size in the responder group (n=12). This study also affirmed an improved prognosis in patients with HPV(+), with higher expression of TUBB3, and with positive expression of PD-1 in the tumors. Co-occurrence of multiple deleterious mutations were associated with patients who did not respond to therapy. Therefore, combination rescue therapies that can target multiple pathways to abrogate the mutational effects can aid/enhance therapeutic benefits in HNSCC patients.

#4024

Phase 1/2a LYM1002 study of ibrutinib (ibr) + nivolumab (nivo): Exome and gene expression profiling (GEP) analyses by histology and responder status.

Brendan Hodkinson,1 Michael Schaffer,1 Joshua Brody,2 Wojciech Jurczak,3 Cecilia Carpio,4 Dina Ben-Yehuda,5 Irit Avivi,6 Rao Saleem,7 Muhit Özcan,8 John Alvarez,1 Rob Ceulemans,9 Nele Fourneau,9 Sriram Balasubramanian,1 Anas Younes10. 1 _Janssen Research & Development, Spring House, PA; _2 _Division of Hematology and Medical Oncology, Icahn School of Medicine at Mount Sinai, New York, NY;_ 3 _Department of Hematology, Jagiellonian University, Krakow, Poland;_ 4 _Department of Hematology, University Hospital Vall d'Hebron, Barcelona, Spain;_ 5 _Department of Hematology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel;_ 6 _Department of Hematology and Bone Marrow Transplantation, Tel Aviv Sourasky Medical Center and Sackler Faculty of Medicine, Tel Aviv, Israel;_ 7 _Bristol-Myers Squibb, Lawrenceville, NJ;_ 8 _Department of Hematology, Ankara University School of Medicine, Ankara, Turkey;_ 9 _Janssen Research & Development, Beerse, Belgium; _10 _Lymphoma Department, Memorial Sloan Kettering Cancer Center, New York, NY_.

A phase 1/2a study (LYM1002 [EudraCT 2014-005191-28]) of ibr (560 mg once daily) + nivo (3 mg/kg on a 14-day cycle) demonstrated acceptable safety and promising efficacy vs single-agent ibr in Richter's transformation (RT), follicular lymphoma (FL), and diffuse large B-cell lymphoma (DLBCL). We examined potential biomarkers of treatment (tx) response using archived biopsy samples.

GEP was used for DLBCL subtyping and to assess proportions of 22 distinct immune cells. Exome data were generated from 72 formalin-fixed paraffin-embedded samples, and sequencing analysis was used to identify mutations in genes of interest and assess somatic mutation burden. The correlations of immune cell proportions and gene variants were evaluated by investigator-assessed responses in each histology and by ongoing responses in DLBCL patients (pts; progression-free survival [PFS] > 24 months, n = 7 vs not, n = 20).

In pts with available GEP and response data, overall response rates were 29.6% (8/27) for DLBCL, 43.5% (10/23) for FL and 81.3% (13/16) for RT. Proportions of CD8 and follicular helper T cells, M1 macrophages, and resting dendritic cells were higher in DLBCL responders vs nonresponders, while the proportion of regulatory T cells was decreased. These subsets did not differ by response in FL although an increase of CD4 memory resting T-cells was noted in responders. The trend toward increased follicular helper T cell and resting dendritic cell proportions in DLBCL pts was also associated with longer survival (PFS > 24 months) vs not.

Gene variant data and responder status were available for 26 pts with DLBCL, 26 with FL, and 17 with RT. Comparison between responders and nonresponders showed that DLBCL pts with RNF213 (4/10 [40.0%] vs 1/16 [6.2%]) and KLHL14 (3/10 [30.0%] vs 0/16) mutations were more likely to respond to ibr + nivo. Conversely, nonresponders were associated with variants in EBF1, ADAMTS20, AKAP9, SOCS1, TP53, and genes in BCR pathways such as TNFRSF14, MYD88, and NFKB1B. BCL2 mutation in FL (9/12 [75.0%] vs 4/14 [28.6%]) and ROS1 mutation in RT (0/13 vs 2/4 [50.0%]) were associated with response; both are involved in the NF-kB pathway.

In DLBCL, the most frequent gene mutations were RNF213, NBPF1, and BCL2 in pts who had PFS > 24 months (3/7 [42.9%] each), and KMT2D (8/20 [40.0%]) and CSMD3 (8/20 [40.0%]) in pts who did not. Somatic mutation burden was lower in responders vs nonresponders, especially in germinal center B-cell-DLBCL, and in DLBCL pts with PFS > 24 months vs not.

In conclusion, we report gene variations among DLBCL, FL, and RT pts associated with response or durable PFS with ibr + nivo. While ibr inhibits Bruton's tyrosine kinase-dependent pathways, we identify

alternative gene pathway variants that may impact tx outcomes. Immune cell infiltration into the microenvironment relates to differential tx response with this immune combination and is histology dependent.

#4025

STEAP4 ISH and IHC diagnostics for Tarloxotinib activation in EGFR/HER2 mutant cancers.

Hui Yu,1 Shevan Silva,2 Adriana Estrada-Bernal,3 Chris Rivard,1 Fred Hirsch,1 Maria Abbatista,2 Jeff Smaill,2 Robert C. Doebele,3 Adam Patterson,2 Avanish Vellanki,3 Vijaya G. Tirunagaru3. 1 _University of Colorado, Aurora, CO;_ 2 _The University of Auckland, Auckland, New Zealand;_ 3 _Rain Therapeutics Inc., Fremont, CA_.

Background: Tarloxotinib is a clinical-stage prodrug that releases a potent, irreversible EGFR/HER2 inhibitor (Tarloxotinib-E) selectively in severely hypoxic regions of tumors. One-electron reduction leads to loss of the trigger moiety under oxygen-deficient conditions, releasing the neutral, diffusible 'warhead' Tarloxotinib-E. CRISPR/cas9-mediated disruption of the metalloreductase STEAP4 has demonstrated its role as the dominant plasma membrane reductase that facilitates one-electron reduction of Tarloxotinib.

Methods: Cancer cell lines, PDX and resected tumor tissues processed in Formalin Fixed Paraffin Embedded (FFPE) blocks were evaluated. RNAScope technology was used to develop a method to assess STEAP4 mRNA expression by in situ hybridization using specific probes for human STEAP4. mRNA ISH technology for STEAP4 was optimized on a Leica Bond RX autostainer for detection of STEAP4 in FFPE specimens. STEAP4 antibodies were evaluated for Immunohistochemistry (IHC) analysis. STEAP4 IHC was employed as cross-reference, using an anti-STEAP4 antibody validated against STEAP4 knockout and over-expressing cell line and tumor models. Staining data was compared to quantitative PCR results for mRNA ISH and Western blot analysis for IHC. STEAP4 mRNA expression was assessed in FFPE sections of various EGFR/HER2 mutant tumor xenografts and EGFR/HER2 mutant patient tumor specimens.

Results: The STEAP4 mRNA ISH assay was optimized and validated using FFPE sections from cell lines and STEAP4 overexpressing tumor blocks and compared to wild type tumors with minimal expression. High level of STEAP4 mRNA was observed in STEAP4 overexpressing tumors. STEAP4 mRNA ISH analysis revealed variable levels of expression in EGFR/HER2 mutant xenograft tumors. STEAP4 mRNA and protein expression level correlated with Tarloxotinib conversion to Tarloxotinib-E. STEAP4 mRNA expression in EGFR/HER2 mutant patient tumor specimens will be presented.

Conclusion: STEAP4 reduces Tarloxotinib and leads to the conversion to the active drug, Tarloxotinib-E, in hypoxic tumors. Assessment of STEAP4 expression in individual patients is important to guide patient stratification in Tarloxotinib clinical trials and represents an attractive biomarker to enroll tarloxotinib-responsive patients in clinical trials.

#4026

Assessment of microsatellite instability (MSI) status in colorectal cancer tissue using a next-generation sequencing-based tumor profiling assay.

Kevin Z. Qu, Suzzette Arnal, Sirisha Sunkara, Mohammad R. Sheikholeslami, Zhong J. Zhang, Felicitas L. Lacbawan, Charles Ma, Sugganth Daniel. _Quest Diagnostics Nichols Inst., San Juan Capistrano, CA_.

Background: MSI status, along with PD-L1 expression and tumor mutational burden (TMB), is an important predictive biomarker for immune checkpoint inhibitors. Recent studies demonstrated that next-generation sequencing (NGS)-based MSI assays can simultaneously detect MSI status and variants in other target genes. We developed and validated an NGS-based MSI assay that utilizes data from a 49 gene panel used for solid tumor profiling. Here we report the concordance of this NGS-MSI assay with 2 commonly used PCR fragment-based methods for MSI status. We also evaluated the relationship between NGS-MSI status and TMB as assessed with the 49-gene solid-tumor profiling assay.

Method: This study used de-identified, formalin-fixed, paraffin embedded tumor tissue and adjacent normal tissue from 55 patients with colorectal cancer. All specimens were tested with a hybrid capture-based 49-gene NGS assay on the Illumina NextSeq500 platform. Components of the MSIsensor software v0.2 and a Fisher's Exact test were used to discriminate the repeat distributions between the tumor and normal specimens on the following MSI markers: BAT25, BAT26, NR21, NR24, and NR27. Specimens demonstrating instability in 2 or more the 5 markers were considered MSI positive (MSI-H), and others were considered MSI negative (MSI-L/MSS). NGS-MSI results were compared to those of the Bethesda NCI panel (BAT25, BAT-26, D5S346, D2S123 and D17S250) (55 patients) and the Promega MSI test (BAT25, BAT26, NR21, NR24 and Mono27) (52 patients). TMB was compared between NGS-MSI-positive and NGS-MSI-negative specimens.

Results: The NGS-MSI assay showed strong concordance with both the Bethesda NCI panel and the Promega MSI test. Concordance between the NGS-MSI and Bethesda-MSI test was 97% (53/55): 26 specimens were concordant for MSI positive and 27 were concordant for MSI negative, while 2 were MSI negative by NGS-MSI but MSI-H by Bethesda-MSI. Concordance between the NGS-MSI and Promega-MSI tests was 98% (51/52): 25 specimens were MSI positive/MSI-H and 26 were MSI negative/MSS or MSI-l. The discordant specimen was MSI-negative by NGS-MSI and MSI-H by Promega-MSI. The mean TMB was higher in MSI-positive (11.3 mutations/specimen) than in NGS-MSI-negative specimens (2.5 mutations/specimen). The mutations and its relationship with MSI status in 4 DNA mismatch repair genes (MLH1, PMS2, MSH2 and MSH6) were under analysis.

Conclusion: The NGS-based MSI assay, performed as part of a 49-gene NGS tumor profiling test, demonstrated strong concordance with the Bethesda and Promega MSI tests. As expected, MSI-positive tumor specimens had higher TMB than did MSI-negative samples.

#4027

**XNA-Based OptiSeq** TM **lung and colorectal cancer mini panel, a high sensitivity method for cancer early detection.**

Michael Y. Sha, Wenjing Feng, Qing Sun, Ke Zhan, Michael J. Powell, Aiguo Zhang. _DiaCarta, Inc., Richmond, CA_.

The identification of genetic variants with low allelic frequency using next-generation sequencing method is confounded by the complexity of human genome sequence and by bias that arise during library preparation, sequencing and analysis. Herein, we introduce a novel molecule, Xenonucleic Acid (XNA) for NGS application. XNA is able to selectively suppress primer directed amplification of DNA with wild type alleles and only amplify DNA target templates containing mutant alleles. Mutants with low allelic frequency will be easily detectable without deep sequencing after enrichment by adding XNA in multiplex PCR. The 17 actionable mutants related to lung or colorectal cancer diseases at different VAF% were investigated in this study. Upon XNA blocking of wild type alleles, enriched variant allelic frequency (VAF) can be increased by ~32 fold from 10 ng of gDNA samples containing mutants as low as 0.10% VAF. Analytical sensitivity of Limit of Detection (LOD) is about 0.10% VAF. These 17 actionable mutants were tested and verified by using tissue biopsy (FFPE) and liquid biopsy (cfDNA) of lung or colon cancer patient samples. Clinical sensitivity for FFPE sample is about 100% for lung cancer and colorectal cancer samples respectively, comparing to without XNA NGS about 85.7% for lung cancer and 70% for colon cancer. For cfDNA sample its clinical sensitivity is about 100% for lung and colon cancer, but without XNA mediated enrichment NGS is only about 70% for lung cancer and undetectable for early colon cancer. This method provides a simple, robust, accurate, faster turnaround, highly sensitive and low cost alternative compared conventional NGS employing deep sequencing. This technology will greatly enhance the adaptability of NGS as a clinically useful platform for every modern pathology laboratory.

#4028

Tenascin C in gastric cancer stroma is a predictive marker for postoperative prognosis.

Hirotoshi Kikuchi, Tomohiro Murakami, Sanshiro Kawata, Amane Hirotsu, Tomohiro Matsumoto, Yusuke Ozaki, Yoshihiro Hiramatsu, Kinji Kamiya, Yoshifumi Morita, Takanori Sakaguchi, Hiroyuki Konno, Hiroya Takeuchi. _Hamamatsu University School of Medicine, Hamamatsu, Japan_.

Background: Tenascin C, a glycoprotein in the extracellular matrix is reportedly associated with poor clinical outcome in several malignancies. We have previously reported that tenascin C in colorectal cancer stroma is a predictive marker for liver metastasis (Br J Cancer 117:1360-70, 2017). In this study, we evaluated the clinicopathological and prognostic significance of tenascin C expression in gastric cancer stroma.

Materials and methods: Expression levels of tenascin C protein were immunohistochemically analyzed in 176 primary gastric cancer samples. Tenascin C staining intensity and relativity were classified into four scores and two groups, respectively. The correlation of the tenascin C staining intensity and relativity with clinicopathological factors, postoperative overall survival (OS) and disease-free survival (DFS) were analyzed.

Results: There were positive correlations between the tenascin C staining intensity or relativity and tumor depth, lymphatic invasion, venous invasion, lymph node metastasis, pathological stage, high preoperative carcinoembryonic antigen (CEA)>5ng/ml, and high preoperative carbohydrate antigen (CA19-9) >37U/ml. Multivariate analysis revealed that high tenascin C staining intensity significantly correlated with tumor invasion into the muscularis propria (p=0.015) and venous invasion (p=0.042), and that high tenascin C staining relativity correlated with venous invasion (p=0.013). Gastric cancer patients with high tenascin C staining intensity had significantly shorter postoperative OS (p<0.001), whereas tenascin C staining relativity did not correlate with postoperative OS (p=0.05). High tenascin C staining intensity and relativity significantly correlated with shorter postoperative DFS (p<0.001 and 0.005, respectively). Cox proportional hazard model identified high tenascin C staining intensity as an independent prognostic factor for poor postoperative OS (hazard ratio (HR): 13.16, 95% confidence interval (CI): 1.362-125.000, p=0.026) and DFS (HR: 6.02, 95%CI: 1.538-23.810, p=0.010).

Conclusions: Tenascin C protein expression in primary tumor stroma was identified as a predictive marker for postoperative prognosis in patients with gastric cancer.

#4029

Analysis of DLL3 and ASCL1 in surgically resected small cell lung cancer (HOT1702).

Megumi Furuta,1 Jun Sakakibara-Konishi,1 Hajime Kikuchi,2 Hiroshi Yokouchi,3 Hiroshi Nishihara,4 Hiroyuki Minemura,5 Masao Harada,3 Shigeo Yamazaki,6 Kenji Akie,7 Yuka Fujita,8 Kei Takamura,2 Tetsuya Kojima,9 Toshiyuki Harada,10 Yoshinori Minami,11 Naomi Watanabe,12 Satoshi Oizumi,3 Hiroyuki Suzuki,5 Masaharu Nishimura,1 Hirotoshi Dosaka-Akita,1 Hiroshi Isobe9. 1 _Faculty of Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan;_ 2 _Obihiro Kosei Hospital, Obihiro, Japan;_ 3 _National Hospital Organization Hokkaido Cancer Center, Sapporo, Japan;_ 4 _Keio University School of Medicine, Tokyo, Japan;_ 5 _Fukushima Medical University, Fukushima, Japan;_ 6 _Keiyukai Sapporo Hospital, Sapporo, Japan;_ 7 _Sapporo City General Hospital, Sapporo, Japan;_ 8 _National Hospital Organization Asahikawa Medical Center, Asahikawa, Japan;_ 9 _KKR Sapporo Medical Center, Sapporo, Japan;_ 10 _JCHO Hokkaido Hospital, Sapporo, Japan;_ 11 _Asahikawa Medical University, Asahikawa, Japan;_ 12 _Sunagawa City Medical Center, Sunagawa, Japan_.

Background:Delta-like protein 3 (DLL3) is a Notch ligand that has an important role in the tumorigenesis of small cell lung cancer (SCLC). Recently, rovalpituzumab tesirine (Rova-T), a DLL3-targeted antibody-drug conjugate, has been developed for treating SCLC and has shown improved clinical response in patients with high DLL3 expression. DLL3 is a transcriptional target of the achaete-scute homolog-1(ASCL1) transcription factor, which is involved in pulmonary neuroendocrine cell development. However, the relationship between DLL3 and/or ASCL1 expression and the clinical features of SCLC remains unknown, especially for early-stage resected SCLC. This study aimed to investigate the expression of DLL3 and ASCL1 in resected SCLC samples using immunohistochemical analysis.

Materials and Methods:We collected 95 formalin-fixed and paraffin-embedded SCLC samples, which were surgically resected at 11 institutions participating in either the Hokkaido Lung Cancer Clinical Study Group Trial (HOT) or the Fukushima Investigative Group for Healing Thoracic Malignancy (FIGHT) between 2003 and 2013. Immunohistochemistrystaining was performed to investigate the correlation between the expression of either DLL3 or ASCL1 and clinicopathological features of study patients.

Results:Seventy-seven (83%) of 93 immunohistochemically evaluable samples were positive for DLL3 (expression in ≥ 1% of tumor cells), and DLL3-high expression (≥ 75%) was observed in 44 samples (47%). Sixty-one (64%) of 95 samples were positive for ASCL1 (expression in ≥ 5% of tumor cells). A positive correlation was observed between DLL3 and ASCL1 expression. DLL3 and ASCL1 expression were not associated with survival in SCLC patients. DLL3 and ASCL1 were more prevalent in patients with advanced clinical disease and pure SCLC, respectively.

Conclusion:DLL3 and ASCL1 were highly expressed in surgically resected SCLC patients.

#4030

Case classification with tumor antigen presenting and TGF-β signaling biomarkers to predict anti-PD-1 outcome in GI tract tumors using automated quantitative fluorescence multiplex IHC.

Xiangxue Wang,1 Shizen Moh,1 Antony Hubbard,1 José L. Muñoz-Rodríguez,1 Mehrnoush Khojasteh,2 Jim Martin,2 Qingfeng Zhu,3 Robert Anders,3 Luis Diaz,4 Lidija Pestic-Dragovich,1 Lei Tang,1 Wenjun Zhang1. 1 _Roche Tissue Diagnostics, Tucson, AZ;_ 2 _Roche Tissue Diagnostics, Santa Clara, CA;_ 3 _John Hopkins University Hospital, Baltimore, MD;_ 4 _John Hopkins University Hospital, Baltimore, CA_.

Introduction: Anti-PD-1/L1 immune checkpoint blockade results in tumor stabilization or shrinkage in only 15-40% of patients. Predictive biomarkers are crucial in identifying responsive patients while excluding others from the toxicities of immunotherapies. Major histocompatibility complex class I (MHC-I) downregulation is one of the most frequent mechanisms of tumor escape from the host's immune system, but little attention has been devoted to MHC-I expression in studies of the PD-1/L1 blockade. Recently, Tauriello and Mariathasan revealed stromal transforming growth factor (TGF)-β signaling in CD8+ T lymphocytes exclusion as a key determinant of resistance to PD-1/L1 blockade in colorectal and urothelial carcinomas. We present here a multiplex panel IHC of MHC-I, β2-microglobulin (B2M), CD14, TGF-β receptor 2 (TGFBR2), and pan-cytokeratin (panCK) in tumor micro-environment, and their predictive values to anti-PD-1 treatment.

Methods: With the multiplex panel, 51 pre-pembrolizumab treatment patient specimens were stained, including pancreatic, colorectal and cholangiocarcinoma (33 non-responders: 17 PD, 13 SD, 3 NE; 18 responders: 14 PR, 4 CR). Pathologists annotated tumor areas on whole slide scans. HALO High-Plex FL module was used for image analysis. Epithelial tumor (panCK+) and stroma (panCK-) were masked with HALO's random forest classifier. Spatial location, count, intensity, and percent abundance of each marker were identified. 43 features were designed based on the rationale of hypothesized biological significance. MATLAB was used for feature selection, ranking, and prediction of responses to anti-PD1 treatment.

Results: There was a trend of higher MHCI expression on tumor cells in the responders than non-responders to pembrolizumab treatment. Heterogeneous MHCI expression of tumor cells, and fraction of TGFBR2+ CD14+ cells in stroma were the top features ranked by Relieff k-nearest neighbor (k=30) for the prediction of the response to pembrolizumab treatment. Using Quadratic Discriminant Analysis (QDA) with five-fold cross-validation, the prediction accuracy was 76.5%. Independent validation was not performed due to small sample size.

Conclusions: Deep immune characterization of tumor microenvironments using high dimensional feature spaces derived from multiplex IHC staining may provide insightful directions on finding and validating predictive markers for various immunotherapy regiments (ex. PD-1/L1 blockade; dual TGF-β and PD-1/L1 blockade; combination of PD-1/L1 blockade with other treatments that enhance MHC-I molecules on tumor cells).

Acknowledgement: We thank Nick Cummins, Jorge Lozano, and John Hurley for their technical assistance.

#4031

The dielectric properties of brain tumor tissue.

Martin A. Proescholdt,1 Amer Haj,1 Christian Doenitz,1 Alexander Brawanski,1 Zeev Bomzon,2 Hadas Sara Hershkovich2. 1 _University Regensburg Medical Center, Regensburg, Germany;_ 2 _Novocure Ltd., Haifa, Israel_.

Introduction: Recently, tumor treating fields (TTFields) have been established as for the treatment of newly diagnosed GBM1. One of the most crucial parameters defining the treatment efficacy of TTFields is the electric field intensity. The electric properties of the normal intracranial compartments are well established, allowing the prediction of the electric field distribution. In contrast, there is no data available about the electric properties of tumor tissue. In this study we determined the dieclectric properties of brain tumors by analyzing resected tissue following a fast acquisition protocol. To account for the intratumoral heterogeneity, different regions of the tumor were sampled and analyzed separately.

Methods: A cohort of thirty patients with tumors of different histology and malignancy grade have been recruited (meningioma: n=11; brain metastases n=7; low grade glioma n=6; glioblastoma n=6). Tissue probes were acquired whenever possible from the vital tumor area, and perinecrotic compartment identified intraoperatively using neuronavigation, intraoperative ultrasound and fluorescence guidance. From each region, five tissue probes were sampled. After acquisition, the tissue was measured immediately to avoid artifacts induced by temperature change, differences of fluid composition as well as post resection ischemia. A fragment was dissected from each tissue sample and was placed into a cylindrical cell with a known diameter. Two parallel electrodes were placed on both sides of the sample and the thickness of the tissue was measured using a micrometer. The impedance was recorded at frequencies 20Hz-1MHz using a software specifically developed for this study, which controls the LCR meter (Keysight Technologies, Santa Rosa, USA). The measured impedance was translated into dielectric properties of the sample (conductivity and relative permittivity) based on the parallel plate model, the recorded complex impedance and the geometry of the samples. Each tissue probe was fixed, H&E stained and histologically analyzed for tumor cell count and specific tissue features.

Results: After receiving a positive ethics votum, the first 30 patients were recruited. As a reference, grey and white matter tissue samples from mouse brains were used. We found significant differences between the tumor entities with meningiomas showing the lowest and GBM tissue the highest conductivity values. Consistently, the perinecrotic areas displayed lower conductivity values compared to the solid tumor compartment. Also, we found a significant heterogeneity in the dielectric properties within one tumor. Conclusion: The dielectric properties of intracranial tumors appear to be depending on histological class and malignancy grade and show significant intratumoral heterogeneity. These results may allow a more precise modelling of electric field intensity distribution within the Tumor.

#4032

The alternative NF-kB pathway activity in refractory and relapsed Hodgkin lymphoma.

Angélica María Gamboa-Cedeño,1 Mariángeles castillo,1 Victoria Otero,1 Natalia Schutz,1 Dorotea Fantl,1 Federico Jauk Vitali,1 Hernán García Rivello,1 Myriam Nuñez,2 Stella Maris Ranuncolo1. 1 _Hospital Italiano de Buenos Aires, Buenos Aires, Argentina;_ 2 _Universidad de Buenos Aires, Buenos Aires, Argentina_.

The refractory and relapsed disease is currently the challenge in Hodgkin Lymphoma (HL) treatment. There is no specific therapy but rescue chemotherapy schemes, which fails in 50% of the cases and associates with high risk severe toxicity. This highlights the need to deeper understand the HL molecular biology and the screening for therapeutic directed-targets.

We have previously reported that HL relies on the alternative NFkB pathway, mediated by Rel-B and NIK, to survive. Its constitutive activation seems to be involved in the refractory and relapsed disease.

To determine the specific Rel-B target genes we performed ChIP-Seq in UH01 and L1236 human HL cell lines for Rel-A, Rel-B, cRel, p50 and p52. We found 29,414 Rel-B peaks genome-wide distributed. To further identify genes with higher probabilities of being directly regulated by Rel-A, Rel-B, and/or cRel we concentrated on the peaks that localized within a +/- 2 kb window, relative to the gene transcription start site. The +/- 2 kb Rel-B peaks were distributed on 4,509 genes; meanwhile the +/- 2 kb cRel peaks were on 1,994 genes and the Rel-A peaks were on 830 genes. Out of the 4,509 genes with the Rel-B peaks, only 6% overlapped with the Rel-A peaks and 11% with the cRel peaks. The Rel factors distribution showed a Rel-B DNA-binding hierarchy in HL.

The ChIP-Seq data were merged with gene expression arrays results. The gene expression assays were analyzed by comparing the signal from cells infected with noninduced shRNAs for the Rel factors to that from cells infected with induced shRNAs. Differentially expressed genes were identified via ANOVA analysis. Genes that were downregulated more than 2-fold with a p<0.001 were considered significant. We found that the Rel-B downstream controlled gene set was enriched for cell cycle and cell death regulation and DNA damage and repair signatures, among others.

One of the exclusively Rel-B target genes was BCL2. We showed that exogenous BCL2 was able to partially rescue HL cells from dying in response to Re-B depletion. We also found that BCL2 was useful as a prognosis marker in terms of overall survival in a cohort of 96 HL patients [Log Rank Test (p=0.002)]. In this retrospective study [follow up period 47,4 (6-136) months], BCL2 was able to identify at diagnosis patients that were refractory to conventional first line treatment and patients that relapsed. The HL cell lines U-H01, L1236, SUPDHL1, KM-H2 and L540 were sensitive to the BCL2 inhibitor venetoclax. The HDM-2 cell line, which does not express BCL2, was a good control of target specificity since it did not die in response to venetoclax.

In summary, we found that the alternative NFkB pathway plays an important role in the refractory and relapsed Hodgkin disease, being BCL2 a key downstream target. BCL2 performed well as a prognosis marker identifying refractory patients and those that would relapse. We propose BCL2 expression assessment in the lymph node biopsy at diagnosis and BCL2 as a directed therapy.

#4033

RAC1 **genomic aberrations as predictive biomarkers for head and neck squamous cell carcinoma (HNSCC).**

Hoi Lam Ngan, Yuchen Liu, Peony Hiu Yan Poon, Vivian Wai Yan Lui. _The Chinese University of Hong Kong, Hong Kong, Hong Kong_.

RAC1 is a small GTPase with known oncogenicity. Mutations of RAC1 are highly relevant to melanoma. Yet, little is known about RAC1 aberrations in other human cancers, including HNSCC. Pan-cancer analysis of 32 cancer types from the TCGA reveals that RAC1 mutations only affect 12/32 cancer types and HNSCC is the 2nd most frequently affected (2.64%; 14/510 cases), after melanoma (4.59%, 22/479 cases). 28/34 cancer types harbor RAC1 copy number alterations, including HNSCC (2.08% cases, all are amplifications). Among all 14 HNSCC-associated RAC1 mutations found in the TCGA cohort, RAC1 p.A159V mutation (residing in the highly conserved G5 Box of RAC1) appears to be particularly prevalent in HNSCC (6/14 cases), suggesting a likely mutant-specific role in HNSCC. HNSCC patients with somatic RAC1 aberrations [gene amplification/copy number gain/hotspot mutations (p.P29, p.K116 and p.A159)] have poorer overall survival (OS)(P=4.555e-5; median survival of 30.91 months vs 68.43 months) and disease-free survival (DFS) (P=5.49e-5; 27.89 months vs unreached median) vs. RAC1-unaltered HNSCC patients. RAC1-mutated tumors are also associated with a higher rate of TP53 mutation (P=2.07e-6), and a lower rate of HPV infection (P=7.397e-6; 7.7% vs. 87.2%; Fisher's Exact test). Further, RAC1-altered patients are of more advanced T clinical staging (T3/4 vs T1/2; P=0.0312) and with more gross/microscopic extension (P=0.0111; 59/119 vs 54/201). These evidences strongly suggest that HNSCC with RAC1 aberrations are more aggressive and may serve as a prognostic biomarker for HNSCC. To further elucidate the biological function of RAC1 mutations in HNSCC, we analyzed the mutational profile of RAC1-mutated HNSCC tumors which showed enriched mutations of 3 tumor suppressor genes: FAT1, FAT4 and CSMD3 (P=0.0001, P=0.0038 and P=0.0467 respectively). Gene set enrichment analysis (GSEA) of RAC1-mutated HNSCC also demonstrates significant dysregulation of immune-related gene sets (vs. RAC1-WT tumors) including increased interleukin-6 (IL-6) production (P<0.0001), a pro-tumorigenic inflammatory cytokine known to be involved in HNSCC tumorigenesis and progression. A previous study showed that overexpression of RAC1 activating mutation (p.V12) could upregulate IL-6 production in a cervical cancer model, HeLa, which is supportive of a RAC1/IL-6 axis in HNSCC. This is further supported by our RNAseq finding that RAC1-mutated HNSCC tumors do have significant upregulation of MYD88 (P=0.0436), a gene that has been shown to induce IL-6 secretion in HNSCC cells. Subsequent Tumor Immune Estimation Resource (TIMER) analysis shows that RAC1-mutated HNSCC primary tumors have higher infiltrating neutrophil levels than RAC1-WT tumors (P=0.019). Our findings may uncover a novel tumorigenic mechanism by RAC1 mutations in HNSCC, first linking IL-6 dysregulation to an oncogene mutation in HNSCC.

#4034

DCLK1 is upregulated in melanoma and it is a novel predictive marker for survival and response.

Dongfeng Qu, Nathaniel Weygant, Parthasarathy Chandrakesan, Kamille Pitts, Randal May, Adam Asch, Alexandra Ikeguchi, Courtney Houchen. _Univ. of Oklahoma Health Sciences Ctr., Oklahoma City, OK_.

Melanoma is the deadliest form of all skin cancer, and the incidence of melanoma has been rising for the last 30 years. There are only a few reported biomarkers for prediction and prognosis for melanoma, therefore, there is an unmet medical need to identify predictive and prognostic biomarkers. Doublecortin-like kinase 1 (DCLK1) marks a population of tumor-initiating cells in colonic and pancreatic cancers, and it is overexpressed in many solid tumors. Our preliminary studies suggest that DCLK1 is overexpressed in serum samples of melanoma patients and it maybe a novel biomarker for melanoma. In this study we examined DCLK1 expression in melanoma tumor tissues and further evaluated whether its levels in serum could be quantitatively assessed in melanoma patients before and after therapy treatment. Immunohistochemistry (IHC) analysis was performed to detect DCLK1 in a tissue microarray with 60 tumor and normal adjacent tissues (NAT) from melanoma patients. ELISA assay were performed to detect DCLK1 levels in the serum samples of melanoma patients before and after treatment. Western blotting was also performed to detect DCLK1 protein in the serum samples of melanoma patients. Additionally, analysis of DCLK1 and correlative gene expressions was performed using TCGA Melanoma dataset. Here we report that the intensity of DCLK1 staining in melanoma tumor tissues are significantly increased compared to NAT. DCLK1 levels in the serum were elevated in melanoma patients (n=65) compared to healthy volunteers. Patients with higher levels of serum DCLK1 had reduced overall survival compared to patients with lower levels of serum DCLK1. We also compared DCLK1 levels in 10 patients for whom pre- and post- treatment samples were available. Among these patients, seven had elevated DCLK1 before treatment and had a significantly decrease (about 30-50%) in serum DCLK after treatment. Three patients had no change in DCLK1 levels after treatment. Interestingly, the patients with decrease level of DCLK1 post-treatment demonstrated significant clinical responses to therapy, however, the patients with no change in DCLK1 level post therapy showed no clinical improvement (p<0.002). Our study suggested that serum DCLK1 may be an independent prognostic factor, and treatment-induced reduction in serum DCLK1 was associated with excellent clinical response. These data taken together suggest that DCLK1 maybe a novel biomarker for melanoma for prognosis and response purposes.

#4035

Protein tyrosine kinase 7 (PTK7) biomarker analysis in patients (pts) treated with PF-06647020, a PTK7 antibody-drug conjugate (ADC), in a phase I dose expansion study.

Amy Jackson-Fisher,1 Navi Mehra,2 Roberto Gianani,2 Pamela Whalen,1 Pamela Vizcarra,1 Shibing Deng,1 Stephen McComish,1 Xiaohua Xin,1 Eric L. Powell1. 1 _Pfizer Early Oncology Development & Clinical Research, La Jolla, CA; _2 _Flagship Biosciences, Inc, Westminster, CO_.

Background: PF-06647020, an ADC comprising a humanized monoclonal antibody against PTK7, a cleavable valine-citrulline linker, and an auristatin payload, is being investigated in an ongoing Phase I clinical trial in pts with advanced solid tumors. We hypothesized that response to a PTK7-directed ADC would correlate with PTK7 expression in the pts tumor. We report preliminary baseline PTK7 expression in pts tumors and clinical response using a novel CLIA validated laboratory developed test (LDT).

Methods: We developed and validated an LDT comprising an anti-PTK7 immunohistochemistry assay with digital tissue analysis using Flagship Biosciences Inc's cTA® platform to detect membrane-associated PTK7 in formalin-fixed paraffin embedded (FFPE) epithelial malignancies of ovary, breast and lung. Tumor samples were immunolabeled with the LDT and image analysis was applied to derive a digital H-score. Specifically the digital image annotation included desired regions of analysis (ROA) and excluded regions not of interest. The cTA® platform identified each cell in a given ROA and quantified an overall H-score for malignant epithelial cell membranes immunolabeled for PTK7. Non-small cell lung cancer (NSCLC) pts with H-scores ≥ 56.8 were eligible for enrollment whereas ovarian cancer (OVCA) pts were enrolled unselected. Triple negative breast cancer (TNBC) pts were initially enrolled unselected, but after review a requirement for H-scores ≥ 92.8 was implemented for enrollment.

Results: PTK7 IHC was performed on baseline FFPE samples from 69 pts and digital H-scores were generated (Table 1). An additional 7 pt samples were not amenable to analysis with the cTA® platform.

Conclusions: A novel CLIA validated LDT was developed and implemented to assess PTK7 baseline levels in FFPE tumors from pts treated with PF-06647020. Clinical responses tended to correlate with H-scores that were higher than the mean for each tumor type.

Table 1. PTK7 digital H-scores in Phase I dose expansion

---

|

Number of Samples | Minimum H-score | Maximum H-score | Mean H-score | Mean H-score of Non-responders | Mean H-score of Responders

OVCA | 31 | 1 | 200 | 89.2 | 80.2 | 111.2

NSCLC | 19 | 93 | 287 | 167.8 | 159.2 | 200.3

TNBC | 19 | 53 | 258 | 159.2 | 147.0 | 224.3

#4036

The efficacy of immune checkpoint inhibitors in patients with EGFR mutated non small cell lung cancer in retrospective analysis.

Tadaaki Yamada,1 Soichi Hirai,1 Akihiro Yoshimura,1 Satoshi Watanabe,2 Junji Uchino,1 Koichi Takayama1. 1 _Kyoto Prefectural Unversity of Medicine, Kyoto, Japan;_ 2 _Niigata University Graduate School of Medical and Dental Sciences, Japan_.

Background The treatment with EGFR-TKIs leads to initial response in most patients with EGFR mutated non-small cell lung cancer (NSCLC). In contrast, little is known the subpopulation of NSCLC patients with EGFR mutations who had clinical outcomes to treat for immune checkpoint inhibitors (ICIs). Therefore, to seek for the eligible cases to treat with ICIs, we retrospectively analyzed the correlation between clinical feature and the efficacy of ICIs in patients with EGFR mutations.

Methods/Materials and methods We analyzed a retrospective study of patients with advanced NSCLC harboring with EGFR mutations who were treated with ICIs after the resistance to EGFR-TKIs between February 2016 and April 2018 at six institutions in Japan. The association between clinical outcome and the efficacy or ICIs was investigated.

Results We enrolled 27 patients who have EGFR activating mutation. The objective response and disease control rates were higher in patients with uncommon EGFR mutations than in those with common EGFR mutations (71% versus 35.7%, and 57% versus 7%, p= 0.14 and p < 0.01, respectively). Patients with uncommon EGFR mutations or without T790M mutations was associated with a significantly longer median progression-free survival than those with common EGFR mutations or with T790M mutations (p = 0.003, 0.03, respectively).

Conclusions Patients with uncommon EGFR mutations and without T790M mutations are associated with best outcomes for the treatment with immunotherapy among EGFR mutated NSCLC in the retrospective analysis. Further research is needed to validate for the clinical biomarkers involved in ICI responders with EGFR mutation.

#4037

Analytical and inter-observer comparability of 4 clinically developed programmed death-ligand 1 (PD-L1) immunohistochemistry assays in advanced clear cell renal cell carcinoma (CCRCC).

Ulrich Sommer,1 Markus Eckstein,2 Johannes Ammann,3 Till Braunschweig,4 Stephan Macher-Goeppinger,5 Kristina Schwamborn,6 Stefanie Hieke-Schulz,3 Bernd Wullich,2 Manfred Wirth,1 Wilfried Roth,5 Ruth Knüchel,4 Wilko Weichert,6 Gustavo Baretton,1 Arndt Hartmann2. 1 _Universitätsklinikum Carl Gustav Carus, Technische Universität, Dresden, Germany;_ 2 _University Hospital Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany;_ 3 _Roche Pharma AG, Grenzach-Wyhlen, Germany;_ 4 _Uniklinik RWTH Aachen, Aachen, Germany;_ 5 _Universitätsmedizin Mainz, Mainz, Germany;_ 6 _Technische Universität München, Munich, Germany_.

Background: PD-L1/programmed cell death-1 (PD-1)-targeting checkpoint inhibitors have shown clinical activity in advanced and metastatic RCC, which correlates with PD-L1 expression on tumor cells (TC) and/or tumor-infiltrating immune cells (IC). We examined the analytical performance and inter-observer agreement of 4 clinically developed assays for PD-L1 expression on IC and TC in locally advanced CCRCC.

Methods: Archived formalin-fixed paraffin-embedded nephrectomy specimens from 30 patients with locally advanced (T2a+) CCRCC were selected from 201 cases based on PD-L1 status per VENTANA SP142 (IC <1%, 1-5%, or >5%; 10 cases each). All selected cases were stained for PD-L1 using VENTANA SP142 and SP263, and DAKO 22C3 and 28-8, each at one site, according to manufacturer protocols. Stainings were blinded with respect to both assay and sample information and scored at 5 sites for PD-L1 expression on IC (% per tumor area) and TC (% of tumor cells). All readers had been trained in scoring IC per tumor area.

Results: Adjusted means of IC staining for PD-L1 varied from 4.00 to 4.90%, and TC from 1.32 to 10.68%, depending on the assay used (Table). Pairwise comparisons of adjusted means between assays revealed small, non-significant differences for IC (-0.9 to 0.3%), while for TC there were significant differences for SP142 v other assays (-9.4 to -5.7%) and for SP263 v 28-8 (-3.7%), and small, non-significant differences for other comparisons (-2.9 to -0.8%). The pre-specified allocation to binary cutoffs (1% or 5%) for IC or TC alone predominantly showed moderate-to-substantial Kappa agreement scores (0.4 to 0.8) for IC and TC between assays for each reader.

Conclusions: This is the first multicenter analytical comparison study of PD-L1 assays in CCRCC. Good-to-high concordance rates across all assays were achieved between trained readers for scoring PD-L1 on IC and for some assays for PD-L1 on TC.

Assay | PD-L1 on IC, % (95% CI)* | Reader ICC† | PD-L1 on TC, % (95% CI)* | Reader ICC†

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

VENTANA SP142 | 4.10 (3.33 to 4.86) | 0.725 | 1.32 (-2.14 to 4.79) | 0.142

VENTANA SP263 | 4.26 (3.49 to 5.02) | 0.494 | 6.98 (3.52 to 10.45) | 0.823

DAKO 22C3 | 4.00 (3.23 to 4.76) | 0.701 | 9.87 (6.41 to 13.34) | 0.758

DAKO 28-8 | 4.90 (4.14 to 5.60) | 0.422 | 10.68 (7.21 to 14.14) | 0.778

* Sample adjusted means for each assay; † Intra-class correlation per assay between 5 readers.

## IMMUNOLOGY

### Biomarkers and Immune Monitoring

#4038

In-depth PD-L1 scoring reveals potential correlations between tumor-intrinsic PD-L1 and myeloid-inflamed profiles in head and neck cancer.

Takahiro Tsujikawa, Tsutomu Kawaguchi, Kanako Yoshimura, Junichi Mitsuda, Akihito Aarai, Shigeru Hirano. _Kyoto Prefectural University of Medicine, Kyoto City, Kyoto, Japan_.

Programmed cell death protein-1 (PD-1) inhibition has been showing encouraging results for recurrent/metastatic head and neck squamous cell carcinoma (HNSCC), which simultaneously requires the development of predictive biomarkers to guide selection of conventional and immune checkpoint therapies. Most recently, PD-L1 IHC scoring of tumor and inflammatory cells, but not of tumor cells alone, has been reported as effective metrics for selection between PD-1 inhibition and standard combination therapy of platinum/5-FU/cetuximab for HNSCC. Toward establishment of PD-L1 score-based biomarkers, the present study is aimed to evaluate in-depth PD-L1 profiles of tumor and immune cells in association with immune complexity profiles of HPV-positive and negative HNSCC via 12-marker multiplex immunohistochemistry (IHC) and image cytometry. As previously reported (Tsujikawa et al. Cell Reports. 2017), oropharyngeal HNSCC cases were divided into three clusters of lymphoid-, hypo-, and myeloid-inflamed profiles based on cell densities of immune cell lineages. Averages of PD-L1+ percentages in immune cells were 5.3, 7.1, and 12.8 in lymphoid-, hypo-, myeloid-inflamed groups, respectively. High PD-L1 expression on immune cells was observed in CD163+CSF1R\+ tumor-associated macrophages and mature dendritic cell populations particularly in intra-tumor regions. Interestingly, averages of PD-L1+ percentages in tumor cells were 0.17, 0.64, and 5.3 in lymphoid-, hypo-, myeloid-inflamed groups, respectively, suggesting that tumor-intrinsic PD-L1 expression without lymphoid-inflamed profiles was potentially associated with myeloid-inflamed profiles. To further evaluate the prognostic significance of PD-L1 expression with low lymphoid-inflamed profiles, we evaluated the the Cancer Genome Atlas (TCGA) HNSCC cohort (N = 528), and observed that high PD-L1 expression with low CD8+ T cell cytolytic activity was associated with short overall survival. Immune complexity analysis for longitudinal specimens from a nivolumab-resistant case with PD-L1 expression on tumor cells revealed the potential involvement of myeloid-inflamed status, suggesting the presence of differential PD-L1 profiles between tumor and immune cells in the context of lymphoid/myeloid-inflamed status. Those findings together indicate that combination of in-depth PD-L1 scoring and immune complexity profiling may contribute to biomarker development toward optimized management for HNSCC.Acknowledgement: This project was supported by the Japan Society for the Promotion of Science Grant-in-Aid (17H07016).

#4039

Simultaneous quantification of activated immune cells and PD-L1 expressing circulating tumor cells (CTCs) in peripheral blood of cancer patients receiving checkpoint inhibitor therapy.

Rachel Krupa, Robin Richardson, Priscilla Ontiveros, Joseph Schonhoft, Jiyun Byun, David Lu, Sean Nisperos, Yipeng Wang, Mark Landers, Ryan Dittamore. _Epic Sciences, San Diego, CA_.

Purpose: Activated immune cell subpopulations and circulating tumor cells in patients receiving checkpoint inhibitor therapy were characterized with the aim of developing more sensitive diagnostic tools for prediction of immunotherapy response or resistance.

Methods: Blood samples from bladder, kidney, and prostate cancer patients undergoing checkpoint inhibitor therapy were collected at baseline and on-therapy at convenience and sent to Epic Sciences for processing. Red blood cells were lysed and nucleated cells were plated onto glass slides and stained with CTC (pan-CK, CD45, PD-L1 and DAPI) and immune panels (CD4, CD8, Ki-67, and DAPI). 2-3 million cells per patient were imaged using high throughput digital pathology pipelines and changes in immune cell sub-populations and changes in circulating tumor cell (CTC) burden with PD1/PD-L1 immunotherapy were assessed.

Results: A method to profile activated (CD8+Ki-67\+ and CD4+Ki-67+) leukocyte subpopulations and PD-L1+ CTCs in a single blood sample was developed and feasibility was demonstrated in cell lines, healthy donor (HD), and patient blood. CTCs were detected in 73% (24/33) of patients tested. Of the 33 baseline samples tested, 12% (4/33) had PD-L1+ CTCs detected. No PD-L1+ CTCs were detected in on-therapy samples. Of 14 patients with matched samples, 57% (8/14) patients had an increase in activated CD4+ leukocytes and 36% (5/14) patients had an increase in activated CD8+ leukocytes in on-therapy samples compared to baseline. A decrease in activated CD4+ leukocytes was observed in 21% (3/14) patients and a decrease in activated CD8+ leukocytes was also observed in 7% (1/14) patients with therapy.

Conclusions: We developed a liquid biopsy-based platform that can simultaneously measure immune related biomarkers in CTCs and leukocytes at single cell resolution from ~1 mL of blood. The platform has extreme sensitivity over conventional methodologies and immune cell populations existing in less than 1% of the total are readily quantified. Efforts to identify morphological sub-populations within each canonical leukocyte category and comparison of leukocyte and CTC biomarker panels to patient outcomes are ongoing.

#4040

**PD-L1 expression level and CD8** + **/FoxP3** + **T cell ratio in breast cancer and prognosis.**

Masaya Hattori, Galina Khramtsova, Lise Sveen, Toshio Yoshimatsu, Dezheng Huo, Olufunmilayo I. Olopade. _The University of Chicago, Chicago, IL_.

Introduction: PD-L1 can bind many receptors, including PD-1, which is expressed on activated T cells and dendritic cells. This interaction contributes to the suppression of the T cell-mediated anti-tumor response. While CD8+ T lymphocytes play a key role in tumor-specific cellular-adaptive immunity, their actions may be counteracted by FoxP3+ cells, a major suppressor of CD8+ T cells. The aims of this study were to examine the expression level of PD-L1 as well as quantification of CD8+ and FoxP3+ cell infiltration in breast tumors and their associations with prognosis.

Methods: Tumor samples from 96 breast cancer patients were obtained from the University of Chicago Breast Cancer tissue bank, and were classified into four molecular subtypes based on immunohistochemical (IHC) staining: luminal A (ER+/PR+, HER2-), luminal B (ER+/PR+, HER2+), Her2-enriched (ER-, HER2+), and basal-like (ER-, HER2-, EGFR+ and/or CK5/6+). IHC for PD-L1, CD8 and FoxP3 was performed on tissue microarrays. PD-L1 expression was scored as negative, weak positive, moderate, and strong. CD8+ and FoxP3+ cells were counted manually and recalculated for 1 mm2. The Kaplan-Meier method was used to estimate probabilities of overall survival (OS), breast cancer-specific survival (BCSS), and distant disease-free survival (DDFS).

Results: The study cohort was comprised of 64.6% luminal A, 7.3% luminal B, 5.2% Her2-enriched, and 22.9% basal-like cases. The mean age at diagnosis was 58.2 years and 58% had node-negative breast cancer. 15% were grade 1, 42% grade 2, and 35% grade 3. PD-L1 expression was scored as follows: negative 9.4%, weak positive 50%, moderate 31.2%, and strong positive 9.4%. PD-L1 expression was not significantly associated with OS, BCSS and DDFS in the total cohort, but was associated with OS in the basal-like subtype (p=0.03). PD-L1 high-expressing tumors had, on average, greater FoxP3+ cell infiltration. The highest FoxP3+ cells infiltration was observed in the basal-like subtype and the lowest was in the luminal B subtype (p=0.02). The CD8+/FoxP3+ ratio was lowest for the basal-like subtype (mean: 2.3), but there was no significant association with OS, BCSS and DDFS for the total cohort or for each subtype.

Conclusions: The PD-L1 expression pattern and CD8+/FoxP3+ T cell ratio were different among the breast cancer molecular subtypes and were not statistically associated with the prognosis in the total cohort. The presence of and interactions among immune cell subsets in the tumor microenvironment for each breast cancer subtype are important in understanding the immunologic response and its effect on outcome. Obtaining a more comprehensive picture of the tumor immune microenvironment and maximizing the

information from tumor tissues require a multiplexed three-dimensional (3D) staining approach. Future studies will evaluate multiplex 3D immunofluorescence of multiple immune biomarkers in breast tumors.

#4041

Coupling neoantigen specific T cell clonotypes and their molecular phenotypes at the single cell level in resectable anti-PD-1 treated NSCLC.

Justina X. Caushi,1 Zhicheng Ji,1 Jiajia Zhang,1 Margueritta El Asmar,1 Valsamo Anagnostou,1 Tricia R. Cottrell,1 Hok Yee Chan,1 Haidan Guo,1 Taha Merghoub,2 Jedd D. Wolchok,2 Jarushka Naidoo,1 Kristen A. Marrone,1 Jamie E. Chaft,2 Matthew D. Hellmann,2 Edward Gabrielson,2 Janis M. Taube,1 Julie R. Brahmer,1 Victor E. Velculescu,1 Ni Zhao,1 Ludmila Danilova,1 Leslie Cope,1 Patrick M. Forde,1 Drew M. Pardoll,1 Hongkai Ji,1 Srinivasan Yegnasubramanian,1 Kellie N. Smith1. 1 _Johns Hopkins University, Baltimore, MD;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY_.

We recently completed a phase II study evaluating the safety and efficacy of neoadjuvant anti-PD-1 in non-small cell lung cancer (NSCLC). To evaluate anti-tumor T cell responses, we used the recently described MANAFEST (Mutation Associated Neoantigen Functional Expansion of Specific T cells) assay, which links antigen specificity with unique CD8+ TCR Vβ CDR3 identities, in 7 patients from the trial. We then carried out single cell sequencing of tumor infiltrating T lymphocytes (TIL) to enumerate the genome wide digital gene expression of each single cell (VDJ+DGE analysis), and more particularly those with Vβ CDR3 regions identical to those identified by MANAFEST. Neoantigen-specific TCRs were detected in all patients with major pathological response (MPR) and in 3 of 4 patients without MPR. VDJ+5'DGE single cell sequencing demonstrated differential phenotypes of TIL obtained from surgical resection, and these T cells had varying degrees of peripheral perturbations during treatment. In PBMC from patient MD043-011, the MANAFEST assay detected a T cell response specific for a CARM1 R208W mutation, despite no evidence of pathological response in this patient. These neoantigen-specific T cells possessed a single TCR Vβ CDR3 at the aa level and represented 3.4% of TIL. Upon analysis of TIL with the VDJ+DGE platform, the same CDR3 was detected in 32 TIL, all of which possessed the identical TCR Vα at the aa level. However, analysis of CDR3 sequences at the nucleotide level revealed 3 distinct clones encoding this antigen-specific TCR. Differential gene expression profiling demonstrated a unique and discriminating expression profile for each clonotype, with one clone expressing CTL activation markers such as granzyme k, 4-1BB, and PD-1, the second clone expressing IL10 family member IL26 and genes that inhibit effector and memory CTL function, such as CISH, TOX, and SLAMF1, and the third clone expressing no known activation markers. These findings demonstrate the existence of different intratumoral T cell clones with distinct functions yet identical specificity for a tumor neoantigen. We propose that these differential expression programs are induced because these clones were differentially activated under different conditions in the TME, primed in distinct anatomical locations, or at different times during disease progression despite recognizing the same antigen. Analyses to evaluate the prevalence of this phenomenon in the larger cohort are ongoing. In conclusion, the coupling of MANAFEST with single cell VDJ+ DGE analysis enabled the first comprehensive phenotypic profiling of neoantigen-specific T cells using the TCR as a molecular barcode. Ultimately, this integrative approach may provide key insights in predicting and understanding not only clinical response to neoadjuvant PD-1 blockade in NSCLC, but also other early stage cancers agnostic of tissue of origin.

#4042

Longitudinal monitoring of neoepitope-specific T cell repertoires in patient blood following cancer immunotherapy.

Songming Peng,1 Benjamin Yuen,1 Joanne Tan,2 Fangfang Yin,2 Robert Bao,1 Zheng Pan,1 Olivier Dalmas,1 Duo An,1 Boi Quach,1 Michael Yi,1 Michael Bethune,1 Stefanie Mandl,1 Matt Walters,2 Juan Jaen,2 Alex Franzusoff1. 1 _PACT Pharma, Hayward, CA;_ 2 _Arcus Biosciences, Hayward, CA_.

T cells targeting neoepitopes derived from mutations exclusive to the tumor are one of the main drivers of cancer immunotherapy efficacy. Tracking these neoepitope-specific T cells during cancer immunotherapy has been hampered by the impracticality of repeated sampling from the tumor, and by the low frequency of neoepitope-specific T cells in peripheral blood. An ultra-sensitive and high-throughput technology (imPACT) has been developed for the identification and isolation of neoepitope-specific T cells from peripheral blood. Subjects with colorectal cancer, endometrial adenocarcinoma, nasopharyngeal carcinoma and other solid tumors were treated with AB122 (anti-PD-1 antibody) as part of an ongoing dose-escalation clinical trial. Pre-treatment blood samples were analyzed to identify the basal repertoire of neoepitope-specific T cells. Evolution of this repertoire during AB122 treatment was monitored to enable immune phenotyping and correlation with clinical outcomes. In addition, transcriptional profile changes were monitored at the single-cell level for each neoepitope-specific T cell. These data will enable us to analyze T cells targeting neoepitopes and identify driver mutations that correlate with and may be responsible for therapeutic benefit. More broadly, this platform technology promises to significantly advance our understanding of T cell-mediated mechanisms of cancer immunotherapy.

#4043

Application of high complexity flow cytometry for Monitoring activated T-cells, T and B regulatory subsets in combination immunotherapy trials, melanoma case study.

Jennifer Tsau,1 Michael Abadier,1 Brittney Atzmiller,1 Christine Vaupel,1 Naveen Dakappagari,1 Ghanashyam Sarikonda,1 Ahmad Tarhini2. 1 _Navigate BioPharma Services, Inc., a Novartis subsidiary, Carlsbad, CA;_ 2 _Cleveland Clinic, Cleveland, OH_.

Background

Ever increasing knowledge of the interplay between immune composition and a novel drugs' treatment efficacy or the lack thereof leads to continual advancements in immunotherapies. Given the exponential increase in combination therapies, tools to determine the most promising regimens are critical.

Immunotherapeutic agents either reverse T-cell inhibitory pathways (e.g., PD-1) or activate the immune system to induce cytotoxic T-cell activation and proliferation through agonistic stimulation (e.g., Cytokines). These treatment strategies have proven to be efficacious by enhancing the balance between immune-effector and suppressive/regulatory cells that can directly affect the outcome of immunotherapy.

To concurrently monitor multiple immune cell subsets responsible for either immuno-suppression or activation, we developed a high-complexity (13-color) flow cytometry panel to reliably enumerate activated T-cells, Tregs (A & B subsets) and Bregs for clinical trial application.

Methods

The panel includes CD3, CD4, CD8, CD19, FoxP3, CD25, CD127, CD45RA, HLA-DR, CD38, CD24, and CD27 to monitor total Tregs, Treg-subsets, Bregs and T-cell activation. After robust analytical method validation following industry guidelines for exploratory biomarker assessments, we confirmed the clinical utility of this panel in stage III or IV melanoma patients (N= 14) enrolled in a randomized phase II clinical trial receiving high-dose of IL-2 with or without Afliberecept (VEGF inhibitor).

Results

We observed up-to 20-fold increase in activated CD8+ T-cells following treatment. Interestingly, while no discernible change was observed in the naive Treg-A cells (CD45RAhiFoxp3lo), a remarkable 5-fold expansion of effector Treg-B cells (CD45RAloFoxp3hi) was observed following treatment. Additionally, frequencies of Bregs (CD24hiCD38hi) remained unchanged after treatment.

Conclusions

In summary, we demonstrated the utility of a powerful high complexity clinical flow cytometry tool for precisely monitoring immune cell subsets involved in tumor escape such as Tregs and Bregs or those involved in tumor killing like CD8+ cytotoxic T-cells. More notably, this tool may be utilized to deprioritize agents that cause immunosuppression and focus on combination agents that lead to additive immune activation.

#4044

The immune phenotype in serial biopsies from metastatic TNBC undergoing chemo-immunotherapy.

Jiehui Deng,1 Suhagi Shah,1 Aatish Thennavan,2 Michelle Krogsgaard,1 Charles M. Perou,2 Kwok-Kin Wong,1 Sylvia Adams1. 1 _NYU Langone Medical Center, New York, NY;_ 2 _Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC_.

Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer with poor prognosis when metastatic. Immunotherapy, especially in combination with taxane chemotherapy has recently been shown to improve outcomes with PD-L1 as predictive biomarker for survival. To understand the mechanisms of response/resistance to chemo-immunotherapy for TNBC, we obtained serial core biopsies of TNBC patients (n=30) undergoing nab-paclitaxel and pembrolizumab treatment in a prospective trial at three time points, including baseline (before treatment), after 1 cycle (nab-paclitaxel treatment without immunotherapy), and after 2 cycles (nab-paclitaxel plus pembrolizumab treatment). fluorescence-activated cell sorter (FACS). We performed 10x genomics single-cell RNA sequencing (scRNA-seq) of immune cells isolated from these fresh, serial tumor specimens from two patients, a patient with subsequent RECIST response and a patient with rapid disease progression. Results were further validated by FACS and immunohistochemistry analyses. ScRNA-seq showed significant baseline heterogeneity as well as perturbations of the immune cell infiltrates with chemotherapy and chemo-immunotherapy. In the responder patient there was a significant expansion of T cells, including both CD4+ and CD8+ T cells. Before the pembrolizumab treatment, there are 1.84% of CD4+ T cells and 6.45% of CD8+ cells among total tumor infiltrating leukocytes (TILs). After the treatment, the percentage of CD4+ and CD8+ cells increased to 33.32% and 23.42% among TILs, respectively, as determined by FACS analysis. Cytotoxic T lymphocytes (CTLs) showed enrichments for transcripts of tissue-resident memory T cells (Trm). In contrast, tumors from a patient with rapid disease progression showed persistence of a myeloid compartment that suppress anti-tumor immunity. Although T cells were initially present, they mainly consisted of a naïve T cell phenotype and did not expand even after treatment. No Trm cells were detected within TILs by scRNAseq from this patient after pembrolizumab treatment. When comparing baseline samples from these two patients, we found there is a distinct tumor cell population in the non-responder with differentially expressed genes related to oxygen transport and proliferation. The association between this population and the response to chemo-immunotherapy remains to be explored. Our study provides proof of concept that single cell analyses of serial biopsies are a feasible tool to understand or even predict responses to chemo-immunotherapy for TNBC patients.

#4045

**Single cell RNA sequencing of serial tumor and blood biopsies from lymphoma patients undergoing** in situ **vaccination.**

Tanaya Shree, Anuja Sathe, Hanlee Ji, Ronald Levy. _Stanford University, Stanford, CA_.

The critical determinants of effective antitumor immune responses, whether pre-existing or induced by therapy, remain poorly understood due to the complexity and plasticity of the immune system. To better profile and track these responses, we have performed single cell RNA sequencing for paired gene expression and immune repertoire analysis on tumor fine needle aspirates and peripheral blood of lymphoma patients undergoing immunotherapy on one of two clinical trials (NCT02927964, NCT03410901).

In these in situ vaccination studies, patients with low-grade lymphoma receive local low-dose radiation and a TLR9 agonist intratumorally to one site of disease, along with either ibrutinib or an OX40 agonist. Tumor fine needle aspirates (FNAs) and peripheral blood samples are obtained prior to treatment, and at one week and six weeks after treatment start. We performed single cell RNA-sequencing to an average targeted depth of 50,000 reads/cell for gene expression libraries and 5000 reads/cell for TCR sequencing. Identification of variable genes, principal component and/or canonical correlation analysis, graph-based clustering and differential expression analysis was performed using the Seurat algorithm. Single-cell TCR repertoires were analyzed using TCR-specific analysis software. Sequencing libraries have been prepared from 67 longitudinal samples from 9 patients thus far.

We have successfully generated single cell gene expression and TCR libraries from 3,000-10,000 cells per sample from tumor fine needle aspirates and peripheral blood, with excellent sequencing quality metrics obtained. Analyzing 23 samples sequenced thus far, we consistently identify many B and T cell subsets of interest within tumors and reliably distinguish tumor B cells from normal B cells. In two responding patients, we confirm shrinkage of the tumor B cell compartment and find specific increases in CD8 and CD4 effector cells with decreases in T follicular helper cells and/or regulatory T cells at the treated site, results that are corroborated by multiparameter flow cytometry performed in parallel. Comparing pre-and post-treatment tumor FNAs from the same sites, we find evidence of increased cytotoxicity and interferon response following treatment. We also find distinct transcriptional shifts in tumor B cells both at the treated and at the distant untreated sites. Sample collection, sequencing, and analysis are ongoing.

Deep profiling of serial biopsies during immunotherapy using single cell RNA sequencing promises to illuminate underlying cellular dynamics, and paired with clinical outcome data, determinants of response. Ultimately, this may provide a roadmap for successful translation of single-cell genomics into the clinic for treatment monitoring and response prediction.

#4046

Immune repertoire sequencing reveals tumor microenvironment and tracks clonally expanded B cell and T cell in blood.

Chen Song, Pingfang Liu, Andrew Barry, Bradley W. Langhorst, Fiona J. Stewart, Salvatore Russello, Theodore B. Davis, Eileen T. Dimalanta. _New England Biolabs, Ipswich, MA_.

The study of complex immunological diseases and tumor microenvironments has progressed through recent developments on sequencing of the immune repertoire. Using this approach, the interrogation of disease progression is facilitated through analysis of millions of V(D)J combinations from B cell antibodies (Igs) and T cell receptors (TCRs). One major challenge of immune repertoire sequencing is to accurately capture the structural and sequence complexities of antibodies and TCR genes. We have developed a method for accurate sequencing of full length immune gene repertoires of B cells and T cells. RNA extracted from tumor tissues containing tumor infiltrating lymphocytes, as well as matched peripheral blood mononuclear cells (PBMCs) were used to generate full length Ig and TCR libraries. Unique molecule index (UMI) was used to discretely barcode each mRNA molecule, enabling absolute quantitative ranking of antibody/TCR clone abundance. Full length immune repertoire sequencing facilitates detection of distinct and shared clones in tissue and blood samples, enabling identification of disease specific clones to evaluate immunotherapy effects. Highly expanded clones in tumor samples are also found in blood samples. RNA-seq libraries were also constructed from the same RNA for Ig and TCR libraries. The expression level of IGH/IGL/IGK and TRA/TRB in the immune sequencing libraries highly correlates with the RNA-seq data. In addition, both Ig and TCR libraries can be constructed in one tube to obtain a whole immune repertoire profile.Our immune repertoire sequencing approach allows accurate clonal determination for both Ig and TCR. This technique is applicable for investigation of lymphocytes infiltration of tumor microenvironments, tracing expanded B cell and T cells in blood samples, and monitoring of minimal residual disease.

#4047

Tumor-infiltrating lymphocytes in renal cancer patients demonstrate a diverse PD-1 expression and characteristic Treg classification.

Daisuke Sugiyama, Tomoaki Muramatsu, Yoichi Kobayashi, Naoto Sassa, Shoichi Maruyama, Momokazu Goto, Yoshiki Akatsuka, Hiroyoshi Nishikawa. _Nagoya University Graduate School of Medicine, Aichi, Japan_.

Immunotherapy has been shown to be effective for a proportion of chemoradiotherapy-refractory cancer patients. In particular, immune checkpoint inhibitors such as anti-CTLA-4 and anti-PD-1 antibodies are more effective for patients with high tumor mutation burden (TMB) or high expression of immune checkpoint molecules. In this study, we investigated immune responses in patients with renal cancer (RCC). Previous studies have demonstrated that the clinical effect of immune checkpoint inhibitors was 20% to 30%, and TMB was moderate (high; melanoma and lung cancer, low: astrocytoma and acute lymphocytic leukemia). We performed immune profiling of lymphocytes in the tumor tissue, normal tissue, and peripheral blood from 13 RCC patients by flow cytometry. Firstly, we examined the difference of CD4+ T cells, CD8+ T cells, and regulatory T (Treg) cells among 3 individual sampling sites. The frequency of CD8\+ T cells in the tumor and normal tissues was higher than that of CD4+ T cells in the peripheral blood. In contrast, the frequency of CD4+ T cells in the peripheral blood was the highest, and that of effector-Treg (eTreg) cells (defined by CD3+CD4+FOXP3highCD45RAlow) in the tumor tissue was the highest. Additionally, we found that the RCC patients were arbitrarily classified into three groups based on the frequency of eTreg cells (i.e. high: > 10%, moderate: > 5%, and low: > 0%). Although this simple eTreg classification has not yet been shown to be associated with patient prognosis, this pattern is distinct from that of other cancer types. Secondly, we examined the expression of immune checkpoint molecules in the CD8+ T cells and eTreg cells. The CD8+ T cells in tumor tissue showed various degrees of PD-1 expression. An extremely high PD-1 expression in CD8+ cells was correlated with a moderate to high frequency of eTreg cells. The tumor-infiltrating CD8+ T cells showed a low expression of other checkpoint/accessory molecules such as LAG-3, CTLA-4, and TIGIT. In contrast, tumor-infiltrating eTreg cells showed high TIGIT and CTLA-4 expression, moderate PD-1 expression, and no LAG-3 expression. With regards to other immune-related molecules, the eTreg cells showed high CCR4 and low ICOS expression. These preliminary data implicate that the clinical benefit of immune checkpoint inhibitors for RCC patients could be associated with the high level of PD-1 expression in CD8+ T cells and the magnitude of eTreg cell infiltration into their tumor tissues. By analyzing the mechanism of high PD-1 expression in the CD8+ T cells and assessing the eTreg classification in RCC patients, we speculate that it is possible to develop a new treatment strategy by combining the anti-PD-1 antibody and Treg-depletion therapies. These data, along with additional measurements of samples from further patients, will be presented at the meeting.

#4048

REAlease® technology: Controlled release of antibody-fluorochrome conjugates for maximal flexibility in flow sorting and fluorescence microscopy applications.

Jennifer Pankratz, Sabine Schmachtenberg, Nicole Jansen, Marie Hansen, Susanne Krauthäuser, Ali Kinkhabwala, Karolina Schlegel, Svenja Meiler, Christiane Siewert, Alina Bartholomäus, Dmytro A. Yushchenko, Christian Dose. _Miltenyi Biotec, Bergisch Gladbach, Germany_.

Multi-parameter immunofluorescent labeling is the method of choice for the flow-based sorting of targeted cell populations from heterogeneous samples. However, existing antibody-fluorochrome conjugates used for flow sorting limit the types of downstream analyses that can be applied to the isolated cells, as their continued presence on the cells blocks specific cellular epitopes and restricts the choice of downstream fluorescence detection channels. Herein, we present a new type of antibody-fluorochrome conjugates that facilitates a highly specific multi-parameter cell staining for flow sorting as well as subsequent complete release of the conjugate probes from the sample. These new conjugates rely on recombinantly engineered antibody fragments that individually possess low epitope binding affinities. A tailor-made covalent conjugation chemistry allows for their multimerization as well as fluorochrome labeling, generating fluorescent probes comparable to conventional antibody conjugates. Importantly, upon addition of a release reagent, the antibody-fluorophore conjugates can be rapidly released from the cell surface. Accordingly, previously blocked epitopes are free for re-labeling, which provides maximal flexibility for renewed epitope targeting in downstream applications. As many targeted cell populations require multi-parameter detection of several antigens for their isolation, the recombinant engineered antibodies were conjugated with different fluorochromes. The versatility of this technology is presented in multi-parameter panels enabling clear distinction and discrimination of different cell populations of different starting material, e.g. peripheral blood and tumor cells. The applicability of the new REAlease® antibody fluorochrome conjugates is also demonstrated by flow sorting of human naïve regulatory T cells. In addition, we show that the fast and efficient releasability of the new conjugates can serve as an optimal basis for cyclic immunofluorescence microscopy using a new MACSima™ imaging platform, which permits cell analysis with potentially unlimited number of parameters.

#4049

Cellular phenotypes of spleen cells in cancer patients targeted for adoptive cellular therapy.

Kathryn Cole, Quan Ly, Jesse L. Cox, Michael A. Hollingsworth, James C. Padussis, Jason M. Foster, Luciano M. Vargas, James E. Talmadge. _Univ. of Nebraska Medical Ctr., Omaha, NE_.

The spleen is a rich resource of large numbers of lymphocytes, with potential for use in adoptive cellular therapy (ACT). This is of particular relevance to patients with a distal pancreatic ductal adenocarcinoma (PDAC) who frequently undergo a pancreatectomy that required a splenectomy to clear the nodal basin. In this study we compared the phenotypes of eight paired sets of peripheral blood (PB) and spleen cell populations to assess the frequency of myeloid derived suppressor cells (MDSC) and lymphocyte subpopulations. Because a positive clinical response to ACT requires a low frequency of T-regs and MDSC's, both systemically and in the tumor microenvironment these cells were one of the foci for our studies. A high frequency of these cells negatively correlate with survival and positively with tumor burden. Indeed, in this study we observed a significant decrease in the frequency of granulocytic, monocytic and immature MDSC's in the spleen vs PB (4.8±1.6 vs. 54.4±6.1; 0.2±0.1 vs. 0.9±0.4; and 0.6±0.1 vs. 2.3±0.8 percent respectively) but not T-regulatory (T-reg) cells (0.8+0.5 vs. 0.1+0.04 percent) supporting the potential superiority of spleen cells relative to PB as a cellular source for ACT. In addition to a significantly higher number of spleen cells as compared to the PB for expansion/infusion, spleens have a higher frequency of T-cells as compared to the PB including CD3+ (35+5 vs. 16+4%), CD4+ (19+2 vs. 10+2%) and CD8+ (10+1 vs. 5+2%) T-cells. The frequency of transitional memory (Ttm) T-cells, which have a high proliferative potential were also significantly increased in the spleen vs. PB (37.5±6 vs. 26±3.0%) as was the frequency of PD1+CD8+ T-cells (8+2 vs 1+0.5%), which have been shown to have tumor specific cytotoxicity in cancer patients. In summary the increased frequency of PD-1+CD8+ cells, a Ttm memory phenotype and low levels of an exhaustion phenotype (TIM3 and Lag3 expression) in spleen cells vs the PB is supportive of the potential utility of these cells for expansion and therapeutic utility for these cells.

#4050

Molecular characterization of residual triple-negative breast cancers after neoadjuvant chemotherapy identifies immune composition and features associated with clinical outcome.

Caroline Nebhan,1 Paula I. Gonzalez-Ericsson,1 Roberto Salgado,2 Jennifer Bordeaux,3 Ju Young Kim,3 Christine Vaupel,3 Henry Gomez,4 Justin Micah Balko1. 1 _Vanderbilt University Medical Center, Nashville, TN;_ 2 _Peter MacCallum Cancer Centre, Melbourne, Australia;_ 3 _Navigate Biopharma Services, Inc., Carlsbad, CA;_ 4 _Instituto Nacional de Enfermedades Neoplásicas (INEN), Lima, Peru_.

Although neoadjuvant chemotherapy (NAC) induces complete response in 30-40% of triple-negative breast cancers (TNBC), patients with residual disease at surgery have poor prognosis and limited treatment options until recurrence. Tumor-infiltrating lymphocytes (TILs) in the residual disease are a positive prognostic factor, but the composition of TILs has not been explored in granularity, and how specific immune composition of the tumor guides outcome is unclear, resulting in a lack of understanding of how to employ immunotherapies in the adjuvant setting. We assessed multiple immunologic biomarkers in a series of 100 residual TNBCs after NAC. Immune markers were assessed by multiple methods, including H&E TILs analysis, standard immunohistochemistry (IHC; HLA-A, CD4, CD8, LAG-3) and multiplexed immunofluorescence (mIF; HLA-DR, GZMB, CD4, CD8, PD-L1, pan-CK). TILs and IHC were scored by clinical research pathologists and IF was scored by AQUA® analysis. Where multiple markers were multiplexed together, subset analyses was performed. Association among parameters were assessed (n=83) including clinical outcome after surgery (n=79). As previously demonstrated, TILs in residual tumors after NAC predicted both RFS and OS. H&E-scored TILs were correlated to both CD4 and CD8 as scored by IHC or IF (Pearson's r range 0.41-0.55; all p<0.001). CD4 and CD8 measured by IHC and IF were highly correlated (r=0.68 and 0.71, respectively; p<0.0001). CD4 and CD8 were also highly correlated to one another (r=0.67; p<0.0001). Likewise, infiltration by CD4 or CD8 were each associated with improved recurrence-free (RFS; coxPH p=0.004 and 0.007, respectively) and overall survival (OS; p=0.003 and p=0.005, respectively). Tumor and stromal PD-L1 (E1L3N) staining was associated with marginally poorer RFS (p=0.06) but not OS (p=0.94). Colocalizing GZMB and CD8 cells by mIF yielded a paradoxical finding that cytotoxic CD8 cells were associated with significantly worse RFS and OS (p=0.004 and 0.0004, respectively). In a multivariate model, total PD-L1, total CD4, and GZMB+CD8+ T cell infiltration each remained significant, suggesting independence, and provided a strongly predictive signature for RFS (p=4.6e-06). In the same analysis to predict OS, only GZMB+CD8+ T cells remained significant (p=0.0002). High GZMB+ CD8+ T cells were not associated with PD-L1 expression, but were associated with low LAG-3 expression (p=0.0009). Although the results of this study remain to be validated in larger cohorts, the paradoxical finding that GZMB+ CD8+ T cells are a negative prognostic factor for long term outcome in NAC-treated TNBC is an intriguing result. We hypothesize it may be these patients whose T cells are poised for cytotoxic activity but remain restricted through high PD-1 expression, and thus may benefit from adjuvant immunotherapy.

#4051

First step for development of checkpoint inhibitors immune monitoring panels in human whole blood and peripheral mononuclear cells.

Laila-Aicha Hanfi. _Caprion Biosciences Inc., Montreal, Quebec, Canada_.

Cancer immunotherapies represent more than half of treatments approved for oncology, as well as those currently in development. Among the key targets of these therapies are immune-checkpoint molecules (i.e. PD-1, LAG3, PD-L1, CTLA-4). Novel immunotherapies targeting these molecules are designed to impact tumor microenvironment immune-suppression. The characterization and monitoring of circulating and tumor infiltrating immune cells by flow cytometry is a critical aspect of pre-clinical and clinical drug development. In order to generated robust data to support drug development, flow cytometric methods must be fully optimized. In this presentation will discuss the critical aspects of assay optimization for flow cytometric methods with an emphasis on screening monoclonal antibody clones. The importance of the data is illustrated in the differences in assay performance when the correct clones are identified.

Methods: Four different anti-PD-1 obtained from different providers were screened on activated human PBMC. Additionally, four PD-L1 and five LAG-3 monoclonal antibody clones were screened on activated or rested PBMC spiked in whole blood. All antibodies were all titrated to obtain saturating concentration and optimal titers of different clones were compared.

Results: On CD4+ T cells from thawed cryopreserved peripheral blood mononuclear cells (PBMC), the best performance was observed with EH12.2H7 or EH12.1 clones which identified 30-33% of CD4+ T cells as PD-1 positive and up to 55-60% of CD8+ T cells as PD-1 positive. Clone eBioJ105 staining of the same samples on the same day identified 14% PD-1 positive in CD4+ T cell and around 40% PD-1 positive in CD8+ T cell, while clone MIH4 staining revealed low level of PD-1 expression on CD4+ T cells and CD8+ T cells. After ex vivo stimulation with staphylococcus enterotoxin B (SEB), EH12.2H7, EH12.1 and eBioJ105 clones all yielded similar results with 76-85% PD-1 positive on both CD4+ T cells and CD8+ T cells but fewer positive cells were identified with MIH4 clone.

Out of the four PD-L1 antibodies tested, only two were able to distinguish a clear PD-L1 population on PBMC rested for 4 days and spiked in whole blood used as positive control for PD-L1 induction.

For LAG-3 antibodies, all five were able to reveal the induction of this checkpoint molecule on cells activated by SEB and spiked in whole blood with % of LAG3+ cells in lymphocytes ranging from 13% to 25%.

Conclusions: These results illustrate the importance of thorough reagent evaluation in order to achieve a sensitivity coherent with the information-rich promises of flow cytometry.

#4052

Cytokines and chemokines production after radiosphere treatment for uveal melanoma patients with hepatic metastases.

Mizue Terai,1 Emma Link,1 Carin F. Gonsalves,2 David J. Eschelman,2 Rober D. Adamo,2 Meggie Danielson,1 Marlana M. Orloff,1 Takami Sato1. 1 _Sidney Kimmel Medical College of Thomas Jefferson University, Philadelphia, PA;_ 2 _Jefferson University Hospital, Philadelphia, PA_.

Uveal melanoma (UM) is the most common primary intraocular malignancy in adults. Despite successful treatments for primary UM, up to 50% of patients subsequently develop systemic metastasis, often in the liver. Treatment options are limited after development of hepatic metastasis, and the median survival of patients is reported to be 6 to 12 months. Immune-checkpoint blockades have not proved effective in the management of metastatic UM. We have conducted a phase 2 clinical trial using 90Y resin microspheres (radiosphere) (SIR-Spheres, Sirtex Medical) for uveal melanoma patients with liver-dominant metastasis [NCT NCT01473004]. In this clinical trial, patients with liver dominant metastasis were enrolled to arm A (n=24): no previously liver-direct treatment, and arm B (n=23): second line treatment after failing immunoembolization (IE). Blood samples were collected and processed before therapy (baseline), 1 hour after completion of therapy, at 1 month, and 3 months after therapy. Serum levels of cytokines and chemokines were analyzed by Bead-based Multiplex assay using the Luminex technology (MilliporeSigma). All of the samples were tested using a panel of 11 cytokines: interleukin (IL)-10, IL-1 beta, IL-6, IL-8, IP-10, MCP-1, TNF-alpha, TGF-beta1, CXCL9, HMGB-1, and HGF by Luminex FLEXMAP 3D. IL-8 levels were increased at 1 and 3 months after radiosphere (RE) treatment in both arms compared to baseline (paired T-test, P<0.05). Pretreatment IL-8 levels were 8.75 ± 2.1 pg/mL in arm A and 22.02 ± 8.3 pg/mL in arm B, respectively. IL-8 levels at 1 and 3 months were 20.1 ± 6.5 and 16.2 ± 4.2 pg/mL in arm A and 31.8 ± 9.3 and 40.2 ± 11.2 pg/mL in arm B, respectively. IP-10, MCP-1 and CXCL9 were also increased at 1 or 3 month after treatment in both arms, compared to the baseline levels (paired T-test, P<0.05). The pattern of these cytokine productions were similar in both arms suggesting that this is most likely related to chronic inflammation after RE treatment rather than previous therapies. We also analyzed the correlation of cytokines level with clinical responses in all patients. There was no significant difference in cytokines levels in partial response (PR), stable disease (SD), and progress disease (PD) except HMGB-1 levels prior to treatment between PR (15.6 ± 2.7 pg/mL) and PD (30.5 ± 6.4 pg/mL) (unpaired T-test, P<0.05). There is no significant difference in any cytokines and chemokines levels at baseline between arm A and B. In addition, there were no significant treatment-related change in other inflammatory cytokines, such as IL-6, TNF-alpha, and IL-10. In contrast to IE, RE did not produce robust inflammatory cytokine responses. However, baseline HMGB-1 and increase in IL-8 and IFN-gamma-related chemokines after RE treatments might be used to predictive clinical response and development of chronic inflammation after radiosphere.

#4053

Genetic and molecular profiling of ICOS hi CD4 T cells demonstrates clonal expansion of Th1 effector cells following JTX-2011 treatment in subjects with solid tumors.

Christopher Harvey, Amanda Hanson, Martin Fan, Lara McGrath, Daniel Felitsky, Calvin Johnson, Sean Lacey, Heather Hirsch, Ellen Hooper, Ty McClure, Elizabeth Trehu, Deborah Law, Haley Laken. _Jounce Therapeutics, Cambridge, MA_.

Background: Inducible T cell Co-stimulator (ICOS) is a costimulatory molecule expressed primarily on T lymphocytes that is upregulated upon cell activation. JTX-2011 is a first-in-class ICOS agonist antibody whose primary mechanism of action is the stimulation of primed CD4 T effector cells. Clinical and biological activity of JTX-2011 was assessed in the advanced solid tumor setting in the Phase I/II ICONIC trial (NCT02904226). Clinical responses to JTX-2011 were associated with the emergence of an ICOS hi CD4 T cell population, which was subsequently demonstrated to be due to activity of JTX-2011 and not a PD-1/L-1 inhibitor.

Methods: Assessment of phenotype and function of ICOS hi CD4 T cells was conducted using serial collections of peripheral blood mononuclear cells (PBMCs), whole blood, and tumor samples from a subset of evaluable subjects treated with JTX-2011 at 0.1mg/kg and 0.3mg/kg monotherapy and in combination with nivolumab. Biological activity was assessed through ex vivo functional assays, flow cytometry-based profiling, and genomic analysis including assessment of changes in T cell clonality. Mutational analysis of tumor samples was used to identify neoantigens for functional assessment of antigen specificity.

Results: Flow-based phenotyping revealed ICOS hi CD4 T cells as T effector cells of primarily the Th1 lineage. Analysis of changes in clonal abundance in the peripheral blood T cell receptor repertoire identified significant on-treatment expansion of clones in 18/22 (~82%) of subjects following JTX-2011 treatment, including those from monotherapy. Expanded clones detected in the periphery were tumor-associated clones present in archival tumor samples, suggesting that JTX-2011 may function to enhance cell-mediated anti-tumor immunity. Assessment of tumor mutational burden identified several potential MHC Class II neoantigens, again raising the potential of agonism of CD4 effector cell immunity. In independent experiments using tetanus based priming of donor PBMCs, ex vivo stimulation by soluble JTX-2011 was active only if ICOS hi CD4 T cells were already present, with JTX-2011 inducing potent polyfunctional cytokine responses characterized by a 4-fold average increase in both IFNγ and TNFα.

Conclusion: Emergence of a distinct ICOS hi population of peripheral CD4 T cells correlates with response to JTX-2011 treatment. Independent characterization of ICOS hi CD4 T cells induced by recall antigens demonstrate potent effector function induced by JTX-2011, but only if the cells were already ICOS hi, which is hypothesized to contribute to biological activity. The emergence of this population is being used to guide clinical development by informing sequencing of combination approaches that maximize the potential for a combination agent to induce ICOS hi CD4 T cells, followed by subsequent agonism with JTX-2011.

#4054

Mass cytometry approaches to biomarker discovery via high-dimensional antigen-specific T cell identification and profiling.

Mathew Cobbett,1 Michael Fehlings,1 Evan Newell2. 1 _immunoSCAPE, Singapore, Singapore;_ 2 _SiGN, Singapore, Singapore_.

immunoSCAPE leverages the high-dimensional immune profiling capabilities of mass cytometry combined with a unique technology for the identification of antigen-specific T-cells to support the development of immunotherapy strategies in cancer and immune-related diseases. In cancer, there is now strong evidence that immunotherapy-mediated tumor rejection can rely on tumor-specific (neoantigen-specific) CD8+ T-cells. Consequently, the discovery of neoantigens becomes valuable for personalized cancer immunotherapies. Although in silico pipelines exist, that are capable of predicting non-synonymous mutations potentially giving rise to tumor-specific neoantigens, it is not clear how accurate these methods are in identifying immunogenic and therapeutically relevant epitopes, since T-cell epitope usage can be influenced by many factors. Moreover, analysis of T-cells in cancer patients is challenging as it requires detecting rare antigen-specific T-cell populations in samples that are usually limited in volume and availability.

By applying cytometry by time of flight in conjunction with combinatorial peptide-MHC tetramer staining and high-performance dimensional analysis tools, we are able to map broadly MHC-class I epitope with a high sensitivity for rare antigen-specific T-cells and perform concurrently in-depth characterization of these cells. We will show here the application of this technology in the context of immunotherapy, through the example of a murine in vivo tumor model responsive to checkpoint blockade inhibitors, as well as through the analysis of different human cancer samples.

Together, by providing insights into the nature of neoantigen-specific T-cells, immunoSCAPE's unique target discovery and high-dimensional immune profiling platform is a valuable tool for the development of novel diagnostic biomarkers and therapeutic strategies at different stages of drug development.

#4055

Late-differentiated effector neoantigen-specific CD8+ T cells are enriched in non-small cell lung cancer patients responding to atezolizumab treatment.

Mahesh Yadav,1 Michael Fehlings,2 Suchit Jhunjhunwala,1 Bill O'Gorman,1 Priti Hegde,1 Leesun Kim,1 Alessandra Nardin,2 Susan Flynn,1 Hermi Sumatoh,2 Marcus Ballinger,1 David Shames,1 Boon Heng Lee,2 Evan Newell2. 1 _Genentech, South San Francisco, CA;_ 2 _immunoScape, Singapore_.

There is strong evidence that immunotherapy-mediated tumor rejection can be driven by the reinvigoration of tumor-specific CD8+ T cells recognizing neoantigens derived from tumor somatic mutations (citations). Thus, it is possible that the relative abundance and/or characteristics of these tumor-reactive, mutation-specific CD8+ T cells can be used as predictive biomarkers of response to immunotherapy. However, only a fraction of these potential neoantigens are usually immunogenic and in addition, these tumor-reactive, mutation-specific CD8+ cells are present only at low frequencies in blood, making it difficult to reliably identify these effector cells.

Here, mass cytometry and highly-multiplexed combinatorial tetramer staining together with cellular barcoding was used to profile immune cells in longitudinally collected PBMCs from 14

non-small cell lung cancer (NSCLC) patients treated with anti-PD-L1 (atezolizumab) antibody to compare patients with objective response (n=8) and progressive disease (n=6). Although no significant phenotypic differences were detected in bulk CD8+ T cells, greater insight was gained from a parallel analysis using highly multiplexed peptide-MHC multimer staining to screen and profile antigen-specific T cells.

A longitudinal analysis was performed using peripheral blood CD8+ T cells for 800 candidate tumor neoantigens and 73 known viral-derived control peptides across all patient samples. In addition to virus antigen-specific T cells, a total of 20 different neoantigen-specific T cell populations were detected and their high dimensional profiles were compared. We found that neoantigen-specific T cells were more frequently detected in responding patients and their phenotypes were almost entirely distinct from non-responding patients. Neoantigen-specific T cells from responding patients showed a differentiated effector phenotype with high expression of KLRG1, 2B4 (CD244) and CD57, similar to CD8+ T cells associated with CMV and some types of EBV infection. In contrast, more memory-like phenotypic profiles, with high CD27 and CD127 expression, were observed for neoantigen-specific CD8+ T cells from patients with progressive disease.

In addition to the utility of this approach for the ex vivo identification, characterization, and longitudinal tracking of rare tumor-specific T cells, this study supports further research into assessing whether the presence of late-differentiated neoantigen-specific T cells could be used as a predictor of response to checkpoint blockade.

#4056

Correlation analysis between PD-L1 expression, TMB and clinical characteristics in Chinese non-small cell lung cancer.

Yang Yu,1 Xinglong Fan,2 Yucheng Wei,3 Zimin Liu,3 Zuping Lian,4 Hongxia Han,5 Ming Yao,5 Kai Wang6. 1 _Shandong Provincial Hospital Affiliated to Shandong University, Jinan, China;_ 2 _Qilu Hospital Qingdao Branch, Qingdao, China;_ 3 _The Affiliated Hospital of Qingdao University, Qingdao, China;_ 4 _Ruikang Hospital Affiliated to Guangxi University of Chinese Medicine, Nanning, China;_ 5 _OrigiMed, Shanghai, China;_ 6 _OrigiMed & Zhejiang University International Hospital, China_.

Background: Immunotherapy has played an increasingly important role in the treatment of non-small-cell lung cancer (NSCLC) and it was approved for the first-line treatment of advanced lung cancer. PD-L1 expression and TMB are two important immunotherapy biomarkers, however, the relationship between PD-L1 expression, TMB and clinical features is still unclear.

Methods: A total of 205 Chinese NSCLC patients (pts) including 120 males and 85 females with a median age of 61.5 years (range 31-84) were enrolled. Tumor stage was evaluated according to the 8th edition of the AJCC/UICC TNM staging system for NSCLC. FFPE tumor and matched blood samples were collected for NGS based 450[[Unsupported Character - Codename &shy;]]gene panel assay. TMB was assessed by standard NGS algorithms. TMB data (median: 5.4 muts/Mb, range 0.8-68.9 muts/Mb) from 206 lesions were provided for analysis. 172, 16 and 18 lesions tissues were analyzed for PD[[Unsupported Character - Codename &shy;]]L1 expression by IHC with 22C3, 28-8 and ZR3 antibodies, respectively.

Results: According to TNM staging system, pts were divided into stage I (44 pts), stage II (20 pts), stage III (40 pts) and stage IV (101 pts). The pathological types of pts are adenocarcinoma (69.4%), squamous cell carcinoma (18.4%) and other types (12.1%). 34.5% of lesions were positive for PD-L1 expression (TPS≥1%), including 25.7% PD-L1(TPS≥10%) and 16.5% PD-L1(TPS≥50%) respectively. Significant differences between males and females were observed in PD-L1 expression (TPS≥1%: 40.8% vs. 25.6%, p<0.05; TPS≥10%: 30.8% vs. 18.6%, p<0.05; TPS≥50%: 20.8% vs. 10.5%, p<0.05). In addition, males had higher TMB than females (median: 8.5 vs. 3 muts/MB, p<0.001). A higher percentage of males than females were identified with TMB≥10 muts/Mb (45.0% vs. 12.8%, p<0.001) and TMB≥20 muts/Mb (11.7% vs. 2.3%, p<0.05). The percentage of PD-L1 positive in advanced pts was higher than in stage I pts (TPS≥1%: 39.5% vs 19.6%, p<0.005; TPS≥10%: 30.7% vs 12.4%, p<0.005; TPS≥50%: 16.1% vs 5.2%, p<0.05). Squamous cell carcinoma had higher TMB than adenocarcinoma (median: 8.5 vs. 3.8 muts/MB, p<0.001). Squamous cell carcinoma had a higher percentage of TMB≥10 muts/Mb than adenocarcinoma (44.7% vs. 23.1%, p<0.01). A low correlation was observed between PD-L1 expression and TMB (p <0.001, r=0.2). Pts with PD-L1 TPS≥10% had higher TMB (median: 10 vs 3.8 muts/Mb, p<0.001) and pts with TMB≥10 muts/Mb had higher percentage of PD-L1 positive expression (50.8% vs 27.0%, p<0.001).

Conclusion: In this study, we found the most commonly used two biomarkers for immune checkpoint inhibitors, PD-L1 expression and TMB, may be correlated with gender, stage and pathological type of NSCLC patients. There is also a weak correlation between these two biomarkers. Exploring the relationship between immune biomarkers and clinical features may better guide in screening more suitable patients for immune checkpoint blockade.

#4057

**Measurement of myeloid derived suppressor cells (MDSC), T regulatory cells (Treg), CD8** + **and CD4** + **T-cells, and cytokines/chemokines in patients with metastatic melanoma treated with pembrolizumab and propranolol.**

Shipra Gandhi, Manu Pandey, Kristopher M. Attwood, Cheryl Allen, Joseph Tario, Shin La Shu, Maria Levis, Suzanne M. Stack, Paul Wallace, Paul Bogner, Igor Puzanov, Mark J. Bucsek, Elizabeth Repasky, Marc S. Ernstoff. _Roswell Park Comprehensive Cancer Center, Buffalo, NY_.

Introduction: Immunotherapy has recently emerged as the cornerstone of metastatic melanoma treatment. Monotherapy with PD1 inhibitors results in response rate (RR) ~30%, while RR with combination PD1 + CTLA-4 inhibitors is ~60% but associated with high-grade toxicities in 50% patients (pts). We have shown in a mouse model that adrenergic receptor signaling blockade with propranolol (PRO) enhances anti-tumor activity of PD1 inhibition. Thus, we designed a phase Ib/II clinical trial to improve the activity of pembrolizumab (P) by combination with PRO at different doses recruiting 3 pts at each dose (10, 20 & 30 mg BID).

Methods: 3 pts with metastatic melanoma were treated with P 200 mg every 3 weeks + PRO 10 mg BID. Biomarkers analysis were planned to identify PRO dose which should be tested in phase II. Blood samples were collected at baseline, day 8 & prior to each cycle. MDSC & its subtypes, Treg, CD8+ & CD4+ T cells & 29 cytokine/chemokines were measured at each time point. Ratios of CD8+/m-MDSC, CD8+/e-MDSC, CD8+/PMN-MDSC & CD8+/Treg were analyzed.

Results: Trend toward an increase in CD8+T cell & MDSC subsets with decrease in above ratios was observed. There was a significant time-effect of CD8+/ PMN-MDSC (p=0.039). The 6 and 30 wk levels were significantly lower than baseline (p=0.046; 0.008). The 30 wk value of CD8+T cell/m-MDSC was also significantly lower compared to baseline (p=0.037). There was a significant time-effect for IP-10 (p=0.007) where the expression increases from baseline, peaking around 3 wk (Table 1).

Conclusion: We see a decrease in CD8\+ T cell/MDSC (all subtypes) & increase in IP-10 over time. The inverse relationship of high IP-10 with decreasing CD8+/MDSC & CD8+/Treg populations would suggest potential role of IFN-γ & trafficking of CD8+ T-cells into tumor/tissue. We plan to recruit more pts in the study to analyze these trends & confirm our findings.

Table 1

---

Variables | Baseline | 1 week | 3 weeks | 6 weeks | 9 weeks | 12 weeks | 30 weeks | p-value

CD8+T cell%/Eosinophilic(e)-MDSC% | 31.1±19.2 | 15.9±7.1 | 45.8±37.1 | 13.9±4.6 | 19.3±6.8 | 13.9±2 | 13±1.5 | 0.532

CD8+T cell %/monocytic(m)-MDSC% | 290.4±287.2 | 5.8±2.1 | 8±3.3 | 7.2±4.2 | 4±1.5 | 6±2.9 | 2.5±1.1 | 0.170

CD8+T cell%/Polymorphonuclear(PMN)-MDSC% | 3544.2±2900.3 | 208.3±77.7 | 485.6±368.7 | 110.3±76.1 | 231.9±116.4 | 168.3±76.3 | 39.5±14.9 | 0.039

CD8\+ T cell%/Treg% | 8.3±3.2 | 5.8±1.7 | 6.8±2.4 | 6.9±1.9 | 7±2 | 6.5±1.9 | 7.1±0.2 | 0.65

Interferon gamma induced protein-10(IP-10) pg/ml | 576.1±83.9 | 1009±295.1 | 1865±593.5 | 1464.7±400.3 | 1353.4±445.9 | 1307.2±357.8 | 934.2±227.3 | 0.007

#4058

The miRNA expression profile of inflammatory tumors reveals a unique immune cell profile and potential companion targets for checkpoint blockade immunotherapy.

Bjarne Bartlett, Vedbar Khadka, Mark Menor, Youping Deng. _JABSOM Bioinformatics Core, University of Hawaii, Honolulu, HI_.

Background: Extrinsic factors such as expression of PD-L1 (programmed death-ligand 1) in the tumor microenvironment (TME) have been shown to correlate with responses to checkpoint blockade therapy. More recently two intrinsic factors related to tumor genetics, microsatellite instability (MSI) and tumor mutation burden (TMB), have been linked to high response rates to checkpoint blockade drugs. These response rates led to the first tissue-agnostic approval of any cancer therapy by the FDA for the treatment of metastatic, MSI-H tumors with anti-PD-1 immunotherapy. Our team sought to explore the biology of such tumors further and suggest potential companion therapeutics to current checkpoint inhibitors.

Methods: A CRC patient cohort (n=549) from TCGA was used for analysis. MSI was assessed with the MicrOSAtellite Instability Classifier (MOSAIC). TMB was assessed using Mutect2 and a 5% cutoff for allele frequency. Tumors with >20 somatic mutations per megabase were classified as high mutation. Expression of PD-L1 was assessed by quantifying gene expression -- FPKM values from TCGA were used for this. Immune cell populations were analyzed using 3 algorithms: CIBERSORT, TIMER, and xCell. Pathway analysis was conducted with miRbase, an online tool for analyzing gene targets for miRNAs.

Results: Analysis by Pearson Correlation revealed 41 miRNAs correlated with mutation burden, 62 miRNAs correlated with microsatellite instability, and 17 miRNAs correlated with PD-L1 expression. Fifteen of these miRNAs were correlated with all 3 tumor features and chosen for further analysis. let-7i, mir-1266, mir-132, mir-146b, mir-155, mir-212, mir-22, mir-223, mir-511, mir-625, and mir-629 were associated positively associated with the 3 tumor features while mir-335, mir-552, and mir-92a-2 were negatively associated with all 3. None of these miRNAs were associated with overall survival. Two cellular pathways known to influence immunological function were predicted by miRbase to be targeted by the miRNAs: PI3K-Akt (p<1e-16) and TGF-beta (p=1.52e-14). Three immune cell deconvolution algorithms were employed to further explore the link between PI3K-Akt/TGF-beta and the TME. A significant link was found between the M1 macrophage polarization and tumors displaying the 3 features indicating favorable responses to immunotherapy. All 3 features were associated the proportion of M1 macrophages: PD-L1 expression (p<2.2e-16), MSI status (P=.001), and TMB (P=1.5e-06). Similar results were found with both TIMER and xCell.

Conclusions: Exploring miRNA targets as companions to treatment by immune checkpoint blockade revealed 15 potential miRNA targets predicted to impact 2 important cellular pathways: PI3K-Akt and TGF-beta. M1 polarization of macrophages was also associated with tumors predicted to respond to therapy by immune checkpoint blockade.

#4059

Identification of pre-existing neoantigen-specific T cells in patients receiving checkpoint inhibitor therapy using a deep learning antigen prediction model.

Christine D. Palmer,1 Brendan Bulik,1 Aaron Yang,1 Meghan Hart,1 Matthew Davis,1 Joshua Francis,1 Fujiko Duke,1 Rita Zhou,1 Jennifer Busby,1 Tyler Murphy,1 Andrew Clark,1 Lauren Young,1 Mike Zhong,1 Kamilah Caldwell,1 Jesse Abhyankar,1 Alison Cook,1 Tommy Boucher,1 Raphaël Rousseau,1 Cynthia Voong,1 Naiyer Rizvi,2 Mark Frattini,2 Roman Yelensky,1 Karin Jooss,1 Mojca Skoberne1. 1 _Gritstone Oncology, CAMBRIDGE, MA;_ 2 _Columbia University, New York, NY_.

Immune Checkpoint Blockade (ICB) has revolutionized the treatment of patients with cancer but provides durable clinical benefit to only a minority of patients. Neoantigens, antigens derived from tumor mutations, are emerging as key targets of T cells in patients with clinical responses to ICB whose tumors exhibit high mutational burden. T cells specific to some neoantigens appear to be naturally generated in these patients and can be further expanded upon ICB. Accurate neoantigen identification may fuel therapeutic advances by driving potent anti-neoantigen immune responses through therapeutic immunization or adoptive cell therapy. However, identification of neoantigens and neoantigen-reactive T cells remains a significant challenge. Current methods are time consuming and labor intensive and require non-routine clinical specimen collections. Here, we apply deep learning to a large HLA peptide and genomic dataset from NSCLC patients to create a computational model of minimal epitope presentation for neoantigen prediction. We demonstrate that our model allows rapid identification of neoantigens and neoantigen reactive T cells using routine clinical biopsies and blood specimens. This methodology does not require HLA-specific reagents and enables identification of T cell responses for every patient. To identify neoantigen-specific T cells, selected peptides, prioritized by our prediction model, were synthesized as minimal epitopes and added to short-term in vitro stimulation (IVS) cultures to expand neoantigen-reactive T cells. The presence of antigen-specific T cells was assessed two weeks later using IFN-gamma (IFN-g) ELISpot against pools of the prioritized neoantigens. Patient-specific peptide pools and IFN-g ELISpot enabled identification of neoantigen-reactive T cells in 5/9 (56%) patients on ICB. Deconvolution yielded an average of 2 recognized neoantigens per patient, with increased responses over time observed in one patient where multiple time points were available. Neoantigen responses were confirmed in repeat IVS cultures where additional samples were available. Importantly, expansion of HLA-matched healthy controls in the presence of patient peptide pools did not result in any responses by IFN-g ELISpot. In addition, antigen-reactive T cells were sorted for single cell sequencing on the 10x platform using CD137 as an HLA-independent T-cell activation marker. In summary, pre-existing neoantigen specific T cells and TCR clones can be rapidly identified utilizing routine clinical specimens, through (i) a powerful neoantigen prediction model, (ii) short-term in vitro T-cell expansion followed by IFN-gamma ELISpot, and (iii) HLA-independent selection and single cell sequencing. This methodology may allow rapid design of neoantigen-targeting vaccines or selection of TCRs for cell therapeutic approaches.

#4060

A plasma immune-related microRNA-signature classifier (MSC) predicts prognosis in advanced NSCLC patients treated with immunotherapy.

Mattia Boeri, Diego Signorelli, Claudia Proto, Giuseppe Lo Russo, Massimo Milione, Carla Verri, Mavis Mensah, Giovanni Centonze, Ugo Pastorino, Marina Garassino, Gabriella Sozzi. _Fondazione IRCCS Ist. Nazionale dei Tumori, Milan, Italy_.

Purpose: Immune-checkpoint inhibitors (ICIs) have improved the survival of advanced NSCLC patients. However, only a subset of patients benefit from ICIs, and the role of PD-L1 as predictive biomarker is still debated. We identified a plasma microRNA-signature classifier (MSC) that reflects the tumor-host interaction and exhibits prognostic value in lung cancer screening trials. In the present exploratory study, we tested the efficacy of the MSC as prognostic marker in advanced NSCLC patients treated with ICIs.

Experimental Design: The MSC risk level was prospectively assessed in a consecutive series of 140 NSCLC patients before starting treatment with ICIs. Overall response rate (ORR), progression-free (PFS) and overall survival (OS) in strata of PD-L1 and MSC alone or combined were evaluated by log-rank test and Cox-proportional-hazards models. Multiple plasma samples to monitor MSC risk level during treatment were also profiled. CIBERSORT analyses were run on gene expression data of 37 lung primary tumors.

Results: Adequate tissue and plasma samples were available from 111 (79%) and 104 (75%) NSCLC patients, respectively. MSC risk level was associated with ORR (P=0.0028), PFS (multivariate HR=0.31; 95%CI:0.17-0.56; P=0.0001) and OS (multivariate HR=0.33; 95%CI:0.18-0.59; P=0.0002). The combination of MSC and PD-L1 stratified patients into three risk groups having 39%-18%-0% one-year PFS (P<0.0001) and 88%-44%-0% one-year OS (P<0.0001), according to the presence of 2-1-0 favorable markers. During treatment, MSC risk level decreased or remained low until tumor progression in patients with responsive or stable disease. Decreased numbers of cytotoxic CD8 and CD4 Tcells and increased numbers of Tregs characterized the tumor immune contexture of MSC high risk patients.

Conclusions: The plasma MSC test, reflecting an impaired tumor immune contexture, could supplement PD-L1 tumor expression to identify a subgroup of patients who do not benefit from immunotherapy.

#4061

Investigating the Indoleamine 2,3-dioxygenase 1 (IDO1) responsive gene expression panel in predicting outcome in stage matched colorectal cancer patients.

Ravindra Kolhe, Chance Bloomer, Pankaj Ahluwalia, Chetan Pundkar, Saleh Heneidi, Ashis Mondal. _Medical College of Georgia at Augusta University, Augusta, GA_.

Colorectal cancer has emerged as a third most commonly diagnosed cancer in males and second in females. There is need for new tools in the form of prognostic biomarkers which could assist in tailoring of individualized treatment. Indoleamine 2,3-dioxygenase (IDO), a single chain oxido-reductase that serves as a catalyst for the degradation of tryptophan into kynurenine, has merged as a key player in T-cell suppression and in the induction of immune tolerance to tumors. IDO inhibitors have entered the clinical arena with a promise to restore immune functions in breast cancer patients. We identified through extensive literature search a list of IDO1-responsive genes. These gene along with IDO1 interferes with cancer control pathways and leads to cancer progression. The expression of following genes was quantified on Nanostring platform - IDO1, CTLA4, IL4R, PDL1, CTLA4, IL4R, HIF and IL6. Under an approved IRB protocol we identified 750 colon cancer patients at the Georgia Cancer Center with a 5 year follow-up. These patients were then divided into different experimental groups based on tumor stage (AJCC stages 1-4), survival (more or less than 3 years), tumor grade (AJCC grades 1-3), and age (older or younger than 68). A total of 88 patients fitted in our inclusion criteria on the basis of survival duration after diagnosis. The FFPE blocks were acquired from Medical College of Georgia, Augusta. Total RNA was isolated and quantified through Nanodrop method. The statistical analysis of data was performed using student t-test and Pearson correlation. IDO1 had significantly expressed at higher levels in stage IV patients (p = 0.01*). CTLA4 and IL4R were significantly expressed in stage IV patients with higher survival (p = 0.03*) and (p = 0.04*) respectively. PDL1 expression was higher in stage III patients with higher survival (p = 0.04*). CTLA4 was expressed at higher levels in patients with higher survival (>5 years). IDO1 was expressed at higher levels in males (p = 0.23*). In smokers, HIF and IL6 expression was higher as compared to non-smokers, (p = 0.02*). Preclinical data have shown that IDO1 inhibitors such as 1-Methyl-D-Tryptophan (1-MT) can synergize with chemotherapy in killing tumors. Therefore the inhibition of IDO as an approach to immunotherapy has a high amount of potential. Combining this new agent with current therapies may boost their efficacy and improve survival in TNBC patients. Furthermore, despite the rapid progress made in cancer immunotherapy, very few biomarkers are available for predicting the responsiveness and effectiveness of immunotherapy agents. Therefore, there is an urgent need to identify and develop such predictive biomarkers. These findings points to the clinical significance of IDO responsive genes and tested its importance in a gene expression based prognostic biomarker panel in CRC patients.

#4062

Activation of innate and adaptive immunity using intratumoral tilsotolimod (IMO-2125) as monotherapy in patients with refractory solid tumors: a phase Ib study (ILLUMINATE-101).

Hani M. Babiker,1 Vivek Subbiah,2 Orla Maguire,3 Shah Rahimian,4 Hans Minderman,3 Cara L. Haymaker,2 Chantale Bernatchez,2 Erkut Borazanci,5 James Geib,4 Srinivas K. Chunduru,4 Peter M. Anderson,6 Igor Puzanov,3 Adi Diab2. 1 _University of Arizona Cancer Center, Tucson, AZ;_ 2 _University of Texas MD Anderson Cancer Center, Houston, TX;_ 3 _Roswell Park Comprehensive Cancer Center, Buffalo, NY;_ 4 _Idera Pharmaceuticals, Exton, PA;_ 5 _HonorHealth Research Institute, Scottsdale, AZ;_ 6 _Cleveland Clinic, Cleveland, OH_.

While checkpoint inhibitor therapy has transformed treatment of multiple tumor types, many patients remain refractory. Tilsotolimod, a toll-like receptor 9 agonist, has been shown in preclinical models to activate plasmacytoid dendritic cells and increase T cell infiltration to the tumor microenvironment. Preliminary results of a phase 1/2 study suggested that intratumoral injection of tilsotolimod in combination with ipilimumab may revive an immune response in patients with immune checkpoint inhibitor-resistant metastatic melanoma. To further explore the role of tilsotolimod in modulating the tumor immune microenvironment, we conducted a Phase Ib monotherapy trial (ILLUMINATE-101). Adults with histologically or cytologically confirmed diagnosis of cancer not amenable to curative therapies received intratumoral tilsotolimod in doses escalating from 8 mg to 32 mg into a single lesion at weeks 1, 2, 3, 5, 8, and 11. Objectives of the dose evaluation portion included characterizing safety and pharmacokinetics, and evaluating alterations in the tumor microenvironment. Blood samples and tumor biopsies of injected and distal lesions were obtained at baseline and on treatment. Immune analyses included evaluation using Nanostring and/or flow cytometry of activation of the type 1 interferon (IFN) pathway, IFN gamma levels, activation of dendritic cell subsets, and changes in T cell status. As of November 7, 2018, 41 patients have been enrolled, including 38 patients into the dose evaluation portion and 3 patients into a melanoma expansion cohort. No dose-limiting toxicities or treatment-related adverse events have been observed. Within 24 hours, fresh tumor biopsies showed significant increases in IFN gamma levels, activation of the type 1 IFN pathway, induction of an antigen processing gene signature (a measure of the MHC class I antigen presentation pathway), and maturation of dendritic cells as measured by expression of HLA-DR (MHC class II), compared to pretreatment biopsies. Of 25 evaluable patients who received at least 1 dose of tilsotolimod and had at least 1 post-baseline disease assessment, 12 (48%) had a RECIST v1.1 disease assessment of stable disease. For patients with at least one disease assessment following documentation of stable disease (n=8), duration of stable disease ranged from 0.53 to 4.2+ months, with 3 patients ongoing. These results demonstrate that single agent tilsotolimod was well tolerated and induced robust alterations in the tumor microenvironment.

#4063

Single cell immune profiling of patients with advanced biliary cancers treated with combination checkpoint inhibition and GM-CSF reveals diverse T cell and myeloid cell mechanisms of action.

Bridget P. Keenan, Whitney Tamaki, Eric Liu, Brandon Chen, Alexander Cheung, John D. Gordan, Brenna Sheldon, Li Zhang, Robin K. Kelley, Lawrence Fong. _University of California San Francisco, San Francisco, CA_.

Background: Advanced biliary cancers (ABC) including cholangiocarcinoma and gallbladder adenocarcinoma are rising in incidence with limited standard treatment options. While checkpoint inhibition (CPI) achieves durable tumor responses in subsets of patients across many malignancies, less than 10% of ABC patients respond to single agent PD-1-targeted therapy. Combination immunotherapy aims to overcome pre-existing and adaptive resistance to immunotherapy. GM-CSF is a cytokine that activates and matures antigen-presenting cells, suggesting the potential to enhance immune responses. The exact mechanisms of action of this cytokine have not been defined in cancer patients. The combination of GM-CSF and anti-CTLA-4 CPI demonstrated safety along with inducing clinical responses in melanoma and prostate cancer. We conducted a phase II trial of the novel combination of GM-CSF and pembrolizumab (Pembro) in patients with ABC which has resulted in durable clinical responses in greater proportion than previously reported with anti-PD-1 monotherapy.

Methods: We assessed peripheral blood mononuclear cells (PBMC) from patients on Pembro monotherapy and combined Pembro/GM-CSF by mass cytometry (CyTOF) and T cell receptor (TCR) sequencing. We explored for the differences between clinical responders versus non-responders.

Results: We find that the addition of GM-CSF to Pembro leads to a higher frequency of myeloid cells overall; however, certain sub-populations of classical monocytes and conventional dendritic cells decreased in peripheral blood following upfront Pembro followed by GM-CSF. GM-CSF did not seem to change the phenotypes or relative frequencies of circulating T cell subsets. We also find that clinical responders possess specific circulating populations of classical monocyte and conventional dendritic cells prior to treatment. Responders also had higher percentages of CD8+ T cells expressing CD39 following Pembro treatment compared to non-responders. Combination therapy with Pembro/GM-CSF led to different effects on the T cell repertoire compared to Pembro alone.

Conclusions: The addition of GM-CSF to Pembro leads to dynamic shifts in myeloid cell subsets in the peripheral blood of ABC patients treated with immunotherapy, whereas Pembro alone led to changes in the activation states of T cell subsets. The TCR repertoire shifts reflect distinct mechanisms of action for Pembro monotherapy versus Pembro/GM-CSF. Future studies will explore the mechanisms driving the increased response rate seen with combination immunotherapy in comparison to Pembro alone in ABC patients, both through the study of peripheral immune responses as well as via immune-profiling of tumors from sequential biopsies of patients on Pembro monotherapy versus combined Pembro/GM-CSF.

#4064

Association of molecular signatures, mutations, and sTILs, with pCR in breast cancer patients in NRG Oncology/NSABP B-52.

Katherine L. Pogue-Geile,1 Ying Wang,1 Huichen Feng,1 Corey Lipchik,1 Rim S. Kim,1 Reena S. Cecchini,2 Samuel A. Jacobs,1 Ashok Srinivasan,1 Joseph P. Costantino,2 Eleftherios P. Mamounas,3 Charles E. Geyer,4 Priya Rastogi,5 Peter C. Lucas,6 Soonmyung Paik,7 C. Kent Osborne,8 Norman Wolmark,9 Mothaffar F. Rimawi10. 1 _NRG Oncology/NSABP, Pittsburgh, PA;_ 2 _NRG Oncology/NSABP, and The University of Pittsburgh, Pittsburgh, PA;_ 3 _NRG Oncology/NSABP, and UF Health Cancer Center at Orlando Health, Orlando, FL;_ 4 _NRG Oncology/NSABP, and The Massey Cancer Center, Virginia Commonwealth University, Richmond, VA;_ 5 _NRG Oncology/NSABP, and The University of Pittsburgh Cancer Institute, Pittsburgh, PA;_ 6 _NRG Oncology/NSABP, and The University of Pittsburgh School of Medicine, Pittsburgh, PA;_ 7 _NRG Oncology/NSABP, and The Yonsei University College of Medicine, Pittsburgh, PA;_ 8 _NRG Oncology/NSABP, and The Baylor College of Medicine/Dan L Duncan Comprehensive Cancer Center, Houston, TX;_ 9 _NRG Oncology/NSABP, and The Allegheny Health Network Cancer Institute, Pittsburgh, PA;_ 10 _NRG Oncology/NSABP, and The Baylor College of Medicine/Dan L Duncan Comprehensive Cancer Center, Pittsburgh, PA_.

Background: NRG Oncology/NSABP B-52 neoadjuvant clinical trial was conducted to test if the addition of estrogen deprivation (ED) would improve the pCR rate in HER2+/ER+ breast cancer pts treated with docetaxel, carboplatin, trastuzumab, and pertuzumab (TCHP). A numerical increase in the pCR rate was observed with ED (46.1% v 40.9%), but the difference was not statistically significant. The purpose of this study was to determine the utility of using stromal tumor infiltrating lymphocytes (sTILs), mutations, established, and novel signatures, to assess their value for predicting pCR, particularly in the TCHP + ED arm.

Methods: Whole transcriptome RNA-Seq and Ampli-Seq libraries were sequenced on the Ion Torrent platform. Mutations were assessed with a custom Ampli-Seq panel of 117 genes, including HER2-activated pathways and/or trastuzumab (T)-resistance markers. The 8-gene T-benefit signature was prospectively tested for association with pCR, and was previously validated in the adjuvant setting in B-31 and NCCTG N9831. Hot-spot mutations, sTILs, and signatures for immune cells, intrinsic subtypes, risk of recurrence proliferation (RORP), and MammaPrint, were also tested for associations with pCR. RNA-Seq data was also used to identify ED-predictive genes. Differential expression was assessed in normalized RNA-Seq data using DESeq2 and each sample was subtyped with the AIMS classifier. Wilcoxon two-sided test, chi-square, or Fisher's exact tests were used to assess associations with pCR.

Results: Subtypes were determined with RNA-Seq data from pretreatment biopsies (N=230). The 8-gene T-benefit signature was associated with pCR. The high-, medium-, and low- T-benefit groups had pCR rates of 55%, 44%, and 6.8%, respectively (p=1.3e-13). Intrinsic subtypes were associated with pCR by comparing HER2E to all other subtypes combined (69% v 28%, p=3.9e-08). Hot spot mutations in PIK3CA alone (p=0.014), or combined with hot spot mutations in ERBB2, ERBB3, AKT1, PTEN, and MAP3K1, were associated with no pCR (p=0.0007) and may be useful as resistance markers. sTILs, MammaPrint, RORP, and some immune cell signatures also showed statistically significant associations with pCR but did not identify a subset of pts with an increased pCR rate in the TCHP + ED arm. In contrast, expression of the SH3BP2 gene was associated with pCR only in the TCHP + ED arm (interaction p=0.03).

Conclusion: The previously validated 8-gene T-benefit signature identifies a subset of pts with very low pCR rate (6.8%) with TCHP. PIK3CA and other activating mutations were associated with no pCR. These findings may identify pts with resistant disease who may require a different treatment. Exploratory analyses suggest that SH3BP2 expression may identify pts who may benefit from TCHP + ED, but validation is required for clinically utility.

Support: BCRF, U10CA180868, -180822; UG1CA189867, Genentech, PUMA Biother

#4065

Prognostic and predictive role of tumor mutation burden and copy number alterations across metastatic cancer: Immunotherapeutic implications.

Xin-Ran Tang, Zhong Yi Dong, Dehua Wu. _Nanfang Hospital of Southern Medical University, Guangzhou, China_.

Background: Cancer immunotherapy has provided durable responses and improved survival in a subset of patients with advanced disease. Tumor mutation burden (TMB) and copy number alterations (CNA) have been shown to be associated with response to immune checkpoint inhibitors (ICI). However, the prognostic and predictive role of TMB and CNA across different types of advanced cancer has not yet been clarified.

Methods: We analyzed the prognostic value of TMB and CNA respectively and in combination across different types of advanced cancers from MSK-IMPACT sequencing cohort. For predictive analysis, non-small cell lung cancer (NSCLC, n=240) and skin cutaneous melanoma (SKCM, n=174) patients from previous studies were analyzed for efficacy to ICIs by TMB and CNA respectively and in combination. Kaplan-Meier curve and log-rank tests were used to estimate correlations of these molecular classifiers with progression-free survival (PFS), and overall survival (OS).

Findings: Patients with low TMB level had longer OS in most cancer types including NSCLC, head and neck squamous cell carcinoma (HNSCC), colon and rectal adenocarcinoma (COAD) and prostate adenocarcinoma (PRAD) compared with patients with high TMB level. Similarly, patients with low CNA level had longer OS in most cancer types including NSCLC, bladder urothelial carcinoma (BLCA), breast cancer (BRCA), PRAD and glioblastoma (GBM) compared with patients with high CNA level as well. Furthermore, the combination of TMB and CNA level could serve as a better prognosis biomarker in almost all of the cancer types above. In predictive cohort, high TMB (p=0.009) level and low SCNA (p=0.052) level was associated with longer PFS in NSCLC patients receiving anti-PD1/L1 treatment respectively. For SKCM cohort, patients with low CNA level (p<0.001) had longer OS receiving anti-CTLA4 treatment, while TMB level was not associated with treatment outcome (p=0.272). More importantly, when we combined the TMB and CNA, those with TMBhigh CNAlow had the strongest objective response rates and favorable response in both NSCLC (ORR, 0.032; PFS, p=0.003) and SKCM (ORR, p<0.001; OS, p=0.012) patients. Analysis of MSK-IMPACT database indicated that the joint stratification of TMB and CNA might be associated with diverse prognosis and potential sensitivity to ICIs across 24 cancer types.

Interpretation: This study shows that TMB and CNA represent both a prognostic and predictive factor of outcomes. Furthermore, the combination of TMB and CNA can jointly stratify metastatic cancers into groups with different prognosis and clinical responses to ICI treatment. It might guide immunotherapeutic decisions for patients with metastatic cancer.

#4066

Development and validation of a gene mutation-based signature to predict response to PD-1 inhibitors in nonsquamous NSCLC.

Zhong-Yi Dong, Xin-Ran Tang, Li Liu, De-Hua Wu. _Nanfang Hospital, Southern Medical University, Guangzhou, China_.

Background:

Genetic variations in nonsquamous NSCLC displayed significant impact on immune microenvironment and response to programmed cell death protein 1 (PD-1) blockade immunotherapy. We undertook an unbiased analysis to develop a gene mutation-based signature (GMS) and predict the efficacy of anti-PD-1 treatment.

Methods: Two independent cohorts (MSK-IMPACT and CheckMate 012) consist of 265 nonsquamous NSCLC patients treated with anti-PD-1 were analyzed for gene mutation via next-generation sequencing. A GMS was built in a randomly selected 123 samples from MSK-IMPACT training cohort, using multivariate cox analysis of high-frequency mutation genes (≥10%) associated with progression-free survival (PFS) after anti-PD-1 treatment. We then validated our findings in the remaining 83 samples (internal validation cohort) and in CheckMate-012 external validation cohort (n=59).

Results: A GMS that consisted of 6 genes (KRAS, EGFR, TP53, STK11, PTPTD and KMT2C), was generated to classify patients into high and low GMS groups in the training cohort. Patients with high GMS in the training cohort had longer PFS (p<0.001) compared with low GMS. We noted equivalent findings in the internal validation cohort (p=0.004) and in the external validation cohort (p=0.001). The GMS was an independent predictive factor for anti-PD-1 treatment (Table 1). Furthermore, GMS can successfully predict PFS in anti-PD-1 monotherapy (p<0.001) as well as combined treatment (p<0.001), and in high PD-L1 (p=0.004) as well as low PD-L1 (p=0.006) expression subgroups. When combined the GMS and PD-L1, those with GMS high/PD-L1high had the strongest objective response rates and favorable survival than other subgroups.

Conclusion: Our study highlights the potential predictive value of GMS for immunotherapeutic benefit in nonsquamous NSCLC. The combination of GMS and PD-L1 may be a feasible and promising biomarker in guiding treatment decisions for anti-PD-1 therapy.

Table 1

Univariate and multivariable Cox regression analysis of predictive factors

---

|  | |  | |  | |

|

Univariate analysis | |

Multivariate analysis

Variable | HR | 95%CI | P-value | |

HR | 95%CI | P-value

MSK-IMPACT cohort

GMS (High vs. low) | 0.36 | 0.19-0.68 | 0.002 | |

0.38 | 0.20-0.74 | 0.004

PD-L1 (High vs. low) | 0.52 | 0.31-0.86 | 0.011 | |

0.57 | 0.33-0.96 | 0.034

TMB (High vs. low) | 0.59 | 0.36-0.99 | 0.046 | |  | |

Gender (Male vs. female) | 0.81 | 0.49-1.35 | 0.423 | |  | |

Age (≥ 65 vs. <65 years) | 1.09 | 0.65-1.82 | 0.753 | |  | |

Smoking (Ever vs. Never) | 0.50 | 0.28-0.91 | 0.022 | |  | |

CheckMate-012 cohort

GMS (High vs. low) | 0.27 | 0.11-0.65 | 0.003 | |

0.27 | 0.11-0.65 | 0.003

PD-L1 (High vs. low) | 1.44 | 0.74-2.83 | 0.284 | |  | |

TMB (High vs. low) | 0.40 | 0.21-0.79 | 0.008 | |  | |

Gender (Male vs. female) | 1.04 | 0.53-2.05 | 0.902 | |  | |

Age (≥ 65 vs. <65 years) | 0.87 | 0.45-1.68 | 0.671 | |  | |

Smoking (Ever vs. Never) | 0.60 | 0.28-1.28 | 0.183 | |  | |

1

#4067

PD-L1 expression, CD8 cells and MHC-class-1 Beta-2-microglobulin expression in advanced non-small lung cancer.

Lucie Hijmering-Kappelle, Jeroen Hiltermann, Birgitta Hiddinga, Anthonie van der Wekken, Nils 't Hart, Harry Groen, Wim Timens. _UMCG, Groningen, Netherlands_.

Introduction: PD-L1 expression has been approved as predictive biomarker for immune checkpoint inhibitors (ICI) in non-small cell lung carcinoma (NSCLC). In case of lack of PD-L1 expression in tumor biopsies, 8-10% of patients will respond to ICI. However, in case of > 50% PD-L1 tumor expression half of patients show no response. Tumor immune escape phenotypes are characterized by loss of MHC-class-I and a lower grade of CD8 T-cell infiltration, despite upregulation of PD-L1 expression. At least one of the four phenotypes of MHC-class-I and PD-L1 co-expression is considered responsible for non-responsiveness. We investigated the frequency of PD-L1, MHC-class-I β2-microglobulin (β-2M) co-expression and presence of CD8 cells in advanced NSCLC.

Methods: Analysis was performed on formalin fixed, paraffin embedded (FFPE) transbronchial or transthoracic tumor biopsies of 27 patients with advanced NSCLC. Immunohistochemistry was performed using Ventana Benchmark Ultra immunostainer. PD-L1 was detected using Ventana PD-L1 (SP263) Assay (CE IVD), CD8 by Ventana CONFIRM anti-CD8 (SP57) antibody and MHC-class I β2-M by Agilent-Dako polyclonal rabbit anti-human beta-2-Microglobulin. PDL1 expression was scored as percentage positive tumor cells according to the manufacturers guideline, β2-M was scored as positive or negative in tumor cells and CD8 cells as low (+), intermediate (++) or high (+++) numbers in tumor or peritumoral stroma.

Results: In 27 patients IHC staining was performed. PDL1 expression was scored as <1%, 1-49% and ≥50% in 17, 3, and 7 patients, respectively. PD-L1 positive (>1%) patients had positive β-2M in 10 of 10 cases. PD-L1 negative tumors showed presence of β-2M in 10 of 17 cases. Seven out of 27 cases (26%) were β-2M negative; all of them were PD-L negative cases. Low CD8 score was seen in 7 patients and 2 of these patients have a PD-L1 expression >1% and positive β-2M.

Conclusion: In this ongoing study one quarter of advanced NSCLC patients had no tumor MHC-class-1 β-2M expression. In our cohort β-2M negative phenotype was associated with lack of PD-L1 tumor expression.

### Combination Immunotherapies 3

#4068

Radiation, immune checkpoint inhibition, and modulation of the tumor immune microenvironment promotes immunologic rejection of established HPV-associated tumors.

Jared M. Newton,1 Aurelie Hanoteau,1 Hsuan-Chen Liu,1 Angelina Gaspero,1 Robyn D. Gartrell,2 Thomas D. Hart,3 Damya Laoui,4 Falguni Parikh,1 Yvonne M. Saenger,2 Andrew G. Sikora1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _Columbia University Medical Center, New York, NY;_ 3 _Columbia University, New York, NY;_ 4 _Vrije Universiteit Brussel and VIB, Brussels, Belgium_.

Immune checkpoint inhibitors (ICI), including those targeting cytotoxic T-lymphocyte-associated-antigen-4 (CTLA-4) and programmed cell death receptor-1 (PD-1), have shown tremendous potential against solid tumor malignancies; however, response to ICI remains unpredictable with 60-90% of patients receiving minimal to no benefit. Lack of efficacy is commonly attributed to inadequate tumor-specific T cell generation and the immunosuppressive effects of the tumor immune microenvironment (TIME). Thus, we hypothesized that a combinatory treatment strategy aiming to enhance antigen presentation and revert the immunosuppressive TIME could improve response rates of ICI in established solid tumors. Using a syngeneic tumor model of HPV-associated head and neck cancer (mEER) established to 60-75 mm2 in size, we found that CTLA-4 and/or PD-1 inhibition only minorly delayed tumor growth and flow cytometry profiling revealed that the TIME maintained a "cold" or immunosuppressed state similar to untreated tumors. When PD-1/CTLA-4 inhibition was combined with a weekly dose of tumor-directed radiation (10 Gy x 2), we observed upregulation of antigen presentation molecules in the draining lymph node but the combination remained incapable of generating long-term survival benefit. This lack of efficacy was attributed to the immunosuppressed and lymphodepleted TIME, a common consequence of radiation. Thus, to improve the TIME, we optimized an immune-stimulating drug combination previously developed in our lab combining cyclophosphamide (CTX) and a small molecule inducible nitric oxide synthase (iNOS) inhibitor L-n6-(1-iminoethyl)-lysine (L-NIL). When we combined CTX/L-NIL immunomodulation, PD-1/CTLA-4 checkpoint inhibition, and radiation (collectively called the "CPR" regimen), we observed complete rejection of approximately 70% of established tumors in a CD8 T-cell dependent manner and potent development of immunologic memory against tumor-associated antigens. Tumor immune profiling after treatment revealed a "cold to hot" transition of the TIME, including increased levels of myeloid and lymphoid subsets associated with anti-tumoral immune responses (i.e. CD8 T cells, dendritic cells, M1 macrophages) and downregulation of immunosuppressive cellular subsets (i.e. T regulatory cells, granulocytic myeloid derived suppressor cells). We observed strong lymphoproliferation effects in the tumor-draining lymph node which resulted in significant TIME improvements including a 15-fold increase in the CD8 to regulatory T cell ratio. Thus, we have demonstrated that the rational combination of TIME immunomodulation, localized radiation to enhance antigen presentation, and immune checkpoint inhibitors to prevent T-cell exhaustion can promote the immunologic rejection of established solid tumors.

#4069

Development of PSB205, a bifunctional MabPair product that targets PD-1 and CTLA-4.

William C. Fanslow, Zhi Liu, Zhonghua Hu, Hua Liu, Min Shen, Yufeng Peng, Cristina Domeier, David Treiber, Ron Schoner, Wei Yan. _Sound Biologics, Bothell, WA_.

Here we describe a novel platform that enables the production of two full length IgG molecules from a single production cell line. This technology was achieved using a molecular engineering approach to precisely control the cognate pairing of antibody heavy chains and light chains during the assembly of two antibodies in the same cell line. This technology platform, which has been designated as MabPair, can be applied for the development of therapeutic antibody products that contain a mixture of two different antibodies. The antibody cocktail is produced by the same cell line in a predefined ratio and can be manufactured using a conventional antibody production process. When addressing multiple targets, MabPair products can offer significant advantages and flexibility over a bispecific antibody. MabPair technology represents a novel approach for developing antibody combination products. This approach is exemplified by the development of PSB205, our lead MabPair product candidate. PSB205 targets two immune checkpoint pathways, PD-1 and CTLA-4. The development of PSB205, as a potential anti-cancer immunotherapeutic, is discussed.

#4070

Combination of Interleukin-15 with a STING (Stimulator of Interferon Gene) agonist: A potential immunotherapy for prostate cancer.

Ana M. Esteves, Efthymia Papaevangelou, Prokar Dasgupta, Richard A.G. Smith, Christine Galustian. _Kings College London, London, United Kingdom_.

Introduction & Objectives: Prostate cancer (PC) is the second most commonly diagnosed cancer in men and the eighth leading cause of cancer-related mortality in the world. The only successful immunotherapy for the disease (ProvengeTM) only extends life by a few months. Therefore the finding of immunotherapeutic agents for the treatment of PC is of major interest. It has been previously shown by our group that Interleukin-15 (IL-15), unlike other therapeutic cytokines such as IL-2 and IL-12, can stimulate expansion and activity of CD8 and NK cells in vitro when they are exposed to prostate cancer cells, while studies in mice have shown a reduction in tumor size by 50% with no apparent toxicity. In this study, we aim to examine potencies of IL-15 in combination with a cyclic dinucleotide (CDN) that activates the Stimulator of Interferon-Gene (STING) receptor. CDNs have previously been shown to activate both T cells and Dendritic cells through STING. We hypothesize that combination of CDNs and IL-15 can additively increase NK and T cell activity as they act to increase type 1 interferons through STING activation and IFN gamma through IL-15.

Material & Methods: Peripheral blood mononuclear cells (PBMCs) were isolated and co-cultured with cancer cells (LNCaP, ATCC® CRL-1740™) in the presence of either IL-15, the CDN 2'3'-c-di-AM(PS)2, or a mixture of IL-15 with CDN. Control experiments were also prepared in which IL-15 was replaced by PBS and the CDN by a linear dinucleotide. Co-cultures were incubated for 48 hours at 37°C and 5% CO2. After this period, all cells from each well were collected and stained with fluorophore conjugated anti-CD3, -CD8 and -CD56 to identify CD8 T cells and NK cells, and anti-NKG2D and -perforin to analyze activation of NK and T lymphocytes. Tumor cell death was also evaluated and quantified using Live/Dead staining. All samples were acquired on FACS Canto. Results We observed that 1µg/ml of 2'3'-c-di-AM(PS)2 in combination with 2.5 ng/ml of IL-15 caused 81% of LNCaP killing in prostate cancer-lymphocyte co-cultures, while IL-15 by itself led to approximately 45% of cancer cell death. Little or no cancer cell killing was seen without IL-15 or CDN. Interestingly, the mixture of IL-15 with the CDN did not expand NK cells more than IL-15 alone. This suggests that the activity of NK cells is increased despite lack of their expansion. Preliminary data suggests that the level of perforin is increased by up to 35% in NK cells cultured with the mixture of both therapeutic drugs when compared to IL-15 alone in the cocultures. Of note, cell culture of either LNCaP or PMBCs with 1µg/ml of CDN did not affect cell growth.

Conclusion: The combination of two immunotherapeutic drugs, IL-15 and 2'3'-c-di-AM(PS)2, lead to an increase in the activity of NK cells, with an apparent increase in the levels of perforin and doubling of cell death mediated by immune effector cells compared with IL-15 alone.

#4071

A novel combination therapy of high intensity focused ultrasound and PDL1 blockades against advanced breast cancer.

Shinya Abe, Takuya Osada, Kensuke Kaneko, Pei Zhong, Herbert K. Lyerly. _Duke University, Durham, NC_.

Background: Previous studies have reported that tumor debris and inflammation made by high-intensity focused ultrasound (HIFU) therapy induced the antitumor immune response, however, HIFU as a monotherapy is still not potent enough to eradicate tumors and treat distant metastasis. We have reported that mechanical HIFU (M-HIFU), that will mechanically destroy tumor cells/tissues through acoustic cavitation, induced stronger antitumor immune response compared to conventional thermal HIFU (T-HIFU) that will cause thermal ablation of tumors. In the present study, we established an immune based combination therapy of M-HIFU and immune checkpoint blockade to enhance systemic antitumor immune response and treated distant/metastatic tumors in murine breast cancer models.

Methods and Results: HER2 oncogene-dependent murine breast cancer cell line, MM3MG-HER2, was established in our lab. In mice with bilateral implantation of MM3MG-HER2 tumors, M-HIFU monotherapy induced stronger HER2-specific cellular response and inhibited the growth of HIFU-untreated distant tumors as well as HIFU treated-tumors, more potently than T-HIFU. Flow cytometry and immunohistochemical analysis of tumor microenvironment revealed significantly stronger accumulation of activated T cell and NK cells in M-HIFU-treated tumors. On the other hand, M-HIFU induced stronger expression of programmed death-ligand 1 (PD-L1) on various immune cells in both sides of tumors than T-HIFU therapy or no treatment control. Based on these findings, we investigated the combination therapy of M-HIFU and PD-1/PD-L1 axis blockades, and found significantly enhanced tumor-specific cellular immune response compared to each monotherapy, which resulted in improved therapeutic effect against distant tumors as well as HIFU treated-tumors. Furthermore, immune cell depletion studies demonstrated that both CD8+ T cells and NK cells played an essential role for the antitumor efficacy in this combinatory therapy against both HIFU-treated tumors and untreated distant tumors.

Conclusion: this study provides strong rational evidence that M-HIFU combined with PD-1/PD-L1 axis blockades could be a promising treatment strategy against advanced breast cancer with metastatic lesion.

#4072

A combinational therapy of oncolytic virus and nature killer cells for melanoma treatment.

Lin Chen,1 Xianju Chen,1 Xiankui Tan,1 Meng Li,1 Fang Hu,1 Haiyan Zhang,2 Xiaoyuan Wang3. 1 _Hangzhou ConVerd Co., Ltd, Hangzhou, China;_ 2 _Nanjing Legend Biotech, Nanjing, China;_ 3 _Shanghai Bendao Genentech, Shanghai, China_.

Immunotherapy has obtained substantial impact on the clinical treatment of melanoma which is largely resistant to current therapies as chemotherapy and radiotherapy. Oncolytic virotherapy of melanoma is an appealing approach for inducing not only tumor cell death but also antitumor immunity. Furthermore, NK cells, a lineage of innate lymphocytes, not relying on peptides presented in an HLA class I context, have gained more and more attention as anti-melanoma effector cells. However, the combination of oncolytic adenovirus and NK cells has not yet been explored in melanoma. In this study, the combinational efficacies of oncolytic virus H101 and NK cells was tested in vitro. The combination of H101 and NK cells resulted more efficient killing of tumor cells (HT29, A549 and HepG2) compared to the monotherapies, and displayed no significant cytolytic effect of normal cells (HUVEC and MRC-5). In addition, we present a patient with right maxillary sinus malignant melanoma who was treated with the combination of H101 and adoptive NK cells. We observed intratumoral injections of H101 without dose-limiting toxicity. H101 injections stimulated self-immunity response with transient whole body fever. Furthermore, NK injections caused tumor temperature rise although contralateral cheek temperature was normal and body temperature was normal. Data are consistent with the hypothesis that virus-infected cancer cells attracted NK accumulation within tumor. The homing NK cells attacked targeted cancer cells, causing local temperature rise. Two courses of combination therapy in conjunction mediated the significant durable regression of melanoma, which had been ongoing for >22 months, and it represents a new immunotherapy approach for the treatment of these patients.

#4073

Novel BETi NHWD-870 synergizes with immune checkpoint inhibitor in preclinical model of non-small cell lung cancer.

Yongchang Zhang, Ziyi Sun, Nong Yang. _Hunan Cancer Hospital, Changsha, China_.

Background: Small molecular inhibitors targeting bromodomain and extraterminal domain (BET) protein is promising in cancer therapy. NHWD-870 was a highly selective BETi and has been proved effective in lung cancer according to our previous findings. Combination of immune checkpoint inhibitors with BETi is now under investigation for lung cancer treatment.

Method: We set up a fast track preclinical model with C57 mouse. Growth rate and survival data were measured in subgroups receiving single NHWD-870, single PD-1 antibody and combined treatment respectively. Finally, pathology sections, immunohistochemistry and inflammatory cell infiltration was applied to evaluate the pathological response.

Result: Millions of mouse lung cancer cells were harvested and injected to the lateral leg. Lung cancer xenografts were visible in 3-5 days. And the total survival time was within 30 days without any treatment. Tumor volume was measured for effective evaluation. During the three groups, NHWD-870 combined with anti-PD-L1 showed robust efficacy in inhibiting tumor growth. And the survival time was also prolonged significantly. Pathological examination showed the lung cancer cells were filled with necrosis. Inflammatory cell infiltration and immunohistochemistry showed that PD-L1 was down regulated and CD4+/ CD8+ positive lymphocyte cells increased significantly.

Conclusion: We established a useful method to evaluate tumor response to immune checkpoint inhibitor. And our data proved BETi plus immune checkpoint inhibitor to be a promising treatment of NSCLC.

#4074

Heat shock protein 90 inhibitors alter pancreatic stellate cell cytokine production and enhances the efficacy of immune checkpoint blockade in pancreatic cancer.

Mohammad Zaidi, Yuchen Zhang, Michael B. Ware, Matthew R. Farren, Hannah Komar, Brian Olson, Ganji P. Nagaraju, Mehmet Akce, Olatunji Alese, Shishir Maithel, Juan Sarmiento, Walid Shaib, Christina Wu, Bassel El-Rayes, Gregory B. Lesinski. _Emory University, Atlanta, GA_.

Pancreatic ductal adenocarcinoma (PDAC) has a prominent fibrotic stroma, which is a result of interactions between tumor, immune and pancreatic stellate cells (PSC). Prior work from our laboratory has defined a role for stroma-derived cytokines such as IL-6 as a significant barrier restraining immunity against PDAC. Our group is pursuing novel approaches to target pathways in the tumor microenvironment (TME), in an effort to improve access of effector immune cells to PDAC and response to immunotherapy. Heat shock protein-90 (Hsp90), is a chaperone protein and a versatile target in pancreatic cancer. Hsp90 regulates a diverse array of cellular processes of relevance to both the tumor and the immune system. However, to date the role of Hsp90 in PSC has not been explored in detail. We hypothesize that targeting Hsp90 can modulate the TME, through its ability to target inflammatory signaling and cytokine production by PSC and enhance the efficacy of immunotherapy. Treatment of immortalized and primary patient PSC with the Hsp90 inhibitor XL888 led to decreased IL-6 at the transcript and protein level in vitro. XL888 directly limited PSC growth, and reduced expression of alpha-SMA, Jak/STAT and MAPK signaling intermediates as determined via immunoblot. Combined therapy with XL888 and anti-PD-1 was efficacious in C57BL/6 mice bearing syngeneic subcutaneous (Panc02) or orthotopic (KPC-Luc) tumors, as compared to treatment with either agent alone. The treatment was well-tolerated, with no difference in body weight observed in either model. Laboratory studies are assessing immune and stromal biomarkers to define the impact on PSC, cytokines and T-cell biomarkers in the TME. Finally, we have completed the dose escalation phase of a Phase Ib/II clinical trial of XL888 (Hsp90i) and pembrolizumab (anti-PD-1) at our institution. Expansion cohorts of patients with metastatic pancreatic cancer (n=16) or colorectal cancer (n=16) are now accruing, with paired biopsy and peripheral blood samples to address the impact of Hsp90 inhibition on anti-PD-1 mediated T cell proliferation, cytokine production, and PSC-derived cytokine signatures.

#4075

Overcoming glioma immunoediting and MHC class I loss during adoptive cellular therapy.

Tyler J. Wildes, Catherine T. Flores, Kyle Dyson, Connor Francis, Adam Grippin, Bayli Divita Dean, Duane A. Mitchell. _University of Florida, Gainesville, FL_.

Introduction: Adoptive T cell immunotherapy (ACT) leads to 30% long-term cures of intractable models of malignant glioma. While promising, glioma-bearing hosts succumb to disease for unclear reasons. Therefore, we studied glioma escape variants after immunoediting by ACT. Since the escaped tumors displayed marked gene loss making them distinct from original tumor, we anticipated that successful future strategies would need to be tailored to the new antigenic signature while using combinatorial approaches to overcome other mechanisms of escape. We therefore studied the hypothesis that a polyclonal population of ex vivo-expanded T cells specific for the total tumor RNA of glioma escape variants could successfully retarget glioma escape variants, despite immunoediting pressure.

Methods: RNAseq revealed that tumor escape variants from KR158B (TOGA1.1/1.2) displayed a loss of ~80% of the genes that were present in KR158B and GL261 glioma. We regenerated polyclonal T cells against TOGA using total tumor RNA from TOGA tumors. We used restimulation co-cultures of T cells with tumor and IFN-γ ELISAs for T cell activation. In vivo therapeutic models required re-implantation of TOGA tumors intracranially after gross dissection and passaging in vitro. Flow cytometry determined marker expression in tumors.

Results: TOGA1.1 or TOGA1.2-specific T cells recognized cognate TOGA1.1 or TOGA1.2 cells but not KR158B or the opposite glioma escape variant. When we utilized TOGA-specific T cells in vivo for TOGA-bearing mice, median survival was prolonged from 24 to 33 days (p=.0003). However, all animals succumbed to disease. We then evaluated additional mechanisms of escape. At endpoint, MHC class I on tumors was ~50% of its original expression after antigen-matched ACT (KR158B-bearing: 70% to 40% post-ACT; TOGA-bearing: 22% to 10%). Additionally, KR158B and TOGA-specific T cells express ~50% PD-1 both post-expansion ex vivo and in the tumor-infiltrating lymphocytes post-treatment while tumor-infiltrating NK cells express 30% PD-1. Given the role of NK cells in targeting MHC Ilo tumors, and the recent discoveries that PD-1 checkpoint blockade works on T cells and NK cells, we investigated the impact of PD-1 and antigen-matched ACT in KR158B or TOGA-bearing animals. This revealed that the combination generates 71% long-term cures in KR158B primary glioma while promoting an increase in activated NK and T cells. Treatment of TOGA-bearing animals is underway and will include the depletion of either CD8 T cells or NK cells to determine the relative contributions of each cell type.

Conclusions: Glioma escape variants retain expression of immunogenic antigens. Generation of ACT with specificity for escape variants provides exquisite antigen-specific recognition of the immunoedited tumors. Combinatorial use of antigen-specific ACT with PD-1 blockade provides a flexible and specific platform for treatment of primary and escaped tumors.

#4076

**Finding the optimal strategy of incorporating immune checkpoint inhibitor in** ALK **-positive non-small-cell lung cancer using** EML4-ALK **transgenic mouse model.**

Kyoung-Ho Pyo,1 Sun Min Lim,2 Ha Ni Jo,3 Jae Seok Cho,3 Jae Hwan Kim,1 Ji Min Lee,1 Chun-Feng Xin,1 Sung Eun Kim,1 Chae Won Park,1 Wongeun Lee,3 Hye Ryun Kim,4 Byoung Chul Cho4. 1 _Yonsei University College of Medicine, Seoul, Republic of Korea;_ 2 _CHA Bundang Medical Center, Seongnam-si, Republic of Korea;_ 3 _JEUK Co, Gumi-City, Republic of Korea;_ 4 _Yonsei Cancer Center, Yonsei University College of Medicine, Seoul, Republic of Korea_.

Introduction: EML4-ALK is a distinct molecular entity that is highly sensitive to ALK tyrosine kinase inhibitors (TKIs). Although the role of oncogenic and native ALK in modulating the immune responses has been suggested, immune checkpoint inhibitors (ICIs) have not proved efficacy in ALK-positive non-small cell lung cancer (NSCLC) so far. In this study, we evaluated comprehensive immunomodulatory effect of ALK TKI and aimed to suggest optimal method of incorporating ICIs in ALK-positive NSCLC using an EML4-ALK transgenic mice model.

Methods: Ceritinib or anti-PD-1 was treated in EML4-ALK transgenic mice, and tumor response was evaluated using magnetic resonance imaging. Progression-free survival (PFS) and overall-survival (OS) were measured to compare the efficacy in mice. To examine the dynamics of immune response, flow cytometry of tumor region, cytokine-ELISA of bronchoalveolar lavage (BAL) fluid were performed at baseline and at the time of disease progression. Sequencing of ALK kinase domain was performed to identify acquired ALK mutations.

Results: Upfront ceritinib was clearly more efficacious than anti-PD-1 upfront, as shown by the median PFS was 139 days vs.13 days (P < 0.05). Use of ceritinib in the first line resulted in a prolonged OS compared to the use in the second line (OS 203 days vs. 135 days, P< 0.05). The efficacy of ceritinib and anti-PD-1 combination was not more effective than ceritinib alone in the first line. Of note, we identified 1 mouse which showed a complete response to anti-PD-1 after progression on 1st line ceritinib. Tumors which progressed on ceritinib showed increased proportion of CD8\+ tumor infiltrating T cells among total CD3\+ T cells, and increased expression of IFN-γ in BAL fluid, representing Th1-dominant immune phenotype. In tumors which acquired resistance to ceritinib, we identified known ALK kinase domain mutations such as G1202R with a concomitant increased tumor mutational burden compared to ceritinib-naïve tumors. Ceritinib-resistant tumors also had significantly increased memory cytotoxic T cells and PD-1-expressing helper T cells in total CD3\+ population. On the contrary, we noted decreased expressions of central memory T cells, effector memory T cells, IFN-γ in tumors which progressed after ceritinib and anti-PD-1 combination therapy.

Conclusion: Our study confirmed that combination of ALK inhibitor and ICI is not efficacious, as evidenced by low infiltrating T cells and memory T cells. We concluded that tumor progressing on ceritinib could be sensitive to anti-PD-1 due to increased neoantigens derived from acquired ALK mutations, increased tumor mutational burden and increased number of memory T cells and PD-1-expressing helper T cells.

#4077

ATOR-1144 is a tumor-directed CTLA-4 x GITR bispecific antibody that acts by depleting Tregs and activating effector T cells and NK cells.

Sara Fritzell, Mattias Levin, Ida Åberg, Maria Johansson, Magnus Winnerstam, Karin Enell Smith, Peter Ellmark, Christina Furebring, Per Norlén, Anne Kvarnhammar. _Alligator Bioscience, Lund, Sweden_.

ATOR-1144 is a human CTLA-4 x GITR bispecific IgG1 antibody generated by fusing a high affinity CTLA-4 binder, derived by FIND® optimization of the CTLA-4 binding domain of CD86, to an agonistic GITR antibody isolated from the human antibody library ALLIGATOR-GOLD®. CTLA-4 and GITR are highly expressed on Tregs in the tumor microenvironment, and GITR is also expressed on NK cells and certain tumor cells. ATOR-1144 was designed to induce a tumor-directed immune activation for treatment of solid tumors and hematological malignancies. The in vitro activity of ATOR-1144 in terms of Treg depletion and activation of T cells and NK cells was investigated using purified cells from healthy human donors. ATOR-1144 induced depletion of primary Tregs in an ADCC assay with NK cells. T-cell activation in terms of IL-2 and IFN-γ release was demonstrated in the presence of CTLA-4 and Fcγ receptor crosslinking. NK cells treated with ATOR-1144 released IFN-γ, perforin and granzyme B, and displayed enhanced cytolytic killing of tumor cells. Moreover, NK cell-mediated depletion of CTLA-4-expressing cells was significantly improved upon stimulation with ATOR-1144 compared to monospecific anti-CTLA-4, indicating that NK cells activated via GITR acquire an enhanced capacity to induce ADCC. In conclusion, ATOR-1144 is a first-in-class bispecific tumor directed antibody targeting CTLA-4 and GITR. ATOR-1144 acts through several mechanisms, including depletion of Tregs, activation of effector T cells and activation of NK cells for enhanced Treg depletion and tumor cell killing.

#4078

Enhanced anti-tumor activity through a combination of intravenous injectable TLR7 agonist, DSP-0509 and immune checkpoint inhibitors.

Yosuke Ota, Takeshi Otsubo, Masashi Goto, Yasushi Matsuki. _Sumitomo Dainippon Pharma Co., Ltd, Osaka, Japan_.

Toll-like receptors (TLRs) are a family of pattern-recognition receptors (PRR) that recognize pathogen-associated molecular patterns (PAMPs). TLR7 is mainly expressed in plasmacytoid dendritic cells (pDCs) and recognizes virus derived single-stranded RNA (ssRNA). TLR7 stimulation in pDCs induces type I interferon secretion, which results in innate immune activation. In this preclinical study, the characteristic changes in the tumor microenvironment (TME) after the combination treatment of DSP-0509 with immune checkpoint inhibitors (ICIs) in DSP-0509-responsive and -resistant tumors were assessed. Mutation rates of microsatellite loci were measured by 3130 xl Genetic Analyzer. Anti-tumor activity was evaluated in syngeneic mouse models along with flow cytometric analysis of tumor infiltrating lymphocytes (TILs). Gene expression profiles in tumor tissues were measured by qPCR arrays. Mutation rates of 5 microsatellite loci and basal levels of CD8+T cell infiltration were examined in tumor tissues derived from 10 syngeneic tumor-bearing mouse models. Intravenous administration of DSP-0509 significantly suppressed tumor growth in some tumor-bearing mouse models with high mutation rates and CD8+T cell infiltration including the CT26 (P<0.05), but not in any models with low mutation rates and CD8+T cell infiltration including 4T1. Gene expression analysis revealed that the CT26 tumors highly expressed T cell inflamed genes including Ifng, Fasl and Il15 at the baseline, whereas the 4T1 tumors highly expressed immune suppressive genes including Il10 and Csf3. In contrast, the combination with anti-PD-1 antibody suppressed the tumor growth not only in the CT26 (P<0.0001) but also in the 4T1-bearing model with immune suppressive TME (P<0.05) and increased the expression of IFN-gamma signature genes including Ifng, Cxcl10 and Gzmb in both models. Similarly, the combination with anti-CTLA-4 antibody suppressed the tumor growth in both models and increased IFN-gamma signature genes. The frequency of PMN-MDSC (CD11b+Ly6g+) in 4T1 tumor was significantly decreased after the combination treatment of anti-PD-1 antibody (P<0.05). Anti-tumor activity of DSP-0509 was associated with tumor mutation rates and the presence of CD8+ T cells within the tumor. The treatment of DSP-0509 in combination with anti-PD-1 antibody showed anti-tumor activity with increased expression of IFN-gamma signature genes and decreased PMN-MDSC even in the 4T1-bearing mouse model with immune suppressive TME (i.e., "cold"). In addition, the combination treatment with anti-CTLA-4 antibody also showed the anti-tumor activity with the increased IFN-gamma signature gene expression. These results suggest that combination of ICI with DSP-0509 resulted in anti-tumor activity by changing immunogenic "cold" to "hot" TME.

#4079

EVT801: a selective VEGFR3 inhibitor with the potential for combination with immune-checkpoint therapies, preclinical evidences and plans for first-in-human evaluation.

Pierre Fons,1 Michael Esquerre,1 Michael Paillasse,1 Virgile Visentin,1 Frederique Dol,1 Jerome Meneyrol,1 Gaelle Badet,1 Eric Cogo,1 Lionel Vidaud,1 Antoine Alam,2 Isabelle Blanc,2 Francoise Bono,3 Joanna Lisztwan,1 Muriel Poublanc,4 Thomas Filleron,4 Ian Hunneyball,5 Jean-Pierre Delord,4 Mark Whittaker5. 1 _Evotec, Toulouse, France;_ 2 _Evotec, Lyon, France;_ 3 _Onxeo, Paris, France;_ 4 _Institut Claudius Regaud, IUCT-Oncopole, Toulouse, France;_ 5 _Evotec, Abingdon, United Kingdom_.

We have identified a highly selective VEGFR3 inhibitor drug candidate, which strongly inhibits angiogenesis without inducing hypoxia, reputed as one of the main causes of cancer-associated immunosuppression. Combination of EVT801 and Immune Checkpoint Therapy (ICT) could be synergistic as hypoxia-induced-immunosuppression should be avoided. We have demonstrated that EVT801 is a narrow spectrum inhibitor of the VEGFR3 tyrosine kinase with residual activity against VEGFR2 and TAK1 and with an activity on VEGFRs different from Fruquitinib and Lenvatinib. We then showed that EVT801 inhibited human endothelial cell proliferation in vitro and tumor angiogenesis on ex-vivo mouse ring aortic assay. EVT801 was active in vivo in the RIP1-Tag2 mouse model during the angiogenic switch. In comparison to sorafenib, EVT801 in 2 different tumors mouse model decreased tumor development without inducing increase of hypoxia which is in favor to a sustained blood vessel tumor normalization. In orthotopic 4T1 mammary carcinoma, we demonstrated that EVT801 in combination with PD1 mAb was significantly superior than single agent treatment and a decrease of lung metastasis spreading was observed. We observed that decrease of MDSCs in blood in response to EVT801 was correlated with decrease in tumor weight. By IHC analysis, we showed that treatment with EVT801 increases CD8+ T-cells infiltration inside the tumor. Taken together, these results indicated that EVT801 represents an innovative anti-angiogenic drug for cancer immunotherapy which may improve the frequency of response to ICT. Clinical trial design including patient stratification and biomarkers of activity has been proposed in order to be ready for first-in-human evaluation by mid-2019.

#4080

Expansion of CX3CR1+ cytotoxic CD8+ T cells provides a rationale for IL-15 in combination cancer therapy with PD-1 antibody.

Henan Zhang,1 Xin Liu,1 Siyu Cao,1 Lingling Chen,1 Susan Harrington,1 Ying Li,1 Aaron Mansfield,1 Sean Park,1 Yiyi Yan,1 Roxana Dronca,2 Haidong Dong1. 1 _Mayo Clinic, Rochester, MN;_ 2 _Mayo Clinic, Jacksonville, FL_.

Introduction:

IL-15 has been used in clinical trials with PD-1 antibody to overcome cancer resistance to PD-1 monotherapy in cancer patients. However, it is not clear what T cell population would be expanded to mediate tumor control in IL-15 in combination with PD-1 antibody. We recently found CX3CR1+ cytotoxic CD8+ T cells, which increased in responders compared with non-responders after anti-PD-1 in melanoma patients, demonstrated an increased transcription of CD122 (IL2RB). Since CD122 is a subunit of IL-15 receptor, we hypothesized that IL-15 in combination with anti-PD-1 therapy may have the ability to expand CX3CR1+ cytotoxic CD8+ T cells for tumor control.

Methods and findings:

To examine whether IL-15 plus anti-PD-1 expand CX3CR1+ cytotoxic CD8+ T cells in tumor tissues, we treated tumor-bearing mice with IL-15/IL-15Rα complexes alone or with PD-1 antibody and measured the frequency of CX3CR1+Granzyme B+ CD8+ T cells. As only the combination therapy induced dramatic tumor regression, we found the highest increase of CX3CR1+Granzyme B+ CD8 T cells in tumor tissues in the group treated with IL-15 plus anti-PD-1 (28.87±3.19% vs. 7.91±1.32%, p<0.01) compared with groups treated with either IL-15 alone or anti-PD-1 alone. In contrast to wild type mice, IL-15 plus anti-PD-1 did not suppress tumor growth in CX3CR1-KO mice. To determine whether IL-15 alone can also expand human CX3CR1+ cytotoxic CD8+ T cells, we cultured peripheral blood mononuclear cells (PBMCs) isolated from healthy donors (n = 5) with IL-15 for 24 hours in vitro. We found IL-15 (5 ng/mL) only modestly increased the frequency of CX3CR1+ Granzyme B+ CD8+ T cells than those cultured with no IL-15 (39.9±6.44% vs. 27.56±4.98%). To test whether this increase is due to enhanced proliferation of this subset of CD8+ T cells, we measured the expression of Ki-67, a cell proliferative marker, in CX3CR1+ CD8+ T cells by intranuclear staining. We found IL-15 modestly induced more proliferating CX3CR1+ CD8+ T cells in vitro culture compared with control group without IL-15 (6.25±0.54% vs. 0.91±0.25%).

Summary:

Taken together, we found IL-15 combined with anti-PD-1 therapy has the ability to significantly expand CX3CR1+ cytotoxic CD8+ T cells, however IL-15 alone may have limited ability to significantly expand this effector cell population. Our studies provide a new rational for combination therapy of IL-15 and PD-1 antibody to overcome cancer resistance for patients who did not respond to initial PD-1 therapy.

#4081

Efficacy of MERTK inhibitor in combination with pembrolizumab in lung cancer.

Kyoung-Ho Pyo,1 Ha-Ni Cho,1 Chun-Feng Xin,1 Jae Seok Cho,1 Han Na Kang,2 Jiyeon Yun,1 Hye Ryun Kim,3 Byoung Chul Cho3. 1 _Severance Biomedical Science Institute, Seoul, Republic of Korea;_ 2 _JEUK Institute for Cancer Research, Republic of Korea;_ 3 _Yonsei Cancer Center, Yonsei University College of Medicine, Seoul, Republic of Korea_.

Introduction: MERTK is thought to play a role in cancer immune evasion mechanism based on M2 type macrophage-driven immunosuppression in tumor microenvironment (TME). Thus, MERTK is a highly promising target for anti-cancer therapy under high tumor associated macrophages (TAMs) condition. We evaluated the anti-tumor effects and immune reaction of combination or single MERTK inhibitor in two lung squamous patients derived xenograft (PDX) tumor engrafted Hu-CD34-NSG models.

Methods: The aPD-1 sensitive (YHIM2004) and aPD-1 non-sensitive (YHIM2009) squamous cell carcinoma PDX models were selected from pre-test with pembrolizumab. The selected two models were composed four groups; control, pembrolizumab, MERTK inhibitor, and combination. We applied pembrolizumab as aPD-1 blockade (10 mpk, q2w, i.p. inj.). The dose of MERTK inhibitor was 50 mpk, q1d by orally. The treatment was started when tumor size reached 100 mm3. Whole exome sequencing (WES), microarray (mRNA), flow cytometry, and multispectral image analysis were performed on the tumors. To elucidate the involvement of MERTK inhibitor in tumor, the tumor mutation burden, tumor microenvironment and immune cell phenotype were deconvoluted.

Results: The TMB of YHIM2004 and YHIM2009 was 3.23 and 2.64 (mutations/Mb) respectively. The PD-L1 and TIL level of YHIM2004 were relatively higher than YHIM2009. Both two models were observed high macrophage in tumor. The two PDX-models, YHIM2004 (aPD-1 sensitive), YHIM2009 (aPD-1 non-sensitive) were observed anti-tumor effects in MERTK inhibitor single treatment groups (TGI = 44.23%, 67.40% respectively, p < 0.05, t-test). Especially, YHIM2004 tumor was reduced after combination therapy more than single treatment. Of note, the NOG-PDX model demonstrated no anti-tumor effect in MERTK inhibitor monotherapy, suggesting that this drug involves anti-cancer immune responses. Flow cytometry data showed increased PD-1+ and granzyme B+ T cells (p <0.05, t-test) in the combined group (YHIM2004) and MERTK inhibitor monotherapy group (YHIM2009) as much as the tumor suppressive effect.

Conclusion: We concluded that MERTK signaling involves regulation of anti-cancer immune responses. Our results suggest an anti-cancer therapeutics of MERTK selective inhibitor on aPD-1 sensitive or non-sensitive tumors under high TAM tumor microenvironment.

#4082

Preclinical assessment of external or targeted radiotherapy in combination with immunotherapies and co-development of companion imaging diagnostic.

Olivier Raguin,1 Céline Mirjolet,2 Claire Bernhard,3 Peggy Provent,1 Bertrand Collin,2 Franck Denat,3 Fabrice Viviani,1 Alexandre Cochet,2 Cyril Berthet1. 1 _Oncodesign S.A., Dijon Cedex, France;_ 2 _Centre Georges François Leclerc, Dijon, France;_ 3 _Université de Bourgogne Franche-Comté, Dijon, France_.

Immunotherapies have proven to be highly efficient for cancer treatments and combination with either internal vectorized or external radiotherapies can enhance this efficacy through notably increasing tumor immune infiltrate. The clinical evaluation of such combination therapies requires expertise and exquisite processes in multiple fields, immunotherapy, radiotherapy and nuclear medicine. Similarly, this applies into preclinical settings for assessing proof of concept of novel combinations. Herein, we will present our preclinical evaluation process to combine external or internal (i.e. lutetium-177 radiolabeled molecule) radiotherapy with immunotherapies that include radiochemistry, target expression in tissue by autoradiography, in vitro binding evaluation, in vivo tolerance and activity based on single or fractionated treatment doses. The targeted radiotherapy (TRT) is a systemic target-based approach combining a high-affinity receptor-binding ligand radiolabeled with a high-energy beta-emitting radionuclide whereas external 3D image guided radiotherapy targets the shape of the tumors very precisely. Combination studies with immunotherapies in preclinical setting require the selection of appropriate and relevant syngenic or humanized mouse models, driven by target expression, radioresistance (i.e hypoxia), and tumor immune infiltrate (lymphocyte CD8, macrophage etc.). In addition, the therapeutic evaluation should take into consideration the tolerance related to ionizing radiations, the scheduling of treatment (cumulated dose, fractionation), antitumor immune response and monitoring of immune checkpoint target expression. As nuclear imaging is a powerful tool to monitor and predict in vivo target expression and treatment efficacy, TRT is systematically developed in parallel of a PET or SPECT companion diagnostic to visualize and quantify the target expression prior to the treatment with a therapeutic radiolabeled drug. It necessitates the development of radiolabeling processes for either therapeutic or diagnostic purposes and is then performed concomitantly using appropriate bioconjugation strategies suited for theranostic pairs of radionuclides .We will present our recent results that highlight the importance to optimize the external radiotherapy schedule to improve the efficacy and synergistic effect of the association radiotherapy/immunotherapy such as anti-PDL1. Similarly, it is of high interest to combine 177Lu-labeled molecule with immune checkpoint inhibitors in various solid tumors. Transversal skills are mandatory to perform such models and experiments, all being conducted in a dedicated facility for external 3D image guided radiotherapy (SARRP), radiochemistry (hot cells, 177Lu, 68Ga, 64Cu, 89Zr, 111In) and pharmaco-imaging (PET, SPECT, MRI, CT, and optical).

#4083

Immunogel - a novel intratumoral sustained release immunotherapy platform for eradication of large established tumors.

Trine B. Engel,1 Lars Ringgaard,1 Jennifer S. Jørgensen,1 Fredrik Melander,1 Sophie B. Jensen,1 Julianna Thuróczy,2 Lajos Balogh,3 Frederikke P. Fliedner,4 Martin Bak,1 Linda M. Bruun,1 Jonas R. Henriksen,1 Andreas Kjaer,4 Anders E. Hansen,1 Thomas L. Andresen1. 1 _Technical University of Denmark, Kgs. Lyngby, Denmark;_ 2 _Animal Health Center Budafok, Budapest, Hungary;_ 3 _National Public Health Center, Budapest, Hungary;_ 4 _Rigshospitalet & University of Copenhagen, Copenhagen, Denmark_.

The introduction of immunotherapy is revolutionizing the field of anti-cancer therapy, however, many immunotherapeutic agents are associated with an increased risk of systemic dose-limiting side effects. To circumvent these, we have developed a flexible drug delivery platform that provides local sustained release of immunotherapeutic agents at the target site (Immunogel). The constituents of the drug delivery platform have been validated in experimental porcine models, spontaneous canine cancers and human cancer patients for radiotherapy guidance. The Immunogel is injected intratumorally with high precision through small gauge needles and has the potential to increase the drug concentration at the target site, while reducing systemic toxicity. In the present work, an in vitro and in vivo drug release kinetic characterization of the Immunogel as well as a preclinical evaluation in large established syngeneic tumor models is included. The therapeutic efficacy focused on combining Immunogel formulated with the TLR7/8 agonist R848 and immunogenic cell death inducing chemotherapy and low-dose radiotherapy. Intriguingly, the combined therapy resulted in cancer eradication and rejection in 60% of the treated mice compared to 0% in control treatment groups. To investigate the underlying mechanisms, the tumor microenvironment (TME) was evaluated by flow cytometry. Immunogel in combination with chemotherapy improved the cytotoxic T cell (cT) to regulatory T cell ratio more than nine-fold compared to the untreated group. Additionally, a high increase of strongly activated CD137+ cTs as well as increased infiltration of central memory cTs was identified in the TME. In summary, the combined treatment polarized the TME in a pro-immunogenic direction and induced a potent tumor-specific cytotoxic T cell mediated immune response. It is well known, that multimodal approaches improve the successful outcome of anti-cancer immunotherapy. In line with this, TLR7/8 Immunogel therapy was combined with inhibition of either PD-1 or TGFβ. These combinations were able to completely eradicate 100% of all large established tumors in combination with low-dose radiotherapy. Additionally, Immunogel with sustained release of TLR7/8 agonist and anti-TGFβ in combination with low-dose radiotherapy was able to delay the formation of lung metastasis in an aggressive metastasizing breast cancer tumor model. Recently, the TLR7/8 Immunogel has undergone preliminary testing in healthy beagle dogs and the results displayed a high tolerability yet clearly evident immune stimulation during a three-week evaluation period. The Immunogel represents a novel drug delivery platform with promising results.

#4084

Radiation enhances efficacy of TEM1-specific cancer vaccination.

Stefano Pierini, Mireia Uribe-Herranz, Renzo Perales-Linares, Silvia Beghi, Francesca Costabile, Sergei Pustylnikov, Andrea Facciabene. _University of Pennsylvania, Philadelphia, PA_.

Tumor endothelial marker 1 (TEM1 or CD248) is a protein found in both tumor vasculature and stroma. In prior experiments, we showed that immunizing with Tem1-TT plasmid-DNA (Tem1 cDNA fused to the minimal domain of the C fragment of tetanus toxoid) was effective in controlling tumor progression in three mouse tumor models in a T cell-dependent manner. Moreover, effective Tem1-TT vaccination reduced tumor vascularity without impacting normal angiogenesis. Radiation therapy (RT) is an established curative and palliative cancer treatment regimen which uses high-energy radiation to kill cancer cells. RT modulates the tumor microenvironment - by increasing MHC-I expression, inducing immunological cell death, and damaging the tumor-associated vasculature - potentially synergizing with anti-cancer immunotherapies. Here, we combined RT and Tem1-TT heterologous vaccination and showed that combination of the two therapies was more effective in controlling CT26 and TC1 tumor progression than either single therapy alone. Interestingly adding RT to Tem1-TT vaccine resulted in a stronger vaccine-specific immune response and increased epitope spreading toward gp70, a TAA expressed by CT26 tumor cell line. Further analysis of the tumor microenvironment revealed a significant increment of PD-L1 expression in both tumor cells and tumor vasculature after single or either combination therapy. This led us to test whether blocking the PD1/PD-L1 axe would further improve the efficacy of RT plus Tem1-TT therapy. Our results demonstrate that administration of anti-PD-L1 antibody further synergized with RT plus Tem1-TT and improved mice survival. In conclusion, triple combination of Tem1-TT vaccine, RT and anti-PD-L1 can offer a therapeutic advantage for TEM1-positive tumors which also express PD-L1.

#4085

DDR2 inhibition enhances response to anti-PD-1 immunotherapy.

Megan M. Tu,1 Francis Y. Lee,2 Robert T. Jones,1 Abigail K. Kimball,1 Elizabeth Saravia,2 Robert F. Graziano,2 Brianne Coleman,1 Krista Menard,2 Jun Yan,2 Erin Michaud,2 Han Chang,2 Hany A. Abdel-Hafiz,1 Andrii I. Rozhok,1 Jason E. Duex,1 Neeraj Agarwal,1 Ana Chauca-Diaz,1 Linda K. Johnson,1 Terry L. Ng,1 John C. Cambier,1 Eric T. Clambey,1 James C. Costello,1 Alan J. Korman,3 Dan Theodorescu4. 1 _University of Colorado Anschutz Medical Campus, CO;_ 2 _Bristol-Myers Squibb, NJ;_ 3 _Bristol-Myers Squibb, CA;_ 4 _Samuel Oschin Comprehensive Cancer Institute, CA_.

Therapies targeting PD-1 are used in multiple cancer types. While a fraction of patients show durable therapeutic responses, most remain unresponsive, highlighting an urgent need to better understand and improve these therapies. Using an in vivo screening approach with a customized shRNA pooled library, we identified DDR2 as a leading target for the enhancement of response to anti-PD-1 immunotherapy. Using isogenic in vivo murine models across five different tumor histologies, bladder, breast, colon, sarcoma and melanoma, we show that DDR2 depletion increases sensitivity to anti-PD-1 treatment compared to monotherapy. Combination treatment of tumor-bearing mice with anti-PD-1 and dasatinib, a tyrosine kinase inhibitor of DDR2, also led to tumor load reduction and in some cases, complete clearance. RNAseq and CyTOF analysis revealed higher CD8+ T cell populations in tumors with DDR2 depletion and those treated with dasatinib when either was combined with anti-PD-1 treatment. Our work provides strong scientific rationale for targeting DDR2 in combination with PD-1 inhibitors.

#4086

Preclinical evaluation of pharmacokinetics, pharmacodynamics and efficacy of the dual CD73-A2AR Inhibitors.

Dinesh Chikkanna, Kishore Narayanan, Sunil Panigrahi, Garima Priyadarshini, Megha Goyal, Jiju Mani, DS Samiulla, Sagar dadhania, AB Aravind, Girish Daginakatte, Thomas Anthony, Kavitha Nellore, Susanta Samajdar, Murali Ramachandra. _Aurigene Discovery Technologies Limited, Bangalore, India_.

As a potent immunosuppressor adenosine is essential for maintaining tissue homeostasis and preventing an overzealous immune response during inflammation and infection. However, adenosine generated within the tumor microenvironment by the action of ecto-nucleotidases including CD73 hinders the immune reaction towards cancer cells by signaling through adenosine receptors such as high affinity A2AR expressed on immune cells. Inhibitions of either adenosine generation or signaling by inhibiting CD73 or A2AR have been shown to be effective therapeutic approaches. Interestingly, the co-blockade of CD73 and A2AR results in a more pronounced anti-tumor activity than blockade of either, likely due to increased CD73 expression upon A2AR inhibition and compensatory activity of other adenosine receptors such as A2BR. In view of this, we sought to discover and develop small molecule inhibitors that dually target CD73 and A2AR with oral bioavailability for ease of administration and use in combination with other anti-cancer therapies. Aurigene's dual inhibitors exhibit potent inhibition of both A2AR and CD73 in respective biochemical and cellular assays. High potency translated into rescue of NECA or AMP induced repression of IFNΥ and IL2 in human PBMCs. Lead compounds exhibited desirable drug-like properties including solubility, permeability, lack of CYP inhibition, and pharmacokinetic exposure. In Syngeneic tumour model, treatment with lead compounds resulted in significant tumour growth inhibition while correlating well with tumour drug levels and modulation of pharmacodynamic markers. In summary, we have identified first-in-class dual inhibitors with good drug like properties, which showed significant antitumor efficacy. Evaluation of these lead compounds in additional tumour models and in vivo toxicity studies is currently under way.

#4087

Efficacy of three checkpoint Inhibitors, five chemotherapeutic agents, and the experimental drug thiarabine against MC38 colon cancer expressing human PD-L1 in transgenic C57BL/6 mice expressing human PD-1 and PD-L1 checkpoint genes.

Murray A. Stackhouse,1 Ted Green,1 Jay Liu,1 Charlotte Hammond,1 LaJuana Durbin,1 Ana Chen,2 Jerry Zhou,2 Mike Koratich1. 1 _Southern Research, Birmingham, AL;_ 2 _Nanjing Galaxy Biopharma, Nanjing, China_.

Human checkpoint genes PD-1 and PD-L1 are the target of extensive research for treating human cancers. Checkpoint inhibitor therapy shows great promise in the clinic. Combining checkpoint inhibitors with standard chemotherapeutic agents is an important for these inhibitors. Animal models that use human clinical antibodies and chemotherapeutic agents will be beneficial for evaluating new therapies. A novel transgenic mouse model is described that expresses human PD-1 and PD-L1 in place of the murine genes. Additionally, a murine MC38 colon tumor cell line was modified to express human PD-L1. We initially evaluated early treatment efficacy of human check point antibodies nivolumab, pembrolizumab, and atezolizumab in the transgenic animal model with modified MC38 colon cancer cells. Five hundred thousand transgenic MC38 cells were implanted in the C57BL/6 PD-1/PD-L1 knock in mice and allowed to grow for three days. Tumor-implanted mice were treated with nivolumab and pembrolizumab at 100 µg per animal and atezolizumab at 1 mg per animal on Days 3, 7, 10, and 14. Treatment with nivolumab and pembrolizumab caused tumor regression by Day 17. Growth inhibition was 84%, 58%, and 94% on Day 17 for nivolumab, atezolizumab, and pembrolizumab, respectively, compared to the control animals. There was no significant body weight loss and no signs of toxicity in any of the treated animals. To extend the utility of the model we implanted mice and allowed the tumors to grow to 63 to 126 mg before treatment initiation (Day 10). Similar results were observed in established tumors as for early treatment with the PD-1 inhibitors nivolumab and pembrolizuman. In contrast there was little activity from the PD-L1 inhibitor atezolizumab in established tumors. To prepare for combination studies, we also tested cisplatin, irinotecan, gemcitabine, decitabine, rucaparib, and thiarabine near the MTD for each agent with various treatment schedules against the established tumors. The doses for cisplatin, irinotecan and decitabine resulted in excess animal deaths and require reduced doses for combination therapies. Gemcitabine, decitabine, and thiarabine (in house development drug) were too active with complete regressions observed; the doses for combination treatment will be reduced. Due to the effectiveness of the PD-1 checkpoint inhibitors the number of doses will be reduced from four to two treatments in combination studies; no change for atezolizumab. We have shown that PD-1 inhibitors are effective in treating genetically modified MC38 colon tumors in transgenic mice in early treatment and in established tumors while the PD-L1 inhibitor was only effective in early treatment. We also have identified doses and schedules of five chemotherapeutic agents to use in future combination studies in this model.

#4088

Th1 cytokines and oncodriver inhibition: A combination treatment strategy in TNBC.

Amrita Basu, Yongsheng Jia, Krithika Kodumudi, Doris Wiener, Brian Czerniecki. _Moffitt Cancer Center, Tampa, FL_.

Triple Negative Breast Cancer (TNBC) is characterized by lack of hormone receptors and targeted therapies, resistance to existing treatment and poor survival outcome, highlighting the need for novel therapy development. Oncogene addiction in breast cancer (BC) cells to drive malignancy up presents oncodrivers as promising therapeutic candidates in BC. Our group has shown loss of anti-oncodriver CD4+-T-helper-type-1 (Th1) response in TNBC patients. Here, we studied effects of combination treatment with Th1 cytokines (IFN-γ+TNF-α) and oncodriver blockade in human and mouse TNBC cells (MDA-MB-231, MDA-MB-468, HCC1143, BT549, 4T1). We measured expression of 3 oncodriver proteins: EGFR, HER3 and cMET and observed multiple oncodrivers in majority of the cell lines, suggesting combined oncodriver inhibition may have stronger antitumor effects. Based on oncodriver status, we tested combination treatment with Th1 cytokines and a) monoclonal α-EGFR antibody cetuximab, b) cMET RTK inhibitor crizotinib and c) siRNA inhibition of three oncodrivers. No significant difference in proliferation after IFN-γ or IFN-γ+TNF-α treatment suggested IFN-γ is the primary effector cytokine. IFN-γ alone reduced proliferation >50% in majority of TNBC cells. In EGFRhigh/HER3pos/cMETlow MDA-MB-468, IFN-γ and cetuximab treatment significantly increased apoptosis compared to untreated cells. Combined EGFR and HER3 siRNA inhibition, followed by IFN-γ treatment, increased apoptosis in those cells. In EGFRpos/HER3low/cMEThigh MDA-MB-231 and EGFRlow/HER3high/cMEThigh 4T1, IFN-γ and crizotinib combination significantly decreased proliferation (p<0.0001) compared to single agent. In EGFRpos/HER3pos/cMETpos HCC1143, combined siRNA inhibition of 3 oncodrivers followed by IFN-γ treatment increased apoptotic signal compared to control. Protein expression showed increased STAT1Tyr701 phosphorylation as primary mechanism of IFN-γ mediated proliferation inhibition. Western blot after cetuximab/crizotinib and Th1 cytokines treatment suggests altered phosphorylation of signaling effectors, PI3K/AKT, p44/42MAPK and STATs 1, 3 and 5 may contribute to anti-proliferative, apoptotic effects of combination treatment. siRNA inhibition of one oncodriver resulted in a compensatory increase in expression of the other oncodriver (increased HER3 level in siEGFR transfected MDA-MB-468 cells and vice versa), suggesting a complex signaling crosstalk between oncodrivers in TNBC. We are currently investigating effects of combination treatment on cellular distribution of oncodrivers, % of ALDHpos stem-like cells, cytokines and chemokines secretion profile and protein expression in TNBC cells. Our study suggests combination treatment with Th1 cytokines and oncodriver inhibition in TNBC may contribute to improved therapeutic benefit and clinical outcome.

#4089

Antitumor activity of eribulin in combination with anti-PD1 antibody in a mouse syngeneic breast cancer model.

Taro Semba, Kimiyo Tabata, Yoichi Ozawa, Saori Watanabe Miyano, Junji Matsui, Yasuhiro Funahashi. _Eisai Co., Ltd., Tsukuba-shi, Japan_.

Eribulin is a synthetic analog of halichondrin B, which is isolated from the marine sponge natural product, as the first halichondrin class of microtubule dynamics inhibitors. Its clinical formulation is currently approved for advanced breast cancer (BC) and advanced liposarcoma in numerous countries. Eribulin shows antitumor activity through cell death induction by an antimitotic effect, and modulation of tumor microenvironment that include vascular remodeling, and reversal of EMT in some of preclinical subcutaneous xenograft models. Effects of eribulin on tumor microenvironment led us to test combination of eribulin with anti-PD1 blockades, and Phase2 clinical study of eribulin in combination with pembrolizumab for metastatic BC patients is in progress. In this study, we investigated antitumor activity of eribulin in combination with anti-PD1 mAb in preclinical mouse BC model using 4T1 cells. Because parent 4T1 cells express P-glycoprotein (Pgp) and eribulin is a substrate of Pgp, we established Pgp-knockout (Pgp-KO) mouse 4T1 BC cells to examine combination antitumor activity of eribulin with anti-PD1 antibody. We used a clone 31 of Pgp-KO 4T1 (#31) cells, which maintains similar tumor phenotypes to parent 4T1 orthotropic transplantation (OT) models, such as growth rate and formation of metastasis. Eribulin inhibited in vitro proliferation of Pgp-KO 4T1 (#31) cells with a lower concentration compared to parent 4T1 cells. Eribulin were intravenous administered at 1 mg/kg with either Q7Dx2 or Q4Dx3 in the combination with anti-PD1 mAb, 200 µg/head, Q3Dx10 in the Pgp-KO 4T1 (#31) OT model (6 mice per a treatment group). Anti-PD1 mAb did not clearly show in vivo antitumor activity. Treatments of eribulin by Q4Dx3 showed enhanced antitumor activity in the combination compared to each single treatment, and all tumors became non-palpable in the combination group. On the other hand, weekly treatments of eribulin did not enhance antitumor activity in the combination with anti-PD1 mAb, although eribulin single treatment significantly inhibited in vivo tumor growth of Pgp-KO 4T1 (#31) tumors. These preclinical results provide preclinical rationale for clinical testing of eribulin in combination with anti-PD1 blockades in patients with BC, and further preclinical analysis of MOA of combination antitumor activity of eribulin with anti-PD1 antibody is warranted.

#4090

Defeating primary checkpoint resistance: SRTβ1-Ab3 is a first-in-class, fully human antibody that renders resistant tumors sensitive to anti-PD-1.

Thomas Schürpf, Constance J. Martin, Christopher Littlefield, Christopher Chapron, Stefan Wawersik, Ashish Kalra, Allison Simpson, Francis Danehy, Christopher Boston, Anastasia Nikiforov, Susan Lin, Justin Jackson, Gregory J. Carven, Alan Buckler, Abhishek Datta. _Scholar Rock Inc., Cambridge, MA_.

Despite the clinical breakthroughs achieved by checkpoint blockade therapy (CBT), a majority of patients treated with PD-(L)1 inhibitors fail to respond due to primary or acquired resistance. TGFβ signaling has recently been implicated as a mechanism of primary resistance to CBT, very likely via mechanisms that include immune exclusion. However, therapeutic targeting of the TGFβ pathway has been hindered by dose-limiting cardiotoxicities, most likely due to inhibition of signaling from multiple TGFβ isoforms. Upon secretion, TGFβ growth factor is held dormant in a latent complex with its non-covalently associated prodomain. TGFβ activation is triggered by extracellular events, such as integrin binding or proteolytic cleavage, that release the growth factor from this latent complex. We have demonstrated that isoform-specific inhibition of TGFβ activation can be achieved by targeting the prodomain to stabilize the latent TGFβ complex. We recently identified TGFβ1 as the predominant isoform in many human cancers, especially those for which CBT is approved for therapeutic intervention. SRTβ1-Ab3 is a fully-human antibody against latent TGFβ1 that inhibits its activation without binding or inhibiting latent TGFβ2, latent TGFβ3, or the active TGFβ1 growth factor. In syngeneic tumor models of primary CBT resistance, pharmacologic blockade of TGFβ1 activation with SRTβ1-Ab3 is sufficient to sensitize TGFβ1-predominant tumors to PD-1 inhibition. Mechanistically, combination treatment with anti-PD-1/SRTβ1-Ab3 overcomes immune exclusion in these models, induces CD8+ T cell infiltration into the tumors, and results in a reduction of myeloid immunosuppressive cells. In contrast, monotherapy with either anti-PD-1 or SRTβ1-Ab3 alone has only modest effects on these cell populations. Gene expression profiling of single vs. combination treated tumor samples provides a molecular view of effects on signaling pathways, cell populations, and activation status of these cells. These data demonstrate the efficacy of TGFβ1-specific inhibition in combination with anti-PD-1 in multiple mouse models of primary checkpoint resistance. Taken together, these synergistic effects at the tumor, cellular, and molecular levels, the preclinical safety profile, and the pharmacokinetic properties of SRTβ1-Ab3 establish a strong rationale for advancing the development of SRTβ1-Ab3 toward clinical application in cancer immunotherapy.

#4091

Evaluating the efficacy of a STING agonist in a murine model of prostate cancer.

Jonathan Gurkan, Jae Eun Choi, Jean Tien, Marcin Cieślik, Luigi Franchi. _University of Michigan Rogel Cancer Center, Ann Arbor, MI_.

Despite the promise of checkpoint inhibitor therapy, prostate cancer has remained resistant to this treatment. Innate immune agonists, however, have been shown to have anti-tumor effects in various cancer types, such as melanoma and colon cancer, and have been nominated to be used in combination with approved anti-PD1/PD-L1 drugs. For instance, STING agonist + anti-PD-1 combination therapy has been demonstrated in a syngeneic mouse model of melanoma. Other innate immune agonists, such as TLR7/8 and TLR9 have also been shown to have potential anti-tumor effects. We hypothesized that innate immune agonists may be effective in the MycCaP syngeneic mouse model of prostate cancer. To compare the efficacy of three innate agonists targeting the STING (3'3' cGAMP), TLR7/8 (CL097), and TLR9 (ODN2395) pathways, FVB mice were first injected bilaterally with MycCaP subcutaneous tumors. Upon establishment of tumors, mice were administered 3 doses of a single-sided intratumoral injection of each drug. The injected tumors had responses of 50% (6/12), 23% (3/13), and 8% (1/13) for the STING, TLR7/8 and, TLR8 agonists, respectively. Contralateral tumors showed no significant regression.

Published data have reported inherent resistance of MycCaP tumors to anti-PD-1 treatment in vivo. Given our results, we hypothesize that the addition of a STING agonist could enhance efficacy of anti-PD-1 therapy in this model. Mice with MycCaP tumors were administered anti-PD1 alone (n=10) or in combination with bilateral injections of the STING agonist (n=14). Combined therapy resulted in significant reduction in tumor size (71%) compared to anti-PD1 alone (9%). Additionally, there was a significant increase in the Ifnb1 (p=.04), Ifng (p=.008), Tnf-α (p=.006), IRF3 (p=.003) and II6 (p<.0001) gene expression in the combination group showing enhancement of the STING pathway genes. Combined, these findings suggest that targeting the STING pathway may have a modest local anti-tumor effect when compared to other innate immune pathways in prostate cancer. The efficacy of anti-PD-1 therapy was significantly enhanced when combined with a STING agonist, possibly through enhancement STING pathway genes. Our data suggest that combination therapy with a STING agonist and anti-PD1 therapy may have potential anti-tumor efficacy in prostate cancer. Future studies will examine these effects on both local and abscopal tumors.

#4092

CD40L/4-1BBL virotherapy enhances efficacy of checkpoint blockade therapy in a resistant melanoma model.

Jessica Wenthe,1 Sedigheh Naseri,1 Ann-Charlotte Hellström,1 Emma Eriksson,1 Angelica Loskog2. 1 _Uppsala University, Uppsala, Sweden;_ 2 _Uppsala University, Lokon Pharma AB, Uppsala, Sweden_.

Despite the impressive effects of checkpoint blockade antibody therapy seen across indications, many patients remain resistant to treatment. PD-1/PD-L1 blockade aims to restore the function of anergic tumor-infiltrating T cells, thereby inducing an efficient anti-tumor response. Thus, a prerequisite to benefit from checkpoint blockade is the presence of T cells in the tumor. One promising strategy to prime non-inflamed tumors for checkpoint blockade is the use of oncolytic viruses, which can additionally be engineered to express immunostimulatory molecules to further induce immune activation and T cell infiltration. In this study, we are investigating the use of the oncolytic adenovirus mLOAd703 expressing a murine trimerized membrane-bound (TMZ)-CD40L and 4-1BBL in the syngeneic murine B16 melanoma model. Adenoviruses cannot replicate in murine cells, but the immunostimulatory effect of the murine transgenes TMZ-CD40L and 4-1BBL can still be evaluated. LOAd703 (serotype 5/35) targets human CD46 for viral entry. Thus, a B16 cell line expressing human CD46 was used to allow infection.

Upon infection with mLOAd703, B16-hCD46 cells not only expressed the transgenes TMZ-CD40L and 4-1BBL, but also changed their phenotype to become more immunogenic by upregulating co-stimulatory molecules CD80 and CD86, as well as MHC molecules. Moreover, expression of the death receptor Fas was increased, altogether potentially making the tumor cells more susceptible to T cell-mediated killing. Strikingly, two markers (CD44 and beta-3 integrin) associated with metastasis were reduced upon virus infection. Previous in vivo studies in immunocompetent C57BL6 mice with a predecessor of mLOAd703, which only expresses murine TMZ-CD40L, led to an increase of CD8+ T cells and CD11b+MHCII+ antigen presenting cells in the tumors. However, these cells also upregulated PD-1 or PD-L1, respectively, which warrants a combination with P-D1/PD-L1 checkpoint blockade. To evaluate this combination, mice with established B16-hCD46 tumors were treated with mLOAd703 (intratumoral 6x, 1x10e9 IU/mouse/injection), checkpoint blockade antibody (anti-mouse PD-1 or PD-L1, i.p. 6x 5mg/kg/mouse/injection; BioXcell), or treated in combination with mLOAd703 plus anti-PD-1 or mLOAd703 plus anti-PD-L1. Monotherapy with checkpoint blockade antibodies did not reduce tumor growth in this model. However, mLOAd703 reduced tumor growth as a monotherapy, but this reduction was even further enhanced in combination with both anti-PD-1 and anti-PD-L1 therapy.

In conclusion, mLOAd703 could infect B16 cells expressing CD46, leading to TMZ-CD40L and 4-1BBL transgene expression. In addition, infected tumor cells became more immunogenic and reduced molecules involved in metastasis. In vivo studies revealed that mLOAd703 therapy sensitized PD-1/PD-L1-resistant mice to checkpoint blockade therapy.

#4093

Synergy of CTL tumor cytotoxicity with myxoma oncolytic virotherapy.

Meixuan Chen, Ana L. Matos, Padhmavathy Yuvaraj, Laura Belmont, Grant McFadden, Karen S. Anderson. _Biodesign Institute, Arizona State University, Tempe, AZ_.

Background: Checkpoint blockade (CKB) are in clinical trials in breast cancer, but responses may be limited by the low tumor micro-immune profile and somatic mutation burden. Oncolytic viruses may function by activating the immune microenvironment, and improve clinical responses when combined with CKB against cancers like melanoma. In this study, we evaluate the synergy of cytotoxic T lymphocytes (CTLs) combined with oncolytic myxoma virus in an in vitro model of human breast cancer.

Method: BMLF1-CTLs were generated from human HLA-A*02 PBMCs, which were stimulated with antigen presenting cells pulsed with the EBV BMLF1 HLA-A*02 peptide (GLCTLVAML). MCF-7 was engineered to express the full-length BMLF1 gene from EBV. To assess the impact of myxoma virus infection on CTL cytotoxicity, target MCF-7 cells (+/- BMLF1) were co-cultured with BMLF1-CTLs (E:T ratio=5:1) and myxoma virus vMyx-M135KO-GFP (vMyx135KO), at multiplicity of infection (MOI) ranges from 0.1 to 10 ffu/cell, in vitro for 48 hours. Cytotoxicity was measured by flow cytometry with propidium iodide (PI). Cell surface expression of MHC class I and class II were measured by flow cytometry. Statistical comparisons were performed using an unpaired t-test and variation among and between groups was calculated using ANOVA. Statistical significance was defined as p<0.05.

Results: At the highest MOI (10 ffu/cell), cell surface MHC class I expression on MDA-MB-231 was decreased 38-fold (p<0.01), compared to untreated control. MCF-7 was more sensitive to MHC class I downregulation (MOI 1 ffu/cell, 37.3-fold reduction, p<0.002). No significant change of cell surface expression of MHC class II was detected in either breast cancer cell lines at any MOI. In MCF-7-BMLF1- cells, baseline cytotoxicity remained low in both CTL and vMyx135KO (MOI=0.1 ffu/cell) only treatments (14% vs. 10%). With antigen expression (MCF-7-BMLF1+), CTL cytotoxicity increase to 21% (p=0.02). At a MOI of 0.1 ffu/cell, vMyx135KO viral infection further sensitized cells to CTL cytotoxicity (21% vs. 36%, p<0.001)

Conclusions: Oncolytic myxoma virus infection can decrease the MHC class I expression in human breast cancer cell lines, but only at high multiplicities of infection. At low level of virus, myxoma virus infection can enhance CTL cytotoxcity of breast cancer cell line.

#4094

**The TLR4 agonist G100 enhances rituximab-mediated ADCC** in vitro **and has synergistic anti-tumor effects with anti-CD20 mAb** in vivo **.**

Hailing Lu, Alec Betancur, Jan ter Meulen. _Immune Design, Seattle, WA_.

Background: G100, which contains the synthetic TLR4 agonist Glucopyranosyl lipid A (GLA) formulated in a stable oil-in-water emulsion (SE) for intratumoral therapy, is currently in clinical development in combination with pembrolizumab for the treatment of B-cell follicular lymphoma (FL). The current study was undertaken to investigate the interaction of G100 with rituximab, the anti-CD20 mAb that is standard-of-care treatment for FL.

Methods: In vitro studies were performed using peripheral blood mononuclear cells (PBMC) from healthy human donors to investigate the effect of GLA-AF (an aqueous formulation of GLA) on natural killer (NK) cell activation. IFNγ production in NK cells after activation with plate-bound anti-CD16 or target K562 leukemia cells was measured by intracellular cytokine staining (ICS). Rituximab-mediated antibody-dependent cellular cytotoxicity (ADCC) was measured using pretreated or untreated PBMC and rituximab-coated Raji Burkitt's lymphoma cells as target cells. In vivo studies were carried out in Balb/c mice with bilateral A20 lymphoma. Mice received G100 alone (10μg, intratumoral, 3 injections/week), anti-CD20 alone (clone 5D2, provided by Genentech, 200μg, intraperitoneal, 1 injection/week), or G100+anti-CD20 for a total of 3 weeks.

Results: Incubation of PBMC from 3 different donors with GLA-AF (1 μg/mL) resulted in NK cell activation as demonstrated by increased IFNγ production, as well as enhanced response to the K562 target cells and anti-CD16. When PBMC from 5 normal donors were used in rituximab-mediated ADCC assay, pretreated PBMCs had significantly enhanced lysis of target Raji cells compared to untreated PBMC (p<0.05). In mice with bilateral A20 tumors, the combination of G100 with anti-CD20 resulted in significantly better tumor control and prolonged survival (n=5 per group, p<0.05).

Conclusions: G100 enhances NK cell activity and rituximab-mediated ADCC in human PBMC. Combination of G100 and anti-CD20 mAb has synergistic anti-tumor effects in a mouse lymphoma model. These data support clinical testing of G100 with rituximab in patients with FL.

#4095

Adding fuel to the fire: Augmenting the efficacy of adoptive T-cell therapy with pro-oxidants.

Nada Aboelella, Gang Zhou, Kateryna Fesenkova, Zhi-chun Ding. _Augusta University, Augusta, GA_.

Cancer cells face increased oxidative stress driven by oncogene activation, tumor suppressor gene mutation and increased metabolism. Even though cancer cells can resist this redox imbalance by activating various antioxidant defense mechanisms, they are more susceptible to further redox disruption by pro-oxidants. Our previous study indicated that adoptive T-cell therapy (ACT) leads to heightened oxidative stress in tumor cells reflected by depletion of cellular antioxidant glutathione (GSH), accumulation of reactive oxygen species (ROS), and presence of oxidative DNA damages. Based on this observation, we propose that intensifying oxidative stress in tumor with pro-oxidants can sensitize cancer cells to ACT and lead to improved therapeutic outcomes. In this study, we used clinically relevant ACT models to screen a panel of pro-oxidant agents for their ability to potentiate the antitumor effects of ACT. We identified several clinically applicable pro-oxidants, including some commonly used non-steroidal anti-inflammatory drugs (NSAIDs), that can benefit the infusion of tumor-specific CD4+ T cells or CD19-targeting CAR T cells. Our data provide a rationale for combining selected pro-oxidants with ACT to achieve synergistic antitumor effect.

#4096

Clinical study of personalized neoantigen-based cancer vaccine in the treatment of advanced SCLC patients with brain metastasis: A case report.

Song Gao,1 Yongchao Li,2 Zhenyu Ding,3 Heng Xu,3 Jiaqian Wang,2 Xianling Guo,1 Juemin Fang,1 Guochao Wei,2 Huanlong Qin,4 Yuquan Wei,3 Qing Xu,1 Li Yang3. 1 _Department of Oncology, Shanghai Tenth People's Hospital, Tongji University, Shanghai, China;_ 2 _YuceBio, Shenzhen, China;_ 3 _Department of Biotherapy, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu, China;_ 4 _Tongji University Cancer Center, Shanghai, China_.

Background: Tumor neoantigens arising from somatic mutations are less likely to be subjected to central immune tolerance and therefore the attractive targets for designing cancer vaccine. The personalized neoantigen-based cancer vaccine, a passive immunotherapy based on the theory of adaptive immune responses, has been developed rapidly in recent years.

Case Presentation: A 75-year-old male small cell of lung carcinoma (SCLC) patient with brain metastasis, who have experienced multiple chemotherapy resistance, and disease relapse after PD-1 therapy, was enrolled in this study. Whole-exome sequencing (WES) and RNA-seq were used to evaluate patient's mutation burden and to predict the potential neoantigens. Twelve neoantigen peptides were selected and then were synthesized to generate autologous dendritic cell (DC)-based neoantigen vaccine. At last, 1.5 × 108 DC-based neoantigen vaccine was subcutaneously injected into the patient's body to evaluate the treatment efficacy. By following-up to 10 months after vaccination, we observed an objective response with all brain metastases regressed. The immunogenicity of each of the 12 neo-epitope administered in this study was analyzed by IFNγ ELISpot in CD4+ and CD8+ T cells in pre- and post-vaccination blood samples. Responses were detected against 66.67% (8/12) of the predicted neo-epitope.

Conclusions: The autologous DC-based neoantigen cancer vaccine can effectively trigger a positive immune response to patients' tumor and prolong patients' survival. The vaccination can also augment the anti-tumor response of T cells by targeting other nonsynonymous mutation-derived neoepitopes. Therefore, these data demonstrated that the autologous DC-based neoantigen cancer vaccine is safe and effective in eliciting a broad antitumor immunity. Moreover, our work has introduced a framework for testing neoantigen-based vaccine combinatorial strategies in patients with advanced recurrent cancers.

#4097

TMV vaccine inhibits growth of squamous cell carcinoma of head and neck in mice.

Ramireddy Bommireddy,1 Luis Munoz,1 Chrstopher D. Pack,2 Sampath Ramachandiran,2 Shaker JC Reddy,2 Janet Kim,1 Georgia Chen,1 Nabil F. Saba,1 Dong M. Shin,1 Periasamy Selvaraj1. 1 _Emory University, Atlanta, GA;_ 2 _Metaclipse Therapeutics, Atlanta, GA_.

Introduction: Head and neck cancer is a leading cause of cancer related deaths accounting for approximately 3% of all cancer related mortalities in the US. Currently, there is no cure for the advanced squamous cell carcinoma of the head and neck (SCCHN) thus development of efficacious therapies is urgently needed. To test whether vaccine-induced immunity inhibits tumor growth, we investigated efficacy of a tumor membrane-based vaccine immunotherapy in a murine SCCHN (SCC VII) model.

Materials and Methods: The SCC VII tumors grown subcutaneously in C3H/HeJ mice were harvested to generate tumor membrane vesicles (TMVs). TMVs were then protein transferred with glycolipid-anchored immunostimulatory molecules mouse GPI-B7.1 and mouse GPI-IL-12 to generate the TMV vaccine. Mice were vaccinated with TMV vaccine either before (prophylactic) or after SCC VII tumor cell challenge (therapeutic) and tumor growth was monitored every 3 days. Survival was then assessed using a Kaplan-Meier survival curve and significance determined using a Log-rank test for comparison analysis.

Results: SCCVII cells express MHC I, CD44, CD47 and respond to IFN-γ in vitro. The therapeutic TMV vaccine inhibited tumor growth and improved the survival of mice challenged with SCCVII tumor cells. Tumor-free mice remained protective from rechallenge with SCCVII cells.

Conclusions: These observations suggest that tumor tissue-based vaccines can be harnessed to develop an effective immunotherapy for SCCHN.

Funding: Supported by Head and Neck SPORE pilot funding from Emory Winship Cancer Institute. 

### Immune Checkpoints 2

#4098

A heavy chain only antibody against CTLA4 with outstanding preclinical efficacy and safety profile.

Xin Gan, George Liu, Yiping Rong, Yun He. _Harbour Biomed, Shanghai, China_.

Anti-CTLA-4 antibodies are one of the only three types of immune checkpoint inhibitors (together with anti-PD-1 and anti-PD-L1 antibodies) with proven monotherapy value in cancer therapy. Despite demonstrated efficacy, their broad application in monotherapies and combinational therapies are hampered by the safety profiles. Evidence in animal models suggests that efficacy of anti-CTLA-4 antibodies is at least partially due to depletion of regulatory T cells (Treg) via ADCC function, whereas clinically adverse events are positively correlated with systemic exposure.

Here we embarked to develop the next generation anti-CTLA-4 antibody with better efficacy and safety profile. Fully human Heavy Chain only antibody (HCAb) 4003-2 was generated using the Harbour HCAb transgenic mouse platform. 4003-2 specifically binds to CTLA-4 and blocks its binding to B7.1/B7.2. It was optimized to have enhanced ADCC function that affords superior ability to deplete Treg cells both in vitro and in vivo. 4003-2 showed in murine tumor models significantly more potent anti-tumor activity than other CTLA4 antibodies, at least partly driven by substantially enhanced depletion of intra-tumoral Treg cells. In addition, due to its shorter serum half-life, 4003-2 has less systemic drug exposure in vivo at efficacious doses, suggesting potentially improvement of safety profile in clinical applications.

The augmented efficacy and safety profile of 4003-2 present it as an excellent candidate for the development of next generation anti-CTLA4 therapy.

#4099

Intrinsic Tumor Cell Consequences of Acquired Resistance to PD-L1 Blockade.

Yuhao Shi,1 Michalis Mastri,1 Melissa Dolan,1 Michael Oberst,2 John Ebos1. 1 _Roswell Park Comprehensive Cancer Center, Buffalo, NY;_ 2 _MedImmune, Buffalo, NY_.

The use of immune checkpoint inhibitors (ICIs) that block programmed cell death protein ligand 1 (PD-L1) binding to PD-1 have led to durable responses in multiple cancer types; however, a subset of patients eventually stop responding. Currently, few preclinical models faithfully recapitulate clinically-relevant acquired ICI resistance that could help identify second-line treatments. Here we describe the in vivo derivation of cells from orthotopically implanted mouse mammary EMT-6 primary tumors in mice that were initially responsive to PD-L1 inhibition but eventually developed resistance. Transcriptomic comparisons of in vivo-derived parental (EMT6P) and anti-PD-L1 resistant (EMT6PDR) cells revealed alterations in multiple signaling pathways controlling secretory profiles that govern metastasis and angiogenesis. Using a small molecule screen of ~1500 FDA approved cancer drugs, we found that anti-proliferative activity in EMT6PDR cells was decreased for ~120 compounds, including numerous receptor tyrosine kinase inhibitors (RTKIs) currently being tested in ICI-refractory patient populations. Molecular analysis indicated resistance-mediated secretory changes may be linked to intrinsic functions of PD-L1 on the resistant tumor cell which, in turn, may explain differential RTKI sensitivity. Subsequent studies show that EMT6PDR secretory changes can be interferon (IFN) mediated. Together, these results reveal that acquired resistance to PD-L1 blockade can result in PD-L1-regulated intrinsic tumor cell changes involving secretory proteins and controlled, at least in part, by IFN signaling. Current studies seek to assess the impact of these cellular changes on RTKI responsiveness to identify secondary treatments after ICI failure.

#4100

Receptor occupancy and neo-epitope specific T-cell repertoires in patients with solid tissue tumors following anti-PD-1 therapy.

Devika Ashok,1 Songming Peng,2 Benjamin Yuen,2 Matt Walters,1 Stephen W. Young,1 Lisa Seitz1. 1 _Arcus Biosciences, Inc., Hayward, CA;_ 2 _PACT Pharma, Hayward, CA_.

Background:

In patients undergoing chemotherapy and/or immune therapy, endogenous T cells within the tumor micro-environment (TME) and in the periphery are subject to dynamic changes. Particularly, exhausted T cells in the TME and peripheral blood mononuclear cells (PBMCs) isolated from whole blood express the inhibitory receptor, Programmed Cell Death Receptor 1 (PD-1). A small but significant number of these T cells can recognize neo-epitopes (neoE) arising from patient specific mutations. We determined receptor occupancy (RO) in patients treated with monoclonal anti-PD-1 antibody AB122, which is currently in clinical development.

Methods: Multi-color flow cytometry was used to determine RO post-AB122 administration using a directly-conjugated competitive antibody to AB122. In addition, we determined RO values using the established method for nivolumab using a biotinylated anti-human IgG4 to detect bound anti-PD-1. The assays were also optimized to determine RO in whole blood from patients with solid tumors treated with AB122. Receptor occupancy using both methods were compared. Further, whole exome sequencing from archival formalin-fixed, paraffin-embedded (FFPE) tissue was performed and T cells that recognized patient-specific neoE resulting from tumor-specific mutations were identified in patient blood.

Results:

In the dose-escalation study for AB122 monotherapy arm, a variety of dosing regimens have been evaluated, including Q2W, Q3W and Q4W. The number of doses received ranged from 1 to 20. Data from patients in the 80 and 240 mg Q2W cohorts showed that AB122 serum concentrations ≥1.5 μg/mL (equivalent to 10 nM) are associated with full receptor occupancy. The RO associated with AB122 dosing is maintained when dosed in combination with the potent A2aR/A2bR dual adenosine receptor antagonist AB928 in an ongoing clinical trial.

Conclusions:

Full RO is observed with both cohorts of AB122 monotherapy (80 and 240 mg Q2W) and in the 240 mg Q2W combination with AB928. Study of peripheral target engagement and expansion of neoE-specific T cell populations in a longitudinal study of solid tumor patients treated with AB122 could provide insight towards designing successful combination immune therapy regimens in future clinical trials with AB122.

#4101

Spatial heterogeneity of tumor mutational burden (TMB) counts in pulmonary adenocarcinoma: Separating biological effects from technical artifacts.

Volker Endris, Michael Allgäuer, Jan Budczies, Jonas Leichsenring, Anna-Lena Volckmar, Martina Kirchner, Olaf Neumann, Regine Brandt, Eugen Rempel, Carolin Plöger, Moritz von Winterfeld, Alexander Harms, Mark Kriegsmann, Roland Penzel, Peter Schirmacher, Daniel Kazdal, Albrecht Stenzinger. _Heidelberg University, Heidelberg, Germany_.

Introduction: Tumor mutational burden (TMB) is an emerging biomarker to identify patients more likely to benefit from immuno-oncologic therapy. Therefore, academia- and industry-driven endeavors are underway to establish mostly panel-based sequencing solutions. Aside from various unsettled technical aspects, tumor biology itself might play an important role in determining a tumor's TMB. Intratumor heterogeneity (ITH) is frequent in pulmonary adenocarcinoma (ADC) and seems to be a key factor regarding response failure and resistance development during therapy. In addition, ITH might contribute to inconsistent distribution of a biomarker and therefore an assay's readout might depend on the region of tumor which is submitted for testing. Therefore, the assessment of ITH is an essential step in the evaluation of new diagnostic applications.

Experimental Set-up: In order to investigate potential spatial differences in the TMB of a tumor, we analyzed a comprehensively morpho-molecularly characterized cohort of pulmonary ADC by sequencing 3-4 sections per tumor including lymph node metastases. So far 10 tumors were included in the ongoing analysis. None of the selected tumors had an oncogenic EGFR mutation, six were KRAS positive and four had no known driver mutation. TMB was determined applying the TruSightTM Oncology 500 targeted sequencing panel (TSO 500), covering 500 genes, with a NextSeq 500 sequencing system (both Illumina Inc., San Diego, CA, USA).

Results Summary: The current sequencing analysis using the TSO 500 panel covered an average genomic coding region of 1.26 Mbp with a mean read depth of x658. The average TMB of all samples was 8.55 mut/Mbp (range 0.79 - 18.95). Comparing different regions within a given tumor, we detected a considerable variance of TMB (± 5 mut/Mbp) in about half of the analyzed cases, with an overall mean absolute deviation of 2.2 and a maximum difference of 12.63 mut/Mbp. However, in some samples an in depth evaluation of morpho-molecular characteristics revealed several factors impairing TMB read out. Aside from technical issues like insufficient depth of coverage in combination with unadjusted automated variant calling, sample characteristics like tumor cell content had a prominent influence on TMB values. Often, tumor regions with lower TMB turned out to have lower tumor cell content, which led to decreased allele frequencies of tumor specific mutations and subsequently compromised mutation detection.

Conclusions: Our data demonstrate spatial heterogeneity of TMB in pulmonary ADC, which can impact clinical decision making and therapy. However, spatial genetic heterogeneity reflecting tumor biology needs to be carefully dissected from technical conditions resulting in pseudo-heterogeneity.

#4102

DNA methylation and repressive histones in the promoters of immune checkpoints in tumor tissues and peripheral blood of breast and colorectal cancer patients.

Eyad Elkord, Varun Sasidharan Nair, Salman M. Toor. _Qatar Biomedical Reseach Institute, Doha, Qatar_.

Higher expression of immune checkpoints in tumor microenvironment plays significant roles in inhibiting anti-tumor immunity, which is associated with poor prognosis and progression of cancer patients. Major epigenetic modifications in both DNA and histone could be involved in upregulation of immune checkpoints (ICs) in cancer. We investigated transcriptomic expression of different ICs and some ligands including PD-L1 and galectin-9 in the tumor microenvironment (TME) and in circulation of primary breast cancer (PBC) and colorectal cancer (CRC) patients. We found that transcriptomic expressions of PD-1, CTLA-4, TIM-3 and LAG-3 in PBC and PD-1, CTLA-4, TIM-3, TIGIT, PD-L1 and galectin-9 in CRC were significantly upregulated in tumor tissues (TT), compared with normal tissues (NT). Additionally, expressions of TIM-3, TIGIT, and PD-L1 were significantly upregulated, while LAG-3 was downregulated in peripheral blood of both PBC and CRC patients. We then investigated the epigenetic modifications beyond this upregulation of immune checkpoint genes. Interestingly, we found that CpG islands in the promoters of PD-1, CTLA-4 and TIM-3 in PBC, and CTLA-4 and TIGIT in CRC patients were significantly hypomethylated in tumor compared with normal tissues. Notably, CpGs in PD-L1 promoter were completely hypomethylated in PBC and CRC in both TT and NT. In addition, PD-L1 was hypomethylated in in peripheral blood of both PBC and CRC patients, while TIGIT was hypomethylated only in circulation of CRC patients, compared with healthy donors. Next, we checked the abundance of repressive histones (H3K9me3 and H3K27me3) in the promoter regions of ICs/ligands. We found that in PBC TT, distribution of both H3K9me3 and H3K27me3 was lower in the promoters of PD-1, CTLA-4 and LAG-3, while distribution of only H3K27me3 was lower in TIM-3 promoter, compared with NT. Furthermore, in CRC TT, the distribution of H3K9me3 was lower in PD-1 and TIGIT promoters, and H3K27me3 was lower in CTLA-4 promoter, while both H3K9me3 and H3K27me3 were lower in TIM-3 promoter, compared with NT. Our data demonstrate that both DNA methylation and repressive histones are involved in upregulation of ICs and their ligands in the tumor microenvironment and peripheral blood of breast and colorectal cancer patients. These epigenetic modifications could be useful as diagnostic/prognostic biomarkers and/or therapeutic targets in breast and colorectal cancers.

#4103

Blockade of intrinsic PD-1 promotes human colon cancer cells survival, chemo resistance and in vivo tumor growth of human colon cancer cells.

Caterina Ieranò,1 Crescenzo D'Alterio,2 Maria Napolitano,1 Luigi Portella,3 Giuseppina Rea,4 Gelsomina Monaco,4 Piera Maiolino,4 Antonio Luciano,4 Antonio Barbieri,4 Giuseppe Palma,4 Claudio Arra,4 Roberto Pacelli,5 Stefania Scala4. 1 _Istituto Nazionale per lo Studio e la Cura dei Tumori, Fondazione "G. Pascale"- IRCCS, Italy;_ 2 _Istituto Nazionale per lo Studio e la Cura dei Tumori, Fondazione "G. Pascale"- IRCCS,, Italy;_ 3 _Istituto Nazionale per lo Studio e la Cura dei Tumori, Fondazione "G.Pascale"- IRCCS, Italy;_ 4 _stituto Nazionale per lo Studio e la Cura dei Tumori, Fondazione "G.Pascale"- IRCCS, Naples, Italy;_ 5 _Federico II University School of Medicine, Naples, Italy_.

A considerable proportion of colorectal cancer patients develops local recurrence and distant metastasis within 5 years after surgical treatment. The anti-PD-1, Nivolumab, is indicated for treatment of patients with metastatic colorectal cancer carrying DNA mismatch repair-deficient (dMMR)/microsatellite instability-high (MSI-H) whose disease has progressed on fluoropyrimidine, oxaliplatin, and irinotecan. Thus only a minority of patients with CRC will benefit from the immunotherapy. Beyond immune cell expression, recent evidence demonstrates that PD-1 is expressed on human cancer cells, such as melanoma and NSCLC cells. It is possible to speculate that colon cancer immune resistance could partially reside on intrinsic colon cancer cells PD-1 overexpression. PD-1 expression was evaluated through RT-PCR and flow cytometry in human colon cancer cell lines (HT29, HCT116, SW620, Colo205, LOVO). PD-1 was robustly expressed in HT29 (74,4%), in HCT116 (57,5%) and in SW620 (62,9%) while the ligand PD-L1 was weakly expressed in these cell lines. Recombinant PD-L1 fusion protein significantly decreased HT29 and HCT116 proliferation, while Nivolumab protects the cells by PD-L1 effect. To investigate on the effect of PD-1 on chemo sensitivity, cytotoxicity assays were conducted with Oxaplatinum (OXAL) or 5-Fluoruracil (5-FU) in the presence of Nivolumab. Unexpectedly, Nivolumab significantly protected cancer cells from OXAL and 5-FU (HT29 cells were 2.02- and 1.77-fold more resistant to 5FU and OXAL in the presence of Nivolumab and HCT116 were 1,7- and 2,1-fold more resistant to 5FU and OXAL in the presence of Nivolumab). Moreover, Nivolumab protected HT29 cells from apoptosis 5-FU-OXAL induced at 72 hrs. In vivo, Nivolumab accelerated the growth of subcutaneous HT29 tumor growth and reduced the efficacy of chemotherapy. Mechanistically, preliminary results demonstrated that Nivolumab increased the mTOR target p4EBP1, pErk and p38 signaling. Taken together, these results demonstrate that, while intrinsic PD-1 axis activation decreases human colon cancer cell proliferation, Nivolumab protects the human colon cancer cells PD-1 overexpressing. These effects need to be considered in the evaluation of immunotherapy efficacy in colon cancer.

#4104

Expression of the immune checkpoint receptor TIGIT in seminoma.

Maximillian Lennartz, Niclas C. Blessin, Andrea Hinsch, Ronald Simon, Martina Kluth, Kristine Fischer, Claudia Hube-Magg, Wenchao Li, Tim Mandelkow, Nicolaus F. Debatin, Doris Höflmayer, Guido Sauter, Jakob R. Izbicki, Sarah Minner, Franziska Büscheck, Ria Uhlig, David Dum, Till Krech, Andreas M. Luebke, Corinna Wittmer, Frank Jacbosen, Eike Burandt, Stefan Steurer, Waldemar Wilczak. _Univ. Medical Ctr. Hamburg-Eppendorf, Hamburg, Germany_.

A characteristic feature of testicular seminoma is the abundance of immune cells in the tumor microenvironment, raising the possibility that immune checkpoint inhibitors could be a therapeutic option in these tumors. TIGIT (T cell immunoreceptor with Ig and ITIM domains) is an inhibitory immune checkpoint receptor in analogy to PD-1 and drugs targeting TIGIT are currently tested in clinical trials. Little is known about the expression of these proteins in testicular seminomas. Here, we employed immunohistochemistry to determine the relative abundance of TIGIT and PD-1 in relation to the total CD3+immune cell infiltration in a tissue microarray (TMA) constructed from 78 seminoma patients. The fraction of TIGIT+and PD-1+lymphocytes was highly variable in individual cancers and ranged from 2.3% to 69.4% (mean:32.2±14.7%) for TIGIT and from 0.8% to 56.5% (mean:21.6±13.2%) for PD-1. The same high degree of variability was also found for the ratio of PD-1 to TIGITpositive cells that varied from a dominance of TIGIT (PD-1:TIGIT ratio=0.02) in 74% of patients to a predominance of PD-1 (PD-1:TIGIT ratio=12.5) in 23% of patients. In summary, the immune checkpoint receptors TIGIT and PD-1 are abundantly expressed in human seminomas. Once available, anti-TIGIT antibodies, possibly in combination with anti-PD-1 drugs, may be a promising therapeutic option for clinical studies in this cancer type.

#4105

CD8 positive tumor infiltrated lymphocytes as prognostic factor in sinonasal squamous cell carcinoma.

ROCIO GARCIA-MARIN,1 CRISTINA RIOBELLO,1 VIRGINIA N. CABAL,1 LAURA SUAREZ-FERNANDEZ,1 SARA REDA,2 FERNANDO LOPEZ,2 JOSE L LLORENTE,2 MARIO HERMSEN1. 1 _ISPA, OVIEDO, Spain;_ 2 _HUCA, OVIEDO, Spain_.

Introduction

Sinonasal squamous cell carcinoma (SNSCC) patients have a poor survival rate and treatment options are limited. We evaluated CD8-positive tumor-infiltrated lymphocytes (TILs) in 57 SNSCC and the relation to PD-L1 expression. Furthermore, we analyzed PD-L1 mutations and CMTM6 expression in relation to enhanced ability to inhibit TILs. Our aim was to identify patients that may benefit from therapy with immune checkpoint inhibitors.

Experimental Procedures

Four tissue microarray blocks were constructed including three 1 mm cores from different areas of 57 SNSCC. Each block also contained normal mucosa samples as controls. The presence of CD8\+ TILs, tumoral PD-L1 and CMTM6 expression was evaluated by immunohistochemistry. The stainings were evaluated by 3 independent observers and results were correlated to clinico-pathological characteristics and follow-up data. Co-localization of CD8+ TILs and PD-L1+ cells was performed in 2-μm-thick FFPE sections by immunofluorescence. Exon 6 of the PD-L1 gene, which encodes the DTSSK motif, was amplified by PCR and products were sequenced by Sanger sequencing.

Results

High level presence of intratumoral CD8+ TILs occurred in 11/57 (19%), low level in 39/57 (69%) and absence in 7/57 (12%) cases. Five-year disease-specific survival (DSS) was 34% of cases with intratumoral CD8+ versus 71% in cases CD8- (P=0.03). Five-year DSS was 0%, 24% and 43% in high level, low level and absence of intratumoral CD8+ TILs, respectively (p=0.006). PD-L1 expression was observed in 26/57 (46%) of cases and did not correlate with clinico-pathological parameters or DSS. All patients with high intratumoral CD8+ expression were PD-L1 positive

and 75% of patients with strong PD-L1 staining showed high intratumoral CD8\+ expression. All intratumoral CD8+/PD-L1+/- patients died of disease before 5 years compared to 24% CD8-/PD-L1\- and 48% CD8-/PD-L1\+ patients who survived up to 5 years (P=0.003). Tumors CD8+/PD-L1+/- had a significant worse DSS than CD8-/PD-L1+/- cases (p=0.015). No mutations were found in the DTSSK domain of PD-L1 and CMTM6 showed expression in 43/57 (75%) of patients while 17 of those 43 (40%) were also PD-L1+.

Conclusion

Our data showed that SNSCC are immunogenic tumors with up to 88% of cases harboring some level of CD8+ TILs. The presence of intratumoral CD8+ TILs was correlated to worse survival, as has been observed previously in other tumor types. We also found 46% of tumors to express PD-L1. Together these results indicate that SNSCC patients could benefit from immunotherapy releasing the PD-1/PD-L1 immune checkpoint and reactivating the already present cytotoxic CD8+ TILs to exercise their antitumor activity. Pembrolizumab and nivolumab have already received FDA approval for clinical application in recurrent or metastatic HNSCC and may also be considered for treatment of recurrent SNSCC.

#4106

Outcomes of combined ipilimumab and nivolumab therapy following progression on nivolumab monotherapy in renal cell carcinoma: A retrospective cohort study.

Roy M. Elias, Nirmish Singla, Isaac A. Bowman, Payal Kapur, Raquibul Hannan, Hans Hammers, James Brugarolas. _University of Texas Southwestern Medical Center, Dallas, TX_.

Background: Nivolumab was the first immune checkpoint inhibitor to gain FDA approval for use in the treatment of renal cell carcinoma (RCC), however only a quarter of patients respond to nivolumab monotherapy (Nivo). More recently, the combination of ipilimumab and nivolumab (Ipi/Nivo) gained approval as first-line therapy in RCC with better outcomes than previously described with Nivo. Whether Ipi/Nivo can rescue RCC patients who progress on Nivo is an important question that remains unanswered. We sought to evaluate the oncologic benefit of treating metastatic RCC patients with Ipi/Nivo following progression on Nivo at our institution.

Methods: A systematic medical record search was conducted to identify patients with RCC who had been treated with Ipi/Nivo following progression on Nivo from 2013 to Jan 31, 2018. Clinical benefit after starting Ipi/Nivo was measured by time to next therapy (TNT), overall survival (OS), and objective responses defined by RECIST v1.1. Kaplan Meier methods were used to analyze event-free survival.

Results: 90 RCC patients were treated with Nivo from 2013 until Jan 31st, 2018. 17 (19%) of these patients received Ipilimumab following progression. Baseline characteristics of this subgroup were similar to the entire cohort. 13 (76.5%) had intermediate or poor risk disease by the Heng prognostic risk score. 6 (35.3%) patients had one prior line of therapy and 11 (64.7%) had two or more prior lines of therapy. The median duration of nivolumab monotherapy was 11.9 (95% CI; 4.4 - 13.0) months. Following the addition of ipilimumab at progression, the median TNT was 3.9 (95% CI; 3.3 to 7.0) months and the median OS was 20.3 (95% CI; 12.1 - 22.1) months. There were no objective responses to Ipi/Nivo following Nivo alone. Stable disease was the best overall response in 5 (29%) patients, and the remainder demonstrated progressive disease. One patient had stable disease lasting 11 months. Immune related adverse events were observed in 8 (47%) of patients resulting in cessation of therapy for 1 patient.

Discussion: In this analysis, Ipi/Nivo did not confer substantial treatment benefit to patients who had already progressed on Nivo alone. The were no objective responses, and most patients were started on the next line of therapy at their first set of imaging studies. These patients still demonstrated approximately a 2-year OS suggesting that alternative agents, such as TKIs, remain effective treatments in this population. Prospective evaluation is warranted to further evaluate the utility of Ipi/Nivo following progression on Nivo alone.

#4107

Predictive value of soluble PD-1, PD-L1, VEGFA, CD40 ligand and CD44 for nivolumab efficacy in advanced non-small cell lung cancer.

Manuela Tiako Meyo,1 Anne Jouinot,1 Etienne Giroux-Leprieur,2 Elizabeth Fabre,3 Pascaline Boudou-Rouquette,1 Audrey Bellesoeur,1 Jennifer Arrondeau,1 Hélène Blons,3 Audrey Mansuet-Lupo,1 Diane Damotte,1 Michel Vidal,1 François Goldwasser,1 Jérôme Alexandre,1 Benoit Blanchet1. 1 _Cochin Hospital, Paris, France;_ 2 _Ambroise Paré Hospital, Boulogne Billancourt, France;_ 3 _Georges Pompidou European Hospital, Paris, France_.

Context Anti-PD1 therapy nivolumab has been approved for the treatment of advanced non-small cell lung cancer (NSCLC). However, a large inter-individual variability in its efficacy has been observed. Thus, the search for reliable factors to predict anti-PD1 efficacy represents a major challenge, particularly in NSCLC patients. The aim of this prospective study was to assess the correlation of five immunity-related plasmatic biomarkers, soluble PD-1 (sPD-1), soluble PD-L1 (sPD-L1), VEGFA, soluble CD40L and soluble CD44, with survival in nivolumab-treated metastatic NSCLC patients.

Method This study included patients from the CERTIM prospective cohort (Immuno-modulatory Therapies Multidisciplinary Study group, Cochin Hospital, Paris, France), treated with nivolumab for a metastatic NSCLC between July 2015 and June 2017. Plasma levels of the five biomarkers were assayed at baseline and after two cycles of nivolumab using commercial ELISA kits. Due to their inconsistent expression, a cut-off of positivity for sPD-1, sPD-L1 and sCD40L expressions was defined as a plasma level above the lower limit of quantification (0.156ng/mL). The primary and secondary endpoints were progression-free survival (PFS) and overall survival (OS), respectively.

Results Fitfty-one patients were included (43% female, median age 66 years old): 40 patients (78%) had an adenocarcinoma, and 35 (69%) received nivolumab as a second-line regimen. Median [interquartile range] follow-up was 804 days [553 - 1112]. Baseline sPD-1, sPD-L1 and sCD40L were positive for 15 (29.4%), 27 (52.9%) and 18 patients (50%), respectively. Baseline positivity of sPD-1 and sPD-L1 did not independently correlate with PFS and OS in multivariate analysis. We defined a composite criteria (sCombo) corresponding to the positivity of sPD-1 and/or sPD-L1 for each patient. Patients exhibiting baseline sCombo positivity experienced shorter PFS (median [95% confidence interval]: 78 days [55-109] vs. 658 days [222-not reached]; HR 4.12, 95%CI [1.95-8.71], p=0.0002) and OS (median [95% CI]: 367 days [167 - 501] vs. not reached [402 - not reached]; HR: 3.99, 95%CI [1.63-9.80], p=0.003). The multivariate analysis including clinical factors and tumor cell PD-L1 expression showed that positivity of baseline sCombo was independently associated with a shorter PFS (HR: 2.66, 95%CI [1.17-6.08], p=0.02) but not with OS (HR: 2.17,95%CI [0.86-5.45], p=0.10). An increased or stable sPD-1 level after two cycles of nivolumab independently correlated with longer PFS (HR: 0.23, 95%CI [0.09-0.62], p=0.004) and OS (HR: 0.16, 95%CI [0.05-0.52], p=0.002). VEGFA, sCD40L and sCD44 did not correlate with patients' survival in this cohort.

Conclusion We propose a composite biomarker using soluble PD1 and soluble PDL1, predictive of nivolumab efficacy in NSCLC patients. A larger prospective validation study is warranted to confirm these results.

#4108

Preclinical evaluation of half-life extended Affimer® biotherapeutics targeting the PD-L1 pathway.

Amrik Basran, Emma Jenkins, Estelle Adam, Floriane Laurent, Michele Writer, Assa Oumie, Jyrki Sivula, Maureen West, Emma Stanley, Jennifer Hillman, Viviana Robles-munoz. _Avacta Life Sciences, Cambridge, United Kingdom_.

Programmed Cell Death-Ligand 1 (PD-L1) binds to its receptor, Programmed Cell Death protein (PD-1) and is expressed on a range of activated immune cells and is upregulated by tumor cells and by immune cells in the tumor microenvironment. PD-L1 immunotherapies have emerged as effective treatments both as monotherapies and in combinations, providing long term responses in a subset of patients.

Phage display selections have identified a range of potent Affimer biotherapeutic antagonists to both human and mouse programmed death-ligand 1 (PD-L1). We have demonstrated when formatted as bivalent molecules they have improved sub nM affinity, half-life extension in a mouse PK study, blocking activity in vitro and in vivo efficacy in syngeneic or xenograph mouse models.

Affimer biotherapeutics are a 14kDa monomeric scaffold protein based on the human protease inhibitor Stefin A, lacking post-translational modifications such as disulfide bonds and glycosylation. Large diverse phage display libraries have been created by engineering in two nine amino acid peptide loops into the scaffold backbone. We have identified a range of affinity competitive binders to mouse or human PD-L1 confirmed by performing competition ELISA and SPR using Fc formatted antigen. Half-life extension of lead Affimer proteins was achieved by fusing to human IgG1Fc or as an in-line fusion (ILF) fused to serum albumin binding Affimers. We demonstrated avidity for binding to target antigen, binding to cells in vitro and blockade of the PD-1L/PD-1 interaction using Promega cell based assay. The PK properties of the formatted Affimers in mouse showed half-life extension from 40-120 hours depending on the clone and half-life extension technology used.

We have demonstrated that the Affimer scaffold has the necessary properties to be developed as an immunotherapy. The anti-PD-L1 Affimer scaffold proteins were formatted as Fc fusion and in-line fusion formats which showed in vivo efficacy. These molecules will be developed further for both bispecific and drug conjugates to generate new novel biotheraputics that will give greater efficacy in the clinic.

#4109

Functional profiling of 17,000 open reading frames identifies regulators of PDL1 expression in HNSCC.

Jacqueline Mann, Samantha Devenport, Aditi Kulkarni, Rebecca Hoesli, Susan Foltin, Nicole Michmerhuizen, Alexey Nesvizhskii, Chad Brenner. _Univ. of Michigan, Ann Arbor, MI_.

The interaction of Programmed Death Ligand 1 (PD-L1) with the PD-1 receptor serves as an immune checkpoint, dampening anti-tumor immunity. While immunotherapies targeting this pathway (Pembrolizumab and Nivolumab), were approved for use in recurrent and metastatic head and neck squamous cell carcinoma (HNSCC) in 2016, overall response rates are low and few biomarkers have been identified to differentiate responders. We expect a more detailed understanding of PD-L1 checkpoint regulation in HNSCC to provide insight toward addressing these deficits. Thus, we developed a high- throughput screening protocol to discover signals modulating PD-L1 expression. We have employed a lentiviral open reading frame (ORF) library for overexpression of over 14,000 different genes in a pooled format. UM-SCC-49 cells transduced with this library were stained for cell surface PD-L1 and sorted by flow cytometry to select cells with enhanced PD-L1 expression. Sequencing of the selected sub-population was performed to determine ORFs enriched over an unsorted pool. To validate a role for individual target genes in modulating PD-L1 expression, monoclonal populations were expanded from the sorted pool and evaluated for PD-L1 expression. We confirmed increased PD-L1 expression in 7 of these cell lines over wild-type and LacZ overexpressing controls via western blotting and flow cytometry. Furthermore, we selected 5 ORFs of interest overrepresented in the sorted pool for further validation using stably expressed V5 tagged constructs. Target gene expression was confirmed by western blot, and enhanced cell surface PD-L1 expression was validated via flow cytometry for 4/5 cell lines. Thus, we provide evidence for successful identification of signals regulating PD-L1 using a novel high-throughput overexpression screen. Future experiments will aim to clarify the mechanism of PD-L1 induction in response to the signals identified here, and may ultimately provide a more thorough understanding of genetic factors modulating immunosuppression and response to immunotherapy in HNSCC patients.

#4110

Identification of PD1 B cell mimotopes with functional PD1-PDL1 blocking capacity: New strategy for cancer immunotherapy.

Joshua Tobias, Claire Battin, Annika De Sousa Linhares, Baier Karin, Katharina AMbroye, Mirjana Drinic, Erika Garner-Spitzer, Christoph Zielinski, Michael Kundi, Peter Steinberger, Ursula Wiedermann. _Medical University of Vienna, Wien, Austria_.

Immune checkpoint (ICP) blockade has become a major focus in cancer immunotherapy, and the application of monoclonal antibodies (mAbs) as ICP inhibitors shows impressive results in the therapy against cancers like melanoma, renal cell carcinoma or non-small cell lung cancer. Active immunization with B cell epitopes/mimotopes of ICP inhibitors rather than application of the corresponding mAbs for passive immunotherapy may, however, provide advantages such as induction of antibodies by the patient's own immune system and overcoming the costly treatment of the mAbs.

Applying a platform for bacterial surface-display of overlapping peptides spanning the extracellular domains of human PD1 (hPD1) and mouse PD1 (mPD1), as well as in vitro solid phase-based ELISA assay and cellular-based assays involving human Jurkat T cells expressing human/mouse PD1 and/or PDL1, B cell epitopes/mimotopes of Nivolumab and an anti-mPD1 mAb (with a blocking capacity) were identified. The mimotopes were not only shown to inhibit the binding of the corresponding mAbs but also to revert the PD1-PDL1 blocking inhibitory capacity of the respective mAbs. More importantly, to proof the antitumor activity of antibodies against PD1 mimotopes in vivo, rabbits were immunized with the mimotope of anti-mPD1 mAb to generate mimotope-specific IgGs. The specific IgGs not only inhibited the interaction of mPD1 and mPDL1 in vitro, but also significantly reduced tumor growth in a syngeneic model of mice grafted with cells expressing the breast cancer-associated tumor antigen Her-2/neu.

Our results show systematic identification and characterization of mimotopes of anti-PD1 ICP inhibitor B cell mimotopes conferring in vivo anti-tumor activity, and also indicate the potential of such mimotopes for active immunization in cancer therapy. Based on the results, enhancement of our second generation of anti-Her-2/neu vaccine (HerVAXX; Tobias et al, 2017*) by combining with mimotopes of ICP inhibitors is also proposed, which may potentially pave the way to a paradigm change in active immunotherapy against Her-2/neu overexpressing cancers using an effective multi-level vaccine.

The study is granted by Imugene Limited, Australia, and Medical University of Vienna.

* Tobias J. et al. BMC Cancer, 2017, 17:118.

#4111

Novel modules of effector T cell cytolytic activity are prognostic in multiple solid tumors and are predictive of response to immune checkpoint blockade therapy.

Chaitanya R. Acharya, Herbert K. Lyerly. _Duke University, Durham, NC_.

The prognostic and predictive value of tumor infiltrating lymphocytes (TILs) for immune checkpoint blockade has been recognized in a variety of tumor types. Nonetheless, our understanding of the mechanistic aspects of T cell activation remains incomplete. We hypothesize that a specific effector phenotype of T cell cytolytic activity (ECA) is a consistent feature of epithelial tumors, possibly varying by tumor types with a range of inflammatory features.

We first evaluated 5,174 purified CD3+ single cells from human primary triple negative breast cancer and computed sample set enrichment scores of a set of previously published immune metagenes. Following unsupervised clustering of the enrichment scores of the entire single cell population, two subgroups of cells with highest and lowest average enrichment score of T cell cytolytic activity formed a basis for detecting modules. ECA high group is characterized by enriched dendritic cells, NK cells, chemokines, MHC class I molecules and an enhanced T cell activation. On the other hand, ECA low group is enriched for CD4+ regulatory and helper T cells. Spectral decomposition and Jackstraw analysis estimated eight modules with overlapping sets of genes. Each gene expression module was then used to train a Random Forest classifier of ECA phenotype.

Each classifier was validated as a prognostic biomarker in about 6,000 samples including triple negative breast tumors, melanoma, lung adenocarcinoma and squamous cell carcinoma, head and neck cancer, colorectal cancer, renal cell carcinoma, bladder cancer, hepatocellular carcinoma, serous ovarian carcinoma, endocervical adenocarcinoma, glioblastoma, uterine cancer, prostate adenocarcinoma and low grade gliomas. Expectedly, univariate cox models suggest that in many of the tumor types, an increased ECA is associated with favorable prognosis. Interestingly, we found that increased ECA is associated with poor prognosis in both clear cell and papillary renal carcinoma, prostate, glioblastoma and low grade gliomas. These findings are not isolated and were previously documented.

Additionally, we investigated the association between enhanced ECA and response to immune checkpoint blockade therapy. We evaluated our modules in pre-therapy tumor samples of fifty-one advanced melanoma patients treated with Nivolumab, who previously either progressed on ipilimumab or were ipilimumab-naïve. Our ECA module was able to identify the responders with 75% accuracy (P = 0.04) and was associated with progression-free survival (log-rank P = 0.02; HR: 0.28 [0.05-1.5]). Patients, previously exposed to ipilimumab, were identified with an even higher accuracy of 80% (P = 0.008).

Effector T cell cytolytic activity modules have potential as prognostic and predictive immunotherapy biomarkers. Further evaluation in prospective clinical samples is warranted.

#4112

Programmed death-1 ligand 2 (PD-L2) on dendritic cells protects T cells from cellular exhaustion during melanoma.

Rebecca J. Faleiro,1 Deshapriya S. Karunarathne,1 Ji Liu,1 Glen Boyle,1 Gunter Hartel,1 Nick Hayward,1 Helmut Schaider,2 Nic Waddell,1 Chris Schmidt,1 Michelle Wykes1. 1 _QIMR Berghofer Medical Research Inst, Brisbane, Australia;_ 2 _The University of Queensland Diamantina Institute, Brisbane, Australia_.

T cells are an important effector cell population which can provide protective immunity against cancers such as melanoma. However, these T cells develop a state known as cellular exhaustion, where they are unable to offer protection mediated by expression of a crucial negative regulator Programmed cell death-1 (PD-1). Thus, tumors escape immune surveillance. PD-L1, a ligand for PD-1 is expressed by both tumor and immune cells, is known to mediate this cellular exhaustion. In contrast, the role of PD-L2, the other PD-1 ligand, is less clear. To better understand the contribution of PD-L2 to melanoma immunity, we studied dendritic cells in humans and in mice with melanomas. In humans, microscopy of melanoma biopsies revealed a loss of PD-L1 and PD-L2 expression on CD11c+ cells with progression of disease form early to late stage disease with a corresponding increase in cellular exhaustion CD8+ T cells. Flow cytometry of blood DCs confirmed this observation and showed a negative correlation with between expression of PD-L2 and T cells expressing exhaustion markers, indicating PD-L2 was protective. Finally, to determine if PD-L2 was protective in vivo, we used a pre-clinical melanoma model (B16-F0). For this, B16-F0 melanoma cells were injected intra-dermally on the back of recipient mice which were treated with either control IgG or mouse PD-L2 attached to human immunoglobulin-Fc fusion proteins commencing on day 3. Soluble PD-L2 treated mice reduced the tumor progression compared to the control group. Analyses of the tumor infiltrating cells showed a significantly lower incidence of exhausted CD8+ T cells, identified as PD-1Hi cells as well as LAG3 and TIM3 expression. Overall, these studies show PD-L2 protects T cells from exhaustion during melanoma, and has therapeutic potential.

#4113

Quantitative cell-based bioassays to advance immunotherapy programs targeting immune checkpoint receptors.

Jamison Grailer, Julia Gilden, Pete Stecha, Denise Garvin, Jun Wang, Michael Beck, Jim Hartnett, Gopal Krishnan, Frank Fan, Mei Cong, Zhi-jie Jey Cheng. _Promega Corp., Madison, WI_.

The human immune system is comprised of a complex network of immune checkpoint receptors that are promising new immunotherapy targets for the treatment of a variety of cancers and autoimmune disorders. The Nobel prize for Medicine in 2018 was awarded for seminal studies on the role of immune checkpoint targets in T cell activation and furthermore, combining different strategies to release the brakes on the immune system with the aim of eliminating tumor cells even more efficiently. Immunotherapies designed to block co-inhibitory receptors (e.g. PD-1, CTLA-4, TIGIT) are showing unprecedented efficacy in the treatment of cancer. However, not all patients and tumor types respond to this approach. This has resulted in broadening of immunotherapy research programs to target additional co-inhibitory (e.g. LAG-3, TIM-3) and co-stimulatory (e.g. 4-1BB, GITR, OX40, ICOS) receptors individually and in combination.

A major challenge in the development of antibody-based biologics is access to quantitative and reproducible functional bioassays. Existing methods rely on primary cells and measurement of complex functional endpoints. These assays are cumbersome, highly variable and fail to yield data required for drug development in a quality-controlled environment. To address this need, we have developed a suite of cell-based functional bioassays to interrogate modulation of immune checkpoint receptors individually (e.g. PD-1, LAG-3, TIM-3, GITR, 4-1BB) and in combination (e.g. PD-1+CTLA-4, PD-1+LAG-3). These assays consist of stable cell lines that express luciferase reporters driven by response elements under the precise control of mechanistically relevant intracellular signals. Thus, the bioassays reflect mechanisms of action for the drug candidates designed for each immune checkpoint receptor and demonstrate high specificity, sensitivity and reproducibility. Here we describe the application of MoA-based immune checkpoint receptor bioassays as tools for biologics drug discovery, development, potency and stability studies.

#4114

NPM1 upregulates the transcription of PD-L1 and suppresses T-cell activity in triple negative breast cancer.

Qian Long,1 Yizhuo Li,1 Ge Qin,1 Changlin Zhang,1 Dingbo Shi,1 Miao Chen,1 Shuihan Shi,2 Kefang Zhang,2 Wuguo Deng1. 1 _Sun Yat-sen University Cancer Center,Key State Laboratory of Oncology in South China, Guangzhou, China;_ 2 _Global Life Care Institute, Guangzhou, China_.

Programmed cell death protein-1 (PD-1)/programmed death ligand-1 (PD-L1) interaction plays a crucial role in tumor-associated immune escape. Here we verified that triple-negative breast cancer (TNBC) had higher PD-L1 expression than other subtypes. We then discovered that nucleophosmin (NPM1) bound to PD-L1 promoter specifically in TNBC cells and activated PD-L1 transcription, thus inhibiting T cell activity in vitro and in vivo. Furthermore, we demonstrated that PARP1 suppressed PD-L1 transcription through its interaction with the nucleic acid binding domain of NPM1, which was required for the binding of NPM1 at PD-L1 promoter. Consistently, the PARP1 inhibitor olaparib elevated PD-L1 expression in TNBC and exerted a synergistic effect with anti-PD-L1 therapy. Together, our research has revealed NPM1 as a novel transcription regulator of PD-L1 in TNBC, which could lead to potential therapeutic strategies to enhance the efficacy of cancer immunotherapy.

#4115

Naturally circulating anti-LAG3 antibodies exist in cancer patients.

Ilya Tsimafeyeu,1 Pavel Ershov,2 Leonid Kaluzhskiy,2 Jean-Philippe Couture,3 Hugo Gagnon,3 Vladimir Kuznetsov,4 Aleksei Kalachev,5 Alexis Ivanov,2 Gennady Bratslavsky1. 1 _ILGen Inc., Wilmington, DE;_ 2 _Institute of Biomedical Chemistry, Moscow, Russian Federation;_ 3 _PhenoSwitch Bioscience Inc., Sherbrooke, Quebec, Canada;_ 4 _Stolichnaya Diagnostica Clinic, Tuchkovo, Russian Federation;_ 5 _Kidney Cancer Research Bureau, Moscow, Russian Federation_.

To date there was no evidence of existence of the naturally circulating antibodies to checkpoint receptors in humans. LAG3 checkpoint is crucial in modulation of immune response that is altered in humans with inflammatory and malignant conditions. We have previously described novel methodology in identification of diagnostic and therapeutic antibodies (US Patent #9810694). Here we report identification of novel potentially therapeutic fully human anti-LAG3 antibodies in cancer patients.

Anti-LAG3 antibody presence was evaluated in 25 individuals: 11 healthy donors (controls) and 14 cancer patients in two different laboratories blinded to clinical information. Surface plasmon resonance analysis was done using the optical biosensor BiacoreT200 where LAG3 protein was covalently immobilized on the optical chip and biosensor signals from human serum samples were analyzed. Western Blotting used recombinant LAG3 loaded on a 10% SDS PAGE followed by blotting with plasma samples followed by biotinylated anti-human IgA/IgG/IgM and IrDye 800 streptavidin. Plasma anti-LAG3 IP utilized recombinant LAG3 crosslinked to MagResyn Carboxyl beads followed by incubation with plasma, followed by a biotinylated anti human IgA/IgG/IgMand IrDye streptavidin. No anti-LAG3 antibodies were detected in control group.

Among three assays there was complete correlation for presence of anti-LAG3 antibody in 3 cancer patients. One of these patients had sufficient plasma quantity and anti-LAG3 concentration (21 - 33 µg/ml) that should allow for the antibody purification and de novo sequencing. The novel fully human anti-LAG3 antibody will be identified, purified, and the sequence will be determined.To our knowledge this is the first report of checkpoint antibody presence in humans. Further study with cloning and evaluation of therapeutic properties of novel fully human anti-LAG3 antibody in xenograft models will be performed.

#4116

Immune cell checkpoint profiling of solid tumors by multiplex immunofluorescence.

Amanda Finan, Nicolas Goulange, Manon Motte, Maroua Tliba, Alexandra Mace, Jean-Philippe Coton, Domenico Lazzaro, Renaud Burrer. _Histalim, Montpellier, France_.

Immune checkpoint proteins are important regulators in self-tolerance. However, these same molecules also allow cancer cells to evade immune destruction. Checkpoint inhibitor (CKI) blockade therapies that aim at restoring antitumoral immunity have resulted in long-lasting remission in patients with cancers that were previously seen as incurable. However, only a fraction of the patients respond to these treatments. Research is currently focusing on identifying biomarkers that can predict a response to CKI therapies and stratify patient populations. As it is likely that several CKI need to be targeted at once to restore immune responses, numerous clinical trials associating more than one CKI are under evaluation. We have developed a multiplex immunohistochemistry panel, validated by in-depth image analysis, for immune cell checkpoint profiling. The base of the panel consists of CD3/CD8/PD-1/PD-L1 with the capability of adding additional targets including other immune checkpoint (e.g. TIGIT, SIRPα, CTLA-4) or tumor markers. Here we present our validation assay which uses the base panel and the ability to interchange the additional targets. Assay conditions for the individual biomarkers, including antigen retrieval, antibody concentration, panel position, and efficiency of stripping, are optimized. Simplex protocols are analyzed to ensure accuracy and specificity of the staining, success of stripping, and a balance of signal intensities. Validation includes the comparison of staining (% positive cells or % positive area) between a reference protocol and the position of interest. Multiplex optimization consists of staining serial, single stained slides of the individual biomarkers and the multiplex slide in parallel. The staining concordance (% positive cells) is evaluated with imaging software. Repeatability and robustness of the panels are validated by image analysis. This technique gives clinicians the potential to determine candidate patients for CKI single or double therapies and their progress during treatment. The established, rigorous workflow that we have developed allows our customers to adapt the panels to their needs and to have the highest confidence in the consequent images and results related to the profiling of immune cell checkpoints in solid tumors.

#4117

Novel bead-based multiplex assay for the quantification of human immune checkpoint biomarkers.

Jason Lehmann, Priyanka Rughwani, Amy Zhao, Weiping Jiang, Shaoquan Ji, Binggang Sun. _BioLegend, San Diego, CA_.

Immune checkpoint molecules are important regulators of immune activation. This regulation is critical to maintaining immune homeostasis, and is driven by a complex interplay of both co-stimulatory and inhibitory signals. These checkpoints are key to preventing autoimmunity in healthy individuals; however, cancer cells often exploit this system by modulating the expression of these molecules in order to suppress anti-tumor responses during host immuno-editing. The ability to accurately quantify expression levels of checkpoint molecules is critical to ongoing biomedical research efforts exploring potential therapeutic interventions to overcome tumor-mediated immune-evasion.

To further aid these ongoing research efforts, we have developed a new bead-based multiplex assay panel specifically designed to quantify several human oncology-related immune checkpoint biomarkers including sCD25 (IL-2Ra), GITR, 4-1BB, sCD27, B7.2 (CD86), free active TGF-β1, CTLA-4, PD-L1, PD-L2, PD-1, TIM-3, LAG-3, Galectin-9. The panel utilizes fluorescence-encoded microsphere technology and is designed for use on commonly available flow cytometers. The panel has been extensively validated for specificity, dilution linearity, cross-reactivity, and inter- and intra-assay precision.

This multiplex panel is a robust tool for measuring the concentrations of several immune checkpoint mediators in human serum, plasma, and cell culture supernatant samples, and offers greater efficiency and broader dynamic ranges compared to conventional ELISA-based quantification methods.

#4118

Molecular cytometry for immunotherapy trials.

Woodrow E. Lomas,1 Aidan F. Winters,1 Jason Alexandre,1 Jody Martin,2 Nidhanjali Bansal,2 Margaret Nakamoto,2 Brent Gaylord,2 Suraj Saksena,2 Pratip K. Chattopadhyay1. 1 _NY School of Medicine, New York, NY;_ 2 _BD Biosciences, San Jose, CA_.

The ability to simultaneously query both protein and mRNA expression on tens of thousands of single cells has emerged only recently, with the development of CITE-seq, REAP-seq, and Ab-seq platforms. Each technology relies on antibodies conjugated to oligonucleotide tags, followed by capture of antibody-stained cells for single cell RNA-sequencing. The technologies have important advantages over flow cytometry, chiefly in that they allow study of a limitless number of parameters, and single cell RNA sequencing, which cannot identify cell populations with as much verity as protein/antibody-based analysis. We characterized the newest molecular cytometry technique (Ab-seq, BD Biosciences) by titrating nearly 100 antibodies (in a single staining experiment and sequencing run), and demonstrated that oligonucleotide antibodies exhibit the same staining characteristics and patterns as flow cytometry antibodies. We also comprehensively examined cells discordant for protein and mRNA expression, in order to discriminate true features of immune cell biology from artifacts caused by drop-out in sequencing assays. Importantly, we also found that mRNA expression could guide the discrimination of cells expressing a protein versus those lacking expression, and define a remarkably low limit of detection for the technology: 1-5 molecules. This remarkable sensitivity will allow detection of cells expressing even the lowest levels of immune exhaustion and checkpoint molecules in cancer immunotherapy trials, and could enable better biomarker discovery than existing technologies. Moreover, this approach allows more detailed characterization of the tumor microenvironment and periphery than ever before. Indeed, in a lymphoma patient, we show that cells expressing both PD1 and CTLA4 carry a unique and specific signature that includes markers never revealed by flow cytometry or other technologies, which might represent new targets for immunotherapy. In sum, this presentation will characterize this new technology and demonstrate the power and promise of new molecular cytometry tools.

#4119

Atezolizumab and Bevacizumab attenuate cisplatin resistant ovarian cancer progression synergistically via suppressing epithelial-mesenchymal transition.

Ying Chen, Lei Zhang, Fangxuan Li, Lewen Bao, Wenxin Liu. _Tianjin Medical University Cancer Hospital and Institute, Tianjin, China_.

The AURELIA trial demonstrated that adding Bevacizumab (BEV) to chemotherapy significantly improved progression-free survival (PFS) for platinum resistant recurrent ovarian cancer. Recently, immunotherapy also presented potential anti-tumor effects in several malignant solid tumors. This study aimed to investigate whether combining anti-PD-L1 Atezolizumab with BEV may have a synergistic effect and enhance the efficacy of both treatments in cisplatin resistant epithelial ovarian cancer (CREOC). We retrospectively analyzed 124 epithelial ovarian cancer (EOC) patients from Gynecologic Oncology Department of Tianjin Cancer Hospital between January 2013 and June 2018, who all were diagnosed with cisplatin resistance due to progressing < 6 months after completing platinum-based therapy. Based on responding to at least 2 cycles of BEV-containing chemotherapy (BC), these patients were divided into BC response group and BC non-response group. Immunohistochemistry was used to detect that PD-L1 expression and tumor angiogenesis-related proteins (VEGF and Semaphorin4D) in tissues from 124 patients with CREOC. The positive expressions of PD-L1, VEGF, and Semaphorin4D (SEMA4D) were found in 58.73%, 50.79%, and 71.43% of the 63 cases CREOC tissues with BC response, respectively, which were significantly higher than that in the 61 cases BC non-response group (P<0.05). PD-L1 expression correlated with SEMA4D and VEGF positively (ρ=0.344 and 0.363, P<0.001). Over-expressions of PD-L1, VEGF and SEMA4D are associated with more malignant clinicopathologic characteristics of CREOC Patients. In survival analysis, patients' response to BC was the independent factor for evaluation of PFS and overall survival (OS). Furthermore, cell functional assays showed that Atezolizumab in combination with BEV inhibited the proliferation, migration, and invasion of cisplatin resistant ovarian cancer cell line A2780cis in vitro synergistically, which maybe associate with BEV suppressing the epithelial-mesenchymal transition (EMT) and PD-L1 expression by targeting STAT3. These findings suggest a novel therapeutic strategy for cisplatin resistant recurrent EOC and its mechanism warrants further study.

#4120

Dose selection and dosing optimization of immune checkpoint inhibitors.

Jennifer Sheng, Kinjal Sanghavi, Shivani Srivastava, Akintunde Bello. _Bristol-Myers Squibb, Princeton, NJ_.

Background: The differential mechanisms of action of immune checkpoint inhibitors and targeted therapies versus chemotherapy warrant new thinking regarding the conventional maximum-tolerated dose (MTD) approach. Dosing optimization of immune checkpoint inhibitors via flat-dosing compared with body-weight-based dosing reduces dosing errors, provides benefits for patients, and is advantageous for healthcare providers. A review of the strategies used for dose selection and optimization for approved checkpoint inhibitors is presented.

Methods: Clinical pharmacology data on dose selection and optimization for FDA-approved immune checkpoint inhibitors from FDA Summary Basis of Approval documents, published research papers, and scientific meeting/congress presentations were summarized.

Results: Of the 7 immune checkpoint inhibitors approved by the FDA and/or the EMA, none had an MTD determined during their phase 1 dose-escalation programs. Dose selections were based on the integration of preclinical and clinical information, including in vitro and/or in vivo receptor target engagement, preclinical and clinical pharmacokinetic/pharmacodynamic data, and efficacy and safety data. Chosen regimens were confirmed by exposure–response analyses using phase 3 data. Post-approval optimization strategies were employed to refine the dosing schedules (eg, provide beneficial flexibility to patients and caregivers). Interestingly, 6 of the 7 currently approved immune checkpoint inhibitors use a flat-dosing regimen, with 4 changing prescribing information to flat-dosing after the initial approval of dosing by body weight, and 3 of them with fixed dosing in the initial submission.

Conclusion: The new era of oncology therapy, epitomized by the advent of immuno-oncology and molecular targeted agents, has been accompanied by a shift from the MTD-based approach developed for cytotoxic drugs to those that aim to achieve optimized doses and regimens using the totality of preclinical and clinical data. Close collaborations between discovery and biomarker scientists, clinicians, clinical pharmacologists, and pharmacometricians facilitate the selection of optimal regimens. As the number of indications covered by immune checkpoint inhibitors increases, and clinical experience accumulates, it is likely that the transition from body-weight-based to fixed dosing will move to earlier stages of drug development. These efforts enable the early identification of the optimal dosing regimens, reducing development time and increasing the probability of success. Importantly, the utilization of optimized dosing development paradigms enables the development of oncology drugs with improved efficacy and tolerability profiles, thus providing increased benefits to patients and healthcare providers.

#4121

Preclinical characterization of CS1003, a novel clinical-stage PD-1 monoclonal antibody.

Fu Li,1 Jingrong Li,1 Ke Yin,1 Juan Zhang,1 Zhenhu Li,1 Liang Lu,1 Yuanwu Bao,1 Dan Pu,1 Zhen Qin,1 Yong Zheng,2 Baotian Yang,2 Jing Li,2 Xinzhong Jon Wang1. 1 _CStone Pharmaceuticals Co., Ltd., Shanghai, China;_ 2 _Wuxi Biologics Co. Ltd., Shanghai, China_.

Background: The programmed cell death protein 1 receptor (PD-1) is an immune-checkpoint protein that negatively regulates the immune system to avoid collateral damage to self-tissues. Tumors hijack this mechanism as a way to avoid immune detection and destruction. Presently, the FDA has approved three anti-PD-1 antibodies for the treatment in more than ten cancer types. CS1003 is a novel anti-PD-1 mAb recognizing a unique epitope developed to disrupt the PD-1 interaction with PD-L1/PD-L2 to restore or improve T-cell function as stand-alone therapy or in combination with other approaches.

Methods: A panel of rat PD-1 mAbs were generated through conventional hybridoma technology. Several mAbs with favorable characteristics were further humanized. CS1003, a humanized, hinge-stabilized IgG4 mAb, was selected as our lead candidate for further characterization including antigen binding, biophysical properties as well as functional characterization.

Results: CS1003 binds mouse, cynomolgus monkey and human PD-1 with similar affinity. CS1003 bound PD-1-expressing cell lines and chronically-activated T cells, blocked PD-1 interactions with PD-L1/PD-L2, resulting in inhibition of PD-1 signaling, and enhanced T cell cytokine secretion and proliferation to levels comparable to those observed with nivolumab or pembrolizumab reference molecules. CS1003 showed significantly anti-tumor activity in conventional MC38 as well as in hPD-1 knock-in mice. CS1003 showed no unexpected cross-reactivity in human tissues, with specific staining observed primarily in lymphocytes of lymphoid organs. In a pharmacokinetic (PK) study in cynomolgus monkeys following single intravenous administration at multiple dose levels, PK properties were linear with the proportionally increasing exposures from 2-18 mg/kg. In a repeated-dose toxicity study, CS1003 was well tolerated in cynomolgus monkeys and the major finding was mononuclear infiltration in multiple organs, which was consistent with its pharmacologic activity. CS1003 demonstrated a favorable safety profile with the highest non-severely toxic dose (HNSTD) of 100 mg/kg.

Conclusion: CS1003 is a novel anti-PD-1 mAb with favorable preclinical characteristics and safety profile in cynomolgus monkeys. The unique feature of CS1003 is its cross-reactivity with both human and murine PD-1. This should greatly facilitate further evaluation its potential for combination therapy in various syngeneic mouse tumor models. A phase I study for CS1003 is being conducted to evaluate the safety, tolerability, PK and anti-tumor activity in patients with advanced tumors [NCT03475251].

#4122

The regulatory mechanism of PD-L1 level through ER-associated degradation.

Jong-Ho Cha, Wen-Hao Yang, Weiya Xia, Yongkun Wei, Li-Chuan Chan, Chia-Wei Li, Heng-Huan Lee, Mien-Chie Hung. _University of Texas MD Anderson Cancer Center, Houston, TX_.

Programmed death ligand-1 (PD-L1) is an important immune checkpoint molecule that allows cancer cells to evade immune surveillance. Further, the PD-L1 level is relatively higher in tumor tissues compared to normal tissues. Thus, the PD-L1/PD-1 axis has served as primary target for cancer immunotherapy. However, the regulatory mechanisms of PD-L1 level are not fully understood.

Here, we demonstrated that the ER-associated degradation (ERAD) mechanism of PD-L1 through cross-talking between phosphorylation and glycosylation. We discover that AMP-activated protein kinase (AMPK) activated by metformin directly binds with PD-L1 in the ER lumen and phosphorylates Ser 195 of PD-L1. The Ser 195 phosphorylation induces abnormal mannose-rich glycan structures of PD-L1 through excessive ER trimming process. PD-L1 with abnormal glycan structures is occupied by ER quality check components in the ER and finally degraded through ERAD pathway.

We provide new insights to understand cancer immune checkpoints by identifying a new regulatory mechanism of PD-L1 and these findings can be used to improve the efficacy of previous cancer immunotherapy for the PD-L / PD-1 axis.

#4123

Clinical significance and the difference between programmed death-1 ligand-1 and ligand-2 in patients with esophageal cancer.

Kazuo Okadome, Yoshifumi Baba, Taisuke Yagi, Yuki Kiyozumi, Kojiro Eto, Yukiharu Hiyoshi, Yohei Nagai, Takatsugu Ishimoto, Masaaki Iwatsuki, Shiro Iwagami, Yuji Miyamoto, Naoya Yoshida, Hideo Baba. _Kumamoto University, Kumamoto, Japan_.

Background: Immunotherapies targeting the PD-1/PD-L1 pathway have emerged as promising therapeutic strategies for many cancers. PD-L1 and PD-L2 are ligands of PD-1 on T-cells and prevent T-cells activation and proliferation. There have been reported that PD-L1 and PD-L2 are expressed on not only antigen-presenting cells but also some types of tumors. Clinical and prognostic features of the PD-L1 have been widely reported for many types of cancers but those of PD-L2 remain unclear in esophageal cancer. The aim of this study was to analyze the clinical significance and the difference between PD-L1 and PD-L2 in esophageal cancer.

Methods: Using a database of 437 curatively resected esophageal cancer from April 2005 to March 2016, we evaluated PD-L1 and PD-L2 expression in tumor cells by immunohistochemical staining and analyzed the association between those with clinicopathological features. Double immunohistochemical staining for PD-L1 and PD-L2 was performed to confirm the localization of each positive cell.

Results: Out of 437 cases of esophageal cancer, 69 cases (15.8%) showed PD-L1 positive and 71 cases (16.2%) showed PD-L2 positive expression. There was no significant relationship between PD-L1 and PD-L2 positive cases (p=0.18). In both PD-L1 and PD-L2 positive cases, double immunohistochemical staining for PD-L1 and PD-L2 showed that there was a heterogeneity for PD-L1 and PD-L2 positive tumor cell. PD-L1 positive cases were significantly associated with tobacco use (p=0.013), pathological stage (p=0.001), and preoperative therapy present cases (p=0.015). In the other hand, PD-L2 positive cases were significantly associated with younger age (p=0.029). Thus PD-L1 and PD-L2 patient characteristics were likely different. Overall survival rates of the patients with PD-L1 or PD-L2 positive were significantly worse than those with PD-L1 or PD-L2 negative (p=0.009 and p=0.013, respectively).

Conclusion: There was no significant relationship between PD-L1 and PD-L2 expression, and patient characteristics of those were different in patients with esophageal cancer. But both PD-L1 and PD-L2 expression were significantly associated with prognosis, supporting their roles as prognostic biomarkers.

#4124

Expressions of co-inhibitory / co-stimulatory molecules may impact immune checkpoint therapies.

Pirouz M. Daftarian,1 Marybeth George,1 Eden Kleiman,1 Wushouer Ouerkaxi,1 Amy Yamamura,2 Zhongliang Li,2 Mingfa Zang,2 Derron Yu,2 Eunmi Hwang,2 Annie X. An,2 Ann E. Lin,2 Henry Li2. 1 _JSR Micro, Inc., Sunnyvale, CA;_ 2 _Crown Bioscience, La Jolla, CA_.

Screening patient peripheral blood using our proposed non-invasive method can boost the accuracy in selecting proper candidates for immune checkpoint therapy in the clinical setting, thereby leading to higher success in treatment. The immune system is regulated by a sophisticated network of modulatory molecules. In chronic infections and cancers, T cell exhaustion can arise when clearance of antigens is incomplete due to sustained expression of co-inhibitory molecules. T cell exhaustion has been exploited by some cancers, and also described in chronic infections with latency. Such exhausted T cells may be reversed with the right immune checkpoint blockade, thereby restoring effector T cell function. We have optimized a T cell Exhaustion Recall Antigen Assay, demonstrating T cell exhaustion may be reversed by immune checkpoint blockades in certain hosts. We hypothesize that understanding of the capacity and expressions of co-inhibitory molecules assist in predicting responders for specific immune checkpoint therapies. We have used a flow cytometry-based approach to examine changes in an array of immune checkpoint molecules on activated T cells and antigen presenting cells. We first tested the effect of anti-PD1 on tumor antigen-specific activated T cells using splenocytes from PMEL T cell receptor (TCR) transgenic mouse, which expresses mouse homologue of human premelanosome protein, or gp100. Activated CD3+CD8+ gp100-specific T cell population and IFNγ production were measured by flow cytometry. Secondly, we evaluated the expression levels of various immune checkpoint molecules post anti-CD3 stimulation of human peripheral blood mononuclear cells (PBMC) from characterized healthy hosts. Surprisingly, we found high expressions of co-inhibitory and co-stimulatory molecules including CTLA-4, PD1, PDL-1/PDL2, TIGIT, LAG3, TIM3, GITR, are associated with poor response to immune checkpoint blockades. Levels of activated T cells and cytokine production in donors with high checkpoint receptors showed lower T cell activations and cytokine production. Screening patient peripheral blood using our proposed non-invasive method can boost the accuracy in selecting proper candidates for immune checkpoint therapy in the clinical setting, thereby leading to higher success in treatment.

#4125

Precise and reproducible measurement of tumor mutational burden using panel sequencing: the role of panel size and of mutation types.

Jan Budczies, Eugen Rempel, Michael Allgäuer, Daniel Kazdal, Volker Endris, Peter Schirmacher, Albrecht Stenzinger. _Heidelberg University Hospital, Heidelberg, Germany_.

Tumor mutational burden (TMB) is an emerging biomarker for patient stratification for immune checkpoint inhibition. Implementing TMB measurement by panel sequencing, experimental setup and bioinformatics pipelines need to be optimized to minimize sources of error and variance. To gain insight into contributors to TMB variance, combinatorial calculations were performed based on the hypothesis that each base in the coding sequence was mutated with the same probability. Simulations of sequencing panels in the TCGA data were performed to check for additional contributors to the variance. A narrow definition of TMB including only missense mutations was compared to a broader definition additionally including other mutation types. The quantity under investigation was TMB measured in relatively to the size of the panel (per Mbp). The combinatorial contribution to the coefficient of variation (CV) of TMB turned out to be inversely proportional to the square root of the panel size and inversely proportional to the square root of the TMB. For a tumor with TMB of 10 muts/Mbp, the combinatorial CV increased drastically for panels sizes smaller than 1.5 Mbp. The combinatorial variance represents a persistent contribution to the error of TMB calculation that cannot be avoided. Simulations in WES data confirmed the combinatorial variance as main contributor to the imprecision of TMB estimation. In many instances, the fold change between the number of all mutations in the coding sequence and the number of missense mutations was similar for tumors of the same cancer type. However, the fold changes were different in specific cancer types for example significantly higher in melanoma (1.66) compared lung adenocarcinoma (1.48). In particular, the former tumors were observed to include a higher proportion of synonymous mutations than the latter. The combinatorial source of error implies that a panel of least 1.5 Mbp is needed to precisely determine TMB for tumors ranging around typical currently used diagnostic thresholds (10 muts/Mbp). Additional sources of error might even favor larger panel sizes such as 3 Mbp. The combinatorial error can be reduced by including all mutations types instead of only missense mutations and simultaneously adjusting TMB with a cancer type specific factor.

#4126

Inferring target occupancy from fitting nonlinear-PK data with mechanistic PKRO model for Pembrolizumab.

Fei Hua, Lin Lin, Lore Gruenbaum, Bjorn L. Millard, John M. Burke, Joshua F. Apgar. _Applied BioMath, Concord, MA_.

Purpose: Pembrolizumab (Pembro), an anti-PD-1 antibody, has been approved for several cancer indications at 200 mg or 2 mg/kg IV every 3 weeks (Q3W). Understanding the percent target receptor occupancy (RO) for PD-1 at this approved dose can help with dose selection for other anti-PD-1 drugs in development. However, RO can be challenging to determine through direct experimental measurements. A mechanistic PKRO model was developed to fit to Pembro phase 1 PK data and was then used to predict RO at 2 mg/kg IV dosing Q3W.

Methods: A mechanistic PKRO model describes linear clearance of Pembro, tissue distribution of Pembro, binding to PD-1, binding to soluble PD-1 (sPD-1) and clearance of drug-PD-1 complex, clearance of drug-sPD-1 complex as well as drug distribution to tumor and binding to PD-1 in the tumor compartment. Binding between Pembro and PD-1 was fixed to 29 pM (Pembro BLA, Pharmacology Review) and half-life of internalization of drug-PD-1 complex was set to 2 hours.

Results: Since a wide range of sPD-1 concentration has been reported, different sPD-1 concentration was used to fit to the PK data. The model predicted PD-1 expression level in circulation to be approximately 10.8 pM with no soluble PD-1. In the presence of soluble PD-1, PD-1 expression is predicted to be higher to capture the non-linear PK. The model was then used to predict RO at 2 mg/kg IV dosing Q3W and > 98% RO was predicted in the tumor compartment with a range of sPD-1 concentrations from 0 to 3000 pg/mL tested. The impact of Kd and drug-PD-1 turnover rate on predicted RO was explored. When either of the parameters was varied by 10-fold higher or lower from its nominal value, the model still predicted greater than 90% RO at 2 mg/kg dose.

Conclusion: A mechanistic PKRO model was built to capture Pembro phase I PK data and then predicted near complete PD-1 target receptor occupancy at the approved dose. This approach with expanded model scope has the potential to be used to inform dose selection for other anti-PD-1, or I/O therapeutics, as a single therapy or in combination.

### Novel Immunomodulatory Agents 2

#4127

RX-5902 exhibits direct and immunomodulatory anti-tumor activities in melanoma PDX models.

Andrew Eisen. _Rexahn Pharmaceuticals, Rockville, MD_.

Background: Nuclear β-catenin promotes deleterious processes in tumors and their microenvironment. Diminishing nuclear β-catenin attenuates these oncogenic processes. RX-5902 is an oral, small molecule inhibitor of β-catenin nuclear translocation mediated by phosphorylated p68 (DDX5). RX-5902 exhibits potent anti-cancer activity in many cancer cell lines, xenograft models in immunodeficient mice and immune competent mice and in patients. This study examines the relative contribution and context of the direct and immunomodulatory effects on the anti-tumor activities of RX-5902.

Methods: Four patient-derived melanoma xenograft models were implanted into either immunodeficient nude mice or NOG mice "humanized" with HLA unmatched CD34+ stem cells with average humanization of 36%. The PDX models were derived from 2 tumors that were clinically unresponsive to an anti-PD-1 agent and 2 tumors obtained prior to any immune treatment. In each category one tumor had BRAF WT and one a V600E mutation. Mice were randomized to receive either vehicle or RX-5902 at 5, 15 or 50 mg/kg po QDx5/Off x2 when implanted tumors reached 100-120 mm3 and began treatment when tumors reached 200mm3. Treatment lasted for 28 days and animals were observed for an additional 14 off therapy.

Results: The tumor growth inhibition data at 50 mg/kg is found in the table below. There was evidence of an immune contribution to the TGI in those tumors derived from subjects who did not benefit from I/O (Nivolumab) therapy. In each category the response was greater in tumors with WT BRAF.

Conclusion: RX-5902 provided greater TGI in the two melanoma PDX models derived from subjects who were resistant to I/O therapy compared to the models from I/O naïve subjects and, regardless, is greater in BRAF WT vs mutant tumors. The RX-5902 immune-mediated anti-tumoral activity may benefit the majority of melanoma subjects with limited response to BRAF-directed and/or I/O therapy.

#4128

Identification and characterization of LHC165, a TLR7 agonist designed for localized intratumoral therapies.

Jonathan A. Deane,1 German A. Cortez,1 Chun Li,1 Nora Eifler,2 Shailaja Kasibhatla,1 Nehal Parikh,3 Shifeng Pan,1 Steven Bender1. 1 _GNF/Novartis, San Diego, CA;_ 2 _Novartis, Basel, Switzerland;_ 3 _Novartis, East Hanover, NJ_.

Checkpoint inhibition has transformed immunotherapy by alleviating T cell exhaustion in a subset of patients. However, an important component of effective immune targeting to expand the benefit of immune response requires engagement of both innate and adaptive responses. Our understanding of safe and effective engagement of the innate immune system is evolving, with multiple preclinical and clinical agents targeting pathways such the Toll-like Receptors. Here we disclose the structure and preclinical activity of LHC165, a benzonapthyridine TLR7 agonist that is adsorbed to aluminum hydroxide. The interaction between LHC165 and aluminum hydroxide allows for a slow release from the injection site resulting in improved efficacy in mouse models compared with free LHC165. This localization allows for immune activation at the site of the tumor and also results in lower systemic exposure and cytokine induction. Intratumoral studies in syngeneic preclinical studies show single agent activity and a benefit when dosed in combination with checkpoint blockade. LHC165 as a single agent and in combination with PDR001 is currently enrolling patients with advanced malignancies in CLHC165X2101.

#4129

Novel small molecule TLR7/8 agonists for enhancing NK cell-mediated ADCC.

Vidhi Khanna, Hyunjoon Kim, Wenqui Zhang, Peter Larson, David Ferguson, Jayanth Panyam. _University of Minnesota, Minneapolis, MN_.

Antibody‐dependent cell‐mediated cytotoxicity (ADCC) is a key mechanism of action for some therapeutic antibodies. ADCC involves killing of an antibody‐coated target cell by an effector cell through the release of cytotoxic or cell death‐inducing molecules. ADCC is triggered through interaction of Fcγ receptors present on the effector cell surface with the Fc region of the target-bound antibody. Natural killer (NK) cells are one of the primary effector cells that mediate ADCC. There is significant interest in designing therapeutic agents that can enhance ADCC because this can result in improved clinical responses with approved antibodies. We have developed a suite of highly substituted imidazoquinolines, which activate TLR 7 and/or 8 and induce significantly higher levels of cytokines compared to the FDA-approved TLR7 agonist imiquimod. In the current study, we evaluated our series of TLR7-specific, 8-specific and 7/8 dual selective agonists for their ability to improve ADCC with Cetuximab. We investigated NK cell activation in the presence of these compounds, as well as NK cell mediated ADCC against an EGFR expressing lung cancer cell line, A549. In addition, we also measured cytokine induction in human peripheral blood mononuclear cells in response to these compounds. Our studies show dual TLR 7/8 and 8-specific agonists induce robust pro-inflammatory cytokine secretion and activate NK cells. however, mixed agonists also induce greater immunosuppressive cytokines compared to TLR8-specific agonists. Further, these agonists also significantly enhanced Cetuximab mediated ADCC in vitro. In vivo studies examining the anticancer efficacy of the combination of selected TLR7/8 agonists and Cetuximab are ongoing.

#4130

Anti-cancer efficacy of systemic and Intratumoral YS-ON-001 as monotherapy or in combination with checkpoint inhibitor in syngeneic cancer models.

Zhongkai Shi, Yuan Liu, Yi Zhang. _Yisheng Biopharma, Rockville, MD_.

Background: YS-ON-001 is a complex containing synthetic double-strand RNA, a toll-like receptor 3 (TLR3) agonist. TLR3 agonist stimulates antigen-presenting cells, increases T, NK, NKT cells and decreases regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), reprogrames macrophages from protumorigenic to anti-tumor. YS-ON-001 is being evaluated for systemic and intratumoral use alone or combined with checkpoint inhibitor to treat cancer.

Method: The anti-tumor efficacy of systemically or intratumorally (it) injected YS-ON-001 alone or in combination with PD-1 antagonist was assessed in several syngeneic tumor models including pancreatic cancer, hepatocellular carcinoma. Tumors were implanted into left and/or right flanks while only one tumor was injected with YS-ON-001. YS-ON-001 was administered by IM injection every other day or it injection twice a week. Murine PD-1 antibody was infused intraperitoneally (ip) once a week. In addition to body weight, tumor volume was monitored on both flanks. Surviving mice with unnoticeable tumor were rechallenged. Flow cytometry was used to analyze immune cells from the tumors including T, NK, NKT cells, macrophages 1 and 2, Tregs, and MDSCs.

Results: Within tumor microenvironment YS-ON-001 IM monotherapy resulted in increased infiltration of CD4+ T cells and CD8+ T cells, NK cells and NKT cells, reduced Tregs and MDSCs, and increased the M1/M2 ratio, leading to significant tumor growth inhibition in more than 10 cancer models. Combination of YS-ON-001 im with anti-PD1 ip resulted in greater anti-tumor efficacy compared to anti-PD1 monotherapy in both 4T1 breast cancer and LLC2 lung cancer models. Rejecting tumor rechallenge in surviving mice from the initial challenge treated with IM YS-ON-001suggested long-term anti-tumor immunity. Monotherapy of YS-ON-001 it or anti-PD-1 ip significantly suppresses tumor growth not only at the primary injection site, but also at uninjected site (p<0.05). Combination of YS-ON-001 it with anti-PD-1 ip significantly enhanced suppressive effect of anti-PD1 at both primary injected and distant uninjected sites.

Conclusions: these experiments demonstrated systemic or intratumoral delivery of YS-ON-001 can modulates immune responses in tumor microenvironment by increasing anti-tumor cells in T, NK , NKt cells and decreasing pro-tumor cells in Treg, MDSC, leading to significant tumor growth inhibition. Combination of YS-on-001, IM or It with PD-1 antibodies significantly enhanced the anti-tumor efficacy of PD-1 antibodies. These results provide a strong rationale and support clinical development as standalone therapy or in combination with other modalities such checkpoint inhibitors.

#4131

Overcoming aryl hydrocarbon receptor mediated tumor immunosuppression.

Jeremy Tchaicha, Silvia Coma, Meghan Walsh, Jill Cavanaugh, Marta Sanchez-Martin, X. Michelle Zhang, Alfredo Castro, Jeff Ecsedy, Mark Manfredi, Karen McGovern. _Kyn Therapeutics, Cambridge, MA_.

Checkpoint inhibitors (CPIs) have become the cornerstone of immune-based oncology therapy; still many cancer patients do not benefit from these agents. Resistance to checkpoint inhibitors is due in part to the presence of immunosuppressive molecules which prevent immune activation despite T cell checkpoint inhibition. Aryl Hydrocarbon Receptor (AHR) is a transcription factor that mediates the immune response in multiple innate and adaptive immune cells subsequent to binding of a diverse set of endogenous and exogenous ligands. One such AHR agonist is kynurenine. Kynurenine, metabolized from tryptophan by indoleamine-pyrrole 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase 2 (TDO2), binds to AHR and leads to a net immunosuppressive tumor microenvironment.

Given that kynurenine can be synthesized by both IDO1 and TDO2 and that AHR is activated by endogenous ligands other than kynurenine, AHR inhibition provides a novel approach to reverse immunosuppression in a broad range of tumor types. We demonstrated that AHR antagonism affects multiple immune cell types and can lead to pro-inflammatory phenotypes in human T cells and myeloid cells in vitro and in murine tumor models. AHR antagonism inhibits growth in the B16-IDO, CT26 and MC38 models and reverses the immunosuppressive microenvironment as indicated by changes in T cell and myeloid cell populations. Immune signature changes characterized via Nanostring in multiple tumor models treated with AHR antagonists were also assessed.

Oral dosing of AHR antagonists led to tumor growth inhibition as a single agent and increased anti-tumor activity when combined with checkpoint inhibitors. In addition, beneficial anti-tumor activity occurred with AHR antagonists combined with chemotherapy or with radiation therapy in syngeneic mouse tumor models. Our data indicate that reversing AHR-mediated immune suppression in the tumor microenvironment drives anti-tumor activity alone and in combination with other therapeutic modalities.

Overall, our data demonstrates that AHR is an attractive target for reversing immune suppression in tumors. Therefore, we are developing AHR antagonists and translational insights to treat patients most likely to benefit from this approach.

#4132

Preclinical characterization of TP-018, a novel ligand of AhR with potent anti-tumor activity.

Suoming Zhang, Luqing Yang, Guodong Li. _Shanghai Tangrun Pharmaceuticals, Shanghai, China_.

The aryl hydrocarbon receptor (AhR) is a cytoplasmic receptor and transcription factor, activated by structurally diverse compounds from the environment, diet, microbiome, and host metabolism. Emerging evidence suggests that AhR is a key sensor allowing immune cells to adapt to environmental conditions, and changes in AhR activity have been associated with autoimmune disorders and cancer. Furthermore, both exogenous and endogenous AhR ligands impact immune disease outcomes, identifying AhR as a potentially actionable target for immunotherapy. Here we report the results with TP-018, which exerts a potent anticancer effect via the activation of AhR pathway. TP-018 strongly suppressed H22 liver tumors established in syngeneic model with balb/c mice. In identifying this agonist, we synthesized a series of small-molecule agonists of the human AhR. In the cell-based luciferase reporter assay, TP-018 showed activation of human AhR at a low nano molar EC50. To further evaluate their effect of AhR activation on immuno-modulation and tumor growth, we selected several compounds from our series, including TP-018, a highly potent agonist of AhR, for further evaluation in vivo. Dosed at 10-80mg/kg (ip, QD) in the H22 liver syngeneic mouse model, TP-018 resulted in a reduction of tumor growth. Interestingly, the combination of TP-018 and a PD-1 immune checkpoint inhibitor significantly reduced tumor growth. Immune studies are undergoing to elucidate the molecular and cellular mechanisms. Our results indicate that TP-018 is a potential clinical candidate and targeting AhR activation could emerge as the next paradigm in cancer immunotherapy.

#4133

**The** in vitro **melanoma tumor microenvironment conditions macrophages to an immunosuppressive M2-like phenotype, which is reversible by oncolytic virus ORCA-010 with immune modulators.**

Ioanna E. Milenova,1 Rieneke van de Ven,2 Wenliang Dong,1 Victor W. van Beusechem,2 Tanja D. de Gruijl2. 1 _ORCA Therapeutics, Amsterdam, Netherlands;_ 2 _Amsterdam UMC, VUmc, Amsterdam, Netherlands_.

The melanoma tumor environment suppresses the immune reaction, favoring tumor escape from the immune system. This suppressive environment is in part sustained by tumor associated M2-like macrophages. In order to activate the immune system, one approach is macrophage-targeted therapies, that repolarize M2-like macrophages to M1-like macrophages. ORCA-010 is a potency enhanced, replication selective oncolytic adenovirus, with strong anti-tumor activity.

As a model of macrophage polarization in the tumor microenvironment, human melanoma cell lines were co-cultured in vitro with CD14+ monocytes. The effects to induce M1-like macrophages by addition of lipopolysaccharide (LPS) and interferon (IFN)-gamma, JAK2 or p38 MAPK inhibitors combined with ORCA-010, was assessed in the co-cultures. Cell phenotypes were characterized by flow cytometry.

Our data demonstrate that ORCA-010 significantly reduces M2-like macrophage marker CD163 on CD45+ cells in co-cultures, and increases co-stimulatory M1-like macrophage markers CD80 and CD86 expression on macrophages. CD80 is significantly up-regulated by ORCA-010 with LPS and IFN-gamma. Next, the macrophage polarizing effects of combining ORCA-010 with p38 MAPK and JAK2 inhibitors, known for their favorable myeloid M1 and dendritic cell skewing properties, were assessed in the melanoma co-cultures. The inhibitors JAK2 (AG490) and p38 MAPK (LY2228820) both significantly reduced CD163 levels, however did not induce increased CD80 and CD86 by themselves. Only when combining these with ORCA-010 did we observe a CD86 up-regulation.

In a B16.OVA immunocompetent mouse model, ORCA-010 appeared to enhance recruitment of CD8+ T cells to the tumor. Interestingly, combining ORCA-010 with anti-PD-1 resulted in a significant increase of recruitment of CD8+ T cells at the tumor, which could be further enhanced by co-treatment with a p38 inhibitor.

Our data suggest that ORCA-010 induces a repolarization of the M2-like macrophages to a favorable M1-like phenotype. This can be further enhanced by the combined administration of ORCA-010 with a TLR4 agonist and IFNγ, or with a JAK2 inhibitor.

This work is supported by the European Union Horizon2020 ITN-EID grant 643130 "VIRION".

#4134

Reversal of adenosine-mediated immune suppression by CB-708, an orally bioavailable and potent small molecule inhibitor of CD73.

Clarissa C. Lee, Deepthi Bhupathi, Roland J. Billedeau, Jason Chen, Lijing Chen, Ethan D. Emberley, Matthew I. Gross, Tony Huang, Weiqun Li, Yong Ma, Andrew L. Mackinnon, Amani Makkouk, Gisele M. Marguier, Silinda Neou, Francesco Parlati, Natalija Sotirovska, Timothy F. Stanton, Susanne M. Steggerda, Jing Zhang, Winter Zhang, Jim Li. _Calithera Biosciences, Inc., South San Francisco, CA_.

Introduction: High adenosine (ADO) in the tumor microenvironment suppresses the immune response against cancer cells by inhibiting immune effector functions and promoting the development of immunosuppressive cells. Extracellular ADO can be generated from ATP released by cells undergoing stress or death through the combined actions of the ecto-nucleotidases CD39 (ATP to AMP) and CD73 (AMP to ADO). Inhibition of ADO production via CD73 is a promising therapeutic approach for the treatment of cancer.

Methods: The potency of CB-708 was evaluated against recombinant CD73 and CD73-expressing cells using a malachite green assay. Selectivity against related ecto-nucleotidases was also assessed. Inhibition of CD73 in plasma was measured using LC/MS to assess conversion of 15N5-AMP into 15N5-ADO. The ability to reverse AMP-mediated immune suppression of human CD8+ T cells was determined by adding exogenous AMP during T cell activation. T cell proliferation was assayed by flow cytometry and cytokine levels were measured by ELISA. The EG7 syngeneic tumor model was used to assess the therapeutic effect of CB-708.

Results: CB-708 is an orally bioavailable small molecule inhibitor that can potently inhibit both soluble human CD73 (IC50 = 170 pM) and cell-bound human CD73 (IC50 = 210 pM), but does not inhibit human CD39 (IC50>10 µM), ENTPD2 (IC50>10 µM), nor ENTPD3 (IC50>10 µM). CB-708 retained high potency in the presence of plasma and reversed AMP-mediated suppression of human CD8+ T cell proliferation and production of IFNγ (EC50 = 4.5 nM) and granzyme B (EC50 = 5.6 nM) in vitro. Orally administered CB-708 had dose-dependent single-agent tumor growth inhibition in the EG7 mouse syngeneic tumor model and that was associated with pharmacodynamic inhibition of plasma CD73. Enhanced tumor growth inhibition was observed when anti-PD-L1 was combined with a highly related analog of CB-708 in the EG7 model.

Conclusion: CB-708 is an orally bioavailable and highly potent small molecule inhibitor of CD73. CB-708 reverses the immunosuppressive effects of AMP-derived ADO in vitro and in vivo and leads to anti-tumor activity as a monotherapy. CB-708 is expected to enter clinical development in 2019.

#4135

**Novel dual A** 2A **A** 2B **adenosine receptor antagonists for cancer immunotherapy: in vitro and in vivo characterization.**

Michal Galezowski, Paulina Węgrzyn, Aneta Bobowska, Katarzyna Dziedzic, Joanna Szeremeta-Spisak, Marcin Nowogrodzki, Grzegorz Satala, Alicja Obara, Iwona Lozinska-Raj, Marcelina Dudek, Anita Janiga, Jacek Reus, Marek Wronowski, Magdalena Zastawna, Grzegorz Statkiewicz, Maciej Rogacki, Magdalena Ziembik, Karolina Grycuk, Foteini Soukou, Kinga Michalik, Agnieszka Adamus, Karolina Wiatrowska, Natalia Lewandowska, Aniela Golas, Olga Haberkiewicz, Rod Porter, Krzysztof Brzozka, Mateusz Nowak. _Selvita S.A., Krakow, Poland_.

Adenosine is important messenger molecule that is involved in signal transmission in central nervous, cardiovascular and immunological systems. The immunosuppressive role of adenosine attract attention of many researchers recently and reversal of that effect is addressed by several different approaches i.e.: inhibition of enzymes that are producing the adenosine in tumor microenvironment like CD39 and CD73 but also the antagonization of adenosine receptors mainly A2A and A2B subtypes. Here we present a novel series of unsurmountable, dual A2A/A2B antagonists that retain its nanomolar potency in tumor-like adenosine-rich environment. This advantageous profile comes mainly from long residence time in adenosine receptors which transfers into high receptor occupancy even after full clearance of the compound from plasma. We are demonstrating how our inhibitors restore adenosine depleted immune functionality in the series of functional in vitro assays in primary cells. This includes the cytokine release by activated CD4+ and CD8+ human T-lymphocytes (driven by A2A) and dendritic cells (driven by A2B). Most importantly presented compounds show improved in vivo pharmacological profile in comparison to adenosine inhibitors currently tested in clinical trials manifested by high inhibition of CREB phosphorylation in mouse model.

#4136

Direct intratumoral microdosing via the CIVO® platform reveals anti-tumor immune responses induced by the SUMO inhibitor TAK-981.

Beryl A. Hatton,1 Marc Grenley,1 James Garnsey,2 Vaishali Shinde,2 Dennis Huszar,2 Connor Burns,1 Sally Ditzler,1 Angela Merrell,1 Joyoti Dey,1 Emily Beirne,1 Richard A. Klinghoffer1. 1 _Presage Biosciences, Seattle, WA;_ 2 _Takeda Oncology, Cambridge, MA_.

The complex interplay between a drug, the tumor and the surrounding microenvironment is a critical determinant in how a patient will respond to their selected cancer therapy. Yet to date, this complex response has been difficult to evaluate prior to late stage clinical investigation. To enable a means for testing investigational agents earlier in the development process but still directly in human patients, in a way that limits the risk of adverse effects and provides an indication of efficacy, Presage Biosciences developed a technology called CIVO. This platform allows for simultaneous assessment of multiple drugs or drug combinations directly in a single solid tumor, in the context where the drugs are ultimately intended to be used, directly in the patient. Here we demonstrate the potential for using the CIVO platform in Phase 0 microdosing studies to detect complex responses to investigational agents. In this study, we used the CIVO platform to assess the impact of TAK-981 on the native tumor immune microenvironment of animal models. TAK-981 is a novel and selective small molecule inhibitor of the SUMOylation enzymatic cascade currently in Phase I clinical trials. SUMOylation is a reversible post-translational modification that regulates protein function by covalent attachment of a small ubiquitin-like modifier (SUMO) protein to protein substrates. TAK-981 was microinjected into tumors from a syngeneic mouse model of B cell lymphoma and responses assessed via immunohistochemistry and in situ hybridization following tumor resection. An early inflammatory response was evident by 24 hours, including the accumulation of neutrophils, inflammatory macrophages and a Type I interferon response. The chemokine IP10 was secreted around TAK-981 injection sites and was accompanied by the accumulation of cytotoxic T lymphocytes, likely recruited from the local tumor microenvironment. A localized cell death response was observed proximal to TAK-981 injection sites by 72 hours and was likely induced by the granzyme B-bearing cytotoxic T cells enriched at TAK-981 sites. Abscopal studies demonstrated that the local immune modulation induced by TAK-981 translated into an adaptive immune response. The results from this study were consistent with findings from systemically dosed in vivo mouse efficacy studies carried out at Takeda, demonstrating that the local responses to agents microdosed intratumorally via CIVO are predictive of responses induced by systemic drug exposure. These studies highlight the unique capability of TAK-981 to promote antitumor immunity, which may be further evaluated using the CIVO platform in a Phase 0 trial in human solid tumor patients.

#4137

Circulating TGFâ and TNFá in metastatic breast cancer patients treated with eribulin. The TRANSERI project.

Ornella Garrone,1 Cristiana Lo Nigro,1 Andrea Michelotti,2 Fillippo Montemurro,3 Anna Maria Vandone,1 Andrea Abbona,4 Matteo Paccagnella,4 Antonella Falletta,4 Elena Genua,3 Claudia De Angelis,2 Federica Tonissi,4 Paola Vanella1. 1 _S. Croce & Carle Teaching Hospital, Cuneo, Italy; _2 _AOU Pisana, Ospedale S. Chiara, ITT, Pisa, Italy;_ 3 _Candiolo Cancer Institute FPO-IRCCS, Candiolo, Torino, Italy;_ 4 _ARCO Cuneo Foundation, Cuneo, Italy_.

Background: Eribulin (E) is approved for the treatment of metastatic breast cancer (mBC) patients (pts) after failure of antracyclines and taxanes. E interferes with microtubule leading to apoptosis and G2/M cell cycle arrest. In human cancer cell lines and in mice, it reverses epithelial mesenchimal transition (EMT) and reduces metastases in mice. TGFβ, an immunosuppressive cytokine, acts as growth factor for cancer-associated fibroblasts (CAFs) and promotes EMT. TNFα synergizes with TGFβ to promote EMT. The TRANSERI study investigates the modifications of TGFβ and TNFα levels in 40 mBC pts treated with E.

Methods: Plasma levels of TGFβ and TNFα were determined by ELISA assay at baseline, before cycle (C) 3, 5 and at disease progression in mBC pts treated with E at 1.23 mg/m2, d 1-8 every 21 d. Statistical analysis of the changes in the longitudinal samples was performed by GraphPad 5. Clinical outcome was monitored according to standard internal follow up procedure.

Results: We report analyses in pts who completed 5 C or progressed before C 5. So far, we have evaluated TGFβ level in 34 pts and TNFα in 26 pts. The median (M) basal TGFβ value was higher in pts than in 9 healthy volunteers (201.9 pg/ml vs 115.1 respectively). At C 5, 14 pts had a decrease in TGFβ with a M value approaching the one of healthy controls (161.9 pg/ml vs 115.1 pg/ml respectively). 19 pts progressed before C 5. Overall 26/33 pts experienced progression. The M value of TGFβ in pts at progression was 293.4 pg/ml. A significant difference of TGFβ was observed between non progressed vs progressed pts (p=0.021). The M TNFα value at baseline was higher in pts than in healthy volunteers, even if in both groups it was close to the lower sensitivity cut-off of the assay. Altogether 20/26 pts experienced progression, that occurred before C 5 in 14 out of them.Intriguingly, at C 5, the TNFα / TGFβ ratio was significantly lower in pts who did not progressed compared to the value at progression (p=0.048).

Conclusions: TGFβ levels changed during treatment with E and correlate with outcome. We are evaluating the role of tumor burden in modulating the TNFα / TGFβ ratio. Definitive data will be presented.

#4138

Understanding the effects of NF-kappaB signaling on macrophage phenotype to inform therapeutic approaches.

Evan B. Glass,1 Alyssa Hoover,1 Whitney Harris,1 Zahra Mirafzali,2 Todd D. Giorgio,1 Andrew Wilson,3 Fiona Yull1. 1 _Vanderbilt University, Nashville, TN;_ 2 _Encapsula Nanosciences, Brentwood, TN;_ 3 _Vanderbilt University Medical Center, Nashville, TN_.

It is recognized that modulation of macrophage phenotypes within the tumor microenvironment has the potential to achieve anti-tumor outcomes. However, formulating a strategy to achieve the benefits of such an approach in a cell-specific manner while minimizing off-target or uncontrolled deleterious side effects remains a challenge. We hypothesize that modulation of Nuclear Factor Kappa-B (NF-kappaB) signaling within macrophages in the proximal tumor microenvironment represents an effective approach. However, there remains controversy whether NF-kappaB signaling in macrophages induces pro- or anti-tumor outcomes. Understanding the mechanisms by which this signaling pathway defines predominant macrophage characteristics is therefore critical in order to use strategic interventions to obtain therapeutic benefits. Our goal is to gain insight into how to modulate NF-kappaB signaling in macrophages to generate anti-tumor phenotypes and to use this information to develop new treatments. We use immortalized bone marrow-derived macrophages or ex vivo macrophages in cell culture approaches to investigate impacts of modulation of NF-kappaB signaling on macrophage phenotypes. We have three methods to modulate signaling: doxycycline inducible transgenic mice, liposomal delivery of the bacterial cell mimic MTP-PE, and optimized polymeric nanoparticle-mediated delivery of siRNA targeting the NF-kappaB inhibitor. In addition to our in vitro assays, we also employ in vivo murine models of ovarian and breast tumor progression. This multi-faceted approach is providing insights into how altering NF-kappaB signaling in macrophages impacts their interactions with tumor cells and other cells in the tumor microenvironment. Our data suggests that high levels of NF-kappaB signaling in macrophages induce both direct tumor cell-killing and immune-stimulating responses. These studies are designed to better understand the mechanisms by which NF-kappaB regulates macrophage functions to inform development of a novel macrophage-based immunotherapy that will be effective across a wide spectrum of solid tumors and metastatic disease.

#4139

Q702, selective Axl, Mer and CSF1R triple kinase inhibitor with dual potentials leading to tumor regression: Immuno-oncology therapy and targeted cancer therapy.

Yeong-In Yang,1 Hwankyu Kang,1 Dongsik Park,1 Yeejin Jeon,1 Jeongjun Kim,1 Baejung Choi,1 Jaeseung Kim,1 Jinkyung Rho,2 Jaecheol Lee,2 Kiyean Nam1. 1 _Qurient Co., Ltd., Seongnam-si, Republic of Korea;_ 2 _Asan Medical Center, Seoul, Republic of Korea_.

Functional composition of tumor micro-environment (TME) has been regarded as one of the most important factors in tumor progression. Cancer cells induce immune suppressive condition in TME in order to evade immune system as well as to promote metastasis. Axl, Mer and CSF1R play important roles in making tumor friendly TME by increasing regulatory T cells (Tregs), M2 macrophages, myeloid-derived suppression cells (MDSCs) and suppressing antigen presentation. On the other hand, in cancer cells, overexpression of Axl is reported to have correlation with poor prognosis of patients through promoting epithelial-to-mesenchymal transition (EMT) and inducing drug resistance to chemo or targeted therapy. Q702 is an orally available selective Axl, Mer and CSF1R triple kinase inhibitor under IND enabling studies. In this poster, we present dual potentials of Q702 leading to tumor regression through: 1) immune stimulating activities of Q702 through decreasing Tregs, M2 macrophages, MDSCs population and promoting antigen presentation, 2) direct cytotoxic activity in tumor models including acute myeloid leukemia (AML) and EGFR TKI resistant non-small cell lung cancer (NSCLC).

#4140

Discovery and characterization of novel highly potent A2A adenosine receptor antagonists for cancerimmunotherapy.

Kyungik Lee, Seungah Jun, EunYoung Byun, Hosun Lee, Yongtaek Lee, MiJin Moon, Yu-Yon Kim, Hyun Jeong Kang, YoungGil Ahn, YoungHoon Kim, Kwee Hyun Suh. _Hanmi Pharm. Co., Ltd., Hwaseong-si, Gyeonggi-do, Republic of Korea_.

Introduction: Extracellular adenosine produced at high concentrations within the tumor micro-environment (TME) and suppresses immune function via inhibition of immune cell activation. Targeting adenosine receptors has emerged as a novel method to activate anti-tumor immunity. In particular, A2A adenosine receptor (A2AR), one of the G-protein-coupled-receptors, exhibits immunosuppressive functions. Herein, we suggest novel A2AR antagonist lead compounds as a highly potent antagonist of A2AR for immunotherapy.

Materials and Methods: Novel A2AR antagonist lead compounds were designed using CADD and synthesized as the active biologic inhibitory compound. The protein preparation and molecular docking were performed using Glide (Schrödinger). A radioligand binding assay was performed to evaluate the affinities of A2AR Antagonists for the human adenosine A2AR. The potencie of A2AR antagonists were determined by cAMP assay in HEK293-hA2AR cells and cAMP-mediated pCREB assay in human CD8+ T cells from whole blood. In vivo CT26 and MC38 syngeneic tumor models were used to assess the therapeutic effect of A2AR antagonists.

Results: A2AR antagonist lead compounds showed strong binding affinities toward human A2AR. They also potently inhibited the NECA-mediated production of intracellular cAMP in HEK293 cells expressing human A2AR. Elevated intracellular cAMP following A2AR activation results in the phosphorylation of CREB (cAMP response element-binding protein). A2AR antagonist lead compounds treatment inhibited pCREB in NECA-stimulated HEK293-hA2AR and human CD8+ T cells. A2AR antagonist lead compounds inhibited tumor growth of mouse syngeneic tumor models as a single agent and combination with anti-PD-L1. In combination with anti-PD-L1, A2AR antagonist lead compounds had remarkable antitumor activities in multiple mouse tumor models, including restoration of immune responses in models that incompletely responded to anti-PD-L1 monotherapy.

Conclusion: These results showed the potencies of A2AR antagonist lead compounds with high capability of A2AR inhibition. Blockade of the adenosine signaling pathway may be vital for enhancing anti-tumor responses in solid tumors that show an incomplete response to anti-PD-L1 therapy. A2AR antagonist lead compounds demonstrate a novel approach to anti-cancer immunotherapy.

#4141

The induction of iNKT cells and CIK cells toward anti-tumor phenotypes.

Adisak Wongkajornsilp, Khin Su Su Htwe, Nathawadee Sawatpiboon, Sunisa Duangsa-ard, Kanda Kasetsinsombat. _Mahidol Univ. Faculty of Medicine Siriraj Hospital, Bangkok, Thailand_.

Natural killer T (NKT) cells and cytokine-induced killer (CIK) cells contain features of both T cells and NK cells that occasionally brought confusion to several studies. These cells have been reported to possess either anti-tumor or immunosuppressive properties. NKT cells could be directly isolated from the peripheral blood, while CIK cells require ex vivo culture of peripheral blood lymphocytes for 2-3 weeks with IFN-γ, IL2 and anti-CD3. NKT type I cells carry invariant TCRαβ recognizing α-Galcer loaded CD1d on antigen-presenting cells. We investigated whether NKT type I cells, as well as CIK cells, could be induced toward Th1 phenotype that should elicit anti-tumor activity. The inducers included ODN, checkpoint inhibitors and CDKi. We observed the tipping toward Th1 phenotype in both cell types, especially on CIK cells. It is concluded that both NKT type I cells and CIK cells could be induced toward the anti-tumor Th1 phenotype.

#4142

Physical and functional interactions between MNK and mTOR signaling regulate the activation and differentiation of T cells.

Craig R. Stumpf, Vikas K. Goel, Rajesh K. Sharma, Gary G. Chiang, Peggy A. Thompson, Kevin R. Webster. _eFFECTOR Therapeutics, San Diego, CA_.

The role of the PI3K/mTOR and MAPK signaling pathways in regulating T cell activation and differentiation is well established. It has been shown that mTOR acts as a sensor of the T cell metabolic state, coordinating diverse inputs to determine the balance between effector versus memory cell fates. Similarly, the MAPK interacting kinases, MNK1 and MNK2, are key downstream effector kinases that mediate post-transcriptional gene regulation of critical mediators of the T cell response, including immune checkpoint proteins and cytokines, via the phosphorylation of specific RNA binding proteins such as eIF4E and hnRNPA1. eFT508 is a potent and selective inhibitor of both MNK1 and MNK2, which enhances anti-tumor immune responses by decreasing the expression of immune response modulators including PD-1, PD-L1, LAG3, TIM3 and IL-10. Furthermore, eFT508 treatment boosts memory T cell populations while increasing the effectiveness of cytotoxic T lymphocytes. Phosphoproteomic analysis of the effects of eFT508 on early T cell activation identified novel eFT508 sensitive phosphopeptides, including a subset that overlaps with mTOR-dependent phosphorylation sites. In vitro biochemical analysis has shown that eFT508 is not a mTOR kinase inhibitor and that most of these sites are not direct MNK substrates. These results suggest inhibition of MNK by eFT508 can have significant effects on mTOR signaling. Consistent with these findings, we observe a physical association between MNK and mTOR that is disrupted by either eFT508 or the allosteric mTOR inhibitor rapamycin. Our phosphoproteomic analysis also identified novel MNK-dependent phosphorylation sites within the translation initiation factor eIF4G1 that regulate the ability of mTOR to recognize eIF4G1 as a substrate. In addition, phosphorylation of the mTOR target 4EBP1 is decreased upon treatment of T cells with eFT508, although 4EBP1 itself is not a direct substrate of MNK in vitro. These data are consistent with a role for MNK in regulating mRNA translation, not only through phosphorylation of eIF4E, but also by modulating the activity of mTOR toward specific substrates. Given the role of mTOR in controlling memory T cell differentiation, our results provide a potential mechanistic basis to explain the impact of MNK inhibition on T cell differentiation. Ongoing studies are further characterizing the consequences of eFT508 regulation on MNK/mTOR signaling and T cell differentiation. This work significantly expands our current understanding of eFT508's mechanism of action as well as its ability to promote a prolonged anti-tumor immune response.

#4143

The novel hexavalent human GITR agonist HERA-GITRL promotes anti-tumor efficacy independent of Fc-functionality and shows superior activity compared with the monoclonal anti-GITR antibody TRX518.

Matthias Schröder, Viola Marshall, Meinolf Thiemann, David M. Richards, Christian Merz, Jaromir Sykora, Julian P. Sefrin, Mauricio Redondo-Müller, Karl Heinonen, Katharina Billian-Frey, Oliver Hill, Christian Gieffers. _Apogenix AG, Heidelberg, Germany_.

Glucocorticoid-induced TNFR-related protein (GITR, TNFRSF18, CD357), a TNFR-SF member, is a co-stimulatory receptor that increases anti-tumor T cell activation. Based on Apogenix TNFR-SF agonist HERA-technology, we created a fully human hexavalent GITR ligand fusion protein - HERA-GITRL - intended for T cell costimulatory approaches in immuno-oncology (IO) therapy. HERA-GITRL is composed of a trivalent single chain GITRL-receptor-binding-domain fused to an IgG1-derived silenced Fc-domain serving as dimerization scaffold. The unique design that combines a molecular mimic of the endogenous GITRL with a silenced Fc-domain, allows the study of pure GITR agonism in contrast to Fc-mediated mixed modes of action. Here we report in vitro and in vivo properties of our novel HERA-GITRL construct. For functional characterization of HERA-GITRL in vitro, human immune cells isolated from healthy-donor blood were profiled by multicolor flow cytometry and real-time cell analysis. Stimulation of unfractionated human T cells or purified naïve CD4+ T cells by anti-CD3 antibody was further augmented by HERA-GITRL. This effect was accompanied by increased proliferation, differentiation and elevated levels of TNF-α and IFN-γ. Importantly, HERA-GITRL-mediated T cell activation increases tumor cell killing by PBMCs in vitro and showed in vivo anti-tumor efficacy as a single agent in a subcutaneous syngeneic colon cancer model (CT26wt) in mice. This anti-tumor effect is independent of its Fc functionality, as murine HERA-GITR ligands with functional Fc- or silenced Fc-domains show similar tumor growth inhibition. TRX518, an anti-human GITR monoclonal antibody currently investigated in a clinical Phase I study, was used in a direct in vitro comparison with trivalent GITRL and our hexavalent HERA-GITRL. Without crosslinking HERA-GITRL showed superior agonistic activity over trivalent GITRL and TRX518. Crosslinking increased the activity of trivalent GITRL while the residual activity of TRX518 was even decreased. We constructed a CHO cell line stably expressing fully human HERA-GITRL with high purity and yield. The resulting research cell bank is ready to be used for subsequent GMP process development. By clustering the receptor chains in a spatially well-defined manner, HERA-GITRL induces potent agonistic activity without being dependent on additional Fc-mediated crosslinking. A comparison of HERA-GITRL with the anti-GITR antibody TRX518 showed superior agonistic activity of our HERA construct in vitro with and without cross-linking. The HERA-ligand concept has also been successfully translated to HERA-TRAIL (now in Phase I), -CD40L, -CD27L,-LIGHT and -4-1BBL.

#4144

**Water extract from** Chlorella vulgaris **cell membrane fraction enhances host antitumor immune responses and inhibit colon carcinoma growth in mice.**

Susumu Ishiguro,1 Nicole Robben,1 Deepa Upreti,1 Paige Cote,1 Sarah Greenway,1 Riley Burghart,1 Ayaka Nakashima,2 Masaaki Tamura1. 1 _Kansas State Univ., Manhattan, KS;_ 2 _euglena Co. Ltd., Japan_.

Natural products obtained from various resources are often used as new compounds or seed compounds of effective drugs for disease including cancer. The water extract partially purified from the cell membrane fraction of Chlorella vulgaris (CMWE) was evaluated on its antitumor and immunomodulatory effects in cell culture and a colon carcinoma mouse model. The CMWE was filtered through 0.22 μm pore size membrane for all experiments. In two-dimensional cell cultures, the CMWE treatment inhibited cell growth of both murine and human colon carcinoma cells in a dose- and time-dependent manner. In contrast, the cell proliferation of mouse splenocytes (SPLs) and bone marrow cells (BMCs) were stimulated by the treatment with CMWE. The treatment with CMWE also increased specific subpopulations of the cells in BMCs: antigen presenting cells (CD19+ B cells, 33D1+ dendritic cells and CD68+ macrophage), CD4+ and CD8+ T cells, whereas LY-6G+ neutrophils were decreased. In a three-dimensional spheroid culture, spheroid growth of CT26 cells co-cultured with Jurkat cells (human T lymphoblasts) was significantly attenuated by CMWE treatment via induction of apoptosis. In a two-dimensional cell culture of Jurkat cells, mRNA levels of TNFα, IFNγ and granzyme B were significantly increased by CMWE treatment. In a mouse CT26 colon carcinoma peritoneal dissemination model, intraperitoneal injection of CMWE (30 mg/kg/day, started from 3 days after CT26 inoculation for 9 days, every other day) significantly attenuated the growth of CT26 colon carcinoma nodules in syngeneic mice by CMWE treatment-associated apoptosis without any noticeable adverse effect. Analysis of subpopulation of intraperitoneally infiltrating immune cells collected from CT26 cell tumor-bearing mice, indicated that CMWE treatment increased CD4+ and CD8+ T lymphocytes, CD19+ B cell, and CD68+ macrophage populations. In contrast, LY6G+ neutrophil population was decreased as compared to PBS-treated group. The present study suggests that CMWE inhibits colon carcinoma growth via direct cell growth inhibition and a stimulation of the host antitumor immune responses. Taken together, the current study suggests that water extract obtained from the cell membrane fraction of Chlorella vulgaris contain significant bioactive materials that inhibit colon carcinoma growth. This study was supported by 2016EUGLENA-RC1 (MT), 2017EUGLENA-RC2 (MT), Kansas State University Johnson Cancer Research Center 2018CRA (NR and MT), and NIH grant P20 RR017686 (MT).

#4145

**A novel form of 4-1BB agonist shows robust immune protection against various tumor types through CD4** + **memory-like T and NK cell axis.**

Haval Shirwan, Hampartsoum B. Barsoumian, Lalit Batra, Pradeep Shrestha, William S. Bowen, Hong Zhao, Nejat K. Egilmez, Jorge G. Gomez-Gutierrez, Esma S. Yolcu. _Univ. of Louisville, Louisville, KY_.

Background: Costimulation through 4-1BB (CD137) receptor has been targeted for cancer immunotherapy using agonistic antibodies, which have been shown to cause liver toxicity in preclinical and clinical settings. We hypothesized that this might not be a bona fide feature of 4-1BB signaling, but rather an antibody-mediated off-target effect. To test this hypothesis, we have generated a novel oligomeric form of the natural ligand, SA-4-1BBL, with robust costimulatory activity as a soluble protein. Pretreatment with SA-4-1BBL as a single agent protected mice against challenge with various tumor types, a feature that was not shared by an agonistic 4-1BB antibody.

Study Design: C57BL/6 Mice were treated subcutaneously once or twice (two weeks apart) with various doses of SA-4-1BBL protein or an agonistic antibody (3H3) to 4-1BB, followed by challenge with various tumor cell lines subcutaneously into the left flank and monitored for tumor growth. For immunological studies, animals were treated with SA-4-1BBL twice, two weeks apart and then euthanized 7 days later to harvest various lymphoid tissues for immunophenotyping using flow cytometry. Antibodies against CD4+ T, CD8+ T, B, and NK cells were used in vivo to deplete individual cell types to assess their contribution towards the observed immune protection.

Results: Pretreatment with SA-4-1BBL protected mice against 3 different tumor types, two lung cancer and one cervical cancer cell lines without detectable toxicity. The conferred protection against tumors was dose-dependent and evolved within two weeks of the first treatment. Importantly, the response was not tumor type-specific and lasted more than 8 weeks. In contrast to the protection conferred by SA-4-1BBL, pretreatment with the agonistic 4-1BB antibody had no impact on the tumor growth. Pretreatment with SA-4-1BBL expanded CD4+CD44+4-1BB+ T cells and NK cells producing IFN-γ. Our immune-cell depletion studies proved CD4+ T and NK cells to be indispensable for the observed anti-tumor effect. In marked contrast, depletion of CD8+ T cells or B cells had no significant impact on tumor growth. Also, in vivo blockade of IFN-γ negated SA-4-1BBL-conferred protection against tumor challenge.

Conclusion: This study demonstrates unique and unexpected immunomodulatory features of SA-4-1BBL that bridge innate and adaptive immune responses with preventive efficacy against cancer. The tumor type-independent protection conferred by SA-4-1BBL is significant with important clinical implications for primary and secondary cancer prevention modalities. The safety profile of SA-4-1BBL and its cancer immunoprevention attributes are fundamental characteristics that are not shared by agonistic antibodies to the 4-1BB receptors. This highlights and emphasizes SA-4-1BBL's potential for cancer immunoprevention and therapy. Funded in parts by NIH R41CA199956 award.

#4146

Calcineurin represses IL-12 secretion in acute lymphoblastic leukemia.

Alisha D. Hunter,1 Jairo Fonseca,2 Jodi Dougan,2 Lori A. Gardner,3 Jennifer Rabe,3 Manali Rupji,4 Bhakti Dwivedi,4 Curtis J. Henry,5 Christopher C. Porter5. 1 _Cancer Biology Program, Emory University, Atlanta, GA;_ 2 _Department of Pediatrics, Emory University School of Medicine, Atlanta, GA;_ 3 _University of Colorado School of Medicine, Aurora, CO;_ 4 _Winship Cancer Institute, Atlanta, GA;_ 5 _Department of Pediatrics, Emory University School of Medicine; Aflac Cancer and Blood Disorders Center, Children's Healthcare of Atlanta, Atlanta, GA_.

Acute lymphoblastic leukemia (ALL) remains a leading cause of disease-related deaths in children. Although immuno-therapeutics have become a powerful strategy for the treatment of ALL, the mechanisms by which the leukemia cells avoid recognition and clearance by the immune system have not been clearly defined. Previous studies by our group defined leukemia-cell calcineurin (Cn) as a critical mediator of immune evasion in an aggressive, clinically relevant, murine model of BCR-ABL1+ B-ALL, as knocking it down in leukemia cells extended survival in immunocompetent, but not B and/or T cell-deficient mice (P=0.001).

To identify downstream mediators of Cn-dependent immune evasion, we performed a cytokine array that demonstrated increased levels in chemokines and cytokines in the supernatant of Cn-deficient leukemia compared to the control. To validate this finding, ELISA was performed to quantify cytokine production and secretion. IL-12 secretion in the supernatant of Cn-deficient leukemia was significantly increased compared to the control. Ex vivo analyses demonstrated that T cell stimulation with the supernatant of Cn-deficient leukemia cells led to CD8+ T cell activation and CD4+ T cell differentiation into Th1 cells, as determined by measuring intracellular levels of IFN-γ and TNF-α. Furthermore, we found that the enhanced T cell activation by factors secreted from Cn-deficient leukemia was ablated by IL-12 neutralizing antibodies. Notably, analyses of publicly available gene expression profiles demonstrate that children with ALL in which either IL-12A or IL-12B is expressed at high levels have better outcomes, suggesting that IL-12 has clinical relevance.

Importantly, treatment of mice with recombinant IL-12 (rIL-12) dramatically reduced the leukemia burden and prolonged survival in WT mice with parental leukemia as compared to vehicle treated WT controls (P=0.002). Successfully treated mice survived a second challenge with leukemia, suggesting the establishment of immunologic memory in these mice. In addition, rIL-12 treated Rag1-/- mice with leukemia had a similar reduction in leukemia burden, and transient prolongation of survival as compared to vehicle treated Rag1-/- mice (P=0.003). Given that Rag1-/- recipients lack mature B and T-cells, these results suggest that IL-12 responsive innate immune cells, such as NK cells, also play a major role in the effective suppression of leukemia cells. To begin to address this hypothesis, WT, Rag1-/-, IL2rg-/-, and double knockout (DKO) mice lacking B, T and NK cells (Rag1-/-/IL2rg-/-) were engrafted with calcineurin-deficient leukemia. A trend was observed in which DKO mice showed the highest rate of leukemia progression compared to the other groups. Overall, these data suggest that Cn-mediated inhibition of IL-12 secretion contributes to evasion of both innate and adaptive immunity by leukemia cells, and that novel strategies for therapeutic IL-12 delivery are needed.

#4147

**EOS100850, an A** 2A **receptor antagonist with prolonged pharmacodynamic activity, mediates the generation ofspecific durable immune responses in a murine breast cancer model.**

Romain Pirson, Anne-Catherine Michaux, Diane Jamart, Julie Preillon, Kim Frederix, Paola Basilico, Chiara Martinoli, Xavier Leroy, Gregory Driessens, Erica Houthuys, Scott Chappel, Stefano Crosignani, Reece Marillier. _iTeos Therapeutics, Gosselies, Belgium_.

We and others have shown that extracellular adenosine (Ado), acting predominantly through the A2A receptor (A2AR), mediates immunosuppression through different modes of action. These include suppression of Th1 responses and cytotoxicity as well as increasing the activity of Tregs and MDSC. We therefore developed EOS100850, a novel, non-brain penetrant and highly selective inhibitor of A2AR with sub-nanomolar Ki. As compared to other A2AR antagonist in development, EOS100850 maintain its potency in high Ado environment (>10uM).

To better characterize the Ado levels found in the tumor microenvironment, we first assessed extra-cellular Ado concentrations by microdialysis. Concentrations measured in mouse and human tumors were elevated compared to normal skin and confirmed that immune cells infiltrating the tumor microenviroment are confronted with elevated suppressive Ado levels.

To demonstrate and compare the ability of EOS100850 to inhibit A2AR signaling in vivo with other molecules in development, we measured the in vivo pharmacodynamic (PD) modulation of A2AR activity by investigating its ability to inhibit CREB phosphorylation (pCREB) following activation of the Ado receptor by NECA, an A2AR agonist. While EOS100850 demonstrated complete inhibition of pCREB 30 minutes after oral gavage at a dose as low as 0.1 mg/kg, other A2AR inhibitors required much higher concentrations to achieve a similar effect. Remarkably, 24 hours after dosing, more than 60% of inhibition of pCREB was still observed, at a dose of 10 mg/kg when the EOS100850 antagonist was no longer detectable in the plasma. These results demonstrate that EOS100850 has a PD activity that extends well beyond its PK, which can be explained by a long residence time.

To demonstrate the in vivo antitumor efficacy of EOS100850, we used the murine syngeneic breast cancer model, EMT-6. EOS100850 administered in combination with anti-CTLA-4 mAb increased the number of complete responders (CR) and significantly reduced tumor growth compared with anti-CTLA-4 single agent treatment. Furthermore, the CR were protected against re-inoculation with EMT6 but not irrelevant CT26 tumor. Lastly, flow cytometry and RNAseq analysis, performed on tumors taken during the course of treatment, confirmed the potential of EOS100850 as single agent or in combination with anti-CTLA4 to induce a pro-inflammatory response that correlate with tumor regression.

This best-in-class profile of EOS100850 A2A receptor antagonist supports its progression into clinical development.

#4148

An orally bioavailable small molecule antagonist of TIM-3 signaling pathway shows potent anti-tumor activity.

Pottayil G. Sasikumar, Sudarshan S. Naremaddepalli, Raghuveer K. Ramachandra, Nagesh Gowda, Srinivaskumar Devarapalli, Sreenivas Adurthi, Jiju Mani, Rashmi Nair, Amit A. Dhudashiya, Dodheri S. Samiulla, Nagaraj M. Gowda, Murali Ramachandra. _Aurigene Discovery Technologies Limited, Bangalore, India_.

Activation of anti-tumor immune response by specific inhibition of PD1 pathway using monoclonal antibodies has now become of the mainstay in cancer therapy as evidenced by its widespread use in an expanding list of indications. Although these antibodies show impressive durable clinical activity, low response rates are witnessed in a large number of cancers, including colorectal cancer that remain largely refractory to PD-1 blockade. Upregulation of alternative immune checkpoints such as T cell immunoglobulin and mucin-domain containing-3 (TIM-3) and VISTA contributes to the lack of response in patients not responding to therapies with anti-PD-1/PD-L1 antibodies. TIM-3 is a co-inhibitory receptor expressed on IFN-γ-producing T cells, FoxP3+ Treg cells and innate immune cells. Synergistic effects in restoring the anti-tumor immunity in preclinical models upon dual blockade of TIM-3 and PD-1 has provided a strong rationale for developing TIM-3 agents for use in combination with PD1 agents in the clinic. We sought to discover and develop an orally available small molecule antagonist targeting TIM3- signaling pathways. Unlike antibodies an oral agent potentially offers the convenience, flexibility to adjust dose and schedule to address any emergent adverse events and ease of combination therapy. Because TIM-3 shares sequence and structural similarity with the B7 family ligands, a focused library of compounds mimicking the interaction of checkpoint proteins of B7 family was screened towards the functional antagonism of TIM-3. Further optimization of the hit compounds resulted in lead compounds targeting TIM-3 pathway with desirable potency and selectivity. Lead compounds exhibited potent functional activity comparable to that obtained with an anti-TIM-3 antibody in rescuing the effector functions in human PBMC-based assays. Additionally, an advanced lead compound exhibited desirable drug-like properties including solubility, metabolic stability and pharmacokinetics with good oral bioavailability. In a syngeneic tumor models, once a day oral dosing of the advanced lead compound resulted in significant tumor growth inhibition as a single agent and in combination with anti-PD1 antibody that correlated well with immune PD in the tumor. The findings reported here support the development of the oral TIM-3 antagonist for use in the clinic.

#4149

**Extensive characterization of the adenosine pathway in human solid tumors gives a hint on cancer indication selection for the A** 2A **receptor antagonist EOS100850.**

Noemie Wald, Chiara Martinoli, Marjorie Mercier, Annelise Hermant, Florence Nyawouame, Joao Marchante, Charlotte Moulin, Vanesa Bol, Stefano Crosignani, Veronique Bodo. _iTeos Therapeutics, Gosselies, Belgium_.

Extracellular adenosine is an immunosuppressive metabolite generated by sequential action of the two ectonucleotidases CD39 and CD73. Tissue hypoxia, which is frequently observed in neopleastic tissues, increases CD39 and CD73 expression, and is a major driver of extracellular adenosine accumulation. High levels of adenosine, predominantly signaling through the A2A receptor (A2AR), dampen innate and adaptive immune cell responses, leading to suppression of antitumor immunity. We therefore developed EOS100850, a potent and highly selective A2A receptor antagonist to treat a wide range of tumor types. With the aim to support selection of cancer indications for EOS100850, we used immunohistochemistry and computer-assisted image analysis to extensively characterize the expression and spatial distribution of markers of the adenosine pathway and the extent of lymphocyte infiltration in ten tumor types. First, 318 primary tumor specimens included in disease-specific tissue microarrays from nine cancer indications were screened for expression of CD39, CD73 and infiltration of A2AR+, CD8+ and Foxp3+ cells. CD39 and CD73 were expressed by immune cells, endothelial cells and other stromal cells as well as by tumor cells, and their expression was increased in hypoxic tumor regions. Expression levels of the two adenosine-generating enzymes were variable among the analyzed cancer types, with lung and gastric tumors displaying the highest levels of expression of CD39 (78% and 95% of samples with high CD39 expression, respectively) and gastric, uterine and lung cancers displaying the highest levels of CD73 (>70% of samples with high CD73 expression). A2AR staining was observed on different immune cell populations, and significantly correlated with CD8+ and Foxp3+ cell densities (p values <0.0001). A2AR+ immune cells were frequently detected in lung, gastric and head and neck tumors (>80% positive samples) but not in kidney and prostate tumors (<20% positive samples). To encompass the inherent heterogeneity of tumor tissues, the expression and spatial distribution of CD39, CD73, A2AR and lymphocyte populations were further analyzed in several lung, colorectal and breast primary tumor resections. These tumor types display high, medium and low levels of the adenosine cloud markers, respectively. CD39 expression as well as densities of A2AR+, CD8+ and Foxp3+ cells were all higher in the tumor surrounding stroma than in the center of the tumor in the three cancer types. Moreover, CD39 and A2AR were significantly correlated with CD8+ and Foxp3+ cell densities in the tumor center but not in the stroma. Altogether these data strongly support the relevance of targeting A2AR and the adenosine pathway in immuno-oncology, and pave the way to the identification of the cancer types that will benefit more from EOS100850.

#4150

An HPK1 inhibitor CMPD0431 is a novel immuno-oncology agent that induces anti-tumor effects.

Seungmook Lim, A Yeong Park, Misoon Kim, Gyooseung Jung, Suyeon Jo, Keonseung Lim, Gwibin Lee, Heekyoung Yang, Hyonam Kim, Hyeongjun Kim, Minwoo Lee, Jamie Jae Eun Kim, Jinhwa Lee. _1st Biotherapeutics, Inc., Seongnam, Republic of Korea_.

Hematopoietic progenitor kinase 1 (HPK1, MAP4K1) is a serine/threonine kinase and a member of MAP4K. HPK1 is prominently expressed in subsets of hematopoietic cell lineages. HPK1 is a newly identified as a critical negative regulator in the activation of T lymphocytes and dendritic cells. It has been recently demonstrated that the important roles for kinase activity of HPK1 in anti-cancer immunity as a new intracellular checkpoint molecule as well as potential advantages of combination therapy with current checkpoint regimens. HPK1 inhibition is expected to have dual functions, 1. prolonged activation of T cells; 2. enhanced APC functions by dendritic cells. This dual targeting may synergistically work together for efficient immune responses in tumor microenvironment. We have developed a series of small molecule inhibitors of HPK1 with a lead compound of CMPD0431, which are orally available with good physicochemical and pharmacokinetic profiles. CMPD0431 and its series demonstrated good potency for HPK1 with kinase IC50 values of ~20 nM or under. Treatment of T cells with CMPD0431 successfully blocked serine 376 phosphorylation of SLP76, a critical biomarker substrate for HPK1 and also for downstream signaling in T cell activation, leading to sustained activation in human T cells. CMPD0431 was shown to increase the CD3/CD28-induced proliferation of human primary T cells with enhanced production of pro-inflammatory cytokines such as IFN-γ and IL-2. Furthermore, CMPD0431 enhanced the functional activity of antigen presentation with increased level of pro-inflammatory cytokine productions in mouse bone marrow-derived dendritic cells. All together, we have confirmed that HPK1 inhibition indeed play a dual function in T cells and dendritic cells to effectively promote cancer immunity. In the CT-26 syngeneic mouse model, we showed that CMPD0431 inhibited tumor growth with increased intratumoral infiltration of CD3\+ lymphocytes and with increased pro-inflammatory cytokine production, demonstrating promoted cancer immunity with a potential for conversion of cold tumor into hot tumor. A subset of mice with high dose treatment of CMPD0431 showed a tumor regression with no further tumor regrowth for 20 days without compound treatment. The cured mouse group did not grow tumor even after re-challenging with CT-26 without compound treatment, demonstrating the effective immunological memory by CMPD0431. Those immune memory mouse group displayed increased secretion of IFN-γ by splenic T lymphocytes with enhanced response to the stimulation by CT-26 specific antigen, AH1 peptide. All together, these results support further development of CMPD0431 and its derivatives, first-in-class and advanced leads of HPK1 inhibitors with novel mechanism of action to be applied as a single-agent or combinational therapy with the current checkpoint inhibitors.

#4151

Siglec-15 is a novel immunomodulatory effector of adaptive immune evasion in acute lymphoblastic leukemia.

Claire Pillsbury,1 Jairo A. Fonseca,1 Jodi Dougan,1 Lori Gardner,2 Jennifer Rabe,2 Christy Gearheart,2 Chris C. Porter1. 1 _Emory University, Atlanta, GA;_ 2 _University of Colorado School of Medicine, Aurora, CO_.

Despite advances in therapy for acute lymphoblastic leukemia (ALL), it remains a leading cause of illness-related death in children and has poor long term disease-free survival and prognosis in recurrence in adults. Recent developments in immune-based therapies, such as CAR T cells, have shown great promise, but immune evasion mechanisms of leukemia remain poorly understood, and alternative strategies to engage the immune system are needed.

Our group has characterized a role for calcineurin (CN) as a driver of a potent immune escape mechanism in an aggressive murine model of BCR-ABL+ ALL. We performed RNA-seq analysis of control and CN-deficient ALL cells to identify downstream mediators of this effect. Siglec-15 emerged as one of the most differentially expressed genes, being preferentially downregulated in the more immunogenic CN-deficient cells. The role of this Siglec-family immunomodulator is yet unstudied in the context of leukemia and could represent a novel mechanism for immune evasion.

We first validated lower expression of Siglec-15 in CN-deficient leukemia cells by RT-qPCR. We next mined publicly available databases, which revealed higher expression of Siglec-15 in primary B-ALL and AML leukemias as compared to normal PBMCs, as well as in post-chemotherapy MRD samples as compared to at-diagnosis samples. Western blot analysis of human leukemia cell lines demonstrated upregulation of Siglec-15 expression in B-ALL, some AML, as well as DLBCL, as compared to normal PBMCs.

To determine if Siglec-15 mediates immune evasion in vivo, we knocked down Siglec-15 in the murine model of BCR-ABL+ ALL, which resulted in rapid suppression of the leukemia following engraftment in immunocompetent mice, an effect lost in immunocompromised counterparts. Preliminary studies with cells in which Siglec-15 was knocked-out using CRISPR/Cas9 revealed similar results. Importantly, Siglec-15-deficient cells do not have any changes in proliferation or susceptibility to DNA damage-induced apoptosis.

To address the capacity for therapeutic targeting of Siglec-15, mice with parental leukemia were treated with a monoclonal blocking antibody against Siglec-15, yielding an overall reduction in leukemic burden and prolonged survival. Taken together, these results suggest that Siglec-15 is a novel immunomodulatory effector and has a therapeutically exploitable role in immune evasion of ALL.

#4152

Irreversible electroporation induces immunogenic cell death and mediates durable response in orthotopic PDAC model in combination with anti-PD-1.

Jun Zhao,1 Xiaofei Wen,2 Tingting Li,1 Marites Melancon,1 Sanjay Gupta,1 Weiyi Peng,3 Chun Li1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _The Fourth Hospital of Harbin Medical University, Harbin, China;_ 3 _University of Houston, Houston, TX_.

Immunotherapy has only limited efficacy against pancreatic ductal adenocarcinoma (PDAC) because of the presence of an immunosuppressive tumor-associated stroma. Here, we showed that combined IRE and anti-PD-1 immune checkpoint blockade significantly suppressed tumor growth and prolonged the lives of immunocompetent mice bearing well-established orthotopic PDAC. Remarkably, more than 35% of mice had a durable response, and were found to be free of residual tumor upon necropsy. All mice that survived for 60 days after IRE and anti-PD1 treatments rejected tumor cell re-challenge. Analyses of splenocytes confirmed that these long-term survivors developed an anti-tumor memory T cell response. Further mechanistic studies unveiled that the efficacy of IRE + anti-PD-1 could be attributed to multiple factors, including rapid release of danger associated molecular patterns, activation of DCs, and alleviation of immunosuppressive tumor microenvironment. Neutralization of CD8+ T cells by anti-CD8 antibody nullified the antitumor effect of combined IRE and anti-PD-1, suggesting that tumor-infiltrating CD8+ T cells played a key role in mediating the anti-tumor efficacy of IRE + anti-PD-1.Given that both IRE and anti-PD-1 antibodies are already used in the clinic, our results support the translation of this combination as a promising approach for treating patients with PDAC.

#4153

Therapeutic targeting autophagy to sensitize cancer immunotherapy in various cancer types.

Yuanyuan Qiao,1 Jae E. Choi,1 Josh N. Vo,1 Jean C. Tien,1 Lisha Wang,1 Lanbo Xiao,1 Stephanie A. Simko,1 Andrew D. Delekta,1 Nathan B. Hodge,1 Parth Desai,1 Kristin Juckette,1 Alice Xu,1 Fengyun Su,1 Rui Wang,1 Xuhong Cao,1 Xiaoju Wang,1 Xiaoming Wang,1 Javed Siddiqui,1 Zhen Wang,2 Amélie Bernard,3 Ester Fernandez-Salas,1 Nora M. Navone,4 Ke Ding,2 Eeva-Liisa Eskelinen,5 Elisabeth I. Heath,6 Daniel J. Klionsky,7 Weiping Zou,1 Arul M. Chinnaiyan1. 1 _Univ. of Michigan Medical School, Ann Arbor, MI;_ 2 _School of Pharmacy, Jinan University, Guangzhou, China;_ 3 _Université de Bordeaux, Bordeaux, France;_ 4 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 5 _Institute of Biomedicine, University of Turku, Turku, Finland;_ 6 _Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI;_ 7 _Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI_.

Although cancer immunotherapy has revolutionized cancer treatment, patient response to immunotherapy remains varied. Despite progress, the mechanisms limiting cancer immunotherapy are not yet fully understood. A low number of tumor infiltrating T cells (cold tumor) is one of the limiting factors for cancer immunotherapy. Agents that enhance immunotherapy by shifting cold tumors to hot tumors will greatly benefit cancer immunotherapy. In prostate cancer, the majority of tumors are known to be cold and hence, cancer immunotherapy is not the ideal treatment option. Here, we use a prostate cancer model as an example to demonstrate that modulating the tumor microenvironment through altering autophagy will change the tumor cytokine secretion profile, which in turn attracts immune lymphocytes into the tumor microenvironment. In tandem, we have identified a candidate compound known as ESK981 for such a purpose.

Method A small molecule library was used for screening autophagy activity and cytokine secretion. Various types of human cancer cell lines (prostate, renal, bladder, breast etc.) and multiple syngeneic mouse lines were examined for autophagy activity as well as an in vitro response to interferon stimulation with or without ESK981. A syngeneic mouse prostate cancer was used for the in vivo examination of autophagy as well as the anti-tumor effect by ESK981 monotherapy and/or in combination with anti-PD-1 therapy.

Conclusion We have discovered a robust, novel autophagy-modulating small molecule, named ESK981, for the treatment of various cancer types as a monotherapy. In addition, we have demonstrated that autophagy has an essential role in the anti-tumor effect of immunotherapy, especially for anti-PD-1 in syngeneic prostate cancer model. Therefore, the use of a small molecule such as ESK981 to target autophagy can enhance immunological infiltration induced by cancer immunotherapy, such as immune checkpoint blockade, for non-immunogenic tumors.

Conflict of Interest Statement A.M.C. is a co-founder and serves on the Scientific Advisory Board of Esanik Therapeutics, Inc. which owns to the rights to the clinical development of ESK981. Esanik Therapeutics, Inc. did not fund or approve the conduct of this study.

Funding This project is supported by Prostate Cancer Foundation.

#4154

**EOS100850 inhibits** **A** 2A **receptor signaling in human whole blood: Two pharmacodynamic assays to monitor EOS100850 activity in clinical studies.**

Chiara Martinoli, Margreet Brouwer, Erica Houthuys, Marjorie Mercier, Noemie Wald, Bruno Gomes, Stefano Crosignani, Veronique Bodo. _iTeos Therapeutics, Gosselies, Belgium_.

High levels of extracellular adenosine in the tumor microenvironment promote tumor immune evasion. We and others have shown that adenosine, predominantly through the A2A receptor, suppresses innate and adaptive immune cell responses leading to suppression of antitumor immunity. We therefore developed EOS100850, an A2A receptor antagonist specifically designed as a potent, highly selective, non-brain penetrant, orally administered agent to treat a wide range of tumor types.

To monitor A2A receptor engagement by EOS100850 in clinical studies, we developed two pharmacodynamic assays that allow to quantify the inhibition of adenosine signaling in cancer patients dosed with this compound. The assays were developed and optimized to: 1) minimize the amount of patient blood required for each test; 2) minimize sample manipulation; 3) allow marker quantification in cryopreserved samples.

The first assay is based on the quantification of phosphorylated cAMP response-element binding protein (pCREB) as a proximal readout of A2A receptor signaling. Whole blood is ex vivo stimulated with the A2A receptor agonist CGS-21608, or with dimethyl sulfoxide or phorbol-12-myristate-13 acetate and ionomycin as controls, and pCREB in CD4+ and CD8+ cells is analyzed by flow cytometry. After having confirmed that pCREB was rapidly induced after incubation with CGS-21680 (median fold-induction in 24 healthy donors was 5.4 and 4.0 in CD4+ and CD8+ cells, respectively), we showed that EOS100850 inhibited CGS-21608-induced pCREB in a dose-dependent manner, with an IC50 of 9.7 nM and 11.2 nM, respectively, for CD4+ and CD8+ T cells, and 83-87% signal inhibition in the presence of 40 nM EOS100850 (data from 10 donors).

The second assay is based on the quantification of soluble mediators as a distal readout of A2A receptor signaling. Stimulation of whole blood with lipopolysaccharide (LPS) normally induces the release of innate cytokines and chemokines, but in the presence of the A2A receptor agonist CGS-21680, LPS activity is impaired. In this assay, whole blood is ex vivo stimulated with LPS in the presence or in the absence of CGS-21608 using TruCulture tubes (Myriad RBM), and secreted mediators are quantified using the human multianalyte profile immunoassay platform. After having confirmed that CGS-21680 induced a profound (>50%) alteration of cytokine secretion in LPS-stimulated blood, we showed that EOS100850 restored IFN-γ, IL-8, IL-10 and TNF-α secretion in a dose-dependent manner, with a complete rescue at 40-100 nM of compound (data from 3 donors).

Both the assays were qualified in terms of precision, robustness and stability using blood of healthy volunteers, applied to blood of five cancer donors and are now ready to be used for the clinics.

#4155

Identification and characterisation of Affimer Proteins that are able to bind and agonise human co-stimulatory cell surface targets.

Amrik Basran. _Avacta Life Sciences, Cambridge, United Kingdom_.

Despite the impressive success of PD-1 pathway and CTLA-4 targeted immunotherapies, there remains a large proportion of cancer patients with many tumor types, that fail to benefit or acquire resistance during therapy. Co-stimulation of the tumor microenvironment may prove to enhance current checkpoint inhibitor therapies and kick start T-cell activity in tumors that have poor immune infiltrate. CD40 is a co-stimulatory protein and Glucocorticoid-induced TNFR-related protein (GITR) is a checkpoint inhibitory protein, both a member of the TNF-receptor superfamily. CD40 is expressed on a range of antigen presenting cells (APCs) binding to its ligand CD40L expressed on CD4+ T-cells, whilst GITR is expressed on T-cells. CD28 is also expressed on T-cells and provides co-stimulatory signals for T-cell activation and survival. CD40, GITR and CD28 are currently being investigated to provide complementary immune-modulation of the tumor micro environment alongside checkpoint inhibitor blockade therapy.

Affimer biotherapeutics are a new protein scaffold with great potential for the generation of biotherapeutics. Based on the human protease inhibitor Stefin A, the scaffold is small (14kDa), lacks any post translational modifications such as disulfide bonds and expresses at high levels recombinantly. The Affimer biotherapeutic platform which is based on the human protein Stefin A, has been used to generate binders to members of the TNFR superfamily (CD40, and GITR) and CD28 using phage display. Selections were carried out with an Affimer phage library of size 6x 1010 made with two random loops each of size nine amino acids. With the Affimers raised against huCD40, we were able to encode those molecules as DNA with a membrane anchor sequence. These molecules were expressed transiently in HEK293 cells in vitro, shown to be correctly folded and anchored on the cell surface. The engineered cells were shown to be able to agonise huCD40 in the HEK Blue reporter gene assay. GITR binding Affimer proteins were able to agonise huGITR in a NF-κB Luciferase assay and huCD28 binding Affimer proteins were able to agonise CD28 in a IL-2 Luciferase gene reporter assay.

We have demonstrated that Affimers can be used to agonise important co-stimulatory cell surface targets, either as recombinant protein or when displayed on a cell surface. Encoding the genes for these Affimers and delivering them directly to a tumour for expression (e.g. using an oncolytic virus) could offer a novel method of safely agonising these important co-stimulatory targets in cancer patients without generating the toxicity seen with using mAbs administered systemically. 

## EPIDEMIOLOGY

### Cancer Predisposition

#4156

Multiple rare germline variants in a thyroid cancer family.

Taina T. Nieminen,1 Daniel F. Comiskey,1 Yanqiang Wang,1 Sandya Liyanarachchi,1 Pamela Brock,1 Paivi Peltomaki,2 Huiling He,1 Albert de la Chapelle1. 1 _The Ohio State University, Columbus, OH;_ 2 _University of Helsinki, Helsinki, Finland_.

Thyroid cancer is the 12th most common cancer in the USA. Four main types exist: the majority (85-90%) is papillary thyroid cancer (PTC). Familial form of PTC represents 5 to 15 % of all thyroid cancers. We have access to 155 PTC families including clinical data and biological samples. Our aim was to search for highly penetrant gene variants in 17 PTC families. Data from one of the families in which PTC is segregating in three generations are described here. Whole genome sequencing was performed in three family members affected by PTC. Sanger sequencing at germline DNA and RNA levels followed by TOPO cloning was used to study the effects of mutations. We found 4 different either novel or ultra-rare mutations in the ITGAD, AARS and PDPR genes on chromosome 16 (Table). All mutations segregated perfectly with PTC in family members (n=4) and were absent in unaffected members (n=3). We Sanger sequenced cDNA from two of the PTC patients and cloned the products into the pCR 2.1-® TOPO vector. Based on the cDNA sequencing and cloning results, all of the mutations will create a premature stop codon leading to a truncated protein. We will verify the effects of the mutations by protein analysis as well. In addition, structural variation analysis will be done to exclude large genomic rearrangements. The germline events leading to PTC are neither well known nor extensively researched. When occurring in families PTC regularly shows autosomal dominant inheritance with incomplete penetrance. Only a few of probable mutations have been reported. Nevertheless, our finding of the truncating mutations in 3 genes on chromosome 16 that are cosegregating with PTC in a 3-generation family is surprising. Even more so: is the localization of the three genes in one chromosome coincidental? Preliminary findings in some of the other 16 PTC families suggest that the occurrence of more than one truncating mutation is not a unique event. We look forward to answer the question whether combination of two or more mutant genes can be responsible for one disease segregating in families.

#4157

Idenitificaiton of pathogenic germline variants of patients with double primary cancer of stomach and colon.

Yoon Young Choi,1 Ji Soo Park,1 Seung-Tae Lee,1 Su-Jin Shin,2 Jae Eun Lee,1 Jeong-Hyeon Jo,1 Eun Ji Nam,1 Taeil Son,1 Hyoung-Il Kim,1 Woo Jin Hyung,1 Sung Hoon Noh,1 Jae-Ho Cheong1. 1 _Yonsei University Health System, Seoul, Republic of Korea;_ 2 _Hanyang University College of Medicine, Seoul, Republic of Korea_.

Purpose: The incidence of multiple primary cancers has increased as survival of cancer patients has improved. The most common type of multiple primary cancers is the combination of stomach and colon cancer in Korea. Patients affected by two primary cancers at early age would be mainly related to their own genetic risk, however, germline variant of patients with double primary cancer have not been well evaluated.

Methods: Patient who is with pathologically confirmed cancers in both stomach and colon in Severance hospital between January, 2000 and December, 2016 were enrolled in this study. The patients were classified into two groups: early-age group was defined as both cancers were diagnosed before 55-years, and others were considered as late-age group. The overall early-age group (n=19) and randomly selected a fourth of late-age group (n=36) were enrolled in this study. The DNA was extracted from an archived formalin fixed paraffin embedded normal mucosa that was retrospectively reviewed. Target sequencing analysis focused on 65 genes that are known to be related to hereditary cancer was conducted. The characteristics of both cancers and family history were also evaluated.

Results: Overall 11 pathogenic/likely pathogenic germline variants (PGVs) were detected in nine patients (16.4%, 9/55, MLH1 [n=7], BLM [n=1], BRCA1 [n=1], MSH6 [n=1], and MSH2 [n=1]). The incidence of PGVs was 36.8% (7/19) and 5.6% (2/36) in early and late-age group, respectively (p<0.001). The early-age (Odds ratio[OR]: 9.92, 95% confidence interval [CI]: 1.81-54.49, p=0.008), Amsterdam_II criteria (OR: 24.29, 95% CI: 2.45-241.36, p=0.006), multiple lesions either gastric and colon cancer (OR: 13.13, 95% CI: 2.48-69.55, p=0.002) and mucinous/poorly differentiated histology of colon cancer (OR: 10.75, 95% CI: 1.48-78.06, p=0.019) were significant risk factors of PGVs in patients with double primary cancer at stomach and colon.

Conclusions: The Lynch syndrome related PGVs were identified in patients with double primary cancer at stomach and colon. Patients with double primary cancers at stomach and colon related to early age, multiple lesions, family history especially Amsterdam_II criteria, and poorly differentiated histology of colon cancer would be good candidate for genetic evaluation.

#4158

Dissecting the role of zygosity and lineage in Lynch Syndrome-associated microsatellite Instability.

Preethi Srinivasan,1 Chaitanya Bandlamudi,2 Jinru Shia,2 Alicia S. Latham,2 Philip Jonsson,2 Alexander Penson,2 Sumit Middha,2 Jackie Hechtman,2 Ahmet Zehir,2 Allison Richards,2 Shweta Chawan,2 Yelena Kemel,2 Diana Mandelker,2 Liying Zhang,2 David Hyman,2 Marc Ladanyi,2 Mark Robson,2 Kenneth Offit,2 David Solit,2 Barry Taylor,2 Michael Berger,2 Zsofia Stadler2. 1 _Weill Cornell Graduate School, New York, NY;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY_.

Lynch syndrome (LS) is an inherited condition leading to an increased risk of developing colorectal cancer and other select malignancies. LS patients have germline alterations in mismatch repair (MMR) genes and their tumors often demonstrate microsatellite instability (MSI). With the advent of immune checkpoint blockade, this is of both prognostic and therapeutic significance. Nevertheless, the factors that dictate the presence of the MSI phenotype in tumors diagnosed in LS patients are poorly understood. We integrated germline pathogenicity with somatic alterations in 17,152 prospectively sequenced advanced cancer patients to determine how zygosity and lineage shape the presence and intensity of the somatic MSI phenotype in LS patients. Overall, we identified 117 LS patients (0.68%) with one of 46 distinct cancer types, 111 of which had sufficient purity for somatic assessment. The tumors of 72% (80/111) of these patients had biallelic inactivation of the germline allele as assessed by integrating high-precision mutant allele fractions with allele-specific DNA copy number analysis or, in select low purity tumors, confirmatory immunohistochemistry. Among patients with biallelic inactivation, 73% (58/80) had MSI positive tumors whereas, only 1/31 (3%) of heterozygous tumors were MSI positive, indicating that biallelic inactivation of the germline allele was obligate to drive the somatic MSI phenotype (p-value=5.4e-12). Lineage appears, however, to condition this dependence of MSI status on biallelic inactivation. Leveraging the prevalence of germline MMR mutations in our cohort, zygosity enrichment, and literature curation of MMR penetrance, we classified cancer types as either conventionally Lynch-associated or not. Notably, despite the presence of biallelic inactivation, MSI was not seen in LS patients whose cancers were not conventionally Lynch-associated such as breast, lung, and thyroid cancers among others. The nature and degree of the MSI phenotype also varied as a function of underlying genotype in these patients. Despite comparable mutational burdens, MSH6 germline carriers that were somatic biallelic had lower MSI scores, lower rates of frameshift indels and higher rates of missense mutations than patients with biallelic mutations in other MMR genes. Collectively, these data suggest that the presence of MSI is dictated by lineage-dependent selection for biallelic inactivation in LS tumors, and emerges to differing degrees driven by the underlying genotype, which together has implications for the immunogenicity of resulting tumors and the biomarker of greatest response to immune checkpoint blockade. By expanding our prospective cohort to >30,000 patients with advanced cancer and integrating clinical response to immunotherapy, we will further explore gene-specific variability in MSI patterns observed in LS patients and its effects on outcome and therapeutic response.

#4159

**Gonadal mosaicism in a family with** TP53- **associated Li-Fraumeni syndrome.**

Payal P. Khincha,1 Kristine Jones,2 Kedest Teshome,2 Belynda Hicks,2 Phuong L. Mai,3 Sharon A. Savage1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _Cancer Genomics Research Laboratory, Bethesda, MD;_ 3 _University of Pittsburgh School of Medicine, Pittsburgh, PA_.

Li-Fraumeni syndrome (LFS) is an autosomal dominant cancer predisposition syndrome with exceptionally high lifetime cancer risks, caused primarily by germline pathogenic variants in TP53. Sarcomas, pre-menopausal breast cancer, brain tumors and adrenocortical carcinomas are the 'core' LFS cancers among a multitude of other cancers that can occur. Individuals with LFS are also at risk of developing multiple primary cancers over their lifetime. Clinical criteria have been established to denote LFS and 'Li-Fraumeni like' (LFL) families to facilitate genetic testing for TP53. De novo pathogenic germline variants in TP53 are estimated to cause between 7-25% of LFS. Gonadal mosaicism is an alternate explanation for presumed de novo variants and is yet to be described in LFS.

We present a proband who developed WHO grade II astrocytoma at age 18 years, who had a deceased brother with osteosarcoma diagnosed at age 13 years, and a healthy older sibling. The only other cancer history was leukemia at age 62 years in the paternal grandmother, who also had polycythemia vera. The family meets Eele's criteria for LFL. The proband's germline genetic testing revealed a heterozygous TP53 c.743G>A p.R248Q pathogenic variant. Subsequent clinical genetic testing of the parents failed to reveal the variant, and the proband was presumed to have a de novo variant. Given the characteristic LFS-associated cancers in the brothers, we investigated further and identified the same TP53 pathogenic variant in the brother's osteosarcoma tissue and pathology-confirmed benign liver tissue. TP53 sequencing of the brother's tumor did not show loss of heterozygosity. Parental relationships to the proband and brother were confirmed through STR-based identifiler profiling. Ultra-deep sequencing (>50,000x) of DNA derived from the parents' blood, saliva and father's benign colon polyp did not identify the variant, thus ruling out somatic mosaicism in the parents. We were unable to test gonadal tissues from the parents. In the setting of two siblings with the same pathogenic variant in TP53 which is absent in both parents, this family presents a case of assumed gonadal mosaicism resulting from a post-zygotic event.

The possibility of gonadal mosaicism presents complexity in genetic counselling of families with inherited disorders, and a conundrum in syndromes such as TP53-associated LFS where the presumed de novo rate is high and cancer risks are significantly elevated. The presented family suggests that further investigation of individuals with presumed de novo variation, especially in the setting of suspicious family cancer history, is required to accurately identify family members at-risk.

#4160

PZP as a new gene associated with increased breast cancer risk.

Ekaterina Sh Kuligina,1 Anna P. Sokolenko,1 Rohit Kumar,2 Ilya V. Bizin,1 Aglaya G. Iyevleva,1 Kirill Zagorodnev,3 Syed K. Hasan,2 Aleksandr A. Romanko,1 Ashok K. Varma,2 Evgeny N. Imyanitov1. 1 _N.N. Petrov Research Inst. of Oncology, St. Petersburg, Russian Federation;_ 2 _TATA Memorial Centre, Navi Mumbai, India;_ 3 _St. Petersburg Pediatric Medical University, St. Petersburg, Russian Federation_.

Germline mutations in known breast cancer (BC) predisposing genes account for only 20 - 25% of hereditary breast cancer susceptibility. In this study, we anticipated that application of exome analysis to a series of genetically homogeneous Slavic BC patients will allow to identify novel recurrent BC predisposing mutations. 49 BRCA1/2-negative BC patients with evident clinical signs of the hereditary disease (family history and/or BC bilaterality and/or young onset) were subjected to Illumina-based whole exome sequencing. Two patients were found to carry a rare nonsense mutation in pregnancy zone protein (PZP) gene (p.Arg680*, rs145240281). This mutation was further analyzed in a case-control study involving 1046 high-risk BRCA1/2-negative BC cases, 1608 consecutive BC and 1082 middle-aged tumor-free women. Distribution of PZP p.Arg680* alleles was in agreement with its putative BC-predisposing role: 4/1046 (0.38%) and 9/1608 (0.56%) BC cases vs. 1/1082 (0.09%) controls, producing risk estimates in consecutive BC group equal to 6.08 (95% CI 0.770 to 48.096, P = 0.087]. 5/15 heterozygous for PZP p.Arg680* variant patients were diagnosed with multiple primary tumors (2 bilateral BCs, BC + colorectal cancer, bilateral BC + gastric cancer, BC + basal-cell like skin cancer). To the best of our knowledge, no reports associating gene PZP to cancer pathogenesis are available. In order to evaluate the functional effects of PZP p.Arg680*, we obtained two heterozygous mutant clones of CRISPR/Cas9-modified MCF7 cells containing 10-bp and 4-bp deletions adjacent to the desired targeted location. The real-time quantitative PCR confirmed significant down-regulation of PZP transcript in the clones carrying CRISPR/Cas9 induced deletions. Since this mutation results in a truncated protein, we also performed shRNA mediated knock-down of PZP. To study the effects of PZP mutation on cell proliferation and apoptosis, CRISPR/Cas9- and shRNA-modified clones were treated with tamoxifen and etoposide for 72 hours. The results have shown a significantly higher inhibitory concentration (IC50) for both drugs and higher clonogenic potential in CRISPR/Cas9-modified PZP clones compared to control MCF7 cells. Apoptosis induction was quantified using AnnexinV/PI staining; the reduced apoptosis in PZP-defective clones was documented. Taken together, obtained data argue that PZP may function as a tumor suppressor gene, and truncating variant p.Arg680* can represent a moderate-risk breast cancer susceptibility allele. This work has been supported by the Russian Science Foundation (grant 17-15-01384) and DST/INT/RUS/RSF/P-11

#4161

Germline mutational profileof Chinese patients with colorectal cancer and diagnosed lower than 70 years.

Feng Wang, Qi Zhao, Teng-jia Jiang, Ying-nan Wang, Fu-rong Liu, Jia-jia Hu, Pei-rong Ding, Zhi-zhong Pan, Jian-yong Shao, Rui-hua Xu. _Sun Yat-sen University Cancer Center, Guangzhou, China_.

Background/Aims: Colorectal cancer (CRC) is a common cancer worldwide with increasing morbidity in China. Approximately 5% CRC are caused by well-defined hereditary CRC syndromes, whose inherited susceptibility account for nearly one-third of CRC predisposition. Most of hereditary CRC syndromes were diagnosed at the age of lower than 70 , whereas little is known about the mutational profiles Chinese patients and their correlations with clinical features.

Method: In our present research, we retrospectively collected 731 patients who were diagnosed as CRC from Sun Yat-sen University Cancer Center, with ages lower than 70. All of them received genetic mutation detection consisting of a set of 14 specific inherited genes, which used target area capture and the second-generation high throughput sequencing technology. The characteristics of he patients with or without mutations were compared. We applied several comparison analysis to investigate the germline mutations distribution of genes that associated with the defined hereditary CRC syndromes in Chinese cohort.

Results: 397 patients (54.3%) harbored functionally variants of the 14 genes in CRC patients that diagnosis lower than 70. Intriguingly, we observed no statistical difference for clinical factors such as age, sex, smoke, drink, family history, pathology grade, clinic stage and overall survival between groups. In Mut group, the mutation frequency of each genes are MLH1(21.4%), MSH2(17.6%), MSH6(15.1%), PMS2(9.3%), EPCAM(8.3%), MLH3(8.1%), AXIN2(7.1%), PMS1(4.0%), MUTYH(12.8%), APC(12.3%), STK11(3.5%), BMPR1A(1.8%), SMAD4(0.3%), PTEN(1.0%). Genome mutual exclusivity analysis has identified a set of genes such as MLH1, MSH2, MUTYH, MSH6, APC and PMS2, for which most pairwise combinations are found rarely cooccurred. In correlation analysis between mutational genes and clinical features, pathology grade correlates strongly with PMS1 and SMAD4(P<0.05). Tumor location correlates strongly with APC, MLH1, MSH2 and MSH6(P<0.05). In mutation group, the ratio of Lynch syndrome and non-Lynch is 76.8% and 23.2%, respectively.

Conclusion: The clinic features of two groups have no statistic difference, which suggested other genes undiscovered may play a partial role in pathogenesis of patients of non-MUT group. In summary, we conducted a retrospective study to lead a comprehensive comparison of mutational profiles between MUT and non-MUT hereditary CRC patients, which supplemented the limited data of hereditary CRC in China.

#4162

Prevalence of germline mutations and variants of uncertain significance (VUS) among Hispanics with suspected hereditary colorectal cancer syndromes.

Julyann Perez-Mayoral,1 Nicole Cruz-Reyes,2 Andrea Rosa-Vazquez,3 Maria Gonzalez-Pons,1 Marcia Cruz-Correa1. 1 _UPR Comprehensive Cancer Center, San Juan, PR;_ 2 _University of Puerto Rico Cayey Campus, Cayey, PR;_ 3 _University of Puerto Rico Rio Piedras Campus, San Juan, PR_.

Background: Colorectal cancer (CRC) is one of the leading causes of cancer death in Puerto Rico (PR) and U.S. Approximately 5-10% of all CRC cases are due to hereditary CRC syndromes. Some of the most common hereditary colorectal cancer syndromes include: Familial Adenomatous Polyposis (APC), Lynch Syndrome (MLH1, MSH2, MSH6, PMS2) and MUTYCH (MYH). Germline genetic testing is the gold standard for diagnosing these syndromes. The implications of obtaining a Variant of Uncertain Significance (VUS) in germline genetic testing results remains a clinical challenge especially among minority Hispanic populations. The aim of this project was to determine the prevalence of germline pathogenic mutations and VUS in CRC-risk genes; as well as the phenotype of Hispanic patients with suspected Hereditary CRC syndromes.

Methods: Germline genetic testing from patients with suspected hereditary CRC syndromes seen at the UPR Cancer Genetic Clinic during the period of 2015-2018 were evaluated. Sociodemographics, clinical, tumor and genetic testing data were accessed through the subject's medical record. Descriptive statistics and frequency distribution tables were generated with the subject's sociodemographic and clinical information, as well as germline genetic testing results.

Results: A total of 91 patients (61.5% female; mean age 51 years) with suspected hereditary CRC syndromes were tested for germline mutations in relevant CRC-risk genes. The most common malignancies reported were CRC (80%) and endometrial cancer (12%). The majority of patients (n=60) did not have germline mutations in hereditary CRC-risk genes. Mutations in CRC-risk genes were detected in 22% of patients (50% MYH, 45% MMR, 5% APC), while VUSs were detected in 12% (18% MYH, 45% MMR, 36% APC). There were no statistically significant differences with regards to gender, age, personal history of cancer or 1st degree family history of cancer across study groups. All patients with VUSs in MMR-genes had microsatellite stable tumors; while those with mutations in MMR-genes had microsatellite unstable tumors.

Conclusions: VUSs were present in a high-percentage of the patients evaluated for hereditary CRC syndromes. Furthermore, there were no phenotypical differences between patients with pathogenic mutations and those with VUSs. This presents a clinical challenge as non-European populations including Hispanics have genetic variances, which may in fact carry an increase risk for cancer. Future research is warranted to examine the significance and/or reclassify VUSs to improve assessment of genetic test results in admixed populations.

#4163

Genomic variation in PDAC-predisposing genes identified using the MCW germline exome panel.

Jenica Abrudan, Susan Tsai, Wendy Demos, Michael T. Zimmermann, Michael Tschannen, Jennifer Geurts, Angela Mathison, Gwen Lomberk, Douglas Evans, Raul Urrutia. _Medical College of Wisconsin, Milwaukee, WI_.

Recent evidence suggests that genomic variations in cancer predisposition genes are found at higher prevalence in the general population. However, currently, only a handful of genomic variants in these genes are classified as pathogenic with certainty, leaving a significant number classified as variant of unknown significance (VUS) in terms of their potential pathogenicity. Addressing this problem is of significant relevance for improving cancer screening, genetic counseling, and in some cases therapy. Consequently, the GOAL of the current study was to evaluate genomic variations in pancreatic ductal adenocarcinoma (PDAC) predisposition genes from affected patients. We developed and validated the MCW PDAC Germline AmpliSeq Panel, which assessed variation in 53 cancer predisposition genes, including those from hereditary cancers, chronic pancreatitis, DNA damage repair and metabolism. The target DNA panel was amplified and a library was prepared according to the AmpliSeq for Illumina Custom Panel kit. Paired-end, 2x300bp read sequencing was performed at a depth of at least 500X using the MiSeq platform. Variant calling was performed with the Genome Analysis Toolkit (GATK) v3.7 Haplotype Caller. The protein coding effect of genomic variants was annotated using SNPEff and Ensembl canonical transcripts. We annotated variants with their population allele frequency from GnomAD, cancer incidence from COSMIC, and hereditary disease association from ClinVar and HGMD, using the BioR toolkit.A total of 5,658 variants were found in 545 patients representing 4,087 single-nucleotide variants (SNVs) and 1,146 insertion/deletion variants (indels). Among these variants, we found that 483 were seen previously in the COSMIC database, most of which were associated with 'carcinoma' primary histology. When considering all variants regardless of their impact, the genes with variants in the highest number of subjects was POLE, followed by APC, BARD1, BRCA2, ATM, and PMS2. APChad the highest number of coding variants across all samples, followed by PMS2, BRCA1, ATM, BRCA2, and BARD1. Lastly, 18 variants (0.31%) have been previously identified as pathogenic, likely pathogenic, or disease-causing variations for pancreatic cancer, while 231 (4.08%) represented VUS. In summary, ourfindings support the existence of wide genomic variation in PDAC-predisposition genes. Identified variants include some of uncertain significance, which warrant future functional studies to better understand their pathogenic potential. Combined, these results raise the possibility that some VUS may predispose to PDAC.

#4164

Comprehensive analysis of germline variants in patients with hereditary breast and ovarian cancer susceptibility from 4 countries of Latin America.

Felipe Vaca-Paniagua,1 Rosalia Quezada-Urban,1 Clara Estela Díaz Velásquez,1 Eva María Gómez García,2 Claudia Fabiola Méndez Catalá,1 Javier Oliver,3 Alejandra Franco,3 Cecilia Frecha,3 Cecilia Riggi,3 Claudia Carranza,4 Carlos Castañeda Altamirano,5 Ana Milena Gómez,6 Ana Lorena Montealegre,6 Sandra Gaitán Chaparro,7 Juan Javier López,7 Sandra Perdomo8. 1 _Facultad de Estudios Superiores Iztacala, Mexico;_ 2 _Instituto de Seguridad Social del Estado de México y Municipios, México, Mexico;_ 3 _Hospital Italiano, Argentina;_ 4 _Instituto para la Investigación Científica y la Educación Acerca de las Enfermedades Genéticas y Metabólicas Humanas, Guatemala;_ 5 _Instituto Nacional de Enfermedades Neoplásicas, Peru;_ 6 _Hospital Universitario San Ignacio, Colombia;_ 7 _Clínica Universitaria Colombia-Colsanitas, Colombia;_ 8 _Universidad El Bosque, Colombia_.

Hereditary breast and ovarian cancer syndrome (HBOC) is an autosomal dominant disease mainly associated to high-risk pathogenic alleles in the BRCA1 and BRCA2 genes, but only in ~25% of HBOC cases. With the emergence of broader multi-gene panels, population-based studies have revealed that 3-4% of high-risk individuals have germline pathogenic variants in cancer risk genes other than BRCA1 and BRCA2, including ATM, CHEK2, PALB2, PTEN, TP53, and others. One of the understudied population with HBOC is the Latin American, and in this study, we aimed to identify the prevalence of high, moderate and new pathogenic alleles within two panels of cancer predisposition genes (143 and 86 genes) in 217 Latino breast cancer patients that met the NCCN criteria for panel testing in 4 countries. The Latino population was integrated by 68 Mexicans (31.3%), 19 Guatemalan (8.8%), 56 Argentinians (25.8%) and 74 Colombians (34.1%). We detected (i) 19.4% patients with pathogenic variants (42/217; Mexicans: 12, 5.5%; Guatemalans: 3, 1.4%; Argentinians: 10, 4.6%; Colombians: 17, 7.8%); (ii) 36.9% harbored variants with unknown clinical significance (VUS) (80/217; Mexicans: 24, 11.1%; Guatemalans: 6, 2.7%; Argentines: 26, 12%; Colombians: 24, 11.1%) and (iii) 43.7% were negative (95/217; Mexicans: 32, 14.7%; Guatemalans: 10, 4.6%; Argentines: 20, 9.2%; Colombians: 33, 15.2%). Moreover, most of the pathogenic variants (66%, 28/42) were found in high-risk genes including BRCA1/2, MSH2, MSH6 and PALB2. Our findings show the high locus heterogeneity of HBOC in the Latin American population. To establish the level of risk and the ultimate clinical utility of the variants detected in expanded panels tests, further international efforts are necessary in a population-based context.

#4165

Unstable microsatellite instability proportion among unselected ovarian and endometrial cancer patients among Korean population.

Min Kyu Kim. _Samsung Changwon Hospital, Seoul, Republic of Korea_.

Background: There are several availability of microsatellite instability(MSI) among ovarian and endometrial cancer for detection of Lynch syndrome and biomarker of immune chemotherapeutic treatment. We investigated to know proportion of abnormal microsatellite instability (MSI-L/H).

Methods: Through retrospective search,we identified 51 cases of MSI samples from ovary and endometrial cancer visiting hereditary gynecologic cancer center.

Results: We found 34 ovary cancer ,13 endometrial cancer and 4 colon cancer patients. Median age was 56.6(18~78). About thirty percent have family cancer history among patient relatives (14/45(31.1%)). Early stage cancer (I~II) (4/25,16.0%) have higher incidence than late stage cancer (III~IV) (2/28,7.1%). Abnormal MSI have three serous type and endometrioid type pathology. Among abnormal MSI, we found two MSI-L and four MSI-H (6/51(11.8%)). We found one MSI-H ovary cancer ,3 MSI-H endometrial cancer patients (1/34(2.9%),3/13(23.1%)).

Conclusions: There are about 10 percent of abnormal MSI in this study group

#4166

Hereditary cancer syndromes in Guatemalan population.

Claudia L. Carranza,1 Mariela Guerra,1 Claudia Osorio,1 Vanessa Zamora,1 Vivian Herrera,2 Jose Gil,2 Luis Alvarez1. 1 _INVEGEM, Santa Lucía Milpas Altas, Guatemala;_ 2 _Roosevelt National Hospital, Guatemala, Guatemala_.

BACKGROUND The majority of cancer are sporadic, only around 5 % are considered hereditary. Although it is a low percentage, the study of these cancers are of vital importance, since they require a special clinical monitoring and management. These kind of cancers are caused by the presence of an inherited gene mutation, which increase the risk of developing a tumor. The mutations associated with these tumors are germline mutations, this means that are present in all the patient´s cells, including the egg cell an sperm; this is the reason why this type of cancer can transmit to the future generations. There are many syndromes that increase the risk of hereditary cancer development, the most frequent syndromes are: Familial Breast and Ovarian cancer (BRCA1 and BRCA2 gene mutations), Lynch syndrome (MSH2, MLH1, MSH6 and PMS2 mutations), Li-Fraumeni syndrome (TP53 gene mutations) and Familial adenomatous polyposis (APC gene mutations). The aim of this study is to screen the most common hereditary cancer syndromes in Guatemalan population.

METHODS We analyzed 199 blood samples of patients with different types of tumors. We extracted DNA from the samples and then analyzed the gene mutations by next generation sequencing.

RESULTS AND DISCUSSION We have included in the present study, 93 cases with Familial Breast cancer and 106 with another type of tumors. Of all cases, we have found 17 cases with hereditary cancer syndromes (8%). The most frequent is the Breast and Ovarian hereditary syndrome caused by BRCA1 and BRCA2 (12 patients). The other hereditary cancer syndromes are shown in Table 1. The detection of this germline mutations, have a large impact in the familial cases, because these patients and their families could be monitored and evaluated more frequently. This is the first report of these syndromes in our population.

CONCLUSION We reported 6 different syndromes and 17 cases, related to hereditary cancer risk and predisposition in Guatemalan population.

Frequency of hereditary cancer syndromes

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SYNDROME | NUMBER OF PATIENTS | CANCER TYPE | AGE | SEX | GENE MUTATION

Breast and ovarian hereditary syndrome | 7 | Breast cancer | 29-48 | F | BRCA1

Breast and ovarian hereditary syndrome | 5 | Breast cancer | 29-71 | F | BRCA2

Lynch syndrome | 1 | Breast cancer | 80 | F | MSH6

Li-Fraumeni syndrome | 1 | Breast cancer | 58 | F | TP53

Breast hereditary cancer | 1 | Breast cancer | 57 | F | CDH1

Peutz-Jeghers syndrome | 1 | Pancreatic cancer | 15 | F | STK11

Familial adenomatous polyposis | 1 | Colorectal cancer | 13 | M | APC

#4167

Germline variants in women with inflammatory breast cancer (IBC).

Steven Van Laere,1 Jianming Pei,2 Jacqueline N. Talarchek,2 Jennifer S. Winn,2 Elias Obeid,2 Katherine Alpaugh,2 Massimo Cristofanilli,3 Sandra V. Fernandez2. 1 _University of Antwerp, Antwerp, Belgium;_ 2 _Fox Chase Cancer Center, Philadelphia, PA;_ 3 _Northwestern University, Feinberg School of Medicine, Chicago, IL_.

Inflammatory breast cancer accounts for 15% of breast cancer-related deaths in US and is characterized by a young age at diagnosis, aggressive tumor features, high metastatic potential, and poor overall survival compared with non-IBC breast cancer. In order to better understand the etiology of this aggressive phenotype, we performed a study to evaluate the genomic alterations in 33 IBC patients using cell free DNA (cfDNA) from blood, including 19 triple negative, 9 with ER+ Her2-, 3 with ER+ Her2+ and 2 ER- Her2+. In 20 patients, mutation analyses were also studied in tumor tissues and/or tumor cells from pleural effusions. From the 33 IBC patients (25 W with 1 patient being Jewish and 1 with Ashkenazi Jewish ancestry; 5 AA; 2 Asian and 1 Hispanic), the median age at diagnosis was 48 years- (32- to 66-years); the survival time from diagnosis was 38 months and, 4 patients survived more than 7 years from diagnosis (alive). Next generation sequencing and a panel of 93 breast cancer related genes (Qiagen) were used; the data was analyzed using the GeneGlobe portal and interpretation was performed with QCI- Interpret (Qiagen). Interestingly, some variants were observed with high allele frequencies (VAF), ~50% or ~100%, indicating they were germline variants and classified as:

• Pathogenic: BRCA2 (A1327fs*4, 1/33), BRCA1 (E23fs*17, 1/33), PALB2 (F440fs*12, 1/33), TP53 (G266R, 1/33), CHEK2 (1/33)

• Likely Pathogenic: BRCA2 (R2659K, 1/33), RAD51D (E233G, 2/33), RAD51C and AR (1/33), RB1 (V45fs*21; 1/33), ATM (R2032K, 1/33), CASP8 (1/33) and MUTYH (1/33)

• Variants from Uncertain Significance: PALLD (M224T; 18/33), BARD1 (V507M; 10/33); SYNE1 (K4050S; 3/33) and RET (R982C; 2/33), and others (1/33): BRCA1, BRIP1, XRCC2, RAD50, APC, ESR1, RB1, ERBB2, MYC, MAP3K1, CDKN2A, ATM, KMT2C, and IRAK4

• Benign: BRCA1 (P871L; 24/33) and BRCA1 (I379M; 1/33) both entailing loss/decreased of BRCA1 functional activity.

Five patients had at least one pathogenic variant, 7 others had at least one likely pathogenic variant, 20 had at least one variant of uncertain significance and one African-American patient had only BRCA1 (P871L and I379M) benign variants at 100% frequency. Interestingly, seven patients showed both germline variants BARD1 (V507M; uncertain significance) and BRCA1 (P871L; benign). Most of the patients (29/33) had a family history of cancers including breast, prostate, colon, pancreatic, melanoma, CNS cancer and blood cancers. The variants in IBC patients correspond to proteins involved in DNA damage repair (BRCA1, BARD1, BRCA2, PALB2, RAD51D, RAD51C, XRCC3, XRCC2, MUTYH, ATM, CHEK2, BRIP1), control cell cycle (RB1, SYNE1, MYC, CDKN2A, TP53) and cell shape and migration (PALLD). In conclusions, our results suggest a high incidence of germline variants in patients with IBC that could be related with the disease. The data, if validated in larger cohort, may suggest that genomic analysis could identify women at high-risk of developing IBC in need of monitoring.

#4168

Identify the predisposition genes to endometrial cancer.

Yu-Cheh Chiang,1 Ting-Fang Lee,2 Yung-Feng Lin,2 Hui-Ying Weng,3 Shih-Feng Tsai,4 Cheng-Wen Wu5. 1 _Institute of Microbiology and Immunology, Nnal Yang-Ming University, Taipei, Taiwan;_ 2 _Institute of Molecular and Genomic Medicine, National Health Research Institutes, Miaoli, Taiwan;_ 3 _VYM Genome Research Center, National Yang-Ming University, Taipei, Taiwan;_ 4 _Institute of Genetics, National Yang-Ming University, Taipei, Taiwan;_ 5 _Institute of Clinical Medicine of National Yang-Ming University, Taipei, Taiwan_.

Endometrial carcinoma is the most dominant gynecological tumor worldwide. Familial risk of endometrial cancer has been reported but little is known about the predisposition genes. Here we focused on a family with high incidence of cancer, aiming to identify potential markers with low allele frequency and high disease-causing probability. Whole-genome sequencing was performed with available DNA from 4 family members: one healthy control and 3 endometrial cancer patients. We analyzed the non-synonymous variations in the coding region expressed in 3 of the patients but not in the healthy control. The variations with high allele frequencies in Taiwanese population were excluded. Five potential candidates, KMT2D, PCDHA8, COG5, RAPGEF3 and PFDN1, were identified as heterozygous mutations predisposed to endometrial cancer. In addition, we demonstrated that PCDHA8 and COG5 genes play crucial roles in cell proliferation in endometrial cancer cells. Notably, KMT2D (lysine methyltransferase 2D) is highly mutated in the tumors of endometrial cancer. An interaction between mutated KMT2D and other candidate genes may explain the high risk of cancer formation in this family. These results validate the potential markers of endometrial cancer and create opportunities for therapeutic applications and personalized prevention.

#4169

Population-based breast cancer risk estimates associated with cancer predisposition gene mutations from 32,298 breast cancer patients and 31,869 matched unaffected controls from the CARRIERS study.

Fergus J. Couch,1 Chunling Hu,1 Steven N. Hart,1 Rohan Gnanaolivu,1 Jenna Lilyquist,1 Kun Y. Lee,1 Chi Gao,2 Bruce Eckloff,1 Hoda Anton-Culver,3 Paul Auer,4 Leslie Bernstein,5 Mia Gaudet,6 Christopher Haiman,7 Julie Palmer,8 Amy Tentham-Dietz,4 Song Yao,9 Susan Domchek,10 Jeffrey N. Weitzel,5 David E. Goldgar,11 Katherine L. Nathanson,10 Peter Kraft,2 Eric C. Polley1. 1 _Mayo Clinic, Rochester, MN;_ 2 _Harvard University, Cambridge, MA;_ 3 _University of California, Irvine, CA;_ 4 _University of Wisconsin, WI;_ 5 _City of Hope, CA;_ 6 _Emory University, GA;_ 7 _USC, CA;_ 8 _Brown University, MA;_ 9 _Roswell Park, NY;_ 10 _University of Pennsylvania, PA;_ 11 _University of Utah, UT_.

Clinical germline genetic testing of cancer predisposition gene panels is used to identify women at increased risk for breast cancer. Pathogenic mutations in cancer predisposition genes are associated with established risks of breast cancer and are actively used for risk management of breast cancer for women qualifying for genetic testing because of a family history of breast and/or ovarian cancer or young age of diagnosis. However, the risks of breast cancer associated with mutations in these genes have likely been overestimated for many women in the general population. The goal of the "CAnceR RIsk Estimates Related to Susceptibility" (CARRIERS) study was to estimate breast cancer risks associated with mutations in hereditary cancer panel genes. Germline DNA from 32,298 breast cancer patients and 31,869 matched unaffected controls enrolled in cohorts and population-based case-control studies were sequenced to identify pathogenic mutations in 37 cancer predisposition genes.The average age at diagnosis was 61.2 years and the average age of last follow-up was 62.1 years. DNA was subjected to dual bar-coded QIAseq multiplex PCR-based amplification of 1733 target regions in 37 predisposition genes. Products from 768 samples were pooled and sequenced in a single lane of a HiSeq 4000 system. Mutations were called by GATK Haplotype Caller and Vardict.High quality sequence data was obtained for 99.3% of target regions. Pathogenic mutations in 12 known breast cancer predisposition genes were identified in 6.2% of all breast cancer cases and 2.1% of controls; and in 6.7% of African American breast cancer cases and 1.8% of controls. In case-control association analyses adjusted for age at diagnosis or last follow up and family history of breast and ovarian cancer, mutations in BRCA1, BRCA2, and PALB2 were associated with high risks of breast cancer (odds ratio (OR)>4.0). Mutations in CHEK2 and BARD1 were associated with moderate risks of breast cancer (OR>2.0), whereas mutations in ATM had lower clinical relevance (OR=1.6). Stratification by ER, PR, and HER2 status of tumors found that BRCA1, BRCA2, PALB2 and TP53 were associated with high risks of studies of triple negative breast cancer (TNBC), and that BARD1, BRIP1, FANCM, and RAD51C were associated with moderate risks of TNBC. Overall, results from the CARRIERS study establish that mutations in predisposition genes are associated with lower risks of breast cancer in the general population than in high-risk families. The age-related estimates of breast cancer risk for each of the hereditary cancer panel genes in this study may inform on the use of cancer screening and other risk management strategies for women in the general population with mutations in predisposition genes.

#4170

Combination of germline and tumor testing yields a high rate of loss-of-function variants in non-mucinous epithelial ovarian cancer patients.

Florentia Fostira,1 Despoina Kalfakakou,1 Myrto S. Papamentzelopoulou,1 Aggeliki Delimitsou,1 Paraskevi Apostolou,1 Andromachi Vagena,1 Christos Papadimitriou,2 Gerasimos Aravantinos,3 Georgios Fountzilas,4 Drakoulis Yannoukakos,1 Irene Kostantopoulou1. 1 _NCSR Demokritos, Athens, Greece;_ 2 _National and Kapodistrian University of Athens, Athens, Greece;_ 3 _Agii Anargiri Cancer Hospital, Athens, Greece;_ 4 _Hellenic Foundation for Cancer Research/Aristotle University of Thessaloniki, Thessaloniki, Greece_.

Non-mucinous epithelial ovarian cancer diagnosis is a stand-alone criterion for genetic testing referral, irrespectively of family history or age at diagnosis. Identification of at least BRCA1 & BRCA2 mutations are fundamental for clinical decision making of ovarian cancer (OC) patients. The purpose of our study was to evaluate the prevalence of damaging variants that can lead to defects of DNA repair by homologous recombination (HR) in an unselected cohort of epithelial non-mucinous OC patients. Identification of such variants can indicate ideal candidates for Poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitor therapies.We therefore comprehensively analyzed genomic DNA from 578 epithelial non-mucinous OC patients for mutations in 94 genes that are involved in DNA repair, implementing a commercially available gene-panel. Additionally, 121 tumors were collected from OC patients that had negative germline testing and were assessed for somatic BRCA1 & BRCA2 mutations. Tumor cell content and necrosis were evaluated prior to tumor DNA extraction and were above 50% and below 20%, respectively.Overall, 25.4% (147/578) of the patients carried germline loss-of-function (LoF) variants, distributed in 18 genes namely, BRCA1, BRCA2, RAD51C, ATM, FANCL, CHEK2, FANCM, BRIP1, TP53, NBN, FANCA, RECQL4, MLH1, MSH2, MSH6, PMS2, ERCC2 and SLX4. After BRCA1 & BRCA2, which accounted for 72.1% of the total LoF variants, RAD51C LoF variants were the most frequent, accounting for 4.8% of the total LoF variants. Interestingly, the vast majority of LoF variants (136/147; 92.5%) involved homologous recombination/Fanconi anemia genes, detected in 23.5% of the total OC patients tested. Notably, 4.5% of the pathogenic variants were detected in genes involved in Mismatch Repair (MMR) pathway. Subsequently, tumor analysis resulted in the identification of damaging BRCA1 & BRCA2 variants in 11.5% of the ovarian tumors tested. Variant allele frequencies varied from 9%-47%. Altogether, approximately one in three of OC patients in our cohort could be good candidates for therapies targeting defective mechanisms of DNA repair, including HR and MMR.Our study highlights the high prevalence of LoF variants in HR genes, when combining germline and tumor testing in unselected, non-mucinous epithelial OC patients. These genetic defects can predominantly lead to HR deficiency, the ultimate biomarker for therapeutic intervention by PARP inhibition leading to tumor-cell death.

#4171

Identification of individuals with colonic polyposis in a highly inbred region.

Tirzah Braz Petta Lajus, Ana Rafaela Timoteo. _UFRN, Natal, Brazil_.

Familial adenomatous polyposis (FAP), also known as colonic polyposis, is a hereditary autosomal dominant disease, responsible for only 1% of colorectal cancer (CRC) in the population. FAP patients with a germline mutation in the APC gene have a 100% risk of developing CRC. MAP (Mutyh associated polyposis) is the polyposis associated with biallelic mutations in the MUTYH gene, and carriers are at risk of developing CRC. Few studies propose heterozygous Mutyh mutation is associated with high risk for breast cancer, but the results are conflicting. In the city Caicó, Brazil, we identified 8 non-related breast cancer patients with the same mutation c.536A>G, p.Tyr179Cys. In this region we find high frequencies of consanguineous marriages, which varied from about 9% to 32%, suggesting a direct association to genetic diseases. We hypothesize the founding mutation MUTYH_c.536A>G was established during Dutch colonization in Brazil between 1620 and 1650. For this, we are developing a population-based, case-control study of all newly diagnosed breast cancer in this city. In our group we have one oncologist, one psychologist and one geneticist. We recruited the patients (index-cases) from the archives in the Laboratory of Pathology at the Hospital Liga contra o Cancer and we invited the individuals to participate in the research.

#4172

Identification of familial Hodgkin lymphoma predisposing genes by whole genome sequencing.

Sara Giangiobbe,1 Aayushi Srivastava,1 Abhishek Kumar,2 Nagarajan Paramasivam,2 Dagmara Dymerska,3 Wolfgang Benisch,4 Mathias Witzens-Harig,4 Mathias Schlessner,2 Jan Lubinski,3 Kari Hemminki,2 Asta Försti,2 Obul R. Bandapalli2. 1 _German Cancer Research Center-University of Heidelberg, Heidelberg, Germany;_ 2 _German Cancer Research Center, Heidelberg, Germany;_ 3 _Pomeranian Medical University, Szczecin, Poland;_ 4 _University Hospital of Heidelberg, Heidelberg, Germany_.

Hodgkin lymphoma (HL) originates from germinal center B-cells and accounts for about 10% of newly diagnosed lymphomas and 1% of all de-novo neoplasms worldwide. Though familial risk for HL is among the highest of all cancers, not many genetic risk factors have been identified besides associations with the human leukocyte antigen (HLA) complex and few germline variants in familial classical Hodgkin lymphoma and nodular lymphocyte predominant Hodgkin lymphoma.

With the aim of identifying novel predisposing germline variants in familial Hodgkin lymphoma, we performed whole genome sequencing (WGS) on seven affected and nine unaffected family members from three HL-prone families (families I, II and III). WGS identified a total of 98564, 170551 and 113654 variants in families I, II and III, respectively at ≤0.1% minor allele frequency. Variants were prioritized using the Familial Cancer Variant Prioritization Pipeline version 2 (FCVPPv2) developed by us. Pedigree-based filtering reduced the number of variants to 18158, 496 and 26851. Non-synonymous SNVs were the most represented exonic variants. Application of FCVPPv2 criteria resulted in prioritization of 19, 6 and 13 potentially causative exonic variants in families I, II and III, respectively. Variants affecting promoters, enhancers, super-enhancers, transcription factor binding sites and microRNA seed sequences were identified using FANTOM5 (Functional Annotation of the Mammalian Genome 5), SEA (Super-Enhancer Archive), SNPnexus and TargetScan tools. Pathway analysis of the involved genes using Ingenuity Pathway Analysis showed enrichment of B-cell receptor signaling, PI3K signaling in B lymphocytes and protein kinase A signaling pathways. Implementation of FCVPPv2 on data from family I resulted in confirmation and functional validation of a novel heterozygous missense variant (c.T5133G: p.I1711M) in the tumor suppressor gene DICER1 as potential HL predisposition factor. In order to identify the causal variants in families II and III, the variants short listed based on our pipeline are being investigated for segregation, frequency in large cohorts and functional consequences.

In conclusion, WGS data analysis of three HL-prone families allowed us to prioritize coding and non-coding variants and identify enrichment of B-cell activating pathways. We aim to select and validate novel cancer predisposing variants that can facilitate genetic counseling and personalized therapy in these families as well as screening of other individuals at risk of developing Hodgkin lymphoma.

#4173

Previously identified common glioma risk SNPs are associated with familial glioma.

Quinn T. Ostrom,1 Georgina Armstrong,1 Christopher I. Amos,1 Jonine L. Bernstein,2 Elizabeth B. Claus,3 Jeanette E. Eckel-Passow,4 Dora Il'yasova,5 Christoffer Johansen,6 Daniel H. Lachance,4 Rose K. Lai,7 Ryan T. Merrell,8 Sara H. Olson,9 Joellen H. Schildkraut,10 Sanjay S. Shete,11 Richard S. Houlston,12 Robert B. Jenkins,4 Margaret R. Wrensch,13 Beatrice Melin,14 Jill S. Barnholtz-Sloan,15 Melissa L. Bondy1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _Memorial Sloan Kettering Cancer Center, Houston, NY;_ 3 _Yale University, New Haven, CT;_ 4 _Mayo Clinic College of Medicine, Rochester, MN;_ 5 _Georgia State University, Atlanta, GA;_ 6 _The Danish Cancer Society Research Center, Copenhagen, Denmark;_ 7 _University of Southern California, Los Angeles, CA;_ 8 _NorthShore University HealthSystem, Evanston, IL;_ 9 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 10 _University of Virginia School of Medicine, Charlottesville, VA;_ 11 _University of Texas MD Anderson Cancer Center, Houston, TX;_ 12 _The Institute of Cancer Research, Surrey, United Kingdom;_ 13 _University of California, San Francisco, San Francisco, CA;_ 14 _Umeå University, Umeå, Sweden;_ 15 _Case Western Reserve University School of Medicine, Cleveland, OH_.

BACKGROUND: Approximately 5% of gliomas occur in individuals with a family history of glioma, and first-degree relatives of brain tumor cases have a two-fold increase in risk of brain tumor. Recent somatic characterization has shown that tumors from familial cases are indistinguishable from sporadic cases, suggesting that familial cases may arise through similar mechanisms of gliomagenesis, and therefore may be associated with common variants as well as rare mutations. In this analysis, we assessed whether previously identified common risk variants are associated with familial glioma.

METHODS: Data were obtained from the Glioma International Case Control (GICC) and Gliogene studies for 448 cases with reported family history, 4,405 cases without reported family history, and 3,288 controls. We assessed 25 risk loci previously identified by glioma GWAS, and odds ratios (OR) and 95% confidence intervals (95%CI) were calculated using an additive genetic logistic regression model adjusted for age, sex, and the first two principal components for familial cases versus unaffected controls, and non-familial cases versus controls. Results were considered significant at p<0.002 (Bonferroni correction for 25 tests).

RESULTS: Significant associations were detected at 5/25 loci, including TERT, EGFR, CCDC26, CDKN2B, and RTEL1. The strongest association was at rs55705857 (CCDC26, OR=2.7, p=7.49x10-17). For GBM (222 familial cases), significant associations were detected at 6/26 loci (TERT, EGFR, CDKN2B, TP53 and RTEL1), while in non-GBM (205 familial cases) significant associations were detected at 3/25 loci (LRIG1, CCDC26, PHLDB1). These SNPs were further examined using a case-only approach comparing familial to non-familial cases, and there was no significant difference in allele frequencies by family history status. There was a strong correlation between log(OR) for familial cases only versus non-familial cases (adjusted R2=0.88).

CONCLUSIONS: In this analysis we identified a significant association between familial glioma and five common risk loci previously identified by glioma GWAS. This provides further evidence of shared pathways of genetic risk and gliomagenesis between familial and non-familial glioma. Further exploration is necessary to determine the overall contribution of common genetic variation to risk of familial glioma.

#4174

Familiality of ovarian cancer histotypes in the Utah Population Database.

Mollie E. Barnard, Nicola J. Camp, Jennifer A. Doherty. _Univ. of Utah Huntsman Cancer Inst., Salt Lake City, UT_.

Introduction: Ovarian cancer is a heterogeneous disease; each histotype has distinct molecular origins, risk factors and outcomes. While it is well established that ovarian cancer has a familial risk component, the familiality of ovarian cancer histotypes has not been well described. The Utah Population Database (UPDB) captures over 10 million individuals, including more than 5 million with a minimum of three generations of genealogy data. By linking records from the UPDB to the Utah Cancer Registry (UCR) and other statewide data sources, we sought to describe the familiality of ovarian cancer histotypes.

Methods: Our study includes all ovarian cancer cases in the UPDB and leverages clinical data reported to UCR for the case histotypes. Cases were matched to controls on age, birth cohort and at least three generations of UPDB data versus not. We quantified familiality by comparing the odds of ovarian cancer occurrence among family members of cases to the odds of ovarian cancer among family members of matched controls. We calculated odds ratios for first-degree relatives, second-degree relatives, and first cousins.

Results: As of September 2018, the UPDB included 3,989 ovarian cancer cases. We observed an increased risk of epithelial ovarian cancer among first-degree relatives (OR=1.62, 95%CI=1.36-1.93), second-degree relatives (OR=1.53, 95%CI=1.34-1.74), and first cousins (OR=1.38, 95%CI=1.26-1.53) of ovarian cancer cases. When we analyzed epithelial ovarian cancers by histotype, familiality was most evident for high-grade serous, mucinous, and unclassified cancers. For example, we observed a 1.62-fold increased odds (95%CI=1.14-2.29) of high-grade serous ovarian cancer among the first-degree relatives of high-grade serous cases, and a 5.59-fold increased odds (95%CI=2.09-14.92) of mucinous ovarian cancer among first-degree relatives of mucinous ovarian cancer cases. Familiality patterns were less clear among endometrioid and clear cell cases.

Conclusions: The UPDB has the potential to contribute greatly to family-based studies of the genetics of ovarian cancer histotypes. Despite the possible variations in quality of histotype data across generations, preliminary results suggest some histotypes may be particularly familial and good candidates for gene mapping studies. Work is ongoing to refine the histotype assignments for cases by performing pathology review using the 2014 World Health Organization guidelines. Analyses are also in progress to stratify analyses by BRCA mutation status, and to consider the potential for shared familiality across ovarian cancer histotypes and with cancers at other sites.

#4175

Spectrum of heritable mutations in 43 known and candidate breast cancer susceptibility genes in African American women with breast cancer.

Kristen Purrington, Gregory Dyson, Douglas Craig, Julie Boerner, Julie Madden, Jennifer Beebe-Dimmer, Ann G. Schwartz, Michael Simon. _Wayne State Univ. School of Medicine, Detroit, MI_.

Mutations in twenty genes have been associated with heritable breast cancer (BCA), yet the prevalence and clinical implications of most of these genes have not been well described in African American women (AAW). AAW who harbor inherited mutations in BCA susceptibility (BCS) genes are less likely to be identified and thus less likely to receive standard of surgical and preventive care for heritable BCA. A better understanding of heritable mutations in AAW with BCA would ultimately help to define guidelines for clinical management of high risk AAW. We performed targeted sequencing for 288 AAW diagnosed with invasive BCA unselected for family history, age, or BCA subtype. Participants were enrolled in the Detroit Research on Cancer Survivors (ROCS) cohort study or the Karmanos Cancer Institute Biobank. A custom sequencing panel captured the coding sequence of 43 genes (Known: ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, MRE11A, MSH6, MUTYH, NBN, NF1, PALB2, PMS2, PTEN, RAD50, RAD51C, RAD51D, STK11, TP53; Candidate: MLH1, MSH2, PMS1, SEC23B, BLM, ATR, BAP1, BBC3, CDKN1A, FAM175A, FANCA, FANCC, FANCI, FANCL, FANCM, GEN1, RAD51B, RBBP8, RECQL, RINT1, TP53BP1, XRCC1, XRCC3). Samples were sequenced using the Illumina MiSeq platform multiplexed for 100X mean depth of coverage. Intronic or synonymous exonic variants and those with <40 reads, or alternative allele frequency >3% in African populations in the 1000 Genomes, ExAC, or NHLBI ESP6500SI databases were filtered. Only variants in known BCS genes could be considered definitively pathogenic (DP), defined as missense variants categorized as pathogenic in ClinVar and all frameshift, nonsense, and splice site variants. All other variants were defined as variants of uncertain significance (VUS). We identified 54 women harboring DP mutations in 15 known BCS genes. Mutations in BRCA1/2 accounted for 32% of all DP mutations. Notably, nearly 17% of DP mutations were in MSH6, a gene only recently described as associated with increased risk of BCA in the general population. Among women with these DP mutations, nearly 25% had no high-risk characteristics. Ten VUS in known BCS genes were identified, where 4 were reported in ClinVar (BARD1, RAD5C, BRCA2, PMS2) and 6 were novel (PMS2, BRCA1, MUTYH, MSH6, NBN, MRE11). Among the candidate BCS genes, 63 VUS were identified: 51 frameshift variants, 5 nonsense variants, 3 splice site variants, and 4 missense mutations predicted to be pathogenic by ≥8/9 algorithms. Nearly 60% of participants had neither a DP mutation or VUS. These data suggest that AAW with BCA have a unique mutation spectrum and may benefit from BCS gene sequencing regardless of family history, age, or subtype. Additional sequencing in AAW will lay the foundation for identification of germline factors associated with BCA risk and prognosis specifically in AAW and offer opportunities for personalized risk assessment.

#4176

Familial lung cancer exhibits multiple novel linked haplotypes within pedigrees.

Anthony Musolf,1 Haiming Sun,1 Bilal A. Moiz,1 Claudio W. Pikielny,2 Mariza de Andrade,3 Diptasri Mandal,4 Colette Gaba,5 Ping Yang,3 Yafang Li,2 Ming You,6 Richard K. Wilson,7 Elena Y. Kupert,6 Marshall W. Anderson,6 Ann G. Schwartz,8 Susan M. Pinney,9 Christopher I. Amos,10 Ramaswamy Govindan,11 Joan E. Bailey-Wilson1. 1 _NIH NHGRI, Baltimore, MD;_ 2 _Dartmouth College, Lebanon, NH;_ 3 _Mayo Clinic, Rochester, MN;_ 4 _Louisiana State University Health Sciences Center, New Orleans, LA;_ 5 _University of Toledo, Toledo, OH;_ 6 _Medical College of Wisconsin, Milwaukee, WI;_ 7 _Nationwide Children's Hospital, Columbus, OH;_ 8 _Karmanos Cancer Institute, Detroit, MI;_ 9 _University of Cincinnati, Cincinnati, OH;_ 10 _Baylor College of Medicine, Houston, TX;_ 11 _Washington University, St. Louis, MO_.

Lung cancer (LC) kills more people in the United States each year than any other cancer. While it is well known that a variety of environmental factors (particularly tobacco smoke) strongly increase the risk of LC, there are multiple associated genetic variants with small contributions to risk. High aggregation of LC within rare individual families suggest that there are high-risk genetic variants as well. However, these genetic risk factors for LC are under studied due to the rapid fatality of LC. We studied 28 highly aggregated extended high-risk familial lung cancer (HRFLC) families collected from eight different sites across the US. Whole exome sequencing was performed on 290 individuals from these families to identify potential risk variants for HRFLC using genetic linkage analysis. Quality control was performed on the sequence data, filtering on parameters such as read depth, genotype quality, missingness, and Mendelian inconsistencies. Identity-by-descent (IBD) values were also calculated to verify correct familial relationships. Quality control procedures left approximately 400,000 SNVs and indels for analysis.Parametric two-point linkage analysis was performed assuming an autosomal dominant mode of inheritance. Disease allele frequency was set to 1% with a penetrance of 80% for carriers and 1% phenocopy rate. While we did not identify any genome-wide significant variants across the 28 families, multiple suggestive variants were identified. The largest cluster of suggestive variants was located at 14q32 in the CATSPERB gene. Given the likely locus heterogeneity in LC (combined with the lack of power in some families), it is not surprising that none of the variants were significant across the families; looking at the individual family results proved more informative. Long haplotypes linked to LC risk were identified in multiple families. These long runs of positive linkages, which have little to no negative linkage evidence across them, are characteristic of true linkage signals in these types of analysis. Two of the most interesting linked regions were at 7p36.1 and 4q21.23-28.23. The 7p signal (observed in a single family) was genome-wide suggestive and located within the SSPO gene. SSPO has been implicated in breast and skin cancer (melanoma); it is a novel lung cancer signal. The 4q linkage (again observed in a single family) covers a large chunk of 4q and contains multiple potential candidate genes, however, the best candidate gene is PTPN13, a gene implicated in lung cancer but never in familial lung cancer. We are currently evaluating the individual family results of several other pedigrees and plan to perform additional analyses to confirm these linkages.

#4177

The joint effects of polygenic risk scores and pathogenic variants in cancer predisposition genes on breast cancer risk in the general population: results from the CARRIERS study.

Chi Gao,1 Chunling Hu,2 Steven N. Hart,2 Rohan Gnanaolivu,2 Kun Y. Lee,2 Jenna Lilyquist,2 Nicholas J. Boddicker,2 Bruce Eckloff,2 Raed Samara,3 Josh Klebba,3 Paul Auer,4 Leslie Bernstein,5 Mia Gaudet,6 Hoda Anton-Culver,7 Amy Trentham-Dietz,8 Julie R. Palmer,9 Song Yao,10 Christopher Haiman,11 Janet E. Olson,2 Susan Domchek,12 Jeffrey Weitzel,13 David Goldgar,14 Katherine L. Nathanson,15 Eric C. Polley,2 Fergus J. Couch,2 Peter Kraft1. 1 _Harvard T.H. Chan School of Public Health, Boston, MA;_ 2 _Mayo Clinic, Rochester, MN;_ 3 _Qiagen, Hilden, Germany;_ 4 _UWM Joseph J. Zilber School of Public Health, Milwaukee, WI;_ 5 _UConn Health, Farmington, CT;_ 6 _Rollin School of Public Health, Atlanta, GA;_ 7 _University of California, Irvine, CA;_ 8 _University of Wisconsin, Madison, WI;_ 9 _Boston University School of Public Health, Boston, MA;_ 10 _Roswell Park Comprehensive Cancer Center, Buffalo, NY;_ 11 _Keck School of Medicine, Los Angeles, CA;_ 12 _University of Pennsylvania School of Medicine, Philadelphia, PA;_ 13 _City of Hope Comprehensive Cancer Center, Duarte, CA;_ 14 _University of Utah, Salt Lake City, UT;_ 15 _University of Pennsylvania, Philadelphia, PA_.

Background: Understanding the joint effect on breast cancer risk of rare pathogenic variants in cancer predisposition genes and polygenic risk scores (PRS) from common variants can enable a precision approach to breast cancer management. Previous work found that PRS were associated with breast cancer risk in BRCA1 and BRCA2 mutation carriers recruited from cancer genetics clinics (Kuchenbaecker et al. JNCI 2017). The joint effects of pathogenic variants and PRS have not been studied in samples drawn from the general population. There is also no existing study evaluating the effect of PRS in mutation carriers in genes other than BRCA1/2. Here we evaluate the joint effects of PRS and pathogenic mutations in established cancer predisposition genes in a population-based case-control sample.

Method: We analyzed 53,199 European-ancestry individuals (20,730 controls and 21,272 cases) drawn from 7 cohorts and 2 population-based case-control studies in the CAnceR RIsk Estimates Related to Susceptibility" (CARRIERS) consortium. Targeted sequencing was performed using dual bar-coded QIAseq. Mutation calling was conducted with Haplotype Caller and Vardict. We sequenced 18 breast cancer predisposition genes: ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CDKN2A, CHECK2, FANCC, MLH1, MSH2, MSH6, NF1, PALB2, PTEN, RAD51C, RAD51D and TP53. The PRS was calculated based on 128 common variants using effect estimates from the largest published breast cancer GWAS. The PRS was standardized to a mean of 0 and standard deviation of 1. Logistic regression was performed to assess the combined effect of identified mutation and common variants (including main and interaction effects for carrier status and PRS).

Results: A total of 1,770 pathogenic mutations were observed. About 1% of the study sample had mutations in either BRCA1 (n=209) or BRCA2 gene (n=305), and 2.2% had mutations in other genes (n=1,176). The effect of PRS in BRCA1 carriers was OR= 0.97 (95%CI: 0.62, 1.52); however, this is not statistically significantly different from the PRS in non-carriers [OR=1.23 (95%CI: 1.20, 1.25)] or previous estimates in carriers [OR: 1.14, 95%CI: 1.11-1.17]. The effect of PRS in BRCA2 carriers was OR=1.87 (95%CI: 1.31, 2.78) which was statistically significantly different from that of the non-carriers. Among the mutation carriers in genes other than BRCA, the effect of PRS on breast cancer was OR=1.00 (95%CI: 0.88,1.15).

Conclusion: Consistent with previous studies, we did not find evidence that the effect of the PRS among BRCA1 carriers was statistically significantly different than non-carriers. We found some evidence that the PRS effect may be larger among BRCA2 carriers than non-carriers. Our results also suggest that PRS may not have a strong effect on breast cancer risk in mutation carriers of other predisposition genes; further work is needed for verification.

#4178

**Germline mutations in** BRCA2 **and pediatric/adolescent non-Hodgkin's lymphoma: A report from the St. Jude Lifetime (SJLIFE) and Childhood Cancer Survivor Study (CCSS) cohorts.**

Zhaoming Wang,1 Ti-Cheng Chang,1 Carmen L. Wilson,1 Chimene A. Kesserwan,1 Todd M. Gibson,1 Nan Li,1 John Easton,1 Heather L. Mulder,1 Gang Wu,1 Michael N. Edmonson,1 Michael C. Rusch,1 James R. Downing,1 Kim E. Nichols,1 Smita Bhatia,2 Gregory T. Armstrong,1 Melissa M. Hudson,1 Jinghui Zhang,1 Yutaka Yasui,1 Leslie L. Robison1. 1 _St. Jude Children's Research Hospital, Memphis, TN;_ 2 _University of Alabama at Birmingham, Birmingham, AL_.

Pathogenic or likely pathogenic (P/LP) monoallelic germline mutations in BRCA2 increase risk of developing breast, ovarian, prostate, and pancreatic cancers. In a prior report from the SJLIFE study, BRCA2 was the third most frequently mutated gene (14 occurrences) among 3006 survivors of childhood cancer with the highest number observed among lymphoma survivors (7/586). To further investigate BRCA2 as a potential predisposition gene for pediatric/adolescent lymphoma, we analyzed 781 additional lymphoma survivors in the SJLIFE and CCSS cohorts with whole-genome sequencing (30X coverage). In the combined set of 1367 survivors (808 Hodgkin's lymphoma [HL], 559 non-Hodgkin's lymphoma [NHL]; 54% male; median age at diagnosis 12.6 [range 1.1-22.7] years), 13 P/LP mutations in BRCA2 were identified, with 7 mapped to the breast or ovarian cancer cluster regions defined by the Consortium of Investigators of Modifiers of BRCA1/2. Compared to reference controls in the Genome Aggregation Database (gnomAD) (Table 1), a significant association was observed between lymphoma and mutations in BRCA2 (odds ratio [OR], 3.1; 95% CI, 1.7-5.5) but not BRCA1. When stratified by diagnosis, the association was significant for NHL (OR, 4.8; 95% CI, 2.0-9.6) but not for HL. BRCA2 mutation carriers included a broad spectrum of NHL histological subtypes. Our findings support inclusion of pediatric/adolescent NHL in the spectrum of cancers associated with germline BRCA2 mutations. Approximately 1.4% of survivors of pediatric/adolescent NHL are carriers of a P/LP mutation in BRCA2, which may be the underlying etiology of their primary diagnosis. Clinically, counselling regarding BRCA2 mutation status should be considered for pediatric/adolescent NHL patients. Large scale genetic studies of newly diagnosed pediatric/adolescent lymphoma patients are warranted to replicate and refine diagnosis-specific risk estimates.

Table 1. Comparisons of Mutation Carriers for BRCA1/2 Genes Between Lymphoma Survivors and gnomAD Controls

---

|  | Lymphoma Survivors | gnomAD Controls

(Hu et al. JAMA 2018) | Cancer Risk

(Fisher''s Exact Test)

Gene | Cancer

Diagnosis | Carriers | Non-Carriers | Carriers | Non-Carriers | Odds Ratio

(95% CI) | P Value

BRCA2 | HL+NHL | 13 | 1354 | 313 | 102426 | 3.1 (1.7-5.5) | 0.00045

|

HL | 5 | 803 | 313 | 102426 | 2.0 (0.7-4.8) | 0.11

|

NHL | 8 | 551 | 313 | 102426 | 4.8 (2.0-9.6) | 0.00041

BRCA1 | HL+NHL | 3 | 1364 | 208 | 103914 | 1.1 (0.2-3.3) | 0.76

|

HL | 1 | 807 | 208 | 103914 | 0.6 (0.02-3.5) | 1.0

|

NHL | 2 | 557 | 208 | 103914 | 1.8 (0.2-6.6) | 0.31

#4179

Identification of BRCA1 and BRCA2 mutation spectrum and their founder effect in epithelial ovarian cancer from Middle East.

Abdul K. Siraj, Rong Bu, Kaleem Iqbal, Nabil Siraj, Maha Alrasheed, Laila Ghazwani, Tariq Masoodi, Sandeep Kumar Parvathareddy, Khawla S. Al-Kuraya. _King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia_.

Epithelial ovarian cancer (EOC) remains the most fatal gynecological malignancies. Germline mutations in Breast Cancer susceptibility gene 1 and 2 (BRCA1 and BRCA2) have previously been estimated to contribute to 13-18% of all EOC. The prevalence and influence of BRCA1 and BRCA2 mutations in EOC in Middle Eastern population is not fully explored. Ethnic differences of ovarian cancer genomics have prompted us to investigate the spectrum of BRCA1 and BRCA2 mutations among our Middle Eastern EOC patients. To characterize the prevalence of BRCA mutations in our patients, BRCA mutation screening was performed in over 400 unselected ovarian cancer patients using Capture and/or Sanger sequencing. Seventeen short tandem repeat (STR) markers were used for founder mutation analysis. A total of 19 different types of pathogenic mutations were identified in 50 cases (40 mutant cases in BRCA1 and 10 in BRCA2). Nine mutations were recurrent accounting for 80% (40/50) of all mutant cases (9.8% (40/407) of the entire cohort). Haplotype analysis carried out on all unrelated cases with these mutations revealed only two (c.4136_4137 delCT and c.1140 dupG) sharing the same haplotypes thus representing founder Saudi mutations. Remarkably, lifetime risk estimation for these founder mutations appears to approach 100% given their complete absence in healthy controls. BRCA1 mutant cases were significantly associated with positive family history (p <0.0001), grade 3 tumors (p = 0.0003) and loss of BRCA1 protein expression (p <0.0001). 85.0% (34/40) of all BRCA1 mutant cases were of serous histology. BRCA2 mutant cases were associated with loss of BRCA2 protein expression (p=0.0102) and a trend towards younger age (p=0.0505). Identification of the mutation spectrum, prevalence and founder effect in Middle Eastern population facilitates genetic counseling, risk assessment and development of cost-effective screening strategy.

#4180

Association between genetic polymorphisms of DNMT genes and breast cancer in a nested case-control study of African Americans.

L Joseph Su,1 Hui-Yi Lin,2 Tung-Chin Chiang,1 Gail Runnells,1 Lora Rogers,1 Ping-Ching Hsu,1 Ronda Henry-Tillman,1 Susan Kadlubar1. 1 _University of Arkansas for Medical Sciences, Little Rock, AR;_ 2 _Louisiana State University Health Sciences Center, New Orleans, LA_.

Introduction/Purpose. DNA methylation plays a significant role in both the development and progression of various cancers. Although aberrant DNA methylation of CpG islands is a common epigenetic alternation found in cancers, the interpretation of observed DNA methylation and cancer risk remains a challenge. DNA methylation is typically mediated by DNA Methyltransferases (DNMTs). This study took advantage of the infrastructure and existing data and samples of Arkansas Rural Community Health (ARCH) Study cohort to conduct a pilot nested case-control study of breast cancer among African Americans examining the relationship between breast cancer and DNMT single nucleotide polymorphisms (SNPs).

Methods: ARCH Study cohort consists of 26,387 women aged 18 to 85 recruited from all 75 counties in Arkansas between 2007 and 2014. All cohort participants donated a saliva sample for DNA and completed a short questionnaire at baseline. 309 African American breast cancer cases and 530 age-group, race, and recruitment year frequency-matched controls were selected for this analysis. Genotyping of DNMT1, DNMT3A, and DNMT3B were conducted using Tagman method. Multivariable logistic regression (odds ratio (OR), 95% confidence interval (CI) was used to examine the association between breast cancer and each SNP while considering the factors of age, education, and status of obesity, child birth, breast feeding, and menstrual-stop as potential confounding variables. Hardy-Weinberg equilibrium for the loci examined were evaluated. Haplotype analyses were conducted.

Results: Among 16 SNPs evaluated, heterozygous genotypes rs7575625 in DNMT1 and rs7605753 in DNMT3A were positively associated with breast cancer risk; rs12991495 in DNMT3A and rs17123590 and rs2424910 in DNMT3B were inversely associated with breast cancer. Haplotype AGA of rs8101626, rs2290684, and rs11880388 in DNMT1, which accounts for 43.9% population, differed significantly as a risk factor between cases and controls after adjusting for confounders (OR = 2.05; 95%CI=1.34, 3.12). Haplotype AGGGGT of rs2304429, rs12991495, rs7605753, rs11892646, rs7575625, and rs10196635 in DNMT3A differed significantly as a risk factor between cases and controls (OR = 4.06; 95%CI=1.93, 8.52); while haplotype rs2424905, rs2424910, rs17123590, rs6088008, and rs6058896 in DNMT3B was inversely associated with breast cancer (OR = 0.02; 95%CI=0.001, 0.29).

Conclusions: Despite the knowledge of DNMTs on DNA methylation, very few studies examined its role in breast cancer, in particular among underserved and minority population. This study took advantage of the existing sample and data of African Americans in a cohort population and demonstrated DNMT genetic polymorphisms are associated with breast cancer. Further evaluation of methylation patterns could provide knowledge on the mechanistic pathways for the association observed.

### Descriptive Epidemiology: Risk, Outcomes, and Disparities

#4181

Awareness of human papillomavirus (HPV) infection among men and women with oral cancer.

Assia Miller, Latrice Rollins, Ananya Banerjee. _Morehouse School of Medicine, Atlanta, GA_.

Background There is a high level of public awareness of association human papillomavirus (HPV) infection and genital cancer. Though recent research showed that 26 million Americans have oral HPV infection, the information on awareness of association HPV infection with oral cancer among men and women is limited.

Methods The objective of our study was to assess population awareness of and knowledge about the association between HPV and oral cancer among men and women. We obtained cross-sectional data from the National Cancer Institute's Health Information National Trends Surveys (HINTS 2007-2015, N=25,410). We applied a Wald chi-square test to examine the association of HPV awareness in participants with oral cancer compared to participants with other forms of cancer stratified by gender.

Results: Among men with oral cancer (N=10) 16 % were aware of HPV while among men with other forms of cancer (N=3557) 44% were aware of HPV (p=0.0924). Among women with oral cancer (N=5) 56 % were aware of HPV while among women with other forms of cancer (N=5631) 64% were aware of HPV (p=7971).

Conclusion: There is decreased awareness and knowledge of HPV in men with oral cancer promoting opportunities to target communication about a vaccine.

#4182

Community and cancer registry collaboration to reduce persistent stomach cancer disparity in Korean Americans: Asian American Stomach Cancer Task Force.

Eunjung Lee,1 Juanjuan Zhang,1 Sohee Park,2 Amie E. Hwang,1 Lihua Liu,1 Haeseong Park,3 Sang-Hoon Ahn,1 Anna H. Wu,1 Dennis Deapen1. 1 _USC Norris Comprehensive Cancer Ctr., Los Angeles, CA;_ 2 _Yonsei University, Seoul, Republic of Korea;_ 3 _Washington University in St. Louis, St. Louis, MO_.

South Korea and Japan have the highest stomach cancer incidence rates worldwide. Using 1988-2012 data from the California Cancer Registry, we have previously reported a striking stomach cancer disparity in Korean Americans, marked by 5 times higher incidence than in non-Hispanic whites (NHWs) and two times higher incidence than in Japanese Americans, the racial/ethnic group with the second highest incidence. The majority of Korean American adults were born in Korea while the majority of Japanese Americans adults were born in the US. Although Korean Americans had a more favorable stage distribution than other Californians in our earlier study, Korean Americans had a worse stage distribution compared to populations in Korea or Japan, where population-based screening is available. Data from South Korea demonstrated benefits of screening endoscopy in improving stage at diagnosis as well as mortality from stomach cancer. Bi-annual stomach cancer screening is now recommended for South Korean adults aged 40 or above, with an estimated participation rate of 75%. The United States lacks well-defined stomach cancer screening guidelines. In this study, we used the 2010-2014 Surveillance, Epidemiology, and End Results (SEER) data and compared stage distribution in Korean Americans in Los Angeles County, other areas in California, and SEER regions outside California. The proportion of early stage disease was highest in Korean Americans in Los Angeles County (49%), followed by other areas in California (41%) and SEER regions outside California (36%). The earlier diagnosis in Los Angeles County compared to other SEER regions was not observed for Japanese Americans or NHWs. It is reasonable to speculate that the increased public and physician awareness particularly in areas with a large Korean community helped Korean Americans somewhat overcome the disparity coming from the high incidence. However, the proportion of localized disease among Los Angeles County Korean Americans was still much lower than the proportion in Korea during the same time period (60-66%), indicating the need for targeted screening or well-defined guidelines for diagnostic endoscopy. Dissemination of these cancer registry findings led to a collaborative partnership between a number of Korean American community physician organizations and Los Angeles County SEER cancer registry, the Los Angeles Cancer Surveillance Program, establishing the 'Asian American Stomach Cancer Task Force' (AASCTF). The overarching goal of AASCTF is to reduce stomach cancer disparity in Korean Americans and other immigrants from high risk regions by promoting stomach endoscopy through community campaign and supporting research activities to help the medical community and policy makers to make evidence-based decisions.

#4183

Recruiting African-American prostate cancer patients for biobank research.

Jennifer Damonte,1 Siddhartha Roy,2 Carmen Benson,1 Kala Jamison,1 Hyun Park,1 Allan Pollack,3 Shahla Masood,4 Changdong Yeo,5 Young-chul Kim,1 Jasreman Dhillon,1 Kosj Yamoah,1 Julio Pow-Sang,1 Thomas A. Sellers,1 Clement K. Gwede,1 Jong Y. Park1. 1 _Moffitt Cancer Center, Tampa, FL;_ 2 _Penn State University, Hershey, PA;_ 3 _University of Miami, Miami, FL;_ 4 _University of Florida at Jacksonville, Jacksonville, FL;_ 5 _The Catholic University of Korea, Seoul, Republic of Korea_.

Significant racial disparities exist in prostate cancer (PCa) incidence and mortality rates. Exploration of the basis for disparities would be enhanced by access to data and biological specimens on men of African Ancestry (AA). Unfortunately, AA men remain underrepresented in cancer biorepositories. To address this gap, we have embarked on an effort to create a state-wide biospecimen bank focused on AA men with PCa in Florida. The objective is to collect and manage patients' data, outcome information, and biospecimens (i.e., tumor tissue samples and saliva) from diverse AA men for research. The objective is to collect and manage patients' data, outcome information, and biospecimens who have been diagnosed with PCa in Florida. Self-identified AA PCa patients, diagnosed between 2013 and 2017, who were living in Florida at the time of diagnosis were identified through the Florida State cancer registry. Potential participants were mailed packets describing the study, with follow-up by telephone to answer questions and obtain informed consent. Interested patients were screened for eligibility and asked to complete a questionnaire, provide a saliva sample, and provide permission to obtain their tumor tissue sample. Patients were offered up to $30 for participating in the study. The Florida Cancer Registry reported a total of 7,960 AA PCa cases during the ascertainment period. Information packets were sent to 4,898 AA. A total of 116 were found to be ineligible mainly due to deceased or non-English speaking patients. To date, 796 have consented to participate, 1,175 declined ether by mail (n=185) or phone (=985), 740 could not be located, and 2,033 have not responded to the initial information packet/phone after total of 5 attempts. This yields a participation rate of 16.3% (796/4,898). The adjusted participation rate, or the percentage of participants who consented to participate out of those who had communication with the research team, is 41% (796/1,959). Primary reasons for declining include patients stating that they are not interested (72%), too sick (8%), or too busy (5%). A significant inverse association was found between current age and participation rate (p<0.0001) but not insurance status. Older AA PCa patients were found to be less likely to participate in the study. A significant association was also found between Gleason score and participation rate. AA men who had a more aggressive form of PCa (higher Gleason score) were more likely to participate in the study (p<0.01). Our results demonstrate that recruiting Blacks/African-American PCa patients in the biobank study using a cancer registry is feasible, yet difficult. Despite prevailing recruitment challenges, we implemented a diverse of recruitment methods to increase reach. These recruitment methods helped identify additional avenues for targeting this population to increase participation and, ultimately, address cancer disparities among this population.

#4184

Tumor and risk factor characteristics among breast cancer patients from different geographic regions in Peru.

Valentina Zavala,1 Tatiana Vidaurre,2 Katie Marker,3 Jeannie Vásquez,2 L Tamayo,4 Renzo Florez,2 Sandro Casavilca,2 M Calderon,2 J Abugattas,2 H Gómez,5 H Fuentes,2 C Monge-Pimentel,2 S Song,1 D Cherry,6 Laura Fejerman1. 1 _University of California San Francisco, San Francisco, CA;_ 2 _Instituto Nacional de Enfermedades Neoplasicas, Peru;_ 3 _University of California Berkeley, CA;_ 4 _University of Chicago, IL;_ 5 _) Instituto Nacional de Enfermedades Neoplasicas, Peru;_ 6 _University of California San Diego, CA_.

There are few Latin American cohorts with available biospecimens that include women of high Indigenous American ancestry. The Peruvian population is characterized by a high degree of Native American (NA) ancestry, with this ancestral component varying between 56 to 100% on average, depending on the region. We have collected 1199 Peruvian samples from the Instituto Nacional de Enfermedades Neoplasicas in Lima. This cohort of patients represents a unique opportunity to study the molecular characteristics of breast cancer in the NA genetic and genomic background. Here we present a basic description of the women in the study and a comparison of tumor subtypes distribution and risk factor information of the patients by place of birth and residence.

We explored differences in tumor subtype distribution and risk factors in relation to place of birth or residence in the three main geographical region of Peru. To test differences in proportions we used Chi2 or Fisher-exact tests. To test differences in means for continuous variables we used ANOVA or t-tests. Genetic ancestry was estimated using genome wide genotypes and the program ADMIXTURE. Tumor subtypes were defined using the following criteria: ER+/PR+/HER2- as luminal A, ER+/PR+/HER2+ as luminal B, ER-/PR-/HER2+ as HER2+ and ER-/PR-/HER2- as triple negative.

Overall, the patients included in the study were relatively young (50 yrs, SD=11.0). The average number of full-term pregnancies was 3 (SD=1.8), the average age at first pregnancy 22 (SD=5.7) and age at menarche was 13 (SD=1.8). The tumor subtype distribution was 31% of Luminal B tumors, 24% luminal A, 12% HER2 and 12% triple negative and did not differ by place of birth or residence. We found that patients from the Coastal region were heavier and taller than those born in the Andean region (p<0.005). Women born in the Coastal region had the lowest age at menarche and a lower number of full-term pregnancies (p<0.0001). Similar trends were observed when we compared women by place of residence. Patients born in Lima, the Capital of Peru, smoke more (p<0.05), were heavier, had lower age at menarche, lower number of full term pregnancies and were diagnosed at a younger age, compared to women born outside the city (p<0.05). The distribution of NA genetic ancestry also varied by place of birth: patients born outside Lima had higher proportion of NA ancestry (78% SD=0.15 vs. 74%, SD=0.18, p<0.05).

The distribution of tumor subtypes among women in the Peruvian breast cancer cohort did not differ by place of birth or residence. However, we found that for some breast cancer risk factors, exposures differed between women from different regions. Finally, given the relatively low observed values for reproductive and lifestyle related exposures and the high proportion of Indigenous American ancestry of Peruvian women, this cohort is likely to be particularly informative to study genetic predisposition to breast cancer.

#4185

Establishing sustainable research collaborations in the Caribbean through the utilization of multiple technology platforms.

Elizabeth Blackman,1 Kimlin Tam Ashing,2 Camille Ragin,1 Marshall Tulloch-Reid,3 J. Robert Beck,1 Althea Bailey,3 Ian Hambilton,4 Natalie Guthrie-Dixon,3 Georgia Williamson,3 Andrea Forman,1 Robin Roberts5. 1 _Fox Chase Cancer Ctr., Philadelphia, PA;_ 2 _City of Hope Medical Center, Duarte, CA;_ 3 _University of the West Indies-Mona Campus, Kingston, Jamaica;_ 4 _University of the West Indies-Cave Hill, Barbados;_ 5 _University of the West Indies- School of Clinical Medicine and Research, Nassau, Bahamas_.

Introduction: International research collaborations are a critical strategy in tackling immigrant and global prevention and control challenges. Available communications and data technology facilitates collaborations to address global cancer inequities. The purpose of the African Caribbean Cancer Consortium (AC3) is to develop and sustain partnerships to investigate and respond to the increasing cancer vulnerability among African descended peoples worldwide. In the Caribbean, cancer incidence and mortality are rising in persons of African ancestry. We will present preliminary outcomes from our NCI-P20 award.

Methods: Our goal is to establish a Caribbean Regional Center of Research Excellence (CRCRE). The project started in Jamaica in partnership with the University of the West Indies-Mona. The CRCRE is building a sustainable research center at the Mona campus that will spread to other campuses throughout the University system. Using ZOOM, WhatsApp, YouTube, GoogleDocs, and REDCap, we will develop a sustainable research infrastructure in the Caribbean.

Results:

Regional Needs Assessment- Using REDCap, we were able to conduct a research needs assessment across the Caribbean. A survey was developed by the research team and administered within the Mona campus and Caribbean-wide through the AC3 member listserv. We had over 150 respondents representing key stakeholders who identified top priorities to develop sustainable research in the region.

The REDCap database for the CRCRE will also house training webinars readily available for users.

Mentor-Mentee Program was implemented to match junior faculty in the Caribbean to senior faculty within the AC3 network. Among Caribbean researchers, we are building capacity for basic, translational, clinical and behavioral research. Mentors and mentees will utilize ZOOM, e-mail, Googledocs, WhatsApp and YouTube to facilitate training, mentoring and study processes.

Genetic Counseling Training was conducted between genetic counselors at Fox Chase Cancer Center (FCCC) and 3 Caribbean nurses based at Princess Margaret Hospital, Nassau, Bahamas. Nurses received virtual training via NCCN and ZOOM lectures. Nurses then shadowed FCCC genetic counselors. They will utilize ZOOM, WhatsApp and YouTube to facilitate ongoing training, mentoring, and supervision.

Discussion: Using communication and data technologies are effective and cost effective platforms for developing and sustaining international cancer prevention and control research in low and middle-income countries including the Caribbean. Caribbeans account for 10% of all immigrants and 50% of all immigrant Blacks. Therefore, attending to and addressing cancer biologic, genetic, environmental and behavioral risk and disparate outcomes among Caribbeans will benefit the Region as well as inform strategies to reduce cancer disparities within U.S. Black population.

#4186

Aggressive prostate cancer among men in Pennsylvania: Differences between Appalachian and non-Appalachian counties.

Alicia C. McDonald,1 Emily Wasserman,1 Eugene J. Lengerich,1 Jay D. Raman,2 Nathaniel Geyer,1 Raymond Hohl,3 Ming Wang1. 1 _Penn State Univ. College of Medicine, Hershey, PA;_ 2 _Pennsylvania State Milton S. Hershey Medical Center, Hershey, PA;_ 3 _Penn State Cancer Institute, Hershey, PA_.

Introduction: The incidence of prostate cancer (PC) is known to vary within Appalachia, with Northern Appalachia and Southern Appalachia having higher and lower rates, respectively. However, the geographical disparity for aggressive PC has not been investigated. The objective of this study was to determine whether having more aggressive PC was associated with residing in an Appalachian/non-Appalachian county and rural/urban community.

Methods: A cross-sectional study was conducted among men, aged > = 40 years, who had a primary, clinical PC diagnosis identified in the Pennsylvania Cancer Registry between 2004 and 2014. PC aggressiveness was defined as less aggressive (Gleason score 6 or 7 [3+4] and clinical/pathologic tumor stage T1-T2) and more aggressive (Gleason score > = 7 [4+3], clinical/pathologic tumor stage T3-T4, or distant metastasis) PC. Logistic regression analysis was used to examine the association between having aggressive PC (less vs more aggressive PC - dependent variable) and residing in a geographical region (Appalachian versus non-Appalachian county - independent variable), stratified by rural/urban, after adjusting for age, race, ethnicity, insurance status, marital status, serum PSA, positive lymph node status, and cancer treatment.

Results: There were 94,264 PC cases, aged 40 to 105 years, included in the study. The majority of the PC cases were white (83.9%) followed by black race (10.7%). PC cases from urban Appalachian counties (35.42%) had the highest frequency of aggressive PC followed by rural non-Appalachian (34.18%), rural Appalachian (32.12%), and urban non-Appalachian counties (32.08%). In urban areas, Appalachian PC cases were more likely to be aggressive compared to non-Appalachian PC cases, after adjusting for confounders (odds ratio [O.R.] =1.16; 95% confidence interval [C.I.], 1.13-1.21). Conversely, in rural areas, Appalachian PC cases were less likely to be aggressive compared to non-Appalachian PC cases, after adjusting for confounders (O.R. =0.91; 95% C.I., 0.85-0.98).

Conclusion: Differences in having more aggressive PC was observed for Appalachian populations by rural/urban status, with urban Appalachian PC cases more likely and rural Appalachian PC cases less likely to have more aggressive disease, compared to their non-Appalachian counterparts. Further investigation on the underlying mechanisms of aggressive PC in urban Appalachia in PA is warranted.

#4187

Racial disparities in kidney cancer incidence across South Texas, Texas, and United States using TCR, SEER and NPCR registries (2004-2014).

Casey Perez, Pankil K. Shah, Aaron Mathews, Edgar Munoz, Alex Bokov, Dharam Kaushik, Joel Michalek, Amelie Ramirez, Ronald Rodriguez, Dimpy P. Shah. _UT Health San Antonio, San Antonio, TX_.

Introduction: Despite overall decrease in incidence of cancers in U.S., kidney cancer is one out of the only seven cancers with increasing incidence. Limited research has been conducted using national, state-wide, and regional data, especially comparing racial and ethnic disparities in incidence and outcomes for kidney cancer.

Methods: Kidney cancer incidence and outcomes from 2004 to 2014 were compared using Texas Cancer Registry, National Program for Cancer Registries, and Surveillance, Epidemiology and End Results. Age-adjusted incidence rates (per 100,000 population) and temporal trends, reporting sources, diagnosis, patient and tumor characteristics between Hispanics and Non-Hispanic Whites (NHWs) were compared for each state. The Kaplan-Meier method was used for relative survival analyses, which were limited to Texas due to data availability.

Results: A total of 566,716 patients were identified. The temporal trend shows a sustained increase in kidney cancers in U.S. and a consistently higher incidence in TX and South Texas (STX) over the past decade, with Hispanics in particular having the highest annual percent change (APC) among the 20-29 year old age group (5.3 in U.S. and 7.7 in TX, respectively). Age-adjusted kidney cancer incidence was 26.2 in STX, 24.5 in TX and 21.8 in U.S. Both males and females had a higher incidence in STX and TX compared to U.S. (35.1, 32.5 vs. 29.6 for males and 18.8, 17.9 vs.15.2 for females). Overall, Hispanic males in STX had the highest kidney cancer incidence (37.7) throughout U.S. The difference in incidence rate in Hispanics compared to NHWs was significant in STX (4.9) and TX (3.4), compared to U.S. (-0.9). Tumor grade was only available in 60% of cancers in TCR compared to 70% in U.S. Despite localized tumor stage being the most common presentation for all regions, the 5-year survival was lower in Hispanics compared to NHWs in STX (68% vs. 73%) and TX (69% vs. 71%).

Conclusions: Kidney cancer rates were higher in STX and TX when compared to U.S., especially among Hispanic males. Relative 5-year survival between Hispanics and NHWs were similar in TX and STX. Improvement in cancer reporting and detailed studies examining causes for racial/ethnic disparities in incidence in the top 15 states with highest disparities, especially TX, are needed.

#4188

Registering cancer recurrence in a population-based registry: The value of pathology data.

Leah Moubadder, Audrey Chang, Kevin C. Ward, Timothy L. Lash. _Rollins School of Public Health, Emory University, Atlanta, GA_.

INTRODUCTION No population-wide cancer registry systematically records recurrence. It is often requested of U.S. registries, but cannot be provided. To fill this gap, the Georgia Cancer Registry (GCR) has embarked on a five-year project to add recurrence data to the registry for early-stage breast, prostate, colorectal cancer and lymphoma patients. Multiple data streams will be used to provide signals of recurrence, which will then be validated by a Certified Tumor Registrar from the registry staff. Electronic pathology data are submitted in real-time to the GCR and could be a key data stream to signal recurrences.

METHODS We created cohorts of early-stage breast and colorectal cancer patients treated at Commission on Cancer (CoC) facilities in Georgia and for whom the CoC facility both reported their pathology data to the GCR electronically and had documented a recurrence in the patient's record. A randomly selected sample (n = 60) from each cohort was linked to electronic pathology (E-Path) reports in the registry to evaluate a) the proportion of patients with a report available in the GCR within 10 days of the documented recurrence and b) the proportion of patients whose E-Path report included the specific term(s) "recurrent", or "recurrence(s)". All pathology reports were manually reviewed to make the above determinations.

RESULTS All 60 cases from each cancer site had E-Path reports available for the incident cancer that was diagnosed. Upon manual review, 67% of breast cases and 63% of colorectal cases had reports in the registry within 10 days of the recurrence date reported by the CoC facility. When examining all reports for a given patient, regardless of date, for use of the specific term(s) "recurrent", or "recurrence(s)", only 18% of breast cases and 9% of colorectal cases contained these exact terms. CoC facilities also attempt to document the location of the recurrence in addition to the date. For cases with reports available in the registry within 10 days of the recurrence reported by the CoC facility, confirmation of the recurrence location was documented in the pathology report for the majority of patients (78% for breast and 89% for colon).

DISCUSSION E-Path reports will be an important source of information to signal recurrences, but the specific use of recurrence terminology was limited in the sample of reports selected for this study. Use of the terms "metastatic" or documentation of other organs as "positive for malignancy" was much more common. Not all recurrences will be signaled by E-Path reports, so the use of additional data streams as planned by this project will be critical and validation of these signals by trained staff will establish the gold standard of true recurrences. This ambitious project is just launching, with the aim of providing the first-ever descriptive data about recurrence rates for four common cancers over up to ten years of follow-up.

#4189

Limited-versus-extensive staging system for small cell prostate cancer.

Salman Syed,1 Faisal Ali,1 Spyridon Basourakos,2 Zimu Gong1. 1 _Presence St. Joseph Hospital - Chicago, Chicago, IL;_ 2 _New York-Presbyterian Hospital, New York, NY_.

Introduction: Small cell prostate cancer (SCPC) is one of the most common extrapulmonary small cell cancers as well as one of the most common histology type among non-adenocarcinoma prostate cancers. While small cell lung cancer follows a limited-versus-extensive staging system which is different from the stage I to IV system used for non-small cell lung cancer, the prostate adenocarcinoma staging system is used in SCPC despite its distinct clinical feature and aggressive disease course. Limited by the rarity of SCPC, the optimal staging strategy for SCPC remains unknown. The present study aimed to investigate the prognostic value of limited-vs-extensive staging system in SCPC patients.

Method: The Surveillance, Epidemiology, and End Results database was used to analyze the incidence and outcome of small cell prostate cancer. Patients diagnosed between the year 2004 through 2015 were included. Limited stage is defined as those without metastasis regardless of degree of local invasion. Extensive stage is defined as any metastasis to locoregional lymph node, to distant lymph node, or to distant organs.

Result: A total of 364 SCPC patients are included in the study cohort, accounting for 0.05% of all prostate cancer cases (n=665,054). Age adjusted incidence of SCPC was 0.959 per 1,000,000 per year over the whole study period. The median age at diagnosis of SCPC is 71 years. The median overall survival is 9 months. In the 322 patients with known metastasis status, 243 (75.5%) patients are in extensive stage, whereas 79 patients are in limited stage. Patients in extensive stage are more likely to have PSA level greater than 20 than those in limited stage (26.3% vs 6%), but a similar proportion of patients in both groups have PSA level less than 4 (44.3% vs 40.0%). Compared to patients with extensive stage, those with limited stage disease had a significantly better overall survival (17 months vs 9 months, p<0.001) as well as disease specific survival (28 months vs 11 months, p<0.001). Limited stage patients with different T stage had a similar overall survival (p=0.43). Similarly, all extensive stage patients had similar outcome despite different types of metastasis (p=0.40). Multivariable analysis of treatment showed that only chemotherapy is associated with improved survival in extensive stage disease with HR of 0.46.

Conclusion: SCPC patients can be staged to limited stage and extensive stage based on the absence versus presence of metastasis. T stage in limited stage patient and type of metastasis in extensive stage patient is not associated with different outcome.

#4190

Impact of comorbidities and nonclinical factors associated with sinonasal cancer all-cause mortality in the United States.

Premal B. Desai,1 Aleksandr R. Bukatko,1 Matthew C. Simpson,1 Eric Adjei Boakye,2 Jason W. Greenberg,1 Greg M. Ward,1 Ronald J. Walker,1 Jastin L. Antisdel,1 Nosayaba Osazuwa-Peters1. 1 _St. Louis University School of Medicine, Saint Louis, MO;_ 2 _Southern Illinois University School of Medicine, Springfield, IL_.

Introduction: The anatomical complexity of the sinonasal region contributes to the poor prognosis and relative rarity of sinonasal cancer. While several studies have focused on the major clinical factors associated with survival, the burden of comorbidities and its impact on all-cause mortality of sinonasal cancer is unknown. Objective: This study aimed at describing the impact of comorbidities and nonclinical factors on all-cause mortality of sinonasal cancer in the United States. Methods: In this retrospective cohort study (n=10769), adult cases of sinonasal cancer (International Classification of Diseases for Oncology, third edition typology codes: C30.0, C30.1, C31.0-C31.3, C31.8, C31.9) diagnosed from 2004 through 2013. The outcome of interest was all-cause mortality. Independent variables included treatment type, time to treatment, staging, comorbidity scoring, histology, HPV test status, age, gender, race, facility type, distance to facility, insurance, local income, and urban level. Survival analysis was conducted via multivariable extended Cox regression with Heaviside adjustment for time-varying component of treatment variables. Results: In this cohort, 79% were whites, 60% males, and mean age of diagnosis was 63.5 years. Approximately 1-in-5 patients (18.6%) had a major comorbidity (Charlson-Deyo score ≥ 1) at the time of sinonasal cancer diagnosis. In the final model, after controlling for clinical factors including stage of presentation, treatment modality, HPV status, and histological subtypes, increasing Charlson-Deyo comorbid score was associated with a corresponding increase in hazard of mortality [(aHR score of 1 = 1.25; 95% CI 1.16, 1.35), (aHR score of 2+ = 1.62; 95% CI 1.42, 1.84). There were significant effects of race and socioeconomic status on all-cause mortality. Males (aHR = 1.11; 95% CI 1.05, 1.18); blacks (aHR = 1.12, 95% CI 1.02, 1.24); those uninsured (aHR = 1.40; 95% CI 1.21, 1.62) or covered by Medicaid (aHR = 1.48; 95% CI 1.31, 1.66); and patients living in zip-codes with lower median income quartile (aHR <$30,000 = 1.17; 95% CI 1.06, 1.29) all had greater hazard of all-cause mortality. Additionally, treatment at community cancer programs was associated with increased hazard of mortality (aHR = 1.14, 95% CI 1.01, 1.28) compared with being treated at an academic/research program. Conclusions: Besides clinical factors such as stage of presentation, severe comorbid disease is most associated with all-cause mortality among patients with sinonasal cancer. Additionally, other nonclinical factors associated with sinonasal cancer all-cause mortality included race, insurance status, income, histological subtypes of the cancers, and comorbidity scores. Understanding the impact of these factors on mortality could help clinicians in disease prognostication and management of patients with sinonasal cancer.

#4191

The worldwide female breast cancer incidence and survival, 2018.

Zoubida Zaidi, Hussain Adlane Dib. _University Hospital of Setif, Setif, Algeria_.

Background: this communication presents the latest international descriptive epidemiological data for invasive breast cancer amongst women, including incidence and survival in the worldwide.

Methods: the incidence statistics presented here for cancers worldwide were taken from the International Agency for Research on Cancer IARC: * The Cancer Incidence in five Continents Vol XI.* GLOBOCAN 2018. *The datas of Cancer survival are taken from: * Cancer survival in five continents, a worldwide population-based study or Concord Study Version 1, 2 and 3. These Concord studies included 101 population-based cancer registries in 31 countries for the period 1990-1994 and followed up to 1999 for the Concord Study 1, 279 population-based cancer registries in 67 countries for the period 1995-2009 for the Concord Study 2 and 412 cancer registries in 85 countries for the period 2000-14 for the Concord Study 3

Results: breast cancer is by far the most frequent cancer among women with an estimated. 2 million new cancer cases diagnosed in 2018 (23% of all cancers), and ranks second overall (10.9% of all cancers). It is now the most common cancer both in developed and developing regions.* Incidence rates vary from 19.3 per 100,000 women in Eastern Africa to 89.7 per 100,000 women in Western Europe, and are high (greater than 80 per 100,000) in developed regions of the world (except Japan) and low (less than 40 per 100,000) in most of the developing regions.For women diagnosed during 2010-14, the range of survival estimates is still wide in each continent, apart from North America and Oceania with 5-year net survival approached 90%. **Age-standardised 5-year net survival was 85% or higher in 25 countries, Costa Rica, Martinique, Canada and the USA, Israel, Japan and 16 European countries, Denmark, Finland, Iceland, Norway, Sweden, UK, Austria, Belgium, France, Germany, the Netherlands, and Switzerland and Italy, Malta, Portugal and Spain. **5-year survival was in the range 80-84% in 12 countries, three countries in Central and South America Argentina, Peru, and Puerto Rico, five Asian countries (Singapore, China, Hong Kong and Taiwan and Turkey and four European countries the Czech Republic and Latvia and Slovenia.**Survival was in the range 70-79% in 12 countrie, Cuba and Ecuador, Kuwait and Mongolia and eight countries in Europe, Estonia, Lithuania, Croatia and Bulgaria and Poland. **Breast cancer survival remains lower in Eastern Europe and Africa.

Conclusion: the future worldwide breast cancer burden will be strongly influenced by large predicted rises in incidence throughout parts of Asia due to an increasingly westernised lifestyle. Efforts are underway to reduce the global disparities in survival for women with breast cancer using cost-effective interventions.

#4192

Increasing incidence of EBV-related nasopharyngeal carcinoma in the United States.

Ilona Argirion,1 Katie R. Zarins,1 Julie J. Ruterbusch,2 Patravoot Vatanasapt,3 Hutcha Sriplung,4 Erlene K. Seymour,2 Laura S. Rozek1. 1 _University of Michigan, Ann Arbor, MI;_ 2 _Wayne State University, Detroit, MI;_ 3 _Khon Kaen University, Khon Kaen, Thailand;_ 4 _Prince of Songkla University, Songkhla, Thailand_.

Purpose: Incidence of nasopharyngeal carcinoma (NPC) is highly geographically variable and historically rare in the US. While etiological factors differ by histological subtype, Epstein-Barr virus (EBV) is generally accepted as the primary risk factor for non-keratinizing NPC. In light of the changing epidemiology of HPV-associated oropharyngeal cancer, it is important to evaluate temporal incidence of NPC in the US.

Methods: Incidence and survival data from 1973-2015 were obtained from the Surveillance, Epidemiology and End Results (SEER) Program. Stratified analyses were conducted to assess temporal trends in NPC by histological subtype, sex and race. The data were analyzed using SAS 9.4 and Joinpoint Regression Software to determine age-adjusted incidence rates, trends in annual percent change (APC) as well as calculate 5-year relative survival estimates and Kaplan-Meier curves.

Results: Although overall NPC incidence is decreasing in the US, the non-keratinizing differentiated subtype appears to be starkly increasing with an APC of approximately 4% among white males (95%CI: 2.5, 5.2), white females (95%CI: 1.9, 6.2), and black males (95%CI: 2.0, 5.7), 2.7% among black females (95%CI: 0.8, 4.6) and 1.8% among women of other race (95%CI: 0.4, 3.3). When compared to other histological subtypes, patients with keratinizing squamous cell carcinoma had the worst survival (log rank p<0.001). Additional survival disparities were noted, with black males having consistently poorer survival across all histological subtypes, and those individuals in the "other" race category, particularly females, experiencing the highest 5-year relative survival estimates.

Conclusions: Although NPC remains relatively rare in the overall US population, there is evidence to suggest that the EBV-related differentiated subtype is increasing across all genders and races.

#4193

Epidemiological and clinical description of head and neck cancer cases in Bogotá, Colombia.

Sandra P. Perdomo,1 Paula A. Rodriguez,2 Jose A. Hakim,2 Yubelly Avello,2 David A. Suarez,2 Alberto Escallón,2 Vanessa Ospina,2 Nicolás Useche,2 Alvaro Muñoz,2 Margarita Baldion,2 Mauricio Palau2. 1 _El Bosque University, Bogotá, Colombia;_ 2 _University Hospital Fundación Santa Fe de Bogotá, Bogotá, Colombia_.

In 2018, almost 36,477 new head and neck cancer (HNC) cases were estimated to occur in South America. Colombia presents an intermediate age-standardized rate per 100,000 of 178.8 for both sexes. However, the incidence has not decreased in the las 10 years. Here, we present a description of the epidemiological and clinical characteristics of HNC patients studied in a private institution in Bogotá, Colombia. Between November 2014 and October 2018, 73 HNC cases were enrolled in the InterCHANGE- project: HNC in Latin America (http://interchange.iarc.fr/index.php). All cases had information on sociodemographic and lifestyle factors and complete clinical data. Stage at diagnosis was based on the 8th TNM AJCC classification. Inmmunohistochemical HPV studies included evaluation of p16INK4a (Clone E6H4). Mean age of diagnosis was 66 (SD 11.8), 66% of cases were male, 64% identified themselves as mestizos, 66% had superior complete educational level. 41% were nonsmokers, 56% former smokers and 52% current drinkers. 64% reported ever practiced oral sex. Tumors were mostly localized in the oral cavity (41.1%) and the oropharynx (41.1%), followed by larynx (16.4%) and hypopharynx (1.3%). 45% of cases were diagnosed at stage I and surgical treatment of primary tumor was performed in 66% of cases. Overall, 45% of cases were p16 positive. Interestingly, 93% of oropharyngeal cases were p16 positive, majority male (82%) with a mean age of diagnosis of 63 (SD 11.8) and among them, only 34% were nonsmokers. After a 2-year period of follow-up 82% of all cases were alive.In our population alcohol consumption and HPV infection are the major lifestyle risk factors associated to HNC. Oropharyngeal tumors were predominantly associated to HPV infection. Stage at diagnosis and percentage of survival is higher from what has been reported in other South American populations.

#4194

Rare cancer's 'valley of death': Critical gaps in data, research, and funding.

Robert L. Treuting, Sean Murphy, Niall Murphy, Bovey Rao, Anekha Goyal, Nico Tuccillo, Ryan Ki-Hoon Kim, Dylan Parker, Katherine Arline. _Shepherd Therapeutics, Cambridge, MA_.

Over 370 forms of rare cancer compose at least 29.7% of all U.S. cancer diagnoses, or over 500,000 Americans per year. And yet, available data, research, and public funding related to these indications is insufficient in comparison with the 20 forms of non-rare cancers. Rare cancer is defined by the American Cancer Society as a cancer having an incidence of <6 per 100,000 per year. A review of the AACR Annual Proceedings 2018 showed that at least 150 rare cancers were not the focus of any abstract. Non-rare forms of breast cancer, expected to represent 11.9% of new cancer cases in 2018, had almost the same coverage (1081 abstracts, 25.6%) as all rare cancers combined (1155 abstracts, 27.3%). Rare cancers which were included in AACR 2018 were the focus of an average of 5 abstracts each, while the 20 non-rare cancers were the focus of an average of 154 each. That gap in basic science is also visible in broader data sources. A computational review of the 10,000 most recent cancer-related publications in PubMed showed that over 100 rare cancers had no publications, while that was true of only 1 non-rare cancer. Included rare cancers had an average of 3.2 mentions in PubMed abstracts, while non-rare cancers had an average of 115. Adequate patient-derived tumor gene expression data is critical for targeted drug development. Analysis of the Gene Expression Omnibus (GEO) repository demonstrated that, while all 20 non-rare cancers had DataSets, over 100 rare cancers had none. On average, each rare cancer included in GEO had 1.04 DataSets, while each non-rare cancer had 22.8. Government grants to companies are an important tool for bridging the drug development "valley of death" between basic science and the clinic. Between 2013 and 2017, 781 SBIRs and STTRs related to cancer research, development, or treatment were identified. 489 of them related to a specific cancer or cancers. Of those grants, 41.5% were related to breast, lung, or prostate cancers. 22.3% were related to rare cancers. Among rare cancers, glioblastoma was the specific focus of the most grants (18). 356 forms of rare cancer were not the specific focus of any grant. Pediatric cancers, which are all rare, were the focus of 8 grants (1%). These data highlight a significant gap in rare cancer-focused data, research, and public funding which contributes to lower availability of clinical trials and therapeutics for over half a million U.S. patients per year.

#4195

Clinical, endoscopic and histological aspects of colorectal cancer at the CHU de Cocody.

Serge Franck Roland Fonkoua Fohom. _CHU de Cocody, Abidjan, Côte D'Ivoire_.

Introduction: The colorectal cancer (CRC) is the 4th cancer in Côte d'Ivoire (WHO 2014). Its overall prevalence appears to be advancing in sub-Saharan Africa over the years. The CRC frequently occurs at an early age and is very often discovered at an advanced or even late stage, with a bad prognosis.

Aims: To assess the clinical, endoscopic and histological aspects of CRC.

Patients and methods: An observational retrospective study was conducted from the reports of lower GI (LGI) endoscopies performed at the GI Endoscopy Unit between 2008 and 2018. The diagnosis of CRC was retained on the basis of the macroscopic and/or the histological aspect of the tumor. The following parameters were collected : number and indication of LGI endoscopies, age, gender, macroscopic appearance, location and histological type of the tumor.

Results: Over the study period, 2118 LGI endoscopies were carried out. We found 145 CRCs, corresponding to an endoscopic prevalence of 7%. The annual prevalence was 18,13 cases. A CRC was diagnosed every 14,6 LGI endoscopies performed. The sex ratio was 1,42. Median age was 56 years, with extremes of 14 and 92 years. Patients younger than 40 years (with a median age of 32 years) accounted for 18% of cases. Rectorrhagia was the main cause of LGI endoscopies found in 48 patients (33,1%), followed by an imaging discovery (ultrasound and/or abdominal CT scan) of a colorectal tumor in 38 patients (27,2%), abdominal pain or constipation in 15,3% of cases (21 patients), weight loss in 13 patients (9,5%), abdominal or anorectal mass in 8% of cases (11 patients), chronic diarrhea in 8% of cases (11 patients), metastatic tumor liver in 9 patients (6,6%), melaena in 4,4% of cases (6 patients) and anemia in 3 patients (2,2%). Macroscopically, rectal localization was the most common with 42,36% of cases, followed by sigmoid (36,1%), cecum (16,7%), right colon (11,8%), colon transverse (7%) and left colonic angle (2,8%). The ulcerative-budding form was the most common (88,65%), with strictures in 57,5% of cases and necrotic-hemorrhagic in 48,23% of cases; followed by the polypoid form (4,25%). There were no ulcerative and infiltrative form. The histological type was found in 30 patients (4,83%). Adenocarcinoma was the most common (93,3%). Stromal tumor was found in 3,3% of the cases.

Conclusion: This work confirms that the prevalence of CRC has increased in Côte d'Ivoire, that it occurs at an early age and that it is discovered at an advanced stage. This work highlights the importance of screening for CRC as early as possible in front of a Rectorrhagia, even in young patients.

#4196

Survival differences between males and females diagnosed with childhood cancer.

Lindsay A. Williams, Logan G. Spector. _University of Minnesota, Minneapolis, MN_.

Childhood cancer survival differs by sex, but few reports exist characterizing this relationship using modern tumor type classifications for main cancer types. Using the SEER 18 registries (2000-2014), we estimated survival differences between sexes among children aged 0-19 years. We used Kaplan Meier survival curves and Log-Rank p-values to characterize overall survival differences. Cox proportional hazards models were used to estimate hazard ratios (HR) and 95% confidence intervals (95% CI) as the measure of association between sex and risk of death by tumor type classified using the International Classification of Childhood Cancer 3rd edition. We used an inverse odds weighting method to determine whether the association between sex and risk of death by tumor type was mediated by stage of disease for solid tumors with significant total effects.

Males and females had similar racial distributions by tumor type. Males were more frequently diagnosed with distant stage of disease than females for Hodgkin lymphoma (33% female, 40% male), Non-Hodgkin lymphoma (45% female, 49% male), neuroblastoma (49% female, 56% male), and osteosarcoma (20% female, 24% male). Males had lower 5-year survival than females for acute lymphoblastic leukemia (ALL) (88% female, 85% male), ependymoma (78% female, 71% male), neuroblastoma (78% female, 74% male), hepatoblastoma (82% female, 77% male), osteosarcoma (71% female, 64% male), and Ewing tumor of the bone (71% female, 67% male). Males had worse overall survival for ALL, ependymoma, neuroblastoma, and osteosarcoma (all Log-Rank p-values<0.02). Males had a higher risk of death than females for ALL (crudeHR:1.24, 95% CI:1.12-1.37), ependymoma (crudeHR:1.36, 95% CI:1.05-1.77), neuroblastoma (crudeHR:1.28 , 95% CI:1.09-1.51), and osteosarcoma (crudeHR:1.29, 95% CI:1.08-1.53). Results were similar after adjusting for age at and year of diagnosis, and stage of disease. Among solid tumors with a significant total effect, the association between sex and the risk of death was mediated by stage of disease for neuroblastoma (indirectHR:1.12, 95% CI:1.05-1.19) and was suggestive for osteosarcoma (indirectHR:1.06, 95% CI:1.00-1.12), but not ependymoma. Results were similar in mediation analyses adjusted for age and year of diagnosis.

Sex differences in survival, particularly for ALL, neuroblastoma, osteosarcoma, and ependymoma, may depend to some degree on sex differences in tumor biology or response to treatment. ALL, ependymoma, neuroblastoma, and osteosarcoma account for 1/3 of new cancer diagnoses and ~40% of deaths among children and adolescents. If the five-year survival percentages were the same between sexes for these four cancers, ~18% of male deaths and ~11% of total deaths due to these cancers could be avoided; therefore, studies with detailed molecular tumor and clinical data will be important for identifying mechanisms underlying the observed sex differences in survival.

#4197

Long-term follow-up of a racially and ethnically diverse population of patients with localized prostate cancer who did not undergo initial active treatment.

Jeff Slezak,1 Stephen Van Den Eeden,2 Kimberly L. Cannavale,1 Gary W. Chein,1 Chun R. Chao1. 1 _Kaiser Permanente Southern California, Pasadena, CA;_ 2 _Kaiser Permanente Northern California, Oakland, CA_.

Background: Long-term studies of men who were not initially treated for prostate cancer have been limited, especially among ethnic minorities. We sought to provide detailed long-term outcomes of a large, diverse cohort of men diagnosed with prostate cancer between 1997 and 2007 in a large managed care organization.

Methods: Incident prostate cancer cases diagnosed at localized stage were identified using a SEER cancer registry. Treatments were identified by the cancer registry and patients' medical records. Men were excluded if an initial course of treatment was started within 6 months of diagnosis. All patients were followed to identify later initiation of treatment, suspected prostate cancer metastasis or death. Suspected metastases were manually reviewed and confirmed. Death due to prostate cancer (PCD) was identified using the underlying cause of death on the death certificate. Competing risks methods were used to estimate the cumulative incidence of treatment initiation or metastasis (naive of treatment), and the incidence of death due to prostate cancer or other causes.

Results: A total of 6207 men were included: 60.4% white, 18.9% Black, 15.2% Hispanic and 5.5% Asian or Pacific Islander (API). At 19 years after diagnosis, the estimated incidence of treatment initiation, metastasis, PCD, and death due to other causes was 39.1%, 13.4%, 12.0% and 52.9%, respectively. Rates of treatment initiation at 19 years were highest in Hispanic (44.5%) and Black (44.7%) men, compared to 35.4% in white and 38.6% in API men. Rates of treatment-naïve metastasis as well as PCD were highest in Blacks (19.3% and 14.1%, respectively) and lowest among Hispanics (7.7% and 9.9%, respectively). API men had low rates of PCD through the first 10 years after diagnosis (2.4% compared to 5.6-6.9% for other groups), after which rates increased and at 19 years their estimated rates of PCD (12.0%) approximated those of whites (11.7%).

Conclusions: Significant risks of metastasis and prostate cancer death remains even 15 years after diagnosis among men who were initially untreated. Notably, there is an apparent increasing risk of PCD in API more than 10 years after diagnosis.

#4198

Epidemiological and molecular analysis of acute lymphoblastic leukemia in the pediatric population of Puerto Rico.

Ingrid M. Montes-Rodriguez,1 Carlos R. Torres-Cintron,2 Marievelisse Soto-Salgado,3 Erick Suarez,3 Joel Rivera-Concepcion,3 Carmen L. Cadilla,3 Luis A. Clavell1. 1 _University of Puerto Rico Comprehensive Cancer Center, San Juan, Puerto Rico;_ 2 _Puerto Rico Central Cancer Registry of the University of Puerto Rico Comprehensive Cancer Center, San Juan, Puerto Rico;_ 3 _University of Puerto Rico School of Medicine, San Juan, Puerto Rico_.

Background: Acute lymphoblastic leukemia (ALL) accounts for 80% of leukemias diagnosed in children. In United States (US), ALL accounts for 27% of cancers diagnosed in children 0 to 19 years old, disproportionally affecting children between 2 and 5 years. Even though ALL age patterns are consistent across racial/ethnic groups, the incidence and mortality of this malignancy is highly variable. For example, Hispanics have a higher incidence of ALL and lower survival rates when compared to non-Hispanic patients, with higher likelihood of recurrence. However, Hispanic is a broad category used to refer to subpopulations of Mexican, Cuban, Puerto Rican, South or Central American, Dominican, or individuals with other Spanish descent. Hence, the cancer data that is reported for Hispanics is aggregated, masking important differences between these subpopulations that, due to genetic admixture over 500 years, have unique biologic and non-biologic factors associated with disease outcome.

Objective: The objective of this study was to assess the racial/ethnic group differences in children diagnosed with ALL in Puerto Rico (PR) and to compare incidence and mortality risk with US Hispanics (USH), non-Hispanic-white (NHW) and non-Hispanic-black (NHB). To access these differences, we estimated the Standardized Rate Ratio (SRR) for 2010-2014. Furthermore, we aimed to characterize the ALL genetic subtypes in the PR population.

Methods: Secondary data-analysis of the Puerto Rico Central Cancer Registry (PRCCR) and the National Cancer Institute's Surveillance, Epidemiology, and End Results program (SEER) databases was performed for the 2010-2014 period. We also described the childhood ALL sub-types more prevalent in PR for the years 2014-2015 using the pathology reports at the PRCCR.

Results: Our data shows that PR children have lower ALL incidence and mortality risk than USH, lower incidence but higher mortality risk than NHW, and higher incidence and mortality risk than NHB. The most prevalent ALL subtypes in Puerto Rico (n=50 for the years 2014-2015) were B-ALL (n=42) and T-ALL (n=8). The most common chromosomal aberration was the ETV6-RUNX1 translocation, found in 8 of 50 (16%) of the cases, followed by Hyperdiploidy and IGH-gene rearrangements, each found in 4 of 50 of the cases (8%).

Conclusion: The observed differences in ALL incidence and mortality among PR and USH children may be related to their genomic background, environmental exposures, and/or gene environment interactions. Future research is warranted to elucidate the factors contributing to this racial/ethnic disparity.

#4199

HPV prevalence of cervical cancer in the United States 2014-2015.

Jacqueline M. Mix,1 Elizabeth Unger,1 Troy Querec,1 Trevor Thompson,1 April Greek,2 Thomas Tucker,3 Charles Lynch,4 Edward Peters,5 Mona Saraiya1. 1 _Centers for Disease Control and Prevention, Atlanta, GA;_ 2 _Battelle Memorial Institute, Seattle, WA;_ 3 _University of Kentucky, Lexington, KY;_ 4 _University of Iowa, Des Moines, IA;_ 5 _Louisiana State University, Baton Rouge, LA_.

INTRODUCTION: To determine the prevalence of human papillomavirus (HPV) types in invasive and in situ cervical cancers identified in three US population-based cancer registries (PBCRs) approximately 10 years after HPV vaccine implementation.

METHODS: Archived tissue from invasive cervical cancer (ICC) and cervical carcinoma in situ (CCIS) diagnosed between 2014 and 2015 were identified from three PBCRs in Iowa, Kentucky, and Louisiana. HPV genotyping was performed using Linear Array (Roche Diagnostics); follow-up testing of HPV-negative samples was done with INNO-LiPA HPV Genotyping Assay (Innogenetics). Type-specific weighted HPV prevalences among ICC and CCIS were estimated using the hierarchical proportion to HPV 16 and 18 oncogenic genotypes included in all vaccine formulations (16/18) and protection from five additional types in the 9-valent vaccine (31/33/45/52/58). Comparisons were made with a similar study of seven PBCRs with cervical tumor tissue obtained from diagnoses between 1994 and 2005.

RESULTS: A total of 363 ICC and 350 CCIS were successfully genotyped. Prevalence of HPV16/18 and HPV31/33/45/52/58 types were: ICC: [16/18: 69%, 31/33/45/52/58: 10%] and CCIS: [16/18: 68%, 31/33/45/52/58: 22%]. HPV prevalence in the 1994-2005 study was: ICC (n=777): [16/18: 67%, 31/33/45/52/58: 14%] and CCIS (n=481): [16/18: 61%, 31/33/45/52/58: 22%]. No overall changes were observed in the prevalence of HPV16/18 and HPV31/33/45/52/58 between the two studies.

CONCLUSIONS: Given the long latency period of HPV-related cancers, continuing to monitor HPV prevalence in invasive and in situ cervical cancers will be important for evaluating the impact of the HPV vaccine on genotype prevalence.

#4200

Racial differences in multiple myeloma cases with anemia.

Riddhi Modi, Luciano J. Costa, Elizabeth E. Brown. _UAB, Birmingham, AL_.

Introduction. Multiple Myeloma (MM) is a hematologic malignancy defined, in part, by prolonged survival and accumulation of clonally expanded plasma cells in the bone marrow microenvironment and extramedullary sites and the presence of hypercalcemia, renal insufficiency, anemia or bone involvement, collectively known as end organ damage. MM is the most common hematologic malignancy affecting Blacks in the US, with standardized incidence rates that are 2 to 3-fold higher than Whites. A rationale for the disparity remains unclear.

Methods. Using participants enrolled in the Molecular And Genetic Epidemiology (iMAGE) study of myeloma (515 MM cases; 54.1%White, 41.9% Black), we examined the clinical and laboratory features associated with the presence of anemia in MM cases at diagnosis and differences by race to provide a rationale for the observed disparity. Risk estimates were calculated using odds ratios and corresponding 95% confidence intervals from logistic regression adjusted for sex, age at diagnosis, presence of other end organ damage and race, where appropriate. Sensitivity analyses were conducted in MM cases with only anemia as the end organ damage.

Results. Overall, anemia was present in 16.7% of MM cases as the only end organ damage at diagnosis and in 56.8% of MM cases as one or more end organ damage. In the combined population, no notable differences by sex or age were observed among MM cases with anemia; however, the presence of anemia was significantly elevated in Blacks compared to Whites (OR=2.11, 95% CI 1.36-3.27; P=0.001) and among Blacks, women were significantly more likely to present with anemia at diagnosis (P=0.002). Overall, compared to MM cases without anemia at diagnosis, significantly more MM cases with anemia exhibited clonal bone marrow plasma cells ≥ 60% (OR=2.43, 1.58-3.76; P<0.0001), high serum lactate dehydrogenase (LDH >300 units/L; OR=5.03, 95% CI 1.47-17.27; P<0.0001), serum total monoclonal protein ≥ 3 g/dL (OR=2.85, 95% CI 1.76-4.62; P<0.0001), involved to uninvolved serum free light chain ratio ≥ 100 (OR=2.20, 95% CI 1.33-3.62; P=0.002) and among chromosomal abnormalities, translocation 4;14 (FGFR3; OR=7.27, 95% CI 1.54-34.28; P=0.012), deletion 13q (OR=1.96, 95% CI 1.14-3.34; P=0.013) and chromosome 1q amplification (OR=2.76, 95% CI 1.31-5.80; P=0.007) after adjusting for confounders. In contrast, MM cases with anemia were significantly less likely to present with serum albumin ≥ 3.5 mg/dL (OR=0.30, 95% CI 0.19-0.47; P<0.0001) and bone involvement (OR=0.23, 95% CI 0.13-0.42; P<0.0001). No notable differences in clinical or laboratory features with the presence of anemia at diagnosis were observed by race. Findings were confirmed in sensitivity analyses.

Conclusions. The excess risk of MM observed in Blacks and the variation in clinical features observed in MM patients according to the presence of anemia at diagnosis may contribute to disparate trends in incidence yet similar overall survival rates.

#4201

Patient-level predictors and hysterectomies among women with autoimmune diseases.

Zahra Bahrani-Mostafavi, Larissa R. Huber. _Univ. of North Carolina at Charlotte, Charlotte, NC_.

Introduction: Hysterectomy is the second most common surgical procedure performed among reproductive aged women. The primary reason for hysterectomy among women 35-54 years is uterine fibroids and for older women is uterine prolapse or gynecologic (GYN) cancer. According to the National Center for Health Statistics, an estimated 600,000 hysterectomies are performed annually in the U.S. with an annual cost of $5 billion, which makes hysterectomy a major public health concern. Autoimmune diseases (AD) are among the top ten leading causes of death among U.S. women age 65 and under. This high mortality rate among women with AD has been linked to cancer and other causes. Considering hysterectomy procedures as are invasive and costly procedures, a comprehensive study to investigate the association between patient-level predictors and hysterectomy procedures among AD patients in the U.S. is imperative.

Methods: 2007-2013 data from Florida State Inpatient samples of the Healthcare Cost and Utilization Project were used. The study population (n=699,280) was restricted to women who had any AD. The outcome variable was performance of hysterectomy, identified by CPT codes. Patient-level factors including race/ethnicity, age, insurance type, income level, and other comorbidities were analyzed as explanatory variables. Logistic regression analyses were used to identify independent predictors of hysterectomy procedures.

Results: Among AD subjects, the highest rate of hysterectomy was in Caucasian women (64.64%) and the lowest rate was in minorities other than Black and Hispanic women (3.0%). The odds of hysterectomies are were increased in older women who were older (0.74, 0.70-0.78), women, black (1.45, 1.36-1.54) women, have had private insurance (2.30, 2.14-2.45), and are fromhad lower income levels (1.10, 1.02-1.19), and had with more comorbid conditions, specifically GYN cancer.

Discussion: GYN complications may lead to hysterectomy, and ADs are major diseases among women. Because of the high prevalence of AD among women, more elucidation on the association between hysterectomy procedures and AD is essential for GYN complications management. Any unique combinations of characteristics that describe patients at risk for hysterectomy among AD patients could be used as a potential risk assessment for better management of GYN complications, and in particular GYN cancer.

#4202

Identifying predictors of racial disparities in mortality among Blacks and Whites diagnosed with breast cancer in South Carolina.

Oluwole Adeyemi Babatunde, Swann A. Adams, Jan M. Eberth, Tisha M. Felder, Robert Moran, Samantha Truman, Christian Alvarado, James Hebert. _Univ. of South Carolina, Columbia, SC_.

Introduction: Mortality among White breast cancer (BrCA) patients is 7% lower in South Carolina (SC) and is 29% higher among Black BrCA patients in SC compared to the national average. The aim of this study was to identify predictors of racial disparities in mortality among Black and White BrCA patients in SC.

Methods: Breast cancer cases were obtained retrospectively from the SC Central Cancer Registry, linked with administrative data from a private payor insurance and Medicaid Plan from 2002 to 2010. The main outcome variable was mortality which was operationalized by vital status and total survival time. The main exposure variable was patient race (White vs Black). Cox proportional hazard analyses were conducted to compare hazard ratios among Blacks and Whites to identify predictors of mortality. In assessing the relationship between race and mortality, we examined marital status, urbanicity, region, and Best Chance Network enrollment as potential effect modifiers. The multivariable model was stratified by the effect modifier variables and a backward elimination process was utilized to obtain the best fitting model among models with the following covariates: age, year of diagnosis, hormone-receptor status, stage, and grade.

Results: A total of 2155 breast cancer patients (nWhites=1557; nBlacks= 598) were reported in the study period. Adjusting for cancer stage, the hazard ratio of mortality was 3.45 (1.64,7.25) among Black women who lived in the Low Country region of the state compared with White women who lived in the Low Country region of the state. In the other three regions of the state (Midlands, PeeDee and Upstate); there were no statistically significant differences between Blacks with Whites. Adjusting for cancer stage and grade showed that the hazard ratio of mortality was 1.53 (1.04,2.26) among Black women who lived in urban areas compared with White women who lived in urban areas. There were no differences between Blacks and Whites who lived in rural areas.

Conclusions: Mortality was higher among Blacks who lived in the Low Country region of the state and among Blacks who lived in urban areas. To reduce racial disparities in survival, efforts directed at early detection and linkage to timely breast cancer treatment should be focused on black women living in the Low Country region of the state and in urban areas.

#4203

Race and triple-negative breast cancer survival: A population-based study.

Ying Liu, Min Lian, Graham A. Colditz. _Washington Univ. School of Medicine,, St. Louis, MO_.

Tripe-negative breast cancer (TNBC) is characterized by lacking the expression of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2, and thereby has poor prognosis. A higher incidence of TNBC in Black women than in White women has been well documented. However, it remains uncertain if there are racial differences in TNBC outcomes. Using the Surveillance, Epidemiology, and End Results dataset, we identified 23,077 women diagnosed with non-metastatic invasive TNBC, including 5,839 non-Hispanic Blacks and 17,238 non-Hispanic Whites, between 2010 and 2015. Cox proportional hazards regression was used to estimate relative risks (RRs) of breast cancer-specific death. During a median follow-up of 29 months, 2,494 cases died from breast cancer. The racial difference in estimated 5-year breast cancer-specific survival was 6.8% for younger (<65 years) women and 4.7% for older (≥65 years) women. The RR of breast cancer-specific death was 1.22 (95% CI 1.10-1.34) in Black women compared with White women after adjustment for age, insurance status, cancer registries, tumor characteristics, treatment, neighborhood socioeconomic deprivation, and rural locations. The race-associated higher risk was observed in women under 65 (RR=1.29, 95% CI 1.14-1.45), in less socioeconomically deprived counties (RR=1.33, 95% CI 1.18-1.49), and in urban counties (RR=1.27, 95% CI 1.15-1.41). The higher risk in Black women did not significantly vary by health insurance within age strata; the RR was 1.34 (95% CI 1.06-1.69) in younger patients having no insurance or Medicaid, 1.27 (95% CI 1.10-1.47) in younger patients having non-Medicaid insurance, 1.10 (95% CI 0.67-1.81) in older patients having no insurance or Medicaid, and 1.09 (95% CI 0.90-1.33) in older patients having non-Medicaid insurance. Black women with TNBC had worse survival compared with White counterparts, which was unlikely attributed to insurance coverage and was not evenly distributed across geographic areas. This warrants further research to investigate nonbiological and biological mechanisms for racial differences in TNBC outcomes, particularly in women under 65.

#4204

Racial difference in the risk of secondary bladder cancer following radiation therapy among prostate cancer patients.

Lu Zhang, Mei-Chin Hsieh, Qingzhao Yu, Xiao-Cheng Wu. _Louisiana State University Health Sciences Center, New Orleans, LA_.

Purpose: Radiation therapy has been found associated with increased risk of secondary bladder cancer diagnosis among prostate cancer patients. It is unknown whether this association varies by race. This study aimed to investigate whether the association between radiation and secondary bladder cancer differs for white vs. black prostate cancer patients.

Methods: Data on white or black men who were diagnosed with primary localized prostate cancer in Louisiana between 2004 and 2013 and received radiation only or surgery only were obtained from Louisiana Tumor Registry. We excluded those who developed secondary bladder cancer within 180 days of the prostate cancer diagnosis or had follow-up less than 180 days or with unknown tumor size or Gleason score. Every patient was followed up until the end of 2016. The exposure variable was treatment (radiation only vs. surgery only). The outcome variable was the diagnosis of secondary bladder cancer and the time from prostate cancer diagnosis to secondary bladder cancer diagnosis or to loss to or end of follow-up. Covariates included age at diagnosis, marital status, insurance, census tract level poverty, tumor size, and Gleason score group. The competing risk survival analysis was applied. Death during follow-up was considered as competing event. Analysis was stratified by race.

Results: Of 10,861 white and 5,117 black prostate cancer patients, 52.7% and 56.8% received only radiation respectively. Regardless of race, patients who only received surgery were younger, more likely to be married, privately insured, live in low poverty census tract, and have smaller or lower grade tumor (P <0.0001 for each in both racial groups). Among white prostate cancer patients who received surgery only vs. radiation only, 31 (0.60%) vs. 127 (2.22%) of them developed secondary bladder cancer (P < 0.0001). Among black surgery only vs. radiation only patients, 14 (0.63%) vs. 29 (1.00%) of them developed a secondary bladder cancer (P = 0.16). The median follow-up time was 8.9, 8.0, 8.3, and 7.6 years for white surgery, white radiation, black surgery, and black radiation patients, respectively. After adjusting for covariates, the hazard ratio (95% confidence interval) [HR (95% CI)] of developing secondary bladder cancer was 2.70 (1.58, 4.61) for white radiation patients compared to white surgery patients; 0.95 (0.31, 2.89) for black radiation patients compared to black surgery patients. Compared to white surgery patients, the HR (95% CI) of developing secondary bladder cancer was 1.16 (0.61, 2.22) and 1.38 (0.74, 2.56) for black surgery and black radiation patients, respectively.

Conclusions: Radiation is associated with increased risk of secondary bladder cancer among white prostate cancer patients, but not among black patients. Early screening of secondary bladder cancer need to be emphasized among white prostate cancer patients who received radiation therapy.

#4205

Pan-cancer molecular study of disparities among African Americans.

Olivia D. Lara,1 Ying Wang,1 Wei Hu,1 Lin Zhang,2 Alejandro Rauh-Hain,1 Anil Sood1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _University of Pennsylvania, Philadelphia, PA_.

Objective: Several factors have been proposed to contribute to racial disparities in gynecologic cancers, however there are no comprehensive studies that have investigated inherent molecular features that may be racially unique. Our objective was to perform a pan-cancer clinical and molecular analysis of African-Americans (AA) and European American (EA) patients.

Methods: We performed cross-platform analyses utilizing cancer registry and molecular databases [Surveillance, Epidemiology and End Results (SEER) and The Cancer Genome Atlas (TCGA)] to evaluate differences in AA vs EA patients. To characterize genetic ancestry we utilized the recently established The Cancer Genetic Ancestry Atlas (TCGAA). We further evaluated epigenetic modifications through non-coding RNA expression in these tumors.

Results: A total of 2,045,500 patients (87.5% EA, 12.5% AA) with 64 primary tumor types were evaluated using SEER to evaluate clinical outcome differences. Compared to white race, AA race had higher rate of death in 45 cancer types (HR>1.0, CI 1.00-5.23). 19 cancer types were then identified to have the largest differences in overall survival (HR>1.2). Utilizing TCGA, we analyzed molecular data of 7028 tumors (88.9% EA, 10.9% AA) having miRSeq data and genetic ancestry data. We identified differentially expressed miRNA's in five cancer types: breast invasive (300 miRs, p<0.068, FDR<0.1), renal cell (408 miRs, p<0.205, FDR<0.1), prostate (102 miRs, p<2.68e-01, FDR<0.1), thyroid (63 miRs, p<1.28e-.02, FDR<0.1), and uterine corpus endometrial (177 miRs, p<8.18e-02, FDR<0.1) carcinomas. Differentially expressed miRs among all cancer types were associated with canonical pathways of cancer drug resistance by drug efflux, altered T- cell function, and regulation of epithelial-mesenchymal transition using Ingenuity Pathway Analysis (IPA) software. We identified miR-130, miR-180, miR-146, and miR-8 as commonly dysregulated miRs in these cancer types which may represent a molecular signature unique to African Americans.

Conclusions: Our results identify molecular and genetic features unique to African Americans that may contribute to worse survival outcomes. While the cause of cancer disparities is multifactorial, a focus on molecular signatures unique to AA may identify actionable molecular abnormalities.

#4206

Examining genetic ancestry in diffuse large B-cell lymphoma (DLBCL).

Michelle J. Lee,1 Camber Ileen Jhaney,2 Christopher R. Flowers2. 1 _Emory University, Atlanta, GA;_ 2 _Winship Cancer Institute, Atlanta, GA_.

Background: There are significant racial differences in incidence and outcomes for DLBCL in the United States, and African American patients (pts) present at a younger median age (54 vs. 65 years), more commonly at advanced stage, and have inferior 5-year survival compared to white pts (Shenoy et al., 2011). It is unclear if there are inherent genomic differences contributing to the survival disparities in African American pts compared with white pts.

Methods: Using whole exome sequencing data of 1,001 DLBCL pts (Reddy et al., 2013), we performed quality control of samples (heterozygosity, missingness >10%) and single nucleotide variants (SNVs; missingness >20%) with PLINK. For global ancestry estimation, we used unsupervised model-based ADMIXTURE with K=3 postulated ancestral populations and examined correlations with 21 pts (17 African-American, 2 White, and 2 Hispanic) with self-reported race. Pt characteristics (age, gender, serum lactate dehydrogenase, tumor stage, ECOG performance status, number of extranodal sites of disease, B symptoms, International Prognostic Index score, RNA-sequencing based molecular subtype) and survival were compared across ancestry groups. We examined ancestry differences in mutational patterns and frequency of top 20 driver genes. Chi-squared tests and ANOVA were used for the analysis of pt characteristics and clinical pathological features across ancestry groups. Multivariate Cox regression analyses were used to evaluate prognostic factors.

Results: ADMIXTURE global ancestry prediction had 100% correlation with self-reported race and identified 619 pts with >90% European ancestry, 81 with >90% African ancestry, and 43 with >90% Asian ancestry. DLBCL pts with >90% African ancestry had an age at diagnosis 10 years younger (p < 0.001), more commonly had B symptoms (p < 0.001), more commonly had elevated LDH (p < 0.001), more commonly had extranodal sites of involvement (p = 0.001), and more commonly had advanced stage diseases (p = 0.007) compared to patients with >90% European ancestry. There were no statistical differences between these ancestry groups in gender, ECOG, IPI score, and molecular subgroup (ABC/GCB/Unclassified). The total variant distribution (missense, truncation, CNV gain, CNV loss) was similar between patients with >90% European ancestry and >90% African ancestry, and only HIST1H1E copy number loss and MGA missense mutations were significantly more common in patients with African ancestry.

Conclusions: Additional studies examining the differentially mutated genes or other factors in African American DLBCL pts may explain comparatively adverse features and outcomes for African American pts.

#4207

Elucidating drivers of the Hispanic paradox in non-small cell lung cancer.

Madelyn Klugman, Xiaonan Xu, Mindy Ginsberg, Thomas Rohan, H. Dean Hosgood. _Albert Einstein College of Medicine, Bronx, NY_.

The Hispanic paradox refers to findings in the United States (US) showing similar or better health outcomes in Hispanics than in non-Hispanic whites (NHWs) despite lower average socioeconomic status (SES). This paradox has been reported for non-small cell lung cancer (NSCLC) using data from national cancer registries, with Hispanics experiencing lower mortality rates than Non-Hispanics (NHs). However, these registries often lack critical covariate data such as smoking. To this end, we developed the Lung Cancer Clinical Cohort at Montefiore Medical Center (LC3MMC) in the Bronx, NY, to describe the growing but understudied US Hispanic population and to further elucidate factors in lung cancer survival. Subjects in LC3MMC were ≥18 years old with no prior cancer history who were diagnosed with incident primary lung carcinoma of any histology and of stage 1-4, between 2004-2017. Demographic and clinical data were obtained from MMC's clinical systems, and tumor-related information was obtained from MMC/Einstein's Cancer Registry. Of the 5102 subjects in LC3MMC, the mean age at diagnosis was 68 (±12) years, 50% were male, 80% were ever-smokers, 72% had known ethnicity [NHW: n=1278, Non-Hispanic Black (NHB): n=1222, Hispanic: n=855], and 80% had known histology (NSCLC: n=3554, SCLC: n=505). Based on log-rank tests, overall survival was greater in Hispanics compared to NHs (all subjects: p=0.01, NSCLC only: p=0.01) and did not differ between NHWs and NHBs (all: p=0.26, NSCLC: p=0.21). Cancer subtype (χ2: NSCLC vs. SCLC: p=0.18) and treatment rates (yes vs. no) [χ2: surgery (all: p=.08, NSCLC: p=0.12), radiation (all: p=.85, NSCLC: p=0.55), chemotherapy (all: p=0.26, NSCLC: p=0.23)] did not vary by ethnicity. Multivariate Cox proportional hazards modeling compared survival between Hispanics and NHs while adjusting for age; gender; stage; adenocarcinoma histology; smoking status; receipt of surgery, chemotherapy, radiation, and palliative care; marital status; and SES. In NSCLC patients, Hispanic ethnicity was associated with decreased risk of death (HR=0.86, 95% CI=0.74-0.99). When limiting the model to Hispanics, stage (regional: HR=2.81, 95% CI=1.72-4.57; distant: HR=6.78, 95% CI=4.24-10.85; unknown: HR=4.17, 95% CI=2.42-7.18), receipt of surgery (HR=0.53, 95% CI=0.39-0.72) and receipt of chemotherapy (HR=0.74, 95% CI=0.57-0.97) were associated with survival. Hispanic ethnicity was not associated with better survival in SCLC subjects (adjusted HR=0.97, 95% CI: 0.68-1.39). We have observed the Hispanic paradox in NSCLC in one of the largest US academic medical centers. Clinical and social factors do not fully explain the improved survival in this group. Our results provide the basis for detailed exploration of the demographic, cultural, and molecular drivers of lung cancer survival disparities among different racial/ethnic groups in the Bronx.

#4208

Integrated molecular approach to identify biological factors contributing to breast cancer disparities in Chicago.

Ashlie M. Santaliz Casiano,1 Zeynep Madak-Erdogan,1 Oana Danciu,2 Hariyali Patel,2 Landan Banks,2 Jermya Buckley,2 Garth Rauscher,2 Anita Fareeduddin,3 Elonia Martin,3 Archana Bargaje,3 Carlos Garcia,3 Lauren Schulte,4 Deanna Taiym,4 Julie Kim,4 William Gradishar,4 Scott Hegerty,5 Jonna Frasor,2 Kent Hoskins2. 1 _University of Illinois, Urbana-Champaign, IL;_ 2 _University of Illinois, Chicago, IL;_ 3 _Mercy Medical Center, Chicago, IL;_ 4 _Robert H. Lurie Cancer Center of Northwestern University, Chicago, IL;_ 5 _Northeastern Illinois University, Chicago, IL_.

BACKGROUND: African American (AA) women have a 4-5 fold greater risk of death from estrogen and/or progesterone receptor positive (ER/PR positive) breast cancer compared to non-Latina white (white) women, even after controlling for stage at diagnosis, treatment, and other prognostic factors. The purpose of this study is to examine whether there are racial differences in biologic mechanisms typically activated in ER/PR positive breast cancer and whether they might lead to a higher rate of distant metastases and/or resistance to endocrine therapies.

METHODS: Eligible cases -AA and white women, aged 20-79, with a new diagnosis of stage I-III ER+ breast cancer- are being recruited from 3 cancer institutions in Chicago. Control subjects -AA and white women presenting for a screening mammogram without breast symptoms and no history of cancer- have been recruited from the corresponding mammography centers. We are collecting serum from AA and white cases and controls in order to conduct a metabolomic analysis to identify oncometabolites that might promote aggressive phenotypes in ER/PR breast cancer cells. Serum collection is taking place prior to breast surgery or initiation of any neoadjuvant or adjuvant therapies to ensure that our findings are not due to treatment effects on metabolites.

RESULTS: To date, we have enrolled 175 out of 300 planned study participants. Recruitment will be completed by January 2019, and preliminary metabolomic data will be presented. We will explore associations between candidate oncometabolites and neighborhood socioeconomic deprivation as well as individual patient and tumor characteristics typically related to poor outcomes in AA women with BC. Analyses will adjust for tumor intrinsic subtype (Luminal A vs. Luminal B) to account for known differences in molecular pathways by tumor subtype. We hope to identify biochemical differences in serum that could help to explain the unanswered question regarding why AA patients with ER/PR positive breast cancer or more likely to die from the disease compared to their white counterparts.

#4209

Codon 72 polymorphisms of p53 are associated with obesity, diabetes, and race in breast cancer.

Michael Behring, Bunyamin Ozaydin, Upender Manne. _University of Alabama Birmingham, Birmingham, AL_.

Background: As a tumor suppressor and a factor in glucose metabolism, p53 is an alluring point of focus in the study of obesity and cancer. Population-based studies show that single nucleotide polymorphisms (SNPs) at codon 72 of p53 are individually linked with development of breast cancer, high BMI, and type II diabetes. In this study cohort of African American (AA) and Caucasian (CA) women with breast cancer, we assessed the relationships between arginine (Arg) and proline (Pro) phenotypic variants and obesity and type II diabetes, accounting for confounding molecular and demographic influences. We also evaluated the prognostic relevance of these factors.

Methods: DNA samples isolated from breast tumor tissues collected from 190 (AA=90 and CA= 100) women were genotyped to detect SNPs at codon 72 of the p53 gene and were sequenced by using exon-specific primers (4 through 9). These exons were selected because more than 90% of mutations are located in these exons of p53. For all women, information on BMI and type II diabetes status was collected from electronic health records. Obese was defined as BMI ≥ 30. The closest measure of BMI within 5 years of the date of the diagnostic sample was used, and the time difference between BMI and sample date was included as a confounding variable in adjusted analyses. Only pre-tumor sample diagnoses of type II diabetes were considered. Clinical and demographic variables were evaluated for their association to p53 SNPs, using χ2 tests for categorical analysis and F-tests for continuous variables. Linear regression models were used to examine interactions with BMI. Cox proportional hazard regression models were used for all time-to-event analyses. Separate models were built for each BMI-defined category of weight (underweight, normal, overweight, or obese).

Results: Both SNP72 and BMI were strongly associated with race. For AAs, 68% were obese, and 49% were Pro/Pro phenotype. CAs were less obese (24%), and most were Arg/Arg (60%). Most cases of type II diabetes (78%) were AAs, but 23 total patients with diabetes was insufficient for BMI-stratified analysis. As determined in a linear regression model, there was an interaction between race, SNP72 and BMI. AAs with Arg/Arg had significantly higher BMI values than other SNP/race groups (P=0.003). In a Cox model of obese-only patients, adjusted for race, p53 somatic mutation, time of BMI measure, tumor stage, and molecular subtype, Arg/Arg women had 3.81 times higher hazard of death from cancer than Pro/Pro women (95% CI: 1.08,13.48). Type II diabetes apparently did not influence survival, but this observation may be due to insufficient sample size.

Conclusions: The association between SNP 72 and BMI varied according to race. African American women who exhibited Arg/Arg had an average BMI of 35. For obese patients, Arg/Arg had a negative influence upon survival, which was independent of established risk factors, including race.

#4210

Ethnic differences in omega-3 polyunsaturated fatty acid intake.

Sachelly Julian-Serrano,1 Kevin W. Dodd,1 Ivonne Anglero,2 Rachael Stolzenberg-Solomon,1 Nancy J. Emenaker1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _University of Puerto Rico, San Juan, PR_.

Background: In the US, over 1.7 million new cancer cases are forecast in 2018, with highest incidence in non-Hispanic blacks and lowest in Asians, and with non-Hispanic whites having higher cancer incidence than Hispanics. Omega-3 polyunsaturated fatty acids (n-3 PUFA), particularly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), may play a role in reducing risks for some diseases, including cancer. Ethnic dietary intake patterns are known to affect dietary intake habits affecting individual macronutrient and micronutrient consumptions. Some previous population-based intake studies suggest differences in total fat consumption patterns including n-3 PUFA across Hispanic ethnic groups.

Objectives: This study aims to determine if mean n-3 PUFA dietary intakes of EPA and DHA differ across race/ethnic groups in the National Health and Nutrition Examination Survey (NHANES) 2011-2014 and to describe the main EPA and DHA food sources consumed. We hypothesize n-3 EPA and DHA dietary intakes differ across ethnic groups based on ethnocentric dietary intake patterns.

Methodology: Dietary intake data collected from the Day 1 of the 24-hour recall in the NHANES 2011-2014 was used to estimate mean daily EPA and DHA intake and identify food sources contributing to n-3 dietary intakes in adults across race/ethnic groups. We estimated mean EPA and DHA intake in grams (g) with 95% confidence intervals (CI) in Hispanics, non-Hispanic whites, non-Hispanic blacks, and non-Hispanic Asians. For major food sources across ethnic groups, the fractions of total intake (and corresponding 95% CIs) from each food item were also calculated.

Results: A total of 9,848 individuals were included in this analysis, including 21% Hispanics, 44% non-Hispanic whites and others, 23% non-Hispanic blacks, and 12% non-Hispanic Asians. Non-Hispanic blacks reported higher total PUFA intake (Mean: 19.60g; 95% CI: 18.99-20.22) and non-Hispanic Asians reported the lowest intake (Mean: 16.57g; 95% CI: 15.86-17.27). However, non-Hispanic Asians reported an intake 3x higher of EPA (Mean: 0.07g; 95% CI: 0.06-0.07) and 2x higher of DHA (Mean: 0.12g; 95% CI: 0.11-0.14) than other ethnic groups. Baked or broiled salmon was the largest contributor of EPA and DHA across race/ethnic groups. For non-Hispanic whites and non-Hispanic blacks, salmon cake or patty was their second largest source of EPA. Hispanics had a higher intake of foods with lower DHA content.

Conclusions: Our results suggest EPA and DHA intake differs across race/ethnicity and the dietary sources to obtain these n-3 PUFA shows substantial heterogeneity. Epidemiologic studies of cancer and other disease outcomes should employ nutritional assessment tools that consider ethnic-specific sources of n-3 PUFA intake. 

## PREVENTION RESEARCH

### Clinical Prevention, Early Detection, and Interception 2

#4211

Breast cancer risk factors by mode of detection among screened women in the Cancer Prevention Study-II.

Mia M. Gaudet,1 Emily Deubler,1 W. Ryan Diver,1 Samantha Puvanesarajah,1 Alpa V. Patel,1 Ted Gansler,1 Dana Flanders,1 Mark Sherman,2 Susan Gapstur1. 1 _American Cancer Society, Atlanta, GA;_ 2 _Mayo Clinic, Jacksonville, FL_.

Identifying risk factors for women at high risk of symptom-detected breast cancers that were missed by screening would enable targeting of alternative prevention strategies. To identify breast cancer risk factors by mode of detection, we examined these associations in heavily-screened women from the Cancer Prevention Study (CPS)-II Nutrition Cohort. Among 76,406 women followed for a median of 13.8 years, 2,469 screen-detected and 1,189 symptom-detected breast cancer cases were diagnosed. Multivariable-adjusted associations were estimated using joint Cox proportional hazards regression models with person-time calculated contingent on screening. The mean (standard deviation) age at baseline was 62.0 (6.6) years for non-cases, 61.6 (6.1) for screen-detected cases, and 61.5 (6.2) for symptom-detected cases. Women with symptom-detected tumors were diagnosed at a slightly younger age (mean=69.4) and less likely to be diagnosed with a localized tumor (65.3%), than women with a screen-detected tumor (mean age at diagnosis=70.4 and localized tumors=83.3%). Factors associated with higher risks of symptom detected vs. screen detected breast cancer, included: current combined menopausal hormone use (HR=2.04, 95% CI 1.70 - 2.46 vs. HR=1.43, 95% CI 1.25 - 1.63), current estrogen only menopausal hormone use (HR=1.41, 95% CI 1.15 - 1.72 vs. HR=0.94, 95% CI 0.82 - 1.09), and history of benign breast disease (HR=1.89, 95% CI 1.67 - 2.13 vs. HR=1.49, 95% CI 1.36 - 1.62). The opposite pattern of higher risk of screen detected vs. symptom detected breast cancer was observed for greater adult weight gain (HR=2.32, 95% CI 1.91 - 2.82 vs. HR=1.42, 95% CI 1.10 - 1.84) and alcohol intake (HR=1.39, 95% CI 1.24 - 1.64 vs. HR=1.20, 95% CI 0.92 - 1.58). Our results suggest that understanding risk factors for symptom-detected cancers may help identify women who would benefit from more intensive screening to facilitate early detection.

#4212

A pilot clinical trial to study the effects of Angelica gigas dietary supplement (CognI.Q) on human immune cells (NCT03630328).

Deepkamal N. Karelia,1 Jeremy S. Haley,1 Todd D. Schell,1 Diane Hershock,1 Junjia Zhu,1 Cheng Jiang,1 Junxuan Lu,1 Jinhui Zhang2. 1 _Penn State Univ. College of Medicine, Hershey, PA;_ 2 _Food and Drug Administration, Silver Spring, MD_.

Korean Angelica gigas Nakai (AGN)-containing products are marketed as dietary supplements for memory improvement, pain relief and women's health especially for menopausal symptom management. Decursin (D) and its isomer decursinol angelate (DA) are the major marker compounds in the ethanol extract of AGN root. In rodent models, we and others have shown that D/DA are rapidly converted to decursinol (DOH) after gavage or i.p. injection. Our multi-omic analyses of the TRAMP neuroendocrine carcinomas suggest immune enhancement by AGN. We have published a 20-subject single dose-PK study (NCT02114957) in men and women volunteers in Amarillo, TX (age 21-58 years) with AGN dietary supplement CognI.Q (purchased from Quality of Life Laboratories). For dosage, each person swallowed 4 vegicaps (800 mg AGN) at time 0, the recommended daily dose by manufacturer. The human AUC0-48h for DOH (27579 h.nmol/L) is much higher than for DA (335 h.nmol/L) and D (37 h.nmol/L), supporting extensive conversion from D/DA to DOH (Zhang, J. et al, PLOS One, 2015). Significantly, as secondary endpoint parameters, the neutrophil counts in these human subjects were increased by 71% at 24h post-dose vs. pre-dose. The natural killer cell (NK) mRNA signature in their peripheral blood mononuclear cells (PBMC) was increased by 60~90%. Neutrophils (myeloid lineage) fight against bacterial infection whereas NK cells (lymphoid lineage) not only kill virus-infected cells but also recognize and kill cancer cells, serving as a crucial innate immune surveillance against malignancy in our body. Therefore, we hypothesize that daily intake of AGN dietary supplement (CognI.Q) increases the number and/or activities of neutrophils and NK cells, and in turn may boost human innate immune function against bacterial and viral infections as well as cancer risk. As an initial effort to test our hypothesis, the goal of this pilot clinical trial is to delineate the CognI.Q supplement-specific innate immune enhancement activity in healthy men. We will use a double-blinded, placebo-controlled and crossover trial design so that each man will serve as his own comparison for the immune cell responses to CognI.Q and placebo. Supplement period of 3 weeks will be interspersed with a 2 week washout period. We expect to detect an increase in both neutrophil and NK cell counts compared to placebo. Positive data from our proposed trial will be the first of its kind to support enhancement in healthy US residents of these innate immune cells by an AGN dietary supplement. Detailed study design and interim study analysis will be discussed at the meeting.

#4213

Stool-derived eukaryotic RNA (seRNA) assay for noninvasive detection of colorectal cancer and high-risk adenomas.

Erica K. Barnell,1 Yiming Kang,1 Katie M. Campbell,2 Kimberly R. Kruse,3 Andrew R. Barnell,3 Elizabeth M. Wurtzler3. 1 _Washington University School of Medicine, Saint Louis, MO;_ 2 _University of California, Los Angeles, Los Angeles, CA;_ 3 _Geneoscopy, Saint Louis, MO_.

Colorectal cancer (CRC) is the second leading cause of cancer related deaths in the United States. The high mortality rate for CRC is largely attributable to the frequency of late-stage diagnoses, caused by low patient compliance with screening guidelines. A novel nucleic acid extraction method that isolates stool-derived eukaryotic RNA (seRNA) has permitted development of a colorectal cancer stool diagnostic test (CRC-SDT) that uses a fecal immunochemical test (FIT) and 11 seRNA transcripts to serve as a reliable and noninvasive screening alternative to lower mortality associated with CRC. Stool samples were obtained from 190 individuals prior to undergoing a screening colonoscopy. Patients were diagnosed as either having colorectal cancer, having high-risk adenomas (high-grade dysplasia, villous growth pattern, or >1.0cm in size), or healthy (low-risk adenomas, benign polyps, and/or no findings on a colonoscopy). FITs were obtained for each sample. Samples that had a positive FIT were considered positive for the CRC-SDT. Samples that had a negative FIT underwent seRNA isolation, library preparation (Illumina TruSeq Targeted RNA) and subsequent sequencing (Illumina NextSeq 550). A random forest model was built using 11 transcripts that were differentially expressed (log2 fold-change > 1; ANOVA p < 0.05) between diseased patients (CRC and high-risk adenomas) relative to the healthy cohort. Internal 10-fold cross-validation was performed for the training set and a receiver operating characteristic (ROC) area under the curve (AUC) determined model accuracy for cancer and high-risk adenomas relative to the healthy cohort. Model predictions for an independent hold-out test set were used to determine model accuracy for colorectal cancer and high-risk adenomas. Internal cross-validation for 154 samples attained a ROC AUC of 0.82. The trained model was employed on the independent hold-out test set of 36 samples and model predictions were compared to colonoscopy results. The model attained a 100% sensitivity for CRC (n = 7), a 60% sensitivity for high-risk adenomas (n = 5), and an 88% combined specificity for the healthy cohort (n = 24). CRC-SDT uses 12 biomarkers (FIT and expression from 11 seRNA transcripts) to accurately detect CRC and high-risk adenomas. Use of seRNA biomarkers dramatically improves detection of high-risk adenomas relative to existing noninvasive screening methods for CRC.

#4214

A microfluidic platform to capture and detect cancer cells in Leukemia patients.

Rolf Muller,1 Rachel Toughiri,1 Alena Bartakova,1 Paul Diaz,1 Craig Carson,1 Paul Armistead,2 George Fedoriw,2 Mathew Foster,2 Maryam Zomorrodi,1 Jennifer Barber-Singh,1 Veronica Cheung,1 Emily Mirkin,2 Roksolana Melnychuk,1 Elizabeth Fabio,1 Sangeetha Purushotham,1 Judy Muller-Cohn1. 1 _BioFluidica, San Diego, CA;_ 2 _University of North Carolina, Chapel Hill, NC_.

Introduction : BioFluidica has developed a platform, The Liquid Scan™ to analyze circulating cancer cells in whole blood. The Liquid Scan is unique in its performance metrics compared to existing platforms by employing fully automated instrumentation in conjunction with a highly sensitive and a specific cell detection microfluidic chip. This technology can be applied to a broad variety of tumors, including liquid tumors such as leukemias and lymphomas. The unique design of the microfluidic chip permits the capture and analysis of live circulating leukemic cells (CLCs) from whole blood, even when these cells appear undetectable by other methods.

The Liquid Scan has been applied to capture CLCs from patients suffering from acute myeloid, as well as acute lymphoblastic leukemia (AML and ALL, respectively). These leukemias are acute diseases of the blood and bone marrow, with mutational changes and clonal expansion of abnormal myeloid and lymphoblastic precursors, respectively claiming a great number of lives every year. Bone marrow biopsies/aspirations have been the standard methods for reliably diagnosis and monitoring these diseases. These procedures are extremely painful for patients, necessitate specialized centers, and trained personnel, which increases the overall cost of treatment.

Methods: We have applied the Liquid Scan™ to capture CLCs and characterization, using specific antibodies to capture known clones of leukemic blasts. As a model, we use Kasumi-1, KG-1 and HL-60 cells for AML, and SupB15 cells for ALL, spiked in healthy whole blood.

Results: We characterized the ability of the The Liquid Scan™ to capture and release circulating leukemic cells from whole blood, by using cell line models spiked-in whole healthy donor blood and applied to the microfluidic chip. Our results are directly applicable to patient in different stages of the disease, both as an initial diagnostic tool, and as a way to monitor recovery and diagnose relapse.

Conclusions: Precision medicine (PM) is significantly changing the treatment of cancer and is bringing new hope for patients. Diagnostic testing for circulating tumor and leukemic cells are promising prognostic and predictive tools for selecting optimal therapies based on the patient's genetic content. Highly sensitive, and selective BioFluidica's system is highly sensitive and selective with the capability of rapidly delivering purified and concentrated circulating cancer cells compatible with downstream molecular diagnostics.

#4215

Patient and screening characteristics associated with positive lung cancer screening examinations.

Louise M. Henderson,1 Samantha Sites,1 Tina Tailor,2 Sara C. Bearden,1 Roger Huamani,1 Allison Throneburg,1 Max Nagle,1 M Patricia Rivera1. 1 _UNC Chapel Hill, Chapel Hill, NC;_ 2 _Duke University, Durham, NC_.

Introduction. The likelihood of having a positive low dose computed tomography (LDCT) lung cancer screening (LCS) examination that requires workup may vary according to patients' characteristics and risk factors. The purpose of this study is to evaluate patient characteristics associated with positive LCS exams.

Methods. We utilized data from 1684 LDCT exams conducted for LCS at five academic and community sites from 2015-2018. During the screening visit, patients were asked to complete a comprehensive questionnaire including socio-demographics, smoking history, occupational and environmental exposures, family history of lung cancer, and comorbid conditions. Information on the LCS examination, including the radiologists' Lung Reporting and Data System (Lung-RADS) assessment, was obtained from the radiologist report. We dichotomized Lung-RADS into negative (Lung-RADS 1 (negative) or 2 (benign appearance or behavior)) and positive (Lung-RADS 3 (probably benign), 4A (suspicious), or 4B (suspicious)) based on the management recommendation of continuing with annual screening in 12 months or requiring follow-up before 12 months. We compared Lung-RADS results (positive versus negative) by patient characteristics using chi-square tests and examined predictors of positive LCS exams using multivariate logistic regression, reporting adjusted odds ratios (aORs) and 95% confidence intervals (95%CI).

Results. Screened patients ranged in age from 50-78 years, with 46.7% less than 65 years and 53.3% ages 65 or older. Approximately 53.3% were male and 46.7% were female; 84.5% were White, 11.8% were African American, and 3.7% were other races. The majority (76.5%) of screened patients were overweight or obese. Two-thirds (68.5%) of patients had more than one LCS exam. Most (85.0%) LCS exams were negative while 15.0% were positive. There were no significant differences in Lung-RADS assessment by race, gender, education, or body mass index. Lung-RADS were significantly more likely to be positive in those ages 65 and older versus those ages less than 65 (aOR=1.47, 95%CI: 1.03-2.10) and in baseline versus subsequent screening exams (aOR=0.43, 95%CI: 0.30-0.62).

Conclusion. Predictors of having a positive LCS exam requiring work-up before the next recommended annual screening test include being aged 65 and older and having a baseline LCS examination.

#4216

The microbiota associated to cervical and anal HPV infections in a Hispanic population.

Frances Vázquez-Sánchez,1 Gilmary Ortiz,1 Ana P. Ortiz,2 Filipa Godoy-Vitorino1. 1 _University of Puerto Rico, School of Medicine, San Juan, PR;_ 2 _University of Puerto Rico Comprehensive Cancer Center, San Juan, PR_.

Anogenital human papillomavirus (HPV) are the world's most commonly diagnosed sexually transmitted infections and high-risk types are linked to dysplasia. Susceptibility to HPV infections is related to the microbial communities of these genital surfaces, which are interfaces between the host and environment. Microbes are a predicted cause of malignancies, revealing a tremendous potential of microbiome-related processes for cancer prevention and diagnostics. The cervix and anus share a susceptible transformation zone, characterized by a metaplastic epithelial site. In fact, in women, anal and cervical squamous intraepithelial lesions tend to occur concurrently, an observation that could be explained by the fact that anal and cervical HPV infections are strongly correlated. We hypothesized that bacterial communities may differ in the anus and cervix and may reveal populations associated to HPV infections in these two body sites. To test this hypothesis we characterized the microbiota of a cross-sectional population-based sample of Puerto Rican women (self-sampled) from the San Juan metropolitan (n=300 samples) and related them to anogenital HPV infections (HPV typing performed by PCR). The microbiota of resident cervical and anal bacterial communities was performed through sequencing of the 16S ribosomal RNA V4 region with the Illumina platform. Data was analyzed at first with QIIta, using the SILVA database as taxonomic reference, and community analyses were performed in QIIME and R. Significant differences in community structure (betadiversity) were found between cervical and anal communities (p-value = 0.001), but not according to HPV (p-value = 0.212) in any of the body sites. Indeed, overall anal communities were dominated by OTUs within Prevotella, Bacteroides and Clostridiales, while cervical samples were dominated by Lactobacillus and Bifidobacteraceae (Gardnerella). Despite the common core communities at each body site, cervical samples of HPV positive patients revealed an enrichment of Ureaplasma and Prevotella while Peptinophilus was associated with anal HPV infections. Although more analyses are needed, our data suggests cervical and anal bacteria are associated to HPV infections which could pave the way to the development of early detection methods or novel probiotics for in women living in Puerto Rico.

#4217

Tumor mutational burden (TMB), T cell-inflamed gene expression profile (GEP) and PD-L1 are independently associated with response to pembrolizumab (Pembro) in patients with advanced melanoma in the KEYNOTE (KN)-006 study.

Antoni Ribas,1 Caroline Robert,2 Jacob Schachter,3 Georgina V. Long,4 Ana Arance,5 Matteo S. Carlino,6 James Larkin,7 Andrea L. Webber,8 Jared Lunceford,8 Qing Zhao,8 Razvan Cristescu,8 Michael Nebozhyn,8 Chunsheng Zhang,8 Wendy Blumenschein,8 Clemens Krepler,8 Nageatte Ibrahim,8 Adil Daud,9 Jean-Jacques Grob10. 1 _University of California, Los Angeles, Los Angeles, CA;_ 2 _Gustave Roussy and Paris-Sud University, Villejuif, France;_ 3 _Sheba Medical Center at Tel Hashomer, Ramat Gan, Israel;_ 4 _Melanoma Institute Australia, The University of Sydney, Mater Hospital and Royal North Shore Hospital, Sydney, Australia;_ 5 _Hospital Clinic de Barcelona, Barcelona, Spain;_ 6 _Westmead and Blacktown Hospitals, Melanoma Institute Australia, and The University of Sydney, Sydney, Australia;_ 7 _Royal Marsden Hospital, London, United Kingdom;_ 8 _Merck & Co., Inc., Kenilworth, NJ; _9 _University of California, San Francisco, CA;_ 10 _Aix Marseille University, Hôpital de la Timone, Marseille, France_.

Background: Biomarkers may aid in identifying pts who can benefit from checkpoint blockade therapies. Biomarkers of response to anti-PD-1 therapy include TMB and inflammatory biomarkers such as PD-L1 expression and T cell-inflamed GEP. Effects of TMB and GEP on clinical outcomes were evaluated for Pembro (anti-PD-1) and Ipi (anti-CTLA-4) monotherapies in the KN-006 study.

Methods: In this phase 3, controlled, open label study, 834 pts were randomized to receive Pembro 10 mg/kg every 2 or 3 wks or 4 doses of Ipi 3 mg/kg every 3 wks. Data were available for analysis of TMB (whole exome sequencing) in 216 pts (Pembro n=151 and Ipi n=65), for GEP (RNA, NanoString nCounter) in 641 pts (Pembro n=461 and Ipi n=180) and 134 pts had both TMB and GEP data available. All pts with TMB data and 458 pts with GEP data had PD-L1 expression data (PD-L1 IHC 22C3 pharmDx, MEL score). Statistical testing was prespecified prior to merging biomarker and clinical outcome data. Relationships between biomarkers and best overall response (BOR) were assessed by logistic regression and by area under the ROC (AUROC) curve; progression free survival (PFS) and overall survival (OS) by Cox proportional hazards regression. Prespecified cutoffs were -0.318 for GEP and 175 (mutations/exome) for TMB. Clinical data cutoff was Dec 04, 2017.

Results: TMB, GEP and PD-L1 MEL were significantly associated with BOR, PFS and OS in the Pembro arm (p<0.050). Response rates to Pembro therapy were greater in pts who had high levels of both TMB ≥175 and GEP ≥-0.318 or TMB ≥175 and PD-L1 MEL ≥2 compared with those with low levels of these biomarkers (54% vs 14% and 51% vs 33%, respectively). TMB, GEP and PD-L1 MEL remained significantly associated with response to Pembro for BOR and PFS, and in general for OS, when assessed in joint models. TMB showed low correlations with GEP (0.14; p=0.057) and PD-L1 (0.22; p=0.001); GEP and PD-L1 were moderately correlated (0.60; p<0.001). In Ipi-treated pts, TMB was not significantly associated with BOR (p=0.428) and trended toward significance for PFS (0.098) and OS (p=0.099); GEP was also not significantly associated with BOR (p=0.188) but was significantly associated with PFS (p=0.012) and OS (p<0.001). AUROCs (95% CI) were 0.64 (0.55, 0.73) and 0.53 (0.35, 0.70) for TMB, and 0.64 (0.59, 0.7) and 0.56 (0.45, 0.67) for GEP with Pembro and Ipi. Note the observed TMB relationship with Ipi is based on an assessment of limited data in that arm and definitive conclusions cannot be made.

Conclusion: TMB and inflammatory biomarkers (GEP and PD-L1) showed statistically significant and independent associations with BOR, PFS and OS in Pembro-treated pts with advanced melanoma. Only GEP showed significant associations with PFS and OS for Ipi, potentially indicating a prognostic value.

#4218

Validation of a new blood-based biomarker strategy for the early detection of lung cancer.

Michael N. Kammer, Amanda K. Kussrow, Sanja L. Antic, Rina Nguyen, Heidi Chen, Darryl J. Bornhop, Pierre P. Massion. _Vanderbilt University, Nashville, TN_.

Rationale: The management of indeterminate pulmonary nodules (IPNs) remains a challenging problem. Our new assay methodology, the Free Solution Assay (FSA), measured by the Compensated Interferometric Reader (CIR), consisting of a diode laser, capillary and CCD, provides 40-fold lower limits of quantitation (LOQ) than ELISA for known candidate serum protein biomarkers, while speeding assay development, accuracy, and sensitivity. We hypothesized that lowering the LOQ of previously investigated biomarkers can increase the biomarker discriminatory power, enabling patients in an intermediate risk group (15-80%) to be reclassified into a low (<15) or high (>80) risk group. Methods: In this retrospective case-control study, FSA-CIR was used to measure the serum concentration of CYFRA 21-1 in two patient cohorts, a training cohort (N=274) of patients with IPNs collected at Vanderbilt University Medical Center and an external validation cohort (N=103) collected at the University of Pittsburgh Medical Center. Patient malignancy was determined by tissue biopsy or 2-year follow-up CT scan showing no signs of nodule growth. Baseline risk for cancer was calculated using nodule size. The added value of CYFRA 21-1 was assessed by comparing the difference in risk for lung cancer after incorporating CYFRA 21-1 into the model. Results: The limit of detection (LOD) of 6pg/mL and an average LOQ for standards was determined to be 60pg/mL. Patient samples in the training cohort were found to have CYFRA 21-1 concentrations ranging 100 pg/mL to 10 ng/mL, with a median of 0.79 (0.28-1.22, interquartile range) ng/mL in the control population and 1.90 (1.33-3.35) ng/mL in the case population, providing a ROC-AUC of 0.86 across three histological subtypes (adenocarcinoma, squamous cell carcinoma, and small cell lung cancer). The CYFRA 21-1 + nodule size risk model correctly reclassified 28 (25%) [DB1] of intermediate-risk benign nodules into the low-risk group and 28(24%) of intermediate-risk malignant nodules into the high-risk group. The independent validation cohort's controls had a median of 0.35 (0.20-0.56) ng/mL, while cases had 0.97(0.66-1.22) ng/mL, providing a ROC-AUC of 0.84 and correct reclassification of 12(34%) of intermediate-risk benign nodules and 6(15%) of intermediate-risk malignant nodules. The CYFRA 21-1 + nodule size risk model correctly classified 90.6% in the low-risk group and 87.0% in the high-risk group. Conclusions: FSA-CIR measurements requiring only a few microliters of serum allowed for reclassification of patients in the intermediate risk group in both the training and validation cohorts. The results suggest that CYFRA 21-1 measured by FSA-CIR represents a strong candidate biomarker for risk stratification of patients with IPNs.

#4219

Mitochondrial DNA damage: A promising tool to screen cervical cancer risk.

Balaji Sadhasivam, Camille C. Gunderson, Elizabeth H. Hahn, Sarah E. Johnston, Yan D. Zhao, Vengatesh Ganapathy, Lurdes Queimado. _University of Oklahoma Health Science Center, Oklahoma City, OK_.

Background: Cervical cancer is the 4th most common female cancer, accounting for 500,000 new cases and 200,000 deaths across the world. 99% of cervical cancer is due to infection by human papillomavirus (HPV). The factors leading to HPV integration and induced carcinogenesis are not entirely clear, but high DNA damage levels have been proposed to play a role in HPV integration. Chronic HPV infection increases DNA damage and reduces DNA repair, leading to even higher DNA damage levels. Recently, we showed that the levels of nuclear DNA (nDNA) damage are a potential biomarker predictive of cancer risk. Mitochondrial DNA (mtDNA) damage has been reported to be more persistent than nDNA damage. We have previously demonstrated a good correlation between mtDNA and nDNA damage. Herein, we performed a pilot study to assess whether the levels of mtDNA damage can be used as a potential tool to screen cancer risk.

Aims: (1) To measure determine whether the amount of mtDNA damage differs between cervical samples with distinct pathologies. (2) To determine whether the number of mtDNA lesions correlates with the grade of cervical dysplasia.

Methods: Ethics Committee approval and written informed consent was obtained from all participants. Samples from 25 patients were collected from the endocervix during a pelvic exam in the Dysplasia Clinic. Samples for DNA damage studies were collected by cytobrush and DNA extracted as previously reported. Demographic and risk factor data were collected through detailed questionnaires. Clinicopathologic data was abstracted from the medical chart. The amount of mtDNA damage was quantified by a long-run real-time PCR-based DNA-damage quantification (LORDQ) assay. Data were analyzed by Student's t-tests. Logistic regression analysis was done modeling the probability of having present pathology risk.

Results: Histopathological reports indicated that 9 cervical samples were normal (C0) and 16 had cervical dysplasia. Dysplasia samples were as follows: 9 low-grade squamous intraepithelial lesions (LSILs or C1) and 7 high-grade squamous intraepithelial lesions (HSILs or C2/3). The amount of mtDNA lesions per 10,000 bases were increased two-fold in LSILs or C1 cases and three-fold in HSILs or C2/3 cases when compared to C0. Regression analysis showed no significant correlation between age, race, smoking and drinking status compare with pathology risk.

Conclusion: Our data show that mtDNA damage is the lowest in patients without cervical dysplasia and increases progressively with higher grades of dysplasia and correspondent cancer risk. This pilot study shows the feasibility of the approach and stresses the potential of using mtDNA damage to predict cervical cancer risk. Measuring mtDNA damage detection by LORDQ is less time consuming and more cost-effective than the measurement of nDNA damage. Larger population studies are urgently needed to fully assess the potential of this approach for cervical cancer risk.

#4220

Evidence of bovine leukemia virus genes detected in Colombian women with and without breast cancer: A zoonotic infection.

Nury N. Olaya-Galán,1 Sandra P. Salas-Cárdenas,2 Adriana P. Corredor-Figueroa,2 Gertrude C. Buehring,3 HuaMin Shen,3 Manuel A. Patarroyo,1 Ma, Fernanda Gutierrez2. 1 _Universidad del Rosario, Bogota, Colombia;_ 2 _Pontificia Universidad Javeriana, Bogota, Colombia;_ 3 _University of California, Berkeley, CA_.

Bovine leukemia virus (BLV), is an oncogenic virus that infects cattle worldwide and is the causative agent of leukemias, lymphomas and persistent lymphocytosis. BLV has also been found in humans and has recently been proposed as a risk factor for developing breast cancer in the USA and Australia. In Colombia, there is evidence of infection in women but no correlation with breast cancer. This study was aimed at comparing the presence of the virus in breast tissue from different sources: necropsies of women without tumor development (normal breast tissue), and surgeries of benign and malignant tumors, to better understand the role of BLV in Colombia. A cross-sectional study was designed in which 315 participants were included. Paired samples of breast tissue and blood were obtained from surgeries and necropsies in Bogotá city. The presence of BLV was determined by nested PCR and in situPCR targeting different viral genes. For the nested PCR, DNA was extracted from fresh tissues and blood samples, and human GAPDH amplification was carried out to assess DNA quality for the PCRs. Afterwards, different BLV genes were detected by nested PCR. In situ PCR was directed to the tax region of the virus and was done to FFPE sections of the same samples. Positive samples were considered when at least one of the techniques was positive for the virus and were confirmed by Sanger sequencing. Correlation with other risk factors related with breast cancer (HER2, PR, ER and KI67) and the presence of the virus were determined. From the overall population, 40% of the samples were positive for BLV. In the breast cancer population, 37% of the samples were infected with the virus; similar distributions were found in the other groups (pre-malignant, 33%; benign tumors, 27%). Interestingly, almost 60% of the samples were infected in the no-tumor group. 22% of the samples were positive for both breast tissue and blood from the same patients. All the positive samples were confirmed to have BLV by Sanger sequencing of different gene regions of the virus. When correlating viral presence with other risk factors of breast cancer, it was found that most of the positive BLV samples were also positive for progesterone (79%) and estrogen (93%) receptors and were negative for the HER2 mutation. It could thus be possible that the hormonal profile could be linked with the presence of the virus. This study shows that irrespective of the breast pathology, BLV can be found in Colombian women both in breast tissue and blood. Sanger sequencing showed that the virus found in humans is similar to previously reported sequences obtained from cattle with high identity percentages. Even when the biology of the virus remains unknown in humans, it is a big concern to find the presence of an oncogenic virus coming from animals. Further studies are needed to understand the viral mechanisms related with cancer in humans.

#4221

Development and evaluation of cell line-derived FFPE reference material for MSI assay validation.

Takahiro Matsusaka, Eva-Maria Surmann, Sian Constantine, Hannah Child, Julie Wickenden. _Horizon Discovery Limited, Cambridge, United Kingdom_.

Microsatellite Instability (MSI) is defined by variance in the repeat count of microsatellite motifs and occurs in cells that are deficient in one or more mismatch repair proteins. MSI is present in varying cancer types, but is most commonly found in colorectal, gastric and endometrial cancer. Patients with early stage colorectal cancers that display MSI have a better prognosis and show a better response to chemotherapy compared to those with microsatellite stable tumors. MSI alongside additional markers such as tumor mutational burden is also a positive predictive biomarker for immune checkpoint inhibition Identification of MSI tumors in the diagnostic laboratory is traditionally performed by fluorescent multiplex PCR, evaluating the microsatellite length of two mononucleotide repeats and three dinucleotide repeats (recommended NCI Panel) or a commercially available kit, consisting of five mononucleotide markers alongside IHC staining for the four MMR proteins MSH2, MSH6, MLH1, and PMS2. With the increase in MSI testing related to the recent FDA approval of Keytruda®, several companies and laboratories are now developing newer and better PCR-based and next-generation sequencing assays to assess MSI. To control for error, we have developed a pair of cell line-derived MSI/MSS reference samples covering the most commonly used MSI biomarkers. MSI/MSS cell lines were mixed at biologically-relevant ratios and processed into FFPE to serve as a whole-process control. Our data support the suitability of this material on a variety of different platforms and with a high degree of consistency throughout various FFPE batches. In conclusion, our cell line-derived reference samples represent a commutable control to support MSI assay development and validation.

#4222

Omega-3 fatty acids produce DNA methylation changes in inflammation-related genes and pathways with implication of toll-like receptor signaling.

David E. Frankhouser,1 Sarah Woelke,2 Michael Sovic,2 Ralf Bundschuh,2 Pearlly Yan,2 Lisa D. Yee1. 1 _City of Hope, Duarte, CA;_ 2 _The Ohio State University, Columbus, OH_.

Introduction Omega-3 polyunsaturated fatty acids (n-3 PUFA) have been studied for potential health benefits in many diseases including breast cancer. The preventive effects of n-3 PUFAs may relate in part to inhibition of chronic, low-grade inflammation. Of interest is the role of n-3 PUFAs in modulating pro-inflammatory gene expression via epigenetic factors such as DNA methylation (DNAm).

Methods DNA methylation of PBMCs obtained at 0 and 6 months of n-3 PUFA supplementation was assessed via reduced representation bisulfite sequencing (RRBS). PBMC samples were obtained from women at high risk for breast cancer during a randomized clinical trial investigating the effects of different n-3 PUFA doses. The dosing arm selected for this study was 5 g of EPA+DHA/day, with baseline and 6 month samples (n=10). DNAm was quantified using Bismark from trimmed, pass filter reads and analyzed with MethylKit to determine n-3 PUFA treatment specific DNAm changes.

Results Global DNAm showed no change after 6 months of n-3 PUFA treatment; however, we detected 30,974 differentially methylated CpGs (DMCs) across the genome. DMCs, both genome-wide and in gene promoters, where DNAm can regulate gene expression, were significantly enriched for hypermethylation events after treatment. Focusing the analysis on pro-inflammatory signaling mediators led to identification of candidate gene promoter DMCs involved in several inflammation-related pathways. Four pathways were significantly enriched for both DMCs and DMC hypermethylation events even when accounting for the genome-wide trend toward hypermethylated DMCs (hypergeometric test p-value < 0.001). The Toll-Like Receptor Pathway (TLR) genes harbored the most DNAm changes post n-3 PUFA treatment.

Conclusion Our findings demonstrate that n-3 PUFA supplementation can result in inflammation-related changes stemming from PBMC methylome perturbation. The results suggest the TLR pathway as a potential mechanism for the anti-inflammatory effects of EPA and DHA. Functional studies are needed to confirm our current observation.

#4223

Conducting community oral cancer screening among South Asians in British Columbia.

Leigha D. Rock,1 Madhurima Datta,1 Denise M. Laronde,1 Anita Carraro,2 Jagoda Korbelik,2 Alan Harrison,2 Martial Guillaud2. 1 _University of British Columbia, Vancouver, British Columbia, Canada;_ 2 _BC Cancer, Vancouver, British Columbia, Canada_.

Introduction: Globally, more than 300,000 cases of oral cancer are diagnosed annually. South Asian countries, such as India, bear the brunt of this disease due to rampant use of chewing tobacco, betel quid and areca nut. BC has a high proportion of South Asian immigrants. Oral cancer has a high mortality rate (~50% 5-year survival) due to the advanced stage at which it is often diagnosed. It is purported that the majority of oral cancers develop from oral potentially malignant lesions (OPML). While lesions can be easily detected by oral health care providers, it can be challenging to differentiate benign lesions from OPML. DNA aneuploidy has been shown to be an effective marker to predict malignant transformation in OPML. Quantitative cytology (QC) studies DNA content (ploidy) and nuclear morphometric changes within the cell. The aim of this project is to assess the need for oral cancer screening in South Asians in BC and to validate QC as an adjunct screening device in a predominantly South Asian community screening setting to assess its effectiveness in identifying high-risk lesions among visually suspicious lesions.

Methods: Demographic information (gender, age, country of birth, ethnicity, risk habit information and dental usage) were collected at the time of screening. Extraoral, intraoral and fluorescence visualization (FV) examinations were conducted. Buccal mucosal brushings were collected from each participant. Brushings were also collected from lesions or areas that had a loss of fluorescence. Thin-layer cytology slides were prepared and stained using Feulgen Eosin. Slides were scanned using the Cancer Imaging Scanner at BC Cancer using machine learning classification algorithms to identify single, in-focus epithelial nucleus. Cells are classified based on DNA ploidy and malignant associated changes.

Results: 307 participants were screened of which 303 were eligible. More than 99% of the participants were South Asian or Asian. 104 (34%) lesions were documented: 45 (15%) were high risk (white or red lesions, lichen planus (LP)) and 59 (19%) were low risk (trauma, candidiasis, aphthous ulcers). Twenty participants (7%) were suspected to have high-risk OPMLs (not LP): 12 were referred directly to our Next Gen Oral Dysplasia clinic for biopsy, while 8 were reassessed at 3 weeks. Chewing tobacco was found to be strongly associated with lesion presence (p<0.01). QC analysis is ongoing for 320 samples. To date, 5 biopsies have been performed resulting in 1 mild dysplasia, 1 severe dysplasia and 3 hyperplasia.

Conclusion: South Asians in BC were found to be at high risk for OPMLs. QC may help to improve the sensitivity and specificity of oral cancer screening by distinguishing false FV positive inflammatory lesions from high-risk lesions.

#4224

SGLT2 is a diagnostic target for early stage lung adenocarcinoma.

Brendon Villegas, Eva Koziolek, Jie Liu, W. Dean Wallace, David Elashoff, Denise R. Aberle, David B. Shackelford, Jorge R. Barrio, Jane Yanagawa, Steven M. Dubinett, Claudio Scafoglio. _University of California, Los Angeles, Los Angeles, CA_.

Lung cancer is the leading cause of cancer-related death, and lung adenocarcinoma (LUAD) is the most frequent histology. Early diagnosis and treatment are essential; however, there are no targeted diagnostic and therapeutic approaches for early LUAD. An important hallmark of cancer is the increased glucose uptake via GLUT transporters. GLUT activity is imaged in cancer by positron emission tomography (PET) with 2-[18F] fluorodeoxyglucose (FDG). FDG PET is a standard tool for staging advanced lung cancer, but has low sensitivity for early-stage LUAD. This is considered a consequence of slow growth and low requirement of glucose. However, we discovered a novel system of metabolic supply not detected by FDG PET: the sodium glucose transporter 2 (SGLT2)1. SGLT2 activity can be measured in vivo with the PET tracer methyl-4-[18F] fluorodeoxyglucose (Me4FDG). We identified high expression of SGLT2 in human lung premalignancy and early-stage LUAD. Me4FDG detects early-stage, FDG-negative LUAD in mouse models. Targeting SGLT2 with FDA-approved inhibitors significantly reduces tumor growth and prolongs survival in genetic and patient-derived murine models, confirming an important role of SGLT2 in early-stage LUAD2. The reliance of early stage LUAD on SGLT2 opens exciting diagnostic and therapeutic opportunities. The National Lung Screening Trial showed a 20% reduction in lung cancer mortality in high risk individuals using low-dose helical computed tomography (CT). CT is highly sensitive for detecting lung nodules, but is limited by low specificity, especially for LUAD. On CT, LUAD may appear as solid or subsolid nodules. Most subsolid nodules are not cancer, and many will remain stable or resolve; however, subsolid lesions can represent premalignant lesions or adenocarcinoma in situ. These lesions in the early spectrum of LUAD may persist for months to years before transforming into invasive disease. As a result, current standard of care is to follow these patients with CT imaging to monitor these indeterminate lesions for radiologic signs of malignant progression. The identification of novel biomarkers to predict the malignant potential of these nodules at their initial identification is of paramount importance. 1Scafoglio et al. Proc Natl Acad Sci U S A 2015;112(30):E4111-4119 2Scafoglio et al. Sci. Transl. Med. 10, eaat5933 (2018)

#4225

Is biomarker-driven precision medicine possible by using high dimensional augmented intelligence assisted analysis of cancer immune responses.

Carsten Krieg,1 Luis Cardenas,1 Silvia Guglietta,1 John Wrangle,1 Mark Rubinstein,1 Mark Robinson2. 1 _Medical University of South Carolina (MUSC), Charleston, SC;_ 2 _University Zurich, Zurich, Switzerland_.

Checkpoint inhibitors have significantly accelerated cancer treatment but still a majority of patients do not respond. Biomarker driven patient stratification early to the right immunotherapeutic might enhance response and patient survival. Here we used high-dimensional mass cytometry (CyTOF) combined with machine-learning bioinformatics for the in-depth characterization of immune responses before and during anti-PD-1 immunotherapy. CyTOF allows us to monitor protein expression of 34 markers on a single cell while running 20 samples simultaneously. The analysis is data driven, can be adapted to high throughput approaches and can model arbitrary trial designs such as batch effects and paired designs and is quantitative over millions of events. Using CyTOF as a precision medicine tool we could predict response to anti-PD-1 using liquid blood biopsies. Biobanked peripheral blood mononuclear cells (PBMCs) from 51 patients with stage IV melanoma before and after 12 weeks of anti-PD-1 therapy was analyzed. We observed a clear T cell response on therapy. The most evident difference in responders before therapy was an enhanced frequency of CD14+ CD16+HLA-DRhi classical monocytes. We validated our results using conventional flow and found a clear correlation of enhanced monocyte frequencies before therapy initiation with clinical response such as lower hazard and extended progression-free and overall survival. In a second study we used CyTOF to monitor immune response in 21 non small cell lung cancer (NSCLC) patients that initially responded and then progressed under anti-PD-1 to a novel combination immunotherapy of anti-PD-1 plus an IL-15 super-agonist (ALT-803). In this phase Ib clinical study a response in the CD8+ T cell compartment was observed. Unexpected our high dimensional unbiased analysis was able to detect and characterize a strong expansion of innate tumor-reactive effector NK cells starting around day 4 of therapy. Taken together, our unbiased artificial intelligence driven immune workflow might support patient selection prior to therapy, and serve as a novel tool for precision medicine to select the right drug combination and identify new drug-able cell populations.

#4226

TNFRSF14 is a cell surface marker of MITF expression in melanoma.

Ken Dutton-Regester, Nicholas K. Hayward. _QIMR Berghofer Medical Research Institute, Brisbane, Australia_.

Background: Transcriptional 'cell states' (defined as the genes expressed in a cell in any given point in time) can determine response to targeted and immune-based therapies in late-stage melanoma treatment. MITF and AXL are common markers of drug-sensitive and drug-resistant 'cell states', respectively; and extensive heterogeneity of both cell populations have been observed in melanoma cell lines and tumors. Understanding the mechanisms controlling these 'cell-states' may lead to therapeutic strategies to overcome resistance and improve patient survival.

Problem: To date, certain experiments assessing cell population heterogeneity dynamics in melanoma (such as live cell sorting applications) has been limited by the lack of surface markers defining the MITF 'cell-state'. Furthermore, existing antibodies against MITF commonly detect multiple expressed isoforms which can limit the accuracy of downstream experimental assays, such as in-cell western blot detection.

Methods and Results: To find a suitable cell surface marker defining the MITF 'cell state', we used the PARIS GenePattern module with data from the Cancer Cell Encyclopedia. TNFRSF14, our highest ranked gene, was highly correlated to MITF transcript expression in melanoma cell lines. We confirmed this at the protein level through western blot detection and flow cytometry. Using flow cytometry, we could accurately follow 'cell-state' population dynamics of AXL and MITF following shRNA knockdown of MITF and during acquired drug resistance in culture.

Outcomes: We have identified TNFRSF14 as a robust cell surface marker of MITF in melanoma. Our TNFRSF14 and AXL live cell flow protocol will be useful for those exploring 'cell-state' population dynamics and heterogeneity in melanoma and could contribute to new mechanistic insights of drug resistance.

#4227

**Salvage genetic testing on buccal swab samples using liquid** in situ **amplification identifies genetic mutations from previous test failures.**

Glen J. Weiss,1 Andrew Ford,2 Charmaine Brown,2 Elizabeth Caver,2 Matthew Vallejo,2 Claribel Adeyemi,2 Forrest Collins,2 Chen-Hsiung Yeh2. 1 _Beth Israel Deaconess Medical Center/Harvard School of Medicine, Cambridge, MA;_ 2 _Circulogene, Birmingham, AL_.

Background: NCCN guidelines version 2.2019 address BRCA1/2 testing for men with a personal history of prostate cancer limited to Gleason ≥7 and specific family history features. Inherited genetic mutations have been identified in up to 12% of metastatic prostate cancer, primarily in DNA repair genes. Here we report liquid in situ amplification (LISA) technique followed by hereditary testing on buccal swab samples that had previously failed to yield a genetic testing result.

Methods: Previously failed buccal swab samples from another CLIA-certified laboratory were re-prepared using LISA. Qubit quantification following LISA revealed goodDNA concentrations even for these poor-quality swab samples ranging from 5 to 70 ng/uL, which is sufficient for next-generation sequencing analysis (NGS). Coding regions of ATM, BRCA1, BRCA2, CHEK2, PALB2, and RAD51D genes were amplified by a panel of specific primer pools followed by massively parallel sequencing for the identification of germline mutations. Minimal sequencing coverage was set as 50X, and pathogenic, likely pathogenic, and variants of unknown significance (VUS) were classified and reported.

Results: Buccal swab samples from 29 men with at least intermediate-risk prostate cancer were received as low-yield, low-quality DNA (n=12), pre-cut swabs in preservation solution (n=6), and aged swab samples (n=11). LISA and subsequent NGS testing was able to salvage results for 24/29 (82.8%) (failed samples included 4 aged swab and 1 pre-cut swab). 1 patient had a pathogenic BRCA1 mutation (c.4309delT) and 3 patients had VUS (BRCA1 c.4776C>G, BRCA1 c.1846_1848delTCT, and RAD51D c.268G>A). The remaining 20/29 (69.0%) samples have either benign or likely benign variants.

Conclusions: LISA applied to these previously failed samples was able to salvage results for over 80% of cases, including the identification of a pathogenic BRCA1 mutation which led cascade hereditary testing for immediate family members. It also provided confirmation for nearly 70% of cases about the absence of hereditary risk for the genes on the panel.

#4228

Integrated drug high-throughput screening and quantitative phosphoproteomics rationalizes combination therapy for mutant KRAS pancreatic cancer.

Joseph Capri, Thuc Le, Caius Radu, Timothy Donahue. _UCLA, Los Angeles, CA_.

Pancreatic ductal adenocarcinoma (PDAC) is a highly recalcitrant malignancy for which better therapies are urgently needed. KRAS is the most frequently altered gene in PDAC and it orchestrates a number of effector pathways that ultimately drives PDAC growth and proliferation. The recent development of allele-specific inhibitors of oncogenic KRAS G12C, such as ARS-1620, provides an unprecedented opportunity to explore direct targeting of oncogenic KRAS signaling in PDAC. Nearly 3% PDAC patient harbor KRAS G12C, corresponding to ~1,600 new cases of PDAC/year in the US alone for which currently there are no robust targeted therapies. Even if the inhibitor effectively block KRAS function, compensatory mechanism will probably be triggered to overcome the KRAS-mutant PDAC.To better position the direct mutant KRAS inhibitor, we have developed a platform that includes: a high-throughput screen anchored with ARS-1620 to identify companion inhibitor to further dial down the oncogenic KRAS signaling, quantitative mass spectrometric method to map out the adaptive signaling mechanisms of this combination therapy, and bioinformatics pipeline to reveal and rank targetable kinases that drive the adaptive resistance mechanism. To do so, we will apply our platform on a panel of KRAS G12C primary PDAC models. We will profile the proteomic/phosphoproteomic changes of cells treated with combination therapy to identify adaptive signaling pathways that could be potentially targeted.High-throughput chemical-genomics screening identified receptor tyrosine kinases (RTKs) to be synergistic to the KRAS inhibitor, ARS-1620. In particular, multiple epidermal growth factor receptor (EGFR) inhibitors restrict growth of patient-derived PDAC, XWR200, when combined with ARS-1620. When profiling the proteomic/phosphoproteomics changes induced by the combination therapy, we quantified nearly 9600 protein groups and 44,000 phosphopeptides with 36,000 class 1 phosphopeptides having quantitation in all groups (FDR < 1%). We interrogated the Drug Signature Database (DSigDB) using the ~2,400 significantly-altered proteins (FDR < 0.1%) and found the protein gene set to be highly correlated to genes downregulated by topoisomerase inhibition. To identify adaptive signaling pathways, we performed kinase-substrate enrichment analysis (KSEA) on the phosphoproteomic dataset containing ~8,000 significantly altered phosphopeptides (FDR < 1%). KSEA identified hyperactivation of several G2/M checkpoint kinases such as Aurora kinases A and B. This integrative platform has revealed synergistic drug combinations with ARS-1620 and the phosphoproteomics has prescribed possibly the third drug of the combination therapy.

#4230

Real-world utilization of PD-L1 IHC testing and results across multiple tumor types.

Gabriel S. Krigsfeld,1 Emily Prince,1 Kim Zerba,1 Vladislav Chizhevsky,2 Josette William Ragheb,2 James White1. 1 _Bristol-Myers Squibb, Princeton, NJ;_ 2 _NeoGenomics Laboratories, Inc, Aliso Viejo, CA_.

Background: A number of programmed death ligand 1 (PD-L1) immunohistochemistry (IHC) diagnostic (Dx) tests have been approved by the FDA to guide treatment (Tx) with programmed death-1/PD-L1 inhibitors. Here, we evaluate the utilization of the Dako PD-L1 IHC 28-8 and 22C3 pharmDx and Ventana PD-L1 (SP142) assays on real-world samples across multiple tumor types, characterize PD-L1 testing practices among physicians who order a PD-L1 test, and investigate how PD-L1 test results impact physicians' Tx decision-making and use of immuno-oncology (I-O) Tx.

Methods: 55,652 samples with clinical characteristics were provided by Symphony Health Solutions. NeoGenomics Laboratories, Inc assessed PD-L1 expression between October 2015–April 2018, according to manufacturers' protocols at time of study. Clinical characteristics were matched to PD-L1 test results using unique identifiers for the 4714 patients (pts) whose diagnoses and treatment could be determined.

Results: Across tumor types, 56% of pts received a test prior to first-line (1L) Tx. Most lung cancer (57%) or melanoma (MEL; 65%) pts had a PD-L1 test prior to 1L Tx, while most squamous cell carcinoma of the head and neck (70%) or urothelial carcinoma (73%) pts had a PD-L1 test after Tx initiation, in line with drug and complementary Dx approvals for these tumor types during the testing period. The percentage of lung cancer pts whose PD-L1 expression was tested prior to 1L Tx rose from 32% during Q4 2015–Q3 2016 to 63% during Q4 2016–Q1 2018, in line with FDA approval of a companion PD-L1 IHC Dx for non-small cell lung cancer. Regardless of PD-L1 test used, most lung cancer (73%) or MEL (93%) pts received I-O Tx, defined as nivolumab (NIVO; + ipilimumab [IPI] for MEL only), pembrolizumab (pembro) ± chemotherapy, atezolizumab, or IPI alone. Moreover, the majority of lung cancer pts who had ≥1% PD-L1 expression or MEL regardless of PD-L1 expression received 1L I-O Tx irrespective of the PD-L1 test used. Analysis of PD-L1 expression, Dx test used, and Tx received showed that, for MEL pts tested with 22C3, 28%, 17%, and 0% of pts received NIVO or IPI, while 28%, 43%, and 43% received NIVO+IPI, at the expression cutoffs of 0%, 1–49%, and ≥50%, respectively. Lung cancer pts with 1–49% PD-L1 expression, as determined by a 22C3 test, received NIVO or pembro monotherapy at similar rates (12–18%). Of those who had ≥50% PD-L1 expression, as determined by the 28-8 test only, 52% received pembro monotherapy vs other Tx.

Conclusions: Since FDA approval of the first PD-L1 IHC Dx in 2015, analyses of PD-L1 testing practices and physicians' Tx decision behavior at a US national reference laboratory have shown that physicians' adoption of PD-L1 testing is responsive to FDA approvals and that most testing for lung cancer and MEL occurs before 1L Tx initiation. In lung cancer or MEL pts who had a PD-L1 test result available, Tx decisions do not appear to be tied to the specific intended use of the PD-L1 assay that was ordered.

#4231

Association between age of first full-term pregnancy and subsequent breast cancer risk.

Neha Tabassum. _Imperial College London, London, United Kingdom_.

Introduction

Amongst the various risk factors for breast cancer (BC), age at first full-term pregnancy is still a complex problem. Early first full-term pregnancy (before 25 years of age) is believed to confer protection towards the development of BC after menopause compared to the risk in nulliparous women but is also correlated with a transient increase in the risk of developing BC soon after pregnancy. On the other hand, a late first pregnancy (after 35 years of age) is believed to increase the risk of developing breast cancer after menopause, equating or increasing the risk compared to nulliparous women.

Material and Methods

In our project, currently, we aim to do a pilot study with 4 fresh frozen normal samples of nulliparous and early-and late-parous women from Komen tissue bank (USA) and to create a mathematical model to determine how rates of both drivers and passengers mutations are affected by pregnancy and how the risk can be determined in parous and nulliparous women. We plan to measure low-frequency mutations from fresh frozen normal breast samples from patients with different reproductive stages using both Whole Genome Sequencing in this pilot group,and later on to expand the findings with Exome Sequencing on the remaining cohort of 55 normal samples.

Results and Discussions

We are currently in the process of analysing the data from the whole genome sequencing and whilst we do this, we are also collecting DNA from laser-capture microdissected epithelial and stromal cells, for the remaining samples. Bioinformatics tools are used to eliminate germline mutations from the stroma and to determine where the mutations are located within the genome, and how their frequency changes within the sample cohort.

Conclusion

While this study is aimed to understand the scientific basis of breast cancer and therefore it is not clinically applicable in the immediate future, understanding the cancer risk associated with age of pregnancy will provide future patients, in particular from at-risk categories, with an informed choice when deciding to plan a pregnancy and a surveillance programme could also be implemented to closely monitor any changes in the breast after a late pregnancy and improve early detection. Furthermore, we could hypothesise that in the long-term future, with the wider application of mammary ductoscopy, breast samples could be analysed in the clinic to calculate the mutation rate of the patient and identify individual risk. This risk stratification along with available predictors such as the 'Gail model' could be applied in breast cancer surveillance programmes.

## BIOINFORMATICS AND SYSTEMS BIOLOGY

### Applications of Cancer Computational Biology 2

#4232

Spatially constrained tumor growth affects the patterns of clonal selection and neutral drift in cancer genomic data.

Kate Chkhaidze,1 Timon Heide,1 Benjamin Werner,1 Marc J. Williams,2 Weini Huang,2 Giulio Caravagna,1 Ann-Marie Baker,2 Trevor A. Graham,2 Andrea Sottoriva1. 1 _Institute of Cancer Research, London, United Kingdom;_ 2 _Barts Cancer Institute, Queen Mary University, London, United Kingdom_.

Quantification of the effect of spatial tumor sampling on the patterns of mutations detected in next-generation sequencing data is largely lacking. Here we use a spatial stochastic cellular automaton model of tumor growth that accounts for somatic mutations, selection, drift and spatial constrains, to simulate multi-region sequencing data derived from spatial sampling of a neoplasm. We show that the spatial structure of a solid cancer has a major impact on the detection of clonal selection and genetic drift from bulk sequencing data and single-cell sequencing data. Our results indicate that spatial constrains can introduce significant sampling biases when performing multi- region bulk sampling and that such bias becomes a major confounding factor for the measurement of the evolutionary dynamics of human tumors. We present a statistical inference framework that takes into account the spatial effects of a growing tumor and allows inferring the evolutionary dynamics from patient genomic data. Our analysis shows that measuring cancer evolution using next-generation sequencing while accounting for the numerous confounding factors requires a mechanistic model-based approach that captures the sources of noise in the data.

#4233

Improving the diagnostic yield of a 124 gene pediatric solid tumor panel through somatic copy number variation analysis.

Raghu Chandramohan, Jacquelyn Reuther, Ilavarasi Gandhi, Horatiu Voicu, Karla R. Alvarez, Kevin E. Fisher, Dolores H. Lopez-Terrada, Donald W. Parsons, Angshumoy Roy. _Baylor College of Medicine, Houston, TX_.

In cancer genomes, both large-scale CNVs (>100 kb) spanning chromosome arms, and smaller CNVs limited to a few genes are prevalent. While large CNVs are readily detected with existing methodologies (e.g. SNP array), gene-level CNVs require a higher resolution not routinely available in current tools, including next generation sequencing (NGS)-based clinical cancer mutation panels. We sought to leverage NGS data generated by one of these panels: the TCH Pediatric Solid Tumor panel (124 cancer genes), and built a clinical-grade analytic pipeline for detection of somatic CNVs.

After reviewing literature, CNVkit was selected for its ability to perform unmatched (requiring no matched normal specimen) CNV analysis, customize segmentation algorithms, and provide superior visualization. CNVkit performs circular binary segmentation on the log2 difference of binned read depths (on- and off-target) from tumor and pooled-normal blood samples to identify CNVs. Although the panel captures only 124 genes (~1Mbp) at > 300X coverage, the low background coverage of 0.05X (off-target) due to imperfect hybridization capture allows us to detect chromosome-arm level changes. As a pilot study, we optimized CNVkit to detect gene-level and chromosome arm-level CNVs in a reference aneuploid colon cancer cell line (HT-29) characterized by aCGH and observed high correlation (r = 0.89) between the aCGH and CNVkit fold change. Through in-silico and bench experiments we performed limit of detection analysis, by diluting different proportions of CNV positive tumor samples with normal sample, to set thresholds for calling amplification and losses. We show the ability of the pipeline to detect shallow and deep deletions if they are present in at least 40% and 80% of the sample sequenced, respectively. During validation, we used 24 pediatric cancer samples with diverse histology to confirm and detect numerous CNVs of clinical significance, including deletions of SMARCB1, PTEN, BRCA2, RB1, and ARID1B, and amplifications of MYCN, MYC, CCND1 and KRAS.

As the panel densely tiles tumor suppressors, we are also able to infer apparent intragenic deletions in BRCA1, BRCA2, ATRX, RB1 and PTEN, highlighting the theoretical resolution of this tool for detecting intragenic events over other methods. We have also developed a Python based interactive CNV Viewer for assessing the copy number analysis results from NGS data. The browser like interface allows the user to zoom, link out to UCSC Genome Browser, hover for information, take high-quality screenshots, etc. Hence, the CNV pipeline we have developed will allow more comprehensive evaluation of the existing integrated DNA and RNA analysis pipeline, increasing the diagnostic yield of mutation panel testing for childhood cancer patients.

#4234

Computational analysis of immune-associated genomic and transcriptomic elements differentiating papillary thyroid cancer subtypes.

Jaideep Chakladar, Megan Chu, Aditi Gnanasekar, Kristen F. Rosenberg, Joseph C. Tsai, Lindsay M. Wong, Weg M. Ongkeko. _UCSD, La Jolla, CA_.

Approximately 600,000 patients live with papillary thyroid cancer (PTC) in the United States, with its incidence rising from 4.8 to 14.9 cases per 100,000 people from 1975 to 2012. Despite being the fastest growing cancer, the therapeutic options for PTC has remained unchanged and are primarily limited to surgery, radioactive iodine, and chemotherapy. Considering the untapped potential of immunotherapy for thyroid cancer, this study explores the genomic and transcriptomic features of PTC subtypes in order to identify dysregulated immune-associated (IA) genes and IA pathways that may serve as therapeutic targets. Using data from The Cancer Genome Atlas (TCGA), the three most common histological subtypes of PTC, including the classical (CPTC), the follicular variant (FVPTC), and the tall cell (TCPTC), were analyzed computationally and compared to normal tissue to identify differentially expressed IA genes. These dysregulated IA genes were further filtered for prognostic significance using MACIS scores. To investigate the molecular mechanism for their dysregulation and explore the possibility of genome-based patient stratification, we profiled the correlation of genomic alterations, including mutation and copy number variation, in the various PTC subtypes to IA gene expression using the information theory-based algorithm REVEALER. Our analyses revealed that the expression profiles of CPTC and TCPTC were the most similar from an immunological perspective, with 360 IA genes similarly dysregulated in both of those subtypes. Additionally, TCPTC had the most unique immunological profile, with 300 IA genes dysregulated exclusively in TCPTC, compared to 107 in FVPTC and 22 in CPTC. Specifically, gene such as RAET1E, FGFR11, and MUC1, which are associated with innate and adaptive immune responses, cell survival and proliferation, and TP53-transcription respectively, were dysregulated only in TCPTC. We also studied gene expression regulators and determined that microRNAs had a limited role in the gene dysregulation we observed. However, the regulatory pathways and mutational features unique to certain subtypes were indicative of pathways differentiating cancer prognosis between PTC subtypes. Most notably, genes in the glycosylation pathway mediated by MUC1 and B3GNT3 are upregulated in TCPTC but are downregulated in the less aggressive FVPTC and CPTC subtypes, suggesting a possible mechanism for the aggressive progression of TCPTC. In summary, our findings indicate that subtypes of PTC have both common and unique dysregulated IA genes and pathways, which may be exploited to develop novel immunotherapy targets or biomarkers predictive of prognosis for the different subtypes.

#4235

Phosphorylation of Serine 861 in the ECT2 guanine nucleotide exchange factor activates Rho family GTPases and is a biomarker for several tumor types.

Durui Wang, Xiaolan Qian, Sera Eng, Shivani Nellor, Alex G. Papageorge, Douglas R. Lowy. _NCI-NIH, Bethesda, MD_.

The Rho family of GTPases, which cycle between inactive GDP-bound and active GTP-bound states, have high activity in many cancers. One mechanism for their activation is by guanine nucleotide exchange factors (GEFs), which stimulate the replacement of GDP-bound Rho with GTP-bound Rho. In this study, we report phosphorylation of Epithelial Cell Transforming 2 (ECT2), which is a Rho family-specific GEF, is a biomarker in several tumor types, based on bioinformatic analysis and biological assays. Our analysis of the NCI Clinical Proteomic Tumor Analysis Consortium (CPTAC) database found that Serine 681 (S681) in ECT2 is more highly phosphorylated in several tumor types compared to its phosphorylation in the relevant normal tissue. S681 phosphorylation is 2.1-fold and 2.4-fold higher in colon cancer and ovarian cancer, respectively, and high S861 phosphorylation in breast cancer is correlated with a poorer prognosis. Phosphorylation of S681 is especially high in triple-negative breast cancer, as it is 2.3-fold higher in this type compared with the other types of breast cancer. Increased phosphorylation of S861 is associated increased ECT2 mRNA and protein levels in colon cancer and breast cancer. In experimental studies, siRNA knockdown of endogenous ECT2 in two triple-negative breast cancer lines resulted in lower levels of RhoA-GTP, Rac-GTP, and Cdc42-GTP, and led to reduced cell migration, cell proliferation, and anchorage-independent growth, suggesting that ECT2 is a key regulator of cellular signaling and oncogenic growth in the triple-negative lines. Conversely, ectopic expression of wild-type ECT2 induced increased levels of RhoA-GTP, Rac-GTP, and Cdc42-GTP and promoted migration, proliferation, and anchorage-independent growth. Compared with wild type ECT2, ectopic expression of ECT2 carrying a phospho-defective (S861A) or phospho-mimic (S861D) mutation, respectively, reduced or enhanced ECT2 GEF and biological activities. Our results indicate that phosphorylation of S861 enhances the pro-oncogenic activity of ECT2, which is correlated with the increased phosphorylation of S861 in the three cancer types, the increased expression of ECT2 in two of these cancer types, and the prognostic significance of S861 phosphorylation in breast cancer. * Dunrui Wang and Xiaolan Qian are Equal contributors

#4236

Evaluating alterations in mRNA biogenesis with TCGA breast cancer sequencing data.

Thomas J. Yan,1 Matthew E. Burow,1 Elizabeth C. Martin2. 1 _Tulane University School of Medicine, New Orleans, LA;_ 2 _Louisiana State University, Baton Rouge, LA_.

Alternative splicing leads to the production of multiple mRNA isoforms of a gene transcript with distinct functional characteristics. These events are frequently observed in rapidly dividing cancer cells and are broadly recognized as important signatures for oncogenesis pathways and treatment options. In this study, we utilized data from The Cancer Genome Atlas (TCGA) to identify differences in isoform expression between estrogen receptor-positive and estrogen receptor-negative breast cancer cell lines. RNA sequencing analysis combined with clinical annotation data revealed alterations in isoform expression that were associated with patient survival. These global changes in isoform expression occurred independently of total gene expression in both estrogen receptor-positive and -negative breast cancer. Furthermore, we identified multiple underrepresented isoforms including CDKAL1 and CSTF3 that were associated with favorable prognostic outcomes despite a lack of association observed with their traditional transcripts. These data suggest an underlying role mediated by alternative spliced isoforms in driving clinical outcome in breast cancer, warranting further investigation in delineating isoform expression profiles in previously established cancer pathways.

#4237

CC chemokines are differentially expressed in breast cancer and are associated with racial disparity.

Jeronay K. Thomas, Hina Mir, Neeraj Kapur, Shailesh Singh. _Morehouse School of Medicine, Atlanta, GA_.

Despite recent advances in diagnosis and treatment Breast cancer (BrCa) still impacts women's lives and this impact is disproportional in African Americans (AA) compared to European Americans (EA). Addressing socioeconomic and behavioral status has not reduced the race-specific gap in BrCa outcome, suggesting contribution of biological differences in BrCa disparity. We have previously shown involvement of chemokines in the pathogenesis of BrCa. In this study, we have determined the expression of CC chemokines in BrCa tissues and normal tissues utilizing the ONCOMINE database. Further, we determined clinical parameters associated with the CC chemokines overexpressed in BrCa tissues using The Cancer Genome Atlas (TCGA). Additionally, using Bc-GenExMiner and KMplotter we determined the effects of CC chemokines on reoccurrence, overall and relapse-free survival. Our analysis shows overexpression of CCL5, CCL7, CCL11, CCL17, CCL20, CCL22 and CCL25 mRNA in BrCa tissues compared to normal. Overexpression of CCL21 and CCL22 were significantly correlated with metastatic recurrence. High mRNA levels of CCL7, CCL8, CCL17, CCL20 and CCL25 predicted a decrease in overall survival and CCL7 and CCL8 expression showed a trend in decreased relapse-free survival. Furthermore, overexpression of CCL17 and CCL25 was associated with decreased overall survival in AA whereas in EA, CCL8 was associated with a decrease in overall survival. Interestingly, expression of CCL5, CCL8, CCL17, CCL20 and CCL25 were highest in TNBC tissues. Expression of CCL11 and CCL22 were associated with HER2. In addition to these, there was an association with CC chemokines expression with age in BrCa. Higher CCL11 expression was noted in younger women and CCL8, CCL20, CCL22 and CCL25 expression was higher in older women. Furthermore, CCL25 was significantly elevated and CCL8 was nearly significant in AA tissues when compared to EA. Expression of CCL7, CCL8, and CCL25 were associated with stage. In conclusion, our analysis suggests an association of CC chemokines in BrCa progression, overall survival and observed disparity in disease outcome between AA and EA patients.

#4238

DNA-methylation based epigenetic signatures predict and deconvolute somatic genomic alterations in gliomas.

Jie Yang,1 Qianghu Wang,2 Ravesanker Ezhilarasan,1 Lihong Long,1 Benedikt Wiestler,3 Wolfgang Wick,4 Yinsen Miao,5 Erik Sulman6. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _Nanjing Medical University, Nanjing, China;_ 3 _Technical University of Munich, Germany;_ 4 _University of Heidelberg, Germany;_ 5 _Rice University, Houston, TX;_ 6 _NYU Langone School of Medicine, New York, NY_.

PURPOSE: Genomic alterations classify cancers into subtypes with distinct clinical management and prognoses. Molecular classification of gliomas, the most common and lethal primary brain tumor, define tumors into distinct biologic and clinical entities. Mutation of isocitrate dehydrogenese (IDH) is associated with hypermethylation of CpG sites in gene promotors. Other alterations, including telomerase (TERTp) mutation, ATRX mutation, chromosome 1p/19q co-deletion (1p19q codel), and gene expression subtype (Classical/CL, Mesenchymal/MES, Proneural/PN), have yet to be associated with an epigenetic signature. We hypothesized that DNA methylation signatures would classify gliomas based on genetic alterations and give insight into the development of each subtype. The resulting platform functions as a unified diagnostic (UniD) with high processivity applicable to clinical diagnosis and generalizable across molecular oncology.

METHODS: Machine learning algorithms were applied to whole methylome data to build classifiers for IDH, TERTp, and ATRX mutations; 1p19q codel and gene expression subtype. Models were validated with data from a phase III trial of anaplastic gliomas.

RESULTS: Individual models were generated and prediction accuracies for IDH, TERTp, and ATRX mutations, and 1p19q codel were 100%, 98.3%, 90.48%, and 99.21% in test set and 89.9%, 82.8%, 92.47%, and 89.99% in the validation clinical trial data. The prediction model for gene expression subtype, a previously reported classifier enriched for characteristic somatic alterations, achieved 72% accuracy. Analysis of the misclassified cases revealed that the characteristic alterations associated with the expression subtypes were more correctly classified by methylation than by gene expression. Methylation-determined CL subtype showed high EGFR (p-value = 0.04) and amplification (p-value = 0.00019) compared to transcriptional MES samples, and low expression (p-value = 0.08) and amplification (p-value = 0.056) in methylation-determined MES but transcriptional CL samples.

CONCLUSION: Distinct DNA methylation signatures were associated with key somatic genomic alterations in gliomas. It improved the existing classifiers based on gene expression and provided a unique clinical diagnostic platform for rapid determination of glioma subtype at the time of patient diagnosis. The extensive and significant relationship between cancer epigenetic signatures indicates that this approach would have broad applicability to other tumor types and lead to similar unified diagnostic platforms. A R package (UniD) is provided for implantation of this diagnostic platform.

#4239

Development of pan-cancer transcriptional signatures that predict chemosensitivity.

Jason D. Wells, Todd W. Miller. _Dartmouth College, Lebanon, NH_.

Background: Despite increasing understanding of the molecular characteristics of cancer, chemotherapy success rates remain low for many cancer types. Studies have attempted to identify patient and tumor characteristics that predict sensitivity or resistance to different types of conventional chemotherapies, yet a concise model that predicts chemosensitivity based on gene expression signatures across cancer types remains to be formulated. We attempted to generate a pan-cancer chemosensitivity predictive model using publicly available data from multiple sources. Such a model may increase the likelihood of identifying the type of chemotherapy most likely to succeed for a given patient based on the gene expression signature of their tumor.

Methods: Data used to build predictive models were obtained from the Genomics of Drug Sensitivity in Cancer (GDSC) database, consisting of gene expression profiles from 962 cancer cell lines via RNA sequencing (RNA-seq) and drug sensitivity profiles reported as ln(IC50). Predictive gene signatures were generated using a cross-validated generalized linear model using elastic net penalization parameters. Models were generated for individual chemotherapy drugs, and accuracy of the models was determined by multi-class area under the curve (AUC). Models were then validated using publicly available data from Cancer Cell Line Encyclopedia (CCLE) and NCI-60 as well as publicly available human tumor datasets such as The Cancer Genome Atlas (TCGA). As the training data used to generate the models were from RNA-seq and some of the validation data were generated using microarray technology, feature-specific quantile normalization was used to enable cross-platform analyses.

Results: We developed pan-cancer models to predict chemosensitivity to 8 individual chemotherapy drugs from GDSC data. For most models, multi-class AUC ranged from 0.63 to 0.68 when considering all available cancer types. Other models, specifically for etoposide and methotrexate, showed poor performance overall (AUC 0.54-0.55) but performed well when predicting chemosensitivity for bone cancers (AUC 0.70-0.73), which proved difficult with models for other drugs. When considering how well the models predicted chemosensitivity within cancer types, accuracy was improved in some cancer types (e.g., thyroid cancer), with more heterogeneous cancer types (e.g., breast cancer) giving less accuracy.

Conclusions: Our results show that these models can predict chemosensitivity across cancer types with clinically useful levels of accuracy, with some cancer types resulting in a high rate of accuracy across several classes of chemotherapy. The inclusion of future clinical datasets, particularly from those cancer types in which chemosensitivity has been difficult to predict, may provide opportunities to strengthen model accuracy as well as decrease the numbers of genes needed to assess chemosensitivity.

#4240

Rare candidate variants shared among affected family members in the African American Hereditary Prostate Cancer Study families.

Deyana D. Lewis,1 Shukmei Wong,2 Angela S. Baker,3 Isaac Powell,4 John D. Carpten,3 Joan E. Bailey-Wilson,1 Cheryl Cropp5. 1 _National Human Genome Research Institute, Baltimore, MD;_ 2 _Translational Genomics Research Institute, Phoenix, AZ;_ 3 _University of Southern California, Los Angeles, CA;_ 4 _Wayne State University, Detroit, MI;_ 5 _Samford University, Birmingham, AL_.

Purpose: Prostate cancer is the most common cancer in males, but also exhibits a ∼1.5-2-fold higher incidence in African American men compared with whites. Epidemiologic evidence supports a large heritable contribution to prostate cancer, with over 100 susceptibility loci identified to date that can explain ∼33 % of the familial risk. A portion of the undefined risk may be due to rare susceptibility variants. The African American Hereditary Prostate Cancer (AAHPC) Study, established in 1997, enrolled 77 AA families from seven clinical sites across the United States (Ann Epidemiol, 2000). The aim of this study is to identify rare, predictive, deleterious variants through exome sequencing of 99 cases from 26 families selected from the AAHPC families (2 to 3 affected men sequenced per family) and three female 1000 Genome controls.

Methods: To explore the contribution of rare variation in coding regions of 38 known cancer-causing genes to prostate cancer risk (PCa), we sequenced the exomes of 99 AAHPC cases at a mean coverage of 30x. Post-variant calling quality control (QC) was implemented using Golden Helix SVS 8 software with filters set for removal of variants with Read Depth >10, Quality Score >10, and Quality Score: Read Depth Ratio > 0.5. Mendelian inconsistency was checked using PLINK. Prioritization of all candidate genes/variants were evaluated using online databases including 1000 Genomes and ANNOVAR for non-reference allele frequency and predictions of functional impact.

Conclusions. Through exome sequencing of 99 AAHPC cases and 3 female 1000 Genome controls, we identified 37 non-synonymous single nucleotide variants that are considered damaging by at least one predictive scoring tool in our 38 candidate genes. However, most of these variants were common and thus unlikely to be causal. We observed three rare variants that were considered damaging by three predictive scoring tools in two known PCa genes, PCNT and ZFHX3. These 3 variants were each observed in a single different family (in 1 of 3 affected individuals). Interesting rare candidate variants (MAF ≤ 0.01) rs190517526 & rs114692729 were found in known cancer susceptibility loci (CD82 and EZH2). The CD82 variant was observed in all 3 sequenced affecteds in one family and the EZH2 variant was observed in 2 of 3 sequenced affecteds in a different family. These predicted damaging variants in these four genes represent potentially novel causal candidates for some of the 26 AAHPC families sequenced here. Future work will carefully analyze the remainder of the genome in the other 21 sequenced families including planned sequencing of additional family members.

#4241

Molecular profile of tumor-associated adipose in obese women with breast cancer.

Niel Infante,1 James Coad,2 Donald Primerano,3 James Denvir,4 Linda Vona-Davis2. 1 _West Virginia University School of Public Health, Morgantown, WV;_ 2 _West Virginia University Cancer Institute, Morgantown, WV;_ 3 _Marshall University School of Medicine, Huntington, WV;_ 4 _Marshall University School of Medicine, Morgantown, WV_.

Breast tumors have a direct physical and vascular interface with white adipose tissue, making them ideal model systems to study the adipose-tumor microenvironment, especially within the context of obesity. As a first initiative to understanding how adipose tissue contributes to breast tumor etiology, we explored cancer-associated adipose tissue in patients with invasive breast cancer. We hypothesized that obesity leads to greater alterations in cancer-associated adipose tissue by expressing genes involved in cellular growth, angiogenesis, and invasion, thus providing support for breast cancer progression. We also proposed that adipocytes adjacent to tumors express a genotype unique to obese breast cancers. Adipose tissue samples (n=10) were obtained from obese and lean women with breast cancer. Within individuals, tissue was sampled proximal and distal (2 cm) to the tumor. Using next-generation whole transcriptome sequencing, we examined genes from cancer-associated adipose tissue in both groups. A heat map of normalized expression values was generated with unsupervised clustering and the fifty most informative genes. Samples from obese individuals with breast cancer tended to cluster together. There were significant gene differences between adipose collected proximal versus distal from the tumor in obese but not in lean women, such as genes associated with myosin heavy chains MYH1, 4, 8, and 11 (adj. p < 0.05). In examining obese women, there were 16 Hallmark pathways that were statistically significant (adj. p < 0.10); whereas a subset of these (11 pathways) were significant in lean women. Of interest, pathways that were significant in both were pathways that regulate EMT, adipogenesis and TGF beta signaling. The molecular signature of cancer-associated adipocytes is different between lean and obese patients. (Supported by NIH P20GM103434 and NIGMS U54GM104942)

#4242

Systematic transcriptome analysis of small cell carcinoma of esophagus suggests a possibility for immunotherapy.

Qi Zhao, Yan-xing Chen, Ying-nan Wang, Qi-nian Wu, Hui Sheng, JIa-jia Hu, Yun-xin Lu, Ze-xian Liu, Zhi-xiang Zuo, Feng Wang, Rui-hua Xu. _Sun Yat-sen University, Guangzhou, China_.

Background/Aims: Small cell carcinoma of the esophagus (SCCE) is a rare and highly deadly malignancy with rapid disease progress and extensive metastasis, whereas present therapy for the cancer is limited, making the identification of a new treatment strategy an area of intense research. We previous reported the genomic characteristics and alterations of the SCCE, but the transcriptome abnormality of the cancer has not been investigated yet.

Method: we performed an RNA Sequencing on nine paired samples from patients with SCCE and conducted a series of comprehensive analysis on the data. DESeq2 and GSEA were applied on transcriptome of SCCE tumor tissue and their para-normal to identify differential expressive genes and associated pathways. Besides, with MCP-counter and a list of immunity related genes, we also compared immune signal of the expression data in pair. By applying a rigorous batch removing strategy, including preprocessing raw data in identical method and RUVSeq, transcriptome data of healthy esophageal mucosa from GTEx was involved as control samples in comparison. In addition, we applied CIBERSORT to calculate the fraction of different tumor infiltrated leukocytes (TILs) of SCCE and compare the number with those of other cancers that have been proven to be potentially responsive to immunotherapy.

Results: We identified 1486 and 1782 genes were significantly up-regulated and down-regulated in SCCE tumor tissue against their para-normal, respectively. Pathway analysis revealed the E2F Target and G2M checkpoint are significantly enriched in tumor tissue. Immune signal analysis across the samples in pair showed that in 3 out of 9 paired sample, immunity reaction upregulates in tumor tissue. T-SNE cluster across transcriptome data of SCCE tumor tissue, their para-normal and healthy esophageal mucosa showed the para-normal tissue, namely the normal tissue adjacent to tumor, is more similar to tumor tissue than the healthy. CIBERSORT analysis showed the difference in TILs fraction between SCCE and the compared cancers. For example, the relative fraction of plasma cell of SCCE is higher than those of ESCC(P=0.040), CIN type of STAD(P=0.040), while lower than those of SCLC(P=0.020). M2 macrophages is higher in SCCE than ESCC(P=0.002), EAC(P<0.001), CIN type of STAD(P<0.001), HNSCC(P<0.001), while Tregs of SCCE is lower than EAC(P=0.012) and CIN type of STAD(P=0.015). The result reflects the robust but suppressed immunity in tumor microenvironment of the cancer. Cluster with all TILs across SCCE and the compared cancers also showed the similarity of immuno-microenvironment between SCCE and small cell lung cancer.

Conclusion: In summary, our analysis provides transcriptome evidence that immunotherapy might be an efficient treatment strategy for small cell esophagus cancer.

#4243

Genetic variants frequently detected in Egyptian breast cancer tumors: Comprehensive cancer panel by ion torrent DNA sequencing technology.

Abdel-Rahman N. Zekri,1 Osman Mansour,1 Samah A. Loutfy,1 Mohamed M. Hafez,1 M. Gomaa,1 Abeer Bahnassy,1 Mai M. Lotfy,1 Amira S. Youssef,1 Ola S. Ahmed,1 Mohammed Abouelhouda,2 Auhood Nassar1. 1 _National Cancer Inst. Cairo Univ., Cairo, Egypt;_ 2 _Cairo University, Cairo, Egypt_.

Breast cancer is the leading cause of cancer-related death in women worldwide. In Egypt, it is the most common cancer among females and its incidence is progressively increasing with a great tendency to occur with advanced stages in younger ages. Due to the heterogeneity of breast cancer, it was classified into different subtypes, each exhibits a unique gene mutation profile, based on biological characteristics and on gene expression pattern. Therefore, further research is needed to investigate the unknown genetic mutations involved in the progression of that disease. This study aimed to sequence 409 exons of tumor suppressor genes and oncogenes to identify the frequency of the detected genetic mutations in breast cancer using Ion Comprehensive Cancer Panel. Forty-eight tissue samples of various breast cancer subtypes were collected from National Cancer Institute (NCI) outpatient clinic, Cairo University. Analysis revealed 191 exonic and splicing variants. In this paper, we will address the most frequently detected Egyptian genetic variants (in 31.25 % of cases or more) as well as other deleterious variants commonly associated with breast cancer. Most of the detected genetic variants were checked in 1000g, dbSNP and Exac All databases. Other variants were found at known hotspot sites. We reported fifty-one somatic and germline mutations in thirty-two genes; AKAP9, BUB1B, RPS6KA2, AURKB, FANCA, RNF213, FGFR4, KAT6B, NLRP1, KAT6A, PER1, ERBB4, IL6ST, PIK3CA, P53, AURKA, WRN, PALB2, PTEN, GATA3, AKT1, ERBB2 and KRAS. Only KAT6B incurred non-frameshift deletion and only GATA3 had frameshift insertion while KAT6A, ERBB4 and PTEN had frameshift deletion. All the identified variants were detected with different frequencies in each breast cancer subtype. Each sample harbored at least four mutations and the maximum number of mutations per sample was twelve. The current data showed that gene panels analyzed

by Next-Generation Sequencing (NGS) identifies large number of germline and somatic mutations that is crucial for understanding cancer predisposition and developing personalized or combination therapies that efficiently target the individual breast cancer-specific mutations.

Key words: Breast cancer, Somatic mutations, Germline mutations, Ion torrent sequencing, Targeted sequencing.

#4244

Novel expressed long non-coding RNAs in uveal melanoma.

Daniel A. Rodriguez, Jeffim N. Kuznetsov, Margaret I. Sanchez, Stefan Kurtenbach, J. William Harbour. _Bascom Palmer Eye Institute, Sylvester Comprehensive Cancer Center, and Interdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, Miami, FL_.

Uveal melanoma (UM) is a highly aggressive eye cancer that leads to metastatic death in up to half of patients. UMs can be divided into two prognostic groups based on their gene expression profile (GEP). The class 1 GEP is associated with low metastatic risk and the class 2 GEP with high metastatic risk. Class 1 tumors are associated with EIF1AX or SF3B1 mutations while Class 2 tumors are associated with inactivating mutations in the tumor suppressor BAP1. Class 1 and Class 2 UMs have been shown to differ in their expression of numerous known micro-RNAs and long non-coding RNAs (lncRNA). Here, we sought to identify novel differentially expressed lncRNAs using a publicly available RNA-Seq database. Raw RNA-Seq fastq files from 80 TCGA UM samples were obtained from the Cancer Genomics Hub (CGHub), quality controlled using FastQC (v0.11.3), and trimmed using trim-galor (v0.4.1). Sequences were aligned to the human genome (GRCh38) and accompanying general transfer format file (gtf) (Gencode v28) using STAR (v2.5). Transcript discovery was performed using Cufflinks (v2.2.1). Protein coding probability was calculated using CPC (v2.0), and transcripts predicted to be non-coding with transcript length >200bps were retained. 1671 novel transcripts were added to the gtf file, and fastq files were realigned with the new annotation. Estimated counts for all known and novel transcripts were generated using RSEM (v1.3.0) after STAR alignment. A cutoff of RPKM > 1 in at least 35% of tumors was used as a threshold for transcripts of interest, resulting in 61 novel transcripts, 32 of which were differentially expressed at FDR < 0.05 between Class 1 and Class 2 tumors, including 7 upregulated and 25 downregulated in Class 2 tumors. Further work is underway to elucidate the function of these novel transcripts in UM pathogenesis.

#4245

**Drug response metrics and pharmacological profiling using the OncoPanel** ™ **cell-based profiling service.**

Charles R. Wageman, Lee R. Cavedine, Vanessa Norman, Natiya Robinson, Tracy Lu, Victoria McBain, Joseph Murphy, Kayla Stehle, Steven M. Garner, Alyssa M. Croff, Brogan A. Epkins, Kristin Dempsey, Alastair J. King, Jesse J. Parry. _Eurofins Panlabs, Inc., St. Charles, MO_.

High-throughput, cell-based drug screening platforms are essential to guiding drug discovery programs and can rapidly generate large amounts of pharmacological information about a candidate drug. Drug response data combined with genomic and phenotypic information enables an exploratory analysis that may reveal potential associative biomarkers and deepen the pharmacological profile of a drug. The choice of metric is an important consideration as different metrics will reflect different underlying biological mechanisms. Normalized growth rate inhibition (GR) metrics offer an alternative summary of drug response, and may eliminate bias associated with experimental factors that contribute to variation in growth rate. In these sets of experiments, we used drug response data, generated with the Eurofins OncoPanel cell-based profiling service, to investigate a variety of standard of care compounds against a cell line panel selected for variation in growth rate, cancer indication, cancer hallmark mutational status, and baseline apoptosis rate. Seeding density and incubation times were systematically varied to investigate the relationship between end-point confluence, kinetics, genotype, apoptotic priming, and the robustness of different dose response metrics. Based on these results, select cell lines and standard of care compounds were included in a series of drug-drug combination studies to further investigate the biological underpinning of synergy, at the same time exploring how alternative metrics influence pharmacological profiling experiments. Compounds were added to cells in 10 half-log dilutions, in triplicate, and allowed to incubate for 1, 3, 5, or 10 days. Initial cell density conditions were varied to allow for a final targeted confluence of 30, 80, or 100%. A time-zero plate was fixed at compound addition to allow for GR calculations. Following incubation, the cells were fixed, stained with DAPI, and imaged with a high-content imaging system. Cell proliferation dose response curves were fitted using a 4-parameter log-logistic model with custom curve-fitting software. These data were re-analyzed using the GRmetrics package in R to determine the GR metrics. Summary parameters, including IC50, GR50, and GRmax, were compared in subsequent analyses. For the combination analyses, a 9 x 9 concentration matrix design was used, and Bliss analysis was conducted to assess synergy. Single parameters are typically used to summarize drug responses in cell lines, which are used to classify cell lines as sensitive or resistant to the drug, impacting the interpretation of downstream analyses. We examined the robustness of different parameters, under various growth conditions for select standard of care compounds against a panel of cancer cell lines, and investigated their interpretation on biomarker and drug-drug combination analyses.

#4246

Association of transcriptional levels of folate-mediated one-carbon metabolism related genes in cancer cell lines with drug treatment response.

Julia Krushkal, Suleyman Vural, Dong-Joon Min. _National Cancer Institute, Rockville, MD_.

Folate-mediated one-carbon metabolism (OCM) is essential for growth and survival of cancer cells. It affects biosynthesis of nucleotides and amino acids, regulation of redox status, methylation of nucleic acids and proteins, and genome maintenance. We investigated whether the response of cancer cells to antitumor therapy treatment may be partially influenced by variation in expression of one-carbon metabolism genes. We used publicly available gene expression data and drug response measures for 251 antitumor agents in 635 cancer cell lines with matching information from the Cancer Cell Line Encyclopedia and the Genomics of Drug Sensitivity in Cancer resources. We examined whether pre-treatment expression levels of OCM-related genes were associated with drug response. Among the 34 OCM genes examined, expression of GART, TYMS, SHMT2, MTR, ALDH2, BHMT, MAT2B, MTHFD2, NNMT, and SLC46A1 showed modest correlations with response to treatment with a variety of antitumor agents. Higher expression levels of the SLC46A1 transporter gene were associated with resistance to multiple drugs, whereas elevated expression of GART, TYMS, SHMT2, MTR, BHMT, and MAT2B was associated with chemosensitivity to multiple antitumor agents. NNMT expression was bimodally distributed and showed different directions of association with various agents. Correlation of increased NNMT expression with sensitivity to dasatinib was validated in the NCI-60 cancer cell line panel. Expression of several OCM genes was strongly associated with expression of multiple components of drug target pathways. For example, expression of both TYMS and GART was strongly positively correlated with BRCA1 expression, NNMT expression was associated with expression of multiple drug target genes including EGFR and ABL2, and the expression of TYMS, DHFR, and SHMT1 was positively correlated with AURKB expression. Pretreatment expression levels of many OCM genes including DHFR, TYMS, ATIC, GART, AHCY, MTHFD1, SLC19A1, and multiple other genes were positively correlated with each other, suggesting their co-regulation. In contrast, NNMT expression was negatively correlated with expression of several other OCM genes. Further studies could investigate whether correlations of expression levels of individual OCM genes with drug response are related to specific metabolic roles of these genes or whether such associations may be due to the general increase in cellular growth and proliferation and tumor progression, which may affect sensitivity to cancer treatment.

#4247

Predicting the prognosis for cancer patients with interleukins gene expression level.

shrikant pawar,1 Aditya Stanam2. 1 _Georgia State University, Atlanta, GA;_ 2 _University of Iowa, Iowa City, IA_.

Introduction:

The human genome encodes more than 50 interleukins (IL) and related proteins The majority of IL'S are synthesized by helper CD4 T lymphocytes, as well as through monocytes, macrophages, and endothelial cells. They promote the development and differentiation of T and B lymphocytes, and hematopoietic cells [1]. Interleukin 1 alpha and interleukin 1 beta (IL1A and IL1B) are cytokines that participate in the regulation of immune responses, inflammatory reactions, and hematopoiesis [2]. Similarly, few other important IL pathway genes like IL1R1, IL1R2, IL1RAP, IL1RN, and MYD88 have been found to be expressed highly in different solid tumors [1, 2, 3]. We hypothesize that these selected IL pathway genes can be used to predict the survival in different cancers.

Materials and Methods:

The microarray data of four different cancer types is analyzed in this study obtained from the Gene Expression Omnibus database (GEO). These specimens were used to establish cancer prognostic biomarker model and validation model. Associated clinical information like patient gender, clinical stage, neoplasm histologic grade, vital status, patient race, and patient's days to death was also retrieved. The gene expression levels were measured for each cancer type with its respective reference as a control. Finally, Kaplan-Meier survival (KM) curve was applied to detect the differences of survival time for the index genes. Functions "Surv" and "survfit" were used for making survival objects and a survival fit [4]. All the code repositories for analysis can be found on authors GitHub account at https://github.com/spawar2/Interleukin_Pathway_Survival_Gene_Expression_Analysis

Results:

All the patients selected in the study for lung, ovarian and head and neck cancer showed high expression of all the selected IL pathway genes. While 2, 1, 0, 58, 0, 51 and 15 pancreatic patients showed up-regulation of IL1A, IL1B, IL1R1, IL1R2, IL1RAP, IL1RN, and MYD88 genes. So we separated these patients as high expression cohort and remaining as low expression group patients. We further did a Kaplan-Meier survival (KM) curve comparing survival probability of patients with high and low six gene expression index in pancreatic cancer patients. Subsequent survival fits were plotted on same plot for ovarian, lung and head and neck cancer patients.

Conclusion and Discussion:

In our study, a cancer prognostic biomarker model constituted by few RNAs was established to stratify the risk hazards for these patients. Our results display a significant fold expression of all the IL pathway genes in ovarian, lung and head and neck cancers. The comparison of high and low expression of IL pathway genes in pancreatic patients reveal a difference but with an insignificant P-value. The reasons for which can be limitations in sample size, inter-sample variability etc.

#4248

Recurrent somatic mutation pair occurrence patterns reflect clinical outcome for chemo- & immunotherapy treatments.

Jonathan M. Friedman. _Can-Bas, Ltd, Numazu-shi, Japan_.

Exhaustive analysis of somatic mutation pairs (MC3, [1]) in The Cancer Genome Atlas (TCGA,[2]) revealed significant relationships between the efficacy of drug treatment (PFS/DSS from TCGA-CDR, [3]) and the presence of particular somatic mutation-pairs for each [cancer type + drug treatment] (CANxDRG). For each combination of [CANxDRG + somatic gene mutation-pair], patients were split into 8 categories based on the efficacy (HIT, H) or inefficacy (MISS, M) of the drug treatment (i.e. in the top or bottom ≈33rd percentile of PFS/DSS over all drugs for a given cancer type) and on the presence (1) or absence (0) of mutations in each gene of the pair. Analysis of the resulting 4x2 table of patient counts for each [CANxDRG + gene-pair] provided a probability that the frequency of appearance of patients differed between each [mutation-presence x HIT] class and the corresponding [mutation-presence x MISS] class. Mutations for some critical genes, when considered individually for a CANxDRG, led to patient outcomes mixed between the HIT and MISS classes, but patient outcomes were almost entirely in only one of the HIT or MISS classes on considering the combined presence of a second gene. (e.g. CAN=coad {colon adenocarcinoma} DRG=5-Fluorouracil, "class:pat.count": // APC&KRAS H00:7 M00:3, H10:26 M10:1, H01:4 M01:0, H11:12 M11:6 // APC&TTN H00:8 M00:1, H10:17 M10:6, H01:3 M01:2, H11:21 M11:1 // KRAS&TTN H00:17 M00:2, H10:8 M10:5, H01:16 M01:2, H11:8 M11:1 // ) Some single genes appeared often in such critical gene-pairs across many CANxDRGs, but some single genes, while clearly outcome-determining, were critical for a smaller number of CANxDRGs:["GENE ID","count of CANxDRGs with GENE ID in top-appearing, critical gene-pairs for cases categorized as Stage 3 or Stage 4 cancer": TP53 33; TTN 33; MUC16 14; APC 12; CSMD3 12; RYR2 12; PIK3CA 10; SYNE1 8 . . . ] Also, some gene classes with many gene isotypes had a high collective rate of significance across isotypes of the gene class, although each individual isotype might be present in top-appearing critical gene-pairs for many fewer CANxDRGs. ["GENE CLASS ID", "count of CANxDRGs with GENE CLASS ID in top-appearing, critical gene-pairs": TP# 33; TTN 33; cadherins (CDH# + PCDH# + PCDHA# + PCDHB# + PCDHGA# + FAT#) 35 total / 21 unique; CSMD# 19; MUC# 17; DNAH# 16; ZNF# 16; APC 12; RYR# 12; . . . ] Gene pairs found to be critical in TCGA for a CANxDRG, when present (or absent) in GDSC database [4] cell lines, sometimes indicated relative drug sensitivity, but sample sizes were small. Top critical genes are consistent with those inferred by others [5] from the ratios of non-silent to silent mutation frequencies. References: [1] Ellrott et al., 2018, Cell Systems 6, 271-281; [2] TCGA Research Network: http://cancergenome.nih.gov/; [3] Liu et al., 2018, Cell 173, 400-416; [4] Yang et al., 2013, Nucleic acids res. 41, (database issue) D955-61; [5] Lawrence et al., 2013, Nature 499, 214-218.

#4249

Tumor-infiltrating B lymphocytes may serve as a prognostic biomarker for head and neck squamous carcinoma.

Yuchen LIU, Vivain W. Lui. _The Chinese University of Hong Kong, Hong Kong, China_.

B lymphocytes exert multifaceted functions to produce antibodies in humoral response, present antigen in cell-mediated immunity and suppress immunopathology via anti-inflammatory cytokines. Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancers in world. The development and progression of HNSCC is closely related to the dysregulation and evasion of immune system, which enables cancer cells to proliferate and metastasize. In HNSCC, high CD8+ T cell infiltration predicts better patient survival, and the suppressive T cells (Treg) correlates with tumor progression. Nevertheless, the precise role of tumor-infiltrating B lymphocytes (TIL-B) in HNSCC is poorly defined.

With a recently published bioinformatics approach, Tumor Immune Estimation Resources (TIMER), comprehensive pan-Cancer analysis was performed on 25 cancer types using the publicly available TCGA database with both survival and RNA-Seq information. Univariate COX regression demonstrates that in lower grade glioma (Hazard Ratio (HR)=830, P=1.3E-06), diffuse large B-cell lymphoma (HR=130, P=0.01), and papillary renal cell carcinoma (HR=15, P=0.02), TIL-B levels are associated with poor survival. Whilst in HNSCC (HR=0.082, P=0.0015), breast invasive carcinoma (HR=0.12, P=0.042), uterine corpus endometrial carcinoma (HR=0.045, P=0.039), cervical carcinoma (HR=0.00049, P=0.022,) and thymoma (HR=1.6E-05, P=0.026), TIL-B levels are associated with better survival. Note that among these 5 cancer types, HNSCC has the lowest P-value, suggesting TIL-B level is likely a good prognostic biomarker for patient outcome in HNSCC. In other 17 cancer types, TIL-B are not related to patient survival (P=n.s.).

In HNSCC, clinicopathological analysis shows that the extreme high TIL-B level (top 15% patients with the highest TIL-B level vs. the bottom 15%) is associated with living status (P=0.0047), early AJCC pathological T stage (P=2.71E-04), high differentiation grade (P=5.00E-05), and the absence of perineural invasion (P=8.52E-04). Furthermore, Kaplan-Meier survival curves based on TIL-B level indicate that patients with high TIL-B (top 15%) show longer survival compared with those with low level (bottom 15%; P=0.0005). The median overall survival time is 67.81 months in TIL-B high and 35.91 in TIL-B low group.

In conclusion, this study reveals that TIL-B may serve as independent prognostic biomarker for overall survival of HNSCC patients.

Acknowledgements:

VWYL receives funding supports from the Research Grant Council, HK (General Research Fund: #1711484; #17121616; #14168571; Theme-based Research: T12-401/13-R), Health and Medical Research Fund (HMRF #15160691), the University-Industry Collaboration Program (UIM/329; Innovation and Technology Fund, HK), and the Hong Kong Cancer Fund, HK.

YL receives funding (Postdoctoral Hub of UIM/329) from the Innovation and Technology Fund, HK government)

This study also receives funding from Lee Hysan Foundation Research Grant & Endowment Fund Research Grant Schemes 2018-2019.

#4250

Signatures of rearrangements across 2800 cancer genomes.

Kevin Hadi, Xiaotong Yao, Marcin Imielinski. _Weill Cornell Medicine, New York, NY_.

Genomic instability is a hallmark of processes driving cancer evolution. With publicly available whole cancer genome sequences, we observe large scale somatically acquired rearrangements, at or near base pair resolution. While discovery of single nucleotide variant (SNV) signatures have contributed much insight into exogenous and endogenous exposures to mutagens, major questions concerning the patterns of structural variants (SVs) throughout genomes of different cancer types remain. By understanding SV signatures, we can unravel relationships between novel biological mechanisms behind DNA damage/repair responses and tumor progression. We approach discovery of SV signatures by expanding upon a previous classification of SVs, based on breakpoint size, orientation, and clustering. Using our new graph model of cancer genomes, we add estimated copy number attributed to rearrangements into the feature space for signature discovery. We discover signatures of structural variants from ~3000 cancer genomes across >40 cancer types using Latent Dirichet Allocation, a probabilistic technique to infer classes explaining discrete data. Our results show distinct enrichments of rearrangement contexts in GBM and SARC, tumors heavily burdened by structural variation. Junction centric analysis of SNVs, which involves lifting reference coordinates onto the coordinates of junctions with respect to their orientation, show an enrichment of GC strand coordinated clusters particularly in sarcomas. The co-occurrence of enrichments of structural variant signatures involving high copy rearrangements and GC strand coordinated clusters in sarcomas suggests a novel DNA damage or response process involving APOBEC in this particular cancer type.

#4251

Detecting microsatellite instability in FFPE tissue from CRC Subjects using next generation sequencing.

Hao Wang, Amrita Pati, Dan Klass, Fergal Casey, Alex Lovejoy, Hamid Mirebrahim. _Roche Sequencing Solutions, Pleasanton, CA_.

Background

Microsatellite instability (MSI) is a hypermutable phenotype resulting from DNA mismatch repair deficiency and is observed in up to 10-15% of early-stage colorectal cancers (CRC). It manifests as abnormal lengthening or shortening of DNA repeats at specific genomic loci. Clinical studies have shown that MSI-High (MSI-H) CRC patients respond favorably towards both chemotherapy and immunotherapy and have different prognosis than MSS patients and the FDA has approved Pembrolizumab as the first cancer therapy based on a class of biomarkers rather than a cancer type. Hence, classification of MSS vs MSI-H status may become a routine test in multiple tumor types to predict response to cancer immunotherapy. With increasing options for targeted therapy in solid tumors, being able to combine MSI testing with other cancer biomarkers for approved therapies is beneficial. We show proof of concept of a pan-cancer gene panel using an NGS-based assay and bioinformatics workflow that enables classification of MSI status along with detection of SNVs, fusions, indels and CNVs from tumor tissue samples. Unlike the gold standard PCR-based test, our method does not require a matched normal sample, thus enabling MSI detection based on tumor tissue alone.

Methods

Our NGS based workflow combines whole genome library preparation, hybrid capture target enrichment, high-throughput sequencing and a proprietary analysis algorithm. We expanded the number of genomic loci to improve the sensitivity of MSI detection. The analysis algorithm was trained using DNA from pure and mixed cell lines, tumor tissue and normal samples with known MSI status to classify the molecular alterations between MSI-H and MSS genotypes from sequencing reads. The algorithm leverages repeat lengths in homopolymeric MSI loci in order to predict instability of individual loci followed by aggregation using a statistical framework to make the final call on MSI status.

Results

The above algorithm was evaluated on two cohorts of tissue samples. The first cohort includes 134 stage II and stage III CRC subjects who underwent curative intent surgery. MSI status for these samples was orthogonally tested using a PCR-based MSI Analysis System (31 positives, 103 negatives) and a dMMR system. Using a pre-determined threshold, the algorithm yielded 100% sensitivity and 100% specificity on the above cohort without using matched normal tissue. The second cohort includes 47 CRC patients that are enriched for MSI positive status by virtue of their selection criteria (BRAF positive). Very high concordance on MSI status with an orthogonal method was observed. Data demonstrating high concordance with a PCR_based MSI system will be available at the presentation.

Conclusions

Here we present the feasibility of an NGS-based assay and bioinformatics workflow with robust analytical performance for MSI detection in FFPE tissue samples without a paired normal.

#4252

Detecting microsatellite instability in ffpe tissue from crc subjects using next generation sequencing.

Amrita Pati,1 Hao Wang,1 Hamid Mirebrahim,1 Seng Saelee,1 Joshua Lefkowitz,1 Sean Chien,1 Ashla Singh,1 Fergal Casey,1 Vera Rapoport,1 Xiaoju Max Ma,1 John Lee,2 Alex Lovejoy,1 Daniel Klass,1 Hans-Peter Adams1. 1 _Roche Sequencing Solutions, Pleasanton, CA;_ 2 _Roche Sequencing Solutions (former), Pleasanton, CA_.

Background

Microsatellite instability (MSI) is a hypermutable phenotype resulting from DNA mismatch repair deficiency and is observed in up to 10-15% of early-stage colorectal cancers (CRC). It manifests as abnormal lengthening or shortening of DNA repeats at specific genomic loci. MSI-High (MSI-H) CRC patients have been shown to respond favorably towards both chemotherapy and immunotherapy and have different prognosis than MSS patients and the FDA has approved Pembrolizumab as the first cancer therapy based on a class of biomarkers rather than a cancer type. With increasing options for targeted therapy in solid tumors, being able to combine MSI testing with other cancer biomarkers for approved therapies is beneficial. We show proof of concept of a pan-cancer gene panel using an NGS-based assay and bioinformatics workflow that enables classification of MSI status along with detection of SNVs, fusions, indels and CNVs from tumor tissue samples. Unlike the gold standard PCR-based test, our method does not require a matched normal sample, thus enabling MSI detection based on tumor tissue alone.

Methods

Our workflow combines whole genome library preparation, hybrid capture target enrichment, high-throughput sequencing and a proprietary analysis algorithm. We expanded the number of genomic loci to improve the sensitivity of MSI detection. The analysis algorithm was trained using DNA from pure and mixed cell lines, tumor tissue and normal samples with known MSI status to classify the molecular alterations between MSI-H and MSS genotypes from sequencing reads. The algorithm leverages repeat lengths in homopolymeric MSI loci in order to predict instability of individual loci followed by aggregation using a statistical framework to make the final call on MSI status.

Results

The above algorithm was evaluated on a cohort of 134 stage II and stage III CRC subjects who underwent curative intent surgery. MSI status for these samples was orthogonally tested using a PCR-based MSI Analysis System (31 positives, 103 negatives) and a dMMR system. Using a pre-determined threshold, the algorithm yielded 100% sensitivity and 100% specificity on the above cohort without using matched normal tissue. A second cohort evaluated includes 47 CRC patients that are enriched for MSI positive status by virtue of their selection criteria (BRAF positive). Very high concordance on MSI status with an orthogonal method was observed. Data demonstrating high concordance with a PCR_based MSI system will be available at the presentation.

Conclusions

Here we present an NGS-based assay and bioinformatics workflow with robust analytical performance for MSI detection in FFPE tissue samples without a paired normal.

#4253

Differential expression of protein‐coding and non-coding RNAs in malignant melanoma.

Stephanie Figueroa,1 Raj Tiwari,2 Jan Geliebter,2 Niradiz Reyes3. 1 _Ossining High School, Ossining, NY;_ 2 _New York Medical College, Valhalla, NY;_ 3 _Universidad de Cartagena, Cartagena, Colombia_.

Background: Skin cancer is the most common malignancy in the United States. Although melanoma makes less than 10% of diagnosed skin cancers, it is responsible for most skin cancer-related deaths due to high metastatic potential and therapeutic resistance. Therefore, research is needed to find suitable biomarkers that could improve early diagnosis, prognosis and/or therapy of cutaneous melanoma. In this study, we aim to identify differentially expressed genes in melanoma patients which can serve as potential biomarkers for this disease.

Methods: Gene expression profiling was carried out using GEPIA (http://gepia.cancer-pku.cn/index.html), a bioinformatics research platform for the profiling and interactive analysis of cancerous gene expression based on The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases. Differentially expressed transcripts were classified as protein-coding or non-coding. The list of differentially expressed protein-coding genes was subjected to Functional and Pathway enrichment analysis using the online Database for Annotation, Visualization and Integrated Discovery (DAVID; https://david.ncifcrf.gov). Non-coding RNAs (ncRNAs) were searched in Medline database to determine previous association of specific ncRNAs to melanoma.

Results: A total of 107 transcripts were found upregulated in melanoma compared to normal tissues; 58 were identified as protein-coding RNAs, 48 as ncRNAs, and one as novel transcript. DAVID analysis of differentially expressed protein-coding RNAs demonstrated that upregulated transcripts were significantly enriched in oxidation-reduction processes, melanin biosynthetic process, visual perception, and melanosome organization. To identify novel genes not previously reported in melanoma, a Medline search was performed for transcripts with the highest over-expression in melanoma compared to normal skin tissue. Of the protein-coding RNAs, 33 were previously reported in melanoma, including PMEL, TYR, MLANA, among others, thus validating our results. However, the remaining 25 protein-coding genes were not previously associated to melanoma. In contrast, most of the ncRNAs have not been previously mentioned in melanoma; some of these ncRNAs were specifically expressed in melanoma but not in other cancer types or normal skin. Kaplan-Meier survival analysis of differentially expressed transcripts identified eleven protein-coding and nine non-coding RNAs whose expression levels were significantly associated with overall survival in melanoma patients.

Conclusions: Analysis of differential gene expression between melanoma and normal skin tissue using GEPIA tool to mine data from TCGA and GTEx databases allowed identification of protein-coding and non-protein coding genes that may be involved in cutaneous malignant transformation, and identification of novel genes that may be potential biomarkers for this disease.

#4254

Integrated analysis with MVisAGe identifies concordant and discordant genomic alterations of driver genes.

Vonn Walter,1 Ying Du,2 Ludmila Danilova,3 Michele Hayward,4 D. Neil Hayes5. 1 _Penn State College of Medicine, Hummelstown, PA;_ 2 _Center for Infection Disease Research, Seattle, WA;_ 3 _Johns Hopkins University School of Medicine, Baltimore, MD;_ 4 _University of North Carolina at Chapel Hill, Chapel Hill, NC;_ 5 _University of Tennessee Health Science Center, Memphis, TN_.

Integrated analyses of multiple genomic datatypes are now common in cancer profiling studies. Such data present opportunities for numerous computational experiments, yet analytic pipelines are limited. Tools such as the cBioPortal and Regulome Explorer, while useful, are not easy to access programmatically or implement locally. The MVisAGe R package allows users to quantify gene-level associations between two genomic datatypes in order to investigate the effect of genomic alterations (e.g. DNA copy number changes on gene expression). Visualizing Pearson/Spearman correlation coefficients according to the genomic positions of the underlying genes provides a powerful yet novel tool for conducting exploratory analyses. We demonstrate its utility by analyzing three publicly available datasets from squamous tumors. Our approach highlights canonical oncogenes in chr11q13 that displayed the strongest associations between expression and copy number, including CCND1 and CTTN, genes not identified by copy number analysis in the primary reports. We demonstrate highly concordant usage of shared oncogenes on chr3q, yet strikingly diverse oncogene usage on chr11q as a function of HPV infection status. Regions of chr19 that display remarkable associations between methylation and gene expression were identified, as were previously unreported miRNA-gene expression associations that may contribute to the epithelial-to-mesenchymal transition.

#4255

TET1 survival model molecular mediation experiments in glioma.

Thomas Luechtefeld,1 Nole Lin,1 Jua Kim,1 Channing Paller,2 Joseph Bressler3. 1 _Insilica, Baltimore, MD;_ 2 _Johns Hopkins Hospital, Bethesda, MD;_ 3 _Kennedy Krieger Institute, Baltimore, MD_.

Introduction

A computational experiment to quantify the molecular mechanisms mediating an association between gene expression and patient survival is described and implemented on TET1 in glioma patients.

Methods

TET1 has a strong relationship with patient survival in all glioma types. An approach that chains neural networks for expression and survival was developed to investigate the molecular mediators of this relationship. Models were built using Genomic Data Commons RNA-seq data and patient survival glioma data.

Multitask gene expression networks were built from TET1 associated gene expression inputs and cancer hallmark gene expression outputs. These networks model the expression of cancer hallmark genes as a function of TET1 associated gene expression. TET1 associated genes were defined as those with the greatest differential expression in TET1 deficient relative to TET1 normal glioma cell lines.

Cancer hallmark survival networks were built with RNA-seq expression data inputs and survival data outputs. Input genes were selected from cancer hallmark gene sets. These networks use a cox proportional hazards output layer to train on glioma patient survival data. The models estimate patient hazard as a function of cancer hallmark gene expression.

TET1 associated gene expression is transformed into predicted cancer hallmark gene expression via the multitask expression networks. This predicted expression data is then fed into the associated cancer hallmark survival network. By chaining two independently trained cancer hallmark expression and survival networks a 'mediated concordance' is measured for each cancer hallmark.

Results

A new method for interrogating the processes mediating a gene expression / patient hazard correlation is proposed and demonstrated on TET1 and glioma. Immune response, DNA repair and cell motility score highly as mediators of the TET1 survival effect.

The DNA repair finding is supported by a positive relationship between TET1 expression and mutation counts derived from Mutation Annotation Format files. The cell motility relationship is supported by a glioma cell line experiment showing greater cell mobility in TET1 deficient cell lines.

Conclusions

TET1 and genes affected by TET1 expression effectively modeled patient survival with concordance values of 83% in cross validation of the Genomic Data Commons GBM and low-grade glioma data sets. Neural network mediation experiments demonstrated DNA repair mechanisms (median mediated concordance of ~70%) and immune response (median mediated concordance of ~75%) as the most strongly associated hallmarks with the observed TET1 survival effect. Cell motility was inversely associated with the TET1 survival effect with a median mediated concordance of ~25%.

#4256

Identifying multi-hit combinations of carcinogenic mutations.

Ramu Anandakrishnan,1 Sajal Dash,2 Nick Kinney,1 Robin Varghese,1 Harold Garner,1 Wu-chun Feng2. 1 _Edward Via College of Osteopathic Medicine, Blacksburg, VA;_ 2 _Virginia Tech, Blacksburg, VA_.

Cancer is known to result primarily from different combinations of a small number of genetic defects. However, currently there is no way to determine the specific combinations that are most likely responsible for individual instances of cancer. Current computational approaches focus on identifying driver mutations, which can increase the risk of cancer, but do not result in cancer without additional mutations. We have developed a fundamentally different approach, which focuses on identifying the specific combinations of carcinogenic mutations (multi-hit combinations) that are most likely to be responsible for individual instances of cancer. We have identified a set of multi-hit combinations that differentiate between tumor and normal tissue samples with over 90% sensitivity and specificity on average for seventeen cancer types. With experimental validation, these combinations can be used to identify the most likely cause of carcinogenesis and provide a rational basis for designing targeted combination therapies.

#4257

Integrated network analysis reveals potentially novel molecular pathways mechanism and therapeutic targets of pancreatic ductal adenocarcinoma.

Lijun Cheng, Enze Liu, Li Lang, Xiaolin Cheng, Xiaotian Kong, Korc Murray. _Ohio State University, Columbus, OH_.

Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human cancers and shows insensitivity towards many chemotherapeutic drugs and target-based drugs. There is an urgently need to identify PDAC disease mechanism and detect novelty drug targets. With the large availability of protein interaction networks and microarray data supported, to identify the disease essential genes that have biological significance for potential drug targets is a challenge issue still.

Methods: In this study, protein interaction network PathPPI, DrugBank targets and 374 transcriptome profiles from Gene Expression Omnibus (GEO) by Affymetrix HU 133 Plus 2.0 array test are collected. These microarray data includes 140 PDAC, 92 pancreatic cancer cell-line, 58 human normal pancreas tissues, and 72 patient original xenograft mouse. Based on these datasets, a novelty-integrated algorithm was developed for drug target prioritization by perturbed gene expression and network information. The integration network included data driven reconstruction network of gene regulatory (GRN) by Bayesian Network and protein interaction PathPPI network. The perturbed gene expression was refer to the differential expression genes of tumor verse adjacent normal. A novel hybrid method based on genetic algorithm (GA) is proposed to search the attractor with the maximum network perturbation for candidate drug targets in PDAC.These targets' molecular mechanism was revealed further by network structure comparison of PDAC that is consistent with xerograph mouse model and cancer cells.

Results: Highly accordance 56 genes with high perturbation score both in mRNAs and protein-protein network were identified and recommended as candidate drug targets for PDAC, including 16 novel targets, such as PSMD2, EPOR and PSMB9. Fifty-four essential genes shown strong concordance among tumor-model, patient drive xenograft mouse and pancreatic cancer cell-line by systematically comparison. We identified 1375 dysregulated genes that enriched in 25 pathways in PDAC by Gene Set Enrichment Analysis, among which 244 genes played as hubs in reconstructed PDAC regulatory network.

Conclusion: In the study, we developed a global optimization-based inference of network perturbation to detect attractor for drug-target identification in PDAC tumors. The assembling Bayesian Network-based approach with protein-protein interaction provides a comprehensive information to observe energy transfer of gene perturbation in network to detect global optimum attractor for drug target selection.

#4258

Preliminarily results of the Oncohabitats Study: A multicentre validation of overall survival (OS) estimation of patients with glioblastoma (GBM) using vascular biomarkers.

María del Mar Álvarez-Torres,1 Fuensanta Bellvís–Bataller,1 Javier Juan–Albarracín,1 Elies Fuster–Garcia,1 David Lorente Estellés,2 Gaspar Reynes,3 Fernando Aparici-Robles,3 Carlos Botella,4 Jose Muñoz-Langa,3 Raquel Faubel,5 Sabina Asensio-Cuesta,1 Germán Adrían García–Ferrando,1 Cristina Auger,6 Alex Rovira,7 Jose Pineda,8 Jaime Font de Mora,3 Enrique Mollà-Olmos,9 Antonio José Revert-Ventura,10 Luaba Tshibanda,11 Didier Martin,11 Girolamo Crisi,12 Kyrre Eeg Emblem,13 Paulina Due-Tonnessen,13 Torstein R Meling,13 Juan M García-Gómez1. 1 _Universidad Politécnica de Valencia, Valencia, Spain;_ 2 _Hospital de Castellón, Castellón, Spain;_ 3 _Hospital Universitario de la Fé, Valencia, Spain;_ 4 _Hospital Universitari i Politècnic La Fe -Departament de Salut València La Fe, Valencia, Spain;_ 5 _Universidad de Valencia, Valencia, Spain;_ 6 _Hospital Universitari Vall d'Hebron, Barcelona, Spain;_ 7 _Seccion de Neurorradiologia. Servicio de Radiología Hospital Universitari Vall d'Hebron, Barcelona, Spain;_ 8 _Hospital Clínic de Barcelona, Valencia, Spain;_ 9 _Hospital de la Ribera, Alzira Valencia, Spain;_ 10 _Hospital de Manises, Valencia, Spain;_ 11 _Centre Hospitalier Universitaire de Liège, Liège, Belgium;_ 12 _Hospital de Parma, Parma, Italy;_ 13 _Oslo University Hospital, Oslo, Norway_.

We report preliminarily results of an international retrospective study (NCT03439332) analyzing the prognostic value of the early assessment of vascular architecture of glioblastoma (GBM).

The initial cohort included 300 pts treated at 7 European hospitals. Multiparametric images were processed by Oncohabitats (www.oncohabitats.upv.es) to obtain the cerebral blood volume (CBV) and cerebral blood flow (CBF) from 4 automatically delimited regions of interest (ROIs): high angiogenic tumor (HAT), low angiogenic tumor (LAT), infiltrating peripherial edema (IPE), and vasogenic peripherial edema (VPE). Uniparametric Cox regression models and Kaplan-Meier analysis were developed to test prognostic and stratification capabilities of each biomarker.

115 pre-surgical MRIs passed quality controls and were included in the analysis. Median follow up was 12.9 months (m) (IQR: 9.2-17.6). 73 pts (63.5%) were male. 54 pts (47%) underwent total resection; 84 pts (73%) received adjuvant temozolomide. Median OS was 13.6 m (95%CI:12.1-15.2). Cox regression analysis yielded a significant correlation between OS and maximum rCBV and rCBF in HAT and LAT (Table), independent of the extent of resection. Median OS by Kaplan-Meier analysis in patients with high and low vascularity is shown in the Table.

Our preliminarily results shows that patients with lower vascularity (rCBV or rCBF) in vascular habitats inside enhancing tumor region (HAT and LAT) is associated with higher OS, and could improve patient stratification. Our results are consistent with our pilot study [1], Validation in large multicentric studies is underway. | |  | |  | |

|

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

|

COX REGRESSION ANALYSIS | KAPLAN-MEIER STRATIFICATION ANALYSIS

|

COX REGRESSION ANALYSIS | KAPLAN-MEIER STRATIFICATION ANALYSIS

|

HAZARD RATIO | 95% CONFIDENCE INTERVAL | P-VALUE* | NUMBER OF PATIENTS  | MEDIAN SURVIVAL (months) | P-VALUE

rCBV máximum | |  | |  | |

High Angiogenic Tumor | 10.339 | [1.09- 1.14] | 0.0202* | [29 86] | [16.66 12.78] | 0.004***

Low Angiogenic Tumor | 10.485 | [1.17- 1.31] | 0.0262* | [29 86] | [17.10 12.78] | 0.002***

Infiltrating Peripheral Edema | 0.9835 | [1.30- 1.71] | 0.1208 | [29 86] | [14.60 12.91] | 0.119

Vasogenic Peripheral Edema | 0.7942 | [1.14- 1.63] | 0.6046 | [29 86] | [15.23 12.91] | 0.220

rCBF máximum | |  | |  | |

High Angiogenic Tumor | 0.9997 | [1.02- 1.03] | 0.1077 | [47 68] | [14.93 11.65] | 0.009**

Low Angiogenic Tumor | 10.411 | [1.21- 1.40] | 0.0384* | [29 86] | [15.56 12.75] | 0.016*

InfiltratingPeripheral Edema | 0.8923 | [1.35- 2.04] | 0.2589 | [29 86] | [15.23 12.83] | 0.394

Vasogenic Peripheral Edema | 0.6846 | [1.11- 1.81] | 0.7447 | [29 86] | [14.60 12.96] | 0.508

Table: Results of Cox-regression and Kaplan-Meier analysis. (P VALUE*: False discovery rate corrected.)

[1]: https://doi.org/10.1148/radiol.2017170845

#4259

Defining subtle cancer subtypes using the darkest DNA.

Laxmi Parida,1 Claudia Haferlach,2 Kahn Rhrissorrakrai,1 Filippo Utro,1 Chaya Levovitz,1 Kern Wolfgang,2 Niroshan Nadarajah,2 Stephan Hutter,2 Manja Meggendorfer,2 Wencke Walter,2 Constance Baer,2 Torsten Haferlach2. 1 _IBM Research, Yorktown Heights, NY;_ 2 _MLL Munich Leukemia Laboratory, Germany_.

The confluence of deep sequencing and powerful machine learning is providing an unprecedented peek at the darkest of the dark genomic matter. While deep sequencing uncovers rare tumor variants, the heterogeneity of the disease confounds the best of machine learning (ML) algorithms. Here we set out to answer if the dark-matter of the genome encompass signals that can classify the fine subtypes of disease that are otherwise gnomically indistinguishable. We introduce a novel stochastic regularization, ReVeal, that empowers ML to classify subtle cancer subtypes even from the same 'cell of origin'. Analogous to heritability, implicitly defined on whole genome, we use predictability (F1 score) definable on portions of the genome. In an effort to classify cancer subtypes using dark-matter DNA, we applied ReVeal to a new WGS dataset from 727 patient samples with seven forms of hematological cancers and assessed the predictivity over several genomic regions including genic, non-dark, non-coding, non-genic, dark. ReVeal allowed the classification of all segments of the genome better than standard ML algorithms. The non-genic, non-coding and the dark-matter had the highest F1 scores with dark-matter having the highest level of predictability (F1 = 0.78). Based on ReVeal's predictability of different sectors of the genome, dark matter contains signal significant enough to classify fine subtypes of disease. The agglomeration of rare variants, even in the hitherto unannotated and ill-understood regions of the genome, may play a substantial role in the disease etiology and deserve much more attention.

#4260

Novel insights into immunotherapy by deep mining of big cancer genomic data.

Han Liang. _UT MD Anderson Cancer Ctr., Houston, TX_.

Immunotherapy has revolutionized patient care and cancer research. To gain novel insights into immunotherapy, we performed the pan-cancer analyses of >11,000 tumor samples of 33 cancer types from The Cancer Genome Atlas and CPTAC as well as large amounts of in-house immunotherapy related data. Through the mining on these large-scale cancer genomics and proteomics data, I will discuss three novel ideas that would challenge the current paradigm for developing effective immunotherapy. (I) Checkpoint inhibition: antibody is NOT the only approach. I will discuss the role of enhancers in regulating immune checkpoint. (II) Neoantigen: Somatic mutations are NOT the only source. I will discuss the contribution of RNA editing to proteomics diversity and neoantigen in cancer. (III) Predictive markers: gene-based markers is NOT the only option. I will discuss the utility of a global energy index for predicting the response to anti-PD1 treatment. Collectively, these results suggest novel directions for future immunotherapy development.

## MOLECULAR AND CELLULAR BIOLOGY / GENETICS

### Autophagy

#4261

Upregulation of miR-34b, miR-892a and inhibition of p-glycoprotein play an important role for melatonin-induced apoptosis and autophagy in vincristine-resistant human oral cancer cells.

Shun-Fa Yang,1 Ming-Ju Hsieh,2 Chiao-Wen Lin3. 1 _Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan;_ 2 _Cancer Research Center, Changhua Christian Hospital, Changhua, Taiwan;_ 3 _Institute of Oral Sciences, Chung Shan Medical University, Taichung, Taiwan_.

Multidrug-resistance was resistance of cells to a variety of drugs normally used in tumor treatment. The related mechanism of drug resistance in oral cancer has not been completely explained. Melatonin is an endogenously-produced molecule involved in active biological mechanisms including antiproliferation, oncogene expression modulation, antitumor invasion and migration, and anti-inflammatory, antioxidant, and antiangiogenic effects. Despite these numerous actions, the effect of melatonin on vincristine (VCR)-resistant human oral cancer cells still largely unknown. Present study analyzed the role of melatonin on VCR-resistant human oral cancer cells along with the underlying mechanism. We determined that melatonin induces apoptosis and autophagy of VCR-resistant oral cancer cell; these actions are mediated by AKT, p38, and JNK. Melatonin inhibits ABCB1 and ABCB4 expression in vitro/vivo. Melatonin decrease drug resistance and promotes VCR-resistant oral cancer cell execute apoptosis through upregulation of miR-892a and miR-34b-5p expression. The expression of miR-892a and miR-34b-5p are related with melatonin-induced apoptosis, but not autophagy. Thus, melatonin is a potential novel agent in chemotherapy for VCR-resistant human oral cancer cell lines.

#4262

MiR-873 is the master regulator of autophagy genes through a novel negative feedback mechanism mediated by Elongation factor 2 kinase (eEF-2K) and suppresses tumor growth and progression of triple negative breast cancer.

Hamada Ahmed Mokhlis,1 Nermin Kahraman,1 Seyda Baydogan,1 Abdel-Aziz Hamed Abdel-Aziz,2 Ahmed Ashour,2 Cristina Ivan,1 Gabriel Lopez-Berestein,1 Bulent Ozpolat1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _Faculty of Pharmacy-Al-Azhar University, Cairo, Egypt_.

Autophagy is a highly complex lysosomal degradation process. Recently we demonstrated that autophagy genes encoding Beclin1, ATG7, LC3 promotes cell proliferation, survival, and invasion by promoting Cyclin D1 and Integrin β1/ Src signaling in triple negative breast cancer (TNBC). Studies showed that increased basal autophagy is linked to development of chemoresistance, relapses and metastasis and poor prognosis in TNBC. However, the major molecular mechanisms and the integrated global regulators controlling the autophagic machinery still remains unknown. Here, we identified miRNA-873 as a p53-driven tumor suppressor that is associated with favorable patient survival (TCGA database) and the key factor for regulating the major autophagy genes, including BCN1, LC3, ATG7, ATG16L1 and ATG13 in TNBC. Using in silico prediction algorithms we demonstrated that miR-873 has binding sites on the 3'-untranslated region (3'-UTR) of these genes and directly binds and suppresses their expression using by gene reporter assays. Introduction of mutations to the miR-873 binding sites on 3-UTR of these genes reversed the inhibitory effect of miR-873on these genes. Furthermore, knockdown of Beclin1, LC3 and ATG7 genes significantly suppressed Eukaryotic Elongation Factor‐2 kinase (eEF2K), an unusual alpha kinase, which is highly overexpressed in TNBC patients and associated with poor prognosis. We also found that miR-873 also binds to the 3'-UTR of eEF2K mRNA and regulates its expression and inhibits starvation induced autophagy. Through knockdown and overexpression studies we also demonstrated that eEF2K regulates expression of abovementioned autophagic proteins. Interestingly, we found that BCN1, LC3, and ATG7 also regulates expression of EF2K, suggesting an existence of a novel negative feed-back loop. Lastly, we demonstrated that miR-873 expression is suppressed in TNBC patients and cell lines and restoration of its expression in vivo in MDA-MB-231 and MDA-MB-436 orthotopic xenograft models by systemic injection (I.V, tail vein once a week, 0.15 mg/kg) of miR-873 mimic molecules incorporated in novel single lipid (SLNP)-nanoparticles suppressed tumor growth. Furthermore, in vivo treatment of mice with SLNP-Beclin1, ATG7 or ATG8 siRNAs also completely suppressed TNBC tumor growth. In conclusion, our data suggest that p53/miR‐873/eEF2K axis is a novel post-transcriptional regulator of autophagy and miR-873 functions as a master regulator of the post-transcriptional regulation of the major autophagy genes directly and indirectly through eEF2K dependent dual-suppressor mechanism and modulation of this axis could be used as a potential therapy for TNBC.

#4263

Autophagic ovarian cancer cells exhibit substantially enhanced sensitivity to ALK inhibition.

Alicia M. Blessing, Weiqun Mao, Lan Pang, Philip Rask, Zhen Lu, Robert C. Bast. _UT MD Anderson Cancer Ctr., Houston, TX_.

One of the major factors contributing to poor outcomes for patients with ovarian cancer is the persistence of dormant, drug resistant cancer cells after primary surgery and chemotherapy. The persistent cancer found in positive second look operations is poorly vascularized and autophagy is widespread in more than 80% of cases. Consequently, drugs that regulate survival in autophagic cancer cells may be much more active when administered as maintenance therapy than when used to treat gross recurrent disease. Using unbiased siRNA screens, we have identified target genes that regulate the survival of ovarian cancer cells that are undergoing autophagy-induced by the re-expression of DIRAS3 (ARHI) or by serum starvation. Knockdown of the anaplastic lymphoma kinase (ALK) significantly reduced survival of ovarian cancer cells that were undergoing autophagy. Importantly, FDA-approved ALK inhibitors, including crizotinib, exhibited significantly greater toxicity after induction of autophagy by upregulation of DIRAS3 or serum starvation in 5 ovarian cancer cell lines. Induction of autophagy by upregulation of DIRAS3 or serum starvation in ovarian cancer cells reduced the IC50 of crizotinib and other ALK inhibitors ranging from 1.5 to 20-fold (all p-values <0.05). Crizotinib treatment in autophagic cancer cells enhanced autophagy and apoptosis via a decrease in p-STAT3 and BCL-2 signaling. Selective targeting of p-STAT3 or BCL-2 also resulted in significantly greater toxicity after induction of autophagy by upregulation of DIRAS3 or serum starvation in multiple ovarian cancer cells. Inducible upregulation of DIRAS3 by doxycycline in the first 6 weeks prompted autophagy and dormancy in two separate xenograft models (SKOV3 and OVCAR8). Crizotinib treatment of dormant autophagic SKOV3 xenografts prolonged median survival by 9.7 weeks (p<0.0032) and cured a subset of mice. Thirteen of fifteen mice (87%) bearing SKOV3 xenografts and nine of eleven mice (82%) bearing OVCAR8 xenografts, were tumor free at 200 days compared to the other three control arms (overall survival, p<0.0001 and p<0.05, respectively). Our studies suggest that ALK inhibitors might provide an effective agent to eliminate autophagic, dormant, drug resistant ovarian cancer cells that remain after conventional cytoreductive surgery and combination chemotherapy.

#4264

Lysosome inhibition sensitizes pancreatic cancer to replication stress by aspartate depletion.

Shili Xu,1 Irmina A. Elliott,1 Amanda M. Dann,1 Stephanie S. Kim,1 Evan R. Abe,1 Woosuk Kim,1 Soumya Poddar,1 Alexandra Moore,1 Lei Zhou,1 Jennifer L. Williams,2 Joseph R. Capri,1 Razmik Ghukasyan,1 Cynthia Matsumura,1 D. Andrew Tucker,1 Wesley R. Armstrong,1 Anthony E. Cabebe,1 Nanping Wu,1 Luyi Li,1 Thuc M. Le,1 Caius G. Radu,1 Timothy R. Donahue1. 1 _University of California, Los Angeles, Los Angeles, CA;_ 2 _Harbor-UCLA Medical Center, Los Angeles, CA_.

Objective: Functional lysosomes are required for autophagy and macropinocytosis, the intra- and extracellular scavenging pathways cancer cells engage for nutrient acquisition. Pancreatic ductal adenocarcinoma (PDAC) tumors exhibit high basal lysosomal activity, and genetic or pharmacologic inhibition of lysosome function suppresses PDAC cell proliferation and tumor growth. However, the codependencies induced by lysosomal inhibition in PDAC have not been systematically explored. We hypothesized that identification and targeting the lysosomal inhibition-induced codependencies in PDAC cells would be an effective therapeutic strategy.

Methods: A comprehensive pharmacological inhibition screen of the protein kinome was performed to identified lysosomal inhibition-induced codependency. LC-MS/MS-MRM and LC-MS methods were used to examine nucleotide biosynthesis and amino acid levels, respectively, to understand the mechanism of the identified codependency. A broad panel of PDAC cell lines, primary PDAC culture models, two- and three-dimentional culture models, a PDAC cell/stroma spheroid coculture model, and xenograft and syngeneic PDAC animal models were used to evaluate the treatment efficacy.

Results: We found that replication stress response (RSR) inhibitors were synthetically lethal with chloroquine (CQ) and other lysosomal inhibitors in PDAC cells. CQ treatment reduced de novo nucleotide biosynthesis and induced replication stress. We found that CQ treatment caused mitochondrial dysfunction and depletion of aspartate, an essential precursor for de novo nucleotide synthesis, as an underlying mechanism. Supplementation with aspartate in PDAC cell culture and overexpression of the aspartate transporter SLC1A3 in xenograft PDAC tumors partially rescued the phenotypes induced by CQ. The synergy of CQ and the RSR inhibitor VE-822 was comprehensively validated in a broad panel of PDAC cell lines and primary PDAC cultures, in two- and three-dimentional PDAC cultures, in heterotypic spheroid culture with cancer-associated fibroblasts, and in in vivo xenograft and syngeneic PDAC mouse models.

Conclusion: We discovered a codependency on functional lysosomes and an intact RSR pathway in PDAC, and developed the combination of CQ and RSR inhibitors as a translational therapeutic approach for PDAC.

#4265

Transient versus permanent autophagy inhibition in pancreatic ductal adenocarcinoma.

Jane B. Pearce, Ciara H. O'Flanagan, Stephen D. Hursting. _UNC Chapel Hill, Chapel Hill, NC_.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers worldwide, with a 5- year survival rate of less than 5 percent. KRAS-driven PDACs often exhibit increased dependence on autophagy, a process by which cells degrade internal components to mobilize energy stores. When faced with cellular stress, such as nutrient deprivation, tumor growth may initially slow in response to decreasing supplies of growth factors and cytokines. Under these conditions, the induction of autophagy can enable continued tumor growth even in the presence of nutrient stress. Therefore, we posited that nutrient restriction in combination with autophagy inhibition would synergistically disrupt aberrant metabolic pathways and more effectively stunt PDAC tumor progression.

To determine the impact of transient autophagy inhibition, cells were treated with 10 μM of the pharmaceutical agent chloroquine (CQ) for 48 hours. To modulate growth factor/cytokine signaling as well as nutrient sensing signaling serum and glucose restriction were used in vitro. Next, permanent autophagy ablation was achieved through CRISPR/Cas9 technology, autophagy related 5 (Atg5) was deleted in Panc02 cells generating an autophagy-deficient (Atg-/-) PDAC cell line. Growth kinetics were determined in a range of culture matrixes to determine the proliferation rate, invasiveness, and anchorage independence of cells.

The combination of CQ and nutrient stress elicited an additive effect on cellular proliferation in both murine and human-derived KRAS mutant PDAC cells (Panc02 and MIA PaCa-2 respectively). In contrast, Atg5-/- Panc02 cells did not display any alteration in cellular proliferation in vitro. Atg5 deletion resulted in decreased expression of mesenchymal markers N-Cadherin and Snail as well as decreased invasiveness and anchorage independent colony formation. Upon intrapancreatic injection in a syngeneic model, Atg-/- cells exhibited reduced tumor growth relative to control Atg5+/+cells, indicating the compensatory mechanisms effective in vitro failed to protect these cells in vivo. Thus, our findings reveal a potential synergism between autophagy inhibition and nutrient stress in PDAC.

#4266

Endoplasmic reticulum (ER), a potential therapeutic target for mutant p53 colorectal cancer.

Ashish Tyagi,1 Balaji chandrasekaran,1 Venkatesh Kolluru,1 Becca V. Baby,1 Alatassi Houda,1 Srinivasa Ramisetti,2 Arun Sharma,3 Murali Ankem,1 Chendil Damodaran1. 1 _University of Louisville, Louisville, KY;_ 2 _Penn State Hershey Medical Center, Hershey, PA;_ 3 _Pennsylvania State University, Hershey, PA_.

Introduction: Inactivating p53 mutations contribute to tumor progression and treatment-resistance, resulting in poor patient survival in colorectal cancer patients (CRC). The goal of this study is to identify novel small molecules that therapeutically target mutant p53 in CRC.

Methods: We analyzed the effects of small molecule (ASR458) on p53-wild type (p53-wt; HCT116) and mutant p53 (p53-mut; SW620) colon cancer cells by phenotypic, molecular and in vivo assays.

Results: ASR458 treatment significantly inhibited the proliferation of both HCT116 and SW620 cells at nM concentrations. In p53-wt HCT116 cells, ASR458 caused induction of p53 that resulted in caspase-mediated cell death in both in vitro and in vivo models. On the contrary, ASR458 treatment induced ER-stress signaling (i.e., phosphorylation of ERK and eIF2-α) in p53-mut SW620 cells, which triggered ATF4 activation and subsequent induction of cascade of autophagy events (Atg family proteins, LC3B and Lamp1), causing autophagy-mediated cell death. Silencing ER stress marker ATF-4, a key regulator of autophagy, caused resistance to ASR458 and abrogated autophagy signaling in SW620 cells. This suggested that induction of ER-stress is critical for the cytotoxic effects of ASR458 in p53-mut CRC. Preliminary knockdown studies indicate that silencing of ER markers causes resistance to ASR458 in vitro, further confirming ER-stress as the mechanism of ASR458 action in p53-mut CRC. ASR458 significantly inhibited the growth of SW620 tumors in xenograft. Tissue analysis confirmed the ER-stress signaling observed in vitro.

Conclusion: Our results demonstrate ASR458 as a potential therapeutic agent with distinct targets in p53-wt and p53-mut CRC. This study also suggests that ATF4 mediated autophagy in unmanaged ER stress can reduce CRC pathogenesis. Further investigation into the pharmacokinetics and pharmacodynamics of ASR458 should help clinical translation of this agent.

#4267

mTORC1-dependent tumors have innate vulnerability to autophagic cell death by HDAC inhibitors.

Fuchun Yang, Chenran Wang, Shaogang Sun, Michael Haas, Syn Yeo, Jun-Lin Guan. _University of Cincinnati College of Medicine, Cincinnati, OH_.

mTORC1 plays a significant role in the development and progression of many different types of cancers. Although mTORC1 inhibitors, such as Rapamycin, have anti-tumor effects on some specific cancers, their efficacy is still largely limited to anti-proliferative effects. Histone deacetylase (HDAC) inhibitors have been proven to be effective in cancer treatment including some solid cancers. We have previously established a vascular tumor cell line, Tsc1Δ EC, which was derived from Tsc1 deletion in mouse endothelial cells leading to constitutive activation of mTORC1. Here, we found that a pan-HDAC inhibitor, SAHA, caused Tsc1Δ EC cell death and growth arrest in vitro and in vivo. Our data showed that a selective Class I HDAC inhibitor CI994 treatment also caused Tsc1Δ EC cell death. Unlike mTORC1 inhibitors, the anti-tumor activity of SAHA was dependent on mTORC1 activation but it had little effect on mTORC1 activity in mTORC1 activated tumor cells. We found that Z-VAD-FMK, a pan-caspase inhibitor, had no inhibitory effect on SAHA treatment. Cell death pathway array analysis revealed that SAHA treatment in Tsc1ΔEC upregulated autophagy-related genes. Further experiments revealed that both autophagy gene Fip200 deletion and autophagy inhibition by spautin-1 in Tsc1Δ EC impaired tumor cell death by SAHA treatment. We further found that SAHA could play a role by increasing reactive oxygen species (ROS) via autophagy. Fip200 deletion caused Nrf2 activation through p62 accumulation, which can lead to antioxidation effect through p62-Keap1-Nrf2 pathway. We showed that inhibition of p62 expression re-sensitized Fip200 deleted Tsc1Δ EC to SAHA treatment. Taken together, HDAC inhibitors treatment provides an alternative promising therapeutic strategy for mTORC1-dependent cancers.

#4268

Protein folding pathway modulation upon Hsp70 inhibition in cancer cells.

Sara Sannino,1 Christopher J. Guerriero,1 Amit J. Sabnis,2 Jeffrey J. Bridsky1. 1 _Univ. of Pittsburgh, Pittsburgh, PA;_ 2 _University of California, San Francisco, CA_.

Cancer cells experience acute stress conditions such as low oxygen and energy, and exposure to toxic agents. To survive proliferate without accumulating toxic misfolded proteins, cancer cells constantly modulate protein homeostasis. Thus, it is not surprising that molecular chaperones, like Hsp70, as well as protein degradation pathways are upregulated in cancer cells compared to their normal counterparts. These data suggest that chaperones are potential targets for cancer therapy. We previously demonstrated the dependence of patient-driven rhabdomyosarcoma cell survival on cytoplasmic Hsp70 activity, thanks to the use of a specific Hsp70 inhibitor, MAL3-101. In particular, we discovered that MAL3-101-mediated Hsp70 inhibition activates the PERK arm of the unfolded protein response (UPR) that results in CHOP-dependent cell death (Sabinis et al., 2016). Moreover, by taking advantage of a MAL3-101-resistant cell line (RMS13-R), we recently determined which compensatory mechanism alters MAL3-101-driven cell death. We found that both endoplasmic reticulum-associated degradation (ERAD) and autophagy are upregulated in RMS13-R cells, underlying increased demand on two protein degradation pathways upon inhibition of Hsp70. However, only autophagy inhibition—but not inhibition of ERAD—re-sensitized RMS13-R cells to Hsp70 inhibition, suggesting that autophagy was the key compensatory mechanism for Hsp70 inhibition. Autophagy was further induced by MAL3-101 treatment in RMS13-R cells, as evidenced by an increase in the messages and proteins corresponding to key autophagy components as well as to the accumulation of autophagic-like structures detected by electron microscopy (Sannino et al., 2018). These data highlight a pro-survival role for autophagy induction upon exposure to an Hsp70 inhibitor in cancer, and provide a link between Hsp70, proteasomal degradation, UPR, and autophagy in rhabdomyosarcoma. We next asked if other cancer types might be sensitive to Hsp70 inhibition, and we investigated the potential benefit of combined treatment with autophagy and/or proteasome inhibitors together with MAL3-101. Specifically, we are investigating the effects of Hsp70 inhibition in breast cancer cells, a cancer type in which higher levels of Hsp70 correlate to increased metastasis and poor prognosis in patients. Our preliminary data suggest that HER2-expressing cells are less sensitive to MAL3-101-mediated Hsp70 inhibition and combinatory treatments including, MAL3-101 and autophagy inhibitors promoted HER2-breast cancer cell death. Further investigations will reveal the potential carcinogenic role of Hsp70 inhibitors in breast cancer treatment and highlight which pathways reduce proteotoxicity in different breast cancer subtypes.

#4269

Regulation and functional roles of autophagy in Helicobacter pylori CagA-mediated gastric cancer.

BoRam Hwang, SoDam Lee, ChangYun Jung, ByeongMin Yu, YongChan Lee. _Yonsei Univ. College of Medicine, Seoul, Republic of Korea_.

Autophagy is a catabolic process for the autophagosomic-lysosomal degradation of bulk cytoplasmic components and plays a crucial role in infection, inflammation and various cellular functions. Helicobacter pylori (H. pylori) was recently indicated to be able to induce autophagy. The cytotoxinA (CagA) of H.pylori was demonstrated to be degraded by autophagic pathway, but the the dynamic role of mechanism by which CagA affects autophagic signaling has not been identified. This study was determined to elucidate the functional role and regulatory mechanism of H. pylori induced autophagy in gastric cancer cells.

In this study, we tested that during the H.pylori 60190(CagA+, VacA+) infection, where the nutrient starvation induced a high level of autophagy, in gastric cancer cell lines(AGS, MKN45, MKN28 and GES-1) through CagA and LC3 I/II(autophagy marker) protein expression level. The gastric cancer cells infected with H.pylori 60190, ΔCagA and ΔVacA(60190 isogenic mutant strain) were incubated with a serum free medium containing antibiotic(KM,Kanamycin) to kill extracellular H.pylori, intracellular CagA level decreased and LC3 I converted to LC3 II. The quantification of LC3 by ImageJ increased in the gastric cancer cells. Autophagy induction associated with intracellular CagA stability. CagA was essential and sufficient to induce autophagy in gastric cancer cells, with the induction of autophagy responsible for a decreased in the level of intracellular CagA. The gastric cancer cells infected with or without H.pylori were performed immunofluorescence and detected by confocal microscopy to examine the co-localization of CagA and LC3. CagA located with LC3, therefore, CagA was related to the mechanism of autophagy. The intracellular cagA decreased and LC3 I to LC3 II conversion was detected. To evaluate whether autophagy was specifically associated with H.pylori infection, we used LysoTracker® Red. H.pylori infection affected lysosomal degradation. The autophagy inhibitor (Cychloheximide, Bafilomycin A1, Chloroquine) and PI3K/AKT inhibitor (LY294002), GSK inhibitor (Lithium Chloride) in gastric cancer cells were used to examine the mechanism of CagA degradation. The cagA was affected by those inhibitors. The effects of CagA in promoting autophagy blocked autophagy. The autophagy contributed to CagA degradation in gastric cancer cells. To examine ectopic expression of CagA affects serum starvation induced autophagy, transfection of plasmid vector to AGS were carried out.

Our findings showed that the autophagy play some important roles in H. pylori infected gastric cancer cells by the degradation of intracellular CagA leading to altered epithelial cellular signal transduction pathways. This indicate that studies investigating the host-pathogen autophagic interaction in H. pylori infection will provide the platform for bio-marker research for gastric cancer chemo-preventive strategy.

#4270

**Induction of autophagic machinery upon oncolytic reovirus treatment in colorectal cancer cells and animal** **models.**

Jeeshan Jiffry, Devika Rao, Thongthai Thavornwatanayong, Durvanand Saytoo, Titto Augustine, Sanjay Goel, Radhashree Maitra. _Montefiore Einstein Cancer Center, Bronx, NY_.

Background: Oncolytic reovirus (REO), a promising player in cancer therapeutics targets transformed cells. The molecular mechanisms at play is yet to be defined. We hypothesize that reovirus implements its oncolytic properties by engaging the autophagic machinery. While autophagy inherently is a survival mechanism, it can also lead to programmed cell death if the autophagic machinery is unable to revive the cell. Based on KRAS status we analyzed the relation between REO infection and autophagy induction in colorectal cancer (CRC) cell lines and syngeneic mouse models.

Methods: Six CRC cell lines HCT116, SW620, SW837 (KRAS Mutant), and HKE3, CACO2, LIM1215 (KRAS WT) were infected at 5 MOI (multiplicity of infection) of REO and harvested at 3, 6, 9, 12, 24, 48 and 72hours for Fluorescence Assisted Cell Sorting (FACS) post FITC labelled LC3 antibody staining, western blotting (WB) and quantitative Polymerase Chain Reaction (qPCR). Allograft animal tissue CT26 (KRAS Mutant) and MC38 (KRAS WT) post REO (1x 106 virus particles daily intra-tumoral) treatment were analyzed by WB and qPCR.

Results: WB demonstrated increased LC3A/B expression in REO treated KRAS mutant cells as compared to the wild type at 6 hours (2.9 fold) and 12hrs (1.98 fold) (p<0.05). FACS indicated FITC positive cells expressing LC3A/B were 1.81-fold more in REO treated KRAS mutant cells at 6 to 9 hrs as compared to KRAS wild type (p=0.037). Kinetics of LC3A and LC3B are well studied in autophagy and induction is marked by an increase in total protein with conversion from LC3A to LC3B. WB on protein isolated from CT26 mice indicated significant decrease in LC3A on treatment with REO (p=0.04). qPCR analysis of REO treated CT26 and MC38 indicated similar trend with significant decrease in LC3A in CT26 (0.069-fold, p=0.05) compared to MC38 (0.93-fold, p=0.92). Further, 12 genes from a panel of 88 autophagy related genes namely APOL8, ATG4B, ATG4C, ATG4D, ATG5, BCL2, EIF4EBP2, FKBP12, LAMP3, RAPTOR, SNX30, ULK3 showed significant reversal of expression (p<0.05) between the two groups.

Discussion: Our preclinical models confirmed an overall induction in expression of LC3, a protein integral to the autophagic pathways, upon REO infection with significant upregulation under KRAS mutant condition. Further analysis of different genes involved in autophagy is underway to understand shifts in autophagy's dual role at different time points and/or conditions when subject to REO exposure.

#4271

Induction of STK11-dependent cytoprotective autophagy in breast cancer cells upon Honokiol treatment.

Nethaji Muniraj,1 Marey Shriver,1 Arumugam Nagalingam,1 Sumit Siddharth,1 Sheetal Parida,1 Neeraj K Saxena,2 Dipali Sharma1. 1 _Johns Hopkins Sidney Kimmel Comp. Cancer Ctr., Baltimore, MD;_ 2 _National Cancer Institute, Rockville, MD_.

Background and Aim: Honokiol, a natural phenolic compound isolated from an extract of seed cones from Magnolia grandiflora, is widely known for its therapeutic potential as an antioxidant, anti-inflammatory, antithrombosis, and anti-depressant agent. Our recent studies show that honokiol impedes breast carcinogenesis. Cancer cells undergo cytoprotective autophagy and evade chemotherapy therefore many clinical trials are investigating the efficacy of autophagy inhibition in combination with chemotherapy. In the present study, we investigated the involvement of autophagic process in honokiol-mediated functional networks in breast cancer and explored the efficacy of combination regimens involving honokiol and chloroquine.

Methods: Autophagy studies were conducted utilizing immunoblot, RT-PCR, immunofluorescence analyses, confocal imaging and transmission electron microscopy for autophagy markers. Autophagic flux was analyzed using a plasmid tfLC3B and acridine orange staining. The fusion of autophagosome and lysosome was examined by using GFP-LC3/LysoTracker-red. Functional impact of autophagic process was evaluated using genetic knockout (KO) of AGT7 and BECN1 in MCF7 cells as well as combined treatment with autophagy inhibitors (3-MA, Baf-1 and CQ) and honokiol. Alterations in ATP levels were measured by ATPliteTM luminescence assay. In vivo studies using mammary gland implantation of cancer cells in NOD-SCID mice were conducted to evaluate the efficacy of combination regimen of Honokiol and Chloroquine.

Results: We found that Honokiol induces autophagy flux, increases accumulation of autophagosomes and elevates LC3B-II-conversion. Utilizing tandem-mCherry-GFP-LC3B assay and LysoTracker Red-staining, we observed that honokiol increases the autophagosome/lysosome fusion in breast cancer cells. We found that Honokiol induces autophagic response in a STK11-dependent manner as STK11-null breast cancer cells do not exhibit LC3B-II-puncta in response to Honokiol. Next, we explored the functional impact of autophagy in Honokiol mediated breast cancer inhibition and found that inhibiting autophagosome formation, abrogating autophagosome-lysosome fusion or genetic-knockout of BECN1/Beclin1 and ATG7 effectively increases the efficacy of Honokiol. These results clearly showed the cytoprotective nature of Honokiol-mediated autophagy and put forth the notion that a combined strategy of autophagy-inhibition with Honokiol would be more effective. Indeed, our in vivo studies showed that a combined treatment with Honokiol and Chloroquine can effectively inhibit primary tumor growth as well as metastatic progression.

Conclusion: Together, these results implicate that honokiol is a potent inducer of cytoprotective autophagy and a combined treatment of honokiol and chloroquine is a promising therapeutic strategy for breast cancer.

#4272

Suppression of epigenetic regulator NURF leads to autophagy mediated chemo-and immune sensitization of triple negative breast cancer.

Liliya Tyutyunyk, David A. Gewirtz, Joseph Landry, Nga Dao. _Virginia Commonwealth University, Richmond, VA_.

Dysregulation of the epigenome is implicated in initiation and progression of variety of cancers and their acquired resistance to chemotherapy. As such, targeting epigenetic regulators has the potential to modulate tumor cell biology and reestablish tumor cell sensitivity to chemotherapy and/or radiation. Our studies demonstrate that silencing of the epigenetic regulator Nucleosome Remodeling Factor (NURF) sensitizes breast tumor cells to chemotherapy and enhances the anti-tumor immune response. Compared to controls, NURF KD 4T1 breast tumor cells exposed to doxorubicin (Dox) show increased DNA damage (gamma H2AX staining) and autophagy (acridine orange staining) in vitro and enhanced growth inhibition both in vitro and in vivo. NURF KD also sensitizes 4T1 cells to doxorubicin stimulated Natural Killer (NK) cell antitumor activity ex vivo, which is associated with a cytokine/chemokine secreted by doxorubicin exposed NURF KD cells; potential candidates are CCL5, TNFa and CCL2. The importance of NK cells for the enhanced growth control of doxorubicin treated NURF KD cells was confirmed using a mAb depletion approach. Autophagy may be instrumental in NK cell activation and elimination of tumor cell targets by mediating cytokine secretion. Sensitization of NURF KD 4T1 cells to doxorubicin, both increased autophagy and enhanced NK cell activity, were also observed using a small molecule inhibitor of NURF, suggesting that NURF can also be targeted pharmacologically. Our studies suggest that enhanced doxorubicin induced DNA damage and autophagy (cell autonomous effects) and NK cell cytotoxic activity (cell non-autonomous affects) may be primary contributors to immune sensitization in NURF KD cells. Increased cell autonomous antitumor effects by doxorubicin in concert with increased cell non-autonomous immunogenicity could help to achieve tumor regression, reduce metastasis, and possibly promote long term remission in breast cancer.

#4273

Inhibition of autophagy increases HNSCC sensitivity to cancer therapies.

Yong-Syu Lee, Justin Skiba, Jaimee Kubatzke, Kwangok P. Nickel, Randall Kimple. _University of Wisconsin-Madison, Madison, WI_.

Background: Radiation and EGFR-targeted therapies are commonly used in the treatment of head and neck squamous cell carcinoma (HNSCC). These treatments fail to control a significant number of cancers resulting in a 5-year survival rate that remains around 40-50%. We have shown that both cetuximab and radiation induce autophagy, a pro-survival cellular stress response, in head and neck cancer. In this study, we examine the consequence of autophagy inhibition and investigate the molecular mechanism underlying therapy-induced autophagy.

Methods: Autophagy was assessed using a nano-Luc LC3 reporter (Promega), immunofluorescence for LC3, p62, and acridine orange in HNSCC cell lines. RNAi knockdown of EGFR and LAPTM4B were used to test the involved signaling molecules. Radiation was delivered using a RS225 cabinet irradiator at a dose rate of approximately 3 Gy/min with dose validation by TLD using custom phantoms. Cetuximab was delivered via intraperitoneal injection. Vps34 inhibitor SAR405 and ULK1 inhibitor SBI-0206965 were used to determine whether inhibition of autophagy reduces cell survival or represses cancer cell growth in the clonogenic assay. A flank xenograft model using A253 cells was used to test the combination of autophagy inhibitors and current therapies in vivo.

Results: As previously shown, both cetuximab and radiation induced autophagy by two times. Knockdown of EGFR and LAPTM4B decreased autophagy (62.5% and 65%, respectively) when assessed using the nano-Luc reporter assay. Similar results were seen using IF. Using a clonogenic survival assay, the combination of SAR405 and radiation resulted in complete loss of cell survival suggesting a radiosensitizing effect. In vivo, SAR405 treatment improved tumor control when combined with radiation or cetuximab when compared to either treatment alone.

Conclusions: Therapy induced autophagy is dependent upon expression of both EGFR and LAPTM4B. Inhibition of autophagy resulted decreased cell survival in vitro and resulted in decreased in vivo tumor growth. These results suggest that inhibition of autophagy may be a viable approach to sensitize HNSCC to anti-cancer treatments.

#4274

RNA methylation regulates osteosarcoma growth and progression via autophagy.

Panneerdoss Subbarayalu, Pooja Yadav, Manjeet Rao. _Greehey Children's Cancer Research Inst, UT Health San Antonio, San Antonio, TX_.

Osteosarcoma (OS) is the most common primary bone tumor and third most common cancer in children/teens, after lymphomas and brain tumors. OS is treated by surgery and multi-modal chemotherapy. Even though this treatment regimen has improved the 5-year survival rate to 60-70%, once osteosarcoma metastasize to lung and other bones, the survival is significantly reduced. Moreover, the quality of life for patients who do survive is often substantially reduced due to the toxicity associated with the chemotherapy. Since several decades, there hasn't been any improvement in the osteosarcoma treatment outcome. Therefore, it is important to understand the molecular mechanism of osteosarcoma growth and metastasis and an urgent need to develop effective drug, which can be safe and effective for treating OS patients. We discovered that RNA methylation may regulate OS growth and progression by affecting autophagy. Our studies revealed that RNA demethylase AlkB homolog 5 (ALKBH5) supports OS viability, migration and invasion as well as tumor growth. We discovered several autophagy related genes were significantly altered in ALKBH5-silenced OS cells compared to scrambled control. Importantly, levels of LC3, which is a universal marker for monitoring autophagy, was significantly increased in ALKBH5 depleted OS cells. Furthermore, we found that autophagy initiation complex protein ULK1 level was drastically increased in ALKBH5-silenced cells when compared to scrambled control. To further establish the role of autophagy in osteosarcomogenesis, we treated OS cells with chloroquine, which is a well-known autophagy inhibitor and is currently being investigated for treating adult cancers in clinical trials. Interestingly, silencing of ALKBH5 significantly improved the efficacy of chloroquine as revealed by drastically reduced viability and invasive ability of OS cells. In conclusion, our study is first to shows that RNA methylation plays a critical role osteosarcoma growth and progression by regulating autophagy.

#4275

Gastric cancer stem cells under starvation stress might sustain their stemness via autophagy system.

Shingo Togano. _Osaka City University, Osaka, Japan_.

Background: Cancer stem cells (CSCs) play an important role for the progression of carcinoma. And, CSCs have a potential of resistance to various stresses such as starvation and chemotherapy. Autophagy is an intracellular degradation system which is induced various kinds of cells under various stress. In cancer cells, autophagy is activated and has involved in cancer survival. However, the significance of autophagy in CSCs has still remained to be unknown, especially in starvation and chemotherapy stress. The aim of this study is to clarify the role of autophagy in CSCs under starvation condition.

Materials and Methods: Two scirrhousgastric cancer cell lines, OCUM-2MD3 and OCUM-12 (parent cells) were used. Side population cells (SP cells) which include many CSCs, was known to the fraction of low dyeability of Hoechst 33342 identified by FACScan. Stemness of SP cells, OCUM-2MD3/SP and OCUM-12/SP, were examined by sphere colony assay and RT-PCR using stemness markers, Nanog, Oct3/4, CD44. Then we examined the effect of autophagy in CSCs under starvation condition, as follows.Parents cells and SP cells were evaluated about autophagy. Parents cells and SP cells were incubated in the presence and absence of glucose for various time. The expression level of autophagy molecule, LC3 was examined by western blotting. The effect of an autophagy inhibitor, Chloroquine, to SP frequency was examined by FACScan.

Results: The expression of Nanogand Oct3/4, were significantly higher in OCUM-2MD3/SP rather than parent cells OCUM-2MD3. Also, these stemness markers were significantly higher in OCUM-12/SP cells rather than parent cells. The number of sphere colony was greater in both SP cells rather than that in parent cells. The expression level of LC3 was high in both SP cells, in comparison to that in parent cells under glucose deficient condition.The autophagy inhibitor,Chloroquine, significantly decreased the SP fraction in both cells.

Conclusion: CSCs might have high autophagy activity under starvation stress and sustain their stemness by autophagy system. The autophagy inhibitor,Chloroquine, might be a promising agent for gastric cancer.

#4276

Inhibition of USP14 decrease tumorigenesis via autophagy in lung cancer.

MINSOO PARK, Min Ji Kim, Peter Chang Whan Lee. _Biomedical Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea, Seoul, Republic of Korea_.

The ubiquitin proteasome system (UPS) is the primary pathway through which the levels of regulatory proteins are controlled. Dysregulation of components within the UPS have been linked to both hereditary and sporadic forms of cancer. The deubiquitinating enzyme USP14 is associated with proteasome where it is trimming ubiquitin chain on the substrate. In our current study, we unexpectedly found that USP14 levels were elevated in lung cancer patients. USP14 inhibitor, IU1-47 and siUSP14, significantly decreased cell proliferation, migration, and invasiveness in lung cancer cells. Inhibition of USP14 induced the alteration of LC3-I to LC3-II which leads to autophagic cell death not but apoptosis. Thus our finding suggests that USP14 plays a crucial role in tumorigenesis and USP 14 inhibitor is a potent drug for lung cancer treatment.

#4277

Inhibition of autophagy (Plac8) prevents malignant transformation of cadmium induced prostate carcinogenesis.

Venkatesh Kolluru, Ashish Tyagi, Balaji Chandrasekaran, Murali K. Ankem, Chendil Damodaran. _Univ. of Louisville, Louisville, KY_.

Introduction: Cadmium (Cd) exposure is associated with an increased risk of prostate cancer (CaP). Previously we reported that Cd induces Plac8 expression may be responsible for defective autophagy which facilitates the transformation of prostate epithelial cells. The goal of this study is to decipher the molecular signaling responsible for defective autophagy by plac8 and whether inhibition of plac8 facilitates pro-apoptotic machinery in chronically exposed -Cd in prostate epithelial cells.

Methods: Overexpression or silencing Plac8 in chronically exposed Cd in RWPE-1 cells or cadmium, transformed prostate epithelial (CTPE) cells were subjected to cell viability, apoptosis, autophagy functional studies, Western blot and in vivo analyses. For statistical analysis, data were analyzed using Student's't' test with a p-value less than 0.05 considered significant.

Results: Our results suggest that during cellular transformation, Cd exposure induced Plac8 expression, which caused defective-autophagy that resulted in the proliferation of damaged cells. Inhibition of Plac8 resulted in suppression of pro-survival machinery and concomitantly induced severe endoplasmic reticulum (ER) stress that resulted in caspase-mediated cell death in Cd-exposed cells. More specifically, inhibition of Plac8 by shRNA resulted in induction of ER stress markers (PREK/ATF4) which facilitate caspase-mediated cell death in Cd- transformed prostate epithelial cells. Activation of NFKB is one of the downstream events of Plac8 that transcriptionally regulates NFKB activation in Cd-transformed cells. We identified, Plac8 binding sites in NFKB promoter. Hence inhibition of plac8 resulted in the complete shutdown of NFKB mediated pro-survival signaling in Cd-transformed prostate epithelial cells. While analyzing autophagy signaling network, we found induction of Atg family proteins, however, in the absence of Plac8, the induction of autophagy fails to protect the Cd-transformed cells. Similar results were observed in plac8 silenced cd-transformed cells in xenotransplanted mice, where Cd-transformed cells induced aggressive tumors, and Plac8 knockdown significantly inhibited the tumor growth. While ATF4, PLac8, NFKB were not expressed in normal human prostate tissues, their expression was significantly increased in BPH followed by CaP specimens; normal prostate < BPH << Gleason 6-7 CaP < Gleason 8-10 CaP. Cd levels were much higher in tumor tissues (21.43 μg/g) of CaP patients than in healthy controls (1.12 μg/g). The levels also correlated with Plac8 expression. These results demonstrate the clinical significance of Cd levels and of Plac8 expression as plausible drivers of Cd-induced transformation in prostate carcinogenesis.

Conclusions: These results suggest that Plac8 may be an essential component in the Cd-induced transformation of normal prostate epithelial cells to a cancerous state.

#4278

A comparison of drug sensitivity in isogenic tumor cell lines confirms that cytoprotective autophagy confers intrinsic resistance to cisplatin.

Jingwen Xu,1 Nipa Patel,2 Tareq Saleh,2 Yingliang Wu,1 Santiago Lima,2 David A. Gewirtz2. 1 _Shenyang Pharmaceutical University, China;_ 2 _Virginia Commonwealth University, Richmond, VA_.

Although autophagy inhibition often increases tumor cell sensitivity to chemotherapy and radiation, these observations do not provide definitive proof that autophagy is actually an intrinsic mechanism of resistance since virtually all tumor cells undergo autophagy in response to therapy. To directly address whether autophagy induction can confer chemoresistance, the current studies utilized a paired set of isogenic cell lines in which (cisplatin-induced) autophagy was either cytoprotective (crp53 cells where p53 was knocked out) or nonprotective (parental p53 wt cells) in function, but where the overall extent of autophagy was essentially identical. Cisplatin sensitivity was significantly lower in the crp53 cells where cisplatin-induced autophagy was protective compared to the parental p53 wt cells where cisplatin-induced autophagy was nonprotective, in support of the premise that cytoprotective autophagy could confer a relative degree of intrinsic resistance to chemotherapy. Accordingly, we can draw the following conclusions: (i) radiation and chemotherapy can promote both protective and nonprotective autophagy in tumor cells; (ii) although p53 can act as an autophagic switch, it cannot be reliably predicted whether autophagy will be protective or nonprotective based solely on p53 function; and (iii) even in the case(s) where autophagy is cytoprotective, it cannot be assumed that autophagy will confer drug or radiation resistance, but only that autophagy inhibition may, in select cases, increase drug or radiation sensitivity. These findings highlight the complexity of efforts to exploit autophagy inhibition as a therapeutic strategy to enhance tumor response to chemotherapy and radiation.

#4279

Heterogeneous LC3A expression regulates lung cancer cell plasticity.

Chia-Cheng Miao, Wen Hwang, Yu-Ting Chou. _National Tsing Hua University, HsinChu, Taiwan_.

Tumor metastasis is considered as the main cause that contributes to high mortality during lung cancer progression. Autophagy is a self-cleaning process to maintain cell integrity of intracellular organelles and proteins. To date, the role of autophagy in lung tumor metastasis remains elusive. Here, we report that lung tumors display a heterogeneous expression pattern of the autophagy mediator LC3A, the high expression of which is associated with low metastasis risk. We found a group of lung cancer cells contain differential LC3A expression levels and exhibit plasticity characterized by distinct proliferative and invasive properties. Immunofluorescence staining and immunoblotting assays revealed that LC3A-mediated autophagy is more active in highly proliferative but minimally invasive lung cancer cells than their lowly proliferative but highly invasive counterpart. Clonogenic analysis and cell cycle assays showed that LC3A silencing attenuated cellular growth, causing G1/S cell cycle arrest in highly proliferative lung cancer cells. Highly proliferative lung cancer cells exhibited a higher oxygen consumption rate, accompanied with an elevated oxygen species (ROS) level, compared to their lowly proliferative counterpart. Pharmacological inhibition of autophagy with chloroquine promoted the ROS production in highly proliferative lung cancer cells but not in the highly invasive counterpart. SOX2 expression enhanced LC3A expression and promoted proliferation but inhibited invasiveness in lung cancer cells. Knockdown of LC3A expression in SOX2-high proliferative cells enriched SOX2-low cells, exhibiting decreased proliferation but increased invasiveness. Combined, our findings provide substantial evidence to suggest that LC3A cooperates with SOX2 signaling to regulate lung cancer cell plasticity.

#4280

The cycloartane triterpenoid ADCX impairs autophagic degradation through Akt overactivation and promotes apoptotic cell death in multidrug-resistant HepG2/ADM cells.

NAN YAO,1 Haiyan Sun,2 Maohua Huang,1 Wencai Ye,1 SiBao Chen,2 Dong-Mei Zhang1. 1 _Jinan University, Guangzhou, China;_ 2 _Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China_.

Multidrug resistance is the main obstacle in cancer chemotherapy. Emerging evidence has demonstrated the important role of autophagy in cancer cell resistance to chemotherapy. Therefore, autophagy inhibition may be a promising strategy for conquering drug resistance in liver cancer cells.

Methods: HepG2/ADM cells were treated with ADCX in the presence or absence of relevant inhibitors, apoptosis was measured by flow-cytometry and expression of autophagy and apoptosis related proteins was detected by western blotting. Confocal microscope was used for GFP-LC3 assay, tandem mRFP-GFP-LC3 assay, DQ-BSA method and colocalization of GFP-LC3 and LAMP1. Celluar ultrastructure was observed by transmission electronic microscopy.

Results: ADCX, a natural cycloartane triterpenoid extracted from the traditional Chinese medicine (TCM) source Cimicifugae Rhizoma (Shengma), inhibited autophagic degradation by impairing the maturation of lysosomal cathepsins in multidrug resistant liver cancer HepG2/ADM cells, thereby leading to autophagic flux inhibition. Interestingly, Akt was over-activated by ADCX treatment, which was associated with impairment of cathepsin maturation. Moreover, impairing autophagic degradation promoted ADCX-induced apoptotic cell death in HepG2/ADM cells.

Conclusions: Our study indicates that impairing autophagic degradation may be an effective approach for overcoming resistance and also suggests a novel role for Akt in autophagic degradation.

#4281

LT-IIc, a bacterial type II heat-labile enterotoxin, induces specific lethality in triple negative breast cancer cells by modulaton of autophagy and induction of apoptosis and necroptosis.

Patricia A. Masso-Welch,1 Sofia Girald Berlingeri,1 Natalie D. King-Lyons,1 Lorrie Mandell,1 John C. Hu,1 Christopher Greene,1 Matthew Federowicz,2 Peter Cao,2 Yasser Heakal2. 1 _State University of New York, Buffalo, NY;_ 2 _D'Youville College, Buffalo, NY_.

Despite the recent advances in breast cancer treatment, triple negative breast cancer (TNBC) remains a serious health problem with limited treatment options. Poor prognosis is due to the development of chemoresistance. To discover novel therapeutic approaches to treat TNBC, we screened cholera toxin (CT) and the members of the bacterial type II heat-labile enterotoxin family (LT-IIa, LT-IIb, and LT-IIc) for their capacity to induce cell death in TNBC cells. LT-IIc, but not the other toxins, significantly reduced viability of the TNBC cell lines BT549 and MDA-MB-231 (IC50 = 82.32 nM). LT-IIc had no significant cytotoxic effects on MCF10A (IC50 = 2600 nM), a non-tumorigenic breast epithelial cell line, and minimal effects on MCF7 and T47D, ER+ breast cancer cells, or SKBR-3, HER2+ cells. LT-IIc stimulated autophagy through inhibition of the mTOR pathway, while simultaneously inhibiting autophagic progression, as observed by the accumulation of LC3B-II and p62 proteins. Morphologically, LT-IIc induced accumulation of enlarged LAMP2+ autolysosomes selectively in TNBC cells. Bafilomycin A1, an inhibitor of autophagic flux, blocked the formation and/or retention of these autolysosomes. The increase in caspase 3, 7 activity and annexin V staining indicated that LT-IIc induced an apoptotic response. Co-treatment with necrostatin-1, however, demonstrated that the lethal response to LT-IIc is elicited, at least in part, by concomitant induction of necroptosis. Collectively, these experiments demonstrate that LT-IIc acts bifunctionally, inducing autophagy while simultaneously blocking autolysosomal progression in TNBC cells, resulting in specific cytotoxicity in this breast cancer subtype.

#4282

Crosstalk between apoptosis and autophagy induced by docetaxel-magnetic nanoparticles with different surface in prostate cancer cells.

Masatoshi Watanabe,1 Sanai Takahashi,2 Eri Usugi,1 Kenichiro Ishii,1 Yoshifumi Hirokawa1. 1 _Mie University, Tsu, Japan;_ 2 _Yokohama National University, Yokohama, Japan_.

Although docetaxel-based chemotherapy prolongs life and relieves symptoms in patients with castration resistant prostate cancer (CRPC), this needs to be improved because of limitations such as systemic toxicity, and progression of docetaxel-resistance. Nanotechnology including nanoparticles has served a new role in medical sciences, leading to theranostics. Fe3O4 magnetic nanoparticles (MgNPs) have potential applications in drug delivery, cancer diagnosis and hyperthermia. We have already reported that MgNPs would modify the effect of docetaxel (DTX) in prostate cancer cells by oxidative DNA damage in prostate cancer cell lines. Combined treatment of docetaxel and MgNPs has also shown to induce apoptosis and autophagic cell death in DU145 cells. In this study, we analyzed the combined effect of DTX-carboxyl-modified MgNPs (MgNPs-COOH) on DU145 cells in a view of crosstalk between apoptosis and autophagy. The combination treatment of DTX and MgNPs-COOH more effectively inhibited cancer cell growth and induced apoptosis and autophagic cell death although MgNPs-COOH did exert no ROS production. This combination preferred autophagic cell death than apoptosis. These results suggest that MgNPs-COOH may induce different responses compared with MgNPs, and result in favorable development of current chemotherapy for CRPC. 

### Cell Signaling 2

#4283

The tumor suppressive effects of HPP1 in colorectal cancer are mediated by its EGF-like domain.

Abul Elahi,1 Abidemi Ajidahun,1 Mazher S. Hussain,1 Leah Hendrick,1 Irina Getun,1 Andreas Becker,2 Yan Yang,2 Evan S. Glazer,1 David Shibata1. 1 _University of Tennessee Health Science Center, Memphis, TN;_ 2 _H. Lee Moffitt Cancer Center, Tampa, FL_.

Introduction: We have previously demonstrated that HPP1 is an epigenetically-silenced tumor suppressor gene that exerts its effects via an erbB4-JAK-STAT signaling pathway. Although identified as a secreted multi-moiety transmembrane protein, the specific component responsible for its growth suppressive function has not been elucidated. Notably, HPP1 contains a single Epidermal Growth Factor (EGF)-like domain that differs from EGF by the substitution of arginine (R) for histidine (H) at the critical 299 position. We sought to investigate the isolated biologic effects of HPP1's EGF-like domain.

Methods: Synthetic wild type (WT; H299) and mutant (MUT; R299) peptides were prepared at our crystallographic core facility. The change in amino acid did not alter the predicted 3D structure of the peptide. A total of 50ng/ml of each peptide per well (6-well plate) was used to treat the HPP1 non-expressing HCT116 colon cancer cell line. Cells were starved for 24 hours with a peptide exposure time of 7 minutes. We subsequently examined alterations in relevant erbB-, JAK- and STAT-family proteins as well as associated changes in cell growth in soft agar.

Results: Treatment of HCT116 with WT peptide resulted in increased phosphorylation of erbB4, JAK1, JAK2, STAT1 and STAT2 with a concomitant downregulation of STAT3 activation as compared to untreated controls. Mutant peptide delivery did not result in any alterations in erbB4-JAK-STAT signaling components. WT peptide treatment resulted in a dramatic reduction in colony formation in soft agar as compared to mutant peptide-treated (p=0.0002) and control cells (p=0.0019). Significant increase in colony formation was observed in the mutant vs. control cells (p=0.0047).

Conclusion: The isolated effects of HPP1's EGF-like domain mimic the observed signaling and growth suppressive alterations observed with full-length HPP1. This EGF-like domain along with its histidine residue at position 299 appear critical for HPP1 downstream signaling and its tumor suppressive effects. Further elaboration of these findings may have therapeutic implications.

#4284

Targeting notch signaling in glioblastoma cancer stem cells through modulation of Connexin43 function.

Michael Lunski,1 James Smyth,2 Jennifer Vaughn,3 Zhi Sheng,2 Robert Gourdie,2 Benjamin Purow,4 Samy Lamouille2. 1 _Virginia Tech Carilion Clinic, Ronaoke, VA;_ 2 _Virginia Tech Carilion Research Institute and School of Medicine, Ronaoke, VA;_ 3 _Virginia Teach Carilion School of Medicine, Ronaoke, VA;_ 4 _University of Virginia School of Medicine, Charlottesville, VA_.

Glioblastoma (GBM) is a highly malignant and lethal cancer of the central nervous system for which there has been limited progress in improving patient outcomes despite intensive research efforts. The current multimodal therapy for newly diagnosed GBM patients includes surgical resection, radiotherapy and cytotoxic chemotherapy with temozolomide (TMZ), conferring a median survival time of just 14.6 months. Failure to generate more effective treatment strategies is due to 1) the infiltrative nature of GBM tumor cells preventing complete surgical resection, and 2) the cellular heterogeneity within GBM tumors, which often comprise a sub-population of GBM cancer stem cells (GSCs) characterized by self-renewal characteristics and resistance to chemotherapeutic alkylating agents including TMZ. Multiple signaling pathways, including Notch, participate to the formation and maintenance of GSCs. Recent studies, including our research, have shown that increased levels of gap junction protein Connexin43 (Cx43) correlate with TMZ resistance in GBM cells and inversely correlated with GBM patient survival. Importantly, Cx43 has also been associated with anti-proliferative effects in glioma, and reduced levels of Cx43 protein occur in high-grade gliomas. Therefore, we hypothesized that altering Cx43 localization and/or activity rather than Cx43 expression represents a potent strategy for GBM treatment. Regulating Cx43 function is primarily associated with the multiple sites for post-translational modifications and protein-protein interactions within the Cx43 carboxy-terminus. Our research identifies crosstalk between Cx43 and Notch signaling in GSCs. Using a Cx43 carboxy-terminus mimetic peptide that modulates non-junctional functions of Cx43, we observe a decrease in Notch1 protein expression and reduced transcription of its downstream targets Hes1 and Hey1 in GSCs derived from patient tumors. Most importantly, our Cx43 mimetic peptide decreases cell survival in TMZ-resistant GSCs, limits GSC neurosphere formation in vitro, and inhibits GSC-derived tumor growth in vivo. Our current research aims at dissecting the molecular mechanisms of Cx43 functions on Notch signaling in GSCs using this Cx43 mimetic peptide. In conclusion, we have identified a novel therapeutic opportunity to decrease the tumorigenic potential of GSCs through altering Cx43 activity and Notch signaling to target chemoresistant GSCs in GBM treatment.

#4285

p66Shc/ROS enhances the progression of androgen-sensitive towards castration-resistant prostate cancer cells.

Dannah Miller, Matthew Ingersoll, Arpita Chatterjee, Rebecca Oberley-Deegan, Ming-Fong Lin. _University of Nebraska Medical Center, Omaha, NE_.

Purpose and Background: Prostate cancer (PCa) is the second leading cause of cancer-related death in United States men. Androgen deprivation therapy is the standard-of-care treatment for metastatic PCa; most patients eventually relapse and develop castration-resistant (CR) tumors that currently have no effective treatment. Thus, a useful cell model for analysis of the molecular mechanism of PCa progression is required for developing targeted therapies for CR PCa. In this study, we established a PCa cell progressive model in three independent cell lines, of which androgen-independent (AI) cells were derived from respective androgen-sensitive (AS) cells, recapitulating clinical PCa progression.

Experimental Methods: AS and AI human prostate adenocarcinoma cell lines LNCaP, MDA PCa2b and VCaP were utilized in this study to demonstrate the molecular and signaling alterations seen in CR PCa. Stable p66Shc cDNA transfected subclones were established from LNCaP-AS cells. The tumorigenicity of AS and AI cells as well as AS PCa cells treated with H2O2 and NAC were evaluated via trypan blue exclusion, transwell migration, clonogenic assays and xenograft mouse models. The signaling profiles including phosphoprotein microarray and immunoblotting were conducted.

Results: AI PCa cells have enhanced tumorigenicity, recapitulating the clinical CR phenotype, including AR expression, proliferation and tumorigenicity under androgen-deprived conditions. Further, AI cells exhibit increased ROS levels as well as enhanced signaling of proliferation and survival pathways. We further identified oxidase p66Shc as one of the potential sources of the ROS-mediated phenotypic and cell signaling alterations in AI PCa cells. LNCaP-AI cells and p66Shc subclones have a greater oxidative environment compared to LNCaP-AS cells. Increased ROS via H2O2 enhanced AS cell growth and migration, which was counteracted by antioxidant NAC. Treatment of LNCaP-AS cells with H2O2 resulted in a similar signaling profile to that of LNCaP-AI or p66Shc subclone cells. Further, the ROS-driven alterations of p66Shc subclone cell signaling can be mitigated via p66Shc knockdown or inactive p66Shc mutant. Moreover, LNCaP-AI cells and p66Shc subclones, but not LNCaP-AS cells, develop xenograft tumors with metastatic nodules. Molecular profiling showed that alterations of signaling in LNCaP-AI cells and p66Shc subclones attributed to p66Shc/ROS.

Conclusions: We report the establishment of a PCa cell progression model in three commonly used PCa cell lines that replicates clinical PCa progression from the AS to the AI/CR phenotype. Altogether, the data shows ROS produced by p66Shc promotes PCa tumorigenicity and progression to the CR phenotype. Further characterization of the PCa progressive model will aid in the understanding of advanced PCa progression to help in treatment of this lethal disease.

#4286

TGFβ induced SMAD4 dependent apoptosis proceeded by EMT in colorectal cancer.

Pratheeshkumar Poyil, Abdul K. Siraj, Divya Sasidharan Padmaja, Sandeep Kumar Parvathareddy, Rong Bu, Tariq Masoodi, Yan Kong, Saravanan Thangavel, Saeeda Omer Ahmed, Nasser Al-Sanea, Luai H. Ashari, Alaa Abduljabbar, Samar Alhomoud, Fouad Al-Dayel, Khawla S. Al-Kuraya. _King Faisal Hospital and Research Center, Riyadh, Saudi Arabia_.

Colorectal cancer (CRC) is one of the leading cause of cancer-related deaths Worldwide. In Saudi Arabia, CRC is more aggressive and presents at younger age, warranting new treatment strategies. Role of TGFβ/Smad4 signaling pathway in initiation and progression of CRC is well documented. Current study examined the role of TGFβ/Smad4 signaling pathway in a large cohort of Saudi CRC, followed by in vitro analysis to dissect the dual role of TGFβ on inducing epithelial to mesenchymal transition (EMT) and apoptosis. In this study, we investigated Smad4 alterations and their association with clinicopathological outcomes in a large cohort of CRC samples using targeted capture sequencing, Fluorescent in-situ hybridisation and immunohistochemistry. Our study demonstrated high frequency of Smad4 alterations with low expression of Smad4 protein identifying a sub-group of aggressive CRC to be an independent marker for poor prognosis. Functional studies using CRC cells show that TGFβ induces Smad4 dependent EMT followed by apoptosis. Induction of mesenchymal transcriptional factors, Snail1 and Zeb1 was essential for TGFβ-induced apoptosis. Our results indicate that KLF5 acts as an oncogene in CRC cells regardless of Smad4 expression and inhibition of KLF5 is requisite for TGFβ-induced apoptosis. Furthermore, TGFβ/Smad4 signal inhibits the transcription of KLF5 that in turn switches Sox4 from tumor promoter to suppressor. A high incidence of Smad4 alterations were found in the Saudi CRC patients. Functional study results indicate that TGFβ induces Smad4 dependent EMT followed by apoptosis in CRC cells.

#4287

Peroxiredoxin 2 has a crucial role in the survival of gastric cancer cells by regulating β-catenin and TNF signaling.

Hee Sung Kim,1 Tae Hyeong Lee,2 Dong Hoon Kang,2 Peter Chang-Whan Lee,2 Byung Sik Kim1. 1 _Department of Gastric Surgery, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Republic of Korea;_ 2 _University of Ulsan College of Medicine, Seoul, Republic of Korea_.

Gastric cancer (GC) is globally the fifth most common cancer and third leading cause of cancer death. Functional activation of β-catenin is necessary for early events in gastric carcinogenesis and GC with mutation of APC gene, similar to colorectal tumorigenesis, occur during the early stages of gastric adenoma development. Mammalian 2-Cys peroxiredoxin (Prx) enzymes are overexpressed in most cancer tissues, but the effect of Prx2 expression on GC survival remains unclear. Here, we demonstrate that Prx2 deletion markedly reduces the active β-catenin levels and the expression of β-catenin target genes in GC cells. Furthermore, the TNF α-induced activation of ERK1/2 was inhibited and reciprocally, JNK phosphorylation and cleaved caspase3 was increased in Prx2-knockdown GC cells. Prx2 deletion also can suppress the viability, invasion and colony formation of GC cells. The proof-of-concept experiment using conoidinA as a Prx2 inhibitor shows it can significantly ablates the growth of 5-FU-resistant SNU620. Thus, our findings reveal that Prx2 is a key regulator of β-catenin and TNF signaling pathway in GC, and also suggest a pharmacologic strategy to effectively overcome drug-resistant GC.

#4288

Race specific differences in G-protein decoupling from CCR9 in prostate cancer cells contribute to the differences in docetaxel response.

Neeraj Kapur,1 Hina Mir,2 Shailesh Singh3. 1 _Neeraj Kapur, Atlanta, GA;_ 2 _Hina Mir, Atlanta, GA;_ 3 _Morehouse School of Medicine, Atlanta, GA_.

Current treatment approaches have failed to reduce racial disparity in treatment outcome of prostate cancer (PCa) primarily due to the race-specific difference in the biology of PCa. Therefore, identification of race-specific molecular differences is needed to address the disparity in treatment outcome. In this study, we have determined the race-specific differences in CCR9 signaling. Phospho-proteomic profile of key pro-survival molecules following CCL25 treatment in African American (AA) and European American (EA) PCa cells was determined using antibody microarray and validated by western blot analyses. Coupling and decoupling of G-protein(s) with CCR9 in response to CCL25, which determines downstream signaling of CCR9 was ascertained by immunoprecipitation assay (IPA). Cell viability and apoptosis in response to a taxane drug docetaxel (DTX), with/without CCL25 stimulation and CCR9-blockade, were examined using MTT and flow cytometry, respectively. Levels of pro-apoptotic proteins were determined by western blot analyses. Our data show differential G-protein(s) decoupling from CCR9 in AA and EA cells. CCR9-activation dissociated Gαi2/3/Gαq/ Gα13/Gβ1γ7 –proteins in AA cells compared to Gαs/Gβ2γ9 and Gα13/Gαs/G β1/β3γ7 in EA cells, respectively, contribute to different molecular mechanisms supporting cell survival in cell lines derived from two races. Race-specific G-protein signaling in PCa cells, following CCL25 stimulation, is reflected into hyper-activation of molecules involved in PI3K/Akt and MEK/Erk1/2 signaling, which are associated with PCa aggressiveness in AA cells compared to EA counterparts. Further, activation of CCR9-CCL25 axis significantly influences the therapeutic response of PCa cells against DTX, which is highly compromised in AA cells compared to EA cells following CCL25 stimulation. However, this protective effect of CCL25 was abrogated after CCR9 blockade. Inhibition of MEK/Erk1/2 axis significantly improved DTX-induced cell death in AA cells, suggesting the importance of this axis in reducing drug response. Further, stimulation with CCL25, exclusively downregulated cleaved caspase-7, 8 and Bim in response to DTX in AA cells. In all, differential decoupling of G-proteins from CCR9 controls the downstream signaling and therefore determines disparity associated with PCa outcome and offer novel race-specific therapeutic targets for PCa.

#4289

ERK3 negatively regulates IL-6/STAT3 signaling via SOCS3 in cancer cells.

Astha Shakya, Minyi Chen, Michael Markey, Weiwen Long. _Wright State University, Dayton, OH_.

Extracellular signal-regulated kinase 3 (ERK3) is an atypical mitogen activated protein kinase (MAPK) which is dysregulated in several cancers including lung cancer, breast cancer and melanoma. It has a single Ser-Glu-Gly (SEG) phospho-acceptor motif in its activation loop, instead of the Thr-Xaa-Tyr (TXY) motif conserved in the classical MAPKs. Moreover, compared to the conventional MAPKs, much less is known about the targets of ERK3 signaling. Hence, to elucidate the ERK3-regulated genes and pathways, we performed gene microarray analyses of both A549 lung cancer cells and A375 melanoma cells after treatment with either a siRNA specifically targeting ERK3 or a non-targeting control siRNA. Interestingly, IL-6 target genes were found to be highly upregulated upon ERK3 knockdown. Interleukin-6 (IL-6) is a pleiotropic cytokine which signals via the JAK/STAT3 pathway. Phosphorylation of STAT3 at Tyrosine 705 (Y705) by JAK activates STAT3 in response to IL-6 stimulation. IL-6 signaling is negatively regulated by a feedback inhibitor SOCS3 (Suppressor of cytokine signaling 3), which binds to and inhibits JAK kinase activity. Interestingly, we noted that ERK3 interacts with SOCS3 from a published Yeast-two-hybrid screening. We validated the interaction between ERK3 and SOCS3 by co-immunoprecipitation assay. In addition, we found that ERK3 downregulates the phosphorylation of STAT3 at Y705. In line with its negative regulation of oncogenic STAT3 activity, ERK3 inhibits melanoma cell growth, migration/invasion and colony formation in soft agar. Taken together, we have revealed a novel role for ERK3 in suppressing IL-6/STAT3 signaling via SOCS3 in cancer cells.

#4290

Simultaneous inhibition of Atypical Protein Kinase-C and mTOR impedes bladder canSimultaneous inhibition of atypical protein kinase-C and mTOR impedes bladder cancer cell progression of bladder cancer cell progression.

S M Anisul Islam, Rekha S. Patel, Raja Reddy Bommareddy, Tracess P. Smalley, Mildred Acevedo-Duncan. _University of South Florida, Tampa, FL_.

Despite enormous scientific advancements in cancer treatment, there is still so much to do to combat cancer particularly bladder cancer. Drugs once proved to be effective in treating bladder cancer showed reduced efficacy, hence, cancer recurrence rate is increasing. To overcome this situation, several attempts have been considered such as the development of a new effective drug or modify therapeutic regimens by combining two or more existing drugs. In recent years, atypical protein kinase C (aPKC), a phospholipid-dependent serine/threonine kinase, is considered as a central regulator of various cancer-causing signaling pathways and controls cell cycle progression, tumorigenesis, and metastatic determinant. In addition, the biologically important mammalian target of rapamycin (mTOR) pathway is altered in many cancers including bladder and can stimulate the activation of the aPKC pathway. In this study, we examined whether the concurrent inhibition of aPKC and mTOR using a combination of a novel aPKC inhibitor (ICA-I, an inhibitor of PKC-ι, or ζ-Stat, an inhibitor of PKC-ζ) and rapamycin blocks bladder cancer progression. The cell lines tested were MC-SV-HUCT2 normal bladder and TCCSUP bladder cancer cells. Our observed data showed that the combination therapy induced a significant reduction of human bladder cancer cells' viability, also, individual treatment of aPKC inhibitor and rapamycin brought a similar effect. Moreover, the concurrent inhibition of aPKC and mTOR retards the metastasis of bladder cancer cells. These findings indicate that the administration of aPKC inhibitor together with rapamycin would be a useful therapeutic option in treating bladder cancer.

#4291

p85α alters Gab2 phosphorylation profile in ovarian cancer.

Xinran Li,1 Victor CY Mak,1 Annie NY Cheung,1 Gordon B. Mills,2 Lydia WT Cheung1. 1 _University of Hong Kong, Hong Kong;_ 2 _Oregon Health & Science University, Portland, OR_.

Dysregulation of phosphatidylinositol 3-kinase (PI3K), which is essential for various cellular processes, is a common event in cancers. PIK3R1 encodes the PI3K class IA regulatory subunit p85α, which binds the p110 catalytic subunit forming heterodimeric PI3K complex. The PI3K associates with receptor tyrosine kinase, for example through the docking protein Gab2, to initiate downstream signaling events. Downregulation of p85α is frequently seen in cancers. However, the signaling changes driven by the downregulation remain to be elucidated. Our data herein indicate that reduced p85α expression leads to changes in the phosphorylation status of Gab2 at multiple amino acid residues. PIK3R1 siRNA decreased the levels of phosphorylated Y452 (a site implicated in binding p85α) and S210 (a site that may mediate negative feedback of Gab2 activity). Meanwhile, we observed an increase in phosphorylated S623, which was shown to involve in STAT activation. These findings provide mechanistic insights on the pathway activation upon downregulation of p85α in cancer.

#4292

Molecular mechanism of APIO-EE-7 suppresses cell metastasis by targeting Aurora B in osteosarcoma.

Guoguo Jin, Jitian Li, Zhiping Guo, Junyan Teng, Xiaoling Li, Zhenjiang Zhao. _Hospital, Zhengzhou, China_.

Osteosarcoma is one of the most prevalent malignant bone tumors in children and adolescents and has the characteristics of early metastasis, rapid progression and poor prognosis. Although with the advancement in techniques and treatment increasing the survival rates up to 60%, the prognosis remains poor for most patients with metastatic or recurrent osteosarcoma. Identifying the molecular mechanism and developing therapeutic agents for metastasis is urgent. Aurora B kinase play a key role in mitosis and have been reported to be associated with cancer metastasis. However, the role of Aurora kinase in osteosarcoma is still unclear. In this study, we identified a novel compound, referred to as APIO-EE-7, which inhibits growth and colony formation and induces apoptosis in osteosarcoma. Using computer modeling, we found that APIO-EE-7 can interact with Aurora B at ATP-binding pocket. Treatment with APIO-EE-7 can increase EMT marker expression level of E-cadherin and decrease N-cadherin, vimentin and survivin expression. Importantly, APIO-EE-7 dramatically attenuated tumor incidence and volume in osteosarcoma metastasis mouse model. Our findings suggest that APIO-EE-7 is a potential therapeutic agent against osteosarcoma metastasis by targeting Aurora B, which provides hope for rapid clinic translation.

#4293

Analyzing the redox/fyn/c-Cbl pathway in glioblastoma multiforme: A mechanistic approach.

Neal Shah,1 Jennifer Stripay2. 1 _University of Rochester, Rochester, NY;_ 2 _St. Jude Children's Research Hospital, Memphis, TN_.

Glioblastoma multiforme (GBM) is the most common aggressive tumor of the central nervous system (CNS) with 10,000 newly diagnosed cases annually, and an incidence rate of 3 in 100,000 individuals in the United States. Unfortunately, the current standard of care of radiotherapy with concomitant Temozolomide (TMZ) has not been effective in consistently controlling tumor growth, as the median age of survival for patients is 14.6 months after treatment, with inevitable recurrence beyond this time. One of the difficulties that emerge when treating GBM is tumor heterogeneity. For example, GBM cells upregulate different receptor tyrosine kinases (RTKs). Signaling through RTKs enables these cells to maintain their proliferative status to ensure their survival. The E3 ubiquitin ligase c-Cbl is a negative regulator of RTKs, and our lab has found it to be physically sequestered by ARGHEF7 (Cool-1) in GBM. We identified a combination of drugs, a c-Cbl restorative agent (CRA01) and an oxidizing agent (Tamoxifen [TMX]), that decreased c-Cbl/Cool-1 complex formation and activated c-Cbl through the Redox/Fyn/c-Cbl pathway (RFC) in GBM cells. Treatment with CRA01+TMX led to degradation of RTKs. Little is known about the RFC pathway in cancer regulation or its ability to prevent cell growth in TMZ resistant GBM cells. Here, we examine ways to regulate c-Cbl in GBM cells and seek to identify the mechanism(s) resulting in c-Cbl/Cool-1 complex formation. Furthermore, we aim to use a mouse model of TMZ resistance to determine the effects of CRA01+TMX in vivo. These results will provide important knowledge on the RFC pathway that can help properly identify patients with GBM and different cancers with c-Cbl dysregulation for whom this treatment could prove beneficial.

#4294

C6 Ceramide potentiates paclitaxel and gemcitabine mediated antitumor effects against pancreatic cancer via inhibition of prosurvival PI3k/AKT/mTOR and ERK pathways.

Harold J. Wanebo. _Roger Williams Medical Center, Providence, RI_.

The 5% survival of pancreatic cancer emphasizes need for new treatment strategies. Our studies suggest potential value of C6Ceramide (C6Cer) as a chemotherapeutic adjunct. The current study demonstrates that combination chemotherapy of Paclitaxel with adjunct C6Cer significantly increased cell death (apoptosis) in pancreatic cancer cell lines (L3.6,PANC-1 , MIA (PaCa-2) in vitro as well as in vivo - enhancing tumor regression in SCID mice bearing L3.6 transplants. A similar synergistic effect was observed with addition of C6Cer to cetuximab which is not effective in KRAS mutant Pancreatic or Colorectal cancer and thus is not used clinically. At the molecular level combining C6Cer with Gemcitabine or Paclitaxel resulted in significant decreased p-AKT and p-GSK-a/b(PI3K/AKT (survival)pathway in L3.6 cells.

Conclusion: C6Cer significantly inhibited key cancer survival pathways resulting in significant synergism with chemotherapeutics in therapy of Pan Ca and KRAS mutant CRC.

#4295

Differential lipid raft composition correlates with altered oncogenic properties in the SW13 cell line.

Jacob Moore, Margaux Baatz, Luis Espejo, Kathryn J. Leyva, Elizabeth E. Hull. _Midwestern Univ. - Glendale Campus, Glendale, AZ_.

Introduction: Mutant GOF p53 is an epigenetic regulator which promotes oncogenesis.

Many cancers with a GOF mutation accumulate cholesterol and inhibition of cholesterol synthesis with statin treatment reverses some of the pro-oncogenic properties of GOF p53. Our central hypothesis is that lipid raft signaling is altered by cholesterol accumulation in cells expressing a GOF p53. To address the role of cholesterol in promoting oncogenesis, we use the SW13 cell line bearing a GOF p53 (H193Y). This cell line has two epigenetically distinct cell subtypes that differentially express genes coding for GPI anchors, cholesterol biosynthesis, and sphingolipids, all of which are components of lipid rafts. In addition, the SW13 subtypes have different oncogenic profiles. The SW13(Vim-) subtype has an epithelial morphology and is highly proliferative. The SW13(Vim+) subtype has a mesenchymal-like phenotype and a higher metastatic potential. When treated with histone deacetylase inhibitors (HDACi), SW13(Vim-) appear to adopt a SW13(Vim+) phenotype. Therefore, we treat with HDACi to control the subtype conversion and shRNA to knock-down GOF p53 expression. This experimental approach allows the composition of raft fraction to be correlated to GOF p53 expression and oncogenic properties of each SW13 subtype, with and without p53 expression.

Methods: Proliferation rate, MMP expression, and migration of SW13(Vim-), SW13(Vim+), shRNA SW13(Vim-), and shRNA SW13(Vim+) were quantitated by EdU assays and in situ zymography respectively. We have isolated lipid raft fractions by the detergent-resistant enrichment technique from SW13 cells of each subtype, with and without GOF p53 expression knock-down by shRNA. Proteins by mass spectrometry analysis. Fluorescent cholesterol assays and p53 western blots were used to quantify cholesterol levels and p53 protein expression, respectively.

Results: Data suggest that there is a shift from planar rafts in the SW13(Vim-) to caveolar rafts in the SW13(Vim+) line. Although the contribution of GOF p53 to this shift is under study, evidence suggests that raft composition is altered in the shRNA lines, in which GOF p53 has been knocked down by ~90%. In addition, p53 knock-down increases the proliferation rate in SW13(Vim-) (p = 0.01) but does not affect SW13(Vim+). In contrast, p53 knock-down decreases MMP activity in SW13(Vim+) (p = 0.05) but not SW13(Vim-).

Conclusions: Our data support the hypothesis that lipid raft signaling is significantly altered between the epigenetically distinct SW13 subtypes and that these differences may correlate to oncogenic potential. In addition, statin treatment, which reverses some of the pro-oncogenic properties of GOF p53, may do so, at least in part, by altering lipid raft composition and signaling.

#4296

Identification of cancer specific protein 80 (CSP-80), a novel 80 kDa MAP1a-like protein in ovarian carcinoma cells.

Ahmed Fadiel,1 Viswam S. Nair,1 Peter Aceto,1 Frederick Naftolin2. 1 _USF, Tampa, FL;_ 2 _NYU, NY, NY_.

Background: Although interfering with the actions of microtubule-associated proteins (MAP's) is successful against ovarian epithelial carcinoma (OVCA), knowledge of the MAP's in OVCA cells is lacking. We sought to identify cancer-specific MAP's in OVCA cells.Methods: Four human OVCA cell lines, primary OVCA tissue, ascitic cells and metastases from 9 patients and normal ovarian tissue from six women were compared to control human and rat brain tissue, uterine myomas (benign tumors) and rat and human non-neural organs. Modified Western blotting using panels of anti-microtubule-associated protein monoclonal antibodies were used. An in-vitro brain and OVCA mixing and recovery experiment was carried out to assess possible degradation of MAP1a by OVCA. A paclitaxel-driven tubulin assembly assay was performed to determine whether the OVCA protein has microtubule-associated protein properties. The transcriptome of CSP-80 was sequenced using primer extension sequencing.

Results: (1) In contradistinction to the 350 kDa MAP1a present in control brain extracts, only an 80 kDa molecule with MAP1a immune-reactivity was found in OVCA cell lines and tissues. No immune-reactivity was present in the negative controls. (2)Following the mixing experiments there was no increase of CSP-80; CSP-80 is not a degradation artifact of MAP1a. (3) The paclitaxel-driven tubulin assembly assay showed that CSP-80 bound to microtubules during tubulin polymerization. (4) No CSP-80 was identified in human or rat non-malignant tissues including normal human and rat brain or normal human ovary, placenta or uterine myomas, or normal rat liver, spleen or lung. (5) cDNA sequencing shows a 2603 bp fragment at a 5' terminal of p80 to be homologous with the analogous segment of MAP1a. (6) Further testing showed that CSP-80 was present in other reproductive and non-reproductive carcinomas. Again, no 350 kDa MAP1a was present. Studies are underway to determine whether CSP-80 is a mutated protein or the product of post-transcriptional modification.

Conclusion: CSP-80 is a novel cancer-related protein that appears to be limited to an 80 kDa sequence of the MAP1a gene, or a post-transcriptionally modified product. Since CSP-80 exhibits tubulin-binding activity, this may explain the efficacy of taxane derivatives. CSP-80 may disclose some aspects of carcinogenesis, be a valuable marker of cancer and therapeutic efficacy, and offer new avenues for cancer treatment.

#4297

MYC HIF-2 alpha embryonic stemness genes mediate a transcriptional network to maintain asymmetric self-renewal in oral cancer.

Bidisha Pal,1 Wael Tasabehji,1 Sandhya Sorra,2 Joyeeta Talukdar,3 Bikul Das1. 1 _Thoreau Lab for Global Health, Lowell, MA;_ 2 _Kavikrishna Lab, Assam, India;_ 3 _KaviKrishna Lab, Assam, India_.

Cancer Stem Cells (CSCs) explains therapeutic failure across several tumor types. Despite being evolutionarily successful, the extreme ability to self-renew is asymmetric and limited to CSCs in heterogeneous tumor population. Thus, understanding molecular mechanisms maintaining CSCs asymmetric self-renewal would be therapeutically critical.Here, we identified putative molecular transcriptional network that may regulate CSCs asymmetric self-renewal. We developed 4-NQO (4-Nitroquinoline 1-oxide) induced mouse model of oral carcinogenesis that exhibited similar phenotype as human oral cancer. ABCG2+ CSCs enriched from 4-NQO derived mouse tumors and human oral cancer (n=7) patients indicated high expression of embryonic stemness genes such as Nanog, Sox-2, and Oct-4 in addition to MYC and HIF-2a. Higher engraftment potential was observed when ABCG2+ cells were injected to NOD/SCID mice, while HIF-2a silencing markedly increased mice survival during engraftments. ChIP assay revealed preferential binding of MYC to HIF-2a promoter regions in ABCG2+ vs ABCG2- cells. Nanog and Oct4 facilitated MYC binding to HIF-2a while gene silencing of Nanog and Sox2 led to loss of MYC and HIF-2a expression, indicating the possible regulation by MYC and HIF-2a. Further, we utilized clinical data from The Cancer Genome Atlas (TCGA) and demonstrated significant correlation between HIF-2a/MYC and embryonic stemness genes on Structural Equation Modeling Analysis (SEMA) platform, thereby confirming our in vitro/in vivofindings. Thus,our results may provide evidence of a biologically robust stemness transcriptional network regulating the asymmetric self-renewal of ABCG2+ CSCs.

#4298

Comparative inhibitory effect of different Egyptian Withania somnifera root extracts on PTEN/PI3K/AKT signaling pathway in HepG2 cell line.

Nahla A. Elzefzafy,1 Amira Zaky,2 Wafaa A. Ahmed,1 Ahmad Bassiouny2. 1 _Egyptian National Cancer Institute, Cairo, Egypt;_ 2 _Alexandria University- Faculty of Science, Egypt_.

This study aimed to identify the anti-proliferative/survival/metastatic potential of different Egyptian Withania somnifera "Ashwagandha" root extracts through PI3K/AKT pathway modulation against HepG2 cell lines Background: Liver cancer is the most common malignancy of the digestive system with high death rate; surgical interventions considered as the most effective approach, but only about 20% of patients are suitable for surgery meanwhile, chemotherapeutic drugs for liver cancer have toxic adverse effects which restrict their effectiveness, therefore natural products combination with anticancer treatments could offer an attractive strategy for liver cancer treatment. Experimental procedures: Ethanolic crud extract was fractionated into five fractions according to polarity ex1 ex2 ex3 ex4 ex5 Sulphorhodamine-B assay used to evaluate the anticancer activity of the Withania somnifera factions in HepG2 cells and to calculate the half maximal inhibitory concentration IC50 of each fraction; The potential antitumor activity of the most effective fractions were assessed by investigating their effect on Apoptotic markers Caspase 3 8 9, Antioxidant markers GSH GST MDA, Survival and proliferation pathway PTEN AKT PI3K, Proliferation markers KI67 Survivin. Results: the crud extract and its fractions showed various cytotoxic effects; ex1 ex2 ex3 have more potent cytotoxic effect than the crude extract IC50= 59.6 78 92 140 μg/ml respectively, ex4 has a similar cytotoxic effect to the crude extract IC50=139 μg/ml while ex5 has the lowest cytotoxic effect IC50= 182 μg/ml; these results showed that the crud extract have no synergism between its active constituents. The most interesting finding is that ex2 has the second potent cytotoxic effect and it down regulate the expression level of AKT COX2 IL6 sFas MMP2 survivin Ki67 compared to the control significantly, while ex1 significantly up regulate the expression of PTEN and down regulate the expression of sFas. Interestingly, ex1 and ex2 showed the strongest effect in arresting HepG2 cells in G2/M phase compared to control and other fractions; the arrested cells activate multi pro-apoptotic pathways as they increases the activity of Caspase3 and Caspase 9. On the hand Caspase 8 activity was significantly increased in ex5 treated group which explain that ex5 induce its antiproliferative effect through membrane binding receptor so it has lowest cytotoxic effect Conclusion: The results showed that each extract differently exert its anticancer activity through modulating PI3K AKT signaling pathway at different levels and the most surprising finding was that petroleum ether fraction has potent promising anticancer capacity on HepG2 cells So, further investigation of each fraction is recommended to purify its active compounds which will offer promising opportunities to develop new candidate for targeted cancer therapy

#4299

**Targeting AKT with oridonin inhibits growth of esophageal squamous cell carcinoma** in vitro **and patient-derived xenografts** in vivo **.**

Mengqiu Song,1 Ran Zhao,1 Hua Xie,2 Hanyong Chen,3 Kangdong Liu,4 Ann M. Bode,3 Mee-Hyun Lee,4 Zigang Dong5. 1 _Department of Pathophysiology, School of Basic Medical Sciences, Zhengzhou University, China-US(Hormel) Cancer Institute, Zhengzhou City, China;_ 2 _Division of Anti-Tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China;_ 3 _The Hormel Institute, University of Minnesota, Austin, MN;_ 4 _Department of Pathophysiology, School of Basic Medical Sciences, Zhengzhou University, China-US(Hormel) Cancer Institute, The Collaborative Innovation Center of Henan Province for Cancer Chemoprevention, Zhengzhou City, China;_ 5 _Department of Pathophysiology, School of Basic Medical Sciences, Zhengzhou University, China-US(Hormel) Cancer Institute,The Collaborative Innovation Center of Henan Province for Cancer Chemoprevention, The Hormel Institute, University of Minnesota, MN_.

Esophageal cancer is the sixth leading cause of cancer death worldwide and has low survival rates with poor prognosis. Hyperactivation of AKT has been reported to modulate cell growth, survival, and gene expression in various solid tumors. Oridonin, an inflammatory medical and diterpenoid compound isolated from Rabdosia rubescens, has exhibited various pharmacologic and physiologic properties, including antitumor, antibacterial, and anti-inflammatory effects. In this study, we aimed to investigate the effects of oridonin on the proliferation and growth of ESCC and to elucidate its underlying mechanisms of action. We found that oridonin is an inhibitor of AKT and induces cell cycle arrest and apoptosis in ESCC cells and attenuates growth of patient derived xenograft (PDX) tumors in vivo by interfering with AKT signaling pathways through MTT assay, anchorage-independent cell growth assay and Cell cycle and apoptosis analyses. AKT was developed as a dierctly target of oridonin by in vitro kinase assay, ex vivo and in vitro pull-down assay and computational docking model. Mechanistically, oridonin diminished the phosphorylation and activation of AKT and supressed the downstream of p-GSK-3β, p-mTOR and NF-kB activity. Moreover, a combination of oridonin and 5-fluorouracil or cisplatin (clinical chemotherapeutic agents) enhanced the inhibition of ESCC cell growth. Overall, our study suggests that oridonin can inhibit progression of ESCC tumors in vitro and in vivo by suppressing AKT signaling through its direct targeting of AKT. Thus, this study might provide useful information in the clinical application of oridonin for ESCC chemotherapy.

#4300

Overexpression of PARP is an independent prognostic marker for poor survival in Middle Eastern breast cancer and its inhibition can be enhanced with embelin co-treatment.

Maqbool Ahmed, Abdul K. Siraj, Poyil Pratheeshkumar, Sandeep Parvathareddy, Sasidharan Padmaja Divya, Fouad Al-Dayel, Asma Tulbah, Dahish Ajarim, Khawla S. Al-Kuraya. _King Faisal Hospital and Research Center, Riyadh, Saudi Arabia_.

Patients with aggressive breast cancer (BC) subtypes usually don't have favorable prognosis despite the improvement in treatment modalities. These cancers still remain a major cause of morbidity and mortality in females. This has fostered a major effort to discover actionable molecular targets to treat these patients. Poly ADP ribose polymerase (PARP) is one of these molecular targets that are under comprehensive investigation for treatment of such tumors. However, its role in the pathogenesis of BC from Middle Eastern ethnicity has not yet explored. Therefore, we examined the expression of PARP protein in a large cohort of over 1000 Middle Eastern BC cases by immunohistochemistry. Correlation with clinico-pathological parameters were performed. Nuclear PARP overexpression was observed in 44.7% of all BC cases and was significantly associated with aggressive clinico-pathological markers. Interestingly nuclear PARP overexpression was an independent predictor of poor prognosis. PARP overexpression was also directly associated with XIAP overexpression, with PARP and XIAP co-expression in 15.8% (159/1008) of our cases. We showed that combined inhibition of PARP by olaparib and XIAP by embelin significantly and synergistically inhibited cell growth and induced apoptosis in BC cell lines. Finally, co-treatment of olaparib and embelin regressed BC xenograft tumor growth in nude mice. Our results revealed the role of PARP in Middle Eastern BC pathogenesis and prognosis. Furthermore, our data support the potential clinical development of combined inhibition of PARP and XIAP, which eventually could extend the utility of olaparib beyond BRCA deficient cancer.

#4301

Hypoxia-induced Nur77 activates PI3K-p110a/Akt/mTOR via suppression of Dicer/let-7i-5p to induce epithelial-to-mesenchymal transition in colon cancer cells.

Sally K. Y. To,1 Shan Deng,1 Jin-Zhang Zeng,2 Alice Sze Tsai Wong1. 1 _Univ. of Hong Kong, Hong Kong;_ 2 _Xiamen University, Xiamen, China_.

Cancer cells reside in the hypoxic niches of tumors easily undergo epithelial-to-mesenchymal transition (EMT). We have recently shown that hypoxia-induced EMT to enhance colon cancer cell invasive growth is dependent on the orphan nuclear receptor Nur77 and is mediated by Akt signaling. However, its underlying molecular mechanisms remain elusive. Here we found that hypoxia-induced Nur77 reduced Akt phosphorylation and its downstream mediator mTOR by decreasing the mRNA stability of p110α, the catalytic subunit α of phosphatidylinositol 3-kinase. Further analyses revealed that this effect could be mediated by down-regulation of Dicer, an important microRNA (miR) processor, and decreased biogenesis of the miR, let-7i-5p. Luciferase reporter assays confirmed that let-7i-5p could directly target the 3'UTR of p110α mRNA. Knockdown of Dicer or overexpression of let-7i-5p inhibited Nur77-induced EMT phenotypes. These data uncover a novel mechanistic link connecting Nur77, Akt, and invasive properties of colon cancer in the hypoxic microenvironment.

#4302

AR signaling promotes prostate carcinogenesis.

Siyuan Cheng, Shu Yang, Zachary M. Connelly, Fenghua Chen, Xiuping Yu. _LSUHSC-S, Shreveport, LA_.

Prostate cancer (PCa) is the most common non-skin cancer in American men. Each year, about 164,690 new patients will be diagnosed and about 29,430 men will die from this disease in 2018. Studies have identified several factors that increase the risk of PCa, including age, race, family history, and some diet factors. However, the exact cause of PCa remains unclear. Prostate is an androgen dependent organ and the growth of PCa also dependents on androgen and AR signaling. It is well established that AR signaling is the central player in promoting PCa progression. Here, we explore if AR signaling also plays a role in prostate carcinogenesis. We found that activation of AR signaling in normal prostate epithelial cells suppressed their cell proliferation in vitro but induced carcinogenesis in vivo. To explore the mechanisms that contribute to the differential effects of AR in vitro and in vivo, we examined if AR signaling induces genome instability in prostate epithelial cells, resulting in carcinogenesis and tumor progression. Senescence-associated beta-galactosidase staining results indicated that androgen treatment induced cellular senescence of prostate epithelial cells. Also, we found that the cells that survived the cellular senescence had altered genome ploidy and increased expression of active DNA-PK, suggesting that non-homologous end joining repair pathway is active in these cells. Our data support that AR signaling promotes genome instability in prostate epithelial cells, contributing to carcinogenesis. This may explain the high frequency of cancer developed in hormone-regulated organs such as prostate, mammary gland, and liver.

Funding: This study was supported by: FWCC/Foundation Legacy Funds, DOD New Investigator Award, The Office of Research SEED awards, The Department of Biochemistry & Molecular Biology.

#4303

TanshinoneIIA, an isolated compound from Salvia miltiorrhiza, inhibits cell growth in colorectal cancer via activation of p38MAPK signaling pathway.

Yan Wang, Lihong Zhou, Qing Ji, Hua Sui, Xueqing Hu, Gang Cai, Qi Li. _Cancer Institute of Integrative Medicine, Shanghai, China_.

Tanshinone IIA (TSIIA) is an active ingredient extracted from the root of Danshen (Salvia miltiorrhiza Bunge), a Chinese traditional herb. Recent studies have indicated that TSIIA is a natural anti-tumor agent, however, the underlying mechanisms remain largely unknown. We studied the anti-tumor efficacy of TSIIA on human colon cancer cell lines in vitro and in vivo, and have further demonstrated that the molecular anti-cancer property of TanIIA is dependent on its inhibition of proliferation and induction of apoptotic cell death via p38MAPK signaling pathway.

The results indicated that TanIIA were markedly more effective than control group in preventing tumor growth and increasing survival time. This phenomenon was accompanied by a decrease in cell growth in a concentration- and time-dependent manner. Hoechst 33258 stainning showed an increased number of apoptotic bodies in the cells after treatment with TanIIA. Flow cytometry analysis showed TanIIA could cause an obvious colon cacer cell apoptosis and arrest cells in the G0/G1 phase. After p38MAPK signaling pathway was blocked, the cell apoptotic rates and cell ratio of G0/G1 phase were decreased significantly. In addition, TanIIA increased p38MAPK phosphorylation, and up-regulated the protein expression of ATF-2, The ATF-2 expression was found to be significantly decreased in cells treated with p38MAPK-specific inhibitor. From our results, it is speculated that TanIIA may induce human colon cancer cells apoptosis and arrest cells in the G0/G1 phase involve up-regulation the expression of the ATF-2 mRNA via p38MAPK signal transduction pathway.

#4304

Anti-proliferative activity of nodosin is associated with the regulation of wnt signaling pathway in colon cancer cell.

Eunseo Bae,1 Donghwa Kim,1 Woong sub Byun,1 Sang Kook Lee,1 Young-Won Chin2. 1 _Seoul National University, Seoul, Republic of Korea;_ 2 _Dongguk University, Seoul, Republic of Korea_.

Wnt signaling is a critical mechanism of biological processes, including cell proliferation, tissue homeostasis and development. The aberrant activity of Wnt signaling pathway is highly correlated with the colorectal carcinogenesis. Therefore, the effective compounds targeting Wnt signaling pathway could be applicable for colorectal cancer treatment. The bioactivity of nodosin, a component of Isodon serra, was evaluated in HCT116 colorectal cancer cells. Nodosin exhibited a potential growth inhibition of colon cancer cells. Nodosin also showed an effective inhibition upon wnt/β-catenin reporter gene assay in HEK293 and HCT116 cells. In addition, Wnt target genes such as β-catenin, Axin2, cyclin D1 and survivin were suppressed by nodosin in HCT116 cells. The inhibition of cell proliferation by nodosin was in part associated with the G2/M phase cell cycle arrest in colon cancer cell. These findings suggest that the anti-proliferative activity of nodosin against colorectal cancer cell should be associated with the regulation of Wnt/β-catenin signaling pathway.

This work was supported by a National Research Foundation of Korea (NRF) Grant funded by the Korean Government (MEST) (NRF-2016M3A9B6903499).

#4305

AMPK-RUNX2-mTORC2 axis involved in EMT of MDA-MB-231 breast cancer cells.

Aramati Bindu Reddy, Meher B. Gayatri, Suresh Chava. _University of Hyderabad, Hyderabad, India_.

Metformin is one of the most widely used biguanides for the treatment of type 2 diabetes, whose role as a potent anti-cancer drug has emerged in recent times. Its function as an anti-tumor agent is attributed to its ability to inhibit mTORC1, which is a key player in regulating cell proliferation. However, the use of metformin as an anti-cancer drug is hampered by several limitations. Hence, in order to elucidate the detailed mechanistic properties of metformin in cell growth and metastasis, we investigated the effect of metformin on mTORC2 activity in MDA-MB-231 cells. We report a novel mechanism by which metformin can increase the activity of mTORC2 in a Runx2-dependent fashion, which requires AMPK mediated stabilization of RUNX2. Furthermore, we found that mTORC2 and RUNX2 have a feed forward relation whereby the knockdown of rictor, a key component in activating mTORC2, resulted in loss of RUNX2 through GSK3-β-mediated ubiquitination of RUNX2. Since both mTORC2 and RUNX2 have been found to be involved in activating the EMT, metformin treatment may contribute to this activation by stabilizing AMPK-RUNX2-mTORC2 axis. These results demonstrate that metformin-induced mechanisms can influence metastasis even if they inhibit cell growth, but the genetic heterogeneity of the malignancy may govern cell fate.

#4306

JunD-induced cell proliferation requires MYC signaling in prostate cancer cells.

Bethtrice Thompson (Elliott),1 Ana Cecilia Millena,1 Lilya Matyunina,2 Mengnan Zhang,2 Jin Zou,1 Guangdi Wang,3 Qiang Zhang,3 Nathan Bowen,1 Vanessa Eaton,4 Gabrielle Webb,4 Shadyra Thompson,4 John McDonald,2 Shafiq Khan1. 1 _Clark Atlanta Univ., Atlanta, GA;_ 2 _Georgia Institute of Technology, Atlanta, GA;_ 3 _Xavier University of Louisiana, New Orleans, LA;_ 4 _Spelman College, Atlanta, GA_.

JunD, a member of the AP-1 transcription factor family, is involved in a variety of biological processes including cell proliferation, inflammation, differentiation, apoptosis, and carcinogenesis. We previously showed that JunD is essential for cell proliferation and demonstrated that the knock-down (KD) of JunD in PCa cells resulted in cell cycle arrest in G1-phase concomitant with a decrease in the levels of Id1, c-MYC, and Ki67, but an increased in p21 protein levels. Furthermore, the over-expression of JunD significantly increased proliferation in these cells suggesting that JunD regulates the expression of genes which are required for cell cycle progression. In this present study, employing gene expression profiling, quantitative proteomics, and validation approaches, we demonstrated that JunD KD is associated with distinct gene and protein expression patterns. Comparison integrative analysis by Ingenuity Pathway Analysis (IPA) identified 1) cell cycle control/regulation as the top canonical pathway whose members exhibited a significant decrease in their expression following JunD KD including PRDX3, PEA15, KIF2C, and CDK2, and 2) JunD target genes are involved in cell proliferation, with MYC as the key downstream regulator. Conversely, JunD over-expression induced cell proliferation and the expression of the above JunD target genes including c-MYC. When treated with JQ1, a c-MYC inhibitor, these cells exhibited a significant reduction in cell proliferation and a decrease in JunD target genes. Our data suggest that JunD-mediated cell proliferation involves MYC signaling and inhibiting its target genes may be an effective approach to blocking prostate carcinogenesis

#4307

RAGE is the key receptor for LPA in lung and mammary cancer progression and metastasis.

Nitish Jangde, Rashmi Ray, Vivek Rai. _Institute of Life Sciences, Bhuabaneswar, India_.

Lung and breast cancer signaling are very complex; receptor for advanced glycation end products (RAGE) is highly expressed in various cancers and is correlated with poorer outcome in lung and other cancers. It is an immunoglobulin like membrane receptor shows binding with various ligands sharing structural likeness such as amyloid beta, advanced glycation end products (AGEs), S100B proteins, amphoterin and HMGB1 and mediates numerous inflammatory and cellular events including diseases including various cancers. LPA a RAGE ligand is a biologically active phospholipid involved in cell proliferation, migration and survival via its different G protein couple receptors (GPCRs). LPA and its signalling pathways have been implicated in different pathological conditions of lung tissue, including inflammation, fibrosis and cancer activating various signalling pathways. Here, we have identified RAGE-LPA signaling mechanisms showing AKT, STAT3 and ERK as downstream signaling pathways gets activated upon LPA treatment via RAGE receptor in both lung and breast cancer cells. RAGE knockdown with multiple independent shRNAs in lung and breast cancer cells led to decreased transwell invasion and soft agar colony formation, and proliferation upon LPA stimulation. Our RAGE silencing study in lung and breast cancer cells upon LPA treatment showed upregulation of specific oncogenes Cyclin D1, c-Myc and VEGF via RAGE receptor. RAGE association with LPA induced epithelial to mesenchymal transition (EMT) in both lung and breast cancer cells have also been observed by the RAGE attenuation studies by examining the expression of some EMT markers. The physiological significance of LPA-RAGE axis was exposed by our xenograft study by nude mice intraperitoneal injections of control and RAGE inhibited lung and mammary tumors generated by LPA doses in alternative days, results the formation of tumor foci on peritoneum wall via RAGE receptor. This study provides novel therapeutic insights as the use of RAGE blocking antibodies and antagonists or competitive inhibitors of LPA against RAGE might be useful of breast cancer treatment. This study also suggests that RAGE and LPA can be used as biomarkers for the early detection of lung and breast cancers.

#4308

Phosphatidic acid binds to ERK3 and stimulates phosphorylation of the ERK3 activation loop.

Amanda Myers, Hitham Aldharee, Shimpi Bedi, Weiwen Long. _Wright State University, Datyon, OH_.

Protein kinases modulate the activity of other proteins through phosphorylation and themselves are often regulated by phosphorylation of their activation loop. Extracellular signal-Regulated Kinase 3 (ERK3) is an atypical member of the ERK family of Mitogen-Activated Protein Kinases (MAPK), with a SerGlu-Gly (SEG) motif in its activation loop rather than the canonical Thr-Xaa-Tyr (TXY) motif of ERK1/2. Activation loop phosphorylation of ERK3 is not regulated by the signals known to activate ERK1/2, such as mitogenic growth factors. The cellular stimuli activating ERK3 kinase are virtually unknown. Some kinases, including mTOR and p21 activated kinase 1 and 2 (PAK1/2), are stimulated by binding to certain phospho-lipid signaling molecules, such as phosphatidic acids. Here, we show ERK3 binds to specific phospholipids in vitro, and the C-terminal domain may be important for this interaction. In addition, ERK3 can localize to the cell membrane. Further, phosphatidic acid stimulated phosphorylation of the activation loop in ERK3. Together, these results suggest that ERK3 kinase activity might be upregulated by specific phosphatidic acid species in cells.

#4309

Involvement of pRb-E2F pathway in green tea extract-induced growth inhibition of human myeloid leukemia cells.

Darrell Henry, Sebastien Brumaire, Gabriela Alvarez, Beatriz Alvarez, Xiaotang Hu. _Barry Univ., Miami Shores, FL_.

It has long believed that green tea has many health benefits. However, the clinical research has not provided conclusive evidence on these benefits especially on cancer prevention and treatment. In basic research, there are also many inconsistent reports on the biological activity of green tea extract and its major chemical components (catechins) with both inhibitory and stimulatory effects of EGCG on various cancer cells being reported. The stimulatory or inhibitory activity is often linked to either activation or inactivation of receptor tyrosine kinase. In this study, we present evidence that green tea extract and its major chemical components, Epigallocatechin-3-gallate (EGCG), epigallocatechin (ECG), and epicatechin (EC) inhibit growth of two human myeloid leukemia cells (TF-1a and MV4-11) through the regulation of pRb synthesis and pRb-E2F complexes. Addition of green tea extract to the culture of the myeloid leukemia cells significantly inhibited their proliferation with a substantial portion of cell death and decreased their colony forming cells in soft-agar culture. EGCG, ECG, and EC showed a similar inhibitory effect on these cells with the inhibitory efficiency varied with each catechin and the dose applied. Green tea extract and EGCG had no significant effect on the expression of G1 CDKs and the CDK inhibitors but downregulated the formation of pRb-CDKs examined by Western blot and immunoprecipitation, respectively. Surprisingly, the expression of pRb was markedly upregulated while the phosphorylation of pRb downregulated. The upregulation of pRb was blocked by pre-treatment with cycloheximide, a protein synthesis inhibitor, suggesting a requirement of protein synthesis. In agreement with these results, pRb-E2F complexes were upregulated and E2F DNA binding activity decreased determined by EMSA assay. Since both TF-1a and MV4-11 cell are factor-independent cell lines, the upregulation of pRb-E2F complexes and downregulation of DNA binding activity by green tea extract is most likely through a receptor tyrosine kinase-independent pathway. In summary, this study reveals that green tea extract, EGCG, ECG, and EC inhibited growth in human myeloid leukemia cells via regulating pRb synthesis and the formation of pRb-E2F complexes, leading to the inhibition of the E2F DNA binding activity. In addition, we found that that the stem/progenitor cells derived from the leukemia cell lines tested are more sensitive to the inhibitory effect of green tea extract. We propose that as a beverage one should not consume high amounts of green tea (extract) due to its possible cytotoxic or apoptotic effect, and its significant inhibitory effect on stem/progenitor cells, although the biological effect of green tea extract in vivo could be quite different from in vitro. However, the concentrated green tea extract may have potential for clinical investigation as an inducer of cancer cell death.

#4310

Targeting Wnt signaling pathway using curcumin in metatstatic breast cancer cell lines.

Nancy Ma, Patty Havard, Vinayak Shenoy, Anitha K. Shenoy. _California Health Sciences University, Clovis, CA_.

Metastasis is the major reason for breast cancer-related deaths. The current treatment options for breast cancer include surgery, endocrine targeted therapy, radiation, chemotherapy and/or immunotherapy. However, even after treatment of the primary tumor at an early stage, breast cancer patients are at an increased risk of developing sudden metastases. Wnt signaling is a pivotal oncogenic pathway that contributes to human breast malignancy. High levels of Wnt ligands have been frequently detected in human breast cancers and accumulating evidence indicate a key role for the Wnt signaling pathway in the metastatic process. Curcumin, an active ingredient of turmeric has been shown to alter Wnt signaling as well as suppress metastasis in a variety of cancers. Here, we hypothesize that curcumin inhibits metastatic potential of breast cancer cell lines by targeting the Wnt signaling pathway. An initial dose dependent cytotoxic assay was performed with curcumin on two distinct breast cancer cell lines (epithelial and mesenchymal) to determine the optimum concentration for subsequent experiments. Our results indicate that curcumin imparts a dose dependent cytotoxic effect on both epithelial as well as mesenchymal breast cancer cell lines. Further, we observed that curcumin treatment alters the Wnt signaling pathway genes. Additional studies are warranted to confirm the effect of curcumin on Wnt signaling pathway and its role in combating proliferation and migration of breast cancer cells.

#4311

Mechanisms responsible for the inflammatory polarization of monocytes by subclinical low-dose LPS.

Kisha Pradhan. _Virginia Tech University, Blacksburg, VA_.

Dynamic polarizations of innate leukocytes such as monocytes and macrophages have been increasingly recognized to play key roles in the pathogenesis and/or treatment of human disease. However, mechanisms responsible for distinct polarizations of monocytes are not well understood. Our previous studies indicate that monocytes can be persistently polarized into a low-grade inflammatory state by subclinical super-low dose LPS (bacterial lipopolysaccharide). In this current study, we further characterized the unique polarization of monocytes by super-low dose LPS and its underlying mechanisms. We observed that super-low dose LPS preferentially induced the expression of inflammatory mediators such as MCP-1 while potently suppressed the expression of homeostatic mediators such as Catalase and FPN. Super-low dose LPS activated the inflammatory signaling molecules such as p38, CamKII, and NFkB. In contrast, super-low dose LPS suppressed the basal activation of immune suppressing molecules such as AMPK and SIRT3. Taken together, our data reveal novel insights regarding the persistent polarization of inflammatory monocyte, an emerging key player in various human diseases including cancer.

#4312

Multiplex parallel perturbation of Akt and MAPk signaling cascades.

Andrew R. Wagner, Megan Newby, Kary Oakleaf. _Grace Bio-Labs, Bend, OR_.

PI3K/Akt and MAPk signal transduction pathways are frequently dysregulated in human cancers. Previous work described the molecular basis of pro-survival signals mediated by Akt and MAPk. Mutation in and alteration of these pathways lead to tumorigenesis. However, signal propagation varies across cell type and precise signaling kinetics is less well characterized. The goal of this study is to profile the functional state of the PI3k/Akt and MAPk signaling circuits by coupling cell viability to antibody microarray and immunofluorescence. Simultaneous detection of Akt and MAPk signaling nodes can characterize signaling parameters that correlate to localization patterns across cell type and drug treatment conditions. To examine how signal propagation varied between cell lines, staurosporine was utilized to broadly inhibit protein kinase activity in a cervical cancer cell line, HeLa, and an osteosarcoma cell line, U2-OS, as model systems. To investigate signaling pathways, each cell line was treated and analyzed in parallel by forward phase phospho-specific antibody microarray panels and high content analysis of immunofluorescent staining. Staurosporine inhibited cell proliferation in a time and concentration dependent manner in both cell lines. U2-OS cells had a higher IC50 compared to HeLa cells indicating markedly different signaling parameters between the two models. Our observations suggest detecting cell type specific signaling kinetics can be used as a framework to predict cell sensitivity to specific inhibitors and be used to monitor drug resistance.

### Epigenetic Changes as Molecular Markers

#4313

Identifying and characterizing epigenetic «driver» genes («epidrivers») in regulatory pathways involved in tumorigenesis and tumour cell plasticity.

Andrea Halaburkova,1 Vincent Cahais,1 Cyrille Cuenin,1 Rita Khoueiry,1 Maria Ouzounova,2 Akram Ghantous,1 Zdenko Herceg1. 1 _International Agency for Research on Cancer, Lyon, France;_ 2 _Cancer Research Center of Lyon, Lyon, France_.

One of the ground-breaking discoveries of the international cancer genome sequencing endeavours is the high frequency of mutational and non-mutational changes in epigenetic regulator genes (ERGs), which constitute a "genetic smoking gun" that epigenetic mechanisms lie in the very heart of cancer biology. However, functional importance of disruption of ERGs in tumorigenesis and cancer phenotype is poorly understood. We hypothesise that these genes are candidates to be drivers («epidrivers») of cancer onset and progression, thus regulating mechanisms underpinning cancer development. Moreover, deregulation of epidrivers may play a role in epithelial-to-mesenchymal transition (EMT) and emergence of cancer resilience. The overarching aim of this study was to identify and functionally characterize "Epidrivers" in tumorigenesis and cancer cell plasticity. To this end, we have set up novel epigenome-wide functional screens in human cultured cells (and organoids) combined with state-of-the-art genome-editing approach (based on CRISPR/Cas9 screen) and multiparametric phenotyping. We designed a custom-made lentiviral CRISPR library that consists of 1,649 gRNAs targeting all known ERGs (426 genes). Lentiviral CRISPR library was used to deliver the ERG gRNAs to target cells expressing the RNA-guided DNA endonuclease Cas9. Independent clones (derived from transduced breast and lung cancer cell lines constitutively expressing Cas9) are screened for acquisition of distinct features of transformed cells. The results of the identification of Epidriver genes based on the analysis of deregulation of core cellular processes, epigenome (ChIP-seq) as well as EMT and multiparametric phenotyping, will be presented.

#4314

Reduced m6A modification predicts malignant phenotypes and augmented tumorigenic signaling in gastric cancer.

Cheng Zhang,1 Mengqi Zhang,1 Sai Ge,1 Xiaotin Lin,1 Wenwen Huang,1 Ying Gan,2 Jing Gao,1 Lin Shen1. 1 _Peking University Cancer Hospital & Institute, Beijing, China; _2 _Institute of Urology, Peking University, Beijing, China_.

Purposes: As the most abundant epigenetic modification on mRNAs and long non-coding RNAs, N6-methyladenosine (m6A) extensively exists in mammalian cells. Controlled by writers (methyltransferases), readers (signal transducers) and erasers (demethylases), m6A influences mRNA structure, maturation and stability, thus regulating protein expression in a post-transcriptional manner. Nevertheless, current understandings to m6A's pathological roles in tumorigenesis, especially in gastric cancer (GC) remain to be unveiled. In this study, by incorporating multiple datasets and in vitro experiments, we assessed m6A's clinical-pathological relevance to GC and explored the underlying mechanisms.

Methods: The protein and mRNA expressions of canonical m6A regulators (METTL3/METTL14 as writers, YTHDF1/YTHDF2/YTHDF3 as readers and ALKBH5/FTO as erasers) were merged as signatures to indicate levels of m6A modification. Changes of clinical variables, mutations and molecules were assessed in patients with different m6A indications. Pathway and phenotype changes predicted to be affected by m6A were assessed in vitro with gastric cancer HGC27 and MG803 cell lines.

Results: We discovered that writers and readers were potential tumor suppressive, while erasers were potential oncogenic factors in GC. Low-m6A modification was an indication for adverse clinical outcomes and was correlated with specific cancer driving mutations, including CDH1, AR and GLI3. Oncogenic signaling and phenotypes were selectively enriched in m6A groups. Through in vitro experiments, we proved that reduced m6A (represented by METTL14 knockdown) promoted GC proliferation and invasiveness potentially through activating Wnt and PI3K-Akt signaling, while restored m6A (represented by FTO knockdown) reversed these phenotypical and molecular changes.

Conclusions: Our work demonstrated that m6A is a potential prognostic predictor in gastric cancer and further reveals the reduction of m6A activates oncogenic signaling and promotes gastric cancer malignancy.

#4315

UNC0642 inhibits G9a and induces apoptosis of human bladder cancer cells.

Ruimin Huang,1 Jingya Sun,1 Meiqian Li,2 Yu Dong,1 Yuanheng Zhang,1 Jun Yan2. 1 _Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China;_ 2 _Nanjing University, Nanjing, China_.

Urinary bladder cancer (UBC) is one of the most common malignancies in the world, with the characteristics of frequent recurrence and metastasis despite the standard chemotherapy with gemcitabine and cisplatin combination. Hence, it is needed to develop new drugs to tackle down UBC. Histone modifiers, which are frequently dysregulated in cancer development, are excellent drug targets for cancer treatment. Here, we found that G9a, one of the histone H3 methyltransferases, was overexpressed significantly in human UBC patients, using the expression data from public databases. In the TCGA Provisional dataset, the average expression level of G9a in primary UBC samples (n=408) was 1.6-fold as much as that in normal bladder samples (n=19; P < 0.001). Knockdown G9a by small interfering RNA decreased cell viability and induced apoptosis in human UBC cell lines. Thus, UNC0642, a small molecule inhibitor targeting G9a with high selectivity, low cytotoxicity, and excellent in vivo pharmacokinetic properties, was chosen to test the inhibitory effect on UBC cells. UNC0642 decreased the level of histone H3K9me2, the downstream target of G9a, and increase apoptosis of UBC cells in vitro in a dose-dependent manner. UNC0642 exhibited similar inhibitory effects on the J82 xenografts, without the loss of body weight of mice. Targeting G9a in vivo reduced Ki67 expression and increase the level of cleaved Caspase 3 and BIM protein by immunohistochemistry and Western blot analysis, respectively. Taken together, our data indicate that G9a may be a promising therapeutic target for UBC, and an epigenetics-based therapy by UNC0642 is suggested.

#4316

Targeting LIFR overcomes HDAC inhibitor resistance in ovarian cancer.

Mengxing Li,1 Suryavathi Viswanadhapalli,1 Gangadhara Reddy Sareddy,1 Bindu Santhamma,2 Hui Yan,1 Zhenming Xu,1 Edward Kost,1 Rajeshwar Rao Tekmal,1 Hareesh B. Nair,2 Klaus J. Nickisch,2 Ratna K. Vadlamudi1. 1 _UT Health Science Ctr. at San Antonio, San Antonio, TX;_ 2 _Evestra, San Antonio, TX_.

Ovarian cancer (OCa) is the deadliest of all gynecologic cancers in the United States and a critical need exists for the development of novel therapies for the treatment of OCa. Leukemia inhibitory factor receptor (LIFR) and its ligand LIF play a critical role in cancer progression, metastasis, stem cell maintenance, and therapy resistance. Recent clinical studies showed that cancer cells elucidate feedback activation of LIFR that limits response to histone deacetylase (HDAC) inhibitors. Recently, we developed a first-in-class inhibitor of LIFR, EC359 that directly interacts with LIFR and effectively blocks LIF-LIFR interactions. Here, we examined whether LIFR inhibitor EC359 abrogate the side effects of histone deacetylase inhibitor SAHA (Vorinostat) for the treatment of OCa.

Methods: The effect of EC359 and SAHA as a combination therapy on OCa cell viability was examined by MTT assays. The efficacy of combination therapy on cell survival and apoptosis was determined using clonogenic assays and caspase3/7 assays respectively. The efficacy of combination therapy on OCa stem cells was determined using extreme limiting dilution assays. Mechanistic studies were performed using western blotting, qRT-PCR and Mass Spectrometry analyses. The effect of combination therapy on STAT3 signaling was examined using reporter gene assays. The in vivo efficacy of combination therapy on tumors was examined using ex vivo patient derived explants and mouse xenograft models.

Results: EC359 significantly enhanced the efficacy of SAHA in reducing cell viability, colony formation ability, and apoptosis compared to monotherapy of SAHA in multiple established and primary OCa cells. Further, EC359 enhanced SAHA ability to reduce self-renewal of OCa stem cells. As expected in STAT3 reporter assays, SAHA treatment activated STAT3 reporter and EC359 addition abrogated SAHA mediated STAT3 activation. Mechanistic studies using multiple OCa models and western blot analysis confirmed activation of LIFR signaling pathway upon SAHA treatment and its blockage by EC359 treatment. Treatment of human primary OCa tumor explants with EC359 enhanced ability of SAHA to decrease the proliferation (Ki-67 positivity) compared to monotherapy treated tumors. DIA based Mass Spectrometry analyses identified unique pathways modulated by combination therapy. Treatment of OCa xenografts with EC359 enhanced the ability of SAHA to reduce in vivo tumor growth compared to monotherapy treated tumors.

Conclusions: Our results suggest that EC359 has therapeutic utility in overcoming the limitation of feedback activation of LIFR observed in the treatment of HDAC inhibitors in treating OCa.

#4317

Application of gene methylation analysis for distinguishing primary tumor cells from normal epithelial cells from lung cancer patients in conditionally reprogrammed cell cultures.

Guodong Wu,1 Yangui Xiao,2 Fuyuan Fang,1 Da Yao,1 Ji Liu,2 Yanhua Cao,2 Yu Mao,2 Benjamin Yu,3 Youhui Qian,1 Derek Yu2. 1 _The 2nd People's Hospital of Shenzhen, ShenZhen, China;_ 2 _USK Bioscience Co., Ltd, ShenZhen, China;_ 3 _Northwestern University Feinberg School of Medicine, ShenZhen, China_.

In vitro drug sensitivity assays utilizing "conditionally reprogrammed cells" (CRCs) from different primary tumor types to guide personalized therapy has been emerging. However, this approach has been hindered by the growth of normal epithelial cells in CRC cultures and the lack of effective biomarkers/assays to distinguish them from primary tumor cells. In this study, we developed a DNA methylation-based, real-time PCR assay to investigate the methylation status of SHOX2,PTGER4 and Septin9 gene promotor regions in human lung cancer formalin fixed paraffin-embedded (FFPE) samples. Our results showed increased DNA methylation for at least one of the three genes in 90% (28/31) of tumor samples as compared with the corresponding adjacent normal samples. Then we tested gene methylation in fresh surgical tumors versus normal adjacent tissues from lung cancer patients, and their respective in vitro CRC cultures. Increased methylation for at least one of the three genes was detected in 91.6% of tumor samples (11/12) as compared with the adjacent tissues, and these positive methylation statuses were maintained in 75% (9/12) of CRC cultures. Further results from in vitro assays showed a correlation between higher cell methylation status and increased growth property in 3D culture, as well as differential response to chemotherapy. Together, our results demonstrated that methylation of SHOX2, PTGER4 and Septin9 genes can be used as reliable biomarkers to monitor tumor cells in in vitro CRC cultures.

#4318

**An RNA-helicase DDX21 promotes** MYCN **driven neuroblastoma cell proliferation by up-regulating CEP55 expression.**

Vina Putra,1 Amy Hulme,1 Jane Sun,1 Andrew Tee,1 Nicholas Ho,2 Murray Norris,1 Michelle Haber,1 Tao Liu,1 Pei Y. Liu1. 1 _Children's Cancer Institute, Kensington, Australia;_ 2 _University of Sydney, Camperdown, Australia_.

Neuroblastoma is the most common extracranial solid tumor in early childhood. Approximately 25% of neuroblastoma patients have MYCN oncogene amplification and consequently these patients rarely survive. The MYCN oncogene encodes a transcription factor N-Myc which is involved in many aspects of neuroblastoma development. The RNA helicase DDX21 has recently been found to activate target gene transcription by binding to target gene promoters; however, its role in MYCN oncogene amplified neuroblastomas is not clear. Here we describe a novel mechanism by which N-Myc drives neuroblastoma progression by upregulating DDX21 gene expression.

Through bioinformatics analysis, we found two Myc response element E-Boxes, one is upstream and the other is downstream of the DDX21 gene transcriptional start site. RT-PCR and immunoblot results revealed that knocking down N-Myc by using small interference RNAs significantly reduced DDX21 gene and protein expression. Over-expression of N-Myc in a tetracycline withdrawal-inducible cell line SHEP-21N resulted in a significant increase in DDX21 mRNA and protein expression. Chromatin immunoprecipitation assay showed that N-Myc upregulated DDX21 gene expression by binding to its gene promoter. Affymetrix microarray studies revealed that one of the genes significantly down-regulated by doxycycline-inducible DDX21 shRNAs was CEP55. RT-PCR and immunoblot analyses confirmed that knocking down DDX21 by treatment with doxycycline in neuroblastoma cells stably transfected with doxycycline-inducible DDX21 shRNA considerably decreased CEP55 mRNA and CEP55 protein expression. Luciferase assays showed that knocking down DDX21 expression with doxycycline considerably reduced luciferase activity of the CEP55 promoter luciferase reporter construct. BrdU incorporation and clonogenic assays showed that knocking down DDX21 or CEP55 dramatically reduced cell proliferation and completely abolished colony forming capacity. Importantly, knockdown of DDX21 significantly delayed neuroblastoma tumor progression in mouse model. Analysis of a publicly available RNA sequencing gene expression-patient prognosis dataset, showed that high levels of DDX21 gene expression correlated with high levels of N-Myc and CEP55 gene expression in human neuroblastoma tissues, and independently predicted poor patient prognosis.

In conclusion, our data indicate that DDX21 and CEP55 could serve as novel prognostic markers and therapeutic targets in MYCN driven neuroblastoma.

#4319

Hypoxia regulates m6A-reader YTHDF2 to fuel cancer-promoting inflammation in hepatocellular carcinoma.

Jiajie Hou,1 He Zhang,2 Jun Liu,3 Zhenjun Zhao,2 Jianye Wang,2 Zhike Lu,3 Bian Hu,4 Jiankui Zhou,4 Zhicong Zhao,2 Mingxuan Feng,2 Haiyan Zhang,5 Bin Shen,6 Xingxu Huang,4 Beicheng Sun,1 Mark J. Smyth,5 Chuan He,3 Qiang Xia2. 1 _The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, China;_ 2 _Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China;_ 3 _Institute for Biophysical Dynamics, University of Chicago, Chicago, IL;_ 4 _School of Life Science and Technology, ShanghaiTech University, Shanghai, Shanghai, China;_ 5 _QIMR Berghofer Medical Research Institute, Australia;_ 6 _Nanjing Medical University, Nanjing, China_.

Dynamic N6-methyladenosine (m6A) modification was previously identified as a ubiquitous post-transcriptional regulation that affected mRNA homeostasis. However, the m6A-related epitranscriptomic alterations and functions remain elusive in human diseases such as cancer. Here we show that hypoxia regulates the 'reader' protein YTH domain family 2 (YTHDF2), to ultimately stabilize the methylated oncogene mRNAs in inflammation-associated hepatocellular carcinoma (HCC). YTHDF2 silenced in human HCC cells or depleted in mouse hepatocytes evoked pro-inflammatory responses and accelerated tumor growth and metastatic progression. Under hypoxia, YTHDF2 processed the decay of m6A-containing interleukin 11 (IL11) and serpin family E member 2 (SERPINE2) mRNAs, which were responsible for the inflammation-mediated malignancy. Reciprocally, YTHDF2 transcription succumbed to hypoxia-inducible factor-2α (HIF-2α). Administration of a HIF-2α antagonist (PT2385) restored YTHDF2-programed epigenetic machinery and repressed HCC progression. Hence, our findings provide a new insight into the mechanism by which hypoxia adapts m6A-mRNA editing to promote cancer.

Significance: We show that hypoxia induces downregulation of m6A-reader YTHDF2 and hyper-upregulation of m6A-mRNAs in human HCC, which adapts m6A decoration into a cancer-promoting mechanism. Thus we identify YTHDF2 as a suppressor in tumor growth and metastasis by targeting IL-11 and Serpin E2.

#4320

Enhanced expression of histone methyltransferases increase ERα expression in tamoxifen-resistant breast cancer cells.

Seungsu Kim, Min-Ho Lee, Mi-Ock Lee. _Seoul National Univ. College of Pharmacy, Seoul, Republic of Korea_.

Estrogen receptor (ER)-positive breast cancers depend on ER signaling for growth and progression. Although tamoxifen remains the frontline treatment for such cancers, resistance to this drug limits its clinical efficacy. Nevertheless, the majority of tamoxifen-resistant patients retain ERα expression, providing a rationale for targeting ERα in these patients. Here, we found that the expression of mixed lineage leukemia (MLL) family proteins are increased in tamoxifen-resistant breast cancer cells and patients. Specifically, mixed lineage leukemia 3 (MLL3) and SET domain containing 1A (SET1A) depletion downregulated the expression of ERα protein and mRNA levels in a ER-positive breast cancer cell. Chromatin Immunoprecipitation analysis revealed that the knock-down of MLL3 and SET1A reduce the histone H3K4 methylation status in ESR1 promoter regions. We also observed the positive correlation between ERα level and MLL3/SET1A level in breast cancer clinical data sets. Importantly, MLL3 and SET1A depletion downregulated the ERα and its downstream growth-related protein levels, as well as the growth of both ER-positive and tamoxifen-resistant breast cancer cells. Likewise, MLL3 and SET1A knockdown decreased the proportion of cells entering the cell cycle. Additional fulvestrant treatment resulted in synergistic reduction of ERα levels and growth of the tamoxifen-resistant breast cancer cells. Thus, enhanced expression of MLL3 and SET1A in tamoxifen-resistant breast cancer cells supported ERα-dependent growth of breast cancer cells, suggesting that MLL3 SET1A might provide an attractive target for overcoming endocrine resistance.

#4321

Epigenetic regulation of TP73 expression in HCC and GI cancer.

Zhixing Yao,1 Cristina Di Poto,2 Habtom W. Ressom,2 Zaki A. Sherif1. 1 _Howard Univ., Washington, DC;_ 2 _Georgetown Univ., Washington, DC_.

Tumorigenesis results from an accumulation of mutational and epigenetic changes that alter normal cell growth and survival pathways. TP73 is the homologue of the master tumor suppressor TP53 involved in cellular responses to stress and development. The TP73 gene encodes two different protein isoforms, TAp73 and ΔNp73, and maps to a region on chromosome 1p36 that is frequently deleted in neuroblastoma and other tumors, and is thought to contain multiple tumor suppressor genes. However, the analysis of p73-knockout mice yielded conflicting results with respect to tumor suppression. World-wide efforts in sequencing the TP73 gene in patient tumor samples have not provided evidence for genetic alterations as a common cause of TP73 inactivation in human cancer. The role of TP73 in tumorigenesis has remained elusive to date. In this study, we isolated two stem cell lines from normal young and old human liver tissues to determine TP73 expression in normal human liver stem cell and hepatocellular cancer (HCC) cell lines (HepG2, SNU398, SNU449 & SNU475), gastrointestinal cancer (GI) cell lines (Caco2 & HCT116) and a normal skin fibroblast cell line (HS27). The results show that TP73 only expresses in cancer cell lines. Further immunohistochemistry studies on human normal liver tissues as well as adjacent normal and cancerous liver tissues from HCC patients confirm that TP73 only expresses in the cancerous tissues. Moreover, methylation-specific PCR and bisulfite sequencing studies revealed that TP73 promoter is activated only in cancer cell lines by DNA methylation. Furthermore, ChIP assay results demonstrated that CTCF (a chromosomal networking protein CCCTC binding factor) binds to TP73 promoter and regulates TP73 expression. Our observation may prove significant for the development of future therapeutic and diagnostic applications.

#4322

ONECUT2, encoding a member of the one cut family of transcription factors, is a putative oncogene in gastric cancer and trigger ACSL5, a member of the ACS family.

Eun-Hye Seo, Yong Sung Kim. _KRIBB, Daejeon, Republic of Korea_.

DNA methylation is one of the frequent epigenetic events that play important roles in cancer development. Cancer cells can gain significant resistance to anticancer drugs and escape programmed cell death through major epigenetic changes, including DNA methylation. To date, many studies have focused on both hypermethylation of tumor suppressor genes and global hypomethylation, while less is known about the impact in promoter hypomethylation of oncogenes. To access promoter hypomethylation in gastric cancers, in this study, we performed RRBS and MBD-seq analysis with LCM-derived normal gastric mucosa (GM), intestinal metaplasia (IM) and gastric tumor (GT) cells and found heavily methylated signatures on the promoter regions of ONECUT2 in GM cells, while the methylation signature was decreased in IM and GT cells. Pyrosequencing analysis revealed that ONECUT2 promoter was significantly hypomethylated in 160 gastric tumor tissues and the demethylation was correlated with ONECUT2 upregulation in the tumors. Immunohistochemistry assay with anti-ONECUT2 showed positive staining in IM and GT, but not in GM. Knockdown of ONECUT2 by shRNA inhibited the colony forming, migration, and invasiveness of cell lines that expressed ONECUT2 at relatively high levels and suppressed tumorigenesis in nude mice. We suggest that ONECUT2 is a putative oncogene that is activated in gastric cancers by epigenetic mechanisms. In addition, ChIP-seq and RNA-seq analysis showed that ACSL5, which converts fatty acid to acyl-CoA and is frequently overexpressed in malignant glioma, whereas its functional significance is still unknown, is a direct target of ONECUT2.

#4323

1,25-Dihydroxyvitamin D3 inhibits oral squamous cell carcinoma cell growth via microRNA regulation.

Heba Hussein,1 Wei Wang,2 Liang Shan,2 Karl Thompson,2 Kareem Washington,2 Gihane Madkour,1 Xinbin Gu2. 1 _Cairo University, Cairo, Egypt;_ 2 _Howard University, Washington, DC_.

Objective: Oral squamous cell carcinomas (OSCC) is one of top ten cancers lead to death in USA. This study focused on the impacts of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) treatment on the inhibition of OSCC growth in vitro and the alteration of microRNA (miRNA) expression profile by the treatment.

Materials and Methods: 1,25(OH)2D3-associated apoptosis, cell cycle arrest, and proliferative effect in cultured OSCC cells were assessed using flow cytometry and western blotting methods. miRNA expression profiles were measured using nanoString Sprint Profiller and the Nanostring human miRNA panel. More miRNAs functional pathways were retrieved through bioinformatics using Qiagen IPA system.

Results: 1,25(OH)2D3 was found to inhibit the growth of human JHU-22 OSCC cells in a dose-dependent manner with IC50 of 1.56 x 10-6 M. High levels of apoptotic cells and related biomarkers were obtained when the cells were treated with 1,25(OH)2D3. Over 20 miRNAs were upregulated and 23 downregulated by more than two folds, in 1,25(OH)2D3 treated versus untreated JHU-22 cells. Bioinformatics analysis showed molecular functions of most dysregulated miRNAs. For example, MiR-125b-5p upregulated caspases number 2, 6, 7 in addition to upregulating apoptosis facilitator like BCL-2, BIM, and AIF.

Conclusion: Our data suggested that 1,25(OH)2D3 function as a OSCC inhibiter is through regulating miRNA expression profile and miRNA function directly or indirectly. 1,25(OH)2D3 has potential to be an anticancer agent for OSCC prevention and treatment.

Fold change of top ten unregulated and down regulated miRNAs at nine hour treatment.

---

miRNA | Control (C9) | Vitamin D (D9) | C9/D9

hsa-miR-624-3p | 8 | 28 | 3.5

hsa-miR-521 | 10 | 27 | 2.7

hsa-miR-221-5p | 11 | 29 | 2.64

hsa-miR-1301-3p | 7 | 18 | 2.57

hsa-miR-520a-3p | 8 | 20 | 2.5

hsa-miR-153-3p | 12 | 28 | 2.33

hsa-miR-122-5p | 16 | 37 | 2.31

hsa-miR-1206 | 15 | 34 | 2.27

hsa-miR-125b-5p | 686 | 1547 | 2.26

hsa-miR-450b-3p | 4 | 9 | 2.25

hsa-miR-613 | 21 | 9 | -2.33

hsa-miR-147b | 26 | 11 | -2.36

hsa-miR-525-3p | 12 | 5 | -2.40

hsa-miR-5196-3p+hsa-miR-6732-3p | 18 | 7 | -2.57

hsa-miR-181b-5p+hsa-miR-181d-5p | 21 | 8 | -2.63

hsa-miR-182-3p | 32 | 12 | -2.67

hsa-miR-941 | 22 | 8 | -2.75

hsa-miR-671-3p | 17 | 6 | -2.83

hsa-miR-944 | 18 | 6 | -3.00

hsa-miR-1245b-3p | 24 | 6 | -4.00

#4324

**Prognostic potential of DNA methylation levels of** EPDR1 **and** CHST10 **in colorectal cancer.**

Chun-Ho Chu,1 Shih-Ching Chang,2 Hsiu-Hua Wang,3 Shung-Haur Yang,4 Te-Chang Lee,3 Kuo-Chu Lai5. 1 _National Yang-Ming University, Taipei, Taiwan;_ 2 _Taipei Veterans General Hospital, Taipei, Taiwan;_ 3 _Academia Sinica, Taipei, Taiwan;_ 4 _National Yang-Ming University Hospital, Yilan, Taiwan;_ 5 _Tzu Chi University, Hualien, Taiwan_.

Chromosomal instability (CIN), microsatellite instability (MSI), and the CpG island methylator phenotype (CIMP) are well-characterized molecular features of colorectal cancer (CRC). Aberrant DNA methylation may associate with MSI-high and CIMP-high phenotype in CRC. Studies on aberrant DNA methylation are likely to help in the identification of biomarkers clinically relevant to the process of tumorigenesis. In this study, we explored DNA methylation profile changes in CRC with MSI through genome-wide DNA methylation profiling analysis. Of 34 altered genes, three hypermethylated (pidermal growth factor, EGF; carbohydrate sulfotransferase 10, CHST10; ependymin related 1, EPDR1) candidates were further validated in 75 CRC patients. All validated genes, except EGF, were significantly correlated with MSI status. In addition, methylation of EPDR1 or CHST10 was significantly correlated with better prognosis and mutations in BRAF and TGFβR2. Beside, we found a positive correlation between the methylation of EPDR1 and CHST10. Methylation of EPDR1 was also significantly correlated with node negativity and a lower tumor stage. Negative correlations between mRNA expression and DNA methylation level of both EPDR1 and CHST10 were reported in The Cancer Genome Atlas (TCGA) dataset, revealing that DNA methylation has a crucial function in modulating expressions of EPDR1 and CHST10 in CRC cells. These results suggest that DNA methylation levels of EPDR1 and CHST10 may serve as potential prognostic markers for CRC.

#4325

5-hydroxymethylation captures the heterogeneity in keratinization and cytoskeletal rearrangement processes in head & neck cancers.

Siyu Liu, Katie Zarins, Evan Fernandez, Laura Rozek, Maureen Sartor. _University of Michigan, Ann Arbor, MI_.

Infection with high-risk HPV, or human papillomavirus, has been shown to be a risk factor for head and neck squamous cell carcinoma (HNSCC) development in addition to the classical risk factors of tobacco and alcohol usage. Previous studies have identified epigenetic differences between these two classes of HNSCC, including DNA methylation, histone modifications and non-coding RNAs. 5-hydroxymethylation (5hmC) is an oxidative derivative of 5-methylcytosine (5mC) and is depleted in human cancers of many different origins. However, the roles of 5hmC in HNSCC is still unknown. Our study is the first to characterize 5hmC in HNSCC, which we profiled in 18 HPV(+) and 18 HPV(-) HNSCC tumors. Since previous studies found higher levels of 5hmC in more differentiated cells and lower levels in stem-like cells, we hypothesized an overall higher 5hmC level in HPV(-) tumors, which tend to be more differentiated in nature.

Consistent with our hypothesis, results showed significant hyper-hydroxymethylation in HPV(-) tumors, and detected multiple cancer-related genes with hyper-hydroxymethylation in either HPV(+) and HPV(-) tumors. For example, CDKN2A (p16) and MTAP were the most hyper-methylated genes in HPV(+) tumors, while 5hmC levels of HPV(-) tumors were particularly high in CDH13, TIMP2, and BCAR1. We found that the two HPV(+) subtypes defined by Zhang et al., IMU (heightened immune response, more mesenchymal differentiation) and KRT (more keratinization, more likely viral integration), explained a high level of heterogeneity in 5hmC levels at promoter and enhancer regions. By testing for genes and pathways that demonstrate concordant changes in gene expression and 5hmC levels, we found that keratinocyte differentiation, cell-cell adhesion and adherens junction pathways are both up-regulated and have hyper-hydroxymethylation in HPV(-) tumors. Using predicted enhancers of epidermal keratinocytes, we found a much higher portion of hydroxymethylated regions fall in enhancer regions for HPV(-) samples compared with HPV(+) samples. The higher differential 5hmC in enhancer regions was further validated by stronger histone marks of H3K4me1 and H3K27ac in HPV(-) tumors. Since some of these enhancers can be linked to differentially expressed target genes, this result indicates that the enhancer hydryoxymethylation in HNSCC could also play an important role in downstream gene regulation. We hope that this study could partially explain the different mechanisms and cast light on potential targeted therapies in HNSCC both based on HPV status and different HPV(+) subtypes.

#4326

Epigenetic control of chromosome instability by STAT3 in gastric cancer.

Yu Ming Chuang,1 Sheng-Jou Hung,2 Jiang Liang Chou,3 Wan-Hong Huang,1 Pearlly S. Yan,4 Tsunglin Liu,2 Michael W.Y. Chan1. 1 _Department of Biomedical Science & Center for Innovative Research on Aging Society, National Chung Cheng University, Chiayi, Taiwan; _2 _Department of Biotechnology and Bioindustry Science, National Cheng Kung University, Taiwan;_ 3 _Division of Gastroenterology, Chang Gung Memorial Hospital, Taiwan;_ 4 _Department of Internal Medicine, The Ohio State University, OH_.

Gastric cancer is one of the most common causes of cancer death worldwide. Aberrant activation of JAK/STAT3 signaling is frequently observed in gastric cancer and associated with cancer progression. Our previous studies demonstrated that several STAT3 targets and tumor suppressors such as NR4A3 and GATA6 are found to be epigenetically silenced by DNA methylation in gastric cancer. However, the role of aberrant JAK/STAT3 signaling in the epigenetic changes of downstream targets is not fully understood. In this study, by using Methylation EPIC array, we compared the global methylation changes in gastric cancer patient samples with different STAT3 activation level. In silico analysis was also performed to identify STAT3 binding sites in those differentially methylated loci. Hypermethylated loci which are functionally relate carcinogenesis and mediated by STAT3 expression were examined. Interestingly, SMARCAL1, which was involved in chromosome stability and alternative lengthening of telomeres was identified. Unexpectedly, differentially methylated region (DMR) was not occurred in the promoter, but enhancer region located in the CpG island shore. Additionally, we also found a transcription factor binding site, YY1, which was a methylation-sensitive enhancer-promoter regulator, in this DMR region. By using a statistic shuffling model, we found that STAT3 related methylation changes were significantly associated with H3K4me1 and H3K27me3 at that region. TCGA dataset also indicated that this DMR with dramatic changes was occurred in chromosome instability (CIN) subtype of gastric cancer, whereas the expression of SMARCAL1 was decreased in precancerous lesion in previous published data. Taken together, STAT3-related methylation changes may regulate the activity of enhancer and affect the expression of SMARCAL1, during gastric cancer progression.

#4327

Hypermethylation of miR193b reactivates master drivers of the poor prognosis prostate cancer.

Ying Z. Mazzu,1 Yuki Yoshikawa,1 Subhiksha Nandakumar,1 Joshua Armenia,1 Lina Jehane,1 Gwo-Shu Lee,2 Goutam Chakraborty,1 Philp Kantoff1. 1 _MSKCC, New York, NY;_ 2 _Dana-Farber Cancer Institute, MA_.

Epigenetic silencing of miRNAs is one of the major mechanisms of aberrant miRNA expression in cancer. Several studies showed that hypermethylation of miRNAs contributes to prostate cancer (PC) initiation and progression. Recent screening for epigenetically regulated miRNAs in PC revealed hypermethylation of the miR-193b in tumor, but no related functional studies of miR-193b have been pursued. miR-193b functions as a tumor suppressor in various cancer by targeting multiple oncogenic pathways. Methylation regulated miR-193b silencing has been observed in multiple cancer types. In this study, we aimed to elucidate the function of miR-193b and related molecular mechanisms in PC. In TCGA cohort, we observed a significant correlation between methylation of the miR-193b and its expression level. We further confirmed that some of PC cell lines recapitulate the methylation of miR-193b seen in clinical samples. The phenotypes induced by exogenous miR-193b in PC cell lines showed the tumor suppressive property of miR-193b. Since some of miRNA-target interactions are conserved across organisms, we selected the top 150-downregulated genes by miR-193b in liposarcoma from public available datasets and unraveled the correlation between these genes with PC progression. We further applied the 150 genes to the PC subtype (PCS) signatures. There were 41 genes overlapping with the aggressive PCS1 among 150 genes, indicating miR-193b may affect these key 41 genes that regulate PC progression. The inhibition of the 41 genes by miR-193b was validated in multiple PC cell lines. Furthermore, high expression of these genes was highly correlated with recurrence of PC. The GSEA analysis of 41 genes revealed high enrichment in the FOXM1 target network. We further identified FOXM1 as a direct target of miR-193b and overexpression of FOXM1 is highly correlated with clinical outcomes including metastasis and lethality. Besides FOXM1, we also identified RRM2 as another important target of miR-193b, which is part of the PCS1 signature. Knockdown of RRM2 phenocopied miR-193b in PC cells. Its overexpression is highly correlated clinical outcomes. To explore the therapeutic potential of regulating miR-193b levels, we restored miR-193b expression by using combination treatment with DNMT and HDAC inhibitors (5-azacytidine and mocetinostat), resulting in the inhibition of FOXM1 and RRM2 expression. Interestingly, FOXM1 and RRM2 expression are highly correlated in multiple PC cohorts. All together, we revealed the tumor suppressive function of miR-193b in PC. A 41-gene set was identified and validated as the targets of miR-193b in PC cells, and showed a high correlation with PC progression in multiple PC cohorts. FOXM1 and RRM2 may be the key targets of miR-193b in PC. Our findings suggest that hypermethylation of miR-193b in PC may release the inhibition of some key oncogenes that contribute PC progression.

#4328

Epigenetic modulation of SSTR2 expression provides the potential to broaden PEN-221 treatment population.

Samantha Perino, James Quinn, Jessica Freda, Brian White, Scott Bailey, Viren Bhatia, Beata Sweryda-Krawiec, Mark Bilodeau, Jeffrey D. Bloss, Kerry Whalen, Richard Wooster. _Tarveda Therapeutics, Watertown, MA_.

The expression of somatostatin receptor subtype 2 (SSTR2) is increased in up to 80% of neuroendocrine tumors (NETs), 20-40% of small cell lung cancers (SCLC) and a number of other tumor types. As a cell surface receptor, SSTR2 is a logical target for a drug conjugate. However, marginal, or loss of, expression of SSTR2 in tumor tissues has potential to limit the benefit from such conjugates.

Recent published preclinical studies have demonstrated that epigenetic modulators, such as HDAC inhibitors, can result in increased SSTR2 expression with treatment. Given that the expression of SSTR2 can be variable from patient to patient and in some cases low to absent, we hypothesized that the combination of epigenetic modulators with an SSTR2 target therapy may provide a benefit for patients who are not eligible for these targeted approaches on their own.

PEN-221 is a miniature drug conjugate of an SSTR2 targeting peptide attached to the potent cytotoxic DM1 through a cleavable disulfide linker. PEN-221, currently in Phase 1/2a (NCT02936323), is designed as a potent and selective anticancer agent to treat patients whose tumors express SSTR2. The targeting peptide of PEN-221 selectively binds to SSTR2, triggers receptor internalization leading to the accumulation of the DM1 to provide antitumor activity by disrupting microtubule networks causing apoptosis and mitotic catastrophe. In preclinical studies, single agent PEN-221 treatment leads to complete and sustained tumor regression in xenograft models that over-express SSTR2. We hypothesize that increased expression of SSTR2 as a result of epigenetic modulation from HDAC or other epigenetic modulators, may allow for the broadening of PEN-221 treatable tumors.

To evaluate these hypotheses, we have combined PEN-221 and epigenetic modulators in preclinical models of cancer. Efficacy studies were carried out in models expressing various degrees of SSTR2, and combinations resulted in greater efficacy than that of the single agent. In addition, a pharmacodynamic assessment of epigenetic modulator treatment on SSTR2 expression levels was performed. These data demonstrate that combination of PEN-221 and epigenetic modulators provide greater efficacy than single agent activity alone and support the evaluation of such combinations in the clinical setting.

#4329

Pharmaceutical means of targeting the fusion oncogene EWS-FLI1 in the Ewing family of tumors.

Daniel A. Heisey,1 Timothy Lochmann,1 Marissa Calbert,1 Maninderjit Ghotra,1 Yuki Kato Maves,2 Sosipatros Boikos,1 Cyril Benes,3 C. Patrick Reynolds,4 Anthony Faber1. 1 _Virginia Commonwealth University, Richmond, VA;_ 2 _Crown Bioscience Inc, CA;_ 3 _Massachusetts General Hospital Cancer Center, Boston, MA;_ 4 _Texas Tech University, Lubbock, TX_.

The EWS-FLI1 translocation is the hallmark genomic alteration in the Ewing Family of Tumors (EWFT) a malignancy of the bone and surrounding tissue, predominantly affecting children and adolescents. Although significant progress has been made for the treatment of localized disease, patients with metastasis or who relapse after chemotherapy have less than a 30% five year survival rate. EWS-FLI is currently not pharmaceutically druggable, driving the need for more effective targeted therapies. It is well known that the EWS-FLI1 translocation induces a variety of epigenetic changes which impact tumorigenesis and disease progression. Using high throughput drug screening we have identified a novel epigenetic susceptibility in EWFT to the H3K27 demethylase inhibitor GSK-J4 (GlaxoSmithKline). Subsequent treatment with GSK-J4 leads to a decrease in H3K27 acetylation and ultimately silencing of EWS-FLI1 gene targets. We sought to fully characterize the hypersensitivity of EWFT to GSK-J4 and to elucidate the mechanisms of histone modifications caused by the EWS-FLI1 translocation in EWFT. We are examining key shifts in histone modifications at FLI1 target gene enhancer elements in the presence of GSK-J4 as a means of directly regulating FLI1 tumorigenesis. Additionally, we seek to determine the efficacy of GSK-J4 in combination with a CDK7 inhibitor (THZ1), which have shown synergy in vitro, through the utilization of patient derived xenograft models (PDX) and the identification of biomarkers of sensitivity to rationalize the use of this combination for clinical trials.

#4330

**Activation of** AZIN1 **RNA editing facilitates and promotes invasive potential of cancer associated fibroblasts in colorectal cancer.**

Sho Takeda,1 Kunitoshi Shigeyasu,1 Yoshinaga Okugawa,2 Kazuhiro Yoshida,1 Yoshiko Mori,1 Shuya Yano,1 Kazuhiro Noma,1 Yuzo Umeda,1 Yoshitaka Kondo,1 Hiroyuki Kishimoto,1 Fuminori Teraishi,,1 Hiroshi Tazawa,1 Shunsuke Kagawa,1 Ajay Goel,3 Toshiyoshi Fujiwara1. 1 _Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Kita-ku Okayama, Japan;_ 2 _Mie University Graduate School of Medicine, Japan;_ 3 _Baylor Scott & White Research Institute and Charles A. Sammons Cancer Center, Dallas, TX_.

Colorectal cancer (CRC) is a common malignancy worldwide and remains the second leading cause of cancer-related deaths in Western countries. Emerging evidence demonstrates that such lifestyle choices profoundly impact various epigenetic modifications, and lead to cancer.Adenosine-to-inosine (A-to-I) RNA editing is an epigenetic modification mediated by Adenosine Deaminases that act on the RNA (ADAR), and has recently been discovered to be dysregulated in human cancers. However, the clinical significance and the functional role of RNA editing in cancer-associated fibroblasts (CAFs) remains unclear. By systematically analyzing a large cohort of 627 CRC specimens, we investigated the expression pattern of ADAR1 and the its biological significance on the antizyme inhibitor 1(AZIN1)RNA editing levels, which is one of the most frequently edited genes in cancers. Both ADAR1 expression and AZIN1RNA editing levels were significantly elevated in CRC tissues vs. normal mucosa, and these findings correlated with the increased expression of mesenchymal markers, Vimentin (ρ=0.44) and Fibroblast activation protein (ρ=0.38). ADAR1 was specifically upregulated in consensus molecular subtype 4 (CMS4) CRCs, which have mesenchymal characteristics and associate with poor prognosis.Intriguingly, ADAR1 was overexpressed in both cancer cells and fibroblasts from cancerous lesions. We next assessed whether cancer cells can promote ADAR1 expression and enhance AZIN1RNA editing in fibroblasts. Fibroblasts were cultured in the conditioned medium (CM) derived from CRC cells. Fibroblasts cultured with such CM expressed ADAR1 at significantly higher levels when compared to controls. Likewise, the overexpression of ADAR1 was associated with corresponding increase in AZIN1RNA editing. Overexpression of edited AZIN1RNA enhanced invasiveness of fibroblasts relative to wild-type AZIN1(p=0.0079).Previously the oncogene, ODC, was identified as a downstream target of edited AZIN1. The overexpression of edited AZIN1RNA resulted in upregulation of ODC protein in fibroblasts, confirming that edited AZIN1RNA stabilizes ODC more effectively than its wild-type counterpart in fibroblasts. In the previous study, ODC was reported to be the promoter of invasive potential in fibroblasts. Our results suggest that edited AZIN1RNA may promote invasion of fibroblasts via accumulation of ODC.This study is the first to investigate the clinical significance of ADAR1 expression and the degree of AZIN1RNA editing in colorectal CAFs, along with the determination the functional role of AZIN1RNA editing in this malignancy. Taken together, our study suggests that overexpression of ADAR1 in cancer lesion promotes malignant potential in CRC. We present novel evidence that ADAR1 is overexpressed in fibroblasts and may facilitate invasive potential in cancerous lesions in CRC microenvironment.

#4331

Potential role of the splicing factor SF3B1 in epigenetic regulation and activation of p53 signaling.

Sandra Deliard, Yasuyuki Okamoto, Jozef Madzo, Somnath Pandey, Jaroslav Jelinek, Jean-Pierre Issa. _Lewis Katz School of Medicine at Temple University, Philadelphia, PA_.

To develop novel and improved cancer therapies, we aim to target the epigenome, which is often deregulated in cancer. We identified the splicing factor SF3B1 as a potential epigenetic regulator in a whole genome siRNA screen for reactivation of aberrantly silenced genes alone or in synergy with the DNMT inhibitor Decitabine (DAC). The screen was done in YB5 colon cancer cells which contain a methylated and silenced CMV promoter driving GFP expression, and confirmed in a similar model in HCT116, another colon cancer cell line. SF3B1 has been studied in splicing, but little is known about its effects on gene regulation outside of this function, prompting us to further investigate this. We validated the screen findings by siRNA (siSF3B1) and inhibition of SF3B1 with Pladienolide B (PB). We then performed RNA-Seq and Reduced Representation Bisulfite Sequencing (RRBS) in cells treated with siSF3B1 and DAC, and we performed ChIP-Seq to assess SF3B1 binding. By flow cytometry, we measured a ~7% and ~5% induction of GFP in YB5 cells after siSF3B1 and DAC, respectively. The combination treatment caused a synergistic increase to ~15% GFP positive cells (p < 0.001). siSF3B1 led to 423 up and 338 downregulated genes, while DAC induced 430 up and 135 downregulated genes. With the combination, there was activation of 1190 genes and downregulation of 904 genes (fold change > 2, FDR < 0.1). There were 695, 119, 1584 genes alternatively spliced following siSF3B1, DAC, and the combination treatment, respectively, but there was no significant overlap with the regulated genes (p < 0.05), suggesting a distinct mechanism for gene expression regulation. There were two major subsets of siSF3B1 upregulated genes. A set of genes had low promoter methylation (0-20% methylation) and were p53 activation targets such as CDKN1A and GADD45A. The other set of genes had high promoter methylation (80-100% methylation) and promoters containing TATA-box motifs. RRBS showed global DNA hypomethylation after DAC treatment as expected, with an average methylation of 76.2% compared to 84.7% methylation in siControl cells. Further demethylation occurred in the combination treated cells, which had an average methylation of 72.7% (p<0.001). ChIP-Seq showed differential binding of SF3B1 at the transcription start sites (TSSs) of genes based on their expression. SF3B1 was depleted at TSSs of expressed genes and enriched at nonexpressed genes, resembling the binding pattern of histone H3. Finally, SF3B1 inhibition with PB alone and with DAC also induced reactivation of gene expression and altered DNA methylation. Together, these findings suggest that SF3B1 may be playing a role in gene regulation outside of its role in splicing. In cancer, SF3B1 transcriptional target genes are potentially tumor suppressor genes and upon knockdown or inhibition of SF3B1, their expression is reactivated leading to antitumor effects.

#4332

Targeting MYC overexpressing leukemia with cardiac glycoside proscillaridin through downregulation of histone acetyltransferases.

Elodie Marie Da Costa,1 Gregory Armaos,1 Gabrielle McInnes,1 Annie Beaudry,1 Gael Moquin-Beaudry,1 Virginie Bertrand-Lehouillier,1 Maxime Caron,1 Pascal St-Onge,1 Jeffrey R. Jonhson,2 Nevan Krogan,2 Yuka Sai,3 Michale Downey,3 Moutih Rafei,4 Meaghan Boileau,5 Kolja Eppert,5 Ema Florez-Diaz,6 Andre Haman,6 Trang Hoang,6 Daniel Sinnett,1 Christian Beausejour,1 Serge McGraw,1 Noel J. Raynal7. 1 _Sainte-Justine University Hospital Research Center, Montreal, Quebec, Canada;_ 2 _University of California, San Francisco, CA;_ 3 _Institute of Systems Biology, Ottawa, Ontario, Canada;_ 4 _Universite de Montreal, Montreal, Quebec, Canada;_ 5 _McGill University, Montreal, Quebec, Canada;_ 6 _Institute of Research in Immunology and Cancer Universite de Montreal, Montreal, Quebec, Canada;_ 7 _Université de Montreal, Montreal, Quebec, Canada_.

Targeting MYC oncogene remains a major therapeutic goal in anticancer therapies. Here, we demonstrate that proscillaridin, a cardiac glycoside approved for heart failure treatment, causing Na+/K+ pump inhibition, targets efficiently MYC overexpressing cancer cells. At clinically relevant doses, proscillaridin induced rapid downregulation of MYC protein level, and produced growth inhibition preferentially against MYC overexpressing leukemic cell lines including lymphoid and myeloid stem cell populations. Whole transcriptome analysis with RNA sequencing of acute lymphoblastic leukemia cells showed a downregulation of gene sets involved in MYC pathways and cell replication, and an upregulation of genes involved in hematopoietic differentiation induced by proscillaridin treatment. Gene expression changes were associated with an epigenetic remodeling of chromatin active marks. Proscillaridin induced a significant loss of lysine acetylation in histone H3 (at lysine 9, 14, 18 and 27). In addition, mass spectrometry analysis revealed a loss of lysine acetylation in non-histone proteins such as MYC itself, MYC target proteins, and a series of histone acetylation regulators. Global loss of acetylation correlated with the rapid downregulation of histone acetyltransferase proteins (such as CBP, P300, TIP60 and GCN5) involved in histone and MYC acetylation. Overall, these results strongly support the repurposing of proscillaridin in MYC overexpressing leukemia and suggest a novel strategy to target MYC by inducing the downregulation of histone acetyltransferases involved in its stability.

#4333

GCN5 positively correlates with c-MYC in non-small cell lung cancer.

Lisa Maria Mustachio, Jason Roszik, Aimee Farria, Sharon YR Dent. _MD Anderson Cancer Center, Houston, TX_.

Lung cancer causes the highest mortality in cancer-related deaths. Definition of novel molecular targets is needed since these cancers often become resistant to existing therapies. Epigenetic modifications may provide such targets. Recent reports suggest that the histone acetyltransferase (HAT) module within the transcriptional coactivator SAGA complex plays a role in cancer, creating a new link between epigenetic regulators and this disease. We aim to define the role of the SAGA HAT protein GCN5 in the regulation and function of oncogenes in lung cancers. Since prior work indicates that GCN5 regulates the stability of c-MYC and serves as a coactivator for MYC target genes, we hypothesize that there is a positive relationship between GCN5 and c-MYC in non-small cell lung cancer (NSCLC). We also propose that inhibition of GCN5 will reduce lung cancer formation and progression in mouse models of lung cancer driven by c-MYC overexpression. Our data indicate that both GCN5 and c-MYC proteins are upregulated in NSCLC cells compared to normal lung epithelial cell lines. This trend is observable only at the protein level, indicating that the upregulation of these proteins in NSCLC cells occurs post-transcriptionally. Both GCN5 and c-MYC expression levels are also positively associated in mouse and human NSCLC cell lines. Genetic repression of GCN5 in NSCLC cells reduces cell proliferation, increases the population of necrotic cells, and reduces c-MYC expression. Inhibition of the GCN5 active site using commercially available probes in NSCLC cells significantly (p < 0.05) reduces cell proliferation, increases the percentage of cells undergoing apoptosis, and reduces c-MYC expression. In human NSCLC tissues, GCN5 and c-MYC positively correlate and are associated with MYC target genes. Our ongoing studies will determine whether targeting GCN5 represses multiple oncogenic pathways in cancer and whether it inhibits oncoproteins difficult to directly target, such as MYC. We expect that our findings will highlight the importance of epigenetic regulators in carcinogenesis and extend beyond our current research focus to other cancer types.

#4334

Epigenetic reprogramming of tissue-specific transcription promotes metastasis.

Shuaishuai Teng,1 Yang Li,2 Ming Yang,1 Rui Qi,1 Qianyu Wang,1 Zhi Lu,1 Dong Wang1. 1 _Tsinghua University, Beijing, China;_ 2 _University of California, San Diego, CA_.

Metastasis is the cause of death for 90% of cancer patients, but little is known about how cancer cells adapt to and colonize new tissue environments. Colorectal cancer (CRC) is the third most common cancer and the fourth most common cancer cause of death globally. Here, we investigated how metastatic cancer cells, such as CRC cells, with original-tissue specificity adapt to the environment of a distant tissue, like liver.

By analyzing transcriptome data of multiple cohorts of clinical samples and primary/metastatic cell lines, we found metastatic CRC cells lose their colon-specific gene transcription program and gain a liver-specific gene transcription program as they metastasize in the liver. The elevated expression of a number of liver-specific genes was confirmed in human liver metastatic tumors by immunohistochemistry. Through epigenomic profiling, we revealed this transcription reprogramming is driven by a reshaped epigenetic landscape of both typical and super-enhancers, and chemical inhibition of enhancer activity disrupts the ability of cells to execute this altered transcription program and consequently inhibits metastasis. Further we identified liver-specific transcription factors, FOXA2 and HNF1A, bind to the gained enhancers in liver metastatic cells and activate the expression of liver-specific genes. We also found both FOXA2 and HNF1A are highly expressed in human liver metastatic tumors compared to primary CRC tumors. Loss of function of FOXA2 or HNF1A inhibits liver-specific transcription and impairs the ability of metastatic CRC cells to adapt to liver. Consistently, the gain of function of HNF1A activated liver-specific transcription and enhanced CRC liver metastasis.

Our findings indicate the direct contribution of a reprogrammed tissue-specific transcription program, which is induced by reshaped epigenetic landscape as well as distant-tissue-specific transcription factors, to the adaption and colonization of metastatic CRC cells to a distal organ. Notably, in addition to CRC liver metastasis, this tissue-specific transcription reprogramming is also observed in other multiple distant organs and/or cancer types. In summary, our data suggest that epigenetically reprogrammed tissue-specific transcription promotes metastasis and might have implications for targeted anti-metastasis therapies.

#4335

Epigenetic plasticity potentiates a rapid cyclical shift to and from an aggressive cancer phenotype.

Tong Xu, Hongtao Li, Rong Xu, Jenny Wei, Meng Li, Gangning Liang, Amir Goldkorn. _USC Norris Comprehensive Cancer Ctr., Los Angeles, CA_.

Introduction: Subpopulations of highly tumorigenic, drug resistant cancer stem-like cells (CSCs) play a key role in cancer recurrence and progression. Surprisingly, these aggressive cells can arise repeatedly de novo from bulk tumor cells independently of mutational events. We investigated whether transition to and from a cancer stem-like state is associated with epigenetic alterations, such as DNA methylation and chromatin accessibility.

Materials and methods: Using FACS and Hoechst dye staining, side population (SP) cells marked by high tumorigenicity and drug resistance vs. non-side population (NSP) cells lacking these properties were isolated from bladder cancer cell lines. Global DNA methylation and chromatin accessibility were mapped for SP vs. NSP cells using AcceSssIble assay on the Infinium EPIC platform. Differential gene expression for SP vs. NSP cells was assayed by HuEx array. AcceSssIble and HuEx results were overlapped to generate a subset of genes with differential accessible promoter regions and expression. One such gene, E2F3, was validated by qRT-PCR and analyzed clinically using the Oncomine and Basespace online data mining engines, as well as functionally by lentiviral shRNA depletion in bladder cancer cells for cell migration, invasion and drug resistance. 5-AZA was used to modulate global DNA methylation in bladder cancer cells.

Results: SP and NSP subpopulations exhibited differential DNA methylation and chromatin accessibility at thousands of gene loci by AcceSssIble assay, and a majority of differential chromatin accessibility was independent of DNA methylation. When overlapped with HuEx data, 38 genes exhibited differential expression associated with differential promoter chromatin accessibility. One such gene validated by qRT-PCR was E2F3, a cell cycle regulator that was found to be associated with bladder cancer in the Oncomine and Basespace databases. In vitro E2F3 depletion by lentiviral shRNA resulted in smaller SP subpopulations and reduced cell migration, invasiveness, and drug resistance. Epigenetic interference with 5-AZA blocked the phenotypic transition, reduced SP subpopulations, and lowered E2F3 expression.

Conclusion: Rapid cyclical transition between the SP and NSP subpopulations was associated with dramatic shifts in chromatin accessibility with concomitant changes in gene expression of clinically relevant genes that drove the SP phenotype, cell migration, invasiveness, and drug resistance. These observations implicate epigenetic plasticity as a driver of the switch between cancer stem-like and non-cancer stem-like states, suggesting new potential therapeutic avenues to control cancer resistance and progression.

#4336

Epigenetic regulation of combined hepatocellular-cholangiocarcinoma subtypes.

Kyle M. Schachtschneider,1 Ryan Peter Lokken,1 Yu-Hui Huang,1 Grace Guzman,1 Lawrence B. Schook,2 Ron C. Gaba1. 1 _Univ. of Illinois at Chicago, Chicago, IL;_ 2 _Univ. of Illinois at Urbana-Champaign, Urbana, IL_.

Combined hepatocellular-cholangiocarcinoma (HCC-CCA) is a rare liver tumor comprising histologic features of both HCC and CCA. Due to its heterogeneous nature, treatment of combined HCC-CCA is a significant clinical challenge and prognosis remains poor. Therefore, further understanding of the tumor biology underlying the individual subtypes of this mixed tumor is required to improve treatment stratification and optimize treatment strategies. This study sought to identify epigenetic regulation underlying gene expression patterns in the individual components of combined HCC-CCA. Formalin fixed paraffin embedded tumor specimens from 10 patients diagnosed with combined HCC-CCA were utilized in this study. Hematoxylin and eosin staining was performed for each sample, and regions representative of the individual HCC and CCA components were delineated by a pathologist. Unstained slides were cut and dissected to separate HCC and CCA components. DNA and RNA extraction was performed for each sample for DNA methylation (n = 8 HCC and 7 CCA) and gene expression (n = 8 HCC and 8 CCA) profiling via reduced representation bisulfite sequencing and RNA-seq, respectively. As expected, DNA methylation levels at transcription start sites (TSS) were negatively correlated with gene expression in all samples (Spearman's rho = -0.133 to -0.546; p < 2.2 x 10-16). Samples did not cluster by tumor subtype when comparing genome-wide DNA methylation and gene expression patterns. Interestingly, of the 5 patients with DNA methylation data available for both subtypes, 4 clustered by patient as opposed to cancer subtype, suggesting similar epigenetic regulatory patterns arising from development in the same microenvironment and genetic background. Differential gene expression analysis resulted in the identification of 58 differentially expressed genes (DEGs) between the HCC and CCA subtypes (q-value < 0.05). In addition, a total of 474 differentially methylated regions (DMRs) were identified between the HCC and CCA subtypes (minimum difference > 25%; q-value < 0.05). Of these, 422 DMRs overlapped with 324 known genes, 1 of which (STK38L) displayed increased expression (log2 fold change = 4.39; q-value = 0.01) associated with hypomethylation of 2 regions (-34.86% and -27.47%; q-value < 1 x 10-38) in the CCA compared to HCC group. STK38L encodes a serine/threonine kinase involved in Hippo signaling, a highly conserved signaling pathway that functions as a key coordinator of tissue growth and homeostasis. In all, these results provide insights into the epigenetic regulatory patterns associated with the two components of combined HCC-CCA. Future studies may aim to understand the effects of epigenetic regulation on treatment response for this deadly disease.

#4337

Identifying epigenetic biomarkers in a clinical cohort of individuals with oropharyngeal cancer.

Ryan Langdon. _University of Bristol, Bristol, United Kingdom_.

Oropharyngeal cancer (OPC) shows a distinct epidemiology to other head and neck cancer (HNC) subtypes. Several lifestyle and dietary factors as well as viral infections have been implicated in altering both incidence and prognosisfor OPC, notably smoking, alcohol consumption and HPV infection. We aimed to: 1) assess the impact of these epidemiological factors on epigenome-wide DNA methylation patterns, 2) evaluate whether DNA methylation patterns were associated with survival from OPC,3) ascertain whether novel exposure or prognostic indicators could be derived from DNA methylation patterns and 4)establish whether DNA methylation plays a causal role in mediating the impact of prognostic factors on survival.In a large prospective HNC cohort (Head and Neck 5000) we undertook epigenome-wide association study (EWAS) and differentially-methylated region (DMR) analyses of the aforementioned prognostic factors and mortality, using methylation data on >850.000 CpG sites measured using theIllumina MethylationEPIC on 420 individuals diagnosed with OPC . We performed multivariable linear regression analysis of DNA methylation against the prognostic factors, and Cox regression analysis of survival in relation to DNA methylation. We then determined the extent to which any results mediated the pathway between prognostic factors and OPC mortality using Mendelian randomization (MR).We identified CpG sites and DMRs associated with smoking and alcohol consumption below our multiple-testing threshold, but none with HPV positivity. We also identified 7 CpGs associated with survival (~3 years post-diagnosis), independent of smoking, alcohol consumption and HPV positivity. 18 CpGs across 3 DMRs were found to be associated with both smoking and mortality and 5 CpGswithin 1 DMR were identified in relation to both alcohol and mortality. We hypothesised that for these CpG sites there could be a mediating effect of DNA methylation, whereby DNA methylation mediates the association between the prognostic factor and OPC mortality. Using an inverse-variance weighted (IVW) MR approachto investigate the causal effect of DNA methylation at the identified sites, we found that hypomethylation in CpGslocated within a DMR associated with smoking (Chr2:220325443-220326041; annotating to the SPEG gene) showed some evidence of a causal effect on increased mortality (HR: 1.15, 95% CI: 1.05 to 1.25, P: 1.12x10-3). Within the context of OPC, we found novel epigenetic biomarkers measured by the Illumina MethylationEPIC array to be associated with the prognostic factors of smoking and alcohol, and with mortality, respectively. We also found preliminary evidence of a potential mediating effect of DNA methylation at the SPEG gene, between smoking and OPC mortality. However, longer follow up in Head and Neck 5000 and suitable replication data is needed to strengthen the validity of these findings.

#4338

Enhancer mapping in triple negative breast cancer as a tool for biomarker and oncogene discovery.

Ryan Raisner, Russell Bainer, Karen Gascoigne. _Genentech, Inc., South San Francisco, CA_.

Triple Negative Breast Cancer (TNBC) represents a heterogeneous disease with no common drivers or effective targeted therapies. Here we use ChIP-Seq combined with RNA-Seq to map the gene-regulatory landscape of TNBC in cell lines and primary tumors. Using this approach we identify novel actionable subtypes of the disease based on enhancer activity. We also identify novel tumor-specific enhancers and associated enhancer-driven oncogenes, and confirm enhancer status as a predictive biomarker of gene dependency.

#4339

Critical role of Dnmt3b catalytic activity in prevention of oncogene-induced leukemias and lymphomas.

Katarina Lopusna,1 Pawel Nowialis,1 Staci L. Haney,2 Ajay Abraham,1 Jana Opavska,1 Rene Opavsky1. 1 _University of Florida, Gainesville, FL;_ 2 _University of Nebraska Medical Center, Omaha, NE_.

Cytosine methylation is involved in a variety of biological processes, including development, X-chromosome inactivation, imprinting, and suppressing unwanted transcription. DNA methylation in mammalian cells is catalyzed by several DNA methyltransferases, including DNMT1, DNMT3A and DNMT3B. In addition to DNA methylation catalysis, these enzymes interact with a number of repressive proteins including HDACs, thereby repressing transcription in methylation-independent manner. The extent to which methylation-dependent and independent activities play roles in biological processes remains poorly understood. Here we examined the role of methyltransferase (MT) activity of Dnmt3b in normal mouse embryogenesis and in prevention of hematologic malignancies. Loss of Dnmt3b in mice is embryonically lethal between 10.5-12.5 dpc. Dnmt3b was also identified as tumor suppressor in murine models of lymphoid and myeloid malignancies. To address whether Dnmt3b's catalytic activity is important for mouse embryogenesis and prevention of hematologic malignancies, we generated mice bearing a conventional Dnmt3bCI allele using CRISPR/Cas9 system. This catalytically inactive allele of Dnmt3b (Dnmt3bCI) was generated by a double amino acid substitution in enzyme active center (P656V and C657D) abolishing its ability to perform transfer of methyl group onto cytosine. Surprisingly, Dnmt3bCI/CI mice were born at similar ratios as wild-type littermates and lived over 12 months suggesting that Dnmt3b is dispensable for both pre- and post-natal development. Importantly, MYC-induced T-cell lymphomagenesis was accelerated in the absence of Dnmt3b's methyltransferase activity in MYC;Dnmt3bCI/CI mice. Global gene expression and methylation profiling revealed a number of deregulated events specific for MYC;Dnmt3bCI/CI, resulting in activation of Pak, Fgfr and Igf2 signaling and downregulation of tumor suppressors (e.g. Lrp12, Dusp6 and Marcks). The lack of Dnmt3b catalytic activity also accelerated a development of acute myeloid leukemia induced by MLL-AF9 overexpression in mice. Altogether, our data suggest that Dnmt3b's MT activity is dispensable for mouse development but critical to prevent hematologic malignancies.

#4340

A novel fluoride (ChlA-F) transcriptionally upregulates miR494 via a HuR/JunB axis to inhibit cell invasion in human bladder cancers.

Zhongxian Tian, Xiaohui Hua, Jiheng Xu, Junlan Zhu, Jingxia li, Chuanshu Huang. _Nelson Institute of Environmental Medicine, Department of Environmental Medicine and Urology, New York, NY_.

Bladder cancer (BC) is a commonly occurring cancer and characterized by a low 5-year survival rate particularly in patients with invasive BCs. Here we investigated the chemotherapeutic activity of ChlA-F, a novel C8 fluoride derivative of cheliesisin A (ChelA) with potent anti-neoplastic properties. ChlA-F treatment upregulated miR494 expression and significantly suppressed cell invasion in human BC cell lines. Mechanistically, ChlA-F-induced upregulation of miR494 expression was due to a HuR-mediated increase in JunB mRNA stabilization and protein expression, which led to an increase in miR494 transcription via direct binding to the miR494 promoter region. After ChlA-F treatment, upregulated miR494 was found to bind to the 3' UTR region of c-Myc mRNA, resulting in decreased c-Myc mRNA stability and protein expression. This, in turn, led to decreased transcription of a c-Myc downstream-regulated gene, MMP-2 and ultimately, inhibition of BC invasion. Here we provide the first evidence showing that our newly synthesized compound, ChlA-F, has a profound inhibitory effect on human BC invasion and elucidate the mechanisms underlying this anti-neoplastic activity. Our data suggest that ChlA-F may be a promising therapeutic alternative for treatment of invasive and metastatic human BC in patients. 

### Functional Genomics

#4341

NCI Office of Cancer Genomics: Promoting multidisciplinary research to translate findings into the clinic and advance precision oncology.

Cindy W. Kyi,1 Pamela C. Birriel,1 Tanja M. Davidsen,2 Martin L. Ferguson,1 Patee Gesuwan,2 Nicholas B. Griner,1 Yiwen He,2 Subhashini Jagu,1 Eva Tonsing-Carter,1 Daniela S. Gerhard1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _National Cancer Institute, Rockville, MD_.

The mission of the National Cancer Institute's (NCI) Office of Cancer Genomics (OCG) is to advance the molecular understanding of cancers in order to improve clinical outcomes through precision medicine. Although vast amounts of genomic data are available for many types of cancers, identifying genetic alterations in rare and pediatric cancers is still a challenge. Efficient bioinformatics tools to analyze, manage, store, and access data are also necessary for the research community. To develop effective and targeted treatments, clinically accurate genotypic and phenotypic research models are much needed. OCG's programs focus on addressing these challenges through multidisciplinary, collaborative research efforts.

The four initiatives of OCG support research on structural, functional, and translational genomics, as well as development of next-generation cancer models. The Cancer Genome Characterization Initiative (CGCI) and the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) programs use transcriptomic, genomic, and epigenomic approaches to examine genetic alterations between various tumors and matched normal tissues. CGCI projects focus on HIV-associated and rare cancers such as Burkitt lymphoma, while TARGET focuses on high-risk childhood cancers. The goals of these programs are to attain insights into key mutations that drive tumors and genetic abnormalities specific to cancer subtypes and to develop effective and less toxic therapies for patients. CGCI and TARGET data are available to the research community through data matrices on the OCG website. OCG also recently launched the Pediatric Genomic Data Inventory (PGDI) as a new resource for investigators to access molecular characterization data.The Cancer Target Discovery and Development (CTD2) Network advances cancer research by bridging the knowledge gap between cancer genomics and precision oncology. The Network aims to understand the cancer metastasis, tumor heterogeneity, and drug resistance to develop optimal combinations of small-molecules or immunotherapy with small molecules. As a community resource program, the CTD2 Network develops and provides access to data, tools, methods, and reagents through the Data Portal and the Dashboard. The Human Cancer Models Initiative (HCMI) is an international consortium that is generating novel human tumor-derived culture models from a wide variety of cancer types including rare and understudied cancers. The models, together with related clinical and genomic data, will be available as a resource to the world-wide research community.

OCG's policies on data usage, as well as guides to accessing data, are explained on the OCG website (https://ocg.cancer.gov/). Researchers, potential collaborators, and interested members of the public are encouraged to visit the OCG webpages or contact OCG at ocg@mail.nih.gov.

#4342

**Direct genome editing of patient-derived xenografts for rapid** in vivo **functional genomics.**

Christopher H. Hulton,1 Emily A. Costa,2 Charles M. Rudin,3 John T. Poirier3. 1 _Louis V. Gerstner Jr. Sloan Kettering Graduate School of Biomedical Sciences, New York, NY;_ 2 _Weill Cornell Graduate School of Medical Sciences, New York, NY;_ 3 _Memorial Sloan Kettering Cancer Center, New York, NY_.

Patient-derived xenografts (PDXs) are high fidelity in vivo tumor models that have become valuable tools for cancer research, particularly in the study of emerging cancer therapeutics. However, PDXs are rarely used in mechanistic and functional genomic studies, due in part to the technical barriers imposed by continuous passaging in vivo. We sought to develop a CRISPR-Cas9 platform that would leverage the robust nature of PDXs and enable in vivo functional genomic studies in these cancer models. To this end, we created pSpCTRE-CD4, an all-in-one doxycycline-inducible Cas9 lentiviral vector, which features a novel truncated CD4 selection marker that enables selection of transduced PDX cells by flow cytometry, bypassing the need to culture these cells ex vivo. The inducible TRE3GS promoter controlling Cas9 expression is in the reverse orientation, which reduces potential leaky expression of Cas9 and also places this promoter in close proximity to the constitutive EFS promoter controlling CD4 and rtTA expression. Interestingly, this promoter orientation causes a dramatic ~100-fold increase in CD4 expression over basal levels in the presence of doxycycline and thus provides an indicator for cells that induce Cas9. This property of pSpCTRE-CD4 allows for the study of heterogeneous tumor cell populations without the need to select for single cell clones, which is a common practice in inducible cell line systems. In vitro functional studies of pSpCTRE-CD4 showed that this vector is tightly regulated by doxycycline. We transduced 27 lung cancer PDXs with pSpCTRE-CD4 lentivirus and observed transduction efficiencies ranging from 0-30%. Of these, we successfully enriched 8 Cas9 PDX models derived from multiple lung cancer subtypes. We functionally validated these Cas9 PDXs by introducing an sgRNA targeting the essential gene RPA1 in an in vivo clonal competition assay. For each of the Cas9 PDXs tested, we observed a significant decrease in the sgRPA1:sgNon-targeting ratio for two independent RPA1 sgRNAs in mice treated with doxycycline, indicating a loss of fitness in these PDXs when RPA1 is depleted. Similarly, in JHU-LX55a-Cas9, a KRAS mutant lung adenocarcinoma PDX, we observed a significant decrease in the fitness of cells with an sgRNA targeting KRAS. We are using this system to investigate genetic dependencies within these PDXs and have designed a small sgRNA library targeting the druggable genome to perform CRISPR screens in these models. Additionally, we have designed a recombinant AAV vector that expresses a single sgRNA as well as template to introduce point mutations via homology-directed repair and we are using this system to interrogate variants of unknown significance identified in large-scale tumor sequencing studies. The CRISPR-Cas9 platform we have developed will greatly expand the use of PDXs and enable functional genomic studies to be performed in this robust in vivo model system.

#4343

A focused CRISPR screen to identify synthetic lethal interactions with the novel eIF4A inhibitor eFT226 in KRAS driven NSCLC.

Nathan P. Young, Craig R. Stumpf, Joan Chen, Gary G. Chiang, Peggy A. Thompson, Kevin R. Webster. _eFFECTOR Therapeutics, San Diego, CA_.

Tumor development is often characterized by dysregulated messenger RNA (mRNA) translation of key oncogenic factors that promote increased proliferation, resistance to apoptosis and enhanced metastatic potential. The eukaryotic translation initiation factor 4F (eIF4F) complex, a master regulator of protein synthesis, is comprised of eIF4E (mRNA-cap-binding protein), eIF4A (RNA-helicase), and eIF4G (scaffolding protein), that together orchestrate mRNA recruitment to ribosomal subunits as well as efficient scanning of the mRNA 5'-untranslated region. As a downstream target of growth-promoting signaling cascades, eIF4F also serves as a central node in several important oncogenic pathways including KRAS and PI3K/mTOR. eIF4F subunits are frequently over-expressed in various malignancies; therefore, repressing eIF4F activity has emerged as a promising anti-cancer therapeutic strategy. eFT226 is a potent and selective translational regulator that targets eIF4A1. eFT226 down-regulates the translation of a unique gene set and displays robust anti-tumor activity across multiple models in vitro and in vivo. A functional genomic screen using CRISPR/Cas9 was performed to discover synthetic lethal genetic interactions with eFT226 to enable development of novel drug combination and/or patient selection strategies. The screen was conducted in a panel of non-small cell lung cancer (NSCLC) cell lines driven by oncogenic KRAS and p53 pathway mutations, a tumor subtype with limited therapeutic options. Utilizing a focused guide RNA (gRNA) lentiviral library of ~700 cancer-related targets with diverse roles in growth-factor signaling, metabolism, epigenetics, translation and stress-responses, several genetic perturbations that sensitized tumor cells to eFT226 were uncovered. Interestingly, many of the genes whose loss-of-function enhanced eFT226's activity fall into distinct classes including an oncogenic signaling pathway commonly activated in many cancers as well as redox homeostasis. Moreover, a number of these targets include tumor suppressor genes that are frequently mutated or inactivated through loss of function in certain cancer types suggesting that specific genetic contexts may dictate eFT226 efficacy. Ongoing studies are aimed at confirming these hits from the primary screen. Collectively, these validated targets will represent genomic vulnerabilities to eFT226 that can aid in the design of drug combination and patient selection strategies for NSCLC.

#4344

Integrative functional proteogenomics for unannotated or uncharacterized proteins in cancer.

John R. Prensner,1 Oana Enache,2 Zhe Ji,3 Karsten Krug,2 Karl R. Clauser,2 Xiaoping Yang,2 Federica Piccioni,2 David E. Root,2 Todd R. Golub2. 1 _Boston Children's Hospital, Boston, MA;_ 2 _Broad Institute, Cambridge, MA;_ 3 _Northwestern University, Chicago, IL_.

Introduction: Numerous transcripts annotated as long noncoding RNAs play a central role in cancer biology. For many, their noncoding status is merely a presumption. Genome-wide sequencing of ribosomal footprints has nominated thousands of unstudied open reading frames (ORFs) within lncRNAs, representing an expansion of the proteome. Here, we investigate previously unstudied proteins in cancer cell biology.

Methods: Ribosome profiling data was analyzed with RibORF. 96 hours after infection with lentivirus for selected ORFs, L1000 expression profiling was performed on 4 cell lines. A CRISPR library was screened across 8 cells lines with sgRNA sequencing on days 0, 7 and 21 post-infection.

Results: We analyzed ribosomal profiling data for 14 cell lines (~320 million sequencing reads). We nominated 28530 non-canonical ORFs within annotated protein-coding genes, 6697 ORFs in annotated lncRNAs, and 1252 ORFs in pseudogenes. For further study, we selected 553 candidate ORFs that exhibited compelling features, including DNA conservation, translational efficiency, protein domain, among others.

We validated protein expression for 260 of 553 ORFs (47%): 89 (16%) had supporting peptides in deep-coverage proteomics datasets; 233 (42%) expressed protein after ectopic expression of individual V5-tagged cDNAs; 10 of 30 tested untagged ORFs expressed protein by biochemical in vitro translation.

Ectopic overexpression followed by RNA profiling revealed 259 cDNAs that caused cellular transcriptional changes in at least one of four cancer cell lines (A549, HA1E, A375, MCF7). 137 of the 259 (49%) were validated proteins. As controls, we generated methionine-mutant constructs: 65 of 71 mutant cDNA experiments were unable to cause similar expression changes.

We used a CRISPR library to identify novel ORF dependencies in 8 Cas9-derivatized cancer cell lines (MCF7, A549, A375, PC3, HEPG2, HELA, HA1E, HT29). For 42 ORFs, ≥ 2 targeting sgRNAs produced ≤ -1 log fold depletion in ≥ 1 cell lines. These ORFs were re-tested with a second sgRNA library.

Next, we investigated compelling candidates more deeply with immunoprecipitation with mass spectrometry. For the cancer outlier transcript LINC01314, which encodes a highly conserved 59 amino acid protein harboring a cortexin domain (pfam domain cl12620), we found interactions with IMMT, SAMM50, and CHCHD3, members of a mitochondrial complex. Another example is LINC00116, which encodes a highly conserved 56 amino acid protein that binds the importin-nuclear pore complex.

Conclusion: We establish a framework to discover, validate, and characterize unstudied proteins. About half of tested ORFs generated a detectable protein, and of these, half impacted cellular transcription. We discover novel gene dependencies, and are elucidating mechanisms for several ORFs. Together, our work is the first large-scale attempt to study the role of unannotated proteins in cancer cell biology.

#4345

A large-scale RNA-seq screen to identify regulators of alternative splicing in cancer.

April Lo, Maria McSharry, Alice Berger. _Fred Hutchinson Cancer Research Center, Seattle, WA_.

RNA splicing is dysregulated in a widespread manner in cancers including lung adenocarcinoma. In some cases, splicing changes can be attributed to cis-acting splice site mutations or trans-acting mutations in splicing factors. However, in most cases, the underlying causes of splicing changes are unknown. We hypothesized that upstream signaling inputs to alternative splicing regulation can explain some of these unknowns. Specifically, we studied how oncogenic lung cancer signaling pathways (EGFR/Ras, KEAP1/NRF2, MYC, and others) regulate alternative splicing and the expression and activity of splicing factors. We focus on signaling pathways because they can be readily therapeutically modulated with small molecule inhibitors (e.g. tyrosine kinase inhibitors), offering opportunities for therapeutic suppression of downstream splicing effects.

To experimentally determine how signaling pathway perturbation affects alternative splicing, we perturbed A549 lung cancer cells with each of 82 alleles of 27 genes (n = 4 to 8 biological replicates per allele). Genes and variants were selected based on the occurrence of the variants in lung adenocarcinoma tumors. In total, 417 whole transcriptome profiles were generated using the Smart-Seq v4 method (Clontech) and Nextera XT library preparation (Illumina). We performed differential expression analysis using edgeR and differential splicing analysis using MISO. Using this approach, we identified a high-confidence set of 2430 alternative splicing events differentially spliced in the perturbed samples compared to controls. Of these alternative splicing events, 1219 are skipped exons, 469 are mutually exclusive exons, 235 are alternative 5' splice sites, 255 are alternative 3' splice sites, and 252 are retained introns.

Among the perturbations tested, overexpression of the RBM45 wild-type allele and the RBM45 D434Y variant allele resulted in the greatest number of alternatively spliced events, with 367 and 323 events respectively. A closer look at these events reveals that when the RBM45 wild-type allele is overexpressed, exon 3 of the cyclin gene CCNG1 is skipped. Interestingly, when RBM45 variant alleles (D434Y, M126I) are overexpressed instead of the wild-type, the exon is not skipped, suggesting loss or change of function. These results suggest that, among other roles, RBM45 may regulate cell cycle patterns by regulating alternative splicing.

In sum, our screen identifies splicing events which are regulated by oncogenic signaling pathways. With this information, it will be possible to propose therapeutic options that mitigate aberrant splicing driven by signaling pathway components. Such therapy may already exist in the form of drugs for other targeted uses but could be repurposed to address aberrant splicing. Importantly, therapy of this type has the potential to be more tolerable compared to treatments directly aimed at splicing factors and splice sites.

#4346

PI3K pathway mediated splicing defects in ER+ breast cancers.

Erik Ladewig, Lauren Fairchild, Maurizio Scaltriti, Christina Leslie, Eneda Toska, Jose Baselga. _Memorial Sloan Kettering, New York, NY_.

Activating mutations in PIK3CA, the gene encoding for the catalytic subunit (p100a), are the most common oncogenic alterations in estrogen receptor-positive (ER+). The majority of PIK3CA mutations occur within two hot spots; exons 9 and exon 20 which encode the helical (E545K) and kinase domains (H1047R), respectively. These mutations result in hyperactivation of the PI3K/AKT/mTOR pathway and provide the rationale for the development of inhibitors targeting the PI3K pathway. To this end, PI3K α-specific inhibitors are showing antitumor activity in patients with PIK3CA-mutant, ER-positive breast cancer. Evidence of alternative mRNA regulation and splicing in various cancers has been described in the literature. Although, the majority of events appear to have unknown clinical significance, there is evidence alternative splicing can lead to drug resistance. The role of the PI3K pathway on transcriptional regulation and mRNA processing is not well studied. Through transcriptome analysis and cell assays in mouse MEF and human MCF10A cells we demonstrate splicing defects accrue as a result of the PI3K pathway activation by the PIK3CA H1047R mutation. PI3Ka pathway inhibitors were able to restore wildtype exon inclusion levels in these mutants, suggesting a potential clinical benefit. We propose that PIK3CA H1047R imposes mRNA differential isoform regulation by acting through the most commonly mutated PI3K pathway in ER+ breast cancers and that such mutations are targetable with PI3K pathway inhibitors.

#4347

Differentially expressed genes and molecular pathways in an autochthonous mouse prostate cancer model.

Shiv Verma, Sanjeev Shukla, Mitali Pandey, Gregory T. MacLennan, Sanjay Gupta. _Case Western Reserve Univ., Cleveland, OH_.

Prostate cancer remains a major public health problem and second leading cause of cancer-related deaths in men in the United States. Despite widespread screening efforts and advancement in therapeutic regimens, incidence of prostate cancer remains steady in the past two decades. Although serum prostate-specific antigen (PSA) has been used as a screening test for prostate cancer for over two decades, the practice remains controversial. As a screening test for prostate cancer, PSA lacks sensitivity and specificity as early detection marker. Furthermore, screening may lead to unnecessary biopsies, over-detection, and overtreatment. There is a need for specific and sensitive biomarker(s) which can differentiate between cancer and non-cancer utilized for screening and early detection. The present study aims to comprehend the molecular pathways of prostate cancer which is essential for early detection and treatment. Dorso-lateral prostate from 20 week transgenic adenocarcinoma of the mouse prostate (TRAMP) mice, which spontaneously develops prostate cancer and recapitulates human disease and age-matched non-transgenic littermates were utilized for microarray analysis. Network and pathway analyses were mapped using the Ingenuity Pathway Analysis (IPA), and the database for annotation, visualization and integrated discovery. In total, 136 differentially expressed genes, including 32 downregulated genes and 104 upregulated genes were identified in the dorso-lateral prostate of TRAMP mice, compared to non-transgenic. A subset of differentially expressed genes were validated by QRT-PCR. Among these genes, 36% genes were connected to the nucleic acid binding especially ribosomal proteins which play important roles in protein synthesis, and is the most enriched pathway in the development of prostate cancer. Moreover, the results also suggest deregulation of the metabolic process and an imbalance between extracellular matrix components and adherens junction proteins could induce cancer progression. Taken together, the present study evaluated the underlying pathways and its connection to human cancer, which may further help to assess the risk of prostate cancer detection and its progression to identify potential targets of adjuvant treatment.

#4348

DNA-based fusion detection using a pan-cancer tumor profiling 532-oncogene panel.

Katharine Dilger, Yongming Sun, Kevin Lai, Ushati Das Chakravarty, Nicole Sponer, Kristina Giorda, Patrick Lau, Yu Wang. _Integrated DNA Technologies INC, Redwood City, CA_.

Large-scale cancer profiling using next generation sequencing (NGS) has become instrumental to the discovery and identification of new, targetable cancer alterations. A comprehensive set of 532 oncogene targets were combined to create the new xGen® Pan-Cancer Panel V2 for hybrid capture sequencing. This xGen panel covers 2.2 Mb of the human genome, and allows for the simultaneous detection of copy number variations (CNVs), insertions and deletions (indels), gene rearrangements, and microsatellite instability across a wide range of sample types, inputs, and quality. Using a new library prep workflow optimized for low quality samples and low input, panel performance was first evaluated with 30 ng of input DNA using libraries built from matched samples [formalin-fixed paraffin-embedded (FFPE) tumor gDNA, frozen adjacent normal tissue gDNA, and cell-free DNA (cfDNA)] from five lung cancer donors (n = 15). Sample quality ranged from a mean DIN of 4.4 ±1.1 to 8.3 ±0.9 for FFPE tumor gDNA and frozen normal gDNA, respectively. After subsampling to 200X mean target coverage, 96% of target bases had at least 40X coverage for all libraries. Comparative hierarchal clustering analysis was then used to identify lung cancer mutations shared in all tumor samples. NGS gDNA reference standards from Horizon Discovery (HD753, HD798, HD799, and HD803) with verified CNVs, single nucleotide variations (SNVs), amplifications, and fusions, and were used to evaluate detection rates at different library input masses down to 1 ng. cDNA libraries were also prepared from RNA extracted from FFPE 5-Fusion RNA Multiplex Reference Standards (HD796, HD783). We identified all possible gene fusion events in the positive control using the structural variant caller, LUMPY (https://github.com/arq5x/lumpy-sv). The xGen Pan-Cancer Panel V2 enables a cost-efficient and time-saving approach for the detection of multiple oncogene targets.

#4349

Predicting gene expression from plasma cell-free DNA using both the fragment length and fragment position.

John A. St John,1 Erik Gafni,1 Brandon White,1 Ajay Kannan,1 Loren Hansen,1 Artur Jaroszewicz,1 Anshul Kundaje,2 Nathan Boley1. 1 _Freenome, Inc., South San Francisco, CA;_ 2 _Stanford University, Stanford, CA_.

The ability to use a blood sample to determine the transcriptional state of cells that are releasing DNA into the bloodstream of a patient may be helpful in a variety of clinical applications. Here we present a case study of a gene expression prediction model that uses cell-free DNA (cfDNA) fragment coverage data generated by high-throughput sequencing to predict which genes are highly or lowly expressed in the cells contributing to that cfDNA. We evaluated a number of models, including a convolutional neural network that takes cfDNA fragment information (the density of both fragment midpoint and length by genomic position) over a transcription start site (TSS) as input, and outputs a predicted probability of whether that gene is highly expressed in cfDNA-producing cells. When we trained the convolutional model on a set of 554 genes with TSSs that were either constitutively expressed or unexpressed across leukocyte samples from the NIH Roadmap Epigenome Mapping Consortium, we achieved ~0.97 AUC in cross validation. With other models and splits of the data, we observed AUCs ranging from 0.95 to 0.99 on this gene-expression task. Next, we were interested in whether this trained model could answer specific clinical questions. For example, we hypothesized that we should see an increased influence of colon gene expression profiles in colorectal cancer patients with a higher fraction of circulating tumor DNA. To test this hypothesis, we applied our models to a set of genes with colon-specific expression, which generated a list of probabilities of each gene being expressed in each sample. We then applied simple models on the these lists of probabilities to predict whether a patient had CRC or was healthy. This yielded cross validation AUCs between 0.85 and 0.95 across many of the models we tested in differentiating healthy patients from colorectal cancer patients with tumor fraction over 5%. These results suggest a path forward for modeling transcriptional states using cfDNA sequencing data, which will enable greater insights from cfDNA that could augment those provided by other analytes.

#4350

MAPK1E322K **somatic mutation promotes cell proliferation via positive feedback regulation of EGFR/RAF signaling.**

Jianqing Zhang, Dewey J. Brooke, Jessica P. Blair, Jaques Riby, Akinyemi I. Ojesina. _University of Alabama at Birmingham, Birmingham, AL_.

The RAS-MAPK signaling cascade serves as the central node in the transduction of signals starting from the binding of the epidermal growth factor (EGF) ligand to the membrane receptors (EGFR) through to the nucleus. Recent studies suggested that different defined thresholds of RAS-MAPK activity are required for maintaining homeostasis as opposed to facilitating malignant transformation, and that Ras-oncogenic toxicity is quantitatively dictated by MAPK function. We and others have demonstrated that somatic MAPK1(E322K) mutations in MAPK1 occur in 8% of cervical squamous cell carcinoma (CSCC) and 3% of head and neck squamous cell carcinoma (HNSCC), suggesting that mutant MAPK1 may play an important part in tumorigenesis. To investigate the molecular mechanism involved in the effects of the mutated MAPK1, we transduced MAPK1 wild type and MAPK1(E322K) mutant as lentiviral constructs into the untransformed human embryonic kidney cell line HA1E. We found that cells expressing MAPK1(E322K) exhibited higher rates of proliferation and cellular mobility compared to the cells transduced with wild type MAPK1. However, the cells expressing wild type MAPK1 presented poor cell proliferation compared to the cells transduced with the empty lentiviral vector. Western blot analyses showed approximately two-fold increase phosphorylation of ERK (T202/Y204) following EGF treatment in the cells harboring the mutation compared to the cells expressing the wild type MAPK1. A similar pattern was observed with phospho-EGFR (T669, Y1148, Y1068, Y1148) phospho-cRaf (S259, S338, S289/296/301), phosphor-RSK1, phospho-AKT (S473, T389), phosphor-GSK3β (S9/21) and phospho-STAT5 (Y694), suggesting that MAPK1(E322K) mutation might reinforce the positive/negative feedback loop on EGFR and cRaf. Our data contributes to the understanding of the roles of MAPK1 in the RAS/RAF-MEK-MAPK1 signaling pathway and might help identifying possible therapeutic targets for HNSCC and CSCC patients with tumors harboring somatic MAPK1(E322K) mutations.

#4351

Single-cell RNA sequencing reveals transcriptomic heterogeneity in response to epigenetic inhibitors.

Andrew R. Conery, Sungmi Park, Michael J. Steinbaugh, Barbara M. Bryant, Shruti Apte, Patricia J. Keller, C. C. Yuan, Bill Bradley, Archana Bommi-Reddy, Robert J. Sims. _Constellation Pharmaceuticals, Inc., Cambridge, MA_.

Histone modification is a fundamental regulatory mechanism of gene transcription and is associated with multiple diseases including cancer and inflammatory diseases. The factors that place and read these modifications can be co-opted in pathological conditions to give rise to disease-promoting transcriptional states, and thus represent promising targets for therapeutic small molecule inhibition. A central challenge in the development of epigenetic cancer therapy is the identification of transcriptionally sensitive cells within a heterogeneous population as a way to maximize therapeutic efficacy. To explore this challenge, we used single cell RNA-sequencing to characterize the transcriptional response of intrinsically heterogeneous human cancer and primary cell populations to small molecule inhibitors of epigenetic factors including the BET bromodomains, EZH2 methyltransferase, and CBP/EP300 acetyltransferase. Through clustering and differential expression analysis, we show that bulk transcriptional response in a mixed population of primary or tumor cells can be driven by a profound response in a hypersensitive subpopulation. Further, through the use of trajectory analysis, we show that epigenetic inhibitors can skew cell fate or differentiation status depending on the heterogeneity of the starting population. Overall, our results demonstrate the value of single cell transcriptomics for understanding the response of cell populations to epigenetic small molecule inhibitors and for enhancing the therapeutic potential of targeting pathogenic transcriptional programs.

#4352

Genetic effects on gene expression and survival in patients with multiple myeloma.

Heini M. Natri,1 Austin Gutierrez,2 Bianca Argente,2 Melissa Wilson Sayres,1 Kenneth Buetow,1 Nicholas Banovich2. 1 _Arizona State University, Tempe, AZ;_ 2 _The Translational Genomics Research Institute, Phoenix, AZ_.

Multiple myeloma (MM) is the second most common hematological cancer, accounting for 2% of all cancer deaths. MM is associated with a poor prognosis, with a 5-year overall survival of 50.7%. While the introduction of new therapies in the last decade has nearly doubled the survival rate, most patients still experience a relapse. To this end, The Multiple Myeloma Research Foundation developed the CoMMpass study. CoMMpass is a longitudinal clinical trial aimed at accelerating the discovery of more targeted treatments for MM. Clinical parameters and tumor specimens are collected from each of the 1,147 patients at baseline and through the eight-year observation period. To identify genetic determinants of clinical outcomes, each tumor specimen is characterized by Whole Genome, Exome and Transcriptome sequencing. This work has uncovered a number of somatic mutations and copy number alterations associated with tumor progression and response to therapy as well as a novel classification of MM into 12 distinct subtypes based on gene expression. However, the contribution of germline genetic variation to gene expression changes and MM outcome remains poorly understood.

Genome-wide association studies have identified germline variants associated with MM risk, indicating inherited genetic susceptibility. Furthermore, MM exhibits a disparity in occurrence and mortality between the sexes and ethnicities, men and African Americans being in at a higher risk than women or those of European ancestry. To better understand the genetic and biological basis of MM predisposition, we utilize a systems genomics approach to examine non-coding inherited and somatic genetic effects on gene expression and MM outcome. Data from 607 CoMMpass participants with available WGS and RNAseq from normal blood and baseline tumor specimens were used to map cis-acting eQTLs. We identified 3929 genes with at least one cis-acting eQTL. Furthermore, we discovered a number of regulatory variants with differential effects on tumor gene expression between the sexes and ethnicities, as well as eQTLs overlapping MM GWAS risk loci, providing regulatory mechanisms connecting these loci to MM. Finally, we discovered eQTLs that modulate overall survival in patients with MM. To validate these putatively regulatory loci in vitro, we utilized MM cell lines and CRISPR activation/interference, successfully altering the expression of the target genes. These and analyses integrating germline and somatic variants, gene expression, and clinical outcomes support the development of personalized medicine approaches for the better treatment of MM.

### Metabolic Reprogramming in Cancer

#4353

Stroma drive metabolic reprogramming and induces stemness properties in pancreatic cancer cells.

Kousik K. Kesh, Vineet K. Gupta, Nikita Sharma, Roey Hadad, Vikas Dudeja, Ashok Saluja, Sulagna Banerjee. _University of Miami, Miami, FL_.

Pancreatic Ductal Adenocarcinoma (PDAC) is one of the most devastating human malignancies with poor survival rate owing to chemoresistance and relapse. Small population of Tumor Initiating Cells (TIC) are considered major players driving these traits. Recent literature has demonstrated the importance of microenvironment "niche" in enrichment of these TIC populations. The desmoplastic stroma in pancreatic cancer comprising of stromal cells and immune cells are instrumental in pathogenesis and progression of PDAC. Among many cytokines, IL-6 a pro-inflammatory cytokine plays a major role in it. In this study, we show that stromal cell secreted IL6 altered metabolic reprogramming and increased cancer stemness in PDAC cells. PDAC cells cultured with pancreatic stellate cells condition media (PSC-CM) or recombinant IL6 and stem ness markers expression were investigated. Cytometry Bead Array (CBA) were used for cytokine profiling. Metabolic alteration along with TIC gene expression were measured using flow cytometry and Seahorse. PDAC cells when cultured with PSC-CM exhibited increase glucose uptake along with an enrichment of CD133+ population. An analysis of the conditioned media using CBA reveled that PSC-CM is enriched with cytokine IL-6 and TNF-α. Treatment of PDAC cells with recombinant IL-6 increased expression of self-renewal genes SOX2, OCT4, Nanog along with biochemical marker ALDH. In addition, TIC marker CD133 and invasion marker gene MMP7 expression also increased upon IL6 induction. Moreover, IL6 treatment increased glucose uptake and lactate secretion. Small molecule inhibitor Stattic (inhibitor of STAT3) inhibit glucose uptake and stemness gene expression.Our study shows that stromal IL6 altered metabolic reprogramming in cancer cells as well as increased cancer stemness and invasion. In future, the underlying mechanisms of IL-6 driven metabolic alteration and its impact on stemness and metastasis will be investigated.

#4354

A human bronchial epithelial cell model demonstrates a role for glutamine regulation in genomic instability and oncogenic transformation.

Omar M. Omar, Jamshedur Rahman, Pierre Massion. _Vanderbilt University, Nashville, TN_.

Background: Previous work from our laboratory has investigated the role of solute carrier family A1 member 5 (SLC1A5) as a critical modulator of glutamine (Gln) uptake, cell growth and tumor progression in non-small cell lung cancer (NSCLC). In addition, remarkable metabolic reprogramming was observed in cells from individuals at high risk of lung cancer, including an increased dependence on Gln. However, it remains unclear how these changes in Gln metabolism and other metabolic dysregulations contribute to early events of tumorigenesis such as genomic instability. Given the emerging role of Gln metabolism in cancer and the differential expression of SLC1A5 in NSCLC, we tested the hypothesis that Gln uptake is a key mechanism by which cells with oncogenic potential can transform into a malignant phenotype.

Aims & Strategy: The aim of this study is to evaluate the involvement of SLC1A5 in the process of early oncogenic transformation and survival. To address this aim, we utilized four HBECs immortalized by over-expressing Cdk4 and human telomerase reverse transcriptase (hTERT), along with a series of canonical oncogenic drivers, P53-knocdown, KRAS and MYC overexpression, which drove these cells to exhibit a malignant phenotype. This successful transformation of an in vitro model could be useful in studying the metabolic dysregulation and, in particular, the role of Gln metabolism in contributing to genomic instability in an accessible and employable cell model representative of the oncogenic transformation process.

Results: We found that oncogene driven HBECs exhibited a substantially higher expression of SLC1A5 than unaltered HBEC cells. Inhibition of SLC1A5 by a small molecule inhibitor, gamma-l-Glutamyl-P-NitroAnilide (GPNA), decreased cell survival, Gln uptake and markers of genotoxic stress. The sensitivity to GPNA treatment was significantly correlated with SLC1A5 RNA expression (R2=0.96, P<0.05). We tested the effect of SLC1A5 inhibition on two markers of genomic instability, gamma-H2Ax and 8oxoGuanine, and discovered that GPNA treated cells exhibited higher degree of gamma-H2Ax and 8oxoGuanine staining suggesting that Gln uptake has a protective role against DNA damage. We further investigated the effect of effect of GPNA treatment on apoptotic signaling and found that SLC1A5 inhibition is associated with the activation of Caspase-9.

Conclusion: Overall, Our results indicate that oncogene-transformed HBECs exhibited higher dependence on Gln uptake through SLC1A5 relative to the non-oncogenic clones. Tumor-forming HBECs exhibited higher sensitivity to SLC1A5 inhibition than non-tumorigenic ones. Furthermore, Gln deprivation was implicated in regulation of genomic integrity and apoptosis in this cell model. This works suggests that Gln metabolism regulates aspects of genomic instability and implicates its function in lung tumorigenesis.

#4355

Altered acetate metabolism in cisplatin resistant bladder cancer.

Jayoung Kim. _Cedars-Sinai Medical Ctr., Beverly Hills, CA_.

Introduction and Objective: Cisplatin is an important chemotherapeutic agent against metastatic bladder cancer, but resistance often limits its usage. With the recent recognition of lipid metabolic alterations in bladder cancers, we studied the metabolic implications of cisplatin resistance using cisplatin-sensitive (T24S) and resistant (T24R) bladder cancer cells.

Methods: To test whether there should be differences in metabolism and metabolism-associated pathways between cisplatin-sensitive and resistant bladder cancer cells, we applied the live metabolomics approach that we recently developed to isogenic bladder cancer cell lines T24S (cisplatin sensitive) and T24R (cisplatin resistant). The metabolites generated from 13C-glucose tracer were monitored with 2D 1H-13C HSQC NMR in real time.

Results: Real-time live metabolomics revealed that T24R cells consume more glucose, leading to higher production of glucose-derived acetate and fatty acids. Along with the activation of general metabolic regulators, enzymes involved in acetate usage (ACSS2) and fatty acid synthesis (ACC) and a precursor for fatty acid synthesis (acetyl-CoA) were elevated in T24R cells. Consistently, metabolic analysis with 13C isotope revealed that T24R cells preferred glucose to acetate as the exogenous carbon source for the increased fatty acid synthesis, contrary to T24S cells. In addition, ACSS2, rather than the well-established ACLY, was the key enzyme that supplies acetyl-CoA in T24R cells through glucose-derived endogenous acetate. The relevance of ACSS2 in cisplatin resistance was further confirmed by the abrogation of resistance by an ACSS2 inhibitor and, finally, by the higher expression of ACSS2 in the patient tissues with cisplatin resistance.

Conclusions: Our results may help improve the treatment options for chemoresistant bladder cancer patients and provide possible vulnerability targets to overcome the resistance.

#4356

Menin and ATF4 cooperate to drive serine biosynthesis in Ewing sarcoma.

Jennifer Jimenez, Laurie K. Svoboda, Sudha Sud, Samuel Kerk, Jolanta Grembecka, Costas A. Lyssiotis, Elizabeth R. Lawlor. _University of Michigan, Ann Arbor, MI_.

Ewing sarcoma (ES) is an aggressive bone and soft tissue tumor. We previously reported that the scaffolding protein menin is overexpressed by ES, and that menin inhibition results in impaired ES cell proliferation, survival, and tumorgenicity. Additionally, our recent studies revealed a previously undescribed role for menin in the activation of the serine biosynthetic pathway (SSP), a critical metabolic pathway that is aberrantly activated in many human cancers. The biologic functions of menin are largely determined by its protein-binding partners, the best characterized of which is the histone methyltransferase MLL. In the current study, we are investigating the mechanistic link between menin and the SSP to determine whether menin activates the SSP via epigenetic trithorax complexes.

Our data show that the core SSP enzymes, PHGDH, PSAT1, and PSPH are highly expressed by ES. Moreover, expression of all three genes is highly correlated in primary tumors, suggesting that they may be coordinately regulated by an upstream factor. In other cancer types, activation of the SSP has been shown to result from amplification of the PHGDH locus or activation of the master transcriptional regulator, ATF4. We have found that PHGDH is not amplified in ES but ATF4 is overexpressed, and ATF4 loss of function leads to growth inhibition. Q-RT-PCR and western blot of ES cells following ATF4 knockdown reveals down-regulation of PHGDH, PSAT1, and PSPH. In addition, ChIP-qPCR shows enrichment of ATF4 binding at SSP gene promoters, which is diminished by treatment with MI-503, a menin:MLL interaction inhibitor. Menin inhibition with MI-503 also leads to loss of ATF4 expression, coincident with loss of SSP expression. Additionally, ChIP-qPCR shows enrichment of menin binding at the ATF4 gene promoter, which is associated with H3K4me3 enrichment. Preliminary studies show that ATF4 over-expression via lentiviral transduction may rescue the loss of SSP gene expression induced by MI-503. Together these findings support the hypothesis that ATF4 acts downstream of menin to drive SSP activation in ES. Ongoing studies are assessing whether this is mediated by trithorax-dependent or –independent functions of menin.

#4357

The lactate receptor Gpr81 on non-cancer cells promotes an immunosuppressive phenotype in the tumor microenvironment.

Timothy Brown,1 Sabarish Ramachandran,1 Stefan Offermanns,2 Vadivel Ganapathy1. 1 _TTUHSC, Lubbock, TX;_ 2 _Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany_.

Cancer cells display a unique phenomenon in which, even in the presence of oxygen, cells switch from oxidative phosphorylation to glycolysis as the primary source of ATP with consequent production of lactic acid. This phenomenon, called the Warburg Effect, is a hallmark of cancer. Lactic acid has long been considered as the necessary end product of this metabolic switch, where lactic acid is effluxed out of tumor cells to prevent intracellular acidification. Recent evidence however suggests that lactate and the excess protons in the tumor microenvironment play an active role in tumor growth. In particular, lactate has been shown to function as an agonist for GPR81, a G-protein-coupled receptor expressed on the surface of tumor cells. This autocrine signaling of lactate promotes tumor growth and metastasis, as well as angiogenesis and immune evasion. The present study assesses whether tumor cell-derived lactate has any paracrine role via its receptor in non-cancer cells present in the tumor microenvironment. We generated MMTV-PyMT-Tg mice, a spontaneous model for breast cancer, on Gpr81+/+ and Gpr81-/- backgrounds. The absence of Gpr81 reduced the mammary tumor incidence, delayed mammary tumor progression, and reduced lung metastasis. These data demonstrate the essential role of GPR81 in breast cancer growth and metastasis, but does not differentiate between Gpr81 in tumor cells versus Gpr81 in the tumor microenvironment. We then used the syngeneic transplant of the mouse mammary tumor cell line AT-3 into the mammary fat pads of wild type and Gpr81-/- mice to assess the involvement of Gpr81 in the microenvironment. AT-3 cells are negative for Gpr81, and therefore our model limits Gpr81 expression to non-tumor cells in the host mouse. We found the growth of transplanted tumor cells was significantly reduced in Gpr81-/- mice than in wild type mice. Preliminary RNA-sequencing transcriptome analysis of AT-3 tumors suggest an immunosuppressive function of Gpr81, where tumors grown in a Gpr81-/- background have much stronger gene expression profiles in T-cell signaling pathways. Specifically, AT-3 tumors grown in Gpr81-/- host express significantly higher levels of genes that are specific for T cells and antigen-presenting cells such as MHC-II complex molecules, T-cell co-stimulatory molecules, and transmembrane CD8. It is well established that tumor-cell derived lactate promotes tumor growth in an autocrine manner via Gpr81 expressed on tumor cells; our studies extend the tumor-promoting role of lactate beyond this autocrine function to include paracrine signaling via Gpr81 expressed on immune cells and possibly other cell types in the tumor microenvironment.

#4358

Characterization of primary and metastatic pancreatic tumors in pancreatic ductal adenocarcinoma (PDAC).

Andrés E. Martínez-Muñiz,1 Sharanya Sivanand,2 Matthew Vander Heiden2. 1 _University of Puerto Rico - Mayagüez, Mayagüez, Puerto Rico;_ 2 _Massachusetts Institute of Technology (MIT), Cambridge, MA_.

Pancreatic Ductal Adenocarcinoma (PDAC) is the 5th leading cause of cancer death in the U.S. and Europe. It is a very aggressive form of cancer in which patients have a 5-year survival rate lower than 5%. PDAC arises from alterations in the KRAS and TP53 genes. The cell lines used were derived from 2 different mouse models where KRASG12D was combined with a deletion or point mutation of TP53 (TP53-/-, TP53R172H respectively) to drive oncogenic activation of PDAC. The TP53-/- model exhibits rapid pancreatic tumor growth, thus not allowing metastasis to occur, in contrast to the TP53R172H model, where tumors metastasize to the lungs or liver. TP53R172H cells also show a noticeable dependency on pyruvate availability for proliferation in contrast to the opposing model. Other NAD\+ replenishing molecules such as duroquinone can have similar effects on proliferation. Cell lines from the primary tumors of both models as well as from the metastasized liver tumors were used for in vitro experimentation. Epithelial-Mesenchymal Transition (EMT) is important for metastasis and cell state can be qualified by measuring E-cadherin expression, which has also been shown to be influenced by pyruvate availability. This project had 2 aims: 1) determine the effect of pyruvate availability on EMT by measuring E-cadherin levels and TWIST1 relative expression and 2) measure the effect that availability of both pyruvate and duroquinone have on cell proliferation. Cells were cultured in 6-well plates. After cell adhesion, they were treated with media containing pyruvate and media lacking pyruvate for comparison. Cells were grown for 24-96 hour periods after which they would be recollected for our designated analyses; western blot, total cell count and qPCR. Measured E-cadherin levels showed that there is higher protein expression in the TP53R172H model. Furthermore, it is shown that E-cadherin levels seem to be regulated by pyruvate in this model. Increase in protein quantity with time also suggests that these cells may be shifting towards an epithelial phenotype. Duroquinone did not rescue proliferation, contrary to the hypothesis. Surprisingly, there was a decrease in proliferation in the TP53-/- model. This could be attributed to experimental errors or loss of pyruvate sensitivity in the cell line. Expression levels were measured for Twist (TWIST1), a transcription factor that serves as a negative regulator for E-cadherin. RNA was isolated from cell lysates and reversed transcribed to measure relative expression of Twist by qPCR. A pronounced effect of pyruvate dependency on Twist expression was observed in both models. This may be meaningful since it could offer a possible explanation on how pyruvate could be influencing EMT in these cells.

#4359

Oscillatory HIF-1α induction promotes proliferation of hypoxic cells through a lactate dependent quorum autophagy response.

Kshitiz .,1 Junaid Afzal,2 Yasir Suhail,1 Hao Chang,3 Chi V. Dang,4 Andre Levchenko3. 1 _University of Connecticut Health Center, Farmington, CT;_ 2 _University of California San Francisco, San Francisco, CA;_ 3 _Yale University, New Haven, CT;_ 4 _The Wistar Institute, Philadelphia, PA_.

Intratumoral hypoxia is one of the most important factors present in the tumor microenvironment, regulating various steps in cancer growth and metastasis, including metabolism, vascularization, premetastatic niche formation, and dormancy. Cellular response to hypoxia is highly regulated, and is mediated by hypoxia-inducible factors (HIF), consisting of an oxygen (O2) regulated subunit, among which HIF-1α is the ubiquitously expressed homologue. HIF-1α stabilizes in hypoxia, and transcriptionally activates more than 1500 HIF identified target genes. Changes in HIF-1α activity, can, therefore result in large changes in gene expression, and phenotypic state of the cells. However, there are paradoxes in observations of HIF-1α responses. HIF-1α inhibits cell proliferation, but cells continue to divide in hypoxia. Similarly, HIF-1α induced indirect phosphorylation and inhibition of pyruvate dehydrogenase (PDH) diverts pyruvate away from acetyl-CoA toward lactate synthesis, decreasing respiration; but HIF-1α can also enhance mitochondrial respiration efficiency by controlling the expression cytochrome c oxidase type 4 (COX4) subunits. These paradoxes could potentially be addressed if individual cellular responses to hypoxia may be different than the bulk/average cell responses.Using fluorescent dynamic reporters of HIF-1α activity, we show that hypoxic responses in individual cells can be highly dynamic and variable across the population. These responses fall into three classes, including oscillatory activity. We identify a molecular mechanism that can account for all three response classes, demonstrating that the oscillations of HIF-1a activity and abundance are controlled by the reactive oxygen species-dependent chaperone-mediated autophagy in a subset of respiring cells. Furthermore, we find that the oscillatory response is modulated by the abundance of extracellular lactate in a quorum sensing-like mechanism. Importantly, we demonstrate that oscillatory HIF-1α activity promotes differential gene expression leading to enhanced cell division, which can help reduce the anti-proliferative effect of hypoxia. These results may help resolve the paradox of seemingly contradicting roles of HIF-1α in the control of metabolic cell states and cell proliferation. Further, specific transcription by oscillatory hypoxia suggest that gene regulation can specifically decode oscillatory signals, resulting in novel phenotypes in a subpopulation, with a selective advantage to survive and proliferate under hypoxic stress.

#4360

ALDH2 and the TGF-β/SMAD3 adaptor, Sptbn1 suppress hepatic steatosis and obesity.

Shuyun Rao, Sobia Zaidi, Wilma Jogunoori, Shoujun Gu, Jon White, Bibhuti Mishra, Chu-Xia Deng, Patricia Latham, Lopa Mishra. _George Washington University, Washington, DC_.

Background: Previously we have demonstrated that TGF-β-deficient mutants derived from the loss of Smad3 and the Smad adaptor β2SP (Sptbn1) phenocopy a human cancer stem cell syndrome, are exquisitely sensitive to alcohol, with dysregulation DNA damage repair and have raised levels of aldehyde dehydrogenase (ALDH2). Moreover, β2SP and β2SP/Smad3 mutant mice develop multiple liver abnormalities that include steatosis, chronic hepatitis, inflammation, and cancer. ALDH2 is a key intermediate enzyme for aldehyde and alcohol metabolism. We, therefore, hypothesized that disruption of TGF-β signaling combined with ALDH2 deficiency would increase the susceptibility of liver cancer.

Methods: Aldh2 knockout mice were intercrossed with Sptbn1 heterozygous knockout mice to explore the role of Aldh2 and Sptbn1 in liver steatosis and liver cancer. Conditional knockout of Sptbn1 mice was generated by the intercross of Albumin-Cre mice with mice carrying Loxp sites flanked exons of Sptbn1. Sptbn1 conditional mice were fed with high-fat diet or chow diet. Further analysis included blood lipid panel analysis and tissue profiles including western and RNA analysis, lipidomics and metabolomics analysis of liver tissues from the mutant mice and wild-type controls.

Results: Interestingly, we found that Aldh2‐/‐Sptbn1+/- mice on a normal diet demonstrated early signs of truncal obesity. Within 6 months of age, we observed a greater than 23% increase in body weight, with predominantly abdominal fat accumulation in Aldh2‐/‐Sptbn1+/- mice compared to Aldh2-/- mice or wild type (WT) mice (Aldh2‐/‐Sptbn1+/- vs Aldh2-/- or WT, p<0.05). A marked increase in zone 3 hepatic micro and macro-steatosis with some inflammation was observed in liver tissues of mutant Aldh2‐/‐Sptbn1+/- mice as well as the conditional Sptbn1 knockout (Alb-Cre+Sptbn1F/+) mice in a high-fat diet. In addition to significant increased blood glucose levels in Aldh2‐/‐Sptbn1+/- mice compared with Aldh2‐/‐ mice (165±23 VS 135±13, p<0.05), further analysis revealed that the fatty liver phenotype accompanied with abnormal expression of genes related to glucose uptake, metabolisms and homeostasis as demonstrated by decrease of glucose transporter and increased expression of Ceramide which is known to promote insulin resistance. Moreover, we also observed disruption of lipid metabolism as demonstrated by significantly increased serum and liver triglyceride and cholesterol ester levels in Aldh2‐/‐Sptbn1+/- mice.

Conclusions: Our data suggest that the TGF-β pathway plays an important role in maintaining normal glucose metabolism homeostasis and patients with SPTBN1 and ALDH2 defects may have increased susceptibility to obesity as well as fatty liver (NASH), and cancer.

#4361

Kynurenine - Aryl hydrocarbon receptor axis: A crucial modulator of immunometabolism in cisplatin resistant lung cancer.

Medhi Wangpaichitr, Dan JM Nguyen, Ying-Ying Li, Chunjing Wu, Lynn G. Feun, Niramol Savaraj. _Univ. of Miami/VA Medical Ctr., Miami, FL_.

The effect of tumor metabolism on the tumor microenvironment is not well established. Our previous data have shown that cisplatin resistant (CR) lung cancer cells increased secretion of thioredoxin-1 (TRX1) and kynurenine (KYN). Interestingly, high TRX1 and KYN levels in tumor microenvironment can enhance immunosuppressive environment. We have demonstrated that CR cells possessed lower levels of hypoxia-inducible factor-1α (HIF1α) due to metabolic re-programming. ARNT or HIF1β is a known binding partner of both HIF1α and aryl hydrocarbon (AHR). Importantly, recent study indicates that KYN can serve as an endogenous ligand for AHR in cancer cells. Thus, we hypothesize that in the absence of HIF1α, ARNT is now available to bind and form a new partner with KYN/AHR and initiate the transcription of genes which favor survival/proliferation of CR cells. Four pairs of NSCLC cell lines and their CR variants (ALC, FC, H460R, A549R) were used. Using immunofluorescence technique, our result showed that AHR localized primary in the nucleus of CR cells when compared with parental cell counterparts. We further determined that KYN was specific to AHR activation in CR cells by inhibiting AHR translocation using 10µM of dimethoxyflavone (DMF; an AHR antagonist). AHR was less accumulated inside nuclease after treatment. Importantly, treatment of DMF also resulted in suppression of Indoleamine 2,3-Dioxygenase-1 (IDO1) activities. To further verify these findings, we assayed the expression of AHR-target gene (LAT1 and CYP1B1) with and without adding KYN. Both genes expressions were higher in CR cells and were further augmented upon an addition of KYN. In contrast, we did not find an increase in LAT1 after exposure to KYN in parental cells. Using flow cytometer, we found that CR cells possessed higher surface-LAT1 levels when compared to parental cell counterparts, and treatment of KYN resulted in significant tryptophan uptake in CR cells. Importantly, treatment with IDO1 inhibitor (5.5uM) significantly suppressed LAT1 and CYP1B1 expression in CR cells and reverse cisplatin resistance in CR from 2.5 to 0.75 μg/ml (3.3 fold). Thus, our data strongly indicate that KYN/AHR/ARNT axis plays a unique modulator role in CR cells metabolism. Overall these results will have potential impact on (i) how one can effectively exploit the KP pathway to treat CR tumors, and (ii) improving understanding on how CR cells evade immune surveillance. Supported by Department of Veterans Affairs (BLR&D Merit review and CDA2)

#4362

Glucose transporter 1 regulates cell glycolysis and proliferation in gastrointestinal stromal tumor and its clinicopathological significance.

Masaaki Iwatsuki,1 Hiroshi Sawayama,1 Daisuke Kuroda,1 Yuki Koga,1 Kohei Yamashita,1 Kazuto Harada,2 Shiro Iwagami,1 Kojiro Eto,1 Takatsugu Ishimoto,1 Yoshifumi Baba,1 Naoya Yoshida,1 Jaffer A. Ajani,2 Hideo Baba1. 1 _Kumamoto Univ., Kumamoto, Japan;_ 2 _MD Anderson Cancer Center, TX_.

Background: The unique features that cancer cells prefer glycolysis rather than mitochondrial respiration despite of the presence of oxygen have been a potential target for cancer treatment. GIST is well-known as to demonstrate intense metabolic activity and avid glucose uptake on 18F-FDG PET scanning. In this study, we focused on glucose transporter 1 (GLUT 1), a key glycolytic enzyme which plays critical roles in several cancer type. The aim of this study was to examine the clinicopathological significance of GLUT1 expression in GIST and to characterize the molecular mechanism of cell glycolysis and proliferation by GIST cell.

Methods: We determined the expression level of GLUT1 in 65 GIST cases, correlated those values with clinicopathological features. Furthermore, we investigated that the alteration of GLUT1 can influence on glycolysis, cell proliferation, and cell cycle distribution by extracellular flux analyzer, CCK-8 assay and flow cytometry, respectively in GIST cell in vitro.

Results: Fifty-one (78.5%) cases were placed in the high GLUT1 expression group. In the high GLUT1 expression group, tumor size was significantly larger compared to the low GLUT1 expression group (P=0.02). There are significantly more high-risk cases by modified-Fletcher classification in GLUT1 expression group (P< 0.01). The patients with high GLUT1 expression significantly exhibited a higher SUVmax value (P< 0.01). Compared with the low GLUT1 expression group, the high expression group showed a significantly poorer prognosis in terms of recurrence-free survival. In vitro, GLUT1-specific siRNA suppressed cell proliferation and led to increase in G1 phase arrest. Also, knockdown of GLUT1 cells reduced glycolysis as a function of ECAR by extracellular flux analyzer.

Conclusion: We showed that GLUT1 plays a critical role in GIST by regulating glycolysis and proliferation. Our study suggests that glucose transporter can be a novel target of anticancer therapeutics mediating glucose metabolism in GIST.

#4363

Effects of the small GTPase RhoC on inflammatory breast cancer metabolism.

Laura E. Goo, Joel A. Yates, Andrew C. Little, Daniel Kremer, Ilya Kovalenko, Christopher R. Oliver, Trisha Westerhof, Zhifen Wu, Nathalie Vandecan, Liwei Bao, Peter J. Ulintz, Costas A. Lyssiotis, Sofia D. Merajver. _University of Michigan, Ann Arbor, MI_.

Inflammatory breast cancer (IBC) is an extremely aggressive and rare form of cancer that disproportionally affects African American and younger women. While IBC represents 1-5% of breast cancers, it accounts for 10% of all breast cancer deaths annually in the United States. We have previously shown that the metabolic characteristics of IBC, specifically in the triple negative (TN) and inflammatory breast cancer cell line SUM149, are significantly altered from those of normal breast cancer cells. We have shown previously that RhoC, a member of the Ras superfamily of GTPases, contributes to the metastatic IBC phenotype and acts as a regulator of the metabolite N-acetyl aspartate (NAA) in SUM149 cells. NAA is the second most abundant metabolite in the brain (only exceeded by glutamate) and has been used for the diagnosis of neurodegenerative disorders such as the fatal genetic disorder Canavan's disease, which is characterized by toxic NAA levels due to a deleterious mutation in aspartoacyclase(ASPA). ASPA is the enzyme involved in the catabolism of NAA into aspartate and acetate. NAA has previously been considered a brain specific metabolite and the fundamental role of NAA outside of the central nervous system, especially in the context of cancer, remains elusive but highly intriguing.

We have previously shown that NAA levels are significantly higher in the IBC-derived TN SUM149 cells than TN cancer cell line MDA-MB-231. Interestingly, and independently, recent studies have also implicated high levels of tumoral NAA with significantly worse survival rates in ovarian cancer patients. The synthesis of NAA from acetyl-CoA and aspartate is catalyzed by the enzyme Asp-NAT, which is encoded for by the gene NAT8L. Knockdown of RhoC in SUM149 cells using shRNA significantly decreases both NAT8L expression and NAA metabolite levels. To further understand RhoC's role in the modulation of NAA, we generated RhoC and RhoA knockout cell lines using CRIPSR-Cas9 in a set of inflammatory and noninflammatory breast cancer cell lines. RNA-seq analysis of these show that SUM149 RhoC knockout cells produce the largest amount of differentially regulated genes when compared to wild-type cells. The differentially regulated genes include many of the key genes involved in NAA associated pathways. Furthermore, metabolic studies reveal that the most downregulated metabolites in the SUM149 RhoC knockout cell line are N-acetyl derivates including NAA, N-acetylglutamate(NAG), and N-acetylaspartylglutamate (NAAG). Overexpression and siRNA cell lines for both NAT8L and ASPA have been generated to further elucidate the altered metabolism seen in these cells. Additional molecular studies are ongoing to determine the specific role of NAA in the adapted metabolic pathways of inflammatory breast cancer that may shed light on IBC's increased metastatic potential and decreased survival.

#4364

Oncometabolite L-2-hydroxyglutarate blocks differentiation of renal proximal tubule cells in matrigel.

Mary L. Taub,1 Sunil Sudarshan2. 1 _SUNY at Buffalo, Buffalo, NY;_ 2 _University of Alabama at Birmingham, Birmingham, AL_.

Previously, the Sudarshan laboratory has identified elevations in the level of the oncometabolite L-2-hydroxyglutarate (L-2HG) in human renal cell carcinomas (RCCs). Of particular interest in these regards, both L-2HG inhibits 2-oxoglutarate-dependent dioxygenases (2-OGDs), including histone demethylases, which regulate the epigenetic landscape of cells, and cellular differentiation. Thus, it was of interest to determine whether the accumulation of L-2HG alters the differentiation of the normal renal proximal tubule (RPT), ultimately affecting the phenotype of the RCC. Towards these ends, L-2HG dehydrogenase (L2HGDH) was knocked down in primary cultures of normal rabbit RPT cells, and their capacity for cellular differentiation was examined. Initially, we examined the ability of primary RPT cells to form tubules in matrigel following an L2HGDH knockdown (KD). While tubulogenesis was stimulated by Epidermal Growth Factor (EGF) in primary RPT cells transduced with a control lentiviral vector, tubulogenesis in matrigel was dramatically impaired in primary RPT cells when L2HGDH was knocked down by lentiviral L2HGDH shRNA. In order to determine whether the expression of differentiated transport functions was affected, RealTime PCR (RTPCR) was conducted. The results indicated that an L2HGDH knockdown (80%) resulted in a reduction in the expression of the Na+/Pi cotransporter NaPi2a (81%), the Na+/glucose cotransporter SGLT2 (88%), the water transporter Aquaporin 1 (AQP1) (95%), and the Na,K-ATPase ß1 subunit (atp1b1) (43%), whereas the expression of the p-Aminohippurate transporter OAT1 was not significantly affected. Similar results were obtained when using L2HGDH siRNA and lentiviral shRNA. Not only is tissue architecture critical for the maintenance of functional differentiation, but alterations in tissue architecture are a necessary component of tumor formation. Thus, we are examining the underlying causes for the reduced tubulogenesis in matrigel cultures with an L2HGDH KD. In our initial studies, we examined the expression of differentiated transport functions in matrigel vs. monolayer cultures. Notably, while the expression such transporters as AQP2 increased dramatically in matrigel (as opposed to monolayer cultures), the expression of other transporters such as NaPi2a, SGLT2 and atp1b1 was affected to a much smaller extent. Of particular interest in these regards, is the known role of AQP1 in the migration of renal proximal tubule cells, tumor spread, and wound healing. We are currently studying the effects of an L2HGDH KD on the expression of such genes as AQP1 during tubulogenesis in matrigel.This work has been funded by Grant # 1RO1CA200653-01A1 to Dr. Sunil Sudarshan (PI) with a subaward to Dr. Mary Taub.

#4365

Induction of distinct p53 mutation types differentially influences the control of cellular iron metabolism.

Eshan Dandekar, Aishwarya Srinivasan, Stephen Clarke, Mckale Montgomery. _Oklahoma State University, Stillwater, OK_.

The most commonly mutated gene in all human cancers is the tumor suppressor gene p53, but in addition to loss of tumor suppressor functions, mutations in p53 can also promote cancer progression by altering cellular iron acquisition and metabolism. The primary objective of this work was to determine how p53 mutation status influences the molecular control of iron homeostasis. Though hundreds of p53 mutations have been identified, the majority occur within the DNA binding domain and can be subdivided into two broad classes: contact (e.g., R273H) or conformational (e.g., R175H). By generating cell lines with inducible versions of some of the most common conformational and contact-type p53 mutations we have established that introduction of specific p53 mutation types alone is sufficient to disrupt cellular iron metabolism. We also demonstrate these effects are mediated at least in part due to differences in the responsiveness of Iron Regulatory Proteins (IRP) to cellular iron availability. IRP are considered the master regulators of intracellular iron homeostasis because they coordinate the expression of iron storage (ferritin) and iron uptake (transferrin receptor) genes. In response to changes in iron availability cells harboring either a wild-type p53 or contact (R273H) p53 mutation display canonical IRP-mediated responses, but neither IRP1 RNA binding activity nor IRP2 protein levels are affected by changes in iron status in cells harboring the conformational type mutant (R175H). Yet, both contact and conformational mutants exhibit robust changes in ferritin and transferrin receptor protein expression in response to iron loading and iron chelation, respectively. These findings suggest a novel, IRP-independent mode of iron regulation in cells expressing conformational type p53 mutants. Preliminary proteomics data suggests that lack of IRP responsiveness in cells expressing conformational p53 mutations may result from impaired iron-sulfur cluster biogenesis. These findings are currently being explored as a means of exploiting distinct p53 mutation types to more favorably induce iron-mediated cell death via the activation of ferroptosis. As p53 is mutated in nearly half of all human cancers, and iron is necessary for cancer cell growth and proliferation, the studies have implications for a wide range of clinically important cancers.

#4366

Characterization of diacetylspermine as a metabolic urinary biomarker in breast cancer using patient-derived xenografts.

Thomas J. Velenosi, Kristopher W. Krausz, Frank J. Gonzalez. _National Institutes of Health, Bethesda, MD_.

Breast cancer is a heterogeneous disease and is currently diagnosed using clinical biomarkers to classify tumors and guide therapy. However, drug resistance is a major cause of treatment failure and current methods can only detect resistance after several months following drug therapy. Thus, there is a need for novel biomarkers to track breast cancer prognosis, diagnosis, and to monitor therapeutic efficacy. Polyamines are polycations that support the production of nucleic acids and are necessary for transcription, DNA replication and cell cycle progression. The rate-liming step in polyamine catabolism is the acetylation of spermine and spermidine by spermine/spermidine acetyl-transferase (SAT1). Indeed, diacetylspermine has been identified as a clinical urinary biomarker of early and late stage breast cancer. Despite multiple clinical studies citing the potential for diacetylspermine as a cancer biomarker, the oncogenic events contributing to increased diacetylspermine production are largely unknown. To identify metabolic biomarkers of breast cancer progression and therapeutic efficacy, an untargeted metabolomics study was performed using urine samples from mice implanted with breast cancer patient derived xenografts (PDXs). NSG mice were engrafted with a doxorubicin sensitive (Dox-Sens) or a doxorubicin resistant (Dox-Res) PDX. When tumors reached 100-200 mm3, mice were administered intravenous doxorubicin (2 mg/kg) or vehicle control once weekly for three weeks. Mice were placed in metabolic cages for 24 hours after each treatment to collect urine. At 28 days, a final 24-hour urine collection was performed in the absence of drug treatment followed by euthanization (n = 9). Untargeted metabolomics revealed diacetylspermine as the most significantly altered metabolite in urine samples by two-way repeated measures ANOVA (p = 1.26x10-11, q = 4.82 x 10-8). Urinary diacetylspermine was correlated with growth of the Dox-Sens tumors and undetectable in urine from mice with Dox-Res tumors. Remarkably, when Dox-Sens mice were treated with doxorubicin, there was a further increase in diacetylspermine despite the decrease in tumor size. These results demonstrate the potential utility of urinary diacetylspermine as a biomarker for monitoring tumor growth and doxorubicin treatment efficacy. Furthermore, these breast cancer PDX models have provided an opportunity to characterize the mechanism of oncogenic diacetylspermine production and response to drug treatment.

#4367

**Protein kinase D1 induces metabolic switch in pancreatic cancer** via **modulation of mTORC1.**

Sonam Kumari, Sheema Khan, Murali Yallapu, Subhash Chauhan, Meena Jaggi. _Univ. of Tennessee Health Science Ctr., Memphis, TN_.

Objective: Pancreatic cancer (PanCa) is the third most common cause of cancer-related deaths in the US. Protein Kinase D1 (PKD1) is a kinase molecule which is involved in various important cellular signaling processes. While PKD1 has been reported to play a role in PanCa progression, the molecular mechanisms involved have not been adequately studied. Herein, we investigate the underlying mechanisms which enable PKD1 in enhancing glucose metabolism and further contribute to PanCa aggressiveness.

Methods: PanCa cells with high PKD1 expressing (Panc-1 and AsPC-1) and low PKD1 (HPAF-II and BXPC-3) were used in the study. Immunohistochemistry and confocal immunofluorescence were performed to analyze the expression of PKD1 in pancreatic cancer/normal tissues and cells, respectively. The effect of PKD1 gain/loss-in function was investigated on glucose uptake and lactate production in PanCa cells using commercially available kits. Western blotting and real-time PCR experiments were performed to analyze the expression of protein and mRNA levels. MTT assay was conducted to access the influence of PKD1 on cell proliferation. Boyden chamber migration assay and Matrigel invasion assays were performed to determine the migratory and invasive abilities of PanCa cells. The gene silencing was performed using specific siRNAs in the study.

Results: Our results demonstrate that PKD1 upregulates glucose metabolism in PanCa cells as indicated by enhanced glucose consumption and lactate production. The invasive characteristics of PKD1 expressing cells are augmented in presence of L-Lactate and reduced in presence of 2DG. PKD1 overexpression demonstrated enhanced phosphorylation of mTOR (ser-2448), 4EBP1, s6kinase and AKT, suggesting the role of PKD1 in the activation of mTOR pathway. We further observed that the phosphorylation of mTOR (ser-2448) and ps6kinase was attenuated on silencing PKD1 or in presence of Rapamycin, suggesting the role of PKD1 in activation of mTORC1 complex, both being the main effectors of mTORC1. Additionally, the inhibition in the phosphorylation of mTOR (ser-2448) in presence of kinase dead PKD1 suggests that PKD1 phosphorylates/activates mTORC1 in PanCa. Decrease in glucose uptake and lactate production in presence of PKD1 overexpression was observed on silencing raptor but not rictor in the cells, further suggesting the involvement of mTORC1 in PKD1 induced metabolic reprogramming in PanCa. PKD1 induced enhanced phosphorylation of mTOR resulted in an activation of HIF-1α and Glut-1 proteins, involved in abberant glucose metabolism. Additionally, our results also demonstrate that altered glucose metabolism leads to chemoresistance and silencing of PKD1 expression sensitizes PanCa cells to gemcitabine.

Conclusion: Our studies indicate that PKD1 acts as a key regulator of the glucose metabolism via mTOR activation and facilitates proliferation and invasion of PanCa cells.

#4368

Investigating the role of IDH in normal metabolic and cancer pathways.

Nicholas DiDuca, Sarah H. Roberts, Shane Sturtevant, Marla Tipping. _Providence College, Providence, RI_.

Isocitrate dehydrogenase (IDH) has gained much attention due its frequent mutation in acute myeloid leukemia (AML) and glioblastomas. This enzyme is responsible for converting isocitrate into α-ketoglutarate (α-KG); when this gene is mutated it further converts α-KG into another metabolite, D-2-HG, that competes with α-KG. Production of D-2-HG causes diversion to a new metabolic pathway, as well as inhibits the activity of other enzymes in the cell. We have created flies that express the IDH mutant protein to investigate the other metabolic changes. One of these changes results in an embryo lethality. Additional model systems investigating larval and adult brains have shown glial cell inhibition. We hypothesize that harboring the IDH mutation will have a negative impact on the stable development of progeny.

#4369

Plasma metabolomic biomarkers for an early detection of colorectal cancer.

Anna Brunet-Vega,1 Maria Elisa Quilez,1 Carles Pericay,2 Ismael Macias,2 Laia Vilà,2 Eva Martinez-Balibrea,3 Sergio Lario,1 Guillermo Quintás4. 1 _Fundació Parc Taulí, Universitat Autònoma de Barcelona, Sabadell, Spain;_ 2 _Parc Taulí Hospital, Universitat Autònoma de Barcelona, Sabadell, Spain;_ 3 _Germans Trias i Pujol Reserach Institute, Badalona, Spain;_ 4 _Leitat Technological Center, Barcelona, Spain_.

The aim of this study was to identify a plasma metabolic pattern characteristic of colorectal cancer (CRC) which could be used to assess the effectiveness of CRC surgery procedures and, eventually, support an early detection on CRC recurrence. CRC survival and treatment depends on accurate staging. In the past few years, numerous efforts have been made to discover new non-invasive biomarkers of CRC to enhance current diagnosis and prognostic capabilities. The metabolome is the result of the interaction of different -omic levels (genomic, epigenomic, transcriptional, proteomic) and the set of external interventions. Metabolites are involved in almost every biochemical reaction in the human body and provide a direct meaningful readout of its dynamic biochemical status. Because of that, metabolomics is a highly relevant approach to explore individual phenotypes in systems biology of cancer. This study reports results obtained from the metabolomic analysis of plasma samples collected from 40 CRC patients (stages I-IV) and 20 healthy controls matched by for age and gender. Blood from 20 matched post-CRC surgery cases (stages I-III) was collected 1-2 years after surgery to evaluate the correlation of changes in the plasma metabolome with surgery effectiveness. Metabolomic and lipidomic analysis was carried out by reversed phase LC-MS. The sample set was split into train (CRC pre-surgery (n=30) and Control (n=15) and test (CRC pre-surgery (n=10), CRC post-surgery (n=20) and Control(n=5)) subsets. The train set was used to build a multivariate model to discriminate CRC pre-surgery and Control groups and the test set was exclusively used to evaluate its generalization performance. Results from this exploratory study showed a statistically significant difference between the metabolic profiles of samples collected pre-CRC surgery and those collected post-surgery and from healthy individuals. Among other metabolites, CRC metabolic profile was characterized by lower levels of tryptophan and several carnitines (propionyl-carnitine, methyl-butirylcarnitine) and isoleucyl-leucine. Finally, our results indicate that these metabolites are at similar level than healthy individuals in post-CRC surgery samples. This cohort included only 2 CRC patients with diagnosed recurrence and 18 recurrence-free patients (stages I-III) within 3‐4 years clinical follow-up after surgery and so, more data is required for the estimation of the predictive value of this metabolomic approach for the assessment of the treatment efficiency or disease progression. Conclusions: Our pilot study has identified a pattern of plasma metabolites that correlates with the presence of colorectal tumor. Furthermore, our results indicate that at 1 year post-CRC surgery these metabolites are normalized. Further work is required to establish whether these metabolites could serve as novel disease biomarkers for a minimally invasive diagnosis and monitoring of CRC.

#4370

Knockdown of PKM2 induces autophagic cell death via Akt/mTOR pathway in human prostate cancer cells.

Prasanta Dey, Amit Kundu, Byung Mu Lee, Hyung Sik Kim. _SungKyunKwan University, Suwon, Republic of Korea_.

Pyruvate kinase M2 (PKM2) is crucial for aerobic glycolysis that is highly expressed in the various cancer tissues. Although high expression of PKM2 is observed in prostate tumor tissues, its functional role against affecting cancer metabolism is not clearly understood. Herein, we investigate its role in regulating autophagy and its associated pathways. In the current study, various PKM2-siRNA constructs were used to knockdown the PKM2 expression and observed its cellular pathways on autophagy. Cell viability was significantly reduced in PKM2-siRNA transfected prostate cancer cells, DU145. Acridine orange staining and immunoblotting analysis showed that downregulation of PKM2 markedly increased autophagy cell death. In addition, immunoblotting analysis exhibited the blocking the protein kinase B (Akt) and mTOR1 pathways, which subsequently down-regulated the expression of glycolytic enzymes lactate dehydrogenase A (LDHA) and Glucose transporter 1 (GLUT1). To the best of our knowledge, this is the first study that inhibition of PKM2 changes in cancer cell metabolism and induces autophagy, thus, providing new perspectives into the mechanism of its anticancer activity against prostate cancer, DU145.

#4371

**To investigate the role of heme flux and function in growth and progression of NSCLC** in vivo **.**

Adnin Ashrafi, Poorva Ghosh, Sagar Sohoni, Li Zhang. _University of Texas at Dallas, Richardson, TX_.

Lung cancer is the leading cause of cancer-related mortality and about 85% of the cases are non-small cell lung cancer (NSCLC). Previous research from our lab has shown that the levels of rate limiting heme biosynthetic enzyme ALAS1, heme uptake proteins HCP1 and HRG1 and oxygen utilizing hemoproteins are strongly elevated in NSCLC cells. Furthermore, specific inhibition of heme synthesis using a well-known inhibitor suppresses NSCLC cell proliferation, colony formation and migration compared to normal cells. These results suggest that heme flux and function is important for NSCLC growth and progression.

Lentiviral particles overexpressing eGFP, Alas1, HRG1, and HCP1 were used to transfect NSCLC cell lines. Tumorigenic properties of these cells were analyzed in vitro using invasion, migration and colony formation assay. These cell lines were used to implant subcutaneous xenografts in NOD/SCID mice (n=6). Tumor growth was monitored using Bioluminescence imaging (BLI) and Vernier caliper measurements over a period of five weeks. Mice were sacrificed, tumor tissues were harvested and processed and embedded into FFPE blocks. The tissue sections were then used for histopathological analysis (Hematoxylin and Eosin staining) and immunohistochemistry experiments.

Our data show a significant increase in invasion, migration and colony formation properties of cells lines overexpressing Alas1, HRG1 and HCP1 compared to eGFP overexpressing cell lines (Control) in vitro. At week 5, BLI radiance (total photons/seconds), tumor volumes and masses of Alas1, HCP1 and HRG1 overexpression subcutaneous tumor xenografts were significantly higher compared to eGFP overexpression controls. This was further corroborated by H and E staining.

Our results demonstrate that overexpression of heme synthesis and uptake proteins causes an increase in tumorigenic properties of NSCLC cells in vivo. Thus, heme flux and function plays an important role in growth and progression of NSCLC. Further studies are underway to discern the molecular mechanisms underlying the role of heme in lung tumor growth.

#4372

Effects of high glucose and DCA on ROS production and viability of breast cancer cells with disrupted antioxidant systems.

Radek Buss, Collin Ellenbecker, Emily Minton, Lauren Gray, Mona Nolte, Aysiah Jaeke, Russ Feirer. _St. Norbert College, De Pere, WI_.

A goal of this study was to determine if disruption of protective cellular antioxidant systems would sensitize cancer cells to dichloroacetate (DCA), a compound affecting mitochondrial metabolism (reversing the Warburg Effect). DCA shifts the metabolism of glucose back to mitochondrial oxidative phosphorylation, with subsequent production of reactive oxygen species (ROS) (Bonnet et al., 2007). While cancer cells consume large amounts of glucose to meet energy demands and provide substrates necessary for protein and nucleic acid biosynthesis, they utilize aerobic glycolytic pathways (Warburg metabolism) instead of oxidative phosphorylation. Glutathione (GSH) and thioredoxin (Trx) are among the antioxidants produced to protect cells from oxidative damage; oxidative stress occurs when the production of ROS overwhelms these systems. Biosynthesis of GSH and Trx were inhibited by treating cells with low levels of buthionine sulfoximine (BSO) and auranofin (AUR) to deplete GSH and Trx, respectively. Through its stimulation of ROS production, DCA should enhance the cytotoxic effects in cells with diminished antioxidant systems. A second goal was to investigate the effects of these compounds on cells grown in either low or high glucose media (1 g/L vs 4.5 g/L). Cells grown under high glucose conditions were expected to produce more ROS and exhibit greater sensitivity to the combination of DCA, BSO and AUR. The experiments were conducted using MDA-MB-231 and MCF-7, breast cancer cell lines with known differences in metabolism and responses to chemotherapeutic drugs.

BSO led to a dramatic decrease of GSH levels. AUR, at concentrations above 0.5 µM, significantly reduced cell viability when in combination with BSO. DCA alone reduced viability, but its effects were significantly enhanced when used in conjunction with BSO/AUR. Levels of ROS were quantified using a luminescence-based assay. In low glucose media, BSO/AUR and DCA had no effect on ROS production. In contrast, a high glucose environment led to elevated ROS production under all conditions, with the highest levels being induced by DCA in the cells having disrupted antioxidant systems. In the presence of BSO/AUR, MDA-MB-231 cells produced more ROS than MCF-7 cells. Overall, inhibition of antioxidant systems sensitized cells to DCA, increasing ROS and reducing viability.Previous studies have reported metabolic differences between cell lines (Gatenby and Gillies, 2004). MDA-MB-231 cells rely on aerobic glycolysis while MCF-7 cells are more reliant on mitochondrial oxidative phosphorylation. Therefore, MCF-7 cells have developed increased protection against ROS as compared to MDA-MB-231 cells (Theodossiou et al., 2017). These findings support the observed differences in response to BSO/AUR between the two cell lines. Paradoxically, MCF-7 cells were more sensitive to BSO/AUR than MDA-MB-231 cells in colony formation assays.

#4373

Lack of FGF21 promotes NASH-HCC transition via exosome-mediated carcinogenetic signaling.

Youxi Yu,1 Robert C. Martin,2 Qianqian Zheng,3 Xiaoju Shi,1 Xingkai Liu,1 Wei Guo,1 Suping Li,2 Ping Zhang,1 Yan Li2. 1 _The First Hospital of Jilin University, Jilin University, Changchun, China;_ 2 _University of Louisville, Louisville, KY;_ 3 _Basic Medicine College, China Medical University, Shenyang, China_.

Background: Nonalcoholic steatohepatitis (NASH) is the most severe form of non-alcoholic fatty liver disease (NAFLD) and a potential precursor of hepatocellular carcinoma (HCC). FGF21, with its metabolic benefits, was reported as the liver safeguard to protect from obesity, diabetes, and NAFLD. Recent study showed an exosome based FGF21 inhibition via organs communication to regulate hepatic metabolism. However, the role of the exosome-FGF21 axis in NASH-HCC has never been studied.

Aim: To study the exosome based FGF21 metabolic regulation during NASH-HCC carcinogenetic transformation.

Methods: NASH-HCC model was induced in FGF21 knockout (KO) mice and wild type (WT) control using methionine-choline-deficient diets (MCD) and diethylnitrosamine (DEN). The exosomes releasing, miRNA and protein contents were analyzed in the primary cultured hepatocytes from the liver tissues of NASH-HCC mice (KO and WT) as well as benign controls. FGF21 was knockdown (KD) by shRNA in a mouse hepatoma cell line (Hepa1-6) and a benign mouse hepatocyte cell line (FL83B) to investigate the potential carcinogenetic signaling related to exosome-FGF21 axis.

Result: NASH-HCC model was successfully established with confirmation of the metabolic disorders and liver cancer development. Early HCC detection and increased HCC incidence were found in the KO-MCD mice (P<0.05 versus KO-CD and WT controls). The collected exosomes from primary cultured hepatocytes and cell lines were identified using the antibodies of Tsg101, CD63 and cytochrome C by western blotting and immunofluorescent staining. In the KO-MCD mice, exosomes retained in the cytosol of benign hepatocyte but released increasingly from cancer cell. Similar results were showed in the Hepa1-6KD cells and the FL83BKD cells. Aberrant EMT, and p53 and Wnt signaling was found in the KO-MCD mice as well as Hepa1-6KD cells challenged with free fatty acid.

Conclusion: lack of FGF21 plays critical role for the exosome retention in hepatocytes of NASH mice but not in HCC mice. Abnormal traffic of exosomes contribute to the carcinogenetic signaling. This work was supported by an Institutional Development Award (IDeA) from the NIGMS of the National Institutes of Health under grant number P20GM113226.

#4374

The role of glycine decarboxylase in neuroblastoma.

Ahmet Alptekin, Jane Ding, Bingwei Ye, Han-Fei Ding. _Augusta University, Augusta, GA_.

Neuroblastoma is the most common cancer in infants. While the low and intermediate risk groups of neuroblastoma patients have ≥90% five-year-survival rates, the high-risk group has only 40-50% five-year-survival rates despite multimodal therapies. Genomic amplification of the oncogene MYCN (a member of the MYC family of transcription factors) is a major cause of high-risk neuroblastoma and is strongly correlated with poor prognosis. A key function of MYCN is to promote cell growth and proliferation, which requires increased cellular metabolism to meet the biosynthetic demand for growth. Serine-Glycine-One-Carbon (SGOC) metabolism is particularly important in sustaining cell proliferation by producing amino acids for protein synthesis and one-carbon units for nucleotide production. We investigated the role of glycine decarboxylase (GLDC) in neuroblastoma. GLDC catalyzes the first reaction in glycine cleavage, which generates one-carbon unit and reduced NADH. Analysis of gene expression data from two cohorts of neuroblastoma patients showed that GLDC expression is correlated with advanced stages, high-risk disease and poor prognosis in neuroblastoma. We found that GLDC is required for neuroblastoma cell proliferation as silencing GLDC expression by shRNA induced G1 cell cycle arrest. Consistent with this observation, microarray gene expression profiling revealed that GLDC knockdown resulted in a significant decrease in the expression of E2F target genes. In addition, we obtained evidence that GLDC is a direct target gene of MYCN. Knockdown of MYCN expression reduced GLDC mRNA and protein expression, and overexpression of MYCN increased GLDC mRNA and protein levels. Moreover, chromatin immunoprecipitation with quantitative PCR demonstrated that MYCN binds to the GLDC promoter region. These findings suggest an important role of GLDC in driving neuroblastoma cell proliferation, which could be exploited as a therapeutic strategy against high-risk neuroblastoma with MYCN amplification.

#4375

Overexpression of CD36 promotes colorectal cancer cell proliferation via upregulation of survivin.

James Drury, Naser Jafari, B. Mark Evers Evers, Yekaterina Y. Zaytseva. _University of Kentucky, Lexington, KY_.

Altered fatty acid metabolism has is a hallmark of cancer and continues to be a potential target for therapeutic intervention in cancer. Fatty Acid Translocase (CD36), a multifunctional glycoprotein, has been shown to have an important role in fatty acid metabolism as a fatty acid transporter. Fatty Acid Synthase (FASN), a critical enzyme involved in de novo lipogenesis, has been shown to be upregulated and associated with a poor prognosis in many cancers including colorectal cancer (CRC). However, the role of CD36 in CRC as well as its relation to de novo fatty acid synthesis is not understood. The purpose of our study was: (i) to determine the functional importance of CD36 in CRC and (ii) investigate potential relationships between CD36 and FASN expression in CRC cells.

METHODS. Expression of CD36 and FASN was assessed by immunohistochemistry (IHC) using a CRC TMA with 2 sets of tissues: (i) Primary tumors with matched normal colon tissue; [n=56]; (ii) primary and metastatic tumors (liver [n=12] and lung metastasis [n=5]) with matched normal colon. Cellular proliferation was assessed in control and CD36 shRNA knockdown CRC cells, and in primary CRC cells established from patient derived xenografts treated with CD36 inhibitor Sulfo-N-succinimidyl oleate (SSO) in combination with FASN inhibitor TVB-3664. CD36 knockdown was confirmed by qRT-PCR. Expression of pro-survival and apoptotic markers was assessed via western blot in CD36 knockdown and overexpression CRC cells, as well as CD36+ and CD36- isogenic cells. CD36 localization was assessed via confocal imaging.

RESULTS. We found that CD36 is overexpressed in primary tumors as compared to normal colon mucosa and its expression positively correlates with expression of FASN. Cellular proliferation was significantly reduced when CD36 was inhibited by SSO and a further reduction in proliferation was observed when SSO treatment was combined with TVB-3664. Knockdown and chemical inhibition of CD36 decreased expression of survivin, a member of the inhibitor of apoptosis (IAP) family of proteins, which was shown to promote cancer cell survival in many tumor types. Conversely, overexpression of CD36 increased survivin expression in CRC cells. Additionally, shRNA knockdown of FASN induced CD36 expression in CRC cells. Immunofluorescence imaging of primary CRC cells treated with TVB-3664 showed an upregulation of membrane-bound CD36.

CONCLUSION. Our studies indicate that CD36 upregulation is associated with an increase in cellular proliferation via upregulation of survivin in CRC cells. The correlation between CD36 expression and FASN suggest a connection between CD36 and de novo lipid synthesis. Furthermore, a decrease in FASN expression is associated with CD36 induction, suggesting a possible mechanism of resistance to FASN inhibition. Better understanding of the role of CD36 in CRC may provide new therapeutic approaches for treatment of CRC patients

#4376

Targeting glutamine metabolism disables Warburg physiology by inhibiting proximal glycolysis and Krebs cycle rewiring.

Liang Zhao,1 Matthew Arwood,1 Min-Hee Oh,1 Wei Xu,1 Im-Hong Sun,1 Im-Meng Sun,1 Chirag Patel,1 Robert Leone,1 Jesse Alt,2 Rana Rais,2 Barbara Slusher,2 Jonathan D. Powell1. 1 _Bloomberg-Kimmel Institute for Cancer Immunotherapy, Johns Hopkins School of Medicine, Baltimore, MD;_ 2 _Drug Discovery Program, Johns Hopkins School of Medicine, Baltimore, MD_.

Glutamine plays a critical role in multiple metabolic reactions that support tumor anabolic processes. In spite of this, targeted inhibition of glutaminase, which converts exogenous glutamine into glutamate has met with limited clinical success. We hypothesized that broadly inhibiting glutamine metabolism would more effectively shut down tumor growth. To this end we employed a novel prodrug of the glutamine antagonist 6-diazo-5-oxo-L-norleucine (DON) in a number of syngeneic mouse models of cancer including MC38 (derived from colon cancer), 3LL (derived from lung cancer) and B16 (derived from melanoma). To better understand the anti-tumor mechanisms, we used LC-MS-based metabolomics to profile metabolic flux in these different tumor models with [U-13C]-glucose/glutamine as tracer. We observed that when compared to 3LL and B16, MC38 was highly sensitive to anti-glutamine treatment. As expected, tumor growth inhibition was correlated with inhibition of (the glutamine requiring) purine nucleotide synthesis. Surprisingly however, treatment with the glutamine antagonist markedly inhibited "proximal" glycolytic reactions as determined by inhibition of the generation of glucose-6-P and fructose 1,6-bisphosphate. This inhibition correlated with a decrease in FDG-PET. Likewise, we observed a marked decrease in one carbon metabolism as measured by serine. Strikingly, glutamine antagonism eliminated the glucose-derived succinate. Instead, a dramatic rewiring of the Krebs cycle was identified, which showed an alternative source of succinate was derived from the GABA shunt. In contrast, not only were these pathways upregulated in the relatively resistant B16 and 3LL tumors, glutamine antagonism only minimally affected these pathways. Furthermore, glutamine antagonism led to a marked decrease in kynurenine. Kynurenine is the result of tryptophan metabolism by IDO and is a potent immunosuppressive metabolite and promoter of metastasis. Decreased kynurenine levels were not the result of inhibition of IDO activity by the glutamine antagonist but rather due to a decrease in IDO expression. Interestingly, the hierarchy of susceptibility to glutamine metabolism and the inhibition of these specific pathways (proximal glycolysis, one carbon metabolism and Krebs cycle) correlated with the hierarchy of susceptibility to anti-PD-1 suggesting that tumor specific metabolism might contribute to resistance to immunotherapy. Overall these results accentuate the importance of glutamine requiring metabolic pathways independent of the role of exogenous glutamine uptake. Further, our results highlight the lack of plasticity of highly coordinated tumor metabolic programming; targeting glutamine metabolism inhibited proximal glycolysis, one carbon metabolism and Krebs cycle. In doing so, we identify novel susceptibilities to exploit for inhibiting tumor growth.

#4377

Liver X receptor mediated lipotoxicity represents a treatment option for liver cancer.

Ramona Rudalska,1 Jule Harbig,1 Marteinn Snaebjoernsson,2 Lyudmyla Taranets,1 Florian Heinzmann,1 Stefan Zwirner,1 Wei Ciu Hu,1 Thales Kronenberger,1 Tae-Won Kang,1 Antti Poso,3 Stefan Laufer,1 Mathias Rosenfeldt,2 Nisar P. Malek,1 Bernd Pichler,1 Nikita Popov,1 Almut Schulze,2 Lars Zender,1 Daniel Dauch1. 1 _University of Tuebingen, Tuebingen, Germany;_ 2 _University of Wuerzburg, Wuerzburg, Germany;_ 3 _University of Eastern Finland and University of Tuebingen, Tuebingen, Germany_.

Solid tumors evolve significant changes in metabolic pathways during development by virtue of their specific biosynthetic demands, their particular microenvironment and the potential occurrence of toxic metabolites such as reactive oxygen species. However, the development of cancer treatment approaches that are based on the inhibition of biosynthetic routes is impaired due to the high plasticity of metabolic networks, e.g. resulting in activation of compensatory pathways or in an increased exchange of metabolites between cancer cells and the tumor environment. Therefore, such therapies could not be translated into efficient clinical applications so far.

Here we show that an enhanced lipogenesis, triggered by a pharmacological activation of the Liver X receptor (LXR), represents a new therapeutic strategy for the treatment of liver carcinoma (HCC). A combination of LXR mediated fatty acid synthesis and concomitant Raf suppression results in oxidative stress, induction of a critical ER stress response and subsequently in apoptosis of different murine and human liver cancer cells. Our mechanistic studies identified Raf as an important regulator of lipid metabolism in liver cancer. We found that Raf-1 directly interacts with Stearoyl-CoA desaturase-1 (Scd1), the central enzyme for the conversion of saturated into mono-unsaturated fatty acids and thereby maintains Scd1 protein stability in HCC cells. Thus, inhibition of Raf by Sorafenib diminished Scd1 protein abundance leading to toxic accumulation of saturated fatty acids and metabolic stress in cancer cells under sustained lipogenesis. Treatment studies in autochthonous liver cancer mouse models and xenograft models of human HCC revealed that a combinatorial therapy, consisting of the LXR agonist T0901317 and Sorafenib is highly potent to suppress liver cancer development and to extend the survival of tumor bearing animals. Such a therapy was efficient against hepatic neoplasia with different metabolic phenotypes and well tolerated by mice, even by animals that already suffer from a fatty liver disease.

Taken together, we here propose a pharmacologically induced accumulation of toxic metabolites in cancer cells as a new strategy for efficient metabolic targeting of therapy refractory solid tumors.

#4378

The association of enzymes related to cholesterol esters metabolism with hepatocellular carcinoma.

Zhirong Liu,1 Akram Shalaby,2 Charu Subramony,2 Jinghe Mao,3 Xinchun Zhou2. 1 _Shanxi Medical University, Taiyuan, China;_ 2 _Univ. of Mississippi Medical Ctr., Madison, MS;_ 3 _Tougaloo College, Tougaloo, MS_.

Objectives: The roles of cholesteryl esters (CE) in the pathogenesis of hepatocellular carcinoma (HCC) have not been completely elucidated. This study is aimed to investigate differences in the expression level of enzymes involving in synthesis and degradation of CEs between benign hapatic tissues (BHT) and HCC, and between male and female patients from African American (AA) and Caucasian American (CA).

Methods: TMAs were constructed with 97 BHT and 91 HCC samples from patients enrolled in the University of Mississippi Medical Center from 2010 to 2017. Immunochemistry (IHC) was performed on the TMAs for enzymes involved in CE synthesis (acyl CoA:cholesterol acyltransferase 1, ACAT1 and acyl CoA:cholesterol acyltransferase 2, ACAT2), and in CE degradation (lysosomal acid lipase, LAL and neutral cholesterol ester hydrolase 1, NCEH1) following the manufacturer's instructions. The expression level for each enzyme was expressed as IHC score calculated with a scoring system. Two-tailed Student's t-test was used to compare the difference in the expression level of each enzyme between two groups. Significant p value was set at p<0.05.

Results: The IHC score of ACAT2 was significantly higher in HCC than in BHT in all population (6.21±2.42 vs (4.89.4±2.40, P=0.0006) and in stratified male population (6.15±2.33 vs 5.08±2.32, p=0.046), but not in stratified women, AA and CA populations. There was no significant difference in IHC score for ACAT1 between HCC and BHT in all population and populations stratified by race and gender. The IHC score of LAL was significantly higher in HCC as compared to BHT in all population (3.57±2.31 vs 2.40±1.30, P =0.0001) and in male population (3.33±2.01 vs 2.39±1.16, p=0.015), but not in stratified women, AA or CA population. The IHC score of NCEH1 was not significantly different between HCC and BHT in all studied population and population stratified by gender. However, it was disparate between AA and CA populations: The IHC score of NCEH1 was significantly lower in HCC than in BHT in AA population (5.59±2.04 vs 6.66±1.74, p=0.031) but it was higher in HCC than in BHT in CA population (6.84±1.96 vs 7.12±1.70, p>0.05).

Conclusions: These results suggest that alterations in the expression levels of enzymes involving CE metabolism are associated with pathogenesis, progression and disparities in race and gender of HCC. This study also suggest that ACAT2 predominates CE synthesis as compared to ACAT1, and that LAL plays major role in CE degradation as compared to NCEH1 in association with HCC.

#4379

Prostate cancer cells utilize the GABA shunt to enhance oxidative metabolism.

Erika L. Knott, Sumitra Miriyala, Manikandan Panchatcharam, Nancy Leidenheimer. _LSU Health Sciences Center, Shreveport, Shreveport, LA_.

Although an upregulation of aerobic glycolysis (the Warburg effect) is a canonical hallmark of cancer, it is now recognized that most cancerous cells continue to rely on the TCA cycle as a source of both biosynthetic precursors (DeBarardinis & Chandel, 2016) and the majority of ATP production (Zu & Guppy, 2004). Drug-resistant cancers have been associated with metabolic reprogramming (Rahman & Hasan, 2015) involving an upregulation in oxidative phosphorylation (Wolf, 2014). Two advanced, treatment-resistant variants of prostate cancer are castration-resistant adenocarcinoma (CRPC-adeno) and neuroendocrine prostate cancer (NEPC). Emerging evidence suggests that the γ-aminobutyric acid (GABA) shunt—an evolutionarily conserved pathway for energy production, biosynthetic precursors, and ROS management—may be used by both CRPC-adeno and NEPC. The GABA shunt is a bypass for two steps of the TCA cycle, wherein GABA is synthesized from glutamate by a GAD1-encoded decarboxylase, transaminated into succinic semialdehyde, and metabolized into the TCA cycle intermediate succinate. Analysis of publicly available transcript datasets (via cBioPortal) indicates that GAD1 mRNA expression increases with increasing Gleason score (p < 0.00005, prostate adenocarcinoma TCGA provisional dataset), with higher expression in both CRPC-adeno compared to primary adenocarcinoma (Ippolito and Piwnica-Worms, 2014), and NEPC compared to CRPC-adeno (p < 0.005, Beltran, et al., 2016). Expression of GAD1 transcripts appears to be contingent on low levels of AR transcripts (Trento/Cornell/Broad data set), consistent with the AR independence of the NEPC phenotype. Similarly, GABA levels increase as AR expression is lost during transdifferentiation to NEPC (Solorzano, et al., 2018). GAD67 protein levels in prostate tumors are a more accurate predictor of patient risk for metastasis than Gleason score (Azuma, et al., 2003), and flux through the GABA shunt is critical for the growth of brain tumors in mouse xenograft models (Schnepp, et al., 2017; Neman, et al., 2014). Our experiments show that treatment of an early-stage prostate cancer cell line model (LNCaP) with GABA (100 µM, 24h) induces a shift from aerobic glycolysis to oxidative metabolism. This metabolic reprogramming toward increased oxidative phosphorylation requires at least 6 hours to emerge, and is maximal by 18 hours. During this time period, the presence of GABA in the mitochondrial matrix steadily increases, as shown by our newly-developed GABA-sensing fluorescent receptor, mitoGABASnFR. The observed increases in oxygen consumption are accompanied by GABA-induced decreases in extracellular acidification and lactate levels. In light of these findings, we propose that the GABA shunt represents an understudied pathway for inducing a metabolic shift towards oxidative phosphorylation in advanced prostate cancer, and therefore may be a target for future anticancer therapies.

#4380

LSR promotes cell proliferation and migration subsequent to uptake of fatty acid in epithelial ovarian cancer.

Kosuke Hiramatsu,1 Satoshi Serada,1 Minoru Fujimoto,1 Yutaka Ueda,2 Tadashi Kimura,2 Tetsuji Naka1. 1 _Kochi University, Japan;_ 2 _Osaka University, Japan_.

Background Previously, we identified lipolysis-stimulated lipoprotein receptor (LSR) as a new therapeutic target of epithelial ovarian cancer (EOC), and we reported anti-cancer effect of our newly developed monoclonal antibody (mAb) against LSR-positive EOC in vitro and in vivo. We also demonstrated that LSR took in triglyceride-rich protein and contributed cell proliferation, however, lipid metabolic pathway via LSR is still unclear. Thus, we aimed to reveal the function of LSR on lipid metabolism in EOC cells.

Methods We investigated the activation of beta-oxidation, ATP production, cell proliferation and cell migration via LSR after administration of fatty acid (FA) using LSR-positive EOC cells and LSR-knockout EOC cells. Moreover, we also investigated anti-cancer effect of our anti-LSR mAb against lipid metabolism via LSR.

Results In LSR-positive EOC cells, FA administration increased intracellular lipid accumulation. In glucose restricted environment, LSR promoted FA uptake and promoted ATP production (p < 0.05). In addition, FA promoted cell proliferation and cell migration in LSR-positive EOC cells (p < 0.05). These results suggest that LSR promoted FA uptake in glucose restricted environment and also promoted lipid metabolism including beta-oxidation, TCA cycle and electron transport system leading to ATP production. Finally, our anti-LSR mAb inhibited these lipid metabolic pathways (p < 0.05).

Conclusions Fatty acid uptake via LSR in glucose restricted environment contributed to cell proliferation and migration subsequent to activation of lipid metabolism, and anti-LSR mAb inhibited these processes. LSR might contribute to cancer spread and metastasis of EOC.

#4381

Investigating the reversible MDH1 catalytic reaction in squamous non-small cell lung cancer.

Joi L. Weeks,1 Grace Wells,1 Sati Alexander,1 Christian Metallo,2 Christal D. Sohl1. 1 _San Diego State University, San Diego, CA;_ 2 _University of California, San Diego, San Diego, CA_.

Non-small-cell lung cancer (NSCLC) is a leading cause of death in men and women worldwide. NSCLC relies on glycolysis to support cell proliferation and to synthesize proteins, nucleic acids and lipids. Malate dehydrogenase (MDH) is an enzyme critical for replenishing intermediates in the tricarboxylic acid (TCA) cycle and for helping to drive glycolysis through its reversible coenzyme system NAD+/NADH by interconverting oxaloacetate and malate. The cytosolic form of MDH (MDH1) has been reported to be amplified in squamous cell NSCLC, which has high glycolytic activity. Elucidation of the role of MDH1 in altering cellular metabolism to drive tumorigenic progression has yet to be determined. Thus, our interest lies in identifying the chemical and cellular characteristics required to drive the reversible MDH1 catalytic reaction in squamous cell NSCLC. Previously, we have shown that metabolic dehydrogenases are regulated by microenvironmental changes in pH and oxidation levels, and we are currently extending this work to MDH1. We are also evaluating various cell lines to gain a better understanding of how the MDH1 reaction can be altered to decrease the tumorigenic capacity of squamous cell NSCLC. Experimental knowledge obtained has the potential to increase our understanding of the MDH1 reaction and educate the design of future anti-tumor therapeutics.

#4382

Epigenetic deregulation in glycolysis genes for colorectal cancer.

KuoHsing Chen,1 Liang-Chuan Lai,1 Liang-In Lin,1 Hsien-Fang Chang,1 Yu-Liang Chao,2 Been-Ren Lin,2 Jin-Tung Liang,2 Ann-Lii Cheng,1 Eric Y. Chuang,1 Kun-Huei Yeh1. 1 _National Taiwan University, Taipei, Taiwan;_ 2 _National Taiwan University Hospital, Taipei, Taiwan_.

Background

Energy restriction may have influence on methylation of glycolysis genes and also be associated with decreased incidence of colorectal cancer (CRC) in Dutch Hunger Winter cohort. This study aimed to explore the roles of epigenetic deregulation in energy/glycolysis pathway for CRC.

Materials and methods

To perform a comprehensive analysis of methylation alterations in glycolysis pathway (67 genes in KEGG), we profiled 27 CRCs and 5 adjacent normal colon tissues (NTs) by using a powerful methylation array (Illumina Methylation EPIC Beadchips). We used Wilcoxon rank-sum test (p < 0.05) and β value difference (≧ 0.25 or ≦ -0.25) to define the differential methylated genes (DMGs). We also searched the open datasets to find the shared DMGs in CRC. The shared DMGs were validated by pyrosequencing in our samples.

Results

We found 17 hypomethylation genes and 2 hypermethylation genes involved in

glycolysis pathway in our cohort. After comparing to the methylation profiles of CRC in 3 datasets (GSE42752, GSE25062 and TCGA), only hypermethylation of ALDH1A3 and hypomethylation of HKDC1 remained as shared DMGs. These findings were validated well by pyrosequencing in our cohort. The mRNA expression of HKDC1 and ALDH1A3 was negatively associated with the methylation status of each of them in TCGA dataset. RT-qPCR also showed the corresponding expression of HKDC1 and ALDH1A3 between CRCs and NTs.

Conclusion

Our study reveals that hypomethylation of HKDC1 and hypermethylation of ALDH1A3 exist in CRCs worldwide. Further exploratory study of biologic roles of them in CRC is ongoing. 

### Post-transcriptional and Translational Control of Gene Regulation

#4383

MAPKAPK2: The master regulator of transcript stability in HNSCC progression.

Sourabh Soni, Yogendra S. Padwad. _CSIR-IHBT, Palampur, India_.

Head and neck squamous-cell carcinoma (HNSCC) is the sixth most common cancer globally. The limited understanding of the mechanisms of disease progression in HNSCC has posed huge challenges for the development of new therapeutic strategies. Better understanding will lead to novel therapies and tailoring of existing treatment modalities, thus paving a way for personalized medicine. The p38/mitogen-activated protein kinase (p38MAPK) pathway has been implicated in pathological conditions ranging from inflammation to metastasis. Post-transcriptional regulation of genes harboring adenine/uridine-rich elements (AREs) in their 3'-untranslated region (3'-UTR) is controlled by MAPK-activated protein kinase 2 (MAPKAPK2 or MK2), a downstream substrate of the p38MAPK via its association with RNA-binding proteins (RBPs). A variety of extracellular stimuli lead to MK2 activation which further influences crucial cellular processes and signaling events; regulates inflammatory cytokines and transcript stability. Although MK2 modulates important cellular phenomenon, yet its biological significance in tumor progression has not been well elucidated till date. In our study, we have elucidated that MK2 is the master regulator of RBPs and plays a role in the regulation of transcript stability of HNSCC-pathogenesis linked genes. We studied the role of MK2 in HNSCC progression using patient derived clinical tissue samples and observed that MK2 is reproducibly overexpressed in them. Further, MK2 knockdown (MK2KD) in HNSCC cells resulted in increased stability of p27 and MKP-1 transcripts whereas it led to reduced stability of TNF-α and VEGF transcripts suggesting that MK2 regulates their transcript stability. Furthermore, in vivo heterotropic xenograft mice model established that MK2KD attenuates tumor progression in NOD/SCID mice. Transcriptomics investigations on MK2KD cells and xenograft samples also revealed a clear involvement of MK2 in transcriptional regulation. Simultaneously, we have also been working on unveiling the mechanism of interactions of MK2 with RBPs using both in vitro as well as in silico approaches. Altogether, it could be concluded that MK2 is responsible for regulating the transcript stability and is functionally important to modulate HNSCC pathogenesis.

#4384

Role of EIF4G1 in non-small cell lung cancer pathogenesis and targeted therapy.

Zhiqiang Qin,1 Lu Dai,2 Luis Del Valle2. 1 _University of Arkansas for Medical Sciences, Little Rock, AR;_ 2 _Louisiana State Univ. Health Sciences Ctr., New Orleans, LA_.

Non-small cell lung cancer (NSCLC) accounts for about 85-90% of lung cancer cases, which represents the number one killer among cancers in US. The majority of lung cancer patients doesn't respond well to conventional chemo-/radio-therapeutic regimens and have a dismal 5-year survival rate of ~15%. The recent introduction of targeted therapy and immunotherapy gives new hopes to NSCLC patients, but their outcome/prognosis is far from satisfactory. The translation initiation EIF4F complex has been shown to play important roles in cancer progression. As an important component of the EIF4F complex, EIF4G1 serves as a scaffold and interacts with several other initiation factors such as EIF4E and EIF4A, and help to initiate cap-dependent translation in mammalian cells. EIF4G1 is overexpressed in a variety of cancers, but its functional role, clinical relevance and potential role as a therapeutic target in NSCLC remain largely unknown. Our recent data demonstrate that EIF4G1 expression level is much higher in NSCLC primary tumors than in paired adjacent normal tissues as well as in NSCLC cell lines than in the normal bronchial epithelial cells. Directly targeting EIF4G1 by RNAi or selective small-molecule inhibitors significantly reduce NSCLC cell proliferation, anchorage-independent growth in vitro as well as tumor progression in vivo. Our recent protein array and proteomics analyses indicate that EIF4G1: 1) has a complex interactome network in NSCLC cells; 2) appears to control the expression of a unique subset of the cellular proteome when compared to normal cells, which are closely related to NSCLC cells survival. Taken together, our data support that EIF4G1 plays a critical role in NSCLC survival, oncogenesis and tumor progression, and may serve as a novel and promising therapeutic target for improving NSCLC treatment.

#4385

Eukaryotic translation initiation factor 4 gamma 1 (EIF4G1): A target for cancer therapeutic intervention.

Praveen Jaiswal,1 Sweaty Koul,1 Nallasivam Palanisamy,2 Hari K. Koul1. 1 _LSU Health Sciences Ctr. - Shreveport, Shreveport, LA;_ 2 _Henry Ford Health Sciences Center, Detroit, MI_.

Introduction: Cap-dependent mRNA translation is an essential for translation of key oncogenic proteins at optimal levels and is highly regulated by the rate limiting, initiation step in protein synthesis, thus this pathway could be exploited for therapeutic intervention in oncogene driven tumors. Eukaryotic Translation Initiation Factor 4 Gamma 1 (EIF4G1) serves as the critical scaffold for assembly of cap-dependent translation components in EIF4F complex formation. In the current study, we analyzed the role and expression of EIF4G1 in Pan human cancers panels through various approaches.

Methods: Immunohistochemistry analysis of EIF4G1 protein was done on multi-organ Human Cancer tissue microarray (TMA) derived from patient's samples from different cancers. Western blots for EIF4G1 protein was done for different cell lines in representing the different cancer type. Multiple clinical cohorts were used to analyze the EIF4G1 mRNA expression across human cancers. TCGA data analysis of EIF4G1 was done through Ualcan and c-bioportal web servers. Dependency score was calculated through Cancer Dependency Map.

Results: We analyzed the EIF4G1 protein level by western blot in variety of human cancer cell lines found an elevated level of EIF4G1 protein in these cell lines. We found an increase in EIF4G1 protein levels in tissue sections from different cancer samples as compared to their respective normal tissue. EIF4G1 expression was also elevated across cancer cell lines from multiple organs. Our analysis of the TCGA data revealed the higher expression of EIF4G1 mRNA expression across human cancers. Comparison of EIF4G1 mRNA expression between tumor tissue vs normal tissue in TCGA datasets revealed higher expression of EIF4G1 in cancer tissues. We discovered that alteration frequency in EIF4G1 is prevailed across different human cancers in particular prostate cancer (~25%), ovarian cancer (~15%), Head & neck cancer (~13%) and cervical cancer (~12.5%) were most affected cancer. EIF4G1 Amplification and mRNA upregulation was evident across human cancer. We further analyzed the dependency of EIF4G1 in cancer cell survival based on depletion assay through DepMap and found that EIF4G1 is required for cancer cell survival. Higher EIF4G1 mRNA level was associated with lower survival of patients in Pan Cancer analysis.

Conclusions: This is the first study highlighting a broad role for EIF4G1 across pan cancers suggesting that EIF4G1 may serve as a novel target for therapy in multiple malignancies. However further studies are required to develop these concepts and test usefulness of targeting EIF4G1 in caner utilizing preclinical model systems.

#4386

**Cytoplasmic NAD** + **inhibits mRNA translation in ovarian cancers.**

Sridevi Challa, Keun W. Ryu, Tulip Nandu, William L. Kraus. _UT Southwestern Medical Center, Dallas, TX_.

Introduction: NAD+ is a key metabolite involved in redox reactions and energy production, supporting important biological pathways, such as cell growth, proliferation, and metastasis. In addition, NAD+ regulates important cellular processes, such as gene transcription, by acting as a substrate for enzymes including PARPs and Sirtuins. PARP enzymes are the major consumers of NAD+; they transfer the ADP-ribose moiety from NAD+ to their substrate proteins. We recently showed that nuclear PARP activity is differentially regulated by compartmentalized synthesis of NAD+ by the nuclear and cytosolic NAD+ synthases, NMNAT-1 and NMNAT-2, respectively. While the role of the nuclear PARPs, PARP-1 and PARP-2, in catalyzing polyADP-ribosylation (PARylation) is well understood, little is known about the function of cytosolic PARPs, which catalyze monoADP-ribosylation (MARylation).

Methods: We utilized several biochemical and genomic techniques to understand the role of MARylation in ovarian cancer. We performed cell fractionation and confocal microscopy assays to detect the localization of MARylation in OVCAR3 ovarian cancer cells. We also used a genetically encoded NAD+ biosensor to measure changes in nuclear and cytosolic compartment-specific NAD+ levels upon NMNAT-1 or NMNAT-2 depletion by shRNAs. In addition, we used puromycin incorporation assays to determine the amount of protein synthesis, and Polysome-sequencing to identify the changes in mRNA translation upon NMNAT-2 knockdown. Finally, we used Thioflavin T staining to measure protein aggregation and performed cell growth assays to demonstrate the functional effects of cytosolic NAD+ depletion on ovarian cancer phenotypes.

Results: In this study, we observed that NMNAT2 expression is upregulated in ovarian cancer tissues and that high NMNAT2 expression correlates with poor progression-free survival in ovarian cancer patients. Since cytosolic PARPs predominantly catalyze MARylation of target proteins involved in mRNA regulation, we hypothesized that NMNAT-2 depletion restricts the NAD+ available for the cytosolic PARPs, and alters mRNA function. Indeed, our results show that NMNAT-2 knockdown inhibits MARylation of cytosolic proteins in ovarian cancer cells without affecting nuclear PARylation. Furthermore, NMNAT-2 knockdown results in a higher mRNA association with polysomes, increased mRNA translation, and a paradoxical reduction in cell growth. Intriguingly, we found that the increased load of protein synthesis upon NMNAT-2 knockdown leads to aggregation of proteins, resulting in proteotoxicity.

Summary: Collectively, in this study, we identified a role for NMNAT-2 in the regulation of cytosolic MARylation and ovarian cancer phenotypes. We found that ovarian cancer cells depend on NMNAT-2 to maintain high levels of cytosolic NAD+, which regulate proteostasis by inhibiting protein synthesis.

Supported by grants from the NIH/NIDDK and CPRIT to W.L.K.

#4387

hnRNP Q1 promotes cell proliferation and tumorigenesis by translationally up-regulating cell cycle-related genes through the EGF pathway.

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

Heterogeneous nuclear ribonucleoprotein (hnRNP) Q1, an RNA-binding protein, has been reported as overexpressing in cancer cells. However, the role of hnRNP Q1 in tumorigenesis remains unclear. By using RNA-immunoprecipitation (IP) assay following next-generation sequencing, a group of cell cycle-related genes targeted by hnRNP Q1 were identified, including Aurora-A kinase. Overexpressed hnNRP Q1 can up-regulate Aurora-A and many spindle assembly checkpoint (SAC) proteins, but not alter their mRNA levels, through enhancing translational efficiency by interacting with the mRNA 5'-UTR. Ribosomal S6-IP and ribosomal profiling assay further confirmed the translational regulation of these mRNAs by hnRNP Q1. Stimulation with epidermal growth factor (EGF) enhances both binding between hnRNP Q1 and these mRNAs as well as the efficacy of the hnRNP Q1-induced translation of these mRNAs. EGF/hnRNP Q1-induced translation is mediated by the mTOR and ERK pathways. In addition, hnRNP Q1 overexpression is positively correlated with the levels of Aurora-A and these SAC genes in human colorectal cancer tissues. Taken together, our data suggest that hnRNP Q1 plays an important role in translationally regulating the expression of Aurora-A and a group of cell cycle-related genes, which increases cell proliferation and contributes to tumorigenesis in colorectal cancer. It may thereby contribute to tumorigenesis by translationally up-regulating these genes in colorectal cancer.

#4388

A novel, reversible YAP1-HuR axis promotes pancreatic tumorigenesis.

Samantha Z. Brown, Christopher W. Schultz, Tianyun Li, Aditi Jain, Raymond O'Neill, Wei Jiang, Charles P. Yeo, Jonathan R. Brody. _Thomas Jefferson University, Philadelphia, PA_.

Pancreatic ductal adenocarcinoma (PDAC) is the third-leading cause of cancer-related death in the U.S. In roughly 95% of cases, gain-of-function mutations in KRAS combine with loss of tumor suppressors to progress pre-invasive neoplasms (PanINs) to late-stage ductal adenocarcinoma. Two important mediators of KRAS function, Human Antigen R (HuR) and Yes-associated protein 1 (YAP1), are both highly overexpressed in PDAC. HuR is a RNA-binding protein that facilitates gene expression through the stabilization and increased translation of target pro-survival mRNAs upon stress. YAP1 is a transcriptional co-activator, which associates with a number of transcription factor families to sense and upregulate targets that lead to tumor growth and cellular cross-talk. While the functions of HuR and YAP1 are well known, the events that lead to their overexpression and regulation are poorly understood.

YAP1 was first identified as a HuR target via ribonucleoprotein-immunoprecipitation assays in which HuR-bound mRNAs were run on a whole-transcriptome microarray. YAP1 mRNA was significantly bound to HuR as compared to the IgG isotype control (13.2-fold), and was in line with previously established targets (WEE1, 3.2-fold; PIM1, 13.9-fold). YAP1 mRNA bound to HuR is abolished when treated with a known HuR inhibitor, pyrvinium pamoate, even in the presence of an established HuR stressor (i.e., oxaliplatin). Actinomycin D chase assays demonstrated that YAP1 mRNA stability is significantly dependent on HuR proficiency. We validated that both YAP1 mRNA and protein expression levels are dependent on HuR via real-time quantitative PCR and Western blot analysis. Transcription of downstream YAP1 targets, CTGF and CYR61, was also significantly affected by knockdown of HuR.

Surprisingly, we found that RNA silencing of YAP1 significantly reduced HuR mRNA and protein expression, as well as HuR targets, WEE1 and PIM1. Treatment with small-molecule inhibitors, Verteporfin and CA3, which target the interface of YAP1's transcription-factor binding domain, recapitulated these effects in a dose and time-dependent manner. We are currently using chromatin immunoprecipitation sequencing to identify HuR's promoter/enhancer domains for YAP1-mediated transcription.

Our previous work has shown that overexpression of cytoplasmic HuR correlates strongly with tumor staging. Low-levels of HuR correspond to early PanINs with staining steadily increasing in late-stage PanIN lesions and gross overexpression in invasive adenocarcinoma. Conversely, YAP1 overexpression seems to be most critical for initial development and expansion of the tumor cells, while it converts to a maintenance role once PDAC is fully developed. Ongoing studies will address whether the temporal regulation of these proteins could explain their overexpression patterns in pancreatic pathologic stages as they relate to cooperating with KRAS activity.

#4389

Post-translational modifications of histone demethylase JMJD1A in prostate cancer cells.

Songhui Xu,1 Lingling Fan,1 Xiaolu Cui,1 Fengbo Zhang,1 Arif Hussain,1 Ladan Fazli,2 Martin Gleave,2 Jianfei Qi1. 1 _Univ. of Maryland School of Medicine, Baltimore, MD;_ 2 _University of British Columbia, Vancouver, British Columbia, Canada_.

Castration-resistant prostate cancer (CRPC) is the major cause of death for prostate cancer (PCa) patients. Re-activation of androgen receptor (AR) activity during androgen-deprivation therapy is believed to underlie CRPC development. We have found that histone demethylase JMJD1A primarily regulates AR and c-Myc activity and thus plays a key role in promoting PCa cell proliferation or survival. Unfortunately, specific JMJD1A inhibitors are not yet available. Here, we identified several post-translational modifications of JMJD1A by performing the JMJD1A immunoprecipitation and mass spectrometry analysis. These modifications include the canonical ubiquitination that targets JMJD1A for proteasome-dependent degradation, non-canonical ubiquitination that enhances the recruitment of co-activators, as well as those that can regulate these ubiquitination events. We also found that these modifications could be manipulated by commercially available inhibitors, to reduce the JMJD1A protein level and prostate cancer cell growth. Thus, targeting the JMJD1A modifications may serve as an alternative means to inhibit JMJD1A activity for the anti-PCa therapy.

#4390

Dietary soy isoflavone equol promotes breast cancer progression via regulation of protein synthesis initiation.

Ailed M. Cruz-Collazo,1 Columba De la Parra,2 Robert Schneider,2 Suranganie Dharmawardhane1. 1 _University of Puerto Rico - Medical Sciences Campus, San Juan, PR;_ 2 _NYU School of Medicine, New York, NY_.

We previously reported that dietary daidzein, a soy isoflavone, increased breast cancer progression in a nude mouse model of breast cancer metastasis. Further studies indicated that equol, a metabolite of the soy isoflavone daidzein is responsible for the cancer promoting effects of soy isoflavones in breast cancer cells in vitro. We reported that equol increases the expression of the eukaryotic protein synthesis initiation factor eIF4G1, and the transcription factor c-Myc. Increased eIF4G in response to equol resulted in the non-canonical protein synthesis of pro-cancer molecules such as Cyclin D, Bcl-XL, p120, matrix metalloproteinases, etc., as confirmed by ribosome profiling and eIF4G1 knockdown studies. This study tested the hypothesis that equol promoted breast cancer progression via eIF4G1 upregulation. Immunocompromised nude mice were used to establish mammary fatpad tumors from human metastatic breast cancer cells expressing eIF4G1 knockdowns via a Tetracycline-inducible promoter. Oral gavage of 50 mg/kg BW Doxycycline 3X a week for 4 weeks was enough to knockdown eIF4G1 expression by ~80%. Therefore, one week following tumor establishment, the mice were treated with vehicle (90% corn oil, 10% ethanol), equol, Doxycycline or, equol and Doxycycline. Data show that as expected, equol treatment increased tumor growth relative to vehicle treatment. Moreover, eIF4G1 knockdown abolished the effect of equol on tumor growth. Interestingly, the effect of eIF4G1 knockdown on mammary tumor growth was not evident until 55 days of doxycycline treatment. Therefore, we tested the cell and tumor lysates for potential expression of the eIF4G1 isoform Dap5 when eIF4G1 is knocked out. Dap5 protein was expressed in eIF4G1 knockdown cells and was increased by equol treatment, indicating that Dap5 may compensate for eIF4G1 knockdown by inducing alternative cap-mediated protein synthesis. In conclusion, the cancer promoting effect of equol is abolished in breast tumors where eIF4G1 expression was stably knocked down, thus, validating our hypothesis that equol increases breast cancer progression via upregulation of eIF4G1 dependent protein synthesis initiation.

#4391

**Disruption of the translation initiation complex by eIF4A mutant inhibits prostate tumor growth** in vivo **.**

Puja Singh,1 Hanyong Chen,1 Hanxuan Li,2 Young-In Chi,1 Bin Liu,1 Junxuan Lu,3 Wei Li,2 Zigang Dong,1 Yibin Deng1. 1 _The University of Minnesota Hormel Institute, Austin, MN;_ 2 _The University of Tennessee Health Science Center, Memphis, TN;_ 3 _College of Medicine, Pennsylvania State University, Hershey, PA_.

Translation initiation in cancer cells is the rate-limiting step of oncogenic mRNAs translation/protein synthesis and is primarily mediated by the eukaryotic translation initiation factor 4 F (eIF4F) complex that consists of three subunits: eIF4E, which binds the cap of mRNA; eIF4A, an RNA helicase implicated in unwinding mRNA structure; and a scaffold protein eIF4G. Our prior crystal structure determination of human eIF4A in complex with Heat 1 domain of eIF4GI (722-999) revealed that eIF4G interacts with both N- and C-terminal domains of human eIF4A (eIF4A1-NTD and eIF4A1-CTD). Utilizing structural information gained from this complex, we generated a series of eIF4A mutants (eIF4A1-NTD: E41K, R45D, Y48A and eIF4A1-CTD: T271A, R324D, D296A/T298A) in order to identify the crucial site(s) of interaction with eIF4G Heat 1 domain. Using in vitro binding studies, we demonstrated that amongst various mutants tested, eIF4A-CTD (eIF4A1R324D) mutant disrupted the eIF4A/eIF4G Heat 1 complex formation, mirroring the binding residue E809K mutant of eIF4G that also interrupted eIF4A/eIF4G Heat 1 interaction. Given that eIF4G has an additional eIF4A-binding site named Heat 2 domain, we further investigated whether eIF4A1R324D mutant is able to disrupt binding to full-length eIF4G protein in vivo. Surprisingly, overexpression of Flag-tagged eIF4A1R324D in prostate cancer cell line blocked interaction with exogenous eIF4G as well as endogenous eIF4G. Taken together, these results strongly suggest that eIF4A1R324 residue plays a crucial role in mediating eIF4A/eIF4G complex formation in vivo. Furthermore, overexpression of eIF4A1R324D mutant significantly blocked oncogene c-myc and Bcl-2 mRNA translation thereby inhibiting prostate tumor growth in vivo. To conclude, our studies provide a previously unexplored strategy to identify small-molecule compounds disrupting eIF4A-eIF4G complex by targeting the pocket spanning eIF4AR324 and its binding site residues on eIF4G for cancer therapy.

#4392

Pre-mRNA splicing factors promote cellular plasticity in castration-resistant prostate cancer.

Mark P. Labrecque,1 Ilsa M. Coleman,2 Lisha G. Brown,1 Bryce Lakely,1 Lori Kollath,1 Daniel W. Lin,1 Lawrence D. True,1 Eva Corey,1 Peter S. Nelson,2 Colm Morrissey1. 1 _University of Washington, Seattle, WA;_ 2 _Fred Hutchinson Cancer Research Center, Seattle, WA_.

Background: Metastatic castration-resistant prostate cancer (mCRPC) is a heterogeneous disease with poorly understood drivers of disease progression. We recently characterized five mCRPC phenotypes, including an amphicrine phenotype that co-expresses androgen receptor (AR) and neuroendocrine prostate cancer (NEPC) biomarkers. We determined that loss of RE1-silencing transcription factor (REST), a master regulator of neuronal differentiation, drives the amphicrine phenotype, but is not sufficient for prostate cancer (PC) adenocarcinoma to NEPC conversion. Furthermore, loss of REST through SRRM4-mediated splicing of REST pre-mRNA to REST4 has been suggested to drive adenocarcinoma to NEPC conversion. However, the roles of pre-mRNA splicing factors and REST activity in lineage switching require further investigation.

Methods: Transcriptomic (RNASeq) and immunohistochemical/immunofluorescent analysis (IHC and IF) were conducted on amphicrine and NEPC patient metastases, LuCaP patient-derived xenograft (PDX) models and modified CRPC cell lines. The roles of SRRM3 and SRRM4 were examined using overexpression studies in AR-expressing and AR-null CRPC cell lines.

Results: RNASeq, IHC and IF of metastatic specimens, LuCaP PDX models and VCaP cells confirmed the existence of the amphicrine phenotype in vitro and in vivo. Interestingly, transcriptome analysis of amphicrine patient specimens and LuCaP 77CR revealed that loss of REST repressor activity occurred without SRRM4 expression in a subset of tumor specimens. Indeed, BaseScope analysis using primers specific to REST4 and SRRM4 verified that amphicrine 77CR tumors were positive for REST4 expression but negative for SRRM4 expression, suggesting an alternative mechanism of REST splicing. Notably, overexpression of SRRM4 in AR-expressing C4-2B and AR-null PC-3 cells did not induce REST splicing. Moreover, RNASeq of SRRM4-overexpressing cells displayed heterogeneous transcriptome profiles inconsistent with canonical amphicrine or NEPC gene expression profiles. Interestingly, SRRM3 transcript was expressed at high levels in amphicrine and NEPC patient and LuCaP PDX biospecimens that lacked SRRM4 expression, suggesting an SRRM3-mediated mechanism of REST splicing. Studies interrogating the roles of SRRM3 in REST splicing and CRPC cellular plasticity are ongoing.

Conclusions: Our data highlights an unrecognized mechanism of adenocarcinoma to amphicrine or NEPC conversion that hinges on a SRRM3-REST regulatory axis rather than REST-loss or SRRM4-mediated REST splicing. Identifying the mechanisms that may convert adenocarcinoma to treatment-resistant amphicrine or NEPC phenotypes in mCRPC patients will inform treatment and identify potential molecular pathways for therapeutic intervention.

#4393

Integrative proteomic analysis of prostate cancer reveals distinct regulation of RNA binding proteins during disease progression.

Mauro Scaravilli,1 Annika Kohvakka,2 Pekka Ruusuvuori,2 Ebrahim Afyounian,2 Matti Nykter,2 Tapio Visakorpi,2 Leena Latonen1. 1 _University of Eastern Finland, Kuopio, Finland;_ 2 _University of Tampere, Tampere, Finland_.

To understand the etiology of the disease, and to find novel and more specific drug targets, the driver mutations and expressional changes in prostate cancer have been examined through extensive genomic and transcriptomic characterization. Although significant insight has been gained through these efforts, it is clear that not all molecular alterations influencing the tumor outcome can be captured through these approaches, and that a comprehensive understanding of the molecular events in cancer require thorough investigation of the proteome.

To understand the functional consequences of genetic and transcriptional aberrations in prostate cancer, we aimed to reveal the proteomic changes during disease formation and progression. We performed high throughput mass spectrometry on clinical tissue samples of benign prostatic hyperplasia (BPH), untreated primary prostate cancer (PC) and castration resistant prostate cancer (CRPC). We performed an integrative analysis of the proteomic data with gene copy number, DNA methylation, and RNA expression data from the same samples. Furthermore, proteomic events correlating with the androgen receptor (AR) status of the tumors were analysed.

We uncovered previously unrecognized molecular and pathway events and several novel AR-associated events in the prostate cancer proteomes to study further. We found significant changes in expression of RNA-binding proteins during disease formation and progression. Examining the relationship of RNA binding proteins at the RNA and protein expression level reveal that while many RNA binding proteins exhibit correlation between the expression levels, some seem regulated at the posttranslational level. Two RNA binding proteins, TDP-43 and FUS, which regulated at the protein, but not at RNA level during prostate cancer progression, show opposite behavior during disease progression and correlation with AR status of the tumors. In cultured prostate cancer cell models, we show that these proteins have specific, but divergent interactions with AR at the RNA and protein levels, and that they contribute differentially to AR activity-mediated responses. Thus, these proteins may significantly contribute to prostate cancer molecular evolution and may pinpoint possible targetable pathways in future prostate cancer therapy.

#4394

**Molecular interplay between Tank-binding kinase (TBK1) and Yes-associated protein (YAP1) in** KRAS **mutant NSCLC.**

Biswarup Saha, Neha Jaiswal, Namrata Bora-Singhal, Srikumar Chellappan. _H Lee Moffitt Cancer Center and Research Institute, Tampa, FL_.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality in the United States. Lung adenocarcinomas are highly correlated with smoking and are characterized by mutations in KRAS, EGFR, BRAF and other oncogenes. KRAS mutations are widespread in adenocarcinomas among smokers and known to be a key player in various downstream signaling pathways contributing to the tumorigenesis. Recently, a non-canonical IκB kinase, Tank Binding Kinase 1 (TBK1), has been found to contribute in KRAS mutant cancers. TBK1 has well documented functions in immune response, cell survival and in mitosis. While it has been suggested that TBK1-mediated regulation of Akt signaling might facilitate oncogenesis, the molecular mechanisms underlying TBK1 function downstream of KRAS is not fully elucidated.

Yes associated protein 1 (YAP1) is an oncogenic component of the Hippo signaling cascade which could promote KRAS mediated oncogenesis and could substitute for the loss of Kras in mouse models of pancreatic cancer. In our present study, we demonstrate a unique and novel interplay between TBK1 and the oncogenic Hippo effector molecule, YAP1. YAP1 and its paralog, TAZ are known transcriptional co-activators that function to maintain organ size during development and is often activated in cancers.

We find that TBK1 could physically interact with YAP1 and phosphorylate it at T110, T114, S128 and S131 residues in vitro. Knocking down (KD) or knock out (KO) of TBK1 resulted in a significant elevation of YAP1 expression at the protein level; surprisingly, without any effect at the mRNA level. Interestingly, the upregulation of YAP1 upon depletion of TBK1 was restricted to KRAS mutant NSCLC cell-lines and not in EGFR mutant cell lines. This elevation of YAP1 upon TBK1 KD was mainly observed in the nucleus; notably, there were only minimal changes in the levels of MST and LATS, raising the possibility that these changes occur independent of the classic hippo signaling pathway. Treatment with cycloheximide, an inhibitor of protein-translation, could not diminish the elevated level of YAP1 protein in the TBK1 depleted cells, indicating that the increased YAP1 level is due to enhanced protein stability, probably as a result of post-translational modification(s). Depletion of TBK1 also resulted in the induction of EMT-like features, promoting cell-migration in scratch assays and elevated the proportion of stem-like side-population cells, probably in a YAP1-dependent manner. An unbiased RNA-Seq analysis in A549 and H460 cells indicated that MAP kinase (MAPK) pathway is activated upon TBK1 KD, which might also be involved in the YAP1 protein regulation. Our recent experiments further support this argument. The in-depth molecular mechanism(s) by which TBK1 regulates YAP1 in KRAS mutant cells are under investigation, and we hypothesize that this regulatory event contributes to KRAS mediated oncogenesis in NSCLC.

#4395

The cMYC-associated transcription factor JPO2 is upregulated in taxane resistant prostate cancer cells and interacts with the stress oncoprotein LEDGF/p75.

Greisha L. Ortiz Hernandez,1 Shannalee Martinez,1 Evelyn Sanchez-Hernandez,1 Leslimar Rios-Colon,2 Christina Cajigas-DuRoss,3 Tino Sanchez,1 Nouri Neamati,4 Carlos A. Casiano1. 1 _Loma Linda University, Loma Linda, CA;_ 2 _North Carolina Central University, Durham, NC;_ 3 _Cleveland Clinic, Cleveland, OH;_ 4 _University of Michigan, Ann Arbor, MI_.

Prostate Cancer (PCa) progression leads to an advanced stage called metastatic castration PCa (mCRPC), for which currently there is no cure in spite of advances in treatment with new generation androgen deprivation drugs and chemotherapy with taxanes such as docetaxel (DTX). Our group demonstrated previously that the stress oncoprotein Lens Epithelium Derived Growth Factor of 75 kD (LEDGF/p75) is upregulated in clinical prostate tumors and contributes to DTX-resistance in mCRPC cells. However, little is known about the molecular mechanisms by which LEDGF/p75 promotes taxane resistance. The C-terminus of LEDGF/p75 contains a domain called the Integrase Binding Domain (IBD), which in T cells is responsible for tethering the HIV-integrase complex to transcriptionally active chromatin. In cancer cells, the LEDGF/p75 IBD interacts with oncogenic transcription complexes, such as Menin-MLL and the cMYC binding protein JPO2, to promote cell survival. However, the relevance of these protein-protein interactions (PPIs) in PCa and chemoresistance has never been explored. This study is designed to characterize the interaction between LEDGF/p75 and JPO2 in the DTX-resistant mCRPC cell lines PC3-DR and DU145-DR, and determine if this interaction contributes to drug resistance. Also, we want to target this interaction with repurposed HIV-based small molecule inhibitors (SMI's) of LEDGF/p75, which target the IBD, to abrogate this resistance. We demonstrated by immunoblotting a significant 2-fold co-upregulation of JPO2 and LEDGF/p75 in the DTX-resistant PCa cells. We also observed the nuclear co-localization of these proteins by immunofluorescence microscopy. Using an immunoprecipitation approach, we confirmed that endogenous JPO2 and LEDGF/p75 co-immunoprecipitate in the DTX-resistant PCa cells. In addition, we initiated studies to evaluate SMIs originally designed to target the interaction between LEDGF/p75 and HIV-IN, for their efficacy in sensitizing resistant cells to DTX. After screening over 100 candidate inhibitors, we selected a set of SMIs which demonstrated a 20-30% decrease in viability in DTX-resistant cells when used alone, and 40-60% when used in combination with DTX. Studies are in progress to determine if these SMIs bind to LEDGF/p75 and inhibit its interaction with JPO2 and other oncoproteins. We predict that targeting LEDGF/p75 PPIs with HVI-based SMIs will disrupt transcriptional activity contributing to DTX resistance. Our goals are to establish the contribution of PPIs to LEDGF/p75-mediated transactivation of stress oncoproteins, and target these interactions to overcome PCa chemoresistance.

#4396

Gli, not Androgen Receptor, is the primary driver of prostate cell growth.

Jane Foo, Na Li, Shabnam Massah, Mannan Nouri, Mike Wang, Ralph Buttyan. _Vancouver Prostate Centre, Vancouver, British Columbia, Canada_.

Introduction Hedgehog (Hh) signaling regulates the activity of Gli transcription factors. Gli controls genes needed for development, cell growth and cell motility. Canonical Hh signaling through Smoothened [Smo] suppresses the proteolysis of Gli2 and Gli3, maintaining them in high molecular weight active forms. Previously, we showed that active androgen receptors ([AR] liganded AR full length and AR-V7) recognize and binds Gli3 its Protein Processing Domain. This binding overrides the need for Smo action and provides a means for stabilization and activation of Gli3 in prostate cancer (PCa) cells without Hh. Studies on the effects of AR binding to Gli2 are more challenging because it is expressed at much lower levels than Gli3. Here, we report that AR also affects Gli2 protein stability and show that Gli2, like Gli3, is a driver of PCa cell growth that is overexpressed in castration resistant disease. Method Immunohistochemistry (IHC) using Gli2- and Gli3-specific antibodies was used to assess expression of Gli2 and Gli3 in human PCa tumor microarrays. Western blot were used to identify and quantify levels of active Gli2 and Gli3 in androgen-treated PCa cells (LNCaP, LNCaP-AI, LAPC4). Proximity ligation assays (PLA) was used to visualize in situ and quantify AR-Gli2 complexes in PCa cells. Gli2- and Gli3-specific siRNAs were used to knock down Gli expression in AR+ (LNCaP-AI, LAPC4, 22Rv1) and AR- (PC3) PCa, cells followed by the Gli reporter assay and the cell growth assay. Results IHC outcomes showed that nuclear Gli2 and Gli3 protein expression was significantly higher in castration resistant tumors compared to primary disease. Androgens increased expression of active Gli2 and Gli3 protein expression in PCa cells and stimulated Gli reporter activity. PLA showed the presence of intranuclear complexes of Gli2/Gli3-AR-Full-length in androgen-treated LNCaP and Gli2-AR-V7 complexes in CWR22rv1 cells. Knockdown of Gli2 or Gli3 present significant reduction in Gli transcriptional activity as well as inhibition on growth of both AR+ and AR- PCa cells that were tested. Conclusion AR activity affects Gli2 processing, as it does for Gli3. We can visualize and quantify AR-Gli2/Gli3 complexes in situ in PCa cells. Gli2 suppression, as well as Gli3 suppression, has a profound effect on PCa cell growth but combined suppression has the strongest effects in certain PCa cells. Finally, Gli2 and Gli3 protein expression is elevated in castration resistant disease.

#4397

Gli Activation by Steroid Receptors in Prostate and Breast Cancer Cells.

Shabnam Massah, Na Li, Sarah Truong, Jane Foo, Gail Prins, Ralph Buttyan. _Vancouver Prostate Centre, Vancouver, British Columbia, Canada_.

Background: The nuclear steroid receptor superfamily encompasses a group of proteins best known for their functions as primary transcription factors that are conditionally active when bound to a ligand. Here, we show that prominent members of this family (androgen receptor [AR], estrogen receptor [ER-α] and glucocorticoid receptor [GR]) have a secondary function of coordinately activating the Gli family of transcription factors. Previously, we found that liganded AR recognizes and binds to Gli2/Gli3 proteins at their Protein Processing Domains. This binding prevents their degradation and stabilizes them in their high molecular weight, transcriptionally-active forms. This interaction bypasses the need for signaling through Hedgehog/Smoothened to stabilize Gli. To determine whether other steroid receptors have this activity, we tested the ability of human ER-α and GR to bind to Gli3 and whether they increased Gli activity in exogenous (293FT) and endogenous breast and prostate cancer cell systems. Methods: Human AR, ER-α or GR expression vectors were co-transfected into 293FT cells along with a myc-tagged Gli3. Protein extracts were tested for co-immunoprecipitation of AR-/ER-/GR-Gli3 complexes. 293FT cells were co-transfected with AR, ER, or GR along with a Gli-reporter vector. Luciferase activity was measured in transfected cells upon treatment with vehicle, R1881 (AR ligand), estradiol (E2-ER ligand); or dexamethasone (dex-GR ligand). LNCaP (express AR), MCF7 (express ER) or LNCaP-AI cells (express GR) were transfected with reporter in the presence or absence of R1881, E2, dex or vehicle and luciferase activity was measured. AR-/ER-Gli3 complexes were detected by in situ proximity ligation assays in prostate cancer or breast cancer cells. The effect of AR or ER-α siRNA knockdown in LNCaP or MCF7 cells on Gli3 protein stability was measured on western blot. Results: Pulldowns of AR, ER or GR co-immunoprecipitates with Gli3. Transfection with AR or ER increased Gli reporter activity in the presence of vehicle but was further increased by R1881 or E2 treatment. Gli reporter activity was unchanged by transfection with GR in the presence of vehicle but the presence of 5 or 10nM of dex tripled this activity. Gli-luciferase activity was significantly increased in R1881-treated LNCaP cells, E2-treated MCF7 cells and in dex-treated LNCaP-AI cells. PLA detected the presence of AR-Gli3 and ER-Gli3 complexes in nuclei of LNCaP and MCF7 cells. AR and ER-α knockdown destabilized Gli3 protein in LNCaP and MCF7 cells. Conclusion: Collectively our results established a secondary function (Gli activation) shared by an important evolutionary spectrum of human steroid receptors (AR, ER, and GR). As Gli is oncogenic and regulates the expression of many growth-related genes, our observations may explain the pro-oncogenic effects of steroid receptors in steroid-dependent tumour systems.

#4398

KDM3A and DCLK1 interactions promote stemness and tumorigenesis in PDAC.

Chandrayee Ghosh, Kankaraj Palaniyandi, Santanu Paul, Prasad Dandawate, Sonia Rawal, Dharmalingam Subramaniam, Subhash Padhye, Sumedha Gunewardena, Sufi Thomas, Maura O'Neil, Roy Jensen, Danny Welch, Sally Milisky, Scott Weir, Tomoo Iwakuma, Shrikant Anant, Animesh Dhar. _Univ. of Kansas Medical Ctr., Kansas City, KS_.

Pancreatic ductal adenocarcinoma (PDAC) is the major leading cause of cancer related human death in USA. There is no possible treatment or target available in PDAC. It was proposed that the cancer stem cells (CSCs) can regulates malignancy in PDAC. DCLK1 is one of the quiescent cancer stem cell marker in PDAC that can regulate tumor progression. Identification of the key factors that influence stemness will help to target PDAC. Histone lysine demethylase KDM3A is an enzyme/protein which can regulate stem cell renewability and tumor progression, thereby KDM3A can interact with DCLK1 for tumor progression. KDM3A influences tumor growth and regulates stemness. Therefore, our goal is to find out the the role of KDM3A in tumor progression in PDAC through interactions with DCLK1. We observed expression of KDM3A in both PDAC patients' samples and cells. Knockdown and overexpression of KDM3A were executed by using lentiviral vector. Tumor progression were also observed in orthotopic mice model. ChIP and RNA seq were performed to validate the data. KDM3A was overexpressed in human PDAC patient tissues and human pancreatic cancer cells with concomitant increase of CSC marker, DCLK1. Moreover, DCLK1 and KDM3A was found to be co-localized in patient tissue samples and identified binding sites of KDM3A with DCLK1 using ChIP-seq. Knockdown of KDM3A abrogates oncogenic potential whereas, overexpressed KDM3A in transformed HPNE cells showed malignant properties with enhanced invasive property, pancosphere formation, foci formation and tumor formation in mouse. Moreover, ChIP-seq and RNA seq suggested that KDM3A regulated DCLK1 expression in tumor progression. In conclusion, co-expression of DCLK1 with KDM3A influenced stemness and enhance tumor progression in PDAC.

#4399

Exploring the postmitotic roles of STAG2 mutations in solid tumors.

Mary Guan,1 Mengyao Tan,2 Abhijit Parolia,1 Steve Kregel,1 Marcin Cieslik,1 Arul Chinnaiyan1. 1 _University of Michigan, Ann Arbor, MI;_ 2 _Predicine, Hayward, CA_.

Mutations in the cohesin complex are associated with cancer, in particular Ewing's Sarcoma (EWS) and bladder cancer. The STAG2 subunit of cohesin is essential for its loading onto chromatin and proper function of the complex. In EWS, STAG2 is mutated at a rate of 22% in the form of inactivating loss of function mutations. How STAG2 functions as a tumor suppressor is not fully understood, and few whole-genome experiments have been performed examining the role of STAG2 in EWS. We hypothesize that in EWS, where the EWSR1-FLI1 fusion is pathognomonic, the genetic epistasis of EWSR1-FLI1 and STAG2 is due to the suppressive role of STAG2 on EWSR1-FLI1.

In this study, we used co-immunoprecipitation (co-IP) to examine a ternary interaction between STAG2 and EWSR1-FLI1, as well as STAG2 and EZH2. We then induced KD of STAG2 in EWS cells after 72h of siRNA treatment or 5 days of dox induction with shRNA to examine the phenotypic and transcriptional effects of STAG2 loss. As the H3K27me3 signature was enriched upon STAG2 KD, we performed IC50 drug screens with EZH2 treatment on STAG2 WT vs KD EWS cells. Finally, a STAG2 ChIP-seq was completed in EWS cells.

No physical interaction was detected between STAG2 and EWSR1-FLI1 in A673 EWS cells via endogenous co-IP. In A673 EWS cells, no significant difference in proliferation was observed after 5 days of shNT vs shSTAG2 induction. Turning to the transcriptome, differentially expressed genes after 72h of siSTAG2 KD in three EWS cell lines were identified using RNA-seq and verified using qPCR. No change in expression of EWSR1, FLI1, or EWSR1-FLI1 was seen after siSTAG2 treatment compared to siNT treatment. STAG2 is also found at the borders of topologically associated domains (TADs), and groups have shown that loss of STAG2 increases intra-TAD interactions. One particular GSEA signature common to all three cell lines was enrichment in genes with H3K27me3. This led us to hypothesize that in EWS, loss of cohesin-STAG2 could lead to new intra-TAD interactions via H3K27 methylation by EZH2, destabilizing enhancer-promoter interactions and maintaining the stemness of EWS cells to drive oncogenesis. Western blot of H3K27me3 and EZH2 levels after 5 and 7 days of sh-STAG2 KD did not show differences in total levels of H3K27me3 or EZH2. Treatment with Tazemetostat, an EZH2-inhibitor, resulted in no significant change in IC50 in shNT compared to shSTAG2 A673 cells. ChIP-seq and ChIP-qPCR in EWS cells demonstrated enrichment in known STAG2 targets in mouse, Myc and Sox2.

In summary, our results suggest STAG2 does not physically interact with EWSR1-FLI1 to mediate its expression or expression of its target genes. Instead, STAG2 may act upon chromatin to significantly rewire the cancer transcriptome in EWS in an EZH2 independent manner, thus displaying differential dependency on certain transcriptional regulators. This could shed new light on medical therapy for EWS – which currently does not exist – through targeting of STAG2-activated pathways.

#4400

Epigenetic regulation of CDC16 expression by the ikaros and casein kinase II (CK2) in T-cell acute lymphoblastic leukemia (T-ALL).

Pavan Kumar Dhanyam Raju,1 Shriya Kane,2 Chandrika Gowda,1 Chunhua Song,1 Yali Ding,1 Jonathan Payne,3 Soumya Iyer,1 Nathalia Moreno Curry,1 Mary McGrath,1 Yevgeniya Bamme,1 Bihua Tan,1 Mario Soliman,4 Dhimant Desai,1 Arati Sharma,1 Kimberly J. Payne,3 Sinisa Dovat1. 1 _Pennsylvania State University College of Medicine, Hershey, PA;_ 2 _Georgetown University Medical Centre, WA;_ 3 _Loma Linda University, Loma Linda, CA;_ 4 _University of New Mexico School of Medicine, NM_.

CDC16 is one of several subunits of the anaphase-promoting complex (APC) with crucial role in cell division. Here, we show evidence that expression of CDC16 in T-ALL is modulated at the transcriptional level by oncogenic Casein Kinase II (CK2) via direct phosphorylation of Ikaros, a tumor suppressor protein and transcription factor. Global chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) studies in both cell lines and primary human ALL, shows that Ikaros binds to the promoter of the CDC16 gene. Ikaros functions as a tumor suppressor protein and its deletion is associated with development of T-ALL. Ikaros binding to CDC16 promoter was confirmed via quantitative chromatin immunoprecipitation (qChIP) in primary T-ALL cells. The role of Ikaros in regulating CDC16 transcription in T-ALL was tested using gain-of-function and loss-of-function experiments. Ikaros knock-down with shRNA resulted in increased transcription of CDC16 in T-ALL. Overexpression of Ikaros in human T-ALL was associated with strong reduction in transcription of CDC16. In mice, T-ALL cells that are derived from Ikaros-knockout mice express high levels of CDC16. Transduction of these cells with Ikaros-containing retrovirus results in sharp reduction of CDC16 expression. Since Ikaros function in T-ALL is negatively regulated by pro-oncogenic Casein Kinase II (CK2), we tested whether CK2 can regulate expression of CDC16 in T-ALL. Overexpression of CK2 via retroviral transduction resulted in increased CDC16 gene transcription as measured by qRT-PCR, as well as increased overall expression of CDC16, as analyzed by Western blot. Increased expression of CK2 was associated with loss of Ikaros binding to the CDC16 gene promoter. Molecular inhibition of CK2 using shRNA, as well as pharmacological inhibition with a specific CK2 inhibitor led to reduced expression of CDC16 in primary human T-ALL. Inhibition of CK2 was associated with increased Ikaros binding at the promoter of CDC16. Ikaros knock-down restored high expression of CDC16 in T-ALL cells that were treated with CK2 inhibitors. These data demonstrate that CK2 and Ikaros are major transcriptional regulators of CDC16 transcription in T-ALL and that inhibition of CK2 represses CDC16 transcription via Ikaros-mediated repression. In summary, these results provide evidence that expression of the CDC16 gene in T-ALL is regulated by the CK2 signaling pathway which modulates Ikaros funtion. Targeting CK2 with specific inhibitors has been used as a therapeutic strategy in preclinical models of T-ALL. Presented data reveals a novel mechanism of therapeutic action of CK2 inhibitors – repression of CDC16 expression via Ikaros. These results provide a rationale for the use of novel CK2 inhibitors in T-ALL to target CDC16.

#4401

Implications of STAG2 loss in Ewing sarcoma.

Aparna Gorthi. _University of Texas Health San Antonio, San Antonio, TX_.

Ewing sarcoma is an aggressive pediatric bone and soft tissue cancer that is driven by a translocation event, predominantly involving the RNA binding protein EWSR1 and the transcription factor FLI1. The resultant fusion transcription factor EWSR1-FLI1 is a potent oncogene and drives dramatic changes in the transcriptional landscape of Ewing sarcoma. Our previous work demonstrated that EWS-FLI1 drives aberrant accumulation of R-loops, increases replication stress and suppresses homologous recombinational repair in Ewing sarcoma. Similar phenotypes were also observed with the loss of wildtype EWSR1. However, surprisingly, Ewing tumors have highly stable genomes. The most common mutation is in the STAG2 gene, a component of the cohesin ring complex.

It was recently reported that STAG2-cohesin complexes not only participate in maintaining chromatin architecture but also play a key role in directing tissue-specific gene expression by interacting with transcription factors. In the present study, we evaluate the STAG2 function in R-loop metabolism, replication and repair and attempt to identify the reason for its selective loss in Ewing sarcoma. Using biochemical and biophysical experimental strategies, we observed that STAG2 and EWSR1 both bind R-loop structures with high affinity and are protective against RNaseH-mediated degradation of R-loops to significantly different degrees. We also examine the effect of STAG2 loss on R-loop accumulation and replication stress in Ewing and non-Ewing cell lines. Finally, we evaluate the correlation between STAG2 binding sites and R-loops. We propose that loss of STAG2 alleviates some of the genomic stresses experienced in Ewing sarcoma.

#4402

FGFR1 signaling modulates estrogen-independent ER transcriptional activity in ER+/FGFR1-amplified breast cancer cells.

Alberto Servetto,1 Luigi Formisano,2 Rahul Kollipara,1 Dhivya R. Sudhan,1 Kyung-min Lee,1 Sumanta Chatterjee,1 Ariella B. Hanker,1 Saurabh Mendiratta,1 Ralf Kittler,1 Carlos L. Arteaga1. 1 _UT Southwestern Medical Center, Dallas, TX;_ 2 _University of Naples Federico II, Naples, Italy_.

Background: FGFR1 amplification occurs in about 15% of estrogen receptor-positive (ER+) breast cancers and is associated with resistance to endocrine therapy. In these tumors, nuclear FGFR1 has been shown to interact with ERα. In addition, FGFR1 has been demonstrated to alter gene expression through binding to chromatin. However, the mechanisms underpinning nuclear FGFR1-mediated gene transcription remain unclear. Thus, we sought to elucidate the genomic and non-genomic role of FGFR1 in ER+/FGFR1-amplified breast cancer.

Methods: FGFR1 ChIP-Seq and ERα ChIP-Seq were performed on CAMA1 ER+/FGFR1-amplified breast cancer cells. ChIP-qPCR was employed to quantify DNA binding events. For ChIP-Seq, immunoblot, RT-qPCR, Estrogen Response Element (ERE) luciferase reporter and growth assays, CAMA1 cells were plated in estrogen-free media for 24 hours and then stimulated with 100 ng/ml FGF3 or 1 nM β-Estradiol.

Results: FGFR1 ChIP-Seq detected 2211 DNA binding sites in CAMA1 cells cultured in estrogen-free conditions. Gene Set Enrichment Analysis (GSEA) revealed that the TNFα signaling via NF-KB, MYC targets, G2M checkpoints, ERE early and ERE late response genes (all FDR <0.00001) were among the most enriched gene sets. The majority of binding sites occurred in promoter regions, supporting a role of FGFR1 in regulation of gene transcription. FGFR1 ChIP-qPCR confirmed FGFR1 binding to promoter regions of oncogenes including CCND1, MYC, VEGFA, JUNB and SMAD5. FGF3 stimulation of CAMA1 cells further enriched FGFR1 binding to the CCND1 promoter and upregulation of CCND1 mRNA and protein levels. These effects were ablated upon addition of the pan-FGFR tyrosine kinase inhibitor rogaratinib. Motif analysis revealed CTCF (CCCTC binding factor) as the most enriched motif (p=1e-91). siRNA-mediated knockdown of CTCF inhibited FGF3-induced ERα transcriptional activity and proliferation of CAMA1 cells. These results suggest a role for CTCF in mediating the transcriptional programs regulated by FGFR1. We reported an association of nuclear FGFR1 and ERα in ER+/FGFR1-amplified breast cancer cells. Thus, we next investigated the role of FGF3-induced FGFR signaling on estrogen-independent ERα transcription using ERα ChIP-Seq. FGF3 stimulation of CAMA1 cells resulted in 407 DNA binding sites of which 155 were unique compared to cells in the absence of ligand. GSEA of these 155 peaks revealed enrichment for ERE early (p=3.08e-17) and ERE late (p=2.6e-5) response genes. FGF3-mediated induction of ERα transcriptional program was confirmed by ERE reporter assay and was abrogated by treatment with rogaratinib.

Conclusions: These findings suggest a FGFR1 kinase-dependent role on ER-mediated transcription in ER+/FGFR1-amplified breast cancer cells. We are currently performing mass spectrometry analysis to identify binding partners of nuclear FGFR1 that mediate its transcriptional function.

#4403

Regulation of SATB2 via miR-31 in Ni-induced malignant cell transformation.

Qiao Yi Chen,1 Yusha Zhu,1 Ashley Jordan,1 Jinquan Li,2 Hong Sun,1 Thomas Kluz,1 Max Costa1. 1 _New York University School of Medicine, New York, NY;_ 2 _Wuhan University of Science and Technology, Wuhan, China_.

The special AT-rich DNA binding protein (SATB2) is a nuclear matrix associated protein and an important transcription factor for biological development, gene regulation, and chromatin remodeling. Under normal circumstances, SATB2 is not expressed in most cells, including epithelial cells of the lung. However, aberrant regulation of SATB2 has been found in various types of cancers including lung, colon, prostate, breast, gastric, and liver. Emerging studies have linked the possible role of SATB2 in carcinogenic metal-induced cell transformation. In fact, our previous microarray analysis showed that SATB2 is strongly induced in Ni-transformed cells. As an IARC Class I Human Carcinogen, there is well-established link between chronic nickel exposure and lung cancer; however, despite of considerable research efforts in regards to the epigenetic mechanisms, little is known about the role of microRNAs in Ni-induced carcinogenesis. As post-transcriptional regulators, miRNAs have great importance in maintaining normal cellular development. In particular, miR-31 is one of the most abundant types of miRNAs and commonly studied in disease development, including cancers of the lung, breast, and liver. Previous studies have shown the miR-31 can directly target the homeobox gene SATB, and here we report that miR-31 is capable of regulating SATB2 in Ni-induced tumorigenesis. First, our results confirmed that knocking down SATB2 in Ni-transformed BEAS-2B cells via shRNA significantly reduced the rate of migration, anchorage-independent growth. Notably, compared to scramble control shRNA transfected cells, SATB2 knockdown cells did not generate tumor growth in mice xenograft model. These results highlight the oncogenic role of SATB2 in cell transformation. In addition, our data indicate that both acute Ni-treated and Ni-transformed cells demonstrated significant reduction in miR-31 expression. Furthermore, we show that overexpressing miR-31 in Ni-transformed cells reduced rates of migration, anchorage-independent growth, and cellular invasion, indicating the importance of miR-31 in Ni-induced BEAS-2B cell transformation. Together, our results provide a novel mechanistic pathway for Ni-induced carcinogenesis, and add to the pool of existing research on the implications of SATB2 and miR-31 in tumorigenesis.

#4404

**E-** p **-Methoxycinnamoyl-α-l-rhamnopyranosyl ester (MCR) increases Nrf2 stability by inhibiting ubiquitination in HaCaT cells.**

Jiwon Jeong,1 Lilik D. Wahyudy,1 Heejung Yang,2 Jung-Hwan Kim1. 1 _Gyeongsang National University, Jinju, Republic of Korea;_ 2 _Kangwon National University, Republic of Korea_.

The nuclear factor erythroid-derived 2-related factor 2 (Nrf2) is a key regulator of gene expression during oxidative stress and drug detoxification. Thus, identifying Nrf2 activators to protect from possible cell damage is necessary. In this study, we investigated whether E-p-methoxycinnamoyl-α-l-rhamnopyranosyl ester (MCR), a phenylpropanoid isolated from Scrophularia buergeriana, can activate Nrf2 signaling in human keratinocytes (HaCaT). First, we determined the dose- and time-dependent effects of MCR on the expression and activity of Nrf2. The antioxidant response element-luciferase reporter assay and western blot analysis results showed that MCR markedly induced Nrf2 activity and its protein expression, respectively. Further, MCR increased both the mRNA and protein levels of heme-oxygenase-1, one of the Nrf2 target genes, in the cells. Interestingly, we found that Nrf2 stability was remarkably enhanced by MCR. Furthermore, ubiquitin-dependent proteasomal degradation of Nrf2 was significantly reduced by MCR. Thus, MCR might afford skin protection by enhancing Nrf2 stability or by blocking its proteasomal degradation.

#4405

Acetylation of KLF5 controls the cell fate of basal progenitors during postnatal development and regeneration of mouse prostates.

Baotong Zhang, Jin-Tang Dong. _Emory University, Atlanta, GA_.

Adult stem cells are responsible for lineage specification in epithelial homeostasis, and thus are essential for both postnatal development and tissue regeneration. Genetic changes in adult stem cells have been revealed to promote cancer development. Dissecting the underlying regulators is critical for understanding the intrinsic mechanisms that regulate adult stem cell function, yet whether PTM of a single molecule determines cell fate of adult stem cells during organ development has not been well established, particularly for glandular organs. Using the androgen-dependent mouse prostate as an experimental system, an ideal system for understanding epithelial hierarchy, we examined the Krüppel-like factor 5 (Klf5) transcription factor and its acetylation for their roles in postnatal development and regeneration. The absence of KLF5 eliminated basal progenitor activities in both human and mouse prostate epithelial cells, as indicated by a compromised sphere and organoid formatting capabilities, downregulation of basal cell markers, and decreased basal cells. Klf5 was also necessary for basal progenitor cells to maintain their luminal differentiation capacity, as loss of Klf5 also decreased basal progenitor-derived luminal cells and their proliferation in mouse prostates. Interestingly, the progenitor activity of Klf5 was restricted to deacetylated Klf5 (deAc-Klf5), as indicated by in vitro and in vivo assays. On the other hand, acetylated Klf5 (Ac-Klf5) was essential for proper formation and maintenance of luminal structures in mouse prostates, as deacetylation of Klf5 interrupted the formation of normal luminal structures, almost eliminated the castration resistance in basal progenitor-derived luminal cells, and attenuated the luminal regeneration after a castration-regeneration cycle, even though deAc-Klf5 promoted basal-to-luminal differentiation and the proliferation of luminal cells during postnatal development. Mechanistically, deacetylation of Klf5 increased Notch signaling activity. These findings indicate that Klf5 and its acetylation control the cell fate of prostatic basal progenitors, which have a distinct contribution to postnatal development and regeneration of the prostate. Considering that androgen deprivation therapy (ADT) is currently a major therapeutic strategy in the treatment of prostate cancer, our findings also provide a potential mechanism for prostate cancer progression, i.e., interruption of Klf5 acetylation could sensitize castration-resistant prostate cancer to ADT or slow the recurrence of prostate cancer after ADT.

#4406

TRIB3 stabilizes high TWIST1 expression to promote rapid APL progression & ATRA resistance.

Wu Zhang, Jian Lin, Jie Xu. _Shanghai Institute of Hematology, Shanghai, China_.

Acute promyelocytic leukemia (APL), which accounts for 10-15% of acute myeloid leukemia (AML) cases, is characterized by the t (15; 17) chromosomal translocation and is now highly curable by the combination of granulocytic differentiation induction and the PML-RARα oncoprotein-targeted agents all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). Despite the striking molecular complete remission (CR) and the very few cases of relapse associated with ATRA/ATO-based regimens, mortality events typically result from early fatal bleeding, which remains the most important challenge and the largest obstacle to curing all APL patients. For instance, several studies have reported that the risk of early hemorrhagic death (HD) reaches an incidence of 10-20% during the first month of induction. Importantly, APL patients with a high white blood cell count face an increased risk of early HD. Furthermore, differentiation syndrome and resistance to ATRA/ATO treatment with PML-RARα mutations still endanger the health of a significant proportion of APL patients. Thus, we need to better understand the molecular mechanism of APL pathogenesis and design more effective therapeutic strategies to block the rapid progression of early HD and overcome resistance. Oncogenic transcription factors play an important role in the development of hematological malignancy. The dysregulation of epithelial-mesenchymal transition-inducing transcription factors (EMT-TFs), including SNAI1/SNAI2, ZEB1/ZEB2 and TWIST1/TWIST2, which are closely related to malignant progression in solid tumors, has also been explored in the context of the aggressive invasion, chemoresistance and poor prognosis of AML. In this study, we found through two database (TCGA and E-MTAB-344) gene expression searches and detailed functional verification that the EMT-TF TWIST1 is highly expressed in APL cells and is critical for leukemic cell survival. TWIST1 and TRIB3 were highly coexpressed in APL cells compared with other subtypes of AML. We subsequently demonstrated that TRIB3 could bind the bHLH and WR domains of TWIST1 and stabilize TWIST1 by inhibiting the ubiquitination of the TWIST1 protein. Based on a detailed analysis of a functional screen of synthetic peptides, we discovered a peptide analogous to the previously reported TWIST1 WR domain, which rapidly and specifically represses APL cell survival through disrupting the TRIB3/TWIST1 interaction. Our data also suggest that a peptide similar to the WR domain disturbs the TRIB3/TWIST1 interaction, impairs rapid progression during the early death of APL and reverses resistance to ATRA therapy. These results reveal the important role of a specific oncogenic transcriptional factor, TWIST1, in APL leukemogenesis and suggest a potential peptide-initiated rapid proteolysis therapeutic opportunity to protect against early death and induction therapy resistance in patients with APL.

### Targeting the Cell Cycle: Development of Preclinical Models and Therapeutic Targets

#4407

Next generation CDK2/9 inhibitor CYC065 triggers anaphase catastrophe in diverse aneuploid cancers and markedly inhibits growth and metastasis.

Masanori Kawakami, Lisa Maria Mustachio, Yulong Chen, Zibo Chen, Jason Roszik, Xi Liu, Ethan Dmitrovsky. _MD Anderson Cancer Center, Houston, TX_.

Genetically-unstable cancer cells have supernumerary centrosomes and aneuploidy (a hallmark of cancer). CDK2 antagonism inhibits clustering of excessive centrosomes at mitosis leading to multipolar cell division and apoptotic death. This process is called anaphase catastrophe. Anaphase catastrophe affects preferentially aneuploid cancer cells while sparing normal cells with two centrosomes. CYC065 (Cyclacel) is a next-generation CDK2/9 inhibitor that is undergoing clinical trials. Here, we explored CYC065 activity against aneuploid lung and other cancers. CYC065 substantially inhibited growth, triggered apoptosis, and induced anaphase catastrophe in murine (ED1, LKR13, and 393P) and human (Hop62, A549, and H1299) lung cancer cells. In marked contrast, these effects were largely unseen in bipolar immortalized pulmonary epithelial (murine C10 and human BEAS-2B) cells. Notably, anaphase catastrophe was induced by CYC065-treatment even in pancreatic and colon cancers that are often driven by the KRAS oncoprotein. This suggests that engaging anaphase catastrophe is a general antineoplastic mechanism. Comprehensive expression analysis of nearly 300 growth-regulatory proteins was performed after CYC065 treatment in lung cancer cells using Reverse Phase Protein Arrays (RPPAs). In addition to known CDK targets (such as phosphorylated retinoblastoma protein), FAK phosphorylation and SAC phosphorylation that are known to regulate metastasis were unexpectedly down-regulated. Consistent with this finding, in vitro migration and invasion CYC065 treatment assays markedly inhibited migration and invasion of lung cancer cells. CYC065 anti-neoplastic effects were interrogated in mice. CYC065 significantly inhibited tumor growth in several lung cancer syngeneic xenografts and patient-derived xenografts (PDXs) and reduced metastasis of 344SQ lung cancer cells in a syngeneic tail-vein injection model. Immunohistochemistry (IHC) studies of resected PDX tumors revealed downregulation of p-CDK2 (Thr160) and MCL1 expression after CYC065 treatment which is consistent with CDK2/9 antagonism, respectively. In summary, CYC065 engages anaphase catastrophe and elicits marked antineoplastic effects in diverse cancers. These effects are due to mechanisms that confer after CDK2/9 inhibition, anaphase catastrophe, MCL1 down-regulation and suppression of proteins that regulate metastasis. Taken together, findings reveal that anaphase catastrophe is a general antineoplastic mechanism engaged in aneuploid cancers. Intriguingly, this mechanism is activated after CDK2/9 inhibition of lung and other cancers, including those driven by the KRAS oncoprotein.

#4408

CDK4/6 inhibitor(palbocilcib) efficacy for head and neck squamous cell carcinoma(HNSCC), including both HPV+ and HPV- cell lines.

Jo-Pai Chen,1 Jui-Ying Chang,1 Reuy-Long Hong2. 1 _National Taiwan University Hospital, Yun-Lin Branch, Yun-Lin, Taiwan;_ 2 _National Taiwan University Hospital, Taipei, Taiwan_.

Background:

Betel nut chewing might contribute to (1)strong inflammation, angiogenesis, & invasion; (2)easy recurrence. Our group had found a betel-nuts exposed HNSCC cell line, TW2.6, was resistant to chemotherapies, radiation, and EGFR inhibitors. Defective p53 mutation, p16 loss, and BCL2 overexpression were seen in TW2.6. Astragalus polysaccharides, PI3Kalpha inhibitor, AKT inhibitor, FGFR inhibitor, ALK/IGF1R inhibitor, CDK4/6 inhibitor, BCl2 inhibitor, WEE1 inhibitor, ATR inhibitor, & VEGFR2/ PDGFR/FGFR or VEGFR2/c-MET/Axl triple blockage might be effective on TW2.6 and reverse treatment refractoriness, through the inhibition of mesenchymal transformation, pRB, & PI3K/AKT /mTOR signaling and the modulation of stemness & PD1/PDL1 pathway. TW2.6 might be similar to HPV- EMT subtype in TCGA. CDK4/6 inhibitor was proved to be effective in HPV-/pRB+ HNSCC and had strong immuno-modulatory effects. In our prior works, palbociclib could resensitize TW2.6 to docetaxel, afatinib, & radiation and enhance further response to BYL719 & foretinib(VEGFR2/c-MET/Axl triple inhibitor). Purpose and methods: To test palbociclib efficacy in HNSCC cell lines with different molecular phenotypes. SCC4, SCC9, SCC15, SCC25, FaDu, KB, Cal27, SAS, and TW2.6 evaluated for (1)in vitro sensitivity to palbociclib; (2)synergistic effect of palbociclib with other therapies by MTT assay, colony formation, flow cytometry, and western blotting.

Results:

Cell lines | SCC25 | KB | SAS | CAL27 | FaDu | SCC15 | SCC9 | SCC4 | TW2.6

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

Differ- entiation | Well | Poor | Poor | Poor | Poor | Well | Well | Well | Well, but rapidly replicated, with high hyper-diploidy & complex rearrangements

HPV status | HPV 16/18 | HPV18 | - | - | HPV 16/18 | - | - | HPV 6/11 | -

EGFR status | Medium | Low | High | High | Medium | High | Low | Medium to high | Unknown

Docetaxel sensitivity | +++ | +++ | ++ | ++ | + | ++ to +++ | ++ | - | +

Cisplatin sensitivity | +++ | ++ | +++ | ++ | ++ | + | \- to + | - | \- to +

5-FU sensitivity | +++ | + | ++ | +++ | ++ | - | \+ to ++ | - | \- to +

EGFR inhibitor sensitivity | +++ | \- to + | - | + | ++ | ++ to +++ | +++ | +++ | -

Polo-like kinase Inhibitor sensitivity | +++ | +++ | ++ | ++ | + | ++ to +++ | \- to + | - | \- to +

VEGFR2 Inhibitor sensitivity | - | - | - | - | ++ | +++ | - | - | ++

CDK4/6 Inhibitor response | +++ | \- to + | +++ | ++ to +++ | + | ++++ | + | ++ | ++ to +++

Western blots | Weak p-AKT & VEGF-A, mild PDL1 and BMI-1, Gli-1(+) | Weak p-AKT, mild PDL1 and strong VEGF-A & BMI-1, p16(+) | Moderate p-AKT & BMI-1, high PDL1, mild VEGF-A | High p-AKT & VEGF-A, mild PDL1 & BMI-1 | High VEGF-A, moderate p-AKT & PDL1, weak BMI-1, Gli-1(+) | Weak p-AKT & VEGF-A, mild PDL1 & BMI-1 | Weak p-AKT, VEGF-A, & BMI-1, moderate PDL1 | Moderate p-AKT & VEGF-A, strong BMI-1, mild PDL1, Gli-1(+) | High p-AKT, PDL1, & VEGF-A and moderate BMI-1

Outcomes | Best; like TCGA CL (HPV+) subtype | Like TCGA basal subtype, but responded to particular treatments each | Basal | Basal | Like TCGA mesen-chymal subtype (HPV+) | Like TCGA CL(HPV-) subtype, different characters between these 3 cell lines | CL(HPV-) subtype | CL(HPV-) subtype | Worse; like TCGA EMT subtype (HPV-)

Conclusion: Palbociclib had great efficacy over SCC15(classical HPV- type with EGFR overexpression) followed by SCC25, SAS, TW2.6, & CAL27; but little efficacy over KB. In HPV+ cell lines, palbociclib had promising response on SCC25(classical HPV+ type) and little response on FaDu(HPV+ mesenchymal type) & KB(basal type). SAS & CAL27, also basal types, responded well to palbocilclib. Further NGS analysis may translate these cell lines to represent TCGA subtypes for translational research.

#4409

Prospective identification of RB pathway alterations predict response to SY-1365, a selective CDK7 inhibitor, in a panel of high-grade serous ovarian cancer (HGSOC) patient derived xenograft (PDX) models.

Nan Ke, Liv Johannessen, Nisha Rajagopal, David Orlando, John Carulli, Graeme Hodgson. _Syros Pharmaceuticals, Cambridge, MA_.

Introduction: CDK7, a key regulator of transcription and cell cycle progression, has been implicated in the pathogenesis of ovarian cancer (OC). SY-1365, a potent and selective inhibitor of CDK7, has been shown to induce tumor growth inhibition (TGI), including complete regressions, in OC PDX models, with an association observed between responses and oncogenic alterations in the RB pathway. Here we report on the predictive value of RB pathway alterations in an independent set of OC PDX models.

Methods: Tumor growth inhibition (TGI) was evaluated in 21 independent HGSOC PDX models by comparing growth in SY-1365-treated mice (30-40 mg/kg, twice weekly, 4 weeks) versus growth in vehicle treated mice (n=3 per group). DNA and RNA were extracted from FFPE tumor tissue collected from untreated mice for all 21 PDXs. Gene mutations and copy number (CN) were evaluated by DNA sequencing (OncoCODE410, WuXi). Gene expression was assessed using the Nanostring Pan Cancer Pathway panel. RB pathway changes were defined per The Cancer Genome Atlas analysis of HGSOC - RB1 deletion or mutation, CDKN2A downregulation (DR) or deletion, CCNE1 amplification (Amp), CCND1 Amp, CCND2 upregulation (UR) - which identified these changes in 67% of HGSOC patients. Genes with homozygous deletions (HD), high-level Amp (CN≥6), or deleterious mutations were considered altered; genes with normalized expression values in the upper and lower ranges (Z score±1.5) across all models were considered altered. Models with 1 or more alterations were classified as RB-incompetent and were predicted to respond to SY-1365.

Results: Assessments of SY-1365-induced-TGI and RB pathway changes were conducted independently in a blinded manner. Of the 21 models tested, 12 (57%) showed responses (greater than 50% TGI, range 51-87%). RB pathway changes were observed in 7 (33%) of the PDX models: CCNE1 Amp (n=2); CCND2 UR (n=2); CCND2 UR and CCND1 Amp (n=1); CCND2 UR and CDKN2A DR (n=1); CDKN2A HD/DR and RB1 mutation (L335*) resulting in a premature stop codon (n=1). All RB-incompetent models (predicted responders; n=7) showed anti-tumor responses to SY-1365 (100% positive predictive value). Of the 14 predicted non-responder PDX models, 5 showed responses suggesting the presence of additional undetected RB pathway changes or alternative mechanisms conferring SY-1365 sensitivity. Overall, a balanced accuracy of 79.2% (sensitivity=58.3%, specificity=100%) was observed (p<0.01) for the RB pathway classifier.

Conclusions: RB pathway alterations are predictive of response to SY-1365 in HGSOC PDX models. The results support exploration of RB pathway changes as predictive biomarkers of SY-1365 clinical activity in patients with ovarian cancer. SY-1365 is currently being assessed in a phase 1 trial in adult patients with ovarian and breast cancers (NCT03134638).

#4410

Predictive model of palbociclib response reveals indications in which CDK4/6 inhibitor can be a potential combination partner.

Marc Hafner, Nicholas Dompe, Wei Zhou, Eva Lin, Milena Durrbaum, Anneleen Daemen, Melissa R. Junttila, Ciara Metcalfe, Karl Merrick. _Genentech, South San Francisco, CA_.

CDK4/6 inhibitors are a promising class of targeted therapies, with three molecules (palbociclib, ribociclib, and abemaciclib) approved for advanced hormone-receptor positive (HR+) breast cancers. With hope of building on this success, clinical trials across multiple oncology indications have been initiated. The determinants of response to CDK4/6 inhibitors, beyond pRb-deficiency and Cyclin-E amplification, remain unclear as limited clinical data have been published in indications other than HR+ breast cancers. Here, we measured the response to palbociclib across a panel of tumor cell lines from multiple indications and were able to build a computational model predictive of drug response using the expression of a handful of transcripts. This model revealed cellular states that are related to palbociclib resistance, such as G1-S entry deregulation, or sensitivity, such as competent p53 pathway and high KRAS signaling.

As validation of our model, we compared its predictions with dependency scores from Project Achilles and found good correlations between predicted sensitivity and dependence on CDK4 and CCND1. By applying the predictive model on TCGA data, our analysis uncovered indications, disease subtypes, and genomic alterations that are related to the predicted response to palbociclib in addition to Rb-deficiency and Cyclin-E amplification. As expected, HR+ breast cancer was predicted to be among the most sensitive diseases, along with prostate, thyroid, and some renal cancers. Among non-small cell lung cancers, squamous-cell tumors are predicted to be more resistant than adenocarcinomas. In adenocarcinomas, BRAF and KRAS mutations are predicted to be associated with increased responsiveness. Combining these results with published biomarkers of resistance to standard of care therapies reveals indications in which CDK4/6 inhibitors can be potential combination partners. These results may guide the design of new trials for CDK4/6 therapies and the evaluation of better stratification biomarkers.

#4411

Identification of novel natural compounds as potential inhibitors of cyclin dependent kinases.

Ranjini Sankaranarayanan, Rakesh Dachineni, Siddharth Kesharwani, Ramesh Kumar Dhandapani, Hemachand Tummala, Jayarama B. Gunaje. _South Dakota State University, Brookings, SD_.

Colorectal cancer (CRC) is the second highest cause of cancer deaths in the United States according to CDC (2017). Lifestyle factors including, but not limited to, elevated Body Mass Index (BMI), obesity and diet are proposed to lead to the development of CRC with an estimated annual incidence of 95,520 cases. Increasing evidences suggest that a diet rich in fruits and vegetables will reduce incidences of colorectal adenomas. We hypothesize that the natural compounds present in fruits and vegetables, along with their metabolites generated by the gut microbiota, could be responsible for cancer prevention by targeting the cell cycle proteins. In this study, we investigated the ability of polyphenols [Epigallocatechin Gallate (EGCG), Catechin, Catechin Gallate (CG), and Epigallocatechin (EGC)] and their degradation products [Phloroglucinol; 2,4,6 Trihydroxybenzoic acid (2,4,6 THBA); 3,4 Dihydroxyhydrocinnamic acid (3,4-DHHCA); etc.] to inhibit the CDKs (that regulate cell cycle) activity. Our pilot studies show that the different catechin compounds and their degradation products are capable of inhibiting different CDKs, suggesting their potential role in cancer prevention. We are currently investigating the mechanism of inhibition of CDKs by these compounds utilizing biochemical and molecular docking studies. Elucidation of this pathway will suggest the role of polyphenols in the prevention of CRC as well as assist in the identification of novel compounds that can be used for both cancer prevention and therapy.

#4412

Piperine enhances the efficacy of 5 FU against liver cancer.

David Anwanwan,1 Santosh Kumar Singh,1 James W. Lillard,1 Manoj K. Mishra,2 Rajesh Singh1. 1 _Morehouse School of Medicine, Atlanta, GA;_ 2 _Alabama State University, Montgomery, AL_.

Liver cancer is one of the most devastating malignancies. Hepatocellular carcinoma (HCC) accounts for >90% of primary liver malignancies and is the third-leading cause of cancer-related deaths worldwide. Systemic chemotherapy is one of the most important treatment modalities for advanced HCC. Unfortunately, long-term treatment acquired resistance in HCC due to the activation of drug efflux pumps. A natural compound in combination with chemotherapeutic drugs is a potential approach to combat the side effects. In this study, we have demonstrated whether piperine (natural compound) could increase the effect of 5-FU in HCC cells. The cell viability (MTT) assay was performed to determine the optimal IC50 values of Piperine, 5-FU alone and in combination. Immunofluorescence study confirmed the effectiveness of combined drug; dead nuclei were found more within the cells treated by the combination compared to piperine or 5-FU alone. The combined drug treatment was most effective in inhibiting proliferation and inducing apoptosis; accordingly, it showed noticeable increases in pro-apoptotic (BAK, BID), PARP and decreases in anti-apoptotic genes (BCL-XL) gene in a western blot and RT-PCR analysis. Further, in SK-HEP-1 cells, the combination upregulated the expression of cell cycle inhibitors (p21WAF1/CIP1, p27KIP), which, in turn, inhibited the expression of CDK2/ Cyclin D1 complex. Therefore, it blocks the transition of cells into G0/G1 to S phase. Taken together, we observed that the combination of 5-FU and Piperine resulted in a significant cytotoxicity and apoptosis as compared to alone. Our results suggest that piperine can be a potential agent in enhancing the efficacy of 5-FU in liver cancer treatment. These finding highlights the promising role of natural bioactive compounds and provides the rationale for further transitional researches.

#4413

CDK6 overexpression promotes resistance to CDK4/6 inhibitors in breast cancer.

Qing Li, Pedram Razavi, Zhiqiang Li, Arnaud F. Da Cruz Paula, Edi Brogi, Maurizio Scaltriti, Jorge S. Reis-Filho, Sarat Chandarlapaty. _Memorial Sloan-Kettering Cancer Center, New York, NY_.

CDK4/6 inhibitors (CDK4/6i) are highly active therapies in estrogen receptor-positive (ER+) breast cancer. Unfortunately, tumor relapse on these agents is frequently encountered in clinical practice with limited data on the biologic mechanisms that can mediate resistance. We utilized cell line models, patient derived xenografts and patient samples to interrogate mechanisms of resistance to CDK4/6 inhibitors. A survey of genomic alterations associated with limited benefit of CDK4/6iidentified several alterations involving the Hippo pathway which could potentially impact CDK6 kinase expression. Immunohistochemistry from these patients revealed increased YAP nuclear translocation and high levels of CDK6 protein specifically in the group of patients with short progression-free survival (PFS) on CDK4/6i. A PDX model of acquired resistance to CDK4/6i also showed that prolonged treatment with CDK4/6i led to a subset of tumors that developed resistance, all of which showed Hippo pathway suppression and CDK6 upregulation. Finally, cell lines models exposed to CDK4/6i showed multiple clones that featured upregulation of CDK6 through suppression of the Hippo pathway or amplification of the CDK6 gene. Identifying CDK6 overexpression as a recurrent feature of CDK4/6i resistant tumors, we next investigated the function(s) of CDK6 in our models. We performed knockdown of CDK6 by shRNA and found its overexpression to be necessary for drug resistance in these models while reinforced CDK6 could restore resistance or confer resistance when overexpressed in naïve cells. Given the close homology of CDK4 and CDK6, we asked whether any differences might exist between CDK4 and CDK6 complexes, performing immunoprecipitation and mass spectrometry (IP-MS) and found CDK6 to bind a distinct partition of proteins from CDK4 including INK4 proteins. In vitro kinase assays revealed that INK4 proteins impaired the ability of CDK4/6i to block CDK6 activity. INK4 proteins were found to cooperate with CDK6 in mediating drug resistance as their knockdown restored drug sensitivity to resistant cells, while INK4 overexpression promoted resistance in CDK6-high cells. Taken together, the data revealed that high levels of a CDK6-INK4 complex to be a recurrent mechanism of resistance to CDK4/6i and suggest novel CDK6 inhibitors as a strategy to overcome resistance.

#4414

Inhibition of CDK2 overcomes primary and acquired resistance to CDK4/6 inhibitors.

Claire R. Hall, John E. Bisi, Jay C. Strum. _G1 Therapeutics, Research Triangle Park, NC_.

The cyclin dependent kinase (CDK) family of proteins is associated with cell cycle progression and transcriptional regulation. Abnormalities in the cell cycle, including modifications to the function of CDKs, their regulators (cyclins), or their natural inhibitors, are frequently associated with the formation and growth of tumors. While recent advances in treatments using CDK inhibition have focused on targeting CDK4/6, with regulatory approvals of palbociclib, ribociclib, and abemaciclib, these compounds only target tumors sensitive to CDK4/6 inhibitors. We are focused on developing a novel, potent, and selective inhibitor of CDK2 to treat patients whose tumors are insensitive to CDK4/6 inhibition, either by primary resistance or acquired resistance by prior treatment with a CDK4/6 inhibitor. CDK2, a serine-threonine kinase, is a member of the CDK family that binds two regulatory cyclins, E and A. CDK2 when complexed to cyclin E is involved in the G1 to S-phase transition. However, when CDK2 is complexed with cyclin A, the cell will progress through the S to M-phase. Overexpression of cyclin E has been described in a subset of triple negative breast cancer (TNBC), tumors with acquired resistance to CDK4/6 inhibition such as ER+ Her2- breast cancer, as well as ovarian, lung, and other tumor types. We have utilized medicinal chemistry and structure activity relationship (SAR) modeling, starting from our proprietary scaffold, to generate a series of small molecule CDK2 inhibitors with drug-like properties. These compounds were initially screened for potency and selectivity using an array of biochemical and in-vitro assays. We identified multiple small molecule inhibitors with sub-nanomolar biochemical IC50s for CDK2 when complexed with either cyclin E or cyclin A and evaluated their activity in normal cell lines, a breast cancer cell line resistant to CDK4/6 inhibition, TNBC, and other tumor cell lines. We examined cell cycle analysis, EdU incorporation, cell proliferation, caspase activation, and western blot analysis of genes associated with the downstream targets of CDK2. These CDK2 inhibitors potently arrest cells in the G2/M-phase and inhibit proliferation in a manner dependent on time and dose, with a corresponding decrease in phosphorylated Rb. Our lead compound was also evaluated in mouse xenograft studies for efficacy, investigating tumor growth inhibition in a model with cyclin E overexpression and a model with an acquired resistance to CDK4/6 inhibition. These potent CDK2 inhibitors demonstrate a potentially promising method of treating tumors with primary or acquired resistance to CDK4/6 inhibitors.

#4415

CDK4/6 inhibition with lerociclib (G1T38) delays acquired resistance to targeted therapies in preclinical models of non-small cell lung cancer.

Daniel M. Freed, Claire R. Hall, Jay C. Strum, Patrick J. Roberts. _G1 Therapeutics, Research Triangle Park, NC_.

Tyrosine kinase inhibitors (TKIs) targeting oncogenes such as EGFR, ALK, or RET have dramatically improved anti-tumor efficacy in patients with non-small cell lung cancer (NSCLC). Despite the significant clinical benefit offered by these agents, tumor responses to single-agent TKIs nevertheless remain limited in their magnitude and duration. Here we investigate combination therapy approaches with lerociclib (G1T38), a selective, oral, and potential best-in-class inhibitor of cyclin-dependent kinases 4 and 6 (CDK4/6). Consistent with previous studies reporting frequent cell-cycle gene alterations in NSCLC, we show that lerociclib synergizes with TKIs targeting EGFR, ALK, and RET in lung cancer cell lines and enhances the efficacy of osimertinib and crizotinib in PDX models harboring EGFR or ALK mutations, respectively. Moreover, in a well-characterized murine xenograft model of EGFR-mutant NSCLC, seven days of treatment with a clinically-relevant dose of osimertinib (25 mg/kg p.o. QDx7) resulted in 60% complete tumor responses, versus 100% complete tumor responses in the osimertinib plus lerociclib (25 mg/kg osi + 100 mg/kg lero, p.o. QDx7) group. Strikingly, complete tumor responses in the combination group persisted through an additional 35 days (or more) of observation, by which time all tumors in the osimertinib monotherapy group had re-emerged - suggesting a role for lerociclib in extending the duration of antitumor response. Based on this result, we assessed the ability of lerociclib to delay the emergence of resistance by chronically treating NSCLC cell lines with TKI +/- lerociclib. These experiments revealed that combination treatment with lerociclib significantly delayed acquired TKI resistance in multiple NSCLC cell lines. The same result was achieved in vivo in an EGFR-mutant NSCLC xenograft model known to develop resistance to EGFR TKI therapy, wherein combination treatment with lerociclib delayed the outgrowth of osimertinib-resistant tumors. Studies to characterize the mechanism by which lerociclib delays TKI resistance in NSCLC are ongoing. Finally, we addressed whether combination therapy with lerociclib can treat TKI resistance after it develops. In EGFR-mutant NSCLC xenografts harboring MET amplification - one of the more common mechanisms of resistance observed to EGFR TKIs in the clinic - the combination of lerociclib plus osimertinib caused significant tumor growth inhibition (90%) compared to osimertinib monotherapy (34%) after 46 days of continuous daily dosing. Collectively, our results suggest a compelling rationale for utilizing lerociclib as a backbone therapy for multiple combination regimens in NSCLC. A phase 1b/2 clinical trial evaluating lerociclib plus osimertinib in EGFR-mutant NSCLC is ongoing (NCT03455829).

#4416

Plasma thymidine kinase activity in patients with luminal metastatic breast cancer treated with Palbociclib within the phase II TREnd trial.

Luca Malorni,1 Chiara Biagioni,2 Francesca De Luca,3 Martina Bonechi,3 Giuseppe Curigliano,4 Alessandro M. Minisini,5 Erica Moretti,1 Mattias Bergqvist,6 Karin Mattsson,6 Emanuela Risi,1 Ilenia Migliaccio,3 Stefania Vitale,7 Stefano Gabellini,1 Amelia McCartney,1 Irene De Santo,8 Francesca Galardi,3 Giulia Boccalini,3 Matteo Benelli,2 Lorenzo Rossi,1 Laura Biganzoli,1 Angelo Di Leo1. 1 _"Sandro Pitigliani" Medical Oncology Department, Hospital of Prato, Prato, Italy;_ 2 _Bioinformatics Unit, Hospital of Prato, Prato, Italy;_ 3 _"Sandro Pitigliani" Translational Research Unit, Hospital of Prato, Prato, Italy;_ 4 _Division of Early Drug Development, Department of Haematology and Haemato-Oncology, Istituto Europeo di Oncologia, University of Milan, Milan, Italy;_ 5 _Department of Oncology, Azienda Sanitaria Universitaria Integrata di Udine, Udine, Italy;_ 6 _Biovica International, Uppsala, Sweden;_ 7 _Department of Medical Biotechnologies, University of Siena, Siena, Italy;_ 8 _Department of Clinical Medicine and Surgery, University of Naples Federico II, Naples, Italy_.

Background: The CDK4/6 inhibitor palbociclib (P) is approved for the treatment of luminal metastatic breast cancer (MBC) in combination with endocrine therapy (ET). It leads to reduced phosphorylation of the Rb protein resulting in a decrease in E2F activity and eventual cell cycle arrest. Thymidine kinase 1 is a well-known cancer proliferation marker downstream of the E2F pathway, whose activity can be measured in plasma samples as readout of tumor proliferation. Circulating thymidine kinase activity (TKa) is a prognostic marker in MBC patients (pts) treated with ET, both when measured at baseline and during treatment. TKa has been previously shown to decrease in pts treated with neoadjuvant P+ET for 15 days, which was attributed as pharmacodynamic change on P treatment. The predictive value of TKa changes during treatment with P, as well as the dynamics of TKa changes on P-containing treatments are not yet comprehensively defined. Here we investigate the role of plasma TKa measured at different timepoints in a cohort from the TREnd study (NCT02549430).

Methods: TREnd was a randomized phase II trial that allocated 115 pts with moderately pre-treated luminal MBC to receive single-agent P or P plus the same ET agent that was received in the prior line of therapy. Plasma samples were collected at baseline (T0; n=45), at day 1 of cycle 2 (T1; n=45) and at disease progression (T2; n= 36) from 46 consenting pts. TKa was measured with DiviTum®, a refined ELISA-based assay. Patients were dichotomized as high/low at T0 based on an optimal cut-off (260 Du/L) determined by maximally selected rank statistics, and on the median value at T2 (250 Du/L). Dynamic changes between T0 and T1 were deemed meaningful if >10% of T1 or T0, whichever was greatest. Clinical outcome was estimated using the Kaplan-Meier method.

Results: Median TKa (mTKa) at T0 was 73 Du/L (range 20-4302). As expected, P-containing treatment reduced mTKa levels at T1 (37 Du/L, range 20-4504). Conversely, at disease progression, TKa increased compared to T0 (mTKa at T2, 250 Du/L, range 20-3653). Median time to progression (mTTP) in pts with low TKa at T0 (n= 33) was 8.5 months, compared to 5.6 months in pts with high TKa (n= 12). Interestingly, pts with an increase in TKa at T1 (n=9) had a mTTP of only 3.1 months compared to pts with stable/reduced TKa (N=35), who showed a mTTP of 9 months. Considering the potential significance of TKa measured at disease progression, pts with high levels at T2 (n=18) had a worse outcome on subsequent post-study treatment (both chemotherapy and ET) compared to those with lower levels (n=18) (mTTP at T2, 2.9 vs 8.9 months, respectively).

Conclusions: These data suggest for the first time that TKa may be a useful prognostic biomarker for non-invasive monitoring of MBC in the context of treatment with P. These results warrant further investigation in larger sample sets.

#4417

Synergy of cyclin dependentkinase 4/6 inhibitors with cytotoxic chemotherapy in cholangiocarcinoma.

Mansi Arora, James Bogenberger, Nagalo Bolni, Yumei Zhou, Latha Kannan, Jan Egan, Raquel Yokoda, Daniel H. Ahn, Mitesh J. Borad. _Mayo Clinic, Scottsdale, AZ_.

Purpose: Cholangiocarcinoma (CCA) is a highly lethal malignancy that often presents at advanced stages. Gemcitabine/cisplatin (GP) represents the standard-of-care but provides only a modest response rate and survival benefit. Loss of the CDK4/6 inhibitor CDKN2A is a prevalent genetic event in CCA, thus CDK4/6 inhibitors, FDA-approved in some breast cancer regimens, are hypothesized to have a role in treating CCA. Clinical challenges to target cell cycle dysregulation and exploit cytotoxic chemotherapy include predictive biomarkers for selecting patients most likely to respond. We hypothesized autophagy is involved in therapeutic resistance to CDK4/6 inhibitors, and plays a role in the activity of GP in CCA. Thus, we investigated CDK4/6 inhibitors as monotherapy, and combined with GP in CCA, measuring multiple readouts pertaining to drug synergy, putative pathway biomarkers and autophagy.

Methods: The activity of 3 FDA approved CDK4/6 inhibitors, palbociclib, ribociclib and abemaciclib, as monotherapy (N=24), and combined with GP (N=10), was assessed in CCA cell lines by measuring relative cell number with Cell TiterGlo, and apoptosis with Annexin/PI and cleaved caspase3 quantification via flow cytometry. To characterize RB status for biomarker potential, protein levels of CCND1, CDK4, CDK6, RB, and CDKN2A were measured by western blot. Autophagy was measured using an autophagy flux assay, LysoTracker confocal staining, and by quantifying protein levels of LC3B, p62 and Beclin1A.

Results: 22/24 (92%) of CCA cell lines were found to have at least one defect likely relevant to CDK4/6 inhibition: CDKN2A (92%), Rb1 (63%), CDK4 (23%) and cyclin D1 (32%). Protein levels of CDKN2A and RB1 showed moderate correlation with CDK4/6 inhibitor activity. Abemaciclib was the most potent CDK inhibitor in vitro (IC50≤0.5uM; palbociclib IC50>1uM; ribociclib IC50>5uM). Combined abemaciclib and GP showed higher cytotoxicity and synergy at lower doses compared to chemotherapy alone or single-agent abemaciclib. Compared to CDK4/6 inhibitors or chemotherapy alone, combination therapy enhanced growth inhibition, cell cycle arrest and caspase-dependent apoptotic cell death. Abemaciclib triggered autophagy mediated by significant increases in reactive oxygen species (ROS). Combination therapy reduced ROS production and thus autophagy, as seen by reduced autophagic flux, reduced LysoTracker mean fluorescence intensity, a decrease in the expression of LC3B-II, Beclin1A and an increase in p62.

Conclusion: These results suggest CDK4/6 inhibitors interact synergistically with GP in CCA and mechanistically disrupt autophagy. This suggests potential to further explore CDKN2A and RB as biomarkers for preclinical sensitivity in CCA. Pre-clinical data supports ongoing clinical studies of this concept, as well as planned clinical studies of GP and alternative CDK4/6 inhibitors.

#4418

Pharmacological characterization of a preclinical candidate covalently inhibiting CDK12.

Ramulu Poddutoori, Sujatha Rajagopalan, Subhendu Mukherjee, Sivapriya Marappan, Samiulla D. S., Sasirekha Sivakumar, Shilpa S Nayak, Ravindra M V, Devaraja TS, Srinivas Kondela, Suraj Tgore, Amit A Dhudashiya, Charamanna K. B, Aravind A B, Amith A, Pavithra S, Hema Sankar Pathange, Thomas Antony, Mahaboobi Jaleel, Sanjeev Giri, Girish Daginakatte, Kavitha Nellore, Shekar Chelur, Murali Ramachandra, Susanta Samajdar. _Aurigene Discovery Technologies Ltd, Housr Road, Bangalore, India_.

Cyclin-dependent kinase 12 (CDK12) is a transcription-associated protein that plays a critical role in DNA damage response, splicing, pre-mRNA processing and is crucial for maintaining genomic stability. CDK12 associated with Cyclin K (CycK) regulates transcription elongation by phosphorylating RNA polymerase II (RNAP II) at Serine 2 (pS2) in the C-terminal domain (CTD). Overexpression of CDK12 in various tumor types suggests the possibility that CDK12 has oncogenic properties, similarly to other transcription-associated kinases. Considering its critical role in transcription and RNA processing, CDK12 is emerging as a potential therapeutic target for cancer. Multiple series of potent and selective CDK12 covalent inhibitors were identified by structure-guided and iterative medicinal chemistry approaches. Early lead compounds were optimized towards achieving high on target potency with good selectivity and desirable drug like properties including pharmacokinetic profile to achieve anti-tumour activity. Optimization of early lead compounds from two distinct chemical series resulted in very potent and highly selective CDK12 covalent inhibitors with desirable oral bioavailability. The covalent mode of action for these biochemically potent compounds has been confirmed by CDK12 target engagement assay in the cellular context. These selective inhibitors showed significant anti-proliferative activity in TNBC and other cancer cell lines including those harbouring ETS fusion. Importantly, cell killing is observed in cancer cells but not in normal cells (RWPE1) with short time (2h) and long-time (72h) exposure of these compounds. Anti-proliferative activity is well correlated with the inhibition of pS2 and down-regulation of a number of DNA damage response genes including BRCA1, RAD51, ATM and FANCI. Consistent with the inhibition of genes involved in DNA damage repair, a highly synergistic anti-proliferative activity was observed when treated in combination with cisplatin and PARP inhibitors. Based on the robust efficacy as a single agent in a TNBC mouse xenograft model with one of the optimized leads, the preclinical candidate exhibiting a greater degree of selectivity is being evaluated for efficacy and tolerability in relevant preclinical models.

#4419

Cell cycle analysis during TTF to exploit novel targets for increasing treatment efficacy.

Paul Slangen,1 Mark de Gooijer,1 Olaf van Tellingen,1 Moshe Giladi,2 Gerben Borst1. 1 _Netherlands Cancer Inst., Amsterdam, Netherlands;_ 2 _Novocure, Haifa, Israel_.

Cell cycle distribution and cell death

---

Cell cycle distribution (index,%, t=12hrs)

Treatment | Control | TTF | |

|

S | 10,5 ± 0.4 | 10.8 ± 0.5 | NS | |

G2 | 29.9 ± 1.2 | 48.4 ± 0.9 | p<0.05 | |

M | 6.3 ± 0.6 | 2.0 ± 0.2 | p<0.05 | |

G1 | 53.0 ± 1.2 | 38.0 ± 0.8 | p<0.05 | |

|  | |  | |

Cell death (%)

Treatment | control | Wee1 | TTF | TTF+Wee1

|

Cell count | 100+/-3.2 | 74.4 +/- 4.1 | 50.5 +/- 4.2 | 21.5 +/- 3.2 | Synergistic

Colony formation | 100 +/- 3.2 | 61.6 +/- 6.6 | 75.5 +/- 11.2 | 17.0 +/- 5.3 | Synergistic

|  | |  | |

While the effectiveness of TTFields is initially attributed to the effects of TTFields during cell division, recent studies have shown that TTFields additionally induce DNA damage and replication stress. We want to further exploit the working mechanism of TTFields, its effect on the cell cycle and identify targets to increase its efficacy.

A172 cells were synchronized in S-phase by double thymidine block. Six hours post-release (0h), cells were subjected to TTFields treatment (200kHz; 4,5V/cm pk-pk). Cell cycle analysis was performed by flow cytometry (PI, α-phospho-histone H3Ser10). Treatment of A172 cells synchronized in S phase with TTFields showed that TTFields cause an accumulation of cells in G2 phase, leading to delayed entry into mitosis and, subsequent lower entry into G1 (see table). Because this indication of G2 checkpoint activation we hypothesized the possible synergism between TTFields and Wee1 inhibition. A172 cells were simultaneously treated with TTFields (200kHz; 4,5V/cm pk-pk, 72hrs) and the Wee1 inhibitor (300nM, 72hrs). Cell survival was determined by automated cell counting (including Trypan Blue) and colony formation assay. We observed that abrogation of the G2 checkpoint by Wee1 inhibtion synergistically increases the efficacy of TTFields treatment (see table).

The underlying mechanism of the G2 checkpoint activation is subject of ongoing research. By any means, the combination of TTFields with the Wee1 inhibitor bypasses the G2 activation and causes a synergistic decrease in cell survival.

#4420

Small-molecule inhibition of Wee1 kinase by AZD1775 selectively sensitizes human papilloma virus (HPV)-associated cervical cancer to DNA-damaging therapies.

Osamu Kobayashi, Yuji Ikeda, Yuki Okuma, Nobuki Hayashi, Takahiro Nakajima, Mikiko Asai-Sato, Kei Kawana. _Nihon University, Tokyo, Japan_.

Objective: Wee1 regulate G2-M checkpoint of cell-cycle Inhibition of Wee1 promotes cell mitosis without DNA repair in cases with TP53 deficiency Since human papillomavirus (HPV) E6 involves the inactivation of p53, we aimed to evaluate effects of Wee1 inhibitor on cervical cancer.

Material and Methods: Expression level of Wee1 in cervical cancer was evaluated using Oncomine Cervical cancer cell lines, Caski (HPV16+), SiHa (HPV16+) and C33A (HPV-, P53 mut) and LNCap (HPV-, P53 wt, prostate cancer) were used. After exposure to either Wee1 inhibitor (AZD1775), cisplatin, or both, cell surviving rate was evaluated by MTT assay and expression levels of Wee1 and cell cycle related proteins were evaluated by western blotting cell cycle status in cell lines were observed by flow cytometry.

Results: High expression of Wee1 in cervical cancer was clinically observed among ten cancer species. Stepwise treatment of AZD1775 promoted significant cell death induction, compared with exposure to cisplatin alone in HPV-positive cells (Caski and Siha); however no effect on cell death in HPV-negative cells (C33A and LNCap). In addition, expression level of pCDC2 was reduced after exposure to AZD1775, indicating induction of cell mitosis without DNA repair.

Conclusion: Our results indicate the therapeutic potential of cervical cancer treatment by Wee1 inhibition. Synergistic effect of Wee1 inhibition with conventional treatments (CCRT or chemotherapy) may be a new strategy to improve the prognosis in cervical cancer.

#4421

**SY-5609, an orally available selective CDK7 inhibitor demonstrates broad anti-tumor activity** in vivo **.**

Shanhu Hu, Jason Marineau, Kristin Hamman, Michael Bradley, Anneli Savinainen, Sydney Alnemy, Nisha Rajagopal, David Orlando, Claudio Chuaqui, Eric Olson. _Syros Pharmaceuticals, Cambridge, MA_.

Previously, we reported on a series of highly potent, selective, and non-covalent CDK7 inhibitors that demonstrated antiproliferative activity against triple-negative breast cancer (TNBC) and ovarian cancer (OVA) cell lines and tumor growth inhibition in cell line-derived (CDX) and patient-derived (PDX) mouse xenograft models. Here, we report on the in vitro and in vivo profile of our development candidate, SY-5609. Methods: Kinase inhibition assays at both Km and 2 mM [ATP] were used to assess inhibition of CDK2, CDK7, CDK9, and CDK12. SPR was used to determine the Kd, kon, and koff binding characteristics of SY-5609 to immobilized CDK7/Cyclin H dimer. CDK7 compound occupancy was determined using a biotinylated small molecule probe to pull down free CDK7 following incubating of HL60 cells with SY-5609. Inhibition of tumor cell line growth was assessed following 72 hrs of incubation with SY-5609. Flow cytometry was used to assess apoptosis and cell cycle modulation after 48 hrs of treatment. Effects on DNA damage and repair were assessed by immunofluorescence staining for γH2AX and RAD51 proteins. To assess in vivo effects, mice were implanted subcutaneously and randomized for treatment when tumors reached 150-200 mm3 and dosed orally for 3 weeks by both QD and BID dosing regimens. Collected tumor tissue samples were analyzed for protein levels of MCL1, pCDK2, MYC, and RNA Pol II CTD pSer5 by western blot. Results: SY-5609 bound CDK7/Cyclin H with a Kd of 0.059 nM and occupied CDK7 in HL60 cells with an EC50 of 33 nM. Cell growth inhibition EC50 values were 6-17 nM in a panel of solid tumor cell lines. Selectivity of SY-5609 over CDK12, CDK9, and CDK2 was 2492-, 2508-, and 8068-fold, respectively. SY-5609 led to induction of apoptosis, cell cycle arrest, and inhibition of DNA damage repair in tumor cell lines. Dose-dependent tumor growth inhibition was observed in a panel of CDX and PDX solid tumor models with both QD and BID dosing of SY-5609 with resulting decreases in direct (pCDK2, RNA Pol II CTD pSer5) and indirect (MCL1, MYC) protein biomarkers. In summary, we describe SY-5609, an orally available, potent, and selective CDK7 inhibitor that drives strong responses in CDX and PDX tumor models. These data support the rationale for advancing SY-5609 into IND-enabling studies.

#4422

Molecular and functional characterization of alpha satellite non-coding RNAs.

Rodrigo E. Cáceres,1 Marco A. Andonegui,1 Diego A. Oliva,1 Rodrigo González,1 Fernando Luna,1 Cristian G. Arriaga,1 Alejandro López,1 Diddier G. Prada,1 Clementina Castro,1 Paulina Molina,1 Carlos De la Rosa,2 José L. Reyes,2 Sabrine Hédouin,3 Florent Hubé,3 Claire Francastel,3 Luis A. Herrera1. 1 _Inst. Nacional de Cancerología, Mexico City, Mexico;_ 2 _Instituto de Biotecnología - UNAM, Cuernavaca, Mexico;_ 3 _Université Paris Diderot, Paris, France_.

Eukaryotic cell cycle progression requires the coordination of multiple molecular events in space and time to bring about proper transition from one phase to the next, and to ensure faithful genomic inheritance to daughter cells. The roles of coding transcription, translation, and RNA/protein degradation in cell cycle regulation are straightforward, since they control the abundance of proteins necessary for its progression. However, the contribution of non-coding transcription to this process is poorly understood. Non-coding transcription is notoriously interesting in mitosis for two reasons: First, transcription is repressed in most of the genome, but it is active in the centromere itself during mitosis; second, centromeric transcription produces non-coding RNA molecules which are integral components of the centromere and kinetochore and have functional roles in chromosome segregation. Here, we characterized the centromeric human alpha satellite non-coding RNAs (cencRNAs). We detected that the centromere produces high-molecular-weight (>5 kb) alpha satellite transcripts in sense and antisense orientation in several cancer cell lines and normal human leukocytes. These RNAs have a half-life of ~ 30 min and vary in abundance, but not in size, throughout the cell cycle. By analyzing their abundance under various conditions that promote mitotic arrest, we discovered a new association between proteasome inhibition and cencRNAs overexpression in G2/M HCT116 cells. We also show that their transcriptional inhibition mitigates the mitotic arrest induced by proteasome inhibitors. Furthermore, we demonstrate that exogenous expression of cencRNAs can slightly increase the mitotic index of SW480 cells. Using mass spectrometry-coupled RNA pulldown, we discovered that these RNAs interact with proteins essential for chromosome segregation. Our data show that centromeric transcription is regulated by proteasomes. Proteasomal inhibition promotes abnormal mitotic progression owing to the overexpression of non-coding RNAs at the onset of mitosis and the presumed impairment of their partner proteins functions in mitosis.

#4423

Antitumor activity of the novel oral highly selective Wee1 inhibitor Debio 0123.

Colin O'Dowd,1 Gerald Gavory,1 Frank Burkamp,1 Adam Treder,1 Caroline Boyd,1 Tim Harrison,1 Frederic Massière,2 Astrid Glück,2 Christophe Chardonnens,2 Andrea Zaffalon,2 Stefania Rigotti,2 Robert Mader,2 Grégoire Vuagniaux,2 Anne Vaslin Chessex2. 1 _Almac Discovery, Belfast, United Kingdom;_ 2 _Debiopharm International S.A., Lausanne, Switzerland_.

Background: The Wee1 tyrosine kinase is activated upon DNA damage and regulates the G2-M cell cycle checkpoint. Inhibition of Wee1, in conjunction with additional genetic alterations and/or addition of a DNA damaging agent, results in mitotic catastrophe and apoptosis of cancer cells, offering an attractive approach to treating cancer. The aim of the present study was to characterize the pharmacological properties of the newly discovered, orally available, and highly selective Wee1 inhibitor Debio 0123.

Methods: Profiling of Debio 0123 was performed on 465 selected kinases in a cell-free system. Effects of Debio 0123 on downstream signaling were analyzed by ELISA and western blot in HT29 (colorectal adenocarcinoma) and A427 (lung carcinoma) cell lines. The in vitro growth inhibition activity of Debio 0123 was defined in a broad number of human cancer cell lines after 72h using a proliferation monolayer assay. Debio 0123 in vivo anti-tumoral activity was assessed in an A427 subcutaneous xenograft model in athymic nude mice. Debio 0123 pharmacodynamic effects were also evaluated after oral dosing in surrogate tissues of mice, rats and monkeys by immunohistochemistry.

Results: Debio 0123 is a highly selective ATP-competitive Wee1 inhibitor with an IC50 in the low nanomolar range. Debio 0123 inhibited Cdk1 (Cdc2) phosphorylation at Y15, a direct substrate of Wee1 in cells. Debio 0123 also induced dose- and time-dependent increased mitosis and unrepaired DNA damage shown by increased phosphorylation of histone H3 and γH2AX foci formation. In vitro activity was further studied in a large panel of cancer cell lines. Debio 0123 demonstrated submicromolar cytotoxic activity on multiple cell lines from various histotypes. In vivo, Debio 0123 was shown to inhibit phosphorylation of Cdk1 in tissues of multiple species and induction of DNA damage in a xenograft model. When administered orally once daily for 28 consecutive days, Debio 0123 induced dose-dependent anti-tumor activity and was well tolerated at all doses tested. At 30 mg/kg, treatment with Debio 0123 resulted in tumor regression.

Conclusion: Altogether these results demonstrate that Debio 0123 is a highly selective and potent Wee1 inhibitor able to prevent Cdk1 phosphorylation in multiple species and to induce apoptosis through accumulation of unrepaired DNA damage both in vitro and in vivo that result in tumor regression. The advancement of Debio 0123 into clinical studies may provide improved therapeutic outcomes for patients with cancer.

#4424

Coordinately targeting cell cycle checkpoint functions in integrated models of pancreatic cancer.

Sejin Chung, Paris Vail, Agnieszka Witkiewicz, Erik S. Knudsen. _Roswell Park Comprehensive Cancer Center, Buffalo, NY_.

Cancer cells often have deficiencies in cell cycle control mechanisms and could be dependent on specific cell cycle checkpoints to maintain viability. Due to the documented role of KRAS in driving replication stress, we targeted the checkpoint governing DNA replication using CHK1 kinase inhibitors in pancreatic ductal adenocarcinoma (PDAC) models and examined mechanisms of resistance. Single agent efficacy of CHK1 inhibition was investigated in established and primary PDAC lines. Drug screening was performed to identify cooperative agents. In vitro and in vivo studies were employed to interrogate combination treatment efficacy and mechanisms of resistance. We observed that many PDAC models evade single agent inhibition through mechanisms that allow S-phase progression with CHK1 inhibited. Gene expression analysis revealed FOXM1 as a potential marker of CHK1 sensitivity and defined a form of pancreatic cancer with poor prognosis. Drug screen analysis identified WEE1 as a cooperative agent with CHK1 and was effective in cell culture. In vivo experiments validated the combination efficacy; however, resistance could evolve. Resistance was due to selection of a stable sub-clone from the original PDX tumor, which harbored exceedingly high base line replication stress. In vitro analysis revealed that gemcitabine could eliminate viability in such resistant models. The triplet regimen of gemcitabine, CHK1 and WEE1 inhibition provided strong disease control in all patient-derived xenograft models interrogated. These results demonstrate the therapeutic resiliency of pancreatic cancer and indicate that coordinately targeting cell cycle checkpoints in concert with chemotherapy could be particularly efficacious.

#4425

FCN-437: A novel, potent and selective oral inhibitor of CDK4/6 for the treatment of solid tumors.

Shu Lin,1 Xingdong Zhao,1 Tongshuang Li,1 Huajie Zhang,2 Haohan Tan,2 Xianlong Wang,2 Lihua Jiang,2 Yanxin Liu,2 Jing Sun,2 Li Linghu,2 Qihong Liu,2 Zhifu Li,2 Weipeng Zhang,2 Weibo Wang2. 1 _Fochon Pharma, Inc., San Leandro, CA;_ 2 _Fochon Pharmaceuticals, Ltd., Chongqing, China_.

Cyclin-dependent kinase-4 (CDK4) and CDK6 form a complex with D-type cyclins to phosphorylate Retinoblastoma protein (Rb), leading to a loss of repression of E2F transcription factors, which enables cell cycle progression from G1 to S phase. Loss of cell cycle control caused by aberrations in CDK/Rb signaling pathway are common in solid tumors. Inhibiting CDK4/6 blocks CDK/Rb signaling pathway, which prevents cell cycle progression through G1 restriction point, thus arresting tumor cell growth. Most of the 1st generation of CDK inhibitors lack CDK family selectivity, and thus result in greater toxicity, and poor efficacy in clinical use. The emergence of a new generation of selective CDK4/6 inhibitors, including FDA approved drugs for ER+/HER2- breast cancer (ribociclib, palbociclib and abemaciclib), has shown clinically meaningful prolongation of progression-free survival over endocrine therapy alone. However, the treatment is limited for patients with brain metastases, because the blood-brain barrier (BBB) penetration of approved CDK4/6 inhibitors are either restricted, or lack of clinical assessment. Therefore, it is urgently needed to develop novel CDK4/6 inhibitors with improved effectiveness, better BBB penetration and fewer adverse effects. Here, we developed FCN-437, a novel, selective and orally active inhibitor of CDK4/6. FCN-437 has demonstrated selective inhibitory activities against CDK4/6 over other CDKs. FCN-437 has shown broad-spectrum anti-proliferating activity on Rb+ tumor cells, derived from tissues of various solid tumors. The in vitro potency of FCN-437 is 5-fold more than ribociclib, and comparable to palbociclib. Correspondingly, in tumor xenograft models of breast cancer, colon cancer and glioma, FCN-437 dramatically inhibited tumor growth, comparable or superior to approved CDK4/6 inhibitors. FCN-437 possesses good synergetic effect in combination with letrozole or fulvestrant in vivo. FCN-437 exhibits excellent pharmacokinetic (PK) properties with improved bioavailability. In particular, FCN-437 preferentially distributes to tissues including brain compared to plasma in rats, indicating improved BBB permeability, which provides an opportunity to treat tumors that have metastasized to the brain. In non-clinical studies, FCN-437 has shown preferable safety profiles with low hERG activity and no potential cardiotoxicity. No CYP450 inducing or inhibiting effect was found in FCN-437, minimizing its potential of drug interactions. Overall, FCN-437 exhibits desired drug-like properties, enhanced efficacy, improved PK properties with BBB penetration and preferable safety profiles compared to approved CDK4/6 inhibitors. FCN-437 can be a novel and effective targeted therapy either as single agent or in combination, for patients with advanced solid tumors with brain metastases. A phase 1 clinical trial of FCN-437 has been granted approval by NMPA in 2018.

#4426

Selective inhibition of CDK9 induces apoptosis of TNBC cells independent of Rb status.

Hailey E. Brighton,1 Claire R. Hall,2 Kerry A. Dillon,1 John E. Bisi,1 Jay C. Strum1. 1 _G1 Therapeutics, Inc, Durham, NC;_ 2 _G1 Therapeutics, Inc, Research Triangle Park, NC_.

The family of cyclin-dependent kinases (CDKs) are serine/threonine protein kinases important for cell cycle control and/or regulation of transcription. CDK9/CyclinT1 is a key regulator of transcription in eukaryotic cells and has been shown to be dysregulated at the level of protein and kinase activity in both hematologic and solid tumors. CDK9/CyclinT1 forms the active P-TEFb complex and phosphorylates Ser2 residues in the carboxy-terminal domain of RNA polymerase II (RNApol II) to initiate elongation of mRNA transcripts. CDK9 activity regulates transcription of a variety of short-lived transcripts that promote survival and directly suppress apoptosis in cancer cells, including MYC,XRN2, MCL-1,and XIAP. MYC-driven tumor types with Rb-loss or high expression levels of cyclin E, such as triple negative breast cancer (TNBC), are difficult to treat and are resistant to existing CDK4/6 inhibitors. Since CDK9 is upstream of these oncogenic drivers, inhibition of CDK9 could potentially bypass innate resistance mechanisms and induce cell death in TNBC through blocking expression of MYC and MCL-1, for example. To target CDK9 in TNBC, we used structure-based drug design and developed a library of novel, potent and selective CDK9 inhibitors (CDK9i). The lead CDK9i exhibit single digit nanomolar potency against CDK9/CyclinT1 complexes and good selectivity against other CDK family members in biochemical assays.

In addition, they also display high selectivity over 502 other kinases. Lead CDK9 inhibitors were found to potently inhibit phosphorylation of RNApol II Ser2 in a time and dose dependent manner in TNBC cells and decrease MYC, MCL-1 and XIAP at both mRNA and protein levels. Additionally, treatment of TNBC cells led to a potent G2/M cell cycle arrest, inhibition of cell proliferation and induction of apoptosis within 24 hours, regardless of Rb status of the tumor cells. Primary human bone marrow and normal human fibroblast cells, however, did not undergo apoptosis within 72 hours of treatment with CDK9i, suggesting a potential therapeutic window for CDK9i treatment. Further studies are ongoing to assess the effect of dose and scheduling on tumor efficacy in mouse TNBC tumor models. Selective inhibition of CDK9 presents a novel treatment strategy for difficult-to-treat tumor types, including those resistant to existing targeted therapeutics.

#4427

LS007, a potent and orally bioavailable CDK9 inhibitor, suppresses the growth of triple-negative and estrogen receptor-positive breast cancer.

Muhammed Hamidur Rahaman, Yinghui Zhang, Md Saiful Islam, Gary Heinemann, Abel Tesfaye Anshabo, Hugo Albrecht, Robert Milne, Shudong Wang. _University of South Australia, Adelaide, Australia_.

Treatment of breast cancer represents a major therapeutic challenge, and thus identification of new targeted therapy is of paramount importance. Anti-apoptotic proteins, BCL2 and MCL1, and oncoprotein c-MYC are implicated in evasion of apoptosis and resistance to chemotherapy in both triple-negative breast cancer (TNBC) and estrogen receptor-positive breast cancer (ER+ BC). Transcription of these oncogenic factors requires cyclin dependent kinase 9 (CDK9). CDK9 phosphorylates the C-terminal domain (CTD) of RNA polymerase II (RNAPII) and acts as a rate-limiting step of transcription. Thus, targeting CDK9 can reduce the expression of these proteins effectively and simultaneously, presenting a promising therapeutic strategy for breast cancer. Utilizing extensive medicinal chemistry and biochemical assays, we previously identified and developed LS007 as an orally bioavailable (F = 56% in cynomolgus monkey at 4 mg/kg), and nanomolar inhibitor of CDK9 (Ki = 4 nM). In vitro, inhibition of CDK9-mediated phosphorylation of RNAPII (pRNAPIIS2) by LS007 resulted in downregulation of BCL2, MCL1 and c-MYC and activation of PARP cleavage indicating apoptosis in breast cancer cells (TNBC: MDA-MB-231, Hs578T and ER+ BC: MCF-7, T47D). In vivo, LS007 was well tolerated in mice after oral administration (MTD = 50 mg/kg QD and 100 mg/kg Q3D). In MDA-MB-231 TNBC xenograft, by daily oral dosing at 25 mg/kg and 50 mg/kg LS007 significantly reduced tumor growth (p < 0.001) with T/C of 53 % and 35 %, respectively, on day 21, when compared to the vehicle dosed group. Similarly, in MCF-7 ER+ BC xenograft model, oral dosing of LS007 at 25 mg/kg QD, 50 mg/kg Q2D and 75 mg/kg Q3D showed significant tumor growth inhibition with T/C of 50 %, 60 % and 47 %, respectively, and increased in animal survival (p < 0.001) compared to vehicle control. It was safe in the xenograft models as there was no significant body weight loss or other overt toxicities. In addition, histological analysis of major organs (liver, kidney, heart, bone marrow and intestine) of the mice receiving LS007 showed no drug related toxicities. These results support for development of LS007 towards the clinical trials for the treatment of TNBC and ER+ BC.

#4428

Role of PDGFRA gene and its downstream PI3K signaling pathway in imatinib inducedthrombocytopenia in CML patients.

Sameer Ahmad Guru,1 Ab. Rashid Mir,2 Mamta Pervin Sumi, Imtiyaz Ahmad Najar, Dr. AlpanaSaxena. 1 _Maulana Azad Medical College, New Delhi, India;_ 2 _University of Tabuk, Kingdom of Saudi Arabia, Saudi Arabia_.

A link has been proposed between tyrosine kinases, platelet derived growth factor receptors (PDGFRs) and platelet development. In this study, we explore the role of PDGFRα and its downstream signaling components in the development of thrombocytopenia in imatinib treated CML patients and K562 cell lines.

We studied PDGFRα mRNA expression and its activation (Tyr754; p- PDGFRα) in imatinib induced thrombocytopenic CML patients and K562 cells treated with imatinib and curcumin. The effect of deregulated mRNA expression and its dephosphorylation on the expression of PDGFRα's downstream signaling molecules like PI3K, AKT1 and AKT2 was also studied and compared in thrombocytopenic and nonthrombocytopenic CML patients.

It was observed that there was a significant decrease in PDGFRα mRNA expression and activation (Tyr754; p- PDGFRα) in CML patients who developed thrombocytopenia after imatinib treatment compared to nonthrombocytopenic ones. The effect of PDGFRα decreased activation and expression on its downstream signaling components was also analyzed. However, only mRNA expression of PI3K gene showed a statistically significant down regulation in thrombocytopenic CML patients in comparison to nonthrombocytopenic ones. Though mRNA expression of AKT1 exhibited a reduced expression pattern in thrombocytopenic CML patients compared to nonthrombocytopenic ones but the change was not significant. Moreover, we did not observe any change in mRNA expression of AKT2 gene in thrombocytopenic and nonthrombocytopenic CML patients.

mRNA expression of PDGFRα gene significantly decreased in imatinib and curcumin treated K562 cells compared to untreated cells. The synergistic effect of imatinib and curcumin was also analyzed and it was observed that PDGFRα mRNA expression further decreased in imatinib+curcumin treated K562 cells as compared to imatinib and curcumin cells but the difference was not statistical. Further, reduced PDGFRα mRNA expression and activation (Tyr754; p- PDGFRα) due to imatinib and curcumin treatment resulted in a significant reduction in mRNA levels of PI3K, AKT1 and AKT2 genes. However, we did not find any significant changes in mRNA levels of these genes among treatment groups except in case of AKT2 gene which showed a significant decrease in imatinib treated group as compared to that of curcumin and imatinib+curcumin treated groups.

Down regulation of PDGFRα mRNA expression and its reduced activation due to imatinib treatment may possibly result in reduced levels of its downstream intracellular components which in turn may act as a molecular cause of imatinib associated thrombocytopenia in CML patients receiving imatinib treatment.

#4429

Delivery of miR-143 and miR-506 with novel nano carrier to arrest cell cycle in lung cancer.

A K M Nawshad Hossian, Chandra Mohan Reddy Muthumula, George Mattheolabakis. _University of Louisiana Monroe, Monroe, LA_.

Lung cancer is the leading cause of cancer related deaths worldwide. Although targeting the cell cycle of cancer cells has been a promising approach, no such therapy has translated for the treatment of lung cancer. In our previous work, we identified that the combinatorial treatment of two miRNAs, miR-143 and miR-506, inhibited the cell cycle of lung cancer cells in vitro. These miRs downregulated the CDK1 and CDK4/6 genes, which are involved in the G1/S and G2/M transitions, respectively. Nucleic acid delivery has met formidable challenges, such as short in vivo half-life and rapid degradation in the circulation, as well as limited cell penetration or endosomal escape. We developed a novel nanocarrier, composed of tocopherol (vitamin E), polyethylenenimine, and polyethylene glycol (TPP) in 1:1:1 molar ratio for the delivery of miRNAs. Proper characterization with NMR and FTIR confirmed the synthesis of this molecule. We found it can form nanomicelles, and the positively charged of polyethyleneimine allowed for the complexation of miRNAs, as was detected by a gel retardation assay. Fluorescently tagged TPP micelles were uptaken by cancer cells, and the carrier successfully transfected cancer cells with the combinatorial miRNA treatment inducing gene downregulation. In vivo pharmacokinetics study using a subcutaneous mouse model of lung cancer indicated prolonged accumulation of the TPP-miRNA micelles in the tumor area. In vivo administration of the miR-143/506 in TPP micelles, along with cisplatin co-treatment, inhibited tumor growth in a subcutaneous mouse model of lung cancer. Overall, the promising role of miR-143 and 506 for

the regulation of LC growth may be achieved by this novel TPP-based nanocarrier, which merits further evaluation.

#4430

Simulating delivery of TTFields to the infratentorium in patients with brainstem gliomas.

Marigdalia K. Ramirez-Fort,1 Brittany Cross,2 Ariel Naveh,3 Melissa Mendez,4 Roberto Santiago,5 Sean S. Mahase,1 Jaime Matta,6 Ze'ev Bomzon,3 Christopher S. Lange7. 1 _Weill Cornell Medicine, New York, NY;_ 2 _Novocure, New York, NY;_ 3 _Novocure Itd., Haifa, Israel;_ 4 _SleepNet Neurology and Sleep Center, Bayamon, PR;_ 5 _Auxilio Mutuo, San Juan, PR;_ 6 _Ponce Health Sciences Univeristy, Ponce, PR;_ 7 _SUNY Downstate Medical Center, Brooklyn, NY_.

Purpose/Objective(s): TTFields are FDA-approved for the treatment of recurrent and newly diagnosed Glioblastoma (GBM) in the supratentorial brain. The EF-14 trial showed that combining TTFields with adjuvant chemo-radiation leads to a significant increase in overall survival compared to adjuvant chemo-radiation alone. High-grade gliomas also occur in the infratentorium and brainstem of pediatric and adult patients. Brainstem gliomas have a median survival of 9 to 11 months, necessitating the exploration of novel treatment combinations, such as delivering TTFields to the infratentorial brain.

We recently showed in a simulation-based study that TTFields can be successfully delivered to the infratentorium. Effective delivery was achieved by placement of the arrays on the vertex, bilateral posterolateral occiput, and superior-posterior neck; the array placement results in TTFields at therapeutic intensities throughout the brainstem and cerebellum of realistic computational head models.

The previous simulation-based study was performed using realistic computational models of healthy individuals; it did not provide detailed insight into field distributions within tumor tissue located in the infratentorium. Here, we aim to expand the results of this previous study, by simulating the delivery of TTFields to the infratentorium of adult and pediatric patients with high-grade brainstem gliomas.

Materials/Methods: A realistic computational model was created using MRI data of adult and pediatric patients with high-grade brainstem gliomas. Transducer arrays were placed on the model and the delivery of TTFields to the infratentorial brain was then simulated using a commercial numerical solver. The electric field distribution in the supratentorium, infratentorium, and within the tumor were derived from the simulations, and the dose in the tumor analyzed.

Results: The distribution of calculated isofield lines demonstrates effective delivery of TTFields to infratentorial brain tumors.

Conclusion: Our results provides rationale for clinically investigating the utility of TTFields in treating infratentorial high-grade gliomas.

#4431

Cytotoxic effects of the Jamaican yam tuber in hormone-sensitive cancers and its potential use in green nanotechnology formulation.

Sasha-gay A. Wright. _University of the West Indies, Kingston, Jamaica_.

Historically, plant materials have been widely explored for their anti-proliferative effects against a variety of cancer cells, with many of them proving to be very effective. Additionally, their efficacy has been seen in the specific field of targeted cancer therapy. The use of plants for therapeutic purposes in place of or with traditional medicine is collectively referred to as complementary and alternative medicine (C.A.M). Several Jamaican Yam species have shown to be useful as C.A.M in the treatment of certain medical conditions such as diabetes, hypercholesterolemia and cancer. Hormone-sensitive cancers such as breast and prostate cancer are the most frequently diagnosed malignancy and when combined, is the leading cause of cancer related deaths among the Jamaican populace. These statistics unfortunately, are also reflected in the wider Caribbean region. Currently treatment involves: surgery, radiation and androgen deprivation therapy; however, there are some cases in which mutation causes further growth of the cancer cells regardless of treatment. As such, there still remains a dire need of a more effective and lasting therapy. The aim of this study was to assess the antiproliferative effects of yam material and to also deduce a possible mechanism of action in an effort to formulate a means of targeted therapy. The results showed that an alcoholic extract of the Jamaican yam tuber (Dioscorea spp.) demonstrated anti-proliferative effects in androgen insensitive prostate cancer cell lines (IC50 of 18 ppm, 95% C.I. 24-33ppm) and invasive breast cancer cell lines (IC 50 of 28 ppm, 95% C.I 36-54ppm). Western blot assays also indicated that cell death was possibly induced through the cell cycle regulatory protein, cyclin D. The antiproliferative effects and purported mechanism of action of the yam extract against some hormone-sensitive cancer cells makes it an ideal alternative for the formulation of green nanotechnology for targeted therapy.

#4432

Multi-omics profiling establishes the polypharmacology of FDA Approved CDK4/6 inhibitors and its impact on drug response.

Caitlin E. Mills,1 Marc A. Hafner,1 Kartik Subramanian,1 Chen Chen,1 Mirra Chung,1 Sarah A. Boswell,1 Robert A. Everley,1 Changchang Liu,1 Charlotte S. Walmsley,2 Dejan Juric,2 Peter K. Sorger1. 1 _Harvard Medical School, Boston, MA;_ 2 _Massachusetts General Hospital, Boston, MA_.

FDA approval of multiple drugs with different chemical structures against the same protein target raises the question whether such drugs have sufficiently similar mechanisms of action to be considered functionally equivalent. We compare three recently approved inhibitors of the cyclin-dependent kinases CDK4/6 - palbociclib, ribociclib, and abemaciclib - that have become highly promising therapies for the treatment of breast cancer and potentially other solid tumors. All three drugs have the same nominal targets but differ in selectivity. In humans, abemaciclib uniquely appears to exhibit single-agent activity and has a distinct toxicity profile. We systematically profile targets and activities of these CDK4/6 inhibitors using biochemical assays, mRNA profiling, mass spectrometry-based phospho-proteomics, GR-based dose-response assays, and mouse xenografts. We find that the three drugs differ at a cellular level and that abemaciclib has targets and activities not shared by palbociclib or ribociclib including: induction of cell death (even in pRb-deficient cells), arrest in the G2 phase of the cell cycle, reduced drug adaptation, and unique transcriptional effects in vitro and in vivo. These activities appear to arise from inhibition of CDKs other than CDK4/6 including CDK2/Cyclin A/E and CDK1/Cyclin B. We propose that inhibition of these kinases by abemaciclib target known mechanisms of resistance to CDK4/6 inhibition and thus elicit a response in cell lines that are resistant to palbociclib or ribociclib.

#4433

**Novel and highly selective CDK9 inhibitors suppress proliferation of triple negative breast cancer (TNBC) cells** in vitro **.**

Jean M. Winter,1 Ebtihal H. Mustafa,1 Shudong Wang,2 Luke A. Selth,1 Theresa E. Hickey,1 Wayne D. Tilley1. 1 _Univ. of Adelaide, Adelaide, Australia;_ 2 _Univ. of South Australia, Adelaide, Australia_.

This study evaluates the efficacy of two newly developed selective CDK9 inhibitors (CDK9i) across a panel of TNBC cell lines. MDA-MB-453, MFM-223, MDA-MB-468 and MDA-MB-231 TNBC cells were treated with increasing concentrations of two novel and highly selective CDK9 inhibitors and the effect on proliferation, apoptosis and expression of CDK9 targets determined. MDA-MB-453 and -468 cells showed significant growth inhibition with as little as 150nM of CDK9i, evident 3 days after commencement of treatment. Both MDA-MB-231 and MFM-223 cells were less sensitive to the CDK9 inhibitors, with MDA-231 cells requiring at least 300nM to suppress growth. MFM-223 cells did not demonstrate any growth inhibition after 7 days of culture with CDK9i concentrations up to 1.2uM. Protein expression of CDK9 targets, including RNA Polymerase II (RNAPII), phosphorylated-RNAPII, the proto-oncogene C-MYC, and apoptotic marker cleaved caspase-3, were examined by Western blot after optimal CDK9i exposure across each cell line. CDK9i suppressed phosphorylated-RNAPII, but not total RNAPII, indicative of targeted CDK9 inhibition. The master transcription factor C-MYC, which is highly expressed in TNBC, was downregulated, and cleaved-caspase-3 was upregulated with CDK9i treatment. These data demonstrate cell specific efficacy of novel CDK9 inhibitors in cell line models of TNBC via transcriptional suppression of proto-oncogenes and upregulation of apoptotic pathways. Future studies will identify molecular markers of response to CDK9 inhibition and evaluate these novel inhibitors in TNBC patient derived xenografts.

### Ubiquitin Ligases

#4434

Th1 cytokines promotes E3 ubiquitin ligase Cullin 5 expression via STAT1 signaling cascade and enhance cul5 mediated proteasomal degradation of HER2 in HER2+/ER- breast cancer.

Yongsheng Jia, Ganesan Ramamoorthi, Krithika Kodumudi, Amrita Basu, Doris Wiener, Brian Czerniecki. _Moffitt Cancer Center, Tampa, FL_.

The proto-oncogene HER2/ErbB2 overexpression occurs in 30% of invasive breast cancer patients and its critically associated with aggressive disease, metastasis and tumor recurrence. Despite advances in the treatment of HER2+ breast cancers, resistance and metastatic disease are potential obstacles. Cullin 5 (Cul 5), a scaffold protein mediates formation of the Cullin 5-RING E3 ubiquitin ligase functional complex. This complex acts on protective molecular chaperone heat shock protein (HSP90) complex and subsequently leads to polyubiquitination and proteasomal degradation of its client protein HER2/ErbB2. Reduced expression of Cul 5 in breast cancer patients can abrogates its inhibitory function and favor HSP90 mediated stabilization of HER2/ErbB2. More recently, we showed that Th1 cytokines (IFN-γ and TNF-α) decreased HER2 expression and induced senescence and apoptosis in HER2 overexpressing breast cancer cell lines. However, the role of Th1 cytokines on Cul 5 mediated regulation of HER2/ErbB2 remains unknown. In the present study, low level expression of Cul 5 was observed in various human (SKBR3, HCC1419 and JIMT1) and mouse (Tubo) HER2+/ER- breast cancer cell lines. Interestingly, treatment of HER2+/ER- breast cancer cells with Th1 cytokines increased the expression of Cul 5 and downregulated the expression of HER2. We knocked-down Cul 5 expression by specific siRNA found that treatment of Th1 cytokines failed to decrease HER2 expression in HER2+/ER- breast cancer cells. To investigate whether the mechanism of Th1 cytokines on Cul5 is via downstream signaling molecule STAT-1, we knock-down STAT-1. Silencing of STAT-1 expression prevented Th1 cytokines mediated activation of Cul 5. We also observed no effect on the inhibition of HER2 expression after Th1 cytokines treatment in HER2+/ER- breast cancer cells in which the expression of STAT-1 was knocked-down. Collectively, our findings demonstrate a novel mechanism that Th1 cytokines promote Cul 5 expression via STAT1 signaling and enhance Cul 5 mediated proteasomal degradation of HER2 in HER2+/ER- breast cancer. Enhancing Cul 5 levels may represent a new therapeutic avenue for the inhibition of HER2 overexpression and prevention of metastasis and tumor relapse.

#4435

Molecular mechanism of a novel covalent allosteric inhibitor of SUMO E1 activating enzyme.

Zongyang Lyu, Lingmin Yuan, Katelyn M. Williams, James H. Atkison, Shaun K. Olsen. _Medical University of South Carolina, Charleston, SC_.

Post-translational modification of proteins by ubiquitin and ubiquitin-like proteins (collectively termed Ubls) proceeds through the sequential interactions and activities of parallel cascades of enzymes that are structurally and mechanistically related. E1 enzymes initiate Ub/Ubl conjugation cascades by catalyzing the ATP-dependent activation and transfer of their cognate Ub/Ubl to E2-conjugating enzymes, which then function with an array of E3 ligases to catalyze the formation of an isopeptide bond linking the Ub/Ubl to target proteins. Aberrations in Ub/Ubl modifications are associated with the pathogenesis of a wide range of life-threatening diseases, which has resulted in several Ubl E1 enzymes emerging as attractive targets for the development of small molecule therapeutics. We present the crystal structure of the E1 enzyme for the Ubl SUMO in complex with a small molecule representing a new class of Ubl E1 enzyme inhibitor. The structure reveals that the covalent allosteric inhibitor targets a cryptic pocket distinct from the active site that is completely buried in all previous SUMO E1 structures and that inhibitor binding is accompanied by a network of structural changes that altogether lock the enzyme in a previously unobserved inactive conformation. Altogether our studies provide the molecular mechanisms of inhibition SUMO E1 specificity and establishes a framework for the development of molecules targeting other Ubl E1s at a newly discovered cryptic allosteric site.

Supported by NIH R01GM115568, F30CA216921, T32CA193201, and a Hollings Cancer Center Postdoctoral Fellowship.

#4436

Ubiquitin-specific peptidase 24 decreases c-Myc expression to inhibit lung cancer formation.

Jan-Jong Hung,1 Wen-Chang Chang2. 1 _National Cheng Kung Univ., Tainan, Taiwan;_ 2 _Taipei Medical Univ., Taipei, Taiwan_.

Lung cancer is a malignant lung tumor characterized by high incidence and motility. c-Myc is a transcription factor that plays an important role in oncogenic activation to influence cellular metabolism and proliferation. Ubiquitin-specific peptidase 24 (USP24) is one of the members of USPs family to regulate protein ubiquitination. However, it is lack of evidence to uncover the role of USP24 in cancer formation. In this study, we found that cell proliferation was significantly increased by USP24 knockdown in A549 cells as accompanied by the increase of c-Myc protein. In particular, the RNA level and protein stability of c-Myc were not affected by USP24 knockdown, suggesting that USP24 affects c-Myc through regulating the translational pathway. Furthermore, c-Myc participated in USP24-knocked down-induced proliferation, not migration. To further clarify the mechanism underlying the regulation of c-Myc by USP24, we investigated whether mTOR pathway is involved in c-Myc upregulation by USP24 knockdown. Herein rapamycin, the mTOR inhibitor, prevented c-Myc upregulation caused by USP24 knockdown, suggesting that USP24 affect the protein level of c-Myc by regulating mTOR-mediated translation. We found that the downstream signaling of mTOR is activated by USP24 knockdown, including S6K and RPS6 phosphorylation. In addition, USP24 knockdown induced the binding of activated PRS6 to 5′-UTR of c-Myc, indicating that USP24 regulates c-Myc through mTOR-mediated translation. However, USP24 did not affect the phosphorylation of mTOR at Ser-2448, implying that other phosphorylation residue(s) within mTOR might be regulated by USP24. Since mTOR pathway is critical for many cancers formation, understanding the role of USP24 in regulating mTOR pathway will contribute to develop strategies to fight lung cancer.

#4437

Effects of atypical protein Kinase c inhibitor (DNDA) on lung cancer proliferation and migration by PKC-ι/FAK ubiquination through the Cbl-b pathway.

Raja Reddy Bommareddy, Rekha Patel, Mildred Acevedo Duncan. _Univ. of South Florida, Tampa, FL_.

Lung cancer is the second most common cancer and it is the leading cause of cancer death in both men and women. PKC isozymes play an important role in the development and progression of many cancers by regulating the cell cycle, survival, apoptosis, cell motility and malignant transformation. Our focus is to study the role of atypical PKCs (aPKC) in cell proliferation and migration in lung cancer cell lines. We used a novel non-specific inhibitor of aPKC namely DNDA (3,4-amino-2,7napthalenedisulfonic acid). Our hypothesis is that DNDA inhibits cell proliferation and migration of lung cancer cells. Our data from cell viability and flow cytometry showed significant reduction in cell proliferation and induction of apoptosis with DNDA (10µM) in A549 and H1299 lung cancer cells. Additionally, DNDA showed no toxic effect on BEAS-2B normal lung cells. Elevated levels of Focal Adhesion kinase (FAK) are implicated in the progression of cancer and plays a vital role in the invasion and migration of cancer cells. Western blot results showed that the phosphorylation of PKC-ι and phosphorylation of FAK were decreased in A549 lung cancer cells upon DNDA treatment. Moreover, there was no significant reduction in phosphorylation of FAK in H1299 lung cancer cells upon treatment with DNDA. Immunoprecipitation (IP) data revealed an association of PKC-ι with FAK and FAK with Cbl-b. Ubitest results suggests that PKC-ι regulates the cleavage of FAK through its ubiquination by cbl-b and thus inhibits the migration of A549 lung cancer cells which was evident from scratch assay. Our data indicates that DNDA might inhibit the migration of A549 lung cancer cells by PKC-ι/FAK ubiquination via Cbl-b.

#4438

Role of post translational modification of PAF1/PD2 in gemcitabine resistance of pancreatic cancer.

Sanchita Rauth, Saswati Karmakar, Ashu Shah, Rama Krishna Nimmakayala, Rakesh Bhatia, Sakthivel Muniyan, Sushil Kumar, Samikshan Dutta, Kaustubh Datta, Surinder K. Batra, Moorthy Palanimuthu Ponnusamy. _University of Nebraska Medical Center, Omaha, NE_.

Background: RNA polymerase associated factor 1 (PAF1)/Pancreatic differentiation 2 (PD2) is one of the core subunit of the human PAF1 complex (PAF1C), which regulates various cellular functions such as transcriptional elongation and histone modification. We have previously demonstrated its unique role in oncogenesis and stem cell maintenance. Studies have demonstrated that PAF1/PD2 gene yields a protein of 59.9 Kda (531 amino acids), however it has been found that it always gives a band at 80 Kda. Further, previous studies suggests that the 60 Kda protein represents the precursor, which rapidly process into an 80 Kda mature protein. SUMOylation is a process of reversible posttranslational modification that adds a small ubiquitin-related modifier (SUMO)-1 protein to the target protein. SUMOylation plays various molecular biology function such as transcriptional regulation, protein-protein interaction, and DNA damage repair and in cell cycle. DNA damage happen due to several physiological processes, but can also be caused by genotoxic agents. Promyelocytic Leukemia (PML) is protein that forms nuclear bodies and may be modified by SUMO1 and act as a DNA-damage sensor.

Hypothesis: PAF1/PD2 is interacting with SUMO-1 and PML and, thus, plays an important function in providing gemcitabine resistance to pancreatic cancer cells

Methods: SW1990, F9 (mouse embryonic cells) and CD18/HPAF cells were treated with 2-D08, a potent SUMOylation inhibitor for 24 hours and with siRNA against SUMO1 for 48 hours followed by protein isolation and western blotting. Immunoprecipitation and immunofluorescence were done to show the interaction between PAF1/PD2 and SUMO1 and with PML. To study the effect of PAF1/PD2 on gemcitabine resistance, SW1990 and Capan-1 cells were treated with different concentration of gemcitabine and then the expression of PAF1/PD2 along with SUMO1 was checked through immunoblotting and confocal imaging.

Results: Results shows that inhibiting SUMOylation with both 2D08 and siRNA resulted in a decrease expression of PAF1/PD2 80 Kda protein. Immunoprecipitation and immunofluorescence analysis showed that endogenous PAF1/PD2 interacts with SUMO1. This finding was further verified using ectopically overexpressed Flag- tagged PAF1/PD2 and HA-tagged SUMO1, which showed a physical interaction between PAF1/PD2 and SUMO1. Interestingly, we observed that gemcitabine treatment significantly increased the SUMOylated status of PAF1/PD2 in pancreatic cancer cells. Further our results proved that SUMOylated PAF1/PD2 form nuclear bodies along with PML in pancreatic cancer cells.

Conclusions: Our observation suggest that PAF1/PD2 undergoes SUMOylation and covalently interacts with SUMO1. Treatment with gemcitabine results in enhanced expression of PAF1/PD2 and increased co-localization with SUMO1 and PML, indicates a role of SUMOylated PAF1/PD2 in gemcitabine resistance.

#4439

MLN4924, a protein neddylation inhibitor, elicits antitumor effect via suppression of angiogenesis.

Kuan-Lin Kuo, Shao-Ping Yang, Shih-Ming Liao, Yeong-Shiau Pu, Kuo-How Huang. _National Taiwan Univ. Hospital, Taipei, Taiwan_.

In the process of tumorigenesis, angiogenesis provide oxygen and nutrients for tumor progression. Anti-angiogenesis has become a promising target of cancer therapy. Cullin-RING ligases (CRLs) are a cullin scaffold RING-finger domain containing E3 and are involved in the ubiquitination of specific substrates which regulate important biological processes. The holoenzyme E3 activity of CRLs is controlled by NEDD8. A novel NEDD8-activating enzyme inhibitor, MLN4924, is reported to block the neddylation of cullins and elicits antitumor effect. MLN4924 has been reported to be assoiciated with antiangiogenesis. In this study, we aim to investigate the role of anti-angiogensis in the antitumor effect of MLN4924. Our results showed that MLN4924 induces the cell cycle arrest, apoptosis and suppresses the cell viability and migration in human umbilical vein endothelial cells (HUVECs). Moreover, MLN4924 inhibits angiogenesis in Matrigel plug assay in vivo and tube-formation assay in vitro. Anti-angiogesis was involved in the antitumor effect on human urothelial carcinoma cells (T24) and colon cancer cells in vivo xenograft model. MLN4924 suppressed the VEGF-mediated VEGFR2 downstream activation.

#4440

Genetic knockdown of the E3 ubiquitin ligase Itch increases pancreatic cell line sensitivity to cancer therapeutics: A comparison between different gene editing techniques.

Oliver J. Read, David J. Harrison, Paul A. Reynolds. _University of St Andrews, St Andrews, United Kingdom_.

Introduction: E3 ligases are responsible for tagging ubiquitin onto substrates to either activate, relocate or target the substrate for degradation via the proteasome. Previous studies have shown that genetic knockdown of the E3 ubiquitin ligase Itch caused an increase in sensitivity to gemcitabine in pancreatic cancer models both in-vitro (cell lines) and in-vivo (mouse xenografts). Here we test the hypothesis that Itch knockdown sensitises pancreatic cell lines to existing cancer therapeutics, using both transient and stable knockdown methods.

Experimental Procedures: Knockdown of Itch in MiaPaCa2 (p53 mutant) and Capan 2 (p53 WT) cells was performed using both siRNA and CRISPR-Cas9 techniques; validated by western blot and RT-PCR. Alongside parental and scrambled controls, cells were treated with either doxorubicin, gemcitabine or γ-radiation. For doxorubicin and gemcitabine treatment, cells were exposed to the drug for 24 hours before it was removed. In the case of irradiation; cells were subject to 0-4Gy of radiation before being passaged onto plates. Cell viability was assessed in 96-well plates using a combination of a Sulforhodamine B assay (SRB) to quantify live cells 4 days post-treatment, and automated cytometry analysis (Celigo) to assess well confluence over each of the 4 days. Clonogenicity was also assessed by aliquoting cells at low initial density and treating the cells with distinct doses dependent on mode of treatment and cell line. After 7 days number of colonies using a colony formation algorithm on Celigo.

Data Summary: Transient knockdown of Itch in p53-/- cells was sufficient to decrease cell viability. This decrease in cell viability was further potentiated by the application of gemcitabine, doxorubicin or radiation. Stable knockdown using CRISPR-Cas9 increased MiaPaCa2 cell sensitivity to low-dose doxorubicin and radiation (5-10nM and 0.5-1Gy respectively). Whilst statistically significant, the change in response was not as large as that observed with the transient knockdown. In p53 WT cells there was no significant difference in cell sensitivity upon Itch knockdown.

Conclusions: The data from this investigation support the hypothesis of Itch being a possible target for sensitising pancreatic cell lines to cancer therapeutics. However the discrepancies in response to treatment between the transient and stable Itch knockdown scenarios raises the possibility that, in the case of the stable knockdown, the cells have adapted to the absence of Itch. This presents a potential weakness in using CRISPR-Cas9 in long-term experiments where the downstream consequences of gene silencing are not immediate.

#4441

Discovery of potent and selective inhibitors of USP7 with anti-tumor activity in vitro and in vivo.

Yamini M. Ohol, Michael Sun, Paul Leger, Dennis Hu, Berenger Biannic, Payal Rana, Cynthia Cho, Scott Jacobson, Steve Wong, Jerick Sanchez, Xinping Han, Kyle Young, Akinori Okano, Jack Maung, Gene Cutler, Nick Shah, Lavanya Adusumilli, Deepika Kaveri, Oezcan Talay, Deepa Pookot, Betty Abraham, Delia Bradford, Nathan Kozon, Christophe Colas, Andrea Kim, Jacob Schwarz, David Wustrow, Dirk Brockstedt, Paul Kassner. _FLX Bio, South San Francisco, CA_.

USP7 is a deubiquitinase that regulates the levels of multiple downstream targets with roles in cancer progression and immune response. Inhibitors of USP7 may thus decrease oncogene function, increase tumor suppressor function, enhance immune function and sensitize tumor cells to DNA damaging agents. We have discovered a novel chemical series that potently and selectively inhibits USP7 in biochemical and cellular assays. Our inhibitors reduce the viability of multiple p53-wild type cell lines, including several blood cancer and MYCN-amplified neuroblastoma cell lines, as well as a subset of p53-mutant tumor cell lines in vitro. Further, oral administration of our USP7 inhibitors inhibits MM.1S (multiple myeloma; p53-wild type) and H526 (small cell lung cancer; p53-mutant) tumor growth in vivo. Our work confirms that USP7 is a pharmacologically tractable target and future studies will aim to further understand the mechanism of action of USP7 inhibitors in p53-mutant cancers.

#4442

CUL7 promotes cancer cell survival through promoting Caspase-8 ubiquitination.

Yanjie Kong,1 Zehua Wang,1 Zhongmei Zhou,1 Xiaoyu Mao,2 Ceshi Chen1. 1 _Kunming Inst. of Zoology, Kunming, China;_ 2 _Breast Surgery, The First Affiliated Hospital of China Medical University, Shenyang, China_.

The Cullin 7 (CUL7) gene encodes a member of the cullin family of E3 ubiquitin ligases. Accumulated evidence suggests that CUL7 is oncogenic. However, the mechanism by which CUL7 improves cancer cell survival has not been fully elucidated. Here, we reported that CUL7 confers anti-apoptotic functions by interacting with Caspase-8. CUL7 prevents Caspase-8 activation by promoting Caspase-8 modification with non-degradative polyubiquitin chains at K215. CUL7 knockdown sensitized cancer cells to TRAIL-induced apoptosis in vitro and in nude mice. These results suggest that CUL7 limits extrinsic apoptotic signaling by promoting Caspase-8 ubiquitination.

#4443

Targeting E3 ligase PJA1 via TGF-β pathway in hepatocellular carcinoma.

Kazufumi Ohshiro,1 Jian Chen,2 Wilma Jogunoori,3 Chu-Xia Deng,1 Bibhuti Mishra,1 Shulin Li,2 Lopa Mishra1. 1 _George Washington University, Washington, DC;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 3 _George Washington Univ. Medical Ctr., Washington, DC_.

Background: RING-finger E3 ligases are instrumental in the regulation of inflammatory cascades, apoptosis, and cancer. However, their roles are relatively unknown for specific pathways such as TGF-β/Smad signaling that when disrupted, drive hepatocellular carcinoma (HCC). We, therefore, analyzed the TCGA database for the TGF-β pathway associated 22 E3 ligases, and identified mRNA alterations in 55% of tumors, most prominently for UCHL5 (16.4%), PJA (12.7%), WWP2 (11.8%), SKP2 (9.1%), SMURF1 and SMURF2 (8.2 and 9.1%, respectively), ITCH (6.4%) and KEAP1 (6.4%). We previously uncovered increased PJA1 expression with loss of TGF-β substrates Smad3 and β2SP in TGF-β deficient mice (β2SP+/-/Smad3+/- mice), which spontaneously develop characteristics of a human stem cell syndrome, and HCC by 12-15 months. Here, we report PJA1 oncogenic function in HCC progression. HCC.

Methods & Results: (1) Analyses of primary HCC datasets (91 cases) reveal increased PJA1 correlates with decreased levels of TGF-β/Smad3 and their regulated genes including Smad8 and TGFBR3. (2) To better understand the implications of PJA1-mediated deregulation of Smad3-promoted transcription in HCC, we compared transcriptomes of HepG2 cells silenced by PJA1 shRNA or treated with TGF-β: 1,584 genes including c-FOS were co-up-regulated and 1,279 genes including TERT were co-down-regulated. PJA1 expression in HCC was negatively associated with c-FOS and SERPINE1 expression. (3) PJA1 interacts with the Smad3 MH2 and Linker domains and multiple domains of β2SP to promote ubiquitin-mediated phosphor-Smad3 degradation in a TGF-β dependent manner, resulting in decreased TGF-β target gene activity. (4) We found that PJA1 expression in HCC is negatively associated with c-FOS and SERPINE1 expression. (5) Overexpression of a RING-domain-deleted PJA1 mutant reduced the proliferation rate in HCC cells and PJA1 knockdown significantly decreased anchorage-independent growth and tumorigenicity of HCC cells. (6) Our results also demonstrate that PJA1 promotes liver cancer stem cell formation in Smad3+/- mice.

Conclusions: This study demonstrates that loss of expression of β2SP and Smad3 through PJA1 could play an important role in HCC progression and that PJA1 is a potential novel therapeutic target for this lethal disease. 

## BIOINFORMATICS AND SYSTEMS BIOLOGY

### Convergence

#4444

Deep learning of the immune synapse.

John-William Sidhom, Drew M. Pardoll, Alexander S. Baras. _Johns Hopkins University, Baltimore, MD_.

Artificial intelligence is poised to revolutionize every aspect of human life, finding applications in everything from self-driving cars to diagnosing cancer. In fact, almost any task that involves pattern recognition can be formulated in a way that modern AI algorithms can be used to achieve super-human performance. The immune synapse is a highly complex interaction between several proteins and peptides that allows for a constant surveillance of foreign invaders. However, modeling these interactions is extremely difficult as the combinations of interactions is simply intractable. In immuno-oncology, the study of this interaction is crucial as anti-tumor responses rely on sensitive and specific recognition of tumor-specific antigens. Implications of accurately predicting and modeling these interactions in immune-oncology range from improved and potent vaccine design to biomarkers for predicting response to immunotherapy. Our group has developed a variety of deep learning models to model the signal transmission within the immune synapse. At the core of all architectures designed, convolutional layers, similar to ones used to learn features in images, are used to learn motifs within the sequencing data for a predictive or descriptive purpose.We first present AI-MHC, an applied deep convolutional neural network for class-specific MHC binding algorithm that achieves state-of-the-art performance in both Class and Class II predictions. By incorporating 'meaning' of the allele within the network, we are able to model the interaction of allele and peptide within the context of a neural network. We take these concepts further in the development of DeepMANA, a deep learning framework which combines sequence-specific information about an allele/peptide pairing to not only predict binding affinity for any allele with a known protein sequence but also provide an antigen 'quality' score, based on the "non-self/foreign-ness" of a neoantigen. We observe in three previously published immunotherapy clinical trials, these quality neoantigens are enriched in long-term survivors or responders. Finally, we present DeepTCR, a collection of unsupervised and supervised deep learning algorithms capable of revealing structure in T-cell receptor repertoire. We demonstrate that DeepTCR achieves state-of-the-art performance in clustering antigen-specific TCR's and is capable of learning a predictive signature in TIL repertoire of mice treated with various immunotherapies.These types of AI technologies could yield an entire new area of biomarker discovery as well as improve our understanding of the complex interaction occurring at the immune synapse that is ultimately required for a successful anti-tumor response.

#4445

MHC class II antigen identification for cancer immunotherapy by deep learning on tumor HLA peptides.

Tommy Boucher, Matthew Davis, Christine Palmer, Tyler Murphy, Andrew Clark, Fujiko Duke, Aaron Yang, Lauren Young, Karin Jooss, Mojca Skoberne, Josh Francis, Roman Yelensky, James Sun, Jennifer Busby. _Gritstone Oncology, Cambridge, MA_.

A key goal in immuno-oncology is the identification of tumor antigens recognized by T cells. Significant progress has been made in predicting MHC class I presentation of tumor-specific antigens (peptides) recognized by CD8 reactive T cells. However, predicting antigens recognized by CD4 T cells that are presented by the MHC class II has proven to be more challenging. Studies have shown binding affinity to be less predictive for MHC class II presentation than for class I. Class II antigen-directed therapeutics require accurate antigen identification from patient samples, which remains elusive today.

Methods: We focused initially on antigen presentation by the HLA-DR and generated a dataset of human tumor transcriptomes and HLA-DR immunopeptidomes from resections of B cell lymphomas (N=39). Transcriptomes were obtained by NGS of exome-captured cDNA and immunopeptidomes by immunoprecipitation using the HLA-DR specific Ab L243 and MS/MS. Each sample was typed for HLA-DRB1,3,4,5 using standard methods. Additionally, we obtained published class II mass spectrometry data for two B cell lines, each of which expressed a single common HLA class II allele (HLA-DRB1*15:01 and HLA-DRB5*01:01). RNA sequencing data was not available for either cell line; therefore, we substituted RNA-sequencing data from a different B cell line, B721.221. We combined these data to train a deep learning model of Class II HLA peptide presentation. Our model addressed two key challenges: (1) learning HLA-allele-specific models from tumor and normal data where each sample expressed up to 4 unique HLA-DR alleles and (2) reflecting information about all aspects of HLA presentation, including gene expression, antigen processing and stable binding of peptides to HLA. We evaluated the performance of the model on two independent test datasets. First, we tested the model on HLA peptides from a held-out sample from the lymphoma training dataset. Second, we tested the model on a separate public cell line expressing both HLA-DRB1*15:01 and HLA-DRB5*01:01.

Results: An average of 567 training and 203 testing peptides at q<0.01 and >50M unique transcriptome reads were obtained from the lymphoma samples. From cell-lines, 433 peptides for training and 223 peptides for testing were used. The model demonstrated a significant improvement in prediction accuracy, achieving >10%-point gain in area under the ROC curve (ROC AUC) vs a standard binding affinity-based predictor. On the held-out lymphoma test data, it achieved a ROC AUC of .95 vs the binding affinity model ROC AUC .80. On the cell-line test data, it achieved a ROC AUC of .90 vs the binding affinity model ROC AUC of .79.

Conclusion: We used a large dataset of transcriptomes and HLA peptidomes to train a deep learning model for HLA class II antigen presentation. The new model significantly outperforms standard methods and advances in silico HLA class II antigen selection for personalized cancer immunotherapy.

#4446

Assessment of tissue composition with digital pathology in colorectal cancer.

Enric Domingo,1 Aikaterini Chatzipli,2 Susan Richman,3 Andrew Blake,1 Claire Hardy,2 Celina Whalley,4 Keara Redmon,5 Ian Tomlinson,4 Philip Dunne,5 Steven Walker,6 Andrew Beggs,4 Ultan McDermott,2 Graeme I. Murray,7 Leslie M. Samuel,8 Matthew Seymour,3 Philip Quirke,3 Tim Maughan,1 Viktor H. Koelzer1. 1 _University of Oxford, Oxford, United Kingdom;_ 2 _Wellcome Trust Sanger Institute, Cambridge, United Kingdom;_ 3 _Leeds Institute of Cancer and Pathology, Leeds, United Kingdom;_ 4 _University of Birmingham, Birmingham, United Kingdom;_ 5 _Queens University, Belfast, United Kingdom;_ 6 _Almac Diagnostics, Belfast, United Kingdom;_ 7 _University of Aberdeen, Aberdeen, United Kingdom;_ 8 _Aberdeen Royal Infirmary, Aberdeen, United Kingdom_.

Background: The tumor microenvironment is a key feature to understand cancer biology and may be used clinically. Quantification of tissue composition is usually based either on visual pathological review (VPR) or deconvolution of whole genome molecular data. Although the former is a direct measurement it has modest reproducibility while the latter is an indirect measurement of unclear accuracy, expensive and not always available. Here we test digital pathology coupled with machine learning as a new tool to assess tissue composition.

Methods: As part of the Stratification in COloRecTal cancer (S:CORT) programme, a set of over 500 colorectal cancer (CRC) archival paraffin blocks from resections and biopsies were sequentially sectioned for Hematoxylin and Eosin staining (H&E), RNA extraction, a second H&E and DNA extraction. RNA expression microarrays, targeted DNA sequencing and DNA methylation arrays were applied. Tissue composition from the H&Es was obtained by VPR of expert pathologists and by a deep neural net (DNN) algorithm after supervised training on >1,500 tissue areas from S:CORT, TCGA, TEM and CORGI CRC cohorts. Tumor purity estimates were obtained from RNA and methylation arrays.

Results: DNN estimates including area and cell counts were obtained for tumor, desmoplastic stroma, inflamed stroma, mucin/hypocellular stroma, muscle, necrosis and white space. An average of 6.8x105 total cells (range: 1.2x104-2.8x106) and 1.2x105 (range: 7.2x104-1.8x106) were classified for resections and biopsies respectively. Analyses performed twice on the same H&Es obtained matching results (r=1.0). Comparison of the paired first and second H&E showed very high correlations (r~0.9) and total cell counts correlated with DNA and RNA extraction yields (r~0.6). Tumor purity estimates by VPR mildly correlated with DNN (r~0.5) but they were underestimated and very variable. As a result, copy number adjusted by VPR purity tended to be overestimated compared to adjustment with DNN estimates. The improved performance of DNN is reflected in an accurate capture of non-linear association between area and cell counts in invasive cancer. In contrast, tumor purity estimates derived from RNA or DNA methylation arrays showed better correlations compared with DNN (r~0.6) but both overestimated purity in cases with low cell counts by up to a three-fold difference.

Conclusions: Tissue composition analysis with DNN allows analytical robustness, automatization and standardization and provides very high reproducibility at single cell resolution. DNN-based estimation of tumor purity is more accurate than VPR or extrapolation from molecular data derived from genome-wide omic platforms which tend to under and overestimate tumor purity respectively. DNN could be used to better plan and asses downstream molecular analyses and to investigate tissue-based metrics as potential clinical biomarkers in clinical trials.

#4447

Deep neural network for automatic histopathological analysis of murine lung tumors.

Peter Maxwell Kienitz Westcott,1 Tuomas Pitkänen,2 Sami Blom,2 Thomas Westerling,2 Tuomas Ropponen,2 Nathan Sacks,1 Katherine Wu,3 Roderick Bronson,4 Tuomas Tammela,3 Tyler Jacks1. 1 _MIT Koch Inst. for Integrated Cancer Res., Cambridge, MA;_ 2 _Fimmic Oy, Helsinki, Finland;_ 3 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 4 _Harvard Medical School, Cambridge, MA_.

Lung cancer is the leading cause of cancer-related deaths worldwide, and genetically-engineered mouse models (GEMMs) of cancer provide important mechanistic and preclinical insights into this deadly disease. In particular, the "KP" model enables lung-specific inducible activation of oncogenic Kras G12D, and loss of Trp53, the two most common driver events of human non-small cell lung cancer (NSCLC). Importantly, the KP model is widely used and faithfully recapitulates molecular and histopathological features of the human disease, including progression from early hyperplasia and adenoma to invasive adenocarcinoma. However, the KP model results in multi-focal and heterogeneous tumor burden, and there is a need for improved tools to increase throughput and decrease subjectivity of tumor burden quantification and histopathological analyses.

To this end, we trained a convolutional neural network (CNN) for semantic multi-class segmentation using the Aiforia(R) platform. The CNN was trained to classify and detect lung parenchyma, NSCLC tumors, and NSCLC tumor grades (grade 1-4). For supervised training, we used selected areas from 93 hematoxylin and eosin stained slides. For validation, we analyzed 34 slides completely independent of the CNN training. Tumor grades were manually annotated on the validation slides blinded to the CNN results. The overall F1 score of the CNN in grade classification was 98% and total area error 0.3%. The grade-specific F1-scores were 89%, 97%, 99%, and 98% for grades 1, 2, 3, and 4, respectively. Corresponding grade-specific total area errors were 0.4%, 0.2%, 0.4%, and 0.1%. Manual scoring independent of the training and CNN yielded similar tumor burden and grading results. In addition, the algorithm accurately recapitulates the increased tumor burden and grade seen in KP tumors harboring additional mutation of the tumor suppressor Keap1, and the delayed kinetics of KP tumors harboring a strong T cell antigen, in independent datasets. We have also extended this methodology to identification of tumors in a GEMM of small cell lung cancer, a distinct class of lung cancer with poor prognosis.

In conclusion, we demonstrate that deep neural networks can be used for automated analysis and grading of preclinical models of lung cancer. We anticipate that this powerful technology will increase the throughput, sensitivity and reproducibility of hypothesis-driven studies of factors influencing tumor progression and immune response in mouse models of lung cancer.

#4448

Machine-learning assisted histopathology (HistoMAP) links nuclear membrane instability to disease progression in prostate cancer.

Andries Zijlstra,1 Tatiana Novitskaya,1 Dolores Di Vizio,2 Mariana Reis- Sobreiro,2 Michael Freeman2. 1 _Vanderbilt University Medical Center, Nashville, TN;_ 2 _Cedar-Sinai Medical Center, Los Angeles, CA_.

Introduction: The manifestation of cancer malignancy occurs at the cellular level where individual cells escape tissue confinements and disseminate. Identifying this malignant behavior at a cellular level is necessary in order to deconvolve the tumor heterogeneity, identify disease progression and predict cancer-related morbidity and mortality. To achieve that goal, we established a unique computer-assisted single-cell histopathology analysis of prostate tissue that evaluates nuclear-membrane instability and the associated extracellular vesicle (EV) biogenesis to identify malignant potential and assess future disease progression. In recent years cancer EVs have been identified as important mediators of intercellular communication. The potential for using EVs as a liquid biopsy has promoted research on profiling EVs in biofluids. However, the direct analysis of EV biogenesis in tumor tissue has been largely omitted, even thought identifying such cells is likely to be a specific and sensitive measure of disease malignancy. We have previously demonstrated that highly migratory and metastatic cancer cells shed atypically large EVs, known as large oncosomes (LO, Di Vizio et. al., 2012). LO play distinct functions and contain a specific repertoire of molecules that can be used for detection of tumor-derived cargo in plasma (Minciacchi et. al., 2015). The recent discovery that the biogenesis of large oncosomes (LO) in prostate cancer is associated with nuclear membrane instability (Reis-Sobreiro et. al., 2018) offered an opportunity to investigate such biogenesis in patient specimens. We have developed a machine-learning assisted histopathology (HistoMAP) that quantitatively identifies nuclear-membrane instability in prostate cancer tissue and demonstrate a direct correlation between this molecular biogenesis of vesicles and biochemical recurrence after prostatectomy.

Experimental procedures: We developed a multiplex immunofluorescent technique to detect and quantify nuclear-derived LO in formalin fixed paraffin embedded prostatectomy tissues. Using computer-assisted image segmentation and machine-learning we distinguished these LO from all other intracellular compartments and assess malignancy in a cohort of Vanderbilt prostate cancer patients.

Results: LO production was significantly elevated in prostate cancer in comparison to benign tissue and particularly evident in lymph node metastases. Moreover, LO production was associated with the risk of future metastatic disease which re-enforced its relevance in cancer progression.

Conclusions: Our findings demonstrate that, given knowledge of the EV machinery, EV biogenesis can be detected in patient tissues and provide critical information related to patient disease status and assist with the prediction of future clinical performance.

#4449

Designing precision metabolic therapies by identifying vulnerabilities in metabolic pathways due to genomic loss in cancer patients.

Abhinav Achreja, Anjali Mittal, Ziwen Zhu, Justin G. Reinhold, Reva Kulkarni, Deepak Nagrath. _University of Michigan, Ann Arbor, MI_.

Genomic instabilities and metabolic reprogramming are two ubiquitous hallmarks of cancer that are not mutually exclusive. Although, loss of genomic regions containing tumor-suppressor genes (TSGs) are associated with distinct mechanisms of cancer progression, their impact on cancer metabolism has been overlooked. Our previous work has shown that genomic loss can lead to loss of genes that encode essential metabolic genes; However, cancer cells exploit biological redundancies and a complex network of metabolic pathways to compensate for the loss of metabolic function. Current studies are limited to loss-of-metabolic-function for frequently occurring homozygous deletions in TSGs that are well-characterized. As such, genomic loss occurring infrequently or not associated with TSGs there is no formalized platform to identify patient-specific metabolic vulnerabilities emerging due to distinct pattern of genomic loss events in each patient. Our integrated platform presents novel opportunities for precision-based therapeutic intervention by targeting metabolic vulnerabilities in cancer patients. Our novel algorithm utilizes patient genomic and clinical data available in public cancer patient databases to obtain candidate metabolic genes that are lost to genomic deletions. A machine-learning algorithm is employed to stratify patients according to genomic loss events and clinical parameters. We then predict collateral lethal genes involved in compensatory metabolic pathways using an innovative multi-objective metabolic flux analysis approach. The predicted collateral lethal targets are validated using RNA interference (RNAi) screening databases of cancer cell-lines to obtain a tractable number of targets. Our integrated algorithm was able to predict previously unexplored metabolic vulnerability in breast, lung, ovarian cancers and multiple myeloma. These predictions were validated empirically to demonstrate the potential of precision metabolic targets in tumors with distinct clinical and genetic traits.

#4450

Novel transducer array layouts to optimize the treatment of multiple brain metastases with tumor treating fields.

Ofir Yesharim, Ariel Naveh, Ze'ev Bomzon. _Novocure, Haifa, Israel_.

Introduction:

Tumor Treating Fields (TTFields) is an antimitotic cancer treatment approved for the treatment of Glioblastoma Multiforme (GBM). TTFields are delivered using 2 pairs of transducer arrays positioned on the shaved scalp of the GBM patient to optimize the TTFields dose delivered to the tumor. This results in an increased dose of TTFields to the affected tumor region, whilst reducing the intensity of the fields to other regions. Brain metastases, or secondary brain tumors, occur in 10 to 30 percent of adults with cancer. Cancer types most likely to cause brain metastases are lung, breast, colon, kidney and melanoma. When using TTFields to treat brain metastases, it may be desirable to deliver TTFields at therapeutic intensities to the entire brain, in order to treat multiple rather than a single lesion. We investigated novel transducer array layouts designed to deliver a uniform distribution of TTFields to the entire brain.

Methods

Computer simulations were used to calculate the field distributions generated by different array layouts. The simulations utilized a realistic computerized head model of a 40+ years old human male prepared in-house from a T1 MRI series. Using Sim4Life v3.0 (ZMT Zürich), we simulated the delivery of TTFields using pairs of array layouts placed at different locations on the head and neck. To analyze the field distributions, the brain was divided into five regions: (1) the cerebellum, brain stem and other infra-tentorial anatomical regions; and (2-5) the four quadrants of the cerebrum. The mean and median field intensities in the five regions generated by each layout were calculated and compared.

Results

Median intensities between 1.5 V/cm to 1.7 V/cm within all regions were achieved using a layout in which one pair of arrays was placed on the right temple and left scapula, while the second pair was placed on the left temple and right scapula. This layout yielded a uniform intensity distribution within the brain.

Conclusion

We have identified a novel TTFields array layout that could potentially be used to treat the entire brain with sufficiently high intensity to treat for brain metastases. The ongoing Phase 3 METIS study [NCT02831959 is investigating radiosurgery with TTFields for 1-10 brain metastases from non-small cell lung cancer.

## CANCER CHEMISTRY

### Next-Generation Small Molecules: From Hits to Leads to Candidates

#4451

Evaluation of the therapeutic potential of phosphine oxide pyrazole inhibitors in tumors harboring EGFR C797S mutation.

Nicolas Floch, M. Raymond V. Finlay, Ambra Bianco, Sue Bickerton, Nicola Colclough, Darren A. Cross, Emanuela M. Cuomo, Carine M. Guerot, David Hargreaves, Matthew J. Martin, Darren McKerrecher, Daniel J. O'Neill, Jonathan P. Orme, Amar Rahi, Paul D. Smith, Richard A. Ward. _AstraZeneca, Cambridge, United Kingdom_.

Osimertinib is a next-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) with activity against both the activating and the 'gatekeeper' T790M EGFR mutations. An acquired EGFR C797S mutation has been reported to mediate osimertinib resistance in approximately 15% and 7% of patients in second-line and first-line treatment respectively. This percentage in the first-line setting will likely evolve as the first line data mature. The C797S mutation leads to the loss of covalent binding of osimertinib to mutant EGFR. The high affinity of the EGFR triple mutant for ATP presents a challenge for reversible inhibitor design, particularly as the loss of the cysteine at position 797 precludes the previously exploited covalent approaches. We have explored various approaches to address this challenge, including an effort to maximise reversible affinity to target the C797S mutation without requiring a covalent bond. We describe herein the therapeutic potential of reversible phosphine oxide pyrazole inhibitors in tumors harboring C797S.

Using structure-based design, we were able to design a series of phosphine oxide pyrazole inhibitors that displayed exceptionally high biochemical potency against EGFR C797S mutation, which translated into good activity in cell-based assays. Using CRISPR-Cas 9 genome editing technology, we engineered cellular disease-relevant models to express the C797S mutation to evaluate potency in vitro and in vivo.

By modulating the physicochemical properties of our in vitro leads, we were able to achieve good oral exposure of cellularly active EGFR C797S inhibitors such as AZ'7608. We showed that AZ'7608 inhibits signalling pathways and cellular growth of C797S EGFR cell lines in vitro and demonstrated an improved WT EGFR margin. This translated into 52% (p<0.05, at day 14) tumor growth inhibition in vivo when compared to the control group. The efficacy of AZ'7608 is enhanced by its combination with anti-EGFR antibody, showing tumor regression (82%, p<0.001). In addition, we performed pharmacodynamic studies to explore the relationship between efficacy and target/pathway modulation. These studies establish a clear relationship between depth and duration of inhibition of the phosphorylation of EGFR and anti-tumor efficacy.

The work presented herein shows a proof of concept for reversible phosphine oxide pyrazole inhibitors to target tumors harboring C797S. The emergence of the C797S EGFR mutation remains a key area of unmet need and warrants further efforts in drug discovery.

#4452

GNE-0011, a novel monovalent BRD4 degrader.

Robert A. Blake. _Genentech, Inc., South San Francisco, CA_.

The bromodomain and extra-terminal (BET) family of proteins (BRD2, 3, 4 and T) has been the focus of an emerging paradigm in drug mechanism of action, whereby a drug is designed to trigger the proteolytic degradation of the target protein. The current design of protein degraders is typically based on bivalent molecules consisting of a ligand for the target protein and a ligand for a ubiquitin ligase (such as VHL, CRBN or XIAP). The degrader causes the association of the target protein with the E3 ubiquitin ligase and its subsequent ubiquitination and degradation. Due to their bivalent design such drugs typically have a higher molecular weight than classic small molecule drugs and may present some non-ideal properties as drugs. An alternative strategy for designing drugs that act through the degradation of the target protein is exemplified by the group of drugs termed SERDs (selective estrogen receptor degraders), for example fulvestrant. The molecular structures of SERDs are typically designed around an estrogen receptor ligand and a "degradation tail", whose presence results in the degradation of the estrogen receptor. We will discuss the development of GNE-0011, which represents a novel class of monovalent BRD4 degrader that uses a "degradation tail" strategy.

#4453

**Novel, potent and selective small-molecule inhibitors modulating immuno-oncology targets CD73, A** 2A **/A** 2B **adenosine receptors and CSF1R discovered via DNA-encoded library screening.**

Andrew J. McRiner,1 Jannik N. Andersen,2 Lynette A. Fouser,2 Junyi Zhang,1 Kaan Certel,1 John Cuozzo,1 Betty Chan,1 Ragunath Chandran,1 Matt Clark,1 Diana Gikunju,1 Christopher D. Hupp,1 Anthony D. Keefe,1 Julie Liu,1 Yanbin Liu,1 Michael Monteiro,1 Allison Olszewski,1 Moritz Von Rechenberg,1 Daniel Resnicow,1 Heather A. Thomson,1 Dawn M. Troast,1 Zooey Wang,1 Neil Westlund,1 Ying Zhang,1 Fei Zhou,1 Xiaotian Zhu,1 Michael Briskin,2 Diala Ezzeddine2. 1 _X-Chem Pharmaceuticals, Waltham, MA;_ 2 _Xios Therapeutics, Waltham, MA_.

Tumors utilize many different escape mechanisms to evade anti-tumor immune responses. Xios Therapeutics has screened X-Chem's proprietary 200-billion molecule DNA-encoded library against several immuno-oncology (IO) targets addressing T-cell centric, myeloid immunity and onco-metabolite pathways. Adenosine, for example, is a potent immunosuppressive metabolite, and the ecto-5-nucleotidase (a.k.a. CD73), which catalyzes the conversion of AMP to adenosine, is the rate limiting enzyme for the production of extracellular adenosine in the tumor microenvironment. Hence, CD73 and/or the downstream adenosine receptors are considered attractive targets for IO drug discovery. Likewise, the characteristics of tumor-associated macrophages (TAMs) have fueled interest in therapeutically targeting the colony-stimulating factor 1 axis (CSF1R). Here, we exemplify and enumerate the diversity, selectivity and physiochemical properties of selected hit-to-lead compounds identified from our DNA-encoded library screens of CD73, the adenosine A2A receptor and CSF1R.

In the context of the immune-suppressive purinergic pathway, we have integrated structural biology, medicinal chemistry and clinical pathology evaluation of target expression across tumor types and developed both A2A selective and dual A2A/A2B selective receptor antagonists. From a DNA-encoded library screen, we have identified novel sub-micromolar ligands, which binds to CD73 in an 'open conformation' revealed by the co-crystal structure of X6034 (EC50 = 310 nM) in complex with CD73. Interestingly, an inorganic phosphate molecule (Pi) that is structurally shown to be co-present in the active site, illustrates the novelty of these inhibitors, which are chemically distinct from currently reported ADP/AMP substrate analogs. Finally, using tumor tissue microarrays and in situ cell hybridization (RNA-Scope; Advanced Cell Diagnostics), we have explored the co-expression pattern of pathway targets across a subset of immune cells. Informed by the tissue expression pattern of A2AR (primarily CD3+ T-Cells) and A2BR (primarily CD33+ myeloid cells), we compared the ability of equipotent A2A and dual A2A/A2B adenosine receptor antagonists to reverse the effect of NECA, a nonhydrolyzable analog of adenosine, on the maturation and activation of dendritic cells (DC). Starting with the differentiation of human immature DC, we demonstrate the added benefit of dual A2A/A2B inhibitors vs A2A selective inhibitors on relieving immunosuppression of myeloid cells. To conclude, the combination of all these data, together with the productivity of the DNA-encoded library screens for identifying novel, drug like chemical matter on challenging targets like CD73, provide a unique opportunity for potentially harnessing the full power of immunotherapy for cancer.

#4454

Identification of BAY-218, a potent and selective small molecule AhR inhibitor, as a new modality to counteract tumor immunosuppression.

Norbert Schmees,1 Ilona Gutcher,1 Ulrike Roehn,1 Horst Irlbacher,1 Detlef Stoeckigt,1 Benjamin Bader,1 Christina Kober,1 Lars Roese,1 Rafael Carretero,1 Iris Oezen,2 Ludiwg Zorn,1 Michael Platten,2 Ingo V. Hartung,1 Bertolt Kreft,1 Hilmar Weinmann1. 1 _Bayer AG, Pharmaceuticals Division, Berlin, Germany;_ 2 _DKFZ Heidelberg, Heidelberg, Germany_.

Re-constitution of anti-tumor T-cell responses by clinically-approved immune checkpoint inhibitors (ICIs) targeting CTLA4 or PD-1/PD-L1 represents a breakthrough cancer therapy. Nevertheless, a substantial number of patients do not benefit from these new therapeutic modalities chiefly due to local immunosuppression in the tumor microenvironment. In addition, the long circulation time of ICIs restricts options to modify dosing regimens for management of adverse effects. Oral small molecule inhibitors as next generation immune-oncology agents may - in contrast to antibodies - allow targeting of intracellular targets for a defined duration of time. This will permit a fine-tuning of efficacy versus tolerability in single-agent treatment as well as in combination with approved ICIs. The overexpression of indole dioxygenase (IDO1) and tryptophan dioxygenase (TDO2) by many tumors results in increased metabolism of tryptophan (TRP) into kynurenine (KYN), which induces immunosuppression via activation of the aryl hydrocarbon receptor (AhR). Inhibition of AhR was proposed to restore T-cell function and induce tumor rejection. However, it was expected that identification of selective lead candidates for AhR inhibition would be challenging due to the known affinity of the AhR ligand binding site for polyaromatic ligands such as 2,3,7,8-tetrachlorodibenzodioxine (TCDD). A library of 4 million compounds was screened in a cell-based HTS campaign. A thorough hit reduction process was performed based on stringent filter parameters for lead-likeness. This process delivered a hit set of significant chemical diversity. Out of several compound classes with drug-like properties, 1,3-diaryl-pyrazin-6-one-5-carboxylic amides were selected as a preferred lead series. A comprehensive SAR exploration, including in vitro mechanistic and functional validation, was performed. Lead optimization was strongly emphasized on improving lipophilicity efficiency (LLE) to balance potency with a viable PK and CYP450 interaction profile. Several candidates suitable for in vivo profiling were identified and BAY-218 was advanced to in-depth pharmacodynamic and pharmacokinetic in vivo assessments. BAY-218 showed mono-therapeutic efficacy that was comparable to ICI treatment and further therapeutic improvement was achieved by combination with an anti-PD-L1 antibody. We were able to characterize 1,3-diaryl-pyrazin-6-one-5-carboxylic amides as a new and unprecedented class of AhR inhibitors as well as identify the key substitutions that contribute to the overall compound profile.

#4455

**Discovery of AMG 510, a first-in-human covalent inhibitor of KRAS** G12C **for the treatment of solid tumors.**

Brian A. Lanman,1 Jian Jeffrey Chen,1 Longbin Liu,1 Patricia Lopez,1 Alexander J. Pickrell,1 Anthony B. Reed,1 Hui-Ling Wang,1 Pragathi Achanta,1 Jude Canon,1 Daniel A. Erlanson,2 Raymond V. Fucini,2 Joon Won Jeong,2 Christopher Mohr,1 Anne Y. Saiki,1 Victor J. Cee,1 J. Russell Lipford,1 Karen Rex,1 Laurie P. Volak1. 1 _Amgen, Inc., Thousand Oaks, CA;_ 2 _Carmot Therapeutics, Inc., Berkeley, CA_.

The RAS gene family encodes the small GTPase proteins NRAS, HRAS, and KRAS, which play an essential role in cellular growth and proliferation. KRAS is one of the most frequently mutated oncogenes in human cancer, with KRAS p.G12D, p.G12V, and p.G12C constituting the major mutational subtypes across lung, colon, and pancreatic cancers. Despite more than three decades of research, indirect approaches targeting KRAS mutant cancers have largely failed to show clinical benefit, and direct approaches have been stymied by the apparently 'undruggable' nature of KRAS. Cysteine-12 of KRASG12C has recently emerged as a unique vulnerability in KRAS-mutant cancers, and a small number of cysteine-reactive inhibitory tool molecules have been disclosed. We here report independent efforts to identify cysteine-reactive molecules capable of selectively inhibiting KRASG12C. Through iterative screening and structural biology efforts, we identified a novel Cys12-reactive inhibitor scaffold that derived its potency from occupancy of a previously unknown cryptic pocket induced by side-chain motion of the His95 residue of KRAS. Employing a scaffold-hopping approach, we leveraged knowledge of this cryptic pocket to design a series of N-aryl quinazolin-2(1H)-one-based inhibitors that demonstrated significantly enhanced potency relative to prior tool compounds. Extensive optimization of these leads led to the identification of a highly potent, selective, and well-tolerated inhibitor of KRASG12C, which was nominated for clinical development as AMG 510. In preclinical tumor models, AMG 510 rapidly and irreversibly binds to KRASG12C, providing durable suppression of the mitogen-activated protein kinase (MAPK) signaling pathway. Dosed orally (once daily) as a single agent, AMG 510 is capable of inducing tumor regression in mouse models of KRASG12C cancer. AMG 510 is, to the best of our knowledge, the first direct KRASG12C therapeutic to reach human clinical testing and is currently in a Phase I clinical trial evaluating safety, tolerability, PK, and efficacy in subjects with solid tumors bearing the KRAS p.G12C mutation (NCT03600883).

#4456

Discovery of E7766: A representative of A novel class of macrocycle-bridged STING agonists (MBSAs) with superior potency and pan-genotypic activity.

ATSUSHI ENDO, Dae-Shik Kim, Kuan-Chun Huang, Ming-Hong Hao, Steven Mathieu, Hyeong-wook Choi, Utpal Majumder, Xiaojie Zhu, Yongchun Shen, Kristen Sanders, Thomas Noland, Dinesh Chandra, Yu Chen, Karen TenDyke, Kara Loiacono, Donna Kolber-Simonds, Rongrong Jiang, Vaishali Dixit, Janna Hutz, John Wang, Xingfeng Bao, Francis Fang, Nadeem Sarwar. _Eisai Inc., Andover, MA_.

Introduction

STING (stimulator of interferon genes) is an emerging target for cancer immunotherapy. 2',3'-cGAMP, a natural cyclic dinucleotide (CDN) STING agonist, and its phosphorothioate analogs, have drawn broad attention as lead molecules for STING targeted drug discovery. These CDNs, however, lack efficacy in some common STING genotypes disproportionally represented in non-Caucasians. Moreover, such CDNs have not fully addressed liability in chemical/metabolic stability. Here we report our chemistry approach to control STING agonist conformation to enhance binding affinity across all common STING genotypes and broaden the therapeutic potential of such compounds.

Methods

Our SBDD approach started with analysis of the binding pocket and key protein-ligand interactions to prioritize a focused set of analogs for chemical synthesis. Systematic SAR was built upon in vitro assays for STING binding affinity and activation of STING genotypes. X-ray single crystal structures were established for STING and diverse analogs, in free and bound states, to provide structural insight for rational analog design.

Results

Structural modeling was refined to evaluate different binding modes and dynamic conformational changes in the STING-ligand interface. We observed that STING-bound CDNs had the two ancillary nucleobases specifically oriented in close proximity with parallel pi-pi stacking and discovered that covalently linking the nucleobases advantageously pre-organize the bioactive constrained conformation for enhanced STING affinity. Our discovery established a novel class of macrocycle-bridged STING agonists (MBSAs). E7766, a representative of Eisai MBSA platform, shows superior in vitro activity against all the major human STING genotypes over reference CDNs, most distinctly in STINGREF. E7766 co-crystal structures with STINGWT and STINGREF provide structural basis for the added benefit of the topological novelty. The macrocyclic linker bridging the top of nucleobases perturbs the STING lid loop conformation and create new and specific interactions with both genotypes. In twelve subcutaneous tumor models in immune competent mice, single intra-tumoral injections achieved either complete regression or significant tumor growth delay with no serious adverse effect. E7766 also shows excellent chemical and metabolic stability, presumably conferred by conformational rigidity of the unique macrocycle bridge. More biological characterization of E7766 can be found in abstract #.

Conclusion

Eisai successfully discovered E7766, a representative of a novel class of macrocycle-bridged STING agonist topologically distinct from conventional STING agonists. E7766 demonstrated pan-genotypic STING activation, potent anti-cancer activities and excellent chemical and metabolic stability for further development.

#4457

Open science medicinal chemistry: Towards a treatment for DIPG.

Sue Cramp,1 Nicole Hamblin,1 Jeff O'Meara,2 Aled Edwards,3 Owen Roberts,3 Alex Bullock,4 Paul Brennan4. 1 _Charles River Early Discovery, Harlow, United Kingdom;_ 2 _Ontario Institute for Cancer Research, Toronto, Ontario, Canada;_ 3 _M4K Pharma, Toronto, Ontario, Canada;_ 4 _Structural Genomics Corporation, Oxford, United Kingdom_.

Meds4Kids Pharma (M4K)1 is pioneering an open science approach to drug discovery, focussed on the discovery and development of small molecule therapeutics for orphan paediatric cancers. M4K seeks to test the hypothesis that an open science framework can be successfully applied not only to accelerate basic science, using the collective knowledge of the scientific community at large, but also to take an innovative new drug candidate all the way from discovery and clinical proof-of-concept through to product registration, by making use of regulatory data protections and market incentives. Since late 2017, Charles River Early Discovery has been providing in kind drug discovery services to help progress these efforts, including medicinal and synthetic chemistry. The first M4K project aims to generate an orally-available, brain-penetrant therapeutic to treat the diffuse intrinsic pontine glioma (DIPG). DIPG is a rare, aggressive and uniformly fatal childhood brain cancer with a median survival time of 9-12 months and for which there are currently no effective drug treatments. The disease has been shown to be associated with mutations in the ACVR1 gene (activin A receptor, type 1) also known as ALK2 kinase. Early support for the therapeutic hypothesis that an inhibitor of ALK2 kinase would have clinical benefit in DIPG, came from in vivo studies with non-selective ALK2 kinase inhibitors, which both killed DIPG cell lines harbouring the ALK2 mutation and extended lifespan in xenograft mouse models.2 Working collaboratively with M4K and their open science partners, we have made excellent progress towards the identification of potent, selective, brain penetrant ALK2 inhibitors starting from the known inhibitor LDN-214117, previously described for FOP (fibrodysplasia ossificans progressiva).3 Our current lead compound has an excellent in vitro and in vivo profile showing high oral bioavailability and brain penetration in a mouse PK study and is progressing to proof of concept studies. In addition we are progressing with the identification of a back-up series. References: 1. https://m4kpharma.com/ 2. Carvalho et. al. Neuro-Oncology, Volume 18, Issue suppl_6, 1 November 2016, Pages vi154, https://doi.org/10.1093/neuonc/now212.639 3. Mohedas et. al., J. Med Chem, 2014, 57 (19), 7900

## CLINICAL RESEARCH

### Molecular and Cell-Based Circulating Biomarkers to Guide Optimal Anticancer Treatment

#4458

**Clinical significance of** PIK3CA **and** ESR1 **mutations in ctDNA and FFPE samples from the MONARCH 2 study of abemaciclib plus fulvestrant.**

Sara M. Tolaney,1 Masakazu Toi,2 Patrick Neven,3 Joohyuk Sohn,4 Eva-Maria Grischke,5 Antonio Llombart-Cussac,6 Hatem Soliman,7 Lacey M. Litchfield,8 Sameera Wijayawardana,8 Tammy Forrester,8 Valerie M. Jansen,8 George W. Sledge9. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Kyoto University, Kyoto, Japan;_ 3 _UZ Leuven, Leuven, Belgium;_ 4 _Yonsei Cancer Center, Seoul, Republic of Korea;_ 5 _Universitats-Frauenklinik Tubingen, Tübingen, Germany;_ 6 _FISABIO - Hospital Arnau Vilanova, Valencia, Spain;_ 7 _H. Lee Moffitt Cancer Center, Tampa, FL;_ 8 _Eli Lilly and Company, Indianapolis, IN;_ 9 _Stanford University, Stanford, CA_.

BACKGROUND: Mutations in PIK3CA and ESR1 have been implicated in resistance to endocrine therapy in HR+ metastatic breast cancer. Herein, we assessed the clinical significance of PIK3CA and ESR1 mutations from ctDNA and FFPE samples from patients treated with abemaciclib plus fulvestrant and placebo plus fulvestrant.

METHODS: Baseline plasma samples (n = 334) and FFPE tumor samples (n = 434) from 669 patients enrolled in MONARCH 2 were analyzed. Extracted DNA was analyzed by droplet digital PCR for four hotspot mutations of PIK3CA (E542K; E545K; H1047L; H1047R) and ESR1 (D538G; Y537C; Y537N; Y537S). Samples that failed DNA QC or where mutation status could not be determined were excluded from analysis.

RESULTS: PIK3CA mutations were detected in 96 (40.3%) of 238 plasma samples and 133 (39.9%) of 333 FFPE samples. H1047R was the most frequent mutation followed by E545K, E542K, and H1047L. The concordance of PIK3CA mutations in ctDNA and FFPE samples was 62.8%. ESR1 mutations were detected in 190 (64.4%) of 295 plasma samples and 15 (4.4%) of 344 FFPE samples. D538G was the most frequent mutation followed by Y537C, Y537N, and Y537S. The concordance of ESR1 mutations in ctDNA and FFPE samples was 37.1%; this rate of detection of ESR1 mutations in ctDNA and FFPE samples is explained in part by the site of the biopsy (primary vs metastatic). Co-mutations in PIK3CA and ESR1 were observed in 86 (36.1%) plasma and 5 (1.5%) FFPE samples. Finally, we examined the relationship of PIK3CA and ESR1 mutation status and benefit from abemaciclib therapy as shown in Table 1.

CONCLUSIONS: PIK3CA and ESR1 mutations in ctDNA but not in archived FFPE samples correlated with response to abemaciclib, confirming the potential use of ctDNA analysis. The addition of abemaciclib to fulvestrant in MONARCH 2 demonstrated improvement in PFS regardless of PIK3CA or ESR1 status; however, the magnitude of benefit was numerically greater for patients with tumors harboring PIK3CA/ESR1 mutations.

Table 1.  | |  | |

|

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

|

Abemaciclib + Fulvestrant | Placebo + Fulvestrant

|

Mutation Status (Plasma) | n | Median PFS (95% CI) | n | Median PFS (95% CI) | Hazard Ratio (95% CI)

PIK3CA wild-type | 91 | 20 (14, NA) | 51 | 12.7 (7.9, NA) | 0.68 (0.42, 1.09)

PIK3CA mutant | 58 | 15 (9.4, NA) | 38 | 5.7 (3.8, 15) | 0.46 (0.27, 0.78)

ESR1 wild-type | 72 | 16.3 (12.4, 22.8) | 33 | 11.6 (7.4, 23.1) | 0.69 (0.41, 1.18)

ESR1 mutant | 118 | 21.9 (15, NA) | 72 | 10.3 (5.7, 15.6) | 0.49 (0.33, 0.73)

#4459

**Morphologic and genomic characterization of circulating tumor cells in patients with** ERBB2 **mutant HER2 non-amplified metastatic breast cancer treated with neratinib.**

Stephanie Nicole Shishido, Rahul Masson, Lisa Welter, Liya Xu, Anishka D'Souza, Darcy Spicer, James Hicks, Janice Lu, Peter Kuhn. _USC, Los Angeles, CA_.

Metastatic breast cancer patients have a high risk of progression and face poor prognosis overall, with about one third (34%) surviving 5 years or more. In rare instances (2-4% of cases) patients with metastatic breast cancer have ERBB2 (HER2) activating mutations, but are HER2 negative (non-amplified. Neratinib is a potent, irreversible inhibitor that binds HER2 and inhibits downstream signaling. We used the previously validated high-definition single cell analysis (HD-SCA) workflow to investigate the clinical significance of circulating tumor cells (HD-CTCs) in HER2-mutant, HER2 non-amplified, post-menopausal metastatic breast cancer patients starting neratinib and fulvestrant combinational therapy. Genomic analysis of the cell-free DNA (cfDNA) and single cell HD-CTCs was conducted to monitor tumor evolution and identify potential mutational variants unique to the patient's clinical response. A subset of 5 patients presented here were from the MutHER (NCT01670877) and/or SUMMIT (NCT01953926) trials. Patients had an average of 5.4 lines of therapy before enrollment, variable hormone receptor status, and ERBB2 mutations at diagnosis and during treatment. HD-CTC enumeration alone was not sufficient to predict clinical response. Interestingly, treatment pressure was shown to lead to an observable change in HD-CTC morphology and genomic instability, suggesting these parameters may inform prognosis. Single cell copy number variation (CNV) analysis indicated that the persistence or development of a clonal population of HD-CTCs during treatment was associated with a worse response. Hierarchical clustering analysis of the single cells across all patients and timepoints identified distinct aberrant regions shared among patients, comprised of 26 genes that are similarly affected and may be related to drug resistance. Additionally at the cfDNA level, we identified new mutations in ERBB2, PIK3CA, and TP53 that arose due to treatment pressure in a patient with poor response, further informing us on the dynamics of the cancer genome over the course of therapy. The data presented in this small cohort study demonstrates the feasibility of real-time molecular profiling of the cellular and acellular fractions of the liquid biopsy using the HD-SCA platform. Additional insight is warranted to determine the potential use of morphometric and genomic analysis as a prognostic tool to advance personalized oncology.

#4460

Clinical utility of comprehensive cell-free DNA (cfDNA) analysis to identify genomic biomarkers in newly diagnosed metastatic non-small cell lung cancer (mNSCLC).

Natasha Leighl,1 Ray D. Page,2 Victoria M. Raymond,3 Davey Daniel,4 Stephen Divers,5 Karen L. Reckamp,6 Miguel A. Villalona-Calero,7 Daniel Dix,3 Matthew Jackson,3 Justin I. Odegaard,3 Vassiliki A. Papadimitrakopoulou8. 1 _Princess Margaret Hospital, San Diego, CA;_ 2 _Center for Cancer and Blood Disorders, Fort Worth, TX;_ 3 _Guardant Health, San Diego, CA;_ 4 _Tennessee Oncology, Chattanooga, TN;_ 5 _Genesis Cancer Center, San Diego, CA;_ 6 _City of Hope Comprehensive Cancer Center, San Diego, CA;_ 7 _Miami Cancer Institute, San Diego, CA;_ 8 _MD Anderson Comprehensive Cancer Center, San Diego, CA_.

Background: The number of guideline-recommended biomarkers to be assessed in patients (pts) with newly-diagnosed mNSCLC is increasing and includes both predictive (EGFR, ALK, ROS1, BRAF, RET, MET, ERBB2; "Guideline-7"(G7)) and prognostic (KRAS) targets. cfDNA analysis is a viable alternative to tissue genotyping, especially in tissue- or time-limited clinical scenarios. We aimed to demonstrate the non-inferiority of comprehensive cfDNA, relative to standard of care (SOC) tissue genotyping, to identify guideline-recommended genomic biomarkers in pts with mNSCLC.

Methods: Pts with previously untreated non-squamous mNSCLC undergoing SOC tissue genotyping were prospectively enrolled at 28 North American centers between July 2016 - April 2018. Pts submitted a pre-treatment blood sample for cfDNA analysis utilizing a CLIA-certified comprehensive 73-gene next generation sequencing panel (Guardant360®). Turn-around time (TAT) was defined as time from test order to final results.

Results: 282 pts with pre-treatment cfDNA samples were included. Most pts were white (81.9%) and half were female (54.3%). A G7 biomarker was identified in tissue in 60 pts and by cfDNA in 77 pts (21.3% versus 27.3%; p<0.0001). In the 60 tissue-positive pts, the biomarker was identified in tissue alone (12) or concordant with plasma (48). Utilizing cfDNA increased the number of patients with an identified G7 biomarker by 48%, from 60 pts to 89, including those with negative (7), not assessed (16), or quantity not sufficient (QNS)(6) results in tissue. Of 193 pts without a G7 biomarker by tissue or cfDNA, 24 (12.4%) had an activating KRAS alteration identified in tissue alone (3) or concordant with cfDNA (21). cfDNA increased the number of KRAS-positive pts from 24 to 92 (tissue negative=3; not assessed=60; QNS=5). Positive predictive value (PPV) for cfDNA vs. tissue genotyping for FDA approved targets (EGFR, ALK, ROS1, BRAF) was 100%. Median TAT was significantly lower for cfDNA as compared to tissue genotyping (9 vs 15 days; p<0.0001).

Discussion: In the largest cfDNA study in previously untreated mNSCLC, we demonstrate that cfDNA detects G7 biomarkers at a rate similar to tissue, meeting the primary study objective. Additionally, even in this population where tissue genotyping was required, cfDNA rescued 30.2% (85/282) of pts including those who were tissue QNS and those who were incompletely genotyped or negative for the biomarker in tissue. The findings in this prospective, multicenter North American study confirm similar findings in Europe and add to the growing evidence that cfDNA can be successfully used to completely assess and identify G7 biomarkers significantly faster than tissue testing, can rescue G7-positive patients with non-diagnostic tissue results, and demonstrates the clinical utility of cfDNA in newly diagnosed mNSCLC.

#4461

Analysis of circulating tumor DNA (ctDNA) identifies on-treatment genomic alterations in a phase 2 study of veliparib plus FOLFIRI ± bevacizumab vs placebo plus FOLFIRI ± bevacizumab in metastatic colorectal cancer (mCRC).

Cyril Y. Ramathal,1 Erin Murphy,1 Elizabeth Asque,1 Arne Jason Grundstad,1 Peter Ansell,1 Lei He,1 Yan Luo,1 Jordan Berlin,2 Jeff Waring1. 1 _Abbvie, North Chicago, IL;_ 2 _Vanderbilt University, Nashville, TN_.

Changes in plasma ctDNA are used as a surrogate molecular marker of response to determine clinical efficacy and provide insights into potential resistance mechanisms to targeted therapeutics. Detection of genomic alterations in circulating tumor DNA (ctDNA) in colorectal cancer has been widely employed as an approach to measure real-time changes in the tumor genome. In order to understand mechanisms of resistance to the PARP inhibitor, Veliparib (ABT-888), pathogenic alterations in oncogenes and DNA Damage Response (DDR) genes were profiled longitudinally in plasma ctDNA and associated to clinical response to Veliparib concurrent to FOLFIRI +/- Bevacizumab.

Methods: 215 serial plasma samples collected at Baseline, Cycle 2 (28 days post-treatment) and at disease progression (Final Visit) from 78 patients (pts) with mCRC in the NCT02305758 clinical trial were selected for the current study, consisting of ~50% partial responders (PR), ~30% stable disease (SD) pts and 5% Non-responders (PD). Detection of ctDNA mutations was accomplished using a targeted, error-correctible NGS assay encompassing 63 cancer driver genes, tumor suppressors & DDR genes. Paired pre- and on-treatment plasma samples were evaluable from 81% pts selected.

Results: Veliparib (VPRB) pts with longest PFS experienced the largest fold change reductions in total cfDNA concentration by end of treatment (EOT). Interestingly, in both treatment arms, the ctDNA mutant fraction per pt decreased by EOT (median = -41%). In support of this correlation, the ctDNA mutational burden also decreased by 28% in VPRB pts and 39% in PLAC pts, largely driven by PR pts with longer PFS. Overall, tumor-specific mutations detected in ctDNA had 88% concordance with tissue, particularly in common mCRC driver mutations. Baseline KRAS mutations were detected in 46% pts (mostly PR & PD) and are eliminated or diminished in 28% pts (14% VPRB, 14% Placebo) by EOT. 30 DDR & MMR genes were found to be frequently altered in all pts, notably APC & MSH3, at both baseline and EOT. However, by EOT, RAD1, XRCC3, BRCA1 and CHEK1 mutations were acquired or increased, while TP53BP1, POLD1 and ERCC5 mutations were eliminated or reduced, prominently in VPRB PR pts.

Conclusions: Broadly, a reduction in ctDNA fraction and pathogenic mutation burden, notably in KRAS, is evidenced in both treatment arms and appears to trend with longer PFS at least in Veliparib-treated pts. Despite these prognostic ctDNA changes, VPRB-specific DDR mutation changes may provide insight into VPRB mechanism of action and resistance in mCRC and is under further exploration at earlier post-treatment timepoints and in SD pts. These data could add insights into alterations acquired earlier in the treatment regimen and the relationship of these alterations with resistance to PARPi in mCRC.

#4462

Sensitivity and reproducibility of onco-panel sequencing across multiple laboratories and technologies.

Joshua Xu,1 Binsheng Gong,1 Wendell Jones,2 Don Johann,3 Onco-panel Working Group of the Sequencing QualityControl Phase 2 (SEQC2) Consortium. 1 _Food and Drug Administration, Jefferson, AR;_ 2 _Q2 Solutions, Morrisville, NC;_ 3 _University of Arkansas for Medical Sciences, Little Rock, AR_.

Onco-panel sequencing targets sequencing reads to few small regions of the genome and thus is better enabled to detect of rare but clinically relevant sub-clonal mutations. Accurate diagnosis and subsequent tailoring of therapy depends on thorough characterization of tumor mutational spectra. The lack of tumor reference samples, comprehensive approaches to assessing reproducibility and detection sensitivity, and standard practice guidelines is hindering the development of onco-panel sequencing and the realization of its benefits in cancer diagnosis and treatment. As a component of the FDA-led SEQC project phase 2 (SEQC2), the Onco-panel Working Group has designed and characterized a set of reference materials. These materials were created using cell line gDNA pooling and dilution to enable such comprehensive assessments of targeted mutation detection approaches. To this end, a cross-lab evaluation of eight Pan-Cancer panels and five ctDNA liquid biopsy assays is currently underway. Each panel was tested in three independent laboratories. To further evaluate methods to boost reproducibility, synthetic spike-in controls and synthetic plasma were also incorporated into the testing samples. Contrived samples for liquid biopsy testing were prepared through enzymatic fragmentation and size selection to mimic cell-free DNA. The onco-panels tested represent two target enrichment approaches (amplicon based and capture based) and sequencing technologies (multiple Illumina platforms and Ion Torrent). Of note, the liquid biopsy assays employ various molecular barcoding techniques to improve their detection sensitivity of rare mutations. Extensive lab QC data has been collected along with sequencing data. Performance measures have been devised to evaluate reproducibility and sensitivity of the variant/mutation calling results that are produced by the panel providers. Meta-analysis of performance measures will then be conducted for cross panel comparison. This comprehensive study will yield insights into factors underpinning sensitivity and reproducibility of onco-panel sequencing. Quantitative performance metrics and actionable data analysis recommendations will be presented to the targeted sequencing panel community.

#4463

Tumor-derived cell-free DNA from Bronchoalveolar lavage fluid (BALF): A potential Liquid Biopsy Analysis in lung cancer patients.

Yuhua Gong,1 Xinyu Zhang,2 Chun Li,2 weiran wang,1 Maosong Ye,2 Yancheng Zhao,2 Xin Yi,1 Yaping Xu,1 Qin Hu,2 Yanfang Guan,1 Ling Yang,1 Xuefeng Xia,1 Min Zhou,3 Jian'an Huang,4 Hua Zhang,5 Tao Ren,6 Qian Shen,7 Kai Wang,8 Yingyong Hou,2 Xin Zhang2. 1Geneplus-Beijing, Geneplus-Beijing Institute, Beijing, China; 2Zhongshan Hospital,Fudan University, China; 3Ruijin Hospital, Shanghai Jiaotong University, China; 4First People's Hospital, Suzhou University, China; 5Zhengzhou Central Hospital, Zhengzhou University, China; 6Shanghai Sixth People's Hospital, China; 7First Affiliated Hospital of Zhejiang University, China; 8Second Affiliated Hospital of Zhejiang University, China.

Background: Bronchoalveolar lavage (BAL) is the most common technique for sampling the components of the alveolar space. Here, we evaluated the potential use of BAL fluid (BALF) in liquid biopsy in lung cancer.

Methods: This study enrolled 56 lung cancer patients. And 57 BALF (separated supernatant and precipitate) samples and 56 peripheral blood lymphocytes samples were collected. 57 formalin-fixed Paraffin-embedded (FFPE) tissues were also obtain. We utilized a 1021-gene NGS panel in matched tissue DNA, BALF supernatant cfDNA and BALF precipitate DNA to identify somatic mutations with white blood cell DNA as a germline control.

Results: Mutations were identified in 52 (~91.22%) BALF precipitate samples and 55 (~96.49%) BALF supernatant samples, comparing to 53 (92.98%) in tumor samples. EGFR mutations were observed in 22 BALF precipitate samples and 24 supernatant samples. Among these, 23 were also identified in matched tumor samples. One BALF precipitate samples specific and two BALF supernatant samples specific EGFR mutations were confirmed by Cobas assay. In addition, gene fusions including ALK, ROS1 and RET were found in 6 tumors, 4 BALF precipitate samples and 5 supernatant samples, respectively.

In total, there were 195 mutations identified in the tumor samples, of which 147 (~75.4%) mutations were detected in the matched BALF precipitate samples, 168 (~86.1%) mutations were detected in the matched BALF supernatant samples and 145 (~74.4%) mutations were shared in three types of samples. Tumor mutation burden (TMB) were calculated, and the consistency between tissue and BALF precipitate was 90% and the consistency between tissue and BALF supernatant was 94%.

Conclusions: Liquid biopsy using BALF shown high potential to profiling genetic alterations of patients with lung cancer, which might be implemented and standardized into clinical use.

#4464

Tumor derived extracellular vesicles in blood of metastatic breast, colorectal, prostate & non small cell lung cancer patients associate with worse survival.

Afroditi Nanou,1 Leonie L. Zeune,1 Sanne de Wit,1 Craig M. Miller,2 Cornelis J. Punt,3 Harry J. Groen,4 Daniel F. Hayes,5 Johann S. de Bono,6 Leon W. Terstappen1. 1 _University of Twente, Enschede, Netherlands;_ 2 _ClarifyDx Consulting, Clinical Consultant, Quakertown, PA;_ 3 _Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands;_ 4 _University of Groningen and University Medical Centre of Groningen, Groningen, Netherlands;_ 5 _University of Michigan, Rogel Cancer Center, Ann Arbor, MI;_ 6 _The Institute of Cancer Research, Royal Marsden Hospital, London, United Kingdom_.

Circulating Tumor Cell (CTC) counts, as determined by the CellSearch® system, associate with poor overall survival (OS) in castration-resistant prostate cancer (CRPC), breast, colorectal and non-small cell lung (NSCLC) cancer patients. The cut-offs used to discriminate between patients with favorable and unfavorable prognoses are 5 CTCs / 7.5 mL of blood for breast and CRPC, 3 for colorectal and 1 for NSCLC. The low numbers of CTCs frequently limit the accurate discrimination of patients into favorable or unfavorable prognosis groups. We previously reported the presence of CK+, DNA-, CD45- tumor-derived extracellular vesicles (tdEVs) in ~20x higher frequencies in CRPC patients and showed their equivalence to CTC counts in predicting clinical outcome. In this study, we explored the presence of tdEVs in the blood of metastatic CRPC, breast, colorectal, and NSCLC patients, determined the association of tdEVs with OS and whether they aid in stratifying patients with favorable CTC counts. Digitally stored CellSearch® system images obtained from previously reported studies from 190 CRPC, 450 colorectal, 179 breast and 117 NSCLC patients before the initiation of a new treatment were used to enumerate tdEVs automatically using the open-source ACCEPT software (http://github.com/LeonieZ/ACCEPT). tdEV counts highly correlated with CTC counts in all cancer types (Spearman's Rho tests p<0.001) and were present at 0-280-fold (Mean 20, SD 25) higher frequencies. A cut-off tdEV value of 23 (median +2SD of 93 healthy donors) was used to dichotomize patients into favorable and unfavorable groups. Kaplan-Meier analyses showed associations of CTCs and tdEVs with OS with hazard ratios (HRs) of 2.4 & 2.1 in CRPC, 2.7 & 2.1 in breast, 2.3 & 2.0 in colorectal and 1.6 & 2.0 in NSCLC, respectively. 52% of the 43% CRPC patients with favorable CTC counts had elevated tdEVs; 53% of the 50% breast cancer patients with favorable CTC counts had elevated tdEVs; 46% of the 72% colorectal cancer patients with favorable CTC counts had elevated tdEVs and 3% of the 75% NSCLC patients with favorable CTC counts had unfavorable tdEVs. CRPC, colorectal and breast cancer patients with favorable CTC counts could be further stratified using tdEV counts (log-rank tests p < 0.05). CTC and tdEV counts equally predict OS in the different cancer types studied. Use of tdEVs in patients with favorable CTC counts can further stratify patients and thereby contribute to clinical decision making. Funded by the NWO Applied and Engineering Sciences Cancer-ID project #14190, the EUFP7 CTCTrap project #305341 and the EU IMI CANCER-ID project # 115749-1.

## EPIDEMIOLOGY

### Factors Influencing Cancer Outcomes

#4465

The effect of insurance status on childhood and adolescent cancer survival using data from the National Cancer Database.

Xiaoyan Wang,1 Rohit P. Ojha,2 Kimberly J. Johnson1. 1 _Washington University in St.Louis, St. Louis, MO;_ 2 _JPS Health Network, Fort Worth, TX_.

Background

Differences in access, delivery or utilization of health care may impact childhood and adolescent cancer survival. In this study, we evaluated the impact of insurance coverage on survival among children and adolescents diagnosed with cancer, and explored potential mechanisms.

Methods

We obtained data from 58,421 individuals diagnosed with any cancer type at ≤ 19 years old from 2004-2010 from the National Cancer Database. We examined associations between insurance status at diagnosis or initial treatment and stage at diagnosis, treatment received (any vs. none), and all-cause mortality using logistic regression, Cox proportional hazards regression, restricted mean survival time and mediation analyses for all cancers combined and by cancer type. We evaluated the moderation effect of age on cancer death in association with insurance status using the likelihood-ratio test.

Results

Children and adolescents with Medicaid, unknown insurance, and those who were uninsured were more likely to be non-White, from lower income counties, be diagnosed at an advanced stage, and less likely to receive any treatment than those with private insurance. Those with Medicaid, unknown insurance, and who were uninsured had a 27% (95%CI, 1.22 to 1.33), 39% (95%CI, 1.26 to 1.53), and 32% (95%CI, 1.20 to 1.46) higher hazard of death during 5 years of follow-up versus those with private insurance. Survival at 60 months was modestly lower for these groups than those who were privately insured at 1.73 months (95%CI, -2.07 to -1.38), 2.48 months (95%CI, -3.29 to -1.68), and 2.13 months (95%CI, -2.91 to -1.34). The higher mortality risk and lower survival months in non-privately insured cases were observed for most cancer types. In mediation analyses, for Medicaid and uninsured patients, earlier diagnosis for staged cancers was estimated to reduce the survival deficit by approximately one fourth and one half. Treatment did not account for the insurance associated survival difference.

Conclusion

Enhancing existing insurance coverage and ensuring equal and early diagnosis may help to improve survival overall for children and adolescents diagnosed with cancer. Future qualitative studies are needed to understand institutional and family level barriers to diagnosis and care of children with cancer and to further explain the underlying mechanisms for differences in childhood survival by insurance status.

#4466

Somatic mutations within chronic lymphocytic leukemia (CLL) putative driver genes are associated with outcomes beyond the CLL international prognostic index (CLL-IPI).

Geffen Kleinstern,1 Daniel R. O'Brien,1 Brian F. Kabat,1 Kari G. Chaffee,1 Aaron D. Norman,1 Timothy G. Call,1 Sameer A. Parikh,1 Jose F. Leis,2 Wei Ding,1 James R. Cerhan,1 Neil E. Kay,1 Susan L. Slager,1 Esteban Braggio2. 1 _Mayo Clinic, Rochester, MN;_ 2 _Mayo Clinic, Scottsdale, AZ_.

CLL is a clinically heterogeneous disease with wide ranging disease course. A novel CLL-IPI based on Rai/Binet stage, IGHV-mutation status, TP53 mutation/deletion, B2M level, and age was developed to stratify patients into 4 risk groups, with a c-statistic of 0.75. Next-generation sequencing has identified ~60 genes recurrently mutated in CLL, some of which are associated with poor overall survival, whereas the clinical effect of most genes is still unknown. Herein, we examine whether somatic mutations in these putative driver genes are associated with time to first treatment (TTT), and whether they add prognostic value beyond CLL-IPI.

Based on the 2008 International Workshop CLL criteria, we identified 100 CLL and 96 high-count monoclonal B-cell lymphocytosis (MBL) newly diagnosed from the Mayo Clinic CLL biobank. Pre-treatment peripheral blood mononuclear cells were collected <2 years of diagnosis and tumor DNA was extracted from sorted CD5+/CD19+. We sequenced the coding regions of 61 recurrently mutated CLL driver genes using a custom SureSelect panel, with 24 samples per flow cell in Illumina HiSeq 4000. The average coverage depth was >1000X. Somatic mutations were called using MuTect2 in tumor-only mode. To remove germline variants, variants were eliminated based on minor allele frequencies >0.01%, identified in 1000 Genomes Project, ExAC and/or ESP6500 databases, unless present in known mutation hotspots or COSMIC. After filtering, high/moderate impact mutations were analyzed using Cox regression, to estimate hazard ratios (HR) and 95% confidence intervals (CI) to test for associations with TTT.

Among 196 patients the most commonly mutated genes were TP53 (11%), ATM (10%), SF3B1 (10%), NOTCH1 (9%), CHD2 (8%), and BIRC3 (7%). The median follow-up was 8.7 years, and 73 patients were subsequently treated. ATM (HR=3.27, CI:1.8-6.1, P=0.0002) and NOTCH1 (HR=2.41, CI:1.3-4.6, P=0.008) were associated with TTT. When evaluating the total number of mutated genes, we found 32%, 29%, and 39% patients had ≥2, 1, or 0 genes mutated, respectively, and this was associated with shorter TTT (HR=1.74, CI:1.3-2.4, P=0.0005) adjusting for sex and CLL-IPI with a c-statistic=0.8 (CI: 0.75-0.84). When stratified by CLL-IPI, the association held for low (N=99, HR=1.88, CI:1.1-3.4, P=0.03) and intermediate risk (N=54, HR=1.87, CI:1.1-3.2, P=0.03) but not high/very high risk (N=35, HR=1.07, CI:0.6-1.9, P=0.83).

We demonstrated that the total number of CLL putative driver genes with high or moderate impact mutations provided prognostic information in newly diagnosed CLL/MBL beyond CLL-IPI. Moreover, even among those with low or intermediate CLL-IPI risk, the total number of somatic mutations separated those patients who progressed. Sequencing the CLL driver genes at time of diagnosis could be a potential biomarker for outcome prediction.

#4467

Survival of patients with microsatellite instable (MSI) metastatic colorectal cancer (mCRC) upon systemic non-immunotherapy.

G. E. Wensink,1 M. A. Elferink,2 A. M. May,3 L. Mol,2 P. A. Hamers,1 C. J. Punt,4 G. R. Vink,1 J. M. Roodhart,5 M. Koopman1. 1 _UMC Utrecht, Utrecht, Netherlands;_ 2 _Netherlands Comprehensive Cancer Organization (IKNL), Utrecht, Netherlands;_ 3 _Julius Center Research Program Cancer, Utrecht, Netherlands;_ 4 _Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands;_ 5 _UMC Utrecht Cancer Center, Utrecht, Netherlands_.

Introduction: Data on survival of patients with MSI mCRC treated with systemic non-immunotherapy are scarce, which complicates the interpretation of recent immunotherapy trials. Our study examined survival in MSI mCRC patients in a retrospective observational cohort.

Methods: We performed a study involving 186 mCRC MSI patients (n=54 from the phase 3 CAIRO, CAIRO2 and CAIRO3 trials and n=132 from The Netherlands Cancer Registry 2015 and 2016). Overall survival from diagnosis of mCRC (OS), from start of first-line (OS1) and second-line therapy (OS2) to death of any cause were examined. Progression-free survival from start of first line (PFS1) and second-line therapy (PFS2) to progression or death of any cause were examined.

Results: Of the 186 patients, 57% were females, median age 67 years, 19% had liver-only metastasis, 31% peritoneal metastasis and 23% underwent metastasectomy. In 113 patients with known BRAF status, 51% had BRAF mutated tumors. The proportion of patients receiving first, second and third-line systemic treatment was 68%, 27% and 7%, respectively. For all patients, median OS was 13.8 months (95% confidence interval [C.I.] 11.8 - 16.3). In patients receiving at least one line of treatment median OS was 15.6 months (13.8 - 19.3), OS1 was 13.9 months (11.6-16.6) and PFS1 was 6.4 months (5.5 - 9.2). In the 48 patients receiving second-line therapy, median OS was 16.5 months (13.9 - 21.5), OS2 was 6.1 months (5.4 - 9.7) and PFS2 was 2.2 months (1.9 - 4.2). Patients starting second-line treatment with a performance score of 0-1, thus eligible for CHECKMATE-142, had an OS2 of 6.7 months (5.0 - 12.1).

Discussion: A direct comparison between our cohort and the CHECKMATE-142 cohort is not possible due to key differences in the patient populations: CHECKMATE-142 patients often received more than one therapy line prior to inclusion and displayed fewer BRAF mutant tumors. However, the survival from start of second-line in our cohort of approximately 7 months is inferior to the published CHECKMATE-142 results, where median OS was not reached after a median follow-up of 1 year in the nivolumab and the nivolumab and ipilimumab cohorts.

Conclusion: We present data on systemic non-immunotherapy in the largest cohort of trial and population-based mCRC MSI patients to date. Our results suggest that immunotherapy may offer a survival benefit for mCRC MSI patients.

#4468

Role of statins and PSA nadir after androgen deprivation therapy in overall survival of patients with metastatic prostate cancer.

Salma Siddiqui,1 Blythe P. Durbin-Johnson,2 Stanley A. Yap,3 Ralph W. deVere White,3 Paramita M. Ghosh3. 1 _VA Northern California Health Care System., Mather, CA;_ 2 _University of California Davis, Davis, CA;_ 3 _University of California Davis, Sacramento, CA_.

Statins are known to reduce prostate cancer (CaP) aggressiveness in both localized and metastatic disease. Studies have indicated that the use of statin drugs to lower cholesterol levels was associated with decreased risk of localized CaP, less frequent high grade CaP and lower CaP volume, suggesting that statin use has a protective effect against localized CaP. However, less is known about a role for statins in metastatic CaP (mCaP). While it is known that hypercholesterolemia is associated with the development of castration resistant prostate cancer (CRPC) after androgen deprivation therapy (ADT) in patients with bone metastasis, it is not known whether the sequence of statin and ADT affects outcome. Charts of 162 patients with mCaP treated at the VA Northern California Health Care System (VANCHCS) with ADT and statins between 1992 and 2016 were analyzed. Overall survival (OS) was followed until 12/2016. Dates were noted from the medical records entered into the Computerized Patient Record System (CPRS). Time to event outcomes (with the exception of time to Nadir PSA, which was not censored) were analyzed using Cox proportional hazard models. PSA Nadir and time to PSA Nadir were analyzed using linear models, with outcomes log transformed. All models include age at PSA diagnosis and year of metastatic diagnosis as covariates. Of the 162 total patients, 110 were on ADT following biochemical recurrence (BCR) at the time of diagnosis with distant metastasis (ADT before mets, ABM) while 50 were diagnosed with a distant metastatic lesion at the time of ADT administration (mets before ADT, MBA). 46.3% of the patients did not receive statin treatment whereas 41.4% received statins prior to diagnosis with distant metastasis, and 12.3% started statins after metastasis diagnosis (29% prior to ADT treatment and 24.7% after starting on ADT). There was no effect of statins on OS among those who had a diagnosis of metastasis prior to initial ADT (MBA group). On the other hand, among those who developed metastases while on ADT (ABM group), statin use significantly increased OS (based on the time of metastasis diagnosis). However, within this group, the largest benefit was in the patients that received statins after starting ADT (HR = 0.51, p=0.022). The same group (subgroup within the ABM group who initiated statins after ADT) also took longer to reach PSA nadir following ADT compared to those who never received statins (R=1.79, p=0.031) and also took longer to be diagnosed with mCaP following ADT compared to those who had never been (HR=0.45, p=0.003). These advantages were not seen in patients in the ABM group who were already on statins at the time of ADT initiation, and no advantages of statins were seen in the MBA group. Our data indicate that treatment with statins initiated after the patient has undergone ADT is beneficial to patients who develop distant metastases while on ADT treatment.

#4469

Clinical outcomes among patients treated with abiraterone acetate for advanced prostate cancer with pre-existing cardiovascular conditions.

Grace Lu-Yao,1 Nikita Nikita,1 Scott Keith,1 Krupa Gandhi,1 Timothy R. Rebbeck,2 Jennifer Cullen,3 Ginah Nightingale,1 Mark Mann,1 Andrew Chapman1. 1 _Thomas Jefferson Univ. Kimmel Cancer Ctr., Philadelphia, PA;_ 2 _Harvard University, Boston, MA;_ 3 _Uniformed Services University, Bethesda, MD_.

Background: Advanced prostate cancer patients with pre-existing cardiovascular diseases (CVD) are often excluded from clinical trials of abiraterone acetate (AA). Therefore, the clinical outcomes of these patients are uncertain. This study was undertaken to examine their outcomes - changes in the rate of hospitalization and short-term mortality after AA initiation by pre-existing CVD conditions.

Methods: This population-based study identified PCa patients from the linked Surveillance, Epidemiology and End Result (SEER)-Medicare files diagnosed between 1/1/1991 and 12/31/2013 and treated with AA in 2011-2014. The primary endpoints were 6-month overall mortality and changes in hospitalization rates following AA initiation. Major pre-existing conditions are listed in the Table 1. The changes in hospitalization rate were measured by the incidence rate ratios (IRR), post treatment rate divided by pre-treatment rate.

Results: Among 2,845 patients treated with AA (median age 75 years), 1,924 (67.6%) of these patients had at least one pre-existing CVD condition. Compared to pre-treatment period, the hospitalization incidence post-AA increased by 58% to 88%. The crude risk of 6-month overall mortality was between 21.4% to 25.6%.

Table 1: Risk of Hospital Visits and 6-month Overall Mortality among AA Patients by Pre-Existing CVD condition.

Pre-existing CVD condition | No. hospitalizations per 6-months prior to AA | IRR for hospitalizations (95% CI) | Crude 6-month post AA Mortality

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

Acute Myocardial Infraction | 0.65 | 1.88 (1.48-2.40) | 25.6%

Atrial Fibrillation | 0.56 | 1.58 (1.36-1.83) | 24.4%

Congestive Heart Failure | 0.59 | 1.72 (1.54-1.92) | 23.4%

Stroke | 0.52 | 1.67 (1.40-2.01) | 22.1%

Ischemic Heart Disease | 0.47 | 1.78 (1.63-1.95) | 21.4%

No CVD | 0.25 | 1.87 (1.59-2.20) | 15.8%

Conclusion: All patients experienced significant increase in hospitalization after initiation of AA. Our study highlights the importance of carefully monitoring patients after prescribing AA.

#4470

Aneuploidy drives lethal progression in prostate cancer.

Konrad H. Stopsack,1 Charles A. Whittaker,2 Travis A. Gerke,3 Massimo Loda,4 Philip W. Kantoff,1 Lorelei A. Mucci,5 Angelika Amon2. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA;_ 3 _Moffitt Cancer Center, Tampa, FL;_ 4 _Dana-Farber Cancer Institute, Boston, MA;_ 5 _Harvard T.H. Chan School of Public Health, Boston, MA_.

Purpose: Aneuploidy, defined as chromosome gains and losses, is a hallmark of cancer. However, extensive aneuploidy is relatively rare in prostate cancer. We sought to investigate whether extent of aneuploidy is associated with lethal progression in a large cohort of prostate cancer patients, an untested hypothesis.

Methods: To assess chromosome arm gains and losses in archival tumor specimens, we developed a computational method based on normalized gene expression sums of whole-transcriptome profiling in primary prostate tumors from The Cancer Genome Atlas (TCGA) and validated it against DNA copy numbers. We applied this method to tumor tissue from cancer diagnosis from patients diagnosed during prospective follow-up of two cohort studies, the Health Professionals Follow-up Study (HPFS) and the Physicians' Health Study (PHS). These tumors underwent centralized histopathologic review including Gleason grading and immunohistochemistry to characterize proliferation (Ki-67), apoptosis (TUNEL), PTEN loss, and MYC amplification. Patients were followed for lethal disease (metastases and prostate cancer-specific death) on median for 15 years.

Results: Extensive aneuploidy (>=5 gained or lost chromosome arms) was present in 23% of the 333 primary prostate cancers from TCGA. Even 68% of low-risk cancers (Gleason grade <=3+4) had >= 1 altered chromosome arm. Our algorithm detected aneuploidy based on transcriptome profiling with high discriminative accuracy (area under the curve [AUC] for any aneuploidy, 0.83; AUC for >=5 chromosome arms, 0.87). Tumors with greater extent of aneuploidy had higher Gleason scores, but little to no increase in proliferative and apoptotic indices. Among the 404 patients from HPFS and PHS, 113 lethal events occurred. Greater extent of aneuploidy was strongly associated with a higher risk of lethal disease. Patients with >=5 predicted altered chromosome arms had 5.3 times higher odds of lethal disease (95% confidence interval, 2.2-13.1) compared to those without aneuploidy after adjusting for Gleason grade. Aneuploidy was strongly associated with lethal disease even among men with high-risk Gleason grade 8-10 tumors. Chromosome arms 8q and 10q were among the commonly altered arms. The elevated risk associated with 10q deletion and 8q gain was not considerably attenuated when accounting for PTEN loss and MYC overexpression on the respective arms.

Conclusions: The extent of aneuploidy can be estimated from transcriptome profiling, which allows assessment of archival, formalin-fixed paraffin-embedded tumors. Given the complex interactions between genes colocalized on a chromosome arm, the loss or gain of an entire chromosome arm confers aggressiveness beyond affecting well-described tumor suppressors or oncogenes on that arm and beyond altering histologic tumor grade. Our results have clinical implications and point to a key role of aneuploidy in driving aggressive disease in primary prostate cancer.

#4471

Commercial gene expression tests for prostate cancer prognosis provide paradoxical estimates of race-specific risk.

Jordan H. Creed, Anders E. Berglund, Robert J. Rounbehler, Shivanshu Awasthi, John L. Cleveland, Jong Y. Park, Kosj Yamoah, Travis A. Gerke. _Moffitt Cancer Center, Tampa, FL_.

Background: Current National Comprehensive Cancer Network (NCCN) guidelines recommend three gene expression-based tests for prostate cancer (PCa) prognosis in men with low or favorable intermediate risk disease: Decipher, Oncotype DX Prostate, and Prolaris. The three tests feature varying numbers of genes with minimal overlap. Importantly, development and validation efforts for all three panels were undertaken in predominantly European American men (EAM) cohorts, and limited research exists on the value of such signatures in African American men (AAM), who have poorer PCa outcomes. Here, we explored differences in gene expression between EAM and AAM for the three panels recommended by the NCCN for PCa prognosis.

Methods: 232 EAM and 95 AAM patients provided radical prostatectomy specimens. Gene expression was quantified using Nanostring for 60 genes spanning the Oncotype DX Prostate, Prolaris, and Decipher panels. Differential expression and intrapanel co-expression by race were assessed using Mann-Whitney tests and Spearman's correlations, respectively. A continuous expression-based risk score was approximated for each panel with higher scores indicating worse outcomes. Race-specific risks were compared using Mann-Whitney tests and Spearman's correlations.

Results: Clinical and pathologic features were similar between AAM and EAM. Differential expression by race was observed for 48% of genes measured, though the magnitudes of expression differences were small. Co-expression patterns were more strongly preserved by race group for Oncotype DX and Decipher versus Prolaris (integrative correlations of 0.87, 0.73, and 0.62, respectively). Paradoxically, poorer prognosis was estimated in EAM versus AAM for Prolaris and Oncotype DX (p = 0.01 for both), whereas worse prognosis was predicted for AAM versus EAM using Decipher (p < 0.001).

Discussion: Due to observed racial differences across three commercial gene expression panels for PCa prognosis, caution is warranted when applying these panels in clinical decision-making in AAM. Replication of our findings directly on the commercial panels with long-term follow-up is warranted.

## EXPERIMENTAL AND MOLECULAR THERAPEUTICS

### Genomic Correlates of Therapy Response

#4472

The hippo pathway effector TAZ regulates ferroptosis in renal cell carcinoma.

Wen-Hsuan Yang, Jen-Tsan Chi. _Duke University, USA, NC_.

Ferroptosis is a newly appreciated lipid peroxidation-dependent regulated cell death with relevance for many human diseases, which may be a tumor suppression mechanism and may have antitumor potential. Elucidation of the mechanism is therefore important. Here, we reported that ferroptosis sensitivity is significantly influenced by cell density. The effectors of the Hippo pathway, YAP/TAZ, are the molecular sensors of cell density that regulate cell proliferation, survival, and organ size determination. Therefore, we determined the potential role of YAP/TAZ in regulating ferroptosis sensitivities. RCC cell lines and RCC PDX tumor cells mainly express TAZ that undergoes density-dependent nuclear translocation. Importantly, the removal of TAZ confers ferroptosis resistance, while the expression of constructively active TAZ sensitizes cells to ferroptosis. Mechanistically, we found that TAZ knockdown reduced ferroptosis through the repression of its target gene, EMP1. EMP1 affects ferroptosis through the regulation of NOX4 and lipid peroxidation. Collectively, we have shown that the ferroptosis can be regulated by non-genetic factors, such as cell density, through the Hippo pathway and suggest the therapeutic potential of ferroptosis-inducing agent, such as erastin, for TAZ-activated tumors.

#4473

DNMT and PARP inhibitor combination therapy induces an interferon driven homologous recombination defect in triple negative breast cancers and acute myeloid leukemia.

Lena J. McLaughlin,1 Aksinija A. Kogan,1 Eun Yong Choi,1 Rena S. Lapidus,1 Ying Zou,1 Huili Li,2 Stephen B. Baylin,2 Michael J. Topper,2 Feyruz V. Rassool1. 1 _University of Maryland, Baltimore, Baltimore, MD;_ 2 _Johns Hopkins University, Baltimore, MD_.

Poly (ADP-ribose) polymerase inhibitors (PARPis) are effective in a subset of triple negative breast cancers (TNBC) with BRCA gene mutations, which generates homologous recombination deficiencies (HRD), through synthetic lethality. However, PARPis fail for the majority of sporadic TNBC with intact BRCA1/2 genes and other BRCA-proficient cancers, including acute myeloid leukemia (AML). PARPi, Talazoparib (TAL), has potent PARP trapping ability, in both BRCA mutant and proficient TNBCs and AML. We reported that a combination of TAL with low doses of DNA methyl transferase inhibitor (DNMTi) azacytidine (AZA) or decitabine (DAC), increased PARP trapping even further, leading to increased levels of cytotoxic double strand breaks in vitro and decreased tumor burden in vivo. Low doses of DNMTis have been shown previously to reprogram genome wide cancer signaling, including immune and DNA damage/repair pathways. We now tie these two signaling pathways together for events through which DNMTis plus TAL drive HRD.

BRCA proficient TNBC (N=2) and AML (cell lines (N=2) and primary cells (N=4)) treated with low doses of DNMTis (DAC 10nM and AZA 250-500nM), significantly downregulate expression of genes in HR and Fanconi Anemia (FA) pathways, decreasing HR activity, thus generating HRD. HR is further decreased when DNMTis are combined with TAL. We now link DNMTi/TAL induced HRD to a coupled interferon (IFN) response in both TNBC and AML. In these systems, we elucidate DNMTi/TAL potentiated cGAS-Sting and type 1 IFN signaling, culminating in the induction of IFN responsive genes and inflammatory cytokine production. Importantly, in DNMTi/TAL treated cells, increased expression of IFN signaling genes is strongly linked to downregulation of FA/HR genes through protein interaction network mapping. This inverse association between IFN and HR related genes, was further validated in TCGA data sets for AML and invasive BC, suggesting broad applicability of this observed transcriptional program. The acquisition of HRD from IFN signaling specifically, is verified by the observation of reduced HR gene expression and activity in the presence of exogenous double stranded RNA (Poly(I:C)) or IFNβ treatment in both TNBC and AML cell lines. Finally, knockdown of a key connectivity node between IFN and HR related genes, interferon sensitive gene 15 (ISG15), abrogates downregulation of FA gene expression when treated with IFNβ, suggesting mediation of HRD through ISG15. In conclusion, we have identified a novel mechanism through which IFN-sensitive genes can create an HRD phenotype in BRCA proficient TNBC and AML, suggesting that innate immunity and inflammatory processes may be intrinsically linked to DNA repair in cancer. These studies suggest that immune therapies and/or epigenetic therapy are likely to enhance responses to PARPis in BRCA proficient cancers.

#4474

Novel neoplastic RAD51AP1-DYRK4 fusion transcript in aggressive luminal breast cancers.

Chia Chia Liu,1 Jamunarani Veeraraghavan,2 Ying Tan,2 Jin-Ah Kim,2 Xian Wang3. 1 _Hillman Cancer Center, University of Pittsburgh, Pittsburgh, PA;_ 2 _Lester & Sue Smith Breast Center, Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX; _3 _UPMC Hillman Cancer Center, University of Pittsburgh, Pittsburgh, PA_.

Background: Estrogen receptor positive (ER+) breast cancer is known as luminal breast cancer, which can be classified into A and B intrinsic subtypes. While the luminal A tumors have a favorable outcome following endocrine therapy, the luminal B tumors are characterized by higher proliferation index, aggressive clinical behavior, early relapse following endocrine therapy, and high risk of metastatic dissemination. Clinically the treatment options are limited, and it is even difficult to clearly define these deadly tumors. The underlying pathological molecular events remain poorly understood, and recent genome sequencing studies have revealed a paucity of actionable oncogenic drivers, which hinders the development of new treatment strategies.

Experimental design and methods: Large-scale analyses of breast cancer RNAseq data from The Cancer Genome Atlas (TCGA) were performed to identify the driver gene fusions. ER+ breast tumor tissues were screened by RT-PCR. To test the function of RAD51AP1-DYRK4 transcripts in breast cancer, we engineered the fusion cDNA containing the E9-E2 chimeric ORF together with endogenous5' translation start sequences into a doxycycline-inducible lentiviral vector, which was then transduced into T47D luminal breast cancer cells. The overexpression (OE) and endogenous fusion knockdown (KD) models were then subjected to diverse functional assays including MTS, Clonogenic, soft agar colony formation, migration and invasion.

Results: In this study, a large-scale analysis of breast cancer transcriptome revealed a tumor-specific RAD51AP1-DYRK4 fusion transcript preferentially overexpressed in luminal B tumors. Molecular analysis of 200 ER-positive breast cancer tissues detected strong RAD51AP1-DYRK4 expression in 19 tumors (9.5%), which is markedly enriched in the luminal B subtype (17.5%). The fusion encodes c-terminal truncated RAD51AP1 protein fused to an out frame peptide from DYRK4, which leads to the loss of the RAD51 interacting domain. Ectopic expression of RAD51AP1-DYRK4 but not wild-type RAD51AP1 significantly increased invasiveness of luminal breast cancer cells. Further, we have identified the endogenous RAD51AP1-DYRK4 protein in fusion-overexpressing cells, silencing of which leads to decreased cell viability.

Conclusions: In summary, this study identifies the first tumor-specific transcription-induced chimera that is preferentially overexpressed in the luminal B breast cancer, and the underlying mechanism. The results suggest that RAD51AP1-DYRK4 transcript may drive the more aggressive form of luminal breast cancers.

#4475

Targeting c-MET and MAPK pathway in metastatic castration resistant prostate cancer.

Sergio Ruiz,1 María Dolores Fenor de la Maza,1 Juan Francisco Rodríguez Moreno,1 Elena Sevillano,1 Paloma Navarro,1 Sandra Amarilla,1 Teresa García Donas,1 Eduardo Caleiras,2 Juan Carlos Torrego,3 Jesus García Donas1. 1 _CLARA CAMPAL COMPREHENSIVE CANCER CENTRE, MADRID, Spain;_ 2 _CNIO, MADRID, Spain;_ 3 _Campo Grande Hospital, Valladolid, Spain_.

Identification of "agnostic" genetic drivers in cancer is foreseen as a major step forward in precision medicine. Thus, treatment selection could be based on the molecular background of the disease rather than on tumor histology in the near future.

However clinical validation of most alterations is lacking and only preclinical models are available.

We describe a prostate cancer patient refractory to standard therapies that was found to harbor both a c-Met activating mutation and a BRAF fusion that greatly responded to the MEK inhibitor (MEKi) trametinib.

EXPERIMENTAL PROCEDURES

The Next Generation Sequencing (NGS) platforms (Oncomine Assay and Archer Variant Plex) were used to characterize molecularly FFPE tissue from the primary tumor of a 73 years old man with metastatic castration-resistant prostate cancer (mCRPC) refractory to Docetaxel, Enzalutamide and Radium 223.

Immunostaining was performed to evaluate the status of protumoral pathways activated by the identified genomic alterations.

RESULTS

Oncomine assay detected a pathogenic c-MET mutation that affected the splicing of exon 14 and the patient was included in a clinical trial with a c-MET inhibidor. He developed Grade 3 anemia, G3 bone pain which required major opioids, a rise of PSA over 900 ng/ml and progressive disease as best response.

An extended screening using Archer platform detected a SND1/BRAF fusion. Immunohistochemistry revealed homogeneous tumor positive staining for c-MET, and a punctured staining on pERK in FFPE regions with perineural tumor invasion suggesting a critical role of the BRAF fusion in the spread of the disease.

After a comprehensive review of the literature a similar model was identified, where the SND1/BAF function raised in a cell culture of gastric cancer as mechanism of resistance to MET inihibitors (Lee NV et al PLoS One 2012). In preclinical experiments the MEKi trametinib induced significant cell mortality.

After given written consent, the patient was put on trametinib (2mg/day) under compassionate use.

We observed a large decrease of PSA levels to 90 ng/ml after two weeks of treatment, an increase of hemoglobin levels and significant clinical benefit, with no need of analgesics. After two months on treatment the patient continues on tumor response by the time of communication of this abstract.

CONCLUSIONS

According to our knowledge, this is the first published study able to demonstrate that cancer patients harboring c-MET exon 14 splicing mutation and SND1-BRAF fusion genes could benefit from therapies with MEK inhibitors. Further experimental procedures are needed to validate this hypothesis.

1. Lee NV, Lira ME, Pavlicek A, Ye J, Buckman D, et al. (2012) A Novel SND1-BRAF Fusion Confers Resistance to c-Met Inhibitor PF-04217903 in GTL16 Cells though MAPK Activation. PLoS ONE 7(6): e39653. doi:10.1371/journal.pone.0039653.

#4476

Therapeutic exploitation of passenger amplifications in hepatocellular carcinoma.

Christos Patsis,1 Joan Crous-Masó,1 Ilaria Salvato,1 Luise Butthof,1 Rossella Pellegrino,2 Thomas Longerich,2 Matthias Gaida,2 Petros Christopoulos,3 Darjus Tschaharganeh1. 1 _German Cancer Research Center (DKFZ); Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany;_ 2 _Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany;_ 3 _Department of Thoracic Oncology, Thoraxklinik at Heidelberg University Hospital and Translational Lung Research Center Heidelberg (TLRC-H), member of the German Center for Lung Research (DZL), Heidelberg, Germany_.

Hepatocellular carcinoma (HCC) represents the predominant primary liver malignancy and the second most common cause of cancer-related death worldwide. Although several advances have been made in HCC screening and diagnosis, the therapeutic treatment of advanced disease at present relies mainly on sorafenib and regorafenib, two multi-kinase inhibitors, which demonstrate limited clinical efficacy. Recently, several genomic studies have unveiled the underlying genomic changes governing HCC development, consisting of mutations in prominent driver genes, as well as copy-number variations (CNVs), such as amplifications and deletions of whole chromosome regions, which exceed by far the frequency of individual mutations. Interestingly, CNVs are considered to mediate clonal expansion due to the presence of oncogenic driver genes, but generally comprise also neutral bystander genes with no implication in neoplastic transformation. While current therapeutic approaches largely aim at targeting mutation-induced dysregulated pathways, strategies targeting CNVs are not thoroughly pursued. We hypothesized that passenger aberrations in genomic amplifications of tumor cells could be therapeutically exploited by providing actionable molecules on the cell surface. To this end, we performed bioinformatic analyses of TCGA CNV data and uncovered a list of cell surface protein (CSP) genes which are frequently amplified in HCC and could be used as potential candidates for a "Trojan-horse" approach in this tumor entity. More specifically, a set of CSP genes was found to be amplified in more than 60% of HCCs, as well as other solid tumors. Genetic perturbations of their expression levels had no effect on the growth and proliferation of HCC cell lines, suggesting their passenger status in the context of this malignancy. Furthermore, amplification of CSP-genes resulted in increased protein expression, which was localized primarily at the plasma membrane. Interestingly, a wide range of normal tissues displayed absent to low cytoplasmic levels of these CSPs as compared to several solid tumors, which exhibited diverse cell surface-associated expression. In addition, pharmacological inhibition of various oncogenic signaling pathways in cancer cells led to significant CSP upregulation at the protein level in the absence of respective genomic amplifications, raising the therapeutic prospect of targeting these proteins as second hits in combination drug regimens. In summary, we have identified several CSPs as potential targets in HCC, which could achieve high tumor specificity while sparing normal tissue when targeted by anti-CSP radioisotope-conjugated antibodies or CSP-specific CAR-T cells.

#4477

MIK665/S64315, a novel Mcl-1 inhibitor, in combination with Bcl-2 inhibitors exhibits strong synergistic anti-tumor activity in a range of hematologic malignancies.

Ensar Halilovic,1 Maïa Chanrion,2 Prakash Mistry,3 Markus Wartmann,3 Shumei Qiu,1 Sneha Sanghavi,1 Yan Chen,1 Gaëlle Lysiak,2 Ana Leticia Maragno,2 Ulrike Pfaar,3 Felix Huth,3 Marie Schoumacher,2 Audrey Claperon,2 Laurence Kraus-Berthier,4 Sébastien Banquet,4 Alix Derreal,4 Heiko Maacke,3 Frédéric Colland,2 Olivier Geneste,2 Erick Morris,1 Youzhen Wang1. 1 _Novartis Institutes for BioMedical Research, Cambridge, MA;_ 2 _Servier Oncology R &D Unit, Croissy-sur-seine, France; _3 _Novartis Institutes for BioMedical Research, Basel, Switzerland;_ 4 _Servier Oncology R &D Unit, Suresnes, France_.

One of the hallmarks of cancer is evasion of apoptosis. The B-cell lymphoma-2 (Bcl-2) family of proteins represents a crucial point of control of apoptosis. The Bcl-2 family comprises both pro- and anti-apoptotic members, the latter of which (Bcl-2, Bcl-xL, Bcl-w, Mcl-1 and Bcl-2A1) are often overexpressed in cancer cells, supporting their aberrant survival. Thus, these anti-apoptotic proteins have become an attractive target for cancer therapy. BH3 mimetics have been shown to bind to the BH3 binding groove of anti-apoptotic Bcl-2 family members and inhibit their function, resulting in apoptotic cell death, and one such BH3 mimetic, ABT-199 (venetoclax), has recently been approved for treatment of relapsed or refractory Chronic Lymphocytic Leukemia. We have developed two novel and potent BH3 mimetics: MIK665/S64315, a highly selective inhibitor of Mcl-1 and BCL201/S55746, a selective Bcl-2 inhibitor. Both compounds, individually induce apoptosis in hematological cancer cell lines, primary patient samples and demonstrate anti-tumor efficacy in xenograft models. MIK665/S64315 is currently in phase 1 clinical development in AML and MDS (NCT 02979366) and in MM and lymphoma (NCT02992483). Here, we describe the activity of the combination of MIK665/S64315 with BCL201/S55746 or venetoclax, both in vitro and in vivo, across a range of hematological indications (AML, MM and DLBCL). In vitro, a strong synergy was observed with these combinations, resulting in a remarkable induction of cell death in majority of cell lines tested. In vivo, MIK665/S64315 and BCL201/S55746 combinations lead to complete and durable antitumor responses in many different xenograft models in mice and rats. Taken together, these data demonstrate that a combination of MIK665/S64315 and BCL201/S55746 provide strong therapeutic benefit over either monotherapy, and support a rationale for testing Mcl-1 and Bcl-2 inhibitor combinations in patients with hematological malignancies.

#4478

High throughput dynamic BH3 profiling identifies active cancer therapies in solid tumors.

Patrick D. Bhola, Eman Ahmed, Jennifer Guerriero, Ewa Sicinska, Emily Su, Jing Ni, Elizaveta Lavrova, Otari Chipashvili, Timothy Hagan, Kimmie Ng, Andrew Aguirre, Jochen Lorch, Suzanne George, George Demetri, Jean Zhao, Anthony Letai. _Dana Farber Cancer Institute, Boston, MA_.

Phenotypic differences between primary patient tumors and models of tumors that are amenable to chemical screening represents a key barrier to drug discovery and drug repurposing. High-throughput technologies that can accurately identify active drugs using primary patient tumors are therefore valuable additions to the drug development toolbox and may have direct clinical utility as predictive biomarkers. Here we report a method called high-throughput dynamic BH3 profiling (HT-DBP), a robust microscopy-based assay with single-cell resolution that enables chemical screens of hundreds of candidate drugs on freshy isolated tumor cells to identify chemical inducers of mitochondrial apoptotic signaling. HT-DBP requires only 24 hours of ex vivo culture which enables the direct study of fresh primary tumor cells and minimizes adaptive changes that occur with prolonged ex vivo culture.

Using 1650 compounds, we performed HT-DBP on freshly isolated cells from the MMTV-PyMT genetically engineered mouse model of breast cancer to identify drugs that sensitize these tumors for apoptosis. Selected compounds identified by HT-DBP induced regressions in the MMTV-PyMT genetically engineered mouse model of breast cancer in vivo, and in a patient derived xenograft model of breast cancer. To evaluate how chemical vulnerabilities evolve in cell culture, we performed HT-DBP on a cancer cell line from MMTV-PyMT tumors. We observe different patterns of chemical vulnerabilities between freshly isolated tumor cells and in the derived cell lines, which is consistent with the mixed track record of cancer cell line chemical screens.

We demonstrate that HT-DBP can be used to generate personalized apoptotic chemical vulnerabilities (which we refer to as pharmacotypes) for a set of colon cancer PDX models. We find that a PDX model derived from a primary site tumor and a metastatic lesion from the same patient have different pharmacotypes. Using annotations of small molecule targets, we can identify proteins and signaling pathways that represent apoptotic vulnerabilities in colon PDX models. Finally, we apply HT-DBP to primary human thyroid tumors and sarcomas to identify potential active therapies. In sum, HT-DBP can efficiently predict therapeutic sensitivity upon short-term ex vivo drug exposure and may empower functional precision medicine approaches in the clinic.

### Novel Therapeutics

#4479

BT8009: A bicyclic peptide toxin conjugate targeting Nectin-4 (PVRL4) displays efficacy in preclinical tumor models.

Mike Rigby,1 Paul Beswick,1 Gemma Mudd,1 Katerine Van Rietschoten,1 Liuhong Chen,1 Sophie M. Watcham,1 Heather Allen,1 Amy Brown,1 Helen Harrison,1 Gavin Bennett,1 Phil Jeffrey,1 Peter U. Park,2 Maria Koehler,2 Nicholas Keen2. 1 _Bicycle Therapeutics Ltd, Cambridge, United Kingdom;_ 2 _Bicycle Therapeutics Inc, Lexington, MA_.

We have identified a Nectin-4 targeting peptide for delivery of cytotoxic agents to Nectin-4 expressing tumors. Nectin-4 is a cell adhesion molecule, that is highly expressed in certain tumor types, including bladder, TNBC and NSCLC, but has a restricted distribution in normal tissue. Bicycles® are small (1.5kDa) fully synthetic, structurally constrained, peptide drugs that combine the affinity of antibodies with the pharmacokinetic properties of small molecules. The Bicycle phage display platform was utilised to rapidly identify a high affinity (18 nM) and highly selective bicyclic Nectin-4 binding peptide. Synthetic modification of the initial lead peptide improved affinity (0.3 nM), hydrophilicity and stability. The optimised peptide is conjugated through an inert sarcosine spacer chain and a cleavable linker to the toxin MMAE, to form the Bicycle Toxin Conjugate (BTC) BT8009. Once bound to cell surface Nectin-4, the linker system is cleaved by peptidases (e.g. cathepsin B) upregulated in the tumor micro environment. Full structure of BT8009 will be disclosed within the presentation. Fluorescence polarisation and surface plasmon resonance show BT8009 has low nanomolar affinity (3nM) for Nectin-4 and high selectivity (>1000 fold) over Nectins 1-3, and the 5 nectin-like, family members. Good affinity for the cognate native protein is maintained across the preclinical safety species. High content imaging of cultures of MDA-MB-468 cells, demonstrates binding of the BTC to cell membrane. In vivo, BT8009 is well tolerated in mouse and rat and shows regressions across a range of cell and patient derived xenograft models. Efficacy correlates with the expression level of Nectin-4 on the tumor cells, and the dose delivered. In the MDA-MB-468 (TNBC) CDX model full tumor regression was seen with weekly i.v. administration of 3 mg/k g i.v. (4 doses), with no tumor regrowth out to 70 days post last dose. Similar efficacy was seen in two NSCLC PDX models (adenocarcinoma and squamous cell carcinoma) with full regression attained in both. Near full regression was seen in a squamous cell esophageal cancer PDX. Larger tumors show a similar degree of sensitivity to the BTC, with rapid tumor regression from a volume of 800 mm3. As a small peptide BT8009 undergoes rapid clearance from the plasma with minimal exposure to non-targeted organs. After a single dose, MMAE has been shown to be retained within tumor tissue in excess of 60 h, at exposure levels significantly greater than corresponding plasma and other tissue levels. Bicycle Toxin Conjugates represent a novel treatment modality for nectin-4 expressing tumors with excellent efficacy in several mouse xenograft models.

#4480

Preclinical characterization of potent and selective oral PD-L1 small molecule antagonists.

Liang-Chuan S. Wang, Holly Koblish, Yue Zhang, Ashwini Kulkarni, Maryanne Covington, Karen Gallagher, Gengjie Yang, Jonathan Rios-Doria, Christina Stevens, Michael Hansbury, Sybil O'Connor, Yan-ou Yang, Sharon Diamond, Krista Burke, Kaijiong Xiao, Jingwei Li, Wenqing Yao, Liangxing Wu, Peggy Scherle, Gregory Hollis, Reid Huber. _Incyte Corporation, Wilmington, DE_.

Monoclonal antibodies against PD-L1 or PD-1 have been approved for the treatment of multiple tumor histologies by virtue of their ability to restore T cell effector function, increase T cell proliferation and enhance infiltration of tumor-reactive T cells. A small molecule approach to PD-(L)1 axis blockade may offer distinct benefits over the use of monoclonal antibodies including improved tissue penetration, titratability, absence of immunogenicity, ease of administration, and potential for fixed dose oral-oral therapeutic combinations. We have identified a novel class of small molecule PD-L1 antagonists that are capable of functional PD-(L)1 axis blockade by virtue of their ability to induce PD-L1 internalization. In vitro, select small molecules demonstrate high affinity to human PD-L1, potently disrupt the PD-L1:PD-1 interaction (<4nM), and inhibit Src homology region 2 domain-containing phosphatase (SHP2) recruitment to PD-1 (<10nM). As a result, PD-1-mediated suppression of nuclear factor of activated T cells (NFAT) activation is reduced and IFNγ production by T cells is restored. These bioactive small molecule PD-L1 antagonists are shown to reduce surface PD-L1 levels in tumor cells and peripheral blood monocytes with IC50s ranging from 1-250nM, providing an in vivo pharmacodynamic biomarker for compound activity. Using humanized NSG mice bearing MDA-MB-231 tumors, oral administration of small molecule PD-L1 antagonists for 28 days demonstrated a dose-dependent reduction in tumor growth with a concomitant and dose-dependent increase in the number of tumor-infiltrating T cells. These data were concordant with the dose-dependent reduction of surface PD-L1 levels seen on both tumor cells and tumor associated macrophages at the end of the study. Similar data, including dose-dependent tumor growth inhibition, PD-L1 internalization and increase in tumor-infiltrating T cells, were also obtained using a murine MC38 xenograft system genetically engineered to over express human PD-L1. No in vivo activity was observed when tumors were treated in immunocompromised mice, confirming the pharmacologic dependency on a competent immune system. Finally, these oral PD-L1 small molecule antagonists demonstrated equivalent anti-tumor activity in preclinical tumor models when compared head-to-head to clinically approved PD-(L)1 axis targeting monoclonal antibodies. In conclusion, effective PD-(L)1 axis blockade and functional activation of the immune system can be achieved in vivo through this novel series of orally bioavailable small molecule PD-L1 antagonists, supporting the clinical evaluation of the mechanism as a novel approach to immune therapy.

#4481

BT5528, an EphA2-targeting Bicycle Toxin Conjugate (BTC): Profound efficacy without bleeding and coagulation abnormalities in animal models.

Gavin S. Bennett,1 Amy Brown,1 Gemma Mudd,1 Johanna Lahdenranta,2 Nicholas Keen2. 1 _Bicycle Therapeutics Ltd, Cambridge, United Kingdom;_ 2 _Bicycle Therapeutics Inc, Cambridge, United Kingdom_.

Ephrin receptor A2 (EphA2) is part of the Ephrin receptor family of cell-cell junction proteins highly overexpressed in several solid tumours, and is associated with poor prognosis. EphA2 has long been a high-value target for cancer therapeutics, but development of an Antibody Drug Conjugate targeting EphA2 (MEDI-547) was stopped in early clinical development after severe adverse events were seen (Annunziata et al, 2013). We have developed a series of Bicycle Toxin Conjugates (BTC®) comprising a constrained bicyclic peptide (Bicycle®) that binds with high affinity and specificity to EphA2, which is covalently linked to a toxin payload via a cleavable linker. The small size of BTCs offers a significant advantage over other targeted cytotoxic approaches such as antibody-drug conjugates due to rapid extravasation, renal clearance and improved tumour penetration. Bicycle binders for EphA2 were identified using a proprietary phage display peptide technology consisting of highly diverse phage libraries of Bicycles, conjugated to cleavable linkers & toxins to form Bicycle Toxin Conjugates (BTCs). We selected the candidate BTC BT5528 from a panel of >75 BTCs, based on in vivo efficacy, tolerability and drug-like properties. BT5528 is effective in EphA2-expressing xenograft models, with complete tumour regression seen from 0.5mg/kg weekly. Expression of EphA2 is high across a range of cancers of high unmet medical need. A good correlation with efficacy was seen across cell- and patient- derived xenograft models including lung, breast, oesophageal, ovarian, prostate, gastric and sarcoma tumours. No significant efficacy is seen in tumours without EphA2 expression. Rapid tumour penetration, renal elimination and significant bystander effect produce efficacy through a "hit & run" approach, with low systemic exposure to conjugate or free toxin. Efficacy is seen with dosing intervals of 1 or 2 weeks, despite a terminal half-life of ~30min for BTCs across species. In simple xenografts (CDX, ~200mm3), efficacy of BT5528 & anti-EphA2 ADC are comparable. In large & more complex models (heterogeneous PDX, >1000mm3), the ADC shows reduced efficacy, while the efficacy of BT5528 is maintained. Clinical development of MEDI-547 was terminated when patients showed profound bleeding & coagulation toxicity, consistent with the nature of toxicology seen in preclinical studies. In contrast, BT5528 shows no sign of bleeding in toxicology studies in rat and NHP and no changes in coagulation parameters or liver enzymes. The EphA2 targeting BTC BT5528 shows potent anti-tumour activity in a range of solid tumour xenograft models without the limiting toxicity observed with previous Antibody Drug Conjugate approaches. IND-enabling studies for BT5528 are currently underway.

#4482

S64315 (MIK665) is a potent and selective Mcl1 inhibitor with strong anti-tumor activity across a diverse range of hematological tumor models.

Ana Leticia Maragno,1 Prakash Mistry,2 András Kotschy,3 Zoltán Szlavik,3 James Murray,4 James Davidson,4 Gaëtane Le Toumelin-Braizat,1 Maïa Chanrion,1 Alain Bruno,5 Audrey Claperon,1 Heiko Maacke,2 Erick Morris,6 Youzhen Wang,6 Alix Derreal,5 Márton Csekei,3 Attila Paczal,3 Zoltán Szabo,3 Szabolcs Sipos,3 Agnes Proszenyak,3 Balázs Balint,3 Allan Surgenor,4 Pawel Dokurno,4 Natalia Matassova,4 Ijen Chen,4 Gaëlle Lysiak-Auvity,1 Anne-Marie Girard,1 Fabienne Grave,1 Frédéric Colland,1 Ensar Halilovic,6 Olivier Geneste1. 1 _Institut de Recherche Servier, Croissy sur Seine, France;_ 2 _Novartis Institutes for BioMedical Research, Basel, Switzerland;_ 3 _Servier Research Institute of Medicinal Chemistry, Budapest, Hungary;_ 4 _Vernalis (R &D) Ltd, Cambridge, United Kingdom; _5 _Institut de Recherches Internationales Servier, Suresnes, France;_ 6 _Novartis Institutes for BioMedical Research, Cambridge, MA_.

Mcl-1 is highly expressed in a variety of human cancers (including those of hematopoietic and lymphoid origin) and is exploited by cancer cells to evade cell death and to develop resistance to diverse chemotherapeutic agents. We disclose, for the first time, the structure of S64315 (also named MIK665) a highly potent and selective inhibitor of Mcl-1 with improved potency over its predecessor S63845 (Kotschy et al, Nature, 2016). S64315/MIK665 is currently in phase 1 in AML (Acute Myeloid Leukemia) and MDS (Myelodysplastic Syndrome) (EudraCT 2016-003768-38, NCT 02979366) and in MM (Multiple Myeloma) and lymphoma (NCT02992483). A fragment-based, structure-guided drug discovery effort led to the identification of S64315/MIK665 that binds to human Mcl-1 with a sub-nanomolar affinity (Ki 0.048 nM) and selectively over other anti-apoptotic Bcl-2 family members. It has similar affinity for human, rat, dog and monkey Mcl-1 but about a ten-fold lower affinity for mouse Mcl-1. S64315/MIK665 causes dose-dependent activation of the intrinsic apoptosis pathway in a Bax/Bak-dependent manner, as measured by increased caspase activity and cleaved PARP. S64315/MIK665 shows strong cell killing activity in a diverse panel of human hematological tumor cell lines, including AML, lymphoma and MM. The activity profile of S64315/MIK665 is distinct from that of venetoclax, a selective Bcl2 inhibitor. In vivo, S64315 as single agent demonstrated potent and dose-dependent apoptotic and antitumor response after intravenous administration in several human hematological tumor models grafted in immuno-compromised mice and rats. Complete regression of established tumors, at well tolerated doses, was achieved using different intravenous dosing regimens in rats as well as in mice. Finally, dual BH3-mimetic targeting approach combining S64315/MIK665 with BCL2 inhibitors showed strong and durable antitumor responses in several hematological tumor models both in vitro and in vivo.

#4483

Novel small molecule antagonists of the PD-1/PD-L1 axis that mediate cell surface PD-L1 dimerization and internalization.

Phillip C.C. Liu, Richard Wynn, Liangxing Wu, Alla Volgina, Nina Zolotarjova, Luping Lin, Pramod Thekkat, Alex Margulis, Ronald Klabe, Wenqing Yao, Kaijiong Xiao, Jingwei Li, Xin He, Mark Rupar, Hong Chang, Paul Waeltz, Yanlong Li, Peggy Scherle, Reid Huber, Gregory Hollis. _Incyte, Wilmington, DE_.

Blocking the PD-(L)1 immune checkpoint axis with therapeutic antibodies against either the receptor or the ligand has proven to be an effective treatment modality for multiple cancer histologies. We describe the identification and characterization of novel small molecule antagonists of the PD-(L)1 axis that function by inducing dimerization and subsequent internalization of the PD-L1 protein, effectively depleting the ligand from the cell membrane and preventing PD-1 activation on T cells. Compound-dependent PD-L1 dimerization was characterized using several biophysical techniques including fluorescence resonance energy transfer (FRET) measurements, size exclusion chromatography and thermal shift analysis. Experimental evidence demonstrates compound-dependent dimer conformation with slow dissociation kinetics and significantly enhanced thermal stability. Many of the PD-L1-directed small molecules blocked binding of soluble PD-1 to either native PD-L1 expressed on cancer cell lines or PD-L1 expressed in CHO cells with low nanomolar potency. However, only a subset of the small molecules caused loss of cell surface PD-L1 in a time- and concentration-dependent manner. Importantly, there was a strict correlation between the promotion of PD-L1 internalization secondary to dimerization and the induction of an NFAT response element-luciferase reporter gene. Strikingly, only those small molecules that could produce a specific dimeric PD-L1 conformation as measured using FRET were associated with functional activity in cells, suggesting that PD-L1 dimerization was necessary but not sufficient for internalization and cellular activity. A cell-active tool compound (cell binding IC50 <5 nM, internalization EC50 <10 nM) was fluorescently labeled to enable direct visualization of intracellular trafficking. Confocal microscopy with this PD-L1 antagonist showed time-dependent increases in intracellular fluorescence in PD-L1 expressing, but not PD-L1 deleted, cells. The internalized antagonist showed punctate staining coincident with markers of the early endosome, and independent studies confirmed that the internalized PD-L1 also trafficked to the early endosome. By disrupting the suppressive activity of PD-L1 on PD-1, these inhibitors result in functional activation of T cells in ex vivo cellular assays in a manner equivalent to antibodies directed against either PD-1 or PD-L1. In summary, we have identified a series of potent, small molecule PD-L1 antagonists that induce dimerization of the protein; inhibitors that trigger an appropriate dimeric conformation can also induce PD-L1 internalization thereby alleviating PD-L1-induced suppression of T cell activation.

#4484

**Discovery and** in vitro **characterization of AMG 510 - a potent and selective covalent small molecule inhibitor of KRAS** G12C **.**

Anne Y. Saiki,1 Kevin Gaida,1 Karen Rex,1 Pragathi Achanta,1 Tisha San Miguel,1 Neelima Koppada,2 Dhanashri Bagal,3 Brian A. Lanman,1 Robert S. Foti,2 John D. McCarter,1 Laurie P. Volak,1 Jude Canon,1 Victor J. Cee,1 J. Russell Lipford1. 1 _Amgen Inc., Thousand Oaks, CA;_ 2 _Amgen Inc., Cambridge, MA;_ 3 _Amgen Inc., South San Francisco, CA_.

Activating mutations in RAS represent the most common oncogenic driver mutation in cancer. The single amino acid substitution of cysteine for glycine at position 12 (KRASG12C) is frequently found in solid malignancies, particularly in lung adenocarcinoma (~13%), colorectal adenocarcinoma (3%), and pancreatic adenocarcinoma (~1%). Recently it has been demonstrated that KRASG12C can be targeted with covalent small molecule inhibitors which react with the mutant cysteine adjacent to the switch II pocket (SIIP), locking KRAS in its inactive GDP-bound state. We describe here the discovery and in vitro characterization of AMG 510, a covalent inhibitor of KRASG12C possessing potent biochemical and cellular activity, as well as robust in vivo efficacy. AMG 510 inhibited SOS1-catalyzed nucleotide exchange of recombinant mutant KRASG12C/C118A but had minimal effect on KRASC118A, which is wildtype at position 12. The observed rate constant (kinact/Ki) of covalent modification of KRASG12C by AMG 510 was determined biochemically by mass spectrometry as well as in the cellular context (kobs/[I]). Cysteine proteome analysis of cells treated with AMG 510 revealed that only the G12C-containing peptide of KRAS was covalently modified. AMG 510 inhibited KRAS signaling as measured by ERK phosphorylation in all KRAS p.G12C cell lines tested, but did not inhibit phosphorylation of ERK in cell lines lacking the KRAS p.G12C mutation. Cellular occupancy of KRASG12C by AMG 510 was determined by mass spectrometry and correlated well with inhibition of ERK phosphorylation. AMG 510 also selectively impaired the viability of KRAS p.G12C mutant lines. Combination treatment of AMG 510 with inhibitors of other cellular signaling pathways exhibited evidence for synergistic effects on cell viability. Treatment of KRAS p.G12C lines with covalent KRASG12C inhibitors increased the expression of HLA. To test the impact of KRASG12C inhibition on immune surveillance in vivo, we generated a syngeneic tumor cell line that is suitable for testing AMG 510 in combination with checkpoint inhibitor therapies and characterized this line in vitro. AMG 510 is currently being evaluated in a Phase I study in patients with solid tumors harboring KRAS p.G12C mutations.

#4485

Enhancing anti-tumor immune responses with clinical BET bromodomain inhibitor RG6146.

Simon J. Hogg,1 Lisa Wellinger,2 Daniel Rohle,2 Ricky W. Johnstone1. 1 _Peter MacCallum Cancer Centre, Melbourne, Australia;_ 2 _Discovery Oncology, F. Hoffmann-La Roche, Basel, Switzerland_.

The BET family of proteins bind to acetylated lysine residues on histone proteins and transcription factors to co-activate gene expression. BET proteins regulate the expression of oncogenes and can control the activity of various oncogenic transcription programs and have thereby emerged as therapeutic targets for the treatment of cancer. RG6146 is a novel non-covalent inhibitor of BET proteins that is in early phase clinical trials for the treatment of haematological and solid malignancies. The anti-tumor activity of BET inhibitors has primarily been attributed to tumor cell intrinsic effects, however increasing evidence suggests BET inhibitors modulate anti-tumor immune responses. Here, we examined the anti-solid tumor activity of RG6146 and evaluated the ability of RG6146 to enhance anti-tumor CD8+ T-cell responses.

To model anti-tumor CD8+ T-cell responses in vitro, syngeneic colon and breast tumor cells expressing ovalbumin (Ova) antigen were co-cultured with activated CD8+ T-cells derived from OT-1 transgenic mice. RG6146 functionally increased the activity of both wild-type and perforin-deficient OT-1 T-cells, leading to significantly enhanced T cell-mediated tumor cell death in a time- and dose-dependent manner. Mechanistic studies revealed that enhanced tumor cell death induced by RG6146 was dependent upon CD8+ T-cell derived tumor necrosis factor-α (TNF-α), independent of perforin/granzyme-dependent granule exocytosis. As RG6146 did not increase TNF-α production in CD8+ T-cells, we hypothesized RG6146 may sensitize tumors cells to TNF-α. Indeed, screening of cell lines revealed that BET inhibition significantly enhanced TNF-α-induced cell death in solid tumors of diverse origin. Underlying this response, we demonstrate using RNA- and ChIP-sequencing that BET inhibition suppresses transcription of pro-survival NF-kB target genes to elicit a potent pro-apoptotic phenotype. Finally, using syngeneic solid tumor models, we demonstrated that the adaptive immune system promotes the efficacy of RG6146 and evaluated the ability of RG6146 to therapeutically augment cancer immunotherapies in vivo.

Taken together, these data demonstrate that RG6146 is a potent BET bromodomain inhibitor with multi-faceted anti-cancer activity against solid tumors. We have identified a novel immunological TNF-α-dependent mechanism of bystander tumor killing by which BET inhibitors promote anti-tumor responses in vivo. Finally, we provide evidence that BET inhibition will augment the activity of cancer immunotherapies, establishing a strong rationale to evaluate these combinations in the clinic.

## IMMUNOLOGY

### Tumor Immune Microenvironment

#4486

Androgen-deprivation therapy promotes immune suppression in a murine model of prostate cancer.

John J. Krolewski, Kai Sha, Kent L. Nastiuk. _Roswell Park Cancer Inst., Buffalo, NY_.

Most prostate cancer (PCa) deaths are due to castration resistant PCa (CRPC), following failure of androgen deprivation therapy (ADT). ADT is the standard of care for patients with advanced PCa but nearly universal progression to CRPC occurs 2-3 years after ADT is initiated. Immunotherapy with checkpoint inhibitors has not been effective in most prostate cancers, perhaps because such cancers lack functional CD8 T-cells. This may be caused by infiltration of myeloid cell populations into the tumor immune cell microenvironment (TIME). Recently, we found that in a PTEN-deficient mouse PCa model, castration induces an immunosuppressive state within the tumor that is concurrent with tumor recurrence. The response to castration/ADT is tri-phasic: a pro-apoptotic regression phase when tumor shrinks, followed by selection for a residual population of resistant tumor cells and finally recurrent growth as CRPC. Using PCa cell lines to model the first two phases of the response to ADT, we have shown that ADT induces apoptosis, thereby enriching for an ADT-resistant stem/progenitor population that we propose is the in vivo source of TNF. Mechanistically, in our model system the response to ADT is driven by the soluble mediators TNF and CCL2, which facilitate communication within the TIME. Specifically, a TNF-CCL2-CCR2 paracrine loop is induced between prostate cancer cells and non-tumor cells in the microenvironment: TNF produced by tumor cells acts on myofibroblasts to induce CCL2 production, which in turn recruits CCR2+ tumor-associated macrophages (TAMs). To investigate the ADT response within the TIME in an in vivo model of prostate cancer, we employed a prostate-specific PTEN-deficient mouse model (PbCre4 x PTENf/f). Castration caused the tumors to regress, consistent with initial phase of the response that is seen in the human disease. At late times post-castration (5-6 weeks), corresponding to the selection phase, we observed a coordinate increase in the stem/progenitor tumor cell population, as well as TNF and CCL2, within the TIME. Immunohistochemical staining of tumors 5 weeks post-castration revealed an increase in TAMs, and a decrease in CD8 T cells, consistent with an immuno-suppressive or immuno-evasive state. This phenotype was reversed by a soluble receptor that binds TNF (etanercept). Thus, following ADT, TNF derived from an ADT-resistant stem/progenitor epithelial tumor cell population promotes an immunosuppressive state via CCL2 in the TIME. Analysis of public human PCa data sets show the transcripts for TNF, as well as gene signatures for stem/progenitor tumor cells and M2 TAMs are increased in CRPC, consistent with our hypothesis that ADT drives the development of myeloid immuno-suppressive state via a TNF-CCL2-CCR2 axis. Our results set the stage for the future development of immunotherapies that could improve the efficacy of ADT.

#4487

Association of the tumor-immune microenvironment with response to niraparib and pembrolizumab in relapsed, platinum resistant ovarian cancer.

Anniina Farkkila,1 Jia R. Lin,2 Julia Casado,1 Huy Nguyen,1 Yinghui Zhou,3 Julie R. Graham,3 Bruce J. Dezube,3 Steven Waggoner,4 Pamela Munster,5 Gini F. Fleming,6 Sandro Santagata,7 Ursula A. Matulonis,1 Peter K. Sorger,2 Elizabeth M. Swisher,8 Alan D. D'Andrea,1 Panagiotis Konstantinopoulos1. 1 _Dana-Farber Cancer Institute, Brigham and Women's Hospital, Harvard Medical School, Boston, MA;_ 2 _Harvard Medical School and Ludwig Center for Cancer Research at Harvard, Boston, MA;_ 3 _Tesaro, Waltham, MA;_ 4 _University Hospitals of Cleveland, Cleveland, OH;_ 5 _Helen Diller Family Comprehensive Cancer Center, San Francisco, CA;_ 6 _University of Chicago, Chicago, IL;_ 7 _Brigham and Women's Hospital, Laboratory for Systems Pharmacology, Boston, MA;_ 8 _University of Washington, Seattle, WA_.

Introduction: TOPACIO/Keynote-162 is a multi-center, open-label, single-arm phase 1/2 trial of Poly-ADP Ribose Polymerase (PARP) inhibitor niraparib combined with an anti-PD-1 antibody pembrolizumab. One cohort enrolled patients with recurrent ovarian cancer (OC). As tumor BRCA1/2 mutation (tBRCA), Homologous Recombination Deficiency (HRD) test (Myriad), or PD-L1 testing by immunohistochemistry were unable to predict clinical responses, we set out to examine additional DNA repair and immune biomarkers for response.

Methods: We analyzed pre-trial Formalin Fixed Paraffin Embedded (FFPE) tumor samples from 49 patients with recurrent OC. The samples were collected either at diagnosis, or a median of 4.3 months (range 1.5 - 91.1) after the diagnosis. The median time from diagnosis to trial entry was 35.8 months (range 11.4 - 136.5). To further assess HRD we performed BROCA sequencing of 69 DNA repair genes as previously reported. NanoString gene expression analysis was performed using an IO360 panel complemented with 30 DNA repair genes and analyzed with the NSolver software. Findings were correlated with clinical response evaluated based on RECIST v1.1.

Results: Overall, the confirmed objective response (OR) rate was 18% with a clinical benefit (complete response + partial response + stable disease; CB) rate of 65%. HRD as assessed by BROCA sequencing did not associate with OR or CB. In the Nanostring mRNA expression analyses, the samples taken at the time of diagnosis (n=23) had significantly lower abundance score for several immune cell lineages, including cytotoxic cells, macrophages, dendritic cells, and NK-cells compared to samples collected after the diagnosis. In addition, in the at-diagnosis samples, lower abundance scores for overall immune cells (CD45), tumor-infiltrating lymphocytes, CD8+T-cells, and macrophages were associated with clinical benefit. Pathway analysis demonstrated that increased scores for Cytosolic DNA sensing- and interferon pathways significantly correlated with OR, particularly in the platinum-resistant patients. In contrast, the samples collected after diagnosis (n=21), had increased mRNA expression levels of PD-L1, PD-L2 and PD-1, and a higher score for exhausted CD8+ T-cells than the samples collected at diagnosis. In these samples, a low exhausted CD8+T cell/T-regulatory cell score ratio was associated with OR.

Conclusions: Increased interferon signaling, and exhausted T/regulatory T-cell ratio are associated with response to the combination of niraparib and pembrolizumab. We are in the process of performing further profiling of the tumor microenviroment with a highly multiplexed tissue immunofluorescence (tCycIF) providing a comprehensive analysis of cell types, functional states, and spatial interactions at single-cell resolution.

#4488

Effector B cells and tertiary lymphoid structures predict response to immune checkpoint blockade in solid tumors.

Sangeetha M. Reddy,1 Beth Helmink,1 Jianjun Gao,1 Shaojun Zhang,1 Keren Yizhak,2 Moshe Sade-Feldman,2 Rafet Basar,1 Jorge Blando,1 Guangchun Han,1 Vancheswaran Gopalakrishnan,1 Hao Zhao,1 Wenbin Liu,1 Hussein Tawbi,1 Rodabe Amaria,1 Michael Davies,1 Jeffrey Gershenwald,1 Elizabeth M. Burton,1 James Allison,1 Michael Tetzlaff,1 Katy Rezvani,1 Nir Hacohen,2 Padmanee Sharma,1 Linghua Wang,1 Jennifer Wargo1. 1 _University of Texas MD Anderson Cancer Center, TX;_ 2 _Broad Institute of the Massachusetts Institute of Technology, MA_.

Background: Cytotoxic T cell markers, PD-L1, and mutational burden have been identified as biomarkers of response to immune checkpoint blockade (ICB). However, there is a growing appreciation of B cells as biomarkers and mediators of response. We recently conducted a phase II trial of neoadjuvant ICB (NCT02519322) in patients with resectable melanoma, and found increased B cell infiltration in responders (R) versus non-responders (NR).

Methods: We further investigated our B cell findings through whole transcriptomic profiling in samples with high tumor purity and also targeted immune profiling with deconvolution algorithm MCP counter. Spatial organization of the B cells was assessed with singlet and multiplex immunohistochemistry (IHC). Additionally, B cell phenotype was queried through mass cytometry. B cell gene expression signatures were validated in a cohort of renal cell carcinoma (RCC) patients (NCT02210117), and B cell phenotype in a metastatic melanoma cohort, both treated with ICB.

Results: Whole transcriptomic analysis of the neoadjuvant melanoma ICB cohort identified that the most differentially expressed genes by response in baseline (b/l) samples were related to B cells and antibody production (MZB1, JCHAIN, IGLL5, FCRL5, p<0.0001). Targeted profiling via MCP counter confirmed higher B cells by response in all b/l and on-treatment (on-tx) samples (p=0.036 and p=0.038, respectively). This was verified by singlet CD20 IHC stains, which also showed that ICB increased B cells in R (p=0.004) but not NR. The B cells were present in organized tertiary lymphoid structures (TLS) in close proximity to T cells and follicular dendritic cells, and the ratio of area occupied by TLS was higher in R (p=0.037 b/l and 0.002 on-tx). These findings were validated in a RCC cohort, in which B cell lineage scores and TLS marker CXCL13 were higher in R at b/l (p=2.6e-03 and 3.3e-03, respectively). We then investigated the phenotype of the intratumoral B cells in the neoadjuvant melanoma cohort with mass cytometry. Within the tumor microenvironment, we identified naïve, class switched and unswitched memory B cell populations, and plasma cell-like populations (CD27+, IgD-, C38++, CD138-, CD20-). Higher frequencies of class switched memory B cell and plasma-like cell populations were observed in R, whereas NR were characterized by higher naïve B cells. Matching peripheral blood based CyTOF demonstrated non-overlapping clusters of B cell phenotypes. Single cell RNA sequencing in an independent cohort of metastatic melanoma patients also demonstrated class switched, activated B cells in addition to plasma cells, and that activation markers were significantly associated with response to ICB.

Conclusion: Together, these data suggest that B cells, more specifically those with activated effector phenotypes, and TLS predict response to ICB and may also be contributing mechanistically to response.

#4489

IL-10 blockade reactivates anti-tumor immunity in human colorectal cancer liver metastases.

Kevin M. Sullivan,1 Xiuyun Jiang,1 Yongwoo David Seo,1 Heidi L. Kenerson,1 Xiaowei Yan,2 Chris Lausted,2 Changting Meng,2 Neda Jabbari,2 Kevin P. Labadie,1 Sara K. Daniel,1 Qiang Tian,2 Teresa S. Kim,1 Raymond S. Yeung,1 Venu G. Pillarisetty1. 1 _University of Washington, Seattle, WA;_ 2 _Institute of Systems Biology, Seattle, WA_.

Background: Colorectal cancer (CRC) is the 4th most common cancer in the US, and the liver is the most common site of metastatic disease. Immune checkpoint inhibitor therapy has not been successful in achieving a clinical response in most patients with CRC liver metastases (CRCLM). The liver is known to induce tolerance to foreign antigens as a result of immunosuppressive cytokines including IL-10. We hypothesized that blockade of IL-10 signaling in CRCLM would potentiate tumor infiltrating lymphocyte (TIL)-mediated tumor cell death.

Methods: We performed single-cell RNA sequencing (scRNAseq) of CRCLM using the 10x platform to evaluate for expression of IL-10 or IL-10 receptor (IL-10R) RNA within the tumor (n=8). To confirm if the IL-10R protein was present within the tumor microenvironment (TME), we also performed immunohistochemistry (IHC) (n=3). In order to study the functional effects of IL-10 blockade, we utilized a tumor slice culture (TSC) model, which allows for the study of cancers with their intact TME including immune cells. For TSCs, cores (6 mm diameter) were taken from freshly resected sterile human CRCLM and cut to 250 µm thick slices using a vibratome (n=3). Duplicate slices were treated with either IgG control or anti-IL-10 monoclonal antibodies and cultured for up to 6 days. To evaluate for histological evidence of necrosis and cell apoptosis within the tumor slice, we stained slides with either hematoxylin and eosin (H&E) or cleaved-Caspase-3 (CC3). To gain insight into the activation state of TIL after treatment, we measured levels of cytokines within the culture supernatants.

Results: We found by scRNAseq that that IL-10 was expressed by a subset of tumor-associated macrophages, and IL-10R was expressed by both CD4+ and CD8+ T cells as well as macrophages. We confirmed that IL-10R protein was present within the CRCLM TME by IHC, and IL-10R expression was distributed throughout the stroma in non-tumor cells. In TSC treated with anti-IL-10 antibody, CC3+ cells were found to be 82.8% of total cells, compared to 36.1% of control (p = 1 x 10-6) at day 6. These findings were consistent across all human tumor samples treated with IL-10 blockade versus control at all time points examined. Furthermore, IL-10 blockade led to histologic evidence of generalized necrosis compared to an intact TME seen in the control group. Analysis of cytokines released into the media confirmed that IL-10 was present in controls, but absent in slices blocked with anti-IL-10 antibody. We also found increased levels of granzyme B, IL-2, GM-CSF, and IL-18, as well as a reduction in the immune checkpoint receptor TIM3, after one day of IL-10 blockade in culture.

Conclusion: Treatment of human CRCLM TSCs with anti-IL-10 antibody leads to a marked increase in immune-mediated cell death within the tumor. Our data suggest that IL-10 serves as a critical regulator of anti-tumor immunity in the CRCLM TME and may serve as an important immunotherapeutic target.

#4490

PARP inhibitor efficacy depends on CD8+ T cell recruitment via the STING pathway in BRCA-deficient models of triple-negative breast cancer.

Constantia Pantelidou,1 Olmo Sonzogni,2 Mateus De Oliveira Taveira,3 Anita K. Mehta,1 Dan Wang,2 Aditi Kothari,1 Michelle K. Li,2 Tanvi H. Visal,1 Jennifer L. Guerriero,1 Gerburg M. Wulf,2 Geoffrey I. Shapiro1. 1 _Dana Farber Cancer Institute, Boston, MA;_ 2 _Beth Israel Deaconess Medical Center, Boston, MA;_ 3 _A.C. Camargo Cancer Center, Sao Paulo, Brazil_.

Combinatorial clinical trials of PARP inhibitors with immunotherapies are ongoing, yet the immunomodulatory effects of PARP inhibition have been incompletely studied. Here, we sought to dissect the mechanisms underlying PARP inhibitor-induced changes in the tumor microenvironment of BRCA1-deficient triple-negative breast cancer (TNBC). We demonstrate that the PARP inhibitor olaparib induces CD8+ T cell infiltration and activation in vivo, and that depletion of CD8+ T cells severely compromises anti-tumor efficacy. Olaparib-induced T cell recruitment is mediated through activation of the STING/TBK1/IRF3 pathway in tumor and dendritic cells and is more pronounced in HR-deficient compared to HR-proficient TNBC cells. CRISPR-knockout of STING in cancer cells prevents type I IFN production and is sufficient to abolish PARP inhibitor-induced T cell infiltration in vivo. These findings elucidate a novel mechanism of action of PARP inhibitors and provide mechanistic rationale for combining PARP inhibition with immunotherapies for the treatment of TNBC.

#4491

Association of intratumoral immunological profile with overall survival in metastatic pancreatic cancer patients treated with combination immunotherapy with or without PD-1 blockade.

Annie A. Wu,1 Takahiro Tsujikawa,2 Gina Choe,2 Teresa Beechwood,2 Lisa M. Coussens,2 Jennifer N. Durham,1 Elizabeth M. Jaffee,1 Dung T. Le1. 1 _Johns Hopkins Medical Institute, Baltimore, MD;_ 2 _Oregon Health & Science University, Portland, OR_.

Background: Metastatic pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis with a 5-year survival rate of 8%. Single-agent immunotherapies fail to show clinical activity due to a complex tumor microenvironment (TME) and lack of effector T cells. We previously showed in a neoadjuvant clinical trial that an irradiated, granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting, allogeneic PDAC vaccine (GVAX) recruited T cells into tumor and upregulated the PD-1/PD-L1 pathway1. Here we describe the first clinical testing of GVAX prime given with attenuated listeria monocytogenes expressing mesothelin (CRS-207) boost alone or in combination with nivolumab to block PD-1 signaling in metastatic PDAC patients, and evaluated changes in the TME.

Experimental Design: 22 metastatic PDAC biopsy pairs were obtained at baseline and after 2 GVAX prime and 1 CRS-207 boost from 96 vaccinated patients. Nivolumab was administered with each vaccine in patients randomized to vaccine + nivolumab arm. Biopsies containing >30% tumor cellularity were chosen for multiplex immunohistochemistry (IHC) to examine changes in immune cell subtypes and their signaling pathways in tumors. We did a comparative analysis looking at lymphoid and myeloid complexity, immune function, and PD-L1 status of patients with overall survival (OS) <150 days [short OS, n=6], 150-300 days [middle OS, n=11], and >300 days [long OS, n=5] or by treatment arm.

Results: Favorable OS correlated with low CD68+ myeloid cell and high lymphoid cell numbers, which was detected after prime-boost. Evaluation of the functional status of CD8\+ T cells after prime-boost of patients with long OS revealed a 10.2% increase in EOMES+PD1- effector memory and 1.4% decrease in EOMES+PD1+ exhausted T cells. At baseline, fewer CSF1R+ tumor associated macrophages (TAMs), CD68+CD163+ and CD163- myeloid cells in tumors was associated with long OS. We analyzed nivolumab's effect on the TME and found that tumors from nivolumab-treated patients displayed a decrease in CD68+ myeloid cells after prime-boost compared to baseline. Interestingly, CD163+ TAMs in tumors of nivolumab-treated patients expressed higher PD-L1 levels.

Conclusion: This study suggests that induction of lymphoid-inflamed expression profiles with less exhausted and more effector memory CD8+ T cells during treatment is associated with long OS independent of nivolumab treatment. Fewer TAMs in pre-treatment biopsies and a low myeloid: lymphoid cell ratio in post-immunotherapy tumor samples may be predictive of improved survival. Our findings also suggest that nivolumab induces PD-L1 expression on myeloid cells. Continual identification of key tissue-based biomarkers that correlate with OS may be useful for predicting therapeutic response.

1) Lutz E. (2014) Cancer Immunol Res. Jul;2(7):616-31.

#4492

Humanized anti-EGFR antibody panitumumab inhibits tumor growth of inflammatory breast cancer by inducing antitumor immunity.

Xiaoping Wang, Shan Shao, Takashi Semba, Troy Pearson, James M. Re, Debu Tripathy, Naoto Ueno. _UT MD Anderson Cancer Center, Houston, TX_.

Inflammatory breast cancer (IBC) is the most lethal and aggressive form of breast cancer, yet no approved targeted therapies specifically for IBC have been approved. The epidermal growth factor receptor (EGFR) pathway is a promising therapeutic target for patients with triple-negative IBC, with a reported pathological complete response rate of 42%. The tumor microenvironment (TME) is a critical contributor to the aggressiveness of IBC. Therefore, delineating the cross-talk between EGFR-targeted therapy and TME components could lead to more efficient combination regimens and novel clinical trial designs for IBC. Because an immunocompetent IBC animal model is not available for our study, we established an IBC SUM149 immunocompetent mouse model (SUM149-hu-NSG-SGM3) for such studies. Here we report the effects of panitumumab (PmAb), a humanized monoclonal anti-EGFR antibody, on tumor growth and on the TME in the SUM149-hu-NSG-SMG3 mouse model.

Methods: SUM149 cells were mixed with 50% Matrigel and inoculated into mammary fat pads of hu-NSG-SGM3 mice engrafted with human hematopoietic stem cells. SUM149 tumor growth in mice treated with either isotype IgG2 or PmAb was measured. We used flow cytometry to measure the percentages of human immune cells in the peripheral blood and tumor tissues from mice treated with IgG2 and PmAb. The effects of PmAb treatment on the expression and distribution of TME components in tumor tissues was examined by multiplex immunohistochemical staining. Cytokine expression in tumor tissues from IgG2- and PmAb-treated mice was measured by cytokine antibody array, and the changes in cytokines expression were validated with RT-PCR.

Results: PmAb treatment, compared with IgG2 treatment, significantly reduced SUM149 tumor growth. Flow cytometry analysis showed infiltration of lymphocytes in SUM149 tumors with an observed increase in the level of CD8+ T cells and a reduced level of Tregs in both peripheral blood and tumor tissues from PmAb-treated mice compared with IgG2-treated mice. Cytokine array and RT-PCR showed that PmAb treatment increased expression of cytokines that function as a chemoattractant for T cells, natural killer cells, and monocytes, including CXCL10, CCL5, CCL4, and CXCL9. In contrast, PmAb treatment reduced expression of cytokines involved in antitumor immune suppression, including osteoprotegerin (OPG), CCL2, CXCL5, and CCL20.

Conclusion: PmAb treatment reduced IBC tumor growth by increasing cytotoxic T cells and reducing immunosuppressive Treg cells. Our data also suggest that the combination of PmAb with an immune checkpoint inhibitor may potentiate the efficacy of anti-EGFR therapy in IBC. A study of the therapeutic efficacy of the PmAb and anti-PD-L1 combination in the SUM149-hu-NSG-SMG3 mouse model is in progress.

## MOLECULAR AND CELLULAR BIOLOGY / GENETICS

### Deregulated Transcription and RNA Processing in Cancer

#4493

SF3B1 **mutation promotes c-Myc protein stability through aberrant splicing and downregulation of PP2A B56α subunit in chronic lymphocytic leukemia.**

Qimei Han,1 Libin Deng,2 Shuo Tu,3 Lirong Pei,1 Jeong-Hyeon Choi,1 Victor Jin,4 Huidong Shi1. 1 _Augusta Univ., Augusta, GA;_ 2 _Nanchang University, NanChang, China;_ 3 _Nanchang University, Nanchang, China;_ 4 _University of Texas Health Science Center, San Antonio, TX_.

SF3B1 mutation, which occurs in 10-20% of chronic lymphocytic leukemia (CLL) patients, is associated with faster disease progression, shorter overall survival and fludarabine resistance. Mutant SF3B1 utilizes cryptic 3′ splice sites to generate aberrantly spliced mRNAs, half of which may undergo non-sense mediated mRNA decay (NMD), leading to downregulation of the affected genes. To identify potential tumor suppressor genes that are aberrantly spliced and downregulated by mutant SF3B1, we performed RNA-sequencing analysis of SF3B1-mutated primary CLL cells in the presence or absence of cycloheximide (CHX), a translation inhibitor known to inhibit NMD. Our analysis identified PPP2R5A, encoding one of the regulatory subunits of protein phosphatase-2A (PP2A B56α), as one of the key genes that were most consistently affected by an aberrantly spliced junction, and significantly downregulated (>2 fold) in CLL patient samples with various SF3B1 hotspot mutations. Splicing analyses by RT-PCR and Sanger sequencing confirmed that a 13-nucleotides sequence was added before the 5th exon of PPP2R5A via alternative splicing in CLL patients with the SF3B1 mutation. Due to this 13-nucleotides addition, three consecutive premature stop codons were created by frameshift at a position more than 55 bases upstream of an exon-exon junction, which is a canonical feature of the mRNAs degraded through NMD. The down-regulation of PPP2R5A was confirmed by quantitative PCR (p<0.05) and immunoblot at both mRNA and protein levels in CLL patients with SF3B1 mutation as compared to CLL patients without SF3B1 mutation and normal CD19+ B cell samples. Furthermore, overexpression of mutant SF3B1 in K562 and HEK 293 cells resulted in the same aberrant splicing pattern observed in CLL patients with SF3B1 mutation and downregulation of PPP2R5A (p<0.05). Treatment with CHX resulted in an increased abundance of alternatively spliced PPP2R5A transcript, suggesting that the aberrant PPP2R5A transcript was degraded by NMD. PP2A, when incorporated with PPP2R5A as the regulatory subunit, dephosphorylates c-Myc at S62, and results in the degradation of c-Myc. Here, we demonstrated that downregulation of PPP2R5A by mutant SF3B1 led to upregulation of c-Myc through promoting c-Myc protein stability in SF3B1K700E overexpressing cells when compared to SF3B1wildtype overexpressing cells. Knockdown PPP2R5A in MEC1 CLL cells by siRNA also significantly upregulated c-Myc at the protein level and upregulated mRNA expression of c-Myc downstream genes NCL and ODC1. Overall, our results demonstrate that SF3B1 mutation causes aberrant splicing and downregulation of tumor suppressor protein PP2A B56α in vitro and in vivo, leading to upregulation of c-Myc.

#4494

HNRNPH1-dependent splicing of a fusion oncogene reveals a targetable RNA G-quadruplex interaction.

Carla Neckles,1 Robert Boer,2 Nicholas Aboreden,1 Robert L. Walker,1 Bong-Hyun Kim,3 Suntae Kim,1 John S. Schneekloth,2 Natasha J. Caplen1. 1 _Center for Cancer Research, National Cancer Institute, Bethesda, MD;_ 2 _Center for Cancer Research, National Cancer Institute, Frederick, MD;_ 3 _Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD_.

Ewing sarcoma (EWS) is the second most common bone tumor in children and adolescents. The primary oncogenic event in ~85% of EWS is a chromosomal translocation that generates a fusion gene encoding the aberrant transcription factor EWS-FLI1. A third of EWS-FLI1 driven tumors harbor translocations that retain exon 8 of the EWSR1 gene, and in these tumors, skipping of this exon is vital to express an in-frame EWS-FLI1 mRNA. We have shown previously that the heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1) binds to G-rich sequences within EWSR1 exon 8 and facilitates the exclusion of this exon from distinct EWS-FLI1 pre-mRNAs. Guanine-rich nucleic acids may fold into stable tertiary structures, such as RNA guanine quadruplexes (rG4s). Our current studies are focused on the tertiary RNA structure of the G-rich regions within EWSR1 exon 8 and its role in HNRNPH1-dependent RNA processing. Here, we show that the processing of distinct EWS-FLI1 pre-mRNAs by HNRNPH1, but not other homologous family members, resembles alternative splicing of transcript variants of wild-type EWSR1. We demonstrate that guanine-rich sequences within EWSR1 exon 8 can fold into RNA G-quadruplex structures and that this structure favors the recruitment of HNRNPH1. Bioinformatic analysis of transcriptome-wide profiles for RNA G-quadruplexes and RNA targets of HNRNPH1 revealed sequences containing two-quartet G-quadruplex configurations are central for HNRNPH1 binding to exonic elements. Critically, we also demonstrate that the glycine-tyrosine-arginine-rich domain of HNRNPH1 is the minimal domain required for G-quadruplex recognition. Small molecules that can displace HNRNPH1 binding may provide a therapeutic vulnerability in a subset of Ewing sarcoma. To evaluate if this protein-RNA interaction is amenable to small molecules, we performed displacement assays with HNRNPH1 protein and an EWSR1 exon 8 RNA oligomer at varying concentrations of the pan-quadruplex binding molecule, pyridostatin (PDS). PDS disrupts the HNRNPH1-RNA complex with an IC50 of 7 micromolar. Furthermore, treatment with PDS selectivity inhibits the growth of EWS cells harboring EWSR1 exon 8 fusions, decreases EWS-FLI1 activity in cell-based reporter assays, and restores mRNA expression of EWS-FLI1 deregulated transcriptional targets. Our findings illustrate that splicing modulation of EWS-FLI1 pre-mRNA is a promising strategy for future therapeutics against this fusion oncogene expressed in a third of Ewing sarcoma.

#4495

Role of base excision repair (BER) pathway in regulation of KRAS expression in pancreatic cancer.

Suravi Pramanik, Shrabasti Roychoudhury, Hannah Harris, Heyu Song, Kishor K. Bhakat. _Univ. of Nebraska Medical Ctr., Omaha, NE_.

Activating mutations in KRAS proto-oncogene, a signature event driving the development, progression and therapeutic resistance of pancreatic ductal adenocarcinoma (PDAC), remains undruggable. The occurrence of guanine oxidation (8-oxoguanine) in the KRAS promoter and up-regulation of KRAS gene transcription under oxidative stress has been shown to be associated with KRAS expression and cancer development and progression. However, the molecular mechanism by which 8-oxoguanine damage regulates KRAS expression is largely unknown. Here, we show that the base excision repair (BER) pathway, a fundamental DNA damage repair pathway that processes most of the endogenous damages including oxidative base damage is involved in regulation of KRAS expression and survival of PDAC. We show that Apurinic/apyrimidinic endonuclease (APE1), a key enzyme of the BER pathway, is highly elevated in pancreatic cancer tissue samples. To elucidate the role of active BER pathway in the regulation of KRAS expression, we used real-time PCR (RT-PCR) analysis. Inflicting cells with oxidative damage using glucose oxidase increased KRAS gene expression in control cells but not in APE1 downregulated cells. ChIP assay showed enhanced occupancy of APE1 in KRAS promoter upon oxidative stress. Consistent with this, using synthetic oligonucleotide containing the KRAS promoter region, we showed that APE1 could bind and cleave AP site in KRAS promoter. Further, ChIP-qPCR analysis showed decreased occupancy of MAZ, a transcription factor, to the KRAS promoter in APE1 downregulated cells. Down-regulation of APE1 also resulted in decreased KRAS expression and increased sensitivity of pancreatic cancer cells to routinely used chemotherapeutic agents such as Gemcitabine, 5-Fluorouracil, Oxaliplatin, etc., suggesting that targeting APE1 and in turn, BER can sensitize pancreatic cancer cells. Our study suggests that BER pathway or APE1 plays a significant role in the tumor-selective regulation of gene expression and sensitization of cancer cells to chemotherapy, and supports the further investigation of novel treatments that target this pathway for cancer therapy.

#4496

Cytokine-induced posttranslational modifications of FOXA1 affect enhancer selection and estrogen signaling in breast cancer cells.

Shen Li, Raul Mendez-Giraldez, Joseph P. Garay, Kamila Wisniewska, Colby A. Tubbs, Charles M. Perou, Hector L. Franco. _Lineberger Comprehensive Cancer Center and the Department of Genetics at the University of North Carolina Chapel Hill, Chapel Hill, NC_.

The pioneer transcription factor FOXA1 is a critical determinant for estrogen receptor (ER) function in hormone-dependent breast cancers. Upon estrogen stimulation, liganded ER binds to poised enhancer regions across the genome that are demarcated by FOXA1 and histone modifications such as H3K4me1 and H3K27ac. In a recent publication, we show that proinflammatory signaling, caused by the cytokine TNFa, drives FOXA1 to latent enhancer binding sites to promote chromatin accessibility for subsequent ER binding upon estrogen ligation. These latent enhancers, activated by the combined treatment of estrogen and TNFa, induced the expression of a unique transcriptome with clinical significance. The effects of TNFa treatment on FOXA1 chromatin redistribution and subsequent gene expression occur within 40 minutes, which points to a rapid signaling cascade that culminates in either changes in FOXA1's posttranslational modifications (PTMs) or its binding partners. To understand how proinflammatory TNFa signaling can redirect FOXA1 to new sites across the genome, we started by characterizing the posttranslational modifications (PTMs) of FOXA1. We immunoprecipitated FOXA1 from MCF-7 breast cancer cells that were treated by E2, TNFa or E2+ TNFa, and then examined their posttranslational status using semi-quantitative and quantitative mass spectrometry approaches. Several phosphorylation sites and acetylation sites have been identified near the DNA binding domain of FOXA1, and acetylation of lysine 295 (K295) was found specifically enriched in TNFa treatment. To test if acetylation of FOXA1 at K295 changes its binding preference and genomic distribution, we used the programmable properties of CRISPR/Cas9 to create specific knockin mutations to mimic or prevent acetylation of K295 in MCF-7 cells. More specifically, we mutated K295 to glutamine (K295Q) to mimic acetylation and essentially "lock" FOXA1 into a permanently acetylated state and, for comparison, we created another cell line where K295 was mutated to arginine (K295R) to prevent acetylation of FOXA1. Our preliminary data shows changes in the genomic redistribution of FOXA1 in the knock-in cell lines resulting in altered gene expression programs. These data suggest that inflammation-based acetylation of FOXA1 can affect estrogen signaling pathways in breast cancer cells by altering the enhancer landscape of FOXA1 and consequently the estrogen receptor.

Supported by a grant from the NIH/NCI (R00 CA204628) to H.L.F

#4497

Distinct structural classes of activating FOXA1 alterations in prostate cancer progression.

Abhijit Parolia,1 Marcin Cieslik,1 Shih-Chun Chu,1 Lanbo Xiao,1 Takahiro Ouchi,1 Yuping Zhang,1 Xiaoju Wang,1 Pankaj Vats,1 Xuhong Cao,1 Fengyun Su,1 Rui Wang,1 Felix Feng,2 Yi-Mi Wu,1 Robert Lonigro,1 Dan R. Robinson,1 Arul M. Chinnaiyan1. 1 _University of Michigan, Ann Arbor, MI;_ 2 _University of California San Francisco, San Francisco, CA_.

Forkhead box A1 (FOXA1) is a pioneer transcription factor that is essential for the normal development of several endoderm-derived organs, including the prostate gland. FOXA1 is frequently mutated in the hormone-receptor driven prostate, breast, bladder, and salivary gland tumors. In prostate luminal epithelial cells, wild-type FOXA1 delimits tissue-specific enhancers that are transcriptionally activated by AR, and extensively reprograms AR-activity in the transformed prostate epithelia. However, how FOXA1 alterations affect cancer development is unclear, with FOXA1 previously ascribed both tumor suppressive and oncogenic roles. In this study, we assemble an aggregate cohort of 1546 prostate cancers (PCa) and, for the first time, show that FOXA1 alterations fall into three distinct structural classes that diverge in clinical incidence, genetic co-alteration profiles, and oncogenic gain-of-functions. Notably, we find the three classes of FOXA1 alterations to collectively recur at a frequency of 35% in metastatic PCa. Class1 activating mutations originate in early PCa without concurrent ETS or SPOP alterations, selectively recur within the Wing2-region of the DNA-binding Forkhead domain (FKHD), confer enhanced chromatin mobility and binding frequency, and strongly trans-activate a luminal androgen receptor (AR) program of prostate oncogenesis. By contrast, class2 activating mutations are acquired in metastatic PCa, truncate the C-terminal regulatory domain of FOXA1, confer chromatin-binding dominance by increasing DNA affinity, and aberrantly activate the WNT/β-Catenin pathway to enable PCa metastasis. Finally, class3 genomic rearrangements are comprised of tandem duplications and translocations within the highly-syntenic FOXA1 locus. These structural variations amplify or reposition a conserved enhancer element, which we named FOXA1 Mastermind (FOXMIND), to drive overexpression of FOXA1 or other oncogenes, respectively. In summary, our study reaffirms the central role of FOXA1 in mediating AR-driven oncogenesis, and provides mechanistic insights into how different classes of FOXA1 alterations uniquely promote PCa initiation and/or metastatic progression. Furthermore, these results have direct implications in understanding the biology of other hormone-receptor driven cancers where similar FOXA1 alterations are found, and rationalize therapeutic co-targeting of oncogenic FOXA1-activity to extort more potent and durable disease remissions.

#4498

O GlcNAcylation of SOX2 regulates its tumor initiation properties in pancreatic cancer.

Nikita S. Sharma,1 Vineet K. Gupta,1 Patricia Dauer,2 Roey Hadad,1 Kousik K. kesh,1 Vikas Dudeja,1 Ashok Saluja,1 Sulagna Banerjee1. 1 _University of Miami, Miami, FL;_ 2 _University of Minnesota, Minneapolis, MN_.

Background: Pancreatic Cancer is an extremely aggressive disease with a bad outcome owing to its late detection, high rate of metastasis and self-renewal post-surgical and chemotherapeutic intervention. In cancer the balance between differentiation and self-renewal is tightly regulated by SOX2. Over expression of SOX2, as in pancreatic cancer, can tilt the balance towards self-renewal and thus increase the population of these undifferentiated "stem cells".

Methods: A CRISPR based OGT knockout kit was purchased from Origene and a knockout cell line was generated with S2VP10 cells. For the tumor initiation studies OGTi (OGT knockout cells) were implanted subcutaneously at a limiting dilution from 500,000 cells to 500 cells. Site directed mutagenesis was performed using the Quick-change Lightning from Agilent.

Results: In the current study we show for the first time that SOX2 undergoes a type of post translational modification known as O GlcNAcylation. We also show for the first time that in humans this modification happens at Serine 246 and mutating this particular site via a side directed mutagenesis leads to a loss of O GlcNAcylation of SOX2.O GlcNAc modification of SOX2 further leads to its activation which then leads to its stabilization in the nucleus. A CRISPR-OGT knockout in pancreatic cancer cell line S2VP10 resulted in a delayed tumor initiation. We further showed that mutation of this site (S246A) prevents the modification of Sox2 and its downstream activity. Our study also demonstrated that targeting OGT in vivo with a small molecule inhibitor OSMI, results in decreased tumor burden, delayed tumor progression and a decreased expression of SOX2 in pancreatic cancer cells.

Conclusion: Our study highlights for the first time that the O-GlcNAc transferase dependent SOX2 glycosylation has a profound effect on the transcriptional activity of SOX2 and is instrumental in determining self-renewal in pancreatic cancer which can be targeted therapeutically.

#4499

Genomic editing of EWS-FLI1 and its targets, and its therapeutic potential in treatment of Ewing sarcoma.

Sheetal A. Mitra,1 Namritha Ravinder,2 Veronica Magnon,2 Jon Nagy,3 Timothy J. Triche1. 1 _Children's Hospital Los Angeles, CA;_ 2 _ThermoFisher Scientific, CA;_ 3 _NanoValent Pharmaceuticals, MT_.

Ewing sarcoma (ES) is an aggressive bone and soft tissue tumor. These tumors have a low mutational burden and are mainly driven by chimeric transcription factor, EWS-FLI1 or equivalent fusion gene. Tumor dependency on EWS-FLI1 makes it an ideal therapeutic target but targeting this disordered protein has proven to be a challenge. Here we show that CRISPR-Cas9-mediated genomic editing can a) be employed to effectively target EWS-FLI1 and its target genes, both coding and non-coding, and b) be safely delivered to tumors in mice, thus highlighting its potential as a therapeutic agent. All CRISPR-Cas9 reagents (ThermoFisher Scientific) including Cas9 protein, single guide RNA (sgRNA) directed against various gene targets and controls, and LentiArrayTM lentiviruses were obtained and optimized for in vitro and in vivo use. Cas9-sgRNA ribonucleoprotein (RNP) complexes were transfected using CRISPRMAXTM or delivered to ES cells by CD99-targeted nanoparticles. We were able to achieve about 70% genomic cleavage when we targeted the fusion gene, EWS-FLI1, and its coding target, NR0B1, using two different sgRNA delivered as RNP complexes. Loss of EWS-FLI1 slowed down cell growth, but increased cell adhesion, and cell invasion. For EWS-FLI1 target, long non-coding RNA, FEZF1-AS1, we used paired sgRNA targeting two different regions of the RNA to achieve effective genomic editing (70%) and loss of transcript expression. We further used a lentiviral-generated stable Cas9 expressing A673 cell line to effectively knockout FEZF1-AS1 using lentiviruses expressing paired sgRNA. Both the RNP and lentiviral mediated FEZF1-AS1 knockdown did not affect cell growth but significantly decreased cell invasion. Dual knockdown of EWS-FLI1 and FEZF1-AS1 decreased cell growth and cell invasion. Mice with ES tumors in their flanks were treated with intravenous injections of CD99-targeted nanoparticles carrying Cas9-sgRNA RNP. Mice injected with Cas9-scrambled gRNA RNP had regular tumor growth. Cas9-EWSR1 sgRNA RNP-treated mice had nearly completely suppressed tumor growth for the duration of treatment but failed to totally eliminate the tumors. In conclusion, we show that CRISPR-Cas9 mediated genomic editing can effectively target EWS-FLI1 and disrupt its function in ES pathogenesis. We further demonstrate that modified CRISPR strategy can effectively knockout non-coding RNA and that the combined targeting of EWS-FLI1 and its lncRNA target had an added therapeutic response in controlling tumor growth and invasion. The pilot animal studies (more detailed studies underway) emphasize the importance of developing CRISPR-Cas9 as a therapeutic tool provided we can safely deliver it in vivo using vehicles like targeted nanoparticles to help reduce toxicity caused by untoward targeting effects.

### Noncoding RNAs in Cancer Biology

#4500

Mechanisms of tumor associated myeloid cells in modulating host immune microenvironment and metastatic progression.

Hiroki Ishii, Christine Hollander, Tim Greten, Li Yang. _NCI, Bethesda, MD_.

The immune checkpoint inhibitor blockade takes advantage of the specific receptor-ligand interactions between immune and tumor cells. However, the immunosuppressive microenvironment has been largely ignored, which likely contribute to low efficacy and resistance in immune therapies. Our studies demonstrate that TGF-β signaling in tumor-associated myeloid cells isessential in systemic immune suppression and metastatic progression. Using mouse models of breast cancer metastasis, we further uncover that miR130a and 145 are critical in regulating TGF-β signaling in myeloid cells. miR130a and 145 are down-regulated in these myeloid cells, leading to increased TGF-β RII. Ectopic expression of miR-130a and miR-145 in the myeloid cells decrease tumor metastasis. This is mediated through a downregulation of type 2 cytokines in myeloid cells and an increase in IFNgamma-producing cytotoxic CD8 T lymphocytes. Lastly, miR-130a and miR-145 mimics improve systemic anti-tumor immunity and inhibit metastasis in preclinical mouse models. Another important mechanism in myeloid-mediated immune suppression involves a decreased expression of Lamin A/C, nuclear lamina proteins that prominently interact with heterochromatin. Lamin A/C knockout in myeloid cells leads to H3K4me3-mediated upregulation of Gfi1 and C/EBPε,critical transcription factors responsible for granulocytic lineage differentiation. In addition, loss of Lamin A/C attenuates antigen-presentation and compromises host anti-tumor immunity. These mechanistic understandings provide options to reprogram tumor-associated myeloid cells by altering the cytokine milieu and metastatic microenvironment, thus enhancing host antitumor immunity.

#4501

Loss of miR-21 delays myc-driven prostate cancer progression in the hi-myc transgenic mouse model.

Kenji Zennami,1 Fatima Rafiqi,2 Ross Liao,2 Kim Sealover,2 Brian Simons,2 Makoto Sumitomo,1 Ryoichi Shiroki,1 Shawn Lupold2. 1 _Fujita Medical University, Toyoake, Japan;_ 2 _Johns Hopkins University School of Medicine, Baltimore, MD_.

Introduction and Objectives: MicroRNA 21 (miR-21) is overexpressed in virtually all types of cancers including prostate cancer (PCa). We have previously reported that the androgen receptor (AR) induces miR-21 expression, and that elevated miR-21 is sufficient to drive PCa growth and castration resistance. However, the role of miR-21 in PCa initiation and progression has not been fully elucidated. To determine if the absence of miR-21 inhibits or delays prostate cancer incidence or progression, we crossed miR-21 KO mice with Hi-Myc mice. In this study, we identify that loss of miR-21 delays Myc-driven prostate cancer progression in the Hi-Myc transgenic mouse model.

Methods: miR-21 KO mice were backcrossed to FVB mice to generation F10. Resulting miR-21 WT or KO FVB progeny were crossed with Hi-Myc mice to generate Hi-Myc/miR-21 WT or KO mice. Prostates were harvested at 4 weeks (n=10), 3 months (n=10), 5 months (n=58), 6 months (n=25), 8 months (n=20) of age and prostate weight, histology, miR-21 expression levels and protein expression levels were quantified. Proliferation and apoptosis were examined by immunohistochemistry using Ki-67 and cleaved caspase-3.

Results: Cancerous prostates of Hi-Myc mice showed high miR-21 expression at 5m and 8m, when compared to FVB controls, suggesting high miR-21 expression in the Myc-driven prostate cancer. Hi-Myc/miR-21 KO mice demonstrated a marked reduction in adenocarcinoma when compared to Hi-Myc/miR-21 WT mice at 5m. This is apparent in the reduced weight of effected prostate lobes of Hi-Myc/miR-21 KO versus WT mice at 8m. Protein levels of established miR-21 target genes, PDCD4 and PTEN, were up-regulated in the prostates of Hi-Myc/miR-21 KO mice. Also, IHC showed lower Ki-67 and higher cleaved caspase-3 positive cells in miR-21 KO than WT. However, miR-21 KO did not affect prostatic intraepithelial neoplasia (PIN) development.

Conclusions: Our results demonstrate that miR-21 delays Myc-driven prostate cancer progression through reduced proliferation and enhanced apoptosis, and that endogenous miR-21 suppresses prostatic PDCD4 and PTEN protein expression levels. Therefore, targeting of miR-21 or its downstream target genes may be useful for cancer prevention or cancer therapy.

#4502

The role of A-to-I RNA editing in diversifying the microRNA functions in cancer.

Han Liang. _UT MD Anderson Cancer Ctr., Houston, TX_.

Both miRNAs and A-to-I RNA editing, a widespread nucleotide modification mechanism, have recently emerged as key players in cancer pathophysiology. However, the functional impact of RNA editing of miRNAs in cancer remains largely unexplored. Here, we systematically characterized the miRNA editing profiles of ~9000 samples across 20 cancer types from miRNA sequencing data of The Cancer Genome Atlas and identified adenosine-to-inosine (A-to-I) RNA editing hotspots. Focusing on the RNA editing hotspot in miR-200b, a key tumor metastasis suppressor, we found that the miR-200b editing level correlates with patient prognosis opposite to that pattern for the wild-type miR-200b expression. We further experimentally showed that in contrast to wild-type miRNA, the edited miR-200b can promote cell invasion and migration through its impaired ability to inhibit ZEB1/ZEB2 and acquired concomitant ability to repress new targets including LIFR, a well-characterized metastasis suppressor. In another example, an ADAR2-catalyzed RNA editing site within the miR-379 is under-edited in tumors relative to normal tissues, with a high editing level being correlated with better patient survival times across cancer types. We demonstrate that in contrast to wild-type miRNA, edited miR-379 inhibited cell proliferation and promoted apoptosis in diverse tumor contexts in vitro. Importantly, through nanoliposomal delivery, edited miR-379 mimics significantly inhibited tumor growth and extended survival of mice. Our study indicates a role of RNA editing in diversifying miRNA function during cancer progression and highlights the translational potential of edited miRNAs as new class of cancer biomarkers and therapeutics.

#4503

Functional CRISPR screen towards identifying novel conserved long non-coding RNAs with oncogenic activity.

Yajia Zhang, Lanbo Xiao, Mengyao Tan, Yasuyuki Hosono, Arul Chinnaiyan. _University of Michigan Medical School, Ann Arbor, MI_.

Large-scale transcriptome analysis has enabled the systematic profiling of long non-coding RNAs (lncRNAs) in cancer- or lineage-specific contexts. Although a significantly larger portion of the genome is transcribed into lncRNAs compared to proteins, only a small set of them appears to be evolutionally conserved across organisms. In general, functionally essential genes are subject to stronger selective pressure, and are thus more evolutionarily conserved than nonessential genes. However, it remains a question whether the evolutionally conserved lncRNAs play essential roles for cell growth and development. Recently, our lab described THOR, the first evolutionarily conserved lncRNA that is testis/cancer-specific and accelerates tumor growth in preclinical models. Based on these discoveries, we hypothesized that a subclass of conserved lncRNAs that are functionally essential for tumor growth and cancer progression should exist. To test this hypothesis, we first developed a top-down approach to generate a list of cancer-associated evolutionary conserved lncRNAs. By integrating base-pair conservation, best 200-bp conservation, and transcript expression information, we identified a total of 85 ultra-conserved lncRNAs that are expressed in cancer. We then set up a CRISPR-Cas9 based system to screen for functionally essential lncRNAs in various lineages of cancer. Unlike protein-coding genes, whose expression can be efficiently disrupted by conventional single guide RNA-based CRISPR-Cas9 system, lncRNA transcripts are insensitive to read-frame alterations. We thus utilized an lncRNA deletion strategy featured by paired guide RNAs flanking the conserved domains within the lncRNA transcript. We designed a total of 858 pairs of guide RNAs (gRNAs) targeting 242 conserved domains within the 85 ultra-conserved lncRNAs and protein-coding gene controls. Sequencing validation of the library confirms more than 90% coverage of the designed gRNA pairs. Using this library, we have performed functional CRISPR screens in cancer cell line models from various lineages, namely, MIA-PaCa-2 (pancreatic ductal adenocarcinoma), SK-N-Mc (neuroepithelioma), and VCaP (prostate cancer). Notably, we have successfully discovered both novel and previously reported oncogenic lncRNAs (e.g. ZNF503-AS1), which, upon knockout, significantly delayed cell growth (represented by the diminished count of guide RNAs targeting these genes in the cell population). Therefore, from these CRISPR screens, we discovered ultra-conserved lncRNAs with suggestive oncogenic roles in a lineage-specific or pan-cancer manner. Further interrogation of their structures, cellular locations, and interactomes is warranted to reveal their contributions to cancer progression.

#4504

MILIP is a pan cancer-associated long noncoding RNA that links MYC to inactivation of p53.

Yuchen Feng,1 Liu Teng,2 Simonne K. Sherwin,1 Xiaoying Liu,2 Jinming Li,2 Margaret Farrelly,1 Yongyan Wu,3 Wei Gao,3 Zhuanyun Du,2 Yanfeng Xi,3 Jin Liang,3 Ting La,1 Yuanyuan Zhang,1 Hessam Tabatabaee,1 Xu Guang Yan,1 Hamed Yari,1 Tao Liu,4 Rick F. Thorne,1 Su Tang Guo,3 Binquan Wang,3 Xu Dong Zhang,1 Lei Jin1. 1 _The University of Newcastle, Newcastle, Australia;_ 2 _Zhengzhou University, Zhengzhou, China;_ 3 _Shanxi Medical University, Taiyuan, China;_ 4 _The University of New South Wales, Sydney, Australia_.

The involvement of noncoding RNAs (ncRNAs) in cancer pathogenesis has been increasingly appreciated. Although the expression and function of ncRNAs is highly tissue type- and context-dependent, we have found that long ncRNA (lncRNA) MILIP (Myc-inducible lncRNA inactivating p53) is commonly upregulated in TCGA cancer samples across 20 cancer types and that its high expression was associated with poor overall survival of patients. The increase in MILIP expression was subsequently corroborated using in situ hybridization (ISH) in a panel of colorectal cancer and a cohort of non-small cell lung carcinoma samples compared with paired normal tissues and its negative relation to patient survival was confirmed. shRNA silencing of MILIP reduced viability and clonogenicity in various types of malignant cells including A549 lung cancer, MCF-7 and MDA-MB-231 breast cancer, HCT116 colon cancer, U2OS and Saos-2 osteosarcoma and BE(2)C neuroblastoma cells, and retarded A549 and MCF-7 xenograft growth. The inhibitory effect of MILIP silencing on cell viability was reversed by co-introduction of a shRNA-resistant mutant of MILIP, and moreover, introduction of exogenous MILIP promoted cell proliferation. Thus, MILIP functions as an oncogenic driver in diverse types of cancers. Strikingly, transcriptomics analysis revealed that the p53 pathway was mostly enriched in cells with MILIP silenced. Further mechanistic investigations demonstrated that MILIP was located to both the cytoplasm and nucleus, and that both fractions of MILIP contributed to its oncogenic role, as cytoplasmic MILIP accelerated p53 polyubiquitination and proteasomal degradation, whereas nuclear MILIP regulated p53 occupancy of its target promoters. Interestingly, we identified a consensus c-Myc binding site at the proximal promoter of the gene encoding MILIP that was transcriptionally activated upon binding to c-Myc. Indeed, MILIP upregulation appeared to be associated with MYC gene amplification. These results suggest that lncRNA MILIP plays a role in relaying oncogenic signaling of MYC to disabling the tumor suppressor p53. Of note, MILIP silencing also reduced viability of p53-null cancer cells and those carrying mutant p53, indicating that MILIP can also promote cancer pathogenesis independently of its effects on p53. Collectively, these findings identify MILIP as a pan-cancer associated oncogenic driver and implicate that MILIP may constitute a target for treatment of diverse types of cancer.

#4505

miR-122 regulates hepatic glutamine metabolism by directly targeting mitochondrial glutaminase.

Dipanwita Sengupta,1 Teresa Cassel,2 Kun-yu Teng,3 Peng Hu,1 Teresa Fan,2 Kalpana Ghoshal1. 1 _Ohio State Univ. College of Medicine, Columbus, OH;_ 2 _Univ. of Kentucky, KY;_ 3 _City of Hope-Comprehensive Cancer Center, Durate, CA_.

It is well established that the liver specific miR-122, a bona fide tumor suppressor, plays critical role in lipid homeostasis [1]. However, its role, if any, in amino acid metabolism has not been explored. Since glutamine is an important energy source, we monitored glutamine metabolism in the wild type (WT) and miR-122 knockout (KO) mice by SIRM (Stable Isotope Resolved Metabolomics) studies. To this end, 6-8 weeks old mice were injected with [U-13C,15N]-L-glutamine (Gln) (7 mg/mouse, 2x at 15 min interval) and sacrificed 15 min after the last injection. Analysis of polar metabolites from snap-frozen liver tissues by NMR and IC-MS showed elevated 13C-glutamine, -glutamate, -α-ketoglutarate, -isocitrate and -citrate levels without significant changes in succinate, malate or fumarate levels in KO livers suggest enhanced glutaminolysis and a break in the latter half of the Krebs cycle in miR-122 depleted livers. Reduced conversion of [13C,15N-glutamine] to 13C3\- and or/ 13C6-glucose-6-phosphate and -fructose-6-phosphate implicates reduced gluconeogenesis from glutamine in KO livers while that to 13C-glutathiones suggests decreased synthesis and/or increased utilization.

To elucidate the underlying mechanism, we focused on HITS-CLIP and RNA-seq data of the WT and KO mice, which identified functional miR-122 targets in the liver transcriptome [2]. The results showed that Gls is a direct miR-122 target, which was validated by luciferase reporter assay. Importantly, Gls expression was suppressed in glutamine dependent hepatoma cells by ectopic expression of miR-122 mimic. These results explain that the increased conversion of [U-13C,15N]-L-glutamine to glutamate and other Krebs cycle metabolites is likely due to the upregulation of Gls in miR-122 depleted livers. TCGA-LIHC database analysis showed that GLS expression in tumors inversely correlated with miR-122 expression. Upregulation of GLS protein level in primary HCCs relative to the matched benign liver tissues suggests enhanced use of glutamine as energy source by the tumor cells.

Collectively, these results show that miR-122 modulates glutamine metabolism both in vitro and in vivo. These results implicate therapeutic potential of glutaminase inhibitors in patients bearing HCCs that bear low levels of miR-122.

(supported in part, by R01CA193244 grant from NIH and Pelotonia Idea grant)

1. Essential metabolic, anti-inflammatory, and anti-tumorigenic functions of miR-122 in liver. J Clin Invest. 2012 Aug;122(8):2871-83.

2. Argonaute CLIP Defines a Deregulated miR-122-Bound Transcriptome that Correlates with Patient Survival in Human Liver Cancer. Mol Cell. 2017 Aug 3;67(3):400-410.e7.

#4506

MDM2 alternative splicing is regulated by microRNA binding to control p53 activity.

Matias Montes,1 Andrew Goodwin,2 Dawn S. Chandler1. 1 _The Ohio State University and Nationwide Children's Hospital, Columbus, OH;_ 2 _Nationwide Children's Hospital, Columbus, OH_.

Alternative splicing of the MDM2 is an important means by which p53 is upregulated to combat the deleterious effects of genotoxic stress. One splice variant, MDM2-ALT1, is activated in response to genotoxic stress and is comprised of only the two terminal coding exons 3 and 12 and therefore lacks a p53 binding domain. This variant has been identified in a number of human tumors, including invasive carcinoma of the breast and lung, as well as soft tissue sarcomas. Despite its pervasiveness in tumors and the therapeutic possibilities it presents, there is very little known about the regulators of that govern this alternative splicing event.

Our lab has identified a specific microRNA that is expressed at high levels in normal cells but decreased under conditions of genotoxic stress and in rhabdomyosarcoma cells, both of which are characterized by the expression of the MDM2 spliced isoform. Hence, expression of the microRNA is inversely correlated with the splice variant MDM2-ALT1. Though recent studies have shown that splicing factors can be regulated through miRNA-mediated gene silencing and indirectly influence splicing choices, we were unable to identify any splicing regulators of MDM2 to be targeted by the microRNA. Interestingly, the microRNA has been reported as one of the miRNAs that is reimported to the nucleus but has not been ascribed a role in the nuclear space. We therefore wanted to test the hypothesis that the microRNA can directly bind to the MDM2 pre-mRNA to drive splicing choices in a regulated fashion. To this end, we have identified predicted binding sites for the microRNA in the regulated region of MDM2, within and near exons 3 and 12. We have shown that the microRNA binds specifically to these regions in MDM2 in normal cells and has decreased binding after genotoxic stress. Furthermore, we have shown that overexpression of the pre-microRNA can alleviate the induction of the MDM2-ALT1 isoform in response to genotoxic stress. We propose that microRNA binding to the MDM2 pre-mRNA regulates its splicing and plays a role in the DNA damage response and the pathway to tumorigenesis.

Our work implicates for the first time that a miRNA would be acting as a splicing factor, a novel function for this class of non-coding RNAs and a novel method of regulation of the p53-MDM2 axis. Furthermore, our discoveries reveal miR-regulated splicing as a possible strategy to activate p53 and sensitize cancer cells to conventional cancer therapies.

### Oncogenes, Tumor Suppressors, and Carcinogenesis

#4507

Study of CCCTC-binding factor (CTCF) genetic aberrations and dysregulation in head and neck squamous cell carcinoma (HNSCC).

Sze Man Chan,1 Hoi-Lam Ngan,1 Yuchen Liu,1 Wenying Piao,1 Chin Wang Lau,2 Jason Ying Kuen Chan,1 Yu Xiong Su,3 Vivian Wai Yan Lui1. 1 _The Chinese University of Hong Kong, Hong Kong;_ 2 _Yan Chai Hospital, Hong Kong;_ 3 _The University of Hong Kong, Hong Kong_.

CCCTC-binding Factor (CTCF) is a DNA-binding protein that acts as a global genome organizer in higher eukaryotes. CTCF folds the genome into spatial domains that is crucial for regulation of genes, including oncogenes and tumor suppressor genes. Recently, Bornstein et al. has reported that CTCF truncating mutations are associated with progression of Head and Neck Squamous Cell Carcinoma (HNSCC), suggesting a potential role of CTCF and its aberrations in HNSCC progression.

Survival analysis of the US-HNSCC The Cancer Genome Atlas (TCGA) Provisional cohort (N=502) revealed that CTCF mutations are associated with poor overall survival (P = 0.0206, Median survival of 15 months vs 56.9 months) and disease-free survival (DFS; P = 0.0078, median DFS of 12.975 months vs 71.22 months). Best separator analysis was performed to determine the clinical association between patient survival and CTCF mRNA expression levels. Our results showed that CTCF mRNA downregulation (best separator at z-score of 1.93) is correlated with reduced disease-free survival (P =0.0018, median DFS of 13.01 months vs 42.77 months) in HNSCC patients. Gene set enrichment analysis (GSEA) of hallmark gene signatures revealed enrichment of MYC target in HNSCC patients with CTCF mRNA downregulation (<-1.93 z-score; P = 0.0502), and MYC family deregulation is frequently associated with poor clinical outcome in various cancer types, including HNSCC. Intriguingly, RNA-seq analysis of 43 pairs of normal-tumor tissues from the TCGA reveal a tumor-specific upregulation of CTCF mRNA (P < 0.0001). Subsequent Western blot analyses demonstrated at least 1.5-fold CTCF protein upregulation in HNSCC patient primary cultures and HNSCC cancer cell lines relative to normal head and neck squamous cells. Based on these preliminary genomic findings, we hypothesized that CTCF upregulation may have pro-tumorigenic effects in HNSCC. Therefore, cancer cell proliferation was monitored following exogenous overexpression by retroviral introduction of the human CTCF gene or specific knockdown (by shRNA) of CTCF in HNSCC cell lines. We found that exogenous overexpression of CTCF promoted HNSCC cell proliferation by about 40% (N=6). However, with multiple trials, specific knockdown of CTCF only resulted in about 10% growth inhibition of HNSCC cells (N=6) with minimal apparent knockdown of CTCF protein. This unexpected challenge of CTCF protein knockdown could likely be due to potentially long CTCF protein half-life in HNSCC. In fact, we found that CTCF protein is highly stable in HNSCC, with a half-life of around 55 hours (vs control) with cycloheximide inhibition. Our results demonstrated a growth-promoting role of CTCF in HNSCC, and this CTCF protein appears to be highly stable with overexpression in HNSCC, potentially serving as a stable push for tumorigenesis.

#4508

Cyclophilin A as a molecular switch regulating PRLr-Jak2 complex mediated signaling, mammary tumorigenesis and metastasis.

Shawn Hakim,1 Shannon E. Hedrick,2 Justin M. Craig,1 Charles V. Clevenger2. 1 _VCU Wright Center for Clinical and Translational Research, Richmond, VA;_ 2 _VCU Health, Richmond, VA_.

Prolactin (PRL) and its receptor (PRLr) have been implicated in the development and progression of human breast cancer. PRL activates its receptor and induces activation of proximal Janus kinase 2 (Jak2). Jak2 associates with PRLr, phosphorylates c-terminus of the PRLr, which leads to Stat5 recruitment and activation. Cyclophilin A (CypA) is a peptidyl-prolyl isomerase (PPI), which is constitutively bound to the PRLr that catalyze the cis-trans interconversion of proline imide bonds. Treatment with Cyclosporine (CsA) inhibited CypA binding to the PRLr and blocked PRLr-driven activation of Jak2/Stat5. Recently, Waters et al., utilizing Fluorescence Resonance Energy Transfer (FRET) with transfectants expressing CFP and YFP-tagged forms of the growth hormone receptor (GHR) showed that GHR activation induced a rotational movement in C-terminus of the GHR, resulting in a loss of baseline FRET signal. Like the GHR, PRL stimulation of transfectants expressing CFP/YFP-tagged PRLr constructs resulted in a loss of FRET efficiency. In contrast, treatment with NIM811 (a non-immunosuppressive form of CsA) or siRNA knockdown of CypA resulted in a return of FRET signal in the presence of PRL. These studies reveal that ligand stimulation of the PRLr results in a conformational change as measured by FRET signal of the receptor that is reversed by CypA inhibition or knockdown, implicating CypA as the mediator of this conformational change and ligand-induced signaling. To further assess the consequences of CypA inhibition or knockdown on the PRLr/Jak2 mediated signaling/functions, analyses of phospho-tyrosine residues that are believed to be important for interactions/signaling were investigated in breast cancer cells. It was found that NIM811 inhibition or CypA shRNA knockdown significantly reduced prolactin-stimulated phosphorylation of PRLr/Jak2 intermediates in ER+/PR+ T47D cells in a time dependent manner. A microarray analysis revealed that NIM811 inhibited approximately 66% of the top 50 PRL induced genes. NIM811 inhibited ER-/+, and HER2+ breast cancer cell proliferation, survival, migration and anchorage-independent growth. Subsequent pre-clinical testing of NIM811 in relevant mouse mammary cancer models has found that NIM811 treatment of a TNBC xenograft inhibited primary tumor growth, outgrowth of macro-metastasis and induced central tumor necrosis. Furthermore, loss of CypA (by constitutive genetic deletion) in the MMTV-PyMT mouse model demonstrated inhibition of tumorigenesis with significant reduction in lung and lymph node metastasis. Overall, CypA modulates conformational change in the C-terminus of the PRLr through its PPI activity, and alters PRLr/Jak2 complex signaling/functions in breast cancer and mammary epithelium, identifying this isomerase as a novel target for therapeutic intervention as a chemo-preventive and as an inhibitor of metastasis.

#4509

Overexpression of CADM1 enhances malignant features of small cell lung cancer.

Toko Funaki, Takeshi Ito, Yoshinori Murakami. _The Institute of Medical Science, The University of Tokyo, Japan_.

Small-cell lung cancer (SCLC) is one of the representative intractable cancers with the 5-year survival of less than 10 %. Novel approaches to the treatment of SCLC are required because recurrence arises inevitably after the initial chemotherapy. CADM1 was originally identified as a tumor suppressor of non-small cell lung cancer (NSCLC) which encodes a member of immunoglobulin superfamily cell adhesion molecules. CADM1 consists of an extracellular domain with three immunoglobulin-like loops, a trans-membrane domain and a short cytoplasmic domain containing a protein 4.1-binding motif (4.1BM) and a PDZ-II-binding motif. We have reported that CADM1 is highly expressed in 90% of human SCLC cell lines and promotes subcutaneous tumor formation of SCLC cells in nude mice. In this study, we performed clinicopathological evaluation of CADM1 in SCLC and analyzed the molecular mechanisms of malignant progression of SCLC by overexpressing CADM1. Firstly, we performed immunohistochemical analysis of CADM1 expression in high-grade neuroendocrine tumor (HGNEC) of the lung, including SCLC and large cell neuroendocrine carcinoma (LCNEC). We found that CADM1 protein was expressed in 75% (33/44) of SCLC tumors and correlated with the nodal involvement in lung HGENC. To understand pathological significance of CADM1 overexpression in SCLC, we introduced a full-length CADM1 and its mutants in the extracellular or intracellular domain into SCLC cells, SBC5, lacking endogenous CADM1 expression and assessed their malignant features in comparison with control SBC5 cells. CADM1 expression significantly enhanced colony formation in soft agar and subcutaneous tumor formation in nude mice of SBC5, whereas a mutant in 4.1BM did not. Then, we screened chemical library of 170 small molecules of known function to identify compounds which inhibit anchorage-independent growth of SCLC cells enhanced by CADM1. Inhibitors of PI3K, Akt and PKA significantly suppressed cell growth of SBC5, suggesting that CADM1 activates PI3K/Akt and PKA signaling pathways through its 4.1BM and promotes anchorage-independent growth of SCLC cells. We also found that orthotopic injection of NCI-N417 cells overexpressing CADM1 into the lung of nude mice significantly enhanced their metastasis to mediastinal lymph nodes. These results suggest that CADM1 enhances tumor growth and metastasis of SCLC cells probably by activating PI3K/Akt and PKA pathways through its binding proteins at 4.1BM.

#4510

Linking growth factors and immune actors: PTEN/PI3K signaling in cancer and the response to cytokines.

Sanjay Chandrasekaran,1 Maiko Sasaki,2 Brian P. Pollack2. 1 _Emory University Winship Cancer Institute, Atlanta, GA;_ 2 _Emory University, Atlanta VA Medical Center, Atlanta, GA_.

Background: The PTEN/PIK3 pathway plays a critical role in the cellular response to growth factors. Furthermore, aberrant activation of PI3K signaling is a known hallmark of many forms of cancer. While the role of PI3K signaling in the context of growth factor biology has been well studied, less is known about its activity in the cellular response to cytokines such as interferons (IFNs). We examined how PI3K activation by PTEN inhibition would affect interferon signaling pathways using an in vitro experimental model system.

Methods: Cultured human HCT 116 colorectal carcinoma cells and isogenic PTEN (-/-) cells were treated with escalating doses of IFNγ, IFNα2β, and IFNλ. The response to IFNs was evaluated by measuring mRNA levels via RT-PCR and cell surface expression by flow cytometry of MHC Class I and Class II at sequential timepoints. Western blotting analysis was performed to measure levels of total AKT, phospho-AKT (pAKT), PTEN, STAT1, and pSTAT1. Additional pharmacologic studies were performed using cultured HPV-negative SqCC/Y1 oSCC cells treated with IFN, the PTEN inhibitor VO-OHpic, and AKT activator SC79. Further experiments were also done in an isogenic PTEN knockdown BRAFV600E mutant a375 melanoma cell line. Clinical correlates utilizing immunohistochemistry (IHC) on human head and neck cancers were conducted.

Results: IFN-γ- induced surface expression of MHC II in PTEN (-/-) HCT 116 cells was markedly reduced by greater than 50% vs parental cells. PTEN (-/-) HCT 116 cells also expressed lower basal levels of MHC I, with similar attenuation in induction by IFNs. These reductions in surface expression correlated with lower MHC I/II mRNA levels. IHC studies in patient head & neck cancer biopsy samples demonstrated an inverse relationship between pAKT and MHC I expression. Additional studies were performed comparing PTEN knockout and the expression of STAT1, a downstream target of IFN signaling. We observed that PTEN/PIK3 signaling implicates STAT1, and that PTEN inhibition consistently modulated total and phosphorylated levels of STAT1 protein in PTEN (-/-) HCT 116 cells. Similar findings were also observed in melanoma cells.

Conclusions: We aim to further characterize the link between oncogenic signaling and anti-tumor immunity. We demonstrate, using an in vitro model, that genetic and pharmacologic activation of the PI3K pathway modulates the immune response to IFNs and represses MHC induction and expression. Importantly, similar inverse patterns of PI3K signaling and MHC expression were seen in patient biopsy samples. We also present a novel mechanism linking the PTEN/PIK3 and STAT1 pathways to further explain our findings. Additional characterization of this interaction in in vivo models is necessary.

#4511

**Prostate cancer specific enrichment of** TP53 **missense mutations elicit differential, context dependent biochemical and biological outcomes.**

Jennifer J. McCann, Irina Vasilevskaya, Neermala PoudelNeupane, Jeffry Dean, Amy Mandigo, Renee De Leeuw, Chris McNair, Matthew Schiewer, Karen Knudsen. _Thomas Jefferson University, Philadelphia, PA_.

Prostatic adenocarcinoma (PCa) remains the second leading cause of cancer death in men in the United States. While organ-confined disease can be successfully treated with surgery or radiation, patients with advanced disease undergo androgen deprivation therapy (ADT), which targets the androgen receptor (AR) signaling axis. This approach effectively treats the disease as PCa cells are exquisitely dependent on AR signaling for proliferation and survival. Unfortunately, cancer progression resumes after 2-3 years and results in a lethal stage of disease, termed castration-resistant prostate cancer (CRPC). While PCa typically demonstrates low overall mutational burden, TP53 is frequently mutated in both primary and advanced disease. These mutations are most commonly missense mutations in the DNA binding domain of TP53, and can occur in the presence or absence of a wildtype TP53 allele. Interestingly, specific enrichment of certain TP53 mutations frequently occurs in PCa when compared to other cancers, and similar to many other cancers, these mutations occur in the presence or absence of wildtype TP53. Using hormone therapy sensitive and CRPC cells, a panel of cell lines was generated to model these mutations in the presence or absence of wildtype TP53 expression. Defining the TP53 missense mutant-sensitive transcriptomes demonstrated context dependent, differential gene expression between TP53 missense mutants, related to the expression of wildtype TP53 in those cells. This also applied to cistrome analysis, illustrating an expansion of the p53 cistrome upon expression of mutant TP53. Furthermore, expression of p53 mutants also elicited context dependent effects on canonical p53 functions, thereby modulating distinct, downstream biological outcomes. Consequently, these data define the distinct and context dependent roles that mutant TP53 plays in PCa progression.

#4512

Identifying novel mechanisms of p53-mediated tumor suppression.

Nathan H. Leisenring,1 Robert W. Floyd,1 David G. Kirsch2. 1 _Duke University School of Medicine, Durham, NC;_ 2 _Duke University Medical Center, Durham, NC_.

TP53 is the most frequently mutated gene in human cancer. Classically, p53-mediated tumor suppression is thought to occur via transactivation of canonical targets that induce cell cycle arrest and apoptosis. However, this model has been challenged by studies using mouse p53 mutants. The mouse transactivation domain mutant, p5325,26, is unable to activate most canonical p53 targets and fails to induce cell cycle arrest or apoptosis in response to DNA damage. However, it retains the ability to induce cellular senescence and suppresses tumor development in mouse models. Indeed, some human p53 mutants found in Li-Fraumeni syndrome as well as cancer retain transactivation, while some p53 mutants are not found in cancer despite loss of transactivation. The goal of our study is to identify novel p53 functions that drive tumor suppression by evaluating p53 mutations where transactivation is uncoupled from tumor suppression activity.

To identify human TP53 missense mutants with tumor suppressive function dissociated from the ability to activate canonical p53 pathways, we first utilized the results of a 2003 saturation mutagenesis screen performed in yeast which stratified human p53 mutants by their ability to activate 8 canonical p53 targets. We then cross-referenced these results with tumor sequencing data from the IARC, the TCGA PanCancer Atlas, and the MSK-IMPACT Sequencing Cohort. We identified panels of human p53 missense mutants which either 1) retained transactivation ability in yeast, yet had been identified in sequenced human tumors or 2) were unable to transactivate p53 targets in yeast, but had not been identified in sequenced human tumors.

To study these p53 mutants compared to wt p53, we cloned their sequences into doxycycline-inducible pLVX-TetOne-Puro plasmids and puromycin-selected for cell lines stably expressing the construct in p53 null H1299 and HCT116 cells. Using qPCR and western blots we validated the expression of mutant or wt p53 and assessed for transactivation of canonical targets p21, mdm2, puma, gadd45α, and 14-3-3. The IncuCyte Live Cell Analysis system and XTT Cell Proliferation assays were used to evaluate p53-mediated growth arrest of human cancer cells following induction of each mutant. From these experiments, we selected three mutants for further study. Missense p53 mutants E224D and R290H are found in human cancers and in the germline of Li-Fraumeni syndrome patients yet retain the capacity to transactivate p53 targets and arrest tumor cell growth in vitro. In contrast, p53 mutant G262A which is not found in cancer lacks the ability to activate p53 targets and fails to arrest tumor cell growth in vitro.

We are currently using soft agar assays to assess the transformation potential of the identified p53 mutants in FSF-KRAS ; p53fl/+ MEFs. To dissect tumor suppressive function we are developing transgenic mice expressing the p53 mutants. The results of this ongoing study may uncover novel p53 tumor suppressor mechanisms.

#4513

Targeting the DLC1 Rho-GAP tumor suppressor protein to treat solid tumors.

Brajendra K. Tripathi, Meghan F. Anderman, James H. Doroshow, Douglas R. Lowy. _National Cancer Institute, Bethesda, MD_.

Although cancer usually arises from the combined effects of oncogene activation and tumor suppressor gene inactivation, most targeted cancer treatments are focused on the inhibition of oncoproteins, with less consideration given to the possible concomitant effects on tumor suppressors. Here we describe a critical role of the DLC1 tumor suppressor in the growth regulation of cancer and have identified two kinases that directly phosphorylate DLC1 and attenuate its tumor suppressor activity, leading to the hypothesis that reactivation of DLC1 by the relevant kinase inhibitors should be evaluated as a candidate biomarker of clinical response. We determined that the SRC and AKT kinases directly phosphorylate and attenuate the Rho-GAP and tumor suppressor activities of DLC1 by distinct, cooperative mechanisms and that inhibition of the SRC and/or AKT kinases has strong antitumor activity in two DLC1-positive tumor models that have high SRC and AKT activities, the MMTV-PyMT breast cancer model and tumor xenografts from a NSCLC line. In both models, treatment with the kinase inhibitors results in cooperative DLC1 reactivation, which was monitored by the reactivation of the Rho-GAP and tumor suppressor activities of DLC1. Reactivation of DLC1 makes a critical contribution to the antitumor activity of the AKT or the SRC inhibitors in both models, and the combined treatment with inhibitors of both kinases has even greater Rho-GAP and antitumor activities, which is correlated with greater reductions in RhoA-GTP and its downstream signaling. Furthermore, the combined treatment with AKT and SRC inhibitors induced cellular senescence and apoptosis to a greater degree than single agent treatment, as measured by beta-galactosidase and annexin V expression after short-term tumor treatment. Their antitumor activity is much weaker in isogenic tumors that are DLC1-negative or express DLC1 mutants that are not activatable by the AKT and the SRC inhibitors. A limitation of this therapeutic approach is that it is likely to benefit only those tumors that express moderate to high levels of DLC1 protein. However, several types of solid tumors express DLC1 mRNA but have lost DLC1 protein expression through the epigenetic destabilization of the protein. We hypothesize that it could be beneficial to treat this class of solid tumors with a combination of drugs that can stabilize the DLC1 protein and concurrently inhibit the AKT and SRC activities. Our results indicate the feasibility of this approach, as treatment with proteasome inhibitors in several tumor cell lines that lack detectable DLC1 protein but express its mRNA increased the steady state level of DLC1 protein, which enabled SRC and AKT inhibitors to more potently inhibit tumor cell growth and reduce RhoA-GTP and its signaling. Thus, reactivation of the DLC1 tumor suppressor can be a key therapeutic target, in contrast to the usual situation where the focus is principally on inhibiting pro-oncogenic factors.

## TUMOR BIOLOGY

### Invasion and Metastasis 2: Tumor Microenvironment

#4514

Intravital imaging at single cell resolution reveals, for the first time, the mechanism of cancer cell dissemination and metastasis.

Lucia Borriello, Anouchka Coste, Yarong Wang, Maja Oktay, David Entenberg, John Condeelis. _Albert Einstein College of Medicine, Bronx, NY_.

Metastatic dissemination is the major cause of cancer mortality and is responsible for over 1/2 million deaths each year in the U.S. alone. Over the past decades, metastasis has been speculated to be an inefficient process, as the majority of disseminated tumor cells (DTCs) are not believed to complete all the steps of the metastatic cascade. This conclusion has been reached by studies in which TCs are intravenously injected in mice - a process called experimental metastasis (EM) - and then attempt to quantify the number of disseminated cells in the lung over time using low resolution and indirect methods. To date, very little is known about the efficiency of each step in metastatic cascade due to the inability to track metastatic cancer cells in an intact organ over time.

Using a new cutting-edge technology, the Window for High Resolution Imaging of the Lung1, we are able for the first time to (1) track in real-time the fate of each DTC and (2) quantitatively assess the efficiency of each step of the metastatic cascade in the lung, from DTC arrival and retention in the lung vasculature, extravasation, interaction with the host microenvironment, survival, dormancy, to growth into micro-metastases. All steps are studied at single-cell resolution, longitudinally, in the same animal, and using the EM model as well as a more clinically relevant model called Spontaneous Metastasis (SM) in which tumor cells spontaneously disseminate from an orthotopic primary tumor.

A comparative analysis of the two models (EM vs. SM) demonstrated that DTCs in SM have a drastically increased metastatic efficiency compared to DTCs in EM. In particular, we found that DTCs are retained in the lung 10-times more efficiently compared to EM (62% vs. 6%). We further observe that in SM, DTCs extravasate in the lung very rapidly compared to EM (8 hrs vs. 24 hrs) and that the vast majority of DTCs after extravasation died in EM compared to SM where DTCs are dormant and express a stem-like phenotype. These data indicate that dissemination is indeed a very efficient process, but that growth at the secondary site is a rate-limiting step in the SM model.

In conclusion, the ability to observe both spontaneous and experimental metastasis, with single cell resolution, and longitudinally, has provided new insight into the efficiency of each step of the metastatic cascade in the lung. This approach additionally gives the ability to investigate the molecular mechanisms underlying seeding and dormancy of metastatic tumor cells, in the presence of the full tumor microenvironment, as well as to directly evaluate the response of disseminated tumor cells to therapeutic treatment in real time in a live mouse.

(1). Entenberg D, et al., (2017). A permanent window for the murine lung enables high-resolution imaging of cancer metastasis. Nat Methods. 15(1):73-80.

#4515

Utilizing nucleic-acid scavengers (NASs) to inhibit pro-inflammatory and pro-invasive signaling in triple-negative breast cancer.

Elias O. Eteshola,1 Ibtehaj A. Naqvi,2 Ruwan Gunaratne,2 Angelo Moreno,1 Smita K. Nair,1 Bruce A. Sullenger1. 1 _Duke University School of Medicine, Durham, NC;_ 2 _Stanford School of Medicine, Stanford, CA_.

Breast cancers (BC) remain the most lethal malignancies amongst women worldwide and the second leading cause of cancer-related mortalities in the US. Subtype heterogeneity and aggressive invasive potential are believed to be the major contributors of these outcomes. BC lacking canonical histological receptors (i.e. ER/PR/HER2), called triple-negative (TNBC), are notoriously aggressive, difficult-to-treat, and metastatic. It has been observed that the degree of inflammation-driven tumorigenesis tends to correlate with increased levels of cell-free DNA (cfDNA) in cancer patient sera. Our lab had previously shown that nucleic-acid scavengers (NASs) could be used to block the pro-inflammatory and pro-invasive/metastatic signals (e.g. DAMPs) elicited by these cfDNA/RNA likely through innate immune sensors such as of the toll-like receptors (TLRs). Recently, we showed that treatment with the cationic polymer NAS, PAMAM-G3, elicited a drastic reduction in metastatic tumor burden to the liver in an immune-competent murine model of pancreatic cancer (I. Naqvi & R. Gunaratne et al., Molecular Therapy 26, 2018). Ongoing work has shown that both chemotherapy-derived TNBC conditioned media and a TLR9 agonist greatly increased TNBC in vitro invasion and was significantly inhibited upon treatment with PAMAM-G3. To elucidate the mechanism by which this NAS works in these tumor settings, our lab has developed several PAMAM-G3 derivatives, including biotin, IR-, and near-IR fluorophore labeled molecules. These molecules will allow us to conduct DAMP capture and characterization experiments, as well as perform in vitro and in vivo live imaging experiments to gain better insights into NAS PK/PD properties. Mechanistic insight into NAS anti-metastatic and anti-inflammatory capabilities will enhance our basic understanding of metastatic progression and its interplay with the immune system. Moreover, these principles will aid in the development of novel of anti-metastatic therapies to improve TNBC patient outcomes.

#4516

Suppression of metastasis by inhibiting nanoscale physical communication of cancer cell and the endothelium.

Chinmayee Dash, Tanmoy Saha, Sachin Khiste, Shiladitya Sengupta. _Harvard Medical School, Brigham and Women's Hospital, Cambridge, MA_.

The progression of metastasis proceeds through the physical communication of cancer cells with the endothelium via tunneling nanotubes (TNTs). Restriction on the cancer cell and the endothelium communication by inhibiting TNT formation can lead to breakthrough advancement in the treatment of metastasis. However, the mechanistic interpretation about the formation of TNTs between the cancer cells and the endothelium has been poorly defined in literature. Our study integrates the investigation of mechanism behind the TNT formation and introduction of a pharmacological inhibitor for TNT formation.

The nanobridges are primarily composed of cytoskeletal elements such as actin and tubulin, which originates from the energy dependent remodeling of membrane cytoskeletal elements. Here, we propose that the members of Rho-family of GTPases, Cdc42, Ral and Rac along with exocyst complex, are involved in the actin polymerization based on their localization to the nanobridges between the metastatic cancer cells and the endothelium. A significant decrease in mitochondrial transfer and TNT formation has been observed when the inhibitor for Cdc42/Rac1 GTPase inhibitor (ML141), and a geranylgeranyltransferase 1 inhibitor (L-778,123) has been employed in the co-culture. The amount of mitochondrial transfer was evaluated with flow cytometry and the number of TNT formation was counted from imaging, which in case of inhibitors was highly reduced. Simultaneously the involvement of exocyst complex was confirmed through imaging. The proteins involved in the exocyst complex co-localize with the nanotubes and was further validated by siRNA-based knock down study. The nanotubular pathways are the primary mode of communication between cancer cell and endothelium, which leads to the conversion of healthy endothelium to a pathological one. Hindering the TNT mediated communication via pharmacological inhibitors can reduce the chance of metastasis.

Conclusion: Investigation of molecular mechanism behind the TNT formation and employing innovative ways to have restriction over that can be a potential next generation therapeutic module for controlling metastasis.

Reference: Connor Y, Tekleab S, Nandakumar S, Walls C, Tekleab Y, Reiberger T, Husain A, Gadish O, Sabbisetti V, Kaushik S, Sehrawat S, Kulkarni A, Dvorak H, Zetter B, Jain RK, Edelman E, Sengupta S. Physical nanoscale conduits-mediated communication between tumor cells and endothelium enables a metastatic hijack. Nature Communications (2015) 6:8671.

#4517

Tumor associated macrophages are reprogrammed by pancreatic ductal adenocarcinoma cells through tumor induced DNA methylation on metabolic genes.

Xingyi Pan,1 Mengwen Zhang,2 Kenji Fujiwara,1 Noelle R. Jurcak,1 Stephen Muth,1 Lei Zheng1. 1 _Johns Hopkins Medical Institutions, Baltimore, MD;_ 2 _The Second Affiliated Hospital, Zhejiang University, Hangzhou, China_.

Metabolism is shifted toward glycolysis with increased glucose uptake in anti-tumor M1 macrophages, and toward oxidative phosphorylation with significant decreased glucose uptake in pro-tumor M2 macrophages in Pancreatic ductal adenocarcinoma (PDA). Current study aims to investigate the mechanism by which tumor cells reprogram tumor associated macrophages metabolically and functionally. Using mouse bone marrow derived macrophages and human macrophages derived from peripheral blood mononuclear cells for contact and non-contact co-culture experiments with PDA tumor cells, we showed PDA tumor cells, through direct cell-cell contact confirmed by Lucifer yellow dye-coupling assay, induced DNA methylation and downregulation of a panel of glucose metabolism and oxidative phosphorylation genes selectively in M1 but not M2 macrophages by methylation specific PCR, methylation microarray analysis and qPCR, RNA-seq analysis. Glucose-response genes such as IL10 were subsequently activated. Through IL10 and its receptor IL-10R on tumor cells, macrophages enhanced the in vitro migration of tumor cells in trans-well migration assay verified by shRNA knockdown of IL10 and IL10R respectively. Exogenous infusion of both M1 and M2 macrophages promoted PDA metastasis in orthotopic mouse model after resident macrophage depletion, although pretreating with DNA demethylating agent Decitabine prevented M1, but not M2 macrophages in promoting metastasis, suggesting M1 macrophages were reprogrammed metabolically and functionally by tumor cells to become pro-cancerous. To interrogate the mechanism that regulates the tumor-induced DNA methylation in macrophages, we first examined chromatin remodeling complex Polycomb Repressive Complex 2 (PRC2), with its major component enhancer of zeste homolog 2 (EZH2) previously shown to recruit DNA methyltransferases to specific chromatin regions for epigenetic gene regulation. We found EZH2 protein was most significantly increased in M1 macrophages while Ser21 phosphorylated EZH2 (pSer21-EZH2) remained constant, indicating a decreased percentage of pSer21-EZH2/EZH2 in M1 macrophages co-cultured with tumor cells. Further identification of potential F-box containing ubiquitin ligases that interact with pSer21-EZH2 by Co-immunoprecipitation suggested EZH2 may be stabilized in M1 macrophages by tumor cells through inhibiting the ubiquitin-dependent proteolysis of pSer21-EZH2 in a phosphorylation dependent manner. This study thus far reveals a novel mechanism that reprograms M1 macrophages phenotypically into M2-like macrophages and functionally into pro-cancerous macrophages. Identification of cell surface receptors that mediate the direct tumor-macrophage contact are actively being sought, which will lead to the development of therapeutic blockades to reverse the macrophage reprogramming.

#4518

IL-4/IL-13 stimulated tumor associated macrophages enhance breast cancer cell invasion through Rho-GTPase signaling.

Andrew C. Little, Pragathi Pathanjeli, Zhifen Wu, Liwei Bao, Laura Goo, Joel A. Yates, Sofia D. Merajver. _University of Michigan, Ann Arbor, MI_.

The high metastatic potential of inflammatory and other aggressive breast cancer is the major determinant of mortality. Recently, it has become clear that signals from the tumor microenvironment (TME) harbor the capacity to enhance cancer cell dissemination. Of the various cells that comprise the TME, macrophages are the most abundant in solid tumors. Therefore, we aimed to better understand the relationship between macrophage-breast cancer cell crosstalk. Intriguingly, in our previous work we showed that inflammatory breast cancer (IBC) cells are hyper-migratory in response to macrophage conditioned media. Additionally, we found this process is regulated by the Rho-GTPase, RhoC. Recent advances in tumor associated macrophage (TAM) biology have led to the understanding that TAM populations in the TME are diverse, primarily due to directional differentiation promoted by unique secreted components from the various cells in the TME. Therefore, this study aimed to understand which subpopulation of M2-like TAMs had the greatest impact on breast cancer cell aggressiveness. Our data show that stimulation of the triple negative breast cancer (TNBC) cell model, MDA-MB-231, and the IBC TNBC cell line, SUM-149, with media extracts from IL-4/IL-13 stimulated M2 macrophages (M2a) elicit the strongest migratory and invasive responses. Subsequently, we stimulated RhoA or RhoC knockout (CRISPR) MDA-231 and SUM-149 cells with M2a conditioned media to address their role in regulating migratory/invasive responses. In brief, we find that RhoA and RhoC, in part, regulate M2a-TAM induced responses. Secretome analysis of M2a-TAM conditioned media reveals high levels of vascular endothelial growth factor (VEGF) and chemokine (C-C motif) ligand 18 (CCL-18). Functional studies suggest that VEGF and CCL-18 synergistically enhance cellular invasiveness. Moreover, pretreatment with the ROCK inhibitor Y-27632 or GSK429286A drastically inhibited VEGF, CCL-18, or M2a induced migratory responses. These data suggest that the Rho-GTPases regulate M2a-mediated breast cancer cell invasion and potentially offer a novel approach for the treatment of metastatic breast cancer through potential prevention of progression.

#4519

The reciprocal interaction between neutrophil extracellular traps and cancer cells impacts on their malignant potentials.

Hiroki Kajioka, Shunsuke Kagawa, Atene Ito, Kazuya Kuwada, Satoru Kikuchi, Shinji Kuroda, Ryuichi Yoshida, Hiroshi Tazawa, Toshiyoshi Fujiwara. _Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan_.

Although the neutrophil extracellular traps (NETs) are first recognized to function to trap invading microorganisms and eliminate them as a defense mechanism, the involvement of NETs in cancer metastasis is being elucidated. The mechanism of interaction between NET and cancer cells, however, remains to be fully understood. Here, we investigated the direct and indirect interaction between NETs and cancer. NETs were detected in surgically resected metastatic liver cancer tissue specimens. In direct co-culture, NETs actually attracted cancer cells with their web-like structure. Of note, indirect transwell co-culture of neutrophils and pancreatic cancer cells provoked NETs without the other stimuli. In addition, conditioned media derived from cancer cells also induced NETs. The co-culture of NETs and cancer cells dramatically accelerated their migration and invasion abilities. Further investigation revealed that NETs induced mesenchymal markers in cancer cells which was associated with upregulated epithelial to mesenchymal transition (EMT)-related transcriptional factors. Finally, the accelerated cancer migration and overexpression of mesenchymal markers induced via NETs were inhibited by pharmacological blockade of NETs including NADPH oxidase inhibitor, peptidylarginine deiminase inhibitor, and neutrophil elastase inhibitor. These results suggested that cancer cells and neutrophils reciprocally interact when they coexist and NETs drive malignant potentials via EMT, implicating NETs as a candidate therapeutic target.

#4520

Lymphatic vessels regulate exosome trafficking from tumors.

Lea Maillat, Lambert Potin, Witold W. Kilarski, Melody A. Swartz. _University of Chicago, Chicago, IL_.

Prior to metastasis, tumor-draining lymph nodes and distant organs are altered by a number of tumor-derived factors that help to both suppress host immunity as well as form an environment that supports metastatic tumor cells, i.e., a pre-metastatic niche. Recent studies suggest that pre-metastatic niches are induced by vesicles, including exosomes, secreted by tumor cells and cells of the tumor microenvironment. Here, we investigated the contribution of lymphatic vessels, particularly the lymphatic endothelial cells (LECs), in the active regulation of tumor exosome production and trafficking. To track exosomes, we purified and fluorescently labeled exosomes from in vitro cultures of the mouse melanoma cell line B16 F10 and of the human melanoma cell line Me260LN. Upon injection into the mouse ear dermis, fluorescent exosomes were observed within minutes in draining lymphatics, but not in surrounding blood vessels. Using the B16 F10 melanoma model overexpressing the lymphangiogenic growth factor VEGF-C, we found that upon intratumoral injection of exosomes, LECs within the tumor microenvironment abundantly take up exosomes, and at higher levels than blood endothelial cells, delivering exosomes to draining lymph nodes and eventually to the blood. In transgenic mice lacking dermal lymphatics, exosomes injected intratumorally were absent in the lymph nodes and present in far lower amounts in the blood compared to those injected into wild type mice. Additionally, while the density of endogenous blood exosomes increased upon tumor growth in wild type mice, no significant change in blood exosome density was observed in transgenic mice. Using an in vitro model where LECs are seeded onto porous inserts, we found that LEC transport of tumor exosomes occurs preferentially from their basal to apical side, and that some of these exosomes were taken up and stored intracellularly. Together, these findings suggest that lymphatic vessels constitute a major route of tumor exosome distribution to the systemic circulation and that LECs actively regulate exosome transport by active uptake.

### Nonclinical Models of Cancer

#4521

Mutant Kras expression triggers clonal acinar cell expansion as an early step in pancreas cancer.

Steven E. Artandi, Patrick Neuhöfer, Caitlin M. Roake, Stewart Kim, Ryan Lu, Greg Charville. _Stanford Univ. School of Medicine, Stanford, CA_.

Pancreatic ductal adenocarcinoma (PDA) is the third leading cause of cancer-related deaths in the United States with the lowest 5-year survival rate of any major cancer, in part due to late detection of the disease. Studies indicate that pre-invasive lesions - pancreatic intraepithelial neoplasias (PanINs) - progress to PDA. The cell of origin for PDA is still under debate. While lineage-tracing experiments in mice indicate that PanINs originate from acinar cells, cell culture studies suggest that ductal cells can give rise to PanINs. In addition to these important questions regarding cancer initiation, mechanisms of cellular renewal in the exocrine pancreas remain poorly understood. The enzyme telomerase is intimately associated with cancer, and with stem cells and tissue renewal. Telomerase is required for long-term cell proliferation through its critical role in maintaining telomeres. The core enzyme consists of the RNA component TERC and the reverse transcriptase TERT. Moreover, telomerase is activated in over 90% human cancers and genome wide association studies have linked polymorphisms in the Tertgene with an increased risk of pancreas cancer. To analyze the role of TERT-expressing cells in the pancreas during homeostasis, regeneration and tumorigenesis, we generated a mouse line that expresses the regulated CreERT2 recombinase under the endogenous TERT promoter (TertCreERT2/+). Crossing these mice to a reporter strain enables the permanent labeling of TERT-expressing cells and their progeny upon tamoxifen injection. We find that rare TERT+ cells exist throughout the pancreas within the acinar and islet cell populations, but not in ductal cells. We show that rare TERT+ acinar cells clonally expand to renew the acinar compartment during homeostasis and during pancreas injury. To understand how these TERT+ acinar progenitor cells respond to mutant Kras, we crossed TertCreERT2/+mice with KrasLoxStopLox-G12D/+mice. We find that activation of KrasG12D in TERT-expressing cells in adult mice promotes accelerated acinar clone formation and these acinar lesions ultimately convert to PanIN pre-invasive lesions. Our data indicate that TERT-expressing cells represent an acinar progenitor cell population. Furthermore, these progenitor cells respond to mutant Kras first through a regenerative response yielding expanding acinar clones, followed by conversion to ductal PanIN lesions. We propose that acinar cell expansion is an early step in pancreatic tumorigenesis that precedes ADM and PanIN formation.

#4522

**An** in vitro **3D immune exclusion tumor model engineered in a layer-by-layer fashion.**

Kolin C. Hribar,1 Tracy Kleinheinz,2 Joanna Klementowicz,2 Susan Kaufman,2 Robert A. Blake,2 Celine Eidenschenk2. 1 _Cypre Inc., San Francisco, CA;_ 2 _Genentech, Inc., San Francisco, CA_.

Recent immunotherapy failures in clinical trials have elucidated the affects of the tumor stroma on attenuating therapeutic response. In some cancers, the stroma consists of a collagen-rich fibrotic zone on the tumor periphery that traps infiltrating CD8+ T cells, allowing the tumor to evade immune cell-mediated death. Typically, animal models are employed to study complex physiology. However, known challenges to these methods include cost, duration and throughput. Thus, we sought to develop a 3D in vitro model that recapitulates the immune exclusion phenotype in a high throughput manner. We utilized a novel 3D cell culture platform, VersaGel and Symphony, to pattern tumor-stroma-immune compartments in a layer-by-layer fashion in 96-well plates. VersaGel is a chemically modified extracellular matrix that is amenable to light-induced crosslinking using the Symphony apparatus. Moreover, Symphony allows for discrete layers to be formed with any VersaGel/cell mixture, with defined 3D gel thicknesses (eg. 100µm, 250µm, 500µm) and areas in the plate (eg. 2mm, 4mm, or 8mm diameter discs).

GFP-labeled MC38-ova (ovalbumin antigen) colon carcinoma cells were grown in 100µm thick 3D VersaGel with or without CFP-labeled mouse embryonic fibroblasts (MEF) layered on top for 4 days. After, OT-1-RFP CD8+ cells were added to the culture medium to assess their penetration depth and tumor cell death over 3 days. 3D cell analysis through the optically clear VersaGel was performed in situ using high content confocal microscopy, effectively quantifying RFP/CFP/GFP-labeled cells and DRAQ7-labeled dead cells using Z-stack analysis. In the control (tumor-only condition), OT-1 cells fully penetrated the tumor layer and induced cell death. In the experimental condition (with fibroblasts), it was found that the fibrotic stromal layer limited OT-1 penetration to the periphery of the 3D tumor, and, moreover, the total OT-1 cells increased relative to the control. Future analysis may include the utility of wild-type T cells and novel therapeutics targeting the stroma to activate the immune response. The results suggest a novel 3D immune exclusion model that integrates with high content confocal analysis and is amenable to immunotherapeutic screening and translational assessment.

#4523

A novel syngeneic mouse model of prostate cancer bone metastasis: Mechanisms of chemotaxis and bone colonization.

Srinivas Nandana,1 Manisha Tripathi,1 Chia-Yi Chu,2 Haiyen Zhau,2 Stephen Shiao,2 Leland Chung2. 1 _Texas Tech University Health Sciences Center, Lubbock, TX;_ 2 _Cedars-Sinai Medical Center, Los Angeles, CA_.

Background: Bone metastasis in human prostate cancer remains a major clinical problem since no effective therapy exists. The RANKL/RANK pathway plays a predominant role in the interaction between metastasized prostate cancer cells and osteoclasts that increases the bone turnover. The current therapies, including targeting RANKL with denosumab, address the growth of prostate tumor cells that have already colonized the bone, but are largely ineffective in prolonging the survival of human prostate cancer patients with bone metastasis. Further, a major impediment to prostate cancer bone metastasis research is the lack of an animal model that spontaneously recapitulates human prostate cancer bone metastasis in the context of an intact immune system.

Results: To overcome this major limitation, we have developed a novel syngeneic mouse model to study prostate cancer bone metastasis. Both the CXCL12/CXCR4 and RANKL/RANK pathways have been reported to be overexpressed / dysregulated in human prostate cancer bone metastatic samples. Data generated utilizing our immune-intact mouse model shows that the CXCL12/CXCR4 and RANKL/RANK pathways co-operate with each other to drive prostate cancer bone metastasis. Studies have shown that targeting the CXCL12/CXCR4 and RANKL/RANK pathways individually affects the immune system, thereby making our syngeneic mouse model an indispensable tool for studying the critical co-operation between these 2 pathways in the manifestation of human prostate cancer bone metastasis. Extending our earlier findings that RANKL drives PCa metastases in immune-deficient mice (Chu et al., 2014) to immune-intact C57/Bl6 mice, we found that MPC3 mouse PCa cells with RANKL overexpression (MPC3-Luc-GFP-RANKL) develop 70-80% limb and jaw within 4 weeks of intra-cardiac injection in these syngeneic mice. Control MPC3 cells had no bone metastasis. Bone lesions visualized by luciferase imaging and X-ray were confirmed by micro CT and immunohistochemistry. RANKL signaling drove bone and visceral metastases via the downstream CXCL12/CXCR4 signaling axis. MPC3-Luc-GFP-RANKL cells showed increased CXCR4 protein levels by immunohistochemistry. Metastatic bone marrow flush showed dramatically increased levels of CXCL12 mRNA compared with control mice (MPC3-Luc-GFP-EV).

Conclusion: In sum, 1) circulating PCa cells induce a marked CXCL12 elevation after colonizing bone, triggering chemotaxis and recruiting CXCR4-positive PCa cells to migrate to bone; and 2) osteomimetic PCa cells with increased RANKL expression interact with osteoclasts to enhance bone resorption and turnover, releasing additional growth factors and chemokines for PCa cell growth and survival in bone.

#4524

Comparison of PDX, PDC, and PDOrg models from the National Cancer Institute's patient-derived models repository (PDMR).

Yvonne A. Evrard,1 Dianne Newton,1 Biswajit Das,1 Sergio Y. Alcoser,2 Kaitlyn Arthur,1 Mariah Baldwin,1 Carrie Bonomi,1 Suzanne Borgel,1 John Carter,1 Tiffany Chase,1 Alice Chen,2 Lily Chen,1 Nikki E. Craig,1 Vivekananda Datta,1 Emily Delaney,1 Raymond Divelbiss,1 Kelly Dougherty,1 Thomas Forbes,1 Kyle Georgius,1 Joe Geraghty,1 Marion Gibson,1 Michelle M. Gottholm-Ahalt,2 Tara Grinnage-Pulley,2 Kelly Hedger,1 Sierra Hoffman,1 Chris Karlovich,1 Wiem Lassoued,1 Shahanawaz Jiwani,1 Candace Mallow,1 Chelsea McGlynn,1 Mallorie Morris,1 Jenna Moyer,1 Mike Mullendore,1 Matt Murphy,1 Rajesh Patidar,1 Kevin Plater,1 Marianne Radzyminski,1 Nicki Scott,1 Luke H. Stockwin,1 Howard Stotler,1 Jesse Stottlemyer,1 Savanna Styers,1 Debbie Trail,1 Tomas Vilimas,1 Anna Wade,1 Abigail Walke,1 Thomas Walsh,1 P. Mickey Williams,1 Melinda G. Hollingshead,2 James H. Doroshow2. 1 _Frederick National Laboratory for Cancer Research, Frederick, MD;_ 2 _National Cancer Institute, Frederick, MD_.

The National Cancer Institute (NCI) has developed a Patient-Derived Models Repository (PDMR) comprised of quality-controlled, early-passage, clinically-annotated patient-derived tumor xenografts (PDXs), in vitro tumor cell cultures (PDCs), cancer associated fibroblasts (CAFs), and patient-derived organoids (PDOrg). NCI has focused on generating models to complement existing PDX collections and address unmet needs in the preclinical model space. These models are offered to the extramural community for research use (https://pdmr.cancer.gov), along with clinical annotation and molecular information (whole exome sequence, gene expression using RNASeq), via a publicly accessible database. Currently, over 200 PDX models, 50 PDC models, and 100 CAF models are available for distribution to the US research community. Approximately 50 PDOrg models will be released in early 2019. As part of its rare cancer initiative, the NCI is also targeting the collection of infrequently-observed tumor histologies to advance both biological investigations and drug development efforts for under-studied malignancies. Comparison of matched models, models where more than one model type are available (e.g., PDX and PDC), demonstrate a high degree of concordance across the model types. Genetic stability across the models is assessed using multiple criteria including genetic assessment of CNVs and presence of driver mutations. Optimal CNV assessment uses whole exome sequence data corrected for cellularity in the patient specimen using germline reads and corrected for cellularity in the PDX specimens by subtraction of the mouse reads. Histomorphologic comparison of PDXs and cell line xenografts (CLX) generated from in vitro PDCs and PDOrgs also overall show a high degree of concordance, though loss of features and dedifferentiation can be observed in some models. Overall these models demonstrate a high degree of conservation at the genetic and pathologic level when compared to the patient tumor. These models can provide researchers the ability to perform high- or mid-throughput screening in 2D or 3D culture followed by targeted selection of PDX models for in vivo studies. Funded by NCI Contract No. HHSN261200800001E

#4525

**Modulating chemoresistance: Uncovering the role of mutant SMAD4** R361H **in colorectal cancer using PDO and PDX models.**

Ulrike Pfohl,1 Alessandra Silvestri,2 Dirk Schumacher,3 Karsten Boehnke,4 Johannes Haybaeck,5 Sanam Bashir,6 Ralf Kühn,6 Marlen Keil,1 Reinhold Schäfer,3 Wolfgang Walther,1 Christian Regenbrecht2. 1 _Experimental Pharmacology & Oncology Berlin-Buch GmbH, Berlin, Germany; _2 _Cellular Phenomics & Oncology GmbH, Berlin, Germany; _3 _German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany;_ 4 _Eli Lilly and Company, New York City, NY;_ 5 _Otto-von-Guericke University, Magdeburg, Germany;_ 6 _Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany_.

Mutations in the transforming growth factor-β (TGF-β) pathway occur frequently in colorectal cancers (CRC). TGF-β is integral for key cellular processes, including proliferation, migration, differentiation and apoptosis. TGF-β/SMAD4 controls the activation of the pathway. A previously described R361H mutation in SMAD4 has been correlated with decreased overall survival and is suspected to modulate chemoresistance. The aim of the present study is to generate CRISPR-engineered SMAD4R361H CRC organoids and to utilize them as in vitro and in vivo models to investigate the effect of SMAD4R361H mutation on drug response. We have established independent organoid (PDO) and matching patient-derived xenograft (PDX) models from 5 different regions of a chemo-naïve CRC. By targeted amplicon sequencing using a cancer hot spot gene panel, we identified the SMAD4R361H mutation in two of the 5 subpopulations, whereas three of the PDOs were SMAD4wt. Semi-automated compound screening revealed a strong resistance phenotype to afatinib, sapitinib and gefitinib as compared to the wild-type models in vitro. Similar drug response was observed in in vivo testing. To investigate the mechanism of drug resistance, we have engineered isogenic organoids (SMAD4R361H/CRISPR) from SMAD4wt organoids using CRISPR-Cas9 genome-editing. In brief, SMAD4wt organoids were transfected with an all-in-one spCas9-sgRNA-vector and a ssODN as repair template bearing the R361H point mutation. To increase homologous-directed repair, we co-transfected the organoids with a plasmid vector containing i53-bpA, an inhibitor of 53BP1. Single clones were then isolated and genotyped by locus specific PCR to confirm the particular R361H point mutation. We were able to achieve up to 50 % knock-in efficiency in these organoids. Utilizing CRISPR-Cas9, engineered SMAD4R361H/CRISPR organoids were established. These PDOs were subjected with both, native SMAD4wt and SMAD4R361H PDOs, to an in vitro drug screening, allowing for a direct comparison of their drug sensitivity. Based on these results, confirmatory in vivo experiments were designed to investigate how SMAD4 may be involved in modulating resistance in CRC organoids. These models have a prominent role in discovering the connection between genotype and phenotype in complex PDO and PDX models. While this approach is not limited to studying the role of the R361H mutation, it has a broad range of applications in understanding cancer biology, intra-tumor heterogeneity, drug response and may eventually lead to new clinical insights, including design of new therapeutic strategies.

#4526

Tumor invasion and escape from an engineered solid-like aggregate of human breast cancer cells into a cavity.

Andreas P. Kourouklis,1 Usman Ghani,2 Siyang Han,1 Yoseph Dance,2 Allison K. Simi,1 Joe Tien,2 Celeste M. Nelson1. 1 _Princeton University, Princeton, NJ;_ 2 _Boston University, Boston, MA_.

The mechanical properties of the tumor microenvironment (TME) play a critical role on the progression of breast cancer metastasis. However, the complex architecture of the TME conceals the individual effects of different biophysical and biochemical factors on tumor invasion and intravasation. To investigate this question, we engineered a robust breast tumor model of solid-like 3D aggregate of human breast cancer cells with interstitial fluid pressure (IFP), and further integrated it with an empty cavity to emulate the presence of an impaired capillary vessel. In brief, we embed MDA-MB-231 human breast cancer cells in one of two neighboring collagen type I cavities that are molded within polydimethylsiloxane (PDMS) channels. This multicellular aggregate is subject to selected gradients of hydrostatic pressure through opposing reservoirs of culture media that are located at the base (Pbase) and the tip (Ptip) of the tumor. We found that breast cancer cells disseminate from the multicellular aggregate and escape into the proximal cavity under Ptip > Pbase. The separation distance between the aggregate and the cavity influences the features of tumor escape. Tumor models that were seeded within a distance of less than 150 μm from the cavity demonstrated significantly shorter time (t1/2~ 4 days) for the escape of 50% of the tumor population than those seeded between 150 and 300 μm. In contrast, less than 50% of the tumors that were seeded longer than 300 μm apart of the cavity successfully escaped after ~ 2 weeks under Ptip > Pbase. In addition, we found that cells escaped into the cavity through three major modes: a) single-cell migration, b) multicellular invasion, and c) tumor growth. Single-cell migration was the dominant route of escape in collagen gels of low concentration (2.5 mg/ml). In contrast, tumor growth and multicellular invasion were the dominant modes of escape in collagen gels of high concentration (4mg/ml). Moreover, the tumor invasions were found to be preferentially directed normal to the surface of the tumor, and to be drastically eliminated in effect of pharmacological inhibition of matrix metalloproteinases (MMPs). These preliminary findings will be put together with additional quantitative studies to correlate tumor-cavity separation with the different modes of tumor escape. Overall, our engineered breast tumor model composes a unique platform to investigate the biophysical and biochemical mechanisms of the tumor microenvironment that drive tumor invasion and intravasation into the circulatory system.

#4527

Patient derived organoids and xenografts identify neratinib plus HER2 antibody drug conjugate as a synergistic drug combination for HER2 mutated, non-amplified metastatic breast cancer.

Shunqiang Li,1 Highkin Maureen,1 Tina M. Primeau,1 Stephanie L. Pratt,1 Irmina Diala,2 Richard E. Cutler,2 Grace Mann,2 Alshad S. Lalani,2 Cynthia X. Ma,1 Ron Bose1. 1 _Washington University School of Medicine, Saint Louis, MO;_ 2 _Puma Biotechnology, Los Angeles, CA_.

HER2 activating mutations are a novel, druggable genomic alteration in metastatic breast cancer (MBC). These HER2 mutations are predominantly found in HER2 gene amplification negative, hormone receptor positive breast cancers. We have previously demonstrated that HER2 mutations can be potently inhibited by the second generation, irreversible pan-HER tyrosine kinase inhibitor, neratinib (Bose et al., Cancer Discovery 2013). Further, we performed a phase II clinical trial to treat HER2 mutated MBC and we found that neratinib monotherapy produced a clinical benefit rate of 31% and progression free survival (PFS) of 16 weeks in a heavily pre-treated patient population (Ma et al., Clin. Can. Res. 2017). A second clinical trial, the SUMMIT trial (Hyman et al., Nature 2018), similarly showed a response rate of 32% and median PFS of 3.5 months for neratinib monotherapy for HER2 mutated, metastatic breast cancer. The objective of the current study is to explore novel combination strategies to improve the efficacy of neratinib in HER2 mutated breast cancer. In order to accelerate progress on testing multiple drug combinations, we developed organoids from two patient-derived xenografts (PDX's) of HER2 mutated, ER positive metastatic breast cancer from our institution. We found that the ex vivo culture of these patient-derived organoids provides a platform to rapidly perform drug screens and drug combination testing on a scale that cannot be matched by other existing experimental platforms for patient-derived samples. The drug sensitivity of these organoids cultured ex vivo recapitulates the data previously obtained with transfected cell lines and in vivo experiments using PDX's. Further, multiple drug combinations can be tested on these organoids in just two weeks, which is much shorter than the four to six months required for the corresponding slow-growing ER positive, breast cancer PDX's that they are derived from. Strong, single agent activity was seen with neratinib, the HER2 antibody drug conjugate (ADC) ado-trastuzumab emtansine (T-DM1), and the chemotherapy drug vinorelbine. Therefore, we tested combinations of neratinib plus the HER2 ADC and neratinib plus vinorelbine on these patient derived organoids. Neratinib plus HER2 ADC showed a strong drug synergy in both HER2 mutated organoids, as judged by the Loewe model of drug synergy. Prior publications suggest that the mechanism of action of neratinib in this combination is by increasing HER2 ubiquitylation and endocytic degradation, which will increase the uptake of the ADC that binds to HER2. We are now performing 384 well drug screens with these HER2 mutated, ER positive metastatic breast cancer organoids, and the results of the screens will be shown in our presentation. 

# Wednesday, April 3, 2019

## CANCER CHEMISTRY

### Cancer Proteomics

#4528

Quantitative mass spectrometry to interrogate proteomic heterogeneity in metastatic lung adenocarcinoma and validate a novel somatic mutation CDK12-G879V.

Xu Xhang,1 Khoa Dang P. Nguyen,1 Paul Rudnick,2 Nitin Roper,1 Emily Kawaler,3 Tapan K. Maity,1 Shivangi Awasthi,1 Shaojian Gao,1 Romi Biswas,1 Abhilash Venugopalan,1 Constance Cultraro,1 David Fenyo,3 Udayan Guha1. 1 _National Cancer Institute at NIH, Bethesda, MD;_ 2 _Spectragen Informatics LLC, Bainbridge Island, WA;_ 3 _NYU School of Medicine, New York, NY_.

Lung cancer is the leading cause of cancer mortality. Tumor heterogeneity is a major cause of treatment failure. Intra- and inter-metastatic tumor heterogeneity has been demonstrated by next generation sequencing (NGS) studies. However, heterogeneity in the proteome and phosphoproteome has been less studied. Integrated proteogenomics is essential to understanding the intricacies of tumor heterogeneity affecting treatment response. Here, we performed integrated mass spectrometry-based proteogenomics to characterize spatial and temporal heterogeneity of an exceptional responder lung adenocarcinoma patient who survived with metastatic disease for more than 7 years while on combination treatment with HER2-targeted and chemotherapy.

We employed Super-SILAC and TMT labeling strategies to quantify the proteome and phosphoproteome of a lung metastatic site and ten different metastatic progressive lymph nodes from our patient collected across a span of seven years, including at autopsy. To further interrogate the mass spectrometry data, patient-specific database was built to incorporate all the somatic variants identified by NGS. An extensive validation pipeline was built for confirmation of variant peptides. CRISPR-Cas9-mediated gene knockout, cell viability assays, and confocal microscopy were used for further validation of novel variants.

A total of 6214 and 4061 proteins were identified from Super-SILAC and TMT experiments, respectively. 3648 proteins were identified and quantified in both experiments. More than 2000 proteins had catalytic activity, including kinases, phosphatases and metabolic enzymes. We identified 78 and 23 mutant peptides from Super-SILAC and TMT experiments, respectively. Three somatic variants, CDK12-G879V, FASN-R1439Q and HNRNPF-A105T, were confirmed using our variant peptide detection pipeline. Multiple reaction monitoring in a triple quadrupole mass spectrometer successfully identified and relatively quantified two of the variant tryptic peptides harboring the mutations, CDK12-G879V and FASN-R1439Q from the lung and lymph node metastatic sites, respectively. We investigated the consequences of loss of CDK12 function, as predicted from the novel CDK12-G879V mutant, in chemotherapy sensitivity. A549 lung adenocarcinoma cells, upon knockdown of CDK12, exhibited greater chemotherapy sensitivity that was rescued by wild type CDK12, but not by CDK12-G879V mutant.

We demonstrate the importance of integrated proteogenomic analyses to identify variant peptides in mass spectrometry data and studying proteomic heterogeneity affecting treatment response. CDK12-G879V mutation results in a nonfunctional CDK12 kinase and chemotherapy susceptibility in lung metastatic sites, likely explaining the "cure" of lung metastatic sites in this patient.

#4529

Mapping the protein interactome of mitochondrial intermembrane space proteases identifies a novel function for HTRA2.

Aaron D. Botham, Etienne Coyaud, Sanjit Nirmalanandhan, Marcela Gronda, Rose Hurren, Neil Maclean, Jonathan St. Germain, Sara Mirali, Estelle Laurent, Brian Raught, Aaron Schimmer. _Princess Margaret Cancer Center - University Health Network, Toronto, Ontario, Canada_.

Mitochondria possess unique proteases that localize to specific sub-compartments of the organelle. However, the functions of these proteases are largely ill-defined. Here, we used proximity-dependent biotinylation (BioID) to map the interactomes of seven proteases located in the intermembrane space of the mitochondria. The mitochondrial intermembrane space proteases HTRA2, OMA1, YME1L1, LACTB, IMMP1L, IMMP2L and PARL were cloned in-frame with the abortive E. coli biotin ligase BirA*, and expressed in 293 T-REx cells. Cell culture media was spiked with biotin for 24 hrs, the cells lysed, and biotinylated proteins were isolated and identified by mass spectrometry. In total, we identified 342 different proteins as high confidence interactors of the seven mitochondrial proteases. Of these, 272 are assigned a GO mitochondrial annotation, and 230 proteins interacted with only 1 or 2 proteases in our dataset. Validation efforts were focused on high temperature requirement peptidase A 2 (HTRA2). HTRA2 is a serine protease that is released into the cytoplasm during apoptosis where it binds Inhibitor of Apoptosis Proteins (IAPs). However, little is known about the function of HTRA2 in the mitochondria. HTRA2 interacted with 60 mitochondrial, 11 nuclear and 4 cytoplasmic proteins, including its known interactor XIAP, and consistent with its known localization to these cellular compartments. HTRA2 interacted with 8 out of 13 components of the MIB complex, a multiprotein assembly that is essential for proper mitochondrial cristae formation. Knockdown of HTRA2 with shRNA in 293T-REx cells disrupted cristae formation and this phenotype was rescued by expression of an shRNA-resistant HTRA2 cDNA. Compared to normal hematopoietic cells, HTRA2 mRNA expression levels are increased in a subgroup of primary AML cells. HTRA2 knockdown in OCI-AML2 leukemia cells led to a similar disruption of mitochondrial cristae. Knockdown of HTRA2 in OCI-AML2 cells led to increased levels of the MIB subunit IMMT, but not two other MIB complex subunits, SAMM50 and CHCHD3. Finally, in cell-free assays, we demonstrate that recombinant HTRA2 can degrade recombinant IMMT, but not SAMM50 or CHCHD3.Thus, we have mapped the interactomes of the proteases of the mitochondrial intermembrane space. Through this effort, we discovered that HTRA2 regulates protein levels of the MIB complex subunit IMMT and that disruption of this process affects mitochondrial cristae formation.

#4530

An independent validation of a screening test using mass spectrometry for detection of hepatocellular carcinoma.

Sunyoung S. Lee,1 Kristopher Attwood,1 Heinrich Roder,2 Senait Asmellash,2 Krista Meyer,2 Stylianos Kakolyris,3 Carlos Oliveira,2 Joanna Roder,2 Julia Grigorieva,2 Leonidas Chelis,4 Renuka Iyer,1 Devalingam Mahalingam5. 1 _Roswell Park Comprehensive Cancer Center, Buffalo, NY;_ 2 _Biodesix, Boulder, CO;_ 3 _Democritus University of Thrace, Alexandroupolis, Greece;_ 4 _King Fahad Specialist Hospital, Dammam, Saudi Arabia;_ 5 _Northwestern University, Feinberg School of Medicine, Chicago, IL_.

Background: Early detection is critical to improve outcome in hepatocellular carcinoma (HCC). Despite inadequate sensitivity (SS) and specificity (SP), abdominal ultrasound and alpha-fetoprotein (AFP) are considered methods of choice for HCC surveillance. As less than 30% of patients (pts) are diagnosed early enough for resection or transplantation, a test with improved SS and SP is needed. A test to detect HCC in a high-risk population from 10 uL serum, combining MALDI mass spectrometry and AFP data was developed using a dropout-regularized hierarchical machine learning approach designed to minimize overfitting in small development sets. It was previously validated in 293 high risk pts (158 HCC, 135 non-HCC) with SS/SP of 83%/84% in development and 81%/79% in validation across various etiologies and Child-Pugh classification [1].

Methods: The test was applied to an independent validation cohort of 156 pts (97 HCC, 59 non-HCC healthy controls), blinded to clinical data, the performance was assessed by SS, SP. Sub-group analyses were performed by grade and stage. Performance was compared with that of AFP by area under the curve (AUC), using the test output prior to the predefined thresholding which yields the final binary cancer/benign test result.

Results:

Table 1. - HCC patient demographics in the independent validation set

---

Clinical characteristics | Independent validation

|

Patients with HCC (N=97)

Median age (range), years | 62 (38 - 89)

Median AFP (range), ng/mL | 4.0 (less than 1.5 - 10,000)

Gender, male / female | 85% / 16%

Hepatitis B / C / No virus / not available (NA) | 3% / 27% / 20% / 51%

Grade I / II / III / NA | 17% / 22% / 12% / 50%

Stage I / II / III / IV / NA | 12% / 14% / 33% / 26% / 14%

Previous liver directed or systemic therapy yes/no | 44% / 54%

ECOG PS 0 / 1 / 2 / 3 / NA | 13% / 20% / 11% / 5% / 51%

In independent validation, AUC for the test output prior to thresholding was 0.979, significantly better than AFP AUC 0.915 (P<0.001). SS and SP were 88% and 100%. SS in grade I, II, and III was 75%, 76%, and 92% and in stage I, II, III, and IV was 75%, 86%, 94%, and 92%. Conclusion: Results in the independent cohort confirm performance of the test, with better SS and SP than AFP and historical ultrasound. The SS was also high in low grade and early stage disease, indicating the test's potential to improve early detection of HCC when curative approaches are feasible. Data and samples from Data Bank and BioRepository, Roswell Park; funding by the NCI grant P30CA016056 [1] D Mahalingam et al. Hepatology 62(S1): 1135A (2015)

#4531

SWATH-MS profiling identifies prognostic factors for progression-free survival (PFS) In INTEGRATE - A randomized phase II double-blind placebo-controlled study of regorafenib in refractory advanced oesophagogastric cancer (AOGC) - A study by the Australasian Gastrointestinal Trials Group (AGITG).

Sarah A. Hayes,1 Andrew Martin,1 Sonia Yip,1 Viive M. Howell,1 Katrin M. Sjoquist,1 Eric Tsobanis,1 Yoon-Koo Kang,2 Yung-Jue Bang,3 Thierry Alcindor,4 Christopher J. O'Callaghan,5 Niall C. Tebbutt,6 John Simes,1 David Goldstein,7 Nick Pavlakis1. 1 _University of Sydney, Sydney, Australia;_ 2 _University of Ulsan, Seoul, Republic of Korea;_ 3 _Seoul National University, Seoul, Republic of Korea;_ 4 _McGill University Health Centre, Montreal, Quebec, Canada;_ 5 _Queen's University, Kingston, Ontario, Canada;_ 6 _Austin Health, Melbourne, Australia;_ 7 _University of New South Wales, Sydney, Australia_.

Introduction: Gastric cancer is one of the most common cancers worldwide, and a leading cause of cancer death. Advanced OesophagoGastric cancer (AOGC) has a poor prognosis despite treatment. The P2 INTEGRATE multinational 2:1 (active:placebo) randomized trial demonstrated the activity, on progression-free survival (PFS), of the oral multikinase inhibitor regorafenib (REG) in patients (pts) with refractory AOGC (Pavlakis et al JCO 2016), leading to the P3 INTEGRATE II trial (NCT02773524) currently underway. Here, we sought to identify prognostic protein biomarkers in a discovery analysis of a patient subset from INTEGRATE I.

Methods: The discovery analysis set comprised of 40 INTEGRATE I patients (12 placebo; 28 REG) selected using stratified random sampling based on quartiles of observed PFS within each arm of allocation, half from 1st quartile (worst PFS outcome) and half from 4th quartile (best PFS outcome). We profiled the plasma proteome of INTEGRATE pts using data-independent acquisition of liquid chromatography coupled with tandem mass spectrometry (SWATH-MS acquisition). Plasma collected at baseline (10µl) was analyzed with TripleTOF 6600 System (SCIEX, MA, USA). Data was searched against an "extended" spectral library generated using SwathXtend software (Wu et al MCP 2016). Cox proportional hazard regression was used to assess the prognostic value of log2 protein expression level adjusted for treatment allocation.

Results: 437 proteins were identified across all 40 pt samples (>99% peptide confidence). A subset of 27 proteins were identified as candidates for possible further investigation using the verification analysis set on the basis of having p-values <0.05 (no p-value was significant after adjustment for multiple comparisons). These proteins were associated with (i) the immune system, including multiple immunoglobulin variable heavy and light chains, and proteins involved in complement activation (C09, C08G, C4BPA and C05 and others) and two serpins that regulate the acute phase response and promote cancer cell survival (AACT and A1AT); (ii) blood coagulation and angiogenesis (THBS1/PROS1/FBLN1 and others), also instrumental in tumor progression. Other proteins are known to promote local cancer cell adhesion, invasion and distant metastasis.

Conclusions: This is the first time that SWATH has been used to analyse AOGC pt samples, an otherwise challenging biofluid (undepleted plasma) for profiling. These proteins could represent a novel prognostic signature for PFS in AOGC patients, pending validation in the larger verification data set. This work highlights the potential value of incorporating proteomics into risk assessments guiding treatment strategies for patients.

#4532

Differential protein and metabolite regulation in urothelial carcinoma highlights changes in immunity and receptor signaling pathways.

Lee Gethings,1 Adam King,1 Andrew J. Peck,2 Robert S. Plumb2. 1 _Waters Corporation, Wilmslow, United Kingdom;_ 2 _Waters Corporation, Milford, MA_.

Bladder cancer is ranked as one of the tumours posing high morbidity and mortality, with a pathogenesis which is still not well understood. A combination of genetic and lifestyle factors, including smoking, are known to contribute towards increasing the probability of encountering cancer. Here, we describe a multi-omic approach to reveal molecular factors that may be involved in these biomolecular processes. Plasma samples consisting of healthy controls and those from patients diagnosed with bladder cancer were prepared for proteomic, metabolomic and lipidomic analyses. Label-free LC-MS data were acquired using a QTof platform utilizing a data independent (DIA) workflow termed SONAR. Small molecule analysis consisted of using Progenesis QI for data processing to provide normalised values prior to statistical analysis. Unsupervised MVA of the data showed clear distinction between cohorts. OPLS-DA was used to filter for features of significant correlation and covariance prior to identification using metabolite and lipid specific databases. Identifications matching the following criteria, mass accuracy <5 ppm, ANOVA p <0.05, %CV <30 and fold change >2 were considered for further interrogation. SONAR-based analysis indicates that this method of data acquisition enabled over an order of magnitude more specificity than equivalent methodologies with the same resolution. Proteomic data were processed and searched against a Uniprot Homo sapien database, containing reviewed entries and limited to 1% FDR. Additionally, we also searched the data against a spectral library and comparatively analysed the results from both workflows. A number of significant proteins with differential regulation were exhibited for a number of protein groups involved in antigen and lipid binding. Proteins occurring in a minimum of two out of three replicates and with ANOVA p <0.05 were considered. Biological significance of the results was established by merging data from all three omic experiments and performing pathway analysis. A number of significant pathways including complement activation, B cell mediated immunity and receptor signalling were identified as key pathways.

#4533

Plasma and exosome proteomic profiling for prediction of immunotherapy response and toxicity.

Arnav Mehta,1 Gyulnara Kasumova,1 Alvin Shi,2 Marijana Rucevic,3 Markus Sallman-Almen,3 Lina Hultin Rosenberg,3 Emmett Sprecher,3 Jacqueline Ohmura,1 Michelle Kim,1 David Lieb,4 Xue Bai,1 Dennie T. Frederick,1 Manolis Kellis,2 Ryan J. Sullivan,1 Keith T. Flaherty,1 Nir Hacohen,4 Genevieve M. Boland1. 1 _Massachusetts General Hospital, Boston, MA;_ 2 _Massachusetts Institute of Technology, Cambridge, MA;_ 3 _Olink Proteomics, Watertown, MA;_ 4 _Broad Institute of Harvard and MIT, Cambridge, MA_.

Immune checkpoint blockade (ICB) has revolutionized the treatment of many solid tumors, including metastatic melanoma. Despite recent successes, many patients fail to respond or are overcome by severe toxicities that limit further treatment. To date, there are no non-invasive predictors of response and toxicity that can guide treatment decisions. In this work, we perform whole plasma and exosome proteomic profiling to construct a predictive model of immunotherapy response and toxicity, and to glean further biologic insight into the mechanisms underlying resistance to ICB. Whole plasma was analyzed in a cohort of 150 melanoma patients receiving anti-PD1 antibodies (MGH IRB #11-181) at baseline, and on-treatment at 6 week and 6 month time-points. Exosomes were analyzed in 15 of these patients for all time-points. Proteomic analysis was performed using a multiplex proximity extension assay that enabled detection of more than 1000 proteins simultaneously. A linear mixed model with maximum likelihood estimation for model parameters was used to analyze differences between patient groups, and significant differences were determined after Benjamini and Hochberg multiple hypothesis correction. Between plasma baseline and on-treatment time-points, 67 differentially expressed proteins were identified including markers of inflammation such as PD1, CXCL9, CXCL10, CXCL11, IL10, CCL3 and TNFR2. Exosome samples had a distinct protein signature over the treatment period compared to plasma, including differential expression of CXCL16, CCL18, CCL20, and IL6, among others. 41 proteins were differentially expressed in plasma between ICB responders and non-responders including several inflammatory proteins such as CD28, TNFb, MCSFRa and IL8, and others implicated in melanoma resistance, such as MIA and ERBB2. Again, exosome samples had a distinct protein signature between responders and non-responders compared to plasma samples, consisting of CXCL9, CXCL13, CXCL16, CCL19, CD8a, GZMA and CD5 expression. Whereas plasma proteins reflected a myeloid signature, exosome proteins reflected a lymphoid signature, suggesting that the two compartments may capture elements of different immune processes. Integrating data from both plasma and exosome proteomics, we applied machine learning tools to build a predictor of response. Further analysis to look for predictors of toxicity is also currently underway. Overall, our work suggests that plasma and exosome protein signatures are distinct and may reflect unique immunological processes. Proteomic analysis of these compartments may be an effective way for non-invasive liquid biopsy to predict ICB response.

#4534

Label free molecular imaging of tumor sections for two and three dimensional tissue classification and pathway mapping.

Emrys A. Jones,1 Fiona Henderson,2 Matthew Gentry,2 Danielle McDougall,2 James Langridge,1 Adam McMahon2. 1 _Waters, Wilmslow, United Kingdom;_ 2 _University of Manchester, Manchester, United Kingdom_.

Mass spectrometry imaging allows for the presence and abundance of a wide range of bio-molecules to be measured directly from tissue sections without the use of labels or tags. This results in the ability to compare the spatial distribution of hundreds of metabolites, lipids, peptides, and proteins within the same section in a single experiment. Desorption electrospray ionization (DESI) mass spectrometry imaging can differentiate and classify tissue types, and associated disease states, by measuring the unique characteristic molecular fingerprints of the tissues1,2. Furthermore, using histologically annotated databases of molecular profiles, unknown tissues can be classified using the mass spectrometry output coupled to machine learning approaches. A major benefit of DESI is that the analyzed sections are left largely unchanged by the process; the sections can subsequently be stained and the results validated by immunohistochemistry or conventional H&E approaches. During DESI fingerprinting analyses, a full mass spectrum on a cell by cell resolution of 20-50µm pixel size is collected. Chemical changes on very small scales can be distinguished and explored, allowing the chemistry of tumor margins and interface zones to be understood through mapping results onto metabolic pathways. As the data in a DESI experiment is collected and stored, multiple interrogations of the data are possible; as new discoveries are made, previously collected data can be retrospectively mined for additional insights. In this study, the three-dimensional chemical heterogeneity of drug dosed spheroids and tumor xenografts was measured with DESI and compared with MRI and PET imaging modalities and with FT-IR spectroscopic techniques. We demonstrate the power of this multi-analytical approach in glioma models and in colorectal and prostrate tumor samples to uncover metabolic and lipidomic changes during disease progression and to reveal the inherent heterogeneity within a tumor. 1. 1. Calligaris, D.Caragacianu, D.Liu, et al; PNAS, 2014, 111, 15185-151892. Guenther, S., Muirhead, L.J., et al, Cancer Res, 2015, 75, 1828-1837

#4535

The mTOR complex 2 promotes glioblastoma migration via the interactions with multiple actin-binding and microtubule-associated proteins.

Naphat Chantaravisoot,1 Piriya Wongkongkathep,1 Narawit Pacharakullanon,1 Fuyuhiko Tamanoi,2 Joseph A. Loo,2 Trairak Pisitkun1. 1 _Chulalongkorn University, Bangkok, Thailand;_ 2 _University of California, Los Angeles, Los Angeles, CA_.

The mechanistic target of rapamycin complex 2 (mTORC2) is one of the two multiprotein complexes in the mTOR signaling pathway. It regulates several cellular activities such as cell survival, lipid metabolism, DNA damage control, and cell motility. The mTORC2 has been reported to be involved in the promotion of metabolic reprogramming, cancer migration, and cancer invasiveness in glioblastoma. However, underlying mechanisms and roles of this particular complex in glioblastoma migration has not been completely elucidated. Previously, we characterized the mTORC2-associated interactome by affinity purification-mass spectrometry (AP-MS) technique, identifying new RICTOR-interacting partners. In addition, we also investigated the localization of RICTOR under different culture conditions and found that it correlates with migration ability of the cancer cells. When mTORC2 activity is high resulting in increased migration, RICTOR tends to localize around the cell membrane. In this work, we further determined crucial players that interact with mTORC2 to control actin cytoskeleton and microtubule dynamics to promote migration. The results from quantitative proteomic analysis of mTORC2-associated interactome suggested that the amount of certain actin-binding proteins and microtubule-associated proteins significantly changed depending on mTORC2 activity level including Gelsolin, Myosin-9, Plectin, and MAP1B. When mTORC2 signaling was inhibited resulting in reduced migration, these proteins were found to be less interacting with mTORC2. Moreover, the results also indicated that active mTORC2 links both actin cytoskeleton and microtubules together to promote migration in glioblastoma cells. Depletion of RICTOR and inhibition of mTORC2 disrupted the connection between microtubules and cell membrane. Taken together, this work provided a plausible mechanism underlying the regulation of enhanced migration in brain cancer cells.

#4536

Applying immunopeptidomics and machine learning to improve neoantigen prediction for therapeutic and diagnostic use.

Sean Michael Boyle, Datta Mellacheruvu, Nick Phillips, Gabor Bartha, Jason Harris, Robert Power, Rena McClory, John West, Richard Chen. _Personalis, Inc., Menlo Park, CA_.

Background: Neoantigens are increasingly critical in immuno-oncology as therapeutic targets for neoantigen-based personalized cancer vaccines (PCVs) and as potential biomarkers for immunotherapy response. However, identifying which neoepitopes are more likely to provoke an immune response remains an important challenge for improving the effectiveness of PCVs and enabling neoantigens as a biomarker in immunotherapy. In recent years, Immuno-peptidomics has greatly improved in sensitivity and specificity, providing large number of peptides bound to MHC class I alleles in vivo. These advances make it possible to identify processed cell surface MHC bound peptides in an in vivo setting, providing accurate and representative presented peptide data for development of an improved neoantigen prediction pipeline.

Methods: We generated high quality allele-specific training data for development of an accurate predictive algorithm. Mono-allelic HLA class I cell lines were generated by transfecting individual class I HLA alleles into the HLA class I null cell line K562, prioritizing alleles which will ultimately allow for development of a pan-class-I-allele prediction algorithm. Cell surface bound MHC class I peptides were identified for each transfected allele using immuno-peptidomics. We then developed and trained neural networks to predict MHC class I presentation for each assayed HLA allele. The predictive accuracy for each allele was comprehensively validated using immuno-peptidomic results derived from three sources: mono-allelic cell lines, deconvoluted cell lines, and patient derived tumor samples.

Results: We applied immuno-peptidomics to develop a large and highly representative profile of MHC class I peptidomics across 30 HLA class I alleles. We then utilized this dataset to develop a highly accurate HLA class I presentation neural network. Through our work, we have identified thousands of HLA class I peptides bound to each of 30 unique HLA class I alleles, greatly expanding the known mono-allelic space. Our neoantigen prediction algorithm has been extensively validated, consistently achieving a higher overall accuracy across alleles (precision 0.88) than other publicly available tools (precision less than 0.7) based on both in vitro binding data and immuno-peptidomics, when tested on a broad set of peptide sources: mono-allelic cell lines, deconvoluted cell lines, and patient-derived tumor samples.

Conclusions: Effective neoantigen identification can be greatly improved through application of immuno-peptidomics. We have generated extensive mono-allelic HLA class I cell lines and extensively characterized their class I ligandomes. We have used this data to develop and train a novel presentation neural network. Finally, we have extensively validated this tool using multiple newly-derived in vitro and in vivo sources, demonstrating very strong accuracy.

#4537

NCI's clinical proteomic tumor analysis consortium: A proteogenomic cancer analysis program.

Mehdi Mesri, Emily Boja, Alexis Carter, Tara Hiltke, Chris Kinsinger, Annette Marrero-Oliveras, Ana Robles, Henry Rodriguez, CPTAC Investigators. _NIH, Bethesda, MD_.

The National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium (NCI-CPTAC) is an integrative proteogenomic nexus composed of a Proteogenomic Tumor Characterization Program, and a Proteogenomic Translational Research Program. The goal of this multi-faceted program is to: I) comprehensively identify proteins derived from altered genes and related biological processes by applying deep proteogenomic analysis and; II) to determine if novel biological insights can be inferred from this additional facet of molecular information that are not obtained through genomics data alone.

Using high throughput standardized mass spectrometry-based methods, the Proteogenomic Tumor Characterization Program is expanding its deep comprehensive proteogenomic analysis of cancer types with all data and assays to be released to the public. In addition to recently characterized TCGA samples from colon, breast and ovarian cancers, additional prospectively-collected treatment-naïve tumors including clear cell renal cell carcinoma (CCRCC), uterine corpus endometrial carcinoma (UCEC), and lung adenocarcinoma (LUAD) are to be characterized. In the Proteogenomic Translational Research Program, CPTAC is partnering for the first time with NCI-sponsored clinical trials to support clinically-relevant research projects that elucidate biological mechanisms of response, resistance, and/or toxicity. The Proteogenomic Translational Research Program explores triple negative breast cancer (TNBC), high-grade serous ovarian cancer (HGSOC), and acute myeloid leukemia (AML).

CPTAC investigators have recently completed proteogenomic analysis of ~ 200 Colorectal, ~ 230 Breast and ~250 Ovarian tumors comprised of both retrospectively and prospectively collected samples that encompass comprehensive interrogation of the proteome and post-translational modifications including phosphoproteome. Raw and processed mass spectrometry-based proteomic, and genomic data are publicly available on the CPTAC Data Portal (http://proteomics.cancer.gov). CPTAC is also supporting development of new proteogenomic analysis tools that help integrate the proteomic data with existing omics data sets, such as the LinkedOmics (http://www.linkedomics.org). In addition, the CPTAC Assay Portal (http://assays.cancer.gov) is a public resource populated with mass spectrometry-based targeted proteomic assays developed by the consortium for quantitatively measuring proteins of interest, including those discovered through comprehensive characterization. Lastly, well-characterized monoclonal antibodies targeting cancer-specific proteins and peptides are also made available at CPTAC's Antibody Portal (http://antibodies.cancer.gov).

#4538

Global and phosphoproteomic analysis of AML cell line response to phosphatase inhibitor treatment.

Paul D. Piehowski,1 Jason E. McDermott,1 Joshua R. Hansen,1 Samantha L. Savage,2 Cristina E. Tognon,2 Anupriya Agarwal,2 Jeffrey W. Tyner,2 Brian J. Druker,2 Karin D. Rodland2. 1 _Pacific Northwest National Laboratory, Richland, WA;_ 2 _Oregon Health and Sciences University, Portland, OR_.

In the United States, approximately 21,000 people are diagnosed with AML each year and over 10,000 AML related deaths are reported. Genomic analysis has revealed at least 11 genetic classes of AML, and more than 20 subsets can be assigned when considering the differentiation states of Leukaemic blasts. This variability has generated significant interest in the development of targeted therapies for AML. However, complex mutational patterns and a lack of pharmacological agents for most mutational events have challenged the development of effective treatments. Recent results from the Beat AML clinical trial revealed that response to drugs is associated with mutational status, including instances where drug sensitivity is specific to combinatorial mutational events.

To better understand the molecular mechanisms underpinning drug response, we performed global and phosphoproteomic analyses on 4 AML cell lines that carry mutations associated with AML: K562 with a BCR-ABL fusion, MOLM14 with a FLT3 mutation, HL60 with a RAS mutation, and CMK with a JAK3 mutation. These cell lines were each treated with a different kinase inhibitor drug for which they have a known sensitivity. Cell samples were collected prior to treatment and time points of 30 min, 3h, and 16 hrs. Frozen cell pellets were prepared for analysis using the workflow for multiplexed, deep-scale, proteome and phosphoproteome analysis developed by the NCI CPTAC program.

The goal of our Proteogenomic Translational Research Center is to develop drug response signatures allowing the use of baseline proteome and phosphoproteome measurements to predict patient drug response, providing a foundation for rational selection of combination therapies. Preliminary analysis of the data show a dynamic temporal response to drug treatment in both the global and phosphoproteome, Figure 1. Current efforts are focused on combining knowledge-driven and data-driven analysis approaches to identify the pathways associated with drug response.

#4539

Identification of blood-based protein biomarkers for prediction of response to neoadjuvant chemoradiation in rectal cancer.

Delphine Dayde,1 Jillian Gunther,1 Yutaka Hirayama,2 David Weksberg,1 Adam Boutin,1 Hong Wang,1 Hiroyuki Katayama,1 Y. Alan Wang,1 Ronald DePinho,1 Kazuo Hara,2 Masahiro Tajika,2 Yasumasa Niwa,2 Samir Hanash,1 Sunil Krishnan,1 Ayumu Taguchi2. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _Aichi Cancer Center, Nagoya, Japan_.

Rectal cancer represents a third of colorectal cancer and is associated with worse clinical outcome. The current standard of care for patients with locally advanced rectal cancer (LARC) is neoadjuvant chemoradiation (nCRT) followed by surgery. However, the response to nCRT varies among patients and only 11 to 30% of LARC patients achieve a pathologic complete response (pCR) at the time of surgery while ~20% exhibit resistance. Therefore, there is an unmet need of biomarkers to predict response to nCRT at an early time point, allowing to select LARC patients who would or would not have a benefit from nCRT, to reduce toxicity associated with ineffective nCRT, and to provide adequate treatment options. Genetically engineered mouse models of cancer have been shown to recapitulate many of the molecular and biological features of human cancer. To identify plasma proteins that show altered abundance between pCR and non-pCR, we applied in-depth quantitative proteomic analysis to plasma from a mouse model of rectal cancer that harbors an inducible oncogenic Kras allele and conditional null alleles of Apc and Trp53 with chemoradiation treatment. Plasma samples were collected before treatment and analyzed by mass spectrometry, resulting in quantification of 567 proteins. We selected three proteins (VEGFR3, IGFBP4, and CTSB), which were markedly elevated in the plasma from non-pCR mice compared to pCR mice, for validation in human plasma samples. In addition, we explored whether four tissue protein biomarkers (EGFR, Ki67, E-cadherin, and COX2), that were previously associated with response to nCRT, also can have the potential as blood biomarkers. Using immunoassays for seven biomarker candidates as well as CEA on plasma collected before nCRT from 34 patients with LARC (6 pCR and 28 non-pCR), we observed that levels of VEGFR3 (P = 0.0451, AUC = 0.720), EGFR (P = 0.0128, AUC = 0.679) and COX2 (P = 0.0397, AUC = 0.679) were significantly increased in the plasma of non-pCR LARC patients compare to those of pCR LARC patients. Levels of VEGFR3 and EGFR, but not COX2, were significantly decreased after tumor resection. The performance of the logistic regression model combining VEGFR3, EGFR, and COX2 was significantly improved compared with the performance of each biomarker, yielding an AUC of 0.869 (sensitivity 43% at 95% specificity). Through proteomic approaches we identified novel plasma biomarkers that can effectively predict response to nCRT in LARC patients.

#4540

Discover cell signaling pathways using antibody arrays.

Hao Tang, Jianmin Fang, Zhizhou Kuang, Ruo-Pan Huang. _Raybiotech Life Inc., Norcross, GA_.

Cell Signaling pathways are complex networks that are normally regulated and connected by multiple components. Analyzing signaling pathways is essential for understanding the molecular mechanism of human disease and drug development. However, it remains a great challenge to study pathways using traditional methods. Thus, a high-content and high-throughput method for simultaneous detection of multiple targets is necessary. Using a sandwich-based ELISA format, we have developed an antibody array technology which can simultaneously identify relative expression levels or phosphorylation levels of major signaling pathway proteins. This array-based system features a nitrocellulose membrane or glass slide solid support, each spotted with up to 71 different antibodies against a selected panel of classical signaling pathway proteins, including RTK, EGFR, MAPK, AKT, Apoptosis, TGFb, JAK/STAT, NFkB and Insulin Receptor pathways, spanning 182 total proteins. To test our system, we investigated the molecular mechanism of Camptothecin's anti-cancer effect using several pathway arrays. Our array data suggest that Camptothecin-treatment induced DNA double-strand breaks in Jurkat cell activates the DNA damage pathways ATM and Chk2, and then further induces apoptosis through Caspase 3 and BAD. We also studied phorbol 12-myristate 13-acetate (PMA)'s effect on HeLa cells, and found that PMA induces the MAPK pathway through activation of Erk, MSK2, and RSK1. These array results are consistent with previous studies using traditional methods and were further confirmed by western blot analysis. Our studies demonstrate that pathway antibody arrays provide a rapid and efficient approach to study the mechanism of cell events, human diseases and drug effects. With this technology, researchers can quickly screen activation of multiple target proteins in one or multiple pathways in their experimental system.

#4541

Malignant and non-malignant cells from primary tumor and lymph node metastasis ecosystems show different behavior for patient-associated protein signatures in head and neck cancer.

Ariane F. Busso-Lopes,1 César Rivera,2 Barbara P. Mello,3 Luisa L. Villa,3 Wilfredo González-Arriagada,4 Adriana F. Paes Leme1. 1 _Brazilian Center for Research in Energy and Materials - CNPEM, Campinas, Brazil;_ 2 _University of Talca, Talca, Chile;_ 3 _University of São Paulo, São Paulo, Brazil;_ 4 _Valparaiso University, Valparaiso, Chile_.

Although lymph node metastasis is the main prognostic factor in patients with head and neck squamous cell carcinoma (HNSCC), the molecular signals involved in this process are poorly characterized and no molecular markers are currently used in clinical practice. Recently, cancer is being viewed as an ecosystem, in which tumor cells cooperate with each other and host cells in their microenvironment. Herein, we mapped the proteome of primary tumor and matched lymph node ecosystems, isolating malignant and non-malignant cells, to better understand the mechanisms underlying lymph node metastasis. For that, we used formalin-fixed paraffin-embedded (FFPE) primary tumors and paired lymph nodes from 27 HNSCC patients positive (N+, n=13) and negative (N0, n=14) for lymph node metastasis using laser microdissection combined with mass spectrometry-based proteomics. An average of 2,266 ± 216 proteins was identified in primary tumor and lymph node ecosystems. A hundred and five differentially abundant proteins from malignant cells in the primary site (N+ vs N0) were mainly involved in RNA stability and metabolism, while inflammation and splicing processes, respectively, were overrepresented in N+ vs N0 non-malignant cells signatures from primary tumor (N=33) and lymph node (N=13) sites (Student´s t-test; P-value≤0.05), indicating particular lymph node metastasis signatures for each cell population. Interestingly, the protein pattern of malignant cells from the primary site and matched lymph nodes showed an individual patient-specific metastasis profile through hierarchical clustering analysis; however, the profile of non-malignant cells does not represent patient-specific subpopulations and grouped according to the site of isolation (primary tumor and lymph node microenvironment). Our data reveal potential protein signatures associated with prognosis in HNSCC, as well as a non-specificity of protein patterns from host non-malignant cells that can be related with the immunosurveillance failure. Financial support: FAPESP (Process number 2015/19191-6).

#4542

The use of saliva proteins as biomarkers to predict the risk of lymph node metastasis in oral cancer.

Tatiane De Rossi,1 Daniela Campos Granato,1 Ana Carolina Ribeiro,2 César Rivera,3 Henry Heberle,4 Guilherme Pimentel Telles,5 Carolina Moretto Carnielli,1 Thais Bianca Brandão,2 Alan Roger Santos-Silva,6 Márcio Ajudarte Lopes,6 Adriana Franco Paes Leme1. 1 _Brazilian Biosciences National Laboratory, Campinas, Brazil;_ 2 _ICESP, Sao Paulo, Brazil;_ 3 _Talca University, Talca, Chile;_ 4 _Institute of Mathematics and Computer Sciences – ICMC, University of São Paulo – USP, Sao Carlos, Brazil;_ 5 _Institute of Computing – IC, University of Campinas (UNICAMP), Campinas, Brazil;_ 6 _Faculty of Dentistry of Piracicaba, State University of Campinas (UNICAMP), Piracicaba, Brazil_.

Background. The most common oral cancer in the world is squamous cell carcinoma (OSCC), accounting for more than 90% of all cases of cancer in the oral cavity. Thus, the research on molecular markers associated with the development and progress of human diseases has been the subject of intense research. The findings that saliva has molecular profiles indicating systemic diseases urge the study of non-invasive diagnosis using saliva as source of potential diagnosis, prognosis and predictive based on proteomics.

Methods. 204 proteotypic peptides were selected previously based on DDA data from our own studies or retrieved from the SRMAtlas. Heavy labeled synthetic peptides were synthetized and SRM method was developed using Skyline v3.6. Saliva samples were collected from patients with lymph node metastasis (n=26) and without lymph node metastasis (n=14). Saliva proteins were digested with trypsin. Samples were randomized in R environment and analyzed in a triple-quadrupole mass spectrometer (Xevo TQ-XS, Waters). A Peptide Retention Time Calibration Mixture was spiked into the samples. Data comparison between groups was performed using Wilcoxon Mann Whitney test in R environment.

Results. In the SRM method, we monitored 68 proteins and 24 of them were found decreased in patients with lymph node metastasis in comparison with patients without lymph node metastasis. The data analysis showed that at least one protein has, by itself, a higher relative risk in the development of lymph node metastasis in patients with OSCC. Two other panel of proteins presented the same relative risk described before.

Conclusions. This study indicates a panel of potential OSCC marker proteins, which can contribute to the prognostic evaluation, the patient's risk profile and the possibility of recurrence, and guide therapeutic intervention strategies.

#4543

Proteomic characterization of AXL kinase inhibitors and signaling pathways.

Anurima Majumder, Guolin Zhang, Emma Adhikari, Bin Fang, Eric A. Welsh, John M. Koomen, Eric B. Haura. _Moffitt Cancer Center, Tampa, FL_.

AXL is an attractive drug target because of its role in EMT-mediated resistance to EGFR tyrosine kinase inhibitor (TKI) in lung cancer (LC). Lack of genetic alterations and the role of stroma-mediated AXL activation in cancer cells, underscore the need to better characterize AXL TKIs, understand their effects on signaling and phenotype of cells, and develop assays to visualize active AXL signaling complexes.

For this, 25 LC cells were analyzed for total (t) and phosphorylated (p) AXL expression. AXL TKIs, RXDX106, R428 and Cabozantinib, were profiled using western blotting (WB), viability assay and activity-based protein profiling (ABPP). Phosphoproteins (pSTY) altered by RXDX106 were identified using mass spectrometry. Effects of RXDX106 on signaling, viability and migration of LC cells were also evaluated. Cell line models of EMT-mediated acquired drug resistance, treated with a combination of AXL and EGFR TKIs, were analyzed for changes in signaling, cell viability and EMT. Immunoprecipitation (IP) identified adaptors of AXL signaling, and Proximity Ligation Assays (PLA) were developed to detect these active complexes in situ.

H1299 cells, expressing highest levels of p and t AXL among the LC lines screened, was used in this study. RXDX106 and Cabozantinib potently inhibited pAXL in H1299 cells, but did not affect cell viability at these doses. R428 reduced cell viability at doses that did not efficiently inhibit pAXL, suggesting AXL independent phenotypic effects. Our ABPP data shows that apart from AXL, these TKIs target other overlapping and distinct subsets of proteins. R428 has the highest number of off targets and its unique ability to inhibit the FoxO pathway may explain the AXL independent phenotypic effects of R428. The pSTY data shows that RXDX106 deregulates phosphorylation of proteins involved in PI3K signaling, receptor endocytosis and cell migration pathways in H1299 cells. WB and phenotypic assays support these results by showing that RXDX106 inhibits pAXL, downstream pAKT but not pERK, and migration/invasion in these cells. In EGFR TKI resistant cells, EGFR and AXL TKI combination fails to alter downstream signaling, cell viability or EMT. Consistent with the WB and pSTY analyses, IP identifies PI3KR1 as an AXL interactor. PLAs to detect active AXL:PI3KR1 and AXL:pY100 signaling complexes show high basal PLA foci in H1299 and Calu1 cells that are abrogated by AXL TKI. HCC827 cells, which lack ligand independent pAXL, do not show significant labeling by either PLA.

Overall, we demonstrate that different AXL TKIs have distinct target profiles and that inhibition of AXL suppresses downstream PI3K/AKT signaling and migration/ invasion of LC cells. We also show that AXL TKI fails to suppress downstream signaling, cell viability or EMT in EGFR TKI resistant cell lines. We have also established a PLA to annotate AXL adaptor foci that could be developed as a tool to measure drug-targetable active AXL complexes in patient tissues.

#4544

PTP4A3 expression associated with increased invasion in esophageal adenocarcinoma.

Zhuwen Wang. _University of Michigan, Ann Arbor, MI_.

Esophageal adenocarcinoma (EAC) is the most rapidly growing solid malignancy with an overall 5-year survival rate of only 19%. The majority of EACs are diagnosed with advanced locoregional disease requiring neoadjuvant chemoradiation followed by esophagectomy. Understanding the molecular mechanisms associated with locoregional invasion are crucial for improving the treatment outcome.

Protein tyrosine phosphatase (PTP) type IVA member 3 (PTP4A3/PRL-3) is part of the PTP subfamily , which are phosphatases with dual-specificity. PTP4A3 has been shown to be overexpressed in a number of cancer types and often correlates with cell proliferation, migration, invasion, tumor growth, and metastasis. Here, we examine PTP4A3's potential role in the invasive properties of EAC.

Methods: We analyzed PTP4A3 mRNA levels of 122 chemo-naïve EAC samples using Human Gene 2.1 ST Arrays and compared these to available histological features. We also used RNA-seq analysis to examine the expression of PTP4A3 in 68 samples that consisted of Barrett's Esophagus, low-grade dysplasia, high-grade dysplasia, and EAC. PTP4A3 knockdown was achieved by using small interfering RNA and a rhodamine derivative (BR-1) inhibitor and was confirmed by both RT-PCR and Western blot. WST and clonogenic assays were performed to assess cell proliferation in cell lines derived from EAC tumors. Cell invasion and migration were measured by matrigel invasion and cell migration assays.

Results: PTP4A3 was significantly overexpressed in EAC samples compared to a cohort of normal esophageal samples (p<0.001). In addition, the expression of PTP4A3 increased with progression from Barrett's to EAC. PTP4A3 was overexpressed in 15 EACs relative to 26 Barrett's samples on U133A Array, which was confirmed in the 68-sample RNAseq progression cohort (p<0.01). Analysis of 122 EAC samples indicated that overexpression of PTP4A3 was significantly associated with decreased survival (p<0.05) and correlated with poor differentiation, high immune cell infiltration, and a high desmoplastic response (p<0.05). Inhibition of PTP4A3 in EAC cell lines led to a decrease in cell invasion and migration (p<0.01). However, there was no significant change in cell proliferation following knockdown.

Conclusion: PTP4A3 is frequently overexpressed in EAC and is associated with decreased survival. Inhibition of PTP4A3 decreased matrigel invasion and cancer cell migration, suggesting that PTP4A3 may play a role in the invasion and metastases of EAC cells and could serve as a biomarker of more aggressive disease.

#4545

Quantitative profiling of Cytochrome P450 2S1 in colorectal cancer by PRM assay.

Sadr ul Shaheed, Laurence H. Patterson, Klaus Pors, Chris W. Sutton. _University of Bradford, Bradford, United Kingdom_.

Introduction: Cytochromes P450s (CYPs) constitute a superfamily of xenobiotic metabolising enzymes responsible for metabolism of many pharmaceuticals in the liver. Elevated mRNA levels of specific isoforms, such as CYP2S1, are associated with poor prognosis in colorectal cancer (CRC), and represent novel therapeutic targets for biotransformation of prodrugs to potent cytotoxics at the cancer site. In order to understand the expression of CYP2S1 protein, we have developed a parallel reaction monitoring mass spectrometry (PRM MS) assay to screen CRC samples.

Method: Peptides (n=3) uniquely associated with CYP2S1 were synthesized and used as standards to optimise analytical performance (fragmentation conditions, LC retention time, LOQ, LOD, dynamic range) in an Orbitrap Fusion-based PRM MS assay. Levels of CYP2S1 were then determined in protein extracts of CRC, relative to the standards.

Results: The PRM MS assay yielded quantitative data over 3 orders of magnitude. CYP2S1 was detected in C106, CaCO2, HCC2998, HT55 and DLD1 cell lines (0.05-1.08pg) and HT55 and DLD1 xenografts (0.29-0.41pg). CYP2S1 was also detected in patient-specific CRC tissues, with elevated levels in Stage III tumours.

Conclusions: The PRM MS assay provides a specific, sensitive, high throughput method for identifying CYP2S1 compared to established enzyme and immunoassays. CYP2S1 levels vary considerably across CRC sources highlighting the importance of screening to identify the correct models for new drug development and the right patients for subsequent treatment.

#4546

Functional cell surface proteomics of acute myeloid leukemia enables predictive modeling of antibody-drug conjugate cytotoxicity.

Robert Lawrence,1 Robert Thurman,1 Travis Biechele,1 Cristina Tognon,2 Samantha Savage,2 Anna Reister Schultz,2 Jeffrey Tyner,2 William Arthur1. 1 _Seattle Genetics, Inc, Bothell, WA;_ 2 _Knight Cancer Institute, Oregon Health & Science University, Portland, OR_.

Acute myeloid leukemia (AML) remains a significant unmet medical need. The development of targeted therapies for AML is challenging due to the heterogeneity of immunological and genetic disease subtypes and physiological similarities shared by blasts and normal myeloid progenitor cells. The identification of abundant cell surface markers on AML blasts with limited expression on bone marrow progenitors could drive the development of novel antibody-drug conjugates (ADCs) and other antibody-based therapeutics with improved therapeutic windows. Here we used hydrazide chemistry and mass spectrometry-based proteomics to profile the cell surface landscape of 95 primary blast samples from the Beat AML research consortium and normal bone marrow progenitor cells from healthy donors. We measured the abundance of over 5,000 proteins with high reproducibility (R2 = 0.89) and coverage of known cell surface markers (240/371). We also identified more than 2,500 N-glycosylation sites, many of which are predicted by canonical sequence motifs but have not been demonstrated experimentally. These findings thereby expand our knowledge of both the AML and normal hematological cell surface proteome. Additionally, we used pulsed stable isotope labeling coupled with the aforementioned hydrazide method to assess global surface protein abundance and turnover dynamics in a panel of malignant cell lines. We integrated our cell surface characterization with Beat AML's mRNA expression, mutation, and AML subtype classification data to prioritize proteins for further exploration as candidate targets for ADC therapeutics. We generated a library of ADCs using monoclonal antibodies conjugated with Auristatin T, a highly potent membrane impermeable microtubule inhibitor, and measured ADC cytotoxic activity on a panel of AML cell lines as well as primary bone marrow progenitor cells. We used these integrated datasets to more precisely refine AML subtypes and identify patient populations that could benefit from novel AML ADCs. We report the preclinical validation of CD317 as a proof-of-concept for the utility of cell surface proteomics to identify novel targets for ADC therapeutics.

## TUMOR BIOLOGY

### Immune Cells in the Tumor Microenvironment 3

#4547

High dimensional proteomic profiling of immune cell subsets with data-independent acquisition mass spectrometry.

Jakob Vowinckel, Tobias Treiber, Kristina Beeler, Nicholas Dupuis. _Biognosys AG, Schlieren, Switzerland_.

Background Recent success with therapies to activate the immune system has demonstrated the utility of targeting the immune system for control of multiple cancers. These successes have also spurred interest in characterizing immune cell sub-populations to understand mechanisms of activation and suppression, and their relationship to therapeutic response. Currently, antibody-based approaches are commonly used to characterize immune cells, however these methods are limited to 30-40 markers and are driven by previous hypotheses, limiting new discovery. In this context, determination of the surface and cellular proteome of responsive populations will provide a powerful tool for insight into response mechanisms, so far hampered by low sample material availability and insufficient sensitivity of proteomic methodology. Here, we demonstrate how data-independent acquisition mass spectrometry can be used for high-dimensional characterization of immune cells, even with very limited cell numbers. Methods Primary human Cytotoxic CD8+ T cells, CD4+ T cells, CD14+ monocytes and natural killer (NK) cells, isolated from peripheral blood mononuclear cells, were prepared for mass spectrometry using standard sample preparation workflows. All samples were analyzed using 2 and 4 hour LC gradients on a C18 column coupled to a Thermo Scientific Q Exactive HF mass spectrometer in data-independent acquisition (DIA-MS) mode. DIA data was extracted using Spectronaut Pulsar X (Biognosys) both with a library generated using directDIA data searching in Spectromine as well as a Hybrid Library combining the directDIA and a publicly available resource library. Results Cytotoxic CD8+ T cells were evaluated using 100,000 cells of input material, which resulted in more than 3,500 proteins quantified in the primary cells. When combined with a CD8+ T cell resource library, the number of proteins quantified was more than 5,000. In the current experimental setup, 30,000 cells defines the lower limit of detection of CD8A and CD8B. Among other previously characterized proteins associated with CD8+ T cells, CCL5, TBX21, GZMH, PRF1, GNLY, CST7 were all detected at 30,000 cell input. Additionally, Granzyme A and B were also quantified which have classically been used, along with PRF1, as markers of lymphocyte infiltration. Data will also be presented for CD4+ T cells, CD14+ monocytes, and NK cells to further map and compare the immune cell phenotypic landscapes. Conclusions The DIA-MS platform enables deep proteomic phenotyping of sorted immune cell samples, even with limited numbers of cells. These new data sets make available broad and un-constrained biomarker investigation for deconvolution of the processes driving immune cell activation and suppression.

#4548

The chemokine receptor CCR1 is involved in microglia stimulated glioblastoma invasion.

Salvatore J. Coniglio, Poornema Ramasundaram, Neshama Fournier, Danielle S. Hamilton, Gregory Marshall, Keia Smith, Diana Habib, James R. Merritt. _Kean Univ., Union, NJ_.

Glioblastoma Multiforme (GBM) is the most aggressive form of adult brain tumor with a median survival time of twelve months. GBM is highly resistant to conventional therapy which includes surgical resection of the tumor, radiation treatment and chemotherapy. GBM cells are highly motile and invasive resulting in infiltrative tumors with poorly defined borders. GBM tumors are heavily infiltrated with microglia cells which are known to stimulate GBM cell invasion. Our laboratory has previously demonstrated that microglia strongly stimulates GBM invasion both in-vitro and in orthotopic animal models. This interaction was found to be dependent on CSF-1R which is expressed on all tumor infiltrating macrophages/microglia. Blockade of the CSF-1R using compounds such as pexidartinib (PLX3397) can inhibit microglia/macrophage-stimulated GBM invasion in-vitro and in vivo. A variety of chemokines are upregulated in the GBM tumor microenvironment and facilitate "cross-talk" between microglia and GBM cells eliciting a chemotactic response. We have demonstrated that the chemotactic ligand, CCL3, is similarly upregulated in GBM tumors. We postulated that inhibition of CCL3 associated receptors such as C-C receptor 1 (CCR1) might also inhibit GBM invasion, thus, a CCR1 antagonist could prove efficacious for blockade of microglia-induced glioblastoma invasion in vitro. Many potent CCR1 antagonists have been described in the literature. We chose four of these compounds with two distinct structural cores, all with reported IC50's of less than 200 nM for inhibition of CCR1 binding versus CCL3. We examined the ability of these antagonists to block microglia-stimulated glioblastoma invasion using an in-vitro coculture invasion assay. Using quantitative PCR arrays, we also show that expression of chemokines and chemokine receptor genes is greatly altered in GBM conditioned media-treated microglia. Understanding the pattern of tumor-associated macrophage/microglia chemokine secretion in GBM may present additional targets for chemotherapeutic intervention and enhance immunotherapy.

#4549

Macrophage-epithelial metabolic crosstalk impairs chemotherapy in pancreatic cancer.

Christopher J. Halbrook,1 Corbin Pontious,1 Ilya Kovalenko,1 Laura Lapienyte,2 Stephan Dreyer,3 Yaqing Zhang,1 Barbara Nelson,1 Hanna Hong,1 Daivid Chang,3 Jennifer P. Morton,2 Marina Pasca di Magliano,1 Costas A. Lyssiotis1. 1 _University of Michigan, Ann Arbor, MI;_ 2 _CRUK Beatson Institute, United Kingdom;_ 3 _University of Glasgow, United Kingdom_.

Pancreatic ductal adenocarcinoma (PDA) remains a leading cause of cancer related death, contrasting a relatively low incidence rate. A principle barrier in PDA treatment is the physiology of the tumors, characterized by a densely fibrotic stroma, rich with immune cell infiltration including macrophages. Macrophages are polarized by environmental cues which dictate their function. In PDA, macrophages within the tumor (tumor associated macrophages, or TAMs) are strongly immunosuppressive, inhibiting both infiltration and activation of cytotoxic T-cells. Additionally, TAMs have been shown to drive resistance to chemotherapy, though the mechanism for which remains unclear.

Several pathways have been described by which PDA cells recruit and polarize macrophages into TAMs, and the effects that TAMs have on the tumor microenvironment. These pathways have largely focused on signaling proteins, however, metabolic byproducts also influence the behavior of immune cells within the tumor microenvironment. To explore potential metabolic crosstalk, we have profiled the metabolic factors exchanged between PDA cells and TAMs. Among these, we have found that TAMs release metabolites which can regulate the response of PDA cells to chemotherapy. Importantly, this response is consistent across several murine and patient-derived pancreatic cancer cell lines, and this metabolite release appears to be a general property of anti-inflammatory macrophage metabolism. We further validated these findings in vivo, using a combination of pharmacological and genetic models to modulate myeloid cells within the tumor microenvironment. Taken together, these data suggest that further development of interventions which target either PDA-mediated polarization of TAMs or TAM-mediated inhibition of chemotherapy represent opportunities to improve the efficacy of currently available treatment options.

#4550

Correlations between tumor mutation burden, inflammatory profile and histological characteristics of tumor microenvironment in early-stage squamous cell lung carcinoma.

Hui Yu,1 Daniel T. Merrick,1 Ming-Sound Tsao,2 William G. Richards,3 Lucian R. Chirieac,4 Mark A. Watson,5 Christopher J. Rivard,1 David H. Harpole,6 Raphael Bueno,7 Adrie van Bokhoven,1 Aik-Choon Tan,1 Fred R. Hirsch,1 Wilbur A. Franklin1. 1 _University of Colorado Anschutz Medical Campus, Aurora, CO;_ 2 _University Health Network/Princess Margaret Cancer Centre and University of Toronto, Toronto, Ontario, Canada;_ 3 _Brigham and Women's Hospital, Harvard Medical School, Boston, MA;_ 4 _Harvard Medical School, Boston, MA;_ 5 _Washington University School of Medicine, St. Louis, MO;_ 6 _Duke University, Durham, NC;_ 7 _Brigham and Women's Hospital/Harvard Medical School, Boston, MA_.

Background: Anti-PD1/PD-L1 immunotherapy has demonstrated success in the treatment of advanced non-small cell lung cancer (NSCLC). Clinical data have shown that both the expression of PD-L1 in patient tumors and high tumor mutation burden (TMB) predicts the likelihood of a positive response to anti-PD-1/PD-L1 immunotherapy. Also, tumor microenvironment (TME) is the constitutive element in cancer immunity, in which analysis of characteristics reflects the potential existing immune reaction.

Method: Histologic sections from 150 squamous cell lung carcinoma (SqCLC) were evaluated by two pathologists independently for percentage and character of intratumoral inflammatory cells and percentage and character of para-tumoral infiltrate. The ratios of infiltrating inflammatory cells to tumor cells were estimated in 10% increments by microscopic inspection. The proportions of immune cell populations were deconvulated using the CIBERSORT method based on Affymetrix gene expression profiles. PD-L1 protein expression by IHC was evaluated using the Dako PD-L1 22C3 pharmDx kit and scoring was determined according to the Dako tumor proportion score (TPS). Tumor Mutation Burden (TMB) was calculated based on data from targeted genome sequencing. CD4 and CD8 mRNA levels were determined from Affymetrix gene expression data from frozen specimens.

Results: The infiltrates could be divided into intratumoral and paratumoral patterns according to their location in relation to microscopic tumor cell nests. Using the CIBERSORT assay, we confirmed our histological findings by microscopic examination that the SqCLC cohort can be subtyped into plasma cell dominant (74.8%) or other immune infiltrates dominant (such as macrophages), based on the proportions of immune cell populations. We found by regression analysis that TMB had a negative correlation with the percentage of intratumoral inflammatory cells (P=0.014), but did not significantly correlate with paratumoral infiltrates. The TMB demonstrated a significant negative correlation with CD4 mRNA level (P=0.017), but not with CD8 mRNA level. No correlation was determined for TMB and the immune cells dominant subgroup. Interestingly, we didn't find any association for PD-L1 protein expression with the percentage of intra- or para-tumoral infiltrates, plasma cells dominant group and CD4 and CD8 mRNA levels.

Conclusions: TMB was negatively correlated with the percentage of intratumoral inflammatory cells and CD4 mRNA level, which indicate that high TMB may promote an immune suppression environment. In addition, we did not find any association of PD-L1 expression with characteristics of TME in this early-stage SqCLC cohort. Further studies are needed to verify these interesting results.

#4551

Expression profiling-based characterization of immune cell populations in pediatric brain cancers.

Katherine E. Miller, Kathleen Schieffer, James Fitch, Vincent Magrini, Amy Wetzel, Anthony R. Miller, Daniel R. Boue, Jeffrey Leonard, Jonathan L. Finlay, Diana S. Osorio, Mohamed S. AbdelBaki, Christopher R. Pierson, Annie Drapeau, Jonathan Pindrik, Kristen Leraas, Elizabeth Varga, Devon Dishman, Lauren Shoemaker, Nicole Ross, Jeremy Pitts, Julie Gastier-Foster, Peter White, Catherine E. Cottrell, Richard K. Wilson, Elaine R. Mardis. _Nationwide Childrens Hospital, Columbus, OH_.

Cancer treatment strategies targeting the tumor microenvironment and corresponding immune cell infiltrate (i.e., immunotherapy) have recently proven effective against many solid tumors. In pediatric brain cancers, there is little known about the immune tumor microenvironment. We aim to characterize immune cell populations of pediatric brain tumors, which will better inform us about immune cell infiltration in different brain cancer subtypes and may indicate potential for patient response to immunotherapy. The Institute for Genomic Medicine at Nationwide Children's Hospital has recently initiated a translational protocol to evaluate tumor specimens from pediatric patients with rare or refractory brain cancers in a focused N-of-1 manner. We performed RNA-seq and NanoString™ PanCancer Immune expression profiling on extracted tumor RNA from 22 patients comprising eight distinct brain cancer subtypes. We used CIBERSORT algorithm for deconvolution of RNA-seq global gene expression data and NanoString™ expression profiling to investigate methods of quantifying tumor-infiltrating leukocytes (TIL) and expression of immune genes. Our analyses identified a subset of pediatric brain tumors with evidence of immune infiltration. Using CIBERSORT, four gliomas were predicted to harbor significant TIL populations. Overall, we observed high correlation (Pearson R2 = 0.884) between expression values derived from RNA-seq and NanoString™ methods. Similarly, the two methods produced comparable predictions for TIL composition in the glioma tumors. Further genomic analysis identified one patient, with a diagnosis of glioblastoma and Neurofibromatosis type 1 (NF1), with marked infiltration of macrophages and neutrophils. Overexpression of tumor-associated macrophage/microglia genes (CD14, CD163, CD68, ITGAM, PTPRC) and other immune markers (PD-L1, CCL2/MCP-1, IL1B) was observed relative to all other samples. More specifically, overexpression of CD204, CD206, and CD163 suggested the infiltrating macrophages represent the M2/protumor phenotype. CIBERSORT allows more "granularity" in macrophage population prediction and indicated an equal representation of M2 and M0/uncommitted macrophages. Given the M2 phenotype, it is likely that the infiltrating macrophages contributed to tumor progression and radiation resistance observed in this patient. If confirmed, the presence of M0 macrophages and/or overexpression of PD-L1 could indicate potential for immunotherapy in this patient (e.g., oncolytic viral therapy, strategies focusing on M0 to M1/antitumor polarization, or combination targeted therapy with PD-L1 inhibitor). In summary, we identified a patient with progressive glioblastoma tumor growth and significant macrophage tumor infiltration as a likely candidate for immunotherapy.

#4552

BMX-Seq as a new resource for deciphering the transcriptomic hallmarks of tumor plasticity and stromal interactions in brain metastasis.

Emily Wingrove,1 Zongzhi Z. Liu,1 Don X. Nguyen,1 Anna Arnal-Estape,1 Kiran D. Patel,1 Mary-Ann Melnick,1 Katerina Politi,1 Manuel Valiente,2 Harriet M. Kluger,1 Veronica L. Chiang1. 1 _Yale University, New Haven, CT;_ 2 _Spanish National Cancer Research Center, Madrid, Spain_.

Purpose: Brain metastasis remains a major site of relapse for several cancer types. However, the molecular mechanisms of brain metastasis are largely unknown given the complex relationship between tumor cells and the surrounding brain tumor microenvironment (TME). To better characterize the tumor-stromal relationship in the context of brain metastasis, we have developed a Brain Metastatic RNA-Sequencing (BMX-Seq) approach, which leverages xenograft models and can distinguish between the transcriptomes of human tumor and mouse stroma gene expression in vivo. This resource provides an extensive molecular portrait of the co-adaptation of brain metastasis with the brain TME.

Methods: Following intra-arterial injection of the human, lung adenocarcinoma (LUAD), H2030-BrM3 cell model into athymic mice, metastatic brain tumors were macrodisasected, flash frozen and RNA extracted. Healthy brain regions, subcutaneous tumors and H2030-BrM3 cells grown in monolayer were harvested in parallel. Our BMX-Seq pipeline was engineered to analyze bulk xenograft tissue, which includes tumor lesions as well as surrounding stromal tissue. Species-specific Taqman primers and immunofluorescent (IF) staining were used to validate our BMX-Seq results. Models of melanoma and breast cancer brain metastasis were also included in this study.

Summary: In comparing the transcriptomic profiles of tumors grown in the brain as compared to other sites, we identify shifts in epithelial and neuronal-like gene expression programs in malignant cells and show such trends are reversible once tumor cells are removed from the brain TME. Genes involved in WNT signaling and cell projection were also upregulated across melanoma, lung and breast cancer types when cells were grown in the brain versus other sites. In describing the neuroinflammatory response of stromal regions directly surrounding brain lesions, our BMX-Seq analysis revealed increased expression of astrocyte and microglial enriched transcripts. We then utilized IF to confirm the elevated density of both of these cell types in areas directly surrounding and infiltrating tumor regions. We also detect significantly induced expression of stromal TIM3 in areas surrounding tumor lesions and in a model system devoid of T-Cells. Follow-up work revealed TIM3 is largely expressed and induced on tumor-associated macrophages (including microglia) within the tumor region, and we further validate such results in a syngeneic model system as well as in human, brain tissue.

Conclusions: We have developed a highly sensitive RNA-sequencing based approach that can accurately map the transcriptomic adaptation of brain metastatic tumor cells and the surrounding brain TME. We believe these data may serve as an invaluable resource to guide future discovery in CNS metastases.

#4553

Snail1 in primary breast tumors remotely regulates a pro-tumor immune response in the bone marrow.

Mathew Sebastian, Dongjiang Chen, Son Le, Duy Nguyen, Changwang Deng, Dan Jin, Nagheme Thomas, David Tran. _University of Florida, Gainesville, FL_.

Systemic immunosuppression in cancer is a mechanism of immune escape allowing for cancer progression to metastasis and remains a major pathophysiologic barrier to treatment including immunotherapies. The mechanism of this phenomenon remains relatively obscured. Although immune suppressive humoral factors secreted by primary tumors are thought to be a major cause of immune escape, a major question remains whether factors critical for cancer metastasis also dually regulate systemic immunosuppression to prepare peripheral sites for the eventual encounter with metastasizing cancer cells. To that end, we report here that the master cancer epithelial-mesenchymal transition (EMT) initiating factor Snail1, whose expression is restricted to the primary tumor where it promotes the generation of invasive breast tumor-initiating cells, yet also has a direct role in creating a pro-tumor systemic immune environment to promote metastasis. These dual roles for Snail1 in breast cancer progression has not been elucidated. Using both spontaneous genetic murine model of breast cancer and a syngeneic orthotopic breast cancer model we demonstrate that Snail1 is uniquely required for metastatic spread of breast tumor cells while also directly modulates the bone marrow niche, skewing it towards a suppressive immune phenotype with an upregulation of myeloid derived suppressor cells (MDSCs) and a decrease in antigen presenting cell (APC) populations. Importantly, deletion of Snail1 specifically in primary tumors severely abrogated metastasis and simultaneously restored bone marrow immune populations to levels in non-tumor bearing animals. In vitro co-culture assays demonstrated that humoral factors from Snail1-repleted cancer cells promoted MDSC formation and suppressed APCs when compared to Snail1-deficient cancer cells. Mechanistically, Snail1 modulates systemic immunosuppression specifically through 1) upregulating the expression of GM-CSF, CXCL2, and CCL2, factors important for MDSC induction and recruitment, within the primary tumor to saturate the periphery with these factors; and 2) generating metastatic cells that may suppress immune functions within the metastatic niche. Taken together, these data demonstrate Snail1's central role in metastatic progression in breast cancer through its direct generation of metastatic cells and suppressing the systemic immune system to increase the chance that these metastatic cells may survive the periphery.

#4554

IL-17-CXCR2 axis promotes breast cancer metastasis and therapy resistance through facilitating recruitment of neutrophils.

Lingyun Wu,1 Sugandha Saxena,1 Mohammad Awaji,1 Bhawna Sharma,2 Rakesh Singh1. 1 _University of Nebraska Medical Center, omaha, NE;_ 2 _Biology Gilead Sciences, Foster City, CA_.

Breast cancer is one of the most common cancer types that happens to global females. The major challenges for breast cancer include therapy resistance and metastasis. Various factors and cancer-related immune cells are involved in tumor chemotherapy resistance process, including pro-inflammatory cytokine, interleukin (IL) 17, CXCR2 ligands (pro-inflammatory chemokines) and neutrophils. They are the key players for promoting cancer progression, and numerous reports indicated them as a potential therapeutic target for various cancer types including breast cancer. However, the detailed mechanism(s) remains unclear. In the present study, we investigate the role of neutrophils in chemotherapy resistance and metastasis in breast cancer.

In our previous study, we have observed increased levels of CXCR2 ligands in chemotherapy-resistant cells comparing with Cl66 parent cells. In this study, we observed upregulated IL17, IL17 receptor (IL-17R), granulocyte colony stimulating factor (GCSF) by qRT- PCR in resistant cells compared with parent cells. We further evaluated the protein levels of CXCR2 and its ligands, IL17 and its receptor, frequencies of neutrophils and T-helper 17 (Th17) cells in primary and metastatic tumors through using immunohistochemistry method. There were higher levels of IL17R, CXCR2, and CXCR2 ligands in metastatic tumor sites in comparison to the primary tumor sites. Moreover, we observed increased levels of IL17R, CXCR2, CXCR2 ligands together with the higher frequencies of neutrophils and Th17 cells in resistant tumors than Cl66 tumors. At the same time, tumor cells treated with IL-17 increased its proliferation rate and its expression of CXCL1 & CXCL5; and the increase was all in a concentration-dependent manner. In addition, the increase of CXCR2 ligands by IL17 was inhibited when we treated the tumor cells with ERK and NF-ΚB signaling inhibitors, indicating both pathways are involved in the regulation of IL17-induced CXCR2 ligands secretion.

Together, our data demonstrate an IL17-CXCR2 ligands axis plays protumorigenic inflammation, facilitating therapy resistance and metastasis through recruited neutrophils.

#4555

Characterization of the tumor microenvironment with an automated 10-plex IHC panel using UltiMapper™ I/O assays.

Heike Boisvert, Alexis Wong, Gourab Chatterjee, Abdul Mohammed, Douglas Wood, Mael Manesse. _Ultivue, Inc., Cambridge, MA_.

Background: In recent years, identifying and characterizing biomarkers within the tumor microenvironment (TME) has become a major focus for immune-oncology research. The growing field of immuno-oncology necessitates the identification of more biomarkers that help characterize the TME, and development of complimentary processes to rapidly analyze precious tissue samples. Ultivue® has developed UltiMapper™ assays that provide numerous advantages over alternative multiplexed IHC approaches, including high multiplexing in situ, sample preservation, streamlined workflows with staining completed in the same day, and easy integration into any histology lab. In this study we demonstrate a 10-plex immuno-oncology assay in FFPE tumor tissues by combining markers that profile the T-cell infiltrate, antigen presenting cells (APC), and checkpoint expression along the PD-L1/PD-1 axis. This 10-plex is achieved with a single antibody staining step for a fast workflow that is independent of staining order. The combination of 10 different biomarkers allows for characterization of the TME and investigation of PD-L1 expression on tumor and antigen presenting cells on a single slide. Methods: A 10-plex immunofluorescence assay was carried out using the UltiMapper approach on multiple deidentified FFPE tissue samples, including lung, melanoma, colon, and breast. Each sample was stained for the following 10 markers: CK or SOX10, PD-L1, PD-1, CD3, CD8, CD11c, CD20, CD68, CD163, and MHCII. Staining was performed in a single step using the Leica® Bond® Rx autostainer. Whole slide imaging was performed on various tissue scanners including the Zeiss® Axio Scan.Z1 slide scanner, and image analysis was performed using HALO® software from Indica Labs. Results: Cells were characterized and phenotyped based on their expression of specific biomarkers and the abundance of PD-L1 on macrophages, dendritic cells, B cell, cytotoxic T-cells, and tumor cells as measured. PD-L1 expression was observed on both immune and tumor cells with a range of different expression levels. Spatial analysis was employed to measure the distances between immune cells with different phenotypes and tumor cells to further characterize tumors as hot or cold. Conclusions: The multiplexed UltiMapper IHC approach allows for fast and comprehensive analysis of tumor samples necessary to research complex cancer biology. This approach achieves high quality multiplexed data on various tissue types and preserves precious tissue samples. The ability to interrogate a 10-plex I/O panel on a single slide with whole slide imaging presents a significant leap forward for IHC analysis, providing a faster and more thorough approach to characterize the TME and analyze PD-L1 expression on antigen presenting cells and tumor cells.

#4556

Targeting the TAM receptors on prostate cancer tumor-associated macrophages.

Kayla V. Myers, Kenneth J. Pienta. _Johns Hopkins University, Baltimore, MD_.

Tumor growth and progression is influenced by the composition of the tumor microenvironment including host stromal cells and immune cells. In prostate cancer, a large proportion of the tumor volume is made up of macrophages, and high macrophage infiltrate correlates with poor prognosis. Macrophages can be polarized to various subtypes including M1 and M2 in response to stimuli in their environment. M1 macrophages have anti-tumor functions such as immune stimulation. M2 macrophages have pro-tumor functions such as promoting angiogenesis, extracellular matrix remodeling and immune suppression. The majority of prostate cancer tumor-associated macrophages exhibit M2-like characteristics. Thus, targeting M2 tumor-associated macrophages is a promising strategy for cancer therapy. M2 macrophages engage in efferocytosis, or phagocytosis of apoptotic cells. This process is an anti-inflammatory, immunosuppressive event that promotes tissue remodeling and tumor growth. The TAM receptors (Tyro3, Axl, MerTK) are a family of receptor tyrosine kinases whose role in efferocytosis has been well described in the literature. The TAM receptors bind phosphatidylserine on apoptotic cells using their ligands Gas6 and Protein S as bridging proteins. Following binding to phosphatidylserine, TAM receptor signaling induces a cytoskeleton rearrangement to engulf the apoptotic cell. Since efferocytosis is a tumor promoting process, we hypothesize targeting the TAM receptors on M2 macrophages may be a beneficial anti-cancer strategy. We are characterizing TAM receptor expression on M1 and M2 macrophages using two complementary in vitro methods: macrophage polarization of monocytes derived from healthy human donors and of the acute monocytic leukemia cell line THP-1. Cell surface markers of M1 macrophages (CD86) and M2 macrophages (CD163 and CD206) were measured to assess polarization by flow cytometry. We are currently further confirming the degree of polarization by measuring cytokine secretion and mRNA expression of M1- and M2-associated genes. We used flow cytometry analysis to assess TAM receptor expression on the in vitro polarized M1 and M2 macrophages. In the human monocyte derived model, we detected higher levels of expression of MerTK and Tyro3 on M2 macrophages in several biological replicates. We also found that M2 THP-1 macrophages express higher levels of MerTK and Tyro3 than M1 THP-1 macrophages, consistent with macrophages from the monocyte derived model. In conclusion, these data support the TAM receptors as a relevant therapeutic target for inhibiting M2 pro-tumor functions. Further work by qRT-PCR, western blot and immunofluorescence staining will be used to confirm higher levels of the MerTK and Tyro3 on M2 macrophages and further investigate expression of Axl on M1 and M2 macrophages.

#4557

Tumor and CD8 T cells metabolism and consumption in the tumor microenvironment.

Lauranne Poncelet,1 Pierre Levy,2 Rima Ait-Belkacem,1 Maarten Ligtenberg,2 Daniel Peeper,2 Jonathan Stauber3. 1 _Imabiotech, Loos, France;_ 2 _Netherlands Cancer Institute, Amsterdam, Netherlands;_ 3 _Imabiotech, Billerica, MA_.

Introduction: The formation of nutrient-restricted environment is a way tumors can escape immune surveillance. Detailed evidence on the availability and spatial distribution of various nutrients and their metabolites within the tumor microenvironment (TME) as well as their effect on antitumor immune responses are still lacking. Insights into the role of metabolism in tumor development and progression are being used to design new drug targets and cancer therapies. The objective of this study was to explore the TME immuno-metabolic landscape that was recently coined to depict that both types of cells (tumor and immune) largely depend on and compete for the same nutrients. To do so, we developed a workflow to detect and quantify cellular metabolism using a multimodal platform allowing T-lymphocytes (LTs) infiltration staining and label free metabolic hallmarks histological localization and quantification.

Methods: Melanoma D10 cell line was transplanted in immunodeficient (NSG) mice, followed by injection of MART1 TCR transduced CD8 T cells when the tumors reached 200 mm3. Tumors were then harvested, measured and snap frozen at day 0 (control without T cells), 2, 7 and 25 post-T cell injection. 1,5-DAN matrix was sprayed onto tumor sections using the TM Sprayer (HTX Technologies, LLC). Data acquisition was performed using 7T MALDI-FTICR (Bruker Daltonics, Germany) at 50 µm spatial resolution. Acquired data were treated with MultimagingTM software (ImaBiotech). LTs immunostaining was applied on adjacent sections and histological regions were selected prior molecular image overlay, for metabolites relative quantity extraction.

Results: First, the tumor volume was measured showing an increase until 1500 mm3 for the control group, compared to the CD8 injected group (less than 400 mm3). Then, molecular imaging analysis highlighted the molecular distribution of different amino acid, nucleotide, energetic, hexosamine and TCA cycle metabolites. In parallel, CD3-staining showed lymphocytic infiltration starting from day 7 with extensive areas at day 25. After histological and molecular images overlay, metabolites level was measured from different regions. Potential correlation between metabolites/nutrients abundance and tumor/immune cells presence was studied. A decrease of Malate, N-acetylglucosamine, GSH and GSSG was noticed in tumor cells when their level was stable or increasing in LT immune cells. Ornithine and Histidine were increasing in tumor when decreasing in LT immune cells. Finally, ADP, AMP, Inosine, UMP, UDP-N-acetylglucosamine, Phenylalanine and Tyrosine level was stable in tumor when changing in LT cells.

Conclusion: For the first time, we localized and quantified the cellular metabolism of the tumor in its complexity. The combination of all these data extended our understanding on the melanoma D10 TME metabolic hallmarks that could be followed to assess the interplay between tumor and immune cells.

#4558

Mapping tolerized T cell states to exhaustion phenotypes in NSCLC.

Edgar Kozlova,1 Shingo Eikawa,2 Sacha Gnjatic,2 Bojan Losic1. 1 _Icahn School of Medicine, New York City, NY;_ 2 _Tisch Cancer Institute, New York City, NY_.

The promise of checkpoint blockade treatment has yet to be fully realized in part due to a lack of knowledge of immune exhaustion and its efficient characterization, especially in the context of immunosuppressive tumor microenvironments which effectively tolerize the immune system.

We hypothesized that mapping expression signatures defining regulatory T-cell states to previously studied and characterized exhaustion phenotypes of infiltrating T cells in NSCLC would reveal points of contact between T cell tolerization and exhaustion. Concretely, we profiled the gene expression and directly inferred CDR3/1 clonality between tolerized and immunized states in purified CD4+ T-Cells from 10 patients with ovarian or peritoneal and non-small cell lung cancer. The samples were taken before and during immunization therapy for the antigens MAGE-A3 or NY-ESO-1 over a period of 13 weeks, in the presence and absence of adjuvant to emulate the tolerized and immunized states.

Further, by modeling the regulatory T-cell states as an interaction between the tolerized and immunized with time and type of antigen allowed us to identify significant differentially expressed gene clusters and their correlation to clonality. We then projected these signatures onto single cell RNAseq data from NSCLC-TCGA to compare the identified clusters to previously identified exhaustion phenotypes. We show that antigen vaccination is immunostimulating and induces lymphocyte activation, leading to a proinflammatory environment. During an immunized stated an increase in cytokine activity, including IL2, 4, 9, 10, 13 and 17, whereas tolerized T Cells, we observe an increase in clonality, BIRC6 and IL20 apart from alterations in chromosome organization pathways and cell cycle including senescence.

In conclusion, we detail how the differences between tolerized and immunized states help unravel exhaustion mechanisms, which will ultimately identify molecular markers for exhaustion to assist during treatment and prognosis in clinical settings.

#4559

Tumor-derived Galectin-1 promotes tumor growth in an orthotopic C57Bl/6 NB mouse model.

Stefan Fest. _Universitätskinderklinik, Magdeburg, Germany_.

Question

We recently introduced the carbohydrate-binding protein Galectin-1 (Gal-1) as a novel tolerogeneic molecule in neuroblastoma (NB). NB-secreted Gal-1 suppressed the maturation of dendritic cells (DC) and inhibited T cell function. Here, we focused on the question whether tumor-derived Gal-1 is the decisive factor to promote tumor growth in our model for NB.

Methods

C57Bl/6J-derived orthotopic growing NB cells (named NB975A) were used for all culture as well in vivo experiments. Gal-1 knock-down clones (GL) were generated by stable transfection of wt NB975A cells (NBA) with the antisense Gal-1 expression vector (NBA-GL), (kindly provided by Dr. Rabinovich). Blasticidine resistant (5 mikrogram/ml) transfectants were cloned by limited dilution. Protein expression of Gal-1 was determined by Western blot. Orthotopic NB tumors were generated in C57Bl/6J wild type and Gal-1-knock-out (Lgals-/-) female mice by subcapsular tumor cell injection of NBA-GL or wild type NBA (1x106, 0,1 ml PBS) into the left kidney. Primary tumor growth was analyzed by using high frequency ultrasound measurement. Mice were sacrificed 23 days after tumor inoculation and tumor tissue was harvested for further analysis.

Results

NBA-GL showed 50% reduced protein expression of Gal-1 compared to control wild type NBA cells. In vitro viability and proliferation of the tumor cells was not affected by the transfection. Mice inoculated with NBA-GL cells had significantly smaller primary tumors when compared with control animals that received the NBA cells. Gal-1 sufficient tumor cells injected in Gal-1-deficient host mice grew normally.

Conclusion

Tumor-derived Gal-1 and not its expression in host is critical for the promotion of tumor growth.

#4560

In situ **multiplex analysis of regulatory and effector T cells in multiple tumor types.**

Elena Baranova, Maroua Tliba, Manon Motte, Naomi Defort, Amanda Finan-Marchi, Domenico Lazzaro, Fanny Estermann, Jean-Philippe Coton, Renaud Burrer. _Histalim, Montpellier, France_.

Immune-mediated rejection of cancer is prevented in part by the suppression of T effector (Teff) cells by regulatory T (Treg) cells. Favorable survival in numerous types of cancer has been associated with high Teff to Treg cells ratios as determined by flow cytometry or analysis of immunohistochemistry on serial sections. Multiplex immunofluorescence offers a technical advantage by allowing the detection of co-expression and spatial organization of up to 6 targets within a preserved tissue architecture on a single slide. We have designed a multiplex immunofluorescence protocol to identify human and murine Teff and Treg cells in situ. Human tissue microarrays of tumors in a range of tissues were investigated with a multiplex panel consisting of CD3/CD4/CD8/CD45RO/FoxP3/CD25. After multi-spectral acquisition, the T cell populations could be easily phenotyped and localized in all of the tissue types examined. The multiplex panel was then applied to non small cell lung carcinoma tumor and control tissues. Treg and Teff populations were quantified. The approach presented here demonstrates the power of multiplex immunohistochemistry in the identification and quantification of multiple immune cell populations on a single tissue section and the potential application of this method in both preclinical and clinical studies.

#4561

Slit2 inhibits breast cancer growth and metastasis by activating anti-tumor immune response.

Dinesh K. Ahirwar, Nabanita Chatterjee, Sanjay Mishra, Kontestine Shilo, Ramesh Ganju. _Ohio State Univ., Columbus, OH_.

Metastasis is a major cause of mortality in breast cancer patients in part due to the lack of clinically established targeted therapies. Approximately 5%-20% of patients with Stage II, and ~50% of patients with stage III will recur distally and are likely to die from their disease. Metastatic, or stage IV breast cancers, have a 5-year relative survival rate of about 22%. The expression of a tumor suppressor protein, Slit2 has been shown to be downregulated in various types of tumors including breast cancer. The Slit2 acts through Roundabout Homolog1 (Robo1) receptor. Previously it has been shown that ectopic expression of Slit2 inhibits human MCF-7 breast cancer cell line xenograft tumor growth in mice. However, its role in breast cancer metastasis, tumor microenvironment (TME) and anti-tumor immunity has not been studied before. By using genetically engineered human breast cancer cells, spontaneous mammary tumor and pre-clinical mouse models, we evaluated the role of Slit2 in inhibiting breast cancer growth, metastasis by activating anti-tumor immune response. To study the role of Slit2 in breast cancer, we implanted Slit2 overexpressing human breast cancer cell line MDA-MB-231 (231-Sli2) or vector control cells (231-Vec) to the mammary fat-pads of NOD/SCID/gamma (NSG) mice and observed that 231-Slit2 had significantly reduced tumor growth and metastasis to the lungs compared to 231-Vec. To further confirm the anti-metastatic role of Slit2, we treated mouse mammary tumor virus- Polyoma Middle T antigen (MMTV-PyMT) mammary tumor model and MVT-1 orthotopic tumor bearing FVB/J wildtype mice with recombinant Slit2 (rSlit2) that resulted in significantly reduced tumor growth and metastasis to the lungs in both the models compared to PBS treated mice. The ex-vivo immunofluorescence and flow cytometry studies revealed that Slit2 treated tumors possess a very high number of tumor phagocytic macrophages compared to PBS. In-vitro analysis also showed that rSlit2 treated mouse macrophages (RAW264.7) has higher bacterial particle phagocytic ability. Further analysis of tumors elucidated that Slit2 treated tumors recruited higher number of CD4+ and CD8+ T-cells. In addition, The CD8+ cells were also positive for Granzyme-b showing higher number of effector T-cells in the Slit2 tumors compared to PBS. By using human breast cancer tissue microarray (TMA), we have found that Slit2 expression significantly correlates with better overall survival. These observations highlight the ability of Slit2 to enhance tumor phagocytic macrophages and anti-tumor CD8+/Granzyme-b+ T-cells, thereby restricting tumor growth and lung metastasis. These studies suggest that Slit2 could be used as a novel immunomodulatory therapeutic agent.

#4562

Intra-tumor immune activity is linked to genetic diversity of tumor infiltrating lymphocyte and impact clinical outcomes in hepatocellular cancer.

Tstutomu Kawaguchi,1 Li Yan,2 Hideo Takahashi,2 Qianya Qi,2 Xuan Peng,2 Kazuaki Takabe,2 Eigo Otsuji1. 1 _Kyoto Prefetural University of Medicine, Kyoto, Japan;_ 2 _Roswell Park Comprehensive Cancer Center, Buffalo, NY_.

Hepatocellular carcinoma (HCC) remains the second leading cause of the cancer-related death worldwide. Ongoing clinical trials with immune checkpoint inhibitors (CPIs) for HCC have revealed considerable amount of patients do not respond. Hence, to identify the responder is imminent need for immunotherapy in HCC. We aimed to examine the immune landscape of HCC, using the publically available data set The Cancer Genome Atlas (TCGA). Clinicopathological and genomic expression data in 371 patients with HCC were obtained from TCGA. CYT was defined by GZMA and PRF1 expression, and CIBERSORT and TIMER were used to evaluate intra-tumoral immune cell composition. Kaplan-Meier curve for overall survival (OS), disease free interval (DFI), progression free interval (PFI), and disease-specific survival (DSS) were obtained and Cox Progression Hazards model was used for multivariable analysis. Gene Set Enrichment Analysis (GSEA) was also conducted to analyze the gene sets enriched in CYT-High HCC patients. High CYT was associated with high levels of activated CD8+ T cells, gamma-delta T cells, M1 macrophages, and memory CD4+T cells on CYBERSORT. A similar result was also obtained on TIMER. CYT-high HCC patients had significantly improved DFI, PFI, DSS, and OS, compared to CYT-low HCC patients. The levels of immune checkpoint molecules (ICMs), including programmed death-1 (PD-1), programmed death-ligand 1 (PD-L1), PD-L2, cytotoxic T-lymphocyte-associated protein 4 (CTLA4), lymphocyte-activation gene 3 (LAG3), T cell immunoglobulin and mucin domain 3 (TIM3), indoleamine 2,3-dioxygenase 1 (IDO1), and IDO2, correlated significantly with CYT. High-CYT tumors showed increased somatic copy number alterations (SCNA). Gene expression of APOBEC3 family was significantly higher in high-CYT tumors compared to low-CYT tumors. T-cell and B-cell receptors was more diverse in High-CYT tumors compared to low-CYT tumors. Multivariate survival analysis demonstrated high CYT was an independent protective factor for prognosis in patients with HCC. High CYT is associated with significantly improved survival in HCC, secondary to enhanced immunity and increased cytolytic activity by T cell and M1 macrophage. Additionally, high CYT is associated with ICMs, and CYT can be a potential biologic marker for CPIs.

#4563

Profiling the tumor immune microenvironment of adenocarcinoma and squamous cell NSCLC.

Kayla Harmeyer,1 Jiehui Deng,1 Suhagi Shah,1 Ece Bagdatlioglu,1 Aarif Ahsan,1 Christopher Reid,1 Aatish Thennavan,2 Charles Perou,2 Kwok-Kin Wong,1 Harvey Pass1. 1 _New York University Langone Medical Center, New York, NY;_ 2 _University of North Carolina, Chapel Hill, NC_.

The emergence of immune checkpoint inhibitors has changed the treatment paradigm for NSCLC. Recently, immunotherapy in combination with chemotherapy was shown to be a promising first-line treatment strategy for patients with squamous cell lung cancer, where targeted therapy has had little efficacy due to the pathological driver mutations for this disease being poorly understood. However, for both adenocarcinoma and squamous cell NSCLC, use of such immune checkpoint inhibitors has shown to improve the outcome for only a subset of patients with advanced or metastatic disease. Understanding the diversity in the tumor immune microenvironment will be critical to not only advance immune oncology research but to also determine potential clinical distinctions that dictate therapeutic response. Here, we take a multifaceted approach to compare the tumor immune microenvironments of over 50 patients diagnosed with either adenocarcinoma or squamous cell NSCLC. To this end, we performed a comprehensive multicolor flow cytometry analysis to compare tumor immune infiltrates and expression of suppressive markers to adjacent normal lung tissue as well as peripheral blood immune cells from the same patient. This analysis showed differences in the CD4+ and CD8+ T cell, CD19+ B cell, NK cell, and CD14+/CD16+ myeloid cell populations between the tumor and matched tissue and blood. These immune profiles were then compared between adenocarcinoma and squamous cell carcinoma subtypes as well as early versus late stage disease. We then integrated these findings with multiplexed immunofluorescence analysis on a panel of immune cell markers. Finally, 10x genomics single-cell RNA sequencing (scRNAseq) data were used to analyze single-cell transcriptomes from both adenocarcinoma and squamous cell lung tumors. Our result showed great heterogeneity of immune infiltrates across different patient samples, including different subtypes and different disease stages. This detailed comparative analysis will help us further understand the involvement of tumor infiltrating immune cells for different subtypes of lung cancer.

#4564

Tumor infiltrating lymphocytes are associated with p16 protein expression in acral lentiginous melanoma.

Marco A. Galvez Nino, Miluska Castillo, Luis A. Bernabe, sandro Casavilca, Valeria Villegas, Joselyn Sanchez, Jorge Dunstan, Julio Abugatas, Carlos Castaneda, Henry Gomez. _Instituto Nacional de Enfermedades Neoplasicas, Lima, Peru_.

Background: The loss of function of gene p16(INK4a)/CDKN2 is associated with the development of several neoplasm and tumor progression, likewise, the level of tumor infiltrating lymphocytes is a predictive and prognostic biomarker of survival. This study aims to evaluate the association between composition of tumor infiltrating lymphocytes (TILs) and expression of p16 in acral lentiginous melanoma (ALM), and their impact on prognosis.

Materials and methods: A cohort of 148 surgical pathology specimens of ALM was studied. TILs were evaluated by immunohistochemical detection of CD3 and CD8, along with CD20, CD4, CD68, and CD163 in a subset of 43 cases, likewise, we classify the level of TILs in grades by combining density and distribution. p16 protein expression by immunohistochemistry was also investigated in all cases.

Results: The median age was 66 years, median Breslow thickness was 6.0 mm, Grade III of TILs was found in 28.4% and lymph nodes were involved in 44.6%. Breslow thickness (p<0.001), stage I-II (p<0.001), negative lymph nodes (p<0.001) and <10% p16 (p<0.001) were associated with longer survival. Grade III of TILs was associated with thinner Breslow thickness (p=0.008) and lower mitosis (p=0.047). A higher density of CD3 TILs was associated with male gender (p=0.008), thinner Breslow thickness (p=0.047), negative lymph node (p=0.031), early stage (p=0.046), and p16 nuclear expression of >10% (p=0.045). Higher CD8 TIL was associated with >p16 (p=0.03). Survival analysis found that longer survival doesn't associated with high grade of TILs (p=0.094). Levels of CD3+ and CD8+ cells were correlated with those of CD4+, CD20+, CD68+ and CD163+ immune cells.

Conclusions: Loss of expression of p16 is associated with lower levels of CD3+ and CD8+ TIL, indicating a probable relationship between p16 and TILs immune response in ALM.

#4565

The functional role of atypical chemokine receptor 1 in immune cell regulation of breast cancer.

Brittany D. Jenkins,1 Rachel Martini,1 Kevin Gardner,2 Michele Monteil,3 Dorrah Deeb,4 Lisa Newman,5 Melissa Davis5. 1 _Univ. of Georgia, Athens, GA;_ 2 _Columbia University, New York, NY;_ 3 _Univ. of Georgia/Augusta University, Athens, GA;_ 4 _Henry Ford Health System, Detriot, MI;_ 5 _Weill Cornell Medicine, New York, NY_.

Breast cancer (BC) is a heterogeneous disease that leads to varied molecular subtypes and distinct clinical outcomes. Tumor heterogeneity, which is in part influenced by genetic ancestry, contributes to racial disparities in BC. This disparity has persisted for over 30 years, despite improvements in detection, screening, and treatment methods. When investigating race-specific differences in the breast tumor microenvironment (TME), we believe that chemokine receptors, such as atypical chemokine receptor 1 (ACKR1/DARC), play an integral role in regulating chemokine and immune cell migration in circulation and the TME, ultimately influencing tumor progression. ACKR1/DARC specifically plays a role in ancestry-related differences in BC due to an African-specific ancestral allele ("Duffy-null" mutation) that is fixed in Sub-Saharan African populations, and present in 60-70% of African-Americans. Those that harbor the mutation do not express ACKR1/DARC on erythrocytes, ultimately affecting immune cell migration in these populations only. To better understand the effects of this variant in circulation and the TME, we performed immunohistochemistry (IHC) on breast tumors from a cohort of patients with matched blood samples. From our IHC analysis, we quantified levels of ACKR1/DARC, in addition to levels of target pro-inflammatory chemokines, and distinct immune populations of T-cells and macrophages. In addition, individuals were genotyped to determine Duffy-null status, and a correlative Luminex multiplex assay was performed on patient plasma to determine concentrations of pro-inflammatory chemokines in circulation. We determined that those with low expression of ACKR1/DARC in tumors exhibited a unique signature of immune cell infiltrates that would encourage a more aggressive TME. In addition, we observed variable levels of circulating chemokines, where those with the Duffy-null mutation exhibited significantly decreased levels of CCL2. Overall, our analyses support the hypothesis that tumor-specific DARC/ACKR1 plays a vital role in immune cell regulation in women with BC.

#4566

Protumor macrophages are targeted by nanotherapy delivery of c-Myc inhibitor, while preserving antitumor macrophages in breast cancer.

Alison Esser, Michael H. Ross, Xinming Su, Gregory C. Fox, Yalin Xu, Anne H. Schmieder, Xiaoxia Yang, Grace Cui, Katherine N. Weilbaecher, Gregory M. Lanza. _Washington University In St. Louis, Saint Louis, MO_.

Protumor macrophages (M2-like) have been shown to enhance tumor growth in mice and are correlated with worse prognosis for breast cancer patients. Current therapies to reduce tumor-associated macrophages (TAMs) deplete all TAMs however; some subsets of macrophages are necessary for anti-tumor immune responses. We have previously shown that M2-like TAMs express significant levels of integrin β3. We hypothesized that activated αvβ3-targeted perfluorocarbon nanoparticles (αvβ3-NP) could specifically target M2 macrophages. αvβ3-NP use a unique contact mediated drug delivery mechanism. NP bind to target receptors resulting in hemifusion of the NP and outer membrane of the target cell to deliver drug directly to the cytosol, avoiding endocytic uptake and retention. To evaluate the mechanism of drug transfer by αvβ3-NP to macrophages, we used rhodamine labeled αvβ3-NP. We found that M2-like macrophages specifically uptake rhodamine by a phagocytosis independent mechanism compared to unpolarized M0 and M1-like macrophages. We next evaluated protein inhibitors that would specifically affect M2 macrophages if delivered by αvβ3-NP. Recent studies have shown c-MYC is upregulated in IL-4 treated human M2 macrophages and is necessary for inflammatory responses of GM-CSF stimulated mouse macrophages in vitro. We therefore hypothesized that targeted delivery of a c-Myc inhibitor to M2 macrophages could alter polarization and modulate function. We generated a c-Myc prodrug linking the inhibitor to the Sn2 fatty acid to protect against drug loss and metabolism in circulation and to deliver the prodrug (PD) into the target cell cytoplasm. Cytosolic lipases liberate the inactive drug into the cytoplasm as the free active compound. Mice with mammary fat pad tumors established with a BO1 breast cancer cell line integrin β3 CRISPR knockout clone were treated with αvβ3-perfluorocarbon NP loaded with Myc inhibitor prodrug (MI3-PD). M2-like macrophages (CD45+, CD11b+, GR1-, F4/80+, MHCII low, CD206 high) were significantly reduced while M1-like macrophages (MHCII high, CD206 low) were increased. Tumor growth was reduced by bioluminescence imaging but not by tumor weight. To evaluate the effects of Myc inhibition on macrophages, we used transgenic mice expressing yellow fluorescent protein (YFP) driven by the promoter of the pro-tumor myeloid gene, arginase 1. Arginase is upregulated with M2 polarization in mice, serving as a marker for M2 polarization. We found that Myc inhibitor treatment of macrophages derived from arginase-YFP reporter mice and polarized with tumor conditioned media, significantly reduced arginase expression (35%) with no effect on IL-4 polarized macrophages. These data suggest αvβ3-NP targeted with Myc inhibitor can reduce pro-tumor M2-like TAM macrophage number while preserving anti-tumor M1-like macrophage activity in breast cancer.

#4567

**Distribution of CD8** + **cytotoxic lymphocytes in human neoplasms.**

Niclas C. Blessin, Florian Lutz, Patrick Spriesterbach, Wenchao Li, Tim Mandelkow, Vera Nickelsen, Ronald Simon, Claudia Hube-Magg, Florian Viehweger, Maximillian Lennartz, Christoph Fraune, Kristine Fischer, Katharina Möller, Stefan Steurer, Jacob R. Izbicki, Guido Sauter, Sarah Minner, Frank Jacobsen, Andreas M. Luebke, Franziska Büscheck, Doris Höflmayer, Waldemar Wilczak, Eike Burandt, Andrea Hinsch. _Univ. Medical Ctr. Hamburg-Eppendorf, Hamburg, Germany_.

Evidence suggests that the quantity of cytotoxic lymphocytes influences the likelihood for a successful application of immune checkpoint inhibitors. To compare the density of CD8+lymphocytes across various different tumor types, a tissue microarray (TMA) composed of up to 50 tumor samples each from 85 different cancer types and subtypes was analyzed. A total of 2652 cancers and 608 normal tissues were successfully analyzed by CD8 immunohistochemistry followed by automated image analysis of digitized slides. The median number of CD8+lymphocytes ranged from 6 cells/mm2in pleomorphic adenoma up to 1573 cells/mm2in Hodgkin's lymphoma. CD8 counts were generally lower in normal tissues. Blood vessels of the spleen was the only non-lymphatic tissue staining for CD8.In solid tumors, highest CD8 densities (cells/mm2) were found in seminoma (median: 424), Warthin's tumor (median: 425), squamous cell cervical cancer (median 468), medullary breast cancer (median: 657) and thymoma (median: 889).Tumor types approved for therapy with checkpoint inhibitors such malignant melanoma (median: 81), muscle invasive urothelial carcinomas (median: 119), small cell lung cancer (median: 120), clear cell kidney cancer (median: 153), squamous cell cancer (median: 189) and adenocarcinoma of the lung (median: 328) as well as Hodgkin's lymphoma (median:1573) were all ranking among the upper half of our list. Comparably high CD8 densities (cells/mm2) were also found for several rare and aggressive cancer types including Merkel cell carcinoma (median: 70), angiosarcoma (median: 95), anaplastic thyroid cancer (median: 156), anal carcinoma (median: 104), squamous cell carcinoma of the vagina (median: 128) and embryonal carcinoma of the testis (median: 186). The CD8 cell count was highly variable within tumor types. In 73 of 84 analyzed cancer types, the CD8 count at least occasionally exceeded the average CD8 count of tumors for which checkpoint inhibitors have been approved.These data support the concept, that in most tumor types at least some individual cancers may benefit from treatment with immune checkpoint inhibitors.

#4568

The next generation precision medicine tool for durable breast cancer control.

Zuzana Tatarova,1 Oliver Jonas,2 Lisa Coussens,1 Joe Gray1. 1 _Oregon Health and Science University, Portland, OR;_ 2 _Harvard Medical School, Boston, MA_.

In the era of personalized medicine, therapies can be perfectly suited to patients based on tumor molecular characteristics. The critical issue is that only a subset of individuals exhibit clear and durable responses. Achieving durable control will require systemic approach that will i) prevent ''rewiring'' that enables therapeutic escape, ii) block resistance mechanisms that result from bidirectional interaction with the environment and iii) reactivate and enhance immune surveillance. Novel strategies will be needed to counter all of these mechanisms via administration of series of drug combinations that change as the tumors evolve under treatment pressure. We have developed a novel pre-clinical model system that allows for efficient, fast and harmless assessment of tumor cell responses to large numbers of drug (combination)s. Such system is based on the recently developed implantable screening microdevice that permits localized intratumoral drug delivery and provides with the ability to predict the drug efficiency and select the right candidate within few days after application. The unique strength of our approach is that all measurements are performed within a live tissue in the native tumor microenvironment, and with an intact immune system. We combine this technology with multiplexed immunohistochemistry staining to bring in novel insights to mechanisms of response and resistance and propose rational combination treatment in breast cancers. Breast cancers are among the least responsive to otherwise very effective immune-based therapies. This is attributed mainly to low antigenicity and naturally immune-suppressive environment of the tissue. With our new approach, we showed neutrophil infiltrate, immunogenic cell death and increased antigenicity in immediate proximity to the pan-HDAC inhibitor, Panobinostat, suggesting a rational combination with immune checkpoint blocking therapies. We have identified optimal combination strategy enhancing anti-tumor response as well new predictive biomarkers positively correlating with the treatment responses. In the long term, we plan to extend the number of drugs that target distinct cancer signaling pathways and to generate a library of therapeutic signatures for deep understanding of drug response and resistance. This will promote rapid identification of rational combination strategies for durable breast cancer control but also will serve as a baseline for translating the technology towards its clinical use.

#4569

Intra- and inter-run assessment of reproducibility and quantification of UltiMapper™ I/O APC and T-act kits for tissue multiplexing.

Bonnie Phillips,1 Katir Patel,1 Courtney Hebert,1 Aditi Sharma,1 Jamie Buell,2 Sean Downing1. 1 _Ultivue, Cambridge, MA;_ 2 _Ultivue, Framingham, MA_.

Background: The field of immuno-oncology research has enthusiastically adopted multiplex immunohistochemistry (IHC) techniques to establish the spatial relationships between various immune cell populations in a tumor biology in context. Multiplexing enables researchers to gain a deeper understanding and insight into the tumor microenvironment. Unfortunately, many multiplexing technologies face several challenges, specifically in generating highly robust, reproducible, and easily quantifiable data sets. Ultivue's UltiMapper I/O APC and T-act kits utilize InSituPlex® technology, a new method of multiplexed IHC that utilizes a streamlined workflow with single antigen retrieval, staining, amplification, and detection steps, allowing for the completion of the assay in 5 hours. Here we assess these kits for intra- and inter-run reproducibility and quantification.

Methods: Intra-run reproducibility and quantification was accomplished by manually staining 5 serial sections from three tissue types (deidentified samples of tonsil, melanoma, NSCLC) with one set for each of the UltiMapper I/O APC (CD11c, CD20, CD68/CD163, and MHC Class II) and T-act (CD3, Granzyme B, Ki67, and pan-Cytokeratin/SOX10) kits. Inter-run assessment was determined by staining each slide from a set of five serial sections of each tissue type independently. Images were acquired using the ZEISS® Axio Scan.Z1, without the need for linear unmixing allowing for direct whole slide imaging. Analysis was accomplished using Indica Labs HALO® software. Coefficients of variation (CVs) were calculated based on resulting data.

Results: Analysis of intra-run serial section images revealed that cell counts from section to section had CVs of <10% across all markers, in all tissues, for both the UltiMapper I/O APC and T-act kits. This included total cell counts and average signal intensity. Similar results were seen for inter-run comparisons for both kits (<10% CV).

Conclusions: The results presented here indicate that InSituPlex technology is potentially much more reproducible than other tissue multiplexing techniques currently available. Histological standards for coefficients of variation in IHC based assays are typically <15%. Data presented here falls well within that standard indicating the potential for future translational applications. In conclusion, InSituPlex is a highly reproducible and quantifiable multiplexing technology that is able to produce these results across a variety of tissue types and markers.

#4570

Selective depletion of regulatory T cells suppressed neuroblastoma tumor growth in mice.

Sophie von Lenthe,1 Christine Weißenborn,1 Ana Claudia Zenclussen,1 Stefan Fest,1 Leonid Metelitsa2. 1 _Department of Experimental Obstetrics and Gynecology, Medical Faculty, Otto-von-Guericke-University, Magdeburg, Germany;_ 2 _Department of Pediatrics, Texas Children´s Cancer Center, Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX_.

Purpose of the study

Neuroblastoma (NB) is the most common solid extracranial tumor in childhood. Regulatory T cells (Treg) are known to have immune suppressive capacity and to be involved in tolerance establishment. As they support tumor growth and help some tumors to overcome recognition by the host, they are considered to be an important target. Treg are reportedly increased in blood of NB patients. However, there are no scientific proofs to date that they are involved in neuroblastoma growth. To address this important question we employed Foxp3-DTR mice to selectively deplete Treg upon orthotopic application of syngeneic neuroblastoma cells.

Methods

Orthotopic NB tumors were generated in mice Foxp3-DTR mice and in C57Bl/6J controls by subcapsular tumor cell injection of NB975A cells into the left kidney. Primary tumor growth was analysed for 23 days by high frequency ultrasound measurement. To deplete Treg, InjectionDepletion of Diphtheria Toxin (DT, 15 ng/g bodyweight) intraperitoneally every 4 days in Foxp3.DTR mice depleted the Treg pool. Control Foxp3.DTR animals received PBS and control C57B/l6 received DT. All mice were sacrificed 23 days after tumor inoculation. Tumor as wells as immune cells harvested from spleen and inguinal lymph nodes were analyzed by Flow Cytometry, Immunofluorescenceimmunohistochemistry and qRT-PCR.

Results

In control mice bearing tumors, Treg presence was confirmed at the contact zone between the kidney and the growing, inoculated tumor.

Mice devoid of Treg (Foxp3-DTR+DT) showed a significantly reduced tumor growth compared to animals with normal Treg frequencies (Foxp3-DTR+PBS). Tumor growth in wild type controls was not affected by the application of DTR.

Conclusion

Ablation of Treg hindered the growth of NB, strongly suggesting their potential as therapeutic target for this aggressive childhood tumor.

#4571

Pro-inflammatory and pro-angiogenic properties of tumor associated natural killer cells in prostate cancer.

Denisa Baci,1 Matteo Gallazzi,1 Lorenzo Mortara,2 Douglas M. Noonan,2 Antonino Bruno1. 1 _MULTIMEDICA, Milano, Italy;_ 2 _University of Insubria, Varese, Italy_.

Prostate cancer (PCa) represents the second cause of male cancer death worldwide. Immune cells can acquire pro-tumor phenotype and functions as consequence of their plasticity. Natural killer (NK) cells are cellular mediators of the innate immunity, primarily involved in tumor recognition and elimination. Altered NK phenotype and functions have been observed in different tumors including PCa. Here, we provide new insights on phenotype and functional characterization of peripheral blood tumor-associated NK cells (TANKs) from benign prostatic hyperplasia (BPH) and prostatic adenocarcinoma (ADK) patients. We also investigated NK interaction with other components of the tumor microenvironment. NK cell phenotype and functional characterization was performed by multicolour flow cytometry for surface antigens on BPH and PCa TANKs. Interactions of TANKs with other TME components (endothelial cells, macrophages) were investigated using released products from BPH and PCa TANKs for functional studies of angiogenesis, macrophage recruitments and polarization. The effects of TANKS on macrophage polarization state were evaluated by gene expression analysis RT-PCR (qPCR).We found that NK cells from peripheral blood of BPH and PCa patients acquire a pro-angiogenic/ decidual-like phenotype, identified as CD56+CD9+CD49a+CD69+ NKs. These results were confirmed also exposing heathy-donor derived NKs to conditioned media of 3 different prostate cancer cell lines (PC-3, DU-145, LNCaP). In addition, polarization of NKs with the PCa cell line conditioned media resulted in downregulation of IFNγ, TNFα and Granzyme A and increased production of pro-angiogenic factors, such CXCL8, Angiopoietin1 (Angiop1) and Angiogenin (ANG). Secretome analysis revealed the ability of NKs from BPH and PCa patients to release higher level of pro-angiogenic molecules (CXCL8, MMP-1, MMP-9 and uPAR) and cytokines/chemokines involved in macrophage recruitment, CCL1, CCL2, CCL5, CCL13, CXCL1, CXCL11 and GM-CSF. Conditioned media from BPH and PCa NKs were able to promote angiogenesis in vitro, by inducing the formation of capillary-like structures on human umbilical-vein endothelial cells (HUVEC), recruit the THP-1 monocyte cell line and polarize THP-1 differentiated macrophage towards M2-like tumor associated macrophages (TAM).Our data demonstrate that NK cells from PCa patients are switched toward a pro-angiogenic/pro-tumor phenotype and function. Therefore, we propose tumor associated NKs as a new hallmark and potential target in prostate cancer.

#4572

Cross-validation between anti-CD3/CD8/FOXP3/pan-Cytokeratin fluorescent multiplex IHC and chromogenic single IHC by digital image analysis for quantifying tumor-infiltrating lymphocytes in colorectal cancer patient samples.

Daniel Biljes, Philipp Layer, Nickels Winkler, Nadine Fändrich-Dursun, Hartmut Juhl, Bernd Gromoll, Malik Khenkhar, Philipp C. Uhlig. _Indivumed GmbH, Hamburg, Germany_.

A rising need for the overall goal to decipher cancer development and progression is a better understanding of the dynamics of the tumor microenvironment (TME), which are grounded in the interactions and reciprocal manipulation of cancer, stromal, and immune cells. Tumor-infiltrating immune cells influence the TME, and analyses of immune cell types, densities and locations within the TME using fluorescent multiplex immunohistochemistry (mIHC) and digital image analysis appear promising for establishing prognostic indicators and in helping to identify more personalized anti-cancer therapies, but in order to be applied in the clinical setting the relevant assays require a precise validation.

We developed a tyramide signal amplification (TSA)-based fluorescent mIHC assay for the detection of CD3, FOXP3, CD8, and pan-Cytokeratin (pan-CK) in formalin-fixed paraffin-embedded (FFPE) tissue on the Leica BOND RX automated staining platform. Algorithms for digital image analysis of chromogenic and fluorescent mIHC were developed using Visiopharm Oncotopix software, allowing for a distinct quantification of T cell populations within tumor and stroma regions of interest. To ensure the reliability and robustness of the mIHC assay an extensive assay validation was performed on FFPE colorectal cancer (CRC) tissue. Chromogenic IHC of the single markers was analyzed by a pathologist and compared to digital image analysis of chromogenic and mIHC. To ensure a suitable linearity of the assay titration series of the primary antibodies were performed. Furthermore, the repeatability and intermediate precision of the assay were determined. Finally, 50 individual FFPE CRC tissue samples were analyzed by fluorescent anti-CD3/FOXP3/CD8/pan-CK mIHC and digital image analysis.

Good correlations were observed between chromogenic and mIHC, as well as between pathologist and digital image analysis, and an adequate specificity, accuracy, linearity, repeatability and intermediate precision of the assay could be demonstrated. Furthermore, the distributions and ratios of the differently labeled tumor-infiltrating T cell populations in 50 CRC tissue samples were in agreement with published literature and allowed for a classification of the samples regarding their immune phenotype.

The reliable quantification of immune cell subsets in FFPE cancer tissue samples shown here provides an efficient way of analyzing the lymphocyte composition of the TME at a validation level that is comparable to chromogenic IHC and apparently suitable for an application in the clinical setting. The validated combination of mIHC and digital image analysis may therefore enable a classification of the immune status of CRC patient samples and could help to identify new targets for anti-cancer therapy.

#4573

Immune infiltrate profiling for progressive malignant stages of cutaneous squamous cell carcinoma.

Md Maksudul Alam, Alok Khandelwal, Tara Moore-Medlin, Hillary Savage, Xiaohui Ma, Cherie-Ann Nathan. _LSUHSC-Shreveport, Shreveport, LA_.

Cutaneous squamous cell carcinoma (cSCC) is the most common human cancer, affecting more than 300,000 individuals in the United States annually. Although, most cSCC can be treated successfully by surgery and/or radiation, a significant percentage of cSCC are aggressive and often unresectable and/or can metastasize to the lymph nodes. Organ transplant patients are at a significantly higher risk for cSCC due to a compromised immune system. Recently, the PD-1 inhibitor cemiplimab was approved for the treatment of patients with metastatic cSCC or patients with locally advanced cSCC who are not candidates for curative surgery or curative radiation. This suggests a critical role of immune cells in modulation of disease progression in cSCC. Hence, immune profiling of cSCC is important in identifying novel agents that could benefit patients. Our objective was to employ immunohistochemistry (IHC) to characterize the immune cell infiltrate in normal skin and compare it to actinic keratosis (AK), cSCC and metastatic cSCC (cSCC-M), in order to investigate the immune phenotype associated with progressive stages of malignancy. Our results suggest that AK, cSCC and cSCC-M exhibit significantly higher levels of CD3+, CD4+ and CD8+ T-cells compared to normal skin. Although, the number of CD3+ T-cells are comparable in AK, cSCC and cSCC-M, the CD8+ T-cells show higher infiltration in cSCC-M compared with cSCC and AK. The FOXP3+ Regulatory T-cells (T-reg) display higher abundance with progressive stages of carcinogenesis. However, surprisingly no significant alterations were observed in the abundance of CD4+ T-cells among AK, cSCC and cSCC-M. We also found that the CD68+ Tumor-associated macrophages (TAMs) exhibit a trend of increasing abundance with progressive disease stage. Our study suggests that the CD8+ T-cell population in cSCC-M could be potentially exhausted in the immune hostile tumor micro-environment (TME) constituted by T-reg and TAMs. Alternatively, CD8+ T-cells themselves might express FOXP3 and other immune suppressive factors, thereby reaching a state of anergy. Immunotherapy continues to be a burgeoning field and holds exciting therapeutic prospects in the treatment of cSCC. Moreover, transplant patients with aggressive cSCC are a unique patient population that could benefit from immunotherapy.

#4574

Combining multimodal biomarkers as an immunogram to guide immunotherapy use: A proof of concept.

Thomas Sbarrato,1 Lucie Sudre,1 Laurent Vanhille,1 Pernelle Outters,1 Mihaela Angelova,2 Bernhard Mlecnik,2 Angela Vasaturo,2 Gabriela Bindea,2 Tessa Fredriksen,2 Lucie Lafontaine,2 Daniela Bruni,2 Jérôme Galon,2 Jacques Fieschi1. 1 _HalioDx, Marseille, France;_ 2 _INSERM, Laboratory of Integrative Cancer Immunology, Paris, France_.

Our recent understanding of the immune contribution to fight cancer has deeply modified the standard of care of cancer patients. As an example, immunotherapies by immune checkpoint inhibitors (ICI) anti-PD1/PDL1 are now approved in several cancer indications, such as Non-Small Cell Lung Cancers or melanoma. However, ICI are less effective for other high incidence indications like colorectal cancer (CRC). Here, the heterogeneity as well as the relatively low degree of patient response to those immunotherapies have highlighted that factors, present in the tumor microenvironment (TME), may limit or boost the efficacy of treatment. In this context, the comprehensive assessment of these factors could be key to stratify patients and allow the selection of the optimal treatment.

In order to help clinical researchers and biopharmaceutical companies to measure the immune contribution to drug efficacy, HalioDx has developed the Cancer Immunogram, a solution based on Blank CU et al. Science (2016). Our multi-parameter approach encompassing a unique range of immune scoring assays is based on the analysis and the understanding of the immune contexture of tumors and offers a personalized and dynamic "fingerprint" of tumor-immune system interaction.

Here, we provide a Proof of Concept for the Cancer Immunogram in the context of CRC by combining the following technologies and biomarkers: Tumor Mutational Burden (Tumour foreignness), DNA mismatch-repair deficiency (MSI), TCR Sequencing (T Cell Clonality), Immunoscore® Colon (Immune Cell Infiltration), Immunosign® (Tumor Sensitivity to Immune Effectors), Halioseek® PDL1/CD8, Brightplex T-Cell Exhaustion Panel (Immune Checkpoints) and Brightplex MDSC Panel and Treg detection (Immune Suppression). We show that the Cancer Immunogram allows to identify patient specific patterns which might improve the prediction of the response to therapy. We believe that the Cancer Immunogram will help researchers and clinicians to personalise treatments in order to improve patients' outcomes and response to cancer treatment.

#4575

IL17 signaling modulates tumor microenvironment in pancreatic cancer.

Yu Zhang, Erick Marcelo Riquelme Sanchez, Michelle Annabel Zoltan, Florencia McAllister. _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Pancreatic cancer is the one of the most devastating malignancies and the 5-years survival remains below 10% for decades. The pancreatic tumor microenvironment is very complex with tumor cells surrounded by a dense fibro-inflammatory stroma with fibroblasts and immune-suppressive cells which are known to play a pivotal role in the whole process of tumorigenesis, and contributing to the lack of responses to most therapies. We have previously described that IL17, a cytokine secreted from immune cells recruited to the pancreas in response to Kras and inflammation, is involved in the initiation and development of pancreatic premalignant lesions, in part by promoting stemness on the oncogenic epithelium. In this study, we aim to dissect the role of IL17 on the stroma in established pancreatic adenocarcinoma. To this end, we blocked the IL17 signaling pathway with monoclonal antibodies and also used a parallel genetic approach which consisted in IL17RA deletion from the cancer cells using CRISPR-Cas9. RNA sequencing analysis, flow cytometry, immunohistochemistry, and opal-multiplex immunofluorescence were performed on tumors and cellular compartments. We uncovered that neutrophils recruitment was the main pathway altered by IL-17. Using flow cytometry, IHC and opal-multiplex we found that Gr1+ cells were significantly modulated by Il17 in the tumor microenvironment, as not only the numbers of these cells were altered but also their spatial distribution with respect to CD8+T cells, which may contribute to the generation and maintenance of the immunosuppressive microenvironment that characterizes pancreatic cancer. Moreover, we found that reactive oxygen species (ROS) level was also evidently downregulated by IL17 signaling blockage.Therefore, we conclude that IL17 plays an important role in remodeling the pancreatic cancer immunosuppressive tumor microenvironment.

#4576

Study of the immune contexture in advanced pancreatic neuroendocrine tumors reveals tumor-associated macrophages as promoters of poor survival.

Alejandro Francisco-Cruz,1 Naohiro Uraoka,1 Suyu Liu,1 Edwin R. Parra,1 Luisa M. Solis,1 Barbara Mino,1 Arvind Dasari,1 Jaime Rodriguez-Canales,1 Michael J. Overman,1 Jonathan M. Loree,2 James C. Yao,1 Ignacio I. Wistuba,1 Daniel M. Halperin,1 Jeannelyn S. Estrella1. 1 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _BC Cancer, Vancouver, British Columbia, Canada_.

Introduction. PanNETs are characterized by heterogeneous but largely indolent growth, leading to advanced stage at diagnosis, difficulty predicting outcomes, and insufficiently effective treatments. A better understanding of PanNETs immune context is needed for rational immunotherapy strategies. The aim of this study was to characterize immune cell infiltrates within primary tumors, understand the correlation of immune infiltration with genes associated with PanNET development, and clinical-pathological features.

Material and methods. Formalin-fixed paraffin-embedded (FFPE) surgically resected primary tumor specimens from 53 patients with metastatic PanNETs were evaluated for DAXX, ATRX, and immune-cell markers (CD8, CD4, CD45RO, FOXP3, ICOS, OX40, PD-1, LAG3, TIM-3, B7-H3, B7-H4, PD-L1, VISTA, and CD68) by immunohistochemistry (IHC). Intratumoral lymphocyte-enriched areas (LEA), defined by CD8 hot-spots, and macrophage-enriched areas (MEA), defined by CD68 hot spots, were selected for image analysis and cell densities were quantitated. We considered higher densities more than the third quartile value and low density as less or equal than the third quartile value for all the markers. 47 cases from the same FFPE tumor tissue blocks were used for paired-end RNA sequencing (HiSeq 4000 Sequencing System), and exome sequencing (T200 Platform) to MEN1, SETD2, MUTYH, CHEK2, BRCA2, ALT, DAXX, ATRX, PTEN, TSC1, and TSC2 genes. Differences between variables were analyzed by non-parametric t-test and Kaplan-Meier curves for time-to-event using SPSS statistical software version 24.

Results. Overall, higher densities of CD8, CD4, CD68, and B7-H3 were found compared with the other markers. We found a significant correlation between CD8 in LEA with CD4 (r=0.7), FOXP3 (r=0.5), CD45RO (r=0.6), ICOS (r=0.5) and PD-1 (r=0.5) cell densities. In addition, CD68 in MEA had significant and positive correlation with TIM-3 (r=0.6) cell densities. Higher TIM-3 cell densities correlated with higher levels of TIM-3 (P=<0.00001), CD163 (P=0.004), and CSF1R (P=0.02) mRNAs. Patients with high CD68 and TIM-3 densities showed worse disease-specific survival (DSS). The ratio between CD68, TIM-3, B7-H3 and FOXP3 cell densities to CD8 positive cells was significantly higher in patients with loss of ATRX nuclear expression. We observed different gene mutations on those PanNET samples but only PTEN mutant tumors were associated with higher densities of CD68 and worst DSS compared with lowest CD68 and WT PTEN mutant tumors (P=0.002).

Conclusions. TAMs were significantly correlated with inferior DSS in PanNETs. TAM depletion may, therefore, present an appealing and rational target for immunotherapeutic approaches in NETs. This work was funded by a Conquer Cancer Foundation of ASCO Career Development Award, and a grant from the Neuroendocrine Tumor Research Foundation. 

### Immunity in the Microenvironment

#4577

Anti-tumor immunity is a key determinant of small cell lung cancer survivorship.

Farhad Kosari, Prasuna Muppa, Simone Terra, Anurag Sharma, Aaron Mansfield, Marie Christine Aubry, Nafiseh Janaki, Aqsa Nasir, Tobias Peikert. _Mayo Clinic College of Medicine, Rochester, MN_.

Introduction: While most small cell lung cancer (SCLC) patients die within a few months, a sub-group of patients survive for many years. Factors determining long-term survivorship remain largely unknown. We present the first comprehensive comparative genomic and tumor microenvironment analyses of small cell lung cancer (SCLC) between patients with long term (LTS) and expected (EXS) survival times.

Methods: We compared surgically resected tumors of 23 LTS (survival > 4 years) and 18 EXS (survival ≤ 2 years). There were no differences in clinical variables including TNM staging and curative versus non-curative intend surgery between the groups. Gene expression profiling was performed by microarrays and tumor microenvironment analyses were by IHC of prominent immune related markers.

Results: Immune related genes and pathways represented the majority of the differentially overexpressed genes in LTS compared to the EXS. The differences in the immunological tumor-microenvironment were confirmed by quantitative immuno-staining. Increased numbers of tumor infiltrating and associated lymphocytes were present throughout tumors of LTS. Several differentiating patterns of enhanced anti-tumor immunity were identified. While some areas of LTS tumors also harbored higher numbers of suppressive immune cells (monocytes, regulatory lymphocytes, and macrophages), ratios of these suppressive cells to CD3+ lymphocytes were generally lower in LTS tumors indicating a more tumor suppressive microenvironment.

Conclusions: Our data demonstrate that long-term survivorship of SCLC patients is strongly influenced by the presence of anti-tumor immune cells in the tumor microenvironment. Characterization of the anti-tumor immune responses may identify opportunities for individualized immunotherapies for SCLC.

#4578

Macrophages are important source of PD-L1 and PD-L1 expressing on central M2 macrophages leads to the poor prognosis of NSCLC patients: Via macrophage landscape analysis for NSCLC patients with tumor PD-L1 negative.

Lili Cao,1 Xiaofang Che,1 Xiujuan Qu,1 Xueshan Qiu,2 Zhi Li,1 Bowen Yang,1 Shuo Wang,1 Yunpeng Liu1. 1 _Key Laboratory of Anticancer Drugs and Biotherapy of Liaoning Province, Shenyang, China;_ 2 _Department of Pathology, College of Basic Medical Sciences China Medical University, Shenyang, China_.

PD-1 antibody has achieved significant clinical efficacy for advanced non-small cell lung cancer (NSCLC) ,but the ORR is not optimistic for the non-selected patients. PD-L1,as the therapeutic target, is still controversial to be a predictor of efficacy.It was noticed that some patients with tumor cell PD-L1 negative could still benefit from anti-PD-1/PDL1 antibody therapy.Besides the tumor cells, PD-L1 was also known to express in immune cells in TME. Therefore, in this study, we aimed to clarify the factors that affect the PD-L1 expression in TME, and their functions on the NSCLC prognosis and the effect on anti-PD-1/PD-L1 therapy.With the multiple quantitative immunofluorescence staining on 94 tissue specimens of NSCLC patients with PD-L1 expressing negative on tumor cells , we surprisingly found that macrophages,especially M2 macrophages,were the main source of PD-L1 for patients with tumor cells PD-L1 negative.Futhermore, the expression of PD-L1 in central M2 macrophages(M2 macrophages that infiltrate into tumor islets),was important for the poor prognosis of NSCLC patients,suggesting that the expression of PD-L1 in macrophages may also be associated with the benefit of anti-PD-1 therapy, which is an important factor to be considered in evaluating the efficacy of immunotherapy in the future.

#4579

A comparative immune-landscape within tumor-microenvironment: Pattern of distribution of immune cells in tumor versus tumor-adjacent normal tissues.

Xiaoqian Lin, Read Sulaiman, Jennifer Aske, Paul Mayer, Luis Rojas-Espaillat, David Starks, Pradip De, Brian Leyland-Jones, Nandini Dey. _Avera Cancer Institute, Sioux Falls, SD_.

Introduction: Tumor and immune cells can determine each other's fate. Immunotherapy targets either the PD1-PD-L1 axis which modulates immune-checkpoints or the cytotoxic CTLA4-mediated immune invasion of tumor cells. Thus the success of cellular-dynamics based immunotherapy depends on the makeup of the immune landscape within a given tumor as compared to its adjacent normal tissue. Aim: We hypothesize that as the immune-environment directly or indirectly shapes and determines the fate, drug response, and progression of an established tumor, the immune-landscape of the tumor-microenvironment will be characteristically different from that of the tumor-adjacent normal tissue. We studied the distribution pattern of selected immune cells in the tumor and tumor-adjacent normal tissues from patients with cancers of lung and ovary.

Methods: The study was approved by Avera IRB. FFPE tumor and tumor-adjacent normal tissue samples in pairs from surgically resected tissues were used for IHC to identify CD3 (Anti-CD3 [SP7] ab16669), CD4 ([EPR6855] ab133616), CD8 (ab85792), CD68 (Dako. #M0876), CD163 (Cell Signaling #93498), FOXP3 ([236A/E7] ab20034) in immune cells and PD-L1 (Clone 22C3; Dako. #M3653) in tumor cells & tumor-associated macrophages. Tonsil tissue was used for the IHC validation. Tumor and normal cells from paired specimens were compared using H&E stain & Ki-67, pS6RP, cl-caspase 3, CIP2A, and CD31 IHC-stains based on criteria of evaluation for individual protein(s). Anatomic pathological evaluation for the diagnosis of the disease was carried out by Pathologist.

Results: IHC expression of CD3, CD4, CD8, CD68, CD163, and FOXP3 in immune cells and PD-L1 in tumor cells was found heterogeneous. Although each tumor presented an individual pattern of distribution of immune cells, in most tumor samples, the identified immune cells were of tumor-cooperating phenotypes. Tumoricidal cells were rarely observed within the tumor compartment, and barely present in spaces, while tumor-friendly immune cells were frequent. In lung tumors, alveolar macrophages were predominantly of tumor-cooperating M2 types (CD68+/CD163+), whereas tumoricidal M1 macrophages (CD68+) were either absent or rare. The expression of PD-L1 in tumors did not correlate participation of any other immune cells in the tumor-microenviroment. Lung macrophages were highly positive for PD-L1 in which the intensity ranged from punctate to high.

Conclusion: We present for the first time, a tissue-based analysis of distribution pattern of different immune cells in histological subtypes of the lung and ovarian cancers, in the tumor and tumor-adjacent normal tissues. The relationship between the immune-landscape of the tumor and tumor-adjacent normal tissue is being worked out and will be presented in the meeting.

#4580

Primary tumor-induced immunity eradicates disseminated tumor cells in syngeneic mouse model.

Raziye Piranlioglu,1 Eunmi Lee,1 Maria Ouzuonova,2 Riley Rodier,3 Adam Greer,4 Feyzanur Bayraktar,5 Omer Can Durmus,5 Ali S. Arbab,1 Muthushamy Thangaraju,1 Max S. Wicha,6 Esteban Celis,1 Hasan Korkaya1. 1 _Augusta University, Augusta, GA;_ 2 _Cancer research Center of Lyon, Lyon, France;_ 3 _Univeristy of Georgia, Aiken, GA;_ 4 _University of Georgia, Aiken, GA;_ 5 _Bahcesehir University, Istanbul, Turkey;_ 6 _Michigan University, Ann arbor, MI_.

Although clinically apparent metastasis is associated with late stages of cancer development, micro-metastatic dissemination may be an early event. However, the fate of these early disseminated tumor cells (DTC) remains elusive.

Using the syngeneic mouse models, we demonstrated that although both orthotopically-implanted murine 4T1 and EMT6 tumors are capable of disseminating into secondary organs, only 4T1 tumors develop overt metastasis. However, EMT6 tumors induce an anti-tumor immunity in syngeneic mice and that eradicates disseminated tumor cells (DTC) in distant organs. Following the complete removal of primary EMT6 tumors, mice do not develop detectable metastasis and generate an immunological memory that leads to complete elimination of repeatedly injected tumor cells via tail vein. Conversely, these cells readily grow and metastasize in immuno-deficient athymic or Rag2- mice, and when g-MDSCs from 4T1 tumor-bearing mice were co-injected into immunocompetent EMT6 primed mice. In contrast to complete resection, mice with residual tumors following surgery exhibited an enhanced growth of local and concomitant growth of DTCs at metastatic site with increased g-MDSCs accumulation in lung and spleen.

Together, our results suggest that some tumors are capable of inducing anti-tumor immunity against the DTCs when complete resection of primary tumor cures animals. However, in the presence of residual tumors, inflammation induced by surgical procedure promote the growth of DTCs.

#4581

High endothelial venules are associated with immune evasion and hereditary background in mismatch repair-deficient colorectal cancers.

Aysel Ahadova,1 Pauline Pfuderer,1 Alexej Ballhausen,1 Ian Frayling,2 Ann Ager,3 Magnus von Knebel Doeberitz,1 Matthias Kloor1. 1 _University Hospital Heidelberg, Heidelberg, Germany;_ 2 _Institute of Cancer & Genetics, Cardiff University School of Medicine, Cardiff, United Kingdom; _3 _School of Medicine and Systems Immunity Research Institute, Cardiff University, Cardiff, United Kingdom_.

DNA mismatch repair (MMR)-deficient cancers can develop in the context of Lynch syndrome or sporadically. MMR-deficient cancers accumulate a high load of immunogenic frameshift peptide neoantigens as a consequence of insertion/deletion mutations at coding microsatellites (microsatellite instability, MSI). Whether enhanced lymphocyte recruitment contributes to the high immune infiltration and the pronounced local immune response typically observed in MSI colorectal cancers (CRCs) is not known. Here we analyzed the density of high endothelial venules (HEVs), postcapillary blood vessels specialized for lymphocyte trafficking, in MSI CRC of sporadic and hereditary origin. Immunohistochemical staining with rat monoclonal MECA-79 antibody was used to detect HEVs in tumor specimens from MSI (hereditary, n=20 and sporadic, n=28) and MSS CRCs (n=35). Lymph follicle-associated HEVs were normalized per mm2 of tumor and peritumoral area. Mutations of the B2M gene were determined by Sanger sequencing. Significantly elevated HEV densities were detected in MSI (n=48) compared to MSS CRCs (n=35) (0.09 vs 0.02 counts per mm2, p=0.0002). Lynch syndrome-associated MSI CRCs presented with significantly higher HEV densities compared to sporadic counterparts (0.15 vs 0.05 counts per mm2, p=0.025). This difference remained significant when age-matched groups of hereditary and sporadic MSI CRCs were compared. MSI CRCs with B2M mutations (n=12) presented with significantly higher HEV densities than their B2M-wild type counterparts (n=14, p=0.016). Our results demonstrate that a high density of HEVs correlates with the MSI phenotype in CRC, underlining the elevated local immune activation in these tumors. Within MSI CRC, Lynch syndrome was a predictor of high HEV counts, suggesting a likely role of lymphocyte recruitment in immune surveillance of Lynch syndrome CRCs. The observation of high HEV counts particularly in MSI CRCs harboring B2M mutations supports the model that immune evasion occurs particularly in an activated local immune environment. The prognostic value of HEVs in MSI CRCs should be addressed in the future studies.

#4582

Mannose receptor positive macrophage infiltrate correlates with prostate cancer onset and metastatic castration-resistant disease.

Jelani C. Zarif, Javier A. Baena-Del Valle, Jessica L. Hicks, Christopher M. Heaphy, Igor Vidal, Jacob Luo, Tamara L. Lotan, Jody E. Hooper, William B. Isaacs, Kenneth J. Pienta, Angelo M. De Marzo. _Johns Hopkins Univ. School of Medicine, Baltimore, MD_.

M2-Tumor Associated macrophages (M2-TAMs) can suppress inflammation within the tumor microenvironment and have been reported to modulate cancer progression. We and others have previously reported infiltration of M2 macrophages in metastatic castrate-resistant prostate cancers (mCRPC). The objective of this study was to determine whether the extent of M2 macrophage infiltration correlates with prostate cancer aggressiveness, we applied immunohistochemistry to normal prostatic tissue, localized prostate cancer and metastatic castrate-resistant prostate cancer (mCRPC) from 192 patients. To assess M2 macrophage involvement, we analytically validated an IHC assay to detect the human mannose receptor (CD206). Also, we used multiplex immunofluorescent staining to show that a small fraction of CD206 staining co-localizes with endothelial cells of lymphatic vessels, while the vast majority of staining occurs in CD68-positive macrophages. The area fraction of staining for CD206-positive macrophages increased in a stepwise fashion going from normal (i.e. non-inflammation) prostatic tissue, to primary untreated carcinomas, to hormone naive regional lymph node metastases to castration resistant prostate cancer. Complimentary studies using flow cytometry confirmed CD206-positive M2-TAM infiltration. Altogether, this study revealed a progressive increase in CD206-positive macrophages from normal prostate to metastatic CRPC. Given the immunosuppressive nature of these cells and lack of clinical success of immunotherapy for prostate cancer patients, our results provide a rationale for therapeutic development to deplete these cells in the prostate cancer microenvironment as a potential method to augment immunotherapeutic approaches in prostate cancer.

#4583

Versican accumulation and proteolysis in neuroendocrine tumors.

Christopher P. Babiarz, Philip B. Emmerich, Carley M. Sprackling, Cheri A. Pasch, Linda Clipson, Kristina A. Matkowskyj, Fotis Asimakopoulos, Dustin A. Deming. _University of Wisconsin-Madison, Madison, WI_.

Introduction: Neuroendocrine tumors (NETs) are a rare, understudied form of human cancer lacking robust preclinical models. Our laboratory has recently demonstrated the importance of the tumor microenvironment (TME) in T-cell exclusion from tumors. Here we examine the accumulation of the immunoregulatory proteoglycan, versican (VCAN), and its immunostimulatory proteolysis product, versikine (Vkine), produced by VCAN cleavage via ADAMTS proteins. Immunotherapeutics are increasingly being studied in NETs and investigation of the immune permissive nature of their TME is warranted.

Methods: Human NET tissue microarrays (TMAs) were obtained from the University of Wisconsin Carbone Cancer Center TSB Biobank, containing human NET tissues across a broad spectrum of disease sites including the lung and gastrointestinal (GI) tract among others. Immunohistochemistry was performed using antibodies to VCAN, Vkine, and CD8+ T-cells. VCAN and Vkine levels were measured using a four-tiered relative intensity system (0/1/2/3), while CD8+ T-cell infiltration was measured by counting the CD8+ T-cells per high power field (hpf). Experimental groups were binned as low if VCAN or Vkine scored 0 or 1 and high if VCAN or Vkine scored 2 or 3.

Results: Across all samples, high VCAN levels were observed in 20% of samples while high Vkine levels were observed in 55% of samples. Within the lung NET subset, which includes small cell lung cancer, 15% of samples were designated VCAN high and 65% were Vkine high. For the GI NET subset, 34% of samples scored as VCAN high while 50% scored as Vkine high. This study identifies a greater amount of Vkine high tumors present in NETs than we previously observed for colorectal, anal, pancreatic, and breast cancers. The proteolytic predominant phenotype (VCAN low and Vkine high) was present in 55% of lung NETs and 31% of GI NETs. CD8+ T cell infiltration correlated with the VCAN proteolysis predominant phenotype (VCAN low/Vkine High - CD8+ T-cell 16/hpf vs. all other VCAN/Vkine phenotypes 6.5/hpf, p<0.001). This trend persisted across all NET specimen types, including lung and GI primary NETs.

Discussion: VCAN accumulation occurs in a subset of NETs, likely leading to immune exclusion and potential resistance to immunotherapies. Excitingly, we demonstrate that a large subset of these cancers has active proteolysis of VCAN in the TME leading to release of the immunostimulatory fragment Vkine. Those NETs with low levels of VCAN and high levels of Vkine are predicted to have a permissive TME for immunotherapies. Further studies are needed to discern the underlying mechanisms resulting in this proteolysis and the signaling consequences with Vkine production. Additionally, this study indicates that VCAN proteolysis should be investigated as a novel biomarker for immunotherapy response for NETs.

#4584

Malignant pancreatic cancer cells respond to immune selection pressure to foster immunosuppression.

Reham Ajina,1 Annie Zuo,1 Maha Moussa,1 Sarah J. Cooper,2 Yue Shen,2 Quentin R. Johnson,3 Jerry M. Parks,4 Jeremy C. Smith,4 Elana Fertig,5 Marta Catalfamo,1 Sandra A. Jablonski,1 Louis M. Weiner1. 1 _Georgetown University, Washington, DC;_ 2 _University of Tennessee, Knoxville, TN;_ 3 _Oak Ridge National Laboratory, Oak Ridge, TN;_ 4 _UT/ORNL Center for Molecular Biophysics, Oak Ridge, TN;_ 5 _Johns Hopkins University, Baltimore, MD_.

Background:

Host immunity is relevant to pancreatic cancer (PC). Pancreatic tumors are infiltrated with a high number of immune cells, and they play a potential important role in disease evolution. However, there is no FDA approved immunotherapy for PC yet, suggesting that a deeper understanding of immune-cancer interaction is needed. The aim of this study was to investigate the effects of immune selection pressure on malignant pancreatic epithelial cells and on subsequent cancer immune evasion mechanisms.

Materials and Methods:

1 X105 mT3-2D murine pancreatic cancer cells (Kras+/G12D, p53+/-R172H (Boj et al. 2015)) were injected subcutaneously into syngeneic C57BL/6J (WT), B6.CB17-Prkdcscid/SzJ (SCID) and T cell-depleted WT mice (10 mice/group). Tumor growth was monitored over time. 1 cm3 tumors then were harvested, processed and studied. To consider the complex biology of pancreatic cancer immunology, we analyzed tumor specimens in their entirety by histopathological evaluation, multiple color flow cytometry, NanoString nCounter, and global proteomics profiling. Furthermore, to evaluate the influence of immune selection pressure on neoplastic cells, we selectively analyzed malignant epithelial cell gene expression in the cell line, and in cells isolated from tumors grown in WT and SCID mice, respectively, by RNAseq on mT3-2D-GFP\+ fluorescence-activated cell sorter (FACS)-sorted cells. Also, whole exome sequencing was performed on WT tumors.

Results

Here we demonstrate that mT3-2D tumors engrafted in WT mice grow more slowly as compared with syngeneic SCID and T cell-depleted tumors, demonstrating that the reduced mT3-2D tumor growth rate in WT mice is T cell dependent. Putative cancer neoantigens were identified from whole exome sequences with pVACtools. Supporting evidence of tumor immunogenicity came from the observation that WT tumors are moderately infiltrated with T cells, by IHC and flow cytometry. Proteomics and NanoString nCounter analysis demonstrated the selective presence of myeloid suppressive cells in WT tumors, supporting their role in immune evasion. Notably, RNAseq showed selective upregulation of malignant epithelial cell-derived genes known to stimulate this immunosuppressive phenotype, including myeloid- and complement-related genes.

Conclusion

Despite the immunogenicity of the WT tumors and the infiltration of T cells in this PC model, T cell immunity incompletely controls tumor growth. Malignant epithelial cells mediate this cancer immunosuppressive phenotype in response to immune selection pressure, suggesting novel translational opportunities.

References:

Boj SF, Hwang C-I, Baker LA, Chio IIC, Engle DD, Corbo V, Jager M, Ponz-Sarvise M, Tiriac H, Spector MS, et al. 2015. Organoid models of human and mouse ductal pancreatic cancer. Cell 160:324–338.

#4585

IL-25 promotes development of HCC via M2 macrophages expression of CXCL10.

Hua Yunpeng, Li Qiao. _The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China_.

IL-25 promotes development of HCC via M2 macrophages expression of CXCL10 ABSTRACT Objective As an anti-inflammatory cytokine, interleukin-25 (IL-25) promotes type 2 immunity via alternative activation of macrophages, closely associated with inflammation-related diseases, such as fatty liver diseases, even cancers. However, it is not clear which role of IL-25 plays on HCC development. Design IL-25 expression in human HCC and healthy samples was detected by ELISA, western blot and immunohistochemistry. The cell growth, migration and invasion of HCC cell lines were evaluated after co-culturing with IL-25-stimulated macrophages by Brdu proliferation and Transwell assays. Chemokines were measured by reverse transcription PCR and western blot. Antibody neutralization assay of Chemokine CXCL10 was performed to confirm its role on HCC development. The orthotopic-transplanted liver tumor model was made in BALB/c nude mice with portal venous injection of macrophages. Results The expression level of IL-25 was significantly elevated in HCC patients and was negatively correlated with survival rate after hepatectomy. Moreover, there was significantly positive correlation between IL-25 levels and M2 percentage (CD206/CD68) in HCC tumors. Consistently, IL-25 induced the alternative activation of macrophages which promoted the HCC cells migration, invasion and tumorigenesis in vitro and in vivo. Chemokine CXCL10 was increased in the macrophages after IL-25 treatment, and the IL-25-mediated macrophages' effect on HCC cells was reversed by neutralizing CXCL10 in conditional medium (CM) of macrophages. Simultaneously, EMT-related mesenchymal markers (vimentin, Snail, phosph-ERK) were increased, while epithelial marker (E-cadherin) was decreased in HCC cells after co-culturing with IL-25-stimulated macrophages. Conclusion IL-25 promotes HCC development via the alternative activation of macrophages and CXCL10 secretion.

#4586

PLAG enhances macrophage mobility for efferocytosis of active neutrophils via membrane redistribution of P2Y2.

Guen Tae Kim,1 Do Young Lee,2 Ki-young Sohn,2 Sun Young Yoon,2 Jae Wha Kim1. 1 _Korea Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Republic of Korea;_ 2 _Enzychem Lifescience, Jecheon-si, Republic of Korea_.

Neutrophil activity is prerequisite during chemotherapy. The DAMP (Damage Associated Molecular Pattern) molecules generated by chemotherapy could be effectively trapped by activated neutrophil called 'NETosis'. Efferocytosis of macrophages should remove most activated neutrophils including NETosis A timely removal of activated neutrophils is essential for the prevention of abnormal activation of immune response and metastatic activity of cancer cells induced by tumor microenvironment (TME). Particularly, appropriate clearance of the activated neutrophils by efferocytosis should be carried out because activated neutrophils have a detrimental effect on TME. In this research, we investigated the effect of 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) on efferocytosis and its underlying molecular mechanisms. In a co-culture of activated neutrophils with macrophages, PLAG increased the activity of efferocytosis for elimination of activated neutrophils. PLAG accelerated translocation of P2Y2 from lipid rafts to non-lipid-raft plasma membrane domains in macrophages. This repositioning of P2Y2 enables the polarization of the cytoskeleton by association of the receptor with cytoskeletal proteins such as α-tubulin and actin to improve the mobility of macrophages. Through these protein assemble, PLAG encouraged macrophage mobility toward the activated neutrophils. Formation of micelle including PLAG, chylomicron-like structures, was a prerequisite for induction of this macrophage activity. PLAG effect on this activity was not observed in the absence of GPIHBP1, micelle receptor. Taken together, these data showed that PLAG triggered a prompt clearance of activated neutrophils through enhancement of efferocytosis activity. Subsequently, PLAG could have effects on modulation of TME. PLAG could be utilized as an effective lipid-based TME modulator via the prevention of abnormal activation induced by uncontrolled immune response during chemotherapy.

#4587

The N-terminal domain of HARS is a novel NRP2 ligand and can regulate NRP2-dependent macrophage function.

Navatha Shree Sharma,1 Samikshan Dutta,1 Steve Crampton,2 Sanna Rosengren,2 Kaustubh Datta1. 1 _Univ. of Nebraska Medical Center, Omaha, NE;_ 2 _aTyr Pharma, INC, San Diego, CA_.

Tumor-associated macrophages (TAM) are associated with regulation of antitumor immune responses, and neuropilin-2 (NRP2), a pleiotropic receptor with an emerging role in immune responses, has recently been demonstrated to be important for this activity (Roy et al, Cancer Res. 2018). Deletion of NRP2 results in impaired clearance of apoptotic tumor cells through reduced efferocytosis, which plays a role in tumor promotion. We now show that the N-terminal domain of the histidyl-tRNA synthetase (HARS) is a specific binding partner of NRP2, and can regulate the phagocytic function of NRP2 in macrophages. The HARS N-terminal domain, which is found only in higher eukaryotes, is conserved among splice variants of HARS and has evolved to regulate immune cell engagement. Incubation of macrophages with recombinant HARS N-terminal domain, had no effect on phagocytic uptake, but significantly impaired the maturation of phagosomes in a dose dependent manner. This phenotype mimics that of the NRP2 knockout, suggesting pharmacological intervention with this agent to modulate NRP2 driven biology may be possible.

#4588

Characterization of anti-CTLA-4 effects on tumor growth and immune microenvironment in a syngeneic CT26.WT colon carcinoma mouse model.

Jenni H. Mäki-Jouppila, Tiina E. Kähkönen, Mari I. Suominen, Jussi M. Halleen, Jenni Bernoulli. _Pharmatest Services, Turku, Finland_.

Colorectal cancer is the third most common type of cancer and one of the leading causes of cancer related deaths. It is a group of various diseases at molecular level, which can make targeted drug development challenging. However, novel therapeutics including immunotherapies have shown their potential to be utilized in the future also when treating colorectal cancer. One promising example is ipilimumab, a monoclonal antibody targeting CTLA-4 (cytotoxic T lymphocyte antigen-4, also known as CD152). CTLA-4 is a cell surface receptor expressed on activated T and B lymphocytes. It regulates immunological tolerance and downregulates immune responses. The aim of the study was to characterize the effects of anti-CTLA-4 on tumor growth, survival and tumor immune microenvironment of mice subcutaneously inoculated with mouse colon carcinoma CT26.WT cells.

CT26.WT cells were subcutaneously inoculated into female Balb/c mice (n=27). Body weights and tumor dimensions were determined two times a week during the course of the study. The mice were randomized to three groups (n=9/group) with similar tumor volume and variation and dosing was started when an average tumor size of approximately 50 mm³ was reached (day 7). Anti-CTLA-4 antibody (9D9, 10 mg/kg), an isotype control (mouse IgG2b) and vehicle (PBS) were administered (i.p.) twice a week. Tumor growth and survival were followed for 26 days after inoculation of the cancer cells. The mice were sacrificed individually when the maximum tumor volume (1500 mm³) was reached, the tumors were ulcerated or at the latest on study day 26. Tumor samples were collected for histological and immunohistochemical analysis and stained for helper T cells (CD4), cytotoxic T cells (CD8), myeloid-derived suppressor cells (MDSC; CD11b), tumor-associated macrophages (F4/80), and inflammatory monocytes and neutrophils (FCGR1/CD64) for characterization of tumor immune microenvironment.

Maximum tumor volume and tumor ulceration were observed the earliest on study day 17. Anti-CTLA-4 suppressed tumor growth, and all mice in the group receiving anti-CTLA-4 were still in the study on day 26. Only one and four mice were left in the vehicle and isotype control groups, respectively. Immunohistochemical staining suggested an increase of CD4+ tumor-infiltrating lymphocytes (TILs) in anti-CTLA-4 treated mice. Furthermore, more TAMs were observed in tumors of anti-CTLA-4 treated mice, especially in the borderlines of necrotic areas. Large quantity of CD11b+ MDSCs and inflammatory cells were observed in CT26.WT tumors.

CT26.WT subcutaneous model can be utilized for studying the effects of immunotherapies, and anti-CTLA-4 treatment suppressed tumor growth in this model. CT26.WT tumors represent a highly immunogenic hot tumor type with high immune cell infiltration, associated with a good efficacy of anti-CTLA-4 treatment in the model.

### Invasion and Migration 2

#4589

SRC drives invasion of luminal, but not basal, bladder cancer.

Bryan Wehrenberg,1 Andrea Ochoa,1 Woonyoung Choi,2 David McConkey2. 1 _MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, Houston, TX;_ 2 _Johns Hopkins Greenberg Bladder Cancer Institute, Baltimore, MD_.

Preventing progression from non-muscle invasive to muscle invasive bladder cancer (BC) is a major clinical priority. This transition is defined by tumor invasion through the bladder epithelium basement membrane into the lamina propria and muscle wall. Inhibition of SRC, a pro-invasion, non-receptor tyrosine kinase that signals as part of a complex with Focal Adhesion Kinase (FAK), may prevent BC progression. We hypothesize that SRC inhibition will disrupt migration and invasion of BC cells. Using The Cancer Genome Atlas (TCGA) mRNA expression data of human bladder tumors, we generated a classifier to subtype tumors as either "luminal" or "basal" and compared SRC expression between the groups. We then sequenced a panel of 30 bladder cancer cell lines. Each cell line was given a subtype call based on our luminal- basal classifier, and levels of SRC mRNA expression were examined. To determine the impact of SRC inhibition on luminal and basal subtypes, we exposed BC cell lines to the SRC-Family Kinase inhibitor, AZD0530. The impact of SRC inhibition on proliferation was measured by MTT assay. Transwell assays were used to examine the effect of SRC inhibition on migration, collagen invasion, and Matrigel invasion. Phosphorylation of SRC downstream targets after AZD0530 treatment was assessed by western blot. The role of SRC target and signaling partner FAK in migration was determined by siRNA knockdown and transwell assay. Luminal human bladder tumors had significantly greater SRC mRNA expression than basal tumors (p<0.001). SRC expression was also enriched in luminal BC cell lines (p<0.05). Inhibiting SRC with AZD0530 had minimal impact on proliferation with no subtype specific effects. Migration and collagen invasion of all luminal cell lines was significantly reduced by AZD0530 treatment (p<0.05). The majority of luminal models capable of invading Matrigel were also inhibited by AZD0530. In contrast, invasion and migration of the majority of basal cell lines was not significantly reduced by SRC inhibition, and was significantly increased in several cases (p<0.05). Both luminal and basal models exhibited reduced phosphorylation of SRC downstream targets when exposed with AZD0530. siRNA knockdown of FAK had no impact on migration in a basal cell line. Enrichment of SRC expression in luminal tumors and significant reductions in luminal cell line invasion after AZD0530 treatment suggest SRC promotes invasion in luminal BC. These results suggest AZD0530, and other SRC-Family Kinase inhibitors, could serve as effective anti-progression agents in luminal BC.

#4590

Dissecting the biology of leader and follower cells in collective cancer invasion.

Brian A. Pedro,1 Jessica Konen,2 Emily Summerbell,1 Janna K. Mouw,1 Manali Rupji,1 Bhakti Dwivedi,1 Jeanne Kowalski,1 Paula M. Vertino,1 Adam I. Marcus1. 1 _Emory University, Atlanta, GA;_ 2 _The University of Texas MD Anderson Cancer Center, Atlanta, GA_.

Previous work has demonstrated heterogeneity within collectively invading packs of lung cancer cells, including leader and follower cells that cooperate to facilitate invasion into the microenvironment. In order to better characterize the genetic differences between these two distinct cell types, we performed RNA-seq on purified populations of leader and follower cells from the H1299 non-small cell lung cancer cell line. Through this analysis, we identified that leaders and followers each contain distinct mutational and gene expression profiles, despite originating from the same parental cell line. Importantly, we identified 17 point mutations found uniquely in leaders and 18 point mutations found uniquely in followers, thus representing the first known compilation of leader- and follower-enriched genetic variants. Notable leader-enriched mutated genes included NAE1, NUP93 and ACTR3, while notable follower-enriched mutated genes included NADK, NDUFS1 and LEO1, suggesting possible roles for these genes in the biological function of leader and follower cells. After validating these mutations in the genomic DNA of leaders and followers, we sought to determine whether these mutational signatures are correlated with leader and follower gene expression markers within individual cells in a heterogeneous tumor cell population. To this end, we performed single-cell RNA-seq on H1299 parental, leader and follower cells isolated directly from collectively invading packs in a 3-D matrix. Hierarchical clustering and tSNE analysis based upon most variably expressed genes revealed four distinct cell clusters; two expressing higher levels of leader cell genes including MYO10 and JAG1 (clusters 1 and 4), and two expressing higher levels of follower genes including IL13RA2 and HTATIP2 (clusters 2 and 3). Interestingly, clusters 1 and 4 are composed exclusively of cells with leader mutational profiles, while nearly all cells in clusters 2 and 3 contain follower mutational profiles, suggesting that these mutations could be driving the differential gene expression, and ultimately the unique biological behaviors, of leaders and followers. In addition, clusters 1 and 2 display higher levels of proliferative markers compared with clusters 3 and 4, an indication that there are proliferative and non-proliferative subpopulations within the leader and follower populations. Taken together, these data give us new insight into the multiple levels of heterogeneity that exist within an invasive tumor cell population, suggest novel drivers of leader and follower cell biology in collective invasion, and open the door to new potential strategies for targeting and inhibiting metastasis in human lung cancer.

#4591

**Overexpression of p62/IMP2 can promote cell migration in hepatocellular carcinoma (HCC) via activating Wnt/** β **-** catenin **pathway.**

Mengtao Xing, Jianxiang Shi, Pei Li, Jiejie Qin, Xiaojun Zhang, Yangcheng Ma, Xiao Wang, Giulio Francia, Jianying Zhang. _UT El Paso, El Paso, TX_.

There are three members in the family of insulin-like growth factor 2 mRNA binding proteins (IMP): IMP1, IMP2 and IMP3. The IMPs are oncofetal proteins, expressed during embryogenesis and lost in most tissues in adults. However, IMPs were found overexpressed in various cancers. IMPs share similar protein structure in mammals and have similar functions: to bind specific mRNAs, regulate their expression, transportation, and degradation. Although p62/IMP2 was first reported as a tumor-associated antigen in HCC around 20 years ago, we still know little about the role of p62/IMP2 in HCC progression. Our previous studies found that p62/IMP2 was not only overexpressed in HCC tissues, but also overexpressed in HCC cell lines. To explore the biological roles of p62/IMP2 in HCC progression, p62/IMP2 was knockout in two p62/IMP2 positive HCC cell lines (SNU449, HepG2). Due to the low expression level of p62/IMP2 in SNU449, we performed the transfection experiment to overexpress p62/IMP2 in this cell line. The Wound healing assay and transwell migration assay indicated that overexpressed p62/IMP2 in both cell lines showed the ability that could promote the cell migration significantly (p<0.05). On the contrary, the lack of p62/IMP2 expression can reduce the cell migration ability (P<0.05). After analyzing the HCC expression data from Gene Expression Omnibus (GEO), the high and low IMP2 expression groups were set up based on the median expression of p62/IMP2 from GSE 14520. Three genes were selected in differential gene expression analysis with a cancer-metastasis related gene profile, including CTNNB1, ACTR2, VASP (LogFC > 0.3, Adj. p<0.001). Then we performed a western blotting analysis to explore the effect of overexpressed p62/IMP2 on Wnt/β-catenin pathway-related proteins. Overexpressed p62/IMP2 significantly enhance the expression of Wnt and β-catenin, whereas inhibiting the expression of Gsk3b and E-cadherin. Moreover, the migration ability of SNU449 and HepG2 cells were significantly reduced (p<0.05) after cultured with 10mM Wnt/β-catenin signaling pathway inhibitor XAV939 for 24h. In summary, our data showed that overexpressed p62/IMP2 can enhance the migration ability of HCC cells via activating Wnt/β-catenin signaling pathway.

#4592

CD44v6 promotes aggressive phenotypes mediated through activating EGFR pathway via COL11A1/PTPRG axis.

Joanne Jeou-Yuan Chen, Jing-You Guo. _Academia Sinica - Inst. of Biomedical Sci., Taipei, Taiwan_.

Cell surface receptor CD44, an important player in stem cells and cancer biology, is expressed in multiple isoforms via alternative splicing. CD44 isoforms containing variant exon 6 (CD44v6)-encoded sequences are frequently overexpressed in human tumors. In many cancers, CD44v6 serves as a marker of cancer stem cells and accounts for the metastatic susceptibility of the tumors. CD44v6 predicts for poor prognosis in several cancers. However, the actions and underlying mechanisms related to CD44v6-mediated malignant phenotypes remain to be established. In this study, we performed cDNA microarray and Gene Set Enrichment Analysis (GSEA) of the genome-wide expression profiles in cells ectopically expressing the standard form CD44 (CD44s) or CD44v6, and showed that pathways related to cell migration, invasion and metastasis are enriched in CD44v6-expressing cells which are in contrast to cell growth regulatory pathways identified in CD44s cells. In support, overexpression of CD44v6 promoted cell migration and invasion. In silico analyses of genes preferentially regulated in CD44v6-expressing cells against a multi-cancer metastasis-associated gene expression signature dataset and the gene set indicative of embryonic stem cell identity, Collagen XIA1 (COL11A1) was identified as the top candidate in CD44v6-upregulated genes. COL11A1, encoding the α1 chain of the minor fibrillar collagen XI, has been found to be highly expressed in a variety of cancers, and is implicated in the processes of metastasis, angiogenesis, and drug resistance. COL11A1 expression is correlated with cancer aggressiveness and progression, and lymph node metastasis. Using gain- and loss-of-function approaches, we further demonstrated that CD44v6 drives tumor cell invasion in part mediated through the induction of metastasis-related gene COL11A1. Upon examining the involvement of receptor tyrosine kinase pathways, we showed that EGFR is constitutively activated in CD44v6-expressing cells, and that COL11A1 supports CD44v6-mediated EGFR activation via suppressing the expression of PTPRG, an EGFR negative regulator. Our data thus suggest that CD44v6 promotes aggressive phenotypes mediated through activating EGFR pathway via COL11A1/PTPRG axis.

#4593

Inhibition of invasion of HER2-positive breast cancer cells by lysosome targeting drugs.

Malene B. Hansen,1 Maria Postol,1 Davig A. Egan,2 Marja Jäättelä,1 Tuula Kallunki1. 1 _Danish Cancer Society Research Center, Copenhagen, Denmark;_ 2 _Core Life Analytics BV, Netherlands_.

Up to 20% of all breast cancers are over-expressing HER2/ErbB2, leading to malignant and invasive cancers with a poor patient outcome. Though good ErbB2-targeting treatments have been developed, metastasis formation and drug resistance are still major challenges for the long-term cure. This project aims to find out if some of the numerous pre-existing compounds and clinically approved drugs can be repurposed to treat or improve the treatment of HER2/ErbB2-positive breast cancer. We have therefore set up a high-throughput and high-content screen of the Prestwick Chemical Library to identify drugs that can reverse the ErbB2-induced, invasion-promoting, peripheral distribution of lysosomes. Using a breast cancer cell model expressing N-terminally truncated version of ErbB2, we mimic breast cancer that is resistant to the currently used antibodies, trastuzumab and pertuzumab. This cell line shows a highly invasive phenotype with increased activation of the lysosomal proteases, cysteine cathepsin B and L, as well as anterograde movement of lysosomes. This repositioning of the lysosomes enables them to fuse with the plasma membrane and empty their acidic and hydrolyzing content to the extracellular matrix, promoting invasion. Treatment with the clinically used drug lapatinib will reverse this phenotype. The cells ability to reverse the phenotype, through increasing the number of perinuclear lysosomes while removing lysosomes form cellular protrusions, was evaluated for each screened drug, using the ImageXpress Micro Confocal High-Content Imaging System. We have hereby identified eight drugs that can redirect lysosomes from the invadosome-like cellular protrusions to their non-invasive perinuclear position in ErbB2 expressing breast cancer cells. We are currently analyzing the impact of these drugs on breast cancer cell invasion though migration assays and three-dimensional invasion assays. We are also addressing their effect on lysosomal membrane permeabilization, lysosomal pH and investigating their effect on proteins that are known to be involved in lysosomal distribution.

Clinically, our findings may facilitate repurposing or new development of drugs to treat HER2/ErbB2-positive and invasive breast cancers that are resistant to current treatments.

#4594

Cytoplasmic LOXL2 regulates actin cytoskeletal organization in esophageal squamous cell carcinoma progression.

Xiuhui Zhan,1 Jiwei Jiao,1 Haifeng Zhang,2 Jianzhong He,1 Runliu Li,1 Haiying Zou,1 Zhiyong Wu,3 Shaohong Wang,3 Xiue Xu,1 Jianyi Wu,1 Liandi Liao,1 Yinwei Cheng,1 Kai Zhang,4 Gera Neufeld,5 Liyan Xu,1 Enmin Li1. 1 _Shantou University Medical College, China;_ 2 _University of British Columbia, British Columbia, Canada;_ 3 _Affiliated Shantou Hospital of Sun Yat-sen University, China;_ 4 _Tianjin Medical University, China;_ 5 _Technion, Israel Institute of Technology, Israel_.

Background: Lysyl oxidase-like 2 (LOXL2), a copper-dependent enzyme of the lysyl oxidase family, promotes tumor progression and metastasis. We previously revealed that the tumor-promoting role of LOXL2 is mediated by perturbing the architecture of the actin cytoskeleton in esophageal squamous cell carcinoma (ESCC). However, the molecular mechanisms underlying LOXL2-driven ESCC progression remain elusive, and the cytoskeletal proteins that directly interact with LOXL2 remain uncharacterized.

Methods: Knockdown LOXL2 in ESCC cells was used by a lentivirus shRNA targeting LOXL2 and analyzed by RNA-sequencing and bioinformatics analysis. Proteomic analysis following by co-immunoprecipitation and immunofluorescence were applied for identification and validation of interacting partners of LOXL2 and its splicing isoform L2Δ13. Cell migration and invasion were measured by wound healing and transwell assays. By risk score calculations, immunohistochemistry of tissue microarrays were adopted to evaluate the prognosis of ESCC patients.

Results: We found that knockdown of LOXL2 in ESCC cells suppresses cell migration and invasion, whereas re-expressions of LOXL2 and its non-enzymatic splicing isoform L2Δ13 rescue these cell behaviors. Silencing of LOXL2 inhibits filopodia formation, and induces changes in the expression of cytoskeleton-associated genes. Our interactome analysis identified four actin-binding proteins, i.e. ezrin (EZR), facsin (FSCN1), heat shock protein beta-1 (HSPB1) and tropomodulin-3 (TMOD3), as novel interactors of LOXL2 and L2Δ13. These novel LOXL2/L2Δ13 interaction networks carry important prognostic values in ESCC, as molecular signatures of combinations of LOXL2/L2Δ13 and their cytoskeletal interactors are associated with clinical outcome in ESCC patients. Mechanistically, the ezrin-LOXL2 interaction promotes PKCα-stimulated phosphorylation of ezrin at Thr 567 (T567), an essential residue for ezrin activation, which enhances cell motility and cell invasion in ESCC. Correlating with this, double-high expression of LOXL2 and phosphorylated ezrin-T567 predicts significantly shorter overall survival in ESCC patients.

Conclusion: In conclusion, our findings have uncovered a novel molecular mechanism underlying the tumor-promoting role of cytoplasmic LOXL2, which may open new avenues for the therapeutic targeting of LOXL2 in ESCC. (This study is supported by the Natural Science Foundation of China (No. 81472613) and Li Ka Shing Foundation).

#4595

Anti-metastasis traditional Chinese medicine monomer screening system based on PNC analysis in hepatocellular carcinoma cells.

Feifei Liu, Yanning Liu, Guohua Lou, Zhi Chen. _State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Hangzhou, China_.

Background and aims Hepatocellular Carcinoma (HCC) remains lack of effective drugs, especially anti-metastatic drugs. Traditional Chinese Medicines (TCM) monomers have shown anti-hepatoma activity, but few of them are specific for anti-metastasis. Perinucleolar compartment (PNC), which exists in the nuclear of tumor cells, is positively correlated with metastasis of various tumors. In this study, we aimed to establish a screening system for anti-metastasis TCM monomers and then verify its effectiveness.

Methods The PNC prevalence of HCC cell lines (HepG2, Hep3B and Huh7) was detected by Immunofluorescence assay, using PTB monoclonal antibody. The migration and invasion abilities of HCC cell lines were determined by wound-healing and transwell assay, respectively. After exposing HCC cells to the selected TCM monomers for 24 h, the expression levels of epithelial-mesenchymal transition (EMT) related molecules in these HCC cells were analyzed by qRT-PCR and Western Blot assay.

Results We verified that PNC prevalence was positively correlated with the metastasis ability of HCC cells and also found Huh7 cell line has the highest PNC prevalence, followed by Hep3B and HepG2 cell lines. Thus, we choose Huh7 cell line for further screening of anti-metastasis TCM monomers. After two rounds of screening, we found that Camptothecin, Evodiamine and Isoglycyrrhizin were the three most effective compounds to reduce the PNC prevalence in Huh7 cells. The metastasis inhibition effect of these compounds was correlated with their ability of PNC prevalence reduction. Further experiment suggested that Camptothecin could up-regulate the expression of E-cadherin, β-catenin and Claudin-1, and down-regulate the expression of ZEB-1, N-cadherin, Vimentin, Slug and Snail, in a dose-dependent manner, thereby promoting adhesion and reducing metastasis of HCC cells.

Conclusions The screening system based on PNC prevalence can effectively screen out anti-metastatic TCM monomers, and it may provide effective technical assistance for the development of anti-metastasis drugs.

#4596

Synthesis and biological evaluation of small molecules inhibitors targeting heterotrimeric Gαi2 protein.

Silvia Caggia,1 Autumn S. Garrett,1 Aditi Kumar,1 Subhasish Tapadar,2 Adegboyega K. Oyelere,2 Smrruthi Vaidegi Venugopal,1 Shafiq A. Khan1. 1 _Clark Atlanta Univ., Atlanta, GA;_ 2 _Georgia Tech School of Chemistry and Biochemistry, Atlanta, GA_.

Heterotrimeric G-proteins are ubiquitously expressed in cells, and they transduce signals from activated G-protein coupled receptors. It is well known that these proteins are differentially expressed in cancer, thereby activating signals that lead to different biological functions such as proliferation and cell motility. Previously, we have shown that Gαi2 protein is essential for cell migration and invasion in prostate cancer cell lines, and its action is downstream of PI3-kinase/Rac1 activation. We have also shown that in PC3 prostate cancer cells, pre-treated with Pertussis toxin (PTX), a well know inhibitor of Gαi/0, resulted in the attenuation of TGFβ1- and oxytocin-induced migration and PI3-kinase activation, without affecting EGF-induced PI3-kinase activation and cell migration. Here, we synthesized new small molecules inhibitors, GDIs, capable of preventing nucleotide exchange and subunit activation. Compounds #29 and #46 are analogs of the lead compound, lacking the thiophene OH-group and with a thiol- to N-methyl amino-group substitution, respectively. Compound #35, a methyl ether derivative of #46, is designed to test the effect of the modification to the phenolic group on Gαi2 inhibition activity. We pretreated PC3 cells with the small molecules and then stimulated with EGF and oxytocin to activate Gαi2 subunit. Immunoprecipitation of the active Gαi and western blot for Gαi2 showed a decrease in the amount of active Gαi2 that was pulled down compared with the controls. Using cell migration assay, we observed that #29 and #46, both at 10 μM, caused inhibition of migration in different prostate cancer cell lines in response to EGF treatments. In contrast, #35 is inactive in this assay at the same concentration. To determine if the effects of Gαi2 are the same in other cancer cells, we first knockdown Gαi2 protein expression in breast and ovarian cancer cells and performed migration assays. The knock-down of endogenous Gαi2 attenuated cell migration, compared to the control cells. We also performed migration assays in the same cells using compound #29 at 10 μM and observed significant inhibition of migratory behavior in response to EGF- and FBS stimuli compared to the controls. We conclude that the small molecules we used in this study may be considered as potential therapeutic tools, inhibiting the migratory capability of metastatic cells.

#4597

Bladder cancer patients experience circulating tumor cell number surge during intramedullary nailing procedures intended for treating pathological fractures.

Zhongyuan Zhang,1 Liang Dong,1 Stephanie Glavaris,1 Emily Caruso,1 Kenneth Pienta,1 Carol Morris2. 1 _Johns Hopkins University School of Medicine, Baltimore, MD;_ 2 _The Johns Hopkins Hospital, Baltimore, MD_.

Introduction and Objective: The goal of this study is to evaluate if intramedullary nailing procedure of completed or impending pathologic fractures in long bones secondary to metastatic bladder cancer affects the circulating tumor cell (CTC) number.

Methods: Bladder cancer patients who presented with completed or impending pathologic fracture due to metastatic disease and underwent fracture fixation were recruited in this study. During nailing procedures performed by orthopedic oncologists, blood samples were collected from peripheral vein, peripheral artery and central vein at four time points (TP). TP1 was at the time of incision; TP2 was during the passage of the first reamer; TP3 was during wound closure; and TP4 was 24 hours post-operatively. Two CTC capture technologies were used: The FAST disk is a size-selective, clog-free rare cancer cell isolation platform (Clinomics, Ulsan, South Korea). CTCs were captured by a filter and stained with immunofluorescent antibodies. The filter was then scanned using the Metafer5 (MetaSystems, V3.11.8) automated scanning system to enumerate CTCs. The second platform was the AccuCyte-CyteFinder system (RareCyte, Inc., Seattle, WA), a selection-free method to enumerate and characterize CTCs from peripheral blood samples (PB) via immunofluorescent staining and scanning. The criteria for defining a CTC is DAPI positive, CK/EpCAM positive, and CD45 negative.

Results: Three nailing cases from two patients were enrolled in this study (one patient had 2 affected limbs). The surgical sites were the right humerus (2 cases) and the right femur (3rd case). The AccuCyte-CyteFinder system was used for all three cases. FAST was used for the second and the third case. With the AccuCyte-CyteFinder system (TP1/TP2/TP3/TP4), the CTC counts were: 8/3314/7/2 for the first case; 259/3616/322/548 for the second case; 755/2507/917 CTCs for the third case (no TP4 sample). With FAST disk (TP1/TP2/TP3/TP4), the CTC counts were: 5/964/28/9 CTCs for the second case; 27/523/27/13 for the third case. Many CTC clusters were seen for TP2 venous samples, but rarely seen for the other samples.

Conclusions: A surge in CTC number during the nailing procedure was observed in all cases by both methods. Our data suggest that the palliative nailing procedure may contribute to further CTC dissemination. Whether the surge of CTCs results in clinical relevant disease warrants further investigation.

#4598

mTORC2 suppresses GSK3-dependent, but b-TrCP-independent, Snail degradation, contributing to positive regulation of cancer cell invasion and metastasis.

Shuo Zhang,1 Guoqing Qian,1 Qian-Qian Zhang,2 Yuying Yao,2 DongSheng Wang,1 Zhuo G. Chen,1 Lijing Wang,2 Mingwei Chen,3 Shi-Yong Sun1. 1 _Emory University, Atlanta, GA;_ 2 _Guangdong Pharmaceutical University, Guangzhou, China;_ 3 _First Affiliated Hospital of Medical College of Xi'an Jiaotong University, Xi'an, China_.

Mammalian target of rapamycin (mTOR) complex 1 (mTORC1) positively regulates cell invasion and metastasis via enhancing Snail protein translation. The connection between mTOR complex 2 (mTORC2) and cell invasion and metastasis has also been suggested. However the underlying biology or mechanism is largely unknown and thus is the focus of this study. Inhibition of mTOR with both mTOR inhibitors and knockdown of the key components including rictor, Sin1 and raptor of mTORCs decreased Snail protein levels. mTOR inhibitors promoted Snail degradation rate. Moreover, mTOR inhibitor-induced Snail reduction was rescued by proteasome inhibition. Critically, inhibition of mTORC2 (by knocking down rictor), but not mTORC1 (by knocking down raptor), enhanced Snail degradation. Therefore, it is the inhibition of the mTORC2 that induces Snail proteasomal degradation, resulting in eventual Snail reduction. Interestingly, inhibition of GSK3, but not SCF/β-TrCP, rescued Snail reduction induced by mTOR inhibitors, suggesting a GSK3-dependent, but SCF/β-TrCP-independent proteasomal degradation of Snail. Accordingly, mTOR inhibitors elevated E-Cad levels and suppressed cancer cell migration and invasion in vitro and metastasis in vivo. Collectively, the current study has revealed a novel connection between mTORC2 and positive regulation of Snail stability, thus providing a scientific base for mTORC2 in positive regulation of cell EMT, invasion and metastasis. (This study was supported by NIH/NCI R01 CA118450 and CA160522 to SYS and National Natural Science Foundation of China No. 31771578 to QQZ).

#4599

PD-1/PD-L1 axis in cancer cells contributes to cellular EMT in lung adenocarcinoma.

Wen-Pin Su, Li-Chan Chang, Jing-Jou Yan, Wu-Chou Su. _National Cheng Kung University, Tainan, Taiwan_.

Background: Since intrinsic PD-1 receptor functions promote tumor growth was reported, we will investigate the role of PD-L1 in lung adenocarcinoma cells, and their impact on clinical outcome.

Materials and methods: Lung adenocarcinoma CL1-5 cells, derived from CL1-0 cells have more invasive ability. We prepared PDL1-overexpression human lung adenocarcinoma cell line, derived from CL1-0 cells (CL1-0-PD1) and from PC14PE6 cells (PC14PE6-PD-L1). We observed the cellular morphology, and invasiveness; epithelial-mesenchymal transition (EMT) markers and regulators were also evaluated. To explore interaction between PD-1 and PD-L1, we treated the CL1-0, CL1-5, CL1-0-PDL1 cells, PC14PE6 cells, and PC14PE6-PD-L1 cells with anti-PD-1 antibody, and then evaluated cellular invasion. We also suppressed PD-1 to test whether PD-1/PDL-1 interaction contributed to the EMT change. Further, we evaluated cellular proliferation and chemosensitivity by MTT assay and colony formation assay. Finally, we correlated PD-L1 expression with clinical outcome in patients' tumor specimen.

Results: CL1-5 cells possessed higher PD-L1 expression than parental CL1-0 cells. CL1-0 cells with PD-L1 overexpression had more expression of EMT regulators and mesenchymal markers. Overexpression of PD-L1 in another lung adenocarcinoma PC14PE cells (PC14PE6-PD-L1 cells) become more aggressive. On the contrary, down-regulation of PD-L1 in CL1-5 cells and PC14-PE6-PD-L1 cells became indolent. Besides through the observation from confocal microscopy, PD-L1 expression was more co-localized with vimentin and slug, individually in CL1-5, CL1-0-PD-L1 and PC14PE6-PD-L1 cells. Therefore, PD-L1 promoted EMT in human lung adenocarcinoma cells. After adding anti-PD-1 antibody in CL1-5, CL1-0, and CL1-0-PDL1 cells, migration and invasion ability were decreased. Similar phenomenon was found when PD-1 antibody treatment in PC14PE6 cells, and PC14PE6-PD-L1 cells. Silencing PD-1 by siRNA also disrupt the cellular aggressivenss in CL1-0-PD-L1 and PC14PE6-PD-L1 cells. These results indicated PD-1/PD-L1 axis regulated cancer cells migration and invasiveness. PD-L1 expression also decreased cellular proliferation and had little influence on chemsensitivity. Finally, we found that higher PD-L1 expression was correlated with lymph node metastasis in clinical specimen.

Conclusion: Lung adenocarcinoma cells with higher PD-L1 expression promote EMT via PD-1 receptor. PD-L1 expression lowers proliferation rate, but has little impact on chemosensitivity. Lung cancer patients with high PD-L1 expression in tumor have higher lymph node metastasis.

#4600

IL-22 is specifically required for malignancy in breast cancer: A potential target to control cancer metastasis.

Gajendra K. Katara, Arpita Kulshrestha, Sylvia Schneiderman, Safaa Ibrahim, Mahmood Bilal, Valerie E. Riehl, Kenneth D. Beaman. _Rosalind Franklin Univ. of Med. Science, North Chicago, IL_.

IL-22 is important for proliferation and progression of various epithelial cancers. However, in breast cancer, the current knowledge of IL-22 function is based on cell line models and lacks information on stage-specific involvement of IL-22 in multistage pathogenesis of disease. Here, we investigated whether IL-22 expression in the tumor microenvironment (TME) is a stage-specific requirement and if its inhibition can affect that specific stage of cancer progression. We show that the absence of IL-22 in TME did not hinder the initiation and proliferation of cancer but inhibited the invasion and metastasis of cancer. The influence of IL-22 on hyperplasia, adenoma, early carcinoma and late carcinoma stages of cancer was investigated using IL-22-/-/MMTV-PyMT spontaneous breast cancer mouse model. IL-22 levels were evaluated at these stages of cancer progression and validated in stage-specific specimens of human breast cancer. A variety of histopathological, in vivo and in vitro proliferation, migration, invasion and gene expression assays were performed on tumor tissues and purified epithelial cells. Results showed that IL-22 was absent in TME during initiation and hyperplasia stages of breast cancer. It was expressed during early carcinoma and significantly increased as tumor progressed to malignant stage. In human samples, IL-22 was poorly expressed in high and low grade ductal carcinoma in situ. However, it was significantly higher in invasive or metastatic carcinoma compared to ductal carcinoma in situ. Histological examinations revealed an inhibition in the malignant transformation of epithelial cells during invasion stage in IL-22-/- tumors. No difference was observed in initiation and hyperplasia stages of cancer between IL-22-/- and control mice. Ex vivo treatment of IL-22 increased the migration and invasion, but not the proliferation, of purified epithelial cancer cells. RNAseq analysis of premalignant stage epithelial cells revealed down regulation in genes associated with EMT (Epithelial to Mesenchymal Transition) pathway in IL-22-/- mice. These results show that, in breast cancer, IL-22 is not essential for cell proliferation but it is necessary for malignant transformation of cancer cells which is the critical stage for metastasis. Inhibition of IL-22 can hinder cancer cell malignancy and therefore, it can be an effective therapeutic target to control breast cancer metastasis.

#4601

Phospholipid and enzyme antibodies for the evaluation of novel cancer biomarkers.

Cameron Day, Paul Neilsen. _Echelon Biosciences, Salt Lake City, UT_.

Sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) are well characterized bioactive phospholipids with migratory and proliferative effects in cancer progression. Recent studies show that the enzyme responsible for production of S1P, sphingosine kinase (SK), is upregulated in a variety of cancers, S1P is a tumorigenic growth factor, and there is an association between S1P levels in cancer patients and their resulting clinical outcomes. Similarly, LPA has been shown to drive angiogenesis and tumor invasion. LPA receptors and the LPA synthesizing enzyme autotaxin (ATX) have also been shown to be abnormally expressed in at least ovarian and breast tumors. S1P, SK, LPA, and ATX therefore represent potential biomarkers for the detection and staging of cancer. We have now characterized anti-S1P and anti-LPA antibodies for basic immunochemical methods in cells and ovary tissue. These antibodies were previously developed as anti-cancer immunotherapeutic agents. Here we highlight the potential for these reagents as bonafide cancer biomarkers for S1P and LPA.

#4602

Abi1 promotes breast cancer progression by regulating cell-matrix adhesion.

Xiang Li, Angelina Regua, Gennady Bratslavsky, Vladimir A. Kuznetsov, Leszek Kotula. _SUNY Upstate Medical Univ., Syracuse, NY_.

Breast cancer is one of the most common diagnosed cancers in the United States. There will be approximately 266,120 new breast cancer cases in the U.S. and over 40,000 patients are predicted to die from the malignant progression of their breast cancer tumors in 2018. Traditional therapeutic methods of the invasive breast cancer include surgery accompanied by chemotherapy. However, following the initial therapy patients often relapse and eventually die from tumor metastasis. Abl interactor 1, or Abi1, is an adaptor protein which regulates actin polymerization through participation in WAVE (Wiscott-Aldrich syndrome protein family verprolin homologous) complex. The WAVE regulatory complex (also called WRC), regulates actin based cellular behavior, such as cellular adhesion and cell migration, by activating Arp2/3 complex and promoting actin polymerization. Previous studies indicated the direct interaction between integrin α4 and Abi1, which suggests a potential role of Abi1 in regulating cell-matrix adhesion signaling by involved in the inside-out integrin signaling pathways. In order to test the above hypothesis, we developed two genetic engineered Abi1 knocked-out breast cell lines from either HER2+ breast cancer cell line SKBR3 or normal breast epithelial cell line MCF10A by using CRISPR-Cas9 technique. After confirming inactivation of Abi1 gene by Western blotting, DNA and RNA sequencing, cells were harvested and analyzed for integrins levels. Western blotting results indicated dysregulation of multiple integrins in the Abi1 knock-out cell lines. The results from subsequent immunofluorescence experiments of the Abi1 knock-out cell lines also suggested changes in cell-matrix adhesion signaling using staining for scaffold protein paxillin and phosphorylated Focal-Adhesion-Kinase (FAK) as readouts. Our findings suggest that Abi1 might functions as a key regulator in breast cancer tumor progression by playing an important role in regulating signal pathways in cell-matrix adhesion. Supported by the Carol Baldwin Foundation of CNY, and in part by NIH-NCI grant No. R01 CA161018.

#4603

To analyse PIMT function in glioblastoma cell invasion.

Fatima Belkourchia, Richard Desrosiers *. _UQÀM, Montréal, Quebec, Canada_.

The protein L-isoaspartyl (D-aspartyl) methyltransferase (PIMT) is an enzyme able to repair abnormal L-aspartyl residues in damaged proteins. Among examined tissues, PIMT shows the highest level in brain. U87-MG cells are a commonly used grade IV cellular model to study the most frequent brain tumor, the glioblastoma. Previously, we reported that PIMT amount increased when U87-MG cells were detached from the extracellular matrix. Recently, our laboratory also showed that PIMT possessed pro-angiogenic properties. Together, these PIMT features led us to postulate that PIMT could play a critical role in glioblastoma growth. To investigate PIMT involvement during glioblastoma growth, we analyzed its role in U87-MG cell viability, adhesion, migration, invasion, clonogenic properties and the reorganization of the actin cytoskeleton. For instance, PIMT inhibition by siRNA reduced cell migration and invasion by different assays: in wound healing assay, in Boyden chambers coated with gelatin and in Matrigel invasion assay. Conversely, in stably transfected U87-MG cells overexpressing wild-type PIMT, cell migration, invasive capacity and colony formation significantly increased. However, in stably transfected cells with the gene encoding for the inactive PIMT D83V, in spite of its overexpression, the migration and invasion remained similar to that observed in control cells transfected with the empty plasmid. In all these conditions, cell viability was unaffected. Finally, the reorganization of the actin cytoskeleton is regulated by PIMT protein level. Overall, these data enlighten the importance of PIMT level and mainly of its catalytic activity in migration and invasion of malignant glioma U87-MG cells and its possible contribution in cancer cell invasion during glioblastoma growth.

#4604

Kidney injury molecule-1 regulates metastasis in renal cell carcinoma.

Jasper C. Lee,1 Marie A. Sarabusky,1 Audrey Champagne,2 Fabrice Lucien,3 Lakshman Gunaratnam1. 1 _Western University, London, Ontario, Canada;_ 2 _CHU de Québec-Université Laval, Quebec City, Quebec, Canada;_ 3 _Mayo Clinic, Rochester, MN_.

Over 30% of patients with renal cell carcinoma (RCC) present with metastases, with median survival of 2 years. Kidney injury molecule 1 (KIM-1) is a cell-surface glycoprotein expressed by >85% of RCC tumours and may serve as an early biomarker for RCC detection. Here we sought to determine if tumour KIM-1 plays a role in RCC cell extravasation and metastasis to lungs. Methods: We overexpressed KIM-1 in Renca cells (murine RCC) using stable transfection of an expression plasmid, or silenced endogenous KIM-1 in human 769-P and 786-O RCC cells using shRNA. We studied extravasation using the chorioallantoic membrane (CAM) model. We injected ~1x105 Renca or 769-P cells into blood vessels of chicken embryos (n=6), and measured percentage of cells that exited the capillary bed into surrounding spaces after 24 hours. To study lung metastasis, we injected ~5x105 Renca or 786-O cells intravenously through the tail vein of syngeneic BALB/c (n=10/group) or immune deficient Rag1-/- [BALB/c] (n=5/group) mice, and manually counted metastatic nodules in excised lungs after 17 days. Finally, we analyzed the publicly available Cancer Genome Atlas Illumina RNA-Seq dataset to determine if KIM-1 mRNA expression predicts overall survival in patients with clear cell RCC. Results: In CAM experiments, extravasation efficiency was significantly decreased in KIM-1pos 769-P cells compared to KIM-1neg 769-P cells (30.08% vs. 47.94%; p=0.0004), and in KIM-1pos Renca cells compared to KIM-1neg Renca cells (47.78% vs 60.73%; p=0.042). BALB/c mice injected with Kim-1pos Renca cells or Kim-1pos 786-O cells had significantly fewer lung metastases compared to mice injected with Kim-1neg Renca cells or Kim-1neg 786-O cells, respectively (257 vs 429, p=0.02; 131 vs 339 p=0.001). The inhibitory effect of KIM-1 expression in Renca and 786-O cells on numbers of lung metastases was similar in Rag1-/- mice, suggesting that the observed effects were not dependent on adaptive immunity. In the TCGA database, patients with the lowest vs. highest tertile of KIM-1 RCC expression has significantly higher mortality (unadjusted HR 1.82, 95% CI 1.23-2.70; p=0.0034). Conclusion: KIM-1 is a negative regulator of cell extravasation and inhibits the development of lung metastases in mice, and KIM-1 expression in human RCC may predict better survival. Deciphering the underlying mechanisms may lead to novel therapies.

#4605

Exosomes harvested from patient-derived-explants or cells-enhance proliferation and migration in TNBC breast cancer cells and breast epithelial cells.

Jordon Coward,1 Margarite D. Matossian,2 Paige A. Roberts,1 Jasmine Johnson,1 Matthew E. Burow,2 KiTani P. Lemieux1. 1 _Xavier Univ. of Louisiana, New Orleans, LA;_ 2 _Tulane University School of Medicine, New Orleans, LA_.

Triple negative breast cancer (TNBC) is a clinically aggressive breast cancer subtype due in part by high rates of metastasis and poor prognoses. TNBC tumors are molecularly heterogeneous and lack targetable receptors common in other breast cancer subtypes (estrogen receptor, HER2/Neu amplification), hindering the discovery of effective targeted therapies. Our group has designed a series of experiments to gain insight into the role of the tumor microenvironment in TNBC proliferation, migration and invasion. Patient-derived xenografts (PDXs) are translational models that are necessary to use in the laboratory setting when studying the complex tumor microenvironment of TNBC tumors. Recent breast cancer-focused studies show exosomes can be local and systemic cell-to-cell transporters of oncogenic information. Here, we aim to better understand how tumor-derived exosomes harvested from TNBC PDX models affect cell proliferation and migration when cultured with either triple negative breast cancer cell line MDA-MB-231 or non-tumorigenic breast epithelial cell line MCF-10A. PDX tumors or cells were plated in 2D culture conditions, and exosomes were isolated and purified from the cell culture media. Proliferation and migration assays were then performed after co-culture of the exosomes (or RPMI-1640 media as a negative control) with breast cell lines (MDA-MB-231, MCF-10A). The Alamar blue dye exclusion assay was used to measure proliferation, and absorbance was measured to quantify relative proliferation amongst the different treatment groups. To evaluate cell migration, a transwell migration assay was utilized where the breast cancer cell lines were plated with either the exosomal media or standard growth media as the chemoattractant for 48 hours. For invasion experiments, the transwell migration assay was employed. Our data indicate that TNBC PDX-derived exosomes increased cell proliferation in the MDA-MB-231 cell line by an average of approximately 60% compared to the control (p ≤ 0.05). We observed similar patterns of increased cell proliferation in the MCF-10A cell line, with an increase of 34% when cells were exposed to exosomes (p ≤ 0.05) compared to the control. Exosomes from TNBC PDX models increased migration in MDA-MB-231 cells by 4% and in MCF-10A cells by 10 percent. Interestingly, when we screened the exosomes from different TNBC PDX models, we found that one invasive TNBC PDX model contributed to the increased proliferation and migration in both the MDA-MB-231 and MCF-10A cell lines. Exosomes harvested and purified from TNBC patient-derived explants, and 2D culture conditions models increased proliferation and migration, cell line MDA-MB-231, and the non-tumorigenic breast epithelial cell line MCF-10A. Our data suggests that exosomes are key mediators in the progression of cancer.

### Mouse Models

#4606

Optimization of a syngeneic animal model for metastatic melanoma: From ear to lymph node and beyond.

Ariel L. Myers, Kathleen A. Thayne, Rukiyah Van Dross, Gina Murray. _Brody School of Medicine at East Carolina University, Greenville, NC_.

Metastatic melanoma (MM) is the deadliest form of skin cancer in the United States with one person dying from the disease every hour. Metastasis occurs when cells from the primary tumor enter the systemic circulation and grow at local and distant organ sites. The most common sites of melanoma metastasis are the lymph nodes, bone, brain, and lungs. Melanoma that has metastasized is not effectively treated with cytotoxic therapeutics that are currently available, although immunotherapeutic and targeted agents produce more favorable outcomes. Despite the availability of these effective agents, MM is still associated with high mortality rates. In order to develop new and highly effective agents that robustly eliminate MM, current models of metastatic disease in immunocompetent animals must be improved. Several in vivo models focus on injecting tumor cells directly into the circulation via the tail vein to allow lesions to form within the lungs. While these models permit rapid metastasis at a relevant sites, they lack primary tumor formation and therefore, do not mimic the clinical disease. Other MM models employ subcutaneous tumor inoculation to permit primary tumors to spontaneously metastasize. However, the subcutaneous space is not an orthotopic site for melanoma. Therefore, well-established models of spontaneous metastasis from orthotopic sites must be improved to more accurately, recapitulate MM. The goal of the current study is to modify an existing syngeneic, orthotopic model of spontaneous MM to more precisely reproduce clinical features of metastasis. In this model, B16F10 melanoma cells that expressed firefly luciferase were inoculated on the dorsal ear of C57BL/6 mice. Spontaneous metastasis was then facilitated by removing the primary lesion to prolong the duration before humane endpoints were reached. Metastatic tumor formation in the draining lymph node and at distant sites were observed by using the IVIS Lumina imaging system and by gross examination of target organs after euthanasia. According to our preliminary data, primary tumors developed after an average of 2.8 weeks. Metastatic lesions formed in the draining lymph node approximately 8.8 weeks after inoculation and distant metastasis were subsequently observed. Phenotypic characteristics that served as markers of metastasis and determinants of primary tumor removal were also identified. Histopathologic analysis of the primary and metastatic tumors revealed that the morphological appearance of the lesions was consistent with patient melanoma. Further optimization of this model will allow us to mimic MM more precisely. Animal tumor models with greater translational value will provide a more comprehensive understanding of the metastatic process as well as the effects of clinical and experimental therapeutics.

#4607

Androgen signaling is essential for prostate cancer development initiated from prostatic basal cells.

Yongfeng He,1 Erika Hooker,1 Eun-Jeong Yu,1 Gerald R. Cunha,2 Lan Liao,3 Jianming Xu,3 Andrew Earl,1 Huiqing Wu,1 Michael L. Gonzalgo,4 Zijie Sun1. 1 _City of Hope, Duarte, CA;_ 2 _University of California San Francisco, San Francisco, CA;_ 3 _Baylor College of Medicine, TX;_ 4 _University of Miami, Miami, FL_.

Emerging evidence has shown that both prostatic basal and luminal cells are able to initiate oncogenic transformation. However, despite the diversity of tumor initiating cells, most prostate cancer cells express the androgen receptor (AR) and depend on androgens for their growth and expansion, implicating an essential role of androgen signaling in prostate tumorigenesis. Prostatic basal cells express p63 and are able to differentiate into luminal, neuroendocrine, and basal cells. In this study, we used a variety of relevant mouse models and in vivo systems to directly address the significance of androgen signaling in oncogenic transformation and tumor development initiated from prostatic p63-expressing cells. We demonstrate that activating Wnt oncogenic signaling by expressing stabilized b-catenin in prostatic p63-expressing cells is able to induce cell proliferation and the formation of atypical cell clusters in different prostatic lobes at embryonic, prepubescent, and adult stages. Intriguingly, despite the androgen insensitive nature of prostatic p63-expressing cells, androgens are still essential for these cells to grow and develop to androgen-dependent, luminal cell type prostate tumors. These findings are consistent with what have been observed in human prostate cancers, in which the majority of tumor cells are androgen-sensitive and possess luminal cell properties, providing new insight into the molecular mechanisms for prostate cancer initiation and progression.

#4608

Colchicine binding site agents as potent tubulin inhibitors suppressing triple negative breast cancer.

Shanshan Deng, Hao Chen, Raisa Krutilina, Najah G. Albadari, Tiffany N. Seagroves, Duane D. Miller, Wei Li. _University of Tennessee Health Science Center, Memphis, TN_.

Triple-negative breast cancer (TNBC) cases account for about 15% of all breast cancers in the United States and have poorer overall prognosis relative to other molecular subtypes, partially due to the rapid development of drug resistance to chemotherapies and the increased risk of visceral metastasis. One of the standard treatment regimens for TNBC is the use of a taxane-based chemotherapy, such as paclitaxel, which stabilizes microtubules. However, drug resistance and neurotoxicities often limit the clinical efficacy of taxanes. Therefore, there are continuous needs to develop more effective therapies that could overcome resistance to taxanes. In this study, a novel series of structurally related pyridine analogs based on our previously reported lead compound ABI-274, was designed and synthesized to identify a molecule with improved antiproliferative potency. Most of these pyridine compounds exhibited potent cytotoxicity when tested in a panel of melanoma and breast cancer cell lines, with IC50 values in the low nanomolar range. Among them, CH-II-77 is the most potent compound with an IC50 value of 1−3 nM against these cancer cell lines, including paclitaxel-resistant sublines. The high-resolution X-ray crystal structure of CH-II-77 in complex with tubulin protein confirmed its direct binding to the colchicine-binding site. It strongly induced apoptosis and produced G2/M phase cell cycle arrest in TNBC cells in a dose-dependent manner in vitro. In vivo, CH-II-77 inhibited tumor growth in A375 melanoma xenografts and MDA-MB-231 TNBC xenografts in a dose-dependent manner. CH-II-77 was able to induce tumor necrosis and apoptosis in vivo. Collectively, these studies strongly suggest that CH-II-77 is a potent inhibitor of the growth of TNBC in vitro and in vivo. Thus, CH-II-77 and optimization of this analog are promising new generation of tubulin inhibitors for the treatment of TNBC and other types of cancers where tubulin inhibitors are currently being used clinically.

#4609

Establishment of sarcoma PDX models with various subtypes for drug efficacy evaluation.

Jessie Jingjing Wang,1 Mengxiong Sun,2 Likun Zhang,1 Yingqi Hua,2 Henry Qixiang Li,1 Davy Xuesong Ouyang1. 1 _Crown Bioscience, Inc., Taicang, Jiangsu, China;_ 2 _Shanghai General Hospital, Shanghai, China_.

Sarcomas are the neoplasms which arise in bone and peri-osseous soft tissues. Osteosarcoma, Ewing's sarcoma and Chondrosarcoma are the 3 major malignancies of bone sarcoma affecting both children and adults. Among those, osteosarcoma is the most common bone-originated cancer in young ages. Although the primary tumor is often surgically resected and the improvements in therapeutic strategies were achieved in recently years, the outcomes remain poor with 50-60% of 5-year survival rates because of relapses mainly due to the metastases, especially metastases to lung. Soft tissue sarcomas are mesenchymal neoplasms including different histologic subtypes which can arise from viscera such as genitourinary, gastrointestinal or gynecologic organs, or from nonviscera soft tissues such as muscle, adipose, synovium etc. Among various subtypes of soft tissue sarcoma, synovial sarcoma is an aggressive malignancy that accounts for 10%-20% of soft tissue sarcoma in young population. Surgical excision combined with adjuvant or neoadjuvant radiotherapy provides a good treatment of local disease, but the recurrences often occurs with the most common pattern of metastasis to lung. Despite the increasing risk of sarcoma and poor outcome, effective treatments, especially precision therapies, are largely lacking. Breakthrough drug discovery is hindered by limited numbers of suitable in vivo models. In recent decades, patient-derived xenograft (PDX) mouse models are commonly used for drug efficacy evaluation with prominent advantages of similar histopathology and molecular pathology with patient tumors. We have established a series of patient derived xenograft models, recapitulating diverse subtypes of sarcoma, including bone sarcoma and soft tissue sarcoma. More than 30 osteosarcoma PDX models are established with a wide spectrum of age populations and clinical grades. Half of these osteosarcoma PDX models are derived from children and young adults. 2 of these osteosarcoma PDX models are derived from relapse tumors and 5 of them are from metastatic tumors (mainly from lung metastases). Besides, we have also established one Ewing's sarcoma (derived from a 9-years-old child) and 5 Chondrosarcoma PDX models. We have also collected numbers of soft tissue sarcoma samples, of which, we have established 9 synovial sarcoma PDX models, mostly derived from adult patients with 2 of them derived from lung metastatic tumors. Among these PDX models derived from metastasis tumors, we also have established several matched PDX models with their primary tumors. Taken together, our established sarcoma PDX models include different subtypes and different cancer stages (especially primary vs. metastatic tumors), providing a valuable preclinical platform for the understanding of different pathogenesis, as well as supporting drug discovery efforts on different subtypes of sarcomas.

#4610

Prognostic impact of KRAS driver mutations and GSTT1 expression in colorectal cancer to FOLFOX treatment.

Jessie Jingjing Wang, Binchen Mao, Sheng Guo, Davy Xuesong Ouyang, Henry Qixiang Li. _Crown Bioscience, Inc., Taicang, Jiangsu, China_.

Colorectal cancer (CRC) is one of the most common cancer and the leading causes of cancer-related death worldwide. CRCs are neoplasms which have high propensity for metastasis, especially spreading to liver and lung. Liver metastasis accounts for around 50% of CRC patients, usually associated with cancer relapse and worse prognosis of the patient. The combination chemotherapy FOLFOX including 5-flurouracil, oxaliplatin and leucovorin is one of the most common and standard first-line chemotherapy utilized for adjuvant and/or neoadjuvant treatment of patients with CRC, which can reduce the risk of cancer relapse. However, a number of studies reported that chemotherapy alone cannot completely eradicate all the cancer cells, especially metastatic cells. Moreover, the mutations of oncogenes such as KRAS, existed in more than 40% of CRC patients, are often associated with poor response of anti-EGFR therapy either as single agent or in combination with FOLFOX. There is still no direct evidence on the association of KRAS or other oncogenic drivers with the resistance to FOLFOX. To understand if any of the potential biomarkers have the predictive value of FOLFOX treatment for CRC patients, especially later stage diseases, we have selected 16 CRC PDX models derived from patients who are in stage II or later where most of patients' tumors present invasion and metastatic features. We have evaluated the drug efficacy of FOLFOX treatment in vivo for these 16 CRC PDX models and found that 6 of them were sensitive to FOLFOX and 10 models have poor responses. To study the potential biomarkers which affect the efficacy of FOLFOX in CRC PDX models, we analyzed the correlation of tumor growth inhibition (TGI) with the genomic background of these PDX models and identified the genes with significant correlation. Through filtering the genes by their functional relevance to FOLFOX treatment, we found that low expression of GSTT1 was highly related to FOLFOX resistance in these CRC PDX models. GSTT1 deletions was found to enhance the resistance to chemotherapy and a shorter survival in AML patients, which is consistent with our observation. Furthermore, we have also run the driver mutation analysis for these 16 PDX models, and found that models without KRAS driver mutations are more sensitive to FOLFOX treatment. Interestingly, in 32 CRC patients treated with FOLFOX, we found the sensitivity of FOLFOX is correlated with wild type KRAS and high expression of GSTT1, similar to our findings in PDX studies. In conclusion, our studies demonstrated that KRAS driver mutations or low expression of GSTT1 gene may render higher chances of resistance to FOLFOX treatment, these biomarkers may be beneficial for clinicians to choose effective therapies for advance CRC patients.

#4611

Identifying drivers of mammary tumorigenesis & elucidating the mechanisms of cancer initiation in DNA replication defective Chaos3 mice.

Marquita Winters, John Schimenti. _Cornell University, Ithaca, NY_.

Distinguishing commonly deleted genes as drivers or passengers of human cancers are key to delineating mechanisms of tumor initiation. The "Chaos3" mouse model of spontaneous breast cancer carries a missense mutation in a gene called Mcm4 (minichromosome maintenance 4), causing destabilization of the MCM2-7 replicative helicase, which in turn causes elevated genomic instability. Approximately 80% of female mice homozygous for this mutation in the C3HeB/FeJ strain background develop mammary tumors with an average latency of 12 months. Genomic analysis of these tumors revealed a recurrent subset of genes with copy number alterations such as Arid1a and Nf1. This project aims to: 1) test whether the genes Arid1a and Nf1 act as mammary tumor drivers in the Chaos3 breast cancer model; 2) understand the genome-wide effects of Arid1a loss in mammary tumorigenesis; 3) identifying potential therapeutic targets for Arid1a-deficient mammary tumors; and 4) unravel the mechanism by which destabilization of MCM2-7 replicative helicase leads to cancer onset. To achieve these aims, several experiments such as conditional mutagenesis, CUTandRUN, RNA-seq, and replication timing profiling were conducted to address my aims. Preliminary data indicates that heterozygous loss of Arid1a in Chaos3 mammary tumors produces a distinct transcriptional profile compared to mammary tumors without Arid1a deletion. Additionally, Chaos3 primary cells have markedly altered replication timing patterns in certain regions of the genome that might explain increased susceptibility to mutations. My experiments can validate new candidates like Arid1a as a driver of sporadic breast cancer in humans. In addition, these studies will not only lend insight into how key tumor suppressors are deleted in the Chaos3 model, but also potentially reveal features of genomic regions that are particularly susceptible to genetic or environmental perturbations to DNA replication. The Chaos3 sporadic breast cancer mouse model mimics sporadic human luminal breast cancer and can be used as a tool to better understand the biology and genetics of a common disease that plagues women worldwide.

#4612

HOXA13 drives hepatocytes proliferation and liver tumorigenesis in mice.

Gaia Bianco,1 Hesam Montazeri,1 Luca Quagliata,1 Tracy O'Connor,2 Ursula Ehmer,3 Rupert Oellinger,3 Mathias Matter,1 Christofori Gerhard M.,4 Charlotte K.Y. Ng,1 Salvatore Piscuoglio,1 Mathias Heikenwaelder,5 Luigi M. Terracciano1. 1 _University Hospital Basel - Institute of Pathology, Basel, Switzerland;_ 2 _Helmholtz Zentrum Muenchen, Munich, Germany;_ 3 _Technische Universitaet Muenchen, Munich, Germany;_ 4 _Department of Biomedicine (DBM), University of Basel, Basel, Switzerland;_ 5 _Deutsches Krebsforshungzentrum (DKFZ), Heidelberg, Germany_.

Introduction: Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver and the third most common cause of cancer related mortality worldwide. For patients suffering from advanced stage disease, the few therapeutic options available are not curative and improve patient survival by only a few months. Therefore, new molecular targets that can be explored as therapeutic options are highly needed. Class I Homeobox (HOX) genes are fundamental components of embryonic patterning and morphogenesis, with expression persisting into adulthood. They are also implicated in neoplastic transformations. However, the role of HOX genes is poorly understood and the functional relationship between the malignant phenotype and abnormal expression of HOX genes is still unclear. In this study we sought to define the role of the HOXA13 gene in hepatocarcinogenesis using in vivo models.

Methods: To unravel the molecular mechanism of HOXA13 driven tumorigenesis in liver and its direct oncogenicity in vivo, a murine model of HOXA13 overexpression in liver was generated using hydrodynamic injection coupled with a transposase system. This model led to the stable and specific HOXA13 expression in C57BL6\J mouse hepatocytes up to 5 months post injection. Mouse phenotype was followed over time, from 2 weeks up to 1 year post injection. 16 mice (8 for CTRL vector and 8 for HOXA13) were injected and sacrificed for every time point. RNA sequencing was performed to monitor the transcriptomic changes over time.

Results: 1 year post injection 50% (4/8) of the injected mice with HOXA13 developed liver tumors of various histological grades and types, from very well differentiated HCCs to very highly undifferentiated and cholangiocarcinoma like nodules. HOXA13 overexpression in the liver led to highly proliferative hepatocytes after only 2 weeks and the proliferative phenotype was maintained until 5 months post injection, when pre neoplastic lesions began to form. HOXA13 overexpression correlated not only with proliferation but also with the DNA damage marker yH2AX, suggesting a possible mechanism of tumorigenesis driven by genome instability. Gene set enrichment analysis of RNA-seq performed on whole liver extracts of 2 week old mice and tumors showed that the main pathways involved in HOXA13 expression are cell cycle, in particular G2/M transition and mitotic assembly checkpoint, angiogenesis, TP53 pathway, IL6JAKSTAT3 signaling, Notch signaling and epithelial to mesenchymal transition.

Conclusion: Our study highlights the key role of HOXA13 as a potential novel oncogene in HCC development and suggests possible mechanisms through which it drives liver tumorigenesis. We expect that the generated data in vivo, coupled with mass spectrometry and ChIP sequencing experiments performed in vitro, will further help us identify downstream effectors of HOXA13 thus providing new potential therapeutic targets for HCC.

#4613

Mathematical modeling of tumor growth in mouse models.

Chao Zhang,1 Xiaoqian Jiang,1 Sheng Guo,1 Qixiang Li2. 1 _Crownbio Inc, Suzhou, China;_ 2 _Crownbio Inc, Santa Clara, CA_.

A variety of tumor models are used in preclinical oncology drug discovery, including patient-derived xenografts (PDXs), cell line-derived xenografts (CDXs), and syngeneic homografts. Tumors in mouse models exhibit heterogeneous growth patterns with or without drug treatment, which is further complicated by measurement errors. Mathematical modeling of tumor growth helps us better understand such patterns and analyze drug efficacy in transplanted tumor models. Many mathematical models have been proposed to describe tumor growth curves, each with certain assumptions and equations, suitable for specific situations or data sets1-5. It is yet unknown which models are best in fitting the growth curves for a large collection of mouse models. In this study, we systematically evaluated a set of mathematical models including exponential models, logistic and Gompertz model, biphasic models, dynamic carrying capacity model, and power law model on more than 50,000 tumor growth datasets collected from in vivo efficacy studies in PDXs, CDXs and syngeneic models. A set of metrics were used to evaluate the goodness of fit, including Akaike Information Criterion (AIC), root mean square error (RMSE), coefficient of determination, mean absolute relative error, etc. We also proposed a new composite metric to combine many criteria for easy selection of growth models. Our results show that tumor growth patterns are less complex under vehicle treatment, and different mathematical models have relatively small difference in modeling the growth curves. Tumor growth patterns are more diverse under drug treatment. Tumors may shrink or completely remit, though some may grow in the first days after inoculation before drug effect takes place. Still some tumors stop growing or even shrink before drug resistance develops and resume fast growing. Therefore, different mathematical models are needed to describe the different patterns. Finally, we present summary statistics to give an overall picture on the applicability of the mathematical models, and suggest statistical models for subsequent analysis for drug efficacy evaluation and biomarker discovery.

#4614

Establishment of a HER2 positive breast cancer bone metastasis model for validation of novel therapies.

Tiina E. Kähkönen,1 Mari I. Suominen,1 Jenni H. Mäki-Jouppila,1 Jussi M. Halleen,1 Jenni Bernoulli,1 Derek Grant2. 1 _Pharmatest Services, Turku, Finland;_ 2 _Bayer AS, Oslo, Norway_.

Breast cancers with overexpression of human epidermal growth factor receptor 2 (HER2+) have aggressive clinical behavior, and at advanced stages are associated with increased risk for developing metastases to distant organs including bones, brain and lungs. The aim of this study was to establish a systemic metastasis model for HER2+ breast cancer with a special interest in bone metastasis. The effects of cell number, estrogen supplementation and mouse strain on metastasis formation were studied.

In the study, 5-6 weeks old athymic nude and Rag2 mice (n=10-13 per group) were used. Half of the mice received estrogen supplementation (E2-releasing rods 5µg/day) one week before inoculation of the cancer cells. The mice were inoculated intracardially with 1 or 5 x 105 luciferase-labelled triple-positive (ER, PR positive and HER2 overexpressing) human BT-474 breast cancer cells. The formation of metastases was followed by bioluminescence imaging (BLI) at inoculation and once a week for the duration of the study. At sacrifice, X-ray imaging was performed and the bones were collected for histological analysis.

The formation of bone metastases was observed in all study groups, and bone metastases were dominant in the model. Based on BLI, bone metastases developed in nude mice earlier than in Rag2 mice. However, detection of metastases in Rag2 mice was challenging due to dark fur hindering the signal transmittance. The bone metastases appeared between 7 to 36 days after inoculation of the cancer cells in nude mice and between 20 to 43 days in Rag2 mice. The number of bone metastases was higher in nude mice. E2 supplement accelerated the development of bone metastases in both mouse strains. In nude mice with E2 supplement, bone metastases appeared between 7 to 14 days compared to between 20 to 36 days in mice without E2 supplement. In Rag2 mice with E2 supplement bone metastases formed between 20 to 28 days compared to between 36 to 43 days without E2 supplement. The use of higher cell number accelerated the development of bone metastases but had no major effect on their incidence in both mouse strains. Take rate of bone metastases was 90-100% in nude mice with E2 supplement compared to 30-50% without E2 supplement. In Rag2 mice, the take rate of bone metastases was 50% with E2 supplement. X-ray imaging showed estrogen induced bone growth and large tumor-induced osteolytic lesions in the hind limbs in both strains. Due to the extensive bone lesions and occasional fractures, the first mice were sacrificed 50 days after inoculation of the cancer cells. Both mouse strains occasionally developed metastases in soft tissues including brain and ovaries.

In conclusion, a high rate of bone metastasis was achieved in athymic nude mice supplemented with E2. This model can be used to study the efficacy of anti-cancer, such as HER2-targeted, compounds on tumor growth at metastatic locations or on the prevention of metastasis formation.

#4615

Measurement of systemic inflammatory responses in mouse syngeneic tumor models.

Nicolas Solban, Douglas Linn, Cai Li, Razvan Cristescu, Brian J. Long. _Merck, Boston, MA_.

Identifying valid biomarkers that predict for durable responses and benefit from treatment has long been the subject of intense investigation. Following the immense success of cancer immuno-therapies at stimulating anti-tumor immune responses in a subset of patients, a further understanding of systemic inflammatory responses (readily obtained from serum) to predict outcome is urgently needed. Multiple studies have shown that an elevated neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) are associated with decreased overall survival and decreased disease-free survival. We evaluated hematology parameters in mice following inoculation with various mouse tumor cell lines in order to determine if any systemic markers of inflammation could predict for tumor progression. Eleven murine cell lines were individually inoculated subcutaneously in C57BL/6 mice (MC38, B16F10, TC-1, and LL/2 cell lines), BALB/c mice (4T1, CT26, Renca, and EMT-6 cell lines), DBA/2 mice (CM-3 cell line), and CH3 mice (MBT-2 cell line). Animals were euthanized when tumors reached pre-determined sizes (100 mm3, 250 mm3, 500 mm3, 1000 mm3, and 1500 mm3), blood was collect for hematology, and tumors were fixed for histology. The 4T1 cells were also used to establish a metastasis model. Briefly, 4T1 primary subcutaneous tumors are removed 14 days post-inoculation to allow metastasis to develop. On day 4, 11, and 18 following resection, 50 μL of blood was collected for longitudinal hematology measurements. Hematology parameters varied between healthy mice strains. A significant increase in white blood cells (WBC) was measured in DBA/2 mice compared to other strains and a significant decrease in monocytes was measured in C57BL/6 mice compared to other strains. Nonetheless, NLR was strongly correlated with tumor weights across all of the models, with the larger tumors having the highest NLR. On the other hand, NLR did not correlate with body weight loss, suggesting an independent effect from other prognostic factors like cachexia. Overall, NLR was highest in the 4T1 models (subcutaneous and metastatic), consistent with human data where NLR increases with disease stage. Models were also classified based on response to anti-PD-1 treatment. Unresponsive models, 4T1, EMT-6, TC-1 LL/2 had a higher NLR compared to the responsive models (MC38, CM3, and MBT-2). In conclusion, this survey of hematology parameters following mouse cell line inoculation showed a strong correlation of neutrophils, NLR, and lymphocytes with tumor burden. Furthermore, models responsive to immuno-therapies had lowest NLR, similar to observations in humans. This survey could be helpful in modeling clinical responses and potentially identifying novel tumor factors that modulate systemic inflammation.

#4616

Establishment of a metastatic orthotopic model of pancreatic ductal adenocarcinoma (PDAC) for drug development.

Justyna Zdrojewska, Jenni H.E. Mäki-Jouppila, Jussi M. Halleen, Jenni Bernoulli. _Pharmatest Services, Turku, Finland_.

Ductal adenocarcinoma of the pancreas (PDAC) has the worst survival prognosis (<5%) of all common gastrointestinal malignancies. PDAC is typically diagnosed at a very late stage when the tumor has already metastasized to other organs, at which point the treatment can no longer prolong the survival of the patients. To date, surgical resection is the only curative approach present, provided that the cancer is detected at a very early stage. Nonetheless, less than 20% of diagnosed patients qualify for the surgery and majority of these patients will eventually develop recurrence. Despite our advancing knowledge of the tumor biology of PDAC as well as recent improvements in diagnosis, the prognosis remains strikingly poor. The median survival observed after surgery followed by chemotherapy is about 20 months.The aim of this study was to establish an orthotopic model of PDAC that could be used to study efficacy of new potential treatments. Female athymic nude mice (Hsd: Athymic Nude-Foxn1nu) were used in this study. MiaPaCa-2-Luc human PDAC cells were injected to surgically exposed caudal part of the pancreas. At the time of inoculation, the animals were 4-5 weeks of age. To validate the model, the current standard-of-care (SOC) treatment (combination of nab-paclitaxel and gemcitabine) was used. During the study, tumor burden was quantified by imaging the bioluminescence signal emitted by the MiaPaCa-2-luc cells using IVIS Lumina II imaging system. After 30 days in study, tumor growth in the surgical area was observed. The mice were stratified to treatment groups based on similar intensity of the bioluminescent readout. Imaging was performed every second week after inoculation of the cells. The SOC treatment was initiated two weeks post inoculation and was administered twice a week over a period of 4-5 weeks. The observed tumor take rate was 100%. At sacrifice, tumor weight and volume were lower in the group receiving the SOC treatment as compared to the vehicle treated group. Although there were no differences in body weights, mice receiving the SOC treatment gained less weight when comparing the body weights obtained at endpoint relative to baseline. As confirmed by histological analyses at endpoint, the MiaPaCa-2-luc cells induced micrometastasis to other visceral organs including liver. In conclusion, a metastatic orthotopic PDAC model was established successfully and validated with the SOC treatment that slowed down disease progression. Therefore, this orthotopic model provides a promising tool for testing new treatments against PDAC in vivo.

#4617

In vivo **efficacy focused data collection and gaps for preclinical anticancer drug evaluation.**

Taiping Chen, Xiaoqin Sun, Tingting Li. _Effymed Bioscience Ltd, Andover, MA_.

Even though most anticancer drugs have been initially screened, investigated, and evaluated with variety of in vitro platforms, gaps between preclinical nominated candidate and clinical acceptable approval of final drug remain a big challenge for biotech companies all over the world. It is a highly demanding task to establish various animal models which can closely mimic human tumorigenesis in preclinical drug evaluation. Here we summarize several in vivo efficacy models to discuss on various applications in the data collection and interpretation. Currently available models include traditional cancer cell derived xenograft (CDX), patient derived xenograft (PDX), immune component murine tumor models (syngeneic and human gene knock-in), and orthotopic tumor models. Based on different drug mechanisms appropriate models will be initially selected with certain rationale. In order to understand the in vivo mechanisms of action (MOA) and drug target relevance, biomarker profiling and pharmacodynamic changes will be investigated with IHC, and/or FACS analysis particularly when the test articles display clear efficacious responses. Some representative data will be presented to demonstrate that in vivo efficacy will be the key endpoint for further analysis and clinical trial support. Small and large molecules of targeted drugs, antibody drug conjugate (ADC), immune checkpoint therapeutics will be included in the discussion. In sum, the approaches of in vivo data collection here are feasible to support many drug candidates to be evaluated to fill the gaps in the clinical drug development.

#4618

**A novel** Pik3ca **-driven mouse model and syngeneic cancer cell line for the preclinical testing of targeted and immune therapies for anal squamous cell carcinoma (ASCC).**

Glen R. Guerra, Sara Roth, Joseph C. Kong, Rosemary M. Millen, David S. Liu, Shienny Sampurno, Vignesh Narasimhan, Toan D. Pham, Karen G. Montgomery, Alexander G. Heriot, Robert G. Ramsay, Wayne A. Phillips. _Peter MacCallum Cancer Centre, Melbourne, Australia_.

Although many patients with localised anal squamous cell carcinoma (ASCC) initially achieve a complete response with standard chemoradiotherapy, those with persistent, relapsed or metastatic disease (35% of all patients) have limited treatment options and poor outcomes. Recent genomic profiling studies have identified PIK3CA as the most frequently mutated gene in anal cancers. Amplification of the PIK3CA gene and mutations in other PI3K pathway genes, have also been detected providing a strong rationale for targeting the PI3K pathway in these tumours. Similarly, the finding that many ASCC tumours express immune checkpoint receptors including PD-L1, has focussed attention on the potential use of checkpoint blockade in anal cancer. However, the use of targeted and/or immune therapies in the management of ASCC has been hampered by a lack of representative preclinical models for in vitro and in vivo testing of potential new therapeutic approaches. We have used mice with a Cre recombinase (Cre)-conditional knock-in of the Pik3caH1047R mutation and deletion of PTEN, crossed with mice expressing a tamoxifen-inducible Cre under the control of the ubiquitin C promoter, to generate a novel model of ASCC. By applying 4-hydroxy-tamoxifen topically to the anal canal we simultaneously induce expression of Pik3caH1047R and deletion of PTEN specifically in the anal epithelium. This results in anal tumors within 3 weeks with 100% penetrance. As all mice are on a C57Bl/6 background we are able to transplant the tumors subcutaneously into wild type C57Bl/6 mice as a syngeneic graft. To improve the utility of the model, we used tumors from these mice to establish a syngeneic mouse ASCC cell line. This line was then transduced with a human papilloma virus 16 E6/7 lentivirus to recapitulate the human virally-driven form of the disease. The cell line expresses both the Pik3ca mutation and E6/7 oncogenes and is positive for the squamous markers p63 and CK5/6. It had a similar response to 5-fluorouracil, Mitomycin C and radiotherapy, as a panel of human ASCC cell lines (also derived in our laboratory) and was sensitive to a PI3K inhibitor (BYL719). It was tumorigenic in both immunocompetent C57Bl/6 and immunodeficient NSG mice, additionally developing lung metastases in the NSG mice. The syngeneic C57Bl/6 tumors induce a peri-tumoral infiltrate, consisting of a heavy myeloid population, with high PDL1 expression, and a T-cell population expressing PD1; similar to human patients with treatment resistant disease. A major barrier to identifying new treatment options and improving overall survival in anal cancer has been the lack of preclinical models. We have now generated and characterised a novel, relevant Pik3ca-driven mouse model and syngeneic cancer cell line that will facilitate the in vitro and in vivo preclinical testing of targeted and immune therapies for ASCC.

#4619

Development and characterization of a simple animal model of glioblastoma multiforme.

Levi Adams, Yoon-Seong Kim. _University of Central Florida, Orlando, FL_.

Glioblastoma multiforme (GBM), a cancer of the glial cells in the brain, is the most common and aggressive primary brain tumor and even with aggressive and invasive treatment only has a median survival of about a year. There is an urgent unmet need to understand underlying molecular mechanisms and identify and develop robust and effective translational platforms to screen potential therapeutic strategies. Current models rely on immunocompromised animals or cumbersome and complex breeding of multiple transgenic animals, limiting their effectiveness. We report the development of a simple, effective strategy to reproducibly induce tumors in healthy, adult animals. In our laboratory we have developed a single dual-promoter lentiviral construct to be delivered by stereotaxic injection. We incorporated simultaneous ARF and INK4A RNAi and Cre-dependent EGFRvIII overexpression to model mutations most commonly seen human patients. Initial in vitro experiments showed rapid and robust transformation of primary murine astrocytes into tumor-initiating cells. Initial stereotaxic injections of our lentiviral particles into the brain of inducible GFAP-Cre mice showed hypercellular mass formation after just 30 days. We are using our system to explore sub-ventricular zone (SVZ) involvement in GBM. GBM with SVZ involvement is known to have poorer prognosis than distal tumors. The neurogenic SVZ region has recently gained attention as a potential source of GBM, as cells harboring low-level driver mutations were identified in the sub-ependymal region. Our model allows us to precisely target this area and compare SVZ-involved tumors directly to tumors formed in distal regions. Full characterization of these tumors by histology and RNAseq can help shed light on the molecular underpinnings of the prognosis difference and may identify new potential therapeutic avenues.

#4620

Development of orthotopic breast cancer brain metastasis patient-derived xenograft(PDX) model.

Masanori Oshi. _Roswell Park Cancer Institute, Buffalo, NY_.

Background: The vast majority of the mortality of breast cancer results from distant metastasis and pre-clinical models are in critical need for effective drug development. Patient-derived xenograft (PDX) maintains the features of the donor tumors such as intra-tumor heterogeneity; however, PDX model of breast cancer brain metastasis that delivers stable consistent results is not popularized. We demonstrate our novel methods (coined "Oshi"method) used to develop an orthotopic brain metastasis patient-derived xenograft model (PDMOX) for breast cancer brain metastasis via tumor tissue implantation. Methods: Brain metastasis PDX were created using metastatic brain tumors from breast cancer patients and implanting them in the brain of NSG female mice. Tumors of ~1 mm3 were implanted mechanically minced tissue with medium. We implant the tissue through a frontal bone burr hole into the right caudate putamen. Tumor growth was monitored by magnetic resonance imaging (MRI). Results: "Oshi" method utilizes a pipette tip in order to inoculate tumor at the same depth. A minced tumor was instilled using a 23G needle as the "non-oshi" method. One hour post-surgical survival after implantation of minced tumor by "non-oshi" method was only 37.5% (3/8), whereas 100% (40/40) by the "Oshi" method. All tumors were well engrafted in surviving mice after both methods. There was larger variation in tumor growth after "non-oshi" (Median 20±25.0 day, range: 12-24 day. Tumor volume: median 5.6±21.0 mm3, range 2.8-48.7 mm3). One mouse developed ptosis, and 2 out of 3 mice that underwent the "non-oshi" method had sudden death. On the other hand, all mice that underwent the "Oshi" method lived until the tumor grew to 125-200 mm3 without neurological symptoms. The engraftment rate of Mincing tumor were 100% at 1.5 months after implantation, while the engraftment rate of Enzymatic digestion tumors was only 50% at 2 months. Mincing tumor had faster growth speed than the tumor of enzymatic digestion. The engraftment time of enzymatic digestion tumor was slower than other tumor tissue forms. All 3rd generation tumors could be detected at the point of 1month after implantation, on the other hand, it took 2 month to detect 1st generation tumors. The growth of 3rd generation tumors was significantly faster and bigger than others. Conclusion: Various surgical techniques used to generate PDMOX breast cancer models showed major differences in the tumors and outcomes. These novel models are expected to become powerful tools for preclinical studies in metastatic breast cancer.

#4621

Developing genetically modified homograft models of mouse prostate cancer for efficacy evaluation of combinatory immunotherapies.

Jessie Jingjing Wang, Yanrui Song, Annie Xiaoyu An, Likun Zhang, Henry Qixiang Li, Davy Xuesong Ouyang. _Crown Bioscience, Inc., Taicang, Jiangsu, China_.

Despite the early approval of Sipuleucel-T for metastatic castration-resistant prostate cancer, which often perceived as a milestone achievement in cancer immunotherapy, subsequent progress in prostate cancer immunotherapy development has been limited by disappointing results with tumor vaccines and its resistance to immune checkpoint inhibitors, such as PD1 & PD-L1. It is now generally accepted that we need to tackle prostate cancer by combinatorial approaches of chemo-, targeted- and immuno-therapies. Highly relevant preclinical models are very much needed for proof of principle efficacy evaluation. Genetically engineered mouse models (GEMM) recapitulate key aspects of human prostate cancer in both histo- & molecular- pathology. Among those, PTEN loss of function (LOF) in prostate epithelium is one of the central events in human prostate cancer. PTEN haploinsufficiency in mice is sufficient to drive mouse prostatic intraepithelial neoplasia (PIN) formation, while loss of both alleles of PTEN in mouse prostate leads to hyperplasia at 4 weeks, PIN at 6 weeks, and fully invasive adenocarcinoma from 12 weeks of age. PTEN null tumors are also resistant to androgen depletion. In the meantime, although KRAS mutation are not often seen in human prostate cancer, activation of MAPK pathway often happens in advanced tumor. Mutant KRAS or BRAF can robustly promote mouse prostate cancer progression. Prostate specific Pten null and Kras G12D; Pten null mouse prostate cancer models have been well characterized by a number of labs. However, parental GEMM models are difficult to be used for pharmacological studies due to the spontaneous nature of tumor onset and progression. The compound mutant mice are also costly to breed. Here we report generation of transplantable mouse prostate cancer homograft models by passaging the primary tumor subcutaneously in the C57BL/6 mice. These mouse tumors featuring Pbsn-Cre;LSL-KrasG12D/+;Ptenflox/flox or Pbsn-Cre;Ptenflox/flox retain morphological similarity to moderate to poorly differentiated human prostate cancer. These homograft tumors show high levels of M2 macrophage infiltration. Growth of these mouse prostate tumors are resistant to androgen depletion. They are also resistant to the treatment of AR inhibitor enzalutamide, only marginally responsive to docetaxel, but sensitive to treatments with mTOR inhibitor everolimus. When treated with immune checkpoint antibodies, i.e. PD1 and CTLA4, these tumors showed increased T-cell infiltration, but very modest growth inhibition. We are now testing a variety of combinatory therapies with chemotherapies, immune modulators and immune checkpoint antibodies. The results will be presented at the meeting.

#4622

The YUMMER.G mouse melanoma model recapitulates the heterogeneous response to immune checkpoint blockade based on patient sex.

Julie Y. Ramseier, Alexandra Charos, Koonam Park, William Damsky, Marcus W. Bosenberg. _Yale School of Medicine, New Haven, CT_.

Existing murine melanoma models, such as syngeneic transplantation of the B16 melanoma cell line, are aggressive, but poorly immunogenic, and as a result, studying the response to immunotherapy in these models has been challenging. Furthermore, in humans, a heterogeneous response to immune checkpoint inhibitor therapy has been observed between male and female melanoma patients, yet no immunogenic murine cancer model exists to study this sex-related dimorphism. To address these challenges, we created the YUMMER.G model, a melanoma cell line driven by human-relevant Braf activation and loss of Pten and Cdkn2a. YUMMER.G is related to the previously described YUMMER1.7 model; however important differences include: diploid state, female genotype, and Mc1re/e (pheomelanin producing) background. YUMMER.G was created by treatment with UV-light, resulting in a neoantigen rich subclone. Subcutaneous injection of 2 million YUMMER.G cells into C57BL/6J mice resulted in melanoma formation in both male and female mice. Treatment of established tumors with anti-CTLA-4 resulted in a comparable response between male and female mice, with complete, durable tumor regression in 100% of female mice and 88% of male mice. However, when established YUMMER.G tumors were treated with anti-PD-1, 63% of female mice, but only 13% of male mice exhibited complete tumor regression (p = 0.0330). These findings demonstrate the important role of host sex in the response to immunotherapy, and they parallel the sex-related differences in checkpoint inhibitor efficacy that have been observed in human patients. In summary, the YUMMER.G model is a human-relevant, immunogenic mouse melanoma model that will serve as a valuable platform for further investigating the mechanisms that mediate the sex-related dimorphism in anti-tumor immunity and response to immunotherapy.

#4623

Notch3 in lung adenocarcinoma pathogenesis and heterogeneity .

Kieren D. Marini, Stanley Leung, Alex Lee, Alejandro Sweet-Cordero. _UCSF, San Francsisco, CA_.

Lung cancer is the leading cause of cancer related death worldwide. Non-small lung cancer accounts for 85% of diagnoses and the predominant subtype is lung adenocarcinoma (LUAD). LUAD has a less than 20% response rate to conventional chemotherapy and despite identification of several therapeutic targets, the majority of diagnoses continue to lead to death, highlighting the need for better treatments. Notch signaling in mammals is complex, with multiple receptors, ligands and modifiers. Notch ligands at the surface of one cell interact with receptors on an adjacent cell, triggering cleavage and nuclear translocation of the intracellular domain which interacts with Rbpj/Csl to control the transcription of target genes. In LUAD, significant evidence suggests that Notch plays an oncogenic role.

Previously, we identified a population of Tumor Propagating Cells (TPC), which had greater capacity for tumor formation and are enriched after treatment with cisplatin. The TPC population was defined by the expression of CD24, ITGB4 and was Notch high. Further functional validation showed that specifically Notch3 contributes to this propagation phenotype and is key in establishing functional intra-tumoral heterogeneity. However, the TPC population is still heterogeneous and likely only a subset of these cells are true TPCs. Furthermore, the gene expression signature of the mouse TPCs has prognostic significance in human LUAD, however the TPC population hasn't been defined in human patients, limiting our ability to target the Notch pathway therapeutically.

To assess the heterogeneity of the mouse TPC population we performed FACS on tumors from KrasLSLG12D; Tp53flox/flox; YFPLSL twelve weeks after tumor initiation via AdCre. Tumors were sorted for bulk population (YFP+Lin-) and for TPC (YFP+Lin-CD24+ITGB4+Notchhi) population and single cell sequencing was performed using the 10X genomics platform. We hypothesize that aberrant Notch3 signaling is a critical pathway for survival in at least some LUAD and that Notch activation leads to gene expression changes that are important for driving human LUAD maintenance. Patient-derived xenografts (PDX) more closely recapitulate important aspects of human cancer, particularly with regards to the role of intratumoral heterogeneity and self-renewal pathways. To characterize Notch3 expressing cells in human LUAD, we performed FACS on our cohort of LUAD PDXs and sorted the Notch3+ population. We found expression of Notch3 and expression of Notch ligand Jagged2 in all LUAD tumors regardless of driver mutation.

#4624

Profiling antitumor activity, immune cell infiltration, pharmokinetics, and receptor occupancy in a murine colon cancer model following immune checkpoint blockade.

Patrick Allison,1 Hua-Chen Chang,1 Paul Trampont,1 Lindsey Standarski,1 Lauren Urisic,1 Jennifer Rusk,1 Jaydeep Mehta,2 Joshua Fohey,2 Eric Thomas,3 Karan Agrawal,3 Brandy Wilkinson1. 1 _Covance, Greenfield, IN;_ 2 _Covance, Madison, WI;_ 3 _Covance, Indianapolis, IN_.

Clinical success of immune checkpoint blocking antibodies has accelerated the evaluation of immune-related targets for novel anti-cancer therapies. Preclinical testing of immune-targeted oncology agents requires preclinical models with functional immune systems. Utilization of murine syngeneic tumor models provides a robust system to evaluate anti-tumor activity and mechanism of action. This study evaluates the anti-tumor activity and immune response of different immune-oncology therapies in the CT26.WT murine colon cancer model. In addition, an anti-PD-1 pharmacokinetic profile was established and receptor occupancy evaluated.

For this study, C57BL/6JOlaHsd mice were subcutaneously inoculated with CT26.WT cells. Tumor bearing mice were administered anti-CTLA-4, anti-PD-1, anti-PD-L1, anti-OX40, or anti-LAG3 monotherapy twice weekly for 3 weeks. Tumor volume was used to assess anti-tumor activity. Immune response to checkpoint blockade was monitored in a subset of tumors, and lymphocyte and myeloid populations were analyzed following two weeks of dosing. In addition, an anti-PD-1 pharmacokinetic profile experiment was performed using LC-MS/MS on mouse plasma from multiple time points. PD-1 receptor occupancy was also determined by flow cytometry utilizing a saturation/detection method.

Following 3 weeks of monotherapy treatment all immune checkpoint blocking therapies, anti-CTLA4, anti-PD-1, anti-PD-L1, anti-OX40, and anti-LAG3, resulted in significant antitumor response in CT26.WT derived tumors. Phenotypes of tumor infiltrating lymphocytes (TILs) resident in tumors were profiled across check point inhibitor treatments: CD45+ lymphocytes were analyzed for populations of cytotoxic lymphocytes (CTLs, CD8+ cells), T-helper cells (CD4+ cells), regulatory T-cells (T-regs, CD4+/CD25+/FoxP3+ cells). The immune cell responses to checkpoint inhibitors were distinct and varied across immune checkpoint blocking antibodies. Furthermore, an empirical exposure-response analysis of anti-PD-1 in the CT26.WT colon model was demonstrated using a novel LC/MS-MS method of detection. Dose dependent occupancy of PD-1 receptors by anti-PD-1 in CT26.WT tumors was also observed.

Our results demonstrate a significant anti-tumor response across five immune checkpoint blocking antibodies in the CT26.WT colon cancer model; however, immune response profiles are notably different across these blocking antibodies. Importantly, a novel LC/MS-MS method was developed specifically in mouse plasma to determine exposure response. Utilizing these multiple data sets will allow for PK/PD modeling to generate expectations for future dose response. This type of comprehensive analysis constitutes a highly-relevant tool to evaluate efficacy and mechanism of action for novel immune-targeted therapies for oncology.

#4625

Mouse strains for cancer research at The Jackson Laboratory Repository.

Deborah Boswell, Stephen Rockwood, Cathleen Lutz, The JAX Repository Team. _The Jackson Laboratory, Bar Harbor, ME_.

Genetically defined mice have been the cornerstone of cancer research and resources at The Jackson Laboratory since the founding of the laboratory nearly nine decades ago. The Jackson Laboratory distributes more than 10,500 strains to the scientific community, many with applications in cancer research, including mouse models for specific cancers, xenograft models, immunodeficient platforms for PDX studies, multi-purpose "tool strains" (such as CRISPR cas9-expressing lines), recombinase expressing strains, as well as conditional and inducible expression lines (such as Cre-lox). New to the JAX Repository collection is a KrasLSL-A146T strain with inducible mutant Kras A146T for use in research of gastrointestinal tract and hematological cancers. The tamoxifen-inducible model for pancreatic ductal adenocarcinoma (PDAC), KPCX, and the related conditional activatable Kras allele/floxed Trp53 double mutant line, KP, have recently been developed. Inducible expression of mutant human speckle-type POZ protein (SPOP) in one newly acquired line allows study of prostate tumorigenesis. The portfolio of strains useful for engraftment studies has expanded, complementing the versatile humanized NOD scid gamma (NSG) platform lines. For custom generation of mutant strains, the Repository distributes a variety of research tool strains such as cas9-expressing lines, both constitutive expressing and Cre-inducible models, on different defined genetic backgrounds. Other recently added mutants allow research of the role of inflammation in cancer and screening immunomodulatory cancer therapies.

In addition to safeguarding each strain by cryopreservation, the Repository's comprehensive quality control program confirms mutation identity and genetic background, and screens for unwanted alleles (such as stray GFP, cre, lacZ, etc.). Investigators can search for specific strains on the JAX Mice website, or peruse the Oncology Therapeutic Area webpage for cancer research related strains and resources. Researchers can submit their strains to be considered for inclusion in the Repository on The Jackson Laboratory website:

www.jax.org/donate-a-mouse

The Jackson Laboratory Repository is supported by the NIH, The Howard Hughes Medical Institute, and other private charitable foundations.

#4626

Use of integrated genomic analyses in patient-derived tumor model to discover new clinical indications for the multikinase inhibitor drug candidate, DBPR216.

Ching-Chuan Kuo,1 Weir-Torn Jiaang,1 Jing-Jim Ou,2 Chiung-Tong Chen,1 Shu-Ching Hsu,1 Chuan Shih,1 Li-Mei Lin,1 Manwu Sun,1 Yi-Hsin Wang,1 Zih-Ting Huang,1 Jang-Yang Chang,3 Shau-Hua Ueng1. 1 _National Health Research Institutes, Zhunan, Miaoli County, Taiwan;_ 2 _Chang Bing Show Chwan Memorial Hospital, Lukang, Taiwan;_ 3 _National Cheng Kung University, Tainan, Taiwan_.

Developing realistic preclinical models using clinical samples that reflect complex tumor biology is critical to advancing cancer research. Patient-derived preclinical tumor models are the optimal tool for understanding drug action patterns and resistance mechanisms. In order to improve the capability of drug R&D in our institute (NHRI-IBPR), we have generated several patient-derived xenograft (PDX) models and characterized the genomic signature and responsiveness to standard-of-care (SOC) therapy. DBPR216, an orally bioavailable multikinase inhibitor, showed potent effect for treatment of gastrointestinal stromal tumors (GISTs) and acute myeloid leukemia (AML) through targeting of c-KIT and FLT-3, respectively. In order to further discover other indications of DBPR216 for clinical application, we investigated the anti-tumor effect of DBPR216 in several in-house PDX models. Among them, we found that DBPR216 was effectively to suppress PDX tumor growth in the immuno-deficient mice in two colorectal adenocarcinoma PDX models, C008 and C015. These PDX models showed similar genomic features with original tumor samples from patients when test using targeted sequencing of cancer related genes. To further identify if DBPR216 is superior to other kinase inhibitors and SOC therapy, we used in vitro PDX cell proliferation assay to quickly examine the anti-tumor effect of DBPR216 compared to a panel of therapeutic drugs. The result demonstrated that DBPR216 appeared to be superior in potency to kinase inhibitors (Regorafenib, Afatinib, Sunitinib, and Imatinib) and SOC therapy (Irinotecan, 5-FU, and Oxaliplatin). Combining the kinase profiling of DBPR216 and mutational analysis of C008 and C015 PDX models, we proposed that DDR2, FLT1, PDGFRα, PDGFRβ, RET, and SRC may be the potential targets of DBPR216 in these PDX models, and need further elucidation. Taken together, we found that DBPR216 exhibits potent anticancer effect against colorectal cancer and may bring the better opportunity than Regorafenib, a therapeutic agent for metastatic colorectal cancer in clinical. DBPR216 is now under preclinical development for further IND submission.

#4627

Mutant p53 regulation in a Li-Fraumeni mouse model.

Tamara Terzian,1 Molly Plehaty,1 Nema Sobhani,1 Wanida Stevens,1 Stefan Marasligiller,1 Tara Srinivas,1 Rohan Mylavarapu,1 Yvonne Clarke,1 Farinaz Arbab,1 Brendon Podell,2 Roderick Bronson3. 1 _Univ. of Colorado Denver, Aurora, CO;_ 2 _Colorado State University, Fort Collins, CO;_ 3 _Harvard Medical School, Boston, MA_.

The p53 tumor suppressor is maintained mainly at low physiological levels in normal cells by its two major inhibitors, Mdm2 and Mdm4. Mdm2 is a E3 ubiquitin ligase while Mdm4 binds to p53 and inhibits its transactivation activity. Deletion of either inhibitor results in p53-dependent embryonic lethality in mice. p53 is one of the most commonly mutated genes in human cancer. While we have gained considerable knowledge about wild-type p53 regulation and mode of action, our understanding of the oncogenic activity of mutant p53 lags behind. We know that mutant p53 is regulated in the same manner as wild-type p53. In a mouse model of Li-Fraumeni syndrome, Mdm2 binds and inhibits the hot spot mutant p53R172H (p53R175H in humans). Here we show that Mdm4, like Mdm2, regulates p53R172H stability in vivo, and that endogenous Mdm2 cannot compensate for this stabilization. Thus, mutant p53 mice that lack Mdm4 have stable high levels of p53R172H and display accelerated tumor development and an increased range of tumor types, compared to mutant p53 mice. Furthermore, when we compared mutant mice with the loss of two different alleles of both negative regulators, Mdm2 and Mdm4 ,we observed an even higher level of p53. This finding reveals that several tissues can tolerate high expression of oncogenic p53 and indicates a collaborative activity of these inhibitors in regulating mutant p53. The analysis of these mice uncovered new and unexpected functions of mutant p53 that we will discuss for the first time at the AACR 2019 meeting.

#4628

A clinically relevant mouse model to understand how IMiDs modulate the host-tumor immunolandscape in multiple myeloma.

Seth J. Welsh, Meaghen E. Sharik, Victoria M. Garbitt, Link R. Taylor, Daniel L. Riggs, Caleb Stein, Grady Day, Courtney J. Hillukka, Zach J. Hammond, P Leif Bergsagel, Marta Chesi. _Mayo Clinic, Scottsdale, AZ_.

Multiple myeloma (MM) is a predominantly incurable form of plasma cell cancer. Although immunomodulatory imide drugs (IMiDs) such as thalidomide, lenalidomide, and pomalidomide constitute the backbone of most MM therapies, little is understood about how these drugs function to alter the tumor/microenvironment in vivo. Recently, it was demonstrated that IMiDs bind to the substrate receptor protein cereblon (CRBN) leading to the ubiquitination and degradation of multiple neoantigen protein targets, often DNA-binding zinc finger regulatory proteins such as ZFP91, IKZF1 and IKZF3. Interestingly, CRBN differs in amino acid sequence between mice and humans rendering mice non-responsive to IMiDs' effects. This difference has made it difficult to study the complex pleotropic actions of IMiDs in vivo. Consequently, our lab has generated an immunocompetent transgenic mouse that expresses full-length human CRBN under the control of its endogenous regulatory elements (hCRBN+). We crossed this mouse with our well-established Vk*MYC mouse to generate a novel humanized Vk*MYChCRBN+ strain that develops human-like multiple myeloma disease, is sensitive to IMiD therapy, and maintains a fully competent immune system. The purpose of generating our Vk*MYChCRBN+mouse is to determine the mechanism of action of IMiDs on the tumor only, the host only, and to understand the cross-talk between tumor and microenvironment in a clinically relevant in vivo model of MM. We find that hCRBN+ mice are sensitive to IMiD treatment in vivo by displaying degradation of known target proteins such as Ikzf1/Ikzf3 in splenocytes. We also show that non-tumor immune cells such as T and NK cells respond to IMiD treatment by increased proliferation and expression of effector molecules such as IL-2 and IFN-γ. Furthermore, by engrafting IMiD-sensitive Vk*MYChCRBN+ MM cells into an IMiD-insensitive WT host we establish that IMiDs have tumor-intrinsic effects in vivo that are independent of the host's immune system. These tumor-intrinsic IMiD effects synergize not only with dexamethasone, which has been shown clinically, but also with enhancer-targeting BET or p300/CBP inhibitors. Conversely, when engrafting IMiD-insensitive Vk*MYC MM cells into an IMiD-sensitive hCRBN+ host we were unable to demonstrate significant IMiD-induced host immune activation or tumor suppression. We conclude, based on our preliminary data, that IMiD activity is predominantly tumor-intrinsic. Moving forward, we are currently utilizing single-cell RNA sequencing to map the cellular and transcriptional changes across time in both tumor cells and the immune microenvironment during IMiD treatment. Ultimately, our goal is to gain mechanistic understanding in order to optimize clinical practice.

#4629

**FOXA2 controls tumor-associated inflammation in** KRAS **-mutant lung cancer.**

Koichi Tomoshige, Minzhe Guo, Iris Fink-Baldauf, William Stuart, Yutaka Maeda. _Cincinnati Children's Hospital Medical Ctr., Cincinnati, OH_.

Background: Although the role of the transcription factor FOXA2 in lung cancer has been investigated using mouse and human lung cancer cell lines, to our knowledge, the role of FOXA2 in autochthonous lung tumors in GEMM (Genetically Engineered Mouse Model) has not been determined.

Methods: In order to determine the role of FOXA2 in autochthonous lung tumor cells, we deleted FOXA2 in lung epithelia of Kras-mutant lung cancer model mice (Scgb1a1-Cre;[tetO]-Kras4bG12D;Foxa2flox/flox, a GEMM) and assessed survival and lung histology of the GEMM.

Results: Deletion of FOXA2 in autochthonous Kras-mutant lung tumor cells significantly extended the survival of Kras-mutant lung cancer model mice (median survival from 8 weeks to 23 weeks; n>14 for each group). Histological analysis indicated that FOXA2 was required for the growth of lung tumor cells and the recruitment of tumor-associated macrophages.

Conclusion: FOXA2 promotes primary KRAS-mutant lung tumors in part by controlling tumor-associated inflammation.

#4630

In vivo **characterization of the Duffy antigen receptor for chemokines (DARC/ACKR1) in breast cancer tumor progression.**

Rachel N. Martini,1 Brittany D. Jenkins,1 Lisa A. Newman,2 Nancy Manley,1 Melissa B. Davis3. 1 _University of Georgia, Athens, GA;_ 2 _Weill Cornell Medical College, NY;_ 3 _Weill Cornell Medical College, New York, NY_.

In studies of the tumor microenvironment (TME), factors that influence immune cell infiltration are of great interest, as these populations can influence disease prognosis, and potential treatment for patients. Through in silico analysis of TCGA breast cancer (BC) cohort data, we have identified the Duffy antigen receptor for chemokine/atypical chemokine receptor 1 (DARC/ACKR1) as a potential driver of immune cell infiltration in the BC TME. DARC/ACKR1 is an atypical chemokine receptor that modulates levels of chemokine both in circulation through expression on red blood cells, and participates in chemokine transcytosis in tissues through expression on both post-capillary venules of endothelial cells and epithelial cells. As chemokines establish gradients to recruit specific immune cell types, we hypothesize that the function of DARC/ACKR1 in chemokine level modulation also influences tumor-associated immune cell levels. In the TCGA BC cohort, we observe a strongly positive and significant correlation between DARC/ACKR1 expression and total number of tumor-associated leukocytes, specifically populations of B cells, T cell, monocytes and macrophages. To further characterize the role of DARC/ACKR1 in vivo, we have established a novel DARC knock-out BC transgenic mouse model to study DARC/ACKR1 in the BC TME. To develop our target experimental mice, Ackr1-/- female mice were crossed with Ackr1 +/-; C3(1)Tag +/0 to generate the target Ackr1 -/-; C3(1)Tag +/0 experimental mice. Experimental mice and the littermate controls were aged to 3.5 months, at which point they were euthanized. Blood was collected from the mice to characterize the circulating chemokine profile. Mammary glands containing tumors were fixed and paraffin embedded, followed by IHC and immunofluorescent staining for expression of DARC/ACKR1, target chemokines, immune cells, epithelial cells and endothelial cells. The remaining glands were dissociated, followed by fluorescent-activating cell sorting analysis (FACS) to quantify abundance of immune cells in the gland. Using these methods, we have defined the circulating chemokine profile alongside patterns of immune cell infiltration in our target DARC/ACKR1 knockout BC mice compared to littermate controls in mouse mammary tumors.

#4631

Modeling the vascular sarcoma spectrum with genetically engineered mice.

Jason A. Hanna, Casey G. Langdon, Matthew R. Garcia, David Finkelstein, Jerold E. Rehg, Mark E. Hatley. _St. Jude Children's Research Hospital, Memphis, TN_.

Angiosarcomas are highly aggressive vascular sarcomas with an extremely poor prognosis. Angiosarcomas can develop spontaneously or are associated with prior radiation, chronic lymphedema, or exposure to toxic chemicals such as vinyl chloride. Sequencing efforts have identified a number of genetic alterations in angiosarcoma, however the genetic drivers and actionable targets remain unclear. Despite the poor outcome for patients, there are limited resources for studying angiosarcoma, highlighting the need for genetic, in vivo models to better elucidate the underlying biology of the disease. In studying the role of DICER1 and microRNAs in mouse models of tumorigenesis, we found that Dicer1 deletion with aP2-Cre leads to aggressive and metastatic angiosarcoma development with 100% penetrance. ERK and S6 hyperphosphorylation in the aP2-Cre;Dicer1cKO (AD) tumors suggest Dicer1 loss results in activation of the RAS-MEK-ERK and mTOR pathways. To determine if direct activation of the these pathways could similarly transform aP2-Cre expressing cells we first interrogated the combination of oncogenic KrasG12D with Cdkn2a inactivation. We found that aP2-Cre;LSL-KrasG12D;Cdkn2acKO (AKC) mice rapidly develop angiosarcomas providing an accelerated model for assessing cooperating alleles and therapies. To activate the mTOR pathway we tested conditional Tsc1 deletion with aP2-Cre and found that all aP2-Cre;Tsc1cKO (AT) animals develop vascular tumors in the paws. In contrast to the AD and AKC tumors, AT tumors express markers of lymphatic endothelial cells such as PROX1. In addition, the tumors display a distinct nodular spindle cell-like morphology that is consistent with a low grade angiosarcoma resembling human kaposiform hemangioendotheliomas, a vascular tumor occurring predominantly in the extremities of infants. The distinct onset, growth kinetics, anatomic locations, and histologic presentation of the tumors from AD, AT, and AKC mice provide a mechanism to interrogate the drivers of angiosarcoma in less aggressive AT paw tumors and more aggressive AD and AKC tumors. Furthermore, the aP2-Cre driven mouse models provide a platform to study angiosarcoma initiation, progression, and metastasis for the identification of novel therapeutics to improve outcomes for this understudied and devastating disease.

#4632

A new mouse model for rapid identification of key factors driving prostate cancer progression and invasiveness.

Maria Riedel,1 Latifa Bakiri,2 Martin F. Berthelsen,1 Michael Borre,1 Mikkel H. Vendelbo,1 Erwin F. Wagner,2 Martin K. Thomsen1. 1 _Aarhus University, Aarhus, Denmark;_ 2 _National Cancer Research Center (CNIO), Madrid, Spain_.

Prostate Cancer is amongst the most frequently diagnosed malignancies and the second leading cause of cancer-related death in men worldwide accompanied by an increasing incidence. Until now its heterogeneity has been a major challenge in the establishment of good in vivo models for fast validation of potential driver genes.

Classic in vivo models are costly, time-consuming and often target the majority of the prostate epithelium. We successfully introduced a novel prostate cancer mouse model based on CRISPR/Cas9 technology, ensuring multiplexed gene editing.

In this model, specific in vivo gene editing was obtained in murine prostate epithelium cells of a transgenic mouse strain, harboring the CRISPR associated protein 9 (Cas9) endonuclease. Prostate epithelium cells were transduced by an Adeno-associated virus (AAV), carrying multiple single guide RNAs (sgRNA).

Genetically different viral constructs were designed, each expressing different sgRNA combinations against Pten, representing the main driver in prostate cancer, tumor suppressor Trp53 and either one of the AP1 transcription factor subunits Junb or Fos, or tumor suppressor Smad4.

Since viral transduction occurs in only a few cells, edited cell clones can clonally expand and undergo a natural selection process. Furthermore, the simultaneous gene knockouts reflect the human scenario of tumor heterogeneity.

Mouse prostates were isolated and analyzed three to nine months post-injection to obtain insight on whether or not a gene appears crucial for tumor development in a specific tissue and wherein different gene combinations correlate with tumor severity. Histological analysis revealed increased proliferation, increased AKT activation, as well as invasiveness in samples with multiple gene knockouts compared to controls with single Pten knockout. Furthermore, loss of SMAD4 accelerated cancer progression when compared to the loss of the AP-1 transcription factor. Surprisingly, knockout of Trp53 was rarely observed in prostate samples while it occurred frequently in other tumor types in the same model system. This indicates that knockout of Trp53 may not be required for prostate cancer initiation.

Overall, we established an in vivo model system of simultaneous, multiplexed gene editing in the prostate epithelium to address the biological cross-talk between various, altered pathways in prostate cancer initiation and progression.

#4633

Upregulated claudin-2 expression in ulcerative colitis protects from colitis-associated cancer.

Rizwan Ahmad*,1 Balawant Kumar*,1 Narendra Kumar,1 Ishwor Thapa,2 Kiran D. Bastola,2 Mary Kay. Washington,3 Punita Dhawan,4 Amar B. Singh5. 1 _University Nebraska Medical Center, Omaha, NE;_ 2 _University of Nebraska Omaha, Omaha, NE;_ 3 _Vanderbilt University Medical Center, Nashville, TN;_ 4 _University Nebraska Medical Center, VA Nebraska-Western Iowa Health Care System, Omaha, NE;_ 5 _University Nebraska Medical Center,VA Nebraska-Western Iowa Health Care System, Omaha, NE_.

A link between chronic inflammation and colon cancer has long been appreciated. Accordingly, patients with inflammatory bowel (IBD) are at higher risk for developing colitis-associated cancer (CAC). Mucosal barrier dysregulation allies with inflammation and accordingly claudin-2, a tight junction protein, is upregulated in IBD and CAC, and believed to assist in disease progression. Remarkably, recent studies, including ours, using mice manipulated for claudin-2 expression challenge such a belief as claudin-2 transgenic (Cldn-2TG) mice are protected from colitis while Cldn-2KO mice have severe disease. Immune adaptation and mucosal healing appear to help mediate claudin-2 dependent protection from colitis. Of note, claudin-2 expression is restricted to the crypt base and associates with paracellular permeability, differentiation and proliferation. Notably, mucosal healing in IBD patients is associated with decreased risk of CAC. However, a causal role of claudin-2 in progression to the CAC remains unclear. Based on the mice data, we hypothesized that claudin-2 also protects from the CAC.

To test this hypothesis, WT and Cldn-2TG mice were subjected to the murine model of AOM/DSS cancer. Indeed, Cldn-2TG mice demonstrated significant protection against the CAC (p<0.001). Both, tumor incidence and growth (p<0.001) were inhibited. High throughput analysis (RNAseq and Cytokine array) demonstrated significant inhibition of the pro-inflammatory signaling in AOM/DSS-treated Cldn-2TG versus WT-mice. In contrast, growth-promoting signaling was upregulated. An in-vitro model of colitis-injury and recovery further demonstrated positive association between claudin-2 expression and cell viability. Based on these findings that increased colonic claudin-2 expression induces an immune suppressive and growth promoting effect, we further postulated a tumor promoting role for claudin-2 in sporadic colon cancer (CRC). Remarkably, offspring mice from the cross between Cldn-2TG and APCmin mice (APCmin/Cldn2TG) demonstrated significant increases in colon tumor burden and progression. RNAseq analysis from these mice further attested to an immune suppressive and growth-promoting effects of increased claudin-2 expression. To further validate, we subjected the APCmin and APCmin/Cldn2TG mice to DSS in drinking water as it potentiates tumor burden in the APCmin mice. Remarkably, DSS-induced potentiation of colon tumorigenesis was significantly lower in the APCmin/Cldn2 mice versus APCmin mice.

Taken together, we here report a novel and paradigm-shifting role of claudin-2 in protection against the CAC. Moreover, considering the significant role of the immune signaling in CRC and failure of the effectiveness of immune therapy, our murine models can help understand the dynamics of immune and growth signaling in promoting colon cancer, for therapeutic gains.

* Both authors contributed equally

#4634

Mouse model for nodal marginal zone lymphoma.

Victor Yazbeck, Ian McConnell, Joseph Lownik, Ariel Sindel, Roy Sabo, Alden Chesney, Guanhua Lai, Adolfo Mauro, Jamal Zweit, Rebecca Martin, Daniel Conrad, Steven Grant, Jolene Windle, Steven Grossman. _VCU Massey Cancer Center, Richmond, VA_.

Introduction: Indolent B-Cell Non-Hodgkin's Lymphoma (i-NHL) represents a heterogeneous group of lymphoproliferative malignancies, encompassing 40% of NHL, that remains largely incurable. The B-cell receptor signaling pathway is activated in B-cell malignancy and mediates its activity mainly through the Phosphoinositide 3-kinase (PI3K) pathway. Furthermore, novel PI3K inhibitors, such as idelalisib and copanlisib, have shown impressive clinical activity in several indolent lymphomas including marginal zone lymphoma (MZL). This further supports the important role of the PI3K pathway in these tumors.

Methods We generated a genetically engineered mouse model carrying heterozygous knockout alleles of both the tumor suppressor genes Phosphatase and Tensin Homolog (PTEN) and Liver Kinase B1 (LKB1), leading to over-activation of the PI3K-mTOR pathway in all mouse tissues. We closely monitored these mice for tumor formation by at least weekly physical examinations for several months. Upon tumor detection, tumor size was recorded weekly using calipers, with an experimental endpoint of 15-20mm in any dimension. One half of the tumor was immediately preserved in a 4% paraformaldehyde solution and prepared for sectioning, H&E and immunohistochemical staining.

Results: Thirty mice died or were sacrificed due to disease progression, defined as either lymph node enlargement and/or splenomegaly. All mice showed either abnormal lymphadenopathy or splenomegaly. By Kaplan-Meier analysis, we saw a steady decrease in both tumor-free and overall survival after 3 months of age. Utilizing the product limit method, the median survival time was 6 months (95% CI: 6, 8). A total of 51 lymph nodes were sent for IHC and pathological identification. Of the 51 nodes, 61.5% (N=32) showed indolent Non-Hodgkin's Lymphoma, 25% (N=13) were atypical, and 11.5% (N=6) were reactive. All lymph nodes with indolent NHL were Marginal Zone subtype. In order to generate a more specific model of B cells, we used the Cre/LoxP system with CD19-Cre. We detected an increase in the average portion of MZL B cells in the spleen of the homozygous mice compared to wild type (38.3% vs 4.9%, p=0.0216).

Conclusion: Nodal marginal zone lymphoma remains an incurable indolent lymphoma that lacks preclinical models. As novel agents become available, it is important to have a better understanding of the underlying pathogenesis of this malignancy and be able to model it in an immunocompetent mouse with a preserved microenvironment. Our data provides, for the first time, a proof of concept on the role of the PI3K-mTOR pathway in the pathogenesis of nodal marginal zone lymphoma and paves the way for future studies understanding the biology of this disease, and developing rational therapies for this incurable malignancy.

#4635

Newly established mouse oral carcinoma cell lines and their syngeneic tumorigenesis models.

Yi-Fen Chen,1 Chung-Ji Liu,1 Shou-Yen Kao,2 Shu-Chun Lin,1 Kuo-Wei Chang1. 1 _National Yang-Ming Univ. School of Dentistry, Taipei, Taiwan;_ 2 _Taipei Veterans General Hospital, Taipei, Taiwan_.

Oral squamous cell carcinoma (OSCC) is one of the most prevalent malignancies worldwide. Despite the advances in diagnosis and treatment, the survival of OSCC remains to be improved. miR-211 is an oncogenic microRNA frequently disrupted in human OSCC and its high expression is a determinant of patient's poor survival. Herein, we established four murine OSCC cell lines, designated MOC-L1 - MOC-L4 from the tongue tumor tissues induced by 4-nitroquinoline 1-oxide in K14-EGFP-miR-211 transgenic mice. All cell lines appear green fluorescence and express epithelial markers. The gene expression profiles among cell lines are complex and diverse, while MOC-L1 - MOC-L3 cells carry missense mutations in p53 gene. MOC-L1 exhibits tremendous epithelial-mesenchymal transition and the associated aggressive characteristics. On the contrary, MOC-L4 displays the least invasiveness. Both MOC-L1 and MOC-L2 are clonogenic in vitro and tumorigenic when implemented into dermis or tongue in syngeneic recipients. But, only MOC-L1 exhibits high potential for local regional and distal metastasis. Since the expression of miR-196b in MOC-L1 xenografts drastically decreased upon cisplatin treatment, targeting of miR-196b might facilitate tumor abrogation. As these cell lines originate from the C57BL/6 mouse, which is the strain most suitable for transgenic engineering, to approach the interplay of these OSCC cells with other type of genetically modified cells in immune-competent mice would bestow profound insight on OSCC pathogenesis. 

### Mutagenesis, Chemical Carcinogenesis, and Tumor Initiation/Promotion

#4636

Oncogenic Notch triggers neoplastic-tumorigenesis in a transition-zone like tissue microenvironment.

Wu-Min Deng, Sheng-An Yang. _Florida State University, Tallahassee, FL_.

The "seed and soil" hypothesis proposed by Stephen Paget (1889) highlights the importance of tissue microenvironment for secondary tumor formation. The microenvironment that promotes primary tumor origination, however, remains largely unclear. Transition zones, where two types of epithelial tissue meet, have been characterized as high-risk sites for tumorigenesis. Here we show that the Drosophila salivary gland (SG) imaginal ring (ImR) can be used as a model to study tumorigenesis in transition zones. We found that constitutive activation of Notch signaling during the third larval instar drives overproliferation and tumor formation in the SG ImR. Interestingly, tumorigenesis always occurs at the posterior end of the ImR, which is a transitional area between the polyploid giant cells and diploid ImR cells. These Notch-induced tumors display disrupted epithelial organization, grow continuously and can metastasize once transplanted into the abdomen of adult flies, suggesting that they have the characteristics of malignant neoplasms. Further analyses revealed that the SG ImR transition zone possesses endogenously high levels of Janus kinase/signal transducers and activators of transcription (JAK-STAT) and c-Jun amino-terminal kinases (JNK), both of which are necessary for tumor growth. In this region, JNK signaling induces the expression of matrix metalloproteinase 1 (MMP1), which is also required for tumor formation in this model system. Furthermore, we found that ectopic MMP1 expression can transform the anterior end of the SG ImR, which is normally refractory to oncogenic Notch-induced tumorigenesis, into a tumor hotspot. Together, these studies reveal that local endogenous activation of JNK and JAK-STAT signaling creates a niche-like tissue microenvironment that is susceptible to oncogene-induced neoplastic tumor formation.

#4637

Non-stem cell-driven colonic tumors retain an intestinal differentiation hierarchy.

Cherie R. Scurrah, Alan J. Simmons, Eunyoung Choi, Robert J. Coffey, James R. Goldenring, Ken S. Lau. _Vanderbilt University, Nashville, TN_.

Colorectal cancer (CRC) is the third leading cause of cancer mortality in the United States. Cancer stem cells (CSCs) play critical roles in therapeutic resistance, tumor recurrence, and metastasis that lead to patient death. We propose to evaluate the properties of CSCs in the context of the tumor cell-of-origin, the cell that initiates oncogenesis. We hypothesize that the tumor cell-of-origin is directly related to the CSCs present in tumors that consequently arise. To test this hypothesis, the stem-like properties of CSCs from stem cell- and non-stem cell-driven tumors, initiated from the Lrig1 and Mist1 promoters, respectively, were analyzed and compared through the use of lineage mapping and single-cell RNA-sequencing. We observed that although Mist1-expressing non-stem cells do not exhibit stem cell behavior under homeostatic or damage conditions, they can serve as tumor cells-of-origin. These tumors maintain a differentiation hierarchy that is lacking in stem cell-driven tumors. These results suggest that the origin of tumorigenesis influences tumor CSCs and thus tumor characteristics which could be utilized for the development of novel therapies.

#4638

Colorectal adenoma-to-carcinoma progression: The role of miR-17-92 cluster.

Sanne R. Martens-de Kemp,1 Malgorzata A. Komor,1 Rosa Hegi,1 Marianne Tijssen,1 Anne S. Bolijn,1 Gerrit A. Meijer,1 Connie R. Jimenez,2 Remond J. Fijneman,1 Beatriz Carvalho1. 1 _The Netherlands Cancer Institute, Amsterdam, Netherlands;_ 2 _Amsterdam University Medical Center, Amsterdam, Netherlands_.

Background Chromosomal instability plays an important role in the progression of colorectal adenoma to carcinoma. We previously observed that specific non-random DNA copy number changes (8q, 13q, 20q gain, and 8p, 15q, 17p, 18q loss) were already present in the benign adenoma component of malignant polyps (i.e. adenomas with a focus of cancer). This indicates that these specific DNA copy number changes are associated with the progression from adenoma to cancer. DNA copy number dosage affects the expression of different loci, which may play an important functional role in the colorectal adenoma-to-carcinoma progression. We observed that overexpression of the miR-17-92 cluster (located on 13q) is associated with gain of 13q, one of the cancer associated DNA copy number changes. The aim of the present study was to investigate the biological meaning of miR-17-92 overexpression in benign adenomas and its role in adenoma-to-carcinoma progression.

Methods Patient-derived colorectal adenoma organoids were used as model system. Two different background adenoma organoids were transduced with a miR-17-92 expression vector. Overexpression of members of the miR-17-92 cluster in the transduced adenoma organoids was confirmed by real-time quantitative RT-PCR for all individual miRNAs. Both miR-17-92-overexpressing organoids and their counterparts containing empty vectors (controls) were subjected to mRNA sequencing and subsequent differential gene expression analysis and subsequent establishement of a miR-17-92 gene expression signature. Enrichment of the miR-17-92 gene signature was evaluated in an independent series of 52 colorectal adenoma and carcinoma tissue samples. In vitro proliferation rates as well as invasion capacity of the transduced organoids were evaluated by measuring the size of the organoids and by using transwell invasion assays, respectively.

Results We successfully overexpressed the miR-17-92 cluster in the two adenoma-derived organoids. Expression of 42 genes was significantly different (FDR<0.05, >4 fold difference) between organoids transduced with the miR-17-92 cluster and those transduced with the empty vector. In addition, in the series of 52 tumor tissue samples this gene signature could separate adenomas from carcinomas and was enriched in tumours with 13q gain. In vitro functional assays showed that proliferation capacity did not change after overexpression of the miR17-92 cluster in adenoma organoids, but capacity to invade was acquired.

Conclusion We confirmed that overexpression of the miR-17-92 cluster leads to downstream gene expression alterations associated with colorectal cancer. Moreover, we showed that overexpression of this miRNA cluster promotes invasion of adenoma organoids in vitro. These results support a role of this locus in the progression from adenoma to carcinoma.

#4639

SLC7A11 **expression confers cancer stem-like properties in small cell lung cancer cells.**

Shohei Kamenori,1 Kentaro Suina,2 Juntaro Yamasaki,1 Subaru Shintani,1 Yuji Otsuki,1 Yuki Hirata,1 Shogo Okazaki,1 Kenji Tsuchihashi,3 Oltea Sampetrean,1 Yoichiro Mitsuishi,2 Fumiyuki Takahashi,2 Kazuhisa Takahashi,2 Hideyuki Saya,1 Osamu Nagano1. 1 _School of Medicine Keio University, Tokyo, Japan;_ 2 _Juntendo University Graduate School of Medicin, Tokyo, Japan;_ 3 _Kyushu University Graduate School of Medical Sciences, Tokyo, Japan_.

Small Cell Lung Cancer (SCLC) which accounts for 15 % of primary lung cancers is known to grow fast and metastasize easily to whole body at early stages. However, the standard care of SCLC has not been changed for over 10 years and effective treatment remains to be developed. System xc(-) comprises xCT (SLC7A11) and CD98hc subunits and is a major plasma membrane antiporter responsible for the cellular uptake of cystine in exchange for intracellular glutamate. Recently, we studied the impact of xCT inhibitor sulfasalazine (SSZ) in various cancers and found that SCLC cells manifest low level of xCT and are highly sensitive to SSZ treatment compared with non-small cell lines. Furthermore, we found that xCT expression in SCLC cells is regulated by the cellular density and the tumorsphere culture which is known to enhance the cancer stem cell properties also enhances the xCT expression in these cells. To examine the functional relevance of xCT expression to cancer stem-like properties in SCLC cells, we established the stably xCT-expressing SBC-5 cells (SBC5-xCT). Similar to other SCLC cell lines, parental SBC5 cells were not able to proliferate from single-cell level, however, SBC5-xCT cells were found to possess an ability to proliferate from single-cell level. Furthermore, SBC5-xCT cells manifested higher resistance to an anticancer agent cisplatin and higher ability of tumor-initiation than parental SBC5 cells. Our findings establish a new functional role for xCT in the promotion of cancer stem-like properties in SCLC cells.

#4640

Deregulation of MITF in the context of inactive MC1R leads to melanocyte transformation.

Tine Norman Alver,1 Timothy J. Lavelle,1 Karen-Marie Heintz,1 Patrik Wernhoff,1 Vegard Nygaard,1 Geir F. Øy,1 Sigurd L. Bøe,1 Alfonso Urbanucci,1 Eivind J. Hovig2. 1 _Oslo University Hospital, Oslo, Norway;_ 2 _Oslo University Hospital and University of Oslo, Oslo, Norway_.

MC1R, the cAMP pathway and the response to solar UV are all elements involved in the growth, differentiation and survival of melanocytes and in malignant melanoma. Here we present a model of melanomagenesis based on the forced expression of MITF-M in the context of MC1R-inactivating variants in a way not dependent on sun exposure. We demonstrate that lentiviral transduction of MITF-M alone leads to consistent transformation of the Hermes4C cell line with inactive MC1R, but not the Hermes3C cell line having a wild type background. Hermes 4c cells transduced with MITF-M are able to form tumors in mice. We mapped MITF binding to chromatin and found that 4C cells displayed enhanced MITF chromatin binding compared to 3C. Expression analysis revealed that the altered binding pattern leads to enhanced transcription of epithelial to mesenchymal transition genes and consequent repression of key melanocyte-specific genes e.g. involved in pigmentation in the background of inactive MC1R. We observed a marked deregulation of AXL and EGFR, which is accompanied by downregulation of PTEN and increased phosphorylation of ERK and PI3K-AKT pathway. In conclusion, we show that MITF-M is a molecular switch in the context of MC1R-inactivating variant which leads to cell reprogramming and melanomagenesis.

#4641

Metabolic reprogramming of the breast contributes to a cancer promoting milieu.

Natascia Marino,1 Rana German,2 Xi Rao,1 George Sandusky,1 Max Jacobsen,1 Sha Cao,1 Anna Maria Storniolo2. 1 _Indiana Univ.-Purdue Univ. Indianapolis, Indianapolis, IN;_ 2 _Susan G. Komen Tissue Bank at IU Simon Cancer Center, Indianapolis, IN_.

In addition to the accumulation of pro-oncogenic mutations in the epithelial cells, the tumorigenic process involves the dysregulation of the interactions between the epithelial cells and their microenvironment, as well as alterations within the microenvironment itself. The latter is composed of endothelial cells, immune cells, fibroblasts, unaffected epithelium, and adipocytes. Increasing evidences support that cell transformation progresses in a potentially cancer-promoting microenvironment context, also known as field cancerization model. Therefore, it is critical to investigate changes occurring in both the epithelial compartment and the surrounding microenvironment to understand how breast cancer initiates.

In our preliminary study, we analyzed the transcriptome profile of microdissected breast tissue compartments (epithelium, stroma and adipose tissue) from tissue biopsies obtained from women who donated their histologically normal tissue two-to-five years before breast cancer diagnosis (here labeled pre-cancer), and matched healthy control donors. In the pre-cancer breast epithelium we detected a significant increase in lipid metabolism-related genes including lipases (HSL, LPL) and perilipins (PLIN1, PLIN4), which mediate the release of fatty acids from triacylglycerol storage, and ELOVL5 and ELOVL7, which generate oleic acid. Moreover, Acyl-CoA Synthetase Medium Chain Family Member 1, ACSM1, involved in the activation of lipoic acid, an essential cofactor for mitochondrial metabolism, is also upregulated in pre-cancer breast tissue. Upregulation of genes involved in metabolic activation is observed also in the stroma and adipose tissue compartments. Interestingly, the transcriptome profiling of the pre-cancer breast stroma shows the downregulation of genes coding for several immune cell markers as compared with the healthy controls. Immunohistochemical staining of CD45 confirms a significant reduction in immune cells in the pre-cancer versus matched healthy control breasts. This finding suggests the prevalence of an immune-suppressive environment in the breast long before the clinical diagnosis of breast cancer.

Understanding the changes occurring in the cancerized field is critical for elucidating 1) the involvement of microenvironment in cancer initiation 2) the role of histologically normal yet genetically altered surgical margins in risk of disease recurrence upon lumpectomy for early breast cancer diagnosis, and 3) the mechanisms of field cancerization in the contralateral breast.

#4642

RNA interference reveals tumor promoting roles of integrin alpha 6 (ITGA6) in hepatocellular carcinoma.

Guixi Zheng, Carla Zeballos, Hakim Bouamar, Matyas Cserhati, Francisco G Cigarroa, Lu-zhe Sun. _University of Texas Health Science Center, San Antonio, TX_.

Introduction: Hepatocellular carcinoma (HCC) is the sixth most common malignancy worldwide and the third leading cause of cancer related mortality. The incidence and mortality rates of HCC are two times higher in Latinos than in the general population in the US and are the highest in Latinos from the South Texas region. The genetic and epigenetic events associated with the increased incidence of HCC in this population are still unclear. Increasing evidence suggests that integrins are one of the most important receptors for cell metastasis including integrin α6β1 and α6β4. However, few studies have focused on the function of integrin alpha 6 (ITGA6) in the tumorigenesis and progression of HCC. We aim to investigate the expression and potential roles of ITGA6 in HCC.

Materials and Methods: Paired HCC tissues and adjacent non-tumor tissues were collected for RNA sequencing. ITGA6 RNA and protein expression levels were evaluated by RNA sequencing, RT-qPCR, Western blot and immunohistochemistry. HCC cell lines (SNU398 and Huh7) were transiently transfected with two ITGA6 siRNAs and stably transfected with an ITGA6 shRNA lentivector. These cell lines were used for assays testing the effects of ITGA6 knockdown on HCC cell proliferation, migration and anchorage independent growth.

Results: Analysis of RNA sequencing data indicated that the expression of ITGA6 increased 4-fold in HCC tumor tissues compared to adjacent non-tumor tissues, which was validated by RT-qPCR. Western blotting also confirmed increased expression of ITGA6 in the tumor tissues. The knockdown of ITGA6 by siRNAs and shRNA was confirmed with Western blot and qRT-PCR. ITGA6 knockdown significantly decreased proliferation, migration and anchorage independent growth of HCC cell lines.

Conclusions: ITGA6 is upregulated in HCC tumors in Latinos patients. ITGA6 may play a malignant-promoting role in HCC cells. Our studies provided new insights into the molecular mechanisms that drive HCC progression and potential therapeutic targets for treating patients with HCC including South Texas Latino patients.

#4643

Mutant ASXL1 collaborates with HHEX to promote myeloid leukemogenesis.

Shuhei Asada,1 Reina Takeda,1 Daichi Inoue,2 Susumu Goyama,1 Toshio Kitamura1. 1 _Institute of Medical Science, The University of Tokyo, Tokyo, Japan;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY_.

An epigenetic modulator Additional sex combs-like 1 (ASXL1) is recurrently mutated in myeloid neoplasms and its mutations are associated with poor prognosis. Recently, we generated mutant Asxl1 conditional knock-in (Asxl1-MT KI) mice mimicking human ASXL1 E635RfsX15 mutation, one of the most common mutations in myeloid neoplasms (Nagase et al. JEM 2018). Retrovirus-mediated insertional mutagenesis study exhibited susceptibility of Asxl1-MT KI bone marrow cells to myeloid leukemia, and we identified Hematopoietically expressed homeobox (Hhex) gene as one of the common retrovirus integration sites. In this study, we investigated the potential cooperation between the mutant ASXL1 and HHEX in myeloid leukemogenesis. We first performed colony-forming assay and found that forced expression of HHEX enhanced colony replating activity and blocked myeloid differentiation in bone marrow hematopoietic stem progenitor cells (HSPCs) derived from ASXL1-MT KI mice, while it showed only modest effect in normal HSPCs. The synergistic effect between the mutant ASXL1 and HHEX in blocking myeloid differentiation was also observed in human HL-60 cells. We next evaluated the role of endogenous Hhex in the mutant ASXL1-expressing cells. Depletion of endogenous Hhex using CRISPR-Cas9 system ameliorated mutant ASXL1-induced differentiation block in 32Dcl3 cells. Depletion of endogenous Hhex in murine mutant ASXL1-expressing leukemia cells [cSAM cells: cells with combined expression of SETBP1 and ASXL1 mutations (Inoue et al. Leukemia 2015), cRAM cells: cells with combined expression of RUNX1 and ASXL1 mutations (Nagase et al. JEM 2018)] also promoted differentiation and increased apoptosis. Furthermore, Hhex deletion profoundly attenuated the colonogenicity of cSAM and cRAM cells and leukemogenicity of cSAM cells. We then investigated target genes of the mutant ASXL1 and HHEX in myeloid neoplasms using public database and our previous RNA-Seq data. Among the potential target genes of the mutant ASXL1 and HHEX, we found that Myb, Etv5 and Oraov1 genes were upregulated by the mutant ASXL1 and HHEX in murine HSPCs. Conversely, Hhex depletion resulted in downregulation of these genes both in cSAM and cRAM leukemic cells. In addition, depletion of Myb, Etv5 or Oraov1 genes significantly abrogated the colonogenicity of cSAM cells. These data suggest that mutant ASXL1 and HHEX cooperatively induce myeloid leukemogenesis via dysregulating Myb, Etv5 and Oraov1.

#4644

Genetic authentic models in squamous cell carcinoma.

Vicente Planells-Palop, Aaron Tward. _UCSF, San Francisco, CA_.

Head and neck squamous cell carcinoma (HNSCC) accounts for approximately 4% of all primary malignancies in the US. Despite advances in our understanding of the underlying biological mechanisms, we lack targeted biological therapies to treat preneoplastic lesions and their associated malignant counterparts. Genomic studies have made strong predictions of the identities of somatic mutations that are likely to play a pathogenic role in the initiation, progression, and maintenance of dysplasia and HNSCC. However, the number of driver mutations and how they interact to reprogram normal cells into pre-cancerous and cancer cells; remains unknown. Hence, we explored the effect of the most statically significant altered genes in HNSCC by disrupting the function of these genes via CRISPR/Cas9. For this purpose, we transfected primary human keratinocytes with plasmids encoding for CRISRP/Cas9 constructs and GFP. Pure populations of genome-edited keratinocytes were isolated by FACS and assessed by TIDE analysis. Results show that mutations in specific genes such as FAT1, results in proliferative advantages in vitro, while individual candidate driver mutations (TP53, CDKN2A and NOTCH1) are fatal in primary keratinocytes. In contrast, mutations in CDKN2A and/or NOTCH1 in a TP53-null background result in viable keratinocytes that show proliferative advantages in vitro. In addition, TP53-deficient keratinocytes harboring extra loss of function mutations in FAT1, CDKN2A and/or NOTCH1 are capable of selecting themselves against untreated keratinocytes in vitro. Furthermore, we assessed the invasion capacity of these genome-engineered cells by seeding them onto 3D organotypic cultures. Our data shows that primary keratinocytes with a single mutation do not show consistent alterations in organotypic cultures; however, keratinocytes harboring double or triple gene knockouts (including TP53) form stratified epithelium with dysplastic signs (irregular stratification, nuclear pleomorphism, keratin pearls). Finally, we identified the minimal number of events that are required for keratinocyte tumorigenic transformation. Primary keratinocytes harboring mutations in TP53, CDKN2A and NOTCH1 are capable of invading through the basal membrane; thus being able to trigger SCC formation. Our fındings provide important evidence for the understanding of the initiation and development of HNSCC. Mutations in these genes are sufficient for carcinogenic transformation and therefore should be considered as markers for evaluating preneoplastic lesions as well as candidates for targeted therapy.

#4645

**Identification of two methylation sites (CpG 388 and CpG 1664) in the promoter/enhancer region of cytochrome P450 (CYP)1B1: Possible role of promoter methylation in attenuation of CYP1B1 expression and DNA adducts by omega 3- fatty acids in mouse lung** in vivo **.**

Bhagavatula Moorthy, Weiwu Jiang, Lihua Wang, Sudha R. Kondraganti, Guodong Zhou, Chun Chu. _Baylor College of Medicine, Houston, TX_.

3-Methylcholanthrene (MC) and benzo[a]pyrene (BP) are polycyclic aromatic hydrocarbons (PAHs) carcinogens. Cytochrome P450 (CYP)1B1 enzymes plays a key role in the activation of PAHs to carcinogenic metabolites, which initiate carcinogenesis by binding covalently to DNA, and the adducts, if not repaired, could lead to tumorigenesis. In this study we tested the hypothesis that pre-treatment of mice with omega-3-fatty acids, i.e. [eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) will lead to attenuation of PAH-mediated pulmonary DNA adduct formation in part by inhibiting CYP1B1. Twelve week old male and female A/J mice received EPA (60 mg/kg) and DHA (40 mg/kg) from day 1 to day 24. Control mice were treated with vehicle corn oil. On day 3, mice were treated with BP (40 µmol/kg) or 3-methylcholanthrene (MC, 40 µmol/kg) by i.p. In the short-term experiment (DNA adduct studies), 3-5 mice from each group were terminated at day 10 (7 days after BP or MC treatment). EPA/DHA significantly suppressed formation of BP-DNA and MC-DNA adducts in lung and liver of both male and female mice. CYP1B1 expression in lung at protein and mRNA levels was induced significantly by PAHs, but was suppressed by about 70% in the lungs of EPA/DHA-treated mice. We tested the hypothesis that PAHs in part induce CYP1B1 by methylating specific CpG sites in the negative regulatory elements of the promoter. Mouse lung DNA samples were subjected to bisulfite deamination, Methylation-Specific PCR (MSP), and a sequencing primer-flanking nested PCR. ClustalW multiple sequence alignment determined the methylation state of a total of 103 putative CpG sites in the CYP1B1 promoter/enhancer region, which included 3 putative CpG islands. We found that CpG 1664, which is located in a putative CpG island, was methylated by BP, and the extent of methylation was decreased by co-treatment with BP and EPA/DHA. BP partially methylated CpG 388 (83%), while EPA/DHA-treated animals showed significantly decreased methylation (33%) at this site. These results suggest that PAHs induce CYP1B1 in part by inducing methylation of CpG 1664 and CpG 388, which might be putative negative regulatory elements of CYP1B1. We hypothesize that suppression of methylation by EPA/DHA at sites CpG 388 and 1664 leads to repression of CYP1B1 expression, which in turn leads to inhibition of PAH carcinogenesis. This is the first report that links in vivo CYP1B1 promoter methylation to modulation of carcinogenesis by PAHs. Further studies could lead to CYP1B1 as an important molecular target for the prevention and/or treatment of lung carcinogenesis by PAHs in humans.

#4646

Loss of nuclear HoxA10 is associated with proliferation of testicular germ cell tumors.

RuiQi Chen,1 Haolong Li,2 Yinan Li,2 Ladan Fazli,2 Martin Gleave,2 Lucia Nappi,2 Xuesen Dong2. 1 _University of Toronto, Toronto, Ontario, Canada;_ 2 _University of British Columbia, Vancouver, British Columbia, Canada_.

Background: HOXA10 is a key transcriptional factor that regulates testis development as reported from previous transgenic mouse models and human inherited diseases. However, whether it also plays important roles in promoting the development of testicular cancer is not well understood.

Objective: To study the expression of HOXA10 and its regulated signaling pathways in testicular cancers.

Design, Setting, and Participants: A tissue microarray was constructed with benign and cancerous testis. TCam2, NT-2, and NCCIT cell models were applied in this study.

Intervention: Immunohistochemistry and immunofluorescence were performed to measure the expression and cellular localization of HOXA10 in testicular cancer tissues and cell models. Cell proliferation and cell cycling rates were determined by BrdU incorporation and flow cytometry assays. HOXA10 transcriptomes were profiled with Ampliseq RNA-seq in testicular cancer cells. Immunoblotting assays were used to detect HOXA10-regulated signaling.

Results: HOXA10 is a nuclear protein in benign spermatocytes. Reduced nuclear expression and increased cytoplasmic expression of HOXA10 are associated with testicular cancers. These changes are consistent in both seminoma and non-seminoma. Enhanced HOXA10 expression in testicular cancer cell models inhibits cell proliferation and delays cell cycle progression through G2/M phases. These functions of HOXA10 mainly affect the TP53, cKit, STAT3, AKT and ERK signaling pathways.

Conclusions: Loss of nuclear functions of HOXA10 enhances proliferation of testicular cancer cells, suggesting that downregulation of HOXA10 transcription activity may promote the development of testicular cancers.

#4647

Keratinocyte specific deletion of TWIST1 promotes differentiation and inhibits UVB-induced hyperproliferation.

Fernando Eguiarte-Solomon, Okkyung Rho, John DiGiovanni. _University of Texas at Austin, Austin, TX_.

Non-melanoma skin cancers (NMSC) have one of the highest incidences in the United States with over 5 million diagnoses every year. The major risk factor for the development of NMSC is solar ultraviolet radiation consisting of both UVA and UVB. In epidermal keratinocytes, UV radiation results in DNA damage as well as deregulation of both proliferative and survival signaling pathways that contribute to tumorigenesis. The transcription factor TWIST1 has been reported to be essential for the formation and invasiveness of chemically-induced tumors in skin. TWIST1 deletion in mouse epidermis results in arrested cell cycle progression in response to treatment with the tumor promoter, TPA, which in turn prevents skin tumor growth in two-stage skin carcinogenesis assays. However, the impact of the keratinocyte specific TWIST1 deletion on skin carcinogenesis caused by UV radiation has not been clarified. In preliminary experiments, we found that primary mouse keratinocytes with TWIST1 knockout (KO) displayed an increase in the G2/M phase population after a single exposure to 20mJ/cm2 of UVB radiation. Accordingly, protein levels of cell cycle regulator p21 increased while those of pMDM2 decreased after UVB exposure. Moreover, we found that primary mouse keratinocytes with TWIST1 KO displayed an increase in apoptosis as shown by augmented Annexin V staining that corresponded with the reduction in levels of anti-apoptotic regulators Bcl-2 and Bcl-XL and concomitant upregulation of cleaved PARP and cleaved caspase-7 after UVB exposure. Furthermore, deletion of TWIST1 in skin keratinocytes in vivo (using K5-Cre x TWIST1flox/flox mice) led to a significant reduction in UVB-induced epidermal hyperproliferation compared to wild-type mice assessed by both Ki67 immunofluorescence staining as well as BrdU incorporation. Additionally, the protein level for p21 was increased while the levels of c-myc, and Cyclin B1 were reduced in the TWIST1 deficient keratinocytes in vivo. Interestingly, expression of differentiation markers such as Loricrin, Keratin-10, Sca-1, and transglutaminase-1 were also increased in the epidermis of TWIST1 deficient mice. These results from the in vivo model show that TWIST1 deletion results in an interruption of UVB-induced hyperproliferation via cell cycle arrest and induction of keratinocyte differentiation. Collectively, our findings indicate that the deficiency of TWIST1 has significant effects on the proliferation, survival and differentiation of keratinocytes; processes important for UVB skin tumor development. Future experiments will continue to assess the changes in oncogenic signaling in epidermal keratinocytes in response to UVB exposure and further investigate the impact of the deletion of TWIST1 on UV-induced skin carcinogenesis. Research supported by CPRIT 2018 Grant RP150247 and by CONACYT Fellowship 671661.

#4648

Cellular senescence and transformation induced by an oncogenic EML4-ALK fusion gene in normal and immortalized human cells.

Akihiko Miyanaga,1 Izumi Horikawa,2 Masaru Matsumoto,2 Takahiro Oike,2 Jessica Beck,2 Hiromi Tanaka,3 Ana I. Robles,2 Masahiro Seike,1 Akihiko Gemma,1 Curtis C. Harris2. 1 _Nippon Medical School, Tokyo, Japan;_ 2 _National Cancer Institute, NIH, Bethesda, MD;_ 3 _Indiana University School of Medicine, IN_.

Background: Chromosomal inversions involving Anaplastic lymphoma kinase (ALK) and Echinoderm Microtubule Associated Protein Like 4 (EML4) generate a fusion protein EML4-ALK with the constitutive ALK kinase activity in non-small cell lung carcinoma (NSCLC). The basic understanding of EML4-ALK remains insufficient due to the lack of functional studies using normal human cells.

Material and Method: We investigate the activities of EML4-ALK in mortal and immortalized normal human cells.

Results: The expression of EML4-ALK in normal, mortal human fibroblasts caused, through its ALK kinase activity, the early induction of cellular senescence with senescence-associated β-galactosidase activity, upregulation of p16INK4A and p21WAF1, telomere shortening and fusions, and accumulated DNA damage. In contrast, when EML4-ALK was expressed in telomerase reverse transcriptase (hTERT)-immortalized normal human fibroblasts, the cells showed accelerated proliferation and anchorage-independent growth, revealing its transformation activity. No chromosome aberration, no mutation or loss of p53, nor impairment of the p16INK4A response was associated with this transformation, likely reflecting clinical features of EML4-ALK-positive NSCLC. In both mortal and immortalized cells, EML4-ALK induced the phosphorylation of STAT3, which is involved in both cellular senescence and transformation. EML4-ALK was also able to transform hTERT-immortalized human bronchial epithelial cells, a cell type relevant to NSCLC.

Conclusions: Our data not only validate that EML4-ALK functions as an oncogene in human cells, but also suggest that telomerase-mediated immortalization manifests the oncogenic activity of EML4-ALK, switching from its senescence-inducing activity. This study also provides the isogenic pairs of human cell lines with and without EML4-ALK as an in vitro model for screening and testing candidate drugs.

#4649

Duplex Sequencing detects rare subclonal variants that mark early carcinogenesis and preneoplastic clonal evolution.

Charles C. Valentine,1 Mark Fielden,2 Robert Young,3 Jake Higgins,1 Lindsey Williams,1 Tan Li,1 Rohan Kulkarni,3 Sheroy Minocherhomji,2 Rosana Risques,4 Patrick Danaher,1 Jesse Salk1. 1 _TwinStrand Biosciences, Seattle, WA;_ 2 _Amgen, Thousand Oaks, CA;_ 3 _BioReliance, Rockville, MD;_ 4 _University of Washington, Seattle, WA_.

Cancer is a disease of somatic evolution, characterized by the natural selection of genomic mutations that facilitate enhanced cell survival and proliferation. Although many thousands of tumor genomes have now been sequenced, our ability to identify early genetic patterns of clonal selection in both humans and model organisms have been hampered by inadequately sensitive methodologies for identifying mutations during the long period between their occurrence and the final outgrowth of a clinically apparent tumor.

Sensitive molecular tests are capable of measuring minute levels of cancer cells in tissue samples, however no existing method is satisfactory at scaling to high-throughput detection at the rate of one cancer-associated variant per more than a million normal cells. NGS is used to study the genetic variation in cell populations, although the accuracy of standard NGS methods limit our ability to detect sub-clones below ~1%. Duplex Sequencing (DS) is a sensitive NGS error-correction method which increases the accuracy of base calls by more than 100,000-fold. DS enables precise reconstruction of the original double-stranded source molecule while overcoming both chemical and technical artifacts that arise during library preparation and sequencing.

Here we present the use of Duplex Sequencing, in both human and mouse tissues, for measuring sub-clones at allelic fractions less than 1×10-4 for an early glimpse into pre-neoplastic evolution. We illustrate how, merely weeks after mutagen exposure, we observe emerging clones of cells bearing cancer-driving mutations in histologically normal mouse tissue. Furthermore, we illustrate how similar patterns of clonal selection can be seen in multiple otherwise healthy tissues of humans as part of normal aging. We discuss how variations in the pattern and rate of accumulation of rare cancer-associated mutations offers a new preclinical tool for evaluating carcinogenicity of chemicals, as well as a potential clinical tool for assessing life-integrated carcinogenic processes and cancer risk in humans.

#4650

TOPK is a prognostic marker and therapeutic target in skin carcinogenesis.

Eunmiri Roh. _The Hormel Institute, University of Minnesota, Austin, MN_.

Solar ultraviolet (sUV) is linked to development of cutaneous squamous cell carcinoma (cSCC) as a non-melanoma skin cancer (NMSC). Actinic keratosis (AK), induced by sUV irradiation, acts as precursor to cutaneous squamous cell carcinoma (cSCC) and is a pre-malignant skin lesion characterized with an abnormal proliferation of atypical keratinocytes confined to the epidermis of the skin. Therefore, to identify the major signaling molecules in the prevention, development and metastasis of cSCC is a critically important task. Here, we suggested a promising protein target, T-LAK cell-originated protein kinase (TOPK) as an active form of MEK1 continuously phosphorylating downstream targets, which appears to be critical in mediating skin carcinogenesis. We detected high levels of total and phosphorylated TOPK protein expression in SCC, and phosphorylated TOPK levels were also markedly higher in AKs and SCCs from patients compared to matched normal skin. Inflammation is known to contribute to tumor development and thus we examined the effect of a 2-minimal erythema dose (MED) of solar stimulated light (SSL) exposure in clinical human skin samples. Our results indicated that acute SSL irradiation increased epidermal thickness as a feature of inflammation and also enhanced total protein and phosphorylation levels of TOPK in human skin in a time-dependent manner. TOPK phosphorylation in human precancerous HaCaT keratinocytes was also increased by SSL irradiation in a dose- and time-dependent manner. Interestingly, TOPK knockout (TOPK-/-) SKH1 (Crl: SKH1-Hrhr) hairless mice showed no tumors on SSL-exposed skin tissues. In contrast, wild-type (TOPK+/+) control SKH1 hairless mice treated with SSL showed increased tumor volume and number. Furthermore, TOPK-/- mice showed base levels of epidermal thickness similar to the SSL-unexposed control group and the Ki-67 proliferation marker was decreased in SSL-exposed TOPK-/- mouse skin compared to SKH1 TOPK+/+ mice. Notably, levels of the anti-apoptotic protein Bcl-2 were decreased in TOPK-/- mice, compared to SSL-treated TOPK+/+ mice. Moreover, our results indicated that phosphorylated and total TOPK protein levels in SCC metastasized to skin or lymph nodes were significantly higher compared to human SCC primary tumors. These results indicated that phosphorylated and total TOPK proteins are associated with both SCC development and metastasis. Thus, molecular targeting of TOPK could be an effective approach in the clinic to treat or prevent cSCC.

#4651

Polo-like kinase 1 phosphorylates and stabilizes KLF4 to promote tumorigenesis in nasopharyngeal carcinoma.

Jia Mai, Zhuo-Yan Zhong, Xiu-Xing Chen, Yan-Qun Xiang, Xuan Li, Hai-Liang Zhang, Rong Deng, Xiao-Feng Zhu. _Sun Yat-sen University Cancer Center, Guangzhou, China_.

INTRODUCTION Advanced nasopharyngeal carcinoma (NPC) is an aggressive disease with no targeted therapies and poor outcomes. New innovative targets are urgently needed. Krüppel-like factor 4 (klf4, gklf) is a zinc-finger transcription factor involved in a wide variety of cellular processes, including apoptosis, cell cycle progression and stem cell renewal. Emerging data indicate that klf4 dysregulation either facilitates or impedes tumor progression. The function of klf4 in NPC remains elusive.

METHODS we examined the expression of klf4 in human NPC (n=152) with a tissue immunohistochemistry (IHC) assay, and performed klf4 knockdown stable cell line with two different small hairpin RNAs (shRNAs) to evaluate the impact of klf4 on NPC. Global gene expression experiments were performed to explore the molecular mechanisms underlying klf4-dependent tumorigenesis. Small-molecule kinase inhibitor screening was performed to identify potential upstream kinases of klf4. Co-immunoprecipitation assay used to test the interaction between plk1 and klf4. Mass spectrometry analysis was performed to determine the specific site at which klf4 is phosphorylated by plk1. Statistical analyses were conducted using GraphPad Prism, SPSS software and JavaGSEA Desktop Application. A p value of <0.05 was considered to indicate statistical significance.

RESULTS Our investigation showed that high expression of klf4 was correlated with poor prognosis in NPC. Moreover, genome-wide profiling revealed that klf4 directly activated oncogenic programmes, including gene sets associated with KRAS, VEGF, and MYC signalling. We further found that inhibition of polo-like kinase 1 could downregulate the expression of klf4 and that plk1 directly phosphorylated klf4 at Ser234. Notably, phosphorylation of klf4 by plk1 caused the recruitment and binding of the E3 ligase traf6, which resulted in klf4 K32 K63-linked ubiquitination and klf4 stabilization. Moreover, klf4 could enhance traf6 expression at the transcriptional level, thus initiating a klf4-traf6 feed-forward loop. Treatment with the plk1 inhibitor volasertib (BI6727) significantly inhibited tumor growth in nude mice.

CONCLUSION In this study, we revealed that klf4 is upregulated in NPC and is correlated with poor prognoses. Our study demonstrated for the first time that plk1 can directly interact with klf4 and phosphorylate Ser234, alter the affinity of klf4 for the E3 ligase traf6, and promote the K63-linked ubiquitination of klf4 K32. In addition, klf4 can enhance traf6 expression at the transcriptional level and further promote klf4 expression. The resulting increase in klf4 ubiquitination leads to stabilization and upregulation of klf4, which leads to tumorigenesis in NPC. These results expand our understanding of the role of KLF4 in NPC and validate PLK1 inhibitors as potential therapeutic agents for NPC.

#4652

**Repurposing mebendazole in combination with PRIMA1** MET **for ovarian cancer therapy.**

Sugantha Priya Elayapillai, Satish Kumar Ramraj, Doris M. Benbrook, Camille C. Gunderson. _Stephenson Cancer Center, The University of Oklahoma Health Science Center, OKC, OK_.

Background

High-grade serous ovarian carcinoma (HGSOC) is the most aggressive subtype of ovarian cancer. Standard treatment options have a poor survival rate given high risk of recurrence with chemoresistant disease. Hence, there is a need for novel therapy to treat HGSOC. The repurposed drug mebendazole (MBZ) is effective against cancer cells at lower doses. The effectiveness of anti-cancer drugs in treating HGSOC is significantly reduced due to p53 mutations, which are found in most HGSOC (96%) tumors. Chemoresistance may be overcome by combination with p53 reactivator drugs such as PRIMA-1MET. The synergistic drug combination would significantly increase the efficacy of these drugs as well as decrease the overall toxicity by lowering effective doses of drugs. We hypothesize that combination of MBZ and PRIMA-1MET may be a synergistic and effective treatment for HGSOC.

Methods

The combination index (CI) of MBZ and PRIMA-1MET was determined in HGSOC cells with different endogenous p53 mutants (MES-OV R282W, ES2 S241F), exogenously expressed p53 mutants (SKOV3 R273H, SKOV3 R248W), p53 null (SKOV3) and wild type p53 (A2780 WT) cells. The mechanism of drug combination was determined by immunoblotting of intrinsic and extrinsic apoptosis pathways, measurement of soluble/assembled tubulins and reactive oxygen species (ROS) generation analysis. In vivo validation of drug combination is currently being performed using an intraperitoneal tumor model of MES-OV GFP/Luc cells in athymic nude mice.

Results

p53 missense mutant cells (IC50- 1.5 µM) were significantly more sensitive to MBZ compared to p53 null cells (IC50-7.8µM). The combination index of MBZ and PRIMA-1MET indicated synergism (CI= 0.3 to 0.7) in all HGSOC cells except SKOV3 R248W p53, which was nearly additive (CI= 0.90 to 1.10). The average dose reduction index (DRI) of this drug combination is 8.5 fold less than the single dose. The protein level of p53 was increased in both PRIMA-1MET and combination treated groups, and the p21 level was increased in MBZ and combined treated groups. The increased level of cleaved caspase 9 & 3 and cleaved PARP level confirmed that this drug combination is effective in inducing apoptosis in HGSOC cells. Moreover, the decreased level of tubulin polymerization in the MBZ and combination treated groups proves that MBZ affected the microtubule assembly known to lead to mitotic arrest and cell death.

Conclusion

The combination of MBZ and PRIMA-1MET had a synergistic effect and induced apoptosis by modulating microtubule assembly and p53 level in HGSOC cells with different p53 mutations. The drug combination may have a potential benefit to HGSOC patients that recur due to chemoresistance. The significant dose reduction index will allow lower doses of drugs to be used thus reducing the overall toxicity of these drugs.

Acknowledgment

Funded by PHF clinician scientist development grant.

#4653

**Melanocyte specific deletion of** Map3k1 **reveals its role in BRAF** V600E **-driven melanoma.**

Lucas D. Trucco, Piyushkumar A. Mundra, Pablo Garcia-Martinez, Kate Hogan, Nathalie Dhomen, Valeria Pavet, Richard Marais. _Cancer Research UK Manchester Institute, Manchester, United Kingdom_.

BRAF is the most common driver oncogene in cutaneous melanoma. Activating mutations in the BRAF gene are enriched in melanomas associated with intermittent sun exposure, with the most frequent mutations occurring at codon 600, leading to a constitutive activation of the RAF/MEK/ERK signaling pathway. In previous studies, we demonstrated that ultraviolet radiation (UVR) accelerates melanomagenesis in a mouse model of melanoma driven by BRAFV600E. From whole exome sequencing analysis of the tumors we found that Map3k1 was the most recurrently mutated gene across different experimental cohorts. MAP3K1 is unique in having both a kinase and E3 ligase function, allowing it to regulate protein phosphorylation and ubiquitin-mediated proteasome degradation. The vast majority of the mutations in Map3k1 occurred within the RING domain, which contains the E2 binding site for ubiquitin-conjugating enzymes. The most prevalent non-synonymous mutations where present in codon 484 and arose as a consequence of C-to-T transitions at a dipyrimidine site, characteristic of UVR-induced DNA damage. To understand better the role of MAP3K1, we generated Tyr::CreERT2+/o;Map3k1f/f mice allowing us to conditionally delete Map3k1 specifically in the melanocytes upon topical application of tamoxifen to the dorsal skin of juvenile mice. These mice did not develop tumors, but when crossed to mice carrying a Braf+/LSL-V600E allele, all mice develop melanoma, with a shorter latency and an increased multiplicity compared to Tyr::CreERT2+/o;Braf+/LSL-V600E mice. Tumors showed similar histopathological features, but melanomas carrying Map3k1 floxed or mutant variants presented higher levels of phosphorylated ERK1/2 than those with wild-type Map3k1. In addition, analysis of expression data of human cutaneous melanomas from The Cancer Genome Atlas showed an inverse correlation between the expression of MAP3K1 and ERK2 protein levels. Taken together, our results suggest that MAP3K1 loss-of-function cooperates with BRAFV600E to drive melanoma development by increasing MEK-ERK pathway activation.

#4654

Hotspot mutations in the core spliceosomal protein SF3B1 promote breast tumorigenesis.

Bo Liu, Michelle Ki, Omar Abdel-Wahab, Sarat Chandarlapaty. _Memorial Sloan Kettering Cancer Center, New York, NY_.

The core RNA splicing factor SF3B1 is targeted by recurrent hotspot mutations in leukemias and solid tumors with the K700E substitution observed in 2% of unselected breast cancer patients. These mutations promote usage of aberrant intron proximal 3' splice sites. In breast cancer, the links between these aberrant splicing events and the transformed phenotype are not well understood. Here, we integrated findings from multiple genetically modified model systems and human tumor specimens to understand the contributions of mutant SF3B1 to breast cancer pathogenesis. To identify the aberrant splicing events in breast cancers specifically, we first performed RNA-seq analyses of MCF10A breast epithelial cells with CRISPR knock-in of the K700E mutation. Dysregulation of splicing at over 1,000 splice junctions was discovered, most of which involve the use of alternative 3' splice sites. Gene expression analysis revealed upregulation of expression of NFκB pathway genes in the SF3B1 mutant cells. SF3B1 mutant cells showed higher phosphorylation of RelA (p65) and, accordingly, greater migration in wound-healing assays. Mis-splicing of exon 5 in MAP3K7, a gene encoding a central regulator of NFκB pathway, significantly reduced MAP3K7 protein expression in SF3B1 K700E mutant cells. Consistent with these data, pharmacologic MAP3K7 inhibition in SF3B1 wild-type breast cells increased phosphorylation of RelA. To further understand the effects of the K700E mutation on breast cancer development and progression, we generated a conditional knock-in mouse model, in which Sf3b1 K700E mutant allele was activated through MMTV promoter-controlled expression of Cre recombinase in mammary epithelium. Accelerated tumor formation was not identified in the mammary gland of these mice. However, RNA-seq analysis of mammary epithelial cells from the Sf3b1 K700E mutant mice revealed dysregulation of alternative splicing including Map3k7. As we found the majority of SF3B1 k700E mutations to co-occur with activating mutations in the PI3K pathway, we additionally introduced the Pik3ca H1047R mutant allele, by mating the MMTV-Cre Sf3b1 K700E mice with Rosa26-Pik3ca H1047R knock-in line. Markedly decreased survival was observed in double mutant animals, which developed mammary tumors faster, compared with single mutant controls. Overall, these data reveal that breast cancer-associated Sf3b1 mutations induce the NFkB pathway to promote invasion and cooperate with the PI3K pathway to accelerate mammary tumor development.

#4655

Association of a new germline variant in the MUTYH DNA glycosylase gene with colorectal adenoma transformation into malignancy.

Amjad A. Mahasneh, Fawaz Al-Shaheri, Mohamemd N. Banihani. _Jordan Univ. of Science & Technology, Irbid, Jordan_.

Background: Germline mutations in mutY homolog (E.coli) (MUTYH)DNA glycosylase gene have been associated with recessive inheritance of multiple s adenoma. Several studies revealed that germline mutations in this gene are ethnicity related. This study aimed to identify the germ-line mutations in MUTYH gene and determine their prevalence among Jordanian patients with colorectal adenoma.

Methods: 150 colorectal adenoma patients and 150cancer free individuals with no previous history of polyps were recruited. Sanger DNA sequencing of the MUTYH genewas carried out using 3130xL Genetic Analyzer. Sequencing results were analyzed by ChromasPro and mutational effects were predicted by online bioinformatics tools(accession number for the transcript used as reference isNG_008189.1).

Results: Two novel variants, g.87C>T and c.1264G>C, have been identified. G.87C>T was found in 60 (40%) patients and 10 (6.7%) controls.C.1264G>C was found in 90 (60%) patients and 7 (4.7%) controls. Thus, a significant association between g.87C>T and c.1264G>C variants and colorectal adenoma was found (p value for both variants was <0.0001). Moreover, the newly identified germline variant, c.1264G>C, was found to be significantly associated with colorectal adenoma transformation into malignancy (p< 0.0001).

Conclusion: The data showed high prevalence of two germline mutations in MUTYH gene among Jordanians with colorectal adenoma, which may make them as potential early biomarkers for diagnosis of colorectal adenoma.

#4656

TRAF1 is critical for regulating the BRAF/MEK/ERK pathway in non-small cell lung carcinogenesis.

Qiushi Wang, Tianshun Zhang, Ge Gao, Ann Bode, Zigang Dong. _Hormel Institute, Austin, MN_.

Tumor necrosis factor receptor (TNFR)-associated factor 1 (TRAF1) is a unique TRAF protein that can interact directly or indirectly with multiple TNFR family members, regulatory proteins, kinases, and adaptors that contribute to its diverse functions in specific tissues. However, the role of TRAF1 in non-small cell lung cancer (NSCLC) remains unknown. In this study, we report that TRAF1 is overexpressed in human lung cancer cells and tissues. TRAF1 expression level inversely correlated with patient survival probability. Loss of TRAF1 decelerated tumor invasion in a urethane-induced lung carcinogenesis mouse model. Furthermore, TRAF1 expression affected TRAF2-mediated BRAF Lys48-linked ubiquitination, which was followed by the inhibition of growth and differentiation, and the induction of death in lung cancer cells. Overall, our work suggests that TRAF1 plays a novel role in the regulation of the BRAF/MEK/ERK signaling pathway in NSCLC and offers a candidate molecular target for lung cancer prevention and therapy.

#4657

MB21D2 amplification and recurrent mutation exhibit oncogenic activity through centrosome and cilia alteration.

Daniel Esguerra Gracilla, Praveen Kumar Korla, Louis Lai, Jim Jinn-Chyuan Sheu. _National Sun Yat-sen University, Kaoshiung, Taiwan_.

MB21D2 (Mab-21 containing domain 2), a member of the Mab family, is predicted to form protein complexes with different partners, thus performs diverse functions in cells. In silico analyses using TCGA data revealed MB21D2 amplification/overexpression in various types of human cancer and correlated with poor survivals in patients with head and neck and endometrial cancer. More interestingly, a recurrent Q311E mutation was found in the Mab-21 domain critical for protein-protein interactions and downstream signaling. When expressed in oral cancer TW206 and CAL27 cells (low endogenous MB21D2), wild-type MB21D2 moderately increased cell proliferation, migration, and invasion as compared to the vector control. Significantly, the mutant variant (Q311E) showed stronger oncogenic effects on those processes and formed bigger spheres in a 3-D culture condition. Data mining, functional network predictions, and Western blotting confirmed the involvement of MB21D2 in centrosome-associated ciliogenesis via a crosstalk between PIK3CA and PKA (GSK and CREB) signaling pathways. Blockage of such interactome by specific inhibitors against PIK3CA or PKA reduced oncogenic activities mediated by MB21D2. Our data therefore suggest MB21D2 amplification/overexpression or the Q311E mutation as cancer drivers promoting cancer development through alteration of centrosome and primary cilia activity.

#4658

Cigarette smoke extract and benzo(a)pyrene induce osteopontin expression in lung cancer and promote mesenchymal stem cell recruitment.

Ya-Jing Jiang,1 An-Chen Chang,1 Po-Chun Chen,2 Chih-Hsin Tang1. 1 _China Medical University, Taichung, Taiwan;_ 2 _Shin-Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan_.

Cigarette smoking produces many organic compounds, including the carcinogenic polycyclic aromatic hydrocarbon benzo(a)pyrene, (BAP), which is linked to lung cancer. Tumor tissue recruits mesenchymal stem cells (MSCs; undifferentiated multipotent adult stem cells) to increase cancer cell proliferation, invasion and metastasis. We sought to determine the effects of cigarette smoke extract (CSE) and BAP stimulation on lung cancer cells and MSCs. We found that CSE and BAP increased mRNA and protein expression of osteopontin (OPN), a member of the small integrin-binding ligand N-linked glycoproteins (SIBLINGs) family, but had no such effects on any other SIBLINGs proteins. A National Center for Biotechnology Information (NCBI) analysis of data from the Gene Expression Omnibus (GEO) database (GSE31210) identified much higher OPN expression in current and ex-cigarette smokers with lung cancer than in lung cancer patients who were nonsmokers. Our analysis found that stimulating lung cancer cells with CSE and BAP led to the recruitment of MSCs, which adhered to lung cancer cells via OPN production. Next, we used a gene set from the BioCarta database to investigate signaling pathways associated with lung cancer. We found that the MAPK, JAK/STAT and RAS signaling pathways had the highest repeatability in lung cancer patients who were cigarette smokers. We discovered that CSE and BAP only activated the JAK2/STAT3 pathway and that transfection of lung cancer cells with JAK2 and STAT3 small interfering RNAs interrupted CSE- and BAP-induced OPN expression, inhibiting MSC recruitment and adhesion. We are now investigating whether MSCs link back to lung cancer cells and further induce migration and invasion. In summary, our data indicate that CSE and BAP increase OPN expression in lung cancer cells through the JAK2/STAT3 signaling pathway and thereby recruit MSCs and their adhesion to cancer cells.

#4659

Cadmium induced malignant transformation involves activation of the Erk/MAPK pathway.

Pritha Dasgupta,1 Priyanka Kulkarni,1 Nadeem S. Bhat,2 Ravi Gupta,1 Yutaka Hashimoto,1 Sharanjot Saini,1 Altaf A. Dar,3 Varahram Shahryari,1 Soichiro Yamamura,1 Yuichiro Tanaka,1 Marisa Shiina,1 Rajvir Dahiya,1 Shahana Majid1. 1 _UCSF VA Medical Ctr., San Francisco, CA;_ 2 _University of Miami, Maimi, FL;_ 3 _CPMC, San Francisco, CA_.

Cadmium (Cd) is a chemical pollutant of the natural and occupational environment and is reported to be associated with human carcinogenesis. The molecular mechanisms associated with Cd-induced prostate cancer remain elusive. This study provides evidence that Erk/MAPK signaling is a carcinogenic molecular fingerprint for Cd induced prostate cancer. Cd exposed RWPE1 (Cd-RWPE1) cells robustly formed tumors in nude mice. Functionally, chronic Cd exposure of RWPE1 cells promoted cell survival, proliferation and colony formation with inhibition of apoptosis. RT2 PCR array analysis of 84 genes involved in the Erk/MAPK pathway revealed induction of gene expression in Cd-RWPE1 cells compared to RWPE1. This was confirmed by individual TaqMan gene expression analysis. Gene Set Enrichment Analysis (GSEA) for differentially expressed genes in Cd-RWPE1 showed an enrichment of the Erk/MAPK pathway along with other pathways such as KEGG-ERBB, KEGG-Cell Cycle, KEGG-VEGF, KEGG-Pathways in cancer and KEGG-prostate cancer pathway. We randomly selected upregulated genes from the Erk/MAPK pathway and performed profile analysis in a prostate adenocarcinoma data set (n=534) from the TCGA/GDC data base. We observed upregulation of these genes in prostate cancer compared to normal prostate samples. Taken together, these data reveal that Erk/MAPK signaling is a major pathway involved in Cd-induced malignant transformation of normal prostate epithelial cells. Understanding the dominant oncogenic pathways involved in the malignant transformation of normal prostate epithelial cells may help develop optimal therapeutic strategies for prostate cancer.

#4660

Molecular mechanisms by which a bioactivated human carcinogen is transported to target tissues.

Sergei Pomyalov,1 Radha Bonala,2 Robert Rieger,2 Irina Zaitseva,2 Charles Iden,2 John Haley,2 Robert Turesky,3 Francis Johnson,2 Thomas Rosenquist,2 Arthur P. Grollman,2 Gil Shoham,1 Viktoriya S. Sidorenko2. 1 _Institute of Chemistry, Jerusalem, Israel;_ 2 _SUNY at Stony Brook, Stony Brook, NY;_ 3 _University of Minnesota, Minneapolis, MN_.

Aristolochic acid (AA) is a potent human carcinogen and nephrotoxin found in preparations of Aristolochia plants used in Chinese Traditional Medicine. Following biotransformation to form N-sulfonyloxyaristolactam (AL-I-NOSO3), this intermediate undergoes heterolytic cleavage of the sulfate group to generate a reactive cyclic nitrenium ion, the ultimate DNA binding species. Recently, we showed that primary human hepatocytes significantly increase renal toxicity of AA in the integrated human liver-kidney "organs on-chips" model. Therefore, we propose that AA is activated in the liver by forming AL-I-NOSO3, which is transported to the kidney protected from decomposition by binding to serum albumin. We employed mass spectrometric, fluorimetric and X-crystallography based approaches to dissect mechanisms of interactions between human serum albumin (HSA), AA, N-hydroxyaristolactam and AL-I-NOSO3. First, we demonstrate that HSA stabilizes otherwise labile N-sulfonyloxyaristolactam. Quenching of the native fluorescence of HSA due to the presence of a sole molecule of tryptophane-214, allowed us to conclude that all three compounds have similar affinities to IIA drug binding pocket of HSA. Subsequently, we obtained a high-resolution X-ray structure of AA bound to HSA in domain IB (1.9°A, pdb: 6HSC). Since prior to crystallization HSA was enriched with sodium myristate and site IB in circulation is occupied by fatty acids, our results imply that the IB pocket is the primary high affinity binding site for AA and its active forms. To assess whether AL-I-NOSO3 covalently binds protein, we incubated human plasma and purified HSA with this active AA. Immunoblotting of reacted HSA using antibodies that recognize aristolactam(AL)-adducted DNA suggests irreversible covalent adduction of AL to HSA. A combined approach using mass spectrometry instruments and enzymatic digestion revealed that AL is adducted to HSA at the following sites: Trp-214, Tyr-138 and Tyr-141. The former amino acid is located in the IIA drug binding site of HSA, while the latter two can be found in our HSA/AA structure in the site IB in the vicinity to AA molecule, corroborating our X-crystallography and fluoremetric data. Based on these studies we propose that AL-I-NOSO3 has a dual mode of interactions with HSA. If AL-I-NOSO3 decomposes prior to HSA binding, aristolactam will become irreversibly trapped with HSA. This binding to HSA would serve as mechanism of detoxication of AA species. However, if AL-I-NOSO3 binds to HSA prior to decomposition, it should be protected by HSA and transported to target tissues in its intact form.

#4661

Deciphering the causes of the COSMIC mutational signature 17 by combining pan-cancer data with experimental mouse models.

Marketa Tomkova,1 Claire Renard,2 Lara Urban,3 Souad Kolli,2 Maude Ardin,2 Manuraj Pandey,2 Maria Zhivagui,2 Hana Huskova,2 Magali Olivier,2 Hiroyuki Marusawa,4 Benjamin Schuster-Böckler,1 Jiri Zavadil2. 1 _Ludwig Institute for Cancer Research, University of Oxford, Oxford, United Kingdom;_ 2 _Int. Agency for Research on Cancer, Lyon, France;_ 3 _The European Bioinformatics Institute (EMBL-EBI), Wellcome Genome Campus Hinxton, United Kingdom;_ 4 _Graduate School of Medicine, Kyoto University, Kyoto, Japan_.

Gastric and esophageal adenocarcinomas (G/EAC) exhibit a characteristic pattern of somatic variants, known as signature 17. Signature 17 associates with high mutation burden, elevated neoantigen levels and a potential sensitivity to cell cycle inhibitors. Signature 17 correlates with poor survival, and is increased in cancer patients with a history of the common pre-malignant lesion called Barrett's esophagus. We recently showed that oxidative damage during DNA replication is a likely source of signature 17; however this hypothesis remains to be validated. Intriguingly, we observe signature 17 arising in clonally expanded primary mouse cells (Hupki MEF) and in mouse models of liver cancer. Here, we aim to identify the mechanisms underlying signature 17 by mining TCGA and ICGC data, as well as generating genome-wide molecular profiles of relevant experimental cell systems. Mutational signatures were extracted from exome and whole-genome sequencing (WGS) data of over 460 G/EAC tumors. Pathway analysis of RNA-seq data from matching samples was performed using NIH DAVID. Exposure to signature 17 was next assessed with regards to clinico-pathologic data. Lastly, we compared the asymmetry of signature 17 mutations around replication origins and early/late replicating regions in WGS data from MEF cells and G/EAC cancers. Comparing the transcriptomes of signature 17-high and -low tumors implicated cell proliferation, regulation of apoptosis, DNA damage response and repair, oxidoreductase activity and gastric acid secretion as relevant pathways. We found positive associations between signature 17 intensity and Barrett's esophagus history, poorer survival, and microsatellite instability. MEF WGS data revealed signature 17 enrichment in late-replicating genomic regions and on the lagging strand, analogous to observations in human tumors. Importantly, in all cultured MEF clones as well as in most EAC genomes, signature 17 co-occurred with signature 18 which is likely linked to oxidative damage of DNA, supporting the hypothesis that inflammation-driven oxidative DNA damage leads to these human cancers. The replication strand and timing asymmetries were significant in both signature 17 and signature 18 in EAC genomes, while the Hupki MEF clones exhibited significant strand asymmetry in signature 17 but not in signature 18, and showed significant correlation with replication timing in signature 18. Collectively, our results elucidate new features of a modifiable mutagenic activity underlying signature 17 in cancers of the esophagus, stomach, and in model systems. We have also identified novel clinical and molecular associations with signature 17. Investigations using the reported innovative cell-based models will help to identify the precise mechanistic basis of signature 17, with potential implications for primary and secondary cancer prevention and personalized therapy.

#4662

**YYFZBJS ameliorates the progression of colon carcinogenesis in Apc** Min/+ **mice by inhibiting regulatory T-cell generation.**

Hua Sui,1 Kaijuan Gu,2 Junze Ren,3 Haotian Wen,1 Ting Wang,1 Jing Hu,2 Liu Yang,4 Qing Ji,1 Qi Li1. 1 _Shuguang Hospital, Shanghai, China;_ 2 _Preclinical Medicine College of Shanghai University of Traditional Chinese Medicine, Shanghai, China;_ 3 _Changhai hospital of Traditional Chinese Medicine, Shanghai, China;_ 4 _Baoshan Hospital, Shanghai, China_.

Colorectal cancer (CRC) is the third most common solid tumor in the world and shows resistance to several immunotherapies, particularly immune checkpoint blockade which has therapeutic effects on many other types of cancer. T cell (Treg) represents one of the main populations of effector immune cells in antitumor immunity, has been considered as a double-edged sword during the progression of colon carcinogenesis. Our previous studies showed that traditional Chinese herbs had potential anticancer effects in improving the quality of life and therapeutic effect. However, little is known whether the mechanism of TCM formula is depended on the Treg-mediate innate immunity. Here, we used C57BL/6 ApcMin/+ mice, an animal model of human intestinal tumorigenesis, to investigate tumor-associated lymphocyte subsets and the mechanisms of their accumulation into gastrointestinal adenomas, and to evaluate the effects of YYFZBJS on the progression of colon carcinogenesis in vivo and in vitro. Compared to unaffected spleen, intestinal lymphatic, and mesenteric lymph nodes (MLN), accumulated CD4+CD25+ FoxP3 positive Treg cells were reduced after YYFZBJS treatment in ApcMin/+ mice. Treatment with YYFZBJS blocked tumor initiation and progression in the transgenic mouse models (ApcMin/+) with less change of body weight and survival rates. Moreover, YYFZBJS significantly reduced the expression of IL-6, IL-10, TGF-β, and other oncogenes markers such as MMP-2, MMP-9, cyclinD1 and c-Myc in Peripheral Blood and adenomatous tissue. In conclusion, we show that the Treg is involved in CRC development and progression and we identify YYFZBJS as a new potential drug target for the treatment of CRC.

#4663

KRAS **mutations drive adenomatoid odontogenic tumor and are independent of clinicopathological features.**

Carolina C. Gomes,1 Bruna P. Coura,1 Vanessa F. Bernardes,1 Silvia F. de Sousa,1 Josiane A. França,1 Nubia B. Pereira,1 Helder A. Pontes,2 Aline C. Batista,3 Danyel E. Perez,4 Ricardo C. Albuquerque,5 Lelia B. de Souza,6 Manoela D. Martins,7 Marina G. Diniz,1 Ricardo S. Gomez1. 1 _Federal Univ. of Minas Gerais (UFMG), Belo Horizonte, Brazil;_ 2 _Federal Univ. of Pará (UFPA), Belém, Brazil;_ 3 _Federal Univ. of Goiás (UFG), Goiânia, Brazil;_ 4 _Federal Univ. of Pernambuco (UFPE), Recife, Brazil;_ 5 _Universidade Tiradentes, Aracaju, Brazil;_ 6 _Federal Univ. of Rio Grande do Norte (UFRN), Natal, Brazil;_ 7 _Federal Univ. of Rio Grande do Sul (UFRGS), Porto Alegre, Brazil_.

Adenomatoid odontogenic tumor (AOT) is a benign encapsulated epithelial odontogenic tumor that shows indolent clinical behavior and predilection for young individuals. We have recently reported in a few AOT cases mutations in KRAS, which is a proto-oncogene frequently mutated in cancer types such as lung, pancreas and colorectal adenocarcinomas. We aimed to assess KRAS mutations in the hotspot codons 12, 13 and 61 in a large cohort of AOT samples and to test the association of these mutations with clinical (patients' age, tumor site, tumor size, follicular or extrafollicular subtypes) and histopathological parameters. A convenience sample of 38 central AOT cases was included in the study. KRAS codon 12 mutations were assessed by TaqMan allele-specific qPCR (p.G12V/R) and/or Sanger sequencing, and codon 13 and 61 mutations were screened by Sanger. Histological tumor capsule thickness was evaluated by morphometric analysis. In addition, the phosphorylated form of the MAPK downstream effector ERK1/2 was investigated. Statistical analysis was carried out to test the association of KRAS mutations with clinicopathological parameters. KRAS c.35G>T mutation, leading to p.G12V, was detected in 15 cases. A novel mutation in AOT, c.34G>C, leading to p.G12R, was detected in 12 cases and the other 11 were wild-type. Codon 12 mutations were not associated with the clinicopathological parameters tested. RAS mutations are known to activate the MAPK pathway and we show AOTs express phosphorylated ERK1/2. In conclusion, a high proportion of AOT (27/38, 71%) have KRAS codon 12 mutations, which occur independently of the clinicopathological features evaluated. Collectively, these findings indicate that KRAS mutations and MAPK pathway activation is a common feature of AOT and some cancer types. Although it is unclear why different codon 12 alleles occur in different disease contexts and the complex interactions between tumor genotype-phenotype need clarification, on the basis of our results the presence of KRAS p.G12V or p.G12R can favor the AOT diagnosis in challenging oral neoplasm cases.Acknowledgements: This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brazil (CAPES) - Finance Code 001 and CNPq/Brazil.

#4664

Massively parallel sequencing-based analysis of synchronous gastric cancer and lung cancer.

Nandie Wu,1 Rutian Li,1 Yang Shao,2 Lifeng Wang,1 Baorui Liu,1 Jia Wei1. 1 _The Comprehensive Cancer Center, Affiliated Drum Tower Hospital to Medical School of Nanjing Univ., Nanjing, China;_ 2 _Geneseeq Technology Inc., Toronto, Ontario, Canada_.

Synchronous gastric cancer(GC) and lung cancer(LC) have been suggested to represent independent primary tumors rather than metastatic disease. We subjected sporadic synchronous GC/LC from five patients to whole-exome massively parallel sequencing, which revealed a median of and 1174 and 830 nonsynonymous somatic mutations in the synchronous GC and LC repectively. DNA mutations range between 292 to 2421 in lung samples and 287 to 1995 in stomach sample. Common mutations varies between 41 and 145. The substitution of C>A, C>T and T>C accounts for the most common mutations in both lung and stomach cancers. Tumor mutation burden (TMB ) varies from 2.7 to 11.0 Mut/Mb in Lung cancer and from 1.8 to 30.4 Mut/Mb in gastric cancer. We further did pathway analysis based on the list of mutant genes. Top 20 pathways enriched in each patient were analyzed. We found MAPK signaling pathway was most commonly enriched pathways in lung cancer patients, whereas PI3K-Akt signaling and Hedgehog signaling pathway were most commonly enriched pathways in stomach cancer patients. This result suggests the potential benefit of targeted therapeutic treatments involved in these pathways.

#4665

Lineage-tracing technology to understand the molecular events in a mouse model of tongue squamous cell carcinoma (SCC) carcinogenesis.

Marta Melis,1 Tuo Zhang,1 Theresa Scognamiglio,2 Lorraine J. Gudas1. 1 _Weill Cornell Medical College/Weill Cornell Medicine, New York, NY;_ 2 _NewYork-Presbyterian Hospital/Weill Cornell Medical Center, New York, NY_.

Oral cavity squamous cell carcinomas (OCSCCs) account for over 450,000 cases per year worldwide, present a high metastatic potential, and have a survival rate ≤ 50%. There is a high incidence of OCSCC, but the mechanisms of carcinogenesis remain unclear. Therefore, the aim of this research was to delineate the mutational landscape in developing tumors and OCSCCs by combining a lineage tracing approach and a mouse model of tongue carcinogenesis, previously established by our group.

We used K14-CreERTAM; Rosa26LacZ mice, which allowed the long term basal stem cells of the epithelium to be permanently marked at the time of a two-day tamoxifen treatment. We then induced carcinogenesis by treating mice for 10 weeks with the carcinogen 4-nitroquinoline 1-oxide (4-NQO), which recapitulates key phenotypic and molecular features observed in human OCSCC. We performed Laser Capture Microdissection (LCM) to isolate and retrieve high-quality genomic DNA exclusively from each clone of permanently labeled, long-lived LacZ+ stem cells; each clone of cells arises from one LacZ+ stem cell. We then performed exome sequencing of 3 to 5 mice per group, sacrificed immediately after the 10-week treatment with 4-NQO (pre-neoplastic), and mice sacrificed >19 weeks after the end of 4-NQO treatment (SCC). As a reference genome for alignment and germline mutation exclusion, we included 3 mice of the same genetic background that did not receive 4-NQO.

After 10 weeks of 4-NQO treatment the tongues were hyperplastic, with occasional necrosis and mild atypia, whereas all mice evaluated >19 weeks post 4-NQO treatment showed tongue SCCs. The total numbers of somatic mutations and LOH (loss of heterozygosity) identified using the mutation calling tool VarScan were 75% (4615±2170 vs 1148±76) higher in SCC compared to pre-neoplastic tongue samples. Among the mutations predicted as high and moderate impact, the levels of SNVs (single nucleotide variants) vs. indels were similar between SCCs and pre-neoplastic samples, 98.4% and 96.5%, respectively. By detailed analysis of the mutation types we showed that the pre-neoplastic tissues exhibited a much higher proportion of LOH events than the SCCs, 34.4% and 7.6%, respectively. Some of the genes affected by LOH in pre-neoplastic tissues were identified as tumor suppressors. Our findings suggest that a predominance of LOH in pre-neoplastic tongue tissue may result in loss of tumor suppressor genes, potentially defining the early steps of carcinogenesis in tongue SCCs.

In conclusion, following the fate of long term tongue epithelial stem cells has allowed us to identify genomic perturbations occurring in the tongues of 4-NQO-treated mice and to find potential early biomarkers for OCSCC. Grant ID: R01CA205258; Funding Agency: NIH/NCI.

### Signaling Cascades in Cancer Stem Cells

#4666

p21CIP1 promotes mammary cancer initiating cells via activation of Wnt/TCF1/CyclinD1 signaling.

Rachel B. Hazan. _Albert Einstein College of Medicine, Bronx, NY_.

Cancer stem cells (CSCs) generate and sustain tumors due to tumor initiating potential, resulting in recurrence or metastasis. We showed previously that knockout of the cell cycle inhibitor, p21CIP1, in the PyMT mammary tumor model, inhibits metastasis; however the exact mechanism remained unknown. Here, we show a pivotal role for p21 in potentiating a cancer stem cell-like phenotype. p21 knockout in PyMT mammary tumor cells suppressed tumorsphere formation, ALDH1 expression, and tumor initiating potential, that were in turn rescued by p21 overexpression. Consistent with these effects, p21 loss caused mesenchymal to epithelial transition that was accompanied by downregulation of Slug, Sox9, basal keratins and upregulation of luminal keratins and Claudin-3. Furthermore, p21 knockout suppressed Wnt/β-catenin signaling which was due to TCF1 downregulation. In turn, TCF1 knockdown in PyMT cells, attenuated Wnt signaling and tumorsphere formation. Interestingly, p21 rescue in p21 knockout cells, caused dramatic upregulation of Cyclin D1, consistent with known inhibition of Cyclin D1 nuclear export by p21. These data suggest that p21 promotes a cancer stem like phenotype via Wnt-TCF1 signaling along with direct upregulation of Cyclin D1 by p21.

#4667

A synergistic combination strategy for optimal inhibition of colon cancer stem cells: Simultaneous inhibition of Insulin-like growth factor-1 receptor-AKT-mammalian target of rapamycin axis and the mevalonate-isoprenoid biosynthetic pathway.

Chetna Sharon, Rio Boothello, Alberto Vera, Bhaumik B. Patel. _Massey Cancer Center, Richmond, VA_.

Introduction: Targeting cancer stem cells (CSCs) is an attractive new paradigm to achieve long-term remission/cure. The mevalonate-isoprenoid biosynthetic (MIB) pathway is critical in the regulation of colonic CSCs. However, feedback activation of SREBP2 following inhibition of the MIB pathway reduces the efficiency of the latter strategy. We described that Insulin-like growth factor-1 receptor-AKT-mammalian target of rapamycin (InAT) axis inhibits CSCs via inhibition of the MIB pathway at a level distinct from the target of statin - HMGCR, while simultaneously inhibiting SREBP2. Hence, we hypothesized that combining a MIB pathway inhibitor with the InAT axis inhibition will result in optimal inhibition of CSCs.

Methods: Four possible combinations with the MIB pathway inhibitors (Simvastatin or zoledronate) and an InAT axis inhibitors (OSI-906) or Everolimus were tested against spheroids from 12 colorectal cancer cells. Synergistic effect of the combinations was determined using Chou Talalay method (Calcusyn). Most promising combinations were examined in an in vivo model of Dual high CSCs (CD133+/CXCR4+) induced xenografts to assess tumor volume, and ex vivo CSC phenotype (spheroid formation and CSC marker expression) in xenograft-derived cells.

Results: OSI-906 + Simvastatin, as well as Everolimus + simvastatin, showed a robust synergistic effect (Combination index <0.75) in a majority of colon cancer cells which was independent of mutation in PI3K/mTOR or RAS pathways. In fact, both the combinations showed a significantly higher inhibition of Dual high CSCs-induced xenograft volume as well as ex-vivo spheroid formation (2°→4°) and CSC markers (CD133, DCLK1, LGR etc.) than either agents alone or vehicle controls in two model of colon cancer HCT-116 (MSI high, KRAS mut), HT29 (MSS, KRAS mut). Importantly, Simvastatin treatment failed to inhibit SREBP2 levels. However, InAT axis inhibitors caused a significant inhibition of SREBP2 and the MIB pathway genes. In fact, inhibition of the MIB pathway at the level of isopentenyl-diphosphate delta isomerase 1 (IDI1) was critical for simultaneous inhibition of the MIB pathway and InAT axis to attenuate CSC self-renewal as supplementation with DMAPP, and FPP, but not mevalonate or IPP, reversed the effect of the combination on CSCs.

Conclusion: Orally deliverable and combinations of the FDA-approved drugs targeting the MIB pathway and the InAT axis cause optimum inhibition of colonic CSCs through downregulation of SREBP2, achieving maximal inhibition of the MIB pathway.

#4668

Therapeutic efficacy of aryl hydrocarbon receptor agonist in acute myeloid leukemia.

Hee Joo Han,1 Dong-Yeop Shin,2 Sung-Soo Yoon2. 1 _Seoul National University, Seoul, Republic of Korea;_ 2 _Seoul National University Hospital, Seoul, Republic of Korea_.

Background

The presence of leukemic stem cells (LSCs) is known to be a major cause of treatment failure and recurrence in acute myeloid leukemia (AML) patients. Therefore, it is important to develop a treatment method of targeting LSCs. Based on several research, aryl hydrocarbon receptor (AhR) antagonist, StemRegenin 1 (SR1), supports LSC activity. Thus, we speculated that the AhR agonist with the opposite of SR1 could trigger LSC differentiation which may result in the anti-cancer effects. We also hypothesize that the combination of the AhR agonist and ASP2215 targeting FMS-like tyrosine kinase 3(FLT3) mutation, which is known to be present at the LSC level shows the augmented effect on cancer therapy. Here we show the biological significance of AhR and its contribution to leukemic cells, and the therapeutic applicability of this approach.

Methods and Results

The effects of AhR agonists of 6-formylindolo[3,2-b]carbazole(FICZ), and (2′Z)-Indirubin, and AhR antagonist, SR1 were tested on AML cell lines (MV4-11, MOLM14, HL60, NB4, KG-1) using western blot and flow cytometry. AhR modulation was confirmed by changes in AhR and CYP1B1 protein expression levels at 24hours. . When AML cells were exposed to FICZ for 72hours, surface markers for myeloid differentiation, increased significantly. Mononuclear cells isolated from AML patients' leukapheresis blood samples(n=5) were cultured with stem culture medium containing growth factors with agonists or antagonist of AhR to determine the effect on LSC differentiation and stem-ness. The cell surface markers Lin-CD34+CD38- represent the stem/progenitor cell fraction were analyzed by FACS, FICZ and indirubin reduces this cell proportion by 2.4-fold 1.8-fold respectively compared to the control. In addition, multipotential progenitors-like LSCs(Lin- CD34+CD38-CD90-CD45RA-) and lymphoid-primed multipotential progenitors-like LSCs (Lin- CD34+CD38-CD90-CD45RA+) was reduced by 2.8 and 2.6-fold in the FICZ treatment group and 1.8 and 2.2-fold in the Indirubin treatment group, respectively. Colony formation assay was performed to verify the effect of AhR modulating on LSC function. FICZ and indirubin reduced the stem-ness of cultured leukemic cells compared with control and SR1. We investigated whether the AhR agonist, FICZ, has an anti-leukemia effect through a cell viability assay. As a single agent, FICZ did not affect AML cells. However, FICZ improves the cytotoxicity of ASP2215, a potent FLT3 inhibitor, in FLT3 mutant AML cells. And more, the combination enhances the effect of ASP2215 on cancer cell signaling pathways AKT-mTOR and MEK-ERK.

Conclusion

We found that FICZ, an AhR agonist not only results in the LSCs differentiation, but also enhances the inhibitory effect of ASP2215 on cancer cell survival through the AKT-mTOR and MEK-ERK signaling. This cross-talk has the multiple meaning on cancer therapy in that it can simultaneously target cancer cells as well as LSCs.

#4669

Overcoming therapy resistance in stem cell-rich triple negative breast cancer through p38 MAP kinase inhibition.

Petra den Hollander,1 Shruti Shah,1 Xinhui Zhou,1 Abena Redwood,1 Shi-Rong Cai,1 Mary Sobieski,2 David Brunell,2 Clifford Stephan,2 Peter Davies,2 Helen Piwnica-Worms,1 Stacy Moulder,1 W Fraser Symmans,1 Sendurai A. Mani1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _Institute of Biosciences and Technology Texas A &M University, Houston, TX_.

Background: Patients with triple-negative breast cancers (TNBCs) develop chemotherapeutic resistance and have a high mortality rate. Classification of TNBCs based on molecular profiling has enabled the identification of one subtype that is particularly enriched for cancer stem cells (CSCs) and genes involved in epithelial-mesenchymal transition (EMT) pathways. The EMT pathways, as well as CSCs, have been independently associated with chemo-resistance. We have previously demonstrated that cancer cells can acquire CSC properties via the activation of EMT. The FOXC2 transcription factor is a critical mediator for EMT/CSC properties as well as metastatic competence. Activated p38 MAP kinase is also high in EMT/CSC-enriched TNBCs and is important for FOXC2 protein stability, CSC and EMT properties as well as metastasis. We hypothesize that the inhibition of p38 MAP kinase will increase the sensitivity of TNBCs to chemotherapeutic drugs and in combination, it will help reduce tumor recurrence.

Methods: Three clinical candidate p38 inhibitors (LY2228820, VX-702, and PH-797804) were tested next to SB203580, which has shown great efficacy in our pre-clinical models. Effects of p38 inhibition were measured using sphere assay, CD44hi/CD24lo profiles and their effect on FOXC2 protein levels using cells lines as well as cells isolated from PDX. To identify drugs that work in combination with p38 inhibitor, cancer cells were pre-treated with p38 inhibitors and then co-treated with p38 inhibitor as well as NCI Approved Oncology/Custom Clinical Collections of 256 approved drugs and the Broad Informer Collection of about 400 mechanistic compounds.

Results: Inhibition of p38 significantly impaired stem cell properties in a dose-dependent manner. P38 inhibitor worked in combination with Gemcitabine or Epirubicin (currently used for TNBC treatment) and reduced the TNBC cell proliferation. Among PDX samples capable of forming spheres, p38 inhibition reduced their stemness in a dose-dependent manner. P38 inhibition also decreased expression of the stemness marker FOXC2 and increased the expression of the epithelial marker E-cadherin.

Conclusion: P38 inhibition is an effective way to inhibit EMT/CSC properties and it can work in combination with other agents to increase sensitivity in TNBCs.

Acknowledgments: This work was supported by CPRIT-MIRA RP-160710 (SAM, WFS).

#4670

The molecular mechanisms of EpCAM in regulating tumor progression in colon cancer cells.

Kang-Hao Liang, Hsien-Cheng Tso, Shao-Hsi Hung, Mei-Ying Liao, Han-Chung Wu. _Academia Sinica - Inst. of Cell. & Organismic Bio., Taipei, Taiwan_.

Epithelial cell adhesion molecule (EpCAM) is highly expressed in advanced epithelial cancers and tumor-initiating cells, but its role in cancer progression remains to be elucidated. Here, we show that the extracellular domain of EpCAM (EpEX) acts through its EGF-like domain I to bind EGFR and activates downstream ERK1/2 and AKT signaling. Inhibition or shRNA knockdown of EGFR ablated EpEX-induced ERK1/2 and AKT phosphorylation in colon cancer cells. EGFR signaling, stimulated by either EpEX or EGF, then induces regulated intramembrane proteolysis of EpCAM, releasing both EpEX and EpCAM intracellular domain (EpICD). MEK inhibitor, U0126, prevented EpEX-induced EpCAM proteolysis by ADAM17 and presenilin 2 proteases. Furthermore, EGFR inhibitor, AG1478, and MEK inhibitor, U0126, both decreased the production of EpICD, which was found to be necessary for nuclear accumulation of β-catenin protein and expression of HIF1α target genes in vitro and in mouse xenograft models. In clinical samples from colorectal carcinoma patients, high levels of nuclear EpICD predicted metastasis and poor prognosis. Importantly, we also showed that EpAb2-6, an anti-EpCAM neutralizing monoclonal antibody, inhibited EpEX-activated EGFR-PI3K-AKT signaling and induced apoptotic signaling through FOXO3a activation of HTRA2 gene expression. This antibody also inhibited the nuclear translocation of EpICD and oncogenic signaling through β-catenin. Finally, in both metastatic and orthotopic animal models of colorectal cancer, EpAb2-6 therapy exhibited an antitumor effect and markedly extended the survival time of mice. Taken together, the results demonstrate that EpEX contributes to malignancy by functioning as a growth factor, which activates EpICD-mediated signaling, thereby enhancing colon cancer cell survival. Furthermore, the data indicate that EpEX can be considered as a promising target for treatment of colon cancer.

#4671

Novel biological roles of the atypical WNT ligand, Norrin, on glioblastoma stem cells segregate with ASCL1 expression.

Ahmed A. Elsehemy,1 Hayden Selvadurai,2 Arturo Ortin-Martinez,3 Nenad Pokrajac,1 Yasin Mamatjan,4 Katherine Rowland,2 Kenneth Aldape,5 Peter Dirks,2 Valerie Wallace1. 1 _University of Toronto, University Health Network, Toronto, Ontario, Canada;_ 2 _The Hospital for Sick Children, Toronto, Ontario, Canada;_ 3 _University Health Network, Toronto, Ontario, Canada;_ 4 _Princess Margaret Cancer Centre, Toronto, Ontario, Canada;_ 5 _National Cancer Institute, Bethesda, MD_.

Norrin is an atypical WNT ligand that binds the Frizzled-4 (FZD4) and Low-density lipoprotein receptor-related protein (LRP5/6) receptor complex to activate canonical WNT/ β-catenin signaling. Norrin/FZD4 signaling is involved in the regulation of vasculature in several tissues including the retina and for blood-brain barrier function. The role of Norrin in cancer is not very well characterized. Here, we show that NDP, the gene encoding for Norrin, is expressed in a wide range of cancer types, with a particular enrichment in glioblastoma (GBM) and lower grade glioma (LGG). Kaplan-Meier survival analysis of publicly available datasets revealed a significant correlation between NDP expression and survival in GBM, LGG and neuroblastoma. To investigate the function of NDP in GBM, we performed a set of NDP and FZD4 gain and loss of function experiments in patient-derived GBM stem cell (GNS) lines. Recently Achaete-scute homolog 1 (ASCL1) expression was shown to stratify GNS lines into two cohorts with different tumorigenic, proliferation and differentiation dynamics. Surprisingly, we found that NDP manipulation resulted in opposite effects in ASCL1hi versus ASCL1lo lines. NDP inhibited proliferation and sphere formation in ASCL1lo lines through the WNT/ β-catenin pathway, while it stimulated proliferation and sphere formation in ASCL1hi lines independently of the WNT/β-catenin pathway. The NDP/FZD4 effects on growth in both GNS subtypes were recapitulated in xenografts, confirming effects of this pathway on tumor progression in vivo. To gain insight into the cellular and molecular effects of NDP/FZD4 on GNS growth we performed immunocytochemistry (ICC) and RNA-Seq analyses in GNS cells with NDP knockdown. In parallel with our in vitro and in vivo observations, we found that NDP/FZD4 signaling affects GNS proliferation index and cell cycle kinetics and the expression of cell cycle regulatory genes in both GNS subtypes, but in opposite ways. RNA-Seq analysis identified cell cycle regulatory genes and gene sets that are commonly affected by NDP in both GNS types. Interestingly, the analysis also identified several genes and genesets unique to each GNS type, consistent with the divergence of NDP functions between GNS subtypes. Collectively, our results uncover novel functions of NDP in regulating GBM stem cell progression, and that NDP functions in GBM stem cells stratify with ASCL1 expression. Additionally, our results highlight the significance of precision medicine in targeting tumor subtypes based on the molecular signature.

#4672

WDR26 regulates an AKT-Gsk3-Wnt/b-catenin signaling cascade to maintain the breast cancer stem cell population and controls cancer metastasis.

Dharmendra K. Bhargava, Wei Wang, Maddison Lensing, Songhai Chen. _University of Iowa, Iowa City, IA_.

Cancer metastasis is the major cause of tumor mortality and has been attributed in part to the presence of a minority subpopulation of cancer stem cells (CSCs) in the bulk of tumor cells. We showed previously that WDR26, a scaffolding/adaptor protein that is highly upregulated in breast cancer, promotes breast cancer growth and metastasis. Here we show WDR26 was required for maintaining the CSC populations in breast cancer cells and the formation of lung metastases. Downregulation of WDR26 in breast cancer cells impaired the CSC-like activities and reduced the CSC population. Mammary gland-specific deletion of WDR26 in the MMTV-PyMT mouse model of breast cancer had a little effect on primary tumor formation but largely abolished spontaneous lung metastasis. WDR26 promoted β-catenin activation via AKT and GSK3, and the activity of AKT, GSK3 and β-catenin was required for maintaining the CSC population in breast cancer cells. Our results have identified a novel, WDR26-dependent pathway that links breast CSC activities to tumor metastatic potential.

#4673

DCLK1 promotes hepatocellular carcinoma via atypical β-catenin-regulated signaling and immune suppression.

Naushad Ali, Parthasarathy Chandrakesan, Michael S. Bronze, Courtney W. Houchen. _Univ. of Oklahoma Health Sciences Ctr., Oklahoma City, OK_.

Introduction: Genetic lineage tracing in mouse model has revealed that mature hepatocytes are the cell of origin of injury-induced hepatocellular carcinoma (HCC), the second most common cause of cancer-related deaths worldwide. Hepatocyte plasticity, defined as the ability to change cellular identity, plays a critical role in the repair process following liver injury. Inflammatory liver diseases often lead to cirrhosis and HCC. The molecular mechanisms that regulate the decision between hepatocyte injury repair and neoplastic initiation are unclear. Doublecortin-like kinase 1 (DCLK1), a microtubule-associated kinase, is expressed during liver injury, cirrhosis, and HCC, but not in normal liver. Here, we demonstrate a novel mechanism by which DCLK1 regulates injury-associated hepatocyte plasticity and immunosuppression in HCC.

Methods: Cell culture, DEN/CCl4-induced injury in mouse livers, and humanized mouse model were used for analyzing hepatocyte plasticity and DCLK1+ cells-induced changes in hepatic cells and macrophages.

Results: DCLK1-overexpressing primary human hepatocytes formed spheroids in 3D culture. DCLK1-overexpressing hepatoma cells exhibited a 48-kDa active β-catenin with preserved hypophosphorylated N-terminus, which interacted with TCF-4, resulting in enhanced luciferase reporter activity and cyclin D1 expression. Downregulation of DCLK1 using siRNAs inhibited expression of this β-catenin fragment. DCLK1 overexpression increased phosphorylation of GSK-3β at Ser9, thus preventing its complete proteasomal degradation. Liver tissues from patients with cirrhosis and HCC expressed the 48-kDa active β-catenin, DCLK1, cyclin D1, and 80-kDa E-cadherin ectodomain, suggesting a change in epithelial polarity. DCLK1-overexpressing primary human hepatocytes transplanted into FRG mice demonstrated β-catenin activation in the transplanted hepatocytes. Injury-induced DCLK1+ epithelial cells in mouse livers expressed PD-L1. DCLK1+ human hepatoma cells induced CD206 in macrophages in a dual co-culture assay.

Conclusions: DCLK1 enhances hepatocyte plasticity, clonogenicity, and tumorigenic signaling via atypical β-catenin-dependent mechanism. DCLK1+ cells show ability to suppress the host anti-tumor immunity by expressing PD-L1 and inducing macrophage polarization.

#4674

Targeting cancer stem-cells in aggressive variant prostate cancers.

Rama Soundararajan,1 Paul Allegakoen,1 Petra den Hollander,1 Anurag Paranjape,2 Robiya Joseph,1 Sandhya Sundaram,3 Devarajan Karunagaran,3 Peter Shepherd,1 Nora Navone,1 Ana Aparicio,1 Sankar Maity,1 Bradley Broom,1 Christopher Logothetis,1 Sendurai Mani1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _National Institutes of Health, Bethesda, MD;_ 3 _Indian Institute of Technology-Madras, Chennai, India_.

Aggressive Variant Prostate Cancers (AVPC) are lethal variants of the disease and usually occur in the setting of castration-resistant prostate cancer (CRPC). These neuroendocrine tumors are currently incurable, with most patients dying within 12-24 months of diagnosis. AVPCs are androgen-indifferent, and therefore do not respond to Androgen-Deprivation-Therapy (ADT), the mainstay treatment for prostate cancers. We recently discovered that these tumors are enriched with cancer cells with stem-cell properties (CSCs) that lack expression of androgen-receptor (AR) (PMID 26804168). Importantly, we observed that CSCs are generated when prostate cancer cells undergo a cell biological process called epithelial-to-mesenchymal-transition (or EMT). We also demonstrated that it is CSCs that facilitate neuroendocrine trans-differentiation, and promote drug-resistance via p38MAPK signaling. In this project, we aimed to inhibit EMT in patient-derived AVPC tumors (using SB203580, a p38MAPK inhibitor and anti-EMT drug), and identify clinically-relevant biological pathways upon EMT inhibition, using RNA-Seq analyses. We also aimed to develop a diagnostic CSC gene-expression score to identify/predict AVPC tumors. We tested the effect of SB203580 in two well-validated AR-negative PDX models of human AVPC (144-4 and 177-B). These models effectively recapitulate the heterogeneity and complex biology of AVPC. We first determined the appropriate dosing for SB203580 in these models that resulted in statistically significant target (p38MAPK) inhibition. Our data indicated that 10mg/kg SB significantly inhibits p38 activity (as judged by loss in MSK1 phosphorylation - MSK1 is a direct target of p38, and its phosphorylation is dependent on p38 activity). EMT inhibition also resulted in a significant reduction in the expression of SOX2, a known CSC marker for aggressive prostate cancers. In parallel, we observed a simultaneous up-regulation in expression of FOXA1, suggesting a shift to luminal epithelial phenotype upon EMT inhibition. We also developed a novel CSC gene expression score based on p38MAPK signaling components critical for EMT and neuroendocrine trans-differentiation in prostate cancer. We validated it in the Beltran dataset (PMID 26855148), wherein patients are categorized into CRPC-Adenocarcinoma and CRPC-Neuroendocrine type (aggressive tumors displaying extensive RB loss and increased expression of clinical neuroendocrine markers). We observed that our CSC score is highly represented in the CRPC-Neuro group, thus validating our rationale for anti-EMT therapy. This study thus highlights a potentially targetable signaling pathway for treatment of AVPC.

Acknowledgements: We thank Dr. Mahajan (Washington University School of Medicine), Dr. Wistuba, Ms. Mino, Dr. Lin (UTMDACC) and Dr. Tang (Rosewell Park Cancer Institute) for their contributions, & UTMDACC Prostate Cancer SPORE for financial support (RS, SAM).

#4675

Aryl hydrocarbon receptor antagonist enhances cord blood-derived human hematopoietic stem cell expansion and platelet formation.

Dong Chan Kim,1 Sung-Soo Yoon,2 Dong-Yeop Shin2. 1 _Seoul National Univ. College of Medicine, Seoul, Republic of Korea;_ 2 _Seoul National University Hospital, Seoul, Republic of Korea_.

Background: Cord blood transplantation is one of the alternatives for hematopoietic stem cell transplantation (HSCT). However, cord blood transplantation has a crucial problem with the small number of hematopoietic stem cells (HSC) in a single cord blood unit which results in graft failure. To overcome this problem with the paucity of HSCs, here we tested the effect of two aryl hydrocarbon receptor (AHR) antagonists on the HSC ex vivo expansion and explored the mechanism of different effect by analyzing the gene expression profiles related with lineage-specific commitment in expanded human HSPCs.

Methods & Results: For ex vivo expansion culture, we enriched CD34 positive cell proportion up to 90% from cord blood units. We demonstrated ex vivo expansion culture with two AHR antagonist, StemRegenin 1 (SR1) and CH223191. Lin-/CD34+/CD38-, Lin-/CD34+/CD90+ and Lin-/CD34+/CD45RA+cell proportions are increased by SR1 (x6.5, 4.1, 2.9) and CH223191 (x3.6, 2.5, 2.6) at 14 culture days. With repeated analysis, the absolute number of Lin-/CD34+/CD38-/CD90+/CD45RA-cells were increased more than initial seeding cell numbers. We further performed the colony forming assay (CFA) with expanded HSPCs to functionally validate of expanded HSPCs' stemness. The number of each colony distinguished by their morphologies were counted; BFU, CFU-G, -M, -GM, -GEMM. SR1 and CH223191 treatment increased the total number of colonies compared to control, and the number of progenitor cells was increased by antagonists. Fourteen days after CFA culture, colonies were harvested and analyzed by FACS with various hematopoietic lineage cell markers (CD45, CD33, CD235a, and CD19). There were differences between SR1- and CH223191-expanded HSPC by FACS analysis and CFA, though two molecules had the similar ability to expand hematopoietic stem/progenitor cell number with the differentiation capacity. To explore the putative cause of the different effect on HSPC expansion capacities of SR1 and CD223191, we performed total-RNA sequencing (Illumina NextSeq) using expanded progenitor cells and examined results in various ways. By differential expressed gene (DEG) analysis, we selected genes significantly changed their expression. Comparing with control, SR1 and CH223191 decreased the expression of genes which are known to be related with the AhR pathway. On the other hand, we found that CH223191 treatment increased gene expression related to platelet activation and formation, coagulation cascade and complement.

Conclusions: In this study, we show that the blockade of aryl hydrocarbon receptor results in HSPCs expansion. CH223191 induces the expansion of HSPCs and the gene expression which are related to platelet formation. On this point, further investigation for the therapeutic use of CH223191 for expanded cord blood transplant and the treatment amegakaryocytic thrombocytopenia is needed.

#4676

Cancer stem cell induces chemoresistance in breast cancer via macrophage migration inhibitory factor mediated activation of AKT pathway.

Subhayan Das,1 Moumita Kundu,1 Aditya Parekh,2 Deblina Bharadwaj,1 Mahitosh Mandal1. 1 _Indian Institute of Technology Kharagpur, Kharagpur, India;_ 2 _National Centre for Biological Sciences, Bengaluru, India_.

Chemoresistance is one of the significant problems of cancer management. Cellular crosstalk in tumor microenvironment plays a vital role in regulating the chemoresistance in cancer. Cancer stem cell (CSC) is one of the key players in the regulation of chemoresistance in cancer, and they modulate the tumor microenvironment to enhance the resistance to anti-cancer therapy. However, the exact mechanism by which CSC can influence the cellular crosstalk in the tumor microenvironment is mostly unknown. Macrophage migration inhibitory factor (MIF) is attributed to inducing therapeutic resistance in several cancers. We previously found a close relation of MIF secretion with a group of polyploid giant cells in chemoresistant breast cancer.

MIF secretion was increased in the media of the mammosphere of MDA MB 231 cell (231) as well as Doxorubicin (Dox) and 5-Fluorouracil (5FU) resistant 231 cells. MIF secretion also increased upon treating 231 with Dox and 5FU in a time-dependent manner. Addition of recombinant MIF in culture medium increased the resistance of 231 to chemotherapeutic agents, and a similar phenomenon was also observed upon treatment with conditioned medium from CSC. Chemoresistant breast cancer is enriched with the CSC population, and hence we hypothesized that the MIF was secreted from CSC populations and had a significant role in the regulation of chemoresistance in breast cancer. MIF expression was also increased in Chemoresistant breast cancer tissues compare to non-resistant ones. MIF also induced anti-apoptotic protein Bcl2 and Bcl-xl by downregulating Bax and Bad as well as contributed to the chemoresistant phenotype. Conditioned media from CSC and exogenous MIF were unable to induce chemoresistance in parental 231 cell line in the presence of 4-IPP, a MIF inhibitor. The anti-apoptotic effect of exogenous MIF was also reversed by the inhibitor. Thus we confirmed that MIF played a significant role in the regulation of resistant phenotype in breast cancer microenvironment. Upon further analysis of molecular pathways, we found phosphorylation of AKT corresponded with the MIF secretion in CSC as well as chemoresistant 231 cell lines. MIF was able to increase the level of phosphorylated AKT in 231 cell line in a dose-dependent manner. When chemoresistant 231 cells were treated with MIF inhibitor, the AKT phosphorylation was inhibited. Silencing the AKT pathway via siRNA reversed the sensitization of exogenous MIF and reduced the resistance of both parental 231 as well as resistant 231 cells.

So, overall we conclude that MIF plays a crucial role in inducing chemoresistance in breast cancer by upregulating anti-apoptotic protein Bcl2 and Bcl-xl & downregulating Bax and Bad through AKT phosphorylation. The MIF is mainly secreted by CSC population during self-renewal or stress caused by chemotherapy which activates anti-apoptotic pathways in tumor microenvironment.

#4677

Inhibition of 15-PGDH causes Kras-driven tumor expansion through prostaglandin E2-ALDH1 signaling in the pancreas.

Takatsugu Ishimoto, Kota Arima, Tomoyuki Uchihara, Keisuke Miyake, Atsuko Yonemura, Tadahito Yasuda, Rumi Itoyama, Masaaki Iwatsuki, Yoshifumi Baba, Naoya Yoshida, Hideo Baba. _Kumamoto Univ. Graduate School of Medicine, Kumamoto, Japan_.

Chronic inflammation has a crucial role in cancer development and the progression of various tumors, including pancreatic ductal adenocarcinoma (PDAC). The arachidonate cascade is a major inflammatory pathway that produces several metabolites, such as prostaglandin E2 (PGE2). 15-hydroxyprostaglandin dehydrogenase (15-PGDH) participates in the degradation of PGE2 and has attracted attention in recent years. Although inhibition of 15-PGDH is reported to lead to PGE2 accumulation and expand tissue stem cell fraction, the role for pancreatic tumor progression is still unknown. The aim of this study is to elucidate the regulatory mechanism and functional role of 15-PGDH for cancer stem-like cell expansion during tumor progression in pancreas. We found that 15-PGDH expression is frequently down-regulated in PDAC cells and significantly correlated with the number of infiltrating macrophages in human PDAC tissues. Moreover, direct co-culture assay revealed that macrophage derived interleukin-1 beta down-regulates 15-PGDH expression in PDAC cells. Furthermore, pharmacological blockade of 15-PGDH led to PGE2 accumulation, and promoted growth and sphere formation through the expansion of ALDH1-positive cells. We also elucidated the molecular mechanism that PGE2 accumulation by 15-PGDH inhibition increases CYP26A1 expression and subsequently depletes all-trans retinoic acid, and results in ALDH1 up-regulation. Finally, genetic ablation of 15-Pgdh promoted tumorigenesis in KrasLSL-G12D; Ptf1aCre/+ mice through the expansion of ALDH1-positive cells. Our findings highlight the role and significance of PGE2 degradation pathway for PDAC tumor progression.

#4678

Targeting MEK in EGFR amplified glioma stem like cells induces differentiation.

Soon Young Park,1 Yuji Piao,1 Emmanuel Martinez-Ledesma,1 Jianwen Dong,1 Sabbir Khan,1 Sandeep Mittal,1 Ze-yan Zhang,2 Erik P. Sulman,3 Veerakumar Balasubramaniyan,1 John F. de Groot1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _The Laura and Isaac Perlmutter Cancer Center at NYU Langone Health, Houston, TX;_ 3 _The Laura and Isaac Perlmutter Cancer Center at NYU Langone Health, New York, NY_.

The median survival for patients with recurrent glioblastoma is nine to twelve months highlighting the need for better therapeutic strategies for this deadly disease. The revolution in cancer genomics has identified multiple amplification events as potential targets for therapeutic intervention in many cancers including glioblastoma. Multiple factors such as incorrect patient selection, inadequate drug delivery across the blood brain barrier, acquired resistance and drug-target heterogeneity may all lead to clinical failure of targeted therapies. Receptor tyrosine kinase (RTK) signaling mediated by EGFR and PDGFR account for the core RTK signaling alterations in glioblastoma. EGFR-amplification (in ~40%) and PDGFR amplification (in ~12%) are detected in receptor tyrosine kinase- dysregulated glioblastoma. The first generation or second-generation EGFR tyrosine kinase small molecule inhibitors failed to show long term therapeutic benefit in glioblastoma patients. Through an unbiased high-throughput screen utilizing our glioma stem-like cells (GSCs) we identified that glioblastoma cells harboring EGFR amplification are uniquely vulnerable to mitogen-activated protein kinase (MEK) inhibitors. MEK inhibition induces apoptosis in EGFR amplified cells at low concentration. Furthermore, RNA sequence analysis of MEK inhibition revealed upregulation of genes related to differentiation in MEK sensitive glioma stem cells. Based upon these in vitro studies we are currently investigating MEK inhibition in a GBM xenograft mouse model. Overall our data suggest that the MEK inhibition could be a potential therapeutic target in a selective group of glioblastoma patients.

#4679

NRF2/SHH signaling cascade promotes tumor-initiating cell lineage and drug resistance in hepatocellular carcinoma.

Hoi Wing Leung,1 Eunice Yuen Ting Lau,2 Kin Wah Lee1. 1 _Hong Kong Polytechnic University, Hong Kong, Hong Kong;_ 2 _Queen Elizabeth Hospital, Hong Kong, Hong Kong_.

Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide. The long-term prognosis of HCC remains unsatisfactory due to high recurrence rates and chemoresistance. Solid evidence shows that tumor-initiating cells (T-ICs) are the root of tumor relapse and drug resistance, which led to poor prognosis of patients with hepatocellular carcinoma (HCC). Therefore, identification of crucial transcriptional regulator in liver T-IC may improve the prognosis of these patients. Through in vitro liver T-IC enrichment approach, we identified Nuclear Factor (Erythroid-Derived 2)-Like 2 (NRF2) as transcriptions regulator be significantly upregulated in enriched liver T-IC populations. NRF2 regulated liver T-IC properties including self-renewal, tumorigenicity, and liver T-IC marker expression and significantly affected patients' clinical outcome. Furthermore, NRF2 repression led to sensitization to doxorubicin and 5-FU treatment. Furthermore, we found that ROS-induced NRF2 activation regulates sorafenib resistance of HCC cells. Mechanistically, NRF2 was found to physically bind to promoter SHH, which triggers activation of sonic hedgehog pathway. NRF2 was significantly correlated with SHH expression, which was associated with poor patient's survival. The effect of NRF2 knockdown was eliminated upon administration of recombinant SHH, demonstrating that NRF2 mediated the TIC function via upregulation of SHH expression. Our study suggested a novel regulatory mechanism for the canonical sonic hedgehog pathway that may function through NRF2/SHH/GLI signaling axis, thus mediating T-IC phenotypes. Targeting NRF2 mediated signaling cascade alone or in combination with other treatment modalities may be a new a potential therapeutic approach for treatment of HCC.

#4680

KRAS activation in gastric adenocarcinoma stimulates epithelial-to-mesenchymal transition to cancer stem-like cells and promotes metastasis.

Changhwan Yoon,1 Jacob Till,2 Soo-Jeong Cho,3 Kevin Chang,1 Jian-Xian Lin,4 Sandra Ryeom,2 Sam Yoon1. 1 _Mem. Sloan Kettering Cancer Ctr., New York, NY;_ 2 _University of Pennsylvania, Philadelphia, PA;_ 3 _Seoul National University Hospital, Seoul, Republic of Korea;_ 4 _Fujian Medical University Union Hospital, Fujian, China_.

BACKGROUND:

Our previous work showed that in a mouse model of gastric adenocarcinoma (GA) with loss of p53 and Cdh1 that adding oncogenic Kras (a.k.a triple conditional or Tcon mice) accelerates tumorigenesis and metastasis. The Receptor Tyrosine Kinase (RTK)-RAS signaling pathway is altered in 60% of GAs, and KRAS is amplified or mutated in 17% of GAs. There is some evidence that RTK-RAS signaling and oncogenic KRAS are important in the epithelial-to-mesenchymal transition (EMT) and maintenance of cancer stem-like cells (CSCs). We hypothesized that RTK-RAS activation in GA CSCs increases the metastatic potential of these cells.

METHODS:

The RTK-RAS pathway and KRAS were examined in nontransformed HFE-145 gastric epithelial cells, tumor-derived organoids derived from Tcon mice, human GA cell lines (AGS and KATOIII), AGS xenografts, and a tissue microarray of human GAs from 115 patients undergoing surgical resection. KRAS was inhibited using shRNA, and the RTK-RAS pathway was blocked using a MEK inhibitor PD0325901.

RESULTS:

Kras activity was much higher in GA cell lines grown as spheroids compared to GA cell lines grown monolayers. Metastasis following KRAS activation in GA cells results from stimulation of epithelial-to-mesenchymal transition (EMT) to cancer stem-like cells (CSCs). In organoids derived from our mouse model, Kras knockdown decreased spheroid formation, expression of EMT-related proteins, migration, and invasion; similar effects, as well as reversal of chemoresistance, were observed following KRAS knockdown in patient tumor-derived GA cell lines. KRAS inhibition in GA spheroid cells led to reduced flank xenograft growth, loss of the infiltrative tumor border, fewer lung metastases, and increased survival. Supporting clinical relevance, high tumor levels of CD44 (a marker of CSCs) and KRAS activation were independent predictors of worse overall survival in 115 GA patients.

CONCLUSION:

In this study, we show that the addition of oncogenic KRAS into gastric epithelial cells leads to EMT and acquisition of CSC phenotypes. Inhibition of KRAS in GA cell lines and in tumor derived organoids reverses EMT and inhibit CSC phenotypes. We conclude that metastasis following KRAS activation in GA cells likely results from stimulation of EMT and transition to CSCs.

#4681

Functional correlation between cancer stem cells and algorithmic effectiveness of targeted drugs of RAS and PI3K pathways in TNBC.

Jennifer C. Aske, Pradip K. De, Brian Leyland-Jones, Nandini Dey. _Avera Research Inst., Sioux Falls, SD_.

Background: Cancer stem cells (CSCs) are important for the initiation, metastasis, relapse, and chemo/radio-resistance of several tumors including BC (Polyak K et al., 2008, 2010). We reported a method to study a relationship between the characteristic pattern of CSCs (CD24L/CD44H/CD44v6) expression and the mutational status in PTEN-null MDA-MB468, HCC70; RAS/RAF mutated MDA-MB231; BRCA mutated/PTEN-null SUM149 & HCC1937; PIK3CA mutated BT20 TNBC cells. We also tested CD44/CD24 expression in the context of rational combinations of the PI3K-pathway inhibitors in subtype-specific BC (Carlson et al, AACR, 2016; SABCS 2016). Recently, we studied the role of genetic background in determining the effectiveness of the RAS and PI3K pathway(s) targeted drugs in TNBC by testing a combination of the MEK1/2 inhibitor with mTOR kinase inhibitor or AKT inhibitors (Carlson et al SABCS 2018 P2-03-11).

Hypothesis: This study was undertaken to identify the relationship between CSCs and effectiveness of RAS and PI3K pathway(s) targeted drugs in TNBC by testing a combination of the MEK1/2 inhibitor with mTOR kinase inhibitor or AKT inhibitors or PI3K inhibitors.

Methods: TNBC cell lines MDA-MB231, SUM149, MDA-MB468 and BT20 were used for the study. Proliferation (Incucyte) and apoptosis (AnnexinV) were tested following the drug treatment alone or in combination. Clonogenic growth (3D matrigel assay). WB was used to interogate signaling events in the context of proliferation and apoptosis. CSC subpopulations were monitored by flow cytometry following treatment in both 2D and 3D.

Results: The response to treatment with pathway targeted inhibitor is a function of the relative proportion of CSC and the mutational status of TNBC tumor cells. TNBC cells with higher numbers of CSC tend to be more resistant to PI3K inhibition. Rational drug combinations blocked proliferative signals and enhanced apoptosis (cleaved caspase3) as demonstrated by WB. Single agent treatments were mostly cytostatic and temporarily decreased proliferation, while a marked increase in AnnexinV was seen in doublet combinations. Cells with a higher CSC population demonstrated lower sensitivity to treatment in both 2D and 3D models and a smaller change in the CSC population was seen following 3D growth. The mechanistic details of the effect of this combination on the CD44H/CD24L stem cell populations are being worked out to delineate the relationship between CSC and drug response in TNBC models, the results of which will be presented in the meeting.

Significance: As CSCs are considered one of the targets for obtaining a better therapeutic response (Huang R, Rofstad EK, 2017) additionaly normal as well as neoplastic nonstem cells can spontaneously convert to a stem-like state (Chaffer CL et al., 2011), our study reveals the relationship between the effect of targeted therapy and stem cell population.

#4682

TGFB signaling disruption in the formation of oral pre-neoplasia.

Claudia D. Andl,1 Anna L. Means,2 James S. Lewis,2 Alexandra Rothaus,1 Thomas Andl1. 1 _University of Central Florida, Orlando, FL;_ 2 _Vanderbilt University Medical Center, Nashville, TN_.

In humans, tissue homeostasis has to be maintained for decades without significant loss of functionality. Epithelia such as the oral mucosa achieve this through the self-renewing capabilities of stem cells. These life-long residents of the oral mucosa maintain homeostasis of the epithelium and at the same time have to be able to protect themselves from the main threats to longevity, i.e., stem cell exhaustion and malignant conversion and carcinogenesis. A key pathway in mediating cell cycle arrest in oral keratinocytes is mediated by TGFB superfamily signaling. There is good evidence implicating TGFB signaling as a tumor suppressor pathway in oral cancer. We have previously shown that disruption of TGFB signaling and cell adhesion can lead to oral squamous cell carcinoma. In vitro using oral keratinocytes, we found that TGFB regulates the stem cell compartment, inducing gene expression of quiescent stem cell markers MECP2, XPC, ITGB1 and PDPN. Using multiplex immunofluorescence, we have stained a tissue microarray containing 28 cases of squamous cell carcinoma, 4 adenocarcinoma, 4 metastatic carcinoma, 6 hyperplasia, and 5 adjacent inflammatory as well as normal adjacent and normal tissues. The antibodies selected were MECP2, XPC, ITGB1 and K15 in combination with the proliferation marker ki67 to determine if TGFB signaling regulates markers of stem cell quiescence. This quiescent gene signature highlighted a basal niche for slow-cycling cells, reserve stem cells. During oral disease progression, marker expression was abandoned in favor of increased proliferation. In vivo, a mouse model in which KrasG12D expression was induced and the Smad4 gene deleted specifically in the adult epithelium showed invasive cancers in the oral cavity. These Smad4-negative tumors express Sox9, a marker of human OSCCs and a TGFB target, which we have also observed to be upregulated in human premalignant lesions. Sox9-positive areas in the mouse tumors are associated with a downregulation of quiescent oral stem cell markers Krt15 and Mecp2, same as we observed in immunostainings of human tissues. In conclusion, TGFB and SMAD4 regulate quiescent stem cells and loss of this pathways allows invasive cancers to develop in the setting of oncogene expression. Furthermore, we could show that MECP2 in combination with other markers identifies quiescent basal cells in the human oral mucosa and is lost as TGFB signaling is disrupted in tumorigenesis.

#4683

Targeting GPCR signaling in HER2+ breast cancer suppresses cancer stem cell tumorigenicity and sensitizes HER2-targeted therapies.

Wei Wang, Dharmendra Bhargava, Yuan Chao Ye, Songhai Chen. _University of Iowa, Iowa City, IA_.

G protein coupled receptors (GPCRs) are the largest family of cell surface receptors with over 800 members. Aberrant activation of GPCRs has been implicated in cancer initiation and development. However, many of GPCRs are overexpressed in cancer cells, making it difficult to target individual GPCRs for cancer treatment. In this study, we have tested a new approach of targeting GPCRs in HER2+ breast cancer by blocking their common pathways downstream of G proteins. By mammary gland-specific expression of the pertussis toxin (PTx) catalytic subunit, a selective inhibitor that uncouples a subgroup of GPCRs from activating Gi/o proteins, we demonstrated that blocking GPCR downstream signaling delayed the onset of HER2-driven spontaneous mammary tumor formation and suppressed lung metastasis. Moreover, we showed that aberrant HER2 signaling upregulated Gi/o-GPCR expression in breast cancer cells, which in turn activated the ErbB/HER family of protein-tyrosine kinases via Src and PI3K pathways to maintain cancer stem cell tumorigenicity. Targeting Gi/o-GPCR signaling by PTx, Src or PI3K inhibitors sensitizes HER2+ breast cancer to HER2-targeted therapies. Together, our data demonstrate that targeting GPCR downstream signal pathways may represent a new approach to ablate CSCs to block tumor progression and augment HER2-targeted therapeutics.

#4684

Afatinib targets glioblastoma stem cells by inhibiting EGFRVIII-cMet co-activation.

Raghupathy Vengoji, Muzafar A. Macha, Ramakrishna Nimmakayala, Satyanarayana Rachagani, Kavita Mallya, Maneesh Jain, Moorthy P. Ponnusamy, Surinder K. Batra, Nicole Shonka. _Univ. of Nebraska Medical Ctr., Omaha, NE_.

Background

Glioblastoma (GBM) is the aggressive primary brain tumor with a median survival rate of 14.6 months. Currently, first-line treatment includes surgical resection, chemoradiation, and adjuvant chemotherapy with temozolomide (TMZ). However, GBM recurs most often within 6.9 months. Most of the targeted therapies failed in GBM, possibly due to the co-activation of the receptor tyrosine kinases (RTKs). Genetic analysis on GBM tumor revealed that RTKs are dysregulated, with epidermal growth factor receptor (EGFR) representing 57.4% of the deleted/mutated GBM, about 30 - 40% of GBM patients with EGFR amplification carry an oncogenic gene rearrangement EGFR variant III (EGFRvIII) which is constitutively active. In addition, EGFRvIII co-activates cMET RTKs thereby enriches GBM cancer stem like cells (CSCs). CSCs are relatively radio- and chemo- resistant and play a pivotal role in tumor recurrence/progression. We hypothesize that afatinib and TMZ combination would inhibit EGFRvIII co-activation and tumor progression in GBM model.

Methods

GBM cell lines U87MG, U87MG transfected with EGFRvIII (U87 EGFRvIII) and SP/CSC isolated from U87 EGFRvIII cells were treated with afatinib, TMZ alone or in combination and analyzed. The in vivo efficacy of these drugs were also analyzed on U87 EGFRvIII orthograft mouse model.

Results

We observed significantly higher proportion of CSCs in U87 EGFRvIII cells compared to U87MG cells (p = 0.03). While afatinib decreased the percentage of CSCs in both U87MG and U87 EGFRvIII cells (p = 0.02), TMZ only decreased CSC population in U87MG cells. However, combination of afatinib with TMZ significantly decreased the CSCs in both U87MG and U87 EGFRvIII cells (p = 0.02). Our clonogenic (tumorsphere) assay revealed significantly more tumorspheres (p=0.01) formed by U87 EGFRvIII CSCs cells than U87MG CSCs. In addition, we also observed that TMZ significantly decreased the self-renewal properties of U87 CSCs compared to U87 EGFRvIII CSCs. Furthermore, afatinib alone or in combination with TMZ significantly abolished the tumorsphere formation by U87 EGFRvIII CSCs. The underlying mechanism revealed inhibition of cMET RTK co-activation by EGFRvIII using afatinib. Our in vivo studies using U87 EGFRvIII orthograft model revealed significant tumor growth inhibition by afatinib and TMZ combination compared to control and either drug alone.

Conclusion

Our results strongly support the efficacy of afatinib and TMZ combination in inhibiting EGFRvIII-cMET signaling mediated GBM stemness and prevention of tumor progression. This treatment should be tested in EGFR amplified/mutated GBM patients.

#4685

Cancer associated fibroblasts promote ovarian cancer chemoresistance by inducing cancer stem cells through Wnt signaling.

Yiming Fang, Mohamed A. Abd El Aziz, Kartikeya Tiwari, Anirban K. Mitra. _Indiana University School of Medicine, Bloomington, IN_.

Ovarian cancer is the most lethal gynecologic malignancy and the 5th leading cause of cancer related deaths among women in the USA. Most patients eventually succumb to chemoresistant disease and the purpose of this study is to understand the mechanism of development of chemoresistance and disease relapse. Cancer stem cells (CSCs) consist of a small subpopulation in the tumor that are resistant to cytotoxic chemotherapy and cause relapse. The tumor microenvironment can potentially provide an optimal cancer stem cell niche for cancer stem cell growth and maintenance. Cancer associated fibroblasts (CAFs) are one of the main constituents of the tumor microenvironment in ovarian tumors, promoting tumor progression and chemoresistance. We have studied the potential role of CAFs in maintaining CSC population and enhancing chemoresistance with an objective to develop effective approaches to overcome chemoresistance and tumor relapse. Co-culture of high grade serous ovarian cancer cells with CAFs resulted in increased resistance to carboplatin. ALDH1A1 is a well-established marker for ovarian cancer CSCs and the ALDH+ population was significantly increased upon co-culture with CAFs. Similarly, CAFs also enhanced spheroid formation of ovarian cancer cells seeded in ultralow adhesion plates in CSC medium. Interestingly, co-culturing ALDH- ovarian cancer cells with CAFs resulted in the induction of ALDH+ cells within 6 days. Analysis of the signaling pathways activated in ovarian cancer CSCs and the gene expression profiles of ovarian cancer CAFs indicated the potential role of Wnt signaling in the productive cross-talk between CAFs and ovarian cancer CSCs. Treatment with Wnt inhibitors abrogated the induction of CSCs by CAFs. By selectively silencing porcupine, a protein involved in Wnt ligand lipidation and secretion, we further confirmed that CAF derived Wnts are responsible for the induction of CSC. Studies are ongoing to identify the specific Wnt ligand involved in the cross-talk and downstream pathways activated during CSC induction and maintenance by CAFs. Our results indicate that CAF-derived Wnt ligands are instrumental in ovarian cancer CSC growth and maintenance. In the long term, our studies will broaden the understanding of CSC maintenance by the tumor microenvironment and contribute towards the development of novel therapeutic approaches to prevent ovarian cancer chemoresistance and relapse.

#4686

Stem-like cells and bulk cells in breast cell lines express distinct combinations of Eph receptors and ephrin ligands.

Mariana Lucero, Jaspreet Thind, Shayan Senaati, Belinda Jimenez, Tanin Zadeh, Raj P. Kandpal. _Western University of Health Sciences, Pomona, CA_.

Purpose: The maintenance of the breast cancer stem cell niche in mammary tumors has been attributed to aberrant expression of Eph receptors and ephrin ligands. The current study aims to identify Eph receptors and ephrin ligands that may contribute to the tumorigenic and invasive breast cancer phenotypes, and can potentially be used to classify tumor specific stem cells and target them for therapeutic intervention.

Methods: The bulk cells and stem-like cells from MCF10A, MCF7 and MDA-MB-231 cell lines were separated based on CD44+/CD24+ and CD44+/CD24- combinations of cell surface markers, respectively. These cell lines represent non-tumorigenic breast epithelium (MCF10A), non-invasive breast carcinoma (MCF7), and invasive triple negative breast carcinoma (MDA-MB-231). The profiles of Eph receptors and ephrin ligands were determined by qPCR. The changes in selected transcripts were confirmed by immunocytochemistry and western blot analysis.

Results: Our study revealed that Eph receptors and ephrin ligands have distinct expression signatures in CD44+/CD24- tumor-initiating cells relative to the bulk tumor cell population. Such combinatorial expression of these molecules in stem cells and bulk cells may modulate and maintain breast tumor phenotypes. The differential patterns of expression are evidenced by changes in qualitative as well as quantitative combinations of Eph receptors and ephrin ligands. The levels of EphA8 transcript were notably lower in CD44+/CD24- cells as compared to bulk MCF7 cells. However, EphA8 was significantly upregulated in stem cells as compared to bulk cells isolated from MDA-MB-231 cell line.

Conclusion: The relative abundance of Eph receptors and ephrin ligands in stem and bulk cells provides molecular signatures of stem cells specific to tumor phenotypes. The combinatorial expression of these molecules corresponds to invasiveness of breast cancer cells, which can be potentially exploited for diagnostics and therapeutics.

#4687

Gastric cancer stem cell-related marker LINGO2 is associated with cancer phenotype and poor patient survival.

Soo Been Park, Jung Hyun Jo, Chanyang Kim, Hee Seung Lee, Dawoon E. Jung, Si Young Song. _Yonsei Univ. College of Medicine, Seoul, Republic of Korea_.

The expression of leucine-rich repeat and immunoglobulin-like domain-containing nogo receptor-interacting protein 2 (LINGO2), has been reported in Parkinson's disease; however, its role in other diseases is unknown. Gastric cancer is the second leading cause of cancer death. Cancer stem cells (CSC) are a subpopulation of cancer cells that contribute to the initiation and invasion of cancer. We identified LINGO2 as a CSC-associated protein in gastric cancers both in vitro and in patient-derived tissues. We studied the effect of LINGO2 on cell motility, stemness, and tumorigenicity using cells sorted based on LINGO2 expression and LINGO2-silenced cells. Tissue microarray analysis showed that LINGO2 expression was significantly elevated in advanced gastric cancers. The overall survival of patients expressing high LINGO2 was significantly shorter than that of patients with low LINGO2. Cells expressing high LINGO2 showed elevated cell motility and tumorigenicity while LINGO2 silencing reversed these properties. Silencing LINGO2 reduced AKT/MEK/ERK phosphorylation and decreased EMT-associated markers - N-cadherin and Vimentin and stemness-associated markers - OCT4 and IHH. These indicate the involvement of LINGO2 in gastric cancer initiation and progression by altering cell motility, stemness, and tumorigenicity, suggesting LINGO2 as a putative target for gastric cancer treatment. 

### Tumor Evolution and Heterogeneity 3

#4688

Evaluation of tumor heterogeneity in prostate biopsy samples.

Pavithra D. Arachchige, Shannon Carskadon, Gaury Dhamdhere, Mireya Diaz-Insua, Rogers Craig, James Peabody, Mani Menon, Sean Williamson, Nilseh Gupta, Nallasivam Palanisamy. _Henry Ford Health System, Detroit, MI_.

Prostate cancer (PCa) is the second most common cancer among men with more than 25,000 deaths each year. A significant number of PCa patients are known to present with multifocal disease characterized by the presence of more than one tumor nodule. Currently, management decisions for active surveillance are made based on the Gleason grade, tumor stage and volume on initial prostate biopsies. Although morphological heterogeneity has been well recognized, recent studies have revealed that PCa is a heterogeneous disease at the molecular level as well. Importantly, some molecular aberrations have been associated with aggressive disease and clinical outcomes, suggesting that tumor heterogeneity may be a determining factor in the success of active surveillance and other PCa management options. Therefore, we evaluated the incidence of several molecular markers on prostate biopsy samples to understand tumor molecular heterogeneity among PCa cases. A total of 626 biopsy cores were collected from 130 consecutive patients undergoing either standard or double sextant biopsies from July to October 2016 at our institute. Selected cores included benign (n=13), high grade intraepithelial neoplasia (HGPIN, n=124), atypical/ASAP (n=33) and Gleason grade 6-9 tissues (n=447). Of the 626, 119 cores included discontinuous (intervening benign tissue) tumor foci. The presence of the molecular markers, ERG, SPINK1, ETV1 and ETV4 was simultaneously evaluated using a novel, combined approach by dual Immunohistochemistry and RNA in situ hybridization. A total of 281 cores (44.9%) were positive for at least one molecular marker. Of these, ERG+ was more prominent (22.0%), followed by SPINK1+ (14.1%). The incidence of ETV1+ and ETV4+ was low (4.5% and 2.1%, respectively). 253 biopsy cores (56.6%) with Gleason 6-9 cancer were positive for at least one marker. Notably, only 12 cores (9.7%) of HGPIN were positive for any one of the markers while all benign cores were negative. Of note, 13 atypical/ASAP cores (39.4%) were positive for at least one marker, suggesting the potential use of molecular analysis to eliminate ambiguities associated with the diagnosis of cancer in atypical lesions. Of the 119 cores with discontinuous tumor foci, 14 (11.7%) dual marker positive with discordant marker status in each foci. Additionally, 99 cores (83.2%) showed the presence of a single marker either in one or both foci, further emphasizing the independent clonal origin and the presence of distinct driver molecular aberrations in different tumor foci in a subset of PCa cases. Finally, 345 of the 626 cores (55.1%) were negative for all the tested molecular markers indicating hitherto unidentified driver molecular aberrations. In conclusion, our study highlights the presence of significant tumor molecular heterogeneity identified in biopsy samples and emphasizes the importance of considering all tumor nodules in multi focal disease in making clinical decision on active surveillance.

#4689

Subclone-specific evolution of tumor phenotypes -- A framework to study subclone-specific gene expression from a combination of bulk DNA and single cell RNA sequencing data.

Yi Qiao,1 Xiaomeng Huang,1 Samuel Brady,1 Andrea Bild,2 David Bowtell,3 William Johnson,4 Gabor Marth1. 1 _University of Utah, Salt Lake City, UT;_ 2 _City of Hope, Duarte, CA;_ 3 _Peter MacCallum Cancer Center, East Melbourne, Australia;_ 4 _Boston University, Boston, MA_.

Several approaches are now available for subclonal reconstruction of heterogeneous tumor biopsies from somatic variant allele frequencies measured in bulk DNA sequencing datasets, including our own SubcloneSeeker program. However, to understand how chemoresistance, relapse, or metastasis evolves at a subclonal level, we also need to study the molecular phenotype corresponding to each subclone. Single-cell RNAseq technologies now allow one to study the transcriptomic characteristics and phenotypic behaviors of individual cells, but new algorithms are needed to link these cells to the genomically defined tumor subclones they represent.

Here we present a computational approach to make such cell assignments, using a combination of bulk DNA sequencing data (WGS or WES), and single cell RNA sequencing (scRNAseq) collected from the same tissues. Subclones constructed from bulk DNA sequencing data are defined by specific combinations of somatic mutations (SNVs and CNVs). Our algorithm uses the scRNA-seq reads to assess which of these subclone-defining mutations are present in each individual cell, then utilizes a Bayesian probabilistic framework for making the cell-to-subclone assignment. This framework overcomes the challenges of sparse scRNA-seq read coverage, and maximizes the accuracy and efficiency of cell assignment.

We have successfully applied this method to multiple longitudinal and metastatic cancer patient datasets, representing both WES and WGS bulk DNA sequencing, as well as datasets collected using the 10X Chromium and Fluidigm C1 technologies. Our algorithm uses both somatically acquired CNV and SNV events in the tumor for cell assignment. Using our approach, as many of 80% of tumor cells could be assigned to specific subclones, enabling comparative gene expression and pathway analysis across subclones.

#4690

Multi-sample automation of the CLARITY technology for the processing of 3D volumes of tissue.

Sharla L. White, Yi Chen, Qi Shen, Laurie J. Goodman. _ClearLight Biotechnologies, Sunnyvale, CA_.

The current technologies utilized for preclinical and clinical drug development in cancer is largely dependent upon the 2-dimensional (2D) analysis of thin formalin-fixed paraffin embedded (FFPE) tissue sections (5-10 µm). However, the importance of understanding cellular phenotypic information combined with three-dimensional (3D) spatial analysis of tissues has recently evolved. In recent years, several clearing techniques, such as CLARITY, have been developed and modified as a means to image and evaluate these volumetric tissues. Most of these techniques have employed chemical approaches to improve tissue clearing, while inadvertently affecting the tissue integrity on a macroscopic or microscopic level. Our previous work with CLARITY has demonstrated how the tissue-hydrogel matrix (HM) is able to maintain its structural integrity overall. Yet, some of the most noted caveats to employing this technique has been the lengthy processing times, and the lack of robust 3D spatial analysis software. We sought to address these issues through the development of an automated clearing and staining platform for CLARITY processed tissues with a proprietary 3D image analysis employing artificial intelligence and machine learning techniques. All experiments were performed with the CLARITY technique using HM-embedded tissues that were clearing with a SDS/borate clearing buffer. Evaluation of the clearing module was assessed using a passive staining (diffusion-based) approach before sample imaging. The effectiveness of the staining module was assessed using passively cleared tissues that were "actively" stained using the developed respective module, followed by standard imaging. The imaging data was then uploaded into our proprietary 3D software for segmentation, classification, and quantitative spatial analysis. We were able to demonstrate successful clearing and staining in both normal and cancerous tissue samples in a total time of less than one day. Consistent results are obtained for both fresh and formalin-fixed tissues. This was a significant reduction in the time associated with the standard passive clearing and staining procedure. In short, the development of our end-to-end multi-sample clearing and staining platform not only removes the laborious sectioning and sample registration for sample reconstruction, but also maintains the benefits of multiple interrogation of a single sample. Although volumetric clearing and 3D analysis are still in their infancy from a technology perspective, one tissue sample using these novel approaches provides as much volumetric information as 200 FFPE sections, while also maintaining key spatial information.

#4691

Evaluation of the NanoString's Digital Spatial Profiling (DSP) technology in formalin-fixed paraffin embedded (FFPE) cell line mixtures, PBMCs and non-small cell lung cancer (NSCLC) tissues.

Joshua James Rusbuldt,1 Tanesha Cash-Mason,1 Shaozhou Tian,1 Alison VanSchoiack,2 Yan Liang,2 Chandra Rao,1 Denis Smirnov1. 1 _Janssen RD, US, Spring House, PA;_ 2 _NanoString Technologies, WA_.

Evaluation of the NanoString's Digital Spatial Profiling (DSP) Technology in Formalin-Fixed Paraffin Embedded (FFPE) cell line mixtures, PBMCs and Non-Small Cell Lung Cancer (NSCLC) tissues

Purpose: The complex nature of the tumor requires multiplexed, quantitative analysis of tissues which is challenging using immunohistochemistry (IHC). Several novel approaches (including NanoString Digital Spatial Profiling (DSP) Technology) were developed to enable multiplexed analysis of tissues. Here we report on a comparative evaluation of the GeoMx™ DSP platform including a comparison to IHC.

Methods: Performance of DSP methodology was assessed using Raji and CEM/C1 cell line mixtures, as well as healthy donor PBMCs that had been evaluated using flow cytometry for expression of the targeted markers: CD4, CD8, CD68, CD14, CD3, CD45, CD45RO, CD20 and GZMB. DSP technology was compared with IHC by measuring T-cells markers (CD3, CD4, CD8), a monocytic marker (CD68) as well as PD-L1 expression in five FFPE NSCLC tumor specimens. Serial sections were stained by IHC, hematoxylin and eosin (H&E) and regions of interests (ROIs) were selected to sample areas with varied expression of each of the markers (qualified as low, medium and high). Selected ROIs were then assayed on the GeoMx DSP platform in a blinded fashion using a 39-plex immune panel that included CD3, CD4, CD8, CD68 and PD-L1 antibodies and DSP counts were compared to qualitative IHC scoring.

Results: Cell line mixture experiments demonstrated that protein counts were highly correlated to the fractions of cell lines expressing a given marker (r=0.90 - 0.94). The assay also showed good concordance with flow cytometry measurements (r=0.49-0.51 between flow cytometry MFIs and NanoString counts for PMBCs). DSP counts related well to the IHC staining patterns, as higher DSP measurements were detected in ROIs classified as high based IHC staining intensity for the markers investigated. As expected, counts were lower for markers such as CD8 and PD-L1 that were also classified as low expressing by IHC in the analyzed NSCLC tumor samples. Exploratory analysis of co-expression of all 39 markers included in the DSP panel in various areas of the tumors suggested that DSP technology can be used to perform detailed characterization of various areas of the tumor.

Conclusions: Our evaluation affirms that NanoString's GeoMx DSP platform is a promising option for multiplexed analysis of tumor tissues. DSP technology produced measurements comparable to those obtained with flow cytometry and IHC for the evaluated markers. These data show that DSP technology can collect high-parametric proteomics data from FFPE tissue samples which can help to study the complex biology of tumor microenvironment.

#4692

Deconvolution reveals cell-type specific transcriptional effects across cancer types.

Shaolong Cao,1 Rongjie Liu,2 Liuqing Yang,3 Jaeil Ahn,4 Jingxiao Chen,1 Zeya Wang,2 Eleni Efstathiou,1 Daniel E. Frigo,1 Hongtu Zhu,3 Wenyi Wang1. 1 _University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Rice Univeristy, Houston, TX;_ 3 _University of North Carolina at Chapel Hill, NC;_ 4 _Georgetown University, DC_.

Background:

Most tumor samples consist of a variable proportion of malignant and nonmalignant cells including epithelial cells, fibroblasts, and infiltrating immune cells, which confounds biomarker studies of response to treatment. Deconvolution approaches have been developed for transcriptomes to address this heterogeneity in tumor samples. The Cancer Genome Atlas (TCGA) project has generated high-throughput RNAseq in over 11,000 patient samples across 33 cancer types. Using these data, we systematically investigated Pan-Cancer cell-type-specific transcriptional activities using deconvolution tools.

Method:

We established a new deconvolution framework, DeMixT, which deconvolves high-dimensional transcriptome data from mixtures of tumor and stromal components. Besides estimating mixing proportions, DeMixT uniquely provides per-gene per-sample expression levels of each component.

To further address variations observed in real data, we propose a profile likelihood-based prefiltering method to adaptively select a gene set with a high signal-to-noise ratio for proportion estimation, which sequentially improves gene expression estimation for the whole transcriptome.

We applied the pre-filter-enabled DeMixT to 16 TCGA solid primary tumor types: BLCA, BRCA, COAD, HNSC, KICH, KIRC, KIRP, LIHC, LUAD, LUSC, PAAD, PRAD, READ, STAD, THCA, UCEC. In 2 cancer types: PRAD (Prostate Adenocarcinoma) and KIRC (Kidney Renal Clear Cell Carcinoma), we compared results of pathway analyses, clustering, and survival analyses from the original mixed expression data with those from the deconvolved expression data.

Results:

For the 16 cancer types, we obtained tumor purities and deconvolved individual-level gene expression of both tumor and stromal components. Per cancer type, the mean number of genes recovered was 13,239 (sd=886), and the mean number of tumor samples deconvolved was 371 (sd=201).

In PRAD, we identified important pathways that were missed previously but are now statistically significant (FDR<0.1): Epithelial-Mesenchymal-Transition, TP53, NF-κB, Hypoxia, Estrogen Response, Apical Junction, IL2-STAT5 Signaling, and KRAS Signaling. A closer look at Urea Cycle Dysregulation (UCD) genes found augmented downregulation of ASS1 and intensified upregulation of SLC25A13 and SLC25A15 in the deconvolved tumor vs. normal comparison, consistent with the previous report in Lee et al. Cell 2018.

In KIRC, hierarchical clustering of deconvolved expression data resulted in a better enrichment of a PBRM1 mutant in one cluster of patients, who had better survival outcomes. This observation is consistent with the previous report by Kapur et al. Lancet Oncol 2013.

Conclusion:

We provide comprehensive transcriptome deconvolution results for the TCGA Pan-Cancer datasets. These findings will enable novel studies of admixed human tumor samples and improve the understanding of tumor malignancy.

#4693

**Evaluation of prostate tumor molecular heterogeneity using whole mount radical prostatectomy by dual immunohistochemistry and dual RNA** in situ **hybridization.**

Pavithra D. Arachchige, Shannon Carskadon, Gaury Dhamdhere, Mireya Diaz-Insua, Hans Stricker, Craig Rogers, James Peabody, Mani Menon, Sean Williamson, Nilesh Gupta, Nallasivam Palanisamy. _Henry Ford Health System, Detroit, MI_.

Prostate cancer (PCa) remains the second most common cancer among American men. Morphological heterogeneity in prostate cancer is well recognized, however, recent investigations revealed molecular heterogeneity with the existence of unique molecular aberrations among patient sub groups. Molecular heterogeneity may be associated with distinct routes of disease progression, thereby, enabling the use of molecular profiling to facilitate targeted therapy. Importantly, African Americans (AA) are known to develop more aggressive forms of PCa compared to Caucasian Americans (CA). Therefore, the identification of distinct molecular profiles between the two groups may lead to more precise treatment options for PCa patients. Given that a comprehensive molecular mapping, encompassing both AA and CA patients is lacking, we carried out an extensive profiling of multiple molecular markers in a large cohort of AA and CA PCa patients. Whole-mounted post-prostatectomy tissues were obtained from 1117 patients who underwent radical prostatectomy surgery at our institute. The selected cases included 575 (52%) CA and 453 (41%) AA patients. The presence of ERG, SPINK1, ETV1, ETV4 and ETV5 was evaluated using dual IHC and dual RNA ISH. The racial disparity in the molecular marker incidence was analyzed. The incidence of ERG was observed in 52.0% (436/839) of cases while SPINK1 incidence occurred in 45.1% (378/838) cases. The incidence of other ERG family rearrangements, ETV1, ETV4 and ETV5 was low with 9.9% (78/791), 3.8 % (28/733) and 0.6% (2/350) cases respectively. A total of 260 cases showed the incidence of multiple markers on a single prostate. Of these, ERG+/SPINK1+ was prominent with an incidence of 18% (151/838), followed by ERG+/ETV1+ which occurred in 5.2% cases (41/790). In most cases, multiple marker incidence was detected in separate foci, supporting the independent clonal origin of tumor foci in patients with multi focal disease. In a subset of cases, the co-occurrence of ERG and SPINK1 in the same foci was observed, suggesting intra tumor heterogeneity, however, in majority of the cases mutually exclusive pattern of expression was observed, indicating the presence of unique driver molecular aberrations in each tumor foci.. Statistical analysis revealed significant incidence of ERG+ in CA (P=0.00) and SPINK1 in AA patients (P=0.00). Additionally, the incidence of ERG/SPINK1 and SPINK1/ETV1 were more frequent among AA patients compared to CA patients (P=0.0124 and P=0.0417). In conclusion, our study highlights the existence of significant molecular heterogeneity among PCa patients in a racially disparate perspective. Consequently, a thorough understanding of the clinical association between distinct molecular sub groups and disease progression in each racial group will significantly aid the treatment efforts in combatting PCa disease.

#4694

**Evaluation of tumor-associated antigen expression with the MACSima** TM **high-content imaging platform.**

Christoph Herbel,1 Vera Dittmer,1 Manuel Martinez-Osuna,1 Laura Nadine Kuester,1 Daniel Schaefer,1 Jan Drewes,1 Jutta Kollet,1 Werner Mueller,1 Michael Mallmann,2 Peter Mallmann,2 Philipp Stroebel,3 Olaf Hardt,1 Dominik Eckardt,1 Andreas Bosio1. 1 _Miltenyi Biotec, Bergisch Gladbach, Germany;_ 2 _University of Cologne, Cologne, Germany;_ 3 _University of Goettingen, Goettingen, Germany_.

Here we report the use of the MACSima™ imaging platform to perform high-content imaging for the pre-clinical validation of tumor target expression on tumor and healthy human tissues indicative for potential on-target/off-tumor toxicity in vivo.

Major advances have been achieved in cancer therapy in the past decade. In particular, targeted-immunotherapy has progressed and clinical benefits for patients have been achieved. However, on-target/off-tumor toxicity is a potential threat which has been shown to be more pronounced in solid than in liquid tumors. These findings highlight the need for better pre-clinical assays to improve the safety profile of immunotherapies.

On-target/off-tumor toxicity is mainly based on the expression of tumor-associated antigens (TAA) in healthy tissues under physiological conditions. Currently, most prediction methods for on-target/off-tumor expression are based on bulk mRNA expression data of healthy tissue. These prediction models, however, have limitations, mainly poor predictable relation between RNA and protein level. Moreover, it is frequently unclear which cell types are affected by on-target/off-tumor effects.

To overcome these limitations we employ multi-parameter imaging to analyze the expression of TAAs directly at the protein level. Additionally, we gain spatial information about tissue and cellular distribution of TAAs. Notably, our novel high-content imaging technology potentially allows for the analysis of hundreds of markers in a single experiment, paving the way for high-dimensional characterization of cells within complex solid tissues.

We performed high-content imaging with the MACSima™ platform to validate the expression of known TAAs across tumor and healthy tissue samples. Subsequently, we employed unbiased cluster analysis revealing correlation patterns within the datasets to identify cell types at risk. In detail, we analyzed several high-grade serous ovarian carcinoma and pancreatic ductal adenocarcinoma for the expression of known TAAs, described tumor markers, and tissue lineage markers. Additionally, we evaluated the expression of these TAAs across several healthy human tissues. Next we performed pixel- and object-based data analysis for unbiased cluster analysis. Thereby we identified cell clusters that express TAAs in ovarian and pancreatic cancers, as well as in healthy tissues. We found that primarily epithelial cells express the analyzed TAAs in different tissues and that TAA expression shows inter- and intra-tumor heterogeneity.

Taken together, we established a novel workflow for multi-parameter characterization of tissues employing the MACSima™ imaging platform. Our work enables efficient identification of potential tissues for on-target/off-tumor toxicity, paving the way for improved pre-clinical evaluation of TAAs as new targets.

#4695

Development and validation of an accurate exome-scale cfDNA detection platform.

Sean Michael Boyle, Charles Abbott, Robin Li, Eric Levy, Shujun Luo, Robert Power, Rena McClory, John West, Richard Chen. _Personalis, Inc., Menlo Park, CA_.

Background: Neoantigens are increasingly critical as biomarkers for immunotherapy response to checkpoint blockade therapy and as therapeutic targets for neoantigen-based personalized cancer vaccines. Accurate identification of neoantigens requires comprehensive exome and transcriptome sequencing of both a tumor biopsy and the matched normal samples to enable identification of putative neoantigens - which occur across the genome.

Methods: However, as tumor biopsy samples cannot always be obtained, and because tumor heterogeneity can result in an incomplete set of neoantigens from a single biopsy, we developed ImmunoID NeXT circulating tumor DNA (ctDNA) Exome to (1) identify neoantigens in cell free DNA (cfDNA) as a complement to tumor biopsy derived neoantigens and (2) track neoantigens in the cfDNA post immuno-therapy treatment. To demonstrate the utility of the ImmunoID NeXT ctDNA Exome for both identification and monitoring of neoantigens directly from cfDNA, we have performed two extensive studies. Firstly, we obtained cfDNA from 2 healthy donors, mixed them to create somatic variants with AFs down to 0.5% with analytical sensitivity calculated against >10,000 variants. Secondly, we have performed extensive cfDNA testing in cancer patients to assess our capabilities across tumor types. Finally, and very importantly, we utilized multi-location primary and metastatic tumor profiling to demonstrate the ImmunoID NeXT cfDNA platform can be applied to profile tumor heterogeneity.

Results: In our healthy donor mixes, we observed our ImmunoID NeXT cfDNA platform can detect de novo "gold set" SNVs with a sensitivity of 90% down to an allele frequency of 2%. For monitoring applications we are able to detect SNVs with a sensitivity of 92% down to an allele frequency of 0.5%. When tested across a range of tumor types including melanoma, lung, and colorectal, ImmunoID NeXT repeatedly detected high concordance for somatic events shared between tumor and cfDNA. In tumor samples allele frequencies ranged from10% to 100%, and through cfDNA interrogation 60%-98% of these events were accurately detected in the plasma. Finally, we were able to reproducibly detect ctDNA, which were not present in the primary tumor sample, in subsequent primary tumor biopsy curls or metastases in multiple tumor types, demonstrating our ctDNA platform can effectively monitor tumor heterogeneity.

Conclusion: These results show the accuracy of the ImmunoID NeXT platform for detecting and profiling ctDNA somatic events. Further, these results demonstrate the potential of using a comprehensive ctDNA Exome to identify and monitor neoantigens as a complement to the results from sequencing of the tumor biopsy alone.

#4696

**High-throughput single-cell targeted DNA sequencing using an updated Tapestri** TM **Platform reveals rare clones and clonal evolution for multiple blood cancers.**

Nianzhen Li, Daniel Mendoza, Adam Sciambi, Mani Manivannan, Jacob Ho, Kaustubh Gokhale, Jacqueline Marin, Kathryn Thompson, Jamie Yates, Vasu Sharma, Steven Chow, Sombeet Sahu, Shu Wang, Dennis Eastburn, Keith Jones. _Mission Bio, South San Francisco, CA_.

Introduction: The challenge in precision medicine has been improving the understanding of cancer heterogeneity and clonal evolution, which has major implications in targeted therapy selection and disease monitoring. However, current bulk sequencing methods are unable to unambiguously identify rare pathogenic or drug-resistant cell populations and determine whether mutations co-occur within the same cell. Single-cell sequencing has the potential to provide unique insights on the cellular and genetic composition, drivers, and signatures of cancer at unparalleled sensitivity.

Methods: Previously we have developed a high-throughput single-cell DNA analysis platform (TapestriTM) that leverages droplet microfluidics and a multiplex-PCR based targeted DNA sequencing approach, and demonstrated the generation of high-resolution maps of clonal architecture from acute myeloid leukemia (AML) tumors. Here we present an update to the Tapestri Platform which employs new biochemistry and features improved firmware, software, workflow, and data analysis solution resulting in higher throughput, better sensitivity, specificity and unprecedented flexibility.

Results: From cell prep to sequencing-ready libraries, the workflow can be completed within 2 days, and new modifications have doubled the throughput to up to 20,000 genotyped cells per run from 10,000 shown previously. We have validated the performance of an AML (19 genes, 50 amplicons) and a CLL (chronic lymphocytic leukemia) (34 genes, 286 amplicons) panel. We also developed a robust web-based design portal for custom targets. The updated biochemistry enables easy addition of new gene and loci targets into existing panels for improved coverage and updated studies. Using longitudinal AML and CLL samples, we were able to detect rare subclones of <0.1% prevalence, identify mutation co-occurrence, and characterize clonal evolution due to disease progression and drug treatment.

Conclusion: We demonstrate that single-cell DNA sequencing can reveal the heterogeneity of blood cancers and map the clonal architecture and clonal evolution with higher sensitivity than bulk NGS methods. This is critical in patient stratification and drug selection over the entire course of treatment. Besides the catalog AML and CLL panels, the flexibility of system allows for analyzing SNV and indel mutations of any custom cancer DNA targets. Additionally, the system provides capabilities for quality control of gene edited cells, further advancing research into cancer therapies.

#4697

Expression variation analysis for tumor heterogeneity in single-cell RNA-sequencing data.

Emily F. Davis-Marcisak,1 Pranay Orugunta,1 Genevieve Stein-O'Brien,1 Sidharth V. Puram,2 Evanthia Roussos Torres,1 Alexander Hopkins,1 Elizabeth M. Jaffee,1 Alexander V. Favorov,1 Bahman Afsari,1 Loyal A. Goff,1 Elana J. Fertig1. 1 _Johns Hopkins School of Medicine, Baltimore, MD;_ 2 _Washington University School of Medicine, St. Louis, MO_.

Introduction: Tumor heterogeneity provides a complex challenge to cancer treatment and is a critical component of therapeutic response, disease recurrence, and patient survival. Single-cell RNA-sequencing (scRNA-seq) technologies reveal the prevalence of intra- and inter-tumor heterogeneity. Computational techniques are essential to quantify the differences in variation of these profiles between distinct cell types, tumor subtypes, and patients to fully characterize intra- and inter-tumor molecular heterogeneity. To address this, we devised a new algorithm, Expression Variation Analysis in Single Cells (EVAsc), to perform multivariate statistical analyses of differential variation of expression in gene sets for scRNA-seq.

Methods: EVAsc provides a robust statistical test to compare the heterogeneity of transcriptional profiles of genes in a pathway between groups of cells from two phenotypes. Using simulated data, we demonstrated that this method is robust for imputed scRNA-seq data with high sensitivity and specificity to detect pathways with true differential heterogeneity. We then applied EVAsc to public domain scRNA-seq tumor datasets in breast cancer and head and neck squamous cell carcinoma (HNSCC) to quantify the landscape of tumor heterogeneity in several key applications in cancer genomics, i.e. immunogenicity, cancer subtypes, and metastasis.

Results: We demonstrated that immune pathway heterogeneity in hematopoietic cell populations in breast tumors corresponded to the amount diversity present in the T-cell repertoire of each individual. In HNSCC patients, we found dramatic differences in pathway dysregulation across basal primary tumors, indicative of inter-tumor heterogeneity within a single subtype. Within the basal primary tumors we also identified increased immune dysregulation in individuals with a high proportion of fibroblasts present in the tumor microenvironment. Moreover, cells in HNSCC primary tumors had significantly more heterogeneity across pathways than their matched metastatic cells, consistent with a model of clonal outgrowth.

Conclusions: The results of these analyses demonstrate the broad utility of EVAsc to quantify inter- and intra-tumor heterogeneity from scRNA-seq data without reliance on low dimensional visualization. EVAsc is a robust multivariate statistical method to quantify differential variation of pathway gene expression and provides the ability to explore transcriptional variation in numerous disease and normal contexts at a single cell resolution. Accurate characterization of inter-sample variation from scRNA-seq data of tumors is critical to quantify the cellular heterogeneity that drives tumor progression through dysregulation of key cancer pathways. Thus, identifying dysregulated pathways in individual tumors may be an important biomarker for clinical response to immunotherapy.

#4698

**Development of a CRISPR-Cas9** D10A **targetable, high-complexity, single-cell barcoding approach for capture of treatment resistant subclones from heterogeneous cancers.**

Ze-yan Zhang,1 Ravesanker Ezhilarasan,1 Yingwen Ding,1 Qianghu Wang,2 Jie Yang,1 Lihong Long,2 Roel G. Verhaak,3 Erik P. Sulman1. 1 _NYU Langone Health, New York, NY;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 3 _The Jackson Laboratory for Genomic Medicine, Farmington, CT_.

Cancers are composed of heterogeneous cell populations and the clonal evolution of these cells is one of the key reasons for treatment resistance and tumor recurrence. A fundamental challenge in studying clonal evolution in these tumors is the difficulty in capturing the phenotype-associated (e.g. treatment resistant) sub-populations from the heterogeneous population. We developed an approach to individually barcode and isolate specific cell subpopulations by constructing a Cas9D10A and paired-gRNA targetable unique reporter (CAPTURE) barcoding library with up to 36 million unique barcodes. This approach enabled us to uniquely barcode > 1 million cells, track barcode distribution following treatment and then isolate the resistant subpopulation using the subpopulation-specific barcode. Proof of principle studies showed that specific barcoding cells, even at as low as 0.1%, could be efficiently isolated using barcode targeting and cell sorting. Applied our barcoding approach, we found that A375 cell acquired BRAF inhibitor resistance both from pre-exiting and late-emerging mechanisms. Colony formation experiments showed that the isolated subpopulations were indeed resistant clones. Whole exome, transcriptome and methylome analysis were applied to study the captured subpopulations. Our CAPTURE barcoding approach will enable the identification of the both pre-exiting and late-emerging genetic or epigenetic changes driving treatment resistance.

#4699

Enriching tumor purity using a unique flow-sorting approach to elucidate clonal evolution in matched samples of squamous cell carcinoma of the lung.

Arthur Krause,1 Maria R. De Filippo,1 Thomas Lorber,1 Tanja Dietsche,1 Valeria Perrina,1 Christian Ruiz,1 Michael T. Barrett,2 Salvatore Piscuoglio,1 Charlotte K. Ng,1 Lukas Bubendorf1. 1 _University Hospital of Basel, University of Basel, Basel, Switzerland;_ 2 _Mayo Clinic Arizona, Scottsdale, AZ_.

Background:All carcinomas contain a variable proportion of benign stromal and immune cells, limiting the sensitivity in the identification of somatic genetic alterations. The average tumor purity of squamous cell carcinomas (SCC) of the lung is only around 50%. Low tumor content in tumor samples represents a challenge in studying intratumoral genomic heterogeneity in cancer research. Here, we applied flow-sorting to enrich for tumor cell nuclei to investigate the clonal relationship of primary SCC and matched metastases.

Methods:Tumor tissues from 16 patients with primary SCC of the lung and matched metastases were used. We implemented a flow-sorting based approach to enrich for tumor nuclei as followed: after extraction of nuclei from snap-frozen or FFPE tissue, they were flow-sorted by DNA content (ploidy) and cytokeratin expression of the adherent cytoplasm, using a pan-cytokeratin (pCK) antibody. Isolated diploid and aneuploid pCK-positive tumor cell populations were subjected to whole exome sequencing (WES). DNA from diploid pCK-negative populations was used as germline control. Mutational profile and copy number aberrations (CNA) were determined to infer the clonal relationship and evolution between the primary tumors and their metastases.

Results:Our flow-sorting based approach was able to enrich tumor content from 20%-50% to more than 80%-90% and to distinguish between aneuploid and diploid tumor cell populations from bulk tumor tissues. It enabled the identification of somatic mutations and CNA in both, aneuploid and diploid tumor cell populations, including potential subclonal driver mutations in ARID1A and KDM6A at low allelic frequencies. Shared and private mutations were observed in matched longitudinal tumor samples of individual patients and in synchronous distant metastases. Ploidy did not change significantly between primary tumors and relapse or distant metastases.

Conclusion:We present a flow-sorting based method to enrich for tumor cell nuclei to facilitate genomic analysis, which also enabled separate analysis of aneuploid and diploid tumor populations. Our results show that the enrichment approach can be used to decode the clonal evolutionary relationship between primary tumors and their matched metastases, even in samples with low tumor cell content.

#4700

Quantification of intratumoral heterogeneity in individual luminal A breast cancers from whole transcriptome data through semi-supervised learning.

Neeraj Kumar,1 Yash Dharmamer,1 Amit Sethi,2 Peter Gann1. 1 _University of Illinois at Chicago, Chicago, IL;_ 2 _Indian Institute of Technology Bombay, Mumbai, India_.

Introduction PAM50 gene profiling assigns individual breast cancers (BCas) to one of five intrinsic subtypes. However, individual tumors vary in their adherence to the assigned subtype, and a subset of these may show admixture with another subtype. We developed a novel metric to quantify this intratumoral heterogeneity using whole transcriptome expression data of individual BCas and studied specific pairwise admixtures for Luminal A (LumA) cases.

Methods We obtained whole transcriptome expression data and PAM50 labels of 1081 and 1980 BCa cases from TCGA and METABRIC cohorts, respectively. We combined the two datasets, by identifying a common gene set. METABRIC data was pre-processed to normalize and log-transform expression levels and remove batch effects. For each case, the combined cohort had expression data of 11,379 genes, and probabilities of its adherence to each of the four major subtypes were computed using semi-supervised non-negative matrix factorization. The four probabilities obtained for each case were then combined using the proposed Aggregated Differential Sum (ADS) metric. For each subtype, the cases were subsequently stratified into pure, neither and admixed categories by computing ADS tertiles. For every case, we did comparative analysis of the ADS terms to identify the specific alternate molecular subtype for pairwise admixture analysis.

Results Using the proposed ADS criteria, pure and admixed LumA cases spanned tertile 1 (T1) and 3 (T3), respectively. Compared to admixed cases, pure LumA cases were younger and had favorable clinical characteristics including a surrogate LumA profile (ER/PR+ and HER2-), smaller tumor size, lower stage, less prevalence of TP53 mutations, and high likelihood of PIK3CA and CBFB mutations. Kaplan-Meier curves and Cox proportional hazard analysis showed that the admixed cases had worse overall survival and higher mortality risk compared to their pure counterparts. Pairwise admixture analysis revealed that 59% (n=226), 27% (103) and 15% (55) LumA cases in T3 were admixed with Luminal B, Basal and HER2 subtype, respectively. LumA cases admixed with HER2 showed worst tumor characteristics, survival outcomes and highest mortality risk followed by, in order, the admixture with Luminal B and Basal subtypes.

Conclusion Our results demonstrate that stratification of LumA BCas by subtype purity using ADS identifies the admixed cases which usually have worse tumor characteristics and survival outcomes. LumA cases were predominantly admixed with Luminal B subtype followed by Basal and HER2 subtypes. Eventually, we will use the pure cases to train computer vision classifiers for morphological identification of subtype admixture in routine H&E images. This would be a cost-effective complement to molecular subtyping that could take advantage of the spatial information available in whole slide images.

#4701

Single cell mass cytometry analysis distinguishes indolent from aggressive lung adenocarcinomas.

Maria-Fernanda Senosain,1 Yong Zou,2 Deon B. Doxie,1 Aneri Balar,2 Rosana Eisenberg,2 Jonathan M. Lehman,2 Jonathan M. Irish,1 Pierre P. Massion2. 1 _Vanderbilt University, Nashville, TN;_ 2 _Vanderbilt University Medical Center, Nashville, TN_.

Lung adenocarcinoma (ADC) is a heterogeneous group of tumors associated with dramatically different survival rates, even when detected at an early stage. The overarching goal of our research is to identify the cellular and molecular predictors of indolent and aggressive behavior of early lung ADCs. In the work presented here, we hypothesized that mass cytometry, a single cell proteomic approach, would allow the discovery of cellular determinants of early lung ADC behavior. To test this hypothesis, we prepared a mass cytometry panel of 34 labeled antibodies and validated their performance in four lung ADC cell lines (A459, H23, PC9 and H3211), and in peripheral blood mononuclear cells (PBMCs). We then tested our panel in a set of 11 early stage lung ADCs. Based on radiomics features, four of these ADCs were predicted to have long (LS), one intermediate and 6 short survival (SS) establishing the rationale for the comparisons. Tumors were disaggregated into a single cell suspension within one hour after resection and cryopreserved before mass cytometry analysis. We used unsupervised clustering algorithm FlowSOM to identify cellular subpopulations and analyze differences in their distribution both within the tumor microenvironment (TME) and the epithelial compartment. We found that predicted LS tumors had a higher proportion of leukocytes (p-value = 0.027), moderately enriched in CD8+ T cells (p-value = 0.5671), whereas predicted SS tumors had a higher proportion of fibroblasts/mesenchymal cells (p-value = 0.138). Additionally, tumors show high degree of heterogeneity with distinct protein expression profiles among epithelial subpopulations. Epithelial subsets with high vimentin and low HLA-DR, and other subsets expressing p-ERK and p-S6 were mainly present in predicted SS tumors, whereas subsets with a high HLA-DR and low vimentin were mainly present in predicted LS tumors. Also, similarities on the subsets distribution in some samples suggest the presence of ADC molecular subtypes. It remains to elucidate if the observed heterogeneity of the epithelial compartment in combination with a specific composition of the TME could lead to a stronger association with predicted survival. Our preliminary results of mass cytometry in early lung ADCs confirm a distinct cellular profile of epithelial and stromal cells among indolent vs aggressive ADCs. This work deserves further validation at the cellular and molecular level to gain further insights into tumor behavior. [Supported by UO1CA196405 to PPM.]

#4702

Characterization of ultrasound and immunohistochemichal parameters during development of murine tumor.

Jerome Griffon, Delphine Le Guillou-Buffello, Lori Bridal, Michele Lamuraglia. _Laboratoire d'Imagerie Biomedicale (UPMC, CNRS, INSERM), Paris, France_.

Background: We compared the evolution of US index in vivo (morphological tumor size, tissue elasticity and microvascular flow) with whole-slice fluorescent immunohistochemical marker maps at different stages of murine tumor development.

Methods: Murine colorectal carcinoma (CT26) was subcutaneously implanted in 49 mice on Day 0 (D0). Mice were randomized for data acquisition on D7, D10, D14, D16 and D17 (n = 10 each day except for D16 n = 9). Tumors were measured in B-mode along major transverse and longitudinal axes to estimate ellipsoidal volume. Contrast-Enhanced Ultrasound (CEUS) performed with Sequoia 512, 7-14 MHz probe and with syringe-pump-controlled bolus injection in the caudal vein of 40 μL of SonoVue (Bracco Suisse) and Shear-Wave Elastography (SWE; SSI, Aixplorer, SL 15-4 probe) maps were acquired along the longitudinal axis. Whole-slice histological sections were prepared with fluorescent immunohistochemical staining and parametric maps were made with in-house software to estimate the % area of nuclei, apoptosis (AP) and vascular endothelium (VE) marker. Regions with no contrast-enhancement were identified to estimate % perfused area (%P) and to construct perfused (P) and nonperfused (NP) masks that were then co-registered to SWE maps. Mean and standard deviation (SD) of the SWE could be calculated in whole tumor, P and NP zones. Average contrast intensity vs time curves in the P zone were fit to a lognormal model to estimate Area Under the Curve (AUC), Peak Enhancement (PE), Time to Peak (TTP), Mean Transit Time (MTT), Wash In Rate (WIR) and Wash Out Rate (WOR). The non-parametric Wilcoxon rank and Spearman tests were used to test respectively parameter evolution and correlation.

Results: SWE and CEUS parameters did not correlate. SWE did not differ significantly for P and NP zones. Only TTP (RS = 0.47, p < 0.01) correlated positively with tumor volume. Overall, %P was high ( >= 75%) and did not correlate with tumor volume. Individual histological parameters did not change significantly with time or tumor volume, but % area of strong co-marking of VE and AP decreased significantly between D10 and D14/16.

Conclusions: These preliminary results probe the relationship between tumor volume, functional-flow and elastic properties in tumors. One CEUS parameter correlated with tumor growth and thus appears to be sensitive to changes occurring during tumor development. Lack of correlation between SWE and CEUS parameters suggests that they provide complementary information. Nevertheless, it is better to understand the sensitivity of these non-invasive parameters to changes occurring during tumor development in order to obtain a predictive tumor growth parameter. The difference combination of these parameters will change the clinical practice, allowing an early therapeutic adaptation without loss of opportunity to take the good treatment for the patient and without unnecessary therapeutic costs.

#4703

Single Cell CNV End to End workflow to uncover tumor heterogeneity in a murine breast cancer model.

Lee D. Gibbs, Jing Qian, Michelle Webb, Sharon Chang, Zarko Manojlovic, Enrique I. Velazquez, Troy McEachron, David W. Craig, John D. Carpten. _USC Keck School of Medicine, Los Angeles, CA_.

Background: Tumor heterogeneity is a hallmark of cancer and can have significant impact on identifying drivers, including those that may be therapeutically relevant. Although, the traditional sequencing of bulk tumor specimens provides invaluable information, sequencing of individual cells provides a true representation of the cellular heterogeneity that exists within a cancer. Novel technologies such as single cell copy number variation (scCNV) enable copy number profiling at a single cell resolution. Single Cell CNV generates genome wide sequencing libraries from single cells to reveal tumor heterogeneity and evolution of subclonal populations.

Methods: In this study, we utilized a scCNV assay to assess heterogeneity within the 4T1 murine breast tumor model. To accomplish this we have developed an end-to-end workflow including tissue dissociation, single cell library preparation, sequencing and data analysis. We injected 4T1 cells into the mammary fat pad of 4-6 week old BALB/c mice. Mice were sacrificed at 21 days and tumors were removed from the injection site and dissociated using Milteny's Biotec Octo Dissociator. We utilized the 10X Genomics Chromium system to partition single cells and prepare sequencing libraries in parallel such that all DNA fragments produced within a partition are barcoded. Following library construction, the Illumina Novaseq 6000 system was used to sequence single cell libraries. Additionally, single cell libraries from a fraction of the 4T1 cells that were injected into the mammary fat and non-metastatic isogeneic cell line variants, 67NR and 168FARN, were also sequenced.

Results: We observed a pair of amplifications (>8 copies) on chromosome 9 and three segments of amplification on chromosome 15 that were conserved throughout the injection site tumor cells. Interestingly, the amplifications on chromosome 15 encompass one of the most commonly observed breast cancer oncogenes, Myc, and the cell migration regulator, Skp2. Further, we observed three unique cluster of tumors cells which suggests the possibility of detecting major and minor clonal populations using this technology.

Conclusion: Our findings suggest scCNV will help us to improve characterizations of the entirety of a patient's disease to uncover potential clones that are primed to induce metastasis, drug resistance, and relapse.

#4704

Identification of melanoma mutational tumor heterogeneity using BRAF, NRAS and TERT-promoter mutation-detection assays.

Gregory Chang,1 Broderick Corless,1 Nathaniel Fleming,1 Cindy Spittle,2 Farbod Darvishian,1 Anna Pavlick,1 Russell Berman,1 Richard Shapiro,1 George Karlin-Neumann,3 Iman Osman,1 David Polsky1. 1 _New York Univ. School of Medicine, New York, NY;_ 2 _MolecularMD, OR;_ 3 _Bio-Rad Laboratories, CA_.

Purpose: The mutational spectra of melanoma has been well characterized; however, the presence of distinct subclones among multiple tumors from a given patient has been less well described. As mutational heterogeneity has been associated with decreased responses to treatments in other cancers, we sought to estimate the occurrence of distinct subclones within individual melanoma patients by analyzing commonly mutated melanoma genes using a multi-platform mutation-detection approach.

Methods: We analyzed 271 formalin-fixed, paraffin embedded tumors from 99 patients with stage III or IV melanoma enrolled in the NYU Melanoma Biorepository. All patients had two or more available tumor specimens, and complete clinical data. All samples were reviewed for adequate tumor content, and extracted DNA was assessed for mutations at BRAFV600, NRASQ61, and TERT-124C>T and TERT-146C>T using a combination of multiplex SNaPshot assays, Sanger Sequencing, Allele-specific real-time PCR, or droplet digital PCR (ddPCR). Samples undergoing ddPCR analysis for TERT mutations were treated with uracil-DNA glycolase prior to amplification to remove C>T artifacts from formalin-fixation.

Results: Sixty patients had a primary plus one or more metastatic tumors available; 39 patients had multiple metastatic tumors, but no primary tumor available. Overall, 88% of patients had tumors with at least one BRAF, NRAS and/or TERT mutation. We identified inter-tumor mutational heterogeneity in 20/99 (20%) patients, with TERT mutational heterogeneity present in 15 of these patients. Among 14 patients with heterogeneity between their primary and metastatic tumors, 6/14 (43%) had additional mutations in their metastases compared to their primaries. Most interestingly, 8/14 (57%) patients had mutations in their primary that were undetectable in at least one of their metastases; 5 of these patients had TERT mutational heterogeneity. Three patients had a BRAF mutation in their primary that was undetectable in at least 1 of their metastases. One patient had a BRAFV600E/NRASWILD-TYPE/TERTWILD-TYPE primary on the leg and 3 regional metastases lacking BRAF mutations; but carrying NRASQ61K and TERT-124C>T mutations. Another patient had 3 different metastatic tumors, with 3 different mutational spectra. We did not detect any tumors with simultaneous BRAF and NRAS mutations; however, we did detect both TERT-124C>T and TERT-146C>T mutations in 7 tumors from 7 individual patients. Four of these were primary tumors, and metastases from these patients lacked 1 of the 2 TERT mutations identified in the primary.

Conclusion: Clonal heterogeneity in melanoma is fairly common as evidenced by divergent detection of TERT, BRAF and NRAS mutations using high sensitivity multi-platform mutation detection analyses of multiple melanoma tumors from individual patients. Heterogeneity appears to occur in primary tumors.

#4705

Single cell derived 3D organoids recapitulate the tumorigenic features of glioblastoma.

Michelle Chadwick,1 Rachael Mfon,2 Kelly Jara,1 Shabbar Danish,1 Robert Aiken,1 Hatem E. Sabaawy1. 1 _Rutgers-The Cancer Inst. of New Jersey, New Brunswick, NJ;_ 2 _Rutgers-The State University of New Jersey, New Brunswick, NJ_.

In recent years, three-dimensional (3D) models of human brain derived from pluripotent embryonic and iPS cells have emerged as brain organoids, providing established models for brain development and genetic engineering. However, tumor organoids derived from adult glioblastoma multiforme (GBM) patients were seldomly formed. WHO grade IV GBM is a devastating locally invasive brain cancer with a median survival of 14.6 months. Standard of care for GBM includes surgical resection followed by radiotherapy plus concomitant and maintenance temozolomide (TMZ). Resistance to TMZ develops rapidly and neither dose-intensified TMZ nor anti-angiogenic approaches could improve survival. Genomic, transcriptomic and epigenetic profiling allowed classifying adult GBM into three groups: isocitrate dehydrogenase (IDH)-mutant (mut), 1p/19q co-deleted oligodendroglial GBM with best prognosis; IDH-mut, 1p/19q non-co-deleted astrocytic GBM with intermediate outcome; and IDH wild-type (WT) GBM with poor prognosis. Preclinical models that reflect these GBM profiles and heterogeneity are urgently needed to examine new therapies. Patient derived orthotopic xenografts (PDOXs) are thought to better mimic the GBM environment. Genetically engineered mouse models (GEMMs) make gliomas in mice with competent immune systems, but are laborious and expensive requiring compound genetics to mimic human GBM. Here, we describe a 3D serum-free organoid system in low-adherence plates derived from primary and sphere cultures that supports the long-term growth and expansion of GBM organoids for several months. We generated organoids from GBM with IDH-WT or IDH-Mut, EGFR amplification, +7q/-10q genotype, PTEN mutation, and EGFRvIII expression. GBM organoids are comprised of a heterogenous cell populations, thus mimicking the original tumor. GBM organoids could be generated from a single cell, therefore allowing to track intratumor heterogeneity. GBM organoids could be utilized for investigating aspects of GBM biology such as 3D cellular self-renewal showing expression of stem cell and differentiation targets, 3D cell cycle regulation including synchronized growth and phase cycling, 3D cell metabolism including intracellular adenosine triphosphate (ATP) level and protein synthesis rate, 3D cell invasiveness validated with IHC assays, and for evaluation of drug effects in the context of IDH, PI3K and EGFR aberrations. We have demonstrated that GBM sphere cells exhibit a solid growth pattern when implanted orthotopically into the mouse PDOXs. Since GBM organoids form tighter cell-cell contact, oxygen and nutrient gradients, we implanted GBM organoids in mouse orthotopic xenografts to demonstrate their tumor growth and invasive phenotypes. This human GBM organoid platform allows for novel preclinical therapeutic approaches to be assessed and provides personalized therapeutic options for individual GBM patients.

#4706

Temperature-dependent transcription artifacts and cell population biases in scRNAseq data are minimized by tissue dissociation at low temperatures.

Ciara H. O'Flanagan,1 Kieran R. Campbell,2 Farhia Kabeer,1 Allen Zhang,1 Jamie Lim,1 Sohrab P. Shah,3 Samuel Aparicio1. 1 _BC Cancer, Vancouver, British Columbia, Canada;_ 2 _University of British Columbia, Vancouver, British Columbia, Canada;_ 3 _Memorial Sloan Kettering Cancer Center, New York, NY_.

Single cell RNA sequencing (scRNAseq) is a powerful tool, particularly for studying complex biological systems, such as tumor heterogeneity and the tumor microenvironment, which may not be resolved by sequencing of bulk material. Nonetheless, it is not without limitations, which include the technical challenges of generating a high quality single cell suspension. Dissociation of tissue to single cell suspension requires mechanical and enzymatic disruption, and the effect of these methods on gene expression or cellular population bias has not been established. In this study, we examined the effects of enzymatic dissociation on cell population capture and transcriptional changes at single cell resolution in breast and ovarian cancer patient samples, patient-derived breast cancer xenografts and cultured cell lines. scRNAseq data showed that enzymatic dissociation of tissues at 37oC with collagenase resulted in significant induction of heat shock, stress and immediate response genes, which was conserved across all tissues. This gene expression induction was not observed when tissues were dissociated at 6oC with a protease derived from a Himalayan glacier soil bacterium. Moreover, dissociation of patient tumors at low temperature enhanced the abundance of rare cell populations, including B-cells, T-Cells and cytotoxic T-cells, which were significantly depleted following dissociation at 37oC. These biases resulting from standard sample preparation methods could significantly affect biological interpretation of scRNAseq data, and can be minimized by dissociation of tissues at low temperature.

#4707

Quantifying cellular composition using laser microdissection regions of interest via automated digital image analysis of co-registered tissue thin sections.

Lorcan Sherry,1 Mark Anderson,1 Allison Hunt,2 Dave Mitchell,2 Julie Oliver,2 Thomas Conrads,2 Nicholas Bateman2. 1 _OracleBio Limited, Newhouse, United Kingdom;_ 2 _WHIRC, VA_.

Low purity tumors often portend poor disease outcome. Understanding the compositional heterogeneity of complex tumors, such as high-grade serous ovarian cancers (HGSOC) is an area of intense study. Laser microdissection affords decoupled molecular profiling analyses of tumor and non-tumor cells not otherwise possible using routine methods. Automated documentation of LMD collection activities are needed to support the development of high stringency quality control and quality assurance (QA/QC) procedures. This study was conducted to demonstrate that histology combined with digital image analysis can be utilized to quantify tumor and stroma cell content within LMD regions of interest (ROI). A single frozen high grade serous ovarian cancer tissue specimen was acquired under an IRB-approved protocol and thin sectioned onto charged glass or polyethylene naphthalate (PEN) membrane slides followed by staining with hematoxylin and eosin H&E. Tumor and stromal cell populations were harvested from membrane slides in a locoregional manner by LMD (LMD7, Leica) at ~150 µm intervals. Digital whole slide images (WSI) (Aperio ScanScope XT slide scanner, Leica) of HGSOC tissue were provided for analysis consisting of matched reference H&E sections and serial sections following enrichment of tumor (n=15) or stromal cell populations (n=3). Image analysis was performed by OracleBio using the Indica Labs HALO™ platform. Classification algorithms were developed and applied to each LMD image to identify the 'Dissection Area' and 'All Remaining Tissue' as two separate ROIs. A separate cytonuclear algorithm was developed using the H&E stained sections to detect cell nuclei. Each LMD image was co-registered with their respective H&E image to overlay classified ROI before analysis for nuclei detection. Dissected ROIs revealed median tumor cell areas were 27.5% and median stroma cell areas were ~50% of the total tissue area observable across sections. These were consistent with histopathological estimates of tumor (~36%, R2=0.91) and stromal (~52%, R2=0.96) cellularity across sections. Data generated from co-registered post-LMD dissected Area ROI within H&E sections serial to the LMD tissues highlighted that the median number of nuclei per sample within tumor (n=15) and stroma (n=3) ROI was 7,527 ± 665 and 3,470 ± 476 per mm2 respectively. The average size of nuclei detected within tumor and stroma ROI was 24.2 ± 1 and 21.4 ± 0.5µm2 respectively; a difference of ~12% between these cell types. This study highlights the successful use of histology combined with digital image analysis to quantify tumor and stroma cell numbers within HGSOC tissue subjected to LMD. Furthermore, proteogenomic analyses of LMD-recovered tumor and stromal cells will support further understanding of potential correlations between these cell types within the tumor microenvironment of HGSOC.

#4708

Investigating triple negative breast cancer phenotypic heterogeneity of human and patient-derived xenograft samples using imaging mass cytometry.

Amanda L. Rinkenbaugh, Vidya C. Sinha, Xiaomei Zhang, Jiansu Shao, Helen Piwnica-Worms. _MD Anderson Cancer Center, Houston, TX_.

Tumors are highly heterogeneous populations of cells, and measures of intratumoral heterogeneity (ITH) and diversity correlate with worse prognosis in many cancers, including breast cancer. Emerging studies are highlighting functional interactions between subclones, as well as among subclones and components of the tumor microenvironment. However, these studies have largely focused on soluble factors without interrogating the spatial distribution of subclones defined by activated signaling pathways. Previous work in this area has been severely limited by technical restrictions - existing techniques allowing measurement of many biomarkers simultaneously lose all information about the tissue architecture, while those that do retain spatial information can only assay a handful of markers at once. We will circumvent these limitations by undertaking imaging mass cytometry (IMC), which allows for simultaneous measurement of 30-40 antigens while retaining the spatial organization of the sample. Our objective is to dissect the signaling heterogeneity in tumors from patients and patient-derived xenograft (PDX) models of triple negative breast cancer (TNBC), through two main approaches: (1) characterization of signaling heterogeneity in tissue microarrays of human tumors and PDX models of TNBC with IMC and (2) modeling cell signaling heterogeneity in cell line-based models to determine mechanisms of cell-cell interaction and communication. We have constructed an IMC panel of antibodies that combines markers for tissue architecture, tumor and immune cell phenotyping, and signaling pathway activation. Profiling of a diverse panel of TNBC PDX models captures the heterogeneity of human TNBC. Analysis of distinct regions within individual PDX tumors demonstrate unique compositions of cell phenotypes between the edge and core of the tumor. Our PDX collection also includes sequential pairs derived from biopsies taken before and after chemotherapy treatment. Comparison of the pre-/post-chemotherapy pairs indicates emerging patterns of pathway activation. Interestingly, while the tumor cells from these models exhibit distinct phenotypes, the stromal cells are largely indistinguishable from one another, suggesting that these models are capturing tumor cell-intrinsic changes associated with chemotherapy response. Ultimately, we plan to use these results to identify novel vulnerabilities in subclonal interactions that can be targeted therapeutically in TNBC.

#4709

Extensive intratumor proteogenomic heterogeneity revealed by multiregion sampling in a high-grade serous ovarian tumor specimen.

Allison L. Hunt,1 Nicholas W. Bateman,2 Guisong Wang,2 Niyati Parikh,2 Julie Oliver,2 Dave Mitchell,2 Glenn Gist,2 Brian L. Hood,2 Ming Zhou,1 Brian Blanton,2 Kelly A. Conrads,2 Chad A. Hamilton,1 Kathleen M. Darcy,2 Craig D. Shriver,2 Yovanni Casablanca,2 George L. Maxwell,3 Thomas P. Conrads1. 1 _Inova Schar Cancer Institute, Falls Church, VA;_ 2 _Uniformed Services University and Walter Reed National Military Medical Center, Bethesda, MD;_ 3 _Inova Fairfax Medical Campus, Falls Church, VA_.

We generated 200 consecutive thin sections from a high-grade serous ovarian carcinoma (HGSOC) tissue specimen and laser microdissected (LMD) four spatially separated tumor "core" regions throughout the depth of the tissue block to examine proteogenomic intratumor heterogeneity (ITH). Tumor epithelium and stroma were each LMD harvested at 150µm intervals throughout the block and mixed epithelial and stromal (e.g. whole tumor) samples were harvested from additional adjacent thin sections; the remaining tissue was cryopulverized. Mass spectrometry-based proteomics quantified 6,053 proteins and 4,225 phosphosites and RNA sequencing mapped to 20,785 transcripts. Unsupervised hierarchical cluster analysis of the 1,018 and 584 differentially abundant transcripts and proteins (median absolute deviation > 1) demonstrated clustering by LMD sample types collected, with tumor cores and cross-sectional tumor epithelium samples versus mixed epithelium/stroma and stroma samples co-clustering. The cryopulverized proteome classified independently and represented a completely unique sample. Surprisingly, many proteins and transcripts previously classified as ovarian tumor biomarkers, many of which correlate with poor clinical prognosis, were absent in the LMD harvested tumor epithelium, yet were strongly expressed in the LMD enriched stroma. Comparison of our data with existing immunoreactive, differentiated, proliferative, and mesenchymal molecular subtypes, the overall protein and transcript abundance of discrete tumor collections inversely correlated with the mesenchymal HGSOC subtype, which is associated with the worst overall patient survival, whereas the LMD enriched stroma collections were, unexpectedly, most positively correlated. While there was overall concordance between the proteomic and transcriptomic data for each LMD collection type, transcript abundance from LMD enriched stroma was most strongly correlated with the cognate protein products from the mixed epithelial/stromal LMD harvest than to stroma, potentially explainable by the secretory nature of ovarian stromal cells and inclusion of extracellular matrix proteins in mixed LMD collections. Proteins measured from the cryopulverized tissue were overall negatively correlated with LMD harvested tumor epithelium, and notably had limited detection of the ovarian cancer biomarker CA-125. This proteogenomic analysis reveals stark molecular heterogeneity in the cellular admixture of the HGSOC tumor microenvironment and underscores the need to account for compartmental ITH in molecular profiling analyses.

#4710

**Surface protein marker and single cell gene expression profiling of individual tumor cells dissociated from small cell lung cancer pdx mouse models can be correlated with** in vivo **sensitivity to the p70S6K/AKT1/3 inhibitor M2698.**

Warren Porter,1 Eileen Snowden,1 Friedrich Hahn,1 Mitchell Ferguson,1 Frances Tong,1 William S. Dillmore,1 Anderson Clark,2 Hong Zhang,2 Rainer Blaesius1. 1 _BD Technologies and Innovation, Research Triangle Park, NC;_ 2 _EMD Serono, Billerica, MA_.

Small Cell Lung Cancer (SCLC) is characterized by rapid tumor growth and currently, there are few therapeutic options or predictive biomarkers. Patient derived xenograft (PDX) models are capable of recapitulating solid tumor growth including the intra-tumor heterogeneity (ITH) observed in the original patient tumor. The present study aimed to correlate surface marker profiles of SCLC PDX models with previously observed drug responses to M2698, a potent, selective inhibitor of p70S6K and AKT 1/3, and investigate ITH through gene expression profiling of tumor cell subpopulations.

Drug response data had been established in a preclinical screen of 45 PDX models of SCLC, in which two mice were implanted subcutaneously with tumors from each model; one mouse was treated with vehicle while the other was treated with M2698 25 mg/kg QD po until the tumor in the vehicle-treated mouse reached ~1200 mm3. Tumor control (stasis or regression) was seen in 12 (27%) of the models.

We showed previously that cell surface marker profiles of PDX tumor tissue demonstrated high intra-model reproducibility for many surface markers and uniquely associate with each model. Also, distinctly heterogeneous markers were identified that allowed FACS sorting of tumor cell subpopulations with similarly distinct gene expression profiles. To build on these data, a subset of models that were the most and the least sensitive to M2698 (n=7) in the above described screen were selected for implantation into a new set of mice. Tumors were profiled once they reached ~800 mm3. We evaluated 80+ markers commonly used to identify tumor initiating cells (e.g. CD44, CD90, CD133, CD166, CD184), EMT or aggressiveness (e.g. CD166, EphB2, CD324, CD325), poor outcome in SCLC (CD49b, CD221, claudin3) or drug targets (e.g. CD184, EGFR, Her2) to establish extensive marker profiles.

Our data reveal that surface marker profiles in these models allow a meaningful subclassification of SCLC PDX tissue. Correlation of these profiles with efficacy of M2698 (above) suggest that surface markers may have predictive value. Our results prequalify a number of these markers for validation. Furthermore, several models harbor cell subpopulations identifiable by various surface markers. Along with their distinct gene expression profiles this suggests an equilibrium between functionally different compartments within a lesion. In one case, the ratio of two populations shifted concurrently with growth differences, raising the possibility of a dynamic relationship between this equilibrium and the growth stage. Overall, our workflow may provide tools for sample characterization, quality control and elucidation of cellular response markers to varying selective pressures, such as drug challenges.

## EXPERIMENTAL AND MOLECULAR THERAPEUTICS

### DNA Repair and Reactive Agents / HDAC / Demethylating Agents

#4711

PRMT5 selective inhibitor enhances therapeutic efficacy of cisplatin in lung adenocarcinoma cells.

Khuloud Bajbouj, Rakhee Ramakrishnan, Ahmed M.H.Ihmaid, Suhaib Al Haj Ali, Abdulla Alalool, Reem Abdullah, Maha Saber-Ayad, Qutayba Hamid. _University of Sharjah, Sharjah, United Arab Emirates_.

Background: Lung cancer is the most common cancer globally, with an estimate of more than 2 million new cases every year. Protein arginine methyltransferase 5 (PRMT5) is an enzyme that is identified to be involved in multiple cellular events, including gene expression regulation transcriptional regulation and cell division. It has been shown that PRMT5 is highly expressed in cancers including lung adenocarcinoma, hepatocellular carcinoma, and melanoma, raising evidence that PRMT5 might be involved in tumorigenesis. The aim of this study is to examine the potential selective anti-neoplastic activity of AMI-1 and cisplatin on lung adenocarcinoma.

Experimental methods: The effect of AMI-1 and subsequent PRMT5 activity inhibition on lung adenocarcinoma (A549 cell line) behavior with the standard chemotherapeutic agent, cisplatin was assessed, by measuring cell viability using MTT assay, PMRT5/β-catenin protein levels via Western Blotting, the extent of cell migration through wound healing assay, and survival of cancer cells by performing cell cycle progression and Annexin-V staining assays along with both drugs combination potential toxicity that was evaluated in human bronchial epithelial cells (HBEC).

Results and Discussions: Combination treatment with 10 µM AMI-1 and IC50 of Cisplatin (23.4 µM) significantly reduced viable cell percentages at 24 and 48 hours. Treatment with both drugs at 48 hours led to a decreased cells migration rate. G2/M arrest in A549 cells was evident at 24 hours of AMI-1 addition, which was reinforced after drugs combined treatment. Furthermore, 48 hours of both drug administration hindered the ability of A549 cells to recycle again as they G1 occupying cells percentage was increased after 48 hours of combination treatment. There was a minor cell death induction after 48 hours of treatment with both drugs. Western blot analysis showed that there was no evidence change in total PRMT5 and β-catenin protein levels. Nevertheless, there was a reduction in demethylation Histone 4, a PRMT5- downstream target, after 48 hours treatment with AMI-1 alone or in combination with Cisplatin. Interestingly, as combination approach increased anti-survival effect on lung cancer cells, it exhibited cyto-protective abilities on normal epithelial cells.

Conclusion: This study shows a novel selective antitumor activity of AMI-1 and cisplatin in adenocarcinoma cells.

#4712

Selective polo-like kinase 1 PBD inhibitor.

Danda P. Chapagai,1 Merissa Baxter,2 Gurusankar Ramamoorthy,1 Campbell McInnes,1 Michael Wyatt1. 1 _University of South Carolina at Columbia, Columbia, SC;_ 2 _University of South Carolina at Columbia, West Columbia, SC_.

Polo Like Kinase 1 (PLK1) is only expressed in dividing cells and plays a critical role in several stages of mitosis. PLK1 is highly expressed in tumors of various origins while its expression is largely absent in normal tissues. PLK1 inhibition selectively kills cancer cells because cancer cells utterly depend on the mitotic functions of PLK1 overexpression. PLK1 consists of a highly conserved N-terminal catalytic kinase domain and a unique, functionally essential C-terminal Polo Box Domain (PBD). The PBD of each PLK is a phospho-peptide binding motif that determines substrate recognition and sub-cellular localization. PLK1 catalytic inhibitors have advanced to clinical trials but they inhibit other kinases including family members PLK2 and PLK3. This is problematic because PLK3 is a tumor suppressor and partial inhibition of PLK3 could lead to a long-term malignant progression. Moreover, a single point mutation (C67V) in the PLK1 kinase domain confers substantial cancer cell resistance against PLK1 catalytic inhibitors. An alternative approach to developing potent and selective PLK1 inhibitors is to allosterically target the PBD.

We generated fragment ligated inhibitory peptides (FLIPs) through a strategy called REPLACE. This is a computational and synthetic approach that uses structure activity relationships of peptide inhibitors to generate pharmaceutically acceptable lead molecules. Fragments are docked into the crystal structure of a truncated peptide/receptor complex, then prioritized for synthesis with the peptide and subsequently tested in an in vitro binding assay. PBD-interacting protein (PBIP) and Cdc25c are natural substrates of PLK1. We replaced individual amino acid residues in the PBIP (PLHSpTAI) and Cdc25C PBD substrate peptides (LLCSpTPNGL) with benzoic acid fragments. Here we show PLK1 specificity for PBD inhibitors in vitro. A fluorescent polarization (FP) competitive binding assay to the PBD of PLK1 was used to determine binding specificity. A PLK3 counter screen was used to determine the binding selectivity of our PBD inhibitors. To measure the PLK1 inhibitory activity of the FLIPs in cells, phosphorylation of mitotic proteins was measured. Candidate FLIPs induce mitotic arrest, block PLK1 mediated phosphorylation, and inhibit growth of human cancer cells. To make more drug-like molecules, we modified FLIPs to Small Molecule Inhibitors (SMIs). Further, we are optimizing SMIs to improve biochemical selectivity. SMIs bind to the PLK1 PBD as determined by FP assay. Also, SMIs inhibit cancer cell growth as determined by viability measurements and live cell imaging. Overall these results highlight that targeting the PBD of PLK1 has promise as an antitumor strategy.

#4713

Extraction, purification and evaluation of prmt5-inhibitory phytochemical compounds for the treatment of prostate adenocarcinoma.

Oliver H. Richmond, Zhengxin Wang. _Clark Atlanta University, Atlanta, GA_.

The development and advancement of prostate cancer is supported by a plethora of genetic and proteomic abnormalities, including events of post-translational modifications. The protein arginine methyltransferase 5 (PRMT5) enzyme regulates epigenetic events of histone modifications and of post-translational modifications within protein signaling pathways. Under abnormal conditions of overexpression and/or upregulation, PRMT5 functions by constitutively driving the growth and/or proliferation of dysregulated cells. Overexpression and/or upregulation of PRMT5 correlates with disease progression as observed among numerous cancer types, including breast, colorectal, leukemia, lung, melanoma and prostate cancers. We demonstrated previously that PRMT5 localized to the cytoplasm, functions to drive both growth and proliferation of prostatic tumors. Plants naturally produce chemical toxins as mechanisms of defense against microbial and other biological threats. Human exploitation, consumption and/or application of agents isolated from plants for therapeutic intervention dates back throughout the millennia. In this study, we extracted, purified and evaluated natural, small, chemical compounds from plant products that antagonize PRMT5 activity in prostate cancer cells. We found that crude and purified extracts of Dendrobium, Fennel Seed and Magnolia plants attenuated prostate tumor growth and proliferation by selective inhibition of PRMT5 methyltransferase activity. These findings establish the first set of natural PRMT5-specific inhibitors reported.

#4714

Preclinical evaluation of a novel LSD1 inhibitor SYHA1807 for the treatment of small cell lung cancer.

Lingyun Wu,1 Hanyu Yang,2 Lele Zhao,1 Jianjun Sun,1 Jian Liu,1 Yuanfeng Xia,1 Chichung Chan,1 Shuhui Chen1. 1 _WuXi AppTec, Shang Hai, China;_ 2 _CSPC Zhongqi Pharmaceutical Technology Co., Ltd., Shijiazhuang, China_.

Histone post translational modification is an important epigenetic regulation to activate or suppress specific gene expressions. Methylation which is dynamically regulated by methyltransferase and demethylase (e.g. lysine specific demethylase) is one of the mechanisms and is critical for cell cycle and differentiation. Lysine specific demethylase 1 (LSD1) is the first identified histone modifying enzyme that removes methyl from histone lysine and arginine residues. Its overexpression has been found in many human cancers, like blood, lung and breast cancers. Small cell lung cancer is a neuroendocrine cancer characterized as low differentiation, rapid metastatic spread and resistance to regular chemotherapy. Aberration of epigenetic regulation is a common feature in small cell lung cancer, like down regulation of tumor suppression gene and up regulation of oncogenes. LSD1 expression of small cell lung cancer patients is up regulated in tumor tissue but not in normal tissues. Dysregulated histone methylation may contribute to the tumor malignance and treatment resistance. Taken together, LSD1 activity inhibition may provide a promising treatment option for small cell lung cancer. A challenge in LSD1 drug development is how to maintain optimal therapeutic effects while reducing mechanism related hematological toxicity. Through scaffold hopping and thoroughly SAR exploration, a novel LSD1 inhibitor SYHA1807 has been identified. The exposure level of this candidate at lung tissue is about four times higher than in bone marrow. The ideal drug tissue distribution characteristics may provide further safety assurance in clinical application. Detailed ADME/PK, in vitro, in vivo properties and preliminary safety evaluation results will be presented.

#4715

Combination of Tacedinaline and EHMT2 inhibition increases breast cancer cell death involving BIRC5 repression and GADD45A induction.

Pei-Yi Chu,1 Ji-Ching Lai,2 Ming-Feng Hou,3 Chang-Shen Lin4. 1 _Department of Pathology, Show Chwan Memorial Hospital, Changhua, Taiwan;_ 2 _Department of Medical Research, Show Chwan Memorial Hospital, Changhua, Taiwan;_ 3 _Department of Surgery, Kaohsiung Medical University and Hospital, Kaohsiung, Taiwan;_ 4 _Graduate Institute of Medicine, Kaohsiung Medical University and Hospital, Kaohsiung, Taiwan_.

The genomes of cancer cells are different from those of normal cells. These differences include genetic and epigenetic alterations, such as variations of DNA methylation and histone modifications, which cause differential gene expression, increased cell growth/migration capacity, and may induce resistance to anticancer therapy. Thus, epigenetic modulation may have a chance to correct abovementioned cancer cell's aberrations in gene expression and cell behaviors, resulting in eradication of primary and/or suppression of refractory cancers.

Tacedinaline (CI-994) and UNC0638 are specific inhibitors of class I histone deacetylase (HDAC) and histone methyltransferase EHMT2 (G9a), which cause an increase of histone acetylation and a decrease of histone H3 dimethylation at lysine 9, respectively. Here we examined the therapeutic effect of CI-994 and UNC0638 combination on the triple-negative (MDA-MB-231) and estrogen receptor-positive (MCF-7) breast cancer cell lines.

The results showed that the combination of CI-994 and UNC0638 was able to induce more severe cell death than either agent alone. Although the sub-G1 fraction and Annexin V-positive cells were increased, CI-994 and UNC0638 did not significantly induce the activities of caspases 3, 8 and 9, suggesting the involvement of non-canonical and caspase-independent cell death mechanism. Instead, CI-994 suppressed the expression of BIRC5 (Survivin, a member of the inhibitor of apoptosis protein) and, in combination with UNC0638, induced the expression of GADD45A (involved in stress-induced apoptosis).

Mechanistically, combination of CI-994 and UNC0638 resulted in the changes of epigenetic marks, such as acetylation and methylation of histone H3, at the loci of BIRC5 and GADD45A, which were correlated with their altered gene expressions. Genetic knockdown of BIRC5 expression in MCF-7 decreased cell viability, supporting the pro-survival role of BIRC5 in breast cancer cells.

Importantly, the targets of CI-994 (HDAC1, HDAC2) and UNC0638 (EHMT2), as well as BIRC5 were overexpressed in breast cancer specimens, especially those of triple-negative breast cancer (TNBC), based on the analyses of The Cancer Genome Atlas dataset. Meanwhile, the expression of GADD45A was downregulated. BIRC5 upregulation and GADD45A downregulation in breast cancer were associated with patient's overall survival.

These results suggest that Tacedinaline and UNC0638, or targeting the class I HDAC and EHMT2, may be a potential strategy to treat breast cancer including TNBC.

#4716

Therapeutic utility of EC359 for targeting oncogenic LIFR signaling in triple negative breast cancer.

Suryavathi Viswanadhapalli,1 Mengxing Li,1 Yiliao Luo,1 Gangadhara R Sareddy,1 Bindu Santhamma,2 Mei Zhou,3 Shihong Ma,4 Rajni Sonavane,4 Uday P. Pratap,1 Kristin A. Altwegg,1 Annabel Chang,4 Alejandra Chávez-Riveros,5 Kalarickal V. Dileep,6 Kam Y. Zhang,6 Marek Bajda,7 Ganesh V. Raj,4 Andrew Brenner,1 Vijaya Manthati,2 Manjeet Rao,8 Rajeshwar R. Tekmal,1 Hareesh B. Nair,2 Klaus J. Nickisch,2 Ratna K. Vadlamudi1. 1 _UT Health Science Ctr. at San Antonio, San Antonio, TX;_ 2 _Evestra, Inc, San Antonio, TX;_ 3 _Second Xiangya Hospital, San Antonio, TX;_ 4 _UT Southwestern, Dallas, TX;_ 5 _Instituto de Química, Mexico;_ 6 _Center for Biosystems Dynamics Research, RIKEN, Yokohama, Japan;_ 7 _Jagiellonian University Medical College, Cracow, Poland;_ 8 _Greehey Children's Cancer Research Institute, San Antonio, TX_.

Background: Leukemia inhibitory factor receptor (LIFR) and its ligand LIF play a major critical role in cancer progression, metastasis, stem cell maintenance, and therapy resistance. Recent studies in breast cancer have shown that feedback activation of LIFR limits response to histone deacetylase (HDAC) inhibitors and induce resistance. We rationally designed a small molecule (EC359) that emulates the LIF-LIFR binding site and functions as a LIFR inhibitor from a library of compounds. Here, we tested the utility of EC359 as a monotherapy and to effectively block LIF-LIFR interactions in overcoming resistance to HDAC inhibitors.

Methods: We have used multiple triple negative breast cancer (TNBC) models that represent all six types of TNBC. In vitro activity was tested using Cell-Titer Glo, MTT, invasion, and apoptosis assays. Mechanistic studies were conducted using western blot, reporter gene assays, and RNA-seq analysis. Xenograft, patient-derived xenograft (PDX), and patient-derived explant (PDeX) models were used for preclinical evaluation and toxicity.

Results: EC359 treatment exhibited anti-proliferative effects, reduced invasiveness and stemness, and promoted apoptosis in all six TNBC cell lines. The activity of EC359 is dependent on LIF and LIFR expression and CRISPR mediated knockdown of LIFR significantly abolished EC359 activity. Treatment with EC359 attenuated the activation of LIF-LIFR driven pathways including STAT3, mTOR, and AKT. EC359 significantly reduced tumor progression in TNBC xenografts, PDX models and reduced proliferation in patient derived primary TNBC explants. In MTT based cell viability assays, addition of EC359 enhanced efficacy of SAHA compared to monotherapy of SAHA. In clonogenic survival assays, EC359 significantly enhanced ability of SAHA to reduce the colony formation compared to monotherapy. Mechanistic studies using three different TNBC models using western blot analysis and reporter gene assays confirmed activation of LIFR signaling pathway upon SAHA treatment and its blockage by EC359. Treatment of TNBC PDX explants with EC359 enhanced ability of SAHA to substantially decrease the proliferation (Ki-67 positivity) compared to monotherapy treated tumors.

Conclusions: Collectively, these data support EC359 as a novel targeted therapeutic that inhibits LIFR oncogenic signaling as a monotherapy or in combination with HDAC inhibitors.

#4717

Effective preclinical management of angiosarcoma with oral paclitaxel.

Murray J. Cutler, Krista E. Belko, Yahao Bu, Alissa R. Verone-Boyle, David Cutler, Wing-Kai Chan, Rudolf Kwan, Johnson Y. Lau, Michael P. Smolinski. _Athenex, Inc., Buffalo, NY_.

Angiosarcomas are characterized by proliferating anaplastic cells derived from blood vessels. While there is a lack of approved treatments for angiosarcomas, weekly intravenous (IV) paclitaxel has shown some benefit. However, IV paclitaxel therapy is commonly accompanied with hypersensitivity reactions and irreversible neuropathy. Oral paclitaxel ("Oraxol"; paclitaxel co-administered with HM30181A, a non-absorbable P-gp inhibitor) eliminates the risk of hypersensitivity to IV formulations while reducing neuropathy with the potential for improved efficacy. Oral paclitaxel is currently being tested in a pivotal phase III trial for metastatic breast cancer in addition to multiple phase I/II trials for solid tumors. Here, we aim to expand the therapeutic application of oral paclitaxel to the treatment of angiosarcomas.

An MTT assay was utilized to determine the potency of paclitaxel against mouse angiosarcoma cell lines (MS1, SVRA221a, and SVRbag4) and a human endothelium cell line (HMEC-1). The potency (IC50) of paclitaxel was estimated to be 1.8 ± 0.36 ng/mL for MS1, 1.8 ± 1.0 ng/mL for SVRA221a, 3.9 ± 1.0 ng/mL for SVRbag4, and 0.46 ± 0.23 ng/mL for HMEC-1 cells. Immunofluorescence was used to examine the effect of paclitaxel on microtubule polymerization in the angiosarcoma cell lines. Paclitaxel induced polymerization of microtubules accompanied with condensed and rounded morphology starting at 4.2 - 8.5 ng/mL.

To assess the efficacy of oral paclitaxel, MS1, SVRA221a, and SVRbag4 xenograft models were developed. The MS1 tumors grew too slowly while the SVRbag4 tumors did not reach tumor size endpoint due to necrosis and ulceration, so tumor doubling time and tumor growth delay could not be determined. In the SVRA221a angiosarcoma xenograft model, oral paclitaxel administered for three consecutive days, weekly for three weeks, resulted in dose-dependent tumor growth inhibition and subsequent increased survival. Mice treated with 20 mg/kg HM30181A and 30 mg/kg paclitaxel experienced the greatest tumor growth inhibition with 57.5% survival on Day 15, whereas 20 mg/kg and 40 mg/kg paclitaxel administered with 20 mg/kg HM30181A demonstrated 32.3% and 51.6% survival on Day 15 of treatment, respectively. The antitumor effect and increased survival appeared to plateau between 30 and 40 mg/kg, suggesting that increasing the dose above 30 mg/kg provides no additional benefit. Cavernous blood‐filled neoplastic vessels were consistently formed in vehicle and 10 mg/kg paclitaxel-treated groups. However, few to none were present in 20, 30, or 40 mg/kg paclitaxel-treated groups, suggesting oral paclitaxel demonstrated dose-dependent efficacy in this angiosarcoma murine model.

Oral paclitaxel has been granted Orphan Drug Designation by the US FDA for the treatment of angiosarcomas and a pilot study of oral paclitaxel in patients with cutaneous angiosarcoma is planned (NCT03544567).

#4718

SR-4370, a potent and selective inhibitor of class I HDACs, suppresses AR signaling and in vivo prostate tumor growth.

Iqbal Mahmud,1 Guimei Tian,1 Jia Wang,1 Ryan Stowe,2 Zhiguang Huo,1 Yushan Zhang,1 Hamsa Thayek Purayil,1 Eric Helm,1 Theodore Drashansky,1 Dorina Avram,1 Yehia Daaka,1 William R. Roush,2 Daiqing Liao1. 1 _University of Florida College of Medicine, Gainesville, FL;_ 2 _The Scripps Research Institute, Jupiter, FL_.

Androgen receptor (AR) is an androgen-activated transcription factor and drives prostate cancer (PCa) progression. Class I HDACs 1-3 are critical for activating AR-mediated transcription. Thus, targeting these HDACs is a promising strategy for treating PCa. Notably, along with significant adverse effects, several FDA-approved HDAC inhibitors broadly inhibiting different HDACs were ineffective for treating castration-resistant prostate cancer in clinical trials. Significantly, entinostat, an aminobenzamide analog specific to HDACs 1-3, extended overall survival for patients with breast cancer resistant to endocrine therapy. These observations suggest that HDACi selective to HDACs 1-3 may be effective for treating solid tumors including PCa. We have recently discovered the novel benzoylhydrazide class of HDAC inhibitors highly specific to HDACs 1-3. An optimized analog, SR-4370, exhibited low µM to nM potency against HDACs 1-3. SR-4370 markedly suppressed AR signaling, PCa cell proliferation in vitro, and prostate tumor growth in vivo. Gene expression profiling experiments revealed that SR-4370 downregulated AR, AR-Vs and AR target genes as well as the MYC oncogenic network. Chromatin accessibility assay using ATAC-seq showed that SR-4370 altered chromatin states in PCa cells. The chromatins with AR-binding sites became inaccessible on SR-4730 treatment, indicating that altered chromatin accessibility may contribute to the inhibition of AR signaling. Interestingly, SR-4370 sensitized C4-2 cells to enzalutamide. In PCa xenograft models, SR-4370 was effective to suppress tumor growth in vivo. Importantly, SR-4370 was well tolerated and did not cause observable adverse effects as judged by body weight and blood chemistry tests of treated mice. Our data suggest that SR-4370 may be a safe and clinically applicable treatment for advanced PCa refractory to current frontline treatments. (Supported by UFHealth Cancer Center, Florida Breast Cancer Foundation, James and Esther King Biomedical Research Program, and Bankhead-Coley Cancer Research Program, Florida Department of Health)

#4719

Synergistic anticancer activity of triple combinations of eribulin, palbociclib and fulvestrant in hormone dependent patient-derived xenograft (PDX) models of human breast cancer.

Marc Hillairet de Boisferon,1 Elodie Marie Dit Chatel,1 Kenichi Nomoto,2 Bruce A. Littlefield3. 1 _OncoDesign Biotechnology, Dijon, France;_ 2 _Eisai Inc., Woodcliff Lake, NJ;_ 3 _Eisai Inc., Andover, MA_.

Eribulin is a pharmaceutically optimized analog of the marine natural product halichondrin B. As its clinically formulated mesylate salt (Halaven®), eribulin is used for treatment of certain patients with advanced breast cancer and liposarcoma. Mechanistically, eribulin combines cytotoxic tubulin-based antimitotic effects with non-cytotoxic effects on tumor biology, including vascular remodeling, increased perfusion, mitigation of hypoxia and reversal of epithelial to mesenchymal transition (EMT). Reversal of EMT involves cell differentiation pathways that impinge on the G1/S cell cycle boundary. Since estrogenic signaling also impinges on the G1/S boundary, we asked if eribulin could combine advantageously with inhibitors of cyclin dependent kinases and hormonal agents that disrupt estrogenic signaling. Using PDX models of hormone receptor positive (HR+) breast cancer, we previously showed that combining eribulin and palbociclib is considerably more effective than either agent alone, using a "palbociclib holiday" strategy of withholding daily palbociclib doses the day before and the day of weekly eribulin doses to avoid possible cell cycle based antagonism. Here, we ask if the palbociclib holiday is strictly necessary for robust eribulin + palbociclib combination activity, and if triple combinations of eribulin, palbociclib and fulvestrant result in even better anticancer activity than doublet dosing. For the holiday/no holiday comparison, 0.125 mg/kg eribulin was dosed iv Q7Dx3, with palbociclib dosed po either at 150 mg/kg or 107 mg/kg on Q1Dx5[x3 weeks] (holiday) or Q1Dx21 (no holiday) schedules, respectively, resulting in equal palbociclib dose intensities for the 2 schedules. Results showed that synergism was seen in combination with or without palbociclib holiday, but superior results occur with holiday. Using the holiday strategy, we next investigated triple combinations of eribulin, palbociclib and fulvestrant. As single agents at the minimally effective doses selected, eribulin, palbociclib and fulvestrant led to treated/control values (T/C%) of 64%, 63% and 48%, respectively. Combining eribulin and palbociclib led to markedly superior anticancer activity (T/C 23%). Combining eribulin and fulvestrant also led to superior activity (T/C 22%), as did combining fulvestrant and palbociclib (T/C 19%). The triple combination generated the most robust activity at T/C 8%. By mouse RECIST criteria, 10%, 10% and 20% partial responses (PR) were observed for each doublet (fulvestrant/palbociclib, fulvestrant/eribulin, palbociclib/eribulin), respectively. In contrast, 90% PR was seen for the triple combination. These preclinical results support clinical exploration of eribulin, palbociclib and fulvestrant triple combinations for appropriate patients with HR+ breast cancers.

#4720

Ataxia telangiectasia mutated (ATM) kinase inhibitor AZD0156 in combination with 5-fluorouracil and irinotecan in preclinical models of colorectal cancer.

S. Lindsey Davis,1 Marina I. Schlaepfer,1 Stacey M. Bagby,1 Sarah J. Hartman,1 Betelehem W. Yacob,1 Tonia Tse,1 Dennis M. Simmons,1 Jennifer R. Diamond,1 Christopher H. Lieu,1 Alexis D. Leal,1 Elaine B. Cadogan,2 Gareth D. Hughes,2 Stephen T. Durant,2 Wells A. Messersmith,1 Todd M. Pitts1. 1 _University of Colorado Cancer Center, Aurora, CO;_ 2 _AstraZeneca, Cambridge, United Kingdom_.

Background: AZD0156 is an oral inhibitor of ATM, a serine threonine kinase that plays a key role in DNA damage response associated with DNA double strand breaks. Topoisomerase-I inhibitors like irinotecan induce single-strand DNA breaks, which are converted to double-strand breaks during DNA replication. Thus the combination of AZD0156 and irinotecan is a rational combination for clinical use. Irinotecan is used clinically to treat a variety of malignancies, including colorectal cancer (CRC), usually in combination with 5-fluorouracil (5FU) as FOLFIRI. An ongoing phase 1 clinical trial is evaluating AZD0156 in combination with single-agent irinotecan and FOLFIRI in patients with refractory cancers (NCT02588105). The purpose of this study is to evaluate AZD0156 in combination with irinotecan and 5FU in preclinical models of CRC to help inform clinical use.

Methods: Anti-proliferative effects of single-agent AZD0156 and combination therapy with SN38 (active metabolite of irinotecan) and 5FU were evaluated in CRC cell lines using the Cell-Titer Glo assay. Immunoblotting and cell cycle analysis were performed to determine the mechanism of enhanced combination effects. Four CRC patient derived xenograft (PDX) models were treated with AZD0156, irinotecan, and 5FU alone and in combination for assessment of tumor growth inhibition (TGI).

Results: An enhanced antiproliferative effect was observed with the combination treatment over either single agent. A more significant synergistic effect was demonstrated with the combination of AZD0156 and SN38 as compared with the combination of AZD0156 and 5FU. Cell cycle data demonstrated enhanced cell cycle arrest with combination therapy as compared to single agents. Immunoblotting results suggest a decrease in phosphorylated gamma-H2AX in cell lines treated with combination therapies. Increased TGI was observed in CRC PDX models treated with the combination of AZD0156 and irinotecan as compared to single-agent therapy in 3 of 4 models. There was not a significant change in TGI with the addition of 5FU for triplet therapy in the majority of models.

Conclusions: The combination of AZD0156 with irinotecan is synergistic in in vitro models and is associated with increased TGI in CRC PDX in vivo models. The addition of 5FU to AZD0156 and irinotecan did not result in increased TGI as compared to doublet therapy in CRC PDX models, though did not decrease the AZD0156/irinotecan combination effect. An ongoing clinical trial is evaluating this combination in patients with cancers refractory to standard treatments (NCT02588105).

#4721

**Loss of** MTAP **predicts response to GSK3368715 (EPZ019997), a first-in-class Type I PRMT inhibitor, through a tumor intrinsic combination mediated by PRMT5 inhibition.**

Andy Fedoriw,1 Shane O'Brien,1 Sarah Gerhart,1 Satyajit Rajapurkar,1 Melissa Pappalardi,1 Niyant Shah,1 Jenny Laraio,1 Charles McHugh,1 Yan Liu,1 Michael Butticello,1 Susan Korenchuk,1 Nicholas Adams,1 Craig Wagner,1 Francesca Zappacosta,1 Caretha Creasy,1 Nestor Concha,1 Lorna Mitchell,2 Nathalie Rioux,2 Trupti Lingaraj,2 Scott Ribich,2 Smith Jesse,2 Robert Copeland,2 Mike Moyer,2 John Campbell,2 Kim Stickland,2 James Mills,2 Suzanne Jacques-O'Hagan,2 Christina Allain,2 Danielle Johnston,2 Alejandra Raimondi,2 Margaret Porter Scott,2 Nigel Waters,2 Kerrin Swinger,2 Ann Boriack-Sjodin,2 Tom Riera,2 Richard Chesworth,2 Olena Barbash,1 Rab Prinjha,1 Ryan Kruger,1 Helai Mohammad1. 1 _GlaxoSmithKline, Collegeville, PA;_ 2 _Epizyme, Cambridge, MA_.

Type I protein arginine methyltransferases (PRMTs) catalyze the formation of monomethyl-arginine (MMA) and asymmetric dimethyl arginine (ADMA) on numerous proteins, thereby modulating their activity. Mis-regulation and overexpression of Type I PRMTs has been associated with solid and hematopoietic cancers suggesting a rationale for therapeutic intervention. The current report describes the discovery and characterization of GSK3368715 (EPZ019997), a potent, reversible Type I PRMT inhibitor with anti-tumor activity against human cancer cells. Inhibition of Type I PRMTs using GSK3368715 leads to reduced ADMA on numerous substrates and concomitant increases in MMA as well as symmetric dimethyl arginine (SDMA). Co-treatment of cancer cells with GSK3203591, a small molecule inhibitor of PRMT5, a type II PRMT, attenuates the induction of both MMA and SDMA by GSK3368715. Moreover, combined inhibition of both Type I PRMTs and PRMT5 produces synergistic, anti-proliferative effects and converts the cellular response from cytostatic to cytotoxic. Recent reports have demonstrated that PRMT5 can be selectively inhibited by 2-methylthioadenosine (MTA), a natural by-product of polyamine synthesis. Homozygous deletion of the gene responsible for MTA metabolism, Methylthioadenosine phosphorylase (MTAP) can lead to accumulation of MTA and decreased levels of SDMA in tumor cells. Because of its genetic linkage to the tumor suppressor CDKN2A, MTAP is frequently deleted in cancers including 40% of glioblastoma, 25% of melanoma and pancreatic adenocarcinoma, and 15% of non-small cell lung carcinoma. Assessment of over 200 cancer cell lines using a high throughput proliferation screen revealed that MTAP deletion correlates with sensitivity to GSK3368715 indicating that the degree of PRMT5 inhibition through increased MTA levels is sufficient to confer sensitivity to GSK3368715. In pancreatic cancer cell lines, MTAP deletion is associated with cytotoxic responses to GSK3368715, an effect that can be recapitulated by CRISPR mediated disruption of the MTAP locus in a wild-type cell line. Together, these data demonstrate that the anti-tumor activity of GSK3368715 can be enhanced with PRMT5 inhibition, either through use of a small molecule inhibitor or accumulation of MTA in MTAP deficient cancer cells, thereby suggesting a biomarker strategy for patient selection. GSK3368715 has recently entered a Phase I clinical trial (NCT03666988) that will explore the utility of MTAP deficiency as a predictive biomarker of response to this first-in-class Type I PRMT inhibitor.

#4722

Efficacy of a novel EP300/CBP histone acetyltransferase inhibitor in hormone responsive breast cancer.

Archana Bommi-Reddy, Jonathan Wilson, Annissa J. Huhn, Esteban Terzo, Florence Poy, Sungmi Park, Michael J. Steinbaugh, Barbara M. Bryant, Andrew R. Conery, Richard Cummings, Julian Levell, Robert J. Sims. _Constellation Pharmaceuticals Inc, Cambridge, MA_.

Mammalian cell identity or cell fate is governed by transcription factors that recruit co-activators to regulate cell-specific gene expression programs. The histone acetyltransferase (HAT) paralogs EP300 and CREB binding protein (CBP) are master transcriptional co-activators that catalyze acetylation of key histones that mark active enhancers, thereby operating at a fundamental step of transcriptional control. Enhancers are often co-opted by cancer cells to control expression of oncogenic transcriptional programs. Identifying cancer-specific dependencies will greatly help prioritize clinical indications that are uniquely vulnerable to HAT inhibition, allowing for precise targeting of cancer cells while sparing normal tissue to maximize the therapeutic window and clinical efficacy.

Estrogen receptor (ER) signaling is a validated clinical pathway in over 70% of breast cancer. ER-directed therapies are foundational for the treatment of ER+ breast cancer. However, these therapies are limited by residual ER signaling, which remains online through late-stage disease in a significant fraction of patients. EP300/CBP are critical components of the core ER signaling complex, which is recruited to target DNA by ER and other coactivators, to potentiate ER signaling. Furthermore, CBP and EP300 are dependency factors in the context of ESR1 mutations that arise in metastatic disease to facilitate disease progression.

We report for the first time the activity of a novel, potent, selective and in vivo active EP300/CBP HAT inhibitor for the treatment of ER-positive (ER+) breast cancer. CPI-1 has pM biochemical and nM activity in ER+ breast cancer cell lines. Analysis of nascent and steady-state RNA levels facilitated by PRO-seq and RNA-seq technologies revealed a time and dose-dependent suppression of the ER transcriptome. Dynamic gene regulation was coupled with enhancer RNA (eRNA) transcription and enhancer acetylation, to identify enhancers that were hypersensitive to CBP/EP300 HAT inhibition. CPI-1 demonstrates significant tumor growth inhibition in a xenograft mouse model of ER+ breast cancer as a monotherapy and together with an ER targeting agent, the combination displays superior efficacy to either treatment alone. These results highlight the potential of targeting key oncogenic transcriptional nodes with complementary therapeutic mechanisms to achieve superior results and reveals the potential utility of inhibiting the HAT domain of EP300/CBP in molecularly refined cancer populations.

#4723

Combinatorial efficacy of anti-PD1 treatment and selective histone deacetylase 6 (HDAC6) inhibition in chronic lymphocytic leukemia (CLL).

Kamira Maharaj, John Powers, Alex Achille, Eva Sahakian, Javier Pinilla-Ibarz. _H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL_.

A suppressive microenvironment allows CLL B cells to avoid immune surveillance. Coinhibitory antigens, anti-inflammatory cytokines and skewed immune populations support malignancy and contribute to poorer immune responses in CLL patients. Novel approaches to counter immunosuppression are therefore being explored. The HDAC family of proteins epigenetically regulate immune-related pathways in cancer and may be noteworthy immunomodulatory targets. In addition to the recognized role of HDACs in cell survival, HDACi may alter inflammatory status of immune and tumor cells. We have previously reported that HDAC6 regulates IL-10 transcription, and is responsible for T cell anergy in murine and human APCs. IL-10 is secreted by CLL B cells and is thought to influence immunosuppression. Therefore, we hypothesized that HDAC6i could reverse immunosuppression in CLL for antitumor benefit. The purpose of this study was to determine 1) the effects of selective HDAC6i on CLL immune biology and 2) whether combination with HDAC6i could improve efficacy of anti-PD1 for CLL therapy.

To accomplish these aims, the euTCL1 and euTCL1/HDAC6KO adoptive transfer models of murine CLL were utilized. ACY738 (selective HDAC6i) was administered orally in chow at 25mpk daily. Anti-PD1 was administered intraperitoneally 3x per week at 3mpk. CLL surface antigens were analyzed via flow cytometry. Functional mixed lymphocyte assays were performed ex vivo, measuring IFNg production, and Th1/Th2 factors were examined via qRT-PCR in CLL T cells.

Early PD-L1 expression on B cells correlated to CLL burden in euTCL1 mice. Post ACY738 treatment, euTCL1 B cells downregulated expression of PD-L1 and plasma IL-10. In addition, PD-1 and CTLA4 was decreased on CD4+, CD8+, Tregs and effector memory T cells. Splenic T cells from ACY738 mice upregulated TBET and downregulated GATA3, indicative of a less exhausted phenotype. Subsequently, euTCL1 B cells were treated ex vivo with ACY738 and cocultured with T cells. T cell activation (IFNg production) was increased after coculture with treated B cells compared to control B cells. We then hypothesized that ACY738 could be rationally be combined with anti-PD1 mAb for CLL treatment. Mice treated with anti-PD1 accumulated fewer CD5+ B cells, confirming that PD-1/PD-L1 blockade demonstrated anti-CLL benefit. Strikingly, combination of ACY738 and anti-PD1 further delayed the growth of CLL cells compared to either single agent. Consistently, anti-PD1 treatment in euTCL1/HDAC6KO mice further delayed CLL development. In conclusion, we report here that HDAC6i can reinvigorate CLL T cells, and combination of HDAC6i with anti-PD1 increased anti-CLL efficacy in vivo. This work provides evidence that this combination could be clinically investigated to simultaneously reduce tumor burden and improve immune function in CLL patients.

#4724

Regulation of DNA cleavage by the amino and carboxyl domains of human Top2α.

Abhishek Dilip Deshpande,1 Matthew A. Gilbertson,2 Hannah N. Miles,2 Karin C. Nitiss,2 John L. Nitiss2. 1 _University of Illinois at Chicago College of Medicine at Rockford, Rockford, IL;_ 2 _University of Illinois at Chicago College of Pharmacy at Rockford, Rockford, IL_.

DNA topoisomerases are involved in processing DNA structures such as knots and supercoils formed during cellular processes. hTop2α functions by creating transient DNA double-strand breaks, through which a second DNA molecule can be passed. DNA breakage by hTop2α involves formation of a transient covalent DNA-protein intermediate that can be stabilized by small molecules like etoposide and doxorubicin. Therefore, these agents convert Top2 into a DNA damaging agent. The overall architecture of type II topoisomerases has been defined by a combination of biochemical and structural studies. The homodimeric eukaryotic Top2 includes an N-terminal domain that includes an ATP dependent dimerization and ATPase activity, a domain that connects the ATPase domain to the catalytic core, termed the transducer domain, a central breakage/reunion core that carries out metal ion dependent formation of the protein/DNA covalent complex and a C-terminal domain that includes a second dimerization domain. Since drug resistance to Top2 targeting drugs can occur by mutations that greatly reduce enzyme activity, they are of limited usefulness in understanding detailed mechanisms of drug action. We carried out a large-scale screen to identify etoposide hypersensitive mutations in hTop2α. We developed novel approaches to target small regions of the hTop2α coding sequence. As anticipated, we identified several domains of hTop2α that were hotspots for changes leading to etoposide hypersensitivity. These regions included the N-terminal region of the protein (transducer and ATPase domain), the breakage/reunion catalytic core of the enzyme, and C-terminal dimerization domains. For example, we identified a prominent class of mutations including Asp374Gly, Asp374Glu, Asn445Asp, Val415Leu and Thr377Ile in the transducer domain, which confer hypersensitivity to etoposide. We purified and characterized the Asp374Gly mutant protein. In addition to an increased in vitro sensitivity to etoposide, Top2α (Asp374Gly) had a decreased requirement for ATP compared to the wild-type enzyme. The decreased requirement for ATP is unexpected and may suggest an alteration in the communication between the ATPase domain and the catalytic core. In addition, we also isolated etoposide hypersensitive alleles close to the C-terminal dimerization domain. These mutations included Asp1130Asn, Ala1052Gly and Gln1109Lys. Our results suggest at least two distinct controlling modules for Top2 cleavage: the ATPase/transducer module, and the C-terminal dimerization domain. Our results provide additional insight into how etoposide inhibits Top2α and may suggest new domains of the protein suitable for drug targeting.

#4725

Efficacy of a new small-molecule inhibitor of histone deacetylase 6 (HDAC6) in preclinical models of B-cell lymphoma and acute myeloid leukemia.

Lorea Villanueva,1 Montserrat Perez-Salvia,1 Eneko Aldaba,2 Yosu Vara,2 Myriam Fabre,3 Cristina Ferrer,3 Carme Masdeu,4 Aizpea Zubia,4 Eider San Sebastian,4 Dorleta Otaegui,5 Pere Llinàs-Arias,1 Margalida Rosselló-Tortella,1 María Berdasco,1 Fernando Setien,1 Catia Moutinho,1 Alberto Villanueva,6 Eva González-Barca,6 Josep Muncunill,7 José Tomás Navarro,7 Miguel Ángel Piris,8 Fernando Cossio,4 Manel Esteller1. 1 _Cancer Epigenetics and Biology Program (PEBC), Barcelona, Spain;_ 2 _Quimatryx, San Sebastian, Spain;_ 3 _Oncomatryx, Derio, Spain;_ 4 _Basque Country University, San Sebastian, Spain;_ 5 _CIC biomaGUNE, San Sebastián, Spain;_ 6 _Catalan Institute of Oncology (ICO), Barcelona, Spain;_ 7 _Josep Carreras Research Institute (IJC), Badalona, Spain;_ 8 _Fundación Jiménez Díaz, Madrid, Spain_.

Histone deacetylase 6 (HDAC6) is a protein modifier that is an increasingly attractive pharmacological target. Interestingly, the observation that the HDAC6 knock-out mouse is not lethal, in contrast to those undergoing complete loss of class I, II and III HDACs, suggests that specific HDAC6 inhibitors may be better tolerated than pan-HDAC inhibitors or drugs that target the other HDAC classes. In this regard, the compound ACY-1215 (Rocilinostat), the described selective HDAC6 inhibitor, is undergoing clinical trials for the treatment of multiple myeloma. Taking into account the previous information about HDAC6 inhibitor structures, the structural differences between HDAC6 and other HDAC isoforms and also the structural information of other developed HDAC inhibitors, we have previously designed and synthesized a new potential HDAC6 selective inhibitor, QTX125 with growth inhibitory effects in mantle cell lymphoma (MCL) cell lines, mouse models and ex vivo treatment of primary samples obtained from patients with MCL. Herein, we have extended these findings to show that the newly identified HDAC6 inhibitor QTX125 is also able to inhibit the growth of preclinical models of other B-cell lymphomas such as follicular lymphoma and Burkitt's cell lymphoma, but also of acute acute myeloid leukemia. In addition beyond a-tubulin, a well known HDAC6 target, we have developed a pharmacological and proteomic screening to identify other proteins modified by HDAC6 that can contribute to the described lymphoma and leukemia phenotypes.

#4726

Elucidating the pharmacological effects of covalent microtubule stabilizing taccalonolides through functional tagging.

Samantha S. Yee,1 Lin Du,2 April L. Risinger1. 1 _UT Health San Antonio, San Antonio, TX;_ 2 _University of Oklahoma, Norman, OK_.

Microtubule stabilizing taxanes are highly effective in the treatment of solid tumors and continue to be used as single agents and in combination with targeted therapies and immunotherapies. However, limitations of taxane therapy include acquired and inherent drug resistance. The taccalonolides are a novel class of microtubule stabilizers that circumvent clinically relevant forms of drug resistance to the taxanes in vitro and in vivo. The taccalonolides are efficacious in taxane-resistant models due to their distinct mechanism of microtubule stabilization that involves covalent binding to β-tubulin. Pharmacological studies of the taccalonolides have been complicated by their irreversible binding to tubulin. A functional tagged taccalonolide would facilitate a greater understanding of the pharmacological, cellular, and physiological effects of this novel drug class. We have generated and characterized a series of fluorescently tagged taccalonolides that retain microtubule binding and stabilizing activities to serve as functional pharmacological taccalonolide probes. Biochemical tubulin polymerization, live cell fluorescence, immunofluorescence imaging, and the sulforhodamine B cytotoxicity assay were conducted to evaluate the microtubule binding and stabilizing effects of these taccalonolide probes. We identified stable taccalonolide probes that retain the biological activities of untagged taccalonolides and facilitate their detection in live cells and in cell lysates. The rates of taccalonolide uptake and tubulin binding in cells, tumors, and normal tissues were determined. These studies have also allowed for an evaluation of the anatomical distribution of the taccalonolides in vivo, which can inform on their potential for antitumor efficacy in distinct tumor sites. Furthermore, we have generated a cellular model to evaluate the kinetics of taccalonolide binding to β-tubulin mutants that are predicted to influence drug binding based on the crystal structure. These studies have confirmed the covalent interaction between the taccalonolides and D224 on β-tubulin and informed on other residues critical for drug binding. The generation of a functional tagged taccalonolide will continue to be a valuable tool in evaluating the pharmacokinetics of these agents and inform on drug targeting strategies that will facilitate their clinical development.

#4727

Role of SUMOylating enzymes in repair of Topoisomerase II mediated DNA damage.

Bhargavi Ramesh,1 Yilun Sun,2 John L. Nitiss,3 Jay Anand,3 Karin C. Nitiss3. 1 _University of Illinois at Chicago, College of Medicine at Rockford, Rockford, IL;_ 2 _National Cancer Institute, Bethesda, MD;_ 3 _University of Illinois at Chicago, College of Pharmacy at Rockford, Rockford, IL_.

Topoisomerase 2 (Top2) enzymes regulate DNA topology for efficient DNA replication and transcription by introducing transient double strand breaks (DSBs) in the DNA. To generate the DSBs, Top2 forms a covalent intermediate with the DNA termed as the Top2 DNA covalent complex (Top2cc). Top2 is a target of clinically used anticancer agents such as etoposide and doxorubicin. Mammalian cells have two Top2 isozymes, Top2α and Top2β. Top2α is primarily expressed in proliferating cells and essential for cell division. Top2β is expressed in all cells and has been shown to play key roles in transcription. Top2 targeting agents generate anticancer activity by trapping both Top2αcc and Top2βcc. Factors that affect repair of Top2cc are likely to be a key determinant of the clinical efficacy of Top2 targeting agents. Identifying factors that play a role in repair of Top2cc offers a potential strategy for increasing clinical efficacy of Top2 targeting agents. Previous work has demonstrated that an important pathway for removal of trapped Top2 from DNA is initiated through proteolytic degradation of Top2 by the proteasome. Other proteases may also participate in degradation of the protein covalently bound to DNA. Proteasomal inhibition increased Top2cc, especially Top2β, and enhanced the cytotoxicity of Top2 targeting drugs. To study steps involved in proteasomal degradation of Top2cc, we developed a method to purify genomic DNA under conditions that preserve proteins covalently bound to DNA. Levels of trapped topoisomerases can be measured using Top2 isoform specific antibodies. Alternately, after micrococcal nuclease degradation of DNA, the proteins covalently bound to DNA can be characterized for post-translational modifications (PTMs) using antibodies specific for the modifying agents. Etoposide treatment induced SUMOylation of both Top2α and Top2β covalently bound to DNA. Blocking SUMOylation of Top2 by siRNA knockdown of the SUMO E2 conjugating enzyme UBC9 increased levels of both Top2α and Top2β covalent complexes, indicating that SUMOylation is likely involved in the repair of both Top2α and Top2β covalent complexes. We have extended this approach to identify SUMO ligases that directly act on Top2 isoforms. We found that depletion of the SUMO E3 PIAS4 resulted in elevated levels of Top2αcc with minimal effect on levels of Top2βcc. This result suggest that PIAS4 participates in a repair pathway that is specific for repairing Top2α covalent complexes. We have constructed PIAS4 knockouts using CRISPR/Cas9 and are currently examining the roles of other SUMO E3 ligases that may participate in the repair of Top2 damage. Our current results suggest that some pathways for repair of Top2 covalent complexes are likely isoform specific. This allows targeting of specific Top2 isoforms without the need to identify agents that discriminate between Top2 isoforms, and may help alleviate some of the undesirable side effects of Top2 targeting agents.

#4728

IFNγ signaling drives EZH2 degradation to induce MHC-II expression in melanoma.

Jamaal L. James, Susan R. Opalenik, Abigail Toren, Rebecca S. Cook, Justin M. Balko. _Vanderbilt University Medical Center, Nashville, TN_.

Background: The molecular mechanisms governing anti-PD-1 resistance in melanoma are not fully understood. Suboptimal transcriptional and molecular response to interferon-gamma (IFNγ) is commonly identified in tumors with intrinsic or acquired resistance to anti-PD-1. Epigenetic regulators have been shown to reprogram IFNγ responses in some cases, though the mechanism is not clear. The ability of melanoma cells to express MHC-II is a positive clinical predictor to anti-PD-1 therapy, and MHC-II is induced only on cells with robust IFNγ responses. Enhancer of zeste homolog 2 (EZH2), an epigenetic regulator with both methyltransferase-dependent and -independent function, can suppress IFNγ target genes and is frequently overexpressed in melanoma. However, the mechanisms whereby EZH2 interacts with the IFNγ pathway and its role in melanoma-specific MHC-II expression is not known.

Methods: HLA-DR (MHC-II)-proficient (A375, SKMEL28 and SKMEL5) and -deficient (CHL-1 and MEWO) melanoma cell lines were stimulated with IFNγ for up to 48h, and the modulation of EZH2 protein and function were studied temporally. To test the role of EZH2 in MHC-II (HLA-DR) expression in IFNγ responses, genetic (siRNA) and chemical (GSK343) EZH2 inhibition was utilized.

Results: EZH2 was highly expressed in all cell lines tested, regardless of HLA-DR-proficiency. In 2 out of 3 HLA-DR-inducible melanoma cell lines, EZH2 protein expression was downregulated (4-24 hrs) in response to IFNγ stimulation, without effects on EZH2 mRNA expression or downstream tri-methylated H3K27 expression. Blocking proteosomal degradation with MG-132 reversed the IFNγ-induced decrease in EZH2 expression in HLA-DR-proficient cell lines. After siRNA knockdown of EZH2, SKMEL28 cells increased constitutive HLA-DR expression, while A375 cells became more sensitive to IFNγ-induced HLA-DR upregulation. In addition, IFNγ-induced HLA-DR was increased in A375 cells with EZH2 inhibition using GSK343.

Conclusion: This work demonstrates a potential role for EZH2 in suppressing IFNγ responses, including the induction of MHC-II, in melanoma cells. EZH2 is subject to proteosomal degradation in MHC-II-inducible cell lines, which, in part, contributes to the expression of MHC-II.

#4729

Targeting c-MYCdegradation as a novel therapeutic strategy for osteosarcoma: Studies in canine and human osteosarcoma cells.

Ya-Ting Yang, Vilma Yuzbasiyan-Gurkan, Jetze J. Tepe. _Michigan State Univ., East Lansing, MI_.

Osteosarcoma (OS) is the most common bone tumor in both humans and dogs, and has a nearly ten-fold higher incidence in dogs than humans. For the past twenty years, despite advances in treatment of other cancers, the overall survival rates for OS have stagnated. Thus, there is a great need for novel therapeutic strategies. In osteosarcoma, MYC activation is critical for both initiation and maintenance of tumorigenesis and tissue invasion. While targeting c-MYC is a rational strategy to treat OS, c-MYC has evaded therapeutic manipulation to date. In this study, we evaluated using a novel proteasome modulator, TCH-165, which induces c-MYC degradation, and compared it with convention treatments in in vitro studies. We demonstrated dose dependent decrease of c-MYC protein expression with TCH-165 treatment. Utilizing seven canine OS cell lines and three human OS cell line, we determined that the IC50 values for TCH-165 range from 2.2 to 11.6 μM in canine OS, 3.1 to 4.5 μM in human OS cell lines, while IC50 of several primary canine ranged from 22.3 μM to >200μM, suggesting a wide margin of safety. In cell cycle studies, we showed that TCH-165 resulted in G1 arrest after 12 hours of incubation. In addition, we examined the potential of combing TCH-165 with convention therapeutic agents. We selected carboplatin, one of the standard anti-tumor drugs for OS. Combination index calculations using TCH-165 and carboplatin in the canine D17 cell line support synergistic effects of this combination. The data point to potential novel avenues for treatment of OS which can be further examined in proof of concept studies in dogs with OS, serving as a relevant translational model, and accelerate drug development for human OS patients.

#4730

A novel RAD51 inhibitor, CYT01B, shows anti-cancer activity in preclinical models of AID expressing solid tumors.

Melinda Day, Tyler Maclay, Kevin Mills. _Cyteir Therapeutics, Lexington, MA_.

AID, a DNA-directed cytidine deaminase, plays a critical role in somatic hypermutation and immunoglobulin class switching in maturing B-lymphocytes. Unlike site specific recombinases such as RAG1/2, AID is a promiscuous DNA damaging enzyme that deaminates cytidines at sites throughout the genome. AID expressing cells become critically dependent on the homologous recombination factor RAD51 to repair DNA double strand breaks that result from these off target deamination events. We have developed a novel small molecule, CYT01B, which inhibits RAD51 response to DSBs and is potent to a much greater extent in AID expressing cells. Previously, we have shown this molecule to have activity in mouse lymphoma xenograft models that constitutively express AID. Here we present new preclinical characterization data of CYT01B in solid cancer models. We first analyzed the frequency and levels of AID overexpression in solid tumor data in The Cancer Genome Atlas. Multiple solid tumor types displayed ectopic AID expression, including breast cancer, sarcoma, melanoma, pancreatic cancer, lung cancer, and head and neck cancers with about 30% of the patient samples 4-fold above the baseline expression level. We then tested CYT01B in several solid tumor derived human cell lines. We observed a correlation between AID expression and activity. Cell lines with low ectopic expression of AID had EC50 values in the low micromolar range (~2µM), while those without AID expression gave EC50 values of about 5µM and higher. Next, we examined the activity of our small molecule in 14 different PDX models with various levels of AID expression. These models included renal, head and neck, lung, pancreatic, ovarian, colorectal, and breast cancer samples. We observed a wide range of tumor growth inhibition across the different models (0% to 60%+), which tended to correspond with AID expression; the higher the AID expression the greater the observed tumor growth inhibition. Taken together, these data demonstrate that CYT01B shows anti-cancer activity in a range of solid tumor models. Overall, this provides the basis for continued development of CYT01B as an AID/RAD51 synthetic lethal therapeutic for solid cancers.

#4731

Therapeutic targeting of RNA splicing through inhibition of protein arginine methylation.

Jia Yi Fong,1 Diana Low,1 Luca Pignata,1 Kimihito Cojin Kawabata,2 Stanley CW Lee,3 Cheryl Koh,1 Daniele Musiani,4 Enrico Massignani,4 Cheng Mun Wun,1 Pierre-Alexis V. Goy,1 Yudao Shen,5 Heike Wollmann,1 Florence PH Gay,1 Genna Luciani,6 Dalia Barsyte,6 Jian Jin,5 Ari M. Melnick,2 Tiziana Bonaldi,4 Omar Abdel-Wahab,3 Ernesto Guccione1. 1 _Institute of Molecular and Cell Biology (A*STAR), Singapore, Singapore;_ 2 _Departments of Medicine and Pharmacology, Weill Cornell Medicine, New York, NY;_ 3 _Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY;_ 4 _European Institute of Oncology, Via Adamello 16, Milan, Italy;_ 5 _Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY;_ 6 _Ontario Cancer Institute/Princess Margaret Hospital, Toronto, Ontario, Canada_.

Mutations in RNA splicing factors commonly occur in myeloid leukemia and solid tumors. These mutations occur in a heterozygous manner and confer dependency on the wild-type allele. Studies have shown that splicing factor mutant cancers are vulnerable to further perturbation of splicing, by pharmacological intervention that directly targets core splicing factors. Our project identifies the use of PRMT inhibitors, as plausible alternative therapeutic strategy to treat spliceosomal mutant leukemia. The data show that splicing factor mutant leukemia exhibit greater sensitivity to the use of inhibitors against Type I PRMTs and PRMT5, in comparison to their wild-type counterparts. As the need for new therapeutic strategies in cancer treatment increases, this study identifies PRMT inhibitors as a potential therapeutic intervention against cancers with splicing factor mutations.

#4732

CDK4/6 inhibition with lerociclib (G1T38) enhances response to PI3K or ERK inhibitors in high-throughput, ex vivo pancreatic PDX screens.

Bingbing Dai,1 Daniel M. Freed,2 Jessica A. Sorrentino,2 Jithesh Jose Augustine,1 Tara G. Hughes,1 Ya'an Kang,1 Patrick J. Roberts,3 Jason B. Fleming,4 Michael P. Kim1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _G1 Therapeutics, Research Triangle Park, NC;_ 3 _G1 Therapeutics, Research Triangle Park, SC;_ 4 _Moffitt Cancer Center, Tampa, FL_.

Introduction: In pancreatic ductal adenocarcinoma (PDAC), mutations in KRAS and deletion or promoter methylation of the CDKN2A gene - resulting in deregulation of the p16/CDK4/CDK6/RB axis - each occur in over 90% of cases. Therefore, combination therapy approaches targeting KRAS effectors and CDK4/6 may be a promising treatment strategy in a majority of PDAC patients. Lerociclib (G1T38) is an oral and selective CDK4/6 inhibitor in clinical development as a potential backbone therapy for multiple combination regimens in cancer. Here, we used a powerful and reliable ex vivo drug screening platform, the live tissue sensitivity assay (LTSA), to evaluate the ability of lerociclib to synergize with inhibitors of KRAS effectors across a panel of 24 well-characterized PDAC patient-derived xenografts (PDXs).

Methods: PDX tumors were sectioned into uniform tissue slices at 200 µm thickness and arrayed in 96-well plates. For each PDX model, tumor tissue slices were treated with 0.3 µM, 1 µM, and 3 µM of lerociclib, alone and in combination with identical concentrations of a PI3K (pictilisib), mTOR (AZD2014), MEK (trametinib), ERK (ulixertinib), BRAF (vemurafenib) or EGFR (erlotinib) inhibitor for 72 hours. The viabilities of individual tissue slices were measured with PrestoBlue® reagent. A quantitative drug response analysis approach was implemented in which "responder" PDXs were defined as those exhibiting a statistically significant reduction in area under the dose-response curve (AUC) for the combination treatment compared to the most efficacious single agent. Statistical significance was defined as p < 0.05 using Student's t-test.

Results: Treatment of 24 PDAC PDXs using the LTSA revealed a range of sensitivities to each inhibitor as a single agent. When KRAS effectors and CDK4/6 were inhibited in tandem, statistically significant (p < 0.05) enhancements of efficacy were observed when lerociclib was added to pictilisib (21% of PDX models tested), ulixertinib (15%), vemurafenib (10%), erlotinib (5%), trametinib (4%), or AZD2014 (4%). These ex vivo data narrowed the focus of follow-up in vivo work; studies of PDX models in mice treated with lerociclib +/- pictilisib or ulixertinib are ongoing to validate these results. Moreover, differential gene expression and mutational analysis for responders versus non-responders identified through the PDAC PDX LTSA platform are underway, to elucidate molecular factors that predict tumor response or enrich for populations that are sensitive to CDK4/6 and PI3K/ERK inhibitors.

Conclusion: The LTSA platform was used to evaluate drug combinations in viable, PDX tumor tissue. We found that lerociclib significantly enhances the response to PI3K or ERK inhibition in a substantial proportion of the tested PDAC PDXs as measured by AUC analysis in the LTSA platform, supporting future clinical evaluation of these combinations.

#4733

Human functional brain imaging data support preclinical and clinical evidence that marizomib crosses the blood-brain barrier (BBB) to inhibit proteasome activity in the brain.

Daniela A. Bota,1 Kaijun Di,1 David B. Keator,1 Robert G. Bota,1 Matthew Hoffmann,2 C. Dan Dumitru,2 Nancy Levin3. 1 _University of California, Irvine, CA;_ 2 _Celgene Corporation, Summit, NJ;_ 3 _Triphase Accelerator, La Jolla, CA_.

Background: Here we summarize key data suggesting, unlike bortezomib, marizomib (MRZ), an irreversible inhibitor of all proteasome subunits, crosses the BBB, supporting its evaluation in brain malignancies.

Methods: Male Swiss Webster mice with microdialysis probes implanted in the cerebellum (CER) and prefrontal cortex (PFC) were administered MRZ (0.3 mg/kg IV, n = 15), amphetamine (1 mg/kg IP, positive control, n = 4), or vehicle (n = 6). Neurotransmitter levels were measured up to 180 minutes post-dose. Brain samples were collected from MRZ-treated mice at 30, 60, 120 (n = 3 each) and 180 (n = 6) minutes post-dose to determine proteasome activity (chymotrypsin-like [CT-L], trypsin-like [T-L], and caspase-like [C-L] subunits). Proteasome activity was assessed in normal human brain (CER and frontal lobe; n = 6) and glioblastoma tumor samples (GBM; n = 30), and in peripheral blood mononuclear cells (PBMCs) from patients with recurrent GBM (rGBM) prior to and after MRZ administration (MRZ-108 study, NCT02330562). Longitudinal resting-state functional magnetic resonance imaging (fMRI) of whole-brain volumes was acquired before and after MRZ administration to patients with rGBM (n = 6).

Results: In mice, MRZ significantly increased 3,4-Dihydroxyphenylacetic acid (DOPAC) and dopamine levels in the CER and decreased levels of serotonin (CER and PFC), 5-Hydroxyindoleacetic acid (CER and PFC), Homovanillic acid (PFC), and DOPAC (PFC). Proteasome activity of all 3 proteasome subunits (CT-L, C-L, T-L) was significantly reduced in mouse CER and PFC after MRZ administration; CT-L activity was most potently inhibited. In humans, similar CT-L and C-L activity levels were observed in GBM tumor tissue compared with normal brain CER, while activity levels were 3- to 4-fold higher in normal brain frontal lobe. In patients with rGBM, CT-L activity in PBMCs was inhibited 80-100% 1 hour post-MRZ, however, activity levels had returned to baseline prior to the next MRZ infusion 7 days later. Resting-state fMRI data showed, after MRZ exposure, hallucination severity was associated with decreased functional connectivity between left lingual gyrus and both left CER (T[4] = −12.78; P < 0.03 FDR) and left temporal cortex (T[4] = −9.56; P < 0.04 FDR).

Conclusions: MRZ demonstrated rapid, yet transient proteasome inhibition in PBMCs. This activity pattern may be related to proteasome turnover and occur in other nucleated cells, such as glioma cells. Lower proteasome activity levels were observed in CER and GBM tumor tissue compared with frontal lobe tissue. Proteasome inhibition and neurotransmitter level alterations in the mouse brain and functional connectivity changes in the human brain suggest that MRZ crosses the BBB and specifically affects the CER. Taken together, these data suggest GBM tumor tissue, like the CER, may be sensitive to MRZ treatment.

#4734

Response of bladder and colon cancer cells to combined treatment with bromodomain and histone deacetylase inhibitors.

Michael A. Lea, Niyati Patel, Charles desBordes. _Rutgers New Jersey Medical School, Newark, NJ_.

Since bromodomains of proteins bind acetylated histone lysine side chains while histone deacetylase inhibitors increase the level of histone acetylation, it seemed possible that bromodomain inhibitors might counteract some cellular responses to the action of histone deacetylase inhibitors. We have examined this possibility in bladder and colon cancer cells treated with combinations of bromodomain and histone deacetyalase inhibitors. Actions on growth and on the induction of alkaline phosphatase activity by histone deacetylase inhibitors were investigated. Compounds studied were the bromodomain inhibitors JQ1, I-BET763, olinone and RVX208 and the histone deacetylase inhibitors butyrate, RGFP966 and valproate. The cells studied were ones in which alkaline phosphatase activity is induced by histone deacetylase inhibitors. They were bladder cancer lines 5637, HT1197 and HT1376 and colon cancer lines Caco-2 and HT29. The combination that most clearly showed decreased induction of alkaline phosphatase activity by a bromodomain inhibitor was butyrate plus JQ1. If this action represents blocking combination of bromodomain-containing transcription factors with acetylated histones one might anticipate that inhibitory effects of histone deacetylase inhibitors on growth might be mitigated by combination with bromodomain inhibitors. However, the tendency was for additive effects on growth inhibition in accord with some data in the literature. Of the bromodomain inhibitors that were studied, JQ1 was the most effective as a growth inhibitor and olinone was the least effective. The mechanism for additive effects of bromodomain and histone deacetylase inhibitors on growth remains to be defined but antagonistic effects on cell differentiation appear possible.

#4735

The histone deacetylase inhibitor pracinostat modulates the transcriptome of diffuse large B-cell lymphoma cells and is active in combination with several targeted agents.

Afua A. Mensah,1 Filippo Spriano,1 Eugenio Gaudio,1 Chiara Chiara Tarantelli,1 Luciano Cascione,1 Andrea Rinaldi,1 Emanuela Lovati,2 Emanuele Zucca,3 Anastasios Stathis,3 Claudio Pietra,2 Francesco Bertoni1. 1 _Università della Svizzera italiana, Institute of Oncology Research, Bellinzona, Switzerland;_ 2 _Helsinn Healthcare SA, Pazzallo-Lugano, Switzerland;_ 3 _Oncology Institute of Southern Switzerland, Bellinzona, Switzerland_.

Background: Pracinostat (SB939) is a class I, II and IV histone deacetylase inhibitor (HDACi), in phase 3 in combination with azacitidine for acute myeloid leukemia (NCT03151408). We reported a strong preclinical activity as a single agent in lymphomas (Mensah et al, AACR 2018). Interestingly, anti-tumor activity differed between metabolically-defined subtypes of diffuse large B-cell lymphoma (DLBCL): lower in OxPhos than in B cell receptor (BCR) cell lines. Thus, we now report RNA-Seq on OxPhos and BCR DLBCL cell lines treated with pracinostat. We also present results of a combination screening with 9 anti-lymphoma drugs for future clinical development of this HDACi.

Methods: For RNA-Seq, OxPhos (Toledo, Pfeiffer, WSU-DLCL2) and BCR (SU-DHL-4, SU-DHL-6, OCI-LY-1) cell lines were treated with pracinostat or DMSO for 6 hours (h) or 14 days (d). Absolute fold change > 2 with adjusted P <0.01 were used as thresholds.

Combinations (72 h) with 5-azacytidine, ibrutinib, lenalidomide, bendamustine, everolimus, rituximab, idelalisib, bortezomib and copanlisib were evaluated on germinal center B-cell (GCB: SU-DHL-6, VAL) and activated B cell-like (ABC: OCI-LY-10, TMD8) DLBCL using the Chou-Talalay combination index (CI).

Results: At transcriptome level, pracinostat determined a time-dependent modulation of proliferation and cell cycle genes with differences between OxPhos and BCR cell lines.

The more sensitive BCR showed a higher number of modulated genes at both timepoints than the less sensitive OxPhos DLBCL. Moreover, while the upregulated transcripts increased with time in both metabolic clusters [doubled in BCR (1201 vs 591) and almost tripled in OxPhos (330 vs 120)], the opposite was true for downregulated genes in BCR (358 vs 600) but not in less sensitive OxPhos (186 vs 187).

Combinations of pracinostat with ibrutinib [0.37 (0.01 - 0.73)] or lenalidomide [0.29 (0.11 - 0.46)] were synergistic in ABC. Idelalisib [0.67 (0.46 - 0.92)] and everolimus [0.78 (0.57 - 1.11)] were both effective in combination with pracinostat in all ABC and 1 GCB, while rituximab was synergistic in 1 ABC and all GCB [0.61 (0.32 - >1.1)]. Copanlisib [0.87 (0.45 - 0.94)] and 5-azacytidine [0.96 (0.64 - 1.14)] were additive with pracinostat in 3/4 cell lines and were synergistic in the remaining. Combination with bortezomib was beneficial in 3 cell lines [0.94 (0.5 - 1.53)] and bendamustine combined effectively with pracinostat in 2 of the 4 cell lines [1.1 (0.89 - 1.3)].

Conclusions: Modulation of the DLBCL transcriptome by pracinostat occurs already at 6 h, suggestive of a rapid effect of pracinostat on chromatin architecture, but it differs between OxPhos and BCR DLBCL. In terms of combinations, pracinostat addition to other targeted agents was largely beneficial.

#4736

Dose optimization of olaparib plus temozolomide in small cell lung cancer (SCLC) patient-derived xenograft (PDX) models for clinical translation.

Benjamin J. Drapkin,1 Sarah Phat,1 David T. Myers,1 Jun Zhong,1 Beow Y. Yeap,1 Elisabetta Leo,2 Anna Stansiszewska,2 Aaron Smith,2 Elaine Cadogan,2 Maria C. Pietanza,3 Nicholas J. Dyson,1 Anna Farago1. 1 _Massachussetts General Hospital, Boston, MA;_ 2 _AstraZeneca, Cambridge, United Kingdom;_ 3 _Merck & Co, Inc, Kenilworth, NJ_.

Introduction: SCLC is an aggressive high-grade neuroendocrine malignancy in which targeting DNA-damage repair pathways has shown efficacy in pre-clinical and clinical contexts. We previously conducted a phase I/II trial combining the PARP inhibitor olaparib (O) tablets with the alkylating agent temozolomide (T) in patients with relapsed SCLC, in which O and T were both given days (d) 1-7 of each 21 d cycle. The recommended phase 2 dose (RP2D) was O 200 mg PO BID and T 75 mg/m2 PO daily, and the response rate among the first 29 evaluable patients was 41.4%, with the primary toxicities being hematologic (Farago et al., ASCO 2018). Continuous PARP inhibition with O may improve efficacy, though it is unknown how best to incorporate T onto this backbone. Simultaneous clinical testing of multiple dosing schedules for concurrent OT would be impractical. Here we present an optimization of the OT regimen using PDX models generated from patients treated with OT. Methods: SCLC PDX models derived from two patients prior to their durable responses to OT, MGH1518-1B and MGH1528-1, were used for dose optimization. Based on a human-murine comparison of serum Cmax for OT, the estimated murine-equivalents for the RP2D doses were O 25 mg/kg BID and T 6.25 mg/kg/d. The RP2D regimen was then modulated in both dose intensity and duration in a 9-arm PDX trial. The arms included 3 schedule categories, with doses varied within each category: discontinuous regimens (OT d 1-7); continuous O regimens (O d1-21 + T d 1-7); and rapid- or slow-alternating regimens administering O and T on non-overlapping days. Tumor bearing mice were treated when subcutaneous tumors reached a volume of 400-800 cc, enabling measurement of tumor regression (PDX response) and time to volume doubling (PDX TTP). Findings: The RP2D regimen resulted in near-complete response in both models. This degree of regression was recapitulated with nearly all dosing schedules, and was not improved by increasing T to 2x-RP2D. However, the PDX TTP varied. In both models, the longest TTP was seen with continuous O at full- or half-RP2D and d 1-7 T at half-RP2D. This regimen prolonged TTP by 12% in MGH1518-1B and 20% in MGH1528-1, versus discontinuous OT at RP2D. Based on these results, we initiated clinical treatment in a new cohort of patients on OT, with a phase 1 dose escalation starting with O 50 mg BID d 1-21 and T 50 mg/m2 d 1-7 of each 21 d cycle (NCT02446704). At this first dose level, a partial response by RECIST 1.1 criteria was observed in a heavily pre-treated patient, indicating clinical activity of this combination with this new dosing strategy. Conclusions: In PDX models sensitive to OT at our previously determined RP2D, we find that continuous O with intermittent T permits dose reduction of both O and T without loss of efficacy. A clinical trial is ongoing to determine whether this strategy may enable longer-term dosing and improve efficacy in patients.

#4737

Case report: Preclinical testing of NEDD8 and proteasome inhibitors for a treatment-refractory, metastatic high-grade mucinous colorectal cancer.

Erica Torchiaro,1 Consalvo Petti,1 Claudio Isella,1 Enzo Medico2. 1 _Candiolo Cancer Institute, Candiolo, Italy;_ 2 _Candiolo Cancer Institute and University of Torino, Candiolo, Italy_.

Colorectal cancer (CRC) is the third leading cause of death in the world. CRC shows variable phenotypic make-ups; among them, a particularly aggressive histological subtype is the "high grade mucinous" (HGM) adenocarcinoma, highly mucinous, prone to metastasis and typically refractory to treatments. In some cases, HGM is accompanied by a peculiar "signet ring" phenotype of cancer cells. Early stage diagnosis of signet ring HGM is rare, since clinical symptoms tend to occur late and most cases are usually detected at an advanced stage, with a poor overall survival. We previously demonstrated that a transcriptional signature of HGM displays negative prognosis and sensitivity to the NEDD-8 inhibitor pevonedistat, suggesting the involvement of neddylation- and ubiquitination-based mechanisms in these cases (Picco et al., JNCI 2017 djw209). To assess the clinical potential of colorectal cancer HGM identification and targeting by pevonedistat or other inhibitor of proteasome pathway, we recently propagated cells and PDXs from a case of early onset, metastatic CRC in a Lynch syndrome patient, refractory to standard care (FOLFOX6, FOLFIRI-Panitumumab) and, surprisingly, also to nivolumab. The tumor was highly mucinous, with signet ring undifferentiated cells. Mutational analysis on tumor tissue from the first surgery highlighted PIK3CA H1047R mutation and no mutations in KRAS, NRAS or BRAF. Surprisingly, exome analysis on two lesions from a subsequent surgery displayed a different scenario: KRAS G13D but not PIK3CA mutation. Probably these discording results suggest a strong tumor heterogeneity and evolution. Considering the failure of all previous therapies, the lack of actionable genetic alterations and the peculiarity of the phenotypic features, patient-derived models (organoids and 2D cell cultures) were derived from the two latter lesions and tested for sensitivity to the NEDD8 pathway inhibitor pevonedistat and the proteasome inhibitors bortezomib and carfilzomib. Interestingly, all models showed a strong sensitivity to both drugs, in particular to bortezomib. This is an example of a rare case of high-grade mucinous colorectal cancer where the integration of several approaches proves useful to identify possible therapeutic strategies for a patient without further standard treatment options.

#4738

Lipid peroxidation mediates the effects of topoisomerase poisons on their targets.

Amy C. Flor,1 Jing Li,2 Leslyn A. Hanakahi,2 Stephen J. Kron1. 1 _The University of Chicago, Chicago, IL;_ 2 _The University of Illinois, Rockford, IL_.

Our studies characterizing responses of cancer cells to oxidative stress have implicated the topoisomerases TOP1 and TOP2 as key mediators linking reactive oxygen species to DNA damage. Normally, TOP1 and TOP2 maintain chromosome integrity by transiently cleaving and rejoining DNA, thereby releasing topological strain and facilitating transcription, replication, and other processes critical for cancer cell proliferation. The topoisomerase poisons are a structurally diverse group of chemotherapy agents such as camptothecin, doxorubicin and etoposide that trap topoisomerases as cleavage complexes, TOP1cc and/or TOP2cc, where the enzymes remain covalently bound at single or double strand breaks, respectively. Prior studies identified a common mechanism of interfacial inhibition, but the constraints of locking protein onto DNA are hard to reconcile with the diversity of topoisomerase poisons, including simple compounds like H2O2 and 1,4-benzoquinone. Pointing to an alternative mechanism, camptothecin, doxorubicin, etoposide, and other topoisomerase poisons share the potential to markedly increase cellular oxidative stress. Among the most toxic consequences of oxidative stress is lipid peroxidation, leading to formation of lipid aldehydes such as 4-hydroxynonenal that modify and crosslink proteins at nucleophilic residues. In prior work, we showed that sequestering aldehydes blocked cellular effects of topoisomerase poisons while treating cells with 4-hydroxynonenal recapitulated many of their effects. Here, we have examined whether lipid aldehydes also mediate effects of topoisomerase poisons on their protein targets. Thus, we modulated oxidative stress in cells along with directly assaying formation TOP1cc and/or TOP2cc by detection in situ, flow cytometry, and Western blotting. We confirmed that camptothecin, doxorubicin, and etoposide induce lipid peroxidation, leading to production of aldehydes and other electrophiles. Under conditions where TOP1cc and/or TOP2cc are normally formed, blocking lipid peroxidation with butylated hydroxytoluene (BHT) was sufficient to protect cells from topoisomerase poisons. Among candidate mediators, 4-hydroxy-2-nonenal, 1- and E-2-hexadecanal, and 15-deoxy-∆12, 14-prostaglandin J2 could each induce TOP1cc and TOP2cc. The toxicity of the lipid-derived electrophiles was also dependent on the activity of TDP1, which serves a key role in removing TOP1cc and TOP2cc from DNA. Our results suggest a distinct, shared mechanism of action of topoisomerase poisons that depends on oxidative stress, lipid peroxidation, accumulation of lipid aldehydes and covalent modification of surface nucleophiles on topoisomerase enzymes. These covalent modifications may be sufficient to poison topoisomerases on their own and drive formation of TOP1cc and TOP2cc, independent of whether the cleaved conformation is stabilized by interfacial inhibition.

#4739

Induction of cell death by HDAC, Aromatase and MDM2 inhibitors in MCF-7 cells.

Thiagarajan Venkatesan,1 Umamaheswari Natarajan,2 Vijayaraghavan Radhakrishnan,2 Shila Samuel,2 Appu Rathinavelu1. 1 _Nova Southeastern University, Fort Lauderdale, FL;_ 2 _VRR Institute of Biomedical Sciences, Chennai, India_.

The histone deacetylase (HDAC) enzymes are evolving as key enzymes in the regulation of many physiological processes, including chromatin remodeling, genome stability, DNA repair, regulation of transcription, protein secretion, cellular metabolism, cell cycle progression, differentiation, migration, and cell death. Among different regulators of HDACs, suberoylanilide hydroxamic acid (SAHA) is a potent inhibitor and it is known to cause strong growth arrest, differentiation and apoptosis of many tumor types in both in vitro and in vivo experimental models. Interestingly, treatment of MCF-7 breast cancer cells with SAHA was found to trigger necroptosis, which is a caspase independent form of cell death. Typically, stimulation of death domain receptors (DDR) by suitable ligands lead to the activation of RIP1, RIP3 and MLKL proteins which are the critical regulators of necroptosis. It appears that, during SAHA induced necroptosis elevation of p21 may be the main signal that triggers recruitment of RIP1, Phospho-RIP3 and MLKL intracellularly in MCF-7 cells. Also, our results confirmed that the apoptosis marker such as BAX and caspases are not elevated by SAHA in MCF-7 cells after 24 hrs of treatment. In addition, the fluorescence assay conducted with SYTOX Green after 10 and 50 µM necrostatin pre-treatments, confirmed that SAHA treatment activates RIP1 mediated necroptosis pathway in MCF-7 cells. The induction of p21 expression was also down-regulated significantly by 10 and 50 µM necrostatin pre-treatments, which further suggests that p21 is up-stream of the necroptosis executioners in MCF-7 cells. On the other hand, when the MCF-7 cells were treated with Letrazole and RG-7388, BAX appears to be involved in mediating cell death. Interestingly, both Letrazole and RG-7388 were not able to elevate p21 in MCF-7 cells. So far, our results suggest that SAHA can induce BAX independent necroptosis while Letrazole and RG-7388 induce cell death through elevation of BAX and BAK in MCF-7 cells. (The generous support from the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida is gratefully acknowledged). 

### Drug Resistance 6

#4741

Re-evaluating the role of P-glycoprotein in the resistance of high-risk neuroblastoma to standard-of-care chemotherapies.

Caroline Atkinson,1 Carole M. Tactacan,1 Alvin Kamili,1 Federica Saletta,2 Christine Gana,1 Georgina L. Eden,1 Chelsea Mayoh,1 Richard B. Lock,1 Murray D. Norris,1 Michelle Haber,1 Andrew J. Gifford,3 Toby N. Trahair,4 Jamie I. Fletcher1. 1 _Children's Cancer Institute, Randwick, Australia;_ 2 _Children's Cancer Research Unit, Westmead, Australia;_ 3 _Prince of Wales Hospital, Randwick, Australia;_ 4 _Sydney Children's Hospital, Randwick, Australia_.

Background: Neuroblastoma is the most common extracranial solid malignancy in children, comprising 15% of cancer related deaths. Despite intensive treatment, patients with high-risk neuroblastoma (HR-NB) have a survival rate of ~50%, largely due to intrinsic or acquired drug resistance. The multidrug transporter P-glycoprotein (P-gp; ABCB1) effluxes several conventional agents used in HR-NB induction therapy, including doxorubicin, vincristine and etoposide, as well as the ALK inhibitor crizotinib. We observed high P-gp expression in HR-NB cell lines and patient-derived xenograft (PDX) models, so sought to assess the prevalence of P-gp expression and its role in resistance to standard-of-care chemotherapies and relevant targeted agents.

Methods and Results: Using RNA-sequencing, immunohistochemistry and western blot on panels of neuroblastoma tumour samples, PDX models and cell lines, we demonstrated that high ABCB1/P-gp expression is frequent in HR-NB. Analysis of ABCB1 levels in large patient tumor datasets suggests that high expression is attributable to the sympathoadrenal lineage of the disease, that high expression is more common in HR-NB than in most other cancers, and that high relative expression of ABCB1 in HR-NB tumours is associated with poorer outcome, consistent with its multidrug transporter function. We demonstrated that the P-gp inhibitor tariquidar and P-gp knockdown (shRNA) both strongly sensitize cultured high P-gp expressing neuroblastoma cells to vincristine, doxorubicin and etoposide but not to ALK inhibitors. Further, P-gp knockdown sensitized human neuroblastoma xenografts to vincristine, substantially extending survival.

Conclusions: Elevated P-gp expression is common in HR-NB and can be sufficient to confer resistance to standard-of-care chemotherapies in model systems. Our findings suggest that tumour P-gp levels might be used to guide treatment options for individual patients, and to avoid ineffective treatments. The potential of P-gp inhibitors as adjuncts to conventional chemotherapy for HR-NB should be further investigated.

#4742

The influence of cellular developmental state on response to therapeutics in glioblastoma.

Oluwademilade Nuga,1 Ana deCarvalho,2 Douglas Ruden,3 Stephen Brown,2 Indrani Datta,2 Tathianne Malta2. 1 _Wayne State University School of Medicine /Henry Ford Health System, Detroit, MI;_ 2 _Henry Ford Health System, Detroit, MI;_ 3 _Wayne State University School of Medicine, Detroit, MI_.

Glioblastoma (GBM) is an aggressive tumor treated with ionizing radiation (IR) and temozolomide (TMZ). Here we investigate how developmental state of glioblastoma cells: cancer stem cells (CSC) or differentiated cells, influences cellular response to treatment. Tissue from untreated primary GBM tumors representing diverse genotype were dissociated and cultured in conditions selective for CSCs, which were subsequently differentiated into an astrocytic phenotype by culturing in media supplemented with 2% FBS. Stemness index was evaluated using a machine learning predictive algorithm. Sensitivity of both isogenic CSC and serum differentiated cell (SDC) populations to TMZ and IR was determined by non-linear fitting of dose-response curves, using CellTiterGlo to measure cell viability (n=5) . CSCs and SDCs were then subjected to sub-lethal TMZ, IR (4 Gy), or control treatments, in triplicate. Cells were harvested, and DNA was isolated and Illumina 450k DNA methylation array data was analyzed by using the Methylumi package in R software. RNA was isolated for sequencing, and differentially expressed genes were determined by NOISeq R package. Our results show that GBM CSCs are more vulnerable to both TMZ and IR treatment than the isogenic SDCs. Transcriptome profiling show that IR leads to increase in p53 mediated expression of pro-apoptotic genes such as MDM2, PUMA and BCL2 binding protein relative to control in CSCs. In differentiated cells, IR lead to an increase in WNT signaling, DNA-repair activity and histone modification /remodeling proteins. Enrichment analysis using Fisher's exact test identified significant enrichment in lipid, fatty acid and metabolic processes as well as receptor tyrosine signaling. Globally, CSCs exhibited a decrease in protein coding genes and increase in regulatory RNA expression. DNA methylation show a global increase in CpG promoter methylation in differentiated cells relative to isogenic stem-like counterparts. We recovered significant differentially methylated probes between treatment groups (Wilcoxon signed rank test) as well as a global increase in CpG promoter methylation in IR treated cells relative to control. These findings underscore the challenge of treating a highly heterogeneous tumor and challenge a strategy of inducing differentiation to increase GBM sensitivity to treatment.

#4743

Using genome-wide CRISPR screen to understand resistance mechanisms to PCA062, a P-cadherin targeting antibody-drug conjugate.

Angela Tam, Mark Zambrowski, Katherine Seiss, Si-Qi Liu, Tinya Abrams, Giordano Caponigro, William Tschantz, Jennifer Campbell, Tony DAlessio, Qing Sheng. _Novartis Institutes for BioMedical Research, Cambridge, MA_.

P-cadherin (PCAD) is a member of the cadherin family that mediates calcium dependent cell-cell contacts in adherens-type junctions of epithelium. Expression of P-cadherin is high in malignant tumors of epithelial origin, such as breast, esophagus, head and neck cancers, but low in normal tissues. This expression pattern makes P-cadherin a potential good target for antibody-drug conjugates (ADCs). PCA062 is a first-in-class antibody drug conjugate targeting P-cadherin. PCA062 consists of a fully human anti-P-cadherin antibody of the IgG1/κ subtype, a non-cleavable bi-functional linker (SMCC) and a maytansine-derived cytotoxic payload (DM1, with a target average drug to antibody ratio (DAR) of 3.8).

PCA062 activity was examined in a collection of cell lines expressing high level of PCAD. A subset of these PCAD high cell lines are resistant to PCA062 treatment while they remain sensitive to DM1, suggesting defects in the process of PCA062 uptake or the processing and release of DM1 into the cytoplasmic compartment. PCA062 internalization rate was measured by the uptake of a fluorescent dye labeled anti-PCAD antibody (CQY684, the Ab portion of PCA062) in both PCA062 sensitive and resistant lines. PCA062 resistant lines show slower CQY684 internalization as compared to PCA062 sensitive lines, indicating a defect in ADC internalization may contribute to PCA062 resistance. To explore additional resistance mechanisms to PCA062 as well as to find critical components for PCA062 internalization, a genome wide CRISPR screen was performed in PCA062 sensitive HCC1954 and in PCA062 resistant KYSE510 cell line in the presence and absence of PCA062 and DM1 to look for genes that when knocked out may specifically modulate PCA062 sensitivity. The multi-drug resistant gene MRP1 is a strong hit for PCA062 sensitization in both PCA062 resistant KYSE510 and PCA062 sensitive HCC1954 cells. Lysosomal transporter SLC46A3 and Saga transcription complex components are strong rescue hits for PCA062 in HCC1954. These data suggest that in addition to target expression level and cell intrinsic sensitivity to payload, genes involved in ADC internalization and payload cytoplasmic accumulation will also impact tumor cell sensitivity to ADCs.

#4744

A prospective biospecimen collection study from patients with EGFR mutant tumors.

Ivy B. Elkins,1 Jill Feldman,1 Anita Figueras,1 Teri Kennedy,1 Allen Lee,1 Ildiko Medve,1 Colleen Sturdivant,1 Pasi Janne,2 Tony Addario,3 Alicia Sable-Hunt,3 Bonnie Addario,4 David LeDuc,4 Amy Moore,4 Jennifer Jaskowiak,5 Maria Mancini5. 1 _EGFR Resisters, Buffalo Grove, IL;_ 2 _Dana-Farber Cancer Institute, Boston, MA;_ 3 _Addario Lung Cancer Medical Institute, San Carlos, CA;_ 4 _Bonnie J. Addario Lung Cancer Foundation, San Carlos, CA;_ 5 _Champions Oncology, Hackensack, NJ_.

Background: Somatic mutations in the epidermal growth factor receptor (EGFR) are detected in nearly 15% of patients with lung adenocarcinoma. These mutations are critical for tumor development and maintenance; some but not all are sensitive to EGFR tyrosine kinase inhibitors (TKI). EGFR mutations fall into 3 major categories: 1) Exon 19 deletions and L858R which are detected in 85% of patients; 2) Atypical EGFR mutations including G719X, L861Q which are detected in 5 to 8% of patients and 3) Exon 20 insertion mutations which are also detected in 5 to 8% of patients. These different subtypes exhibit varying responses to TKI treatment, with osimertinib being the standard of care for patients with exon 19 deletion or L858R mutations. However, no currently-approved TKI is available for patients with Exon 20/EGFR mutant lung cancer.

Method: Patient advocacy groups representing oncogene-driver mutations in lung cancer (ALK, EGFR, Exon 20, RET, ROS1) have assembled with the objectives of providing patient education/support and driving research into lung cancer's causes and treatments. The EGFR Resisters represent over 775 patients from 26 countries. In an effort to understand mechanisms of resistance and develop new treatments, EGFR Resisters embarked on a collaboration with Dr. Pasi Jänne, the Addario Lung Cancer Medical Institute, the Bonnie J. Addario Lung Cancer Foundation, and Champions Oncology to develop patient-derived xenograft (PDX) models for EGFR mutant lung cancer.

Result: In mid-2018 the EFGR Resisters assembled a coalition of key leaders in the lung cancer space to advance development of research models for EGFR mutant lung cancer. The IRB-approved study, "A Prospective Biospecimen Collection Study from Patients with EGFR mutant Tumors" was launched in November 2018 with a goal of developing at least ten EGFR mutant PDX models with full characterization including whole exome sequencing (WES) and RNA sequencing. Model development will be divided among three cohorts: 1) EGFR T790M patients who have progressed on osimertinib or other third-generation TKIs; 2) EGFR exon 19 del or L858R patients who have progressed on first-line osimertinib; 3) patients with Exon 20 insertion mutations (includes EGFR Exon 20 and HER2 Exon 20). The models will be made available to the scientific community.

Conclusion: Understanding mechanisms of resistance in EGFR mutant lung cancer is high priority as most patients will ultimately develop resistance to currently-approved TKIs. Additionally, there is an urgent need to develop next-generation TKIs since some EGFR mutation subtypes do not respond to current therapies. By leveraging the skills and expertise of diverse partners, the EGFR Resisters have advanced an initiative to create a unique cohort of PDX models that will provide a rich resource for clinical and translational research to understand mechanisms of resistance and develop new therapies for patients with EGFR mutant lung cancer.

#4745

Reversal by inhibiting XBP1 activation of glucose deprivation-induced tolerance to multiple anticancer therapies.

Mini Jeong, Serin Jo, Serkin Park, Jeongwon Sohn, Yun Gyu Park. _Korea Univ. College of Medicine, Seoul, Republic of Korea_.

Tumor microenvironment is harsh condition including low pH, hypoxia and nutrient deprivation. Cancer cells can switch their phenotypes and characteristics dependent on the microenvironment. Most previous researches have focused mainly on the effect of hypoxia on cancer cells plasticity and resistance to anticancer therapies. Despite the accumulation of emerging evidences, the effect of nutrient deficiency to multiple anticancer therapies is still unclear.In this study, human lung adenocarcinoma cells (A549), triple-negative breast cancer (MDA-MB-231), hepatocellular carcinoma cells (HepG2), pancreas adenocarcinoma cells (AsPC-1) were cultured in the medium containing low concentrations of glucose. Low sensitivity to cisplatin or taxol was observed in the cancer cells maintained for 28 weeks or so under the glucose-deprived condition. In addition, the multidrug tolerant cells were also insensitive to cytotoxic T cell (CTL)-based immunotherapy and had increased migration ability compared with control group under normal glucose condition. Reversion to normal glucose condition only for short term period (at least 4 days) made the multidrug tolerant cells susceptible to cisplatin-induced apoptotic cell death. In the unfolded protein response, BiP-XBP1 signaling was increased but PERK signaling was decreased in response to long-term glucose deprivation. Sensitivity to cisplatin treatment and CTL-based immunotherapy was restored by inhibiting XBP1 splicing in glucose deprivation-induced multidrug tolerant cells in various types of cancer.This study demonstrated that glucose deprivation-induced tolerance to multiple anti-cancer therapies is transient and reversible, and requires activation of IRE1-XBP1 pathway.This research was supported by the National Research Foundation of Korea (2018R1D1A1B07045297, 2016R1D1A1A02936945 and 2017R1D1A1B03036008).

#4746

Identifying states of collateral sensitivity during the evolution of therapy resistance in Ewing's sarcoma.

Erin McClure,1 Masahiro Hitomi,1 Jessica Scarborough,2 Jacob Scott1. 1 _Cleveland Clinic, Cleveland, OH;_ 2 _Case Western Reserve University School of Medicine, Cleveland, OH_.

During the evolution of therapeutic resistance many cancers exhibit a phenomenon termed collateral sensitivity, where resistance to one drug aligns with sensitivity to another drug. It has further been shown, in leukemia and non-small cell lung cancer, that this state of collateral sensitivity can vary over time. We hypothesized that this process will also occur in Ewing's sarcoma (EWS), and that these phenotypic states can be identified with molecular signatures based on gene expression. The standard chemotherapy for EWS is repeated cycles of two alternating drug cassettes, one is a combination of vincristine, doxorubicin, and cyclophosphamide (VDC), and a second is a combination of ifosfamide and etoposide (IE). This treatment is highly effective, though advanced stage disease often relapses, and then drug resistance invariably develops, a process following the laws of Darwinian evolution. Finding predictable patterns, or signatures, of collateral sensitivity would provide a useful decision support tool in the clinic to help guide chemotherapy choices. Recent studies in other cancers, bacteria, and in silico simulations have explored and identified optimal timing of switching among the drugs of collateral sensitivity to achieving maximal efficacy. To analyze temporal changes in collateral sensitivity in EWS, we exposed two EWS cell lines, A673 and TTC-466, to half maximal inhibitory concentrations of VDC/IE combinations in vitro. As the cultures recovered from drug exposures, we determined their sensitivities against a panel of drugs including the ones used for the treatment and a new generation of drugs that show efficacy against EWS. The new drugs consist of SP2509, pazopanib, olaparib, dactinomycin, temozolomide, vorinostat, and SN-38. We also collected RNA samples to analyze the transcriptomic alterations which correlate with changes in their collateral sensitivity. Significant findings for the A673 cell line include increased resistance to dactinomycin, doxorubicin, etoposide and vincristine for all treatment replicates, and maximal sensitivity to temozolomide halfway through the treatment schedule for all treatment replicates. Notably, every tumor is different and may develop collateral sensitivity at unique time points. Even in this experiment we observe variation between evolutionary replicates that were originally genetically identical. Future work should include investigating a quick and cost-effective method for testing temporal collateral sensitivity of individual tumors so our findings can be implemented clinically.

#4747

Surviving skills in melanoma cells: An important consideration to both targeted and immunological approach in the treatment of melanoma.

Niramol Savaraj,1 Ying-Ying Li,1 Chunjing Wu,1 Medhi Wangpaichitr,1 Seth Spector,1 Michael Sentome,2 Lynn G. Feun2. 1 _Univ. of Miami/VA Medical Ctr., Miami, FL;_ 2 _Univ. of Miami, Miami, FL_.

While immunotherapy with checkpoint inhibitor in the treatment of melanoma patients (pts) can produce about 40-50 % response rate and with durable response, but 50% of pts still do not respond and other form of treatments are needed. On the other hand, combination of BRAF and MEK inhibitor can produce a rapid response in pts whose tumors harbor BRAFV600E or BRAFV600K mutation, but over 70% of pts will relapse with the duration of response of approximately 12 months. Immunotherapy with checkpoint inhibitor in this group of pts often showed a variable rate of response ranging from 15- 35%. The underlying mechanism for the variable response is not known. We investigated the possible contributory factors to this variation. Five pairs of BRAF mutant and their BRAF and MEK resistant variants (BMR): A375/BMR, A2058/BMR, Mel1220/BMR, SK28/BMR, UACC2/BMR and one pair of A2058v/ BMR were used for the study. A2058v expressed argininosuccinate synthetase and can survive/proliferate in arginine-free media with citrulline supplement. All BMR cell lines were generated from stepwise increasing dosage of BRAF and MEK inhibitor similar to our previous publication in generating BRAF resistant cells (Oncotarget 2016:17665). All BMR cell lines exhibit 3 fold resistance to BRAF inhibitor and 1.8 fold to MEK inhibitor. This panel of cell lines was used to analyze for PD-L1, BCL-2, BCL-XL, MCL1, and NOXA , and AKT/MEK/ERK activation. Our results showed that three cell lines MEL1220, SKmel28 and UACC62 which have high levels of BCL2, do not have high PD-L1 levels both in parental cells and their BMR variants. In contrast, both A375 BMR and A2058BMR exhibit high PD-L1 ( >10 fold). The highest level (>50 fold) was found in A2058v and its BMR variant. All BMR cells have more AKT/MEK/ERK activation. Other anti- and pro-apoptotic expression do not differ significantly. These initial results suggest that the presence of antiapoptotic protein BCL2 may mitigate the necessity to express high PD-L1 and hence lessen the response to checkpoint inhibitor therapy. To further clarify this possibility, we have studied a panel of primary culture from melanoma pts, both treatment naive and those who failed BRAF/MEK inhibitor, as well as BRAF wild type pts . Eight BRAF mutant tumors and seven BRAF wild type were used. Similar correlation existed for both BRAF mutant and wild type primary culture. However, in BRAF wild type pts, two primary culture with high BCL2 still express low levels of PD-L1. Our data suggest that BcL-2 expression and possible other pro-and antiapoptotic proteins may be an additional useful parameter to predict response to checkpoint inhibitor therapy especially when they failed BRAF/MEK inhibitor. Combination with other inhibitors in the apoptotic pathway such as BCl-2 inhibitor may be also useful to improve the therapeutic outcome in these BMR pts. Supported by the VA Merit Review Award(1BX003328 to N. Savaraj)

#4748

**RICTOR signaling modulates the activity of EGFR tyrosine kinase inhibitors (TKI) in** EGFR **-mutated non-small cell lung cancer (NSCLC).**

Ni Fan,1 Yiyu Zou,1 Balazs Halmos,2 Roman Perez-Soler,2 Haiying Cheng2. 1 _Albert Einstein College of Medicine, Bronx, NY;_ 2 _Montefiore Medical Center/Albert Einstein College of Medicine, Bronx, NY_.

Background: Molecularly-targeted therapy has significantly improved the therapeutic efficiency and clinical outcome in a subgroup of NSCLC patients. However, drug resistance inevitably emerges, such as resistance to EGFR TKIs in EGFR-mutated NSCLC. RICTOR is a key component of the mTORC2 complex and plays important roles in cell survival, proliferation, migration and invasion. RICTOR has also been suggested to play a part in the regulation of drug resistance to HER2-targeted therapy in HER2-amplified breast cancer, a niche close to EGFR-targeted therapy. In our current study, we hypothesize that RICTOR modulates the activity of EGFR TKIs in EGFR-mutated NSCLC. Co-targeting RICTOR and EGFR signaling pathways could be a new therapeutic strategy for patients with EGFR-mutated lung cancer.

Results: Genetically modified EGFR-mutated NSCLC cell lines with different RICTOR expression levels have been developed. RICTOR knockdown significantly increases the sensitivity to all three generations of TKI inhibitors (erlotinib, afatinib and osimertinib), whereas overexpression of RICTOR renders the cells more resistant to EGFR TKIs. In addition, RICTOR knockdown restores the sensitivity of erlotinib-resistant HCC827 cells due to AXL overexpression (IC50: 216µM vs. 0.256 µM with RICTOR knockdown, P<0.001). Pharmacological inhibition of the RICTOR signaling pathway with the mTOR1/2 inhibitor (sapanisertib) also improves EGFR TKI sensitivity. Taken together, RICTOR signaling modulates the efficacy of EGFR TKIs in the EGFR-mutated lung cancer models.

Conclusion: Our study provides a new insight into the role of RICTOR signaling modulation in increasing efficacy of EGFR TKI therapy in EGFR-mutated NSCLC. The combination of mTOR1/2 inhibitor and EGFR inhibitor could be a potential novel strategy leading to better therapeutic efficacy.

#4749

Acquired resistance to BET-PROTACs caused by genomic alterations in core components of E3 ligase complexes.

Bridget Riley-Gillis, Lu Zhang, Priyanka Vijay, Yu Shen. _AbbVie, North Chicago, IL_.

Proteolysis Targeting Chimeras (PROTACs) are bifunctional molecules that hijack endogenous E3 ubiquitin ligases to induce ubiquitination and subsequent degradation of protein of interest. Recently, it has been shown that PROTACs with robust in vitro and in vivo activities and, in some cases, drug-like pharmaceutical properties can be generated using small molecule ligands for the E3 ligases VHL and CRBN. These findings stoked tremendous enthusiasm on using PROTACs for therapeutics development. Innate and acquired drug resistance often underlies therapeutic failures, particularly for cancer therapy. With the PROTAC technology progressing rapidly towards therapeutic applications, it would be important to understand whether and how resistance to these novel agents may emerge. Using BET-PROTACs as a model system, we demonstrate that resistance to both VHL- and CRBN-based PROTACs can occur in cancer cells following chronic treatment. However, unlike what was often observed for many targeted therapeutics, resistance to BET-PROTACs did not result from secondary mutations that affect compound binding to the target. In contrast, acquired resistance to both VHL- and CRBN-based BET-PROTACs was primarily caused by genomic alterations that compromise core components of the relevant E3 ligase complexes.

#4750

The role of drug transporters in the acquired resistance to PBD dimer-containing antibody drug conjugates.

Simon Corbett,1 Francesca Zammarchi,2 Philip W Howard,3 Patrick H van Berkel,2 John A Hartley1. 1 _University College London, London, United Kingdom;_ 2 _ADC Therapeutics, London, United Kingdom;_ 3 _Spirogen/MedImmune, London, United Kingdom_.

Antibody drug conjugates (ADCs) delivering interstrand cross-linking pyrrolobenzodiazepine (PBD) dimers are being evaluated clinically in both hematological and solid tumors. These include ADCT-301, ADCT-402, MEDI3726 and rovalpituzumab tesirine (Rova-T) that contain the payload tesirine which releases the PBD dimer warhead SG3199. An important consideration in the future development of PBD ADCs is the development of clinical acquired resistance. The aim was to generate and characterize in vitro acquired resistant cell lines in both a hematological and solid tumor setting. To generate acquired resistant cells to ADCT-301 (targeting CD25) or warhead SG3199, CD25-positive Karpas-299 cells were incubated with an approximate GI50 dose of the drug for 96 h. Cells were washed and returned to fresh medium until normal cell growth was restored. This was repeated until a stable acquired resistance was established. The level of resistance achieved was >700-fold for the ADC, and 3-fold for the naked PBD. Cross-resistance between the agents was observed and also with an ADC delivering a different PBD dimer. Similarly, resistant cells to ADCT-502 (targeting HER2) or SG3199 were established using HER2-positive NCI-N87 cells incubated with GI50 doses for 144 h. The level of resistance achieved was 8 and 4-fold, respectively, and again, cross-resistance was observed. DNA interstrand cross-linking was measured in the parental and resistant lines treated with equi-molar doses of ADC or SG3199. In every case, the resistant lines produced fewer cross-links indicating an up-stream mechanism of resistance preventing SG3199 from reaching its DNA target. In the Karpas ADCT-301-resistant line, a small (15%) decrease in CD25 cell surface expression could not account for the level of resistance. RT2 Profiler PCR Array for human drug-transporters was used to probe the cell lines. In the Karpas ADCT-301-resistant line three gene products were significantly upregulated: ABCG2 (168-fold), SLCO2A1 (4.6) and ABCC2 (3.5). Significantly upregulated in the Karpas SG3199-resistant cells were SLCO2B1 (15.4), ABCC12 (6.4), ATP7A (4.6), SLC16A2 (2.8), ABCG2 (2.8), SLC7A9 (2.5), ABCB4 (2.3) and ABCC11 (2.1). Upregulation of ABCG2, ABCC2 and SLCO2B1 was confirmed by RT-PCR. In NCI-N87 ADCT-502-resistant cells, ABCG2 (91), SLC22A3 (65), ABCC2 (43), SLCO2B1 (30) and SLC7A7 (6.8) and in NCI-N87 SG3199-resistant cells ABCG2 (115), SLC22A3 (61), SLCO2B1 (54), ABCC2 (36), SLC7A7 (8), AQP7 (6.4) and SLC238A3 (2.9) were upregulated. Upregulation of ABCG2, ABCC2, SLCO2B1, SLC7A7 and SLC22A3 was confirmed by RT-PCR and for ABCG2 and ABCC2 also by western blot. ABCB1(MDR1) was not upregulated in any of the lines. These data show that acquired resistance to both PBD-ADCs and SG3199 can involve specific drug transporters. This has clinical implications as potential biomarkers of resistance and for the rational design of drug combinations.

#4751

Overcoming cisplatin resistance of esophageal squamous cell carcinoma by targeting RAC1-regulated Warburg effect.

Ruijie Zeng, Chunwen Zheng, Jinge Gu, Liu Peng, Haixia Zhang, Pan Feng, Yang Chen, Xiue Xu, Liyan Xu, Enmin Li. _Shantou University Medical College, Shantou, China_.

Esophageal squamous cell carcinoma (ESCC) is a highly aggressive malignancy with poor prognosis. Despite treatment advances, development of chemo-resistance remains a major challenge in treating ESCC patients. Ras-related C3 botulinum toxin substrate 1 (RAC1) is essential in regulating cancer progression. However, its role in chemo-resistance of ESCC and the underlying mechanisms are still unknown. In this study, we found that higher levels of RAC1 expression were associated with poorer disease-free survival (p=0.014) and overall survival (p=0.013) by immunohistochemistry in human ESCC samples (n=106). The over-expression of RAC1 was correlated with enhanced cell proliferation, migration, invasion and chemo-resistance in both KYSE150 and KYSE510 cells, while the knockdown of RAC1 produced the opposite results. The combination of RAC1 inhibitor EHop-016 with cisplatin significantly promoted cell viability reduction, cycle arrest at G2/M phase and apoptosis when compared with the single treatment. To examine the in vivo effects of RAC1 inhibition on chemo-resistance, we performed xenograft implantation studies in immunocompromised mice (n=10 per group). In the combination group, the tumor volume was significantly reduced to 21.4% (p<0.001) as compared to the single treatment groups (cisplatin: 62.7%, RAC1 inhibitor: 58.5%). Mechanistically, the RNA-Seq data indicated that the glycolytic pathway was significantly down-regulated in the EHop-016-treated group (p<0.001, Log2FC=1.86) and the combination group (p<0.001, Log2FC=1.76), as compared to the control group. Moreover, the qRT-PCR and western blot results confirmed that RAC1 inhibitor alone or the combination treatment suppressed Akt/FOXO signaling and resulted in inhibition of key enzymes such as PKM2, LDH-A, ALDOA and HK1 for the Warburg effect. Overall, our study suggests that inhibition of RAC1 overcomes cisplatin resistance in ESCC both in vitro and in vivo, and provides a novel mechanism of chemo-resistance in ESCC. Therefore, targeting RAC1 might be a promising and potent strategy for treating ESCC in clinical practice.

#4752

**Exon-16-skipping HER2 contributes to osimertinib-resistance through Src-independent manner in** EGFR **L858R/T790M-positive non-small-cell lung cancer.**

Chia-Chi Hsu,1 Bin-Chi Liao,2 Wei-Yu Liao,3 Aleksandra Markovets,4 Daniel Stetson,4 Kenneth Thress,5 Chih-Hsin Yang1. 1 _National Taiwan University College of Medicine, Taipei, Taiwan;_ 2 _National Taiwan University Cancer Center, Taipei, Taiwan;_ 3 _National Taiwan University Hospital, Taipei, Taiwan;_ 4 _AstraZeneca, Waltham, MA;_ 5 _TESARO, Waltham, MA_.

Osimertinib is current recommended treatment for EGFR T790M-positive NSCLC following progression on prior EGFR tyrosine kinase inhibitor therapy. However, acquired resistance to osimertinib therapy inevitably developed after a period of effective treatment. We had a patient of EGFR L858R/T790M-positive NSCLC who initially responded to osimertinib therapy but eventually developed resistance. Plasma cell-free DNA analysis disclosed exon-16-skipping HER2 (HER2Δ16) in one osimertinib treated patient. HER2Δ16 was regarded as an oncogenic driver in breast cancer and has never been reported in lung cancer before. Therefore, it's intriguing to investigate the role of HER2Δ16 in osimertinib resistance in NSCLC. The purpose of this study is to explore the mechanism of HER2Δ16-mediated resistance to osimertinib and to explore strategies to overcome the resistance.

We constructed and established H1975 cells stably expressing either vehicle, WT-HER2 or HER2Δ16. We observed that without osimertinib treatment, there was no difference in cell growth and the response to EGF-stimulation. However, when treated with osimertinib, H1975-HER2Δ16 cells were more resistant under normal and EGF-stimulating conditions compared to vehicle or WT-HER2 expressing cells. Moreover, osimertinib treatment in H1975-HER2Δ16 cells had less inhibitory effect on phosphor-ERK and phosphor-AKT compared to vehicle or H1975-WT-HER2 cells. Interestingly, co-treatment of osimertinib with Src-kinase inhibitor, dasatinib, failed to reverse the resistance, indicating that HER2Δ16-driven

osimertinib-resistance was independent of Src-kinase. Finally, we revealed that combination of osimertinib with the HER2 small molecular inhibitor, afatinib, can suppress cell growth in H1975-WT-HER2 and H1975-HER2Δ16 cells.

In summary, we have shown that HER2Δ16, may be a resistance mechanism to osimertinib in EGFR mutated NSCLC and that HER2Δ16-driven resistance was Src-kinase independent. Moreover, we found the HER2Δ16-driven resistance could be rescued by combination of osimertinib with afatinib.

#4753

Attenuating colorectal cancer cell growth and enhancing immunogenicity through rational combination of MEK and HDAC inhibition.

Mihailo Miljanic,1 Nisha Holay,2 Anna Capasso,3 Todd Triplett,1 Milad Soleimani,4 Uma Giri,4 Carla Van Den Berg,1 Gail Eckhardt3. 1 _Dell Medical School - UT Austin, Austin, TX;_ 2 _Livestrong Cancer Institutes, Austin, TX;_ 3 _Dell Medical School - UT Austin; Livestrong Cancer Institutes, Austin, TX;_ 4 _Livestrong Cancer Institutes - UT Austin, Austin, TX_.

Background: Studies have shown that MEK inhibitors (MEKi) can reduce tumorigenic growth signaling in colorectal cancer (CRC) through inhibition of the RAS pathway which is upregulated in over 40% of CRC tumors, while HDAC inhibitors (HDACi) lead to cell cycle arrest, inhibit angiogenesis, and induce apoptosis in neoplastic cells. In addition to a direct effect on neoplastic cells, MEKi have also demonstrated the potential to increase immune response to the tumor through upregulation of MHC I on tumor cells, induction of intra-tumoral T-cell infiltration, and enhancement of anti-PDL1 activity. HDAC inhibitors lead to epigenetic alterations which have similarly shown an ability to increase tumor immunogenicity, leading to the hypothesis that these agents may be combined with immunotherapy treatments for enhanced anti-tumor effect. In this study we first analyzed the effect of MEKi and HDACi combination treatment in both resistant and sensitive CRC cell lines to assess for synergy in the attenuation of cancer cell proliferation. We then demonstrated the effect of combination MEKi and HDACi therapy on tumor immunogenicity through increased expression of tumor immune markers.

Methods: We assessed the efficacy of a novel MEKi agent, HL-085, in comparison with trametinib in six different CRC cell lines. Treatment with each agent was separately administered with a maximum concentration of 10 uM. Similarly, five different HDAC inhibitors were tested in each of these cell lines, with a maximum concentration of 1 uM. Optimal combination of MEK inhibitor HL085 and HDAC inhibitor class I/II (OKI-005) were then chosen based on this data. Cellular proliferation was assessed after 72 hours with Cell Titer Glo. Flow cytometry of a panel of immunogenic markers was then performed alongside a viability stain to assess for expression level changes of MCH class I in CRC cells.

Results: Combinatorial effects of dual treatment with MEK inhibitor HL085 and HDAC inhibitor OKI-005 are observed in both resistant and sensitive cell lines with synergistic effect produced in the SW620 and Lovo cell lines (MEKi sensitive) as well as the GP2D (MEKi resistant) cell line. No synergistic effect was observed in the HCT15, DLD1 and LS513 cell lines. Flow cytometry showed an increase in MHC class I expression in neoplastic cells with combined MEKi and HDACi treatment compared to individual agent treatment and control, demonstrating increased immunogenicity of CRC cells with combination treatment.

Conclusions: Combination treatment with a novel MEK inhibitor HL085 and the HDAC inhibitor OKI-005 demonstrates synergy in three out of six colorectal cancer cell lines. We further show increased immunogenicity of CRC cells through enhanced expression of MCH I on tumor cell surface, indicating rationale for further investigation of combination MEKi and HDACi with immunotherapy in the treatment of metastatic colorectal cancer.

#4754

CHTOP is a novel therapeutic target for chemoresistant epithelial ovarian cancer therapy.

Xupeng Bai, Xiaojie Feng, Jie Ni, Julia Beretov, Junli Deng, Ying Zhu, Peter Graham, Yong Li. _Univ. of New South Wales, Kogarah, Australia_.

Background: Chemotherapy is the mainstay treatment for ovarian cancer (OC). Chemoresistance is a major challenge in epithelial ovarian cancer (EOC) therapy. CHTOP was identified as a potential chemoresistant biomarker in chemoresistant EOC cell lines using label-free LC-MS/MS proteomic technique. However, the role of CHTOP in EOC chemoresistance is still unclear.

Aim: In this study, we aimed to investigate whether CHTOP can be used as a therapeutic target in chemoresistant EOC cells and to reveal the mechanism underlying chemosensitization.

Methods: The expression difference of CHTOP was detected in chemoresistant and metastatic EOC cell lines by immunofluorescence (IF) and Western blot (WB). The expression of CHTOP in human EOC tissues was examined using immunohistochemistry (IHC). The effect of CHTOP knockdown (KD) on metastasis was examined using the Transwell® matrigel invasion and wound healing assays. Flow cytometry and TUNEL assay were employed to determine the association of CHTOP with apoptosis, while mammary sphere formation assay and IF were used to evaluate its regulation on EOC-cis cell stemness.

Results: The higher expression of CHTOP was found in EOC-cis (A2780-cis and IGROV-1-cis) and metastatic EOC (SKOV-3 and OV-90) cells as compared to normal epithelial ovarian cells (HOSE) by IF and WB. Also, high expression of CHTOP was found in human EOC tissues and associated with poor prognosis in patients. In contrast, CHTOP KD significantly reduced the metastatic potential of EOC-cis cells and increased their apoptosis at the presence of cisplatin. Furthermore, CHTOP KD decreased the stemness of EOC-cis cells.

Conclusion: Our findings suggest that CHTOP is associated with metastasis, apoptosis, and stemness in chemoresistant EOC cells, the survival in EOC patients, and might be a promising therapeutic target to overcome chemoresistance in EOC treatment.

Keywords: ovarian cancer, chemoresistance, CHTOP, metastasis, apoptosis, stemness

#4755

Clathrin mediated endocytosis as a potential therapeutic strategy for overcoming refractoriness of EGFR TKI.

Boyeon Kim, Young Soo Park, Jae Sook Sung, Jong Won Lee, Saet Byeol Lee, Yeul Hong Kim. _Korea university, Seoul, Republic of Korea_.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) have significantly improved on clinical outcomes in many non-small cell lung cancer (NSCLC) patients. However, therapeutic efficacy of EGFR TKIs is limited on patients who have gefitinib-sensitizing EGFR gene mutations (Exon 19del, L858R). Although, not as sensitive as patients who have these mutations, some patients who have wild type EGFR responded to EGFR TKIs. Therefore, we hypothesized that there are underlying mechanisms that cause most of wild type EGFR NSCLC patients to be refractory. In this study, we demonstrated that intractability of EGFR TKIs in wild type EGFR NSCLC was closely related to clathrin mediated endocytosis (CME), which offered a novel strategy for overcoming EGFR TKI resistance. At first, we classified wild type EGFR NSCLC cell lines into gefitinib sensitive cells and refractory cells according to the IC50 values of gefitinib. Then, we performed immunocytochemistry (ICC) and immunoprecipitation (IP) to examine EGFR levels in early endosome with gefitinib treatment in gefitinib sensitive and refractory cell lines. As a result, EGFR endocytosis proceeded in gefitinib refractory cell lines (SNU1327, H1703) despite of gefitinib treatment, but not in sensitive cell lines (H358, Calu-3). EGFR is known to be internalized through CME and non clathrin mediated pathway (NCE). Then, we treated CME inhibitor, Phenylarsine oxide (PAO), or NCE inhibitor, Filipin III, in gefitinib sensitive and refractory cell lines to vefity if there is any difference in dependency of two main endocytosis pathway. As the ICC and IP data shown, blocking NCE did not inhibit EGFR internalization but blocking CME inhibited EGFR endocytosis in gefitinib refractory cell lines. Interestingly, it was the opposite in the gefitinib sensitive cell lines. Furthermore, we observed that cell survival decreased significantly during CME inhibition and combination treatment with gefitinib in refractory cell lines, but not in sensitive cell lines. To enlighten the elusive pathway, we examined the ratio of EGFR recycling and degradation. As a result, we observed EGFR degradation during CME or NCE proceeded conditions in gefitinib sensitive cell lines. However, in gefitinib refractory cell lines, EGFR protein was accumulated in CME dependent manner. These results implied that CME led EGFR recycling and sustained gefitinib refractory cell lines. For further studies, we are confirming signal molecules activated through CME. In conclusion, our study provides the possibility of new scheme that clathrin-mediated endocytosis inhibition could be a potential therapeutic strategy for the limitation of EGFR TKI therapy. This study was supported by a Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2013R1A1A2057538).

#4756

Activation of AURKA contributes to TKI resistance in EGFR-mutant non-small lung cancer.

Meng-Hsuan Lee,1 Shiao-Ya Hong,2 Yu-Rung Kao,2 Cheng-Wen Wu1. 1 _Institute of Microbiology and Immunology, National Yang-Ming University, Taipei City, Taiwan;_ 2 _Institute of Biomedical Sciences, Academia Sinica, Taipei City, Taiwan_.

The epithelial growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been widely used to treat EGFR-mutant non-small cell lung cancer (NSCLC) in clinics. Several generations of TKI were invented to overcome the unfavorable response and acquired resistance after long-term usage of EGFR TKI. Several mechanisms of acquired resistance have been reported. The well-studied resistant mechanism results from EGFR secondary mutations including T790M and C797S, which are involved in the resistance to first and third generation TKI, respectively. Other mechanisms aside from secondary mutation on EGFR including Her2 and MET amplification or epithelial mesenchymal transition (EMT) also distribute to TKI resistance. However, the resistant mechanisms which are independent to these alterations have not been completely identified yet. Here, we generated resistant cells selected by three different generations of TKI and analyzed their gene expression profiles. We discovered that many signal pathways, especially involved in cell cycle regulation, are commonly upregulated in three TKI-resistant cell lines. Aurora kinase A (AURKA) was the upstream kinase of those pathways; however, neither RNA level nor protein level of AURKA was significant changed. Interestingly, we found that phosphorylation of Aurora A at Thr288 in its catalytic domain is higher in TKI resistant cells. This reveals the possibilities that AURKA activation plays a role in the occurrence of TKI-resistance and that TKI-resistant NSCLC patients have a better response rate to AURKA inhibitor treatments.

#4757

Targeting the mevalonate pathway in HER2+breast cancer to overcome resistance and enhance anti-HER2 therapy efficacy.

Vidyalakshmi Sethunath, Huizhong Hu, Carmine DeAngelis, Jamunarani Veeraraghavan, Lanfang Qin, Martin Shea, Tamika Mitchell, Sarmistha Nanda, Resel Pereira, Susan G. Hilsenbeck, Mothaffar F. Rimawi, Kent C. Osborne, Rachel Schiff. _Baylor College of Medicine, Houston, TX_.

Background: Despite the advent of HER2-targeted therapies, including the monoclonal antibody trastuzumab (T) and the HER1/2 inhibitor lapatinib (L), for HER2+ breast cancer (BC), resistance still poses a major challenge. L and L+T resistant (R) HER2+ cells with inhibited HER2 signaling showed upregulation of RNA levels of the mevalonate pathway (MVA) enzymes (which were inhibited by short term treatment of parental (P) cells with L or L+T) and increased sensitivity to MVA inhibition by statins (e.g. simvastatin (Sim)), suggesting MVA's role as an escape mechanism of resistance. Here we investigated the therapeutic potential of another MVA inhibitor Zoledronate (ZA) and the role of mTOR and YAP/TAZ (Y/T) in mediating this resistance. Lastly, we tested if co-blockade of the MVA or its downstream effectors further sensitizes HER2+ models to anti-HER2 therapies.

Methods: SKBR3 and AU565 P cells and their LR and LTR derivatives were used. The effects of MVA perturbations on cell growth and HER2 or MVA signaling were studied by methylene blue staining and Western blot (WB), respectively. Y/T activity was tested by a luciferase reporter assay and functionally validated by siRNA knockdown and dominant-active (DomA) YAP overexpression. YAP target gene expression was assessed by RT PCR. SCID-Beige mice bearing HER2+ BCM-3963 PDX tumors were treated with vehicle, L, Sim, or L+Sim and monitored for time to complete response (CR).

Results: ZA, like Sim, showed a selective inhibition of cell growth and mTOR signaling in R vs. P cells, which was rescued only by the downstream metabolite geranyl geranyl pyrophosphate (GGPP), but not by the upstream metabolite mevalonate, indicating the on-target effect of ZA. Increased Y/T activity in R models was confirmed, and both Sim and ZA inhibited TAZ levels and induced phospho-YAP levels, which were rescued by the corresponding downstream metabolites. Y/T knockdown inhibited growth and mTOR signaling in R vs. P cells, and DomA YAP negated the mTOR inhibition by Sim. Sim and ZA also significantly decreased levels of the Y/T target gene survivin in R vs. P cells, and the expression was rescued by the downstream metabolites. Inhibition of MVA by Sim or ZA or its downstream signaling effectors, Y/T (by siRNA) and mTOR (by everolimus), enhanced the L sensitivity in P cells. Conversely, DomA YAP reduced the sensitivity of P cells to L. In the presence of Sim or ZA, L treatment more strongly inhibited levels of phospho-S6, a downstream target of mTORC1, compared to L alone. Preliminary in vivo data showed that treatment with L+Sim vs. L alone shortened the median time to CR and numerically increased CR rates.

Conclusions: The MVA pathway mediates anti-HER2 therapy resistance via Y/T, survivin, and mTOR, in some cell models and this resistance can be overcome by Sim and ZA. The potential of MVA pathway inhibition to enhance anti-HER2 therapy efficacy warrants further clinical studies.

#4758

Tumor-stroma interactions as a determinant of drug resistance in BRAF-Mut melanoma.

Rossella Loria,1 Italia Falcone,1 Ursula Cesta Incani,1 Ludovica Ciuffreda,1 Chiara Bazzichetto,1 Fabiana Conciatori,1 Barbara Bellei,2 Marta Di Martile,1 Valentina Laquintanta,1 Donatella Del Bufalo,1 Mauro Picardo,2 Francesco Cognetti,1 Michele Milella,1 Rita Falcioni1. 1 _Regina Elena National Cancer Institute, Rome, Italy;_ 2 _San Gallicano Dermatological Institute, Rome, Italy_.

Background: In BRAF-mut melanoma combined BRAF/MEK inhibition increases survival; however, pharmacological effects on the genetically "normal" tumor microenvironment (i.e. paradox MAPK activation) may set the stage for the development of drug resistance.

Methods: To assess the functional relevance of AXL and Sema6A expression, in the regulation of melanoma/stroma interactions and sensitivity/resistance to pathway inhibitors, we silenced or overexpressed the two proteins by genetic manipulation, and the response of melanoma models to BRAF/MEK inhibitors (alone and combined) has been evaluated in 2D co-culture systems.

Results: Sema6A and AXL expression in a panel of genetically characterized melanoma cell lines, short-term primary melanoma cultures and patient-derived melanoma-initiating cells, were inversely correlated. Furthermore, we also observed an inverse correlation when we overexpressed or knocked down Sema6A and AXL expression in different BRAF-mut melanoma cell lines, by transient/stable transfection of constitutively active gene constructs or RNA interference, as appropriate. Previously, we discovered that HFF significantly protected BRAF-mut M14 melanoma cells from the growth inhibitory activity of BRAF/MEK inhibitors (dabrafenib and trametinib), alone or combined, by "direct cell-cell contact". To ascribe a functional role to Sema6A and AXL in tumor-protective melanoma/stroma interactions, correlative co-culture experiments have been conducted using melanoma cells characterized for high or low/undetectable expression of the protein of interest. In this context, Sema6A silencing, performed in BRAF-mut 2/59 melanoma cells, and/or AXL overexpression, performed in BRAF-mut M14 melanoma cells, abrogated the protective effect derived from melanoma/stroma interactions; viceversa after AXL silencing BRAF-mut SKMEL24 melanoma cells became more resistant to BRAF/MEK inhibitors than control cells.

Conclusions and future perspectives: Our data suggest that tumor-stroma interactions protect BRAF-mut melanoma from MAPK inhibition. Sema6A and AXL are mediators of this interaction and their reciprocal relationships are being further studied in melanoma cell line models. The expression of both molecules is under evaluation on clinical specimens.

#4759

Antitumor activity of eribulin after fulvestrant plus palbociclib treatments in the HR(+) human breast PDx model.

Makoto Asano, Takayuki Nakagawa, Taro Semba, Akira Yokoi, Junji Matsui, Yasuhiro Funahashi. _Eisai Co.,Ltd., Tsukuba, Ibaraki, Japan_.

Fulvestrant (FLV) and Palbociclib (Palb), which have an effect on degradation of estrogen receptor (ER) and inhibition of cyclin dependent kinases 4 and 6 (CDK4/6), respectively are used for treatments to postmenopausal breast cancer patients. However, there is not a clinical rationale to define a standard treatment regimen against breast cancer patients who are resistant to endocrine therapy plus Palb. Eribulin is a fully synthetic analog of the marine sponge natural product halichondrin B. Its clinical formulation is currently approved for advanced breast cancer (BC) and advanced liposarcoma in numerous countries. We examined the antitumor activity of eribulin after endocrine therapy (combination of FLV and Palb) compared with that of Capecitabine (Cape) in the luminal type breast cancer PDx model (OD-BRE-0438). OD-BRE-0438 is a patient derived xenograft (PDx) model developed from patient with luminal B breast cancer. Gene expression analysis by RNA-Seq showed that OD-BRE-0438 highly expressed ER and PR, but not ERBB2. OD-BRE-0438 tumor bearing mice were treated for 2 weeks of FLV (5mg/ head, Q7Dx2, sc) and Palb (10mg/kg, Q1Dx14, po), and treated mice were randomly divided using tumor volume around 350 mm3 as an index (day1). After that, mice were treated with eribulin (0.5 or 1 mg/kg, which are 1/4 MTD or 1/2 MTD in this model, Q7Dx2, iv) or Cape (125 or 250 mg/kg, which are 1/2 MTD or MTD in this model, Q1Dx14, po for 2 weeks). We measured tumor volume of each mouse after treatments (day 15) and analyzed the ratio of tumor volume at day 15 divided by that at day 1 (RTV: relative tumor volume). Treatments of FLV and Palb nearly completely inhibited in vivo growth of OD-BRE-0438 tumors, but didn't cause tumor shrinkage. After two weeks of combination treatment with endocrine therapy, we started treatments with either eribulin or Cape, and compared the antitumor activity. Both drug showed strong antitumor activity with tumor shrinkage. In comparison of antitumor activity with either eribulin or Cape, that of eribulin was significantly greater than Cape. [eribulin 0.5mg/kg (RTV=0.42) vs Cape 125mg/kg (RTV=0.62, p=0.0068), eribulin 1.0mg/kg (RTV=0.32) vs Cape 250mg/kg (RTV=0.59, p=0.0008)]. Further investigation for mechanism of action of eribulin to show clear antitumor activity after treatments of FLV and Palb in the luminal type breast cancer PDx model are warranted.

#4760

RB1 loss accelerates reprogramming of prostate adenocarcinoma to small cell through ASCL1.

Shaghayegh Nouruzi, Daksh Thaper, Soojin Kim, Alastair Davies, Amina Zoubeidi. _Vancouver Prostate Centre, University of British Columbia, Vancouver, British Columbia, Canada_.

Introduction: Resistance to androgen receptor pathway inhibitors (ARPIs), such as Enzalutamide (ENZ), rapidly emerges as they play a role in emergence of a more aggressive, non-AR-dependent phenotype known as treatment induced neuroendocrine prostate cancer (t-NEPC). t-NEPC carries an extremely poor prognosis and treatment remains cytotoxic chemotherapy. Hence, identifying key drivers and developing targeted treatments for this deadly disease are desperately needed.

Previous studies had shown that RB1 is altered in up to ~63% of metastatic castrated resistance prostate cancer (CRPC) patients and loss of RB1 function is associated with poor clinical outcome. Approximately 70% of NEPC patients have homozygous deletion of RB1, suggesting a selection for RB1 aberrations in NEPC patients. Although RB1 loss alone is not sufficient to induce transdifferentiation to NEPC we propose that reduction in RB1 activity/expression may pre-dispose patients to go down the path of t-NEPC under pressure of ARPIs and by facilitating lineage plasticity contribute to ASCL1 induced reprograming of CRPC to NEPC.

Methods: We used shRB1 in our LNCaP derived CRPC cell line (16DCRPC) compared to our in vivo derived NEPC like cell lines 42DENZR and 42FENZR which display RB1 loss signature due to hyper-phosphorylation.

Results: Knockdown of RB1 causes minimal up-regulation in the basal levels of NEPC genes ASCL1 and ENO2 and stem cell genes NANOG, SOX2 and OCT4. Interestingly, while overall EZH2 expression wasn't changed, there was an increase in phosphorylation of the T350 residue on EZH2 with a significant increase in H3K27me3. Moreover, treatment with ENZ drastically accelerated the re-programming process in shRB1 cells with up-regulation of a known driver/marker small cell cancers transcription factor ASCL1. RNA-seq data showed that ASCL1 expression is higher in NEPC patients and CRPC patients with low serum PSA, underscoring its inverse correlation with classic AR activity in clinical samples and re-activation of AR using synthetic androgens (R1881) decreased ASCL1 expression in vitro. Furthermore, targeting AR using ENZ induces expression of ASCL1 in concordance with induction of NE markers such as ChgA, SYP, NCAM and SOX2. Moreover, overexpression of ASCL1 alone induced NE differentiation and targeting ASCL1 using siRNA decreases proliferation and NEPC marker expression in human NEPC cell line, NCI-H660.

Conclusion: This study focuses on delineating the molecular mechanisms behind RB1 loss and the transformation to t-NEPC or t-SCPC observed in patients upon treatment with potent ARPIs. We have discovered that downstream of RB1 loss, several molecular events such as differential activation of EZH2 and up-regulation of transcription factor ASCL1 play a major role in the NE differentiation. Thus, targeting these proteins presents may be a treatment strategy for patients with t-NEPC or t-SCPC.

#4761

Transcriptomic overview of EML4-ALK lung cancer cells resistant to ALK inhibitors highlights a vulnerability to CDK inhibitors.

Athanasios R. Paliouras,1 Peter Magee,1 Sudhakar Sahoo,1 Hui Sun Leong,1 Matthew Krebs,2 Fiona Blackhall,2 Christine M. Lovly,3 Michela Garofalo1. 1 _CRUK MI, University of Manchester, Manchester, United Kingdom;_ 2 _University of Manchester, Christie Hospital, Manchester, United Kingdom;_ 3 _Vanderbilt University Medical Center, Nashville, TN_.

Introduction: EML4-ALK-driven lung cancer represents about 5% of lung adenocarcinomas. Despite the constant expansion of the armamentarium to inhibit ALK, patients typically develop acquired resistance to ALK inhibitors. In the case of resistance mediated by ALK mutations, sequencing of ALK inhibitors is recommended, but in ALK wild-type patients, a better understanding of acquired resistance, as well as new therapeutic strategies are needed. Here, we aimed to characterize the transcriptional network of EML4-ALK lung cancer and used these data to address acquired resistance to ALK inhibitors.

Methods: We utilized EML4-ALK cell lines treated long-term with ALK inhibitors as a model of acquired resistance. Transcriptomic changes were quantified through means of RNA-seq or RT-qPCR.

Results: We have found a dysregulation of several microRNAs in crizotinib-resistant cells. Specifically, the oncogenic miR-25 and miR-30c were upregulated and their inhibition resulted in cell cycle arrest. We analyzed plasma samples from patients with EML4-ALK lung cancer and found increased circulating levels of these miRNAs in 2 patients upon progression with crizotinib. In addition, the known tumour-suppressing miRNAs miR-103a and miR-149 were downregulated in crizotinib-resistant cells and restoration of miR-149 resulted in apoptotic induction. We show that CDK6 is a direct target of miR-103 and an indirect target of miR-149. Moreover, by comparing the transcriptome of parental and crizotinib-resistant cells we uncovered an upregulation of genes implicated in cell cycle. Treatment with Cyclin-Dependent Kinase (CDK) inhibitors led to suppression of cell proliferation and potent induction of apoptosis. Moreover, these inhibitors spared normal epithelial cells while they induced higher levels of apoptosis in EML4-ALK cells compared with other NSCLC cell lines, suggesting a preferential targeting of EML4-ALK cells .Lastly, a pilot xenograft experiment with a crizotinib-resistant cell line showed in vivo anti-tumor activity of CDK inhibition and we are currently expanding this in a larger mouse cohort.

Conclusions: Crizotinib-resistant cells may exhibit several concurrent oncogenic alterations including miRNA- or coding gene-dysregulation, in the absence of ALK mutations. Moreover, the contribution of these alterations to acquired resistance cannot be easily and quickly assessed. A therapeutic intervention which does not rely on identifying the driver of resistance would be of high practical value. We suggest that CDK inhibition may be a new avenue to target relapsed EML4-ALK lung cancer refractory to ALK inhibitors.

#4762

Activation of AXL as a preclinical acquired resistance mechanism against osimertinib treatment in EGFR-mutant non-small cell lung cancer cells.

Kei Namba,1 Kazuhiko Shien,1 Yuta Takahashi,1 Hiroki Sato,2 Takahiro Yoshioka,1 Ken Suzawa,1 Hiromasa Yamamoto,1 Junichi Soh,1 Shuta Tomida,3 Shinichi Toyooka1. 1 _Okayama Univ. Graduate School of Medicine, Okayama, Japan;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 3 _Okayama Univ. Hospital, Okayama, Japan_.

Osimertinib (AZD9291) has an efficacy superior to that of standard EGFR-tyrosine kinase inhibitors for the first-line treatment of EGFR-mutant advanced non-small cell lung cancer (NSCLC) patients. However, patients treated with osimertinib eventually acquire drug resistance, and novel therapeutic strategies to overcome acquired resistance are needed. In clinical or preclinical models, several mechanisms of acquired resistance to osimertinib have been elucidated. However, the acquired resistance mechanisms when osimertinib is initially used for EGFR-mutant NSCLC remain unclear. In this study, we experimentally established acquired osimertinib-resistance cell lines from EGFR-mutant NSCLC cell lines and investigated the molecular profiles of resistant cells to uncover the mechanisms of acquired resistance. Various resistance mechanisms were identified, including the acquisition of MET amplification, EMT induction, and the upregulation of AXL. Using targeted next-generation sequencing with a multi-gene panel, no secondary mutations were detected in our resistant cell lines. Among three MET-amplified cell lines, one cell line was sensitive to a combination of osimertinib and crizotinib. Acquired resistance cell lines derived from H1975 harboring the T790M mutation showed AXL upregulation, and the cell growth of these cell lines was suppressed by a combination of osimertinib and cabozantinib, an inhibitor of multiple tyrosine kinases including AXL, both in vitro and in vivo. Our results suggest that AXL might be a therapeutic target for overcoming acquired resistance to osimertinib.

#4763

Genome-wide CRISPR screening identifies TSC1 as a regulator of sorafenib resistance in acute myeloid leukemia.

Alisa Damnernsawad, Tamilla Nechiporuk, Steve E. Kurtz, Wesley R. Horton, Olga Nikolova, Shannon K. McWeeney, Jeffrey W. Tyner. _OHSU, Portland, OR_.

Hematologic malignancies represent the fifth most common type of cancer and fourth most common cause of cancer-related deaths. These malignancies are characterized by a clonal expansion of hematopoietic progenitor cells that proliferate in the bone marrow, peripheral blood, lymph nodes and other organs. Genetic analyses of these diseases have led to the identification of specific genetic lesions that drive leukemogenesis, which has subsequently led to the development of targeted drugs. Acute myeloid leukemia (AML) is a fast progressing blood malignancy with impaired differentiation and proliferation of myeloid precursors. It is one of the most common leukemias in adults. AML is known for its molecular and biological heterogeneity, and a variety of genetic lesions have been implicated in the disease. FMS-like tyrosine kinase 3 (FLT3) mutations including FLT3 internal tandem duplication (ITD) or point mutations in the tyrosine kinase domain (TKD) are found in around 30% of AML patients.

Sorafenib, a multi-kinase inhibitor that targets FLT3, RAF, VEGFR, FGFR, KIT and RET, is approved for use in hepatocarcinoma, renal cell carcinoma, and thyroid carcinoma treatments. Addition of sorafenib to standard treatment prolonged AML patient survival with or without FMS- like tyrosine kinase 3 (FLT3) mutations, although relapse caused by drug-resistance has limited its usefulness. Understanding the mechanism of resistance to targeted drugs, therefore, is necessary to improve treatment regimens for AML patients. We aim to elucidate resistance mechanisms to sorafenib in AML cells using genome-wide CRISPR screening. Two genome-scale human CRISPR knockout (KO) libraries were used to identify genes whose loss-of-function contribute to lower sensitivity to sorafenib in the MOLM13 AML cells. We verified 10 genes from the top hits showing consistency between the two CRISPR libraries, one of which was Tuberous Sclerosis 1 (TSC1). To validate these findings, TSC1 KO cells were generated using lentiCRISPRv2 system and single sgRNA sequence. Drug sensitivity assays confirmed increase in sorafenib resistance of TSC1 KO cells compared to parental cells or cells harboring non-targeting sgRNA. In addition to the sorafenib resistant phenotype, TSC1 KO cells were resistant to a panel of FLT3 inhibitors, quizartinib, crenolanib, gilteritinib, and UNC2025A. Moreover, RNA sequencing results from 271 AML patient peripheral blood or bone marrow samples revealed that lower RNA expression of Tuberous Sclerosis 1 (TSC1) correlated with lower sensitivity to sorafenib. TSC2 was also observed as a hit gene in both CRISPR libraries and lower TSC2 RNA levels also correlated with lower sensitivity to sorafenib, emphasizing TSC1/2 as an important pathway in sorafenib resistance.

#4764

A global transcriptome analysis of pancreatic cancer cells distinguishes between acute and acquired PARP inhibitor resistance mechanisms.

Aditi Jain,1 Matthew McCoy,2 Lebaron A. Agostini,1 Yuriy Gusev,2 Subha Madhavan,2 Michael Pishvaian,2 Sankar Addya,1 Eric Londin,1 Maria R. Gurevich,3 Chani Stossel,3 Talia Golan,3 Charles J. Yeo,1 Jonathan R. Brody1. 1 _Thomas Jefferson University, Philadelphia, PA;_ 2 _Georgetown University, Washington, DC;_ 3 _Tel Aviv University, Israel_.

Pancreatic ductal adenocarcinoma (PDAC) is the 3rd leading cause of cancer related deaths in the U.S. Recent advances in understanding RNA biology in PDAC have shed light on post-transcriptional regulation of genes and pathways through RNA binding proteins (RBP). Our lab has demonstrated that HuR, an RBP, is overexpressed in PDAC cells and stabilizes pro-survival mRNAs. Additionally, our work and others have demonstrated that this level of gene regulation can support drug resistance in PDAC cells. A synthetic lethal strategy employing Poly-ADP ribose polymerase inhibitors (PARPi) in a subset of patients with DNA repair deficient pancreatic cancers has been gaining interest. However, the success of PARPi is often hindered by the emergence of drug resistance in patients who initially respond. We have published that short-term PARPi treatment of PDAC cells causes activation of HuR where it stabilizes a DNA repair enzyme, PAR-glycohydrolase, and mediates acute PARPi resistance. In this study, we generated olaparib acquired resistant pancreatic cancer cells in vitro and acquired pancreatic patient derived xenograft cell lines (pre- and post PARPi) to understand acute versus acquired resistant mechanism(s). In characterising the acquired resistant model of PARPi resistance, we found that these cells are >20 fold more resistant to olaparib and platinums and >5 fold resistant to other PARPi like rucaparib and veliparib, compared to parental cells. No cross resistance was seen with other chemotherapeutics like gemcitabine. Additionally, we also found acquired resistant cells lost PARP-1 protein expression compared to parental cells. Bioinformatic analyses on HuR-RNA immunoprecipitation-microarray (RIP-microarray) data from acutely treated olaparib cells show enrichment of pro-survival mRNAs. Interestingly, these mRNAs are significantly downregulated in acquired resistant cells compared to control cells (i.e., negative log2 fold changes, p<0.001) in differential expression of HuR and HuR established targets. Interestingly, upregulated gene transcripts in these samples belong to pathways that negatively regulate biosynthetic and metabolic processes, and hence may represent pathways to target. Further, in vitro analyses show that parental PDAC cells are sensitive to combined inhibition of PARP and HuR but acquired resistant cells fail to respond to HuR inhibition. In conclusion, HuR mediates acute resistance to PARPi in PDAC cells and HuR inhibitor therapy could enhance PARPi therapy immediately, yet is most likely not useful in the setting of acquired- resistance. Future studies will explore genetic alterations and novel HuR-independent pathways in PARPi acquired resistant cells. Finally, we have begun a line of investigation of combining PARPi therapy with HuR inhibitors in an effort to optimize upfront therapeutic efficacy

#4765

MERTK is a potential therapeutic target in osimertinib-resistant non-small cell lung cancer.

Dan Yan,1 Rebecca Parker,1 Xiaodong Wang,2 Stephen V. Frye,2 H. Shelton Earp,3 Deborah DeRyckere,1 Douglas Graham1. 1 _Aflac Cancer and Blood Disorders Center, Children's Healthcare of Atlanta, Atlanta, GA;_ 2 _Center for Integrative Chemical Biology and Drug Discovery, Chapel Hill, NC;_ 3 _School of Medicine, Chapel Hill, NC_.

Non-small cell lung cancer (NSCLC) remains a global problem causing more deaths in both men and women than any other cancer worldwide with an urgent need for more efficacious treatments. During the last decade the therapeutic landscape for NSCLC has been profoundly changed with the discovery of activating mutations in Epidermal Growth Factor Receptor (EGFR) (EGFRMT). Osimertinib, a 3rd generation EGFR TKI, was recently FDA-approved as a front-line agent for newly diagnosed EGFRMT NSCLCs due to its superior efficacy relative to earlier-generation EGFR TKIs in the international FLAURA trial. However, unmet clinical needs have arisen in conjunction with osimertinib use, including understanding mechanisms of osimertinib resistance and developing novel approaches to prevent or reverse resistance and/or enhance osimertinib efficacy in responsive patients. Our group previously identified MERTK receptor tyrosine kinase as a potential therapeutic target in NSCLC and developed MRX-2843, a novel small molecule inhibitor with dual MERTK and FLT3 activity, which is currently in Phase I clinical trials. Here, we report upregulation of MERTK and its ligand PROS1 in xenograft tumors derived from the EGFRMT H4006 NSCLC cell line and treated with osimertinib relative to vehicle-treated tumors. MERTK expression was also increased in osimertinib-resistant derivatives of the H4006 and H4011 cell lines generated by culture in escalating doses of osimertinib. However, overexpression of exogenous MERTK in H4006 cells was not sufficient to confer osimertinib resistance. In contrast, stimulation with TAM kinase ligands GAS6 or PROS1 protected H4006 cells from osimertinib treatment, as indicated by restoration of AKT, ERK, and ribosomal S6 phosphorylation in the presence of osimertinib. Together these data implicate MERTK as a mediator of resistance to osimertinib. Indeed, combined treatment with osimertinib and MRX-2843 effectively blocked PI3K-AKT and MAPK-ERK signaling and mediated synergistic inhibition of colony formation in osimertinib-resistant H4006 cells. Thus, MERTK inhibition may be an effective therapeutic strategy to re-sensitize osimertinib-resistant NSCLCs to EGFR TKI treatment.

#4766

Transcriptomic profiling reveals a potential role for JAK/STAT inhibition in CDK4/6 inhibitor-resistant, ER+ breast cancers.

Andrea M. Pesch, Thomas L. Gonzalez, Benjamin C. Chandler, Siqi Sun, Christina L. Gersch, José M. Larios, Wadie S. David, Corey W. Speers, James M. Rae. _University of Michigan, Ann Arbor, MI_.

Purpose: Specific cyclin-dependent kinase (CDK) inhibitors are standard of care for patients with metastatic, estrogen receptor-positive (ER+) breast cancer. CDK4/6 inhibitors have improved rates of progression free survival among metastatic, ER+ patients, but resistance limits their clinical efficacy. Various mechanisms of resistance to CDK4/6 inhibitors have been reported, but a comprehensive understanding of this resistance remains elusive.

Methods: We generated in vitro models of acquired (AR) and intrinsic (IR) resistance to CDK4/6 inhibitors using ER+ breast cancer cell lines (MCF-7, T47D) cultured with either continuous high dose (500nM) or dose-escalated (50nM to 500nM) CDK4/6 inhibition over three months. RNA expression and gene set enrichment analysis (GSEA) was used to nominate potential pathways associated with AR and IR palbociclib resistance. Reverse phase protein array (RPPA) and western blots were used to measure protein and phosphoprotein levels in CDK4/6 inhibitor resistant cell lines to validate nominated pathways. Cellular proliferation assays were performed to calculate the half-maximal inhibitory concentration (IC50) with inhibitors for CDK4/6 and JAK/STAT.

Results: Proliferation assays confirmed that MCF-7 AR and IR cells are resistant to palbociclib (IC50 both >1uM) compared to parental cells (60nM); similar results were observed in the T47D cell lines. Cells resistant to either palbociclib, ribociclib, or abemaciclib demonstrated cross resistance to all three inhibitors. GSEA of transcriptomic data identified 579 genes (from AR cells) and 936 genes (from IR cells) that were differentially expressed between palbociclib-resistant MCF-7s and parental controls. RPPA analyses identified several key pathways that regulate CDK4/6 inhibitor resistance in these models. From GSEA analysis, the interferon (JAK/STAT) signaling pathway was the most differentially expressed pathway identified between palbociclib-resistant and sensitive cells. Western blot analyses showed that baseline expression of phospho-STAT1 is significantly elevated in palbociclib-resistant cells. In cellular proliferation assays, palbociclib-resistant MCF-7s and T47Ds retained sensitivity to JAK/STAT inhibitors like the JAK2-selective compound AZ960.

Conclusions: Our data suggests that overactivation of JAK/STAT signaling may be directly involved in the development of CDK4/6 inhibitor resistance in ER-dependent tumors. CDK4/6 inhibitor-resistant cells retain sensitivity to single-agent JAK/STAT inhibition, suggesting that this may be a viable therapeutic option for patients with CDK4/6 inhibitor-resistant ER+ breast cancer. This work was supported in part by 5T32GM007767-40 (Pesch), the Breast Cancer Research Foundation (N003173 to JMR), the UM Rogel Cancer Center and the Taubman Emerging Scholar funds.

#4767

Distinct resistance profiles of midostaurin and avapritinib in exon 17-mutant KIT.

Beth Apsel Winger, Wilian A. Cortopassi, Diego Garrido Ruiz, Lucky Ding, Na Zhang, Rosaura Esteve-Puig, Ariel Leyte-Vidal, Matthew P. Jacobson, Neil P. Shah. _University of California San Francisco, San Francisco, CA_.

The first several KIT tyrosine kinase inhibitors (TKIs) were ineffective against KIT exon 17 mutations, which favor an active conformation that prevents these TKIs from binding and predominate in several malignancies. The ATP-competitive inhibitors midostaurin and avapritinib, which target the active kinase conformation, were developed to inhibit exon 17-mutant KIT. Because secondary kinase domain mutations are a common mechanism of TKI resistance and guide ensuing TKI design, we sought to define problematic KIT kinase domain mutations for these emerging therapeutics. We demonstrate midostaurin and avapritinib display different vulnerabilities to secondary kinase domain substitutions, and the T670I gatekeeper mutation is selectively problematic for avapritinib. Though gatekeeper mutations often directly disrupt inhibitor binding, our studies unexpectedly suggest T670I confers avapritinib resistance indirectly by inducing distant conformational changes in the phosphate-binding loop. Our findings further suggest combining midostaurin and avapritinib may forestall acquired resistance mediated by secondary kinase domain mutations.

#4768

PARP inhibitor-induced autophagy provides an adaptive mechanism of drug resistance in preclinical models of ovarian cancer.

Janice M. Santiago-O'Farrill,1 Saravut J. Weroha,2 Xiaonan Hou,2 Lan Pang,1 Philip Rask,1 Zhen Lu,1 Robert C. Bast1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _Mayo Clinic, Rochester, MN_.

Poly (ADP) ribose polymerase inhibitors (PARPi) have shown promising activity against recurrent ovarian cancers, particularly in women with germ line mutations of BRCA1/2. The efficacy of PARP inhibitors can, however, be decreased by acquired drug resistance. Autophagy or "self-eating" is one mechanism implicated in cancer progression and therapy resistance. Autophagy is a catabolic mechanism that can degrade organelles and long-lived proteins to provide energy for cancer cells under nutrient poor conditions or in the presence of stress. The energy provided from autophagy is used to fuel the DNA repair process and metabolic needs in cancer cells. Autophagy can protect cancer cells from chemotherapy or can enhance the response to certain drugs. In this study, we have asked whether autophagy contributes to the resistance of ovarian cancer cells to PARPi. Our group has found that four PARP inhibitors (olaparib, niraparib, rucaparib and talazorapib) induce autophagy in 8 ovarian cancer cell lines, detected by LC3A to LC3B conversion on western blot analysis and punctate GFP-LC3 fluorescence. Transmission electron microscopy confirmed the increased autophagosome formation in olaparib treated cells. To test whether autophagy contributes to the resistance of OC cells to olaparib, we determined the impact of autophagy inhibition on the sensitivity of OC cell lines to olaparib by either using chloroquine, hydroxychloroquine or LYS05 to block hydrolysis of proteins and lipids in autophagosomes or using siRNA against ATG5 or ATG7 to prevent formation of autophagosome. Enhancement of PARPi activity was observed in 6 out 8 ovarian cancer cell lines. Moreover, a combination of olaparib and chloroquine proved more effective than either single agent in a xenograft model of the OVCAR8 ovarian cancer cell line and in a patient derived xenograft model. Mechanistically, olaparib decreased PARP activity leading to DNA damage and accumulation of ɣ-H2AX. Olaparib treatment increased reactive oxygen species (ROS) and phosphorylation of ATM, while decreasing the phosphorylation of AKT and mTOR. We also observed an increase in PTEN, a well-recognized negative regulator of AKT. Inhibition of PTEN decreases LC3 conversion, suggesting PTEN as an important regulator of olaparib-induced autophagy in ovarian cancer cells. Taken together, our data demonstrate for the first time that olaparib induces autophagy in ovarian cancer cells and provides an adaptive mechanism of resistance to PARPi. Olaparib-induced autophagy may depend upon activation of ATM and PTEN by ROS, downregulating pAKT/pmTOR signaling. Moreover we have identify unique combinations of therapy with PARPi and CQ or other inhibitors of autophagy that could improve outcomes for ovarian cancer patients.

#4769

Gene expression profiling of response to short- and long-term treatment of LSZ102 in MCF7 cells.

Choi Lai Tiong Yip, Joshua Korn, David Ruddy, Daniel Rakiec, Darrin Stuart, Alex Gaither. _Novartis Institute for Biomedical Research, Cambridge, MA_.

Breast cancer is the most common cancer in women worldwide, and the second most common cause of cancer death in woman in the US. Close to 80% of breast cancer are estrogen receptor (ER) positive and can benefit from antiestrogen treatment. LSZ102 is a novel oral selective estrogen receptor degrader (SERD) in clinical development, which exhibits potent activity in inducing ER degradation, modulating ER-target genes transcription and inhibits the proliferation of breast cancer cells both in-vitro and in-vivo. In this study, we have investigated the effects of short term and prolonged LSZ102 treatment on global gene expression in ER positive MCF-7 breast cancer cells. MCF-7 cells were treated with LSZ102 for 24 hours, 8 weeks and 14 weeks, followed by transcriptome profiling by RNA sequencing. Our findings show LSZ102 effectively inhibited ER transcriptional activity and lead to a sustained suppression of estrogen responsive genes expression in LSZ102-treated cells of all time points. We also observed a cumulative up-regulation of EPAS1, a hypoxia-inducible transcription factor. In addition, we found up-regulation of EGFR and ERBB2 genes and regulated genes in cells with prolonged treatment of LSZ102. In summary, our results suggest a possible role of EPAS1 in eliciting a compensatory growth promoting signals in breast cancer cells adapted to ER inhibition.

### Gene- and Vector-Based Therapy

#4770

Programming tumor-clearing macrophages with targeted in situ gene therapy.

Fan Zhang, Miacheal E. Coon, Neha N. Parayath, Sirrka B. Stephan, Smitha P. Pillai, Matthias T. Stephan. _Fred Hutchinson Cancer Research Institute, Seattle, WA_.

Macrophages (mφs) are key immune effectors that infiltrate into tumor in high numbers. However, within the immunosuppressive tumor milieu, they undergo a switch from an activated tumoricidal (M1-like) state to an immunosuppressive (M2-like) phenotype, which facilitates tumor growth and metastasis. To date, only systemic cytokine blockade using antibodies or small molecule drugs has shown success in this arena; however, because these blockades have low response rate among patients and suppress all mφs in the body, they also induce dangerous side effects.

Instead of ablating mφs through cytokine inhibition, we genetically reprogramed suppressive M2-like mφs in situ into highly effective, tumor-clearing M1-like mφs by delivering genes encoding transcription factors. Specifically, we used nanoparticles to provide mφs with mRNAs encoding master regulators of mφs polarization - IRF5 (IRF5-NPs).

Our in vitro test demonstrated that IRF5-NPs can choreograph immediate, transient, robust IRF5 production, and induce dose-responsive activation of the IRF5 pathway in mφs. To identify the critical gene expression changes associated with NPs treatment, we used genome-sequencing technology to analyze 770 genes in 19 different pathways and processes in myeloid cells. Our results demonstrated that IRF5-NPs treatment on M2-like mφs promoted M1-like signature genes while suppressed M2-like signature genes.

In an ID8 cells-induced syngeneic mouse model of ovarian cancer, repeated intraperitoneal (i.p.) injection of IRF5-NPs regressed tumor growth and significantly increased their median survival from 60 days to 141 days. IRF5-NPs treatment decreased the suppressive mφs population in the peritoneum of ovarian tumor mice and promoted inflammatory mφs/monocytes population. This inflammatory cell population had similar gene profile to M1-like mφs and the ability to produce inflammatory cytokines such as IL-12, TNFα, INFγ. Through biodistribution and toxicity study, we showed that the treatment effect was local and did not impose serious systemic side effects on the healthy mouse. We further showed that in a more aggressive B16F10 lung metastasis mouse model, repeated intravenous (i.v.) injection of IRF5-NPs prolonged the median survival for 30%.

This research is a pioneering effort to treat cancer by genetically reprogramming suppressive M2 mφs. The results of the project could have a transformative effect on cancer therapeutics by providing a foundation for gene-modification systems that would enable physicians to obviate the suppressive tumor milieu.

#4771

The addition of toca 511 and 5-FC to temozolomide improves response in a temozolomide-resistant murine glioblastoma model and correlates with toca 511 dose.

Maria E. Rodriguez-Aguirre, Sophie Viaud, Daniel Mendoza, Tiffany Montellano, Derek G. Ostertag, Harry Gruber, Douglas Jolly, Cornelia Bentley. _Tocagen Inc., San Diego, CA_.

Background: Toca 511 (vocimagene amiretrorepvec), an amphotropic retroviral replicating vector (RRV), can successfully and safely deliver a functional, optimized yeast cytosine deaminase (CD) gene to tumors. Within infected cells, CD converts 5-fluorocytosine (5-FC) to the anti-cancer drug 5-fluorouracil (5-FU). The combination of Toca 511 with oral extended release 5-FC (Toca FC), is currently being evaluated in a randomized phase III clinical trial for recurrent high grade glioma (glioblastoma (GBM) and anaplastic astrocytoma) (NCT02414165, Toca 5). Temozolomide (TMZ), in combination with radiation therapy, is the standard of care used for first-line chemotherapy treatment of patients with GBM, the most common and aggressive form of primary brain cancer. Previously, we have shown that: (1) Toca 511/5-FC treatment provides durable response in a syngeneic murine glioma model and supports anti-tumor immune memory; (2) The combination of TMZ and Toca 511/5-FC had synergistic efficacy in a TMZ-sensitive human glioma nude mouse model; (3) TMZ did not inhibit the efficacy of Toca 511/5-FC in a TMZ resistant murine glioma syngeneic model; (4) Toca 511/5-FC caused significant radiosensitization in a radioresistant murine glioma model.

Results: To assess the interaction of TMZ with escalating doses of Toca 511 (as defined by percent of tumor transduction by RRV), an orthotopic TMZ-resistant murine glioma model, Tu-2449, was utilized. These results show that moderate levels of tumor transduction of Toca 511 (30% - 50%) with 5-FC treatment prolongs survival in the presence of TMZ compared with lower transduction rates (10%). Additionally, mice treated with Toca 511/5-FC and TMZ are being tested for the establishment of anti-tumor immune memory.

Conclusion: These results demonstrate that (1) survival correlates with the transduction levels of Toca 511 when combined with temozolomide in the Tu-2449 orthotopic glioma model and (2) that this combination may support anti-tumor immune memory. These studies along with prior work support evaluation of the combination of Toca 511/5-FC with temozolomide in patients with newly diagnosed GBM (NRG-BN006).

#4772

Evaluation of immune response following ultrasound targeted gene therapy in a murine model of prostate cancer.

Flavia De Carlo, Elliot T. Varney, Pier Paolo Claudio, Candace M. Howard. _University of Mississippi Medical Center, Jackson, MS_.

Human Adenoviruses (hAd) have been broadly used as gene delivery tool in preclinical and clinical studies of cancer gene therapy. The main challenges associated with systemic delivery of hAds are their high immunogenicity and host-specificity. Adenoviruses can elicit strong innate and acquired immune responses that reduce therapeutic gene transfer and expression in malignant sites. Murine cells generally lack some of the receptors necessary for hAd adhesion and internalization making them not permissive to infection and replication and limiting translational studies using murine models. We have developed a gene transfer method, which uses lipid-encapsulated perfluorocarbon microbubbles (MBs) and ultrasound (US) to protect the hAds from the immune system and to deliver them to a targeted tissue, bypassing the necessity for specific receptors. The expression of the Ad-receptors (CAR, Integrins αvβ3 and αvβ5), transduction efficiency of Ad-GFP, and GFP transgene expression were measured in vitro in murine (TRAMP-C2) and human (DU145) prostate cancer cells lines by flow cytometry and immunofluorescence. The activation of the innate and acquired immune system following the injection of Ad-GFP/MBs complexes was evaluated in vivo in C57BL/6 mice healthy or bearing a TRAMP-C2 syngeneic tumor graft. ELISA was used to quantify the inflammatory cytokines TNF-α, IL-6 and measure Neutralizing Antibodies. ELISpot assay was performed to measure the frequency of DBP-specific CD8+ T cells secreting INF-γ. We showed in vitro that murine TRAMP-C2 cells display an expression pattern of Ad-receptors comparable to human DU145 prostate cancer cells. We also demonstrated that both murine and human cells showed a dose dependent increase in the percentage of cells transduced by Ad-GFP at 24 hours. Additionally, we showed that TRAMP-C2 cells efficiently express the GFP transgene at 48 and 72 hours post transduction. To assess in vivo if our gene transfer method could effectively protect the Ads form both the innate and adaptive immunity, we injected C57BL/6 mice with the hAds-GFP/MBs complexes +/-US. Notably we did not observe activation of either innate (secretion of TNF- and IL-6 cytokines) or acquired immunity (neutralizing antibodies and presence of adenovirus-specific CD8+ T cells producing INF-γ). Our data provides evidence that the TRAMP-C2 syngeneic model of prostate cancer is a suitable system to study in immunocompetent mice the capability of the MBs/US to protect the Adenoviruses from the immune system while delivering them to a targeted site bypassing the requirement of specific receptors.

#4773

Development of ONCR-NEP, a lipid nanoparticle delivered oncolytic virus capable of robust in situ amplification resulting in tumor lysis and regression.

Edward M. Kennedy, Jennifer S. Lee, Judy Jacques, Terry Farkaly, Prajna Behara, Peter Grzesik, Brian B. Haines, Agnieszka Denslow, Jacqueline Gursha, Lingxin Kong, Mitchell Finer, Christophe Quéva, Lorena Lerner. _ONCORUS, Cambridge, MA_.

Oncolytic viruses (OV) have shown clinical efficacy for treatment of malignancies when administered intratumorally. Attempts to use OV for systemic treatment of cancer have shown very limited success due to the presence of neutralizing antibodies. Development of an intravenously delivered oncolytic viral therapy that maintains its potency and effectiveness in the presence of neutralizing antibodies is greatly needed for patients with visceral lesions that are metastatic and difficult to inject. Therefore, the OV field is moving toward the design of vector platforms that can be used for systemic therapy to take full advantage of the oncolytic activity and the induction of anti-tumor immunity. Oncorus is developing a new strategy for systemic delivery of OV genomes by using tumor targeted nanoparticles, designed to be immunologically inert systemically, but can initiate a local robust intratumoral oncolytic virus infection.

The Nanoparticle Encapsulated Polynucleotide (NEP) platform is designed for systemic delivery of replication competent OV genomes encoded by a plasmid DNA loaded into tumor-homing nanoparticles. To initiate viral replication intratumorally from a plasmid DNA we engineered a mammalian mRNA cassette that codes for an infectious +sense picornaviral RNA. This RNA is liberated from the larger capped and polyadenylated mammalian transcript by combination of ribozymes and siRNA cleavage, enabling rapid expression of viral proteins and viral replication. We selected Seneca Valley Virus (SVV) as our first candidate virus for the NEP platform. SVV is a potent oncolytic virus in neuroendocrine tumors previously evaluated in clinical trial after IV administration and well tolerated in the patients. The robust initiation of SVV replication from the pDNA expression construct results in cellular lysis in permissive cells when transfected in vitro. In vivo, intratumoral injection of a lipid based formulated SVV plasmid resulted in robust viral replication and dramatic tumor growth inhibition in multiple tumor models. This viral construct has also been modified to express immunomodulatory payloads to enhance the adaptive immune response by recruitment of immune effector cells. Finally, we have enabled miRNA attenuation of his construct to add a genetic tissue selectivity switch for safety, and we have shown that both miR-1 and miR-122 effectively and specifically attenuate viral replication.

To enable ONCR-NEP therapy for intravenously administration, we have begun developing a lipid nanoparticle formulation with an active tumor targeting moiety. We have obtained lipid nanoparticle formulations with favorable physical and functional characteristics that are effective both in vitro and in vivo. We show here for the first time the development of a synthetic virus, a breakthrough therapeutic technology that is highly effective in vivo.

#4774

Engineering an oncolytic adenovirus to target CEA in colorectal cancer.

Jessica C. Schaumburg. _Louisiana State University, Baton Rouge, LA_.

Colorectal cancer is the second leading cause of cancer-related deaths in the United States and the third most commonly diagnosed cancer overall. Screening methods can identify precancerous polyps before they progress, leading to five-year survival rates of 92%. However, if diagnosed in later stages the survival rates decrease to 12%. While treatment options are available for advanced stages, they remain inadequate to affect overall survival. Thus, new therapies are needed to improve treatment outcomes. Oncolytic virotherapy is a unique therapeutic approach in which a virus is genetically modified to target cancer cells while sparing normal tissue. Oncolytic virotherapy takes advantage of the lytic properties of the viral life cycle. These viruses are used replicate inside cancer cells, lyse them and spread to adjacent cells throughout the tumor. The adenovirus is a well-characterized vector that is easily manipulated and produced and is capable of infecting a wide range of cells. However, the endogenous receptor for adenovirus, the Coxsackie and adenovirus receptor (CXADR), is often downregulated in cancers, thus decreasing the ability to selectively infect cancer cells. In this study, an adenovirus was genetically engineered to present the single variable domain (VHH) derived from a camelid heavy chain antibody B2. The B2 antibody recognizes the human carcinoembryonic antigen (CEA), which is often overexpressed in a variety of cancers, including colorectal cancer. Here, the wild-type fiber and knob domain of the adenovirus fiber gene was replaced with a modified T4 fibritin fiber linked to the B2 VHH. To provide for visual tracking of adenovirus infection and biodistribution, a red fluorescent protein (RFP) marker gene was also fused to the capsid protein IX (pIX). This virus was rescued and amplified in the HEK293 F28 packaging cell line with a final round of amplification in HEK293 cells. The virus was purified by CsCl ultracentrifugation and dialysis. The resulting viral stock was titered and characterized for the presence of the modified fiber and knob. Subsequently, the vector was modified to express the human sodium-iodide symporter (NIS) transgene, to allow for noninvasive imaging of the adenovirus uptake and replication as well as radiotherapy using 131I. Further experiments will be completed to confirm viral selectivity for CEA and subsequent lysis of colorectal cancer cells. The results of this study will advance targeting oncolytic adenoviruses to metastatic colorectal cancer.

#4775

Optimizing the potency and dosing design for ARO-HIF2: An RNAi therapeutic for clear cell renal cell carcinoma.

So C. Wong, Anthony Nicholas, Jeff Carlson, Dongxu Shu, Che Liu, Rui Chu, Amanda Frankiewicz, Holly Hamilton, Casi Schienebeck, Aaron Andersen, Matthew Fowler-Watters, Stephanie Bertin, Xiaokai Li, Bo Chen, Josh Schumacher, Julia Hegge, Bruce Given, Zhen Li. _Arrowhead Pharmaceuticals Inc., Madison, WI_.

Background: Clear cell renal cell carcinoma (ccRCC) frequently involves the inactivation of the von Hippel-Lindau (VHL) tumor suppressor. Loss of VHL functions lead to the accumulation of hypoxia-inducible factors (HIFs). HIF2α has been regarded as a key tumorigenic driver of ccRCC and an attractive therapeutic target. Arrowhead has developed a RNA interference therapeutic (HIF2 RNAi) to selectively target and silence HIF2α expression, using a proprietary targeted-RNAi molecule (TRiM™) delivery platform for the treatment of ccRCC. The TRiM™ based Hif2 construct comprises a highly potent RNAi trigger using stabilization chemistries, targeting ligands to facilitate delivery, and structures to enhance pharmacokinetics (PK). The optimization of HIF2 RNAi to enhance the potency and safety profile to maximize the potential clinical success is described.

Methods: Functional optimization of HIF2 RNAi was evaluated in an orthotopic ccRCC tumor xenograft model established with A498 ccRCC cells that stably expresses the reporter gene SEAP (secreted embryonic alkaline phosphatase) as a serum biomarker for monitoring tumor growth. HIF2 RNAi was delivered by intravenous injections. HIF2α gene silencing was evaluated by isolating tumor RNA and measuring relative gene expression by qRT-PCR.

Results: We demonstrate that to achieve deep HIF2α mRNA knockdown (KD), functionalizing HIF2 RNAi with PK enhancement and tumor targeting ligand (TTL) is required. Optimization of the HIF2 RNAi construct enabled a 10-fold improvement in potency. Evaluation of a loading dose regimen improved overall HIF2α mRNA KD compared to a single administration of equal total dosage. Utilizing this strategy, we demonstrated that silencing of HIF2α mRNA (85% KD) resulted in tumor growth inhibition in the A498 xenograft model. Significant improvement in overall survival was also seen in a patient derived xenograft model. Histology evaluation of tumor samples revealed extensive tumor destruction with clusters of apoptotic cells and necrosis. Follow-up studies suggest that loading doses can be administered four hours apart without loss in potency. This allows dosing to be completed in one day and may be more acceptable in clinical settings. The maximum HIF2α mRNA KD after a single dose of HIF2 RNAi was achieved about 7 days after dosing and sustained for about one week in the xenograft model. This suggests that dosing can likely be less frequent in clinical settings. An exploratory toxicity study in rats predicts a wide safety margin.

Conclusions: We demonstrate that the TRiM™ delivery platform can be utilized to deliver a RNAi therapeutic selectively targeting HIF2α for the treatment of ccRCC. This represents a novel therapeutic approach either as a monotherapy or in combination with other therapies in seeking better tolerated and/or more effective treatment for ccRCC.

#4776

Characterizing an oncolytic adenovirus modified with the CXCL12 ligand for breast cancer therapy.

Samia M. O'Bryan, J. Michael Mathis. _Louisiana State University, Baton Rouge, LA_.

Breast cancer is the most commonly diagnosed cancer in women, making up nearly 30% of all diagnosed cancer cases each year. While localized breast cancer is easily treated, advanced cases are difficult to treat resulting in poor five-year survival rates. Thus, new therapies capable of increasing treatment efficacy are needed. Oncolytic virotherapy using human adenovirus is a novel therapeutic approach capable of specifically targeting cancer cells while sparing normal tissue. The adenovirus vector is well-characterized and is uniquely suited for oncolytic virotherapy due to its systemic stability, good safety profile and the ability to infect a broad range of dividing and non-dividing cells. Replication of oncolytic adenoviruses within cancer cells causes the lysis of the cells and subsequent spread of progeny virions within the surrounding tumor stroma. One challenge in targeting cancer cells with adenovirus vectors has been the low expression of the endogenous receptor, the Coxsackie and adenovirus receptor (CXADR), prompting the search for new receptor targets. In this study, we engineered an adenovirus to contain the CXCL12 ligand to target the chemokine receptors CXCR4 and CXCR7 overexpressed in a variety of tumors. Altered expression of these receptors drives tumor progression, migration, invasion and metastasis. Previously, we developed a bispecific adaptor molecule containing the CXCL12 ligand to redirect a replication-deficient wild-type adenovirus to breast cancer cells overexpressing CXCR4. In the current study, we engineered a replication-competent oncolytic adenovirus modified with the CXCL12 ligand to target cancer cells overexpressing CXCR4 and CXCR7. A recombinant gene was constructed using the tail domain of the adenovirus fiber gene fused to the trimerization domain of the T4 fibritin gene followed by the mature CXCL12 sequence. After viral production, the presence of a modified fiber and the CXCL12 ligand were confirmed. Subsequently, the vector was tested in a panel of breast cancer cells for infection and cell killing efficacy in vitro. Receptor knock down and overexpression studies confirmed the specificity of the virus. Together, these studies address the hypothesis that constructing a retargeted oncolytic adenovirus using the CXCL12 ligand provides selective infection and killing of breast cancer cells overexpressing CXCR4 and CXCR7. These results support the rationale for further development of retargeted adenovirus vectors for oncolytic virotherapy in patients. Future experiments will be conducted to assess tumor targeting using a xenograft mouse model of human breast cancer.

#4777

Anti-tumor effect of adenovirus encoding APE1/Ref-1 in breast cancer xenografts: anti-inflammation and apoptotic cell death.

Sunga Choi,1 Yu Ran Lee,1 Yeon Hyang Kim,2 Ki Mo Kim,3 Myoung Soo Park,4 Byeong Hwa Jeon1. 1 _Chungnam National Univ. College of Med., Daejeon, Republic of Korea;_ 2 _Korea Polytechnics, Bio Campus, Nonsan, Republic of Korea;_ 3 _Korea Institute of Oriental Medicine, Daejeon, Republic of Korea;_ 4 _Chungnam National University Hospital, Daejeon, Republic of Korea_.

In persistent inflammatory state involving the chronic activation of the immune system, the interplay between breast cancer and stromal cells is closely associated. We have previously shown that APE1/Ref-1 which could inhibit inflammatory signaling by reductive conformational change of cytokines receptors. In response to hyperacetylation, stimulation by secreted Ac-APE1/Ref-1 binding to RAGE initiated apoptotic cell death in TNBC models, in vitro and in vivo. In the present study, we used Ad-APE1/Ref-1 adenovirus, which can be overexpressed and be secreted into the blood of xenografts. We investigated how the Ad-APE1/Ref-1 influences growth retardation and apoptotic cell death of inflammation associated TNBC in vivo. The Ad-APE1/Ref-1-injection in MDA-MB 231 orthotopic xenografts caused a decrease in the volume and weight of tumors as shown in IVIS images. The treatment of Ad-APE1/Ref-1 also induced an inhibition of tumor growth and development, leading to apoptotic cell death, accompanied by generation of reactive oxygen species. Tumor tissues derived from Ad-APE1/Ref-1 exhibited apoptotic bodies compared with tumors of control or Adβ-galactosidase-injected mice. Moreover, level of inflammatory cytokines was evaluated in plasma of the xenografts; ratio of anti- to pro inflammatory cytokines was comparable with one of normal mice. The PAK1-STAT-3-NFkB axis in inflammatory signaling of tumor tissues derived from AdAPE1/Ref-1 injected mice was significantly inhibited compared to those of control or Adβ-galactosidase-injected mice. These results suggest that regulation of inflammatory signaling with adenoviral mediated APE1/Ref-1 in tumors of MDA-MB-231 xenografts modulates cytokine secretion, thereby inhibiting cancer cell growth.

#4778

Identification of promoters for targeted gene expression in tumors.

Abdul Mohin Sajib, Maninder Sandey, Samantha Morici, Bradley Schuler, Payal Agarwal, Bruce Smith. _Auburn University, Auburn, AL_.

A variety of novel therapeutic approaches to cancer have been proposed in the past several decades. One of the most compelling is the use of gene therapy to treat tumors. Restricting the expression of these genes to tumor avoids toxicity in normal cells and the concurrent side effects that this activity engenders. Promoter sequences can be used as a tool for transcriptional targeting, by restricting the expression of the therapeutic genes, or, in the case of oncolytic virotherapy, the replication of the viral agent. Several tumor-upregulated promoters have been identified in a wide variety of human tumors. Given the recognition of the validity of canine tumors as comparative models of human disease, it is critical to determine the activities of these promoters in the canine system. To achieve this goal, endogenous cSurvivin, cTERT, and cCXCR4 promoter activity was evaluated in a panel of canine normal and tumor cell lines/tissues and primary tumors by RT-Q-PCR. These three promoters, along with an E2F modified Canine Adenovirus 2 E1a promoter (E2F-E1a), were also assessed for their activity when supplied exogenously to tumor cells in an in vitro GFP reporter transfection assay. Endogenous expression of cTERT showed negligible differences between normal canine cells/tissues and tumor cell/tissues. cSurvivin showed elevated endogenous activity in non-hematopoietic tumor cells and moderately elevated activity in some normal tissues and hematopoietic tumors. cCXCR4 activity was elevated exclusively in a T-lymphoma cell line and a primary T-cell lymphoma. Exogenous expression in most cancer cell lines showed enhanced activity with most of the promoters tested. The percentage of cells expressing GFP was elevated in tumor compared to normal with all four tested promoters, when normalized to CMV-GFP expression. However, the relative fluorescence intensity varied significantly between cell lines, while the fluorescence intensity varied less between promoters within a specific cell line. Exogenous expression results did not correlate well with endogenous expression in tumor cells, but they did correlate well in normal cells. This disparity implies that promoter selection for transcriptional targeting of tumors should account for both endogenous and exogenous expression patterns. Additionally, these findings indicate that identification of a pan-cancer promoter is complex and that expression targeting may need to rely on selecting patient-specific promoters to drive the activity of therapeutic genes.

#4779

Coxsackievirus A11 as a novel oncolytic viral therapy for human colorectal cancer.

Hisanobu Ogata,1 Beibei Wang,2 Shoei Miyamoto,3 Yuto Takishima,3 Miyako Sagara,3 Mutsunori Murahashi,1 Hideya Onishi,2 Kenzaburo Tani3. 1 _Kyushu University Hospital, Fukuoka, Japan;_ 2 _Kyushu University, Fukuoka, Japan;_ 3 _The University of Tokyo, Fukuoka, Japan_.

Colorectal cancer (CRC) is a major cause of morbidity and mortality throughout the world. It is the third most common cancer worldwide and the fourth most common cause of death. FOLFOX is known as the effective chemotherapy for stage III and stage IV CRC. Oxaliplatin (L-OHP) is a third-generation platinum agent used among FOLFOX in CRC treatment. Patients with L-OHP-resistant CRC has a worse prognosis. Coxsackievirus is a virus that belongs to a family of nonenveloped, linear, positive-sense ssRNA viruses, Picornaviridae and the genus Enterovirus. Enteroviruses are among the most common and important human pathogens, and ordinarily its members are transmitted by the fecal-oral route. Coxsackieviruses share many characteristics with poliovirus. Coxsackieviruses are among the leading causes of aseptic meningitis. Recently, coxsackievirus A21, which is also called Cavatak, has already been reported to have the oncolytic activity. In this study, we tested whether coxsackievirus A11 (CVA11) has an oncolytic activity or not in CRC. Through our first screening assay, we found that CVA11 displayed the potent oncolytic activities in 4 of 5 various human CRC cell lines including two L-OHP-resistant CRC cells. Mechanistically, the CVA11-mediated cytotoxicity was significantly impaired when PI3K or Wnt inhibitor was added to the CVA11 infection. CVA11 had little effect on the L-OHP-resistant CRC. The pretreatment with L-OHP in L-OHP-resistant CRC cells displayed enhanced oncolytic activity of CVA11 in L-OHP-resistant CRC cells, whereas either L-OHP or CVA11 treatment alone caused slight oncolytic activity. These results suggest that L-OHP pretreatment could sensitize the susceptibility of CRC cells to CVA11 infection. Furthermore, our results of in vivo mouse study showed that intratumoral CVA11 administrations with pretreatment of L-OHP significantly suppressed the outgrowth of L-OHP-resistant xenografts. This combination therapy was more effective than the treatment with only CVA11. Altogether, our findings showed that the combination therapy with L-OHP and CVA11 displayed a remarkable oncolytic activity against L-OHP-resistant human CRC both in vitro and in vivo. Our study also provides the therapeutic potential for L-OHP-resistant CRC by Coxsackieviruse.

#4780

An antisense-based approach to induce anti-tumorigenic variants of MET receptor.

Trushar Rathod, Luca Cartegni. _Rutgers, State University of New Jersey, Piscataway, NJ_.

Rational: Aberrant activity of MET receptor, a receptor tyrosine kinase (RTK), is implicated in various forms of cancer. Moreover, functional crosstalk of MET with other RTKs has been reported in tumors and MET has emerged both as a driver and as a primary mechanism of resistance, thus, making MET an attractive target for cancer therapeutics.

Aim: Recently, we have described natural soluble decoy variants for multiple RTKs (sdRKTs), including MET, which can antagonize RTK signaling by ligand sequestration and non-functional dimerization. These isoforms can be generated via intronic polyadenylation (IPA) of RTK's pre-mRNA in a U1-snRNP dependent manner. Our aim is to activate IPA in MET pre-mRNA using antisense compounds and to increase mRNA variants that express dominant-negative soluble decoy MET (sdMET).

Methods: Identify actionable IPA sites within introns upstream of the transmembrane-coding exon. Express potential sdMET isoforms and characterize their biological properties. For sdMET isoforms with inhibitory functions, anti-sense compounds are utilized to block the upstream U1 binding site, activate IPA sites, and thus increase expression of sdMET variants in MET-dependent cancer cells.

Results: Ectopically expressed sdMET isoforms are secreted into the culture medium. When then added to MET-expressing cells, sdMET variants functioned in a dominant-negative manner over full length MET (FL-MET) and suppressed activation of AKT and MAPK pathways. Moreover, antisense compounds that induce IPA effectively convert oncogenic FL-MET into sdMET variants in multiple cell lines, leading to suppression of downstream pathways and dramatic reduction in cell viability of MET-dependent cells.

Conclusion: Most sdMET isoforms are secreted into extracellular space and have inhibitory function. Our anti-sense approach induces IPA, generates truncated sdMET variants at the expense of FL-MET and efficiently blocks FL-MET's biological activity.

#4781

Preclinical assessment of efficacy and safety of novel oncolytic adenovirus for therapy of disseminated lung cancer.

Svetlana Atasheva,1 Jia Yao,1 Cedrick Young,1 Nelson C. Di Paolo,1 Henry Wyche,2 Dmitry M. Shayakhmetov1. 1 _Emory University, Atlanta, GA;_ 2 _AdCure Bio, Atlanta, GA_.

The majority of lung cancer patients do not respond to current immune-oncology drugs due to low PD1 and PD-L1 expression on immune and cancer cells. Oncolytic viruses are being actively explored as an alternate modality for cancer therapy due to their natural ability to kill infected cells through viral replication. In this study, we analyzed the anti-tumor efficacy and safety profile of AVID-317 oncolytic virus in pre-clinical mouse models of disseminated lung cancer. AVID-317 is a novel oncolytic adenovirus possessing a set of mutations in the virus capsid, which ablate virus interactions with cellular b3 integrins on macrophages and hepatic cells, resulting in virus de-targeting from the liver after intravenous administration. In vitro studies demonstrated that 70% of tested human non-small cell lung cancer (NSCLC)-derived cell lines (N=16) are sensitive to AVID-317 infection, and 85% of AVID-317-sensitive NSCLC lines express no or low PD-L1. Intravenous injection of control parental human adenovirus type 5 (HAdv5) at a dose of 1e11 v.p. per mouse into wild-type mice resulted in release of pro-inflammatory cytokines IL-1b, IL-6, IL-16, and G-CSF into the bloodstream at 6 hours post virus administration. Furthermore, this dose of HAdv5 was lethal due to severe hepatotoxicity within 48 h post virus injection. In contrast, intravenous administration of AVID-317 in the range of doses up to 1e12 v. p. per mouse resulted in no detectible IL-6 and significantly reduced IL-1b, IL-16, G-CSF amounts in the blood. Importantly, AVID-317 administration did not result in mortality. Next, we grafted PD-L1-low human adenocarcinoma A549-Luc-C8 cells into lungs of NCr nude mice to establish an orthotopic model of disseminated lung cancer. When tumor burden reached between 2xe6 to 8e6 RLU, mice were enrolled in four cohorts and treated with AVID-317 at doses of 5e10, 1e11, or 3e11 v.p. per mouse or administered with saline only (control group; N=>10 in each cohort). Mice were euthanized upon detection of 20% weight loss compared to baseline. The median survival in saline-treated group was 57 days post treatment. However, median survival has not be reached for AVID-317-treated groups for the duration of the study (80 days). Taken together, our study demonstrates feasibility of using AVID-317 oncolytic adenovirus for systemic therapy of disseminated lung cancer and its greatly improved safety profile observed upon intravenous virus delivery.

#4782

STACT: A novel tumor-targeting, systemically-administered delivery platform capable of targeting intractable pathways and precise immunomodulation of the tumor microenvironment.

Chris S. Rae, Marina Besprozvannaya, John Faulhaber, Anastasia M. Makarova, Beverly King, Laura Hix Glickman, Christopher D. Thanos, Justin Skoble. _Actym Therapeutics, Inc., Berkeley, CA_.

We have engineered a tumor-targeted, microbial immunotherapy platform called STACT (Salmonella Typhimurium (Attenuated) and Checkpoint Therapy). The platform strain has been engineered to increase tumor-specific enrichment, reduce immunosuppressive inflammation and enhance tolerability. Upon phagocytosis by tumor-resident immune cells, the microbe delivers plasmid DNA, which can either encode inhibitory microRNAs to knockdown immune targets of interest, or encode immuno-modulatory cDNA expression cassettes, alone or in specific combinations.

The STACT platform strain is over 10,000-fold attenuated for virulence due to disruptions in the msbB and purI genes, that result in the detoxification of the surface LPS and purine/adenosine auxotrophy, respectively. The STACT platform strain required addition of exogenous adenosine or purines at concentrations found in the tumor micro-environment, but not in healthy tissue for replication in vitro. The STACT platform strain was unable to replicate significantly in infected macrophage cell lines, but was able to colonize tumors in mice and deliver functional plasmids. This strain was further engineered by precise genome deletions of the fljB and fliC genes, encoding the bacterial flagellar subunits that are strong TLR5 agonists and induce inflammasome-mediated pyroptosis in macrophages. Deletion of the flagellin genes prevented bacterial cell motility but did not affect tumor-specific colonization after IV administration, and tumor enrichment was observed at levels over 100,000 times greater than in spleens. A plasmid maintenance system was engineered by deletion of the chromosomal asd gene and complementation of the asd gene on a copy-number optimized plasmid to ensure plasmid maintenance in vivo. The asd system allowed for significantly improved plasmid retention in vivo without antibiotic selection. Furthermore, the vector incorporates immunostimulatory CpG motifs into the strain to help promote a TLR9-mediated adaptive immune response to tumor antigens. A library of inhibitory RNAi's against a set of immuno-modulatory targets, including TREX1, PD-L1, and TGF-beta were screened for optimal knockdown of gene expression by qPCR and western blot. Pairwise combinatorial knockdown of specific targets was also observed in human cells. IV administration of STACT encoding TGF-beta RNAi demonstrated significant tumor growth inhibition in a subcutaneous tumor model.

STACT is a highly attenuated microbial immunotherapy platform engineered to deliver immunomodulatory molecules to phagocytic cells in the tumor microenvironment after systemic administration. This platform can be engineered to knock down combinations of immune checkpoints or express immunostimulatory genes in a single therapeutic modality.

#4783

Highly efficient, non-viral precision genome engineering for the generation of personalized neoepitope-specific adoptive T cell therapies.

Kyle Jacoby, Robert Moot, William Lu, Diana Nguyen, Barbara Sennino, Andrew Conroy, Bhamini Purandare, Adam J. Litterman, Fabrizia Urbinati, Susan P. Foy, Theresa Hunter, Albert Tai, Michael T. Bethune, Songming Peng, Olivier Dalmas, Alex Franzusoff, Stefanie J. Mandl. _PACT Pharma, South San Francisco, CA_.

Methods used to engineer cells for adoptive cell therapies (ACT) utilizing receptors that are constant across many patients (CAR or shared Ag TCRs) typically rely on Lenti-, retro-, or adeno-associated virus to deliver specificity-altering sequences to T cells. However, for personalized therapies such as the generation of neoepitope-specific TCR T cell therapies, use of viral vectors is not feasible due to long manufacturing timelines and prohibitive per-patient costs. PACT Pharma has developed a highly efficient, DNA-mediated (non-viral) proprietary precision genome engineering approach to engineer neoepitope-specific primary human T cells. This method can be widely utilized to generate T cells at research scale, as well as for ex vivo manufacturing.

Briefly, genomes of individual primary human CD8 and CD4 T cells are engineered with site-specific nucleases in a single-step transfection process to yield efficient, targeted replacement of the endogenous TCR with the therapeutic neoTCR sequences. In this way, the expression of the endogenous TCR is abolished ensuring natural expression and regulation of the inserted neoTCR. The precision of neoTCR-T cell genome engineering was evaluated by Targeted Locus Amplification (TLA) for off-target integration hot spots or translocations, and by next generation sequencing based off-target cleavage assays and found to lack evidence of unintended outcomes.

Engineered neoepitope-specific T cells are highly functional as demonstrated by antigen-specific proliferation, killing and cytokine production. Phenotype and detailed functional characterization of PACTs neoTCR-P1 T cells were performed and are described in a separate abstract.

PACT's precision genome engineering approach enables highly efficient generation of bespoke NeoTCR T cells for personalized adoptive cell therapy for patients with solid tumors. Furthermore, PACT precision genome engineering method is not restricted to the use in T cells and has also been applied successfully to other primary cell types, including natural killer and hematopoietic stem cells.

#4784

Combination of sorafenib with liver cancer-targeted gene therapy exerts synergistic efficacy against hepatocellular carcinoma.

Huei-Yue Dai, Long-Yuan Li. _National Chung Hsing University, Taichung, Taiwan_.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. Sorafenib, a multikinase inhibitor, is the first-line target therapy for patients with advanced HCC, but the response rate is low. Thus, development of more effective therapeutics for HCC is needed. Previously we have established a HCC-targeted BikDD gene therapy vector driven by a liver cancer-specific α-fetoprotein promoter/enhancer and transcriptional amplification module. Systemic administration of liposome-loaded HCC-targeted BikDD gene therapy vector effectively suppressed tumor growth and prolonged survival in multiple orthotopic HCC mouse models with no toxicity. Here, we further investigated whether combination of sorafenib with gene therapy could enhance anti-HCC activity. The results showed that combination of BikDD gene therapy with HCC targeted therapeutic agent sorafenib synergistically killed a panel of liver cancer cells but not normal cells in vitro by WST-1 assays and analysis of combination index according to the Chou and Talalay method. Moreover, a low dose of sorafenib combined with BikDD gene therapy augmented tumor inhibition and better mice survival compared with separate treatment alone in xenograft and syngeneic orthotopic HCC mouse models. These results suggest that sorafenib combined with HCC-targeted gene therapy may improve the therapeutic efficacy and may be a potential alternative therapeutics for HCC.

#4785

Mimicking virotherapy-elicited human cytokine storm using humanized mice.

Satya Pathi,1 Maria Anna Zipeto,2 Annie An,1 Michelle Solomon,1 Jayant Thatte,1 Henry Li3. 1 _Crown Bioscience, San diego, CA;_ 2 _Crown Bioscience, La Jolla, CA;_ 3 _Crown Bioscience, San Diego, CA_.

Cytokine release storm (CRS) is characterized by the systemic release of inflammatory cytokines, predominantly TNF-α and interferon-γ (IFN-γ), followed by interleukin-6 (IL-6) and IL-10 and, in some cases, IL-2 and IL-8. CRS has been seen with several therapeutic biologics such as monoclonal antibodies (mAbs), including, rituximab, muromonab-CD3 and TGN1412. In the case of TGN1412, CRS was life threatening with organ failure in all the healthy volunteers. This episode, along with the mortality of one young patient due to multiorgan failure during a viral vector based gene therapy trial at the University of Pennsylvania, underscore the need for additional models more representative of human CRS for acute toxicity testing of biologic therapeutics. Preliminary observation suggested that hCD34\+ Hematopoietic Stem Cells (HSC) humanized mice can support the identification of aspects of safety profiles for biologic agents, such as viral vectors. Administration of a specific replication competent viral vector resulted in acute toxicity, evidenced by sudden and significant body weight loss and mortality in such humanized mice, reflective of typical clinical symptoms of CRS, therefore suggesting a potential CRS model of human dysfunctional immune system. In the current study, we are performing comprehensive investigation of this potential CRS system by testing a variety of parameters, including dose titration, multiple routes of administrations (intraperitoneal, intravenous as a representation of slow and rapid infusion respectively, and intratumoral), safety assessments, including survival and clinical signs (appearance, behavior and body weight), as well as lymphocyte activation markers, laboratory tests of plasma cytokines, and viremia levels, etc. We believe that immune deficient mice with a reconstituted human immune system have the potential of becoming a powerful preclinical tool for the assessment of safety of biologic test agents, therefore enabling more successful clinical trials and most importantly, reduced risks for the patients.

#4786

BP1003, a novel liposome-incorporated STAT3 antisense oligodeoxynucleotide inhibitor.

Bingbing Dai,1 Ana Tari Ashizawa,2 Jithesh J. Augustine,1 Michael Kim,1 Jason Fleming3. 1 _The University of Texas M.D. Anderson Cancer Center, Houston, TX;_ 2 _Bio-Path Holdings, Inc., Bellaire, TX;_ 3 _Moffitt University, Tampa, FL_.

Background: Signal Transduction and Activator of Transcription-3 (STAT3), though typically inactive in normal cells, is aberrantly active in cancer cells. Activation of STAT3 has been found in many types of cancers, including non-small cell lung cancer (NSCLC), acute myelogenous leukemia (AML), and pancreatic ductal adenocarcinoma (PDAC). Activation of STAT3 correlates with poor clinical outcome, high grade disease and metastasis, and has been linked with resistance to chemotherapy, including gemcitabine, which is one of the standard of care agents for advanced PDAC. Therefore, inhibition of STAT3 in combination with chemotherapy is expected to produce clinical benefit. We have developed BP1003, a novel liposome-incorporated STAT3 antisense oligodeoxynucleotide (oligo) as a specific inhibitor of STAT3.

Methods: Four candidate antisense oligo sequences directed against STAT3 mRNA were initially identified. They were manufactured as nuclease-resistant P-ethoxy oligos and incorporated into neutral dioleoylphosphatidylcholine liposomes. Cell viability tests and Western blots were conducted to determine the inhibitory effects of liposome-incorporated STAT3 antisense oligo on NSCLC and AML cells. Ex vivo live tissue sensitivity assay (LTSA) was performed with a panel of 20 PDAC patients-derived xenografts (PDXs) to study the overall activity of BP1003, alone and in combination with gemcitabine. Using previous defined criteria, tissue slice viability inhibition greater than 30% and p<0.05 was considered to be a response. For validation of ex vivo results, PDAC PDX tumor bearing mice were administered with BP1003 and gemcitabine twice a week for 28 days. Tumor volumes were monitored for up to 49 days.

Results: The most potent liposome-incorporated STAT3 antisense sequence in decreasing NSCLC cell viability was selected as the candidate BP1003 drug. Further validation in AML cells demonstrated that BP1003 inhibited cell viability and STAT3 protein expression. In the ex vivo LTSA assay, BP1003, at a dose of 10 µM, significantly inhibited the tissue slice viability of 9 out of 18 PDAC PDXs by over 30% (p<0.05). Combination of BP1003 and gemcitabine further enhanced ex vivo efficacy of BP1003 in a subset of PDXs. In the in vivo study with PDAC PDX models, combination of BP1003 and gemcitabine caused tumor regression during the 28-day drug treatment period. This anti-cancer activity was maintained for another 21 days, even when drug treatment had ceased.

Conclusions: Liposome-incorporated STAT3 antisense oligo, BP1003, shows promising in vitro and in vivo preclinical activity as single and combination agent and might be a novel therapeutic strategy in patients with advanced tumors and/or metastasis.

### Novel Antitumor Agents 3

#4787

New and potent small molecule as EF2K inhibitor: A novel EF2K inhibitor.

Ferah Comert Onder,1 Mehmet Ay,2 Serdar Durdagi,3 Bulent Ozpolat,1 isik Kantarcioglu3. 1 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Canakkale Onsekiz Mart University, Canakkale, Turkey;_ 3 _Bahcesehir University, Istanbul, Turkey_.

Triple-negative breast cancer (TNBC) which constitutes 15-20% of breast cancers, is the most aggressive and chemo-resistant subtype of breast cancer and associated with the highest mortality rates of mortality among all breast cancer subtypes. TNBC is characterized by a lack of molecular targets (e.g., estrogen, progesterone, and HER2 receptors. Current treatment options for TNBC are still limited to conventional chemotherapies such as anthracyclines (e.g., doxorubicin) and taxane-based therapeutics. Identification of new molecular targets is critical for development of novel targeted treatments to improve poor patient prognosis and survival. We recently found that expression of eukaryotic elongation factor-2 kinase (EF2K), an unusual alpha kinase, is commonly upregulated in the majority of TNBC patients and associated with poor prognosis. We showed that EF2K is a potential molecular driver by promoting cell proliferation motility and invasion and drug resistance and validated it as a molecular target by RNA based therapeutic (i.e, siRNA and microRNA) in various TNBC models in mice (Tekedereli 2012, Hamurcu 2017, Bayraktar 2017). Several inhibitors of eEF2K have been described in the previous studies. However, these inhibitors have been neither specific nor potent. Small molecule inhibitor was synthesized in many steps by using different synthesis methods and also performed all of the structural analysis (FT-IR, 1H-NMR, 13C-NMR and LC-MS-MS). Computational studies were performed by using Schrödinger 2015 programme to investigate of in vitro activity of the compounds. By performing in vitro assays we identified a potent inhibitor of EF2K which inhibited p-EF2 at 1 μM (2 hours) detected by Western blots. Treatment of TNBC cells with this compound starts to inhibits cell proliferation and colony formation at 5-10 μM concentrations. The further studies are still ongoing. In conclusion, our results suggest that our newly designed small molecule may be potent EF2K inhibitor and can be used for targeting EF2K in TNBC.

#4788

Novel hybrid benzothiazole screening in U87-MG glioblastoma cell line.

Priscilla Kyi, Desmond H. Murray, Denise L. Smith. _Andrews University, Berrien Springs, MI_.

Benzothiazole is a heteroaromatic compound known for its wide range of bioactivities including anticancer, antiviral, antimicrobial, anti-inflammatory, anticonvulsant, antidiabetic, antihelminthic, and antitubercular activities. Research has shown that derivatives of benzothiazole exhibit inhibition of proliferation via apoptosis in various human cancer cell lines, such as liver cancer (Wang, et. al., 2010). In this study, a series of novel hybrid benzothiazole α-cyanostilbene derivatives and styrylbenzothiazole derivatives containing boronic acid and non-boronic acid pharmacophores were synthesized and their anti-cancer properties on U87-MG glioblastoma cells were investigated in vitro. U87-MG cells were incubated with synthesized novel hybrid compounds at varying concentration to determine the inhibitory concentration 50 (IC50) of the compounds. Most hybrid compounds displayed inhibitory effects on cell growth and the IC50 of the compounds varied depending on the nature of the pharmacophores. Moreover, boronic acid-containing compounds exhibit lower IC50 than nonboronic acid-containing compounds. Cell mobility has been investigated and we have found that treated cells were less mobile than untreated cells. In the future, we will be further screening for mobility, invasiveness, and mode of death of these novel hybrid benzothiazole treated glioblastoma cells.

#4789

Predicting sensitivity to Lantern Pharma's pipeline drug candidate LP-184 using the Response Algorithm for Drug Positioning and Rescue.

Aditya Kulkarni, Umesh Kathad, Yuvanesh Vedaraju, Barry Henderson, Gregory Tobin, Panna Sharma, Arun Asaithambi. _Lantern Pharma, Dallas, TX_.

LP-184 is a DNA Damage Repair inhibitor being developed by Lantern Pharma primarily as a non-hormone, non-chemotherapy option for the growing indication of taxane- and hormone-resistant metastatic prostate cancer. LP-184 is a next-generation analog of Irofulven with broad anti-tumor activity that counteracts multi-drug resistance pathways and is potentially synergistic with many classes of anticancer agents. LP-184 has a favorable therapeutic index and pharmacokinetic profile. Knowledge about its shared mechanism of action with Irofulven and potential biomarkers implicated in induction of bioactivation and synthetic lethal interactions is available. To advance LP-184 into clinical stages and achieve accelerated approval, Lantern Pharma is employing its proprietary Artificial Intelligence (AI)-driven technology. Lantern Pharma has developed a technology platform termed RADRTM that can be used to construct responder/ non-responder profiles based on gene expression signatures and predict true responders before conducting a clinical trial in order to achieve higher success rates. RADRTM is an Al-based machine learning approach for candidate biomarker identification and patient stratification. RADRTM is a combination of three automated modules working sequentially to generate drug- and tumor-specific gene signatures predictive of response. RADRTM emphasizes the integration of biological knowledge, data-driven feature selection, and robust Al algorithms to derive biomarkers in a hypothesis-free manner. In analytic demonstrations, RADRTM was able to achieve an average accuracy of 85% in validation tests using preclinical datasets associated with selected solid tumor indications and approved drugs. As part of RADRTM drug model building and development, we used a dataset showing preclinical efficacy of our pipeline oncology candidate LP-184. We obtained baseline cell line gene expression profiles covering more than 18,000 transcripts per cell line and corresponding LP-184 sensitivity records from the NCI60 cancer cell line panel. Using RADRTM, we derived a panel of 10 genes whose expression levels are predictive of overall response at an accuracy of 100%. Thus, RADRTM was able to identify the top 10 genes for prediction of either drug sensitivity or insensitivity, demonstrating the hypothesis-free identification of biomarkers with biological relevance and statistical rigor and having highest possible prediction accuracy. Genes from the final 10 predictive list were found to be functionally involved in LP-184-specific induction of bioactivation and are in agreement with its mechanism of action. These preliminary biomarker analyses will be further validated using LP-184 sensitivity and pre-treatment gene expression data derived from ex vivo models of fresh prostate tumor biopsy samples.

#4790

Identification and characterization of ATP-mimetic choline kinase inhibitors.

Paola Gnocchi, Francesca Quartieri, Alessandra Badari, Roberta Bosotti, Elena Casale, Emiliana Corti, Cinzia G. Cristiani, Ulisse Cucchi, Fabio Gasparri, Laura M. Gianellini, Laura M. Giorgini, Marisa Montemartini, Giuliana Mion, Marcella Nesi, Christian Orrenius, Claudia E. Re, Daniele Donati, Eduard R. Felder, Arturo Galvani, Antonella Isacchi. _Industry, Nerviano (MI), Italy_.

Introduction: Choline kinase alpha (ChoKα), the first enzyme in the Kennedy pathway that catalyzes the phosphorylation of free choline to phosphocholine (PCho), is responsible for the de novo biosynthesis of phosphatidylcholine (PC), the major phospholipid of cellular membranes. Aberrant choline metabolic profiles and concomitant ChoKα upregulation have been described in most human malignancies (i.e. breast, lung, ovary, liver) and have been found to correlate with advanced histological tumour grade. ChoKα, depletion by siRNA or shRNA inhibits growth and migration of different tumor cell lines both in vitro and in vivo, which is not observed for the ChoKβ isoform. Choline mimetic inhibitors of ChoKα (i.e. MN58b) have been shown to have antitumor activity in preclinical models, although their efficacy is hampered by a significant toxicity, possibly due to cross-reactivity with other choline-dependent proteins (transporters, enzymes). At NMS a high throughput screening (HTS) was performed with the objective to identify ATP-mimetic ChoKα inhibitors potentially less toxic than choline-mimetic compounds.

Methods: Hits from different classes were characterized for biochemical activity on ChoKα and ChoKβ and biochemical mechanism of inhibition. The binding site of selected ATP competitive inhibitors was confirmed by co-crystallization with ChoKα. On-target mechanism of action in cells was confirmed by analysing inhibition of PCho formation.

Result: Structure-based chemical expansion of Hits from one of the prioritized classes resulted in compounds with biochemical potencies in the single digit nanomolar range displaying selectivity vs ChoKβ as well as a diverse panel of protein kinases. In several tumor cell lines, the compounds were able to inhibit the formation of PCho at a concentration in agreement with that required to achieve anti-proliferative activity. The most potent compounds were tested on a panel of 24 representative breast cancer cell lines which showed differential sensitivity towards ChoKα inhibition. Analysis of genomic (DNA and RNA) and proteomic (>50 markers) expression profiles of the breast cancer cell lines is ongoing to identify predictive biomarkers of response.

Conclusion: Medicinal chemistry expansion of a novel class of compounds identified by HTS allowed the development of potent ChoKα ATP-mimetic inhibitors able to modulate PCho levels in cells, which can be used to identify preferential sensitivity contexts. Medicinal chemistry activities are ongoing to further improve their potency and optimize their ADME/PK properties.

#4791

Impact of folate transport redundancies on the therapy with tumor-targeted and untargeted antifolates.

Zhanjun Hou,1 Carrie O'Connor,1 Changwen Ning,1 Adrianne Wallace-Povirk,1 Josephine Frühauf,1 Md. Junayed Nayeen,2 Aleem Gangjee,2 Larry H. Matherly1. 1 _Barbara Ann Karmanos Cancer Inst., Detroit, MI;_ 2 _Duquesne University, Pittsburgh, PA_.

Folates participate in one-carbon (C1) transfer reactions in normal and cancer cells. Antifolate therapeutics disrupt cytosolic C1 metabolism, and have long been a mainstay for the therapy of a number of cancers. There are three major folate transporter systems in human tissues and tumors, including the reduced folate carrier (RFC), folate receptors (FRs) and proton-coupled folate transporter (PCFT). RFC is broadly expressed in tissues and tumors and is characterized by a neutral pH optimum. PCFT has more limited tissue distribution but is widely expressed in human solid tumors and exhibits an acidic pH optimum. FRα is expressed in a subset of solid tumors including epithelial ovarian cancer (EOC) but shows limited expression in most normal tissues. We previously discovered novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl compounds typified by AGF94 which show selective cellular uptake by FRα and/or PCFT over RFC, in contrast to the classical antifolates methotrexate (MTX) and pemetrexed (PMX) which are transported by both PCFT and RFC, and to a lesser extent by FRα. We showed that AGF94 had enhanced antitumor activities by targeting cytosolic C1 metabolism to PCFT-expressing tumors but lacking RFC, reflecting contraction of cellular tetrahydrofolate pools. In EOC cells, AGF94 showed substantial in vitro inhibition of cell proliferation independent of FRα expression, as long as PCFT was present. We systematically explored the impact of folate transporter redundancies among PCFT, FRα and RFC in determining anti-tumor efficacy of novel tumor-targeted and classical antifolates. Towards this goal, we engineered cell line models from PCFT-, FR-, and RFC-null HeLa cells to express doxycycline (DOX)-inducible FRα or RFC. We used these models to constitutively express PCFT together with DOX-inducible FRα or RFC. These were characterized for transporter expression and function, as well as intracellular folate levels, with/without DOX induction. The relative contributions of RFC, PCFT and FRα to transport function were evaluated from pH 5.5 to 7.4, with radiolabeled MTX and AGF347 which targets both cytosolic and mitochondrial C1 metabolism. We assessed antiproliferative activities of classical antifolates (PMX, MTX, PT523) versus tumor-targeted compounds (e.g., AGF94, AGF102, AGF347) in these models with/without DOX. Our results establish that co-expression of the major folate transporters can have variable and surprisingly disparate and substantial impacts on anti-tumor efficacies of both classical and tumor-targeted antifolates. Our study identified critical determinants of anti-tumor activity with classical and tumor-targeted antifolates, including relative levels of folate transporter expression and transporter specificity, and the impact of intracellular folate levels and extracellular pH.

#4792

Azetidine functionalized small-molecules potently inhibit STAT3 signaling and block tumor growth in human breast cancer xenografts.

Peibin Yue,1 Francisco Lopez-Tapia,1 Wenzhen Fu,1 Christine Brotherton-Pleiss,1 Marcus Tius,2 James Turkson1. 1 _University of Hawaii Cancer Center, Honolulu, HI;_ 2 _University of Hawaii at Manoa, Honolulu, HI_.

The development of inhibitors of Signal transducer and activator of transcription (STAT) 3 for clinical application has faced significant challenges and to date there are no suitable STAT3 small molecule inhibitors in the clinic. We report a novel class of azetidine-based small molecules that potently and preferentially inhibit STAT3 DNA-binding activity in vitro, with IC50 of 300-800 nM, compared to IC50 of 17 μM or higher against STAT1 DNA-binding activity. In the human breast cancer MDA-MB-231 and MDA-MB-468 cells treated with the azetidine compounds, including H182, constitutive STAT3 DNA-binding activity and tyrosine phosphorylation were strongly inhibited in both time- and dose-dependent manner. By contrast, the azetidine compounds showed little effects on EGF-induced STAT1 activity, Ras-Raf-MEK-ERK or other STAT3-independent signaling pathways, or on other SH2 domain-containing proteins. Furthermore, H182 and other azetidine compounds strongly inhibited viability, anchorage-dependent and -independent growth, and colony survival of the human breast cancer cells, with IC50 of 1.0 - 1.9 μM, compared to the IC50 of 3.8 - 8.1 μM against the viability and growth of the immortalized breast epithelial MCF-10A cells. The migration of breast cancer cells in vitro were similarly inhibited by treatment with low concentrations of H182 for 18 h, without changes in viable cell number. Analysis of STAT3 downstream genes show that the expression of VEGF and survivin were significantly suppressed in breast cancer cells treated with the azetidine compounds. In vivo evaluation of antitumor efficacy shows H182 inhibited growth of human breast tumor xenografts. Studies together identify the azetidine-functionalized small molecules as potential candidate compounds for clinical development as novel therapeutic agents for human breast and other cancers that harbor aberrantly-active STAT3.

#4793

Preclinical evaluation of the combination rogaratinib and copanlisib in HNSCC and HCC in preclinical in vitro and in vivo models.

Oliver Politz,1 Sylvia Gruenewald,1 Isabel S. Jerchel,1 Alexander Walter,1 Peter Ellinghaus,1 Pascale Lejeune,1 Jens Hoffmann,2 Konrad Klinghammer,3 Dominik Mumberg1. 1 _Bayer AG, Berlin, Germany;_ 2 _EPO-Berlin-Buch GmbH, Berlin, Germany;_ 3 _Charité Campus Benjamin Franklin, Berlin, Germany_.

Rogaratinib is a potent small molecule pan-FGFR inhibitor that leads to downregulation of MAPK and PI3K signaling (1). In a recent Phase I study rogaratinib demonstrated good efficacy in urothelial bladder cancer (UBC) tumors with FGFR mRNA overexpression (2).

Here, we explored the anti-tumor activity of rogaratinib in 2 indications, hepatocellular carcinoma (HCC) and head and neck cancer (HNSCC), where FGFR signaling plays an important role. In HCCs around 80% of tumors overexpress at least one FGF and/or FGFR (3), with main alterations in FGFR3 and FGFR4/FGF19. Head and neck cancer (HNSCC) displays FGFR1 amplification in about 35% of cases (4) and FGFR inhibition resulted in strong anti-tumor efficacy in HNSCC xenograft models with high FGFR1-3 mRNA expression levels.

In vitro, HCC and HNSCC cell lines were profiled for inhibition of cell proliferation by rogaratinib or copanlisib. We furthermore tested the combination of FGFR inhibition by rogaratinib and PI3K inhibition by copanlisib in selected HCC cell lines showing synergy in three models with combination indices (Ci) below 0.8.

In vivo, two HCC patient derived xenograft models with elevated levels of FGFR3/4 or FGFR4 showed strong monotherapy efficacy of rogaratinib treatment which was clearly superior to established therapies with multikinase inhibitors.

Using a mouse-clinical trial set-up we profiled a set of patient derived HNSCC xenograft models for their sensitivity to cetuximab, rogaratinib and copanlisib in monotherapy as well as in combinations. The anti-tumor responses observed in selected models in monotherapy or in combination treatment support further investigation for the potential of FGFR and / or PI3K inhibition, especially in cetuximab resistant models.

In conclusion, rogaratinib showed potential of tumor growth inhibition in xenograft models of HNSCC and HCC with overexpression of at least one FGFR subtype and warrants further investigation in these 2 indications. The combination with the PI3K inhibitor copanlisib provides options to improve efficacy. The importance of the specific molecular background will need further analysis.

1. Gauglhofer, C., S. Sagmeister, et al. (2011). Hepatology 53(3): 854-64.

2. Jerchel, I. S., A. Kamburov, et al. (2018). Cancer Research 78(13 Supplement): 4781-4781.

3. Joerger, M., P. A. Cassier, et al. (2018). J. Clin. Oncology 36 (suppl.)(abstr. 4513).

4. Tillman, B. N., M. Yanik, et al. (2016). Head Neck 38 Suppl 1: E1646-52.

#4794

**Targeting mitochondrial and cytosolic one carbon metabolism of pancreatic adenocarcinoma via the proton-coupled folate transporter with novel 5-substituted pyrrolo[3,2-** d **]pyrimidine analogs.**

Changwen Ning,1 Aamod Dekhne,1 Md. Junayed Nayeen,2 Jade M. Katinas,3 Jennifer Wong,3 Josephine Frühauf,1 Xun Bao,1 Carrie O'Connor,1 Adrianne Wallace-Povirk,1 Jing Li,1 Charles E. Dann,3 Aleem Gangjee,2 Larry H. Matherly,1 Zhanjun Hou1. 1 _Barbara Ann Karmanos Cancer Inst., Detroit, MI;_ 2 _Duquesne University, Pittsburgh, PA;_ 3 _Indiana University, Bloomington, IN_.

Pancreatic cancer (PaC) represents the 4th leading cause of cancer-related deaths in the US with a mortality rate of 99%. The 5-year overall survival rate for PaC is currently 8%. One-carbon (C1) metabolism is frequently altered in cancer. For PaC, TCGA data sets show that elevated expression of key enzymes involved in cytosolic [5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFTase) and serine hydroxymethyltransferase (SHMT)1] and mitochondrial [SHMT2 and methylene tetrahydrofolate dehydrogenase 2 (MTHFD2)] C1 metabolism is associated with poor survival. Antifolate therapeutics disrupt cytosolic C1 pathways required for syntheses of thymidylate, purines, and certain amino acids, and are a mainstay for therapy of several cancers. Antifolate uptake into tumors and tissues involves the reduced folate carrier, the major tissue folate transporter, and the proton-coupled folate transporter (PCFT), which shows a more limited tissue distribution but is widely expressed in human solid tumors and is active only at acidic pHs characterizing the tumor microenvironment. We discovered novel 5-substituted pyrrolo[3,2-d]pyrimidine analogs (AGF347, AGF359) with PCFT transport that potently inhibited proliferation of PaC cell lines (AsPC-1, BxPC-3, CFPAC-1, HPAC, MIA PaCa-2 and PANC-1), of which HPAC (KRAS mutant) and BxPC-3 (KRAS wild-type) cells were most sensitive. The PaC cell lines all expressed PCFT transcripts and proteins that were active for PCFT transport with 3H-AGF347 at acid pH. When HPAC cells were incubated with 3H-AGF347 over 48 h, drug accumulated in both cytosol and mitochondria. 3H-AGF347 was extensively metabolized to polyglutamates. Treatment of PaC cells with AGF347 and AGF359 inhibited proliferation by inducing glycine and adenosine auxotrophy that was rescued by excess glycine and adenosine. This implied that both mitochondria and cytosolic C1 metabolism was inhibited. Inhibition of mitochondrial SHMT2 and cytosolic SHMT1, glycinamide ribonucleotide formyltransferase and/or AICARFTase was confirmed by in vitro targeted metabolomics and assays with purified enzymes. Tumor cell killing was confirmed (with HPAC and BxPC-3) by colony-forming assays with AGF347 and AGF359 and drug-induced apoptosis with AGF347 was demonstrated (with HPAC) by annexin V-PI staining and flow cytometry. AGF347 and AGF359 depleted purine nucleotides and inhibited mTOR signaling via S6K1 at least in part (for BxPC-3) via activation of AMPK, likely due to elevated ZMP accompanying suppression of AICARFTase. Collectively, our studies identify first-in-class inhibitors and establish the considerable therapeutic potential of dual-targeting mitochondrial and cytosolic C1 metabolism in PaC independent of KRAS mutation status and reflecting cellular uptake by PCFT.

#4795

Oxidative stress by proguanil suppresses breast tumor growth.

Nehal Gupta,1 Sanjay Srivastava2. 1 _Texas Tech Univ. Health Sciences Ctr., Amarillo, TX;_ 2 _Texas Tech Univ. Health Sciences Ctr., Abilene, TX_.

Breast cancer is one of the most common malignancies and the second leading cause of cancer related mortality among women in U.S., thus developing new strategies to control breast cancer is an important mission. Repurposing of old drugs as new anti-cancer drugs is important as it can save time and cost of drug development. Proguanil is used in combination with atovaquone for the treatment of malaria. In this study, we determined the anticancer effects of proguanil in breast cancer cells. Proguanil significantly reduced the viability of MDA-MB-231, HCC1806 and MCF-7 breast cancer cells with IC50 of 42μM, 44μM and 40μM respectively after 72 h of treatment and induced apoptosis as exhibited by FITC/Annexin assay and cleavage of caspase 3 as well as PARP. Proguanil also reduced the survival of several patient-derived cells with IC50 in the range of 30-40μM. The anti-cancer effects of proguanil were associated with the generation of ROS and persistent disruption of mitochondrial membrane potential. ROS generation by proguanil was about 2-3 fold more as compared to control in MDA-MB-231 and HCC1806 cells. The generation of ROS was inhibited when the cells were pretreated with a general antioxidant N-acetyl cysteine (NAC). Moreover, exposure of MDA-MB-231 and HCC1806 cells to proguanil was also associated with increased expression of Bax, p-H2AX, c-caspase9 and down-regulation of bcl-2 and survivin. We further evaluated the oxygen consumption rate with proguanil treatment in breast cancer cells using flux analyzer. Our results showed significant decrease in the mitochondrial respiration rate and ATP production rate after proguanil treatment. Furthermore, 20mg/kg proguanil when given orally suppressed the growth of 4T1 breast tumors in female Balb/c mice by 55%. Tumors from proguanil treated mice demonstrated increased apoptosis, which was related to the increased expression of p-H2AX and Bax. Taken together, these results show that proguanil is an effective inhibitor of in vitro and in-vivo growth of breast cancer cells. These findings provide the rationale for further clinical investigation of proguanil against breast cancer.

#4796

**EG-011 is a novel small molecule with** in vitro **and** in vivo **anti-tumor activity against lymphoma.**

Eugenio Gaudio,1 Filippo Spriano,1 Chiara Tarantelli,1 Matilde Guala,2 Eugenia Riveiro,3 Gaetanina Golino,4 Antonio Lupia,4 Giosuè Costa,4 Roberta Rocca,4 Luciano Cascione,1 Silvia Jenni,5 Yi-Chien Tsai,5 Beat Bornhauser,5 Stefano Alcaro,4 Francesco Paduano,4 Francesco Trapasso,4 Emanuele Zucca,6 Anastasios Stathis,6 Natalina Pazzi,2 Franco Cavalli,6 Francesco Bertoni1. 1 _Università della Svizzera italiana, Institute of Oncology Research, Bellinzona, Switzerland;_ 2 _Chimete, Tortona, Italy;_ 3 _Early Drug Development Group, Paris, France;_ 4 _University "Magna Græcia" of Catanzaro, Catanzaro, Italy;_ 5 _Children's Hospital Zurich, Zurich, Switzerland;_ 6 _Oncology Institute of Southern Switzerland, Bellinzona, Switzerland_.

Introduction: Despite the improvements, still too many patients die for their lymphomas and novel compounds are needed. We present a new small molecule, EG-011 (PCT/EP2018/057678), with in vitro and in vivo anti-cancer activity in lymphoma models.

Methods: Lymphoma and solid tumor cell lines were exposed to a large range of concentrations of EG-011 as single agent for 72h, followed by MTT proliferation assay and IC50 calculation. Cell viability of twelve acute lymphoblastic leukemia (ALL) primary patient cells from different high-risk subgroups (VNN2+, E2A-HLF, refractory T and IKZF plus) co-culture with marrow-derived MSCs were assayed after 72h of incubation with EG-011 and controls. Apoptosis assay was measured with annexin V by FACS. Xenografts were established s.c. into the left flanks of female NOD-SCID mice; treatment (200 mg/kg, i.p. 5 days per week) started with already established tumors. Combinations were evaluated with Chou-Talalay combination index (CI): synergism (<0.9), additive (0.9-1.1), antagonism/no benefit (> 1.1) after 72 hr treatments.

Results: EG-011 presented a median IC50 of 2.25 μM in 62 lymphoma cell lines (95% C.I. 1-5μM). A higher activity was observed in a group of 21 cell lines that had a median IC50 of 250 nM (95% C.I. 40-600 nM). Among these there were 11 germinal center B cell (GCB) diffuse large B cell lymphomas (DLBCL) (sensitive n=11/21, resistant n=9/41, P < 0.05), 4 mantle cell lymphoma (MCL) (sensitive n=4/21, resistant n=6/41, P n.s.), 3 marginal zone lymphoma (sensitive n=3/21, resistant n=2/41, P n.s.). EG-011 did not show any anti-proliferative activity in a panel of 25 solid tumor cell lines (IC50s > 10 μM), Among 12 primary ALL samples, 7 were sensitive to EG-011 with IC50 values between 0.3-4.6 µM after 72h, 5 displayed IC50 higher than 20 µM.

A dose-dependent increase in cell death (20-55%) was observed in lymphoma cell lines (OCI-LY-19 and REC1) (500 nM and 2 μM; 72h). No cytotoxicity was seen in PBMCs from two healthy donors after treatment at 1 and 10 μM for 24h and 48h.

In an in vivo xenograft experiment with the MCL REC-1 cell line, EG-011 delayed tumor growth (Day 6, Day 7, Day 9, P < 0.05) and tumor weight. EG-011-treated tumors were 2.2-fold smaller than controls (P < 0.001).

Combinations were tested in DLBCL (OCI-LY-1, OCI-LY-8, TMD8) and MCL (REC1, MINO). EG-011 was synergistic with rituximab, bendamustine, venetoclax, ibrutinib and lenalidomide in all tested cell lines.

Conclusion: The selective anti-lymphoma activity, in both in vitro and in vivo models, and the observed in vitro synergisms with FDA approved targeted agents make EG-011 a novel intriguing new drug candidate deserving further preclinical studies.

#4797

A novel Diiminoquinone targets colorectal cancer stem cells.

Alissar Monzer,1 Nayri Jabotian,1 Jie S. Zhu,2 Mark J. Kurth,2 Wassim Abou Kheir,1 Makhlouf Haddadin,1 Hala Gali-Muhtasib1. 1 _American University of Beirut, Beirut, Lebanon;_ 2 _University of California at Davis, Davis, CA_.

5-Fluorouracil (5-Fu) remains the standard chemotherapy for metastatic colorectal cancer, but drug resistance and unpredictable cardiotoxicity limit its effectiveness. The high recurrence rates and common resistance are thought to be due to a population of self-renewing cancer stem cells (CSCs). In this study, we synthesized four novel heterocyclic compounds that are similar in structure with quinones and tested their anticancer activity against HCT116 human colon cancer cells in 2D monolayer and 3D sphere cultures. In 2D, all compounds caused significant inhibition of colon cancer cell viability at concentrations non-cytotoxic to normal human FHs74Int intestinal cell lines. In 3D cultures, these heterocycles eradicated the self-renewal ability of the highly resistant cancer stem cells and inhibited colon sphere formation in first generation (G1), as well as subsequent generations. This study represents the first documentation of the activity of these novel heterocyclic compounds, particularly compound 6a, abbreviated as DIQ-3, which we propose to be an effective treatment strategy to prevent colon cancer recurrence by targeting colorectal CSCs. Our findings provide the basis for suggesting these non-toxic and stable compounds for additional testing against cancer.

#4798

**The Ashitaba (** Angelica keiskei **) chalcones 4-hydroxyderricin and xanthoangelol suppress melanomagenesis by targeting BRAF and PI3-K.**

Tianshun Zhang,1 Qiushi Wang,1 Hitoshi Ashida,2 Ann Bode,1 Zigang Dong1. 1 _Hormel Institute University of Minnesota, Austin, MN;_ 2 _Kobe University, Kobe, Japan_.

Malignant melanoma is an aggressive tumor of the skin and still lacks effective preventive and therapeutic approaches. In melanoma, both the BRAF/MEK/ERK and PI3-K/AKT signaling pathways are constitutively activated though multiple mechanisms, which result in cell cycle progression and prevention of apoptosis. Therefore, the development of novel strategies for targeting BRAF and PI3-K are of utmost importance. In the current study, we found that the Ashitaba (Angelica keiskei) chalcones, 4-hydroxyderricin (4HD) and xanthoangelol (XAG), suppressed melanoma carcinogenesis by directly targeting both BRAFV600E and PI3-K, which blocked the activation of downstream signaling. This led to the induction of G1 phase cell cycle arrest and apoptosis in melanoma cells. Importantly, 4HD and XAG dramatically attenuated tumor incidence and volume in the BRAF-activated Pten-deficient melanoma mouse model. Our findings suggest that 4HD and XAG are promising chemopreventive or potential therapeutic agents against melanomagenesis that act by targeting both BRAF and PI3-K, providing hope for rapid clinical translation.

#4799

Inhibition of STAT3 in pancreatic ductal adenocarcinoma and immunotherapeutic implications.

Rafal Zielinski,1 Izabela Fokt,1 Stanislaw Skora,1 Edward Felix,1 Krzysztof Grela,1 Jayakumar Arumugam,1 Radj Venugopal,1 Midan Ai,2 Genevieve Hartley,1 Michael Curran,1 Waldemar Priebe1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _Carolina BioOncology Institute, Houston, TX_.

Introduction: Signal Transducer and Activators of Transcription 3 (STAT3) plays a pivotal role in carcinogenesis, chemo- and radio-sensitivity, metastasis and immune evasion in multiple malignances including Pancreatic Ductal Adenocarcinoma (PDAC). Our drug discovery program focused on modulators of transcriptional activity led us to identify small molecules that potently inhibits tyrosine 705 phosphorylated STAT3 (p-STAT3). Compound WP1066, currently being evaluated in a Phase I clinical trial (NCT01904123) as orally administered agent and its novel, analog WP1732 suitable for IV administration, were selected as promising, potent p-STAT3 inhibitors with drug-like properties for further development as lead compounds. The purpose of these study is to perform a preclinical evaluation of WP1066 and WP1732 aiming at their future application for treatment of PDAC.

Materials and Methods: The chemical synthesis of WP1066 and WP1732 and their characterization was performed at UT MD Anderson Cancer Center. In vitro efficacy of both inhibitors was assessed using proliferation and apoptosis induction assays in a panel of patient-derived and commercially-available PDAC cell lines. Inhibition of p-STAT3 was investigated using western blot (WB) and immunofluorescence. Acute and multiple dose toxicity of WP1732 was tested in CD-1 mice. Pharmacokinetic parameters of WP1732 after intravenous administration was evaluated in naïve CD-1 mice using Mass Spectrometry LC/MS/MS or rats by liquid scintillation counting (LSC) using radio-labeled agent. Efficacy of both agents alone or in combination with immune checkpoints inhibitors was tested in PDAC tumor models.

Results: Both WP1066 and WP1732 were shown to induce apoptosis and inhibit p-STAT3 and its nuclear localization in all tested PDAC cell lines. Observed IC50 values ranged from 0.5 to 2 µM. WP1732 was well tolerated by mice (LD50 85 mg/kg given IV). Pharmacokinetic and biodistribution studies indicate high plasma levels of the drug and significant accumulation of WP1732 in the pancreas of mice and rats after a single bolus injection of the drug. Importantly, both agents show in vivo efficacy in preliminary experiments when tested alone or in combination with T cell immune checkpoint inhibitors.

Conclusion: WP1066 and WP1732 are inhibitors of p-STAT3 with demonstrated in vitro and in vivo activity against PDAC tumor models. Our preliminary data warrant the further pre-clinical and clinical evaluation of these oncology agents alone and in combination with immunotherapy as a promising new therapeutics for pancreatic cancer.

#4800

Targeting mitochondrial and cytosolic one-carbon metabolism in epithelial ovarian cancer via folate receptor alpha.

Adrianne Wallace-Povirk,1 Carrie O'Connor,1 Aamod Dekhne,1 Zhanjun Hou,1 Md. Junayed Nayeen,2 Khushbu Shah,2 Aleem Gangjee,2 Larry Matherly1. 1 _Wayne State University, Detroit, MI;_ 2 _Duquesne University, Pittsburgh, PA_.

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. Even though most patients initially respond to platinum-based therapy, the likelihood of disease reoccurrence is virtually 100%. Thus, there is an urgent need for new tumor-selective therapies for EOC. One such treatment option involves targeting tumors via folate receptor alpha (FRα) which is overexpressed in up to 90% of EOCs and shows increasing expression with stage and grade of disease. Our laboratory discovered novel 5-substituted pyrrolo[3,2-d]pyrimidine analogs (AGF291, AGF320, AGF347, AGF359 and AGF362) which inhibit mitochondrial one-carbon (C1) metabolism at serine hydroxymethyltransferase (SHMT) 2, with secondary inhibitions at cytosolic enzyme targets including those in de novo purine biosynthesis and SHMT1. Potent inhibition was seen toward isogenic Chinese hamster ovary (CHO) cell lines individually expressing FRα, the reduced folate carrier (RFC, ubiquitously expressed tissue folate transporter) and the proton-coupled folate transporter (PCFT, expressed in a limited number of normal tissues and inactive at normal pH, and several solid tumors at acidic pH including EOC), and with FRα-expressing tumor cells, including KB and IGROV1, a human EOC cell line. Inhibitory potencies were in order, AGF347 > AGF362 >> AGF291 = AGF320 = AGF359. Drug effects were substantially reduced with excess folic acid, confirming FRα-mediated drug uptake. Toward cisplatin resistant SKOV3, TOV112D and A2780 EOC cells, inhibition in the nanomolar range was detected with all compounds. Excess folic acid abrogated drug effects to varying degrees, suggesting significant uptake by the PCFT and/or the RFC, in addition to FRα. Short-term (5 minutes) cell uptake assays with the EOC cell lines and [3H]AGF347 confirmed transport by PCFT and RFC. With sustained exposures resulting in steady state AGF347 accumulations under both physiologic (pH 7.2) and acidic (pH 6.8, approximating the tumor microenvironment) conditions, FRα uptake predominated. [3H]AGF347 treatment of IGROV1 EOC resulted in substantial drug accumulation in both cytosol and mitochondria. Inhibition of cell proliferation for all analogs was reversed by addition of both glycine and adenosine, implicating C1 metabolism in mitochondria including SHMT2 and de novo purine biosynthesis in the cytosol as the targeted pathways; protection by 5-aminoimidazole-4-carboxamide (AICA) with or without glycine was incomplete, implying direct targeting of AICA ribonucleotide formyltransferase (AICARFTase), the second folate-dependent enzyme in purine biosynthesis. Our studies describe first-in-class FRα-selective compounds targeting mitochondrial and cytosolic C1 metabolism, with potent activity against EOC, including cisplatin resistant EOC.

#4801

**2'-Hydroxyflavanone, a diet-derived citrus bioflavonoid, inhibits** in vitro **and** in vivo **growth of breast cancer cells by targeting RLIP.**

Sharad S. Singhal,1 Shireen Chikara,1 Jyotsana Singhal,2 David Horne,2 Ravi Salgia,1 Sanjay Awasthi3. 1 _City of Hope, Monrovia, CA;_ 2 _City of Hope, Duarte, CA;_ 3 _Texas Tech University Health Sciences Center, Lubbock, TX_.

Breast cancer remains one of the major causes of cancer deaths in women. Consumption of citrus-fruits is associated with reduced incidence of breast cancer, the most common cancer diagnosed in women across the globe. In this study, we investigated the anticancer potential of 2-Hydroxyflavanone (2HF) in breast cancer. 2HF, a citrus-bioflavonoid, has demonstrated anticancer properties in various cancers, but its anticancer role in breast cancer has not been well studied. We investigated the in-vitro and in-vivo growth inhibitory effects of 2HF in an array of BC lines and in xenograft mouse models of ER-positive and HER2-positive breast cancer cells. Compared to control, 2HF treatment reduced cell viability of breast cancer cells, while, no growth inhibitory effects were observed in non-tumorigenic breast epithelial cells. Further, 2HF inhibited the expression of RLIP, a stress-defensive and anti-apoptotic protein, which is over-expressed in breast cancer cells and simultaneously reduced proliferation of breast cancer cells. Nude mice bearing MCF7 or SKBR3 breast cancer cells xenografts treated with either 2HF or targeting RLIP by RLIP-antisense or RLIP-antibody treatment had significantly lower tumor-weight as compared to corresponding controls. In addition, Western-blotting and immunohistochemical analysis of tumor tissue from control and treatment group mice showed that 2HF decreased protein expression levels of RLIP, and the decrease was similar to those seen following RLIP-antisense treatment. Furthermore, 2HF decreased expression of Ki67, CD31, vimentin, inhibited phosphorylation of AKT and expression of survivin and Bcl2, and increased levels of Bax, E-cadherin, and cleaved-PARP. Therefore, our results indicate that 2HF may suppress breast cancer growth in-vitro and in-vivo by targeting RLIP, and may serve as a potential adjuvant treatment in breast cancer patients. (This work was supported in part by the Department of Defense grant W81XWH-16-1-0641. Funding from the Beckman Research Institute of City of Hope is also acknowledged).

#4802

In vitro **characterization of TP-2846: a novel tetracycline antileukemia agent.**

Corey Fyfe,1 Joseph Newman,1 Ajay Bhargava,2 Juan Ballesteros,3 Alicia Robles,3 Cuixiang Sun,1 Xiao-Yi Xiao,1 Jacques Dumas1. 1 _Tetraphase Pharmaceuticals, Watertown, MA;_ 2 _Shakti Bioresearch, LLC, Woodbridge, CT;_ 3 _Viva Biotech, S.L., Madrid, Spain_.

Recently published studies suggest two hypotheses for the mechanism of action (MOA) of tetracyclines' anticancer activities: a) disruption of mitochondrial translation (Škrtić, Cancer Cell (2011) 20(5): 10.015); b) regulation of the miRNA-199b-5p-HES1-AKT pathway (Yang, Mol Cancer Ther (2016) 15(3): 1535-7163.MCT-15-0709). Upon treatment with TP-2846, suppressed levels of cytochrome oxidase-1 (COX-1) levels were demonstrated in Western blots. The COX-1 mRNA accumulated approximately 1,000-fold over background levels after 48 h incubation. Furthermore, TP-2846 displayed sub-µM inhibitory activities to mitochondrial translation (IC50 = 0.71 µM) and ribosomal translation both in E. coli and in eukaryotes (IC50 = 0.56 µM and 3.75 µM, respectively).

In addition, TP-2846 led to accumulation of the micro RNA miR-199b in MV4-11 and HL-60 cells after 24 h incubation. TP-2846 had minimal IC50 shift (Resistance Factor (RF) = 1.53) against K-562/ADR, a leukemia cell line overexpressing P-glycoprotein (Pgp), while daunorubicin displayed an RF value of 3.67 in the same assay. TP-2846 was also studied ex vivo using bone marrow samples from 15 AML patients, including those resistant to AraC, and displayed potent activity in all samples (EC50 = 150-250 nM), while anthracyclines had similar activity (EC50 = 100-600 nM) and AraC was 10x less potent (EC50 = 2.3 µM) in the same ex vivo assay.

In summary, TP-2846 exhibits both anticancer MOAs described in the literature for tetracyclines, retains antiproliferation activity in resistant leukemia cell lines, and demonstrates promising ex vivo activity in bone marrow samples from AML patients. This data indicates that further investigations in vitro and in vivo are warranted (see accompanying abstract "In vivo activities of TP-2846: a novel tetracycline antileukemia agent").

#4803

**Limiting heme availability as an effective strategy to target lung cancer growth and progression** in vivo **.**

Poorva Ghosh, Sarada Preeta Kalainayakan, Sanchareeka Dey, Adnin Ashrafi, Li Zhang. _University of Texas at Dallas, Richardson, TX_.

Lung cancer is the leading cause of cancer-related death in the US. About 85% of cases are non-small cell lung cancer (NSCLC). Several chemotherapeutic and targeted therapeutic agents are approved for treating lung cancer, but the five-year survival rate remains low. Thus, alternative approaches for are required for treatment of lung cancer. Several studies demonstrate that enhanced mitochondrial respiration or oxidative phosphorylation (OXPHOS) is a key feature of NSCLC. Heme is a central metabolic molecule that serves as a prosthetic group in several proteins involved in oxygen transport, utilization, and storage. Previous studies from our lab show elevated levels of heme synthesis, uptake, and oxygen utilizing hemoproteins in NSCLC cells compared to normal cells. The results also demonstrate that inhibiting heme synthesis selectively inhibits proliferation of NSCLC cells. We hypothesize that elevated levels of heme are used as fuel for enhanced OXPHOS by NSCLC cells. Therefore, targeting heme flux and function can be an effective way of targeting NSCLC growth and progression.

If elevated heme metabolism is crucial for the tumorigenic functions of NSCLC cells, limiting heme availability may be effective for suppressing lung tumor growth and progression. Therefore, we tried to lower heme availability by utilizing bacterial hemophores. We designed several heme-targeting peptides (HTAs) and tested their ability to inhibit heme uptake in NSCLC cell lines, subcutaneous xenografts, and lung orthotopic xenografts. NSCLC cells expressing luciferase were used to implant subcutaneous xenografts or lung orthotopic xenografts in NOD/SCID mice. The mice were treated with HTA to check if limiting heme availability would delay growth in NSCLC tumor xenografts. Also, mice with lung orthotopic xenografts treated with HTA were treated with a well-known chemotherapeutic agent, docetaxel, to determine if HTA pre-treatment improves survival in these mice. In vivo bioluminiscence imaging (BLI) was used to analyze growth and progression of orthotopic lung xenografts.

Our BLI data show that HTA treatment causes a significant decrease in radiance (total photons/second) in subcutaneous xenografts and lung orthotopic xenografts. This is supported by Hematoxylin and Eosin (H & E) staining in paraffin-embedded tissue sections from lung orthotopic xenografts and tumor volumes of subcutaneous xenograft tumors resected from mice. Furthermore, in mice with lung orthotopic xenografts, HTA pre-treatment leads to a marked improvement in survival of mice further treated with docetaxel as compared to docetaxel treatment alone.

Our results indicate that limiting heme availability can be an effective strategy to limit growth and progression of NSCLC and can be used to increase their vulnerability to chemotherapeutic agents like docetaxel.

#4804

Pre-clinical development of ISOX; A novel therapy for pancreatic cancer identified utilizing system biology approach(s).

Pranita Atri,1 Parthasarathy Seshacharyulu,1 Satyanaryana Rachagani,1 Palanisamy Nallasamy,1 Sanchita Rauth,1 Garima Kaushik,1 Koelina Ganguly,1 Dario Ghersi,2 Moorthy P. Ponnusamy,1 Sukhwinder Kaur,1 Surinder K. Batra1. 1 _University of Nebraska Medical Center, Omaha, NE;_ 2 _University of Nebraska, Omaha, NE_.

Background: Pancreatic Cancer (PC) remains one of the most common causes of cancer-related deaths. The low survival percentage in patients can be attributed primarily to ineffective therapeutic targeting and lack of new potent therapies. Over the years the conventional one target at a time approach for drug development has failed to achieve promising survival and better approaches are needed. Large-scale genomic database connectivity map (CMAP), a collection of perturbagen profiles from >2800 drugs is a potential tool to identify effective therapies for PC by identifying novel drugs that essentially reverse the global gene signature originating due to malignant PC. Considering this information, we hypothesize that "a big data approach using CMAP will lead to the identification of novel drugs for highly lethal PC."

Methods and Results: To generate a meta-gene signature for PC, GEO data for PC cases (N=106) and normal samples (N=68) was extracted, normalized & assessed for differential gene expression using R Bioconductor. Drugs specific to this gene signature across various datasets were identified using CMAP and the top hit ISOX chosen for further assessment. Considering the high mutational heterogeneity of PC, a five-cell line panel with diverse mutations was chosen for the validation studies. Cell line-based assessment for changes to proliferation using MTT and motility using matrigel assisted invasion showed the high potency of ISOX with an IC50 of 2.4nM-1.4µM & up to 90% reduction in invasion supporting initial hypothesis. It was further supported by the 48% induction of apoptosis. Molecular mechanism of ISOX was further assessed using an RNA-seq on ISOX treated PC cell lines followed by ingenuity pathway analyses. Gene ontology pathway analysis indicated SHH-WNT, PI3K-mTOR-AKT, and EGFR signaling as key pathways affected by ISOX. Further, 60% loss of viability was observed in pancreatic tumor organoids at 500nM of ISOX. Furthermore, about 10 fold statistically significant (p-value = 0.014) reduction in tumor weight at 50 mg/kg of ISOX both alone and in combination with 50 mg/kg 5FU (p-value=0.02) was observed in orthotopic mice models.

Conclusion: Our data suggest that ISOX is a potential new therapeutic for PC. In future, we aim to evaluate the potency of newly identified drug in both transgenic mouse models of PC as well as clinical phase I studies.

#4805

**Cannabinoid WIN 55,212-2 induces endoplasmic reticulum stress in prostate cancer cells through CB** 1 **and CB2receptors.**

Domenica Roberto, Laurence H. Klotz, Vasundara Venkateswaran. _Univ. of Toronto, Toronto, Ontario, Canada_.

Cannabinoids have demonstrated anticarcinogenic properties in a variety of malignancies, including prostate cancer. WIN 55,212-2 (WIN) is a highly potent synthetic cannabinoid that binds to cannabinoid receptors (CB1 and CB2). We have previously demonstrated that WIN significantly reduces prostate cancer cell proliferation, migration, invasion, induces apoptosis, and arrests cells in the G0/G1 phase through a cannabinoid receptor 2 dependent manner. We also determined that these effects were mediated though a pathway involving cell cycle regulators p27, Cdk4, and pRb. The current study aims to examine the role of endoplasmic reticulum (ER) stress in apoptosis and investigates whether this effect is modulated by WIN and the cannabinoid receptors.

In this study, we evaluated the effect of WIN and CB receptors on ER stress induced apoptosis in established prostate cancer cells (DU145, PC3). Cells were treated with WIN, cannabinoid receptor 1 antagonist (AM251), and cannabinoid receptor 2 antagonist (AM630). Cell proliferation was determined using MTS assays. Quantitative PCR was used to examine changes in expression of ER stress related genes, including CHOP, TRIB3, and ATF4. Western blotting will be completed to determine changes in the expression of apoptotic markers after treatment with WIN and cannabinoid antagonists. Further studies are ongoing looking at the use of the ER stress inhibitor, Salubrinal, to determine whether ER stress is vital for WIN-induced apoptosis.

Our results reveal that treatment with 20μM WIN resulted in a significant reduction in the proliferation of DU145 and PC3 cells after 24 h compared to control (p<0.05). In contrast, treatment with 5μM of either AM251 or AM630 did not result in any significant changes in cell proliferation. Quantitative PCR studies revealed significant upregulation of ER stress genes CHOP, TRIB3 and ATF4 in WIN treated cells (p<0.05). Expression of ER stress genes were significantly downregulated after blocking CB1 and CB2 receptors (p<0.05).

Interim results suggest that WIN has significant antitumoral activity and modulates ER stress-induced apoptosis in prostate cancer cells, thus, may offer a novel therapeutic strategy in the treatment of prostate cancer.

#4806

Development of a novel antitumor agent NCT compound for the treatment of non-small cell lung cancer.

Seung Yeob Hyun, Huong Thuy Le, Young-Sik Yong, Hye-Young Min, Jeewoo Lee, Ho-Young Lee. _Seoul National University, Seoul, Republic of Korea_.

Despite the development of advanced therapeutic regimens such as molecular targeted therapy and immunotherapy, the 5-year survival of patients with lung cancer is still less than 20%, suggesting the need to develop additional treatment strategies. Here we show the efficacy and biological mechanism of a novel antitumor agent, NCT compound. NCT compound exhibited significant inhibitory effects on the viability and colony formation of non-small cell lung cancer (NSCLC) cells and those carrying resistance to chemotherapy by inducing apoptosis. NCT compound markedly suppressed the migration of NSCLC cells. Consistently, NCT compound significantly suppressed tumor growth in a xenograft model. NCT compound showed minimal effects on the viability of normal cells and no overt toxic effects in mice, suggesting minimal toxicity of NCT compound. Further mechanistic studies revealed that NCT compound downregulated the activation of signaling pathway associated with cell proliferation and survival and the expression of epithelial-mesenchymal transition (EMT)-related markers. These results suggest the potential of NCT compound as an antitumor agent.

#4807

Pimavanserin suppresses pancreatic tumor growth and resistance to gemcitabine by inhibiting Akt/Gli1 signaling.

Sharavan Ramachandran, Sanjay K. Srivastava. _Texas Tech Univ. Health Sciences Ctr., Amarillo, TX_.

Despite major advances in cancer treatment, pancreatic cancer is still incurable and the treatment outcomes are limited. In the current study, we evaluated the anti-cancer effects of pimavanserin tartrate (PVT), a drug used for the treatment of Parkinson's disease psychosis. Our observations indicated that, PVT significantly suppressed the proliferation of pancreatic cancer cells by inducing apoptosis without exerting any cytotoxic effects in normal human pancreatic ductal epithelial (HPDE-6) cells. The colony forming ability of pancreatic cancer cells was significantly inhibited with PVT treatment. Anti-proliferative and apoptosis inducing effects of PVT were mediated by the inhibition of pAkt (Ser473), Akt, Gli1, Oct-4, SOX-2, NANOG and c-Myc. Akt and Oct-4 inhibition by PVT treatment was further validated by immunofluorescence analysis. Moreover, PVT inhibited the formation of tumorspheres in PANC1 pancreatic cancer cells. Pharmacologically inhibiting or genetically knocking out Akt or Gli1 enhanced the growth suppressive effects of PVT in pancreatic cancer cells. Subsequently, we evaluated the effects of PVT in gemcitabine resistant cells. Our results demonstrated that, PVT reduced the survival of MIAPaCa2 gemcitabine resistant cells in a concentration and time-dependent manner. Further mechanistic analysis indicated that, PVT suppressed the phosphorylation of Akt at Ser 473 and inhibited the expression of Gli1, Oct-4 and c-Myc in MIAPaCa2 gemcitabine resistant cells. Moreover, PVT increased the cleavage of caspase-3 and PARP as an indicator of apoptosis. Oral administration of PVT suppressed BxPC3 tumor xenografts by 50% in athymic nude mice. In another in vivo experiment, PVT treatment inhibited the growth of orthotopically implanted PANC1 tumors by 75%. Chronic administration of PVT did not exhibit any general signs of toxicity or behavioral side effects in mice. Taken together, our results indicate that pancreatic tumor growth suppression by PVT is orchestrated by inhibition of Akt/Gli-1 signaling. Since PVT is already available in the clinic with an established safety profile, our results will accelerate its clinical development for the treatment of patients with pancreatic cancer.

#4808

Preclinical studies of the combination of ONC201, radiotherapy and Temozolomide against GBM, DIPG and ATRT cell lines.

Lanlan Zhou, Wafik S. El-Deiry. _Fox Chase Cancer Center, Philadelphia, PA_.

The American Cancer Society predicts, in 2018, there will be ~23,880 new cases and 16,830 deaths caused by primary cancer of the brain and spinal cord, in the US. CNS tumors are the most common cause of cancer-related deaths in adolescents and young adults. First-in-class small-molecule imipridone ONC201 can act as a dual inhibitor of ERK/AKT and can induce an integrated stress response (ISR), pro-apoptotic TRAIL receptor DR5 activation, cancer stem cell depletion, and growth arrest. ONC201 crosses the blood brain barrier and has demonstrated clinical benefits in glioblastoma patients. Radiation therapy is used alone or in combination with surgery and/or chemotherapy such as Temozolomide in the treatment of primary or metastatic brain tumors. We hypothesized that ONC201 may synergize with radiotherapy and Temozolomide in brain tumor treatment. Glioblastoma (GBM), diffuse intrinsic pontine glioma (DIPG) and atypical teratoid rhabdoid tumor (ATRT) cell lines were tested in this study. Cell viability and colony formation assays were performed with ONC201 alone up to 20 μM or in combination with radiotherapy up to 8 Gy and Temozolomide up to 100 μM. Western blots were used to document apoptosis of treated cells. We observed synergy between ONC201 and radiation and between ONC201 and Temozolomide with the best combination indices of 0.51 and 0.21 respectively. Further studies are evaluating the role of dopamine receptors engaged by ONC201, the ISR and TRAIL pathway in the synergistic effect that would support further development of the triple combination therapy in brain tumors.

#4809

**Bromo-ormeloxifene inhibits epithelial mesenchymal transition** via **targeting β-catenin signaling pathways in cervical cancer cells.**

Mohammed Sikander,1 Shabnam Malik,1 Bilal B. Hafeez,1 Sonam Kumari,1 Sheema Khan,1 John Apraku,2 Hassan Mandil,1 Andrew E. Massey,1 Aditya Ganju,1 Parvez Khan,3 Fathi T. Halaweish,2 Subhash C. Chauhan,1 Meena Jaggi1. 1 _Univ. of Tennessee Health Science Ctr., Memphis, TN;_ 2 _South Dakota State University, Brookings, SD;_ 3 _Jamia Millia Islamia, New Delhi, India_.

Background: Cervical cancer (CxC) is a leading cause of mortality and morbidity among women worldwide. Current chemotherapeutic agents for CxC have shown systemic toxicity in CxC patients. Ormeloxifene (ORM) is a non-toxic and non-steroidal drug with well-defined pharmacokinetic and pharmacodynamic properties in humans. Studies have shown its anti-cancer potential in various pre-clinical mouse models. Here, we have synthesized and characterized a novel analogue of ormeloxifene, Bromo-ormeloxifene (Br-ORM), which showed more therapeutic efficacy against CxC in vitro and in vivo model systems.

Methodology: The effect of Br-ORM on CxC cells (CaSki and SiHa) growth and proliferation was determined by colony formation and MTS assays. Molecular docking of Br-ORM with β-catenin was done by AutoDock4 software. Effect of Br-ORM on the expression of epithelial-to mesenchymal (EMT) markers (N-cadherin, slug, snail), MMPs (MMP2 and MMP3) and miR-200a was analyzed by Western blot and qPCR analyses respectively. Apoptosis analysis was done by Annexin-V staining kit. Effect of Br-ORM on β-catenin cellular localization in CxC cells was analyzed by immunofluorescence analysis. The anti-tumor efficacy of Br-ORM was investigated in orthotopic xenograft mouse model of CxC.

Results: Br-ORM (10-20 µM) effectively inhibited growth and proliferation of CxC cells in a dose and time-dependent manner as compared to ORM. Br-ORM efficiently suppressed metastatic phenotypes of CxC cells as determined by significant (P<0.05) decrease in invasion and migration potential of CxC cells. Moreover, Br-ORM showed increased apoptosis, which was observed by enhanced Annexin-V staining and PARP protein cleavage. Br-ORM markedly reduced the EMT process as evident by repression of N-cadherin, slug, snail, MMPs (MMP2 and MMP3) and β-catenin/TCF-4 transcriptional activity. Br-ORM potently reduced the translocation of β-catenin in the nucleus. Bioinformatic analysis revealed that Br-ORM proficiently binds into active site of β-catenin with a minimum energy -7.6 kcal/mol. Br-ORM treatment replenished the expression of miR-200a, which directly targets β-catenin in CxC cells. Br-ORM treatment (250 µg/mouse, three times a week) significantly (P<0.01) regressed the cervical tumor growth in orthotopic xenograft mouse model. Similar molecular effects of Br-ORM were observed in excised tumor tissues.

Conclusion: These results suggest that Br-ORM inhibits the metastatic phenotypes of CxC cells via targeting β-catenin signaling pathway. Br-ORM could be used as a novel therapeutic modality for the treatment of CxC.

#4810

Olive oil-derived (-)-oleocanthal for prevention of breast cancer recurrence.

Abu Bakar Siddique,1 Nehad M. Ayoub,2 Khalid A. El Sayed1. 1 _Univ. of Louisiana College of Pharmacy, Monroe, LA;_ 2 _Jordan Univ. of Science & Technology Faculty of Pharmacy, Irbid, Jordan_.

Despite progress in BC therapy and improved survival rates, several breast cancer (BC) patients have poor recurrence-free rates. More than three million BC survivors are currently at the risk of disease recurrence without feasible preventive options. Surgical excision of early-stage confined breast tumors commonly used to minimize subsequent metastasis. Neoadjuvant and adjuvant therapeutic agents usually cannot prevent BC recurrence. There is a dire need to discover novel recurrence and metastasis inhibitory entities because clinical trials on early-stage cancer patients survival and metastases/recurrence reduction endpoint is not financially feasible and need large patients number. Mediterranean populations have less colon and breast cancers incidence due to their dietary consumption of significant amounts of phenolics-rich extra-virgin olive oil (EVOO). (-)-Oleocanthal (OC) is the most bioactive EVOO phenolic with diverse activities and exceptional in vivo potency. Validated molecular targets of OC in BC include the inhibition of the activation of the RTKs c-Met and HER2 and modulation of estrogen receptors (ER). Met/HER2/ER amplification is implicated in activation of quiescent breast tumor cells, repopulation, subsequent recurrence and relapse. This study reports the potent ability of four-weeks oral 10 mg/kg OC treatments to inhibit more than 90% of local and regional triple negative and hormone-dependent breast tumors recurrence after the surgical excision of orthotopically xenografted primary breast tumors. Oral OC treatments also potently inhibited recurrence after surgical excision of breast tumors subjected to neoadjuvant therapies with either lapatinib (50 mg/kg, 5X/week, 4 weeks) or paclitaxel (4.5 mg/kg, ip, 3X/week for 6 weeks) in athymic orthotopic nude mice models. This was associated with significant reductions of p-c-Met and p-HER2 levels in treated animal tumors by Western blotting. OC-treated mice sera completely lacked the BC recurrence marker CA 15-3, unlike vehicle treated controls which showed significantly high CA 15-3 levels. OC treatment did not show any observable toxicity and did not affect animals body weight over the experiment course. OC is a novel breast cancer recurrence inhibitory dietary supplement lead with excellent clinical applications potential.

#4811

Functional pathway analysis in pancreatic cancer cells treated with a novel anti-cancer agent, copper-tolfenamic acid.

Myrna Hurtado,1 Laszlo Prokai,1 Umesh T. Sankpal,1 Blair Levesque,1 Rajasekhar Maram,1 Jaya Chhabra,2 Deondra T. Brown,2 Raj K. Gurung,2 Alvin A. Holder,2 Jamboor Vishwanatha,1 Riyaz Basha1. 1 _University of North Texas Health Science Center, Fort Worth, TX;_ 2 _Old Dominion University, Norfolk, VA_.

Anti-cancer activity of tolfenamic acid (TA) has been studied using several preclinical cancer models including pancreatic cancer (PaCa). Since the dosage used for the anti-cancer actions of TA is rather high, we investigated Copper-TA (Cu-TA) for anti-proliferative activity using 12 cancer cells (representing 6 cancers) and demonstrated that Cu-TA is more effective than TA (IC50 values are 30-80% less than TA). Recently, we reported that Cu-TA inhibits tumor growth in mouse xenograft (pancreatic tumor) model. The objective of this investigation is to elucidate the underlying mechanisms of Cu-TA using PaCa cell lines. MIA PaCa-2 cells were treated with vehicle (dimethyl sulfoxide) or 29 µM (IC50) of Cu-TA and processed by next-generation sequencing (NGS) to determine the differentially expressed genes using Oncology Biomarker (HTG EdgeSeq) panel. The functional significance of the altered gene expression was determined via Ingenuity Pathway Analysis which identified several networks, regulators, as well as molecular and cellular functions that were affected by Cu-TA treatment. A total of 18 networks were found to be involved with Cu-TA treatment and these networks had a significant overlap. Confirmation experiments assessing the alterations in the expression of selected candidate markers were conducted by qPCR and Western blot analysis using both MIA PaCa-2 and PANC 1 cell lines. The top upstream regulators included tumor protein p53, human epidermal receptor growth factor 2, Sp1 and signal transducer and activator of transcription 3. These regulators were confirmed by Western blot analysis. The top five molecular/cellular functions affected by Cu-TA treatment were cell death/survival, cellular development, cell growth/proliferation, cell cycle and cellular movement. qPCR results of selected genes, Centromere protein F, DNA damage inducible transcript 3 and S-phase kinase-associated protein 2 (differentially expressed genes in sequencing) demonstrated that Cu-TA is efficacious at lower doses than TA. In summary, the NGS and Ingenuity Pathway Analysis identified critical genes or pathways altered by Cu-TA. This investigation demonstrated that the networks and regulators associated with cancer cell survival or apoptosis that are modulated in PaCa cells suggests that Cu-TA is altering genes associated with cancer, thereby demonstrating its potential as an effective anti-cancer agent.

#4812

9-ING-41, a novel inhibitor of glycogen synthase kinase-3beta (GSK-3β), is active as a single agent and within combination therapies in bladder cancer cell lines.

Hiroo Kuroki,1 Tsutomu Anraku,1 Vladimir Bilim,2 Masayuki Tasaki,1 Daniel Schmitt,3 Andrew Mazer,4 Francis J. Giles,3 Andrey Ugolkov,5 Yoshihiko Tomita1. 1 _Niigata University, Niigata, Japan;_ 2 _Kameda Daiichi Hospital, Niigata, Japan;_ 3 _Actuate Therapeutics, Fort Worth, TX;_ 4 _Monopar Therapeutics, Wilmette, IL;_ 5 _Tempus, Chicago, IL_.

Glycogen synthase kinase-3beta (GSK-3β) is a serine/threonine protein kinase that has been established as a therapeutic target in a broad spectrum of human malignancies. We have previously identified aberrant GSK-3β nuclear expression in bladder cancer (BC), and demonstrated that GSK-3β positively regulated BC cell survival and proliferation. Our objective was to evaluate the antitumor effects of the clinically viable agent 9-ING-41, a maleimide-based ATP-competitive GSK-3 inhibitor, which has broad spectrum antitumor activity and marked activity in reversing chemoresistance in a variety of pre-clinical models of human cancers. We used flow cytometry, Western immunoblotting, quantitative RT-PCR, BrDU incorporation and MTS assays to examine antitumor activity of 9-ING-41 in BC (T24, HT1376, RT4 cell lines). Additionally, we studied combination therapy of 9-ING-41 with gemcitabine and cisplatin, standard agents used for the treatment of patients with advanced BC, with cabozantinib, a multi kinase inhibitor, and with chloroquine, an autophagy inhibitor. A dose-dependent decrease in cancer cell proliferation was observed by MTS assay and BrdU incorporation assay with GI50 0.7μM (T24), 4.7 μM(HT1376), 2.2μM(RT4). Treatment with 9-ING-41 induced prominent cell cycle arrest (predominantly G2 arrest) in BC cells. Expression of cell cycle related proteins, including Cyclin D, and anti-apoptotic proteins, such as XIAP and Bcl-2, were significantly decreased as detected by Western immunoblotting and real time RT-PCR. Treatment with 9-ING-41 significantly potentiated the growth inhibitory effect of cisplatin and gemcitabine in cultured cells. In addition, combination therapy with 9-ING-41 and cabozantinib or chloroquine, significantly potentiated the effect of either single agent. Our data proved that a single treatment using 9-ING-41 is effective in BC cells. Combination treatment of 9-ING-41 with standard chemotherapeutic drugs or novel agents may provide a new therapeutic approaches in BC. These data provide a rational for the inclusion of patients with advanced BC in clinical studies of 9-ING-41. 

### Targeted Therapies

#4813

Comparative activity profiling of tyrosine kinase inhibitors (TKIs) against exon 20 insertions and the wild-type form of epidermal growth factor receptor (EGFR).

Richard A. Ward, Ambra Bianco, Nicola Colclough, Darren Cross, Emanuela M. Cuomo, M. Raymond V. Finlay, Martina Fitzek, Nicolas Floc'h, Sladjana Gagrica, Beverley Hammond, Matthew J. Martin, Darren McKerrecher, Daniel J. O'Neill, Jonathan P. Orme, Paul D. Smith, Anna D. Staniszewska, Jelena Urosevic, Nicky Whalley, James W. Yates. _AstraZeneca, Cambridge, United Kingdom_.

Exon 20 insertions (Ex20Ins) have been identified in approximately 5% of epidermal growth factor receptor (EGFR)-mutated lung tumours in patients presenting with non-small cell lung cancer (NSCLC). Several small molecule tyrosine kinase inhibitors (TKIs) have been reported to have pre-clinical activity against such insertions including afatinib, poziotinib, osimertinib, nazartinib, AP32788/TAK-788 and TAS6417. However, there remains a lack of approved treatments for patients with Ex20Ins with early approved EGFR agents appearing to be ineffective in this setting. Poziotinib, osimertinib and AP32788/TAK-788 are undergoing clinical evaluation in patients whose tumours carry Ex20Ins and in some cases clinical responses have been reported giving hope that such insertions can be targeted by small molecules. There is however a need for comparable data across such compounds that would enable understanding of the relative activity of these compounds between Ex20Ins and the wild-type form of EGFR. As many of the Exon 20 insertions are not part of the ATP binding pocket achieving selectivity over wild type EGFR is highly challenging and may limit the clinical utility of agents due to dose limiting EGFR wild-type driven toxicity.

A selection of TKIs were profiled for Ex20Ins and wild-type EGFR activity using biochemical, in vitro cellular phosphorylation and proliferation assays. This has enabled us to differentiate the Ex20Ins versus wild-type EGFR selectivity profiles of a range of pre-clinical, clinical and proprietary compounds. As part of this evaluation we utilized a CRISPR CAS9 approach in H2073 EGFR wild-type NSCLC cell line, where we have established cellular disease models against the most prevalent insertions including D770-N771insSVD (22%). Finally, we will show anti-tumour efficacy data for a selection of these inhibitors along with a potential combination approach of osimertinib and cetuximab. | |

|

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

|

EGFR

D770-N771InsSVD

cell phospho

IC50 (µM) | EGFR WT

(H2073)

cell phospho

IC50 (µM) | Fold-

EGFR WT margin

(cell)

afatinib | 0.006 | 0.0033 | 0.5

osimertinib | 0.094 | 0.41 | 4.4

poziotinib | 0.0034 | 0.0035 | 1

TAS6417 | 0.023 | 0.067 | 2.9

AZ6281 | 0.097 | 2.2 | 23

#4814

Specificity, biodistribution, tumor targeting, and pharmacokinetics of a novel humanized anti-Globo H antibody, OBI-888, for cancer immunotherapy.

Yu-Chi Chen, Ming-Chen Yang, Chi-Sheng Shia, Chun-Yen Tsao, Jiann-Shiun Lai, I-Ju Chen. _OBI Pharma, Inc., Taipei, Taiwan_.

Background: OBI-888 is a humanized monoclonal IgG1 antibody that binds to Globo H (GH), a tumor associated carbohydrate antigen. It is developed as a therapy to treat GH positive cancers. In the present study, we examined the binding specificity, binding epitope, antigen targeting ability, and pharmacokinetics (PK) of OBI-888.

Methods: The binding specificity of OBI-888 was evaluated by cross-reactivity ELISA and titration ELISA with various glycans. Competition ELISA using various truncated structures of GH was conducted to identify the binding epitope of OBI-888. To confirm the antigen targeting ability, OBI-888 labeled with 111In via p-SCN-bn-DTPA chelator were IV injected in mice with tumor bearing GH positive MCF7 cells and no-tumor bearing. 111In-DTPA-OBI-888 at 32 μCi/4 μg and 400 μCi/16 μg were injected for biodistribution and MicroSPECT imaging studies, respectively. PK profile of OBI-888 was evaluated in non-tumor bearing nude mice at 5 mg/kg of OBI-888 via IV injection (n=5).

Results: OBI-888 demonstrated specific binding to GH, with minimal cross reactivities to the other 25 types of glycan tested. Among the Globo series antigens, such as SSEA-3-ceramide and SSEA-4-lipid, OBI-888 showed specific binding to GH-ceramide. In addition, OBI-888 does not bind to truncated structures of GH and instead, requires a full hexasacharide structure of GH. Biodistribution study showed that 111In-OBI-888 was preferentially localized to the tumor site. The uptake of In111-OBI-888 in MCF7 tumor plateaued at 11.71±2.38 %ID/g at 24-hour post injection and remained at similar levels until 72-hour post injection. The tumor/muscle ratio peaked at 11.26±1.90 %ID/g at 72-hour post injection. Besides highly concentrated in the tumor sites, the dynamics and extent of distributions of OBI-888 in various organs of the tumor-bearing mice were comparable to that of non-tumor bearing mice. The radioactivity accumulation of In111-OBI-888 in liver, spleen and kidneys were approximately 10% ID/g at 1 h postinjection and decreased to about 5% ID/g at 24 h postinjection. MicroSPECT imaging studies confirmed the localization of 111In-OBI-888 at the tumor site of MCF7 bearing mice. PK parameters were concluded as follows: half-life (T1/2) = 5.1 days; estimated clearance (CL): 29.1 mL/d/kg; steady state volume of distribution (Vss): 179.3 mL/kg.

Conclusions: The present study identified the epitope of OBI-888 and demonstrated its binding specificity. The observed biodistribution and microSPECT results demonstrated OBI-888's specific targeting to GH positive tumor cells. Pharmacokinetic profiles showed acceptable half-lives in mice. Overall, the pre-clinical data support a first-in-human trial in cancer immunotherapy. Moreover, our findings indicate that the OBI-888 has the potential to be developed into a diagnostic agent for imaging GH-expressing cancers.

#4815

Novel Globo H targeting antibody-drug conjugate with binding specificity and anti-tumor efficacy in multiple cancer types.

Ming-Chen Yang, Yu-Jung Chen, Chi-Sheng Shia, Hui-Wen Chang, Wan-Fen Li, Cheng-Der Tony Yu, I-Ju Chen. _OBI Pharma. Inc., Taipei, Taiwan_.

Background: Globo H, a hexasaccharide, has been reported to be highly expressed in multiple cancers types, but not express or, if at all, express to a lesser extent in normal tissue. Therefore, Globo H may be a potential target for cancer immunotherapy. OBI-999 is an antibody-drug conjugate (ADC) which consists of a Globo H-specific monoclonal antibody OBI-888, conjugated with monomethyl auristatin E (MMAE), a synthetic antineoplastic agent. The present study investigates antigen specificity, drug internalization, anti-tumor efficacy, and pharmacokinetics profiles of OBI-999.

Methods: Globo H expression was surveyed in various human cancer cell lines. The binding specificity and internalization of OBI-999 were determined by flow cytometry and confocal microscopy, respectively. In vivo anti-tumor efficacy was studied in conventional xenograft and patient-derived xenograft models. Pharmacokinetic parameters were evaluated in normal and tumor bearing xenograft mice.

Results: We confirmed positive Globo H expression on the cell surface of multiple cancer cell lines ranging from breast, gastric, lung, to pancreatic cancer. From flow cytometry, OBI-999 showed binding selectivity to the aforementioned Globo H expressing cell lines. In contrast, OBI-999 did not bind to non-Globo H expressing cell lines. When bound to the antigen, OBI-999 was internalized and trafficked to endosome and lysosome within 2.5 to 5 hours, suggesting that the drug payload, MMAE, was cleaved in a lysosome dependent manner. OBI-999 showed significant tumor inhibition in breast, gastric, lung, and pancreatic cancer xenograft and PDX models in dose-dependent manner. At 1 mg/kg of OBI-999, tumor growth inhibition in breast, gastric, and lung cancer model were 77%, 89%, and 68% respectively. At 10 mg/kg of OBI-999, complete tumor growth inhibition in pancreatic cancer model was observed. In vitro serum stability studies revealed that OBI-999 has comparable stabilities to Adcetris® in mouse, rat, monkey, and human sera. Tissue distribution study revealed that OBI-999 is accumulated gradually at the tumor site while the level of OBI-999 showed a time dependent decrease in blood-rich organs. Accumulation of OBI-999 at the tumor site reached its maximum level at 168 hour post treatment. Level of MMAE in tumor mass was approximately 25 folds higher than that in other organs and 250 folds higher than that in serum, suggesting that OBI-999 targets and releases its payload at tumor region.

Conclusion: The preclinical studies of OBI-999 demonstrated its targeting specificity and anti-tumor efficacy in multiple Globo H positive cancer models. OBI-999 also demonstrated a longer retention at tumor site. The preclinical results provided the fundamental basis for OBI-999's clinical application as targeted cytotoxicity therapy for solid tumors.

#4816

Anetumab ravtansine has monotherapy efficacy in mesothelin positive patient-derived NSCLC tumor models and in a syngeneic tumor model in immunocompetent mice.

Anette Sommer,1 Pascale Lejeune,1 Sabine Hoff,1 Annette O. Walter,1 Sandra Berndt,1 Lars Roese,1 Andreas Schlicker,1 Michael J. Wick,2 Cem Elbi,3 Dominik Mumberg,1 Christoph Schatz1. 1 _Bayer AG, Berlin, Germany;_ 2 _START, San Antonio, TX;_ 3 _Bayer Heathcare Pharmaceuticals, Whippany, NJ_.

Chemotherapy and immune checkpoint inhibitors (ICIs) are approved for treatment of non-small-cell lung cancer (NSCLC). However, there still remains a high medical need in NSCLC, eg. in patients non-responsive to ICIs or progressed after treatment with ICIs. Mesothelin (MSLN) is expressed in ~60% of lung adenocarcinomas. Here, we describe the mesothelin targeting antibody drug conjugate anetumab ravtansine (ARav) with the maytansinoid payload (DM4) as a novel treatment option for NSCLC.

In the NSCLC cell line-derived xenograft model NCI-H322, ARav dosed at 2.5 mg/kg or 10 mg/kg, Q3Dx3, i.v., showed significant antitumor activity and was superior to cisplatin (dosed 3 mg/kg, Q3Dx12, ip). In addition, ARav monotherapy also showed antitumor activity in MSLN-positive NSCLC patient-derived xenograft (PDX) tumor models ST1243 and ST1684 at 15 mg/kg, Q2W.

Maytansinoids and maytansinoid-based ADCs have been described to induce immunogenic cell death and immune response in vitro and in vivo, respectively. To explore the effects of ARav alone or in combination with anti PD-L1 Ab on tumor growth and the immune system, the MC38 C57BL/6 mouse colon cancer cell line was stably transfected with human mesothelin (MC38-hMSLN).

MC38-hMSLN cells were transplanted s.c. in immunocompetent C57BL/6 mice to evaluate the anti-tumor activity of ARav. MC38-hMSLN had a high hMSLN expression level as shown by IHC. A dose of 10 mg/kg of ARav (Q3Dx3, i.v.) was highly efficacious with 11/12 animals showing complete regression of the tumor. Tumor free survivors (TFS) re-challenged 80 days after treatment all rejected MC38-hMSLN (11/11 animals), indicating the development of an immune memory response. The specificity of the immune response was further confirmed in an independently conducted experiment, where previously ARav treated MC38-hMSLN TFS mice were re-challenged with B16-F10 melanoma cells, which grew.

Next, a lower dose of ARav (3 mg/kg, Q3Dx3, i.v.) was combined with an anti PD-L1 antibody in the MC38-hMSLN tumor bearing mice. The combination led to increased frequency of TFS compared to each monotherapy (12/12 TFS in the combination versus 7/12 for the anti PD-L1 and 2/12 for ARav). Further studies are currently ongoing to optimize the combination dosing and schedule as well as to characterize the immune cells involved in the response.

In summary, the data supports the development of ARav in NSCLC and further exploration of ARav in combination with immune checkpoint inhibitors in MSLN-positive cancer indications.

Anetumab ravtansine clinical activity is currently assessed at phase I studies in ovarian cancer in combination with pegylated liposomal doxorubicin (Phase 1b, NCT02751918), in multiple indications including NSCLC (Phase Ib, NCT03102320), and in combination with the anti PD-L1 antibody atezolizumab in NSCLC (Phase I /II, NCT03455556).

#4817

**Preclinical evaluation of a new, non-agonist ADC targeting** MET **-amplified tumors with a peptide-linked maytansinoid.**

Katharine C. Lai, Min Li, Kathryn Selvitelli, Surina Sikka, Steven Boulé, L Cristina Gavrilescu, Stuart W. Hicks, Kerry Donahue. _ImmunoGen, Inc., Waltham, MA_.

With cancer among the leading causes of death worldwide, the search for better, personalized treatments is imperative. Novel techniques such as next generation sequencing have identified many assayable genetic biomarkers associated with cancer in patient samples. The tyrosine kinase receptor cMet is one such biomarker that is upregulated in various solid tumors and associated with poor prognosis, disease progression and metastasis. While most patients with elevated cMet show increased levels through protein upregulation, a small population harbors gene amplification. These patients face worse outcomes which could be improved with therapies specifically targeting MET-amplification. Antibody-drug conjugates (ADCs) are a modality designed to selectively deliver highly potent cytotoxic agents to tumors. cMet is an attractive target for ADCs which may address the unmet treatment need for patients with tumors harboring MET amplification. Since dimerization of cMet receptors by ligand HGF leads to agonistic proliferative events, a carefully selected antibody should be chosen to avoid triggering activation. As previously described, we identified and humanized an antibody with minimal agonism. Introducing an additional disulfide in the hinge region while maintaining the IgG1 isotype further reduced agonism as measured in vitro in both cell proliferation and phosphorylation signaling assays, while retaining high affinity to human and cynomolgus cMet, and acceptable expression and biophysical properties. To assess potential toxicity due to normal tissue expression, we measured binding of our antibody to normal hepatocytes from humans and cynos. Here we found very low expression and binding versus tumor cell lines. Next, we demonstrated that the cytotoxic activity of disulfide-cleavable maytansinoid ADCs prepared from the hinge-variant cMet antibody were equivalent to the parental form in in vivo models. In a MET-amplified xenograft model of gastric cancer, Hs746T, both parental and modified hucMet-sSPDB-DM4 ADCs demonstrated tumor eradication and comparable plasma clearance at 5 mg/kg. Similar results were found in the MET-amplified NSCLC model EBC-1 at 2.5 mg/kg. Conjugation to the newly-described dipeptide-linked maytansinoid DM21 further improved anti-tumor activity in both Hs746T and EBC-1 models, with a 2-fold decrease in minimally-efficacious dose. The activity of hucMet27Gv1.3Hinge-L-DM21 was durable, with a single dose yielding full regressions and tumor-free survivors in both models (EBC-1, 1.25 mg/kg dose, 6/6 TFS d49; Hs746T, 2.5 mg/kg dose, 8/8 TFS d55). Taken together, these data demonstrate compelling cMet-targeted activity of hucMet27Gv1.3Hinge-L-DM21 in MET-amplified models of NSCLC and gastric cancer with a wide margin of safety. These data merit further exploration of this ADC as a novel treatment option for patients with MET-amplified tumors.

#4818

Fc-mediated mechanism of action for the novel EGFR-cMET bispecific antibody (JNJ-61186372) in non-small cell lung cancer.

Smruthi Vijayaraghavan,1 Barbara Bushey,1 Lorriane Lipfert,2 Rupesh Nanjunda,1 Eilyn R. Lacy,1 Peter Buckley,1 Sylvie Laquerre,1 Matthew V. Lorenzi,1 Sheri Moores1. 1 _Janssen Research & Development, Spring House, PA; _2 _VWR Catalyst, Spring House, PA_.

JNJ-61186372 (JNJ-372) is an anti-EGFR and cMet bispecific antibody with an active Fc backbone (IgG1) designed to treat non-small cell lung cancer (NSCLC) disease. A first-in-human study is currently being conducted to assess the safety and preliminary efficacy of JNJ-372 in patients with advanced NSCLC. Early data suggests that JNJ-372 can induce partial responses in subjects with diverse populations of EGFR-mutated NSCLC, including Exon 20ins as well as TKI resistance mutations.

Our previous pre-clinical in vivo studies showed that the Fc inactive version (IgG2sigma) of the EGFR/cMet antibody was significantly impaired in its ability to inhibit tumor growth compared to the Fc active JNJ-372. The IgG2sigma variant also reduced the ability of the bispecific antibody to mediate downregulation of EGFR, cMet and downstream signaling components. This suggested that the interaction of the Fc arm with the Fc receptors on the innate immune cells play a crucial role in the mechanism of action of JNJ-372. While JNJ-372 has demonstrated antibody dependent cellular cytotoxicity (ADCC) and antibody dependent cellular phagocytosis (ADCP) in vitro, the potential role of Fc interactions in downregulation of EGFR and cMet was not well understood.

We explored the different Fc-mediated immune effector functions of JNJ-372 and interrogated how they contribute to EGFR and cMet downregulation and overall efficacy. In NSCLC cell lines, JNJ-372 induced Fc-mediated dose-dependent ADCC and ADCP but not complement-dependent cytotoxicity. Further, the presence of isolated human immune cells (PBMC) significantly enhanced JNJ-372 mediated EGFR and cMet downregulation and dose-dependent tumor cell killing. Studies are in progress to better understand which immune cells and which Fc receptor interactions are essential for the drug efficacy through immune cell depletion and FcR blocking studies; such studies may help guide clinical biomarker development. This work elucidates a novel Fc-dependent mechanism of action for the EGFR-cMet bispecific antibody.

#4819

Sacituzumab Govitecan (IMMU-132) in uterine serous carcinoma.

Salvatore Lopez, Chanhee Han, Burak Zeybek, Elena Bonazzoli, Anna Bianchi, Paola Manara, Stefania Bellone, Aranzazu Manzano, Emanuele Perrone, Luca Zammataro, Gary Altwerger, Alessandro D. Santin. _Yale Univ., New Haven, CT_.

Objective: Uterine serous carcinoma (USC) is an aggressive variant of endometrial cancer with poor prognosis. Sacituzumab govitecan (IMMU-132) is a novel antibody-drug conjugate (ADC) targeting trophoblast antigen 2 (Trop-2), a cell surface glycoprotein highly expressed in many epithelial tumors including USC, to deliver SN-38, the active metabolite of irinotecan. The objective of this study was to preclinically evaluate the efficacy of IMMU-132 against primary USC cell lines and xenografts.

Methods: Trop-2 expression in primary tumor cell lines and USC cell viability after exposure to IMMU-132 ADC (hRS7-CL2A-SN-38), non-targeting control ADC (h679-CL2A-SN-38), and naked antibody hRS7 IgG were evaluated using RT-PCR and flow-cytometry-based-assays. Antibody-dependent-cell-cytotoxicity (ADCC) against Trop-2+ and Trop-2- USC cell lines was evaluated in vitro using 4 hr Chromium release assays. Finally, in vivo activity of Sacituzumab govitecan was tested against Trop-2+ USC xenografts by 3 twice-a-week retro-orbital injection of 500 μg of IMMU-132, control-ADC, and hRS7 naked-IgG.

Results: Overexpression of Trop-2 was detected in 67% (8 out of 12) of primary USC cell lines. Primary tumors overexpressing Trop-2 were significantly more sensitive (ie, lower IC50) to IMMU-132 (hRS7-CL2A-SN-38) when compared to control ADC (h679-CL2A-SN-38). Both sacituzumab govitecan (IMMU-132) and the naked antibody hRS7 induced high level of ADCC against Trop2+ USC cell lines while no cytotoxicity was detected against Trop-2 negative tumors. In vivo experiments comparing IMMU-132 activity to control ADC and hRS7 showed a dramatically improved tumor suppression and increased survival in IMMU-132 treated mice when compared to controls (P = 0.0001 and P = 0.0002, respectively).

Conclusion: IMMU-132 demonstrated remarkable antitumor activity against biologically aggressive USC overexpressing Trop-2. Our preclinical results combined with the dramatic clinical response recently reported in an USC patient treated with IMMU-132 (https://doi.org/10.1016/j.gore.2018.05.009) strongly supports further clinical development of sacituzumab govitecan in USC patients with advanced/recurrent disease. (ie, clinical trial IND 140394). Furthermore, due to a cleavable linker that can cause bystander effect, sacituzumab govitecan could be also effective in tumors with heterogenous TROP-2 expression.

#4820

Pediatric preclinical testing consortium evaluation of the CD123 antibody drug conjugate, IMGN632, against xenograft models of pediatric acute lymphoblastic leukemia.

Kathryn Evans,1 Narimanne El-Zein,1 Connor Jones,1 Stephen W. Erickson,2 Yuelong Guo,2 Beverly A. Teicher,3 Sharlene Adams,4 Patrick A. Zweidler-McKay,4 Malcolm A. Smith,3 Richard B. Lock1. 1 _Children's Cancer Institute, Sydney, Australia;_ 2 _RTI International, Research Triangle Park, NC;_ 3 _National Cancer Institute, Bethesda, MD;_ 4 _ImmunoGen Inc, Waltham, MA_.

Introduction: The characteristic expression of CD123 (the alpha subunit of the IL-3 receptor) in multiple hematological malignancies, including acute myeloid leukemia (AML), blastic plasmacytoid dendritic cell neoplasm and acute lymphoblastic leukemia (ALL) has made this antigen an attractive target for the development of new therapeutics. IMGN632 is a CD123 targeting antibody drug conjugate (ADC) comprised of a novel humanized antiCD123 antibody, G4723A, linked to a DNA monoalkylating payload of the indolindobenzodiazepine pseudodimer (IGN) class of cytotoxic compounds. IMGN632 demonstrates potent activity in AML samples at low concentrations with minimal impact on normal bone marrow progenitors, and antileukemia effects in AML xenograft models. A phase I clinical trial of IMGN632 in CD123 positive malignancies is ongoing (NCT033865). Therefore, it was of interest to test the in vivoefficacy of IMGN632 against preclinical models of pediatric ALL.

Methods: Pediatric ALL patient-derived xenografts (PDXs) grew in an orthotopic manner in NSG mice. Engraftment was assessed by the % human leukemic blasts in the peripheral blood (%huCD45+). Treatment commenced when the median %huCD45+exceeded 1%, and mice received IMGN632 or non-binding control ADC (240 µg/kg IV weekly x 3) or vehicle. An event was defined as the %huCD45+>25% or leukemia-related morbidity. The Kaplan-Meier method was used to compare event-free survival (EFS) between treated (T) and control (C) groups. Stringent objective response measures were assigned to each mouse and reported as group medians. Cell surface CD123 levels were expressed as specific antibody binding capacity (sABC). IMGN632 was provided by ImmunoGen, Inc.

Results: IMGN632 was tested against 8 pediatric ALL PDXs (3 B-ALL; 3 MLLr-ALL; 2 Ph+-ALL) in which median cell surface CD123 expression ranged from 510-3375 sABC. NSG mice tolerated IMGN632 well with maximum average weight losses of 0.0-7.7% across treatment groups compared to 0.0-8.1% in vehicle controls. IMGN632 induced significant differences in EFS distribution compared to control in 8 of 8 PDXs. T-C values ranged from 9.8 to 64.8 days (T/C 3.0-9.4) and Maintained Complete Responses were observed in 6 of 8 PDXs. In 6 of 8 PDXs the median EFS of the IMGN632-treated groups extended >5 weeks beyond the end of treatment. The control ADC did not significantly delay the progression of any PDX. There was no apparent correlation between CD123 sABC levels and in vivo activity with IMGN632 efficacy being observed across all CD123 expression levels.

Conclusions: IMGN632 exerted profound in vivo efficacy against PDXs derived from a broad range of ALL subtypes and with a broad range of CD123 antigen levels. These data strongly support the clinical investigation of IMGN632 in acute lymphoblastic leukemia. (Supported by NCI Grants CA199222 & CA199000)

#4821

Development of a potent Trop-2 antibody-drug conjugate, BAT8003, for the treatment of Trop-2 positive gastric tumors.

Weijia Tang,1 Xingxing Mei,1 Ziqiang Ou,1 Jirong Gan,1 Shengfeng Li,2 Jin-Chen Yu2. 1 _Bio-Thera Solutions, Guangzhou, China;_ 2 _Bio-Thera Solutions, Elkridge, MD_.

Trophoblast cell surface antigen 2 (Trop-2) is overexpressed on many epithelial carcinomas while having low levels of expression on normal tissue. Overexpression of Trop-2 has been correlated with poor prognosis in several solid tumors. Trop-2 is overexpressed in ~60% of gastric cancer, which causes more than 500,000 mortalities every year in China. We have developed a Trop-2 antibody-drug conjugate (ADC), BAT8003, that utilizes an anti-Trop-2 IgG1 antibody, engineered for site-specific conjugation, a novel uncleavable linker, and a potent maytansine derivative as the payload. Site-specific conjugation allows for a more controllable drug-antibody ratio (DAR), resulting in a more homogenous product. BAT8003 also incorporates increased ADCC effects through afucosylation to further enhance potency. Here we show that BAT8003 is internalized in Trop-2 positive cells. It inhibits proliferation of a number of different Trop2-overexpressed tumor cells with IC50s of ~1 nM. In a gastric cancer (NCI-N87) mouse xenograft model, BAT8003 demonstrates strong inhibition activity on tumor growth at doses of 5 mg/kg and 15 mg/kg. A multiple dose toxicity study in monkey reveals that the highest non-severely toxic dose (HNSTD) of BAT8003 is 20 mg/kg when dose once every 3 weeks. The preclinical profile of BAT8003 warrants further clinical development for the treatment of gastric cancer as well as other Trop-2 over-expressing cancers.

#4822

Targeting RAS and downstream signaling in high-grade serous ovarian carcinoma with novel RAS inhibitors.

Tyler E. Mattox, Tiffany S. Norton, Adam B. Keeton, Antonio B. Ward, Yulia Y. Maxuitenko, Kristy L. Berry, Bing Zhu, Alla Musiyenko, Elaine Gavin, Veronica Ramirez-Alcantara, Xi Chen, Jacob Valiyaveettil, Jennifer Scalici, Rodney P. Rocconi, Gary A. Piazza, Luciana Madeira da Silva. _University of South Alabama, Mobile, AL_.

High grade serous ovarian carcinoma (HGSOC), which accounts for 70-80% of ovarian cancer deaths, is characterized by TP53 mutations and about half of these tumors present defects in homologous recombination DNA repair pathway genes. PARP inhibitors have now become FDA-approved for the treatment of recurrent and BRCA-associated ovarian cancer. Despite providing a major advance in ovarian cancer treatment in recent years, resistance to PARP inhibitors is often encountered and new treatment strategies must be devised. Oncogenic RAS and constitutively active RAS signaling regulate multiple downstream cascades to increase cell proliferation, survival, and malignant transformation. Despite the absence of RAS mutations, upregulation in the RAS pathway is prevalent in HGSOC, suggesting that these represent key, yet under-explored, inhibition targets for the treatment of ovarian cancer. Further bolstering the importance of RAS pathway inhibition are reports on cytotoxic synergistic effects for the combination of MEK and PARP inhibitors in ovarian cancer. The development of inhibitors directly targeting RAS has been hindered by the lack of suitable surfaces on the protein for small molecule binding and its high affinity for GTP binding. We developed a novel series of indene derivatives that showed highly selective growth inhibitory activity in tumor cells harboring constitutively active RAS vs. cells with low levels of active RAS. Chemical optimization led to a series of compounds that potently and selectively inhibit RAS-dependent tumor cell growth by blocking GTP binding to RAS. Here we examined the effects of RAS inhibition by two novel compounds, MCI-059 and MCI-062, in a panel of HGSOC cell lines with variable baseline levels of active RAS. We observed potent growth inhibitory activity for these novel inhibitors in most of the 15 ovarian cancer cell lines tested (IC50s ~25nM and 7nM for MCI-059 and MCI-062, respectively), with the lesser sensitivity in OV-90 cells correlating with its lowest basal levels of active RAS measured by a pull down assay using GST-RAF1-RBD/ glutathione agarose beads. Time-course experiments revealed that MCI-062 induces apoptosis beginning at 18-24 hours. Furthermore, both MCI-059 and MCI-062 inhibited: i) RAS-RAF1-RBD binding in GTPγS loaded recombinant human KRAS; ii) RAS-RAF1-RBD binding in OVCAR8 and SKOV3ip cells under normal culture growth conditions or under serum starvation followed by EGF-stimulation; iii) EGF-stimulated activation of the RAF-MEK-ERK and PI3K-AKT cascades in OVCAR-8 and SKOV3ip cells. We also confirmed that MCI-062 decreases several components of the RAS downstream signaling pathway via RPPA analysis. In summary, our results demonstrate that MCI-059 and MCI-062 inhibit HGSOC cell growth by blocking RAS-effector interactions, and support further evaluation of our novel RAS inhibitors for the treatment of ovarian cancer.

#4823

Elimination of invasive pancreatic cancer cells by p53-activating oncolytic virotherapy as novel precision medicine.

Takuro Fushimi,1 Hiroshi Tazawa,1 Takeshi Koujima,1 Hiroyuki Araki,1 Takeyoshi Nishiyama,1 Satoru Kikuchi,1 Shinji Kuroda,1 Ryuichi Yoshida,1 Hiroyuki Kishimoto,1 Masahiko Nishizaki,1 Yasuo Urata,2 Shunsuke Kagawa,1 Toshiyoshi Fujiwara1. 1 _Department of Gastroenterological Surgery, Okayama University, Okayama, Japan;_ 2 _Oncolys BioPharma, Inc., Tokyo, Japan, Japan_.

Background: Pancreatic ductal adenocarcinoma (PDAC) is the most lethal disease due to early onset of local recurrence and distant metastasis. Invasive PDAC cells are the main causes of recurrence and metastasis after curative surgery. The precision medicine based on the genetic alterations in the KRAS, p53, CDKN2A, and SMAD4 genes has been recently expected as a promising strategy for the treatment of PDAC patients; however, therapeutic strategy for targeting these genetic alterations in PDAC has not been developed yet. To eliminate p53-inactivated malignant tumor cells, we have developed telomerase-specific replication-competent oncolytic adenovirus OBP-702 that expresses tumor suppressor p53. In this study, we investigated the in vitro and in vivo therapeutic potential of OBP-702 against PDAC cells.

Methods: Four human PDAC cell lines (Capan-1, MIA PaCa-2, BxPC-3, Panc-1) with different invasive property were used. The therapeutic effect of OBP-702 and p53-nonexpressing OBP-301 was assessed in the proliferation, migration and invasion abilities of PDAC cells. The underlying mechanism of virus-mediated therapeutic effect was analyzed on the modulation of p53 signaling and KRAS-MAPK signaling. Subcutaneous and orthotopic BxPC-3 xenograft tumor models were used to evaluate the virus-mediated antitumor efficacy.

Results: OBP-702 induced antitumor effect in association with autophagy and apoptosis more strongly compared to OBP-301 in PDAC cells through activation of p53 expression. OBP-702 inhibited the migration and invasion properties of PDAC cells more efficiently compared to OBP-301 through suppression of KRAS-ERK1/2 signaling, even when ERK signaling is enhanced by nerves and neurosecretory factors. Similar with OBP-702, treatment with ERK1/2 inhibitor or siRNA significantly reduced migration and invasion abilities. Moreover, OBP-702 significantly suppressed tumor growth in subcutaneous and orthotopic BxPC-3 xenograft tumor models.

Conclusions: Our results suggest that OBP-702 is a promising antitumor reagent to eliminate invasive PDAC cells through p53 activation and ERK suppression. Further clinical study is warranted to evaluate the safety and feasibility of OBP-702 as a novel precision medicine based on genetic alterations in KRAS and p53 genes.

#4824

**Targeting CD30 as a novel treatment strategy in** RANBP2-ALK **-rearranged inflammatory myofibroblastic tumor.**

Ashleigh M. Fordham,1 James Blackburn,2 Erin E. Heyer,2 Jinhan Xie,1 Emily V. Mould,1 Andrew J. Gifford,1 Lisa T. Morgan,1 Carol Wadham,1 Mitali Fadia,3 Jamie I. Fletcher,1 Karen L. MacKenzie,4 Toby N. Trahair1. 1 _Children's Cancer Institute Australia, Randwick, Australia;_ 2 _Garvan Institute of Medical Research, Darlinghurst, Australia;_ 3 _ACT Pathology, The Canberra Hospital, Canberra, Australia;_ 4 _Children's Medical Research Institute, Westmead, Australia_.

Rationale: Inflammatory myofibroblastic tumors (IMTs) are a particularly rare type of soft tissue sarcoma comprised of myofibroblastic spindle cells and an accompanying inflammatory infiltrate. There is an unmet clinical need for effective treatment regimens for patients diagnosed with IMT with anaplastic lymphoma kinase (ALK) rearrangement, who relapse following ALK inhibitor (ALKi) therapy or who present with aggressive disease. Fusion of RAN Binding Protein 2 (RANBP2) with ALK in IMT is associated with aggressive disease and has been correlated with tumor cell expression of CD30. This study investigated CD30 as a potential therapeutic target in IMT and the efficacy of the CD30-targeted antibody-monomethyl auristatin E conjugate, Brentuximab Vedotin (BV).

Methods and Results: In a cohort of five recent IMT patients at the Sydney Children's Hospital, RANBP2-ALK fusion was identified in three patients (IMT1, IMT2 and IMT3) by RNA capture sequencing, while patients IMT4 and IMT5 (who did not relapse) harbored CLTC-ALK or SEC31A-ALK fusions respectively. Expression of CD30 was confirmed two of three RANBP2-ALK fusion positive tumors by immunohistochemistry. We established cell cultures and xenografts from malignant ascites of IMT1, at diagnosis (IMT1A) and at relapse (IMT1D) after treatment with ALKi's and low dose chemotherapy. CD30 expression was retained in the cell cultures and xenograft tumors, as demonstrated by flow cytometry and tumor histology. BV was investigated as a potential treatment for IMT with RANBP2-ALK fusion. BV reduced IMT1A and IMT1D cell viability in vitro in resazurin cell viability assays. IMT1A and IMT1D xenograft mice had a partial response to BV which significantly (p<0.0001) prolonged survival compared to untreated controls. However, tumors eventually recurred. Resistance to BV correlated with upregulation of P-glycoprotein and reduced CD30 antigen expression in cells from treated IMT xenograft tumors. The combination of the ALK inhibitor crizotinib with BV has also been investigated. In vivo, the combination of crizotinib and BV resulted in complete resolution of tumor and significantly (p<0.0001) improved survival compared to the individual agents.

Conclusion: CD30 is a promising therapeutic target in RANBP2-ALK-rearranged IMT. BV successfully reduced IMT cell viability in vitro and prolonged survival in IMT xenografted mice, both as a single agent and when given in combination with crizotinib. Since BV is current clinical use for the treatment of Hodgkin lymphoma it may be possible to rapidly translate these findings into clinical practice for the treatment of IMT.

#4825

Preclinical characterization of BAY-924, a first in class ADC targeting CXCR5-positive B-cell malignancies, with a KSP inhibitor as novel payload.

Sarah Johannes, Stefanie Hammer, Stephan Maersch, Hans-Georg Lerchen, Beatrix Stelte-Ludwig, Hannah Joerissen, Oliver von Ahsen, Christoph Schatz, Simone Greven, Christoph Mahlert, Dominik Mumberg, Pascale Lejeune. _Bayer AG, Berlin, Germany_.

Despite recent progress in the treatment of B-cell malignancies, patients are still in need of innovative therapeutic approaches. CXCR5 is a chemokine receptor expressed in a majority of B-cell malignancies including diffuse large B-cell lymphoma (DLBCL), Mantle cell lymphoma (MCL), follicular lymphoma and chronic lymphocytic leukemia. Evaluation of tumor biopsies from relapsed DLBCL patients shows that CXCR5 staining remains high, suggesting altogether that it could be a relevant target to explore for the treatment of non-Hodgkin lymphoma. BAY-924 is a novel first-in-class antibody drug conjugate (ADC) consisting of a humanized anti-CXCR5 IgG1 antibody (Ab) linked to a potent proprietary kinesin spindle protein inhibitor (KSPi). Of importance, the structure of the ADC is optimized for a specific metabolism, matching the KSPi mode of action and enabling a maximal retention of the payload within the tumor cells (Lerchen HG et al.; Angew. Chem. Int. Ed, 2018). Surface plasmon resonance assay showed a high binding affinity of the Ab to CXCR5 (2.5 nM). Affinities of 0.8 to 10 nM were measured by flow cytometry for the ADC in different CXCR5\+ lymphoma cell lines. In vitro, BAY 924 had high and selective anti-proliferative activity in a panel of tumor cell lines with different levels of CXCR5 expression (<0.03 - 2 nM IC50). Efficient internalization and lysosomal co-localization of the Ab was observed in a variety of cell lines including the CXCR5+ MCL REC-1 cells. In vivo, BAY-924 was highly active in several CXCR5+ lymphoma models with a specific accumulation of the payload in tumor versus liver, spleen and kidney and almost undetectable levels in plasma. In the REC-1 model implanted subcutaneously (SC) in mice and treated at large tumor size (500 mm3), long lasting tumor regression was observed after 2 intravenous injections of BAY 0924 at 10 mg/kg, Q7D, whereas the model was insensitive to ibrutinib, a current standard of care (SoC) for the MCL indication. In the advanced ABC DLBCL model OCI-LY1 (SC), a single injection of BAY-924 at 10 mg/kg induced complete responses in 10/10 mice (up to day 95 post-treatment). In this model, head-to-head comparison showed superior activity of BAY-924 compared to the SoCs rituximab (R)-CHOP, R/bendamustine and R/lenalidomide. Also, BAY-924 induced potent antitumor effect with a 4% ΔT/ΔC (day 55) in the ABC DLBCL OCI-Ly3-2b model, expressing weak to moderate levels of CXCR5 in vivo. Given its unique structure, and based on supportive data from other projects, it is anticipated that BAY-924 shows a favorable safety profile, due to the high stability of the ADC and a non-cell permeable free payload which is trapped inside the tumor cells. Overall, these results support further development of BAY-924 as an innovative approach for the treatment of CXCR5+ non-Hodgkin lymphoma.

#4826

Enhancement of PTEN activity via peptidomimetics.

Emily Palumbo, Peng Teng, Prerna Malaney, Jacob Wilson, Fiona Kearns, Michael T. Kemp, Zhi Tian, Vladimir Uversky, Diane Allen-Gipson, Yu Chen, H. Lee Woodcock, Jianfeng Cai, Vrushank Dave. _University of South Florida, Tampa, FL_.

Compromised PTEN function is associated with multiple cancers. As per the continuum model, variable degree of PTEN inactivation drives distinct cancer phenotypes. Loss of PTEN activity, via genomic/non-genomic mechanisms, leads to enhanced oncogenic PI3K signaling. While kinase inhibitors have proved effective in the clinic, they are increasingly met with off-target effects and therapy-resistance due to compensatory feedback mechanisms. Although PTEN restoration therapy has proved promising in experimental models; implementation of these therapies in the clinic remains challenging. To fill this therapy gap, we have targeted endogenous PTEN for activation via peptidomimetics, mitigating PI3K signaling. Our selected peptidomimetics reduced cell proliferation, migration and cell cycle activity after treatment of non-small cell lung cancer cells expressing endogenous PTEN. Computational studies utilizing PTEN crystal structures revealed a binding site at the interface of the Phosphatase Domain (PD) and C2 Domain (C2D). Induced fit docking indicated that a unique functional group is responsible for peptidomimetic-mediated enhancement of PTEN activity. Molecular dynamics analysis revealed energetically favorable binding of our lead peptidomimetic at the PD/C2D interface, which allosterically modulated the conformation of the active site. Binding of our peptidomimetic altered the active site orientation of PIP3, increasing its binding affinity. In summary, we have developed the first-known direct PTEN activators and analyzed their effects in suppressing oncogenic activities in lung cancer. Refinement of our peptidomimetics, complemented with in vivo studies, will provide a novel clinical rationale to reduce doses of standard therapy and their associated toxicities, decreasing morbidity and mortality observed in the clinic.

#4827

**The therapeutic superiority of neratinib in combination with trastuzumab compared to pertuzumab plus trastuzumab in HER2-positive** in vivo **breast cancer models.**

Jamunarani Veeraraghavan,1 Vidyalakshmi Sethunath,1 Martin J. Shea,1 Tamika Mitchell,1 Resel Pereira,1 Lanfang Qin,1 Sarmistha Nanda,1 Carmine De Angelis,1 Kristina Goutsouliak,1 Irmina Diala,2 Alshad S. Lalani,2 Sepideh Mehravaran,1 susan G. Hilsenbeck,1 Chandandeep Nagi,1 Carolina Gutierrez,1 Mothaffar F. Rimawi,1 C. Kent Osborne,1 Rachel Schiff1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _PUMA Biotechnology Inc., Los Angeles, CA_.

Neoadjuvant clinical trials in HER2+ breast cancer showed that lapatinib (L) plus trastuzumab (T), combined with endocrine therapy for ER+ tumors, achieved meaningful complete pathologic response rates without chemotherapy. The irreversible pan-HER kinase inhibitor neratinib (N) has shown greater potency compared to L in the preclinical setting. However, the efficacy of N in combination with T (N+T) and how it compares to pertuzumab (P) +T (without chemotherapy) has not been well studied. We hypothesize that dual HER2 inhibition using N+T will be highly efficacious due to more complete blockade of the HER pathway, with comparable or better potency than P+T. Here, we evaluate the therapeutic efficacy and molecular mechanisms of N, P, and T, either alone or in combination, in cell- and patient-derived xenograft (PDX) models. Immunodeficient mice bearing BT474-AZ cell (ER+/HER2+), and BCM-3963 PDX tumors (ER-/HER2+, wild-type PIK3CA) were randomized to vehicle, N, T, P, N+T, or P+T, with simultaneous estrogen deprivation in BT474-AZ xenograft model. Study endpoints included: (i) treatment outcome - time to tumor regression (TTR) and progression (TTP) (tumor halving/doubling over baseline, respectively), and rate and time to complete response (CR and TCR, respectively); and (ii) biomarker analysis - immunohistochemistry (IHC) and western blot (WB) analysis of tumors harvested 2-4 days post-treatment to assess key biomarkers. In the BT474-AZ model, while tumor regression was observed in 100% of N, P, T, N+T, and P+T treated mice, the tumors treated with N+T regressed faster compared to P (p<0.001), T (p=0.004), and P+T (p=0.044). Further, N+T was superior to N (p=0.018), and T (p=0.007) alone in achieving accelerated CR. In the BCM-3963 model, which was refractory to T, P, or T+P, while CR was achieved in 100% of N and N+T treated mice, the combination of N+T accelerated the attainment of CR compared to N alone (p=0.026). IHC analysis of short-term treated tumors showed that Ki67, pAKT, and pMAPK levels were significantly inhibited by N and N+T, but not by T, P, or P+T. Compared to P+T, N and N+T more potently inhibited Ki67, suggesting the superiority of N-containing regimens in suppressing tumor cell proliferation. Likewise, WB analysis showed that N and N+T markedly inhibited pHER2 (Y1248), pEGFR (Y1068), pAKT (S473), pERK, and pS6 levels, compared to P+T, suggesting a more potent blockade of the HER pathway by N-containing regimens, especially after short-term treatment. In the BT474-AZ model, short-term N+T treatment yielded greater inhibition of pHER2 (Y1248) and survivin levels, compared to N alone. These preclinical findings establish the efficacy of combining N with T for HER2+ breast cancer and support further clinical testing to investigate the efficacy of N+T without chemotherapy in the neoadjuvant setting for patients with HER2+ breast cancer.

#4828

Anti-tumor activity of BAY-943, an anti-IL3RA ADC with a novel KSP inhibitor payload, in CDX and PDX AML models.

Anette Sommer,1 Dennis Kirchhoff,1 Antje M. Wengner,1 Beatrix Stelte-Ludwig,2 Hans-Georg Lerchen,2 Anne-Sophie Rebstock,3 Oliver von Ahsen,1 Lisa Dietz,2 Pascale Buchmann,2 Sandra Johanssen,1 Dominik Mumberg,1 Bertolt Kreft1. 1 _Bayer AG, Berlin, Germany;_ 2 _Bayer AG, Wuppertal, Germany;_ 3 _Bayer SAS, Lyon, France_.

Despite recent progress in the treatment of AML, clinical outcomes have improved only minimally over the past three decades. Therefore, novel therapeutic agents with a high therapeutic window and a favorable tolerability profile are urgently needed to improve the therapeutic outcome for AML patients.

IL3RA (CD123) is the alpha subunit of the interleukin 3 (IL-3) receptor which regulates proliferation, survival and differentiation of hematopoietic cells. IL3RA is expressed at high frequency, with ~84% of AML cases and 59% of classical Hodgkin lymphoma (cHL) cases being positive. IL3RA is expressed in AML blast and leukemic stem cells (LSCs) but not in hematopoietic stem cells (HSCs). In healthy individuals, the IL3RA expression is restricted to myeloid progenitor cells, plasmacytoid dendritic cells (pDCs), basophils and - at low levels - monocytes and B-lymphocyte subsets. This expression pattern suggests that IL3RA could be a clinically relevant target for an antibody-drug conjugate (ADC) approach in treatment of AML, cHL, and MDS.

BAY-943 is a novel antibody-drug conjugate (ADC) consisting of a humanized internalizing anti-IL3RA IgG1 antibody (Ab, EC50 on IL3RA-positive tumor cells in flow cytometry: 2-5 nM) conjugated via lysine residues to a potent proprietary kinesin spindle protein inhibitor (KSPi). The kinesin spindle protein (KSP/Eg5/KIF11) is essential for the proper segregation of duplicated centrosomes during spindle formation in the G2/M phase of the cell cycle, as such it is only active in proliferating cells.

In vitro, in a panel of IL3RA-positive AML and HL cell lines, BAY-943 showed potency in the nano- to subnanomolar range. In IL3RA-positive cell line derived (CDX) AML xenograft models (MOLM-13 and MV4-11) and patient-derived xenograft (PDX) models, BAY-943 dosed at 10 mg/kg given Q7Dx increased survival compared to vehicle treated mice. Tumor burden (percentage of human CD45 positive AML cells) was significantly reduced compared to vehicle treated mice. In the subcutaneous IL3RA-positive cHL CDX model HDLM-2, BAY-943 dosed at 5 and 10 mg/kg Q7Dx2 induced complete tumor remission in 12 out of 13 mice.

In safety studies in Cynomolgus monkeys, BAY-943 (which is cross-reactive with Cynomolgus IL3RA), up to 20 mg/kg single or 10 mg/kg repeat (QWx3) dose were well tolerated with no signs of thrombocytopenia, neutropenia and no liver toxicity, i.e. adverse events observed with ADCs containing other payload classes. As expected, a transient reduction of IL3RA expressing cell types (basophils, pDCs) was observed.

In summary, IL3RA-KSPi-ADC BAY-943 shows efficacy in IL3RA-positive AML and HL models and has a favorable safety profile in monkey repeat dose studies. Overall, the preclinical results support further development of BAY-943 as an innovative approach for the treatment of IL3RA-positive AML.

#4829

Improved safety profile of HER2-KSPi-ADCs compared to T-DM1 in in vitro megakaryocyte assay predictive of thrombocytopenia.

Anette Sommer,1 Pascale Buchmann,2 Hans-Georg Lerchen,2 Beatrix Stelte-Ludwig,2 Christian Bertling,2 Jenny Thoennes,2 Sandra Johanssen,1 Christoph Schatz,1 Dominik Mumberg1. 1 _Bayer AG, Berlin, Germany;_ 2 _Bayer AG, Wuppertal, Germany_.

KSP inhibitors (KSPis) are a versatile new payload class for the generation of highly potent and selective antibody-drug conjugates (ADCs) against different targets. For HER2 (c-ERBB2)- and TWEAKR (Fn14/ TNFRSF12A)-KSPi-ADCs, we have previously shown that they have potent and selective anti-proliferative activity and induce apoptosis in HER2- or TWEAKR-positive cancer cell lines in vitro. Moreover, TWEAKR-KSPi-ADCs induced strong and long-lasting anti-tumor efficacy and complete tumor regression in cell line- and patient-derived xenograft models.

Shortcomings of clinically tested and marketed ADC payload classes are the off-target / on-toxophore dose-limiting toxicities observed in the clinic, in particular neutropenia and thrombocytopenia. Thrombocytopenia is a common side effect of 3 of 4 approved ADCs (including T-DM1, Kadcyla). Thrombocytes are generated by proliferation, differentiation and fragmentation of specific megakaryocyte (MK) progenitors. As ADCs can be taken up by differentiating hematopoietic stem cells the released toxic payload can inhibit MK proliferation/differentiation and prevent generation of platelets resulting in thrombocytopenia.

The potential to induce thrombocytopenia of KSPi-based ADCs with different effector chemistries (ECs) was compared with the clinically approved ADC T-DM1 (with a non-cleavable SMCC linker) by evaluating their impact on in vitro megakaryocyte differentiation. To this end, the HemaToxTM MK assay (Stemcell, Cologne, Germany) was used which allows to assess the impact of compounds on proliferation/differentiation of CD34+ stem cells into GPIIb/IIIa (CD41) and CD45 double positive megakaryocytes by flow cytometry.

Whereas the anti-HER2 Ab trastuzumab had no impact on MK differentiation, T-DM1 elicited MK toxicity in vitro. HER2-targeted or isotype control KSPi-ADCs with a legumain (LGMN) cleavable EC showed a comparably benign profile in the MK assay as trastuzumab, and a further improved profile versus KSPi-ADCs with a non-cleavable EC. Both features, the LGMN specific cleavage of KSPi-ADCs after internalization and cellular trafficking to the lysosome, and the released, non-cell-permeable KSPi payload, may contribute to an improved safety profile compared to T-DM1. This is further supported by the safety profile of a KSPi-ADC with a LGMN cleavable EC was also studied in vivo: In a repeat dose Cynomolgus study with the IL3RA-KSPi-ADC (BAY-943) dosed up to 10 mg/kg (QWx3), thrombocytopenia was not observed.

These results indicate that KSPi-ADCs with specifically designed effector chemistries containing LGMN cleavable linkers have an improved safety profile in the MK assay with first evidence that this may translate also in a better safety profile with regard to lack of induction of thrombocytopenia in vivo.

#4830

ABX9xx: A bispecific centyrin that synergizes to attenuate intracellular signaling in Met/EGFR positive tumors.

Russ Addis, Robert Kolakowski, Swapnil Kulkarni, Josh Gorsky, Rebecca Meyer, Karyn ONeil. _Aro Biotherapeutics Company, Philadelphia, PA_.

ABX9xx is a bispecific Centyrin targeting the Met-EGFR axis, an emerging cancer treatment paradigm. Centyrins are small, chemically simple proteins based on a human FN3 domain that can be engineered to have high affinity for a selected target and are easily linked to form multi-specific binders. The standard of care for patients with EGFR mutant non-small cell lung cancer (NSCLC) is treatment with small molecule tyrosine kinase inhibitors (TKI) including recently approved third generation molecules (ie. osimertinib). Despite initial promising responses with TKIs, most patients regress within 12-14 months due to resistance. A predominant resistance mechanism dependent on Met amplification and signaling has been described from both in vitro experiments and clinical data. Targeting Met and EGFR with a bispecific ligand blocking inhibitor that attenuates intracellular signaling may provide a significant efficacy advantage and reduced side effect profile compared to small molecule TKI combinations. Exploiting the potential for avidity on tumor tissue, a bispecific ligand blocking inhibitor of Met and EGFRis also anticipated to provide improved selectivity for tumor tissue that overexpress both receptors compared to normal tissue with lower receptor expression. To enable sustained exposure while maintaining small size for good tumor penetration, ABX9xx includes a domain comprising an albumin binding Centyrin. ABX9xx activity on Met, EGFR and downstream signaling proteins was confirmed on several lung cancer cell lines in vitro including those carrying clinically observed mutations in Met and EGFR. ABX9xx inhibited EGFR phosphorylation independent of EGFR mutational status with higher potency than osimertinib. In addition, ABX9xx inhibited Met phosphorylation with higher potency than Crizotinib, an ALK inhibitor with cross reactivity to Met, currently used to treat Met positive patients. The pharmacokinetic properties of ABX9xx in animal models suggest a suitable dosing paradigm in patients. Together the data provides a strong rationale for advancing ABX9xx into clinical development for NSCLC and other cancers where EGFR and MET are drivers of tumor progression.

#4831

**Using reporter-based bioassays and engineered TNFalpha and VEGF** + **target cells to measure Fc-mediated ADCC and CDC activity of anti-TNFá and anti-VEGF therapeutic antibodies.**

Denise Garvin, Jamison Grailer, Rich Moravec, Jim Hartnett, Chris Heid, Frank Fan, Mei Cong, Jey Cheng. _Promega Corporation, Madison, WI_.

Fc receptor-mediated effects contribute to the therapeutic response in terms of efficacy and safety of anti-TNF antibodies. Measurement of Fc-mediated antibody-mediated cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) during antibody drug discovery and development is not only important for antibodies that harness ADCC and/or CDC as their primary mechanism of action (e.g. rituximab, trastuzumab), but also for antibodies designed to target and block soluble ligands such as TNFα and VEGF. We previously reported the development of a cell-based reporter bioassay platform which has been used to measure ADCC and ADCP mediated through FcgRI, FcgRIIa and FcgRIIIa. These reporter bioassays exhibit the specificity, accuracy, precision, and robustness necessary for qualification according to ICH guidelines and have been used extensively to characterize and measure the potency of antibody-based biologics drugs that target cell surface immune receptors. In the current study, we sought to evaluate Fc-mediated ADCC and CDC activities of therapeutic antibodies designed to target and block soluble ligands including TNFa and VEGF. To measure ADCC activity of anti-TNFa and anti-VEGF blocking antibodies, we developed engineered target cells that express either membrane-bound TNFα or VEGF. When used as target cells with reporter-based effector cells expressing a relevant FcgR, ADCC activity of adalimumab (anti-TNFa) and bevacizumab (anti-VEGF) was detected in a specific and dose-dependent manner. Similarly, when used in a luminescence-based CDC assay, the engineered target cells elicited an appropriate FcgR-mediated response. The assay signals demonstrated IgG isotype specificity as IgG4 variants showed minimal activity in both ADCC and CDC assays. Our results demonstrate that the combined use of cell-based reporter bioassays with target cells engineered to express membrane-bound soluble ligands can provide a simple, specific, and quantitative platform to measure Fc-mediated effector functions of therapeutic antibodies targeting soluble ligands.

#4832

Preclinical evaluation of neratinib plus T-DM1 in orthotopic PDX models of HER2-positive breast cancer brain metastases.

Jing Ni,1 Yanzhi Wang,1 Irmina Diala,2 Sheheryar Kabraji,1 Rachel Freedman,1 Nancy Lin,1 Jean Zhao1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Puma Biotechnology, Los Angeles, CA_.

Up to half of patients with metastatic HER2-positive breast cancer will develop brain metastases. Breast cancer brain metastases (BCBM) are a major cause of morbidity and mortality, despite multimodal management including surgery, radiotherapy, and systemic therapies. Therefore, there is an urgent need to develop novel, efficacious therapies. Neratinib is an orally bioavailable, irreversible pan-HER tyrosine kinase inhibitor that is FDA-approved in the extended adjuvant treatment setting for HER2-positive, early breast cancer. Neratinib has only modest activity as a single agent in clinical trials of patients with HER2-positive brain metastases. Though the combination of neratinib and capecitabine results in CNS responses in up to half of patients, patients eventually develop drug resistance, and toxicities have been a concern-thus exploration of alternative neratinib combinations is of significant clinical interest. Ado-trastuzumab emtansine (T-DM1) is an antibody-drug conjugate with reported single-agent activity against HER2-positive BCBM. Here, we used HER2-positive orthotopic patient derived xenograft (PDX) models of BCBM to test if combining neratinib with T-DM1 could improve tumor response. PDX cells are labelled with luciferase to allow tumor growth measurement in vivo. We found that neratinib is able to reduce phosphorylated HER2 in an orthotopic PDX tumor derived from HER2-positive BCBM, indicating that neratinib can cross the BBB and inhibit HER2 activation in BCBM PDX tissues. However, in the HER2-positive DF-BM354 PDX model, single agent neratinib did not block orthotopic tumor growth compared to vehicle control as monitored by bioluminescence measurements. In contrast, combined treatment of neratinib with T-DM1 significantly reduced tumor growth compared to single agent treatment of neratinib or T-DM1 alone at earlier time points. At later time points, the combined treatment is comparable to T-DM1 alone. These data warrant further testing of neratinib alone or in combination with T-DM1 in additional BCBM PDX models to better understand drivers of resistance and susceptibility to HER2-inhibitors in HER2-positive BCBMs. Furthermore, they support the launch of a prospective clinical trial (NCT01494662) to test the efficacy and tolerability of T-DM1 in combination with neratinib in patients with progressive HER2-positive BCBM.

#4833

Antibody-drug conjugate targeting glypican-1 shows tumor growth inhibition in cholangiocarcinoma.

Keiichiro Yokota, Satoshi Serada, Shigehiro Tsujii, Kosuke Hiramatsu, Tsutomu Namikawa, Ichiro Murakami, Kazuhiro Hanazaki, Tetsuji Naka. _Kochi University, Kochi, Japan_.

Cholangiocarcinoma is one of the most highly malignant cancers. Many patients need systemic chemotherapy for tumor development and recurrence, but their prognosis is poor. Therefore new treatment options are urgently required. We confirmed that the expression of glypican-1 (GPC1) was enhanced in cholangiocarcinoma. GPC1 is a cell surface membrane protein and has been reported as a poor prognostic factor in pancreatic cancer. In this study, we aimed to develop a new therapy for cholangiocarcinoma by the antibody-drug conjugate (ADC) targeting GPC1. By immunohistochemical analysis, enhanced expression of GPC1 was observed in clinical specimens of cholangiocarcinoma. KKU-055 and KKU-100 cells, which are cell lines of cholangiocarcinoma, had high expression of GPC1. In the KKU-055 xenograft model when we administered fluorescently-labeled GPC1 antibody to mice, it accumulated in tumor tissue. We developed a new anti-GPC1 monoclonal antibody (mAb) with highly internalizing activity. The anti-GPC1 mAb was conjugated with the cytotoxic agent monomethyl auristatin F (MMAF). GPC1-ADC showed potent antitumor effect toward KKU-055 and KKU-100 cells compared with the control ADC. In the KKU-055 xenograft model, GPC1-ADC had significant and potent tumor growth inhibition in a dose-dependent manner. In summary, our newly-developed GPC1-ADC showed significant tumor growth inhibition against GPC1-positive cholangiocarcinoma cell lines. Our preclinical data demonstrated that targeting GPC1 by ADC is a promising therapy for GPC1-positive cholangiocarcinoma.

#4834

Effective treatment of squamous cell carcinoma of the head and neck (HNSCC) using a combined regimen of tipifarnib and cetuximab.

Lihua Shu,1 Dongsheng Wang,1 Sreenivas Nannapaneni,1 Linda Kessler,2 Dong M. Shin,1 Nabil F. Saba,1 Georgia Z. Chen1. 1 _Emory Univ., Atlanta, GA;_ 2 _Kura Oncology, Inc, San Diego, CA_.

Background: H-RAS driven carcinogenesis has been reported in many types of cancer including squamous cell carcinoma of the head and neck (HNSCC). The mutation rate of H-RAS in HNSCC is 4 - 8%. Tipifarnib is a potent and highly selective inhibitor of farnesyltransferase (FT). It is known that H-RAS, but not K-RAS and N-RAS, is delocalized into cytoplasm and inactivated by farnesyltransferase inhibitors (FTI), such as tipifarnib. Tipifarnib has demonstrated proof of concept activity in H-RAS mutant HNSCC in an ongoing clinical trial (NCT02383927). In this study, we examined H-RAS associated signaling pathway in HNSCC cell lines and PDX models to see if inhibition of wild-type HRAS signaling could re-sensitize cetuximab resistant cells and tumors to inhibition by cetuximab. UMSCC47 and SCC1-C are both H-RAS wild-type HNSCC cell lines, and SCC1-C is resistant to EGFR targeted therapy.

Methods: In vitro cell growth with and without tipifarnib and/or cetuximab treatments were examined by Sulforhodamine B (SRB) and colony formation assay. Patient derived xenograft (PDX) models were then used to study the efficacy of the tipifarnib treatment as a single agent or in combination with cetuximab. Western blot analyses were performed to verify the effect of these treatments on EGFR/ERK/AKT signaling pathways.

Results: Our results showed that tipifarnib could inhibit HNSCC growth, both in vitro and in the PDX model in a dose-dependent manner (control vs the low dose of tipifarnib, p = 0.02; control vs the high dose of tipifarnib, p = 0.001). However, the combined treatment with cetuximab significantly enhanced tipifarnib efficacy in the H-RAS wild type setting, particularly in the PDX model (control vs tipifarnib, p = 0.123; control vs cetuximab, p = 0.022; control vs the combination, p = 0.010). Western blot analyses showed that neither ERK nor AKT phosphorylation was effectively reduced after a prolonged treatment (48 hours) with tipifarnib alone. However, the addition of cetuximab reduced pERK.

Conclusions: Our study demonstrates that cetuximab enhances the anti-tumor effect of tipifarnib in the H-RAS wild type setting through ERK inhibition. This study supports the rationale for combining tipifarnib with EGFR inhibitors as a possible effective therapeutic approach in HNSCC.

#4835

LY3076226, a novel anti-FGFR3 antibody drug conjugate exhibits potent and durable anti-tumor activity in tumor models harboring FGFR3 mutations or fusions.

David Surguladze, Anthony Pennello, Xiaodi Ren, Tim Mack, Alan Rigby, Paul Balderes, Elizabeth Navarro, Nelusha Amaladas, Scott Eastman, Michael Topper, Yung-mae Yao, Chris Moxham, Gregory Plowman, Dale Ludwig. _Eli Lilly and Company, New York, NY_.

Fibroblast growth factor receptor 3 (FGFR3) has been shown to be constitutively activated in bladder, multiple myeloma, non-small cell lung cancer (NSCLC), glioblastoma multiforme, and cervical cancer through overexpression, point mutations, and translocations. In bladder cancer, FGFR3 is typically found to be activated by mutation or over-expression. TCGA data suggests that ~48% of bladder cancer patients harbor FGFR3 mutations such as S249, Y373C and others, as well as fusion proteins of FGFR3 involving transforming acidic coiled-coil gene (TACC3) and FGFR3-BAIAP2L1. LY3076226 is an antibody-drug conjugate (ADC) comprising a fully human anti-FGFR3 antibody, IMC-D11, conjugated to a cytotoxic payload, the maytansine-derivative DM4, via a cleavable linker (sulfo-SPDB). The anti-FGFR3 Ab IMC-D11 recognizes and binds not only wild-type receptor, but also constitutively active mutant receptors and fusion proteins of FGFR3. Preclinical in vitro pharmacodynamics studies have demonstrated that LY3076226 binds to the human FGFR3 protein with high affinity, and following internalization can deliver the potent cytotoxic payload into tumor cells resulting in cell cycle arrest and cell death.

In vivo studies with LY3076226 demonstrated robust antitumor efficacy resulting in tumor stasis or partial or complete tumor responses after 4 weekly doses at 5 mg/kg in bladder cancer cell lines: SW780, RT112 and UMUC-14 that harbor FGFR3-BAIAP2L1, FGFR3-TACC3 fusion or S249C mutation, respectively. We also evaluated over 90 PDXs models using a Modiplex assay and IHC to determine mutational and FGFR3 expression levels. We identified a number of bladder PDX models with R248C, S249 and G370 mutations and a single NSCLC with FGFR3-TACC3, and tested LY3076226 efficacy in these models. Interestingly the incidence of mutations and fusions in these PDX models confirmed prior TCGA results, and confirm that PDX models represent a valuable translational platform for patient tailoring. Similar to RT-112 cell line, LY3076226 significantly inhibited tumor growth in lung PDX model (LXF-2226) with FGFR3-TACC3 fusion resulting in durable complete response in 100% of mice. LY3076226 was also efficacious after 4 weekly treatments at 5 mg/kg, resulting in tumor growth inhibition, tumor stasis or tumor regressions in bladder PDX models with G370C, S249C or R248C FGFR3 mutations, respectively. Additionally, LY3076226 exhibited superior efficacy compared to the naked IMC-D11 Ab or chKTI-ADC controls.

These data support the conclusion that therapy with FGFR3 targeted antibody-drug conjugate LY3076226 may confer tumor control in patients that are FGFR3 positive and harbor FGFR3 mutations or fusion proteins.

#4836

Ly6-neurotoxin1 is a potential target in pancreatic ductal adenocarcinoma cells.

Doaa M. Ali, Nevena Nedyalkova, Michael Zepp, Martin R. Berger. _German Cancer Research Centre, Heidelberg, Germany_.

Ly6/neurotoxin1 (Lynx1) is a cholinergic transmission modulator, which was described as a tumor suppressor in lung cancer. In addition, the expression of Lynx1 was downregulated in rat PDAC cells colonizing rat liver. As cholinergic signaling via the vagus nerve may slow pancreatic tumor progression, we investigated the role of Lynx1 in human PDAC cell lines.

Transient gene knockdown (KD) by siRNA was used to modulate the expression of Lynx1 in BXPC3 and Miapaca cells and KD was confirmed by qRT-PCR and western blot (WB). Resulting effects were determined by respective assays for proliferation, migration and colony formation. Induction of apoptosis was assessed by Hoechst and Annexin V-FITC staining. Induction of autophagy was determined by acridine orange and immunofluorescence (IF) staining for LC3b. Finally, we performed microarrays for gene expression in cells with Lynx1 KD and analyzed by WB cancer pathways of related signaling molecules.

Following successful KD of Lynx1, the response of Miapaca and BXPC3 cells was altered regarding proliferation (-60% and -10%), migration (+200% and -10%) at 72 h after transfection and colony formation (-50% and -40%) at 9 d after transfection. Hoechst staining revealed increased rates of apoptosis in both cell lines. This was confirmed by Annexin V assay in Miapaca cells and these changes were associated with significantly reduced BCL2 levels. Autophagy was increased in both cell lines as were LC3b levels detected by IF. Alterations in mRNA expression, as assessed by microarray, were analyzed by IPA software using a 1.5 fold cutoff. Miapaca cells showed significant activation of 43 pathways including phospholipase C signaling, cholecystokinin/gastrin mediated signaling, and cell cycle regulation. In addition, five pathways showed uniform downregulation, i.e estrogen mediated S phase entry, sirtuin signaling, small cell lung cancer signaling, cyclins and cell cycle regulation, and ataxia-telangiectasia-mutated (ATM) signaling. In contrast to the more sensitive Miapaca cells, BXPC3 cells showed modulation of few pathways only: activation of G protein beta gamma signaling and osteoarthritis pathways, as well as downregulation of the sirtuin signaling pathway. At protein level, the mTOR pathway was downregulated in both cell lines (including phosphorylated forms of mTOR, Rictor, Raptor, PRAS40), as were the upstream regulators PI3K and p-AKT.

In conclusion, KD of Lynx1 caused changes in gene product levels, which are related to cholinergic signal transmission as well as to DNA damage and repair systems. The observed decreased colony formation in Lynx1 KD cells is also indicative of defective cholinergic signaling. Reduced mTOR pathway signaling caused induction of autophagy. These findings suggest that Lynx1 is beneficial for regular cellular functions and its lack will contribute to increased apoptosis, DNA damage and autophagy.

#4837

Role of Notch signaling in human mucoepidermoid carcinoma and combined targeting of Notch and EGFR signaling as an anti-cancer strategy.

Wei Ni, Zirong Chen, Xin Zhou, Rongqiang Yang, Frederic Kaye, Lizi Wu. _University of Florida, Gainesville, FL_.

Mucoepidermoid carcinoma (MEC) is the most common salivary gland malignancy and currently no targeted therapy is available for MEC patients. MEC is characterized by a unique chromosomal translocation t(11;19)(q14-21;p12-13) that generates the CRTC1-MAML2 fusion. Our understanding of CRTC1-MAML2-mediated tumorigenesis and maintenance remains limited. MEC is considered a disease of stem/progenitor cells; however, the regulation of human MEC stem/progenitor cells has not yet been characterized. In this study, we focused on Notch signaling, a pathway important in regulating normal and cancerous stem cells, and investigated its regulatory role in human MEC cancer stem/progenitor cells as well as its potential therapeutic potential. First, we determined the status of Notch signaling activation in MEC cells. Western blotting showed the presence of cleaved active NOTCH1 and the expression of Notch downstream target gene HES1 in human CRTC1-MAML2 fusion-positive MEC cells, indicative of active Notch signaling in MEC. Second, we utilized two approaches of blocking endogenous Notch signaling in human MEC cells in vitro and in vivo, i.e. dnMAML1, a pan-Notch inhibitor that blocks the formation of the Notch transcriptional activation complex, and dibenzazepine (DBZ), a γ-secretase inhibitor that interferes with Notch receptor processing. We observed that Notch signaling inhibition had no effect on the proliferation of bulk MEC cells, but significantly reduced oncosphere forming capacity and ALDH-positive population in human MEC in vitro, suggesting that Notch signaling contributes to the maintenance of MEC stem/progenitor cells. Further, Notch signaling inhibition significantly reduced the growth of human MEC xenografts, indicating an essential role of Notch signaling in MEC growth in vivo. Finally, since we previously showed that the CRTC1-MAML2-induced autocrine AREG-EGFR signaling was required for the growth and survival of human MEC cells, we reasoned that simultaneous targeting of Notch and EGFR signaling will eliminate cancer stem and non-stem cell populations, likely improving therapeutic efficacy. Indeed, concurrent inhibition of Notch and EGFR signaling via respective DBZ and Erlotinib synergistically blocked the colony and oncosphere formation capacity of human MEC cells in vitro and in vivo. Collectively, our data demonstrate that Notch signaling is a critical regulator for maintaining MEC cancer-initiating cells and that the combination strategy targeting Notch and EGFR signaling is a potentially effective approach for MEC treatment.

#4838

Leflunomide inhibits the growth of LKB1-inactivated tumors in both xenograft and immunocompetent genetically engineered mouse models.

Rui Jin,1 Xiuju Liu,1 Wei Peng,1 Yijian Fan,1 Boxuan Liu,1 Shuanying Yang,2 Chun-gen Xing,3 Melissa Gilbert-Ross,1 Adam Marcus,1 Wei Zhou1. 1 _Emory Univ. Winship Cancer Inst., Atlanta, GA;_ 2 _the Second Affiliated Hospital, Xi'an Jiaotong University, Xi'an, China;_ 3 _The Second Affiliated Hospital Of Soochow University, Suzhou, China_.

Introduction: LKB1 is the third mostly frequently mutated gene in lung adenocarcinoma, and is mutually exclusive with EGFR mutations. Furthermore, a recent clinical trial indicated that lung cancers with LKB1 mutations are unlikely to respond to immune checkpoint therapy. Therefore, there is an urgent need for novel therapies for LKB1-inactivated lung cancer. We previously demonstrated that leflunomide preferentially induces apoptosis in LKB1-null lung cancer cell lines in vitro. Here, we evaluated the effect of leflunomide in both a xenograft model and immune-competent genetically engineered mouse model (GEMM).

Methods: LKB1-null H460 xenograft mice were treated with leflunomide (35mg/kg/day) for 23 days through oral gavage and tumor volume was measured every the other day. KrasG12D/Lkb1-/-Luciferase GEMM mice were treated with leflunomide (30mg/kg/day) for 44 days and tumor burden was measured by bioluminescence imaging (BLI) score every week. At the end of experiments, tumors were dissected, weighed, and analyzed by immunohistochemistry.

Results: Although leflunomide is an FDA-approved agent to treat rheumatoid arthritis, its immune suppression function did not facilitate but inhibited the growth of KrasG12DLkb1-/- lung adenocarcinoma. This suppression was also observed in H460 xenografts, and leflunomide treatment did not alter animal weight in either model. Furthermore, distant cancer metastasis was not observed in leflunomide-treated GEMM. In residual tumors from the treated group, cleaved caspase-3 was not detectable but there was a significant decrease in Ki67 staining. This is consistent with our in vitro findings that leflunomide primarily inhibits cell proliferation through the promotion of G1 cell cycle arrest.

Conclusion: Our findings indicate that leflunomide may be a viable reagent for the treatment of LKB1-mutated lung adenocarcinoma.

#4839

Double knockdown of MDM4 and MDM2 modulates the antitumor effects of cytotoxic drugs in colon and gastric cancer cells expressing high levels of MDM4.

Mamiko Imanishi, Yoshiyuki Yamamoto, Shinji Endo, Kenji Yamato, Ichinosuke Hyodo. _University of Tsukuba, Tsukuba, Japan_.

Introduction and Aim: Inactivation of the TP53 tumor suppressor gene is important for cancer development and progression. Wild-type (wt) TP53 is inactivated in various types of cancers including colon and gastric cancers by upregulated murine double minute homolog 2 (MDM2) and MDM4. We previously reported that simultaneous knockdown of MDM4 and MDM2 using synthetic DNA-modified siRNAs revived p53 activity and synergistically inhibited in vitro cell growth in cancer cells with wild-type TP53 and high MDM4 expression (wtTP53/highMDM4), whereas only MDM2 knockdown but not MDM4 knockdown suppressed that of high MDM2 expression cancer cells. In the present study, we investigated the combined activity of MDM4/MDM2 double knockdown and cytotoxic drugs (5-fluorouracil (5-FU), cisplatin, oxaliplatin) to develop a new therapy for gastric and colon cancers with wtTP53/highMDM4.

Experimental procedure: Two colon (HCT116, LoVo) and two gastric cancer cell lines (NUGC-4, SNU-1) were used. Respective synthetic DNA-modified siRNAs against MDM4 (chiMDM4) and MDM2 (chiMDM2) were transfected using Lipofectamine RNAiMAX. Cell viability was determined by WST-8 assays. Expression of p53, p21, MDM2, MDM4, and β-actin were analyzed by immunoblotting. Cell cycle distribution was analyzed by flow cytometry. In vivo antitumor effects were examined using nude mice carrying HCT116 xenograft tumors. siRNAs with atelocollagen were intratumorally injected weekly and 5-FU was intraperitoneally administered three times per week.

Results: MDM4/MDM2 double knockdown with the siRNAs enhanced in vitro antitumor activity of 5-FU in all four wtTP53/highMDM4 cell lines (HCT116, LoVo, SNU-1, and NUGC-4) and cisplatin and oxaliplatin in gastric (NUGC-4) and colon cancer cell lines (HCT116, LoVo), respectively. Exposure to 5-FU increased p53 and its downstream gene products (p21, PUMA, MDM2), and induced cell cycle arrest in the G1 phase. MDM4/MDM2 double knockdown suppressed MDM2, and enhanced expression of p53 and p21 and arrested G1. p53-MDM2 forms a negative feedback loop. Enhanced antitumor effects of 5-FU by MDM4/MDM2 double knockdown may be attributed to blocking this feedback loop, in addition to direct suppression of MDM4/MDM2. Since MDM2 interacts directly with molecules like RB, p21, and E2F1, other than p53, MDM2 knockdown may have some advantages over peptides or small molecular inhibitors of p53-MDM2 interaction in such combination therapies. Intratumor injection of chiMDM4/chiMDM2 suppressed in vivo tumor growth and boosted the antitumor effects of 5-FU in a xenograft model using HCT116 cells.

Conclusion: Combining MDM4/MDM2 knockdown and conventional cytotoxic drugs may be a promising treatment strategy for wtTP53/highMDM4 gastrointestinal cancers.

#4840

Characterization of URST1 as a biomarker and therapeutic target for precision medicine of lung cancer.

Atsushi Takano,1 Yohei Miyagi,2 Yataro Daigo3. 1 _The University of Tokyo, Tokyo, Japan;_ 2 _Kanagawa Cancer Center Research Institute, Yokohama, Japan;_ 3 _Shiga University of Medical Science, Tokyo, Japan_.

This study aims to identify novel biomarkers and therapeutic targets for lung cancer. We initially screened genes that overexpressed in the majority of lung cancers by using our original gene expression profile database. During this process, we identified up-regulated in solid tumor 1 (URST1) as a candidate. Immunohistochemical staining showed that URST1 was expressed in 231 (64.5%) of 358 non-small cell lung cancer (NSCLC) that had undergone curative surgery. Strong URST1 expression was associated with poor prognosis for NSCLC patients (P = 0.0003 by log-rank test). In addition, multivariate analysis showed that strong URST1 expression was an independent prognostic factor for NSCLC patients. Reduction of URST1 expression by siRNA or treatment with selective URST1 inhibitor significantly suppressed cancer cell growth partly through cell cycle arrest at G2/M phase and mitotic cell death as detected by flow cytometric analysis and live cell imaging. Our findings suggest that URST1 could be a potential biomarker and therapeutic target for developing precision medicine of lung cancer.

#4841

Rapid screening of cyclotide-based libraries for the selection of potent E3 ligase antagonists.

Julio A. Camarero. _University of Southern California, Los Angeles, CA_.

We report novel methods for the biosynthesis of natively-folded MCoTI-based cyclotides inside live E. coli cells using a split protein splicing unit. The cyclotide MCoTI-cylotides are potent trypsin inhibitors recently isolated from the seeds of Momordica cochinchinensis, a plant member of cucurbitaceae family. Biosynthesis of genetically encoded cyclotide-based libraries opens the possibility of using single cells as microfactories where the biosynthesis and screening of a particular inhibitor can take place in a single process within the same cellular cytoplasm. The cyclotide scaffold has a tremendous potential for the development of therapeutic leads based on their extraordinary stability and potential for grafting applications. We will show an example, where a large cyclotide-based genetically-encoded library was used to screen for low nanomolar antagonists for the Hdm2-HdmX RING-mediated E3 ligase activity. We will also present different strategies to improve the cellular uptake and pharmacokinetic profiles of bioactive cyclotides.

#4842

Overcoming EGFR and ERK-mediated resistance to enzalutamide in castration-resistant prostate cancer.

Thomas M. Steele,1 Maitreyee K. Jathal,1 Salma Siddiqui,2 Sisi Qin,3 Clifford G. Tepper,1 Ralph W. deVere White,1 Manish Kohli,3 Liewei Wang,3 Allen C. Gao,1 Paramita M. Ghosh1. 1 _University of California Davis, Sacramento, CA;_ 2 _VA Northern California Health Care System, Mather, CA;_ 3 _Mayo Clinic, Rochester, MN_.

The present project was undertaken to determine whether the activation of the EGFR family (EGFR/ErbB2/ErbB3/ErbB4) and its downstream signaling effector ERK1/2 plays a role in enzalutamide resistance and whether treatment with ErbB or ERK inhibitors overcome this resistance. C4-2B (enzalutamide sensitive) and 22Rv1 (partially enzalutamide resistant) cell lines cultured for prolonged periods in enzalutamide, developed resistance to this drug (C4-2B/MDVR and 22Rv1/MDVR, respectively). Whole genome comparison of enza-resistant to parental lines demonstrated that the ErbB signaling network was significantly upregulated in the enza-resistant lines. Comparison of various enza-sensitive and resistant lines demonstrated a strong correlation between phosphorylation at EGFR(Y1068) and enza resistance. Inhibition of EGFR, but not that of ErbB2 or ErbB3 prevented cell viability. The EGFR inhibitors erlotinib and dacomitinib, but not the ErbB2 inhibitor lapatinib, suppressed viability in combination with enzalutamide. We hypothesized that enza-resistant tumors expressing higher levels of EGFR would be more responsive to erlotinib. To test this hypothesis, we compared the effects of erlotinib and enzalutamide, individually or in combination, in organelles from PDX tumors expressing high or low EGFR levels. Significantly, tumors expressing high EGFR, but not those expressing low EGFR, were responsive to the combination. Finally, we investigated the cause for greater efficacy with erlotinib compared to lapatinib in PCa. Comparison of the effects of different ligands revealed that while both lapatinib and erlotinib inhibited EGFR phosphorylation, only erlotinib but not lapatinib was able to inhibit EGF-induced ERK phosphorylation. This indicated an important role for ERK in the mediation of EGFR ligand induced enzalutamide resistance. Continuous culture of enza-sensitive C4 cells in enzalutamide resulted in the development of ERK phosphorylation in these PTEN-null cells that normally do not express phosphorylated ERK, and the newly ERK phosphorylated cells also were more susceptible to erlotinib as well. In support of a role for ERK in mediating the effects of EGFR activation in enza-resistant cells, inhibition of EGFR but not ErbB2 or ErbB3 inhibited ERK phosphorylation, and the ERK inhibitor ulitertinib inhibited viability of enza-resistant lines. We conclude that enzalutamide treatment causes an increase in EGFR ligands that result in the phosphorylation of EGFR at Y1068, which further resulted in phosphorylation of ERK. EGFR inhibitors that prevent ERK phosphorylation were effective in suppressing viability of enza-resistant CRPC cells, whereas ErbB2 inhibitors that did not affect ERK phosphorylation were unable to affect cell viability. These results demonstrate why certain ErbB inhibitors failed to affect enza-resistant CRPC tumors but provide encouragement for others.

### Targeted Therapies and Immunological/Tumor Microenvironment Effects

#4843

TP53-stabilization with APR-246 enhances antitumor effects of immune checkpoint blockade in preclinical models.

Arnab Ghosh, Judith Michel, Lauren Dong, Nathan Suek, Hong Zhong, Sadna Budhu, Olivier de Henau, Jedd Wolchok, Taha Merghoub. _Memorial Sloan Kettering Cancer Center, New York, NY_.

An emerging body of literature suggests a role of TP53 pathway in antitumor immunity including antigen presentation and T cell proliferation. Stabilization of TP53 could therefore alter the immune tumor microenvironment, enabling the immune system to target tumor cells more effectively. APR-246 covalently modifies the core domain of cellular TP53 through the alkylation of thiol groups, leading to reactivation of endogenous TP53 activity. To further study the effect of APR-246 on antitumor immunity we used a B16 pre-clinical melanoma mouse model. In vitro treatment of B16 cells with APR-246 caused intracellular accumulation of TP53, leading to increased apoptosis. However, treatment of B16-melanoma bearing mice with APR-246 monotherapy did not result in a statistically significant change in tumor growth or survival. Analyses of the tumor immune microenvironment showed increased immune potentiating M1 polarized tumor-associated macrophages, and Granzyme B activity in CD8+ T cells, suggesting enhanced anti-tumor immunity. Concomitantly, there was increased PD-L1 expression in the macrophages, PD-1 expression on CD8+ T cells, and Foxp3+ Tregs in tumors from APR-246 treated animals. Therefore, we decided to combine anti-PD-1 antibody (RMP-1) to APR-246 treatment in tumor-bearing mice. The combination led to a significant delay in tumor growth (P < 0.001) and improved survival of B16-bearing mice compared to anti-PD1 or APR-246 monotherapies (P < 0.01). Improved responses were also seen in MC38 colorectal cancer-bearing mice (P < 0.01). The anti-tumor effects of APR-246 and anti-PD1 were T cell dependent and abrogated in hosts lacking T cells. Analyses of the tumor immune microenvironment showed that the combination decreases proliferation but increases cytolytic activity of CD8+ T cells compared to anti-PD1 alone. We next studied the effect of combining APR-246 with anti-PD1+ anti-CTLA-4 (9B9). Combination of APR-246 with dual immune checkpoint blockade using anti-PD1 and anti-CTLA-4 showed further tumor growth inhibition in B16-melanoma compared to monotherapies (P < 0.001). Our studies support a role for restoring TP53 in the tumor microenvironment and provide evidence that reactivation of TP53 by APR-246 can enhance anti-tumor immune responses and inhibit tumor growth in preclinical models.

#4844

**A novel anti-TIGIT antibody (YH29143) enhances T cell activity and suppresses T** reg **cell activity and synergizes with anti-PD-L1 antibody.**

Kwang-Hoon Lee. _Yuhan Corporation, Gyeonggi-do, Republic of Korea_.

TIGIT (T cell immunoreceptor with Ig and ITIM domains) is a T cell coinhibitory receptor, highly expressed on regulatory and memory CD4+, CD8+ T cells and natural killer (NK) cells. TIGIT binding of PVR (poliovirus receptor) on the surface of antigen presenting cells leads to downregulation of CD8+ T cell and NK cell responses in tumors. In this study, we sought to determine the efficacy of TIGIT blockade in antitumor immunity and demonstrate the synergistic effect of treatment with other checkpoint blockade such as anti-PD-L1.

We generated anti-TIGIT monoclonal antibody that binds TIGIT with high affinity and blocks ligand binding. Anti-TIGIT antibody YH29143 was shown to increase T cell activities and suppress Treg cell activities from in vitro cell-based assays. We determined the crystal structure of YH29143 Fab fragment in complex with TIGIT and identified its epitope that overlaps with the PVR-binding interface. We also studied in vivo efficacy of YH29143 whether to inhibit the growth of tumor in two syngeneic colon tumor-bearing mouse models (CT26 and MC38). YH29143 reduced growth of both tumors compared to that of control IgG. In addition to these results, YH29143 showed synergistic efficacy for inhibition of tumor growth in combination with anti-PD-L1 antibody. From these tumor tissues, the ratio of CD8+ T cells to Treg cells was increased by treatment of anti-TIGIT antibody.

Taken together, we evaluated the efficacy of YH29143, a potent anti-TIGIT monoclonal antibody, which enhanced T cell activation and suppressd Treg cell function in vitro, and efficiently inhibited tumor growth in vivo having synergistic effect with anti-PD-L1 antibody. These data also provide the rationale for YH29143 as a potential combination candidate for cancer immunotherapy with other immune checkpoint blockades.

#4845

HERA-CD27L, a true CD27 agonist, is a hexavalent CD27 ligand that enhances T cell activation and induces potent anti-tumor immunity.

Julian P. Sefrin, David M. Richards, Katharina Billian-Frey, Karl Heinonen, Viola Marschall, Christian Merz, Mauricio Redondo Müller, Matthias Schröder, Jaromir Sykora, Meinolf Thiemann, Harald Fricke, Christian Gieffers, Oliver Hill. _Apogenix AG, Heidelberg, Germany_.

Agonistic stimulation of TNFRSF members like CD27 is a promising strategy to boost anti-tumor responses. Although antibodies are effective inhibitors of signaling, they have shown minimal agonistic activity due to their limited binding domains, flexibility and the toxicity mediated by Fc/FcγR interactions. TNFRSF signaling is a structurally well-defined event that takes place during cell contact. The trimeric-trivalent TNFSF-receptor binding domain (TNFSF-RBD) on the conducting cell and the resulting multi-trimer-based receptor clustering on the receiving cell are essential for signaling. In contrast to antibodies, HERA-CD27L mimics the natural ligand and induces potent activity. In order to understand the activity of HERA-CD27L, human T cells were stimulated in the presence of HERA-CD27L, the trimeric CD27L or a clinical benchmark anti-CD27 antibody. In all assays, treatment with the hexavalent HERA-CD27L significantly boosted T cell activation, proliferation and differentiation. Furthermore, the hexavalent molecule was always superior to the trimeric CD27L and bivalent antibody. In fact, treatment with the anti-CD27 antibody resulted in significantly weaker proliferation compared to anti-CD3 antibody alone. To understand early events, we tested CD27 signaling using a reporter cell assay. Treatment with HERA-CD27L and CD27L resulted in high and intermediate, respectively, reporter activity. In contrast, the anti-CD27 antibody failed to show any signaling activity across a wide range of concentrations. Since most T cells express CD27, there is potential for non-specific T cell activation. This was examined by comparing OVA-specific and non-specific T cells in the same environment using the CD8+ "OT-I" T cell adoptive transfer mouse model. Following a single dose of HERA-CD27L, serial blood samples showed a significant and HERA-CD27L dose-dependent clonal expansion of OT-I T cells. OT-I T cells expressed high levels of activation markers, while the endogenous T cells failed to show any response. The potent single-agent anti-tumor efficacy of the hexavalent HERA-CD27L was demonstrated in two different mouse models. With CT26wt, HERA-CD27L also showed superior activity compared to anti-PD-1 antibody. Furthermore, combination of HERA-CD27L and anti-PD-1 antibody showed additive effects. Finally, early treatment with HERA-CD27L significantly increased overall survival, from 19 to 41 days, and tumor-free animals still alive at the end of the study were protected from tumor re-challenge. Various strategies have been proposed for targeting CD27 for cancer therapy. As we have shown here, the hexavalent HERA-CD27L has superior activity compared to bivalent antibodies. Altogether, HERA-CD27L shows single-agent anti-tumor efficacy, is well tolerated by multiple relevant species and the lead candidate is currently ready for GMP cell line development.

#4846

MM-401, a novel anti-TNFR2 antibody that induces T cell co-stimulation, robust anti-tumor activity and immune memory.

Jennifer Richards, Christina Wong, Alexander Koshkaryev, Ross Fulton, Adam Camblin, James Sampson, Lia Luus, James Suchy, Stephanie Grabow, Vinodh Kurella, Sandeep Kumar, James Lulo, James Qiu, Yang Jiao, Lihui Xu, Violette Paragas, Maja Razlog, Marco Muda, Eric Tam, Andreas Raue, Daryl Drummond. _Merrimack Pharmaceuticals, Cambridge, MA_.

TNFR2 has been implicated as a novel target for cancer immunotherapy. While TNFR2 has been linked to enhanced suppressive activity of regulatory T cells (Tregs) in auto-immune models, the effect of TNFR2-targeted therapy in cancer remains unclear. Here we present a novel monoclonal anti-TNFR2 antibody that provides T cell co-stimulation and yields robust anti-tumor activity in in vitro and in vivo models. In syngeneic murine tumor models, treatment with a murine surrogate anti-TNFR2 antibody results in robust anti-tumor activity both alone and in combination with checkpoint inhibitor antibodies targeting PD-1 and PDL-1. Complete responders exhibited immunological memory months after initial tumor clearance. Furthermore, significant anti-tumor activity was observed in anti-TNFR2-treated mice even in a model that proved resistant to PD1-targeted antibody treatment. Depletion studies suggest that CD8+ T and NK cells are required for activity, while TNFR2 knockout models suggest that TNFR2 expression on cancer cells is not required for activity. Using an antibody with a mutant-Fc, we show that activity is dependent on FcγR binding. Studies in FcγR knockout mice, complemented by studies using different antibody-Fc variants, confirm that enhanced agonism via FcγR binding is the dominant mechanism of action. Contrary to antibodies targeting other TNF superfamily receptors, treatment does not lead to strong depletion of TNFR2-expressing cell types such as Tregs. Consistent with its proposed mechanism, long-term dosing of the anti-TNFR2 antibody did not cause toxicity in two inbred mouse strains when compared to an anti-CTLA4 antibody, which caused weight loss, splenomegaly and elevated inflammatory cytokines in serum. Following anti-TNFR2 treatment, we observed a broad reversal of immunosuppression in the tumor characterized by downregulation of suppressive markers. A human anti-TNFR2 antibody (MM-401) with low nanomolar affinity and binding to the same epitope as the murine surrogate antibody has been developed. MM-401 is being developed as a potential novel treatment option for cancer patients.

#4847

Investigating the impact of astrocytes on targeted therapy of melanoma brain metastases.

Joshua Parris,1 Subhasree Basu,2 Thibaut Barnoud,2 Yongkang Zhao,2 Peter Somboonsong,2 Qing Chen,2 Maureen Murphy2. 1 _University of Pennsylvania, Philadelphia, PA;_ 2 _The Wistar Institute, Philadelphia, PA_.

Metastatic melanoma is a devastating disease that leads to the development of brain metastases (MBMs) in 40-60% of patients. Upon diagnosis of brain metastases, patient overall survival is less than 6 months. Poor patient survival is believed to be due to resistance driven by the brain microenvironment. Collectively, these findings underscore the need for novel therapeutic strategies to improve the survival of patients with MBMs. Development of effective therapies relies on an understanding of the interaction between MBMs and different cell types within the brain microenvironment. Astrocytes are the most abundant cell type within the brain and interact with MBMs through direct and indirect mechanisms. Astrocyte interaction with MBMs alters MBM gene expression and promotes metastatic outgrowth and resistance to cytotoxic chemotherapies. Exactly how astrocytes alter MBM response to MAPK inhibition remains unclear. Using conditioned media from primary astrocytes, as well as co-culture of primary astrocytes with either the MBM cell line WM4265-2 or the primary melanoma cell line WM983B, we found that the interaction of melanoma cells with astrocytes increased melanoma sensitivity to MEK inhibition (MEKi). RNA sequencing of WM4265-2 and WM983B cell lines treated with the MEK inhibitor PD0325901 (PD901) showed significant up-regulation of the transcriptional regulator ID3, which is known to play a role in the resistance of melanoma to MAPK inhibition. Interestingly, astrocyte conditioned media prevented the upregulation of ID3. To validate this finding, we silenced ID3 in WM4265-2 and WM983B cell lines, and showed that knockdown of ID3 caused increased sensitivity to MEKi. These combined data support the premise that the increased response to PD901 in MBMs exposed to astrocytes is due to ID3 down-regulation. How ID3 modulates resistance to MAPK inhibition remains unclear. Elucidating the mechanism of ID3 mediated resistance to MEKi may uncover new avenues for improving the treatment of melanoma brain metastases.

#4848

BI 905677, a first-in-class LRP5/6 antagonist targeting Wnt-driven proliferation and immune escape.

Vittoria Zinzalla,1 Barbara Drobits-Handl,1 Alexander Savchenko,1 Jörg Rinnenthal,1 Markus Johann Bauer,1 Michael Sanderson,1 Sophia Maria Blake,1 Norbert Schweifer,1 Robert Gerhardus Jacob Vries,2 Hans Clevers,3 Norbert Kraut1. 1 _Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria; _2 _Foundation Hubrecht Organoid Technology (HUB), Utrecht, Netherlands;_ 3 _Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences and University Medical Center Utrecht, Utrecht, Netherlands_.

Aberrant Wnt/β-catenin signalling has been shown to play a key role in tumorigenesis and resistance to immunotherapy in several tumour indications. Wnt ligand-mediated signals are transduced by two distinct receptor types, the serpentine receptor Frizzled (FZD) and the closely related single-span transmembrane proteins LRP5 and LRP6. Formation of the FZD-Wnt-LRP5/6 trimeric complex induces phosphorylation of LRP5 or LRP6 intracellular domains leading to inactivation of the β-catenin degradation complex, allowing stabilized β-catenin to enter the nucleus, bind to the TCF transcription factors, and act as a transcriptional activator of Wnt target genes. We have developed a first-in-class LRP5/6 antagonist, a bi-paratopic antibody comprising two modules binding to distinct epitopes of LRP5 (or LRP6). BI 905677 is a highly potent blocker of signalling induced by the Wnt family of ligands and shows anti-tumour activity in cancer models harbouring genomic alterations in upstream regulators of the Wnt pathway, such as RNF43 mutations or RSPO fusions. Furthermore, BI 905677 in combination with an anti-PD-1 immune checkpoint inhibitor induces dendritic cell activation and T cell infiltration in tumour tissues leading to complete responses in syngeneic tumour models. A Phase I clinical trial is underway in patients with advanced cancer to evaluate safety, tolerability, pharmacokinetic and pharmacodynamic properties, and efficacy of BI 905677 (NCT03604445).

#4849

CAP-100: first-in-class anti-CCR7 antibody for CLL.

Carlos Cuesta-Mateos,1 Cecilia Muñoz-Calleja,1 Javier Loscertales,1 Fernando Terron,2 Wim Mol2. 1 _La Princesa University Hospital, Madrid, Spain;_ 2 _Catapult Therapeutics, Lelystad, Netherlands_.

The homeostatic CC-chemokine receptor 7 (CCR7) is highly expressed in many hematological malignancies including chronic lymphocytic leukemia (CLL). CCR7 surface expression levels are much higher than those of CD20, and are correlated with poor prognosis. Upon engagement by its ligands (CCL19 and CCL21), CCR7 controls trafficking of CLL cells to locations where these chemokines are expressed, such as the lymph node (LN). In this protective microenvironment CCR7 ligands contribute to CLL cell survival and proliferation. Indeed, high CCR7-mediated in vitro migration of patient CLL cells correlates with LN involvement and adverse prognostic factors. Thus, strategies targeting CCR7 could provide an additional therapeutic approach for CLL.

Therefore, we have generated CAP-100, the first humanized immunoglobulin G1 (IgG1) monoclonal antibody (mAb) that specifically binds to human CCR7 and neutralizes ligand-mediated signaling from both CCL19 and CCL21. The antibody effectively inhibited CLL cells migration and survival. Furthermore, in in vivo pre-clinical studies, CAP-100 was shown to inhibit entry of CCR7-expressing cells to LNs. The antibody showed potent cell killing activity against CLL cells while preserving normal blood cells. This Fc-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) clearly outperformed standard-of-care in CLL, such as rituximab. ADCC and migration inhibition were both independent of prognostic markers for high risk CLL, such as TP53 mutation including 17p deletion and relapse/refractoriness to various standard therapies, including BTK-inhibitors. Finally, when given as monotherapy in disseminated NHL xenograft tumors in SCID mice, CAP-100 exhibited tumor growth inhibition in established tumors and provided significant survival.

Collectively, our results demonstrate that CAP-100, the first-in-class anti-CCR7 mAb, is a potent antagonist with biological activity in CLL primary cells, including those from relapsed/refractory patients. Moreover, these results highlight the relevance of the CCR7-CCL19/CCL21 pathway as a therapeutic target in CLL. CAP-100's unique propensity to block migration of CLL cells to the LN, in combination with its potent cell killing activity provides the biological rationale for use of CAP-100 in CLL, either as monotherapy or in combination with novel agents such as BTK-inhibitors. Clinical trials in CLL and other CCR7-expressing NHL will be initiated soon.

#4850

The relevance of using proper preclinical models when developing therapeutics for localized or bone metastatic prostate cancer.

Tiina E. Kähkönen, Jenni H.E. Mäki-Jouppila, Mari I. Suominen, Jussi M. Halleen, Jenni Bernoulli. _Pharmatest Services, Turku, Finland_.

Early stages of prostate cancer are sensitive to androgen deprivation therapy, but upon progression the disease develops to metastatic castration-resistant prostate cancer, where bone is the dominant metastatic site. Despite of recent advances in drug development, the advanced stage is still incurable. Tumor microenvironment in metastatic locations differs from the primary site and may cause resistance to used therapy and modulate tumor properties. Therefore, it is crucial to use proper models in preclinical drug development. Aim of this study was to establish predictive preclinical in vitro and in vivo prostate cancer models that can be used in drug development when targeting cancer cells or tumor microenvironment at different stages. Androgen receptor (AR) positive human prostate cancer cell line LNCaP was used in all studies. In the in vitro assay, the effects of the antiandrogen enzalutamide on viability of LNCaP cells was determined using CellTiter Glo assay in the presence and absence of a synthetic androgen R1881. In two in vivo studies, male NMRI nude mice were used. In a subcutaneous model, part of the mice received dihydrotestosterone (DHT) supplement prior to cancer cell inoculation. In a bone metastasis model, LNCaP cells were inoculated into tibia bone marrow. The mice were randomized to treatment groups based on similar serum PSA levels and cancer-induced changes in bone determined by X-ray imaging at 6 weeks after inoculation of the cancer cells. The mice were treated with 300 kBq/kg of Radium-223 dichloride or vehicle at 6 and 10 weeks. Bone lesions were followed by X-ray imaging during the study. The study was terminated at 12 weeks after inoculation of the cancer cells and the tibias were removed for histological analysis and AR staining. In the in vitro assay, 0.1 and 0.01 nM R1881 increased LNCaP cell viability, and enzalutamide reduced cell viability in the presence of R1881 compared to the group with only R1881. In the subcutaneous model, tumors formed within 2-3 weeks after inoculation and the maximum tumor volume was reached within 10-12 weeks. LNCaP tumors grew in NMRI nude mice without DHT supplement, but DHT supplement supported tumor growth. In the bone metastasis model, LNCaP tumors induced osteoblastic-mixed bone lesions without additional androgen supplement, and immunohistochemical staining demonstrated strong AR expression. Radium-223 dichloride reduced the progression of tumor-induced bone lesions, decreased serum PSA levels, and decreased tumor area analyzed by histology. This study showcases relevant models that can be used at different phases of preclinical drug development when evaluating efficacy or safety of new therapeutics as mono- or combination therapies. The choice of a preclinical model and key readouts should be carefully selected based on whether developing therapeutics for localized or metastatic prostate cancer.

#4851

Involvement of extracellular vesicles in the abscopal effect induced by oncolytic adenovirotherapy.

Yoshihiko Kakiuchi,1 Shinji Kuroda,1 Nobuhiko Kanaya,1 Kento Kumon,1 Tomoko Tsumura,1 Satoru Kikuchi,1 Masahiko Nishizaki,1 Shunsuke Kagawa,1 Hiroshi Tazawa,1 Yasuo Urata,2 Toshiyoshi Fujiwara1. 1 _Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan;_ 2 _Oncolys BioPharma, Inc., Tokyo, Japan_.

Background: The abscopal effect is a phenomenon that shrinkage of metastatic tumors occurs concurrently with shrinkage of primary tumors by local treatments such as radiotherapy, and is generally considered to be caused through activation of the immune system. Extracellular vesicles (EVs) are recent interesting topics in scientific fields and are known to play important roles in a variety of intercellular communication processes including immune response. We previously developed a telomerase specific oncolytic adenovirus, Telomelysin (OBP-301), which is currently being tested in clinical trials, and have sometimes observed therapeutic effects on metastatic tumors after local injection to primary tumors in immunodeficient mouse models. Here, we hypothesized that EVs derived from OBP-301-treated tumors may be associated with the abscopal effect of OBP-301.

Methods: A human colon carcinoma cell line HCT116 and HCT116 expressing RFP (HCT116-RFP) were used in this study. EVs were isolated by ultracentrifugation from HCT116 cells treated with OBP-301 (Exo-301). Intercellular communication and cytotoxic activity via EVs after OBP-301 treatment were assessed in a non-contact co-culture system in vitro and a bilateral subcutaneous tumor mouse model in vivo.

Results: Exo-301 showed potent cytotoxic effects on HCT116 cells and this cytotoxicity was significantly blocked in the presence of anti-CD9 antibody, which suggested that Exo-301, not OBP-301 itself, was involved in this cytotoxicity. In non-contact co-culture experiments, more RFP was taken up into HCT116 cells after OBP-301 treatment on HCT116-RFP cells than control treatments, which meant that EVs including RFP reached HCT116 cells through membrane after OBP-301 treatment on HCT116-RFP cells. In a HCT116 bilateral subcutaneous tumor model in BALB/c nude mice, intratumoral injection of OBP-301 significantly suppressed the growth of untreated tumors as well as treated tumors, and in a bilateral tumor model with HCT116 on one side and HCT116-RFP on the opposite side, immunohistochemical staining showed the presence of RFP in untreated HCT116 tumors after OBP-301 treatment on HCT116-RFP tumors. These findings suggested that EVs derived from OBP-301-treated tumors was possibly involved in some cytotoxic effects on the opposite untreated tumors.

Conclusion: EVs may be an important factor in the abscopal effect brought by local treatment of OBP-301 besides immune activation.

#4852

Boosting immunity against pancreatic cancer by OBP-702 (Pfifteloxin), telomerase-specific replicative adenovirus armed with wild-type p53 gene.

Hiroyuki Araki, Hiroshi Tazawa, Takuro Fushimi, Takeyoshi Nishiyama, Satotu Kikuchi, Shinji Kuroda, Ryuichi Yoshida, Hiroyuki Kishimoto, Masahiko Nishizaki, Yasuo Urata, Shunsuke Kagawa, Toshiyoshi Fujiwara. _Okayama Univ, Gradatuate School of Medcine, Okayama, Japan_.

Background: Pancreatic ductal adenocarcinoma (PDAC) is the most lethal disease with a 5-year survival rate of less than 10%. Although immune checkpoint blockade has recently emerged as a novel antitumor therapy, PDAC is less sensitive to immunotherapy due to small number of tumor-infiltrating T cells. Recently, oncolytic virotherapy has been shown to stimulate the immune system as an immunogenic antitumor therapy. Activation of p53 has been also known to enhance antitumor immunity. In this study, we investigated the potential of telomerases-specific p53-expressing oncolytic adenovirus (OBP-702, Pfifeteloxin) for inducing the immunogenic cell death in PDAC cells.

Methods: OBP-702 (Pfifteloxin) is a telomerase-specific oncolytic adenovirus, in which the human telomerase reverse transcriptase (hTERT) promoter drives the expression of the viral E1 gene for tumor-specific replication, that expresses the wild-type p53 by inserting the human p53 cDNA at the deleted E3. OBP-301 (Telomelysin) is an original telomerase-specific adenovirus lacking p53. OBP-502 is an RGD mutant fiber-containing OBP-301. We used 4 human PDAC cell lines (MIA PaCa-2, Capan-1, BxPC-3, Panc-1) and mouse PDAC cell line (Pan02). In vitro antitumor effect of these viruses was evaluated using a XTT assay. The molecular mechanism of virus-mediated cell death was investigated by western blot analysis. The virus-induced immunogenic cell death was assessed by analyzing the level of extracellular ATP and high-mobility group box protein B1 (HMGB1) using ELISA assay. The comparative in vivo antitumor efficacy was also investigated using a syngeneic Pan02 subcutaneous tumor model.

Results: Telomerase-specific oncolytic adenoviruses induced the antitumor effect in all PDAC cells in a dose-dependent manner. The antitumor effect of OBP-702 was superior to that of OBP-301 or OBP-502. OBP-301 and OBP-502 mainly induced autophagy, whereas OBP-702 induced autophagy and apoptosis at 72 h after infection. The concentration of extracellular ATP and HMGB1 was significantly increased in OBP-702-infected PDAC cells compared to OBP-301- or OBP-502-infected cells at 24 and 48 h after infection. In mice carrying subcutaneous Pan02 murine PDAC tumors, intratumoral injection of OBP-702 resulted in a significant inhibition of tumor growth. Moreover, the number of tumor-infiltrating CD8+ T cells was significantly increased in OBP-702-treated groups compared to mock-treated group.

Conclusion: Our data suggest that telomerases-specific p53-expressing oncolytic adenovirus OBP-702 (Pfifteloxin) induces profound immunogenic cell death to boost the immune responses in PDAC. Clinical trials with OBP-702 and immune checkpoint inhibitors for PDAC are warranted.

#4853

M6123 is an ADCC-enhanced and selective monovalent antagonist of FGFR1 showing antitumor activity in preclinical lung cancer models.

Christina Esdar,1 Christel Iffland,2 Astrid Zimmermann,1 Angela Lim,3 Michael Sanderson,1 Andree Blaukat1. 1 _Merck KGaA, Darmstadt, Germany;_ 2 _Ligand Pharmaceuticals, Inc., San Diego, CA;_ 3 _EMD Serono Research and Development Institute, Billerica, MA_.

Despite the considerable progress made with immunotherapies and targeted therapies, lung cancer remains a devastating disease with a high medical need for more effective and safer therapeutic options. Targeted therapies have delivered meaningful benefit to subsets of lung adenocarcinoma patients harboring specific oncogenic driver alterations but have not yet been extensively explored in other lung cancer subtypes. In the case of lung squamous carcinoma and small-cell lung cancer, amplification and overexpression of FGFR1 have been described as potential oncogenic drivers.Several small molecule tyrosine kinase inhibitors of the FGFR family are in clinical development but initial reports have suggested limited therapeutic benefit of these agents when applied as monotherapy in FGFR1-amplified lung cancer patients. To overcome toxicities associated with pan-FGFR inhibition, such as dysregulation of FGF23-mediated phosphate homeostasis, M6123 was developed as an FGFR1-selective biological antagonist. An immunization campaign in OmniRats® led to the identification of the fully human, potent and highly selective FGFR1 antibody A08 which was converted into the monovalent FGFR1 antagonist M6123 based on the proprietary SEED platform. Furthermore, M6123 was engineered for enhanced ADCC effector function. In contrast to bivalent FGFR1 antibodies, M6123 was well tolerated in mice, with no signs of body weight loss. Of note, M6123 did not interfere with hormonal FGF23 signaling in mice, a severe side effect of FGFR kinase inhibitors. Concentration-dependent inhibition of FGFR1 auto-phosphorylation was demonstrated in FGFR1-overexpressing lung cancer cells both in vitro and in vivo. Moreover, M6123 monotherapy had strong antitumor activity in specific FGFR1-expressing cell line- and patient-derived xenograft models, whilst combination with chemotherapy significantly enhanced efficacy. In summary, M6123 is a unique and clearly differentiated selective FGFR1 antagonist which warrants further testing in preclinical models to refine a predictive biomarker strategy for selection of potentially sensitive lung cancer patients.

#4854

A translational multiplexed hypothesis generating study on circulating cytokines in patients with cancer cachexia.

Mª Teresa Agullo-Ortuño, Virginia Pardo-Marques, Elena Prieto-Garcia, C. Vanesa Diaz-Garcia, Irene Otero, Jose A. Lopez-Martin. _Hospital Universitario 12 de Octubre, Madrid, Spain_.

Cancer cachexia is a frequent pathophysiological state associated to terminal stage cancer patients, characterized by a significant reduction in body weight and resistant to nutritional interventions. It is an unmet medical need, and its underlying mechanisms are not completely understood.

Objectives: To measure multiple soluble cytokines in patients with cachexia, to develop new hypotheses on pathways and/or eventual therapeutic targets.

Methods: This is part of a pilot, observational, cross-sectional, case-control, IRB-reviewed translational research in cancer patients with/without cachexia. Anthropometric, clinical and biochemical data from consenting eligible cancer patients were collected. Blood samples were collected from all the participants, and plasma was separated and immediately stored at -20°C until analysis. For this substudy, human 63-plex kits were purchased from eBiosciences and used according to the manufacturer's recommendations. Plates were read using a Luminex 200 instrument, and the assay was performed in the Human Immune Monitoring Center at Stanford University (CA, USA). Data were analyzed by univariate and multivariate methods, using error adjustments for multiple testings (False Discovery Rate, FDR).

A total of 15 subjects were included in this study, 8 cachectic and 7 non-cachectic patients. Median age in cachectic group was 61.5 (36-81) years versus 63.9 (48-80) in non-cachectic group. The respective relative weight loss within the prior 3 months was 17.6% and 1.0%. Clinical, analytical and metabolomics from these patients have been reported elsewhere (doi: 10.1002/jcsm.12270). Of the 63 cytokines tested in our panel, 18 have been selected for fold-change (FC) analysis: for 12, their log2(FC)>1 (IL1RA, ENA78, IL8, GROA, IL23, MCP1, BDNF, PDGFB, IL18, IL21, RESISTIN, IL6), being the FC > 10x for IL1RA, ENA78, IL8, GROA and IL23. For the remaining 6, their log2(FC) < -1 (LEPTIN, GMCSF, IL12P40, IL1B, CD40L, IL5). The cytokines selected for their FDR were: LEPTIN, LIF, GMCSF, RESISTIN, TNFA, IL1B, IL12P40, IL4, IL12p70, BDNF, IL2, IL5, IL23, IL17A and IL13. We integrated these results with prior metabolomics research and performed pathway analyses that enabled us to identify a significant enrichment in several pathways related with infections, immune response, inflammation, energy homeostasis and cancer. This study, despite the size constraints, is able to capture known players in cancer cachexia (leptin, IL6, IL1). We show a potential role for BDNF (brain derived neurotrophic factor), which and has been described as a master coordinator of the physiological mechanisms that regulate food intake and energy expenditure, and identified as a player in murine models for cancer cachexia.

Results from this study will be integrated with data from additional research in the same patients (clinical, analytical, metabolomics and proteomics).

#4855

AMC303 inhibits tumor growth and metastasis in animal models by targeting CD44v6, a co-receptor of multiple oncogenic receptor tyrosine kinases.

Tina Heumann, Vanessa Al-Rawi, Thorsten Läufer, Martin Augsten, Alexandra Matzke-Ogi, Oliver Coutelle. _amcure GmbH, Eggenstein, Germany_.

Introduction: CD44v6, a member of the CD44 family, is an essential co-receptor for c-MET, RON and VEGFR-2. The receptor tyrosine kinases (RTKs) c-MET and RON, are key players in tumorigenic pathways, are activated by the ligands HGF and MSP, respectively and homo- or heterodimerize with each other or can be autoactivated due to amplification. The RTK VEGFR-2 is critical for angiogenesis in solid epithelial tumors. The peptidergic inhibitor AMC303, which allosterically and selectively binds to CD44v6, was investigated for its inhibitory effects on ligand-induced and autophosphorylated c-MET activation, and its impact on tumor growth and metastases in vivo.

Methods: In vitro, tumor cells were incubated with AMC303 for 30 min prior to induction with an RTK-ligand. Analysis of RTK activation was carried out by Western blotting. For in vivo xenotransplantation human pancreatic tumor cells (L3.6pl) were orthotopically injected. Animals were treated with AMC303 i.p. at 0.1, 1, 10 mg/kg QOD or QWK for 3 weeks to address dose-dependency, the optimal treatment schedule was investigated with one, three or five times weekly administrations. Regression of metastases was investigated at 1 mg/kg QOD. Survival was evaluated with a dose of 1 mg/kg QOD. Immunohistochemical (IHC) analysis was performed for cleaved caspase 6, smooth muscle actin and CD31. Reduction of vessel permeability was assessed in Balb/C mice with the Miles assay and quantification of intra-tumoral FITC dextran after i.v. injection.

Results: In vitro, AMC303 inhibited c-MET, VEGFR2 and RON phosphorylation induced by their respective ligands. Notably, AMC303 inhibited c-MET activation in cells with enhanced c-MET signaling caused by the exon 14 skipping mutation, but not c-MET auto-phosphorylation due to gene amplification. In vivo, AMC303 treatment attenuated primary xenograft tumor growth and metastasis and significantly prolonged survival. IHC on treated xenograft-tumors revealed an increase in apoptosis and necrosis along with a reduction in myofibroblast infiltration, angiogenesis and vessel permeability. Pharmacokinetic properties after i.p. administration of AMC303 revealed an terminal half-life of 6h. Administration schedules of AMC303 (1mg/kg) given one, three or five times per week had no significant impact on tumor growth attenuation. AMC303 treatment could be paused for 10 days without incurring significant tumor regrowth.

Conclusions: AMC303 inhibits c-MET in vitro in a ligand-dependent fashion and in vivo significantly improved overall survival in an orthotopic pancreatic xenograft mouse tumor model. AMC303 effectively attenuated both primary and metastatic tumor growth by increasing apoptosis, reduction of angiogenesis and vascular normalization. These findings demonstrate the benefit of targeting multiple oncogenic RTK signaling pathways by AMC303.

#4856

Suppression of PRKCI gene-amplified ovarian cancer using aptamer-delivered siRNA.

Hina Rehmani. _Augusta University, Augusta, GA_.

Ranked fifth in cancer death among women, ovarian cancer cure rates have remained at 30% over the past two decades. Treatment options have steadily followed the same methodology, surgery and then first-line chemotherapy. With a 70% relapse rate within the first two years of diagnosis and limited secondary options, acquiring more targeted treatments is imperative. Copy number aberrations, more than mutations, are commonly found in high grade serous ovarian cancer. Copy number gains of the Protein Kinase C iota (PRKCI) gene are found in over 33% of ovarian cancer patients. According to the TCGA database, patients with PRKCI gene amplification have a significantly poor overall survival than those that do not. With this information and the fact that it is a previously identified oncogene in ovarian cancer, Protein Kinase C iota (PKCi) serves as an excellent target. Unfortunately, the Protein Kinase C iota isoform shares an overall homology of 72% with the Protein Kinase C zeta isoform which makes selective targeting of the iota form a challenge. Previous publications have used gold-based compounds to block atypical PKC family members from interactions that are crucial for apical-basal polarity. We found, however, that these compounds exhibit no specific selectivity for PRKCI gene amplified ovarian cancer cell lines.To specifically target PKCi, we have created an RNA-based aptamer that contains a siRNA sequence against the PRKCI mRNA and EpCAM moieties on either end. The aptamer efficiently becomes internalized after binding to the cell surface EpCAM via clathrin-dependent endocytosis. Once inside, the aptamer is processed by Dicer to release the two unique siRNA duplex intermediates. The guide strand of the siRNA loads into the RNA-induced silencing complex (RISC) complex and Argonaute (Ago) protein family members mediate PRKCI mRNA silencing.We hypothesize that PRKCI gene amplification status offers a unique opportunity to stratify patients into different risk categories and RNA-aptamer targeting of PRKCI can provide therapeutic benefits, by impairing ovarian cancer cell proliferation and tumorigenesis. We expect this approach to provide potent and selective anti-tumor and anti-metastasis activity compared to targeting using other pharmaceutical options.

## CLINICAL RESEARCH

### Advances in Radiation Therapy / Surgical Oncology

#4857

Low dose radiotherapy synergizes with PD-1 inhibitor and maximizes the abscopal effect.

Limei Yin, Mengqian Li, Jianxin Xue, You Lu. _West China Hospital, Chengdu, China_.

Purpose/Objectives: It has been reported that low-dose irradiation (LDI) can reprogram the microenvironment of the radiated tumors and may enable T-cell homing. Lack of preexisting tumor-infiltrating CD8+ T cells may account for the non-response to immune checkpoint inhibitors. Our prior work has shown that hypofractionated radiotherapy (HFRT) can trigger in situ vaccination and subsequent abscopal effects. The goal of this study was to figure out whether LDI could enhance the efficacy of checkpoint inhibitors and whether combination of HFRT to primary tumor and LDI to metastasis could maximize the abscopal effect of HFRT.

Materials/Methods: In experiment exploring the synergy between LDI and immunotherapy, mice bearing CT26 and MC38 colon tumors were randomly divided into 4 groups, respectively: vehicle control, αPD-1 mAb, LDI (2Gy/1f), αPD-1 mAb + LDI. In experiment exploring the abscopal effect, CT26 and MC38 tumor cells were injected s.c. into both right and left hind legs. Subsequently, mice were randomized into 4 groups: sham control, HFRT (24Gy/8Gy/3f, right-side), LDI (2Gy/1f, left-side), HFRT (right-side) + LDI (left-side). Mice were followed for tumor growth and further analyses.

Results: We found that in both tumor models, low-dose irradiation alone could recruit effector CD8+T cells and increase intratumoral chemokines associated with CD8+T cells. Compared to single-therapy modality, a better tumor control was observed following αPD-1 mAb in combination with LDI. IFNγ+CD8+ T cells were augmented in spleens and tumors obtained from mice in αPD-1 mAb + LDI group. Besides, HFRT to primary tumor (right-side) and LDI to the secondary tumor (left-side) could achieve the best secondary tumor control among the four groups, thus maximizing the abscopal effect. In combination group, we found an increase of CD8+ T cells in both peripheral

blood and secondary tumor. Furthermore, the depletion of CD8+T cells deprived the enhanced efficacy in the combination group, indicating that CD8+ T cells played an important role in the efficacy.

Conclusion: These results indicate that LDI may be very useful as a preparatory step to induce T-cell homing. Given its low risk of toxicity and excellent tolerability, LDI may be a clinically applicable response modifier of immunotherapy.

Key words: radiotherapy, immunotherapy, abscopal effect, PD-1/PD-L1

#4858

CNS delivery of VX-970: A selective ATR inhibitor for radiosensitization in GBM.

Surabhi Talele,1 Afroz Mohammad,1 Minjee Kim,1 Danielle Burgenske,2 Ann C. Mladek Tuma,2 Jann N. Sarkaria,2 William F. Elmquist1. 1 _University of Minnesota, Minneapolis, MN;_ 2 _Mayo Clinic, Rochester, MN_.

Glioblastoma (GBM) is an aggressive and infiltrative primary brain tumor with a median survival of 14.6 months following the current treatment strategy of radiation and chemotherapy. Therefore, there is a need to develop strategies to enhance the efficacy of chemo-radiation treatments for GBM. DNA damage response signaling pathways play a critical role in DNA repair and cell survival following radiation therapy and the inhibition of these pathways could augment the cytotoxicity associated with radiation providing a radiosensitizing effect. Ataxia Telangiectasia and Rad3-Related Protein (ATR) is a key regulator of the DNA damage response network and VX-970 is the first potent and selective inhibitor of ATR to enter clinical trials. Preliminary in vitro studies from our lab to determine a dose dependent effect of VX-970 in combination with a radiation dose of 5 Gy on the cell survival indicated that administration of radiation led to an enhancement in the cell death with an increasing dose of VX-970 in the U251 human GBM cell line. We evaluated the BBB penetration of VX-970 and studied the role of efflux transporters on the brain exposure of VX-970 in preparation for efficacy studies in PDX models of GBM. Brain distribution studies were performed in wild-type and Mdr1a/b-/- Bcrp1-/- (triple knockout) FVB mice (n=4) following intravenous administration of 20 mg/kg VX-970. Plasma and brain samples were collected at 7 time points post dosing and were analyzed using LC-MS. The brain-to-plasma (B/P) ratio in transporter (Pgp and Bcrp) knockout mice was 17.5 as opposed to 0.8 in transporter intact wild-type mice. This 22-fold increase in the B/P ratio in the triple knockout mice indicates that Pgp and/or Bcrp play a significant role in the efflux of VX-970 from the brain thereby limiting its brain penetration. Oral administration of VX-970 at a dose of 20mg/kg in transporter intact wild-type mice indicated an oral bioavailability of 38%. Steady state brain distribution studies were also performed in wild-type and triple knockout FVB mice following intraperitoneal implantation of the Alzet osmotic pumps to release VX-970 at a rate of 10ul/hr for 48 hours. We observed that the B/P ratio in transporter (Pgp and Bcrp) knockout mice was 12.6 as opposed to 0.12 in transporter intact wild-type mice We are in the process of conducting additional studies in Pgp and Bcrp single knockout mice to determine a more precise status of the role of efflux transporters in the brain delivery of VX-970. The free fraction of VX-970 was found to be 6.1% in plasma indicating that it has a relatively high unbound fraction as compared to other chemotherapeutics for brain delivery. We are in the process of determining free fraction in the brain and relating the concentration of free drug to a dose range associated with effective radiosensitization through efficacy studies in the PDX models of GBM. This PK-PD relationship will help guide future clinical trials.

#4859

MFP-FePt-GO nanocomposite promotes radiosensitivity of non-small cell lung cancer via activating caspase system and impairing DNA damage repair.

Shan Peng, Yingming Sun, Yuan Luo, Shijing Ma, Yan Gong, Conghua Xie. _Wuhan University, Wuhan, China_.

Recent advances in nanomedicine provide promising alternatives for cancer treatment that may improve the survival and life quality of patients with cancer. A novel nanocomposite (MFP-FePt-GO) based on a lamellar-structure magnetic graphene oxide (GO) and polyethylene glycol drug delivery system was designed, synthesized and functionalized for co-delivery of metronidazole and 5-fluorouracil. The chemical synthesis efficiency was measured by Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy and energy dispersive X-ray spectroscope. The morphology, dispersion and sizes of this nanocomposite were also measured with transmission electron microscope. This new nanocomposite exhibited good solubility, stability and biocompatibility. While no severe allergies, liver and kidney damage or drug-related deaths were observed, MFP-FePt-GO promoted radiosensitivity of non-small cell lung cancer (NSCLC) cells both in vivo and in vitro. MFP-FePt-GO imrpvoed the effects of radiation through activation of the caspase system and impairment of DNA damage repair. This novel nanocomposite also induced a reactive oxygen species burst, which suppressed the antioxidant protein expression and induced cell apoptosis. Furthermore, MFP-FePt-GO prevented the migration and invasion of NSCLC cells. Taken together, MFP-FePt-GO showed a synergistic anti-tumor effect with radiation in eliminating the tumors. With good safety and efficacy, this novel nanocomposite might be a potential radiosensitive agent for NSCLC patients.

#4860

Association between timing of radiotherapy and severity of oral mucositis in head-neck cancer patients.

Fangyi Gu,1 William D. Duncan,1 YingDong Feng,2 Austin Miller,1 Nicolas Schlecht,1 Alan Hutson,1 Anurag K. Singh1. 1 _Roswell Park Comprehensive Cancer Center, Buffalo, NY;_ 2 _University of Buffalo, Buffalo, NY_.

Background: Oral mucositis is a very painful and debilitating complication of radiotherapy, occurring in nearly all head and neck cancer patients. It can severely affect quality of life and lead to treatment interruptions that subsequently impact clinical outcomes. With limited treatment options, there is an urgent need to develop new strategies for mucositis prevention.

Animal studies have shown that sensitivity to genotoxic stress strongly depends on the time of a day therapy is administered. Therefore, radiotherapy could be conducted at optimal times of a day associated with best tolerability, to reduce mucosal damage. This hypothesis is partially supported by two clinical trials that reported less severe oral mucositis in head and neck cancer patients treated in the morning vs. late afternoon; but results were not significant and times of a day not fully covered.

Methods: This study examined the associations between timing of radiotherapy and development of oral mucositis in 219 head neck cancer patients treated at Roswell Park Comprehensive Cancer Center, using data from Electronic Medical Records. Patients' treatment time was consistent throughout a 6/7-week course, and mucositis survey was self-reported weekly during treatment, providing a unique resource for this study. The primary endpoint is "soreness quality score" (SQS), a five-level score "0-no, 1-a little, 2-moderate, 3-quite a lot, and 4-extreme" in assessing patients' soreness severity for mouth and throat in the past 24 hours. We used a generalized linear model approach to assess the association between radiotherapy time and the maximum SQS (MSQS) reported during treatment, adjusting for age, smoking, treatment type, survey week and cancer site.

Results: Multivariate analyses revealed treatment time to be significantly associated with MSQS (p-value=0.025), with lowest MSQS (lsmean=2.26, ste=0.18) seen for patients treated in the early morning (8:30-<9:30am). MSQS increased in patients treated at later times, peaking in the early afternoon (lsmean=2.92 (ste 0.21) and 2.84 (ste 0.18) for patients treated at 12:00-<13:30pm and 13:30-<15:00pm, respectively), then decreased for patients treated in late afternoon (lsmean=2.37, ste=0.25). Among patients treated between 8:30-<9:30am, 43.2% developed severe oral mucositis (SQS grade 3 or 4), compared to 69.2% among those treated between 13:30-<15:00pm.

Conclusion: To our knowledge, this is the first study to find a significant variation of oral mucositis severity by treatment hour of radiotherapy; the variation shows an arc shape with the peak in the early afternoon. Our results suggest that identifying an optimal time of a day for radiotherapy may substantially prevent severe oral mucositis in head and neck cancer patients. Further studies are worthwhile to confirm our findings, and to find optimal treatment times for individual patients.

#4861

Chemotherapy improves overall survival in patients with T2N0M0 non-small cell lung cancer undergoing definitive radiation therapy: An analysis of the SEER database.

Takefumi Komiya,1 Gerard Chaaya,2 Emily Powell1. 1 _Parkview Cancer Institute, Fort Wayne, IN;_ 2 _Tulane University School of Medicine, New Orleans, LA_.

Objectives: Despite relatively routine practice and recommendations by clinical guidelines, an advantage of adding systemic chemotherapy to definitive radiation in patients with early stage non-small cell lung cancer (NSCLC) has never been demonstrated by randomized or large-scale studies. This study evaluates the role of chemotherapy in addition to thoracic radiation in T2N0M0 NSCLC patients who did not undergo surgical resection.

Materials and Methods: Using the Surveillance, Epidemiology, and End Results (SEER) database, we screened for patients with T2N0M0 NSCLC who received radiation therapy without surgical resection from 2004 to 2015. T-staging was defined according to the American Joint Committee on Cancer (AJCC) 6th (Year 2004+) and 7th (Year 2010+) versions. Kaplan-Meier curves and estimated overall survival according to chemotherapy status were determined by Log-rank test. Both univariate and multivariate analyses were conducted for the AJCC 6th (T2), 7th T2a, and 7th T2b groups. All the statistical analyses were performed using JMP software version 13 (SAS Institute, Cary, NC), and significance was achieved when P-value is below 0.05.

Results: A total of 6,075 and 3,138 patients were identified for AJCC 6th (T2; 3cm -7cm) and 7th (T2a; 3 - 5cm, T2b; 5-7 cm) version, respectively. Administration of chemotherapy was associated with younger age, male sex, non-adenocarcinoma, and high pathologic grade. Kaplan-Meier estimates demonstrated that the chemotherapy group had a statistically significant longer five-year overall survival than the non-chemotherapy group in patients with AJCC 6th T2 (19.9% vs 15.8%, p=0.0023) and AJCC 7th T2b (5-7cm, 20.9% vs 13.6%, p=0.0046) but not those with AJCC 7th T2a (3-5cm, 24.3% vs 21.1%, p=0.4369). Multivariate analyses also revealed that the use of chemotherapy was an independent prognostic factor in AJCC 6th T2 and AJCC 7th T2b.

Conclusions: This study strongly suggests that chemotherapy may benefit non-adenocarcinoma patients with primary tumor larger than 5cm (AJCC 8th T3) undergoing chest radiation.

#4862

Effect of irradiation and gold nanoparticle on expression dynamics of cell surface markers in MDA-MB 231 breast cancer cells.

Branislava Janic,1 Fangchao Liu,2 Kevin Bobbitt,1 Stephen Brown,1 Guangzhao Mao,2 Indrin Chetty,1 Benjamin Movsas,1 Ning Wen1. 1 _Henry Ford Hospital, Detroit, MI;_ 2 _Wayne State University, Detroit, MI_.

Cell surface molecules expressed on cancer cells can be used as diagnostic and therapeutic tools. In breast cancer, CD44 and CD24 were identified as cell surface markers characterizing cancer cell stemness that may correlate with prognosis. Breast cancer cells with high CD44 and low CD24 expression have been shown to exhibit proliferative, invasive and metastatic properties that may relate to drug sensitivity and metastatic risk in patients. In addition to stemness, immune evasion is another hallmark of cancer with CD47 "don't eat me" and CD274 "don't find me" molecules playing important roles in antitumor immunity. Although each of these molecules are of interest as therapeutic targets, current therapeutic paradigms involve multimodal approach with radiation therapy often having a central role. Hence, the effect of irradiation, and related radio-sensitizers, on the expression of cancer cells surface markers to be targeted with an adjuvant therapeutic mode must be considered when designing such therapies. The goal of this study was to determine the dynamics of CD44, CD24, CD47 and CD274 expression in radio-sensitized and irradiated MDA-MB 231 breast cancer cells. We have recently reported that gold nanoparticles (AuNPs) sensitized breast cancer cells to irradiation at kV and MV energies. Here, we hypothesize that AuNP may also modulate the irradiation altered expression of cell surface markers. We explored this by using 10 MV energies and 6 Gy radiation dose. MDA-MB-231 cells were incubated for 3 hours with 14nm AuNPs, irradiated and allowed to grow for 24h and 72h after which percenteges of positive cells were determined by flow cytometry. Results are expressed as a percent of control, non-irradiated cells that was set at 100%. At 24h non-irradiated cells pre-incubated with AuNP exhibited a decrease in the percentage of CD24+ and CD44+ cells, while no significant change in the percentage of CD47+ and CD274+ cells was observed. Irradiation with 6Gy at 10 MV induced a decrease in CD24+ and an increase in CD44+ cells and AuNP potentiated this effect. However, 72h post-irradiation, percentages of CD24+ and CD44+ cells significantly increased, compared to non-irradiated controls and this effect was of lesser magnitude in AuNP pretreated cells. Percentage of CD47+ and CD274+ cells also significantly increased 24h post-irradiation, but with less magnitude in AuNP treated cells. At 72h in AuNP treated irradiated cells no change was observed in the percentage of CD274+ cells, while the percentage of CD47+ cells significantly increased in comparison to the irradiated cells not exposed to AuNPs. Preliminary data shown here indicate that expression of molecules important for cancer progression, metastasis and immune evasion undergo changes in response to irradiation, and that these changes are affected by AuNPs. Further studies will shed more light on the mechanisms behind these observed effects.

#4863

Discovery of novel metabolomic plasma biomarkers predictive of radiation induced cardiac toxicity.

Keith Unger,1 Michael Girgis,2 Meena Rajagopal,2 Yeline Ceh,1 Meth Jayatilake,2 Yaoxiang Li,2 Amrita K. Cheema2. 1 _MedStar Georgetown University Hospital, Washington, DC;_ 2 _Georgetown University, Washington, DC_.

Introduction: Although advancements in cancer treatment have led to increased survivorship, radiation induced heart disease (RIHD) remains a major source of acute and long term morbidity and mortality for cancer survivors, for which there are no diagnostic tests in routine clinical use due to lack efficacy or excessive cost. Several studies have documented RIHD; however, none of these investigations have examined the utility of using metabolomics to prospectively identify cancer survivors who are at risk of cardiotoxicity. The overarching goal of this pilot study is to determine whether biomolecules secreted in the circulation by latent yet developing cardiotoxic processes can be leveraged for predicting risk of cardiac dysfunction in cancer survivors.

Approach: We used metabolomics/lipidomics analysis, an emerging field that provides information on biological perturbations based on relative changes in the plasma levels of endogenous metabolites, for a retrospective outcome analysis study. Patients treated at GUH with radiation therapy (RT) for locally advanced esophageal cancer (n = 11 x 3 time points), who underwent serial cardiac MRI's were selected. We identified a subset of patients who developed radiation related heart ischemia and fibrosis in the inferior/ basal segment of the heart associated with the high dose region, as well as associated cardiac functional impairments (n = 6 x 3 time points). We also analyzed plasma and cardiac tissue samples from groups male Sprague Dawley (SD) rats that were either sham irradiated or received fractionated doses (9 Gy per day x 5 days) of targeted X-ray radiation to the heart. Metabolomic analysis was used as a correlative approach for delineation of novel biomarkers associated with radiation induced cardiac toxicity.

Results: Global metabolomics and lipidomics analyses have yielded important data for developing classification algorithms with high predictive accuracy. We are in the process of performing statistical analyses to determine the sensitivity and specificity of the resultant panel, and any influence provided by age and gender on the predictive performance of the biomarker panel. These data will be presented at the AACR meeting.

Conclusion: Cardiac dysfunction following cancer therapy leads to significant morbidity and mortality. While our cohort includes esophageal cancer patients, we believe the cardiac toxicities detailed previously are applicable to other patient populations receiving high dose thoracic radiation therapy. We believe findings from this study are of significant interest to the scientific community since there are critical gaps in knowledge related to mechanisms of cardiac injury, clinical prediction, screening, prevention, and treatment.

Supported by Cancer Center Support Grant (CCSG/NCI/NIH) and funding Ruesch Foundation

#4864

Targeted melanoma therapies as radiosensitizers.

Jessica L. Smith,1 Cecilia Chang,1 Tim Wang,2 Helen Rizos,3 Han Shen,1 Matteo Carlino,2 Eric Hau2. 1 _Westmead Institute for Medical Research, Westmead, NSW, Australia;_ 2 _Westmead Hospital, Westmead, NSW, Australia;_ 3 _Macquarie Univeristy, Macquarie, NSW, Australia_.

Introduction: In BRAFV600 mutant melanoma combination of BRAF and MEK inhibitors is associated with a rapid response in approximately 70% of patients, and an improvement in survival. Radiotherapy is used in the treatment of melanoma in multiple clinical settings including stereotactic radiosurgery for brain metastasis, oligo-metastatic extracranial disease and palliative radiotherapy to symptomatic lesions. Preclinical studies, and a small number of clinical case reports suggest the BRAF inhibitor vemurafenib and the MEK inhibitor trametinib leads to increased radiosensitivity.

Aims: To determine if BRAF Inhibitors dabrafenib, vemurafenib and encorafenib, alone or in combination with their corresponding MEK Inhibitors, are radiation-sensitising in BRAF-mutant treatment naive melanoma cells.

Methods: Three melanoma cell lines harbouring a BRAF V600E mutation were studied: A375, SkMel28 and MM200. The half-maximal inhibitory concentration (IC50) for each cell line was determined by MTS assays for vemurafenib, dabrafenib, encorafenib, and the MEK inhibitors trametinib, binimetinib and cobimetinib. Clonogenic assays were conducted on each cell line. The optimal dose of BRAF and MEK inhibitors was determined from MTS assay and single drug clonogenic assays. Cells were treated with BRAF or MEK inhibitor alone and in combination. Cells were also treated with increasing doses of radiation (X-RAD 320 irradiator, PXI). The mode of cell death and changes in cell morphology were determined by live cell imaging (Wide Field Live Cell Observer, Ziess) for five days following the above treatments.

Results:Using Clonogenic assays, we confirmed that radiation sensitivity varies across different melanoma cell lines, with MM200 being significantly more sensitive to radiation then SKMel28. Clonogenic assays demonstrated that vemurafenib, dabrafenib, encorafenib, trametinib and binimetinib increased radiosensitivity across all three-cell lines. Further, the level of radiosensitsation was greater in combination across all three sets, than with either BRAF or MEK inhibitor alone. Using live cell imaging, we were able to confirm this increase in cell death, and visualise differences in the mode of cell death and morphology with these therapies.

Conclusions: Vemurafenib, dabrafenib, encorafenib, trametinib and binimetinib were shown in this study to be radiosensitisers in BRAFV600Emutant melanoma cell lines. Radiosensitisation was greatest with combined BRAF and MEK inhibition. Further studies are being conducted to determine if this sensitisation continues after BRAF resistance develops.

#4865

Prognostic value of BRCA2 and AR gene alterations in advanced prostate cancer patients treated with PSMA-targeted radionuclide therapies.

Panagiotis J. Vlachostergios,1 Vincenza Conteduca,1 Amy L. Hackett,1 Jyothi Manohar,2 Aileen Lee,1 Aidan Case,1 Michael Sun,1 Muhammad J. Niaz,1 Olivier Elemento,3 Ana M. Molina,4 David M. Nanus,5 Himisha Beltran,5 Neil H. Bander,4 Scott T. Tagawa5. 1 _Weill Cornell Medicine, New York, NY;_ 2 _Englander Institute for Precision Medicine, New York, NY;_ 3 _Englander Institute for Precision Medicine, Meyer Cancer Center, Weill Cornell Medicine, New York, NY;_ 4 _Meyer Cancer Center, Weill Cornell Medicine, New York, NY;_ 5 _Meyer Cancer Center, Englander Institute for Precision Medicine, Weill Cornell Medicine, New York, NY_.

Background: PSMA-targeted radionuclide therapy (TRT) is being developed for treatment of pts with prostate cancer (PC). Several targeting agents were shown to be active and tolerable in early phase studies & randomized trials are underway. Because of the DNA damaging effects of ionizing radiation and the relationship between AR pathway & PSMA expression, we hypothesized that pts with germline or somatic gene alterations in DNA damage repair (DDR) & crosstalk pathways (AR, MYC) treated with PSMA-TRT may demonstrate differential treatment responses & outcomes.

Methods: We examined a single-institution cohort of advanced PC pts with available germline (targeted) or/and somatic (targeted or whole exome) DNA testing, clinical data (Halabi CALGB prognostic factors) & outcome. The Kaplan-Meier method & Cox regression analysis were used to evaluate the associations of mutations/copy number alterations (CNA) with PSA response (≥50%,≥30%,any), radiographic response, PFS and OS. Stepwise forward-selection was used in the multivariable regression model & p for entry was set at 0.1. For final analyses, p≤0.05 was used for significance.

Results: We analyzed 53 pts treated with PSMA-TRT. 16 (30.2%) received 177Lu-J591, 28 (56.6%) 177Lu-PSMA-617, 4 (7.5%) had both concurrently, 2 (3.8%) 225Ac-J591 (3 additional received >1 agent sequentially & are analyzed based upon 1st drug). 6 (11.3%) had pathogenic germline DDR mutations while 31 (58.5%) had ≥1 mutation/CNA in DDR genes. The most frequently affected genes were: TP53 (n=21, 39.6%), BRCA2 (n=14, 26.4%), CHEK2 (n=10, 18.9%), FANCA (n=10, 18.9%), RB1 (n=9, 16.9%), ATM (n=5, 9.4%), ERCC5 (n=5, 9.4%), ERCC3 (n=3, 5.7%), ERCC2 (n=2, 3.8%), BRCA1(n=2, 3.8%), MSH6 (n=2, 3.8%), FANCD2 (n=2, 3.8%), FANCF (n=2, 3.8%). AR amplifications (ampl) or resistance-mutations were found in 22 pts (41.5%), and MYC ampl in 9 pts (16.9%). 19 (35.8%) pts had ≥50% PSA decline, 24 (45.3%) experienced ≥30% decline & 39 (73.6%) had any PSA decline. 4 pts experienced a PR while 18 had SD. BRCA2 inactivating mutations, deletions or losses were associated with any PSA decline (p=0.011). PFS was significantly longer in patients with RB1 deletion or loss (5 vs 3 mos, p=0.003). BRCA2 alterations were predictive of longer OS vs wild-type (49 vs 17 mos, p=0.09). AR ampl or mutations and MYC ampl predicted shorter OS (AR: 13 vs 63 mos, p=0.02; MYC: 8 vs 24 mos, p=0.06). On multivariate analysis after adjusting for Halabi prognostic groups (low vs high risk), BRCA2 & AR alterations retained their significance as independent prognosticators of OS (BRCA2 HR 0.1 [0.02-0.42], p=0.002; AR HR 7.2 [2.09-25.14], p=0.002).

Conclusions: Knowledge of molecular alterations in BRCA2, AR and RB1 genes may have potential utility for prediction of clinical outcomes in pts being considered for PSMA-TRT. These findings merit further testing and validation in larger prospective trials.

#4866

CBX8 regulates radiation resistance in squamous cell carcinoma of esophagus by targeting DNA damage and repair.

Hongcheng Zhu,1 Yixuan Zhang,2 Hui Chen,2 Xueli Yang,1 Xinchen Sun,2 Kuaile Zhao1. 1 _Fudan University Shanghai Cancer Center, Shanghai, China;_ 2 _The First Affiliated Hospital of Nanjing Medical University, Nanjing, China_.

Background: Chromobox 8 (CBX8) contributes to proliferation and invasion of esophageal squamous cell carcinoma (ESCC), but its role and potential mechanisms in radiosensivity is still unknown.

Methods: We used tumor tissue microarray (TMA) to detect CBX8 expression and clinical features and survival of ESCC patients. Lentivirus was used to knockdown of CBX8 gene and stable transmitted cell line of ECA-109 and TE13 were made. In vitro cell counting kit 8 and clone formation assay were used to detect cell viability and proliferation. Radiosensivity was examined by clone formation assay after exposure of 0, 2, 4, 6, 8 Gy X-ray by a medical accelerator of different stable cell lines. Apoptosis, cell-cycle arrest, and γ-H2AX expression were examined with or without irradiation by flow cytometer and immunoflourence. Gene-chips and western blot were used to investigate molecular mechanism. In vivo experiments of xenografts were used to confirm the results.

Results: CBX8 has much higher expression in tumors than adjacent tissues in ESCC patients, and makes a difference in multivariate regression analysis of survival. CBX8 promotes ESCC cells proliferation. Improved radiosensivity was detected in CBX8 knockdown cells. Gene-chips was used in CBX8 knockdown ECA-109 and control cells in normal conditions and irradiation and DNA damage and repair pathways were detected by bioinformatic analysis, which was confirmed by western blot. In xengraft radiosensivity experiments, improved radiosensivity with a reduction of tumor doubling time was observed in CBX8 knockdown tumors. Immunohistochemistry and immunofluorescence of tumor tissue confirmed the molecular mechanism of DNA damage and repair pathway.

Conclusions: Our findings suggest that CBX8 contribute to radiation resistance in ESCC, which may be mediated by DNA damage and repair pathway. Targeting CBX8 may be a potential therapeutic approach in overcoming resistance.

#4867

Radiosurgery consultation to treatment interval, additional brain metastases during the interim and outcomes after therapy.

Federico Ampil, Gloria Caldito, Troy Richards. _Louisiana State University Health Sciences Center, Shreveport, LA_.

Background: Expediting the process of diagnosis and treatment should theoretically improve outcome. Delays in beginning therapy can cause anxiety to the patients and physicians who may be concerned about tumor progression before treatment is initiated. Treatment delays can be characterized as patient-generated or logistical medical infrastructure prolongations.

Objective: The goal of the present study was to determine the impact of stereotactic radiosurgery (RS) consultation to treatment interval (CTI) and the development of additional brain metastases (BRM) during the interim on outcomes after RS.

Methods: Between October 2014 and April 2018, 15 individuals developed more contrast-enhancing BRMs during the CTI. These patients were treated with gamma knife RS (including the six patients who underwent gross resection of metastatic intracranial neoplasm). The median duration of CTI was 19 days (range 5-61 days). A comparison of the ≤19 days CTI (eight patients) to the >19 days CTI (seven patients) was performed to assess the effects of the extent of delay to RS. The total number of BRM for RS was 68. The median target volume was 0.48 cm3, and the median margin dose was 24 Gy.

Results: Most (67%) of the subjects exhibited good performance status. The overall crude survival rate at two years was 27%; acute or late toxicity after RS was not observed. Comparison of the CTI groups revealed: i) A longer CTI was not associated with a poorer prognosis; ii) BRM recurrence after RS was less common in this patient subgroup; iii) Quality of life (QOL) was satisfactory even though the CTI was prolonged.

Conclusion: Acceptable longevity with minimal morbidity satisfying the QOL consideration was found in this audit of cases about CTI and the occurrence of more BRMs during the interim. Decreasing CTI in patients with BRM remains an important goal of quality improvement.

#4868

A preclinical PK/PD model based on a mouse glioblastoma survival model for AZD1390, a novel, brain-penetrant ATM kinase inhibitor, to predict the inhibition of DNA damage response induced by radiation and the human efficacious dose.

Venkatesh Pilla Reddy,1 Andy Sykes,1 Nicola Colclough,1 Stephen T. Durant,2 Lenka Oplustil O'Connor,3 Matthias Hoch,4 Nuria Buil Bruna,4 Serena De Vita,5 Melinda Merchant,5 Martin Pass6. 1 _DMPK, Oncology, IMED Biotech Unit, AstraZeneca, Cambridge, United Kingdom;_ 2 _Bioscience, Oncology, IMED Biotech Unit, AstraZeneca, Cambridge, United Kingdom;_ 3 _Translational Science, Oncology, IMED Biotech Unit, AstraZeneca, Cambridge, United Kingdom;_ 4 _QCP, ECD, IMED Biotech Unit, AstraZeneca, Cambridge, United Kingdom;_ 5 _Oncology TMU, ECD, IMED Biotech Unit, AstraZeneca, Boston, MA;_ 6 _Projects, Oncology, IMED Biotech Unit, AstraZeneca, Cambridge, United Kingdom_.

AZD1390 is a novel, highly selective, brain-penetrant, potent inhibitor of the Ataxia Telangiectasia Mutated (ATM) kinase. ATM is activated in response to double-strand breaks (DSBs) and coordinates cellular responses to ionising radiation and other insults. Radiation is the mainstay of treatment for patients with brain tumors, and glioma cells are exquisitely sensitive to ATM inhibition. Therefore, AZD1390 is in clinical development in combination with radiation therapy for the treatment of patients with Glioblastoma Multiforme (GBM) and brain metastases from solid tumours (NCT03423628). [1] The aim of this work was to develop a translational PK/PD-efficacy model for AZD1390 that would enable the project team to assess the extent/duration of inhibition of target engagement required and help to predict the optimal dose and regimen of AZD1390 to treat patients with brain malignancies in combination with radiation.

Time-course data of pharmacokinetics (PK), pharmacodynamics (PD) and tumor growth inhibition from mouse NCI-H2228 brain tumor orthotopic model studies were used to build a PK-PD/Efficacy or survival model. ATM activation is induced by IR treatment, becoming transiently phosphorylated within minutes of IR exposure and dissipates over a 24-hour period. This model quantitatively and dynamically integrates AZD1390 brain PK to the rate and extent of inhibition of phosphorylation of ATM, a proximal PD marker of ATM kinase activation caused by radiation induced DNA damage in the tumor and rate of induction of cell death (determined by bioluminescence signalling as a measure of tumor growth). In parallel, we also modelled cell cycle profiles in GBM cell lines to complement the in-vivo modelling work. The pATM time-course relative to IR radiation (2 Gy) was modelled using AZD1390 concentrations in the brain tumor and the corresponding pATM inhibition, which was then linked to efficacy by estimating tumor cell kill rate via exponential tumor growth model. The free brain concentration of AZD1390 that resulted in half-maximal inhibition (EC50) of pATM after radiation was estimated to be 0.8 nM (range 0.4 to 1.6 nM). Significant tumor regression in orthotopic tumor model was observed at doses >5 mg/kg. An average pATM inhibition over 24h in the mouse GBM survival model was in the range of 44% (5 mg/kg QD) to 88% (20 mg/kg BD). The % overall survival at 5 mg/kg QD and 20 mg/kg BD dose were 56% and 100%, respectively.

Conclusion: The translation of mouse survival data to clinical schedules is unknown, thus efficacious dose was anchored to pATM inhibition required to deliver significant tumor regression.

References:

1. Durant ST et al., The brain-penetrant clinical ATM inhibitor AZD1390 radiosensitizes and improves survival of preclinical brain tumor models. Sci. Adv. 2018;4.

#4869

Chromosomal and telomeric biomarkers of normal tissue injury to evaluate risk of secondary malignancy following IMRT for prostate cancer.

Jared J. Luxton,1 Miles J. McKenna,1 Lynn Taylor,1 Gregory P. Swanson,2 Susan M. Bailey1. 1 _Colorado State University, Fort Collins, CO;_ 2 _Texas A &M College of Medicine, College Station, TX_.

An overall intent of radiotherapy is to precisely target tumor cells, while minimizing exposures to surrounding normal tissue. Despite successes, there is growing concern that an unacceptably large volume of normal tissue is unavoidably exposed. Chromosome aberrations provide a direct measure of ionizing radiation (IR)-induced DNA damage, as well as an indirect measure of future risk since they are associated with virtually all known cancers. Such structural variants (SVs) include translocations (rearrangements between chromosomes) and inversions (rearrangements within chromosomes), the latter being recently identified as part of a distinctive mutational signature associated with radiation therapy-induced second malignancies. Directional Genomic Hybridization (dGH), is a strand-specific cytogenomics-based methodology for cell-by-cell, high-resolution detection of all SVs, particularly inversions, which when combined with compatible subtelomere probes (Telo-dGH), can be used to distinguish inversions from recombination events (sister chromatid exchange) involving chromosomal termini. Telomeres are critical structural elements that serve to protect the physical ends of chromosomes. Dysfunctional telomeres are associated with instability and carcinogenesis, as well as with a variety of other age-related pathologies (e.g., cardiovascular disease). We are validating Telo-dGH as a prospective "personalized" approach of evaluating normal tissue injury, and therefore future risk, associated with radiation therapy - regardless of tumor type or treatment modality. Here, we report results of monitoring prostate cancer patients before and after intensity-modulated radiation therapy (IMRT) to assess radiosensitivity (toxicity), as well as risk of secondary malignancy and other degenerative late effects. Such a strategy has the potential to better inform patient treatment and management decisions based on predicted individual risk.

#4870

Dedicator of cytokinesis-x promotes chemo and radioresistance of gastric cancer by modulating WNT signaling and cancer stem cell traits.

Hsiang-Cheng Chi, Kwang-Huei Lin. _Chang Gung University, Taoyuan, Taiwan_.

Prognosis of gastric cancer (GC) remains poor and the key players in its molecular pathogenesis are unknown at present. There is therefore a critical need to identify novel prognostic factors of GC. Dedicator of cytokinesis represents a protein family belonging to atypical Rho guanine nucleotide exchange factors (GEF) for Rac and/or Cdc42 GTPases. Dedicator of cytokinesis-x, a member of the Dock-C subfamily, exchanges GDP for GTP for Rac1 and Cdc42, both in vitro and in vivo. Here, we identified Dedicator of cytokinesis-x as an independent biomarker for GC prognosis. Elevated Dedicator of cytokinesis-x expression was associated with significantly shorter cumulative survival in both univariate and multivariate analyses. Moreover, higher dedicator of cytokinesis-x expression is positively correlated with tumor size, depth of invasion, lymph node metastasis, vascular invasion and pathological stage. To establish the downstream effectors of dedicator of cytokinesis-x, we analyzed the genes in relation to dedicator of cytokinesis-x expression using three independent clinical GC cohorts. Genes displaying a positive correlation with dedicator of cytokinesis-x were subsequently subject to gene ontology analysis and shown to be significantly involved in the WNT/β-catenin signaling pathway. Depletion of dedicator of cytokinesis-x suppressed cell proliferation, chemo- and radio-resistance, metastasis, oncosphere formation ability and tumorigenicity as well as expression of gastric cancer stem cell (CSC) markers. Dedicator of cytokinesis-x depletion was accompanied by a concomitant decrease in CD44, CD133 and nuclear β-catenin expressions. Moreover, interactions between dedicator of cytokinesis-x and β-catenin were observed in the nucleus of GC cells. This finding suggested that dedicator of cytokinesis-x may act as a tumor promoter through WNT/β-catenin activation.

#4871

Comparative analysis of tumor treating fields using conventional versus alternative array placement for posterior fossa Glioblastoma.

Edwin Lok, Pyay San, Victoria White, Eric T. Wong. _Beth Israel Deaconess Medical Center, Boston, MA_.

Background: Tumor Treating Fields (TTFields) therapy is an approved treatment modality for glioblastoma. Optimal transducer array placement can be defined by anatomy-based finite element analysis (FEA), which helps to delineate electric field coverage of the tumor. The conventional array placement was designed to provide adequate electric field coverage for supratentorial glioblastomas. There is an alternative array placement configuration for infratentorial tumors, but the extent of coverage to posterior fossa glioblastoma is unknown.

Methods: Patient anatomy-based FEA models were created by segmenting MRI images — using the NewSegment algorithm from Statistical Paramedic Mapping 8 — into tissue "masks" utilizing ScanIP from Simpleware (Exeter, UK). The physical properties of each tissue type were first applied to these masks and then boundary conditions for physics modeling was set up within COMSOL Multiphysics (Burlington, MA). COMSOL generated electric field maps were compared for models using the conventional array placement configuration for supratentorial tumors versus the alternative array placement configuration for infratentorial tumors. Electric field-volume histograms (EVHs) and specific absorption rate-volume histograms (SARVHs) were constructed to numerically evaluate the relative and/or absolute magnitude volumetric differences between models.

Results: The alternative configuration consists of array placement at the vertex, the bi-occipital regions and the upper neck. The highest EAUC was found at the epidural space surrounding the spinal cord and scalp for both types of configurations, whereas the lowest was located at the tongue and orbits. Using the conventional configuration, the gross tumor volume (GTV) had an electric field area under the curve (EAUC) of 40.5 V/m, volume covered with electric field intensity of 150 V/m (VE150) of 0.01%, 95% electric field intensity (E95%) of 30.9 V/m, E50% of 41.1 V/m, and E20% of 46.6 V/m. The GTV also had a SARAUC of 4.0 W/kg, volume covered with SAR of 6 W/kg (VSAR6) of 0%, SAR95% of 0.6 W/kg, SAR50% of 0.7 W/kg, and SAR20% of 0.8 W/kg. The alternative configuration produced EAUC of 52.3 V/m, VE150 of 3.6%, E95% of 29.1 V/m, E50% of 44.7 V/m, and E20% of 58.1 V/m, as well as SARAUC of 0.9 W/kg, VSAR6 of 0.3%, SAR95% of 0.6 W/kg, SAR50% of 0.8 W/kg, and SAR20% of 0.9 W/kg.

Conclusions: The alternative array placement configuration provides a higher coverage of electric field (+29%) to the posterior fossa glioblastoma when compared to the conventional configuration.

#4872

**Disulfiam, a re-positioned aldehyde dehydrogenase inhibitor, enhances radiosensitivity of human glioblastoma cells** in vitro **.**

Hyeon Kang Koh,1 Soo Yeon Seo,2 Jin Ho Kim,3 Hak Jae Kim,3 Eui Kyu Chie,3 Seung-Ki Kim,4 Il Han Kim3. 1 _Konkuk University Medical Center, Seoul, Republic of Korea;_ 2 _Seoul National University, Seoul, Republic of Korea;_ 3 _Seoul National University College of Medicine, Seoul, Republic of Korea;_ 4 _Seoul National University Children's Hospital, Seoul, Republic of Korea_.

Purpose : Glioblastoma, the most common brain tumor in adults, has poor prognosis. The purpose of this study was to determine the effect of disulfiram (DSF), an aldehyde dehydrogenase inhibitor, on in vitro radiosensitivity of glioblastoma cells with different methylation status of O6-methylguanine-DNA methyltransferase (MGMT) promoter and the underlying mechanism of such effect.

Materials and Methods : Five human glioblastoma cells (U138MG, T98G, U251MG, U87MG, and U373MG) and one normal human astrocyte (NHA) cell were cultured and treated with DSF or 6MV x-rays (0, 2, 4, 6, 8 Gy). For combined treatment, cells were treated with DSF before irradiation. Surviving fractions fit from cell survival based on colony forming ability. Apoptosis, DNA damage repair, and cell cycle distribution were assayed by Western blot for cleaved caspase-3, γH2AX staining, and flow cytometry, respectively.

Results : DSF induced radiosensitization in most of the glioblastoma cells, especially, in the cells with radioresistance as wildtype unmethylated promoter (MGMT-wt), but did not in normal NHA cell. DSF augmented or induced cleavage of caspase-3 in all cells after irradiation. DSF inhibited repair of radiation-induced DNA damage in MGMT-wt cells, but not in cells with methylated MGMT promoter (MGMT-meth). DSF abrogated radiation-induced G2/M arrest in T98G and U251MG cells.

Conclusion : Radiosensitivity of glioblastoma cells were preferentially enhanced by pre-irradiation DSF treatment compared to normal cell, especially radioresistant cells such as MGMT-wt cells. Induction of apoptosis or inhibition of DNA damage repair may underlie DSF-induced radiosensitization. Clinical benefit of combining DSF with radiotherapy should be investigated in the future.

#4873

The impact of primary tumor surgery on survival in HER2 positive stage IV breast cancer patients in the current era of targeted therapy.

Ross Mudgway,1 Carlos Chavez de Paz Villanueva,2 Ann C. Lin,2 Maheswari Senthil,2 Carlos A. Garberoglio,2 Sharon S. Lum2. 1 _University of California, Riverside School of Medicine, Riverside, CA;_ 2 _Loma Linda University School of Medicine, Loma Linda, CA_.

Objective: While prior studies evaluating the survival benefit from primary site surgery in stage IV breast cancer patients have provided mixed results, it has been well documented that anti-human epidermal growth factor receptor (HER) therapy for metastatic HER2 positive(+) disease improves outcomes. We sought to examine the impact of primary tumor resection on survival in HER2+ stage IV breast cancer patients in the era of HER2 targeted therapy.

Methods: We conducted a retrospective cohort study of women with HER2+ stage IV breast cancer in the National Cancer Database from 2010 (when mandatory HER2 reporting began) to 2012. Surgical removal of the primary tumor and Cox proportional overall mortality hazard ratios (HR) were assessed. Propensity score matching to diminish selection bias adjusted for demographic, tumor, and treatment variables.

Results: Of 3,231 patients, 71.3% were non-Hispanic (NH) white; 18.4%, NH black; and 5.8%, Hispanic. Bone only metastasis was seen in 25.0% of cases. Treatment included chemo/immunotherapy in 89.4%; endocrine therapy, in 37.7%; and radiation, in 31.8%. Overall, 1,130 (35.0%) underwent primary site surgery and 2,101 (65.0%) did not have surgery. The mean age of those who had surgery was 56.0+13.6 years compared to 59.1+13.7 years who did not (p < .0001). Median follow-up was 21.2 months (range 0-52). Factors associated with increased odds of having surgery included having Medicare/other government or private insurance vs none/Medicaid (OR 1.36, 95% CI 1.03-1.81 and OR 1.93, 95% CI 1.53-2.42, respectively), radiation (OR 2.10, 95% CI 1.76-2.51), chemo/immunotherapy (OR 1.99, 95% CI 1.47-2.70), and endocrine therapy (OR 1.73, 95% CI 1.40-2.14). NH black vs NH white patients (OR 0.68, 95% CI 0.53-0.87) and those treated at an academic/research vs community program (OR 0.67, 95% CI 0.50-0.89) were less likely to have surgery. Overall mortality HR were significantly associated with insurance (Medicare/other government vs none/Medicaid, HR 0.36, p < .0001), receipt of chemo/immunotherapy (HR 0.76, p = .008), endocrine therapy (HR 0.70, p = .0006), and radiation therapy (HR 1.33, p = .0009), NH black vs white race/ethnicity (HR 1.39, p = .002), visceral vs bone only metastases (HR 1.44, p = .0003), and lowest vs highest income quartile (HR 1.36, p = .01). Comorbidities, clinical tumor size, and clinical nodal status were not associated with survival. Propensity score analysis showed surgery was associated with improved survival vs no surgery (HR 0.56, 95% CI 0.40-0.77).

Conclusions: After controlling for covariates, surgery of the primary site in contemporary metastatic HER2+ breast cancer is associated with improved overall survival. If breast surgery is to be considered by patients and providers when deciding treatment strategy, it will be imperative to address significant disparities among patients who are offered surgical therapy.

#4874

Incidental gallbladder cancer: Best step forward.

Saurabh Galodha, Puneet Mahajan. _Indira Gandhi Medical College, Shimla, India_.

Objective: There is a rising trend of Incidental Gall Bladder Cancer (IGBC) with increase in laparoscopic cholecystectomy. Objective of our study was to evaluate the efficacy of further management (completion surgery and adjuvant chemotherapy) in IGBC in terms of long-term survival (> 5 years).

Material and Method: Clinic-pathological and survival data of 145 IGBC patients was analysed from a prospectively maintained database of GBC. Patients investigated with triple phase CT abdomen and if required PET-CT. Diagnostic laparoscopy with Completion extended cholecystectomy performed in patients amenable for resection. Adjuvant therapy consisted of gemcitabine and cisplatin. Patients followed up with OPD visits and telephonic enquiry.

Results: 864 patients of GBC were treated during the period of January 1989 to December 2012. Out of these 145 patients were IGBC [113 women (77%), median age; 52 years)]. 96 patients were detected on histopathology while 49 were diagnosed intra-operatively. Open cholecystectomy was done in 88, laparoscopic in 45 while 12 underwent laparoscopic converted to open cholecystectomy. Commonest T stage was T3 (n=71). All patients were re-evaluated after diagnosis of IGBC. 80 patients (55%) were taken up for surgery of which, 50 (62.5%) underwent completion extended cholecystectomy (rest had metastases or locally advanced unresectable disease). Median time interval between index surgeries to re-exploration was 35 days (0-250). It was longer in patients who could not undergo completion extended cholecystectomy (70 days vs. 49 days). Overall 5-year survival in pT1b, pT2 and pT3 stage was 49.5%, 36.1% and 8.4% respectively, but was considerably better in patients who underwent completion extended cholecystectomy compared with those who did not undergo re-surgery [pT1; 62.5% vs. 29.1%, pT2; 52.2% vs. 15.9%, pT3; 22.6% vs. 7.2%, p<0.001]. Prognostic factors associated with poor prognosis were higher T stage, R1 resection, bile spillage during index surgery, no completion surgery and residual disease on re-exploration on univariate analysis. On multivariate analysis factors portending poor prognosis were higher T stage, R1 resection and residual disease.

Conclusion: Early recognition, early completion radical surgery and adjuvant therapy lends considerable survival benefit to patients of incidental carcinoma gall bladder. Residual disease and R1 resection portends a poorer survival. High index of suspicion should be kept in patients with thick walled GB to avoid missing a malignancy at index surgery.

#4875

Clinical importance and clarification of the WHO subclassification of combined hepatocellular and cholangiocarcinoma.

Hisashi NAKAYAMA, Tadatoshi TAKAYAMA, Yutaka Midorikawa, Masahiko Sugitani. _Nihon University School of Medicine, Tokyo, Japan_.

Introduction: Combined hepatocellular and cholangiocarcinoma, classical type (Comb HCC-CCC) is defined as a single tumor, in which hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) are clearly differentiated. However, combined hepatocellular-cholangiocarcinoma with stem-cell features, cholangiolocellular type (CoCC) was added to the WHO Classification in 2010. This has caused a great deal of confusion and raised many questions for clinicians and pathologists. The aim of this study was to answer these questions and evaluate the clinical importance of the WHO classification. Subjects: The subjects were 1,002 patients with primary liver cancer who underwent hepatectomy from 2010 to 2016, including 931 with HCC, 6 with Comb HCC-CCC, 50 with ICC, and 15 with CoCC. Pathological evaluation using immunohistochemical staining was mainly used for CoCC. Results: The CoCC cases had similar serum total bilirubin and serum albumin to those in other types of hepatic cancer, but with a low ICGR15 level. AFP, PIVKA-II, and CEA were 4.4 (1.3 - 3,689) ng/mL, 18 (9 - 15,933) mAU/mL, and 3.8 (0.2 - 33) ng/mL, respectively. In preoperative CT diagnosis of CoCC, HCC, Comb HCC-CCC, and ICC were diagnosed in 20%, 40%, and 40% of the cases, respectively, suggesting difficulty with this diagnosis. The resected specimen showed the number of tumors (1 [1 - 16]), tumor diameter (4 [1.7 - 12] cm), microscopic-portal venous invasion (43%), microscopic venous invasion (36%), and cirrhosis (22%), suggesting a higher level of invasion to vascular channels compared to other types of cancer. In pathological findings, both transitional HCC and cholangiocarcinoma were found in HE staining, in addition to fibrosis and necrosis in the tumor. Among the subjects, 40%, 100%, 100%, and 100% had biliary cell markers of positive CD117 (c-Kit), positive CD56 (NCAM), positive CK7, and positive CK19, respectively, in addition to high expression of neural cell adhesion molecules. Levels of hepatocytes (10%) and glypican 3 (25%) were low. Cumulative survival was lower than that in other types of liver cancer, with 1- and 3-year survival rates of 67% and 57%, respectively. [Conclusion] The development mechanism of Comb HCC-CCC is thought to be genetic transformation of HCC or ICC, while that of CoCC with stem-cell features is believed to be cancerization of stem cells. However, stem-cell features are just characteristics, and thus do not serve as definitive evidence for stem cell-derived cancer. In addition, since there are various types of CoCC in Comb HCC-CCC, actual classification is extremely difficult in many cases. The clinical importance of clear differentiation between Comb HCC-CCC and CoCC is unclear, and thus the WHO classification of these tumors may be in a transition period.

#4876

Clinical significance of diffuse-type histological type coexistence in Stage II/III gastric cancer.

Hiroaki Tanaka, Kazuya Muguruma, Tatsuro Tamura, Takahiro Toyokawa, Masatsune Shibutani, Tatsunari Fukuoka, HIsashi Nagahara, Masaichi Ohira. _Osaka City Univ. Grad. School of Medicine, Osaka, Japan_.

Background: Signet ring cell carcinoma (sig) and non-solid poorly differentiated adenocarcinoma (por2) are the histological forms of gastric cancer called diffuse type. They are considered to be a tissue type with a poor prognosis. Adjuvant chemotherapy using S-1 is standard therapy for stage II/III gastric cancer in Japan, however, clinical relevance of histological type to outcome of adjuvant therapy remains unclear.

Purpose: The purpose of the present study was to examine the impact of the coexistence of diffuse type on the prognosis of stage II/III gastric cancer.

Patients and Methods: We retrospectively analyzed the clinicopathologic data of 935 gastric carcinoma patients who underwent gastrectomy between 2007 and 2015 at our department.

Results: We observed that there were 62 cases (17%) with sig mixed (dominant 12 cases, inferiority 50 cases) and 104 cases (30%) with por 2 mixed (dominant 19 cases, inferiority 85 cases). The patients with por2 or sig had more peritoneum dissemination compared with intestinal types. In Stage III, the recurrence rate was high in the case with sig mixed, the 3-year relapse-free survival rate was 36% in the mixed case and 55% in the non-mixed case. However, we found no correlation of sig mixture to prognosis of patients with Stage II. Among the patient who received the S-1 adjuvant chemotherapy, the prognosis of sig mixture was relatively poor.

Conclusion: In the case of stage III gastric cancer in which there was mixed signet ring cell carcinoma in the tissue, the recurrence rate was high and the prognosis was poor. Our findings suggested that it should be needed to treat sig mixed cases as a high risk group in the future postoperative adjuvant chemotherapy for gastric cancer.

#4877

Liver resection as part of cytoreductive surgery for ovarian cancer liver metastases in a national cancer hospital.

Jorge Luna Abanto, Luis Garcia Ruiz, Jheff Laura Martinez, Vladimir Villoslada Terrones. _Instituto Nacional de Enfermedades Neoplásicas, Lima, Peru_.

Introduction The current standard of treatment for newly diagnosed ovarian cancer (OC) includes definitive staging or cytoreduction followed by platinum-based chemotherapy. Therefore, studies have explored the implementation of liver resection to achieve complete macroscopic cytoreduction.

Objectives The aim of this study was to describe and evaluate the security of liver resections for OC liver metastases and the benefit in terms of survival as part of cytoreductive surgery.

Methods Data from patients submitted to surgical cytoreduction for OC that included liver resection at Instituto Nacional de Enfermedades Neoplásicas from January 2009 to December 2017 was retrospectively reviewed. The collected information included the patient's age at initial diagnosis, the FIGO staging system, tumor histology and grade, neoadjuvant chemotherapy, associated organ resections, number, dimensions, type and the margin status. Postoperative complications were measured according to Dindo-Clavien score, the length of hospitalization, 30-day mortality, disease-free survival, and overall survival was estimated from the moment surgery. The statistical analysis was made with non-parametric tests; Kaplan-Meyer survival curves were used.

Results 1211 patients were submitted surgical cytoreduction for OC surgery of whom 39 patients had liver resection as part of the surgical treatment of whom 21 had parenchymal metastasis. The mean age of patients was 46 years old, the 87% of patients had stage III/IV OC. 9, 17 and 13 patients had liver resection as part of cytoreductive surgery on primary, secondary and tertiary respectively. 58% of patients had epithelial type ovarian carcinoma, followed by 30% of stromal tumors. 35 patients had single liver metastasis, the mean diameter was 4.38 cm for parenchymal metastasis and 4.55 cm for peritoneal seeding, R0 resection was accomplished in 61% of patients. 33, 3 and 2 patients underwent minor hepatactomy, segmentectomy and major hepatectomy respectively. The margin status was not reported in 76 % of pathology reports. The most frequent associated organ resection was the spleen, omentum and peritoneum. The length of hospital stay was 5 days. 26 and 13 patients had Dindo-Clavien score I and II respectively, the 30-day mortality rate following surgery was 0. The disease free survival was better in patients with peritoneal seeding metastasis, however it was non-significant p:0.127. The overall survival analysis showed no difference p:0.752 between patients who were submitted to parenchymal or peritoneal seeding liver resection.

Conclusions Liver resection for advanced OC is a safe procedure from primary to quaternary cytoreduction and may bring survival benefit. There is a difference in prognosis following surgery between patients with parenchymal or peritoneal seeding liver resection, however further research is needed

### Novel Strategies for Biomarker Identification and Use in Cancer 3

#4878

Prognostic significance of Arginase and CK19 expression in human hepatocellular carcinoma after surgical resection: Correlation with recurrence-free survival.

Ifeyinwa E. Obiorah, Joeffery Chahine, Kyungmin Ko, ByoungUk Park, Jose deGuzman, Bhaskar Kallakury. _Medstar Georgetown University Hospital, Washington, DC_.

Background. Arginase-1 is an enzyme that is responsible for the conversion of arginine to urea in the urea cycle. A previous study has shown prognostic value of arginase expression in HCC following surgical resection. However, clear distinction on the role of arginase-1 as a predictor of recurrence in hepatocellular carcinoma remains unanswered. Occasionally, CK19, a cholangiocytic marker can be expressed in HCC, but the combination of arginase-1 and CK19 expression has never been evaluated. In this study, we investigate the usefulness of arginase-1 and CK19 expression alone and in combination in the determination of tumor recurrence following surgical resection in patients with HCC.

Design: Formalin-fixed paraffin embedded tissue sections from 112 HCCs were immunostained by an automated method (DAKO enVision + Dual Link System-HRP) using mouse monoclonal Arginase-1 (clone SL6ARG, Dako) and mouse monoclonal CK19 (clone RCK108, invitrogen). Nuclear and/or cytoplasmic reactivity was scored based on the intensity and percentage of positive cells in the tumor cells. Arginase-1 expression was separated into high (intense diffuse or regional positivity in > 50% of tumor cells) and low (weak or negative expression). The clinicopathologic variables including recurrence and survival were obtained from the patients charts and were correlated with the immunochemical results.

Results: High arginase-1 expression was detected in 93 patients (83%), whereas CK19 was positive in 19 patients (17%). In the univariate analyses, CK19 positivity (+) in HCC was associated with decreased recurrence free survival when compared with CK19 negative HCC (P=0.001). Arginase-1 expression was not associated recurrence-free and overall survival. However, the combination of arginase-1 and CK19 is associated with decreased recurrence-free survival (p<0.001). Furthermore, Arginase-1/CK19+ combined with TNM staging (p=0.042), vascular invasion (p<0.001) multiple tumor numbers (p=0.02) may play a role as additional predictors of recurrence-free survival. In the multivariate analysis, TNM stage, vascular invasion and CK19 were identified as independent prognostic indicators for recurrence-free survival. In addition, vascular invasion was an independent prognostic predictor of overall survival.

Conclusion: The combination of arginase-1 and CK19 immunoreactivity are potential biomarkers of adverse prognosis in HCC, correlating with presence of multiple tumors, vascular invasion and advanced stage. The results of this study may explain why clear beneficial effects of recombinant human arginase have not been demonstrated in clinical studies. Further study of arginase expression and its potential role as a therapeutic target in HCC appear warranted.

#4879

Poor outcome in hypoxic endometrial carcinoma is related to vascular density.

Casper Reijnen,1 Willem Jan van Weelden,1 Martijn SJP Arts,1 Johan P. Peters,1 Paul F. Rijken,1 Koen van de Vijver,2 Maria Santacana,3 Peter Bronsert,4 Johan Bulten,1 Marc Hirschfeld,4 Eva Colas,5 Antonio Gil-Moreno,5 Amando Reques,6 Gemma Mancebo,7 Fransesc Alameda,7 Camilla Krakstad,8 Jone Trovik,9 Ingfrid S. Haldorsen,9 Jutta Huvila,10 Stefanie Schrouwen,11 Martin Koskas,12 Francine Walker,12 Vit Weinberger,13 Lubos Minar,13 Eva Jandakova,13 Marc PLM Snijders,14 Saskia van den Berg-van Erp,14 Heidi VN Küsters-Vandevelde,14 Xavier Matias-Guiu,3 Frederic Amant,15 ENITEC-consortium, Leon FAG Massuger,1 Johan Bussink,1 Johanna MA Pijnenborg1. 1 _Radboudumc, Nijmegen, Netherlands;_ 2 _UZ Gent, Nijmegen, Belgium;_ 3 _Hospital Universitari Arnau de Vilanova, University of Lleida, Lleida, Spain;_ 4 _University Medical Center Freiburg, Freiburg, Germany;_ 5 _Vall Hebron Institute of Research, Universitat Autònoma de Barcelona, Barcelona, Spain;_ 6 _Vall Hebron University Hospital, Barcelona, Spain;_ 7 _Hospital del Mar, Barcelona, Netherlands;_ 8 _University of Bergen, Bergen, Norway;_ 9 _Haukeland University Hospital, Bergen, Norway;_ 10 _University of Turku, Turku, Finland;_ 11 _University Hospital Gasthuisberg, Leuven, Belgium;_ 12 _Bichat-Claude Bernard Hospital, Paris, France;_ 13 _Masaryk University, Brno, Czech Republic;_ 14 _Canisius-Wilhelmina Hospital, Nijmegen, Netherlands;_ 15 _Center for Gynaecologic Oncology, Amsterdam, Netherlands_.

Optimal identification of endometrial carcinoma (EC) patients at high risk of recurrence is currently lacking. Hypoxia is an important feature of aggressive EC leading to activation of hypoxic and angiogenetic target genes. The present study investigates the prognostic role of hypoxia and angiogenesis in EC. Data and tissues were used from 11 collaborating European Network for Individualized Treatment of Endometrial Cancer (ENITEC) centers. Tumor slides were stained for CAIX as a hypoxic marker and CD-34 for assessment of microvessel density (MVD) as a marker for angiogenesis. Complete slides were digitalized and analyzed using ImageJ software after exclusion of areas without tumor. A cutoff of 1% for the fraction of CAIX positive tumor cells was used. The MVD was assessed according to the Weidner method with the median as cutoff. Correlations with disease-specific survival (DSS), disease-free survival (DFS) and distant disease-free survival (DDFS) were calculated using Cox regression analysis. Sixty-three (16.4%) of 385 ECs showed positive CAIX-expression with high vascular density. Multivariable analysis showed that ECs with combined positive CAIX-expression and high vascular density had a reduced DSS (hazard ratio [HR] 3.71, p = 0.002) and DDFS (HR 2.68, p = 0.009) and a trend for reduced DFS (HR 1.87, p = 0.054). Multivariable analyses with CAIX-expression and vascular density as separate markers, showed that both were independent prognostic markers as well. This study found an impaired DSS and DDFS in ECs with positive CAIX-expression and high vascular density. Differential adjuvant treatment might be indicated for these ECs.

#4880

Parsortix™ system: Analytic performance evaluation using ovarian cancer cell lines.

Arianna Hustler, Gabrielle Wishart, Amy Templeman, Erica Bravo Sales, Sara Arocas Torrecillas, Kyra Mumford, Daniel J. O'Shannessy. _ANGLE Europe Ltd., Guildford, United Kingdom_.

Background: Ovarian cancer has the worst prognosis among gynecologic malignancies, in part due to presentation with later stage disease in a majority of women. Much effort has been expended towards early detection, but it remains a significant issue and challenge. One approach which may have potential in improving the detection of ovarian cancer, particularly in women who present with an adnexal or pelvic mass, is the isolation and interrogation of circulating tumor cells (CTCs) from peripheral blood. The microfluidic Parsortix™ system isolates rare cells from biological fluids, particularly blood, on the basis of cell size and deformability.

Objectives: The primary aim of the present study was to evaluate the performance of a modified Parsortix™ system for the isolation of ovarian cancer cell lines spiked into blood drawn from healthy volunteers (HVs). Parameters such as sensitivity, determined by dilution linearity, reproducibility and stability (time after blood draw) were assessed. In addition, the ability to molecularly interrogate Parsortix™ harvests by RT-qPCR for the CTC-specific genes Epithelial Cell Adhesion Molecule (EpCAM) and CytoKeratin (CK) was assessed.

Methods: Two ovarian cancer cell lines, CaOV3 and SKOV3, were used as model CTCs. Live cells were spiked into HV blood samples collected into K2EDTA vacutainers and stored for up to 120h. The spiked blood samples were then processed using modified Parsortix™ instruments (optimized to speed-up blood processing) and separation cassettes with a 6.5µm critical gap size. Harvests were evaluated for reproducibility, linearity, and stability by counting harvested cells and/or by using an optimized RT-qPCR assay. For experiments where cell counting and percentage recovery were assessed, cancer cells (CaOV3 or SKOV3) labelled with CellTracker™ Green were utilized and counted via fluorescence microscopy. When RT-qPCR was used to assess the recovered cells, unlabeled Parsortix™-isolated cells were lysed in a LiDS buffer, mRNA isolated using Dynabeads™, and cDNA synthesis performed followed by RT-qPCR for selected gene products.

Results: The sensitivity of the Parsortix™ system for the isolation/harvest of CaOV3 and SKOV3 cells was approximately 1 cell/mL blood. There was no appreciable effect of storage of the spiked K2EDTA blood samples on recovery of the pre-labelled CaOV3 cells for up to 120h hours after collection/spiking, with either room temperature or refrigerated (4oC) blood storage. The isolation/harvest of model CTCs was reproducible with a CV of ≤14%. RT-qPCR, while gene dependent, was shown to be linear with model CTC input and showed a sensitivity of approximately 1 cell for EpCAM.

Conclusions: These data demonstrate both the robustness and sensitivity of the Parsortix™ system for the isolation and interrogation of CTCs, and further studies, specifically in the area of ovarian cancer, are ongoing.

#4881

Molecular screening of patients with FGFR alterations for phase 1 (ph1) study with the selective FGFR inhibitor Debio 1347.

Anthony J. Iafrate,1 Darrell R. Borger,2 Ana Vivancos,3 Martin Voss,4 James Cleary,5 Funda Meric-Bernstam,6 Josep Tabernero,7 Keith Flaherty,1 Nobuya Ishii,8 Franck Brichory,9 Hiroaki Tanaka,10 Anna Pokorska-Bocci,10 Jose Baselga,4 Paolo Nuciforo3. 1 _Massachusetts General Hospital, Boston, MA;_ 2 _ODDU Portfolio and Alliance Management, Takeda Oncology, Cambridge, MA;_ 3 _Vall d'Hebron Institute of Oncology, Barcelona, Spain;_ 4 _Memorial Sloan-Kettering Cancer Center, New York, NY;_ 5 _Dana-Farber Cancer Institute, Boston, MA;_ 6 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 7 _Vall d'Hebron Institute of Oncology (VHIO), Barcelona, Spain;_ 8 _Chugai Pharmaceutical Co., Ltd.,, Toyko, Japan;_ 9 _Debiopharm Internataional S.A., Lausanne, Switzerland;_ 10 _Debiopharm International SA, Lausanne, Switzerland_.

Background: Oncogenic alterations in fibroblast growth factor receptors (FGFR) are seen across multiple solid tumors. Debio 1347 is an orally available, highly selective FGFR1,2,3 inhibitor that is undergoing phase I clinical trial evaluation in patients harboring an FGFR genetic abnormality. The availability of tumor tissue for retrospective genotyping, diversity of genetic alterations identified, and concordance with central laboratory testing were evaluated.

Methods: Patients harboring an FGFR1/2/3 gene amplification, mutation, or fusion were identified by local laboratories using different technologies. Patients received escalating doses of Debio 1347 from 10mg up to 150mg daily. Diagnostic tumor tissue was secured for post-hoc analysis at a central laboratory (CL) to confirm the alterations reported on enrollment. Whenever possible, fresh biopsies were collected prior to starting treatment for comparison.

Results: Archived diagnostic samples were available for most patients but material adequacy allowed for post-hoc genotyping to be performed in only 43 of 58 patients. For 23 of these patients, both a diagnostic sample and an on-study screening biopsy were secured and suitable for comparative analysis. Availability of screening material varied across tumor types. Overall, post hoc genetic testing on diagnostic tissue did not confirm the main FGFR alteration in 9 cases (23%): 4 FGFR amplifications, 2 fusions and 3 mutations. Four of FGFR amplifications could not be confirmed: two discordant calls were attributed to the difference in identifying polysomy over focal gene amplification. Two of 11 fusion cases were not centrally confirmed. For one of these cases, the low number of fusion reads for an FGFR3:TACC3 fusion was below the cutoff for CL reporting. When comparing genotypes between diagnostic and screening samples, concordance was 76% between the 2 CL sites. Efforts are ongoing to address all examples of discordance.

Conclusions: The clinical diagnostic landscape proves to be complex. The use of different technologies with different sensitivities and analysis pipelines constitute one of many diagnostic hurdles. Biopsy availability across different cancer types and tumor heterogeneity add to its complexity, as well as tumor evolution over time from initial diagnosis to treatment.

#4882

Expression atlas of FGF and FGFR genes in pan-cancer uncovered predictive biomarkers for FGFR inhibition response in clinical trials.

Yuan Li,1 Long Wu,1 Yanlei Cheng,1 Weiping Tao,1 Dawei Wu,2 Fei Ma,2 Ning Li2. 1 _Renmin Hospital of Wuhan University, Wuhan, China;_ 2 _National Cancer Center, Beijing, China_.

Genomic variation of FGFR has been intensively investigated in cancer for years, however, clinical trials on the basis of FGFR mutation or amplification as a druggable target of FGFR inhibitors have produced disappointing clinical outcomes. Therefore, the identification of predictive biomarkers for FGFR-targeted agents has remained a crucial issue. Expression profiles of FGF and FGFR in 8,111 patients with 24 types of solid tumors and 879 tumor cell lines along with drug sensitivity data were obtained from public databases. Differential expression, survival and correlation analyses were performed to uncover the clinical relevance of FGF and FGFR genes expression in pan-cancer. As results, FGF and FGFR were frequently dysregulated in pan-cancer. Moreover, almost all the FGF and FGFR were significantly associated with overall survival in at least two cancer types, however, mixed prognostic values were seen in pan-cancer. More importantly, tumor cell lines with high FGFR1/3 expression were more sensitive to FGFR inhibitor PD173074, especially in breast cancer, liver cancer, lung squamous cell carcinoma and ovarian cancer. The predicted positive ratios of FGFR1/2/3/4 were generally over 10% in most tumor types, especially in squamous cell carcinoma. High positive FGFR1 or 3 expression ratio were predicted in cholangiocarcinoma (58%), followed by bladder cancer (42%), endometrial carcinoma (35%), ovarian cancer (34%) and head and neck squamous cell carcinoma (34%). FGFR expression were promising predictive biomarkers for FGFR inhibition response in clinical trials and different combination of FGFR genes should be used in screening for patients in certain tumor types.

#4883

A glycan gene signature that robustly predicts prognosis in patients with pancreatic ductal adenocarcinoma.

Priyanka Sharma,1 Raju Kandimalla,1 Jasjit K. Banwait,1 Masayuki Sho,2 Yasuhiro Kodera,3 Ajay Goel1. 1 _Center for Gastrointestinal Research, Baylor Scott & White Research Institute and Charles A. Sammons Cancer Center, Baylor University Medical Center, Dallas, TX; _2 _Department of Surgery, Nara Medical University, Nara, Japan;_ 3 _Department of Gastroenterological Surgery, Nagoya University Graduate School of Medicine, Nagoya, Japan_.

Purpose: Pancreatic ductal adenocarcinoma (PDAC) remains the fourth leading cause of cancer-related deaths in the United States, with an overall 5-year survival rates of ~8%. The currently used clinicopathological factors (e.g. tumor size and grade, lymph node etc.) for determining patient prognosis are suboptimal. The only FDA-approved molecular non-invasive prognostic biomarker for PDAC patients is CA-19-9, which suffers from inadequate sensitivity and specificity. Nonetheless, the clinical use of CA-19-9, along with the growing evidence that altered glycosylation plays an important role in PDAC pathogenesis, provided us with a rationale to explore the clinical significance of glycan-related genes as potential prognostic biomarkers in PDAC.

Experimental design: We performed a comprehensive biomarker discovery (TCGA cohort, n=182), and validation (ICGC, n=80; GSE62452, n=65) to identify a glycan gene signature for predicting overall survival (OS) in PDAC patients. Subsequently, this gene signature was validated in two, independent, clinical cohorts (test cohort, n=103; validation cohort, n=227). The performance of this signature was further evaluated by univariate and multivariate CoxPH analyses. Lastly, using a logistic-regression model, we explored the feasibility of our glycan signature in identifying various molecular subtypes of PDAC.

Results: A comprehensive analysis of 411 glycan genes using Cox-LASSO regression modelling led to the identification of a 12-glycan gene signature, which robustly predicted overall survival (OS) of PDAC patients in the discovery (AUC=0.78), and two validation cohorts (ICGC, AUC=0.72; and GSE62452, AUC=0.70). Subsequent qRT-PCR validation in two in-house clinical cohorts revealed that a 9-gene signature was a robust predictor of OS (Test Cohort, HR: 1.81, 95% CI, 1.22-2.69, p=0.003; Validation Cohort, HR: 2.72, 95% CI, 2.00-3.69, p<0.0001). In univariate analysis, in addition to the 9-gene signature, the nodal status and CA-19-9 levels were significant predictors; while in the multivariate analyses, the gene signature emerged as an independent predictor of OS. A risk-assessment model including the 9-gene signature and the two clinical factors further improved the OS prediction (Test Cohort, HR: 2.71, 95% CI, 1.85-3.99, p<0.0001; Validation Cohort, HR: 2.72, 95% CI, 2.03-3.63, p<0.0001). Intriguingly, our signature was also highly accurate in identifying PDAC subtype with poor survival (i.e. squamous subtype) in the TCGA (AUC=0.87, P<0.0001) and the ICGC cohorts (AUC=0.89, P<0.0001).

Conclusion: Our systematic biomarker discovery and validation efforts led to the identification and establishment of a 9-gene glycan signature that robustly predicts survival in PDAC patients, and can accurately identify poor PDAC subtypes - highlighting its potential clinical significance for the personalized management of PDAC patients.

#4884

Intratumoral high endothelial venules as a surrogate marker for T cell inflamed tumor microenvironment and prognosis in gastric cancer.

Hongjae Chon, So Jung Kong, Joo Hoon Kim, Won Suk Lee, Sewha Kim, Chan Kim. _CHA Bundang Hospital, Seongnam-si, Republic of Korea_.

The absolute number of tumor infiltrating lymphocyte (TIL) have been well-studied for its prognostic impact in various human malignancies and association with therapeutic response to immune checkpoint blockade, but the importance of its distinct histologic pattern is not yet reported in gastric cancer. Also, the prognostic impact of high endothelial venule (HEV), which is a unique vasculature specialized for lymphocyte extravasation from systemic circulation to target tissue, has not gained much attention despite being believed to play a role in T cell infiltration into solid tumors. Here, we comprehensively analyzed the lymphocytic reaction patterns and HEVs using a large cohort of gastric cancer patients, and investigated their relationship with various clinicopathological features and prognosis. This study included 460 patients who underwent surgical resection for gastric cancer. Three representative TIL patterns was scored from 0 to 3. The definitions of the three patterns are: (1) Crohn-like lymphoid reaction (CLR)-nodular lymphoid aggregate with or without germinal center being observed either inside or along the tumor border; (2) Peritumoral lymphoid reaction (PLR)-band-like lymphocytic infiltration along the invasive margin; (3) Intratumoral lymphoid reaction (ILR)-lymphocytic infiltration into the stroma between cancer cells, or lymphocytes coming into direct contact with cancer cells. Immunohistochemistry for MECA-79 was selected as a specific marker of HEV in this study. Prominent CLR (score 2-3) was significantly associated with diffuse type, advanced stage, or more harvested lymph nodes (P < 0.05, all). Prominent PLR was significantly associated with intestinal type or advanced T stage (P < 0.05, all). Prominent ILR was significantly associated intestinal type or upper 1/3 location (P < 0.05, all). High HEV density was observed at significantly higher frequency in female patients and in tumors with diffuse type or early stage (P < 0.05, all). Notably, the number of HEV was associated with all histologic types of TIL patterns, especially with CLR scores (P < 0.001). In terms of disease-free survival (DFS) and overall survival (OS), patients with high score (score 2-3) of either of the three histologic TIL patterns didn't show significant difference in survival compared with those with low score (score 0-1). However, the patients with high HEV density showed longer DFS and OS compared with those with low HEV density (both P < 0.001), and this result was consistent even when other variables such as age, sex, stage were adjusted (both P < 0.001). In conclusion, although the results of this study suggest that all three histopathological lymphocyte infiltration patterns are not associated with patient survival, it presents the HEV density as a potential immune related prognostic factor in gastric cancer.

#4885

AL101 mediated tumor inhibition in Notch mutated ACC PDX models.

Renata ferrarotto*,1 Genia Alpert*,2 Udi Gluschnaider,2 Rami Rauch,2 Adi Mondshine,2 Oz Solomon,2 Bill Kramer,3 Evgeny Izumchenko,4 John Heymach,5 Andrea Vergara-Silva,2 Jon Aster,6 Matti Davis2. 1 _University of Texas M.D. Anderson Cancer Center, Houston, TX;_ 2 _Ayala Pharmaceuticals, Wilmington, DE;_ 3 _Kramer Consulting LLC, North Potomac, MD;_ 4 _Johns Hopkins University, Baltimore, MD;_ 5 _MD Anderson Cancer Center, Houston, TX;_ 6 _Harvard Medical School, Brigham And Women's Hospital, Boston, MA_.

* Equal contribution

Goal: To evaluate Notch inhibitor monotherapy and combination (combo) therapy in Adenoid Cystic Carcinoma (ACC) Patient Derived Xenograft (PDX) models with and without activating Notch mutations (mt). ACC is a rare salivary gland malignancy with no standard of care. Chemotherapy resistance limits treatment to surgery/radiation and ~60% of patients (pts) will recur. ~22% of ACC pts have Notch activating mutations that are associated with a distinct phenotype, aggressive disease and poor prognosis. In a P1 solid tumor study, 2 ACC pts were treated with AL101, an investigational gamma secretase inhibitor (J Clin Oncol 36, 2018 abstract 2515). One pt with an NRR (Negative Regulatory Region) activating mutation had a prolonged partial response, the 2nd pt with a PEST mutation had stable disease. Both had an extended PK profile with a sustained pharmacodynamic response (>50% inhibition of HES1 in peripheral blood). These results prompted us to evaluate the effect of AL101 in tumors that harbor/lack Notch activating mutations.

Design: 4 ACC PDX models were evaluated: ACCx9 (Notch1 mt: NRR activating mutation), ACCx11 (Notch1 mt, tandem duplication), ACCx6 (Notch1 wt), ACCx5M1 (Notch1 VUS, not predicted to be activating). Activated Notch1 (nuclear IHC stain) was confined to the Notch mt models. Tumors were implanted into 6-12 week old athymic nude female mice. Upon reaching 150-300 mm3 tumor volume, mice were randomized to treatment (N=5), or vehicle (N=10) arms. AL101 was dosed at 7.5 mg/kg po (days 1-4 of Q7D) as single agent or in combo with cisplatin (3 mg/kg ip; qw) or everolimus (10 mg/kg; po; qd).

Results: Significant tumor growth inhibition (TGI) was seen with AL101 monotherapy compared to vehicle treatment in both Notch mt models (ACCx9; 110% TGI P<0.0001, and ACCx11; 78% TGI P=0.0441). AL101 had no significant effect on tumors lacking Notch activating mutations (ACCx6: 37% TGI P>0.99, ACCx5M1: 60% TGI P=0.26). Cisplatin and everolimus monotherapy had no significant effect on Notch mt models (cisplatin: ACCx9; 43% TGI P=0.98, ACCx11; 50% TGI P=0.99, everolimus: ACCx9; 51% TGI P=0.6, ACCx11; 54% TGI P=0.99). AL101 with cisplatin or everolimus had no further benefit in the ACCx9 model (both 106% TGI P=0.98 or P>0.99 respectively). The ACCx11 model had a non-significant benefit (104% TGI P=0.17 for cisplatin combo and 107% TGI P=0.18 for everolimus combo). Interestingly, the ACCx6 Notch wt model was sensitive to everolimus (75% TGI P=0.027) with no significant benefit of adding AL101.

Conclusion: AL101 monotherapy had a significant anti-tumor effect in ACC PDX tumors with Notch activating mutations and lacked effectiveness in tumors lacking such mutations. Neither cisplatin nor everolimus monotherapy treatment had a significant effect in Notch mt models, alone or in combination with AL101. These data support the clinical development of AL101 as a targeted monotherapy for ACCs with Notch activating mutations.

#4886

**Patients with** EGFR **amplification but without** EGFRvIII **expression have improved benefit compared to those with** EGFRvIII **expression in samples of the INTELLANCE2/EORTC1410 randomized phase II trial.**

Pim J. French,1 Johan M. Kros,1 Iris de Heer,1 Marica Eoli,2 Juan Manuel Sepulvada,3 Annemiek Walenkamp,4 Jean-Sebastian Frenel,5 Alba Brandes,6 Paul Clement,7 Michael Weller,8 Peter Ansell,9 Jim Looman,9 Earle Bain,10 Lisa Roberts-Rapp,9 Marie Morfouace,11 Thierry Gorlia,11 Vassilis Golfinopoulos,11 Martin van den Bent1. 1 _Erasmus MC, Rotterdam, Netherlands;_ 2 _Carlo Besta, Milan, Italy;_ 3 _Univ Hospital, Madrid, Spain;_ 4 _UMCG, Groningen, Netherlands;_ 5 _Centre R Gauducheau, Saint-Herblain, France;_ 6 _AUSL – IRCCS Institute of Neurological Sciences, Bologna, Italy;_ 7 _UZ Gasthuis, Leuven, Belgium;_ 8 _University Hospital, Zurich, Switzerland;_ 9 _AbbVie, Chicago, IL;_ 10 _AbbVie, Abbott Park, IL;_ 11 _EORTC HO, Brussels, Belgium_.

Background: Depatux-M (ABT-414) is an antibody-drug-conjugate consisting of an antibody (ABT-806) bound to the toxin monomethylauristatin-F. A randomized phase II trial on EGFR-amplified recurrent glioblastomas (GBMs) showed an improvement (p=0.06 in the primary analysis, p=0.024 in follow-up analysis) in overall survival in the Depatux-M+TMZ arm when compared to the control arm (CCNU or TMZ). In this study, we performed targeted next generation sequencing and correlated molecular features with response to treatment in order to better identify patients that benefit from the combination.

Material and Methods: DNA and RNA was isolated from samples, collected at initial diagnosis, and selected for regions with highest tumor content. Target selection was done using the Trusight 170 gene panel (Illumina) which interrogates somatic variants and copy number, RNA levels and fusion genes in a set of known cancer genes. Variant calling was done using the Illumina Basespace sequence hub. For this trial, patients were eligible with centrally confirmed EGFR amplification, defined as EGFR/CEP 7 (centromere) ratio ≥ 2 in 15% of cells (FISH).

Results: DNA and RNA data were generated from 233 and 234 samples respectively (of the 260 study patients). High-copy gene amplification was detected in EGFR (n=202), MDM2 (n=20), MDM4 (n=22), CDK4 (n=24) and CDK6 (n=5) which correlated with high expression levels. With this assay, 17 tumors did not show EGFR copy number (cn) aberrations (cn < 2.8), a further 14 showed copy number changes consistent with trisomy only (2.8< cn < 4). Most EGFR amplified tumors also had additional genetic changes in the EGFR locus including point mutations (111/202), splice variants (132/202, the most common being EGFRvIII n=96) or fusion genes (13/202). Twenty-one samples did not contain additional genetic changes and expressed only EGFRwt. Response (OS) to treatment was not correlated to EGFR gene expression or amplification levels though, since EGFR amplification was a pre-requisite for inclusion, the majority of cases expressed high levels of EGFR (not shown). Preliminary analysis suggests that subjects with EGFR amplification but without EGFRvIII expression had a trend towards superior clinical benefit from Depatux-M +TMZ (median survival 14.3 v. 8.9 and 8.1 months in the Depatux-M +TMZ , TMZ|CCNU and Depatux M arms respectively; HR 0.56, P=0.047 v. Depatux M monotherapy and HR 0.54, P=0.055 v. TMZ|CCNU ).

Conclusion: Depatux-M in combination with TMZ showed a trend towards improved OS in EGFR amplified recurrent glioblastoma. This trend may be greater for subjects with an absence of EGFRvIII expression.

#4887

**Recurrent** NRG1 **rearrangements in Caucasian pulmonary mucinous adenocarcinoma: results from an Italian multi-center cohort.**

Domenico Trombetta,1 Paolo Graziano,1 Angelo Sparaneo,1 Giulio Rossi,2 Antonio Rossi,1 Marcello Tiseo,3 Massimo Di Maio,4 Federico P. Fabrizio,1 Maria C. Manzorra,1 Flavia Centra,1 Leonarda Di Candia,1 Marco Audisio,4 Evaristo Maiello,1 Vito M. Fazio,1 Lucia A. Muscarella1. 1 _Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy;_ 2 _Ospedale Santa Maria delle Croci di Ravenna, Ravenna, Italy;_ 3 _University of Parma, Parma, Italy;_ 4 _University of Turin, Torino, Italy_.

Invasive Mucinous Adenocarcinoma (IMA) is a rare histotype of lung adenocarcinoma associated with an unfavorable prognosis due to the lack of effective treatment. The NRG1 rearrangement is a new subtype-specific molecular feature of IMA and acts as a strong oncogenic inductor of the aberrant tyrosine kinase activity of ErbB2/ErbB3 heterodimers through PI3K-AKT/MAPK cellular cascades. We recently described for the first time the occurrence of NRG1 rearrangements in 31% of Caucasian lung IMAs and highlighted a strong association between NRG1 rearrangements and ErbB3 activation.

Here we extended our lung IMA samples cohort by enrolling a total of 71 patients from three different Italian Centers and collected clinical-pathological information and molecular profile, included the mutational status of KRAS, EGFR and ALK genes. We screened all samples by fluorescent in situ hybridization (FISH) for the detection of putative NRG1 rearrangements and by immunohistochemistry (IHC) for the expression of phosphorylated-ErbB3 (pErbB3) receptor. Finally, we customized a new targeted RNA Custom Panel to detect all 9 NRG1-fusion variants published to date to molecular characterize the NRG1 fusion variants in NRG1 rearranged IMAs.

Results showed NRG1 rearrangements in 32% of lung IMAs, displaying both NRG1 FISH split signals and deletions of the 5' portion of the gene. IHC confirmed our previous findings of association between pErbB3 immunoreactivity and NRG1 rearrangements, and the heterogeneity of fluorescent signal distribution and immunostaining along the tissue sections. The CD74-NRG1 remains the most common fusion variant identified in lung IMA samples. Correlation analysis among clinical-pathological data, pErbB3 expression and NRG1 rearrangements are ongoing.

Our results confirm the usefulness of IHC/FISH combined approach for NRG1 broken tumors identification and highlight the role of NRG1 rearrangement as master molecular marker of lung IMAs, potentially useful to select patients for the emerging target therapies.

#4888

Nuclear localization of antizyme inhibitor may be a marker for aggressiveness of prostate cancer.

Aram Ghalali, James M. Rice, Liangzhe Wang, Chin Lee Wu, Michael S. Rogers, Bruce Zetter. _BOSTON CHILDREN'S HOSPITAL, BOSTON, MA_.

Antizyme suppresses cell cycle by inhibiting the polyamine synthesis through binding to the rate limiting enzyme ornithine decarboxylases (ODC). High levels of ODC have been reported in several forms of cancer, among them prostate cancer. Antizyme Inhibitor (AZIN) binds to antizyme and thereby blocks its inhibitory effect on ODC. Here, we have measured the expression and localization of AZIN in 202 prostate cancer specimens, along with 26 adjacent benign samples and found that nuclear localization of AZIN is associated with significantly lower survival. Upregulation and nuclear localization of AZIN have been observed in several cancers, as has editing of the AZIN1 mRNA. Others have hypothesized that the RNA- edited AZIN (edAZIN) may have an increased affinity to antizyme and that could explain the association of edAZIN to the various cancers. We have studied the mechanism behind the nuclear localization of AZIN and found the single base pair substitution caused by RNA editing is sufficient to result in nuclear localization of the protein in all cell types tested. To determine if the nuclear localization might result from increased antizyme-edAZIN affinity, we developed fluorescent protein FRET sensor for protein-protein interaction using Clover-AZIN and antizyme-mRuby2 fusion proteins. Unexpectedly, we found that the editing event modestly decreases edAZIN affinity for antizyme, notwithstanding increased interaction in vivo. Thus, the data indicate that the change in protein localization to the nucleus may be more important to oncogenic function than the actual degree of binding to antizyme. Other functional differences between edAZIN and AZIN might be explained by altered kinetics of binding, by the contribution of an additional adapter protein which modulates the intracellular antizyme:AZIN complex, or by competition for AZIN binding by other partners whose interaction is affected by the editing event. Finally, we identified AZIN and edAZIN interacting proteins by using tandem affinity purification and LC-MS-MS analysis. Among interacting proteins, we identified a complex containing two isoforms of nuclear actins (ACTG1 and ACTA2) and Myosin-9 that may be the driving force behind the nuclear shuttling of edAZIN.

#4889

**Comparison of tumor mutational burden using the Ion Oncomine™ TML and FoundationOne™ assays with routine clinical FFPE tissue samples to predict durable clinical benefit in lung cancer and melanoma patients - a multivariate analysis integrating PD-L1 and CD8** + **evaluation.**

Simon Heeke,1 Jonathan Benzaquen,2 Elodie Long-Mira,3 Benoit Audelan,4 Virginie Lespinet,5 Olivier Bordone,5 Salomé Lalvé,5 Katia Zahaf,5 Michel Poudenx,6 Pierre-Michel Dugourd,7 Madleen Chassang,8 Thierry Passeron,7 Hervé Delingette,4 Charles-Hugo Marquette,2 Véronique Hofman,3 Marius Ilié,3 Paul Hofman3. 1 _Team 4 IRCAN, Inserm U1081/CNRS 7284, IRCAN, Nice, France;_ 2 _Team 4 IRCAN, Inserm U1081/CNRS 7284, IRCAN, Department of Pneumology CHU of Nice, Nice, France;_ 3 _Team 4 IRCAN, Inserm U1081/CNRS 7284, IRCAN, Laboratory of Clinical and Experimental Pathology, Biobank BB-0033-00025, FHU OncoAge, CHU of Nice, Nice, France;_ 4 _Asclepios Project, INRIA, Sophia-Antipolis, France;_ 5 _Laboratory of Clinical and Experimental Pathology, Biobank BB-0033-00025, CHU of Nice, Nice, France;_ 6 _Department of Oncology, Antoine Lacassagne Comprehensive Cancer Center, Nice, France;_ 7 _Department of Dermatology, CHU of Nice, Nice, France;_ 8 _Department of Radiology, Archet University Hospital, Nice, France_.

Background and objective: Tumor mutational burden (TMB) has emerged in recent clinical trials as a novel biomarker for the stratification of non-small cell lung cancer (NSCLC) patients treated with anti-PD1/PD-L1 antibodies. TMB in trials has been mainly assessed using the FoundationOne™ test (Foundation Medicine, Cambridge, MA, USA), which requires samples to be sent to the authorized testing center for the analysis. Therefore, our aim was to compare an inhouse assessment of TMB using the Ion Oncomine™ TML (Thermo Fisher Scientific, Waltham, MA, USA) and the FoundationOne™ tests. Furthermore, we evaluated PD-L1 expression in tumor cells and the level of tumor infiltrating CD8+ lymphocytes by immunohistochemistry to find the best combination of predictive biomarkers.

Materials and Methods: Late stage lung adenocarcinoma (51) and melanoma (25) patients with first- or second-line treatment with checkpoint inhibitors were included prospectively and selected randomly. Tissue FFPE sections were sent out for TMB and genetic assessment using the FoundationOne™ test, and parallel in-house analysis using the Ion Oncomine™ TML assay and the Ion AmpliSeq™ Cancer Hotspot Panel v2. IHC for PD-L1 (22C3 pharmDx assay; Dako Agilent, Santa Clara, CA), in addition to CD8+ lymphocyte expression analyses (clone SP57; Roche Ventana, Tucson, AZ) were performed.

Results: TMB was assessed by the Ion Oncomine™ TML assay in comparison to FoundationOne™, demonstrating a high correlation in melanoma (R² = 0.95), but a lower correlation in NSCLC patients (R² = 0.88). Prediction of a durable clinical response by receiver operating characteristic (ROC) was comparable between Oncomine™ TML (AUC = 0.70) and FoundationOne™ (AUC = 0.74) and also similar to the CD8+ score (AUC = 0.76) and PD-L1 expression (AUC = 0.72). Combining different biomarkers did not improve the predictive value. In addition, detection of actionable mutations exhibited a good correlation between the different gene panels. TMB analysis using the Ion Oncomine™ TML assay was initially ineffective as elevated deamination rates from formalin fixed samples interfered with the analysis but this could be successfully overcome with bioinformatic filtering and UDG repair. Stringent sample requirements for FoundationOne™ testing prevented the analysis in 22% and 8% of lung cancer and melanoma samples, respectively.

Conclusion: TMB can be assessed using the Ion Oncomine™ TML assay since its predictive value is comparable to that obtained with the FoundationOne™ test. Combining different biomarkers did not improve the prediction of clinical responses in this series with a limited number of patients. Updated clinical data will be presented during the congress.

#4890

Mutational signatures as biomarkers of response to nivolumab in metastatic bladder cancer.

G. Celine Han,1 Peter M. Szabo,1 Abdel Saci,1 Alice M. Walsh,1 Natallia Kalinava,1 Ariella Sasson,1 Bruce Fischer,1 Matthew D. Galsky (co-senior author),2 Padmanee Sharma (co-senior author)3. 1 _Bristol-Myers Squibb, Princeton, NJ;_ 2 _Icahn School of Medicine at Mount Sinai/Tisch Cancer Institute, New York, NY;_ 3 _MD Anderson Cancer Center, University of Texas, Houston, TX_.

Background: Metastatic or surgically unresectable urothelial cancer (UC) is a disease with high unmet need. Nivolumab monotherapy demonstrated clinical benefit in patients with platinum-resistant UC (CheckMate 275; NCT02387996). Because not all patients respond to nivolumab therapy, identifying biomarkers for response is of critical importance. In the CheckMate 275 cohort, higher tumor mutational burden (TMB) was associated with longer overall survival (OS) (1). Previous studies have shown that certain mutational signatures, combinations of mutation types arising from specific mutagenesis processes, were associated with mutational burden and clinical outcome in UC (2,3). We hypothesized that specific mutational signatures may be associated with response to nivolumab in CheckMate 275.

Methods: Whole exome sequencing data and clinical annotations for 139 archival, tumor-normal paired samples from patients enrolled in the CheckMate 275 cohort were collected and analyzed. Sequencing reads were processed and somatic variants called as described previously (4). Percentage of mutations across 30 mutational signatures were generated using deconstructSigs, which infers signature activity with given known signatures (5). Association of the most prevalent mutational signatures with TMB, previously known clinical biomarkers, and clinical efficacy (OS, progression free survival [PFS], and objective response [OR]) were examined.

Results: We identified age-related (signature 1), endogenous mutagenesis-related APOBEC (signatures 2 and 13) and UV-induced (signature 7) as the most prevalent mutational signatures in UC. When examining their correlations with TMB, signature 1 showed negative correlation, signatures 2 and 13 showed high positive correlation, and signature 7 showed low correlation. Higher signature 2 scores were associated with better OS, but were poor predictors for PFS and OR. Overall, while signature 2 added predictive value over previously known clinical biomarkers such as PD-L1 expression, serum hemoglobin level, and presence of liver metastasis, it did not improve on the predictive performance of TMB.

Conclusions: An APOBEC-related mutational signature (signature 2) was associated with OS in patients with advanced UC treated with nivolumab. This signature was correlated with TMB and did not improve upon TMB as a predictive biomarker. Our work demonstrates the importance of including TMB as a covariate when analyzing mutational signatures.

References:

1. Galsky MD et al. Ann Oncol 2017;28(suppl 5). Abstract 848PD

2. Glaser AP et al. Oncotarget 2018;9:4537-48

3. Roberts SA et al. Nat Genet 2013;45:970-6

4. Carbone DP et al. NEJM 2017;376:2415-26

5. Forbes SA et al. Nucleic Acids Res 2017;45:D777-83

#4891

Gene expression analysis of paired baseline (BL) and on-treatment tumor samples from FAIRLANE, a double-blind placebo (PBO)-controlled randomized phase II trial of neoadjuvant ipatasertib (IPAT) plus paclitaxel (PAC) in early triple-negative breast cancer (eTNBC).

Zhen Shi,1 Steven J. Isakoff,2 Cristina Saura,3 Paolo Nuciforo,4 Isabel Calvo,5 Jay Andersen,6 José Luis Passos Coelho,7 Miguel Gil Gil,8 Begoña Bermejo,9 Debra A. Patt,10 Eva Ciruelos,11 Matthew J. Wongchenko,1 Stina M. Singel,1 Na Xu,1 Lorena de la Peña,12 José Baselga,4 Steven Gendreau,1 Mafalda Oliveira3. 1 _Genentech Inc., South San Francisco, CA;_ 2 _Massachusetts General Hospital, Boston, MA;_ 3 _Vall d'Hebron University Hospital, Vall d'Hebron Institute of Oncology (VHIO), and SOLTI Breast Cancer Research Group, Barcelona, Spain;_ 4 _Vall d'Hebron Institute of Oncology (VHIO), Barcelona, Spain;_ 5 _Centro Integral Oncologico Clara Campal (CIOCC), Madrid, Spain;_ 6 _Compass Oncology and US Oncology, Portland, OR;_ 7 _Hospital Beatriz Angelo, Loures, Portugal;_ 8 _Institut Català d'Oncologia, Hospital Duran i Reynals, Barcelona, Spain;_ 9 _Hospital Clinico Universitario, Valencia, Spain;_ 10 _Texas Oncology Cancer Center, US Oncology, Austin, TX;_ 11 _SOLTI Breast Cancer Research Group and University Hospital 12 de Octubre, Madrid, Spain;_ 12 _SOLTI Breast Cancer Research Group, Barcelona, Spain_.

Purpose: In the FAIRLANE trial (NCT02301988) in eTNBC, adding the oral AKT inhibitor IPAT to neoadjuvant PAC led to numerical increases in rates of pathologic complete response (pCR; primary endpoint) and complete response (CR) by MRI in unselected patients (pts), with a numerically greater treatment effect in pts with PIK3CA/AKT1/PTEN-altered tumors [Oliveira et al., AACR 2018]. There was no association between PIK3CA/AKT1/PTEN alterations and response in the PBO arm. Gene expression analysis was performed to evaluate on-treatment changes in the tumor microenvironment and association with response in both treatment arms.

Methods: Gene expression at BL and cycle 1 day 8 (C1D8) was evaluated by RNA sequencing using TruSeq RNA Access (Illumina, Inc., San Diego, CA, USA) at Expression Analysis (Morrisville, NC, USA). Paired samples (pts with RNA-seq at both BL and C1D8) were compared to assess on-treatment changes in gene expression. Using RNA-seq data, in silico cell-type enrichment analysis was performed using xCell to calculate the percentage of infiltrating immune cells [Aran et al., Genome Biol 2017].

Results: Paired samples were available from 87 (58%) of the 151 treated pts; response results in this subset were representative of the overall population. Calculated infiltrating immune cell scores (xCell) increased from BL to C1D8 in both treatment arms, exemplified by increases in multiple immune cell biomarkers (eg CD8A, GZMA, CXCL9, CD274/PD-L1). In the PBO arm, higher C1D8 immune score showed a significant association with better response (pCR or response by MRI); the association was stronger for C1D8 than for BL or change from BL immune score. However, there was no association between immune score (BL, C1D8, or change from BL) and response in the IPAT arm. At C1D8, IPAT-treated pts with a CR by MRI had lower average immune scores than PBO-treated pts with a CR. Among the 14 IPAT-treated pts who achieved a CR by MRI, all 7 pts with immune scores below the median had PIK3CA/AKT1/PTEN-altered tumors compared with only 3 of 7 pts with immune scores above the median.

Conclusions: This exploratory gene expression analysis revealed differential associations of immune infiltration and tumor response depending on treatment. PAC treatment appears to increase immune cell infiltration in the eTNBC setting, and the presence of a T cell-rich environment (at C1D8) is strongly associated with improved outcomes in pts treated with single-agent PAC. However, the addition of AKT inhibition to PAC is especially effective in pts with PIK3CA/AKT1/PTEN-altered tumors, including those with low immune infiltration. Together, the results hint at multiple mechanisms in killing tumor cells when combining IPAT with PAC.

#4892

Pentraxin 3: A stromal derived diagnostic biomarker for pancreatic ductal adenocarcinoma.

Michelle R. Goulart,1 Jennifer Watt,1 Imran Siddique,2 Rita Lawor,3 Thomas Dowe,1 Satyajit Bhattacharya,4 Tatjana Crnogorac-Jurcevic,1 Paola Allavena,5 Aldo Scarpa,3 Hemant M. Kocher6. 1 _Barts Cancer Institute, Queen Mary University of London, London, United Kingdom;_ 2 _University of Lausanne, Lausanne, Switzerland;_ 3 _ARC-NET Research Center for Applied Research on Cancer, University of Verona, Verona, Italy;_ 4 _Barts and the London HPB Centre, The Royal London Hospital, London, United Kingdom;_ 5 _IRCCS Istituto Clinico Humanitas, Milan, Italy;_ 6 _Barts Cancer Institute, Queen Mary University of London, Barts and the London HPB Centre, The Royal London Hospital, Barts Health NHS Trust, London, United Kingdom_.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a dismal prognosis and 5 year survival rate of less than 5%. Desmoplastic reaction, characterized by the proliferation of myofibroblast-like cells (also known as activated pancreatic stellate cells (PSCs)) and the significant deposition of extracellular matrix components, such as hyaluronan, is a prominent pathological characteristic of PDAC and significantly contributes to its chemoresistance. All trans retinoic acid (ATRA) is a pleiotropic agent modulating multiple signaling pathways that renders activated PSCs quiescent. Previous work from our laboratory demonstrated various phenotypic changes in ATRA-treated PSCs and comparative gene expression microarray analysis revealed that the pentraxin-related protein 3 (PTX3) expression is down-regulated in quiescent PSCs compared to activated PSCs. Since PTX3 plays a key role in the formation of the hyaluronan-rich matrix via its interaction with tumor necrosis factor stimulated gene 6 (TSG-6) and hyaluronic acid (HA) chains, we sought to investigate the role of PTX3 in PDAC and to assess it as a potential diagnostic biomarker for patients with pancreatic cancer. Serum PTX3 concentrations were evaluated in 140 patients with PDAC and 138 controls (healthy individuals, patients with other pancreatic diseases or inflammatory conditions) by ELISA. To establish the source of PTX3 secretion we analyzed in vitro cell cultures of PSCs and cancer cell lines. PTX3 specificity was confirmed upon PTX3 siRNA analysis by western blot and qPCR. Three-dimensional organotypic cultures, KPC mice and human tissue were assessed by immunohistochemistry. Expression of TSG-6 and HA were also evaluated using immunofluorescence studies. Our results showed that patients with PDAC had significantly higher serum PTX3 concentrations than patients with other pancreatic diseases and healthy individuals. ROC curve analysis confirmed the reliability of PTX3 serum levels in diagnosing pancreatic cancer with a sensitivity and a specificity of 86%. Western blotting, qPCR and immunofluorescence validated that activated PSCs produce and secrete PTX3 and its expression was down-regulated on rendering these cells quiescent upon ATRA treatment. Organotypic models elucidated that hyaluronan is expressed and secreted by the PSCs and the analysis of human and mouse tissues demonstrated predominant PTX3 expression in the stromal compartment of PDAC. Our study identified that serum PTX3 is a sensitive and specific diagnostic biomarker for PDAC with the ability to separate malignant from other benign conditions of the pancreas. Activated PSCs are the main source of PTX3 secretion and contribute to the formation and stabilization of the PDAC extracellular matrix architecture.

#4893

Combined immunohistochemistry and NGS-based patient profiling for predicting anti-PD-1/PD-L1 therapy response.

Jianjun Yu,1 Jianguo Dong,1 Amy Wang,1 Joseph S. Krueger2. 1 _Predicine Inc, Hayward, CA;_ 2 _Flagship Biosciences Inc, Westminster, CO_.

Introduction: There are several different modalities of predictive tests which support response to anti-PD-1/PD-L1 inhibitors therapy, including PD-L1 expression by immunohistochemistry (PD-L1 IHC), mismatch repair deficiency (dMMR), microsatellite instability (MSI), and recently emerging tumor mutation burden (TMB), and Gene Expression Panels (GEP). Each of these methods capture different facets of the immune system: TMB and MSI evaluates represents mutational/neoantigen load which can stimulate the immune system; GEP establishes a profile of immune response, and whereas PD-L1 IHC directly evaluates the state of checkpoint inhibition in the tumor and tumor microenvironment (TME). We constructed a compound testing paradigm for immune system monitoring called PredicineX, which combines genome analysis which relies on tissue or blood-derived nucleic acids and advanced tissue context analytics based on PD-L1 IHC in solid tissue biopsies to create a comprehensive patient profile to support anti-PD-1/PD-L1 therapy decision making.

Methods: Using Predicine's GeneRADAR technology, we developed a tissue- and blood-based NGS assay to capture genomic alterations in cancer genes including tumor mutation burden (TMB) and microsatellite instability (MSI). Using Flagship Biosciences cTA® tissue image analysis technology, we created an artificial intelligence (AI) based PD-L1 IHC scoring platform which provides both current PD-L1 IHC scoring paradigms and novel computational scores from the rich tissue context data profile created from PD-L1 IHC slides using this technology. We compared biomarker profiles in a cohort of patients using the two technologies and created an integrative data profile to evaluate its association with anti-PD-1/PD-L1 therapy response.

Results: We demonstrate the synergistic value of combining genomic based TMB, MSI, and GEP immune profiles with contextual information from PD-L1 IHC slides in patient biopsies. The high complexity gene profile, combined with the rich tissue context data, provided a novel means to stratify patients into 4 categories: 1)mutation high/immune competent; 2)mutation low/immune deficient; 3)mutation high/immune deficient; and 4) mutation low/immune competent. Notably, a strong association between the stratified groups and anti-PD-1/PD-L1 therapy was identified.

Conclusion: In this study, we have demonstrated the feasibility of a compound test relying on tissue- and blood-based NGS assay and PD-L1 IHC which can ascribe differential phenotypes to patients for clinically relevant and actionable decisions about response to anti-PD-1/PD-L1 therapy.

#4894

Incidence of high tumor mutation burden (TMB) and PD-L1 positivity in breast cancers and potential response to immune checkpoint inhibitors (ICPIs).

Ethan Sokol,1 Lee Albacker,1 Aixa Soyano,2 Ricardo Parrondo,2 Brenda Ernst,2 Emmanuel Gabriel,2 Garrett Frampton,1 Jeffrey Ross,1 Siraj Ali,1 Jon Chung,1 Saranya Chumsri2. 1 _Foundation Medicine, Cambridge, MA;_ 2 _Mayo Clinic, Jacksonville, FL_.

Background: Immune checkpoint inhibitors (ICPIs) have led to dramatic improvement in outcome of several cancers. Program death ligand1 (PD-L1) staining and tumor mutational burden (TMB) have emerged as independent predictive biomarkers of ICPIs in lung cancer. Here we examine the landscape of TMB and PD-L1 expression in breast cancer and present a case of patient with high TMB and PD-L1 negative breast cancer with exceptional response to ICPIs.

Methods: Hybrid-capture based comprehensive genomic profiling of 395 cancer related genes using the FoundationOne assay was performed on 14,867 breast carcinomas sequenced in the course of routine clinical care. Ventana (SP-263) PD-L1 status (n=1425) and hormone receptor status was available for a subset of patients. Subgroup analyses were performed based on histological type [invasive lobular carcinoma (ILC, n=740)], molecular subtypes [ER-positive (ER+; n= 1371), HER2-amplified (HER2+; n=1522), and TNBC (n=917)], patient age (≤45, 46-60, ≥61), and local vs. metastatic disease (n=5241 and 6710).

Results: Consistent with previous reports, the rates of positive PD-L1 staining are highest in TNBC (14%) and lowest in HER2+, ILC, and ER+ disease (6.0%, 5.1%, 2.3%). Interestingly, the rate of PD-L1 positivity, defined as ≥1% tumor staining, was significantly lower in metastatic disease vs. local disease (6.3% vs. 11.1%; p = 0.005). The frequency of high TMB, defined as >10 mutations/mb, was greatest in ILC and HER2+ disease (13.6% and 9.9%) and lowest in TNBC and ER+ disease (7.0% and 6.9%). Rates of high TMB were associated with increased patient age (3.7%, 9.3%, and 12.8% frequency in patients ≤45, 46-60, and ≥61) and were significantly higher in metastatic vs. local disease (11.1% vs 5.3%; p<2E-23). PD-L1 positive and TMB high populations were not significantly co-occurrent (OR = 1.02, p = 0.87). Similar percentages of PD-L1 positivity were observed in both TMB low (9.3%) and TMB high (9.5%). However, among patients with very high TMB (>20 mut/mb), there was a significant association between TMB and PD-L1 positivity (OR = 2.6, p = 0.023). Nevertheless, even with high cutoff, 79% of the TMB high samples were PD-L1 negative.

We also report on a stage IIIb (T4, N2, M0) ER+ HER2- breast cancer patient with high TMB (40muts/mb) and negative PD-L1 IHC who previously progressed on aromatase inhibitor with CDK4/6 inhibitor and chemotherapy but achieved durable complete response of > 1 year with nivolumab in combination with capecitabine.

Conclusions: Predictive biomarkers for ICPIs are critical to identify a subset of breast cancer patients who may respond to immunotherapy. TMB high and PD-L1 positivity do not significantly co-occur and the majority of TMB high cases were PD-L1 negative. Nevertheless, this group of patients may still benefit with ICPIs. Further studies are needed to evaluate this subset of patients.

#4895

Natural history and outcome of patients presenting a metastatic breast cancer with PIK3CA mutation.

Fernanda Mosele,1 Benjamin Verret,1 Amelie Lusque,2 Thomas Filleron,2 Thomas Bachelot,3 Monica Arnedos,1 Mario Campone,4 Florence Dalenc,2 Claudia Lefeuvre,5 Marie Paule Sablin,6 Hervé Bonnefoi,7 Ludovic Lacroix,1 Ivan Bièche,6 Anthony Gonçalves,8 William Jacot,9 Marta Jimenez,10 Amelie Jacquet,10 Fabrice Andre,1 Fabrice Andre10. 1 _Department of Medical Oncology, Gustave Roussy, Villejuif, France;_ 2 _Institut Claudius Regaud, IUCT-O, Toulouse, France;_ 3 _Centre Léon Bérard, Lyon, France;_ 4 _Institut de Cancérologie de l'Ouest, Nantes & Angers, France; _5 _Centre Eugène Marquis, Rennes, France;_ 6 _Institut Curie, Paris, France;_ 7 _Institut Bergonié, Bordeaux, France;_ 8 _Institut Paoli-Calmettes, Marseille, France;_ 9 _Institut Régional du Cancer de Montpellier, Montpellier, France;_ 10 _Unicancer, Paris, France_.

BACKGROUND: Activating PIK3CA mutations occur in 20-30% of patients with metastatic breast cancer (mBC). A recent study showed that alpha selective PI3K inhibitors improve outcome in patients with PIK3CA mutation, HR+/Her2- mBC. There is a need to better understand the natural history of PIK3CA mutant mBC to optimally position of this drug family.

PATIENTS AND METHODS: 649 patients from SAFIR02 trial (NCT02299999), for which mutational profile was available and with clinical data entered in a database, were selected for this analysis. Associations between PIK3CA mutation, clinical characteristics and outcome were analyzed.

RESULTS: 143 patients (22%) harbor PIK3CA mutation in tumor sample. 10% (n=27) and 34% (n=104) of TNBC and HR+/Her2- mBC presented a PIK3CA mutation respectively (p<0.001). In patients with HR+/Her2- mBC, there is no significant association between PIK3CA mutation and site of metastases or number of metastases (p<0.01). In the same group, patients with PIK3CA mutations were less sensitive to chemotherapy (stable disease or response: 51.3% vs. 69.2% in PIK3CA wild type; p=0.005) (Odds ratio multivariate: 0.40 [95% CI: 0.22-0.71] p=0.002). The median overall survival (OS) for patients with PIK3CA mutated HR+/Her2- mBC was of 19.6 months vs. 23.5 for patients with PIK3CA wild type (p=0.048) (HR multivariate: 1.44 [95% CI: 1.02-2.03] p=0.039). HR+/Her2- mBC with PIK3CA mutations presented more frequently a MAP3K1 mutation (16% vs. 4%, p=0.0002). In the TNBC group, the median OS in patients with PIK3CA mutated cancer was of 24.2 months vs. 14 months in the wild type group (p=0.028).

CONCLUSION: Patients with PIK3CA mutated, HR+/Her2- mBC are less sensitive to chemotherapy and present a shorter survival. These data emphasize the need for new therapies in this setting, and to position these therapies earlier than chemotherapy in the treatment sequences.

#4896

A novel approach identifies the potential biomarkers of targeted drug sensitivity in esophageal squamous cell carcinoma.

Dan Su,1 Dadong Zhang,2 Jiaoyue Jin,1 Lisha Ying,1 Miao Han,2 Kaiyan Chen,1 Bin Li,2 Junzhou Wu,1 Zhenghua Xie,2 Fanrong Zhang,1 Yihui Lin,2 Guoping Cheng,1 Jinchao Wang,2 Minran Huang,1 Jing-Yu Li,2 Jianjun Zhang,3 Fugen Li,2 Lei Xiong,2 Andrew Futreal,3 Weimin Mao1. 1 _Zhejiang Cancer Hospital, Hangzhou, China;_ 2 _3D Medicine Inc., Shanghai, China;_ 3 _University of Texas MD Anderson Cancer Center, Houston, TX_.

Purpose: Esophageal squamous cell carcinoma (ESCC) has a high mortality with no effective targeted therapies. Previous studies from Cancer Cell Line Encyclopedia (CCLE) project adopted a commercial human pan-cancer cell line model incorporating genomic landscape to systematically identify drug sensitivity biomarkers. However, the biomarker of drug sensitivity in ESCC is still lack of widely exploring. Here, we established a novel approach combined patient-derived models, targeted deep sequencing and a drug sensitivity evaluation system to investigate potential biomarkers of targeted drug sensitivity in ESCC.

Materials and Methods: Deep sequencing of 365 tumor drug-related genes was performed in tumor tissues and matched blood from an ESCC cohort (n = 161). In order to explore the potential biomarkers of drug sensitivity, we systematically established patient-derived cell lines (PDCs) from an independent cohort including 123 operable ESCC patients. Targeted deep sequencing, RNA sequencing and high-through drug sensitivity were integrated to identify the potential biomarkers of targeted drug sensitivity in ESCC PDCs. Molecular characterization methods and animal models were used to validate the potential biomarker in vitro and in vivo. In addition, patient-derived xenograft (PDX) was further confirmed the results.

Results: The mutational profile of tumor drug-related genes indicated a mean of 9 non-silent somatic mutations (mutation allele frequency >= 0.05) per patient and a high recurrence rate of copy number variation (CNV) were discovered. To explore potential biomarkers of targeted drug sensitivity, we established eight PDCs derived from 123 ESCC patients were successfully established and used for molecular characterization and drug screening. Drug sensitivity evaluation and pharmacogenomics analyses of the eight PDCs revealed prevalent CDKN2A or CDKN2B loss as potentially sensitive biomarkers of the CDK4/6 inhibitors palbociclib and ribociclib, which was neither found in the previous study from CCLE models nor in the present study using commercial ESCC cell lines. Importantly, patient-derived models integrated with DNA and RNA sequencing validated this result in vitro. Moreover, ESCC patient-derived cell line xenografts (PDCX) models with CDKN2A/2B loss are more sensitive to the CDK4/6 inhibitor palbociclib than that in PDCX models with wild-type CDKN2A/2B. Furthermore, mouse model with PDX further confirmed CDKN2A/2B loss as a biomarker of the CDK4/6 inhibitor sensitivity.

Conclusions: These results suggested that patient-derived models combined with deep sequencing and a drug sensitivity evaluation system, incorporating in vitro and in vivo validation platform, could be used as a novel and effective approach for exploring biomarkers of drug sensitivity in ESCC.

#4897

Detection of MET-mediated EGFR tyrosine kinase inhibitor (TKI) resistance in advanced non-small cell lung cancer (NSCLC): biomarker analysis of the TATTON study.

Ryan J. Hartmaier,1 Ji-Youn Han,2 Byoung Chul Cho,3 Melanie M. Frigault,4 Aleksandra Markovets,5 Anne L'Hernault,6 David Duncan,6 Pierre Lao-Sirieix,7 J. Carl Barrett,1 Remy B. Verheijen,8 Dana Ghiorghiu,8 Jonathan Wessen,8 Geoffrey R. Oxnard9. 1 _Translational Science, Oncology, IMED Biotech Unit, AstraZeneca, Boston, MA;_ 2 _National Cancer Center, Goyang, Republic of Korea;_ 3 _Yonsei University Severance Hospital, Seoul, Republic of Korea;_ 4 _Translational Science, Oncology, IMED Biotech Unit, AstraZeneca, CA;_ 5 _Bioscience, Oncology, IMED Biotech Unit, AstraZeneca, Boston, MA;_ 6 _Precision Medicine Laboratories, Precision Medicine and Genomics, IMED Biotech Unit, AstraZeneca, Cambridge, United Kingdom;_ 7 _Companion Diagnostics, Precision Medicine and Genomics, IMED Biotech Unit, AstraZeneca, Cambridge, United Kingdom;_ 8 _Global Medicines Development, AstraZeneca, Cambridge, United Kingdom;_ 9 _Dana Farber Cancer Institute, Boston, MA_.

EGFR-TKIs like osimertinib are widely used to treat advanced EGFR-mutant NSCLC, however tumors inevitably acquire resistance. Amplification of MET occurs in ~20% of EGFR-TKI resistant tumors. Previous studies have used a variety of technologies (FISH, IHC, NGS, ctDNA) with mixed success in identifying MET-driven tumors. Thus, there is an urgent need to better understand the reliability of these assays for the detection of MET-driven EGFR-TKI resistance in NSCLC.

In the TATTON study (NCT02143466), the combination of osimertinib and savolitinib (AZD6094, HMPL-504, volitinib), a potent and selective MET-TKI, has demonstrated encouraging anti-tumor activity in patients with NSCLC and MET-driven EGFR-TKI resistance. During screening, MET testing (central, or local with central confirmation) was performed on tumor tissue collected after the most recent therapy. Informative central MET FISH screening/confirmation results were generated for 254 consented patients. MET overexpression and amplification were further assayed centrally using tissue IHC (n=81), tissue NGS (n=117; Foundation Medicine), and ctDNA NGS (n=199; Guardant Health). Standard NGS provider MET amplification calls were used. Central IHC positivity was defined as 3+ in ≥50% of tumor cells. Central FISH+ was defined as either amplification (MET:CEP7 ratio ≥2) or polysomy (gene copy number ≥5 if MET:CEP7 <2). MET FISH was used as the common comparator across assays.

Central MET FISH+ was found in 123/254 tumors (48%; 75 with amplification, 48 with polysomy), an elevated prevalence likely related to local MET+ prescreening. Comparison of tissue NGS with FISH (n=95) identified high negative-percent agreement (NPA, 98%) but modest positive-percent agreement (PPA, 48%). Further investigation indicated NGS PPA is highly dependent on the FISH result, with higher PPA for amplification (88%) but low PPA for polysomy (4%). Similarly, comparison of ctDNA NGS with FISH (n=112) yielded modest NPA (90%) and PPA of only 25% (43% for amplification; 10% for polysomy). PPA improved to 50% (64% for amplification; 30% for polysomy) when limited to 46 patients with an EGFR mutation detected at >5% allelic fraction in ctDNA. Comparison of IHC with FISH (n=52) identified a 63% NPA and 72% PPA. Notably, of 28 IHC 3+ tumors, 10 (36%) were negative by FISH.

Tissue NGS identifies a subset of MET FISH+ tumors, however MET polysomy is largely missed by NGS assays. MET IHC 3+ staining overlaps extensively with MET FISH+ but also identifies additional potentially MET-dependent tumors. When combined with future clinical efficacy data, this technical comparison will help inform a prospective biomarker strategy for the detection of MET-driven EGFR-TKI resistance in NSCLC.

#4898

Clinical application of quantitative multiplex mass spectrometry-based proteomics in predicting clinical outcomes in locally advanced rectal cancer.

Ho Jung An, Ji-Han Jung, Byoung Yong Shim, Hyung Soon Park, Hyeon-Min Cho, Hyung-Jin Kim, Ri Na Yoo, Sung Hwan Kim, Jonghoon Lee, Kang-Moon Lee, Dae Bum Kim, Ji Min Lee. _St. Vincent's Hospital, Catholic Univ. of Korea, Suwon, Republic of Korea_.

Background: Neoadjuvant chemoradiotherapy (CRT) using 5-fluorouracil (5-FU) is a standard treatment for locally advanced rectal cancer (LARC) to improve clinical outcomes. The pathologic responses after neoadjuvant CRT is a major prognostic factor, thus identification of good or poor responder in advance is important. This study is to find candidate predictive biomarkers for CRT and prognosis in LARC patients using quantitative mass spectrometry (MS).

Materials and methods: 86 patients with stage II/III LARC, received neoadjuvant CRT consisting of 5-FU/leucovorin followed by surgery were included. 14 proteins potentially associated with 5-FU activity or prognosis were evaluated in archived formalin-fixed, paraffin-embedded pre- and post-CRT tumor tissues using MS: DHFR, DPYD, EGFR, hENT1, Her2, MET, OPRT, p16, TK1, TYMP, TYMS, UCK1, UCK2, UPP1. Tumor regression grade (TRG) after CRT was assessed by Dworak criteria: 0, no regression; 1, dominant tumor mass with obvious fibrosis; 2, dominant fibrotic changes with few tumor cells; 3, very few tumor cells; 4, no tumor cells.

Results: TGR was 0 in 2 (2.3%), 1 in 30 (34.9%), 2 in 23 (26.7%), 3 in 20 (23.3%), and 4 in 11(12.8%) patients. Major regression (TRG 2/3/4) was associated with high TK1 (P = 0.040), low TYMS (P = 0.037), p16 (P = 0.055), and UPP1 (P = 0.028) levels. Among the pre-CRT parameters, low CEA level (<5ng/mL, P = 0.013), clinical stage II (P<0.001), tumor differentiation (good, P = 0.052), low EGFR level (<200amol/ug, P=0.006), undetectable TYMS level (P = 0.070) were associated with longer recurrence-free survival (RFS). Among the post-CRT parameters, major pathologic response (P = 0.001), and the absence of lymphatic (P<0.001)/ vascular (P = 0.025)/ perineural invasion (P = 0.001) was associated with longer RFS. For 71 patients with available paired pre- and post-CRT tissues, the changes of protein expression were calculated. The pre-CRT/post-CRT ratio of DHFR (>1.1), Her2 (<1.25), MET (≥0.8), p16 (<2), TK1 (≥0.9) showed trend toward long RFS. Multivariate analysis showed that the presence of lymphatic invasion (HR=3.07; 95% CI, 1.35-7.01; P = 0.008) and pre-CRT high EGFR level (≥200amol/ug; HR=2.23; 95% CI, 1.03-4.83; P = 0.042) were significantly associated with shorter RFS. The presence of lymphatic invasion (P=0.005), positive pre-CRT TYMS level (P = 0.008), and high CEA level (P = 0.015) were significantly related to short survival.

Conclusions: Quantitative MS-based proteomics can facilitate the identification of predictive biomarkers of CRT responses and prognosis in LARC patients.

#4899

**The impact of the** EGFR **-T790M mutation detection by re-biopsy in EGFR mutant NSCLC patients in the retrospective analysis.**

Akihiro Yoshimura, Tadaaki Yamada, Naoko Okura, Junji Uchino, Koichi Takayama. _Kyoto Prefectural University of Medicine, Kyoto, Japan_.

Background: EGFR-TKIs show a good anticancer effect in to most of patients with advanced non-small cell lung cancer (NSCLC) harboring EGFR activating mutations. However, it ultimately acquires resistance to EGFR-TKIs. We currently attempt to detect EGFR-T790M mutation by re-biopsy after disease progression, because the third generation EGFR-TKI osimertinib was effective against the refractory tumors with EGFR-T790M mutation. However, the re-biopsy from tumors is relatively invasive and some cases are impossible to biopsy. Therefore, it is a beneficial if we predict the EGFR-T790M mutation before re-biopsy. In this study, we analyzed the patients with disease progression after initial EGFR-TKIs to address the issues.

Methods: 78 advanced NSCLC patients with EGFR mutations were enrolled from five institutions in Japan. In all patients, re-biopsy samples were obtained successfully after the resistance to initial EGFR-TKI treatment We analyzed the relationship between the emergence of EGFR-T790M mutation and clinical parameters including clinical outcomes with EGFR-TKI treatment and EGFR activating mutation status.

Results: Of 78 advanced NSCLC patients with EGFR mutations, 39 patients were EGFR-T790M positive and 39 were negative based on the re-biopsy samples. Of 39 EGFR-T790M positive patients, 2 patients achieved complete response (CR), 33 partial response (PR), and 4 stable disease (SD) by initial EGFR-TKI treatment. In contrast, 1 patient experienced a CR, 19 a PR, 18 SD, and 1 progressive disease (PD) in 39 T790M negative patients. The objective response rate was higher in patients with T790M positive mutations than those with T790M negative mutations (89.7% versus 51.2%, p< 0.01). There was no statistically significant difference in progression free survival and time to failure treated with initial EGFR-TKIs between both groups.

Conclusion: The response to initial EGFR-TKIs treatment might be one of good predictors for emerging of refractory tumors with EGFR-T790M mutation.

#4900

Dynamic metabolic response of prostate cancer patients treated with ADT and low carb diet.

Stephen Freedland,1 Pao-Hwa Lin,2 Emily Y. Chen,3 Vladimir Tolstikov,3 Jen-Tsan Chi,2 Rangaprasad Sarangarajan,3 Niven R. Narain,3 Michael A. Kiebish3. 1 _Cedars-Sinai, Los Angeles, CA;_ 2 _Duke University Medical Center, Durham, NC;_ 3 _BERG LLC, Framingham, MA_.

Background: Prostate cancer (PrCa) is one of the most common cancers among men and managed through surgery, hormonal therapy, chemotherapy, radiation, and cryotherapy. Depending on the stage of PrCa, androgen deprivation therapy (ADT) is commonly utilized as an intervention. ADT has proven effective in intervening in PrCa progression, although there are several side effects. Recently, the use of low carb diets has been shown to alter patients' metabolic phenotype and as such may reduce the side effects of ADT.

Methods: In this study 35 men, who were beginning ADT, were randomized to low carb diet intervention with recommended exercise or control (no lifestyle change) for 6 months. Primary results have been reported showing significant weight loss at both 3-month (17lb, p<0.01) and 6-month (23 lbs, p<0.01) and improved insulin control at 3-month (HOMA=-19 as compared to baseline, p=0.02) but not at 6-month (HOMA=-4 as compared to baseline, p=0.13). For this exploratory analysis, sera collected at baseline, 3, and 6 months was used for metabolomics analysis utilizing GC/MS TOF, QqQ LC-HILIC-MS/MS, and TripleTOF 6600 LC-RP-MS and lipidomics analysis using TripleTOF 5600+ MS/MSALL workflows to quantify the chemical diversity of the metabolome and lipidome.

Results: Over 450 metabolites and 1000 lipid species were quantified. Metabolomics analysis of sera from the control patients (ADT alone) demonstrated alterations in steroid biosynthesis, androgen/estrogen metabolism, fatty acid metabolism, and lysine degradation. Lipidomics analysis of the control patients demonstrated changes in selective long chain polyunsaturated phosphatidylcholine species. Analysis of the ADT plus low carb patients revealed a change in pyruvate, glucose-alanine cycle, selenoamino acid, phenylalanine/tyrosine, and taurine metabolism. Further, lipidomic analysis revealed changes in several triglycerides, plasmalenylethanolamine, and phosphatidic acid species.

Conclusion: In summary, integration of metabolomic and lipidomic analysis in hormonal and metabolic interventions of PrCa patients revealed dynamic changes providing novel insight for tailoring therapeutic intervention and monitoring.

#4901

p53 expression is a useful predictive marker for recurrence of meningioma.

Atsufumi Nagahama, Masakazu Yashiro, Yuichiro Miki, Hiroki Morisako, Takehiro Uda, Takeo Goto, Takehiro Takami, Kenji Ohata. _Osaka City University Graduate School of Medicine, Osaka City, Japan_.

Introduction: Meningiomas are common intracranial tumor originate from arachnoid cap cells. Meningiomas are classified into three grades (grade I, II, and III) according to World Health Organization (WHO) 2016 criteria. Meningiomas are generally benign tumors with slow growing, and most of them can be treated by one-time resection. However, some of them regrow rapidly and invade into the dura mater widely, which often require additional treatments such as re-operation and irradiation. No useful marker was found to predict the recurrence of meningioma, so far. Then, the aim of this study is to investigate the immunohistochemical (IHC) expression of p53 and to determine the association of this results with the factors which related to the retreatment.

Materials and methods: This retrospective study included 389 patients with intracranial or intraspinal meningiomas which were surgically operated at Osaka City University from 2007 to 2017. The clinicopathological data of each patient was from medical records of Osaka City University hospital. IHC staining of p53 was performed on 389 meningiomas, as follows. The paraffin-embedded sections were de-paraffinized and heated for 10 min at 105°C by autoclave in Target Retrieval Solution. Then sections were incubated with 3% hydrogen peroxide to block endogenous peroxidase activity. The specimens were then incubated with anti-p53 (DO-7) for 1 h at room temperature. The slides were treated with streptavidin-peroxidase reagent and incubated in PBS with 1% (vol/vol) hydrogen peroxide, followed by counterstaining with Mayer's hematoxylin. p53 staining is considered negative when no cell with brown nuclear staining exist. The SPSS software program (SPSS Japan, Tokyo, Japan) was used for the analyses. In all tests, a p-value of less than 0.05 was defined as being statistically significant.

Results: Of the 389 meningiomas, 77cases (19.8%) were p53 positive. Univariate analysis revealed that recurrence of meningioma was significantly correlated with p53 positive (p=0.025), young age (p<0.001), large dural attachment (p<0.001), low height dural attachment ratio (p=0.016), large max diameter (p=0.020) and high Ki-67 index (p<0.001). Multivariate analysis indicated that p53 positive (p=0.037), young age (p=0.002), large dural attachment (p=0.002), low height dural attachment ratio (p=0.046), large max diameter (p=0.001) and high Ki-67 index (p<0.001) were significantly associated with recurrence of meningioma.

Conclusion: p53 expression was correlate with the proliferative activity of meningiomas. p53 might be a useful independent predictive marker for recurrence of meningioma.

#4902

Positive Ki-67 expression in post neoadjuvant chemotherapy (NAC) radical cystectomy samples are associated with lack of tumor downstaging and shorter overall survival (OS) in patients with muscle invasive bladder cancer (MIBC).

Selene Rubino,1 Youngchul Kim,2 Junmin Zhou,2 Charles Peyton,2 Scott Gilbert,2 Wade Sexton,2 Jingsong Zhang2. 1 _USF MCOM, Tampa, FL;_ 2 _Moffitt Cancer Center, Tampa, FL_.

Background: We recently reported single institution data on tumor downstaging and survival outcomes of 332 patients who underwent platinum based NAC followed by radical cystectomy for MIBC [Peyton et al. JAMA Oncol. 2018]. High expression levels of cell proliferation marker Ki-67 are associated with poor outcomes in chemotherapy naïve bladder cancer. Androgen receptor (AR) expression is associated with resistance to cisplatin in bladder cancer cell line models. We therefore studied Ki-67 and AR expression in our cohort of post NAC radical cystectomy samples and correlated them with tumor downstaging and OS.

Methods: Tissue microarrays (TMAs) were constructed from 130 post-NAC cystectomy samples and up to 5 cores were taken from each radical cystectomy sample. Matched lymph node metastases if present were included in TMAs. The expression of Ki-67 and AR were evaluated with immunohistochemistry and the average of the H score was used to represent each radical cystectomy sample and its matched lymph node metastasis.

Results: The median survival of this cohort of 130 patients was 33.4 months (range: 1.13 -127 months). 40 patients (31%) had tumor downstaging and 21 patients (16%) achieved pathological complete response. Using a Cox regression model for OS, positive Ki-67 expression in post NAC radical cystectomy samples is associated with poorer overall survival (hazard ratio=2.412, 95% CI:1.076-5.408, p=0.033), independent of the pathological N stage. Positive Ki-67 was significantly associated with lack of tumor downstage in a multivariable logistic regression model (odds ratio=0.081, 95% CI:0.014-0.464, p=0.004) while adjusting for adjuvant chemotherapy and pathological complete response. No statistically significant associations between AR and OS or AR and tumor downstaging were observed in multivariable analysis. When AR expression is evaluated as a binary variable (AR+, AR-), post NAC AR- expression is significantly associated with tumor downstaging compared to post NAC AR+ samples (Fisher's exact test, p=0.002). When stratified by sex, a trend towards better survival was observed in females (36 patients) compared to males (94 patients) (p=0.089). Post NAC AR- female patients also demonstrated a trend toward better survival (Log rank test, p=0.497) when compared to post NAC AR+ females.

Conclusions: Positive Ki-67 expression in post NAC radical cystectomy sample is associated with poorer OS and lack of tumor downstaging. If confirmed by a larger data set, Ki-67 can serve as a biomarker to select MIBC patients for post cystectomy adjuvant therapy.

#4903

Identification of immune-related mechanisms of cetuximab induced skin toxicity in colorectal cancer patients.

Jin Hyun Park,1 Mi Young Kim,1 In Sil Choi,1 Ji-Won Kim,2 Jin Won Kim,2 Keun-Wook Lee,2 Jin-Soo Kim1. 1 _SMG-SNU Bormae Medical Center, Seoul, Republic of Korea;_ 2 _Seoul National University Bundang Hospital, Seongnam, Republic of Korea_.

Background: Skin rash is well known predictive marker of response to cetuximab (Cmab) in metastatic colorectal cancer (mCRC). But mechanism of skin rash development is not well understood. After EGFR targeted therapies, the association of the IL-8 level changes and the skin rash has been suggested. The aim of this study was to evaluate the association between skin rash and inflammatory cytokine levels.

Material and Methods: Between 2014 and 2017, we prospectively enrolled a total of thirty-eight mCRC patients treated with chemotherapy with either Cmab or bevacizumab (Bmab) at two hospitals. We performed multiplex cytokine ELISA including twenty inflammatory cytokines including E-selectin, GM-CSF, IFN-alpha, INF-gamma, IL-1 alpha, IL-1 beta, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-17A, IP-10, MCP-1, MIP-1 alpha, MIP-1 beta, P-selectin, sICAM-1, and TNF-alpha at baseline before cycle 1, 24 hours after cycle 1, before cycle 2 (=14days), and before cycle 3 (=28days). The cytokine levels were compared by ANOVA test after log-transformation.

Results: Depending on the RAS mutational status,thirty and eight patients were treated with Cmab and Bmab-based chemotherapy, respectively. Of thirty patients who received Cmab plus FOLFIRI, skin rash developed in twenty-three patients (76.6%) after the cycle 1. The mean baseline levels of three groups were described in Table. Only IL-8 levels were significantly different: the mean baseline level of IL-8 in patients with skin toxicity was lower (2.84 ± 0.72) than in patients who did not experienced skin toxicity (3.62 ± 0.51) and received Bmab (3.1 ± 0.8) (ANOVA test, p=0.047).  | Skin toxicity

(n=23) | No skin toxicity

(n=7) | Bmab

(n=8) | ANOVA p-value

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

IL-8 | 2.84 ± 0.72 | 3.62 ± 0.51 | 3.1 ± 0.8 | 0.047

E-selectin | 9.94 ± 0.55 | 9.89 ± 0.39 | 9.91 ± 0.54 | 0.968

GM-CSF | 3.95 ± 0.95 | 4.17 ± 0.53 | 3.63 ± 1.36 | 0.597

IFN-alpha | 1.38 ± 0.72 | 1.55 ± 0.37 | 1.42 ± 0.69 | 0.846

INF-gamma | 4.13 ± 1.46 | 4.55 ± 1.1 | 4.48 ± 1.33 | 0.701

IL-1 alpha | 2.37 ± 0.55 | 2.69 ± 0.41 | 2.26 ± 0.72 | 0.310

IL-1 beta | 2.06 ± 1.1 | 2.33 ± 0.53 | 2.22 ± 1.05 | 0.803

IL-10 | 1.83 ± 0.93 | 2.24 ± 0.79 | 1.66 ± 0.99 | 0.465

IL-12p70 | 3.66 ± 0.79 | 3.84 ± 0.59 | 3.79 ± 0.69 | 0.812

IL-13 | 2.07 ± 0.81 | 2.26 ± 0.22 | 2.08 ± 0.45 | 0.808

IL-17A | 3.04 ± 0.85 | 3.33 ± 0.53 | 3.27 ± 0.78 | 0.613

IL-4 | 4.32 ± 1.11 | 4.66 ± 0.72 | 4.43 ± 0.92 | 0.749

IL-6 | 4.01 ± 1 | 4.17 ± 0.54 | 3.84 ± 0.83 | 0.782

IP-10 | 5.19 ± 1.78 | 5.76 ± 1.08 | 5.71 ± 1.33 | 0.601

MCP-1 | 3.8 ± 0.99 | 4.55 ± 0.39 | 3.95 ± 0.66 | 0.146

MIP-1 alpha | 2.04 ± 0.51 | 2.25 ± 0.25 | 2.22 ± 0.65 | 0.510

MIP-1 beta | 4.78 ± 0.55 | 5.02 ± 0.36 | 4.65 ± 0.57 | 0.387

P-selectin | 9.89 ± 0.65 | 9.8 ± 0.69 | 9.91 ± 0.49 | 0.936

sICAM-1 | 11.99 ± 0.86 | 12.15 ± 0.9 | 12.39 ± 0.85 | 0.527

TNF-alpha | 3.97 ± 0.57 | 4.18 ± 0.28 | 4.05 ± 0.65 | 0.670

Conclusion: This study suggests that the inflammatory cytokines levels might be affected by Cmab exposure and associated with the development of skin rash in metastatic CRC patients. The skin rash was related to lower baseline level of IL-8. Further studies are warranted to evaluate this interaction in patients who treated with Cmab.

#4904

High levels of microRNA-210-3p are associated with increased risk of disease progression in breast cancer patients treated with docetaxel.

BARBARA PASCULLI,1 RAFFAELA BARBANO,1 MICHELINA RENDINA,1 ANDREA FONTANA,1 MASSIMILIANO COPETTI,1 VANNA MARIA VALORI,1 MARIA MORRITTI,1 EVARISTO MAIELLO,1 VITO MICHELE FAZIO,1 MANEL ESTELLER,2 PAOLA PARRELLA1. 1 _FONDAZIONE IRCCS CASA SOLLIEVO DELLA SOFFERENZA, San Giovanni Rotondo, Italy;_ 2 _IDIBELL Bellvitge Biomedical Research Institute, L'Hospitalet, BARCELONA, Spain_.

MiR-210-3p is the most hypoxia-sensitive miRNA showing overexpression in different human cancers, but its role as biomarker in breast cancer (BC) is still controversial. In our study, we performed the expression analysis of miR-210-3p in a retrospective cohort of breast cancer patients with a median follow-up of 91 months enrolled according to the REMARK guidelines (n=284). As controls, 13 normal breast tissues (NBTs) from reductive mammoplasty were also profiled. By this analysis, we found that miR-210-3p levels were significantly increased in tumours as compared with NBTs (P=0.021). According to the surrogate molecular classification, the Triple Negative subgroup showed the highest levels of miR-210, followed by Luminal B tumours, whereas Luminal A, and HER2-amplified tumours showed the lowest expression levels (P=0.0137). The evaluation of the association of miR-210-3p with time-to-event outcomes in patients without synchronous metastases (n=268) demonstrated an association between risk of disease progression (HR 2.13, 95%CI 0.33-3.39, P=0.002) and higher levels of miR-210-3p in the subgroup treated with docetaxel in sequential therapy with anthracyclines (ECD). Moreover, a cut-off value of 20.966 as established by ROC curve analysis allowed to discriminate patients who developed distant metastases with an accuracy of 85% at 3-years (AUC 0.87, CI 0.69-1) and 83% at 5-years follow up (AUC 0.832, CI 0.656-1). Instead, the accuracy in discriminating patients who died for the disease was of 79.6% at both 5- (AUC 0.804, CI 0.517-1) and 10-years (AUC 0.804 CI 0.517-1) follow up. Overall, our data support the putative involvement of miR-210-3p in the response to treatments including docetaxel and suggest its eligibility to become a biomarker of early metastases development in ECD-treated breast cancer patients.

#4905

Comprehensive characterization of MET alterations in a large cohort of 610 advanced non-small cell lung cancer patients.

Cristina Aguado Esteban,1 Cristina Teixidó,2 Ruth Román,1 Ana María Gimenez Capitán,1 Carlos Cabrera,3 Mireia García,2 Clara D. Mayo De Las Casas,1 Ainara Arcocha,3 Sonia Rodriguez,1 Roxana Reyes,3 Elba Marín,2 Ana Perez Rosado,1 Niki Karachaliou,4 Aleix Prat,3 Rafael Rosell,5 Alejandro Martinez-Bueno,6 Miguel Angel Molina-Vila,1 Noemi Reguart3. 1 _Pangaea Oncology, Hospital Quiron Dexeus, Barcelona, Spain;_ 2 _Pathology Department, Hospital Clinic de Barcelona, Barcelona, Spain, Barcelona, Spain;_ 3 _Medical Oncology Department, IDIBAPS and Hospital Clinic de Barcelona, Barcelona, Spain, Barcelona, Spain;_ 4 _Instituto Oncológico Dr. Rosell (IOR), Sagrat Cor Hospital, Barcelona, Spain, Barcelona, Spain;_ 5 _Catalan Institute of Oncology and Institut d'Investigació en Ciències de la Salut, Germans Trias i Pujol, Badalona, Spain, Barcelona, Spain;_ 6 _Instituto Oncológico Dr. Rosell (IOR), Hospital Quirón-Dexeus, Barcelona, Spain_.

Background: A variety of alterations in MET have been described in advanced non-small cell lung cancer (NSCLC) patients, including gene amplification, protein overexpression, splicing variants and point mutations. MET alterations are receiving increasing attention as targets in precision medicine, and several clinical trials of anti-MET agents are ongoing in NSCLC.

Methods: A cohort of 610 patients with stage IIIb-IV NSCLC from two institutions was retrospectively analyzed for MET alterations by next-generation sequencing (NGS) (Ion Torrent PGM® or GeneReader®), nCounter, reverse transcriptase polymerase chain reaction (RT-PCR) or immunohistochemistry (IHC) during a 2-year period. Patients positive for MET amplification by NGS or MET overexpression by nCounter and/or IHC were submitted to fluorescence in situ hybridization (FISH). Representative patients positive for MET exon 14-skipping (METΔex14) mutations by nCounter and/or RT-PCR were confirmed by sequencing exons 13-15 of the MET gene.

Results: Overall, MET alterations were found in 116/610 patients (19%). Some patients had ≥2 MET aberrations. The most frequent finding was MET overexpression (58/333; 17.6%), followed by METΔex14 (31/610; 5.1%) and MET point mutations (3/129; 2.3%). Regarding MET amplification, it was found in 24/157 patients (15.3%). MET positivity by IHC (3+, >50%) showed a 90.8% concordance with MET mRNA overexpression by nCounter, with a 0.768 Cohen's kappa (confidence interval, CI 95% 0.575-0.961). A moderate agreement between RT-PCR and nCounter was found for METΔex14 (Cohen's kappa 0.629; CI 95% 0.434-0.825). MET amplification by FISH was found in the subset of MET-overexpressing patients with the highest mRNA levels by nCounter. Interestingly, a patient with a concomitant EGFR mutation and MET overexpression derived two years clinical benefit from crizotinib.

Conclusions: MET aberrations are present in 19% of advanced NSCLC patients and represent one of the most frequent targetable alterations in this malignancy. A comprehensive testing of MET alterations in advanced NSCLC patients, including NGS and nCounter techniques, is needed to identify a broader number of patients' candidates for targeted therapies.

#4906

Association between low-frequency pretreatment EGFR T790M mutation and tumor immune microenvironment in patients with EGFR-mutated non-small cell lung cancer.

Yoshiya Matsumoto,1 Kenji Sawa,1 Mitsuru Fukui,1 Jun Oyanagi,2 Motohiro Izumi,1 Koichi Ogawa,1 Tomohiro Suzumura,1 Tetsuya Watanabe,1 Hiroyasu Kaneda,1 Shigeki Mitsuoka,1 Kazuhisa Asai,1 Tatsuo Kimura,1 Nobuyuki Yamamoto,2 Tomoya Kawaguchi,1 Yasuhiro Koh2. 1 _Osaka City University, Osaka, Japan;_ 2 _Wakayama Medical University, Wakayama, Japan_.

Background: Involvement of tumor immune microenvironment including programmed death 1 (PD-1) and PD-ligand 1 (PD-L1) in biological properties of EGFR-mutant non-small cell lung cancer (NSCLC) remains elusive. Correlation of EGFR mutational status and PD-L1 expression and its impact on anti-PD-1/PD-L1 therapies have been intensively investigated but the relationship between pretreatment EGFR T790M (preT790M) and PD-L1 status has not yet been tackled. We have previously reported that EGFR-TKI-naïve patients with high preT790M had significantly shorter progression-free survival (PFS) upon EGFR-TKI treatment. In this study we investigated the association between preT790M status of tumor and immune microenvironment and its impact on efficacy of EGFR-TKI.

Materials and methods: We had previously assessed preT790M by droplet digital PCR in 44 advanced NSCLC patients harboring activating EGFR mutations treated with first-line EGFR-TKIs at Osaka City University Hospital between August 2013 and July 2016. For the current study, tumor proportion score (TPS) for PD-L1 and PD-L2 expression was assessed immunohistochemically using 28-8 antibody and D7U8C antibody, respectively. CD8-positive tumor-infiltrating lymphocytes (TILs) in tumor specimens were also evaluated.

Results: PD-L1/PD-L2 TPS and CD8-positive TILs were evaluable in 39 of 44 pretreatment tumor specimens. PD-L1 TPS of ≥50%, 1-49% and <1% were observed in 9 (23.1%), 10 (25.6%) and 20 patients (51.3%), respectively. PD-L2 TPS of ≥50%, 1-49% and <1% were observed in 1 (2.6%), 8 (20.5%) and 30 patients (76.9%), respectively. Significantly more PD-L1 high cases (TPS of ≥50%) were detected in subgroup of tumor with preT790M (41.2%, n=7/17) than in that without preT790M (9.1%, n=2/22) (p=0.026). In non-preT790M subgroup, CD8-positive TILs density was significantly higher in PD-L1 high than in PD-L1 low (median: 1298/mm2 vs. 331/mm2, p=0.035), while in preT790M subgroup, there was no significant difference regardless of PD-L1 level (median: 329/mm2 vs. 226/mm2, p=0.19). The median PFS and response rate for initial EGFR-TKIs were 13.6 months and 95% in non-preT790M/PD-L1 low, 1.6 months and 0% in non-preT790M/PD-L1 high, 11.4 months and 80% in preT790M/PD-L1 low, and 6.7 months and 57% in preT790M/PD-L1 high, respectively. No correlation was observed between preT790M and PD-L2 expression levels.

Conclusion: Results of our study suggest that there may exist the subtypes in EGFR-mutated NSCLC according to PD-L1 status and tumor immune microenvironment. Differential efficacy of EGFR-TKIs based on these subtypes further implies the potential impact of PD-L1 status and tumor immune microenvironment in EGFR blockade along with preT790M.

#4907

Transcriptomic profiling identifies a novel mitochondrial gene expression signature that predicts recurrence in microsatellite stable stage II and III colorectal cancer.

SOUVIK GHATAK,1 Raju Kandimalla,1 Jasjit K. Banwait,1 Hiroyuki Uetake,2 Francesc Balaguer,3 Luis Bujanda,4 Ajay Goel1. 1 _Baylor Scott & White Research Institute and Charles A. Sammons Cancer Center, Baylor University Medical Center, Dallas, TX; _2 _Tokyo Medical and Dental University Graduate School of Medicine, Tokyo, Japan;_ 3 _Hospital Clinic, CIBERehd, IDIBAPS, University of Barcelona, Barcelona, Spain;_ 4 _Instituto Biodonostia, Universidad del País Vasco (UPV/EHU), Centro de Investigación Biomédica en Red de Enfermedades Hepaticas y Digestivas (CIBERehd), San Sebastián, Spain_.

Purpose: Colorectal cancer (CRC) is the third most commonly diagnosed cancer in the United States. Current TNM (Tumor, Node, and Metastasis) classification approach is suboptimal in determining the prognosis of CRC patients. While there is a general consensus that stage II and III CRC patients with microsatellite instability (MSI) exhibit better outcomes, there is a lack of availability of biomarkers that can predict prognosis in microsatellite stable (MSS) patients. Given the emerging evidence that mitochondrial (mt) genomic instability is one of the hallmark features of cancer progression, herein, we systematically explored and established a novel mt-gene panel as a prognostic signature in CRC.

Experimental design: Three genome wide mRNA expression profiling datasets were analyzed for the biomarker discovery (GSE33113, n=65) and validation (GSE14333, n=196; TCGA, n=62) of a mt-gene expression signature that predicts recurrence in stage II/III MSS CRC patients. Subsequently, a 14-gene signature was established, and independently tested (n=257) and validated (n=212) in two clinical cohorts, using qRT-PCR assays. The uni- and multi-variate Cox proportional hazard (CoxPH) regression analyses were performed to evaluate the performance of the mt-gene signature individually, and in combination with various clinicopathological factors (e.g. TNM staging, lymphatic invasion, and tumor infiltration) for predicting recurrence free survival (RFS).

Results: The expression analysis of 568 mt-genes in CRC patients with and without recurrence, resulted in the identification of 32 significantly dysregulated genes (p<0.05). The univariate CoxPH analysis yielded a 14-gene signature that accurately predicted recurrence in the discovery (AUC = 0.89), and two independent validation cohorts (AUC = 0.75 and 0.79, respectively). This 14-gene signature demonstrated significant associations with poor RFS in independent testing and clinical validation cohorts of CRC patients (AUC = 0.87 and 0.73, respectively). Univariate analysis revealed that in addition to the mt-gene signature, TNM-stage (p = 0.007) and lymphatic invasion (p = 0.0004) were significant predictors of RFS; while multivariate analyses demonstrated that our mt-gene signature was an independent predictor (HR = 2.71; P<0.001) of tumor recurrence in stage II/III CRC patients. Intriguingly, a risk-stratification model comprising of the 14-gene signature together with TNM-stage and lymphatic invasion status achieved an even superior AUC of 0.89 and 0.78 in both clinical cohorts.

Conclusions: We have identified a novel, 14-gene signature as an independent predictor of tumor recurrence; which in combination with TNM-stage and lymphatic invasion, offers an easy-to-translate and facile assay for the personalized risk-assessment in stage II/III MSS-CRC patients. 

### Supportive Care and Survivorship Research / Outcome Research

#4908

Defective lipid metabolism as a novel molecular mechanism underlying chemotherapy-induced neurological adverse sequelae.

Xin Zhou, Qiang Zhang, Takashi Shingu, Jiangong Ren, Yiwen Chen, Jian Hu. _UT MD Anderson Cancer Ctr., Houston, TX_.

Up to 75% of cancer patients treated with chemotherapy show cognitive deficits after treatment, and these adverse neurological sequelae(commonly called chemobrain) can last for 2 to 10 years. The cause(s) of adverse neurological sequelae are not clear; however, demyelination is the most consistent neurological change and could explain all manifestations of the sequelae. Intriguingly, a substantial portion (30%-70%) of patients who undergo long-term evaluation develop delayed-onset adverse neurological sequelae, including demyelination, beginning at least 5 months after the completion of chemotherapy. For decades, mature myelin has been considered to be an inert material owing to early experiments with radioactively labeled cholesterol. However, our recent study of mature myelin maintenance led to the dogma-defying discovery that mature myelin is a very dynamic material with rapid turnover of lipid components by knocking out a gene called Quaking (Qk) in mature oligodendrocytes. We discovered that Qki is an essential factor in myelin lipid biosynthesis that controls the transcription of lipid metabolism genes, particularly those for fatty acid (FA) desaturation and elongation, via activating the peroxisome proliferator-activated receptor beta (PPARβ)-retinoid X receptor alpha (RXRα) complex. We reasoned that if mature myelin were an inert material whose components did not turn over, then blocking lipid synthesis in mature oligodendrocytes through deletion of Qk would have no impact on myelin structure. However, we observed that Qk deletion caused massive myelin breakdown within 2 weeks in mature oligodendrocytes of adult Plp-CreERT2;QkL/L mice (postnatal 2-4 months) but had no effect on oligodendrocyte survival, indicating mature myelin is a very unstable material. As with human chemobrain, mice developed delayed-onset demyelination 6 months after receiving a clinically relevant dose of 5-FU, doxorubicin, and paclitaxel. In the corpus callosum of both Plp-CreERT2;QkL/L mice and the mice treated with 5-FU, where demyelination was most prominent, we observed a large reduction in levels of myelin lipids (stained with FluoroMyelin), but not myelin proteins such as myelin-associated glycoprotein. To determine whether demyelination caused by Qk deletion can be alleviated by restoration of lipid supply or lipid synthesis, we treated adult Plp-CreERT2;QkL/L mice with a high-fat diet (HFD), the PPARβ agonist KD3010, or the RXRα agonist bexarotene. We found that all 3 strategies appreciably alleviated the demyelinating phenotype caused by Qk deletion and insufficient lipid synthesis. These data suggest that delayed-onset demyelination after chemotherapy is likely caused by compromised myelin maintenance, particularly lipid biosynthesis, attributable to chemotherapy-induced dysregulation of nuclear hormone receptors such as PPARβ/RXRα/Qki.

#4909

Whole-genome sequencing of childhood cancer survivors treated with cranial radiation therapy identifies 5p15.33 locus for stroke: A report from the St. Jude Lifetime Cohort (SJLIFE) study.

Yadav Sapkota,1 Yin Ting Cheung,2 Wonjong Moon,1 Kyla Shelton,1 Carmen L. Wilson,1 Zhaoming Wang,1 Daniel A. Mulrooney,1 Jinghui Zhang,1 Gregory T. Armstrong,1 Melissa M. Hudson,1 Leslie L. Robison,1 Kevin R. Krull,1 Yutaka Yasui1. 1 _St. Jude Children's Research Hospital, Memphis, TN;_ 2 _The Chinese University of Hong Kong (CUHK), Hong Kong, Hong Kong_.

Long-term survivors of childhood cancer are at increased risk of stroke, which is attributed to the well-established dose-response association with cranial radiation therapy (CRT) dose and risk. While radiation-induced vasculopathy is believed to be the likely mechanism underlying CRT-associated stroke, there is a variation in the dose-risk association, suggesting a potential contribution of genetic factors. We performed analyses of deep-coverage (36.8X) whole-genome sequencing data of 696 childhood cancer survivors of European descent [median (range): 40.4 (12.4-64.7) years old; 54% male] from the SJLIFE cohort treated with CRT, of whom 116 (17%) developed clinically-diagnosed stroke. Analyses of common variants [minor allele frequency (MAF)>0.05] were performed using logistic regression, adjusting for age at cancer diagnosis, sex, age at follow-up, CRT dose and top principal components derived from genotypes. Rare/low-frequency variants (MAF≤0.05) were aggregated by different functional annotations and agnostic 4-kb sliding windows and tested jointly using Burden and SKAT Tests, adjusted for the same covariates. Results identified a genome-wide significant association between a common variant on 5p15.33 locus and CRT-associated stroke [rs112896372: MAFaffected=0.27; odds ratio (OR)=2.93; P=4.66×10-8]. Stratified analyses showed that rs112896372 had the strongest association (OR=4.81; P=4.16×10-4) among survivors treated with CRT dose 25-50 Gray (Gy); survivors treated with CRT dose <25 Gy and those treated with CRT dose >50 Gy had the weaker associations (OR=2.41; P=1.01×10-3, OR=3.01; P=4.91×10-3, respectively). We replicated the association between rs112896372 and stroke in two independent SJLIFE samples: (1) 20 with and 70 without stroke of African ancestry treated with CRT (MAFaffected=0.23; OR=6.47; P=8.99×10-3) and (2) 60 with and 1581 without stroke of European ancestry not treated with CRT (MAFaffected=0.25; OR=1.58; P=0.04). SNP rs112896372 maps to an intergenic region located approximately 155 kb downstream of IRX1 (iroquois homeobox 1), a member of IRX family of transcription factors with established roles in ventricular chamber, conducting system development and heterogeneity of ventricular repolarization. Overall, we identified and independently replicated a novel locus for CRT-associated stroke among survivors of childhood cancer, with a possible CRT dose-specific effect. Considering the large effect size and the relatively high MAF, the 5p15.33 locus may have potential clinical utility for identifying high-risk CRT exposed cancer survivors and guide intervention strategies.

#4910

Neurocognitive effects of central nervous system directed chemotherapy in Non Hodgkin Lymphoma diseased children.

Abeer Abd Elmoneim, Ahlam Eladawy, Mona Abu Elkasem, Soumaya Hadhood. _Sohag University, Sohag, Egypt_.

Background and Purpose. Because the blood-brain barrier serves as a pharmacological barrier, malignant cells can remain in the central nervous system (CNS) despite systemic chemotherapy. Consequently, CNS-directed treatment is an essential part of therapy for Non-Hodgkin lymphoma (NHL). We aimed in this analysis to determine the neurocognitive effects of CNS-directed chemotherapy in children with NHL.

Methods.

In a cross-sectional study 20 NHL diseased children and 10 healthy controls were tested for neurocognitive functions. The selection criteria were: 3 years or more after the end of NHL chemotherapy only regimen, no anaemia, no CNS or systemic diseases. Both patients and healthy controls were examined for global intelligence quotient (IQ), verbal IQ, performance IQ, attention measures, auditory tests, long and short memory, math skills and academic achievement.

Results.

No decline in global IQ in NHL children compared to healthy controls but there was a significant decline of attention, verbal comprehension and performance IQ. Among 20 NHL diseased children 18 had poor recent and immediate memories while remote memory was normal in 16 children. Young age at diagnosis and treatment intensity were the commonest risk factors.

Conclusion.

In absence of cranial irradiation, childhood survivors of NHL experience long-term neurocognitive deficits after chemotherapy treatment. Global IQ is not a sufficiently sensitive measure to detect specific CNS deficits in NHL diseased children. The deficits are mainly present in basic neurophysiologic processes of attention and executive functioning.

#4911

Collaboration on quality of life in myeloproliferative neoplasms: Analysis of body mass index and symptom burden.

Sarah F. Christensen,1 Robyn M. Scherber,2 Martin Goros,2 Jonathan Gelfond,2 Nana Brochmann,1 Christen L. Andersen,3 Esben M. Flachs,4 Hans C. Hasselbalch,1 Ruben A. Mesa2. 1 _Zealand University Hospital, Roskilde Denmark, Roskilde, Denmark;_ 2 _Mays MD Anderson Cancer Center, Mays MD Anderson Cancer Center, TX;_ 3 _University Hospital of Copenhagen at Rigshospitalet, Copenhangen, Denmark;_ 4 _Bispebjerg University Hospital, Copenhagen, Roskilde, Denmark_.

Introduction

BMI increases the risk of Philadelphia-chromosome negative myeloproliferative neoplasms (MPN) development. Patients with MPN often suffer from a severe symptom burden reduced quality of life. The aim of this study was to examine the association between BMI and symptom burden.

Methods

A comprehensive analysis of data from two large cross-sectional surveys of health-related quality of life in MPN patients was done by merging data from the Danish Population-based Study with data from the international Fatigue Study. Both surveys included the validated MPN-SAF questionnaire and questions addressing lifestyle.

Results

Comparing Danish patients with US patients, Danish patients were significantly older (69.2y vs 60.9y, P < 0.001), a smaller proportion were female (56.5%, vs 67.3% P < 0.001) and less patients worked (24.8%, vs 43.3% P < 0.001).

In both the US and the Danish (DK) dataset Normal Weight was most frequent (DK/US=52.4/46.3%), followed by Overweight (DK/US=31.7/30.5%), Obese (DK/US=13.2/21.7%) and least frequent Underweight (DK/US=2.7%/1.5%). Interestingly, we observed a U-shaped association between BMI and Symptom Burden in both datasets with significant higher values of total symptom score for underweight and obese patients relative to normal weight. Furthermore, significant higher scores for Underweight or Obese patients were observed for most individual symptoms (Table 1).

Conclusion

Despite significant differences in demographics, the U-shaped relation between BMI and symptom burden reflects a general pattern of many variables consistent across countries. Weight alteration might be considered as a therapeutic strategy to alter symptom burden in MPN patients, bearing in mind that unintentional weight loss has been demonstrated in some MPN subtypes to be predictive of worsened survival. Further analysis for possible confounders are planned.

Table 1. MPN patients' assessment of symptom severity by BMI category and Country | |  | |  | |  | |  | |

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

|

Denmark (n=2064)

Median [IQR] | USA (n=894)

Median [IQR]

BMI | Under-weight

(<18.5) | Normal Weight

(18.5-24.9) | Over-weight

(25.0-29.9) | Obese

(≥ 30) | P-value | Under-weight

(<18.5) | Normal Weight

(18.5-24.9) | Over-weight

(25.0-29.9) | Obese

(≥ 30) | P-value

Fatigue (BFI) | 3 [0-6] | 3 [0-5] | 3 [0-5] | 3 [1-7] | 0.027 | 8 [5-9] | 6 [4-8] | 7 [5-8] | 7 [6-9] | <0.001

Early satiety | 4 [1-5] | 2 [0-5] | 2 [0-5] | 2 [0-5] | 0.001 | 3 [0-5] | 2 [0-5] | 2 [0-5] | 3 [0-5] | 0.494

Abdominal pain | 0 [0-2] | 0 [0-2] | 0 [0-2] | 0 [0-2] | 0.168 | 1 [0-3] | 0 [0-2] | 0 [0-3] | 1 [0-4] | <0.001

Abdominal discomfort | 1 [0-4] | 0 [0-3] | 1 [0-3] | 1 [0-3] | 0.296 | 3 [1-4] | 1[0-3] | 1 [0-4] | 2 [0-5] | 0.001

Inactivity | 3 [0-5] | 1 [0-4] | 1 [0-4] | 2 [0-5] | 0.003 | 4 [1-6] | 2 [0-5] | 3 [1-6] | 5 [3-7] | <0.001

Headache | 0 [0-2] | 0 [0-2] | 0 [0-3] | 0 [0-3] | 0.401 | 2 [0-5] | 1 [0-4] | 1 [0-4] | 2 [0-6] | <0.001

Concentration problem | 2 [0-5] | 1 [0-4] | 1 [0-4] | 1 [0-5] | 0.311 | 0 [0-7] | 3 [0-6] | 4 [1-6] | 5 [2-7] | <0.001

Dizziness | 3 [1-5] | 1 [0-4] | 1 [0-4] | 1 [0-4] | 0.068 | 0 [0-3] | 1 [0-4] | 1 [0-5] | 3 [0-6] | 0.001

Numbness | 3 [0-5] | 1 [0-4] | 1 [0-4] | 2 [0-5] | 0.001 | 4 [0-7] | 1 [0-5] | 2 [0-5] | 3 [0-6] | 0.003

Insomnia | 2 [0-5] | 2 [0-5] | 1 [0-5] | 3 [0-6] | 0.003 | 5 [3-8] | 3 [1-6] | 4 [1-7] | 5 [2-7] | 0.004

Sad mood | 2 [0-4] | 1 [0-3] | 1 [0-3] | 1 [0-4] | 0.555 | 1 [0-5] | 2 [0-5] | 3 [0-6] | 4 [2-6] | <0.001

Sexuality problems | 1 [0-5] | 2 [0-6] | 3 [0-7] | 3 [0-8] | 0.057 | 4 [0-7] | 3 [0-7] | 4 [0-7] | 6 [1-8] | 0.001

Cough | 2 [0-6] | 0 [0-3] | 1 [0-3] | 1 [0-4] | <0.001 | 0 [0-4] | 0 [0-2] | 0 [0-3] | 1[0-5] | 0.001

Night sweats | 1 [0-4] | 1 [0-4] | 2 [0-5] | 2 [0-5] | 0.021 | 0 [0-2] | 1 [0-4] | 1 [0-5] | 3 [0-6] | <0.001

Itching | 1 [0-3] | 1 [0-3] | 1 [0-4] | 1 [0-4] | 0.010 | 0 [0-5] | 1 [0-4] | 2 [0-5] | 2 [0-7] | <0.001

Bone pain | 1 [0-5] | 0 [0-3] | 0 [0-3] | 1 [0-5] | 0.008 | 1 [0-4] | 0 [0-4] | 0 [0-5] | 3 [0-6] | <0.001

Fever | 0 [0-0] | 0 [0-0] | 0 [0-0] | 0 [0-0] | 0.294 | 0 [0-0] | 0 [0-0] | 0 [0-0] | 0 [0-0] | 0.013

Weight loss | 4 [0-7] | 0 [0-1] | 0 [0-0] | 0 [0-0] | <0.001 | 2 [0-5] | 0 [0-1] | 0 [0-0] | 0 [0-0] | <0.001

QoL | 3 [1-5] | 2 [1-5] | 2 [1-5] | 2 [1-5] | 0.115 | 4 [3-5] | 3 [1-5] | 4 [2-5] | 4 [3-6] | <0.001

TSS | 22 [12-37] | 16[7-30] | 16[7-29] | 21[10-33] | 0.001 | 26[23-41] | 22[12-34] | 26[13-40] | 32[23-44] | <0.001

#4912

A snapshot of myeloproliferative neoplasms in the United States: Analysis of the "myMPN" patient registry.

Robyn M. Scherber,1 Lindsey Whyte,2 Michelle Woehrle,2 Claire Harrison,3 John Mascarenhas,4 Srdan Verstovsek,5 Alison Moliterno,6 Ruben A. Mesa1. 1 _Mays MD Anderson Cancer Center, San Antonio, TX;_ 2 _MPN Research Foundation, Chicago, IL;_ 3 _Guys St Thomas, London, United Kingdom;_ 4 _Mt Sinai, New York, NY;_ 5 _MD Anderson Cancer Center, Houston, TX;_ 6 _Johns Hopkins, Baltimore, MD_.

Introduction: The myeloproliferative neoplasms (MPNs) are an uncommon type of hematologic malignancy which can be accompanied by a medically complex sequalae and severe symptom burden. Patient registries allow for the evaluation and monitoring of clinically meaningful outcomes in rare diseases over time. Although patient registries exist for MPNs, the utility of these registries has been limited by inclusion of only particular institutions and/or regions or the lack of patient reported outcomes, specifically symptoms and quality of life. In September 2017, the "myMPN" patient registry began enrollment as the first MPN patient-centered registry. The purpose of this analysis is to report patient-reported disease features, outcomes, and events uploaded to the registry to date.

Methods: The "myMPN" patient registry was created by the MPN Research Foundation's steering committee and hosted on the Genetic Alliance registry platform. Utilizing previous questions created for MPN populations and validated assessment tools, the myMPN patient registry allows patients to input data disclosures, disease features, treatments, blood counts and symptoms. The registry has been granted independent IRB approval.

Results: Accrual: To date, the registry has 744 participants. Of these, 62% were female and mean age was 61 years (range 18-94). Disease-related information: The registry includes 38% essential thrombocythemia (ET) patients, 36% polycythemia vera (PV) patients, 23% myelofibrosis (MF) patients, and 3% patients who reported an alternative MPN diagnosis. 11.5% of patients were not aware of their mutation status. Disease events: Over the year since study initiation, there have been 2,100 reported disease-related events, which have included 825 blood draws, 298 phlebotomies, 207 MPN medication changes, 144 bone marrow biopsies, 77 transfusions, 39 thrombotic or bleeding events, and 30 genetic testing events. Since registry initiation, 6 patients reported a new ET to MF transformation, and 4 patients reported a PV to MF transformation. Disease Symptom Burden: To date, 400 patients have completed 675 independent symptom assessments. Many have completed two or more symptom assessments. In general, MPN-10 symptom scores are similar to previously published cohorts, but provide data on longitudinal symptom change.

Conclusions: The myMPN patient registry facilitates the research and care of MPN patients by clinicians, researchers, patients, patient-advocates, and caregivers a common platform to interface prospectively. Future goals of the registry are to 1) explore variables related to disease progression/transformation, 2) expand outside of the United states to other English-speaking countries, 3) allow patients to connect with their physicians regarding their registry information, and 4) to develop a compendium medical record and specimen registry.

#4913

Differences in presentation and survival outcomes for African American patients with newly diagnosed multiple myeloma.

Nisha Joseph, Vikas A. Gupta, Craig Hofmeister, Charise Gleason, Leonard T. Heffner, Kafuman L. Jonathan, Lawrence H. Boise, Madhav D. Dhodapkar, Sagar Lonial, Ajay K. Nooka. _Emory University, Atlanta, GA_.

Background: Though the incidence of MM is threefold higher in the African American (AA) population compared to Caucasians, reported long term outcomes are less favorable presumably due to inequities in access to healthcare. We have conducted a retrospective analysis of our institutional data of 1000 patients treated with RVD induction therapy, assessing differences in presentation, disease biology, and outcomes in AA patients.

Methods: A total of 1000 newly diagnosed MM patients were treated with RVD induction therapy from January 1st 2005 until August 31st 2016. Dose adjustments were made based on physician's discretion and patient tolerability. Demographic and outcomes data for the patients were obtained from our IRB approved myeloma database and responses were evaluated per IMWG Uniform Response Criteria.

Results: 56.4%of patients were white (W), and 33.9% were AA, consistent with the demographic representation of the state of GA and our institutional referral population. Median age was 61 years (range 16-83), 57 for AA patients (range, 24-83) compared to 62 (range, 16-81) in white patients, suggesting the onset is earlier among AA. In regard to stage, AA: 73.9% stage I/II, 26.1% stage III; W: 77.1% stage I/II, 22.9% stage III, showing no difference in prognostic staging at presentation. There was no statistically significant difference in the presenting labs between AA and whites except for hemoglobin, with more AA patients presenting with Hgb≤9.9 g/dL (45.7% AA vs 32.5% W, p <.0001). In terms of prevalence of high-risk cytogenetics, there was no significant difference between the two cohorts in: complex karyotype, t(4;14), t(11;14) or del1p. However, there were significant differences found in the rates of: amp 1q 19.2% W/10.6% AA, (p<.0001), del13 28.3% W/19.6% AA (p=.003), and del17p 11.7% W/7.2% AA (p=.019). There was no significant difference in the number of patients who underwent ASCT (84% W vs 82% AA, p=.241), nor in ≥VGPR rates post-induction and 100 days post-ASCT. Median PFS for the entire cohort was 63 months, 62 months (54-69.9) for white patients versus 65 months (53-76.9) for AA patients (p=0.403). At a median follow up of 38 months, median OS has not yet been reached.

Conclusions: This is the largest reported cohort of myeloma patients treated with RVD induction, with one-third of the patients representing the AA population. In our dataset, AAs are diagnosed 5 years younger, with lower hemoglobin at presentation and lower rates of amp1q, del13 and del17p when compared to whites. When offered the same induction regimen and opportunity for ASCT, AAs tend to experience the same survival benefits as their white counterparts. The lack of significant difference in PFS or OS suggests standardization and improved access to care could lead to better long-term outcomes in the AA population.

#4914

Pregnancy and breast cancer outcomes.

Mihong Choi, Jiyeon Han, Bo Ram Yang, Myoung-jin Jang, Miso Kim, Tae-Yong Kim, Seock-Ah Im, Han-Byoel Lee, Hyeong-Gon Moon, Wonshik Han, Dong-Young Noh, Kyung-Hun Lee. _Seoul National University Hospital, Seoul, Republic of Korea_.

Background: Pregnancy-associated breast cancer (PABC) poses a unique challenge as hormonal changes occurring in pregnancy potentially interact with breast cancer outcomes. However, reports on prognosis of PABC have been rare and inconsistent, and most of these studies were from Western countries with only few from Asia. We investigated the outcome of PABC linking a large hospital-based database and the nationwide claims database of Korea.

Patients and methods: We retrospectively studied a cohort of 3687 female patients of reproductive age (<50 years old) who had undergone surgery for breast cancer at Seoul National University Hospital from 2008 to 2015. Data on stage distribution and tumor characteristics were reviewed from the institutional database, and history of delivery or abortion from the nationwide claims database of Korea for comprehensive identification. Breast cancer during pregnancy was defined as delivery within 9 months of the diagnosis of breast cancer, and postpartum breast cancer as breast cancer diagnosis within 12 months of delivery, with both being counted as PABC.

Results: Among 3687 patients, 18 and 45 patients were classified as having breast cancer during pregnancy and postpartum breast cancer, respectively, comprising 63 cases of PABC. Patients with PABC were significantly more likely to have advanced stage and/or hormone receptor negative tumor, and be younger than 35 years old at diagnosis of breast cancer than those without it (P value for T stage, .0001; N stage, .0011; ER negativity, <.0001; PR negativity, <.0001; age at breast cancer diagnosis <35, <.0001). Similar distribution pattern of clinical characteristics was observed with postpartum breast cancer compared to their counterparts (P value for T stage, <.0001; N stage, .0001; M stage, .0421; ER negativity, <.0001; PR negativity, <.0001; age at breast cancer diagnosis <35, <.0001), whereas these characteristics were comparable between patients with and without breast cancer during pregnancy. PABC and postpartum breast cancer were seemingly associated with worse survival with univariate hazard ratio (HR) of 4.53 (95% confidence interval, CI, 2.53-8.13; P value <.0001) and 5.34 (95% CI, 2.91-9.82; P value <.0001), multivariate HR of 1.52 (95% CI, .82-2.83; P value .1841) and 1.57 (95% CI, .82-2.99; P value .1708), respectively. Breast cancer during pregnancy was not associated with worse survival.

Conclusions: PABC and postpartum breast cancer tended to be associated with higher stage, hormone receptor negativity, and thus worse survival, but breast cancer during pregnancy had no adverse effect on survival in patients with breast cancer.

#4915

Optimal frequency of follow up scans on systemictherapy for advanced malignancies.

David J. Stewart,1 Blair Macdonald,2 Sasha Van Katwyk,3 Arif Awan,2 Kednapa Thavorn3. 1 _Ottawa Hospital Regional Cancer Ctr., Ottawa, Ontario, Canada;_ 2 _University of Ottawa, Ottawa, Ontario, Canada;_ 3 _Ottawa Hospital Research Institute, Ottawa, Ontario, Canada_.

Background: Frequency of follow-up scans is specified in trials but there is little evidence to guide optimal frequency in standard therapy. Progression-free survival (PFS) generally follows first order kinetics and PFS half-lives (calculated by nonlinear regression analysis of more than 300 published PFS curves) correlated strongly with PFS medians (Stewart, AACR 2018).

Method: We used the Excel formula EXP(-tn*0.693/t1/2) to calculate proportion of residual patients remaining progression-free at different time intervals, where tn is the potential time of follow-up scans (eg, every 3 weeks, 6 weeks, etc), * indicates multiplication, 0.693 is the natural logarithm of 2, and t1/2 is the PFS half-life in weeks.

Results: Proportion of remaining patients expected to still remain progression-free at each subsequent scan varied with time interval between scans and with PFS half-life for the individual regimen. For example, with a PFS half-life of 4 months (17.3 weeks) and scans repeated every 6 weeks, 21% of the patients would have progressed by the first scan, 21% of the remaining patients would have progressed by the second scan at 12 weeks, etc. With PFS half-lives of 2, 4, 6, 12 and 20 months (for example), the proportion of remaining patients progressing by the time of each subsequent follow up scan if the scans were repeated every 3 weeks would be 21%, 11%, 8%, 4% and 2%, respectively, while with scans repeated every 6 weeks the proportion progressing would be 38%, 21%, 15%, 8% and 5%, by 12 weeks it would be 62%, 38%, 27%, 15% and 9%, and by 15 weeks it would be 70%, 45%, 33%, 18% and 11%, respectively.

Conclusions: For therapies with short PFS half-lives, scanning every 6 weeks may not be often enough, while with long PFS half-lives, less frequent scans may be warranted. We plan analyses to estimate economic implications of varying scan frequency based on PFS half-life, drug and toxicity costs, opportunity costs (where other effective therapies might be offered if progression is detected), etc. We will also assess how this approach might be adapted for PFS curves that follow exponential 2-phase decay due to presence of distinct good vs poor prognosis subgroups, and we are in the process of using published PFS curves to assess the PFS half-lives for multiple different cancer therapies.

#4916

Impact of primary organ site on prognosis in extranodal peripheral T cell lymphoma not otherwise specified.

Taha Al-Juhaishi, Arushi Khurana, Danielle Shafer, Victor Yazbeck. _VCU Health, Richmond, VA_.

Introduction: Peripheral T cell lymphoma, not otherwise specified (PTCL, NOS) is a rare heterogeneous group of diseases that constitutes 25% of T-cell lymphomas. It predominately affects men with a median age of 60 years at presentation. It is known to be an aggressive disease with high risk of relapse and subsequent poor response to systemic therapies. In this study, we sought to investigate the impact of primary site of disease on prognosis in extranodal PTCL, NOS.

Methods: The Surveillance, Epidemiology and End Results (SEER) database was used to identify adult patients (≥20 years) diagnosed with PTCL, NOS between 1973 and 2014. Primary sties excluded were lymph nodes, bone marrow, spleen and skin. All stages of disease were included; treatment effect (surgery, radiation, chemotherapy) was analyzed when available. Sites of disease were grouped into 5 organ systems for convenience. Overall survival (OS) was estimated using the Kaplan-Meier method, and compared using Log-Rank test. Cox proportional hazards models were used for adjusted survival analyses.

Results: Total of 392 patients were included. Median age was 62 years, and majority were male (n=242, 61.7%) Caucasian (n=224, 57.1%), and married (n=231, 58.9%). Gastrointestinal/hepatobiliary site was the most common primary extranodal organ system involved (n=156, 39.8%), followed by head and neck (n=147, 37.5%), thoracic excluding breast (n=42, 10.7%), CNS (n=32, 8.2%), and breasts (n=15, 3.8%). Most patients had stage IV disease (n=116, 29.6%). Total of 137 (34.9%) patients had surgery to the primary site, 94 (24%) had radiation, and 241 (61.5%) received chemotherapy. Median overall survival was 13 (0-252) months, and was statistically different by primary organ system involved (p< 0.001). Gastrointestinal/hepatobiliary group had the worst median survival of only 8 months (95% CI 4.3- 11.6), followed by thoracic [median of 9 months; 95% CI (0.0 - 21.7)], and CNS [median of 12 months; 95% CI (3.5 - 20.4)]. Lymphoma was the most common cause of death (n=166, 42.3%). On univariate analysis, patients who received surgery did not have better survival [HR 0.909; 95% CI (0.70-1.16)]; while patients who received radiation [HR 0.53; 95% CI (0.39-0.71)], or chemotherapy [HR 0.76; 95% CI (0.60-0.98)] had better outcomes. In a multivariate analysis model; stage IV disease and Hispanic race were significantly associated with worse survival; while being married, undergoing radiation or chemotherapy was associated with better survival.

Conclusion: PTCL, NOS is a rare aggressive disease mostly affecting elderly men. Lymphoma involving gastrointestinal /hepatobiliary system is the most common site of primary extranodal disease and portends the worst survival. Patients who received radiation or chemotherapy had better outcomes.

#4917

Efficacy and survival of advanced sarcoma patients enrolled on phase 1 trials in the era of novel targeted and immunotherapies: Signals for the future.

Shiraj Sen,1 Roberto Pestana,2 Kenneth Hess,2 David Hong,2 Filip Janku,2 Aung Naing,2 Gerald S. Falchook,1 Shreyaskumar Patel,2 Funda Meric-Bernstam,2 Vivek Subbiah2. 1 _Sarah Cannon Research Institute, Denver, CO;_ 2 _MD Anderson Cancer Center, Houston, TX_.

Background: Very few effective US FDA approved therapies exist for metastatic soft tissue and bone sarcomas. However, recent advances in our understanding of the molecular drivers and immune microenvironment across sarcoma subsets has fueled novel treatment strategies. Herein we report outcomes of sarcoma patients treated on phase 1 trials with novel immunotherapy and targeted therapy combination approaches in order to identify signals for the future.

Methods: We analyzed clinical and next generation sequencing data from all sarcoma patients treated on phase 1 trials at MD Anderson Cancer Center (MDACC) and performed logistic and Cox proportional hazards regression analyses to evaluate response rate (RR), median time to progression (mTTP), clinical benefit rate (CBR; defined as CR, PR, or SD > 6 months), and median overall survival (OS).

Results: Among the 406 patients with advanced sarcomas (321 soft tissue sarcoma, 85 bone sarcomas) treated on phase 1 trials at MDACC from May 2006 to May 2018, median age was 53 (range 11-84), 48% were female, and patients had a median 3 prior lines of therapy (range 0-9). The most commonly treated soft tissue sarcoma subtypes included leiomyosarcoma (n=66; 16%), liposarcoma (n=52; 13%), GIST (n=44; 11%), UPS (n=14; 3%), and synovial sarcoma (n=11; 3%) and most commonly treated bone sarcomas included osteosarcoma (n=34; 8%), chondrosarcoma (n=28; 7%), and Ewing's sarcoma (n=25; 6%). RR was 7% (95% CI 5, 10), mTTP was 2.9 months (95%CI 2.6, 3.1), CBR was 24% (95% CI 20, 29), mOS was 17.2 months (95% CI 13.8, 20.8). 2 patients had a CR as best response - 1 chondrosarcoma patient treated with an anti-APO2L/Trail agent and 1 patient with Ewing's sarcoma treated with the combination of an IGF1R inhibitor plus mTOR inhibitor. 26 patients (6%) had a PR as best response using novel immunotherapies targeting PD1, PDL1 plus CCR4, CTLA4 plus KIT, and TLR7/8 and novel targeted therapies against TRK, LRRC15, cMET, mTOR, VEGF, MDM2, KIT/PDGFRA, and FGFR. Responses were seen across sarcoma subtypes - ASPS, UPS, myxoid sarcoma, liposarcoma, GIST, carcinosarcoma, clear cell sarcoma, embryonal rhabdomyosarcoma, epitheliod sarcoma, fibrious histiosarcoma, and Ewing's sarcoma.

Conclusion: Our analysis identifies a reasonable survival in heavily pretreated, refractory sarcoma patients on phase 1 trials and highlights signals for future drug development in soft tissue and bone sarcomas. Biomarker analysis is ongoing to help identify the subset of patients who responded to novel targeted and immunotherapy approaches. Advanced sarcoma patients should be considered for molecular profiling and early phase clinical trials.

### Tumor Markers to Assess the Biology and Clinical Course of Cancer 3

#4918

Artificial intelligence-assisted macrophage identification in tumor biopsies.

Kelsey Weigel, Will Paces, Elliott Ergon, Jeni Caldara, Kile McFadden, Cris Luengo, Roberto Gianani, Bharathi Vennapusa. _Flagship Biosciences Inc, Westminster, CO_.

Understanding response to immunotherapy requires accurate and complete characterization of tumor-associated immune cells in order to fully contextualize the immuno-oncology biomarker expression. Current standard practices surrounding enumeration of biomarker-positive immune cells using image analysis necessitate a dual-labeling approach combining the biomarker of interest and immune cell identification assays.

Machine Learning (ML) may be used to distinguish different tissue types in a biopsy (e.g. tumor vs non-tumor), or to identify different cell types (e.g. macrophages vs other cells). A machine learning algorithm obtains statistics for a specific tissue class or cell type based on a training set, given by "ground truth" examples. The algorithm then generalizes from the given examples to "learn" the ability to find the tissue or cell type on the rest of the digital scan of the tissue slide, or other slide scans.

Here we specifically describe ML methods for macrophage identification in digital scans of cancer tissue slides. The described methods can be used independently or in combination and are both based upon the use of artificial intelligence-based computational tissue analysis (cTA®) algorithms. These technologies can recognize macrophages independently of traditional IHC or IF dual-labeling identification methods. This same methodology may also be applicable for other specific subsets of morphologically distinct immune cells.

The first strategy consists of training the cTA algorithm according to pathologist identification of macrophages, without a macrophage-specific staining (e.g., CD68). In this case, the slides are stained only with a PD-L1 assay, and pathologists establish the "ground truth" to teach the ML classifier. The second strategy consists of using CD68 labeling to identify macrophages to teach the ML classifier. Upon successful training and performance testing, the developed macrophage classifier can potentially be applied to new specimens without relying on pathologist annotation or IHC or IF dual-labeling for macrophage recognition. In summary, these two novel approaches demonstrate how ML could be used for characterization of critical immune cells such as macrophages in immuno-oncology tissue evaluations, without the reliance on additional biomarkers to specifically identify the cells. In this study, we evaluated the approach in the context of standard PD-L1 IHC as is used in the Companion Diagnostic (CDx) setting. Successful ML cTA strategies may be applied to other CDx assays to gain additional information about immune cell identification and quantification while only relying on simple monoplex assays. Importantly, these ML strategies would not require a change to the existing clinical practices where higher-level multiplexing would be needed to achieve similar outcomes.

#4919

Required numbers of biopsy samples and tumor cells for the assessment of PD-L1 expression to predict a response to nivolumab treatment in patients with NSCLC.

Jun Sato,1 Tomoyuki Naito,2 Hibiki Udagawa,2 Hidehito Horinouchi,1 Shuji Murakami,1 Yasushi Goto,1 Shintaro Kanda,1 Yutaka Fujiwara,1 Noboru Yamamoto,1 Yuichiro Ohe,1 Yoshitaka Zenke,2 Keisuke Kirita,2 Shingo Matsumoto,2 Kiyotaka Yoh,2 Seiji Niho,2 Noriko Motoi,2 Genichiro Ishii,2 Koichi Goto2. 1 _National Cancer Center Hospital, Tokyo, Japan;_ 2 _National Cancer Center Hospital East, Chiba, Japan_.

Background: PD-L1 expression is a predictive biomarker of response to nivolumab in non-squamous non-small cell lung carcinoma (Non-Sq NSCLC). PD-L1 expression in NSCLC exhibits significant intratumoral heterogeneity. Although multiple biopsy samples are usually obtained during a biopsy procedure, the evaluation of PD-L1 expression is typically performed using only one small sample. The aim of this study was to investigate the required numbers of samples and tumor cells (TCs) to assess PD-L1 expression using biopsy samples to predict the response to nivolumab.

Methods: A total of 225 biopsy samples obtained from 80 Non-Sq NSCLC patients treated with nivolumab between January 2016 and August 2017 were collected. In each patient, the samples were obtained from the same lesion at the same time before nivolumab treatment. The number of TCs and the PD-L1 score after staining with anti-PD-L1 antibody (Dako 28-8) were evaluated. We defined the sample containing the largest number of TCs as Max-TCs, the sample containing the smallest number of TCs as Min-TCs and the sum of all the samples from each patient as Multiple-TCs. The concordances of the PD-L1 scores among the Max-TCs, Min-TCs and Multiple-TCs were assessed. And then, the impacts of the numbers of samples and TCs on the prediction of the efficacy of nivolumab using the evaluation of PD-L1 expression were evaluated.

Results: The median number of samples was 2 (range: 2-6). The median number of TCs was 148 (13-2976) for the Mini-TCs, 570 (29-4826) for the Max-TCs, and 1022 (55-7062) for the Multiple-TCs.The correlation of the PD-L1 scores between the Max-TCs and Multiple-TCs was significantly high (R2 = 0.98). A mismatch of PD-L1 <1%/≥1% was observed between the Max-TC and the Multiple-TC in one patient (1%). The correlation of the PD-L1 scores between the Max-TCs and the Mini-TCs (R2 = 0.83) was weaker than that between the Max-TCs and the Multiple-TCs. A mismatch of PD-L1 <1%/≥1% was observed between the Max-TCs and the Mini-TCs in six patients (8%). The ROC analysis indicated that the required number of TCs to match PD-L1 <1%/≥1% between the Max-TCs and the Mini-TCs was 80 (AUC: 0.62). PD-L1 ≥1% in Mini-TCs containing the ≥80 TCs was associated with a longer progression-free survival (PFS) (median 8.7 vs 1.8 months, HR 0.39, P < 0.01) and a prolonged overall survival (OS) (median 20.9 vs 9.2 months, HR 0.41, P = 0.01) compared with PD-L1 <1%. On the other hand, there were no difference in the PFS (median 3.0 vs 2.4 months, HR 0.77, P = 0.59) and the OS (median 7.7 vs 5.6 months, HR 0.89, P = 0.81) between PD-L1 ≥1% and PD-L1 <1% in the Mini-TCs containing <80 TCs.

Conclusion: Evaluating the PD-L1 score using multiple biopsy samples did not provide any added benefit. One biopsy sample containing ≥80 TCs is required to evaluate PD-L1 expression to predict the response to nivolumab.

#4920

A status of re-biopsy for non-small-cell lung cancer patients after first EGFR-TKI failure and incidence of T790M mutation.

Tetsuya Sakai, Hibiki Udagawa, Yoshitaka Zenke, Keisuke Kirita, Shigeki Umemura, Shingo Matsumoto, Kiyotaka Yoh, Seiji Niho, Genichiro Ishii, Koichi Goto. _National Cancer Center Hospital East, Japan, chiba, Japan_.

Background: Among patients with advanced non-small-cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) mutation, T790M mutation is the most common mechanism of drug resistance to first- or second-generation EGFR tyrosine kinase inhibitors (TKIs). However, the proportion of patients with T790M mutation at re-biopsy is varied by studies or biopsy methods.

Methods: A total of 126 patients who were treated with first- or second-generation EGFR TKIs and had disease progression in our institution from January 2017 to September 2018 were eligible for this study. We retrospectively evaluated re-biopsy methods and the proportion of patients who harbor T790M mutation.

Result: A total of 126 patients (male, 36.5% ; median age, 69 year ) were included. Among 126 patients, 118 patients (93.7%) underwent re-biopsy by any methods: tissue biopsy or cytology sampling in 68 patients and plasma sampling in 50 patients during a first re-biopsy, and 44 patients (37.3%) had T790M mutation. During the first biopsy, in the 68 patients who underwent tissue biopsy or cytology sampling, 32 patients (47%) were positive for T790M mutation, nevertheless there were no tumor samples in 4 patients and only atypical cells in 3 patients and 1 patient was identified transformation to SCLC. In the 50 patients who performed plasma sampling during a first re-biopsy, T790M mutation was detected in only 12 patients (24%). Second re-biopsy was conducted in 19 patients among 74 patients resulted in T790M-negative or unknown after the first re-biopsy, and 10 patients were positive for T790M mutation. Eventually, a total of 80 patients were performed tissue biopsy or cytology sampling, 74 patients (96.1%) were found to have any EGFR mutations and T790M mutation was detected in 39 patients (48.1%). On the other hand, 59 patients underwent plasma sampling and 31 patients (52.5%) were found to have any EGFR mutations and T790M mutation was detected in only 17 patients (28.8%). Totally 54 patients (42.9%) were detected T790M mutation, and 52 patients (41.3%) received Osimertinib subsequently.

Conclusion: After progression of first- or second-generation EGFR-TKIs, about 95% of patients underwent re-biopsy, and T790M mutation was present in approximately half of patients who underwent re-biopsy and almost all patients who were positive for T790M mutation could receive Osimertinib. In patients who underwent tissue biopsy or cytology sampling, high frequency of detecting any EGFR mutations contributed to higher detection rate of T790M mutation compared to plasma sampling. To improve the sensitivity for T790M mutation detection, tissue biopsy or cytology sampling should be performed if possible.

#4921

TGF-beta signaling proteins and CYP24A1 may serve as surrogate markers for progesterone calcitriol treatment in ovarian and endometrial cancers of different histological types.

Ana Paucarmayta,1 Hannah Taitz,1 Yovanni Casablanca,1 Gustavo C. Rodriguez,2 G L. Maxwell,3 Kathleen M. Darcy,4 Viqar Syed1. 1 _Uniformed Services Univ. of the Health Sci., Bethesda, MD;_ 2 _Research North Shore University Health System, Evanston, IL;_ 3 _Inova Fairfax Hospital, Annandale, VA;_ 4 _Uniformed Services Univ. of the Health Sciences and Walter Reed National Military Medical Center, Bethesda, MD_.

Strategies are needed to coordinately block drivers and induce suppressors of cancer to reduce incidence and improve outcomes for individuals with inherited or acquired risk. We previously reported the chemopreventive and therapeutic efficacy of the combination of progestin and calcitriol in transformed and malignant endometrioid endometrial cancer and in ovarian cancer models involving attenuated expression of TGF-β signaling proteins and progestin-mediated inhibition of calcitriol-induced CYP24A1 expression. This study aims to expand the applications for this combination to other subtypes of endometrial and ovarian cancers including those with mutations in ARID1A or PIK3CA, DNA mismatch repair deficiency or BRCA1-null status. The end points for this in vitro investigation included assessments of cell growth and the expression of TGF-β ligands, receptors, SMAD proteins and CYP24A1. Treatment of ovarian clear cell carcinoma, endometrioid carcinoma, papillary serous adenocarcinoma, BRCA1 null, and DNA mismatch repair deficient endometrial cancer cell lines with progesterone alone or in combination with calcitriol inhibited cell growth and expression of TGF-β1, TGF-β2, TGF-Rβ1, TGF-βR2, pSMAD2/3 and CYP24A1. Expression of TGF-βR3, SMAD-4, PR and VDR was not altered in any cell line tested except, ES-2, where VDR expression was upregulated in response to treatment. These results suggest that progesterone alone and progesterone-calcitriol combination have broad application in both chemopreventive and therapeutic settings that merit further development in a wide variety of ovarian and endometrial cancers, including those derived from germline or somatic mechanisms. Moreover, our data suggest that TGF-β signaling proteins and CYP24A1 may be effective surrogate markers indicative of treatment response.

#4922

Utility of evidence-based gene expression panels in predicting outcome in stage matched colorectal cancer patients.

Chance Bloomer, Pankaj Ahluwalia, Chetan Pundkar, Saleh Heneidi, Kimya Jones, Ashis Mondal, Ravindra Kolhe. _Medical College of Georgia, Augusta, GA_.

Purpose: The aim of our study is to create an evidence-based gene expression analysis panel and use it to identify genes that can predict overall survival of stage matched colorectal cancer patients.

Procedure: We performed in silico analysis on 222 colorectal patients in TCGA using the nCounter PanCancer Human Progression Panel (genes n=742) on the cBioPortal platform. After extensive analysis of the individual genes and deep machine learning we selected 8 genes (NFKB1, ELK3, SEMA3E, ADRA2B, WNT5A, MMP14, PKM, and SDC4) associated with decreased survival (p<.05) and 7 genes (VSIG4, SORD, CCR4, VEGFC, SETD2, RUNX1T1, and EPAS1) associated with increased survival (p<.1) to include in our assay. Under an approved IRB protocol, we identified 750 colorectal cancer patients at the Georgia Cancer Center with a 5-year follow-up. The patients were divided into experimental groups based on tumor stage (AJCC stages 1-4), survival (+/-3 years), tumor grade (AJCC grades 1-3), and age(+/-76 years). RNA was isolated from FFPE blocks and analyzed by Nanostring's custom 23-gene multiplex assay (15 experimental and 8 control). Statistical analyses included ANOVAs, Kaplan-Meier survival analysis, and subgroup analysis using Student's t-test.

Results: The CCR4 gene, part of the increased survival panel, showed multiple significant associations with better prognosis. It had higher expression levels in patients with better survivals compared to those with worse survivals (p=.0119*). On survival analysis, higher expression was associated with increased survival (p=.004*). Subgroup analysis showed that patients with stage 4 cancer and better survivals had higher expression levels than those who had worse survivals and stage 4 (p=.0372*) or stage 1 (p=.0389*) tumors. The same trend was found when patients with stage 3 and better survivals were compared to patients with worse survivals and stage 4 (p=.0462*) or stage 1 (p=.0464*) tumors. Analysis of tumor grade plus survival and patient age plus survival gave more associations. Patients with grade 1 tumors and better survivals had higher expression levels than ones with low survival and either grade 1 (p=.0164*) or grade 2 tumors (p=.0026*). Young patients with better survivals had higher expression levels than those with worse survivals who were young (p=.0241*) or old (p=.0115*).

Discussion: These results show a significant correlation between high levels of CCR4 gene expression and longer survivals in patients with high stage tumors, a group rarely associated with such outcomes. The trend between the split survivals of stage 4 is interesting as a potential indicator of prognosis. Associations of age and grade could serve as prognostic indicators as well, especially the split survivals seen in grade 1 tumors and young patients. Future studies should include larger sample sizes as well as investigations into mechanistic explanations for these associations.

#4923

Analysis of companion diagnostic potentials for multifaceted PD-L1 assays.

Jeni Caldara, Joseph S. Krueger, Elliott Ergon, Staci Kearney, Bharathi Vennapusa. _Flagship Biosciences Inc, Westminster, CO_.

In vitro diagnostic (IVD) approvals of qualitative immunohistochemical (IHC) assays offer unique patient selection strategies by equipping pathologists with new tools to assess tumor status including tumor immune landscape. PD-L1/PD-1 checkpoint therapies require accurate diagnostic tools for optimal patient selections, however the interpretation of these tests may be very complicated upon manual assessment. As assessment criteria have increased in complexity, an accurate quantitative approach is needed to objectively and consistently interpret challenging paradigms. Recent IVD tests have multifaceted paradigms which assess both tumor and immune positivity. Although the Ventana PD-L1 SP263 and the Dako PD-L1 22C3 qualitative diagnostics are guiding immunotherapy decisions, interpretation of the results may be subjective and challenging in many cases. This creates a further level of complexity as tumor cell positivity, and immune cell presence and positivity must be assessed in combination.

In this study, we have used Flagship Biosciences' image analysis platform (cTA®) to explore the use of artificial intelligence (AI) and machine learning in the context of complex PD-L1 assays to provide accurate and precise quantification that allows for objective interpretation of the IVD. Non-small cell lung cancer (NSCLC) and urothelial carcinoma (UC) samples were stained with the SP263 and 22C3 PD-L1 assays and image analysis was used to provide an interpretation status based upon the IVD scoring paradigms. A comparison of the cTA results across both tests and tissue indications was also performed to explore how we may further support cross-platform PD-L1 solutions that provide adaptable and objective quantification.

The use of Flagship's cTA platform to identify per-cell biofeatures, using machine learning, may successfully quantify PD-L1 staining in various cell populations. The ability to independently assess these populations allows for a consistent and unbiased method for the assessment tumor status for PD-L1 immunotherapy treatment decisions.

#4924

High ALDH1 expression in colorectal carcinoma could predict early onset of the disease.

Litika Vermani,1 N Senthil Kumar,2 Anuradha Talukdar,1 Monoj Deka,1 Ravi Kannan,1 Rajeev Kumar1. 1 _Cachar Cancer Hospital & Research Centre, Silchar, India; _2 _Mizoram University, Aizawl, India_.

Background: Worldwide there is an increase in the trend of early onset of colorectal carcinoma in individuals who are less than 45 years of age. The composition of young age colorectal cases ranges from 35% to 40% in India. Currently, we do not have the access to any molecular marker that could predict early onset of the disease and its clinical course. Aldehyde dehydrogenase 1 (ALDH1), Twist, E Cadherin and Vimentin are markers for cancer stem cells and epithelial mesenchymal transition. However, there is paucity of data on their clinical significance in terms of their impact on early onset of colorectal carcinoma. This lack of data prompted us to investigate their role in the early onset of the colorectal cancer.

Methods: Immunohistochemisty of ALDH1, Twist, E Cadherin and Vimentin was performed in pre treatment biopsy samples collected from 103 colorectal patients who were further divided into 2 groups: viz, less than or equal to 45 years and more than 46 years.

Results: Over expression of ALDH1, Twist, E Cadherin and Vimentin was observed in 75.7%, 90.3%, 92.2% and 2.9% of pre treatment colorectal carcinoma tissue specimens respectively. ALDH1 expression was found to have strong correlation with early onset of the disease (patients who were less than or equal to 45 years of age (P=0.003). Women patients were found predominantly non smokers (P=0.001) and non tobacco chewers (P=0.004) with respect to men.

Conclusion: High ALDH1 expression correlates with the early onset of colorectal cancer. None of other markers could reach statistical significance with the early or late onset of colorectal cancer.

#4925

Development of a novel PTK7 immunohistochemistry (IHC) assay and analytical validation in epithelial malignancies of lung, breast and ovary.

Pamela M. Whalen, Amy Jackson-Fisher, Pamela Vizcarra, Cory Painter, Steven Pirie-Shepherd, Diane R. Fernandez, Joseph Lee, Eric L. Powell. _Pfizer Early Oncology Development & Clinical Research, La Jolla, CA_.

An immunohistochemistry (IHC) assay was developed to detect protein tyrosine kinase 7 (PTK7) membrane staining in formalin-fixed paraffin embedded (FFPE) human tumors with the intention of selecting non-small cell lung carcinoma (NSCLC) patients, breast cancer (BrCa) patients, or ovarian cancer (OVCA) patients for enrollment in an early stage clinical trial for a PTK7-ADC (antibody drug conjugate) experimental therapeutic. The IHC assay was developed using an anti-PTK7 specific antibody and a panel of five cancer cell lines with a range of constitutive PTK7 expression as characterized by gene transcript and protein based assays. PTK7 messenger RNA (mRNA) and protein levels were highly correlative in the control cell lines in vitro and the corresponding cell line xenografts. A pre-analytic variables study (PAV) using these characterized control cell line xenografts demonstrated that the PTK7 IHC assay was robust to the pre-analytic variables of under-fixation and ischemia. This IHC assay was used to profile PTK7 levels in human lung cancers, breast cancers, and ovarian cancers, and a dynamic range of PTK7 H-scores was detected (see Table 1). The resulting PTK7 H-scores correlated with orthogonal methods for measuring PTK7 protein and gene transcript levels in the human tumors.

Table 1. PTK7 H-scores

---

|

Number of Samples | Minimum H-score | Maximum H-score | Mean H-score | Median H-score

NSCLC | 30 | 34 | 230 | 117.4 | 117

OvCa | 24 | 24 | 240 | 151 | 151

#4926

Loss of deiodinase type 3 expression distinguishes patients with poor prognosis in breast cancer.

Iuri M. Goemann, Vicente R. Marczyk, Mariana Recamonde-Mendoza, Marcia S. Graudenz, Simone M. Wajner, Ana Luiza Maia. _Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil_.

Background: Breast cancer is a highly heterogeneous disease and the identification of biomarkers that predict tumor biological behavior is warranted in improving patient survival. Thyroid hormones (THs) are critical regulators of cellular processes, and TH status alterations are known to contribute to cancer progression through all the hallmarks of cancer. Clinical studies associate THs levels with breast cancer mortality, and THs have been shown to influence breast cancer proliferation, apoptosis, and migration in in vitro models. Type 3 deiodinase (DIO3) is the main enzyme responsible for TH inactivation and disturbed DIO3 expression has been demonstrated in several human neoplasias. Here, we aimed to evaluate expression patterns and the prognostic significance of DIO3 in breast cancer.

Methods: The expression of DIO3 was assessed by immunohistochemistry in a primary cohort of 53 samples of breast tissue and was validated in a second cohort using the RNA-Seq data from TCGA database (BRCA study). We assessed DIO3 expression in both populations according to retrieved clinicopathological information. For methylation analysis, DNA methylation data from the TCGA-BRCA study were obtained. Regulation of DIO3 gene was investigated in MCF-7 and MDA-MB-231 cell lines.

Results: DIO3 expression was present in normal and tumoral breast glandular tissue. DIO3 mRNA expression was found to be reduced in tumor samples when compared to normal tissue (p<0.0001). Unexpectedly, loss of DIO3 expression was associated with increased mortality (HR 4.29; 95% CI 1.24-14.7) in the primary cohort. We then validated this finding in the secondary cohort. Patients with low DIO3 expression exhibited reduced overall survival (OS) when compared to those higher levels of DIO3 expression. Five-year OS rates were 90.4% and 77.4% in the high and low expression groups, respectively (p=0.0002). Among patients with metastasis, five-year OS rate was 70% in the high DIO3 group and 0% in the low DIO3 group (p=0.038). Additional experiments were performed to determine DIO3 regulation in breast cancer. In MCF-7 and MDA-MD-231 cells, DIO3 was subject to T3 stimulation and was under the regulation of MAPK pathway, as MAPK inhibition resulted in a 50% reduction of DIO3 expression (p=0.004). Methylation analysis revealed that DIO3 gene promoter is hypermethylated in tumoral cells when compared to normal tissue (p <0.0001).

Conclusions: DIO3 is expressed in normal and tumoral breast tissue. Here, we demonstrate a role for DIO3 as a prognostic marker in breast cancer, as loss of DIO3 expression distinguishes breast cancer patients with poor overall survival. Moreover, our fındings suggest the utility of DIO3 expression for stratifıcation of patients with metastasis. The finding that DIO3 gene is regulated by the MAPK pathway and that gene promoter is hypermethylated in breast cancer might have therapeutic implications.

#4927

Silencing of the RAD50 gene contributes to enhancing the sensitivity of the triple-negative breast cancer cells to carboplatin.

Kristina V. Havrysh,1 Mikhail V. Bogdanov,2 Ramziya G. Kiyamova1. 1 _Kazan Federal University, Kazan, Russian Federation;_ 2 _University of Texas-Houston Medical School, Houston, TX_.

Triple-negative (TN) breast cancer (BC) is the most aggressive molecular subtype of BC that affected over 250 thousand females worldwide just in 2018. Patients with TNBC have exhibited a higher probability of cancer relapses and poor prognosis of disease outcome which are due to the absence of established and reliable targets for therapy of this subtype of tumors. Only 10% of TN breast tumors have BRCA1/2 mutations associated with high sensitivity of cancer cells to the chemotherapy with platinum salts drugs (e.g., cisplatin, carboplatin (Cb), etc.) and PARP-inhibitors. Recent studies indicated that administration of Cb in neoadjuvant chemotherapy of TNBC increase pCR (pathologic complete response) of patients received Cb in comparison with a control group. Also, Phase II and III of clinical trials have shown that TNBC patients respond differently to adjuvant chemotherapy with Cb. Therefore further studies are required to define subgroups of TNBC patients, which might benefit from carboplatin therapy.

In our previous studies, the RAD50 gene involved in radiation-induced DNA double-strand break repair was identified as a tumor-associated antigen and potential BC marker. We have shown that low expression of RAD50 gene is associated with better TNBC prognosis.

The current study was aimed to evaluate RAD50 as a predictive marker of TNBC cells sensitivity to Cb. The RAD50 gene was silenced in triple negative breast cancer cell lines with wild-type BRCA genes namely MDA-MB-231, MDA-MB-436, and MDA-MB-453 by RNA interference. Then the changes of the cytotoxic sensitivity of these cells to Cb treatment in comparison to the control cells with original RAD50 expression were investigated by using a cell viability assay. Obtained data were analyzed by the Wilcoxon signed-rank test. The statistical analysis was performed using GraphPad Prism 7 software.

Results of this study demonstrated that the knockdown of RAD50 gene expression in human TNBC cells could significantly increase their susceptibility to treatment with Cb (MDA-MB-231: IC50 - 20,65uM (silenced RAD50) and 24,93uM (control), p-value = 0,0098; MDA-MB-436: IC50 - 7,497uM (silenced RAD50) and 10,39uM (control), p-value = 0,0137; MDA-MB-453: IC50 - 7,336uM (silenced RAD50) and 8,711uM (control), p-value = 0,002).

These results strongly indicate that silencing of the RAD50 expression contributes to the sensitization of TNBC cells to treatment with platinum-based drug Cb. Therefore RAD50 could be considered as important TNBC predictive biomarker. Further validation of RAD50 by using the tumors of TNBC patients and animal xenograft models could prove to be essential to use this marker for improving the therapy regimens of TNBC tumors without BRCA1/2 mutations.

This work is performed according to the Russian Government Program of Competitive Growth of Kazan Federal University.

#4928

Relationship between protein biomarkers of potential chemotherapy response and microsatellite status, tumor mutational burden, and PD-L1 expression in patients with cancer.

Mina Nikanjam,1 David Arguello,2 Zoran Gatalica,2 Donald A. Barkauskas,3 Razelle Kurzrock1. 1 _University of California at San Diego, San Diego, CA;_ 2 _Caris Life Sciences, Phoenix, AZ;_ 3 _University of Southern California, Los Angeles, CA_.

Introduction: Chemotherapy and checkpoint inhibitor immunotherapies are increasingly used in combinations. We determined associations between the presence of anti-PD-1/PD-L1 therapeutic biomarkers and protein markers of potential chemotherapy response.

Methods: Data was extracted from a clinical-grade testing database (Caris Life Sciences; Feb 2015 - Nov2017): immunotherapy response markers (microsatellite instability high (MSI-H) and tumor mutation burden-high (TMB-H) (by next generation sequencing (NGS)) and PD-L1 protein expression (immunohistochemistry); and protein chemotherapy response markers (ERCC1, TOPO1, TOP2A, TS, TUBB3, RRM1, and MGMT; immunohistochemistry). Relationships were determined by the Mantel-Haenszel chi-squared test or Fischer's exact tests.

Results: Overall, 28,034 patients (40 tumor types) were assessed. MSI-H was found in 3.3% of patients, TMB-H, 8.4%, and PD-L1 expression in 11.0% of patients. Based on concurrent biomarker expression, combinations of immunotherapy with platinum (ERCC1 negativity) or with doxorubicin, epirubicin, or etoposide (TOP2A positivity), have a higher probability of response while combinations with irinotecan or topotecan (TOPO1 positivity), with gemcitabine (RRM1 negativity), and fluorouracil, pemetrexed, or capecitabine (TS negativity) may be of less benefit. The potential for immunotherapy and taxane (TUBB3 negativity) combinations is present for MSI-H but not TMB-H or PD-L1-expressing tumors; for temozolomide and dacarbazine (MGMT negative), PD-L1 is frequently co-expressed, but MSI-H and TMB-H are not associated.

Conclusion: Protein markers of potential chemotherapy response along with NGS for immunotherapy response markers can help support rational combinations as part of a precision oncology approach.

#4929

Leveraging multi-omics tumor boards for precision medicine in the OHSU SMMART Treatments Program.

Jamie M. Keck, Swapnil Parmar, Ben Kong, Zahi Mitri, Christopher Corless, Annette Kolodzie, Allison Creason, Jeremy Goecks, Patrick Leyshock, Kiara Siex, Brett E. Johnson, Janice Patterson, Laura Heiser, Anastasiya Olson, Matt Viehdorfer, Georgia Mayfield, Jennifer Laverdure, Joe W. Gray, Gordon B. Mills, Raymond C. Bergan. _OHSU Knight Cancer Inst., PORTLAND, OR_.

Comprehensive characterization of an individual's cancer using multi-omic analyses and an expanding list of targeted therapies is providing an opportunity to uncover therapeutic vulnerabilities and rationally target multiple driving alterations in the tumor. To leverage this opportunity, a multi-disciplinary team is required to unravel the complexity of tumor behavior and genomic variant information, integrate data from multi-omic assays, and exploit potential synergies from combination therapy while avoiding toxicity, all of which occurs within the context of the patient's clinical history and comorbidities. We report the preliminary experience of OHSU Knight Cancer Institute's SMMART (Serial Measurements of Molecular and Architectural Responses to Therapy) Treatments Program Multi-omics Tumor Board (MTB). The role of the SMMART MTB, to date, has been to develop and optimize procedures in a feasibility protocol for future application in the clinical setting where we will provide personalized combinatorial treatment suggestions. Here we report our preliminary experience over the last 15 months with multi-disciplinary tumor boards for metastatic breast cancer. A deep analysis of data, in many cases from serial biopsies, enabled identification of personalized, multi-targeted therapy. Clinical and laboratory data are explored individually and collectively by oncologists, oncology pharmacists, molecular pathologists, cancer biologists, and computational biologists. Data from CLIA-certified analytics include, but are not limited to, a 124 gene targeted panel, whole exome sequencing, MSI, gene-fusion, RNAseq, and clinical IHC assays that provide status information about receptors (ER/PR/AR/HER2), proliferation rate, and tumor immunogenicity along with reflexive assays for expression of relevant proteins such as p16, RB, and PTEN. A customized LabKey® system allows for visual display of a patient's clinical timeline with information about diagnoses, treatments, biopsies events, radiographic changes, and blood biomarkers; thereby providing MTB participants with a holistic view of the patient's case summary. Our experience with several example breast cancer case scenarios identified therapeutic vulnerabilities that would not have been considered by a standard clinical tumor board with genomic data alone. Recent cases have included observations such as HER2 status switching, a rare pathogenic germline mutation, high PD-L1 expression, and gained expression of the androgen receptor. These cases further emphasize the value and importance of MTB discussions to oncologists for interpreting and analyzing complex multi-omic data and uncovering therapeutic options for patients. This experience will be utilized in the future for employing SMMART MTBs in a clinical setting as a platform to triage patients into different multi-targeted combinatorial treatment options.

#4930

PAI1, a β catenin transcriptional target, serves as a surrogate biomarker to predict EGFR TKI mediated drug persistence in EGFR mutant NSCLC.

Rajeswara Rao Arasada, Walter Wang, Shankar Suman, David Paul Carbone. _The Ohio State University, Columbus, OH_.

Lung cancer management has been transformed by the discovery and effective targeting of driver mutations such as epidermal growth factor receptor (EGFR). Tyrosine Kinase Inhibitors (TKIs) that block EGFR kinase activity such as erlotinib and osimertinib cause dramatic tumor shrinkage and prolonged responses in patients whose tumors have activating EGFR mutations. However, resistance universally develops. Drug persister cells with properties of cancer stem cell-like cells play a major role in drug resistance. In tumors without pre-existing resistant clones, some tumor clonogens survive, despite expressing the driver mutation, to acquire target reactivation or bypass resistance mechanisms. Identification of biomarkers that are mechanistically linked to EGFR TKI resistant cell populations would have clinical utility in early determination of disease recurrence in EGFR mutant NSCLC patients.

Previously, we have reported that EGFR-TKI treatment activates Notch3, and here we have identified that Notch3 activates β-catenin transcriptional target, PAI1, which could play a role in drug persistence and could be used for early identification of disease recurrence. Our in vitro ELISA studies have identified an increase in secreted PAI1 levels after treatment with the EGFR TKIs erlotinib and osimertinib demonstrate that measuring PAI1 levels could serve as an indicator of EGFR TKI persistence. To further validate PAI1 as a surrogate biomarker for EGFR TKI treatment, we performed a retrospective study in NSCLC patients (n = 42) with EGFR mutant tumors. Using ELISA to detect PAI1 protein in pre- and post-treatment serum samples as a mechanism-based indicator for β-catenin activation, we observed a broad range in pre-treatment PAI1 levels, spanning from 0.8 ng/ml to 134 ng/ml, suggesting a variable baseline β-catenin activation. Doing survival analysis after dichotomizing patients into low and high baseline PAI1 level groups, we found patients with low basal PAI1 levels had a significantly shorter PFS with EGFR TKI treatment compared to patients with high basal levels. Comparing patients with a ≥ 2-fold induction to those with less than 2-fold, there was a similar trend for worsened survival in those who showed significant induction of PAI1.

Overall, we demonstrate that PAI1 expression is a candidate marker for β-catenin activation and the development of drug persisters; thus, it is a potential surrogate marker for poor outcome in patients receiving EGFR TKI therapy and a potential biomarker for selecting patients for β-catenin-targeted therapy.

#4931

Not all tumors are created equal: Evaluating the impact of comprehensive genomic profiling (CGP) on clinical outcomes in esophageal/gastroesophageal cancer (GEJ CA).

Vidya Kollu, Arun Singhavi, Paul S. Ritch, James P. Thomas, Aniko Szabo, Ben George. _FROEDTERT HOSPITAL/MEDICAL COLLEGE OF WISCONSIN, Wauwatosa, WI_.

Introduction:

Systemic chemotherapy plays a pivotal role in the treatment of localized and metastatic GEJ CA patients (pts), however, there are no reliable predictive biomarkers. CGP is increasingly used to guide selection of targeted therapies, but its role in personalizing cytotoxic chemotherapy is not clear. We investigated the correlation between somatic alterations (SAs) and clinical outcome in pts with GEJ CA treated with cytotoxic chemotherapy.

Methods:

Medical records of all patients with GEJ CA who had CGP data available were retrospectively reviewed under an IRB approved protocol. DNA was extracted from formalin fixed paraffin embedded clinical specimens and CGP was performed on hybrid-capture, adaptor ligation-based libraries to a mean coverage depth of >600 unique reads utilizing the Foundation Medicine platform (315 gene panel). The effect of the SAs and clinical covariates on response and survival outcomes were modeled using logistic and Cox regression analyses, respectively. Lasso was used for model selection, with the penalty parameter chosen as providing the lowest error-rate via 5-fold cross-validation.

Results:

Fifty-six patients were identified; median age was 62.5, 51(91%) were male, 39 (70%) had metastatic disease, histology was adenocarcinoma in 41 (73%) and ERBB2 amplification was detected in 11 pts (22%). Median tumor mutational burden (TMB) was 6.1 (0.9-75.7) and one patient had a microsatellite unstable tumor. 46 (82%) pts received a platinum-based combination as first line therapy, mfolfox6 was the most commonly utilized regimen (30%). Complete Response (CR), Partial Response (PR), Stable Disease (SD), and Progressive Disease (PD) were noted in 3 (5.6%), 34 (63.0%), 5 (9.3%) and 12 (22.2%) pts, respectively. Median progression free survival (PFS) and OS was 31.3 and 72.8 months, respectively for patients with localized GEJ CA, while median PFS and OS were 6.3 and 18.7 months, respectively for patients with metastatic GEJ CA. On multivariate analysis, in addition to stage, SA in SPTA1 correlated with improved response (p=0.0026, OR=0.052, 95% CI 0.002-0.378), while SAs in PIK3CG (p = 0.016; HR=-6.7, 95% CI 1.26-30.46) and EGFR (p = 0.041; HR=3.6; 95% CI 0.9-11.8) correlated with worse overall survival (OS).

Conclusion:

We were able to identify SAs that correlated with objective response and survival, however, confirmatory analyses in larger datasets and prospective validation is necessary. The negative prognostic effect of SAs in PIK3CG and EGFR merit further exploration, considering their viability as therapeutic targets. Robust, quantitative and systematic somatic analysis of pre-treatment biopsies, pathway-network analysis and correlation with clinical outcome are essential to gaining mechanistic insights and maximizing therapeutic impact.

#4932

Excision repair cross-complementing group-1 (ERCC1) induction and polymorphism are markers of inferior outcome in patients with colorectal cancer treated with oxaliplatin.

Devika Rao,1 Atrayee Basu Mallick,2 Titto Augustine,1 Cecilia Daroqui,3 Jeeshan Jiffry,1 Amartej Merla,1 Imran Chaudhary,1 Raviraja Seetharaman,4 Arjun Sood,5 Srikanth Gajavelli,6 Santiago Aparo,7 Lakshmi Rajdev,1 Andreas Kaubisch,1 Jennifer Chuy,1 Radhashree Maitra,1 Sanjay Goel1. 1 _Montefiore Einstein Cancer Center, Bronx, NY;_ 2 _Thomas Jefferson University, Philadelphia, PA;_ 3 _IIByT (CONICET-UNC), Cordoba, Argentina;_ 4 _Stempeutics Research Pvt. Ltd., Manipal, India;_ 5 _Northern Light A R Gould Hospital Cancer Center, Presque Isle, ME;_ 6 _Bristol Myers Squibb Inc., Lawrenceville, NJ;_ 7 _Baptist Health Medical Group South Florida, Miami, FL_.

Background: Oxaliplatin confers tumoricidal effect by forming platinum-DNA adducts and is an integral part of the treatment paradigm for patients with colorectal cancer (CRC). The nucleotide excision repair pathway (NERP) plays a crucial role in tissue resistance to the drug. We have previously demonstrated that ERCC1 (part of NERP) is an inducible gene in CRC cell lines on exposure to oxaliplatin, and high levels confer drug resistance. We aimed to translate these findings by using Peripheral Blood Mononuclear Cells (PBMC) as a surrogate and assess ERCC1 as a biomarker of sensitivity to oxaliplatin therapy. Additionally, we assessed prevalence of single nucleotide polymorphisms (SNP) and its relation to patient outcomes.

Methods: In consenting patients with CRC who received oxaliplatin, blood was sampled at 0, 2, 48 hours, and 14 days during any cycle of chemotherapy, and/or DNA from formalin fixed paraffin embedded (FFPE) tissue was isolated. ERCC1 gene expression was quantified by qPCR (quantitative real time polymerase chain reaction) and WB (western blotting). SNPs were analyzed through Sanger sequencing of DNA from blood or tissue. Clinical benefit from oxaliplatin was determined using the parameters of relapse free survival (RFS) for limited stage and progression free survival (PFS) for mCRC.

Results: 54 patients had serial blood sampled; 25 (46.3%) had mCRC and 1 was excluded due to mortality prior to therapy. 13 (52%) had an increase in ERCC1 expression from baseline, while 11 (48%) showed no change or decrease. Median PFS was 190 and 237 days respectively (log-rank test HR 2.35, CI 1.005-5.479; p = 0.0182). In the 29 patients with limited stage disease, 19 (65.5%) had an induction of ERCC. In these patients change in expression did not correlate with RFS. We did not find any significant correlation of PFS with baseline expression of ERCC1 in either group. SNP in rs11615 (AAT>AAC, 97 samples) was assessed. 80/97 had stage 4 disease, with AAC in 69 (86.3%) patients. Median PFS was lower in patients with AAC than AAT (203 vs 684.5 days; log-rank test HR 2.4, CI 1.45-3.97, p=0.0045). On analysis of racial distribution, blacks had the highest prevalence of AAC (100%), followed by Hispanics (81.8%), persons of mixed race (76.9%) and whites (68.8%) (Fisher exact test p=0.0004).

Conclusions: We confirm our hypothesis that the ERCC1 gene is induced in vivo in a sub-population of patients on treatment with oxaliplatin. This induction can serve as a potential marker of drug-resistance in mCRC as evidenced by the significant difference in PFS. Additionally, presence of SNP is an independent marker of drug resistance, which has a significant pattern of distribution among various races. Analysis of a second SNP (rs3212986), also implicated in drug resistance, is underway.

#4933

RAPTOR protein overexpression is predictive of poor clinical outcomes in head and neck squamous cell carcinoma (HNSCC) patients.

Hui Yan Poon,1 Yuchen Liu,1 Yu Xiong SU,2 Jason Y.k. Chan,1 Chin Wang Lau,3 Vivian W y Lui1. 1 _Chinese Univ. of Hong Kong, Hong Kong, Hong Kong;_ 2 _University of Hong Kong, Hong Kong, Hong Kong;_ 3 _Yan Chai Hospital, Hong Kong, Hong Kong_.

Head and neck squamous cell carcinoma (HNSCC) has a high mortality rate, and with rising incidences in Asia. Previously, we demonstrated that the PI3K pathway is the most frequently altered pathway in HNSCC (Lui et al, Cancer Discovery, 2013). However, cumulative PI3K pathway aberrations, nor PIK3CA aberrations, are not associated with clinical outcomes in HNSCC patients (P=n.s.). In this study, we investigate the prognostic significance of RAPTOR (Regulatory associated protein of mTOR), a key component of the PI3K signaling pathway, in HNSCC. To examine the clinical relevance of RAPTOR levels in human cancers, including HNSCC, we performed pan-cancer analyses of RAPTOR protein levels in the Cancer Proteome Atlas datasets (TCPA, USA). Strikingly, across 28 tumor types examined, we found that RAPTOR protein overexpression is most significantly associated with poor patient survival in HNSCC (P=0.00162***), followed by renal clear cell carcinoma (P=0.00198), and stomach cancer (P=0.0443; Log-Rank test; Table 1). We then extracted the original quantification data of RAPTOR protein array of HNC (TCPA, USA), and performed RAPTOR expression-survival correlation analyses on quantitative basis by Kaplan-Meier analysis with Log-Rank test. We found that HNSCC patients with RAPTOR protein overexpression in their primary tumors (mean+5SEM) have significantly poorer overall survival (median OS=27.56 months) than patients with RAPTOR protein underexpression (mean-5SEM; median OS=68.43 months). Moreover, this group of RAPTOR-high patients are found to have a higher risk of recurrences, with an Odd Ratio of 1.75 (P=0.05; Fisher's Exact test). Notably, this poor OS of RAPTOR-high HNSCC is much shorter than that of TP53-mutated HNSCC in the same cohort (45.8 months; TCGA Provisional,N=510). Further, patients with extreme-high levels of RAPTOR protein expression (top 5% cases) have an even worst OS of only 11.56 months vs 48.16 months (the remaining 95% cases). Using our small cohort of Asian HNSCC tumors as a validation cohort, we show that RAPTOR protein overexpression is very common in Asian HNSCC (37.5%; 6/16 cases), suggestive of its likely contribution to HNSCC in Asia as well. In conclusion, RAPTOR-high status is likely to be a useful predictive of very poor HNSCC patient survival, possibly superior than the previously known survival-related biomarker,TP53 mutation. RAPTOR is one of the first components of the PI3K pathway shown to be associated with patient outcome in HNSCC. Acknowledgements:VWYL receives fundings from Health and Medical Research Fund(#15160691), Research Grant Council, Hong Kong(GRF#1711484,#17121616,# 14168571;T12-401/13-R), UICP(UIM/329;Innovation and Technology Fund); Hong Kong Cancer Fund. YCL is supported by Postdoctoral Hub(UIM/329) from Innovation and Technology Fund, Hong Kong government.J YKC supported by Dr Stanley Ho Medical Foundation.

#4934

Modified SWATH MS analysis of serine hydrolase activity in lung adenocarcinoma.

Tatjana Sajic,1 Stephan Arni,2 Walter Weder,2 Rudolf Aebersold,1 Sven Hillinger2. 1 _ETH Zürich, Zürich, Switzerland;_ 2 _Univ. Hospital Zürich, Zürich, Switzerland_.

Lung cancer, especially adenocarcinoma as the most common subtype, is still the most deadly human cancers. Therefore biomarker discovery for implementation of individualised therapies remains of utmost importance. Serine hydrolases (SHs), one of the largest enzyme families, have previously been shown to be implicated in the development of lung cancers. Activity-based protein profiling (ABPP) is a proteomic method that uses active site-directed chemical probes to selectively target the active form of the subsets of the enzymes in question, and then by a combination of a streptavidin-biotin enrichment step and mass spectrometry quantifies the catalytically active amount of the enzyme molecule. In this project, we monitored both forms of serine hydrolase, the catalytically active and inactive, in a patient cohort of lung adenocarcinoma biopsies. For this purpose, we combined the activity-based proteomics for serine hydrolase and SWATH mass spectrometry (MS), which ensures highly reproducible protein quantification in a large panel of clinical samples. Twenty four lung biopsies of long- and short surviving patients with stage IIIA adenocarcinomas and their normal tissue counterparts were available as OCT-embedded tissue. Although OCT represents a non-reactive compound used to preserve tissue morphology in long-term sample storage, this substance is incompatible with mass spectrometry measurements. We optimized and developed an OCT clean-up protocol compatible with enzyme-substrate binding of activity-based chemical probes and the targeted portion of the active enzyme. To prevent digestion on streptavidin beads and MS spectra contamination, we used the reproducible and accurate SWATH MS to indirectly measure the active enzyme form in the biopsy-extract solution which had earlier been depleted for all active enzyme molecules by precipitation with chemical probes attached to the beads. We identified over 4000 lung tissue proteins from a few milligrams of OCT-embedded biopsies, and confirmed good data quality for further SH enzyme quantification. In addition to the analysis of total proteome, 278 distinct proteins were identified on the parallel streptavidin bead samples, with chemical probes being used to deplete the active enzyme from the biopsy-extract. Each SWATH experiment reported the percentage of active enzyme form through the indirectly measured ratio between the inactive SHs (sample depleted for active SHs) and the total SHs (non-depleted sample). We detected around 80 enzymes with the active form comparably measured in all three independent experiments, which amount generally accounted for between 5-55% of the total enzyme concentration. We conclude that with the modified protocol compatible with OCT-embedded tissue biopsies the combination of ABPP and SWATH-MS enables highly reproducible protein quantification in biomarker discovery.

#4935

Identification of GPR115 as a key G-protein coupled receptor as a key prognostic and therapeutic target in pancreatic ductal adenocarcinoma.

Satoshi Nishiwada,1 Tadanobu Shimura,1 Kensuke Yamamura,1 Fuminori Sonohara,2 Takahiro Akahori,3 Kota Nakamura,3 Naoya Ikeda,3 Yasuhiro Kodera,2 Masayuki Sho,3 Ajay Goel1. 1 _Baylor Scott &White Research Institute, Dallas, TX; _2 _Nagoya University Graduate School of Medicine, Nagoya, Japan;_ 3 _Nara Medical University, Kashihara, Japan_.

Purpose: Pancreatic ductal adenocarcinoma (PDAC) remains the fourth leading cause of cancer-related deaths in the United States, and the incidence of this disease continues to rise. Although recent advances in treatment modalities for this malignancy has somewhat improved patient outcomes in the recent years, the overall prognosis of patients with PDAC remains dismal. From a biological viewpoint, while the G-protein-coupled receptor (GPCRs) proteins play a multitude of important functions in tumor pathogenesis, their clinical significance if any in human cancers, especially PDAC, remains unclear. Herein we aimed to perform a comprehensive profiling of all GPCRs, in an attempt to identify the most critical protein(s) that have important biological and clinical significance in PDAC.

Methods: This study included systematic analysis of 796 PDAC patients, which included data analysis from 331 patients from public datasets (TCGA, ICGC and GSE57495) and 465 patients from two, independent, clinical patient cohorts. Computational and bioinformatic analysis of genomewide expression profiling datasets led to the selection of candidate GPCR genes during the discovery and validation steps. The clinical significance of candidate genes were subsequently investigated in two in-house patient cohorts (training cohort: n = 321, validation cohort: n = 144) using qRT-PCR assays. The data were analyzed by performing appropriate statistical analyses to determine patient prognosis. Finally, using a series of functional studies, we confirmed the biological function of the candidate GPCRs in PDAC.

Results: Analysis of all 33 GPCRs, led to the identification of GPR115, as the only significantly up-regulated candidate, with a prognostic significance in all three public datasets (P=0.02, 0.04 and 0.05, respectively). The patients with high-GPR115 expression exhibited significantly poorer postoperative prognosis for OS and RFS, in two, large, independent clinical cohorts (training cohort: P<0.01, P<0.01, validation cohort: P=0.01 and 0.02, respectively). More importantly, multivariate analysis indicated that high GPR115 expression was an independent prognostic factor in both cohorts (HR=1.45; P=0.01, HR=2.25; P<0.01). Using Cox regression, we established a risk-prediction model by incorporating GPR115 expression together with various clinicopathological factors, which was highly accurate in predicting 5-year long-term survival following surgery in PDAC patients. In addition, siRNA-mediated gene silencing of GPR115 significantly inhibited the cell proliferation and migration in human PDAC cells.

Conclusion: We for the first time have identified GPR115 as a key GPCR with important prognostic significance, as well as functional role in tumor progression; providing a rationale that this may be a potential target for therapeutic targeting in patients with PDAC.

#4936

**Development of** NTRK **reference materials for global assay standardization.**

Sebastian Bender,1 Patricia Carrigan,1 Kara O'Brien,2 Catherine Huang,3 Rajeswari Vemula,3 Praveena Kamineni,3 Omo Clement,4 Russell Garlick,4 Bharathi Anekella3. 1 _Bayer AG, Berlin, Germany;_ 2 _Bayer US, LLC, Whippany, NJ;_ 3 _Seracare Life Sciences, Gaithersburg, MD;_ 4 _Seracare Life Sciences, Milford, MA_.

Tropomyosin receptor kinases (TRK) are a family of transmembrane proteins (TRKA, B, and C) encoded by NTRK1, NTRK2 and NTRK3 genes. Fusions involving NTRK genes result in uncontrolled TRK signaling and oncogenesis. Cancers driven by TRK fusions are rare but occur in a broad range of tumor types in both pediatric and adult patients. Accurate testing for NTRK fusions has become critically important because of precision biopharmaceutical treatments, such as Larotrectinib. This drug, which was granted Breakthrough Therapy Designation by the FDA and is currently undergoing review, is a promising treatment option for patients with locally advanced or metastatic solid tumors harboring NTRK fusions.

NGS based assays have the potential to accurately detect NTRK fusions; however, the majority of tests currently available are laboratory-developed tests, which vary substantially in their workflow, chemistry, sensitivity, and bioinformatics analyses. In order to achieve precision diagnostics for personalized therapy, testing standardization is needed worldwide. Towards this end, we developed Seraseq® FFPE NTRK Fusion RNA Reference Material for standardization of NTRK fusion testing by targeted NGS panels.

The reference material contains 15 clinically-relevant NTRK fusions, selected based on prevalence data, in a single reference sample. The five fusion RNAs each for NTRK1, NTRK2, and NTRK3 are all incorporated in the well characterized GM24385 human reference cell line. The reference material challenges detection across multiple break points for each NTRK gene, as well as 12 different amino terminal fusion partners. Each fusion is quantified by digital PCR with concentrations ranging from 150 - 600 fusion copies per nanogram of total extractable RNA.

The reference material was prepared in a formalin fixed paraffin embedded (FFPE) format, which allows evaluation of the full technical workflow. In our laboratory, one 10-micron FFPE curl yields more than 400 ng of extractable RNA when using the Agencourt Formapure® extraction kit. NGS analysis on the RNA-based ArcherDx FusionPlex Solid Tumor assay correctly identified all 15 fusions, demonstrating compatibility with a leading commercial assay.

Highly characterized, patient-like reference materials help standardize testing across clinical trial sites and can speed test adoption, training and validation at clinical labs. The Seraseq FFPE NTRK Fusion RNA Reference Material is designed to aid standardization of NTRK fusion testing to identify patients that will benefit from new precision therapeutics.

#4937

The paradoxical roles of ZEB1 in thyroid cancers: A putative diagnostic and therapeutic target.

Minjun Kim,1 Zhen Xu,1 Seongyoon Ha,1 Sang Gab Yoon,2 Su-jin Kim,3 Kyu Eun Lee3. 1 _Seoul National University, Seoul, Republic of Korea;_ 2 _Seoul National University Hospital, Seoul, Republic of Korea;_ 3 _Seoul National University College of Medicine and Hospital, Seoul, Republic of Korea_.

Objective ZEB1, which is a well-known EMT-inducing transcription factor, has been reported as one of the metastatic inducer in many types of cancers. However, the clinical importance of ZEB1 in thyroid cancers has not been thoroughly investigated. To elucidate the role of ZEB1 in thyroid cancers, we have analyzed the association of ZEB1 with clinicopathological and prognostic factors in thyroid cancers using public databases such as TCGA and GEO, and tested the functional roles of ZEB1 in vitro using thyroid cell lines. Methods We have analyzed 499 well-differentiated thyroid cancer patients (PTC) from TCGA and 37 of undifferentiated thyroid cancer patients (PDTC and ATC) from GEO76039. To validate the results, another dataset GEO33630, which include both 48 of PTC and 11 of ATC, was used. For in vitro analysis, we have used B-CPAP and Nthy/BRAFV600E, which is Nthy-ori 3-1 cell line stably expressing BRAFV600E gene by lentiviral transduction and forms anaplastic thyroid cancer in a xenograft model. To show the roles of ZEB1 in metastatic potentials of the cell lines, we have carried out proliferation, migration, invasion, soft agar colony forming assays after knock-down of ZEB1 using siRNA. Results In well-differentiated thyroid cancers from TCGA, ZEB1 was significantly downregulated in patients with aggressive phenotypes such as a tumor (over normal thyroid), advanced subtype, lymphovascular invasion, BRAFV600E, N1a stage, distant metastasis excluding multiple focality, N1b stage, and survival rate. However, in undifferentiated thyroid cancers, ZEB1 was significantly upregulated in patients with advanced subtype, more mutational burden, TP53 mutation, and N1a/N1b stage. Supporting these results, ZEB1 expression was lower in PTC than in normal thyroid, but higher in ATC than in normal thyroid when analyzed and compared in the same dataset (GEO33630). In in vitro analysis, knock-down of ZEB1 reduced proliferation, migration, invasion, and anchorage-independent growth of Nthy/V600E. However, even though proliferation was inhibited, migration and invasion was promoted by knock-down of ZEB1 in BCPAP. Conclusion Our data show that ZEB1 is strongly associated with clinical outcomes of thyroid cancers, but it depends on differentiated status of thyroid cancers. Downregulation of ZEB1 might be used as a diagnostic factor for aggressive phenotypes of PTC patients. However, ZEB1 increased in undifferentiated subtypes and plays an important role in metastatic potentials of anaplastic thyroid cancer. These results suggest that ZEB1 might be used as a predictive factor for a prognosis of PTC and a potential therapeutic target for undifferentiated thyroid cancers.

#4938

Identification of a subtype of hepatocellular carcinoma with poor prognosis based on expression of genes within the glucose metabolic pathway.

Xiaoli Zhang, Kalpana Ghoshal, Lang Li. _The Ohio State University, Columbus, OH_.

Hepatocellular carcinoma (HCC) is the most prevalent primary cancer and a highly aggressive liver malignancy. Liver cancer cells reprogram their metabolic pathways to meet their needs for rapid proliferation and generation of the biomass for tumor growth. The accelerated aerobic glycolysis in tumor has been considered one of the hallmarks that distinguish cancer cells from normal cells. In the present study, liver hepatocellular cancer dataset of the Cancer Genome Atlas (LIHC TCGA) and datasets from Gene Expression Omnibus (GEO) were used to investigate the alterations in expressions of the genes involved in the glucose metabolic pathways as well as their association with the patient clinical stage and survival in liver cancer. In this study, 97 genes including glucose and lactate transporters, the enzymes involved in glucose metabolism (glycolysis, gluconeogenesis, regulation of glucose metabolism, tricarboxylic acid cycle (TCA), pentose phosphate pathway), and glycogen metabolism (glycogen synthesis, glycogen degradation, regulation of glycogen metabolism) were included. The results showed that the expressions of around 50% of genes involved in the glucose metabolic pathway are altered in liver tumors compared to the corresponding normal tissues (p-values<0.005 and fold change≥1.5). More importantly, the differentially expressed genes are associated with advanced AJCC (American Joint Committee on Cancer) clinical stage (p-values<0.05), and reduced overall survival (OS) and recurrence-free survival (RFS) as determined by multivariate Cox proportional hazard models (p-values<0.05). Unexpectedly but interestingly, supervised clustering with the differentially expressed genes of the LIHC TCGA data identified a subgroup of patients with worse OS compared to those patients that clustered with the normal samples (p-value=0.00015, and 5-year median survival time (months) is 25.50 with 95% CI (20.90, 42.37) vs. 47.43 with 95% CI (40.33, NA)). This group of patients had decreased activation (p-values<0.0001) of FXR/RXR and LXR/RXR involved in lipid, cholesterol and glucose metabolism, but had increased atherosclerosis and leukocyte extravasation signaling (p-values<0.0001) activated during inflammation. This indicates those patients had decreased metabolism and increased inflammation which may contribute to the worse OS. Collectively, this study indicates that expressions of the glucose metabolic genes could be used as potential prognostic markers as well as therapeutic targets for liver cancer.

#4939

Alteration of TGFB signaling pathway predicts worse prognosis in pancreatic ductal adenocarcinoma.

Mao Li,1 Ang Li,1 Xiaochen Zhao,2 Shengzhong Hou,1 Shan Lu,1 Yu Mou,1 Xubao Liu,1 Dan Cao,1 Shangli Cai,2 Bole Tian1. 1 _West China Hospital, Sichuan University, Chengdu, China;_ 2 _3DMedicines Inc., Shanghai, China_.

Background: For pancreatic ductal adenocarcinoma (PDAC), a reliable and practical prognostic biomarker is still in unmet needs. Transforming growth factor beta (TGFB) signaling pathway is one of the core pathways involved in tumor initiation and progression via canonical and non-canonical pathway. The prognostic value of TGFB pathway genes as a functionally related group in PDAC is rarely studied.Methods: 72 PDAC patients who underwent surgery between November 30, 2015 and September 13, 2017 in West China Hospital, Sichuan University were identified and included in this study. Whole-exome sequencing (WES) or targeted next-generation sequencing was performed with tumor tissue from primary tumors. Clinical data was retrospectively collected. Clinicopathologic characteristics and overall survival were analyzed.Results: Genetic alterations were detected in 70 patients (97.2%). While 1 patient (1.4%) had only one genetic alteration, 28 patients (38.9%) had 2-4 genetic alterations and 43 patients (59.7%) had ≥5 genetic alterations. Overall, 25 patients (34.7%) with alteration of TGFB pathway and 47 patients (65.3%) with wild type of TGFB pathway were identified as TGFBm+ group and TGFBm- group respectively. TGFBm+ group had worse overall survival (HR, 1.875, 95% CI: 1.06-4.72; P = 0.04), especially in patients who had radical surgery (HR, 3.11, 95% CI: 1.11-20.3; P = 0.04). While in the subgroup of patients who underwent palliative surgery, OS was not statistically different (HR, 0.93, 95% CI: 0.42-2.04; P = 0.85). Subgroup analysis showed that overall survival hazard ratio favored participants with wild type of TGFB pathway in patients with ECOG performance score >1, abnormal CA199, without metastasis, stage I to II and patients who accepted radical surgery.Conclusion: Alteration of TGFB pathway genes is prevalent in PDAC. Inferior prognosis was observed in PDACs with mutations of TGFB pathway. TGFB signaling might play different role in different tumor stages, which is consistent with previous report about paradoxical effect of TGFB pathway. Genomic sequencing could help screen out patients at risk after surgery and adjuvant therapy might benefit this subgroup of PDACs.

#4940

The clinical significance of MRE11 expression based on location of primary tumor in colorectal cancer patients: an integrative analysis of multi-center data.

Chuanwen Fan,1 Maria Kopsida,1 Youbin Liu,1 Hong Zhang,2 Jingfang Gao,1 Gunnar Arbman,1 Siyuwei Cao,1 Yuan Li,3 Zongguang Zhou,3 Xiaofeng Sun1. 1 _Linkoping university, Linkoping, Sweden;_ 2 _Orebro University, Orebro, Sweden;_ 3 _West China Hospital, Sichuan University, ChengDu City, China_.

MRE11 plays an important role in DNA damage response for maintenance of genome stability and is becoming a prognostic marker. Its role, however, remains controversial in colorectal cancer (CRC) as it we don't know if this difference is caused by the different locations of primary cancer. To figure out the prognostic heterogeneity of MRE11 in CRC, 207 primary CRC samples from Swedish, as well as 596 primary CRC samples from TCGA data were included in present study. The associations of MRE11 expression against tumor-infiltrating inflammatory cells (TIICs) and microsatellite status with overall survival in right-sided CRC (RSCRC) and left-sided CRC (LSCRC) were assessed. The weighed gene co-expression network analysis and ClueGO were adopted to further analyze the different signaling of MRE11-related in RSCRC and LSCRC. The results showd that either high MRE11 expression alone (HR = 0.37, 95% CI = 0.18-0.76, P = 0.007) or the combination of high MRE11 expression with high TIICs was related to a favorable prognosis (HR = 0.150, 95% CI = 0.043-0.524, P = 0.003) in LSCRC. High MRE11 expression associated with a favorable prognosis in LSCRC with microsatellite stability (HR = 0.23, 95% CI = 0.09-0.55, P = 0.001). The relationships furnished above were adjusted for TNM stage, differentiation and/or TIICs, and no such evidence was present in RSCRC. Further analysis of the biological functions and the pathway involving MRE11 was associated with cell cycle process and DNA repair in both RSCRC (cor = 0.55, P = 0.55e-46) and LSCRC (cor = 0.62, P = 3e-64), whereas, activation of the immune response, necrotic cell death specifically correlated with LSCRC (cor = 0.5, P = 8e-38). In Conclusions, High MRE11 expression is associated with a favorable prognosis and the enhanced prognostic potency of combining high MRE11 with high TIICs in LSCRC. The prognostic heterogeneity of MRE11 in CRC may be due to the differential cell signals activated by MRE11 in RSCRC and LSCRC. The prognostic heterogeneity of MRE11 provides a novel rationale for stratifying patients accordingly, in order to obtain a better personalized prognostic evaluation for CRC patients.

#4941

TTF1 negative in non small cell lung cancer adenocarcinoma: A prognostic factor.

Juan F. Cordoba,1 Serafin Morales,1 Joel Veas,1 Alvaro Rodriguez,1 Noemi Reguart,2 Antonieta Salud1. 1 _University Hospital Arnau de Vilanova, Lleida, Spain;_ 2 _Hospital Clinic i provincial de Barcelona, Barcelona, Spain_.

BACKGROUND: Patients with thyroid transcription factor 1 (TTF1) negative lung adenocarcinoma (ADC) have been reported to have a worse prognosis and to lack epidermal growth factor receptor (EGFR) mutations. This study describes a series of sample tumors from patients with clinically confirmed lung cancer.

METHODS: In all lung cancer patients diagnosed in the University Hospital of Lleida from January 2011 to December 2016, a real world data study of TTF1-negative ADC was performed, using TTF1 clone 8G7G3/1(DAKO®). Each patient's clinical history, pathology specimens and molecular results were noted. Two hundred and thirty one patients with TTF1 positive lung ADC formed the control group.

RESULTS: Twenty seven (27) patients were identified with ADC TTF1 negative (74% males). The media age was 66 years. The smoking history was as follows: 55,5% former, 29,6% ex smokers and 14,8% never smokers. The clinical stages were as follows: stage I or II (n = 3 [11%]), stage III (n = 4 [14,8%]) and stage IV (n = 20 [74%]). Patients' mean survival was 7,6 vs 24,4 months in ADC TTF1 negative patients (P= 0,00001). Three hundred and thirty four (334) patients with ADC were found, TTF1 results were available in 258 (77,2%) cases, of them twenty-seven cases (10,4%) had TTF1 negative; when they were compared with the control group, TTF1 negative patients had overall shorter survival (P= 0,00001), regardless of whether patients were treated with radical or palliative intent: 34,4 vs 10, months P=0,00001 (radical treatment) and 14,6 vs 6,7 months P= 0,007 (palliative intent). EGFR mutations were less frequent (P= 0,046) in TTF-negative tumors 5,5% vs 25,3%.

CONCLUSIONS: Patients with TTF1 negative NSCLC ADC, have worse overall survival regardless of treatment intention and a lower frequency of EGFR mutations.

KEYWORDS: lung adenocarcinoma (ADC), non small cell lung cancer (NSCLC), epidermal growth factor receptor (EGFR), prognosis in thyroid transcription factor 1 negative (TTF1) adenocarcinoma.

#4942

Variations in HPV function are associated with patient outcome and identify new candidate therapeutic approaches.

Frederico O. Gleber-Netto, Meng Gao, Xiayu Rao, Christopher P. Vellano, Joseph R. Marszalek, Jing Wang, Faye M. Johnson, Curtis R. Pickering. _UT MD Anderson Cancer Ctr., Houston, TX_.

Human papilloma virus (HPV) is an oncogenic driver for a subset of head and neck squamous cell carcinomas (HNSCC), primarily from the oropharyngeal tissue subsite (OPSCC). These tumors are increasing in incidence and have recently surpassed cervical cancer as the most common HPV-driven malignancy in the United States. Fortunately, these tumors generally respond well to radiation-based therapy (XRT), and long-term (5 yr) survival is around 85%. However, the XRT treatment can generate significant morbidity, including problems with speech and swallowing. There is a clinical effort to reduce the treatment-related morbidity without compromising survival outcomes, through de-escalation treatment protocols. However, there is a subset of HPV+ OPSCC patients who do not respond to the current therapies and should not be given less intense treatment. This has generated the need to stratify patients based on their risk of recurrence or death, but currently no molecular biomarkers are available for risk assessment in OPSCC. By analyzing genomic data from The Cancer Genome Atlas (TCGA) we have identified a gene expression signature associated with expression of HPV genes. This signature identified 2 groups within the HPV+ tumors that demonstrate different levels of HPV function. One group seems to have reduced HPV function and present with intermediate phenotypes between HPV+ and HPV- tumors. Importantly, this signature is also highly prognostic in HPV+ OPSCC (p<0.0001) and significant on multivariate analysis (p<0.01). With the tumors showing reduced HPV function having worse outcomes. This prognostic signature was validated in independent OPSCC (p<0.0001) and cervical cancer cohorts (p<0.0026). The signature is most strongly associated with differential expression in the HPV gene E1^E4 but not with expression of the oncogenic driver genes of E6 and E7 genes. In vitro, we have associated the signature with sensitivity to XRT, suggesting a mechanism for the differences in patient outcome. An in vitro high throughput drug screen has identified candidate druggable targets in both the high and low risk groups, with 2 key pathways being cellular metabolism and proteasome function. Single agent and combination treatments targeting these pathways are currently being evaluated. In conclusion, we have identified a novel prognostic signature for HPV+ tumors that is associated with variations in HPV function among patients. This has the potential to translate into a biomarker assay for risk stratification, for use with de-escalation treatment protocols. Additionally, it has identified key functions of HPV that appear to be targetable and could lead to new therapeutic approaches for the subset of HPV+ patients who do not respond to XRT treatment.

#4943

Leveraging single-cell sequencing to discover novel exhaustion markers of CD8 T cells.

Xiaoshan Shi,1 Savita Jayaram,2 Kayla Lee,1 Keshav Bhojak,2 Vasumathi Kode,1 Mridul Chaudhary,2 Ashwini Patil,1 Ravi Gupta,2 Amit Chaudhuri,1 Papia Chakraborty1. 1 _MedGenome Inc, Foster City, CA;_ 2 _MedGenome Labs Pvt. Ltd., Bangalore, India_.

Therapeutic revival of tumor-specific exhausted T cells using checkpoint blockade antibodies has improved clinical outcomes in cancer significantly. Notwithstanding a high overall response rate to these drugs, long-term benefit is realized by a small fraction of the treated patients. A mechanistic explanation of relapse comes from the recent findings that in both murine and human cancers intratumoral T cells exist in a wide spectrum of functional states – from fully functional at one end of the spectrum, to fully dysfunctional at the other end. Therefore, tumor response to checkpoint inhibitors is determined by the ratio of functional to dysfunctional state of T cells, which in turn is modulated by a wide array of immune-suppressive signals present within the tumor microenvironment. Dissecting the T cell functional states can significantly enhance our understanding of patient responses to immunotherapy drugs and can be used to develop targeted and personalized strategies for restoring antitumor immunity. A key challenge to this problem is that the transcriptional signatures of dysfunctional exhausted T cells are closely intertwined with the activated/effector CD8+ T cell states. This is not surprising, since T cell dysfunction arises following chronic T cell activation. Developing unique markers of T cell exhaustion from in vivo models has not been successful. In this study, we present data to show how single cell transcriptomics on a 10x Genomics platform can identify T cell functional states following antigen-stimulation in an ex vivo T cell activation system. In particular, we identified functional gene clusters and molecular networks that are unique to CD8+ T cell exhausted state. The combined expression of our T cell exhaustion gene signature correlated with poor prognosis when applied to TCGA data of over 1500 tumors from many different cancers. Furthermore, the molecular pathways identified in this study provide opportunities to develop novel therapeutic interventions specially targeting dysfunctional T cells in cancers thereby enhancing the efficacy of checkpoint inhibitors.

#4944

Differential expression of complex immune biology in MSI and MSS colorectal tumor microenvironments using high-plex spatial resolution.

Kit Fuhrman, Sarah Church, Daniel Zolligner, Jason Reeves, Doug Hinerfeld, Christina Bailey, Sarah Warren. _Nanostring Technologies, Seattle, WA_.

Spatial characterization of the tumor microenvironment (TME) including cancer cells, stroma and immune cells is essential for understanding tumor progression and discovering prognostic and predictive biomarkers. However, it has proven difficult to perform such studies in a highly multiplexed manner using limited sample quantity. GeoMx™ Digital Spatial Profiling (DSP) has been developed as an integrated research use platform for hi-plex spatial profiling of mRNA and protein using an optical-barcode read-out without the need for on-tissue molecular biology. In this study, we combined both bulk gene expression analysis using the NanoString PanCancer IO360™ panel and the spatial analysis of 40 proteins and 78 RNAs to characterize microsatellite stable (MSS) or instable (MSI) colorectal tumors to evaluate active and suppressive immune mechanisms in both immune dense regions and tumor versus stroma.

Comparing colorectal tumors characterized by MSS, spatial analysis of RNA and protein expression by DSP was able to differentiate immune hot and cold regions of the tumors despite MSS status. Furthermore, comparison of immune-enriched hot-spots, measured by CD45 dense regions of interest, showed that MSS immune hot tumors had decreased macrophage and tumor proliferation markers compared to MSI immune hot tumors, elucidating potential therapeutic targets. Since there is a subset of patients with MSS colorectal cancer that still responds to immunotherapy, this suggests DSP could ultimately be used to identify unique spatial biology and immune characteristics that might further expand beyond MSS and MSI status and tradition gene signatures to help predict patients' response to mono- or combination therapy.

#4945

Project Survival: Engineering a phenomic and artificial intelligence driven precision medicine biomarker pipeline for pancreatic adenocarcinomas.

Eric Michael Grund,1 Michael A. Kiebish,1 Viatcheslav R. Akmaev,1 Rangaprasad Sarangarajan,1 John J. Crowley,2 Amy Stoll-D'Astice,2 Tori Singer,3 Corinne Decicco,3 Wendy Hori,3 Abena Darkwah,1 Lixia Zhang,1 Valerie Bussberg,1 Leonardo O. Rodrigues,1 Emily Y. Chen,1 Tomislav Dragovich,4 Manuel Hidalgo,3 Niven R. Narain,1 A James Moser3. 1 _BERG LLC, Framingham, MA;_ 2 _Cancer Research and Biostatistics, Seattle, WA;_ 3 _Beth Isreal Deaconess Medical Center, Boston, MA;_ 4 _Banner MD Cancer Center, Gilbert, AZ_.

Pancreatic cancer is a complex and dynamic disorder necessitating a comprehensive clinical design integrated with robust OMICS technologies and AI analytics to identify potential molecular and clinical signatures of diagnosis, progression, and treatment outcomes. Project Survival is a multisite prospective longitudinal study currently in the 4th year of a 6 year initiative of sampling and analysis of subjects in 6 categories: healthy volunteers with a first degree relative with pancreatic cancer (N=39), pancreatitis (N=34), pancreatic cystic neoplasm (N=52), suspicious pancreatic masses with pathology other than pancreatic cancer (N=22), early stage (N=66), locally advanced (N=123), and metastatic pancreatic cancer (N=99). All diseased patients are longitudinally sampled multiple times per year for sera, plasma, buffy coat, saliva, urine, and tumor/adjacent normal tissue. The BERG Interrogative Biology® platform is employed for multi-omic mass spectrometry analysis (metabolomics, lipidomics and proteomics) and applies artificial intelligence (bAIcis®, BERG Artificial Intelligence Clinical Information System) technologies. bAIcis® is harnessed to align the multi-omic profiles with longitudinal clinical information to infer probabilistic cause-and-effect relationships among molecular and clinical variables in a network-based model. Multiple longitudinal time points continue to be collected during the course of the six-year timeline enabling dynamic modeling. The value of this longitudinal study is in the epidemiological assessment of patient type progression to more advanced stages and identification of biomarkers and clinical features that align with the shifts observed in the patient populations. Collectively, we are incorporating patient progression with longitudinal sampling to investigate predictive signatures of disease advancement. Biomarker panels with AUC > 0.7 will be pursued in a further prospective clinical study with a larger subject number. The integration of multi-omic analysis with artificial intelligence has identified several biomarker panels that meet numerous unmet needs for the identification and clinical stratification of pancreatic adenocarcinoma.

#4946

Comprehensive molecular profiling reveals distinct immunemicroenvironment subtypes of oropharyngeal cancers.

Min Hwan Kim,1 Jae-Hwan Kim,2 Dong Min Jung,2 Jae Woo Choi,2 Soo Jin Heo,1 Kyoung-Ho Pyo,2 Min Hee Hong,1 Byoung Chul Cho,1 Hye Ryun Kim1. 1 _Yonsei Cancer Center, Yonsei University College of Medicine, Seoul, Republic of Korea;_ 2 _Yonsei University College of Medicine, Seoul, Republic of Korea_.

Background The oropharyngeal cancer (OPC) constitutes a distinct clinical subset of head and neck squamous cell carcinoma with high prevalence of HPV-associated tumorigenesis. However, the relationship between tumor molecular types and immune microenvironment has not been systematically delineated in OPCs. In this study, we classified the OPCs into three different molecular subtypes regarding their immunological character by comprehensive molecular profiling by RNA-seq and multiplexed immunohistochemistry (IHC).

Methods The 37 surgically resected primary OPC tumors were analyzed, and the HPV status was evaluated by p16 IHC. The transcriptome profiling of tumors by RNA-seq classified OPCs into three molecular subtypes based on t-SNE plotting. The immune microenvironment of each subtype was investigated using VECTRA spectral imaging system in terms of CD8 T cell and macrophage infiltration patterns, as well as T cell exhaustion maker expressions. The 10 OPC patients treated with anti-PD-1/PD-L1 blockade were also classified into three subtypes and their therapy response was compared.

Results We divided OPCs into Immune-rich (IR), mesenchymal (MS), and classical (CL) subtypes by RNA-seq clustering analysis. All IR type tumors were HPV-positive, whereas most CL types (7 out of 8) were HPV-negative. MS type showed mixed HPV status (HPV-positive in 5/14). The IR type showed favorable prognosis compared with poor prognosis of CL type, and MS type showed intermediate survival outcome. The IR type tumors showed high enrichment of adaptive immune response, T cell exhaustion, and cell cycle gene signature implying their highly immunogenic properties. The multiplexed IHC confirmed plenty of PD-1+CD8 T cells and type I macrophages infiltrating tumor nest in IR types. The MS type was characterized by upregulation of muscle, extracellular matrix, and TGF-β signatures with scant CD8 T cell signature expression. The IHC analysis of MS type showed exclusion of CD8 T cells from tumor nest that might be related to their high TGF-β-response signature (TBRS) score. CL types were related to high xenobiotic catabolism signatures, and showed low level of CD8 T cell infiltrations with focal CD73 expression. Among 10 OPC patients with anti-PD-1/PD-L1 blockade treatment, the IR type patients showed durable therapy response (3 of 4 patients), whereas CL type patients showed early progression on the treatment (3 of 4 patients).

Conclusions Our comprehensive analysis reveals that OPCs can be classified into three distinct subtypes based on their immune microenvironment properties. The three molecular subtypes showed distinct therapy response on anti-PD-1/PD-L1 blockade. These findings are pertinent to develop predictive markers on immunotherapy and to design combination immunotherapy strategies in OPC patients.

#4947

Plucked anagen scalp hair: A comparison between different analytical techniques to establish reproducible drug induced transcriptional changes and provide pharmacodynamic biomarker and target engagement information from oncology patients.

Tim Mefo, Elliott Harrison, Alan Murdoch, Benjamin Reed. _Epistem Ltd., Manchester, United Kingdom_.

Plucked anagen scalp hair is an ideal surrogate tissue to monitor drug induced transcriptional changes. High vascularisation of the hair follicle, ease of sampling and high degree of similarity of expression in hair of pathways dysregulated in cancers, make the cellular bulb of plucked human scalp hair an excellent surrogate tissue for the non-invasive monitoring of pharmacodynamic (PD) and mechanism of action (MOA) effects in clinical trials. Using our ex vivo plucked hair culture platform, plucked scalp hair bulbs from several healthy donors were exposed to BEZ235, a dual pan-class PI3K and mTOR inhibitor at different concentrations and durations. Total RNA was isolated from the cellular bulb of anagen hair post-culture and samples were assessed by microarray and RNA-seq analysis to assess dose dependent global transcriptional alterations and develop a gene expression signature indicative of BEZ235 exposure. Two different microarrays, the Affymetrix Clariom D and U133 plus 2.0 arrays, were directly compared to RNA-seq data obtained from the same ex vivo culture samples. Genetic signatures indicative of exposure of plucked scalp hair to BEZ235 were established using all three analytical techniques. The transcriptional readouts from the plucked scalp hair samples revealed strong correlations (0.97-0.99) in the genes expressed in anagen scalp hair between donors. In addition, global gene expression data indicated that assessing pooled scalp hairs was sufficient to detect significant differential expression (p<0.05 & 1.5 fold change) underpinning the low variance in the majority of target genes in plucked hair as a biomarker platform. Gene signatures for compounds targeting the PI3K/mTOR pathway that were established using the ex vivo plucked scalp hair platform were subsequently used to support clinical trials and scalp hair taken from patients receiving treatment showed an excellent post-exposure target signature modulation. In summary, the results of the ex vivo plucked assessment generated a dose dependent and biologically relevant transcriptional signature following exposure to BEZ235. Plucked anagen scalp hair is therefore an ideal non-invasive surrogate tissue to obtain drug-induced pharmacodynamic biomarker and target engagement information from oncology patients.

## IMMUNOLOGY

### Adaptive Immune Cells in the Tumor Microenvironment

#4948

T helper 2 cells block breast cancer promotion.

Margherita Boieri, Anna Malishkevich, Lauren Steidl, Kenneth Ngo, Sowmya Iyer, Mary Awad, Johannes Kreuzer, Wilhelm Haas, Miguel Rivera, Shadmehr Demehri. _Massachusetts General Hospital, Charlestown, MA_.

New immune-based strategies for cancer treatment including the activation of tumor-infiltrating CD8+ cytotoxic T lymphocytes (CTLs) have led to effective therapeutics against late-stage disease. However, the function of the immune system in combating the early-stage cancers remains uncertain. To establish a mechanism that activates immune cells against early carcinogenesis, we have studied the role of an epithelial-derived cytokine, thymic stromal lymphopoietin (TSLP), in early stages of breast cancer development. We have previously discovered that TSLP blocks breast carcinogenesis through the activation of CD4+ T cells that heavily infiltrate the sites of developing cancer in the breast gland without affecting the normal tissue.To investigate the mechanism by which TSLP-stimulated CD4\+ T cells suppress spontaneous breast cancer development, we used MMTV-PyMT transgenic mouse models (PyMTtg) that spontaneously develop breast tumors and resemble the human luminal breast cancer. These mice were crossed into TSLP transgenic mice that produce TSLP under the promoter of a skin-specific keratin 14 promoter (K14-TSLPtg), plus additional knockouts including TSLPR-/- and IL4r𝜶;-/-. Tumor development was arrested in K14-TSLPtg;PyMTtg mice compared to PyMTtg counterparts as their tumors failed to progress to high grade. The tumor suppressive mechanism of CD4\+ T cells was mediated by a block in cancer cell proliferation, but not by cytotoxicity. Polarization of CD4\+ T cells into T helper 2 (Th2) subtype was essential for blocking breast tumor development, and the tumor suppression was achieved by CD4\+ T cells in the absence of CD8\+ T and B cells. Finally, we found the basal levels of TSLP released by premalignant breast epithelial cells to be protective against the early phases of breast cancer development.In conclusion, we demonstrate that CD4+ Th2 cells block early stages of breast carcinogenesis. TSLP stimulated CD4+ Th2 cells induce an arrest in cancer cell proliferation, which represents a novel mode of immunity with great potential for the prevention and treatment of breast cancer.

#4949

Immunological ignorance is an enabling feature of the oligo-clonal T cell response to melanoma neoantigen.

Gerald P. Linette,1 Beatriz M. Carreno,1 Zackary L. Skidmore,2 Jasreet Hundel,2 Obi L. Griffith,2 Malachi Griffith,2 Vincent Magrini,3 Elaine R. Mardis3. 1 _University of Pennsylvania, Philadelphia, PA;_ 2 _Washington University School of Medicine, St. Louis, MO;_ 3 _Nationwide Children's Hospital, Columbus, OH_.

The impact of intratumor heterogeneity (ITH) and the neoantigen landscape on T cell immunity is poorly understood. ITH is a widely recognized feature of solid tumors and poses distinct challenges related to the development of effective therapeutic strategies, including cancer immunotherapy. Cutaneous melanoma is characterized by C->T transitions caused by UV exposure which leads to a large mutational burden encoding putative neoantigens. Here, we performed ultra-deep DNA sequencing of multiple metastases from melanoma patients and observed ubiquitious sharing of clonal and subclonal single nucleotide variants (SNV) encoding putative HLA class I restricted neoantigen epitopes among tumors. MEL66, a reference melanoma patient with a large mutational burden (53 SNV/Mb), provided six resected metastases for study. The spontaneous T-cell response features an oligo/monoclonal T cell-receptor (TCR) repertoire which targets a few clonal neoantigens; most neoantigen-specific T cell clones ignore the tumor. CD8+ T cells to additional HLA class I restricted neoantigens can be elicited by DC vaccination (NCT03092453) with tumor-encoded amino acid substituted (mutated) peptides resulting in a diverse, high frequency TCR repertoire. Isolation of T cell clones by limiting dilution from blood and TIL permits functional validation regarding neoantigen-specificity. Gene transfer of paired abTCR heterodimers specific for clonal neoantigens confirmed correct clonotype assignments based on high throughput CDR3 sequencing. Additional melanoma patients with 2-6 resected metastases were investigated and similar observations were found. Our observations implicate ineffective neoantigen cross-presentation as the basis for immunological ignorance. We postulate that therapeutic vaccination is required to increase the breadth and diversity of neoantigen-specific CD8+ T cells as an adjunct to checkpoint inhibitor treatment for cancer.

#4950

Abnormality of selective autophagic degradation of tenascin-C promotes resistance to T cell-mediated immune attack in triple negative breast cancer.

Rong Deng, Zhi-ling Li, Hai-Liang Zhang, Xuan Li, Jia Mia, Li-Huan Zhou, Yun Huang, Yan Yu, Jun Tang, Xiao-Feng Zhu. _Sun Yat-sen Univ. Cancer Ctr., Guangzhou, China_.

Introduction: Accumulating data has demonstrated the crucial role of immunity in TNBC biology, suggesting the potential involvement of immunotherapy to treat this malignancy. A variety of different immuno[[Unsupported Character - Codename &shy;]]therapeutic strategies, such as anti-PD-1 or anti-PD-L1, are currently being tested in preclinical studies in TNBC patients. However, the response rate (RR) to PD-1 or PD-L1 antibody remains low in TNBC. Autophagy regulates the antitumor immune response. The mechanism that autopahgy stimulates or limits the T cell immune system's attack on tumor cells has not been well understood. Here, we examine the complex role of autophagy in TNBC tumor immunity.

Methods: We depleted Atg5, Atg7 and Beclin1 expression using specific single-guide RNAs (sgRNAs) to construct autophagy-deficient TNBC cell lines. The caspase-3 cleavage assay and lactat dehydrogenase-release (LDH) assay were performed to measuring T-cell-mediated cytotoxicity. SILAC and high-resolution MS procedures were implemented to quantify protein variations between autophagy-competent and autophagy- incompetent cells. Flow cytometry analysis was performed to detect the proliferation and activity of T cells. IHC was performed with antibody against Tenascin-C (TNC) , LC3, CD8 in paraffin blocks of human TNBC breast lesions. The blockade of TNC alone or in combination with immunotherapy was determined. Statistical analyses were conducted using GraphPad Prism, SPSS software and JavaGSEA Desktop Application. A p value of <0.05 was considered to indicate statistical significance.

Results: We found that only in basal tumors but not in Luminal and Her2 tumors, Atg5 and Beclin 1 expression were found to be positively related with the abundance of B cells, CD4 T cells, CD8 T cells, neutrophils, and dendritic cells. The failure of autophagy contributed to the limitation of T-lymphocytes immune system's attack on TNBC cells in vitro and in vivo. We also identified a novel role for TNC as a key regulator of autophagy-deficient-mediated immunosuppression, in which TNC was Lys63 ubiquitinated by the E3 ligase SKP2, , thus promoting its recognition by the autophagy receptorp62 and leading to its selective degradation. The degradation of TNC by the autophagy-lysosome pathway was physiologically significant and clinically relevant in TNBC patients. More importantly, we demonstrated that blockade of TNC sensitized T cell-mediated tumor killing and improved the antitumor effects by PD1 blockade in autophagy-impaired TNBC tumors.

Conclusion: Our studies identify a crosstalk between autophagy and immune compartments as a novel therapeutic strategy for TNBC patients with poor immunotherapy response. Our findings indicates that blockade of TNC may be an effective adjuvant treatment with immunotherapy as a novel management strategy for TNBC patients, especially with autophagy deficiency.

#4951

Obesity-induced changes in baseline immune responses to pre-clinical breast cancer.

Justin Gibson, Prabhakara Nagareddy, Lyse Norian. _University of Alabama at Birmingham, Birmingham, AL_.

Obesity is known to increase morbidity and mortality in breast cancer patients; however, the immunological contributions to such changes remain incompletely understood. Our long-term goal is to investigate the broad impact of obesity on anti-tumor immunity, with a particular focus on understanding the mechanisms underlying obesity-induced immune alterations and how these alterations may influence the use of immunotherapeutics in breast cancer patients. For our studies, we utilized a diet-induced model of obesity (DIO) in which mice were fed either a high-fat or low-fat diet to generate DIO or age-matched lean controls, respectively. Animals were then challenged with the syngeneic E0771 mammary carcinoma cell line. Tumor outgrowth was quantified by caliper measurements, bioluminescent imaging (BLI) via firefly luciferase-expressing E0771 (E0771-fLUC) cells, and endpoint tumor weights. Whole tumor immunogenetic gene expression profiles were evaluated using nanoString and immune populations were assessed via multi-parameter flow cytometry. All measures of tumor outgrowth reveal that obesity significantly increases primary tumor outgrowth and induces a variety of alterations to the immunogenetic profile. Additionally, obesity significantly alters the frequency of intra-tumoral immune populations, including decreases in CD4+ and CD8+ T cells, increases in granulocytic myeloid-derived suppressor cells (G-MDSCs) and myeloid dendritic cells (mDCs), and a trending decrease in total CD45+ tumor infiltrating leukocytes. These alterations are specific to the tumor environment, as they are not present in the spleens of tumor-bearing mice. Collectively these data implicate obesity as a causal agent in impairing anti-tumor immune responses to murine mammary tumors. Furthermore, they suggest several cellular candidates for future mechanistic investigations into obesity-induced alterations in breast cancer immunity.

#4952

**Host lipocalin 2 protects against** Kras **mutant lung cancer development by maintaining an anti-tumor immune contexture.**

Maya Hassane,1 Warapen Treekitkarnmongkol,2 Tina L. McDowell,2 Smruthy Sivakumar,2 Wenhua Lang,2 Joshua K. Ochieng,2 Sayuri Nunomura-Nakamura,3 Casey Finnicum,4 Christel Davis,4 Gareth E. Davies,4 Junya Fukuoka,3 Tina Cascone,2 Florencia McAllister,2 Ignacio I. Wistuba,2 Erik Ehli,4 Seyed J. Moghaddam,2 Paul Scheet,2 Junya Fujimoto,2 Humam Kadara2. 1 _American University of Beirut, Lebanon;_ 2 _The University of Texas MD Anderson Cancer, TX;_ 3 _Nagasaki University, Japan;_ 4 _Avera Institute for Human Genetics, SD_.

Relative to other lung adenocarcinomas (LUADs), Kras mutant LUAD displays dismal prognosis warranting the need for new strategies for early treatment of this lung cancer subtype. Limiting these advances is our poor understanding of events that drive Kras mutant LUAD development. In efforts to fill this void, we recently found that mice with knockout of G-protein coupled receptor 5A (Gprc5a-/-) and after tobacco carcinogen exposure (nicotine-specific nitrosamine ketone/NNK) developed LUADs harboring driver somatic Kras mutations. To understand early events preceding the onset of these Kras mutant LUADs, we performed RNA-sequencing (RNA-Seq) of normal airways in Gprc5a-/- mice and wild type (WT) littermates prior to NNK exposure. We found markedly elevated expression of the antimicrobial lipocalin 2 (Lcn2) gene in normal airways of Gprc5a-/- mice. Also, analysis of publicly available human datasets revealed elevated expression of LCN2 in human LUADs relative to normal lung tissues particularly in KRAS mutant tumors. We then sought to probe the role of Lcn2 in the pathogenesis of Kras mutant LUAD. Mice with knockout of host Lcn2 (Gprc5a-/-/Lcn2-/-) showed increased lung tumor development compared to Gprc5a-/- littermates. RNA-Seq of whole lungs at baseline and at seven months post-NNK (n = 9-10 mice each group) pinpointed differential expression profiles that denoted decreased anti-tumor immunity in Gprc5a-/-/Lcn2-/- mice. Computational estimation of immune infiltrates demonstrated increased levels of neutrophils post-NNK in Gprc5a-/-/Lcn2-/- mice but not in Gprc5a-/- littermates. By flow cytometry analysis of lung tissues, we found elevated numbers of various lymphoid and myeloid immune cells in both groups following NNK exposure and increased neutrophil counts in Gprc5a-/-/Lcn2-/- mice relative to Gprc5a-/- littermates. Histopathological assessment also revealed increased numbers of pro-inflammatory lesions in mice lacking Lcn2. Gprc5a-/-/Lcn2-/- mice at three months post-NNK were also found to exhibit increased production of IL-17A by lung-resident CD4+ and CD8+ T cells relative to Gprc5a-/- littermates concomitant with decreased expression of the anti-tumor Th1/cytolytic immune markers Ifnγ and Gzmb. Preliminary analysis also showed elevated expression of the major immune checkpoint PD-L1 on alveolar macrophages in Gprc5a-/-/Lcn2-/- mice relative to Gprc5a-/- mice with WT Lcn2. Lastly, we preliminarily found, by bacterial 16S rRNA sequencing of temporally collected fecal samples, globally altered microbiome profiles in mice lacking Lcn2. Our findings underscore novel cues in lung oncogenesis and suggest that Lcn2 decreases anti-tumor immunity, perturbs the host microbiome and promotes development of Kras mutant LUAD thereby offering new venues for early treatment of this fatal malignancy.

#4953

Differential expression of ICOS on circulating immune cells and tumor infiltrating lymphocytes in patient with solid tumors.

Richard C. Sainson, Miha Kosmac, Rachael Kimber, Joana Carvalho, Gwenoline Bohris, Anil Thotakura, David Melvin, Matthew McCourt. _Kymab Ltd, Cambridge, United Kingdom_.

Background: The Inducible T-cell costimulator (ICOS/CD278) is upregulated during T cell activation and is an important regulator of the immune response against pathogens and cancer cells. When engaged by its ligand, the activation of ICOS signaling improves the survival of ICOS+ effector (TEff) and regulatory T cells (TReg). In addition, depending on the T cell subtypes expressing ICOS, activation of the signaling pathway induces the expression and secretion of either pro- (by TEff) or anti-inflammatory (by TReg) cytokines. ICOS has been reported to be upregulated in the tumor microenvironment (TME) of most tumor indications. However, the variability in ICOS expression levels on ICOS expressing T cells (TEff and TReg) and their relative abundance in different tumor types is still poorly understood. The aim of this study was to take advantage of multiple analytical platforms to assess the expression of ICOS on immune cells in the periphery and on tumor infiltrating lymphocytes (TILs) from cancers known to express high levels of ICOS, based on previous TCGA analyses.

Methods: We analysed ICOS expression on peripheral blood mononuclear cell (PBMCs) and TILs by FACS, ChipcytometryTM and by single cell RNAseq. The analysis aimed to compare the expression of ICOS on different immune T cell subtypes. This comparison was performed on samples isolated from treatment naive non-small cell lung cancer (NSCLC) patients. In parallel, we established dual immunohistochemistry staining protocols supplemented by digital pathology to quantify and compare the expression of ICOS in the context of either FOXP3 or CD8 positive cells in NSCLC, gastric, esophageal, cervical, bladder and head and neck tumor samples.

Results and conclusion: Using these approaches, we confirmed high ICOS expression in several solid tumor types. ICOS expression was also shown to be higher on immune cells in the TME compared to the periphery. Finally, ICOS expression was found to be more abundant on the surface of intratumoral TReg than on CD8+ T effector cells, confirming the data obtained in preclinical syngeneic tumor models (AACR2018 abstract #2792). Importantly, this work highlighted variability in the abundance and ratio of the different ICOS\+ T cells subtypes (TEff and TReg) between different ICOSHigh tumors. Altogether, the variability in the intratumoral ratio of ICOS positive T cells observed in different tumor types could differentially affect their response to antibodies targeting ICOS. This work suggests that understanding the expression of ICOS in the context of different immune T cell subtypes may improve selection of patients with higher potential to respond to anti-ICOS therapies such as KY1044.

#4954

**NKG2A blockade potentiates CD8** + **T-cell immunityinduced by cancer vaccines.**

Young J. Kim,1 Michael Korrer,1 Nadine van Montfoort,2 Linda Borst,2 Sjoerd van der Burg,2 Thorbald van Hall,2 Pascale Andre,3 Nicolai Wagtmann4. 1 _Vanderbilt University Medical Center, Nashville, TN;_ 2 _Leiden University Medical Center, Netherlands;_ 3 _Innate, France;_ 4 _Dragonfly, MA_.

NKG2A is an inhibiting immune receptor expressed by cytotoxic lymphocytes, including natural killer cells and CD8+T cells. Expression of this receptor on CD8+T cells was predominantly found on CD103+tumour-infiltrating cells and only partly overlapped with other checkpoint receptors. Particularly high frequencies of NKG2A-expressing lymphocytes were detected in tumours with an immune-reactive profile and could be induced by therapeutic cancer vaccination. To examine if NKG2A represented an adaptive resistance mechanism for cancer vaccination, we blocked the receptor with a therapeutic antibody and performed genetic knockdown experiments for its ligand Qa-1, the conserved ortholog of HLA-E. In four mouse tumour models, the modest effect of therapeutic vaccines was greatly potentiated by disruption of the NKG2A/Qa-1 axis. NKG2A blockade operated through CD8+T cells and was even effective in a mouse model refractory to anti-PD-1 therapy. These findings indicate that NKG2A-blocking antibodies might improve clinical responses to therapeutic cancer vaccines. Moreover, the ligand for NKG2A, HLA-E is predominantly expression on tumor infiltrating myeloid cells, suggesting that tumor extrinsic adaptive immune response mechanism accounts for the adaptive resistance mechanism in head & neck cancer patients.

#4955

Impact of PD-1, PD-L1 and EBI3 on prognosis in a cohort of localized high grade undifferentiated pleomorphic sarcoma patients.

Pascaline Boudou-Rouquette,1 Anne Jouinot,1 Virginie Audard,1 Franck Letourneur,2 Béatrice Parfait,1 François Goldwasser,1 Benoit Terris,1 Pierre Laurent-Puig,3 Jérôme Alexandre,1 Karen Leroy,1 Eric Pasmant,1 Odile Devergne,4 Frederique Larousserie1. 1 _Cochin hospital, CARPEM, AP-HP, Paris Descartes, Paris, France;_ 2 _Institut Cochin, CARPEM, Inserm 1016-CNRS 8104, AP-HP, Paris Descartes, Paris, France;_ 3 _HEGP hospital, CARPEM, AP-HP, Paris Descartes, Paris, France;_ 4 _Sorbonne university, INSERM, CNRS, Paris, France_.

Introduction: Studies of soft-tissue sarcomas (STS) and therapeutic outcomes are limited by their rarity and heterogeneity. Despite intensive treatment, including chemotherapy, surgery and radiation therapy, 30% of patients develop recurrent disease, and the outcome of the patients with recurrent or metastatic sarcomas remains poor. The SARC028 phase II, multicenter trial of pembrolizumab reported promising activity in selected histologic subtypes of advanced STS, including undifferentiated pleomorphic sarcoma (UPS). On the other hand, multicentre phase II trial (PEMBROSARC) found that PD-1 inhibition has limited activity in selected STS (UPS and LMS and GIST). The application of immune checkpoint blockade in sarcoma is in its infancy and we do not yet understand which patients will benefit from these therapies.

Methods: We explored the degree of PD-L1, PD-1, and CD8 expression in tumor tissue and microenvironment and their clinical impact in a retrospective analysis of 50 UPS sarcoma samples, at diagnosis. PD-L1 status was assessed on whole sections of fixed tissue. We also studied expression by tumors cells of two heterodimeric cytokines IL-27 (EBI3 / p28) and IL-35 (EBI3 / p35), known for inducing the expression of immune checkpoints. Their expression was investigated by analyzing the expression of their shared subunit EBI3 by immunohistochemistry. Patients presented with grade 3 FNCLCC, localized, sarcoma of the extremities, with a high risk of local and distant recurrence and were treated in a relatively homogeneous way (surgery followed by adjuvant radiation therapy and chemotherapy).

Results: The median overall survival was 63.4 months after a median follow-up of 42.0 months (range, 3.7- 108.3 months). The PD-L1 score on tumor cells (E1L3N, Cell Signaling) was negative in 94% of cases, with only 1 that had more than 10% PD-L1-positive tumor cells. Three semi-quantitative scores were performed on the immune cells: PD-L1 (same antibody): score 0 (62%), 1 (34%), 2 (4%); PD-1 (PD-1 NAT, Abcam): score 0 (52%), 1 (32%), 2 (16%) and CD8 + (C8/144B, Dako): score 0 (4%), 1 (36%), 2 (58%), NE (2%). PD-1 expression was associated with CD 8 infiltration (chi2 p=0.047). The EBI3 score on tumor cells was positive (>30% positive cells) in 6% of cases and correlated with worse overall survival (p=0.04) and progression-free survival (p<0.01), whereas PD1 (expressed by T cells), PD-L1 (expressed by infiltrating immune cells or tumor cells) and CD8 infiltration lacked prognostic significance. We are now integrating data from Affymetrix microarray gene expression and whole exome to investigate the molecular differences on tumors.

Conclusion: This study suggests that targeting immune checkpoints downstream of IL-27/IL-35, different from PD1 may benefit to some UPS patients.

#4956

Oral antibiotics act as potent immunotherapeutic agents against pancreatic cancer.

Vrishketan Sethi, Saba Kurtom, Irina Fernandez, Maria Abreu, Sabita Roy, Sundaram Ramakrishnan, Ashok Saluja, Vikas Dudeja. _University of Miami, Miami, FL_.

Background: Recent reports have elaborated on how the gut microbiota shape vertebrate immune system during ontogeny. Additionally, tumor-promoting inflammation and gut dysbiosis are often associated with multiple cancers including pancreatic ductal adenocarcinoma (PDAC). We sought to investigate the gut microbiota-immune relationship in mouse models of cancer.

Methods: To investigate if cancer-associated dysbiosis modulates cancer per se, neonatal mice with unstable, dynamic gut microbiota (guests) were cohoused with either adult cancer-naive mice or adult pancreatic cancer-bearing KPC (KrasG12D/+;Trp53R172H/+;Pdx-1-Cre) mice. After 4 weeks of co-housing, guest mice were subcutaneously implanted with KPC PDAC and monitored for cancer progression. In a parallel series of experiments, tumor progression was compared between control mice and mice given oral antibiotics and also, between germ free mice and conventionalized mice. Later, oral antibiotics were combined with checkpoint inhibitors in preclinical trials of efficacy. Monoclonal antibodies and knockout strains of mice were used to elucidate mechanism. Pancreatic tumors were probed for bacteria.

Results: Co-housing with KPC mice significantly increased cancer progression in guest mice compared to that in guest mice co-housed with non-cancer-bearing cagemates. Bowel sterilization dramatically reduced cancer burden in subcutaneous, metastatic, and orthotopic models of pancreatic cancer as well as in subcutaneous and metastatic models of melanoma. Similarly, germ free mice had decreased subcutaneous PDAC burden compared to littermate conventionalized mice. Surprisingly and in findings contradictory to previously published reports, oral antibiotics also potentiated the efficacy of checkpoint inhibitors in shrinking tumors. The tumor decreasing effect of antibiotics disappeared in immunodeficient Rag1-/- mice, wild type mice depleted of IL-17a but not in Tlr4-/- mice. Immunophenotyping further confirmed microbiota-mediated infiltration of immunosuppressive cells inside tumor and antibiotics-mediated activation of intratumoral Th1/Tc1 immunity. Interestingly, pancreatic cancer liver metastases revealed a significant presence of 16S rDNA resembling the gut flora and subcutaneous pancreatic cancer tissue grew viable bacteria on culture medium and this was prevented by oral antibiotics, thereby suggesting a gut-to-tumor translocation of bacteria.

Conclusion: Our data suggest that gut microbiota-cancer cross talk is mediated by the immune system and that oral antibiotics may be repurposed as potent anti-cancer immunotherapeutic agents.

#4957

Evidence of clonal T cell expansion in metastatic castration resistant prostate cancer patients with biochemical response to pembrolizumab.

Julie N. Graff,1 Chaojie Wang,1 Zheng Xia,1 Mary A. Wood,2 Reid F. Thompson,2 Amy E. Moran1. 1 _Oregon Health & Science Univ., Portland, OR; _2 _Portland VA Research Foundation, Portland, OR_.

Metastatic castration resistant prostate cancer (mCRPC) is the lethal form of prostate cancer that until recently was thought to be resistant to checkpoint blockade. A recent clinical trial revealed that the PD-1 inhibitor, pembrolizumab, when given in combination with the androgen deprivation therapy (ADT) drug enzalutamide, achieved an unprecedented 18% complete biochemical response rate in mCRPC patients. The mutational burden of prostate cancer is speculated to drive the low response to PD-1 inhibition and, to date, there is a lack of evidence of tumor associated antigen specific T cell responses in the metastatic niche. To investigate potential drivers of response in the immune microenvironment, we performed whole exome sequencing in parallel with TCRβ chain sequencing and single cell RNA sequencing of pre- and post-treatment metastatic biopsies. We found that canonical drivers of immune response such as neoepitope burden and microsatellite instability status were not strongly associated with response to PD-1 inhibition in this cohort. However, we reveal for the first time in mCRPC that patients that experienced clinical benefit (defined as stable disease or biochemical response of 50% or greater), had a more restricted TCRβ chain repertoire prior to treatment. Moreover, TCR clonality was enriched in patients responding to PD-1 inhibition compared to non-responders. Comparison of TCR clonality pre- and post-exposure revealed that tumor associated antigen-specific T cells were expanded by pembrolizumab treatment in a responder. Using single cell RNA sequencing to reveal composition of immune infiltrates, we demonstrated no difference in infiltrating T-cell abundance or other immune population frequencies between responders and non-responders. However, there was strong enrichment of a CD8+ T-cell exhaustion signature among responders compared to non-responders from pre-treatment biopsies (p less than 0.01). Taken together, these data highlight the heterogeneity of the metastatic tumor microenvironment, and suggest that a specific subpopulation of CD8+ T-cells are expanded in clinical responders.

#4958

Alterations in the immune microenvironment during progression from Barrett's esophagus to esophageal adenocarcinoma.

Dyke P. McEwen, Derek J. Nancarrow, Jules Lin, Rishindra M. Reddy, Andrew C. Chang, David G. Beer, Kiran H. Lagisetty. _University of Michigan, Ann Arbor, MI_.

The incidence of esophageal adenocarcinoma (EAC) has increased 500 fold over the past 30 years and is now a top ten leading cause of cancer death. Systemic chemotherapy and targeted radiation have led to modest increases in survival, however 5-year survival is still less than 20%. Immunotherapy has emerged as a promising approach for the treatment of various solid tumors, however very little is known about the EAC tumor immune microenvironment and how it changes during the progression from Barrett's metaplasia to carcinoma. The goal of the current study was to identify key changes in the immune microenvironment during progression from Barrett's Esophagus (BE) metaplasia to the formation of EAC. Tissue samples from 52 patients with various levels of dysplasia (BE, BE w/ low grade dysplasia, BE w/ high grade dysplasia, and EAC) were subject to RNA-Seq analysis and investigated for changes in gene expression of a variety of cytokines and chemokines. Gene expression analysis revealed more than 5-fold increases in expression of multiple cytokines, such as IL8, IL6, CXCR1, and CXCR2, which have been implicated in both immunosuppression and tumor survival. Increases in the expression of immune checkpoint markers, such as PD-L1, TIGIT and CTLA4, were also seen suggesting a worsening immunosuppressive microenvironment during progression from BE to EAC. To further investigate specific changes in the infiltration of CD8 T-cell and T-regulatory cell populations during EAC progression, we performed standard immunohistochemical staining on tissue microarrays from 122 direct-to-surgery EAC patients. Staining for T-cell markers CD3, CD4, CD8, and FoxP3 revealed that only CD8 positive T-cells were increased around tumor cells during progression. Further characterization of individual immune cells was performed through multiplex immunohistochemistry, allowing for the evaluation of multiple immune cell markers within the same tissue section. Multiplex staining for surface expressed markers (CD3, CD8, FoxP3, PD-L1, PanCK, CD163, DAPI), allowed for the quantification and localization of a variety of immune cell types within the EAC microenvironment. In general, the immune cell populations (T-regs, CD8 T cells, and macrophages) increase during progression from BE to BE w/ High Grade Dysplasia. In contrast, EAC tumor sections were relatively immune poor, indicating that the immune microenvironment may often be compromised in cancer. These data collectively demonstrate the EAC microenvironment is characterized by poor infiltration of cytotoxic effector cells, increased expression of immune inhibitory signals from T-regs, inhibitory immune checkpoints and cytokines such as IL-8 and IL-6. These findings suggest an immune suppressive microenvironment which highlights the need for further studies to explore the potential of immune modulating therapy in EAC.

#4959

The gut microbiota contributes to the effectiveness of HER2-targeted therapy.

Martina Di Modica,1 Viola Regondi,1 Giorgio Gargari,1 Arianna Bonizzi,2 Stefania Arioli,2 Beatrice Belmonte,3 Claudio Tripodo,3 Simone Guglielmetti,2 Fabio Corsi,4 Tiziana Triulzi,1 Elda Tagliabue1. 1 _Fondazione IRCCS Istituto Nazionale dei Tumori - Milano, Milano, Italy;_ 2 _Università degli Studi di Milano, Milano, Italy;_ 3 _Università degli Studi di Palermo, Palermo, Italy;_ 4 _ICS Maugeri S.p.A. SB, Pavia, Italy_.

Recently, the composition of the gut microbiota, due to its influence on host immune system, has been linked to the effectiveness of chemotherapy and immunotherapy. Since trastuzumab, besides inhibiting the HER2 signaling, recruits innate and adaptive immune cells that mediate its cytotoxic activity in the tumor, we hypothesized that commensal bacteria can be a source of heterogeneity for the response to therapy in patients with HER2-positive breast cancer (HER2+BC).

The impact of the gut microbiota on anti-HER2 therapy was studied in mice with the intestinal flora alterated by the treatment with vancomycin or streptomycin-two broad spectrum antibiotics poorly absorbed in the intestine. The association between commensal bacteria composition and clinical efficacy of trastuzumab was investigated in a cohort of HER2+BC patients treated with neoadjuvant trastuzumab.

Administration of antibiotics impaired the efficacy of anti-HER2 monoclonal antibodies both in FVB and BALB/c mice bearing syngeneic mammary carcinomas expressing HER2. 16S rRNA gene profiling of FVB mouse feces showed that both antibiotics decreased bacterial α-diversity in the gut as evaluated by Chao1, Simpson and Shannon indices, lowering the abundance of Clostridiales bacteria. Mice transplanted with feces from antibiotic treated mice did not benefit from the anti-HER2 treatment supporting a direct relation between intestinal bacteria and therapeutic efficacy. Analysis by flow cytometry and immunohistochemistry of tumors grown in mice showed that alteration of gut microbiota compromised the recruitment of CD4+ T cells and Natural Killer (CD49b+, GZMB+) cells upon anti-HER2 administration. Fecal 16S rRNA gene sequencing demonstrated a significantly higher microbial α-diversity in patients who achieved a pathological complete response compared to non-responders using several indices. Moreover, a clustering effect by patient's response was observed visualizing the β-diversity. OTUs belonging to the Clostridiales and Bacteroidales orders were reduced and enriched, respectively, in non-responders.

Our data support that the composition of the gut microbiota, especially as regards the abundance of Clostridiales bacteria, has a role in the therapeutic efficacy of trastuzumab both in mice and patients. Therefore, the manipulation of intestinal bacteria may represent a new strategy to improve the cure of HER2+BC patients.

(Supported by Associazione Italiana per la Ricerca sul Cancro).

#4960

Studying the impact of gut microbiome to hepatocellular cancer in an orthotopic murine model.

Xiaoqiang Qi, Ming Yang, Joseph Stenberg, Guangfu Li, Kevin Staveley-O'Carroll. _University of Missouri, Columbia, Columbia, MO_.

Background and Objective: As one of the most important components of human body, gut microbiota play a vital role in human health by participating in metabolomics and maintaining immunological homeostasis. Microbiota dysbiosis is significantly associated with initiation and progression of different diseases. Due to close anatomical and functional connection between gut and liver (gut-liver axis), gut microbiota are found to impact the development of liver diseases including hepatocellular cancer (HCC) without fully understanding mechanisms. The overall objective of present study is to investigate how microbiota modulate HCC growth by regulating intrahepatic immune response.

Methods: Administration of carbon tetrachloride in combination with transplant of oncogenic hepatocytes by intrasplenic injection was used to make an orthotopic mouse model of HCC. A specific antibiotics cocktail (ABX) was selected to inhibit some kinds of gut microbiota. 16s rRNA sequencing was used to characterize gut microbiota profile in mice with the different treatments. Magnetic resonance imaging (MRI) was used to monitor tumor growth. Adoptive transfer of tumor antigen-specific CD8+ T cells following the treatment was used to investigate how ABX administration impacted intrahepatic immune response. Flow cytometry and multiplex cytokine assay were used to detect the alteration of effector CD8+ T cell in phenotype, proliferation, and function. Immunohistochemistry (IHC) was used to investigate immune cell tumor-infiltration.

Results: The established murine model reflects the typical features of human HCC. ABX oral administration significantly changed gut microbiota composition, particularly in abundance of P. aeruginosa and Muribaculaceae. ABX-mediated alteration of gut microbiota significantly retarded HCC progression. Interestingly, ABX treatment was found to prevent adoptively transferred tumor antigen-specific CD8+ T cell from tumor-induced immune tolerance, as higher frequency of effector CD8+ T cell was detected in ABX-treated tumor-bearing mice compared to that in control tumor-bearing mice. Correspondingly, ABX treatment resulted in the increase in expression of CD69 and enhanced production of IFN-γ in tumor antigen-specific CD8+ T cells relevant to that in control mice.

Conclusion: Modulating microbiota with antibiotics is able to shape intrahepatic immune responses and therapeutically suppress tumor growth, providing a new option in HCC control.

#4961

Gut microbiota modulates adoptive cell therapy via CD8α dendritic cells and IL-12.

Mireia Uribe-Herranz,1 Kyle Bittinger,2 Stavros Rafail,1 Stefano Pierini,1 Sonia Guedan,1 Frederic Bushman,1 Carl June,1 Andrea Facciabene1. 1 _University of Pennsylvania, Philadelphia, PA;_ 2 _Children's Hospital of Philadelphia, Philadelphia, PA_.

Adoptive T cell therapy (ACT) is a promising new modality for malignancies. Here, we report that adoptive T cell efficacy in tumor-bearing mice is significantly affected by differences in the native composition of the gut microbiome or treatment with antibiotics, or by heterologous fecal transfer. Depletion of bacteria with vancomycin decreased the rate of tumor growth in mice from The Jackson Laboratory receiving ACT, whereas treatment with neomycin and metronidazole had no effect, indicating the role of specific bacteria in host response. Vancomycin treatment induced an increase in systemic CD8α+ DCs, which sustained systemic adoptively transferred antitumor T cells in an IL-12-dependent manner. In subjects undergoing allogeneic hematopoietic cell transplantation, we found that oral vancomycin also increased IL-12 levels. Collectively, our findings demonstrate an important role played by the gut microbiota in the antitumor effectiveness of ACT and suggest potentially new avenues to improve response to ACT by altering the gut microbiota.

#4962

**Repurposing** bazedoxifene **to suppress gastrointestinal cancer growth.**

Pathum Thilakasiri,1 Tracy Nero,2 Michael W. Parker,2 Matthias Ernst,1 Ashwini L. Chand1. 1 _Olivia Newton John Cancer Research Institute, Melbourne, Australia;_ 2 _Bio21, Melbourne, Australia_.

The interleukin (IL)6 family of inflammatory cytokines, characterized by the shared use of the gp130 receptor, supports the progression of solid cancers. Specifically, gp130 signaling becomes rate limiting for the growth of gastrointestinal tumors arising from oncogenic driver mutations in Apc, but remains dispensable for the homeostatic renewal of the gastrointestinal mucosa. In the present study we investigate the effects of the selective estrogen receptor modulator bazedoxifene, clinically approved for the treatment of osteoporosis, as a putative small molecule inhibitor of gp130 receptor oligomerization and activation. We assessed the effects of bazedoxifene on IL6 and IL11-dependent STAT3 activation and cell proliferation in gastric and colon cancer cell lines. In patient-derived colon cancer organoid cultures, we assessed bazedoxifene effects on IL11-dependent growth. The gp130Y757F, Lgr5CreERT2\- or Cdx2CreERT2-driven Apc flox mouse models of gastrointestinal cancer, were used to examine the effects of bazedoxifene on tumour growth. In silico modeling suggests that the inhibitory activity of bazedoxifene is due to its ability to mimic the interaction between the Site III interface of gp130 receptor and the tryptophan-157 in IL6, or tryptophan-168 in IL11 cytokine. We show that bazedoxifene interferes with IL6 and IL11-dependent proliferation of BAF/03 cells. Bazedoxifene suppresses gastric tumor growth in gp130Y757F mice irrespective of their gender, in which tumors arise through excessive IL11-dependent STAT3 signaling. Strikingly, in sporadic colon cancer models based on aberrant activation of the β-catenin/canonical WNT pathway, bazedoxifene treatment also reduces tumor burden in mice following conditional ablation of the Apc suppressor gene using the Lgr5CreERT2\- or Cdx2CreERT2-driver alleles. Consistent with our observation that nuclear accumulation of β-catenin remains unaffected in colonic tumors of bazedoxifene-treated mice, bazedoxifene treatment of human SW480 colon cancer cells harboring mutant APC did not affect canonical WNT signaling, but suppressed IL11-dependent STAT3 signaling. Our findings provide compelling proof-of-concept for the repurposing of bazedoxifene for the treatment of gastrointestinal cancers that arise from bona fide oncogenic driver mutations.

#4963

A novel intestinal microbiome-derived peptide modulates host T cell activation.

Yuliya V. Katlinskaya, Dhwani Haria, Lily McLaughlin, Sunit Jain, Shoko Iwai, Todd DeSantis, Thomas Weinmaier, Toshi Takeuchi, Anu Hoey, Karim Dabbagh, Kareem Graham, Helena Kiefel. _Second Genome, South San Francisco, CA_.

The microbiome shapes the metabolic and immunological landscape of individuals in health and disease and represents a new robust source of bioactive molecules with therapeutic potential. Second Genome's large and curated microbiome database coupled with its proprietary bioinformatics and machine learning pipeline enables the discovery of novel microbiome-derived drug candidates across multiple disease areas, including immuno-oncology (IO). Microbial genus Bifidobacterium previously showed a positive association with antitumor T cell responses in mouse models and was overrepresented in melanoma patients responding to immunotherapy with anti-PD-1 antibody. Our hypothesis is that bioactive molecules derived from these Bifidobacteria help drive these antitumor responses and could function as cancer therapeutics or adjuvants for cancer immunotherapy. Thus, we nominated protein and peptide candidates with high scores of secretability, uniqueness, stability, and expressability from Bifidobacterium breve and Bifidobacterium longum genomes for screening in cell-based assays. Initial evaluation of more than 50 Bifidobacterium peptides revealed candidates capable of inducing immune activation. Here, we describe a B. breve-derived peptide (termed SG-B) that induced up-regulation of co-stimulatory (OX40 and ICOS) and inhibitory (PD-1) molecules on CD4+ and CD8+ T cells in both: i) a purified human pan-T cell system; and ii) PBMC cultures stimulated with low-dose anti-CD3 antibody. Furthermore, SG-B stimulated secretion of effector cytokines by in vitro-cultured purified T cells (TNF-a, IL-2, IFN-g, and IL-10) and PBMCs (IL-1b, IL-6, and IL-8). These effects were dose-dependent and evident across multiple human blood donors. In the CT26 syngeneic tumor model, peri-tumoral administration of SG-B induced an increase in the proportion of NK cells and CD4+ T cells within the tumor. The peptide also induced up-regulation of activation marker on CD8+ T cells (CD25, Ki67, and OX40). Collectively, these data suggest that SG-B can modulate adaptive immunity via T cell potentiation and may enhance tumor inflammation in vivo. The peptide's ability to up-regulate key co-stimulatory and checkpoint molecules on T cells provides a strong rationale for its potential future use in combination with approved or IO agents in current development. These results validate the capability of the Second Genome drug discovery platform to identify novel microbial agents of potential therapeutic relevance in IO and demonstrate a unique approach that can identify microbial factors involved in modulating immune cell effector functions and/or immune cell differentiation.

#4964

Associations between hepatitis etiology and immune cell infiltration in or around hepatocellular carcinoma.

Yu-Yun Shao, Min-Shu Hsieh, Yun-Ting Tsai, Ying-Chun Shen, Zhong-Zhe Lin, Ann-Lii Cheng, Chih-Hung Hsu. _National Taiwan University Hospital, Taipei, Taiwan_.

Background: Previous studies have demonstrated that the abundance of immune cells may be associated with hepatocellular carcinoma (HCC) prognosis. Because HCC has various hepatitis etiologies, we studied the associations between hepatitis etiology and regional lymphocyte infiltration at and beside the tumor.

Methods: We collected tumor samples from patients who received curative surgery for HCC. Immunohistochemical staining was performed on tissue slides, which were then scanned using TissueFAXS v4.2 and analyzed using HistoQuest v4.0 (both from TissueGnostics Inc.). We calculated the amount of total T cells (CD3 positive), CD8 T cells (CD8 positive), and macrophages (CD68 positive) inside the tumor (IT), at the tumor margin (TM), and outside the tumor (OT). For analysis at IT and TM, the whole area were examined. For analysis at OT, only the five areas with the highest cell density at the low power fields were analyzed. For each area, we defined low and high cell density according to the median values. We then explored the associations of these cell distributions and HCC etiologies or prognosis.

Results: We included HCC samples from 120 patients. Among them, 76 (63%) had hepatitis B virus (HBV) infection, 28 (23%) had hepatitis C virus infection, 14 patients had neither infections, and 1 patient had both HBV and HCV infection. The amount of total T cells (p < 0.001), CD8 T cells (p < 0.001), and total macrophages (p < 0.001) were all significantly higher at TM than at IT. HCV-positive tumors, compared to HBV-positive tumors had more total T cells inside (p = 0.040) and outside the tumors (p < 0.001), but similar total T cells at the margin. On the contrary, the amount of CD8 T cells and total macrophages were similar between HBV-positive and HCV-positive tumors at IT, TM, and OT. Samples of 112 patients included both IT and TM area. Using CD8 cell counts for classification, 40 (33%) were IThighTMhigh, 16 (13%) were ITlowTMhigh, 42 (35%) were ITlowTMlow, and 14 (12%) were IThighTMlow. Patients with ITlowTMlow tumors tended to have poorer overall survival than other patients (p = 0.085), but patients with IThighTMhigh, ITlowTMhigh, and IThighTMlow tumor had similar prognosis (p = 0.770). Hepatitis virus infected tumors were significantly less likely to be ITlowTMlow than hepatitis virus uninfected tumors (33% vs. 69%, p = 0.010); HBV-infected and HCV-infected tumors had similar percentages of ITlowTMlow tumors (68% vs. 65%, p = 0.837).

Conclusions: HCV infection was associated with higher total T cell infiltration inside and outside the tumors. Hepatitis virus uninfected tumors were more likely to have low CD8 cells both inside and at the margin of HCC.

#4965

Extracellular matrix gene expression and cytotoxic T lymphocyte infiltration in the tumor microenvironment in non-small cell lung cancer.

NA LI, Hongzhe Sun, Xin Wang, Zhifu Zhang, Ying Zhou, Courtney Anderson, Xiao-Jun Ma. _Advanced Cell Diagnostics, Inc., Newark, CA_.

Immunotherapy has proven to be a powerful anti-tumor therapy, harnessing the body's own immune system to target and kill tumor cells. However, immunotherapy is not successful in all cancer patients due to both intrinsic non-responsiveness and adaptive resistance. Developing predictive biomarkers and understanding mechanisms of resistance are major goals of the immuno-oncology community. The extracellular matrix (ECM), an important factor for promoting tumor growth, survival, and migration of tumor cells, can also act as a physical barrier to prevent immune cell infiltration and promote tumor immune escape. Components of the ECM such as COL11A1, COL4A1, and LOXL2 have been shown to be associated with cancer progression. Furthermore, new data suggests that TGFβ activation leads to up-regulation of ECM genes in cancer-associated fibroblasts and immune suppression. However, it remains poorly understood which cells in the tumor microenvironment (TME) are the sources of ECM gene expression and how they are related to tumor infiltrating cytotoxic T lymphocytes (CTLs). In this study, we employed a highly sensitive and specific RNAscope in situ hybridization (ISH) duplex assay to directly visualize the tissue distribution of cells expressing COL4A1, COL11A1, LOXL2, and TGFB1 in relation to tumor infiltrating CTLs in non-small cell lung carcinoma (NSCLC). NSCLC tissue microarrays (TMAs) consisting of 63 independent patient FFPE tumor samples were analyzed using this ISH assay with the following probe combinations: Hs-CD8/Hs-IFNG, Hs-CD4/Hs-FOXP3, Hs-LOXL2/Hs-COL4A1, and Hs-TGFB1/Hs-COL11A1. We observed COL4A1 expression in both tumor and tumor-associated stromal cells in different samples. In contrast, COL11A1 was only expressed in tumor-associated stromal cells. Interestingly, high COL4A1 expression was associated with high CD8+ T cell infiltration, whereas high COL11A1 expression was associated with poor CD8+ T cell infiltration. In addition, tumor expression of TGFB1 was positively correlated with COL11A1 expression. These data depict a complex landscape of ECM gene expression and their relationship to T cell infiltration in the tumor and TME. Taken together, these results demonstrate that the RNAscope assay provides a powerful approach to directly examine the interactions between tumor, ECM, and T cell immune infiltration, and offers advantages over immunohistochemistry (IHC) for identifying the cellular sources of secreted proteins such as ECM components in the TME.

#4966

Characteristics of immune repertoires in early lung cancer patients.

Jin Wang,1 Xihao Hu,1 Peng Zhang,2 Xiaole Shirley Liu1. 1 _DFCI, Boston, MA;_ 2 _Shanghai Pulmonary Hospital, Shanghai, China_.

Lung cancer is the leading cause of cancer death in the world, and early lung cancer detection is vital for improving the overall survival. Besides x-ray and CT scan, few genetic tests have been widely used for detecting early lung tumors. We hypothesize that early lung cancer patients have some unique features in the immune repertoires which can be utilized for early cancer detection and profiling disease development. In this study, we performed RNA-seq for paired tumor and tumor-adjacent normal samples from 59 early lung adenocarcinoma patients. We found that early lung tumors have more cytokine activities, higher B cell and CD4+ T cell infiltration, and lower CD8+ T cell infiltration compared to normal lung tissues. We assembled B cell receptors from RNA-seq reads and found more B cell clonal expansion and somatic hypermutations in the tumor than in tumor-adjacent normal tissues, similar to the B cell repertoires in late-stage lung cancer patients. In both early- and late-stage lung tumors, we identified a few cytokines that positively correlated with more IgA isotypes and associated with patient survival. To gain more insights on the B cell binding specificity, we performed motif enrichment analysis for paired tumor and normal samples and validated some tumor-enriched receptor motifs in matched patient blood samples, which implies a new way of early cancer detection. More interesting, we assembled microbial transcripts from RNA-seq data and identified some known lung disease-related species in both tumor and normal samples. The complex interactions between cancer cells, B cells, and microbiota in the early lung tumor development are waiting to be elucidated.

#4967

Neoadjuvant chemotherapy promotes a transient increase of intratumoral T-cell density in colorectal liver metastases.

Kjersti Flatmark,1 audun E. Berstad,1 Vegar Dagenborg,1 Bjørn Edwin,1 Åsmund A. Fretland,1 Krzysztof Grzyb,1 Eirik Høye,1 Marius Lund-Iversen,1 Serena E. Marshall,1 Anne H. Ree,2 Sheraz Yaqub1. 1 _Oslo University Hospital, Oslo, Norway;_ 2 _Akershus University Hospital, Oslo, Norway_.

Background: In metastatic colorectal cancer, only the microsatellite instable (MSI) subgroup of cases has so far demonstrated major responses to immune checkpoint inhibition. Increased infiltration of T lymphocytes in presumed non-responsive tumors has been shown to facilitate response to immune checkpoint inhibition. Cytotoxic chemotherapy might induce immunogenic cell death and cause infiltration of immune cells, turning "cold" tumors into "hot". In this work we investigated whether standard-of-care neoadjuvant chemotherapy (NACT) would influence T-cell infiltration in the tumor microenvironment of colorectal liver metastases (CLM) in a substudy of patients included in the OSLO-COMET trial (n=280; NCT01516710).

Materials and methods: Patients (n=92; bearing 144 metastatic tumors) had resectable CLM, and of these, 45 patients received NACT, in most cases fluoropyrimidine-based, with the addition of oxaliplatin (n=36) or irinotecan (n=8). Quantification of total T-cells (Ttot), cytotoxic T-cells, T-helper cells, and regulatory T-cells was performed by immunohistochemical staining of serial sections with antibodies against CD3, CD8, CD4, and FOXP3, respectively. Hotspots from the invasive margin (IM), intratumoral region (IT), and adjacent liver tissue (NLi) were analyzed. T-cell density was reported as number of T-cells/mm2.

Results: The density of Ttot in the IM region (median 2360 cells/mm2) was 7 times greater than in the IT region (319 cells/mm2) and 13 times greater than in the NLi region (183 cells/mm2), with similar findings for T-cell subtypes. Comparing patients that had received NACT to patients that had not, there was no difference in T-cell infiltration in the IM and NLi regions, but a non-significant trend towards increased T-cell density was detected in the IT region after NACT, exemplified by Ttot 353 vs 292 cells/mm2 (p=0.2). Exploring the influence of the time interval between NACT completion and surgery by receiver operating characteristic curve analysis, 9.5 weeks was identified as the time point that separated the cases most clearly by T-cell density. Interestingly, cases with a shorter than 9.5-week interval had a significantly higher intratumoral T-cell count than cases with the longer interval; with median Ttot 491 vs 236 cells cells/mm2; p<0.0001. Similar findings were detected for all T-cell subsets in the IT region.

Conclusion: The main finding of this work was that NACT administration was associated with a transient increase of intratumoral T-cell density in CLM, potentially promoting an MSI-like tumor immune phenotype. The results suggest that when exploring combinations of cytotoxic therapy and immune checkpoint inhibition, the timing of individual treatment components may be essential. To further optimize treatment schedules, biomarkers reflecting T-cell infiltration may be a useful tool for tailoring personalized therapy.

#4968

Intratumoral heterogeneity and immune checkpoint inhibitor response in the KPC pancreatic cancer model.

Sarah Martinez Roth. _Georgetown University, Washington, DC_.

Cancer cells are subjected to evolutionary selection and adaptation of clonal populations by changes in the tumor and metastatic microenvironment as well as their response to drug treatment. We wished to evaluate how this tumor heterogeneity impacts the efficacy and responses to immune checkpoint blockade. To understand the contribution of clonal subpopulations to the malignant progression and to the response to drugs, we established a model of tumor heterogeneity from six syngeneic, clonal primary cancer cells isolated from a p48-CRE X LSLmutKras X LSLmutP53 mouse pancreatic cancer (KPC). The clonal cell lines were characterized for genomic mutations, gene expression and tumor allografts reconstituted in syngeneic host mice from mixes of the clonal cell lines. Each of the clonal cells formed invasive and metastatic lesions when grafted into hosts. The original tumor and clonal cell lines harbored common mutations in 99 genes demonstrating their common ancestry. Additional unique mutations in the clonal lines were used to identify and quantitate clones in heterogeneous cell pools. The clones showed different levels of MAP kinase signaling, unique morphologies, different growth rates in vitro and tumor growth rates in immune competent mice. Moreover, the sensitivity to ~200 anticancer drugs revealed an up to 25-fold varying in vitro sensitivity of the clones to signal transduction inhibitors and cytotoxic drugs. Drug sensitivity of individual clones when included in a heterogeneous cell population was strikingly different from their drug sensitivity when growing on their own including the sensitivity of different clones to MEK or PI3K inhibition. Furthermore, the sensitivity of clones to an anti-PD1 checkpoint inhibitor was distinct across the clonal cells growing in the heterogeneous mixture: Some clones were resistant and others highly sensitive to the checkpoint blockade. We will discuss pathways and drivers of resistance as well as sensitivity in the different subpopulations. We conclude that malignant progression and selection of checkpoint inhibitor sensitive cancer cell subpopulations is impacted by the crosstalk between clonal cell populations present in heterogeneous tumors and the host environment.

#4969

TIGIT pathway phenotyping sheds light on promising strategies to restore anti-tumor immunity.

Noémie Wald,1 Marjorie Mercier,1 Julia Cuende,1 Florence Nyawouame,1 Shruthi Prasad,1 Margreet Brouwer,1 Erica Houthuys,1 Anne Marie-Cardine,2 Martine Bagot,2 Grégory Driessens,1 Véronique Bodo,1 Catherine Hoofd1. 1 _iTeos Therapeutics, Gosselies, Belgium;_ 2 _Inserm, Gosselies, France_.

TIGIT is a T cell co-inhibitory receptor recently described as a key checkpoint driving tumor cell immunosuppression. It is predominantly expressed on CD4+ Tregs, CD8+ T cells and NK cells from healthy individuals but further upregulated in cancer patients where it frequently co-expresses with exhaustion markers such as PD-1. TIGIT cognate receptors are members of the poliovirus receptors, among which CD155 has the highest affinity for TIGIT. They are expressed on antigen presenting cells but also on tumor cells which provides a strong rationale for blocking TIGIT as a therapeutic approach to reverse T or NK cell dysfunction linked with cancer progression.

To substantiate the importance of TIGIT as an immunotherapy target, we used flow cytometry and immunohistochemistry (IHC) to extensively characterize TIGIT and CD155 expression in both healthy donors and cancer patients.

We initially confirmed TIGIT expression on multiple immune subsets from healthy donors. Similar flow cytometry analysis performed on matched circulating and tumor-infiltrating immune populations from 15 cancer patients highlighted the global overexpression of TIGIT associated with cancer. Interestingly, ex vivo polyfunctional analysis of cytokine release strongly supported the immunosuppressed character of infiltrated TIGIT positive CD4+ and CD8+ T cells. Finally, combining receptor density with frequency of positive cells led to the interesting observation that tumor-infiltrating Tregs represents the population with the highest TIGIT expression, confirming the opportunity to preferentially target that suppressive population within the tumor microenvironment. These findings on Tregs were further confirmed by IHC. TIGIT was expressed in 10 out of the 11 lung adenocarcinoma tissues analysed. Both Tregs and CD8+ T cells express TIGIT, with Tregs showing a higher proportion of TIGIT+ cells.

Finally, direct expression of TIGIT on tumor cells was also found on several haematological malignancies, opening the door for anti-TIGIT therapies to act directly on tumor cells besides revigoration of the immune system.

The presence of TIGIT ligands within the tumor microenvironment was also assessed and confirmed using both flow cytometry and IHC. CD155 expression was analysed by IHC in both normal individuals (n=90) and cancer patient tissues from 9 different cancer indications (n=284). In cancer tissues, CD155 is mostly expressed by tumor cells, ranging from a median percentage of 50 % for pancreatic cancer to 2 % for cervix cancer. Cancer samples with the highest CD155 expression are pancreatic, prostate, kidney, gastric and colon cancers. CD155 expression is always lower in normal tissues compared to their cancer counterparts except for lung.

Together, these data strongly support the relevance of targeting TIGIT in immuno-oncology and pave the way to select the ideal indications where patients could benefit from such a therapy.

#4970

Establishment of a patient-derived platform for preclinical immunooncology studies using autologous tumor and TIL coculture.

Kaede Hinada, Dennis Garland, Dileep Nair, Yong Hu, Thomas Broudy. _BioDuro LLC, San Diego, CA_.

The use of tumor infiltrated lymphocytes (TILs) as an adoptive cell transfer therapy to treat cancer has been conducted in clinic trials for more than two decades. TIL therapy in combination with added immunotherapy treatments, such as IL-2 or anti-CTLA4, has further improved clinical response rates. Isolated from a patient's own tumor specimen, TILs that recognizes tumor-specific known or unknown antigens can be expanded and infused back to tumor-bearing patients to attack autologous cancer cell populations. Despite the progress in clinical research, preclinical and translational models of autologous tumor/TIL coculture have been difficult to establish.

This study has established autologous tumor/TIL coculture methods directly from patient tumor specimens. Models were established from BioDuro's viably cryopreserved tumor & TIL bank, containing more than 100,000 patient biospecimens. Each patient specimen contains a heterogeneous population of cells types, including tumor, TILs and TAFs (tumor associated fibroblasts). These cell populations were expanded under specific growth conditions to then serve as components for the patient-derived coculture systems established.

In this study, TILs and monocytes from a cohort of patient tumor specimens (including kidney, melanoma and lung) were analyzed. Leukocytes, T cells, monocytes and B cells were marked with CD45, CD3, CD14, CD20. Patients' samples were also tested for PD-1 expression levels. The results show significant individual variation on cell subpopulations and PD-1 expression. Established methods were used for expansion of TILs, supporting either CD4 or CD8 phenotypes. Autologous cancer cells were expanded at the same time. TILs and tumor cells were co-cultured and high content fluorescent image cytometry was used to monitor tumor cell killing. This study found that autologous patient-matched TILs showed preferential tumor cell killing compared to patient-unmatched TILs. This response was observed in a dose dependent fashion.

In summary, the BioDuro biobank of viable tumor and TIL patient specimens served as a large and powerful immunooncology resource to establish patient-derived autologous tumor/TIL coculture assays. These assays, and the ability to readily study larger populations of cancer patients, provide a powerful in vitro platform to test immunooncology drug candidates.

#4971

Dominant negative PD 1 armored CAR T cells induce remission in refractory diffuse large B-cell lymphoma (DLBCL) patients.

Tong Chen. _Department of Hematology, Huashan Hospital, Shanghai, China_.

Anti-CD19 chimeric antigen receptor (CAR19) autologous T cell treatment has been demonstrated as effective therapy to relapse/refractory B cell leukemia and lymphoma. However, CAR T shows limited efficacy in treatment of solid tumor or challenging with immunosuppressive characters, such as PD-L1+/hi lymphomas.

PD-1/PD-L1 blockade have been shown as potentially effective anti-tumor therapies. We generated a lenti-vector incorporating an anti-CD19 secondary CAR molecule sequence with 4-1BB co-stimulatory domain and a dominant negative PD-1 sequence (Figure A). Compared with conventional CART cells, these "armored" CART cells show enhanced capability of tumor killing after multiple-round challenging by PD-L1+CD19+ positive tumors ,with a "emory-like" phenotype(Figure B), suggesting dominant negative PD-1 molecule may protect CART cells from inhibition in tumor micro-environment.

Here we report 2 cases of refractory diffuse large B cell lymphomas (DLBCLs) successfully treated with this novel dominant negative PD-1 armored humanized anti-CD19 CART cells. These two patients all failed to achieve response after multiple rounds of chemotherapy and radiotherapy, and infused with autologous CAR T cells at 5.23×10^6/kg and 1.97×10^6/kg during disease progress. Both patients showed significantly tumor mass decrease and SUV max decline in PET/CT results with ongoing response. (From 34.48 to 3.89 at day 27, from 25.02 to 2.38 at day 31, respectively.) (Figure C).

Conclusion: These 2 cases revealed the significant anti-bulky lymphoma response of new type of CAR19 T cells with limited and tolerated cytokine release syndrome and central nervous system toxicity. This novel structure of dnPD1-hCAR19 may augment CART cells persistence in the patient after activation triggered by the lymphoma cells, suggesting that dnPD-1 molecule may enhance efficacy of CAR T cells in treatment of solid tumor. We are continuing to recruit more patients in clinical research.

#4972

Preclinical development of a first-in-class NKp30xBCMA NK cell engager for the treatment of multiple myeloma.

Monia Draghi,1 Jamie L. Schafer,1 Allison Nelson,1 Zach Frye,1 Amanda Oliphant,1 Sara Haserlat,1 jason Lajoie,1 Kenneth rogers,2 Francois Villinger,2 Michael Schmidt,1 Robert Tighe,1 Piotr Bobrowicz,1 Jennifer Watkins-Yoon,1 Thomas Schuetz1. 1 _Compass Therapeutics, Cambridge, MA;_ 2 _New Iberia Research Center, University of Louisiana at Lafayette, New Iberia, LA_.

Multiple myeloma (MM) is characterized by clonal expansion of malignant plasma cells. Although several new drugs for the treatment of MM have greatly improved survival, many patients are known to relapse and become refractory to all presently available therapies or experience treatment-related toxicities. Therefore, MM remains an unmet medical need and the development of additional novel therapies is required. NK cells play a crucial role in the control of multiple myeloma and accumulating evidence shows the presence of highly cytotoxic NK cells in the bone marrow of MM patients suggesting that targeting NK cells could provide a specific treatment modality to leverage NK cell cytotoxicity in myeloma. Furthermore, NK cells are the first lymphocytes population to reconstitute after autologous stem cell transplant (ASCT) providing an opportunity to target minimal residual disease (MRD) shortly after transplant. B-cell maturation antigen (BCMA) is an excellent target in MM because its restricted expression in normal and malignant plasma cells from untreated and relapsed myeloma patients, but absent in all other main bone marrow cell subsets. Our first-in-class NKp30xBCMA NK cells engager, CTX-4419, binds to BCMA on MM cells and to NKp30 and CD16A (FcγRIIIA) on NK cells, specifically redirecting NK cells towards tumor cells expressing BCMA. We demonstrate here that CTX-4419 retains activity in the presence of high levels of BCMA ligands and serum IgG and induces potent NK cytotoxicity against high and low BCMA expressing cell lines as well as patients (autologous) primary myeloma cells. Moreover, CTX-4419 does not require CD16A binding to kill tumor cells, a unique characteristic that overcomes the reduction or loss of activity of CD16A due to receptor shedding or downregulation in the tumor microenvironment. Furthermore CTX-4419 induces NK proliferation and cytokines/chemokines production by NK cells only in the presence of tumor cells providing sustainable anti-tumor specificity to NK cells. CTX-4419 activates in-vitro cynomolgus NK cells in presence of target cells expressing cynomolgus BCMA and, when given intravenously, CTX-4419 decreases plasma cell counts. CTX-4419 represents a novel class of NK-cell engagers that shows strong potency even in the absence of CD16A engagement and induces NK cell proliferation and lysis of tumor cells expressing low amount of antigen. CTX-4419 has strong activity in an autologous setting when tested in bone marrow samples of MM patients and shows efficacy in a non-human primate model of plasma-cell depletion. These data show that CTX-4419 is strongly differentiated from conventional therapeutic antibodies and is a promising candidate for MM treatment with the potential to be used as monotherapy or in combination with adoptive transfer of NK cells and/or other immuno-therapies.

#4973

The immunosuppressive tumor microenvironment of metastatic osteosarcoma inhibits the cytotoxic effect of tumor-infiltrating lymphocytes.

John A. Ligon, Teniola F. Oke, Woonyoung Choi, Megan H. Fong, Adam Levin, Daniel S. Rhee, Carol D. Morris, Nicholas Siegel, Emily H. Hsieu, Christian F. Meyer, David J. McConkey, Robert A. Anders, Drew M. Pardoll, Nicolas J. Llosa. _Johns Hopkins University School of Medicine, Baltimore, MD_.

Background: Patients with metastatic osteosarcoma (OS) have a 5 year overall survival of <25%. We aim to understand the immune architecture of the tumor microenvironment (TME) of OS, with the goal of harnessing the immune system as a major therapeutic strategy for the treatment of patients with OS.

Methods: Immunohistochemistry (IHC) slides from 66 formalin-fixed paraffin-embedded OS tissue blocks were analyzed. Laser capture microdissection and RNA extraction was performed on 13 OS specimens, and gene expression profiles of the tumor interior vs. tumor interface region were compared utilizing RNA seq. Tumor infiltrating lymphocytes (TILs) were isolated from 25 freshly obtained OS specimens and analyzed by multiparameter flow cytometry (MFC).

Results: Digital image analysis of IHC specimens revealed significantly higher immune cell infiltration (CD3+ and CD8+ cells) in the pulmonary metastases compared to primary bone tumors, particularly at the interface between the OS lesion and normal lung tissue. There is increased expression at the interface of the immune checkpoint molecules programmed cell death 1 (PD-1), T cell immunoglobulin mucin-3 (TIM-3) and lymphocyte activation gene 3 (LAG-3). Gene expression profiling showed increased CD8 T cell and resting CD4 memory T cell signature at the interface compared to the tumor interior, and a strong M2 and M0 macrophage signature in both regions. TILs isolated from pulmonary metastases and analyzed by MFC had higher expression of PD-1 and other immune checkpoint molecules, and these TILs were more capable of producing the effector molecules IFN-gamma and granzyme B.

Conclusions: Pulmonary metastatic OS lesions are more highly infiltrated with immune cells than primary bone lesions, particularly at the interface. The fact that these immune cells are shown via MFC to be capable of producing cytotoxic cytokines and proteases suggests that these TILs may be tumor-specific lymphocytes capable of acting against the tumor if they were not being inhibited by multiple immune checkpoint molecules. The inability of these immune cells to penetrate further into the tumor interior may represent an adaptive immune response by the tumor through a combination of repellant cytokines and inhibition of TILs via upregulation of PD-1, TIM-3 and LAG-3. Treatment with a combination of anti-PD-1, anti-TIM-3 or anti-LAG-3 directed treatments may unleash these immune cells and allow them to destroy OS tumor cells.

#4974

Germline prognostic SNPs and tumor-immunity-specific expression QTL (tis-eQTL) in cancer.

Xiaoqing Yu, Xuefeng Wang. _H. Lee Moffitt Cancer Center, Tampa, FL_.

Introduction: Immunotherapy has produced promising results in treating cancers. Recent studies that aim to predict which patients can benefit from an immune checkpoint blocker have been largely focused on tumor molecular profiling such as tumor mutation burden and T cell infiltrations. However, whether the immunity landscape in tumor can be favorably or adversely affected by polymorphisms carried in the germline remains unknown and understudied.

Methods: Here we conduct the largest investigation of tumor-immunity-specific expression QTL to date, or tis-eQTL, to systematically identify germline genetic variants that affect immune landscape in tumor. We develop a reliable statistical method for tis-eQTL mapping using RNA-seq data from heterogeneous tumor samples. We analyze genomic data from 10,380 cancer patients from 33 cancer types to reveal interactions between genetic variants (derived from germline SNP array and WES data) and immune- phenotypes derived from tumor RNAseq data. These phenotypes include immune gene expression (such as CD3E, GZMA, CXCR3 and PRF1), T-cell receptor (TCR) clonality, and antigen presenting scores (APM).

Results: We observed that stratifying patients by the prioritized pathogenic germline polymorphisms exposed distinct tumor immune landscape, implicating new prognostic factors or risk genes of cancer. We further prioritize SNPs that fulfill two criteria: (a) with differential overall survival (b) have a differential expression pattern in immunogenic signatures. In support of these findings, we show that the top-ranked SNPs are associated with treatment responses to anti-PD-1 therapy in melanoma and non-small cell lung cancer. This study creates a unique and pioneering resource of prognostic germline variants with the potential to modulate immune therapy response and cancer risk, opening new avenues for developing next generation therapeutics and for personalizing risk assessment.

#4975

Using humanized B-hSIRPa/hCD47 mouse model to evaluate the efficacy and toxicity of CD47 and SIRPa antibodies.

Yanan Li,1 Chaoshe Guo,2 Jie Xiang,1 Qingcong Lin1. 1 _Biocytogen, Wakefield, MA;_ 2 _Biocytogen, Beijing, China_.

Cancer immunotherapy is one of the most promising research areas in the field of cancer therapy. Along the IO drug development process, production of mouse models for in vivo efficacy evaluation has always been a rate-limiting step. Therefore, we generated a novel humanized knock-in mouse to evaluate the efficacy of human IO antibodies.

CD47 is a transmembrane protein expressed in many human tissues. SIRPa, which is expressed on phagocytic cells, was identified as the receptor of CD47. Engagement of SIRPa by CD47 serves as "do not eat me" signal, inhibiting the phagocytic activity of macrophages. Antibodies interfering with the CD47-SIRPa interaction block inhibition and promote tumor destruction. While CD47 is a potential target for antibody development, side effects associated with CD47 blockade have emerged as a major concern, especially anemia.

To evaluate the efficacy and toxicity of CD47 antibody, we generated a double humanized B-hSIRPa/hCD47 mouse. In this model, exon2 in mouse SIRPa and CD47 were replaced with the corresponding human sequences. In homozygous mice, mouse SIRPa and CD47 was absent and only human protein expression was detected, B-hSIRPa/hCD47 mice paired with genetically modified MC38-hCD47 cancer cells were used to screen CD47 antibodies by balancing their efficacy and toxicity. Antibodies with less toxicity exhibited minimal weight loss, minimal effects on blood cell and platelet production, and reduced ALT/AST elevation in mice. Thus, we demonstrated that these humanized mice could expedite and improve the development of safer CD47 antibodies.

Humanized B-hSIRPa/hCD47 mice can also be used to evaluate the efficacy of antibodies targeting SIRPa. We also generated triple humanized B-hPD-1/hSIRPa/hCD47 and B-hPD-L1/hSIRPa/hCD47 mice that would be useful to develop combination therapies of PD-1/PD-L1 and SIRPa/CD47 antibodies. Humanized B-hSIRPa/hCD47 mice are a promising in vivo efficacy model for the development of CD47 and SIRPa antibodies that can be advanced to human clinical trials.

#4976

Ex vivo **functional** **characterization and** in vivo **efficacy validation of human CD40 agonistic antibodies in the human CD40 knock-in model (CD40 HuGEMM).**

Daniel Xianfei He,1 Lei Zheng,1 Ruilin Sun,2 Annie Xiaoyu An,1 Jian Fei,2 Henry Qixiang Li,1 Davy Xuesong Ouyang1. 1 _Crown Bioscience, Inc., Taicang, Jiangsu, China;_ 2 _Shanghai Model Organisms Center Inc., Shanghai, China_.

CD40 receptor is a member of TNF-receptor superfamily, broadly expressed on the surface of antigen-presenting cells (APC) including B cells, dendritic cells (DC), macrophages, monocytes, and non-APC like platelets, endothelial cells and even some tumor cells. CD40 is essential for APC activation and signalling via ligation of CD40 ligand located on helper T cells and CD40 on APC to mediate licensing (activating) of APC. Ligation of CD40 on DCs, for example, can induce increased surface expression of costimulatory and MHC molecules, production of pro-inflammatory cytokines, and enhanced T cell activation. CD40 ligation on resting B cells can increase its antigen-presenting function and proliferation. In contrast to its critical role in the induction of effective innate and adaptive immune responses, CD40 signalling on certain malignant cells, particularly B cell lymphomas, triggers tumor apoptosis. The dual function of CD40 signalling provide unique opportunities for the use of agonistic CD40 antibodies in cancer immunotherapy. Indeed, several CD40 monoclonal antibodies are now in different stages of clinical development, some shown promising preliminary results in combination with other cancer immunotherapies. However, we are lacking preclinical models to evaluate efficacy of therapeutic CD40 agonistic antibodies, which usually don't cross-bind to mouse targets. To fill this gap, we developed a human CD40 knock-in model (CD40 HuGEMM), of which mouse exon 2-5 are replaced by human counterparts. These KI mice express a chimeric CD40 with human extracellular domain & mouse trans-membrane and intracellular sequences. We have characterized the human CD40 expression of CD40 HuGEMM on a broad spectrum of mouse cells, such as lymphocytes, macrophages, DCs, and endothelial cells. In addition, we confirmed that human CD40 antibodies bind to the chimeric m/hCD40 recombinant protein, and lead to increased expression of MHCII on the B cells. Finally, the in vivo efficacy study indicates that human CD40 antibody treatment lead to robust anti-tumor response and contribute to better survival of the mice. Taken together, our CD40 HuGEMM provides a powerful preclinical model to assess the efficacy of the human-specific CD40 agonistic antibodies. It may also serve as a model for proof of concept studies of CD40 agonistic antibodies with other therapies.

#4977

Dynamic transcriptional programs controlling single NK cell killing dynamics.

Matthew S. Hall, Joseph T. Decker, Emanuelle I. Grody, Rachel B. Blaisdell, Jacqueline S. Jeruss, Lonnie D. Shea. _University of Michigan, Ann Arbor, MI_.

NK cells have a vast heterogeneity in function and phenotype in vivo and their healthy function is critical in the prevention of nascent cancers. NK cell-based immunotherapies, such as adoptive NK cell therapy, are emerging and greater insights into the mechanism of NK cell activation could increase their efficacy. In vitro cytotoxicity assays have revealed that a small subset of NK cells kill the majority of cancer target cells, yet little is known about the dynamic state or triggers of these highly cytotoxic cells. Here, we present methodology for discovery of the molecular precursors and drivers of single NK cell killing events. We study the NK-92 cell line both as a model of primary NK cells and to gain direct insights for improving NK-92 based adoptive cell therapies. In our method, we first performed single cell RNA sequencing on NK-92 cells seeded sparsely on a monolayer of HeLa cancer cells along with a control NK-92 cell population. Clustering cells by principal component analysis revealed groups of NK cells that upregulated genes associated with NK cell cytotoxicity in the HeLa cell activated sample that are absent in the control group. Landmark genes associated with the cytotoxic NK cell clusters included PRF1, GZMK, and ITGB2. Utilizing live cell imaging, we identified categories of dynamic functional phenotypes in NK-92 cells exposed to a HeLa cell monolayer including non-motile and non-killing, highly motile but non-killing, single killing, serial killing, and exhausted. In order to link the systems-level single cell gene expression data to functional NK cell behaviors, we next performed high content live cell imaging of NK-92 cells bearing cas9-based endogenous genetic reporters as they surveilled and killed HeLa cells. Utilizing a custom analysis algorithm, we tracked the motility and killing activity of individual NK-92 cells, and correlated this activity to the dynamic gene expression. Next, we aim to identify the causality and sequence of transcriptional changes associated with NK cell killing and extend our results to the analysis of the critical transcription factor networks governing dynamic NK cell functional phenotypes. We note that the single cell functional discovery strategy that we have shown can be applied more broadly to other model systems of the cancer microenvironment.

### Immunomodulators and Response to Therapy

#4978

Digital spatial profiling of molecular responses to nanoparticle STING agonists identify S100A9 and B7-H3 as possible escape mechanisms.

John T. Wilson,1 Daniel Shae,1 Paula I. Gonzalez-Ericsson,2 Violeta Sanchez,2 JingJing Gong,3 Yan Liang,3 Douglas Hinerfeld,3 Joseph M. Beechem,1 Justin M. Balko2. 1 _Vanderbilt University, Nashville, TN;_ 2 _Vanderbilt University Medical Center, Nashville, TN;_ 3 _NanoString Technologies, Seattle, WA_.

Cyclic dinucleotide (CDN) agonists of stimulator of interferon genes (STING) activate innate immunity to increase tumor immunogenicity. However, the efficacy of CDNs is limited by drug delivery barriers, including inefficient transport to the cytosol where STING is localized. We recently developed STING-activating nanoparticles (STING-NPs), polymer vesicles designed for enhanced cytosolic delivery of the endogenous CDN ligand for STING, 2'3' cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). Intratumorally-administered (IT) STING-NPs significantly enhance the therapeutic efficacy of cGAMP and improve response to immune checkpoint inhibition (ICI) in established murine B16 melanoma tumors. However, the immunologic effects of STING-NPs have not been well characterized in tumors or lymphatic tissue.

We utilized Digital Spatial Profiling (DSP) to identify immunologic responses in the injected tumor, a distal tumor, and both tumor draining lymph nodes (TDLN) 48 hours after a single IT injection of STING-NP or three injections of STING-NP co-administered with systemic ICI (anti-PD-1/CTLA-4). DSP analysis permits simultaneous detection of over 30 protein markers in distinct spatial regions of interest (ROI; n=2-4 per tumor/TDLN) or cell types in tissue sections.

After a single STING-NP injection of B16 tumors, B7-H3, an immunosuppressive T cell checkpoint, was significantly induced in tumors compared to PBS or free cGAMP. More strikingly, in T cell-rich regions of the TDLN, STING-NP dramatically increased B7-H3, GZMB, and S100A9 while suppressing VISTA and CD73 expression. After multiple injections of STING-NP, B7-H3, S100A9, CD73, Foxp3, and beta-catenin were substantially upregulated in the injected tumor, with more modest fold changes in distal tumors. Co-treatment with systemic ICI in part abrogated this effect. In contrast, STING-NP-induced changes in GZMB, CD4, and CD8a expression were observed regardless of ICI treatment, and were generally equivalent in both treated and distal tumors, suggesting an abscopal effect. Both the treated and distal TDLN showed striking S100A9 increases which was preferential, but not exclusive, to T cell-rich vs. B cell regions. This effect was exacerbated with the combination of STING-NP and ICI therapy. B7-H3 upregulation in response to STING-NP (regardless of ICI) was exclusive to treated TLDNs vs. distal TDLNs. B7-H3 was expressed in both B and T cell regions, but was nearly two-fold higher in B cell-rich regions. Upregulation of GZMB (5-10 fold over free cGAMP alone) was observed in both treated and distal TDLNs.

Markers of T cell activation were observed after STING-NP treatment, including moderate abscopal effects in synchronous distal tumors. In TDLNs, STING-NP robustly induced S100A9 and B7-H3, which may be immunosuppressive escape mechanisms that could be targeted to enhance responses.

#4979

Ofranergene Obadenovec (VB-111), an anti-cancer gene therapy, induces immunologic responses in solid tumors transforming cold tumors to hot tumors.

Ronnie Shapira-Frommer,1 Tamar Rachmilewitz Minei,2 Iris Barshack,1 Itzhak Mendel,2 Niva Yakov,2 Yael C. Cohen,2 Eyal Breitbart,2 Dror Harats,2 Richard T. Penson3. 1 _Sheba Medical Center Affiliated to Sackler Faculty of Medicine, Tel Aviv, Israel;_ 2 _VBL Therapeutics, Modiin, Israel;_ 3 _Massachusetts General Hospital, Boston, MA_.

Introduction: Ofranergene Obadenovec (VB-111) is a non-replicating adenovirus 5 (Ad-5, El-deleted) carrying a proapoptotic human Fas-chimera transgene that targets angiogenic blood vessels and leads to vascular disruption. Improved responses seen among patients experiencing post treatment fever suggest that VB-111's mode of action involves induction of a tumor directed immune response. The following experiments were conducted to assess if VB-111 may alter the immunogenic profile of the tumor.

Methods: Biopsies were obtained from 3 patients with recurrent platinum-resistant ovarian cancer treated with intravenous VB-111 1x1013 viral particles (VPs) every 2 months in combination with weekly paclitaxel. H&E and Immunohistochemistry (IHC) were performed for CD8 and CD4 intratumoral T-cells, and results were compared to pre-treatment specimens and to untreated controls. In parallel, in the Lewis Lung Carcinoma (LLC) model, mice were randomly treated with intravenous saline or VB-111 at doses of 1x109 or 1x1011 VPs. Upon sacrifice lungs were harvested and weighted. Tumor burden was assessed, and Immunohistochemistry with anti-CD8 antibody for the presence of infiltrating cytotoxic T-cells was performed.

Results: Specimens from Ovarian Cancer taken before treatment with VB-111 showed no or minimal T-cell infiltration. One month after VB-111 treatment, metastatic lesions demonstrated increase in CD8 (up to 74 CD8+ cells/HPF) and CD4 tumor infiltrating T-cells. At 4.5 months following first drug administration (post 3rd dose) a liver lesion showed necrotic and fibrotic tissue with no viable tumor, lymphocytic aggregate, intensive staining for CD-8 and CD-4 T-cells and pigmented macrophages. In mice induced with LLC tumor and treated with saline, large tumor masses were observed in the lungs, and the calculated average tumor burden was 0.883g. Administration of VB-111 at 1x109 and 1x1011 VPs reduced tumor burden by 42% and 72% respectively. Concurrently, the number of tumor infiltrating CD8 T-cells was increased in correlation with VB-111 treatment dose.

Conclusion: Pre clinical and clinical data suggest that VB-111 induces an Immunotherapeutic effect manifested locally with tumor infiltration with CD-8 T-cells, and evidence of tumor necrosis. The viral vector may promote transformation of the tumor microenvironment from immunologically "cold" to "hot", enhancing immune cell recognition and immune activation.

#4980

Altered pan-Ras pathway and activating mutations in EGFR result in elevated CD73 in multiple cancers.

Akshata R. Udyavar, Daniel DiRenzo, Devika Ashok, Amy E. Anderson, Stephen W. Young, Matt J. Walters, Joanne Bl Tan. _Arcus Biosciences Inc, Hayward, CA_.

Background: Adenosine production mediated by CD73 and/or TNAP is a potential mechanism of immune suppression across many cancer types. We have previously shown that AB928, a dual A2aR/A2bR antagonist, in combination with anti-PD-1 or chemotherapy, rescues the immunosuppressive effects of adenosine in experimental tumor models. Oncogene-driven cancers are targeted with specific tyrosine kinase inhibitors, typically leading to the development of several resistance mechanisms that bypass the kinase signaling. These oncogene-driven cancers tend to be non-responsive to PD(L)-1 inhibition. We hypothesized that oncogenic drivers might be regulating adenosine production machinery as a mechanism to suppress and evade the immune system. Here we show that pan-RAS, BRAF and EGFR alterations drive the expression of CD73, which may contribute to suppressed anti-tumor immunity.

Methods and Results: We used linear models to evaluate the ability of 299 pan-cancer consensus oncogenic drivers (Bailey et al, 2018) to predict CD73 expression independent of tumor type in PanCanAtlas TCGA dataset. We defined a given gene as either wild type or altered if it exhibited a SNV/copy number/fusion event in a given patient. Out of the 299 oncogenes, we identified 20 oncogenes that upregulated and 26 that downregulated CD73 expression (FDR < 0.05). Alterations in KRAS, BRAF and EGFR were the top 3 oncogenes upregulating CD73 expression, followed by other kinases such as RASA1, MET, RET, SMAD4 and CDK4. In KRAS/BRAF/EGFR altered tumors, CD73 expression was significantly higher compared to respective wild-type patients in all cancers combined, as well as in individual tumor types (BRCA, LUAD, LUSC, CRC, PAAD, HNSCC, UBC, CESC, ESCA, STAD, UCEC and LGG). Furthermore, the pan-cancer Ras activation classifier score (Way et.al 2018) was highly predictive of CD73 expression independent of tumor type (p-value < 2e-16). KRAS, HRAS, BRAF, EGFR as well as pan-Ras mutant cancer cell lines exhibited a significantly higher level of CD73 expression than wild-type cell lines, independent of cancer type.

Conclusions: Activating mutations in KRAS and HRAS that result in deregulation of the Ras pathway serve as oncogenic drivers in a variety of cancer types such as CRC, NSCLC, gastric-esophageal, cervical, bladder and HNSCC and upregulate CD73. BRAF mutations are enriched in breast, CRC, NSCLC, and low-grade gliomas that exhibit higher CD73 expression. Finally, EGFR mutations are prominent in RCC, low-grade gliomas and NSCLC, which drive CD73 expression. Oncogene-driven cancers are largely unresponsive to PD-(L)1 inhibition and are targeted with kinase inhibitors resulting in significant but not durable clinical responses. Our data strongly suggests that CD73 inhibition with AB680 and/or A2R antagonism with AB928 might be effective in Ras pathway mutant and EGFR mutant cancers, either in TKI-naïve or relapsed settings.

#4981

HER2-targeted antibody drug conjugates (ADCs) induce host immunity against cancer stem cells and are enhanced by anti-PD-L1.

Leiming Xia,1 Lu Wen,1 Gang Wu,2 Tao Zhang,2 Frank Comer,3 Mary Jane Masson Hinrichs,4 Michael Oberst,3 Steven R. Coats,3 Alfred E. Chang,1 Yuanyuan Liu,5 Yangyi Bao,5 Max Wicha,1 Qiao Li1. 1 _Univ. of Michigan, Ann Arbor, MI;_ 2 _Cancer Center, Wuhan, China;_ 3 _MedImmune, Gaithersburg, MD;_ 4 _MedImmune, Bethesda, MD;_ 5 _The First People's Hospital of Hefei, Hefei, China_.

HER2-targeted antibody drug conjugate (ADC) was only tested in immunocompromised (SCID) host, which doesn't allow the evaluation of the ADC-induced host immune responses. In addition, no potential impact of ADC on cancer stem cells (CSCs) has been reported. Furthermore, tumor cells with PD-L1 expression may escape immune attack by converting PD-1 positive immune effector cells into anergy. We hypothesized that HER2-targeted ADC could modulate host immune response, target HER2high CSCs, and the efficacy of ADC could be enhanced by anti-mPD-L1. To test this hypotheses, we used two immunocompetent murine models: mouse breast tumor D2/F2 and TNBC 4T1 with enforced expression of HER2, D2F2/E2 and HER2-4T1 respectively. Both D2F2/E2 and HER2-4T1 could generate tumor in immunocompetent Balb/c mice, which allowed us to test the efficacy and specificity of HER2-targeted ADC, and to evaluate host immune responses. In D2F2/E2 mouse model, we found that HER2-targeted ADC significantly inhibited D2F2/E2 tumor growth, but not the parental D2F2 tumors, in a dose dependent manner. The specificity was confirmed in HER2-4T1 model. In parallel, we observed that HER2-targeted ADC treatment reduced the number of HER2 positive cells and ALDHhigh CSCs. We found that anti-mPD-L1 mAb significantly augmented the therapeutic efficacy of HER2-targeted ADC in both D2F2/E2 and HER2-4T1 tumor models. Mechanistically, HER2-targted ADC combined with anti-PD-L1 significantly modulated the gene signature of multiple immune factors in tumor microenvironment. At the cellular level, anti-mPD-L1 plus HER2-targeted ADC increased the number of CD3+ and CD19+ tumor infiltrating lymphocytes (TILs). Expanded CD3+ TILs from the tumor subjected to combined treatment killed tumor cells significantly more than mono-treatment. Expanded CD19+ TILs from tumor subjected to the combined therapy produced more IgG than all the controls. Importantly, the IgG could specifically bind to tumor cells, resulting in cytotoxicity of the tumor cells via ADCC. As a result, ADC plus anti-PD-L1 further reduced the number of HER2 positive cells. Importantly, the combined therapy not only reduced the number of ALDHhigh CSCs in the residual tumor, but also significantly paralyzed their tumoriginicity. Together, our data indicate that HER2-targeted ADC could induce significant host immune responses, which was evidenced by the regulation of gene signature and detection of anti-tumor CD3+ and CD19+ TILs. We first show that HER2-targeted ADC could target ALDHhigh cancer stem cells, and such effect was significantly enhanced by anti-mPD-L1 administration.

#4982

Anti-mCTLA-4 treatment results in early and late immune response effects in a murine model of colorectal carcinoma.

David W. Draper, Alden Wong, Stacey Roys, Scott Wise, Maryland Franklin. _MI Bioresearch, Ann Arbor, MI_.

To determine therapeutic mechanism of action (MOA), it is standard to analyze the tumor immune response at a single timepoint. Therefore, to examine the impact of checkpoint inhibition on early and late phases of the immune response, we dosed CT26 tumor-bearing mice with anti-mCTLA-4, or isotype control, and analyzed tumors at day 12 and day 18 post-implant. Tumor measurements at day 17 revealed a 77% growth inhibition in response to anti-mCTLA-4 treatment. To explore MOA, 11 tumor-infiltrating myeloid and lymphoid subsets were quantified and analyzed for various phenotypic and functional markers. We first examined immune cell recruitment in the control group tumors from day 12 to day 18. As expected, dynamic changes were observed in various subsets. Absolute numbers of CD8+ T cells, M1 and M2 tumor-associated macrophages (TAM) increased by 203%, 208%, and 100% respectively. In contrast, a 31% reduction in monocytic myeloid-derived suppressor cells (M-MDSC) was observed. The effects of anti-mCTLA-4 on tumor infiltration was then examined. Enhanced CD4+ helper, CD8+, and regulatory T cell recruitment was observed at both timepoints compared to the isotype control group. While NK and NKT cell recruitment was also enhanced, the increase in NK cells was only transient (130% at day 12). A similar transient increase was observed for M1 TAMs. In contrast, a 33% reduction in M2 TAMs occurred following treatment from day 12 to day 18, compared to controls. Finally, an increase in M-MDSC was observed in the anti-mCTLA-4 group at both timepoints (33% and 58% respectively). To evaluate effects of anti-mCTLA-4 on proliferation, we measured Ki-67 expression as a surrogate marker in CD8+ T cells. A 43% increase in Ki-67 expression was induced by anti-mCTLA-4 compared to controls at day 12. No difference was observed on day 18 suggesting anti-mCTLA-4 increased CD8+ T cell infiltration by triggering an early and transient enhancement in proliferation. Analysis of CD62L and CD44 in the control group demonstrated the pool of memory CD8+ T cells increased in the tumors over time. Anti-mCTLA-4 treatment delayed memory formation resulting in a larger pool of CD8+ T cells with an effector phenotype (CD62L-CD44+). To examine anti-tumor activity in CD8+ T cells, IFNγ production was measured after ex vivo stimulation. Anti-mCTLA-4 enhanced IFNγ responses on day 18 in CD8+ T cells and NKT cells (25% and 17% respectively), but similar responses were observed between groups on day 12. Taken together, these results suggest that checkpoint blockade inhibited tumor growth in part by enhancing early T cell proliferation and recruitment of NK and M1 TAMs. This was followed by a late enhancement of anti-tumor T cell and NKT responses, as well as reduced M2 TAM infiltration. These findings demonstrate that kinetic analysis can help gain insight into therapeutic MOA by revealing dynamic responses that can be missed by analysis at a single point.

#4983

Discovery and characterization of next-generation small molecule direct STING agonists.

Monika Dobrzańska, Stefan Chmielewski, Magdalena Zawadzka, Jolanta Mazurek, Karolina Gluza, Katarzyna Wójcik-Jaszczyńska, Maciej Kujawa, Grzegorz Topolnicki, Grzegorz Ćwiertnia, Aleksandra Poczkaj, Izabela Dolata, Magdalena Mroczkowska, Agnieszka Gibas, Marcin Leś, Sylwia Sudoł, Adam Radzimierski, Kinga Michalik, Magdalena Sieprawska-Lupa, Katarzyna Banaszak, Katarzyna Wiklik, Federico Malusa, Michał Combik, Karolina Wiatrowska, Agnieszka Adamus, Lukasz Dudek, Jose Alvarez, Charles Fabritius, Anna Rajda, Maciej Rogacki, Faustyna Gajdosz, Peter Littlewood, Luigi Stasi, Krzysztof Brzózka. _SELVITA S.A, Kraków, Poland_.

Accumulating evidence highlights an important role of type I interferon response in the immune surveillance mechanisms. IFNβ release by antigen-presenting cells promotes spontaneous anti-tumor CD8+ T cell priming being largely dependent on activation of Stimulator of Interferon Genes (STING). STING agonists promote regression of established tumors and generation of long-term immunologic memory in preclinical animal models. Herein we report the discovery of potent and selective, first-in-class non-nucleotide, non-macrocyclic, small molecule direct STING agonists with molecular weight below 500, structurally unrelated to known cyclic dinucleotide chemotypes with potential for systemic administration. Activation of STING pathway was monitored in THP-1 Dual reporter monocytic cell line as well as peripheral blood mononuclear cells (PBMC) or antigen presenting cells from human and mouse origin. Surface expression of the antigen-presenting cell maturation markers i.e. CD80, CD86, CD83 and HLA-DR was assessed by flow cytometry. Binding affinity was confirmed by three independent assays. RNA sequencing was performed on total RNA isolated from THP-1 cells and PBMC isolated from 2 healthy human donors. Direct binding to both mouse and human STING protein of Selvita agonists have been confirmed in biophysical binding assays (FTS, MST and FP) and by crystallography studies. The compounds have fine-tunable ADME properties with good solubility, permeability and human plasma stability. They selectively activates STING-dependent signaling in both THP-1 reporter assays and in primary cells of human and mouse origin. In addition, RNA sequencing data confirmed selectivity of the Selvita compounds. In vitro functional assays demonstrated their ability to induce cytokine responses (IFNβ, TNFα) in a panel of human peripheral blood mononuclear cell (PBMC), human monocyte derived macrophage (HMDM) and human dendritic cells samples with various STING haplotypes including refractory alleles. Additionally, the compounds efficiently induced cytokine release in mouse bone marrow-derived macrophages and dendritic cells. Pro-inflammatory cytokine profile was accompanied by up-regulation of the maturation markers, i.e. CD80, CD86, CD83 and HLA-DR, on the surface of human antigen presenting cells. These data demonstrate potent, novel, next-generation small molecule STING agonists activating STING-dependent signaling in both mouse and human immune cells to promote potential antitumor immunity. The compounds show good selectivity and in vitro ADME properties enabling further development for systemic administration as a single agent or in combinatory immunotherapies for cancer treatment.

#4984

Development of an improved humanized patient-derived xenograft, Hu-PDX, mouse model for evaluation of antitumor immune response in lung cancer.

Ismail M. Meraz, Mourad Majidi, Feng Meng, RuPing Shao, Min Jin Ha, Shinya Neri, Bingliang Fang, Steven H. Lin, Peggy T. Tinkey, Elizabeth J. Shpall, Jeffrey Morris, Jack A. Roth. _UT MD Anderson Cancer Ctr., Houston, TX_.

Current preclinical models of non-small cell lung cancer (NSCLC) do not recapitulate the human tumor microenvironment. Mice reconstituted with a human immune system and bearing human patient derived xenografts may be advantageous in evaluating human anti-tumor immune response. We developed an improved NOD scid gamma (NSG) mouse model derived from non-expanded CD34\+ stem cells, without CD3\+ T cell contamination, to evaluate antitumor responses to immunotherapy in NSCLC. Using fresh CD34+ from umbilical cord blood reduced humanization time significantly. Human CD45\+ cell reconstitution with increased functional human lymphoid (B, T, monocytes and NK cells) and myeloid (macrophages and MDSCs) lineage repopulation, without the onset of GvHD, was achieved as early as 4 weeks post-stem cell engraftment. Published studies using expanded CD34+ derived humanization reveal compromised purity of CD34+ stem cells with an increasing number of mononuclear cells. Reconstitution of CD8+ and CD4+T cells is not achieved until 12 to 15 weeks post-engraftment at much lower levels than fresh CD34+ humanization. Single cell suspension analysis shows levels of human reconstituted T, B, NK, DC and MDSC cells at 4 weeks, which increased significantly at 6 and 9 weeks in peripheral blood, spleen and bone marrow. Human repopulated T cells were functionally active in secretion of IFN-γ by mitogenic stimuli such as PMA and IL-2 and by allogenic human cancer cells. Antigen specific CTL responses were observed when reconstituted human T cells from PDX bearing humanized mice were challenged with PDX tumor. No non-antigen specific responses were observed when T cells were co-cultured with HLA-matched human bronchial epithelial cells (HBEC). To evaluate the applicability of the humanized mouse in lung cancer translational research, we combined it with Hu-PDX or Hu-xenograft tumors and analyzed tumor growth and treatment response to the anti-PD1 checkpoint inhibitor pembrolizumab. We found that efficient engraftment of PDXs and xenograft tumors were not dependent on donor HLA-status. Similar to the clinical outcome, treatment with pembrolizumab, inhibited tumor growth significantly in both Hu-PDX, and Hu-xenograft mice regardless of donor HLA-types, increasing cytotoxic T cells and decreasing MDSC levels. Pembrolizumab had no effect on the non-humanized NSG controls. In concordance with our previous study with a syngeneic mouse tumor, the antitumor effect of check point blockade was significantly enhanced when combined with nanoparticle systemically deliveredTUSC2, a tumor suppressor and immunomodulatory gene, in a KRAS mutant lung metastasis humanized mouse model. In conclusion, fresh CD34+ are more effective than their expanded counterparts in humanizing mice, do so in much reduced time, and recapitulate the immune response to cancer.

#4985

TLR agonists for anticancer immunotherapy.

Wenqiu Zhang, Hyunjoon Kim, Vidhi Khanna, David M. Ferguson, Thomas Griffith, Jayanth Panyam. _University of Minnesota, Minneapolis, MN_.

Introduction: Toll-like receptors (TLRs) are important pattern recognition receptors through which innate immune cells recognize invasive microorganisms. The immunostimulatory property of TLR agonists allows for activation of dendritic cells (DCs) and enables their use as anticancer vaccine adjuvants. Because TLR 7 and 8 can be activated by synthetic small molecules, they are of particular interest as a target in vaccine adjuvant discovery. However, a key drawback of these synthetic small molecules is that they also induce immunosuppressive cytokines, resulting in immunosuppression.

Methods: We have previously developed a suite of highly substituted imidazoquinolines, which potently activate TLR 7 and/or 8. In this study, we tested some selected TLR7, TLR8 and TLR7/8 dual agonists for their ability to activate DCs without inducing immune suppressive cytokines. Murine bone marrow-derived dendritic cells (BMDCs) were generated from C57BL/6 mice. BMDCs were treated with one of seven TLR agonists for 72 hrs. Cells were collected and stained for flow cytometry analysis, and the culture supernatants were examined for secretion of pro-inflammatory cytokines, IL-12p70 and IFN-γ, and an immunosuppressive cytokine, IL-10, by ELISA. We also investigated activation of T cells by these agonists. Human peripheral blood mononuclear cells (hPBMCs) from healthy donors were incubated with one of seven TLR agonists overnight and cells were analyzed by flow cytometry for the expression of T cell activation marker CD69 and production of IFN-γ.

Results: All the TLR agonists investigated activated BMDCs, as evidenced by the upregulation of costimulatory markers CD40, CD80 and CD86 on DCs. In addition, all the agonists examined induced the secretion of IL-12p70 and IFN-γ. TLR 8 agonists showed the least induction of IL-10 secretion. All the TLR agonists activated both CD4 and CD8 T cells. TLR 8 agonists induced higher production of IFN-γ in both type of T cells than other candidates.

Conclusion: Both TLR 8 and TLR 7/8 agonists activated BMDCs and stimulated strong pro-inflammatory cytokine production. However, TLR8 activation was associated with less immunosuppression. These results suggest that these new imidazoquinoline esters are promising candidates for use as anti-cancer vaccine adjuvants.

#4986

TIGIT directed human antibody modulates T-regulatory and effector cell function.

Alyson Smith, Weiping Zeng, Bryan Grogan, Jane Haass, Amber Blackmarr, Scott Peterson, Shyra J. Gardai. _Seattle Genetics, Bothell, WA_.

T-cell immune receptor with Ig and ITIM domains (TIGIT) is a receptor expressed on activated and memory T cells, immunosuppressive T regulatory cells (Tregs), and NK cells. Engagement with its ligands, CD155 and CD112, drives an inhibitory signal in T cells and supplants ligand binding from the co-stimulatory receptor CD226, collectively resulting in decreased cell functionality. Antibody blockade of TIGIT can release these inhibitory signals, drive T cell activation and augment anti-tumor responses. Additionally, TIGIT antibodies may directly affect Treg cell function and survival. FACS analysis of healthy donor PBMCs confirmed TIGIT expression on NK and T cell subsets with expression most frequent on Temra and Tregs. A fully human TIGIT monoclonal antibody (mAb) that recognizes human, murine, and cynomolgus TIGIT was created to assess TIGIT mAb functionality and antitumor activity. Assessment of a TIGIT directed mAb demonstrated preferential depletion of Tregs in ex-vivo PBMC cultures with NK cell/Treg co-culture experiments confirming Treg loss via ADCC. Anti-TIGIT treatment also supported a memory T cell recall response assessed following PBMC exposure to CMV lysate or pooled viral peptides. The TIGIT directed antibody also modulated memory and new effector CD8 T cell responses in a mixed lymphocyte reaction (MLR). TIGIT antibody treatment increased proliferation and IL-2 production, suggesting naïve and memory T cell activity was bolstered following TIGIT mAb treatment. TIGIT antibodies may be unique in their ability to elicit effector T cell responses as TIGIT mAb treatment of PBMC cultures resulted in activation of innate immune cells and cytokine production suggesting TIGIT mAbs may activate both adaptive and innate arms of the immune system. These in vitro results translated to in vivo antitumor activity in the CT26 colon cancer model, where TIGIT mAb treatment decreased intratumoral Tregs, increased total intratumoral CD8 effector memory T cells, and resulted in curative antitumor responses. These pharmacodynamics changes and anti-tumor responses were associated with generation of antigen-specific immunity and long-term memory T cell responses resulting in complete tumor rejection upon re-challenge. Furthermore, TIGIT mAb treatment of MC38 and A20 syngeneic tumor models resulted in tumor growth delay and up to 66% complete responses in the A20 model. Collectively this data demonstrates the anti-tumor therapeutic potential of a TIGIT targeting mAb and suggests activity comes both from reduction of Treg cells and amplification of naïve and memory T cell responses.

#4987

Age-induced changes in anti-tumor immunity alter the tumor immune infiltrate and reduce response to immuno-oncology treatments.

Suzanne I. Sitnikova,1 Michelle Morrow,1 Viia Valge-Archer,2 Robert W. Wilkinson,1 Simon J. Dovedi,1 Matthew J. Robinson1. 1 _MedImmune, Cambridge, United Kingdom;_ 2 _AstraZeneca, Cambridge, United Kingdom_.

Immuno-oncology research relies heavily on murine syngeneic tumor models. However, whilst the median age for a cancer diagnosis is 65 years or older, for practical purposes the majority of preclinical studies are conducted in young mice, despite the fact that aging has been shown to have a significant impact on the immune response. Using aged mice bearing CT26 tumors, we analysed how aging impacts the immune composition of the tumor, spleen and tumor-draining lymph nodes by flow cytometry. We found many age-related changes between aged (60-72 weeks old) and young (6-8 weeks old) mice, such as a reduction in the naïve T cell population and a decreased CD8/Treg ratio in aged animals. Profiling of co-inhibitory and co-stimulatory receptor expression levels on immune cells in aged versus young mice also identified altered expression profiles in both the periphery and tumor. We hypothesised that these differences may contribute to impaired anti-cancer immune responses in aged mice. To investigate this, we compared the anti-tumor efficacy of immune checkpoint blockade (PD-L1 and CTLA-4) and T-cell costimulation (OX-40) in aged versus young mice. Our data demonstrate that aged mice retained their capacity to generate effective anti-tumor immune responses, albeit often attenuated when compared to the responses observed in young mice. These differences highlight the potential importance of age-related immunological changes in assessing and refining the translational insights gained from preclinical mouse models.

#4988

CD 137 agonists as an adjunct to immune checkpoint inhibitors to overcome resistance in melanoma.

Sreedevi Danturti, Lena Mayer, Christina Twyman Saint Victor. _University of Pennsylvania, Philadelphia, PA_.

Background: Melanoma is currently the fifth most common cancer in men and sixth most common in women with an estimate of 9,320 deaths from the disease in the United States. Anti-PD1, an immune checkpoint inhibitor, is FDA approved to treat metastatic melanoma with durable responses observed in 53% of patients. Studies suggest that combining therapies (e.g. immune checkpoint inhibitors, radiation therapy, and/or agonist antibodies) can be more effective than monotherapy. CD137 is expressed on a variety of cell populations including T cells, dendritic cells (DCs) and macrophages. CD137 agonists have proved effective in multiple murine tumor models and have had modest responses in early clinical trials. We hypothesize that CD137 agonists can augment the anti-PD1 anti-tumor immune response resulting in improved overall survival and decreased tumor burden in melanoma.

Methods: 499 melanoma cells, which were derived from B16-F10, are implanted on both flanks of C57BL6 mice. The mice are treated with anti-CD137 and/or anti-PD1 on day 10, 13 and 16 post-implantation. The anti-tumor response is assessed by both overall survival and tumor burden. Tumors are harvested on day 19 for protein, transcriptomic and immune profiling.

Results: Monotherapy with either anti-CD137 agonists or anti-PD1 antagonists increases the overall survival by 8 days; whereas, combination therapy results in an increase of 25 days. To investigate the mechanisms of response and resistance to combination therapy, tumors were harvested and assessed via a proteomic array that identified 36 proteins that were upregulated in the non-responders. Transcriptomic profiling of these tumors using Nanostring IO 360 corroborated these findings and identified both the myeloid the T cell immune populations are upregulated in responders.

Conclusions: These results suggest a dual role for both the innate and adaptive immune response in treating melanoma. By understanding the mechanism of response and further characterization we can further best apply the combination therapy to overcome resistance after immunotherapy.

#4989

**T-distributed stochastic neighbor embedding (t-SNE) analysis of tumor infiltrating lymphocytes after treatment with a T cell activating therapy identifies a unique population of recruited CD8** + **T cells and novel options for combination immunotherapy.**

Ava Vila-Leahey, Alecia MacKay, Genevieve Weir, Marianne Stanford. _IMV Inc., Dartmouth, Nova Scotia, Canada_.

Immune therapies for cancer seek to alter the immune profile of the tumor microenvironment (TME). A suppressive TME can counteract active immune responses by CD8+ T cells by multiple mechanisms, including induction of checkpoint inhibitor (CPI) expression. DPX™ is a lipid nanoparticle-based platform that can be formulated with cancer targets to induce potent and sustained antigen-specific T cell responses. In preclinical and clinical testing previously published, combination of DPX with low dose intermittent cyclophosphamide (ldCPA) resulted in increased tumor infiltration of antigen-specific CD8+ T cells. These T cells express PD-1, and combination treatment with PD-1 blocking antibody was shown previously to result in better tumor control in preclinical tumor models. In this study, we used tSNE analysis to understand changes to the immune profile induced by DPX/ldCPA treatment in the C3 model. We prioritized the expression of CPIs on tumor infiltrating CD8+ T cells to identify potential combinations with blocking monoclonal antibodies to enhance tumor CD8+ T cell activity. Mice (C57BL/6) bearing HPV16 E7-transformed C3 tumor cells were treated with ldCPA (20 mg/kg/day PO, 7 days), followed by subcutaneous injection with HPV16 E749-57 peptide in a DPX formulation (DPX-R9F). Tumors were collected for analysis 10 days after DPX-R9F treatment and a single cell suspension prepared by digestion. Immune profiling was performed by 12-color flow cytometry, to observe differences in infiltrating immune cells and CPIs. tSNE analysis of flow cytometry data was performed to analyze differences in cellular infiltrates and phenotype with treatment. We found CD8+ T cells that infiltrated tumors in mice treated with DPX-R9F/ldCPA expressed most CPIs tested (including PD-1, Tim-3, Lag-3, ICOS), and fewer expressed CTLA-4. Treatment induced infiltration of a unique population of CD8+ T cells that was PD-1+Tim-3+CTLA-4-, whereas CD8+ T cells present in untreated tumors were primarily PD-1+Tim-3+CTLA-4+. Tumor challenge was performed to evaluate combination of DPX-R9F/ldCPA treatment with anti-CTLA-4 (Clone 9D9) to potentially impact these resident CD8+ T cells. Mice treated with the triple combination had better control of C3 tumor growth compared to mice treated with DPX-R9F/ldCPA or anti-CTLA-4 separately. With tSNE analysis we were able to have a better model to observe differences in the CD8 population with DPX/ldCPA treatment and determine the mechanism for how certain checkpoint targets are impacting tumor infiltrating cells to further sculpt the anti-tumor response to increase efficacy of treatment.

#4990

An in vivo method for determining cancer immunotherapy induced cytokine release syndrome utilizing PBMC humanized mice.

Chunting Ye,1 Mingshan Cheng,1 Michael Brehm,2 Dale Greiner,2 Leonard Shultz,3 James G. Keck1. 1 _The Jackson Laboratory, Sacramento, CA;_ 2 _University of Massachusetts Medical School, Worcester, MA;_ 3 _The Jackson Laboratory, Bar Harbor, ME_.

Monoclonal antibodies (mAbs), as either single agents or in combination, have shown remarkable efficacy for cancer immunotherapy. However the use of antibody-based immunotherapies can result in the development of severe adverse effects for many patients, including cytokine release syndrome (CRS). Two methodologies used routinely for CRS drug toxicity testing are in vitro assays with human PBMC and in vivo testing in animal models. Unfortunately, neither method reliably predicts the immune toxicity in humans. For example, in vitro testing does not mimic the complexities of the biological environment in humans, and testing of human-specific agents in either rodent or non-human primates is limited by the many species-specific differences in immune system function. This significant gap between pre-clinical testing of novel therapeutics and clinical trials demonstrates a critical need for translational protocols that more accurately predict immune toxicity.

We have developed a novel humanized mouse model for testing CRS that is rapid, sensitive and reproducible. This model is based on human PBMC engraftment of NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG; JAX stock number 005557) mice, and assessment of CRS is performed within 6 days of PBMC injection. Within 6 days of PBMC injection, total human immune cell (CD45+) levels averaged 10 to 15% of cells in blood with approximately 70% and 25% of the human CD45+ cells being CD3+ T cells or CD56+ NK cells, respectively. To validate this model, PBMC-engrafted NSG mice were challenged with OKT3 (anti-CD3) by day 6 after PBMC injection, which is a timepoint prior to the development of robust xenogeneic GVHD. Severe clinical symptoms developed rapidly in OKT3-treated mice, including production of human cytokines and a significant drop in body temperature as compared to control PBS-treated mice. Using our validated humanized mouse model, induction of CRS was tested using clinically relevant mAb as either single agents, including pembrolizumab, anti-CD28, and ATG (anti-thymocyte globulin). We observed robust clinical readouts for these mAb treatments, with induction of rapid and distinct cytokine release profiles. Moreover our assay also identified donors that were "high" responders and "low" responders to specific mAb treatments. A direct comparison of our humanized mouse model to an in vitro based PBMC assay revealed several advantages for the humanized assay, including higher sensitivity and more accurate recapitulation of clinical observations. Notably our humanized mouse model also demonstrated utility when evaluating combination therapies, including pembrolizumab/lenalidomide, pembrolizumab/ATG, and anti-CD28/ATG and enabled the identification of unique patterns of CRS.

In conclusion, we have developed a translational humanized mouse model for preclinical assessment of CRS adverse events to mAb therapeutics.

#4991

Hyleukin-7, the Fc-fused interleukin-7, generates anti-tumor activity by modulating both adaptive and innate immune cells in the tumor microenvironment.

Ji-Hae Kim,1 Sung-Wook Hong,1 Young-Min Kim,1 Saet-byeul Jo,1 Man Kyu Ji,2 Yeon Kyung Oh,2 Han Wook Park,1 Sora Kim,1 Donghoon Choi,3 Byung Ha Lee,3 Se Hwan Yang,3 Young Chul Sung,2 Seung-Woo Lee1. 1 _POSTECH, Pohang-si, Republic of Korea;_ 2 _Genexine, Inc., Republic of Korea;_ 3 _NeoImmuneTech, Inc., MD_.

Interleukine-7 (IL-7), a strong candidate for a novel immunotherapeutic agent, plays important roles in the development and homeostasis of T lymphocytes. Recombinant IL-7 has shown positive effects in various models by increasing T cells in both mice and humans; however, the short half-life and stability of recombinant IL-7 has remained a challenge for its clinical application to cancer immunotherapy. Here, we investigated anti-tumor effects of a long-acting form of recombinant human IL-7 fused with hybrid Fc (rhIL-7-hyFc; Hyleukin-7) in mice. rhIL-7-hyFc administration in tumor-free mice generated the cytokine-induced CD8+ T cell proliferation, which altering CD8+ T cell homeostasis by expanding largely the TCM-phenotype CD8+ T cells displaying activation-induced attributes, such as Eomes, Granzyme B, CXCR3, and IFN-γ. When injected into mice with syngeneic tumor graft, rhIL-7-hyFc induced anti-tumor activity in a dose-dependent manner. rhIL-7-hyFc dramatically expands CD8+ T cells in the periphery and recruits effector CD8+ T cells in the tumor, yielding a high CD8+ T/Treg cell ratio in the tumor microenvironment (TME). rhIL-7-hyFc increases Ki-67 and granzyme-B expression but decreases expression levels of immune checkpoint molecules on CD8+ tumor-infiltrating lymphocytes (TILs). Surprisingly, rhIL-7-hyFc reduced myeloid-derived suppressor cells (MDSCs) in the TME, yielding the high CD8+ T/MDSC ratio. Collectively, rhIL-7-hyFc treatment confers anti-cancer activity by inducing a "CD8+ T cell infiltrated-inflamed-immune favorable" TME. The combination treatment of rhIL-7-hyFc with cyclophosphamide and immune checkpoint blockades showed enhanced anti-tumor efficacy in an advanced tumor model. Furthermore, we found that the anti-tumor activity of rhIL-7-hyFc was achieved under lymphopenic conditions by normalizing CD8+ T cell homeostasis. In sum, rhIL-7-hyFc generates an effective anti-tumor response through reconstructing CD8+ T lymphocytes; this activity was highly enhanced by combination therapies with the chemotherapeutics and immune checkpoint blockades. Our data suggests that rhIL-7-hyFc can be applied to various cancer immunotherapy regimens as a monotherapy or in combination partner with conventional and other immunotherapies.

#4992

The occurrence of immune-related adverse events indicates better efficacy of immune checkpoint blockade in solid tumors: A systematic review and meta-analysis.

Xu Yang, Dongxu Wang, Jianzhen Lin, Jianping Xiong, Junyu Long, Yi Bai, Jin Bian, Hanchun Huang, Xiaobo Yang, Yilei Mao, Xinting Sang, Haitao Zhao. _Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical, Beijing, China_.

Introduction: Immune-related adverse events (irAEs) were mostly induced by immune checkpoint blockade (ICB) and were associated with the immune response. Whether irAEs are associated with clinical benefits during ICB treatment in cancer patients remains unclear. This systematic review and meta-analysis aimed to investigate the relationship between irAEs and clinical efficacy.

Methods: We systemically searched and selected irAEs and clinical outcome data from cancer patients treated with ICB in the PubMed, Embase, and Cochrane Central Trial databases up to July 26, 2018. Keywords used for the search included PD-1, PD-L1, CTLA-4, immune checkpoint and immune-related adverse events. The relationships between the occurrence of irAEs and the objective response rate (ORR), progression-free survival (PFS) and overall survival (OS) were assessed using relative risk (RR) or hazard ratio (HR) values and 95% confidence intervals (CIs) in random-effect models.

Results: Nineteen eligible observational articles were included in this systematic review and fourteen studies including 3022 participants (1264 with irAEs and 1758 with non-irAE) were included in our meta-analysis. The occurrence of irAEs was significantly associated with higher ORR (RR = 2.36, 95% CI: 1.85-3.02; P < 0.001), better PFS (HR

= 0.48, 95% CI: 0.26-0.70; P < 0.001), and longer OS (HR = 0.42; 95% CI = 0.18-0.67; P = 0.001) than those in the non-irAE group. Subgroup analysis showed that the effect size of ORR in anti-PD-1 therapy (RR = 2.68, 95% CI, 2.21-3.26) was inferior to that of the anti-CTLA-4 therapy (RR = 1.91, 95% CI, 0.44-8.32). The meta-regression models also found that ICB type (anti-CTLA-4 or anti-PD-1) were associated with significant heterogeneity (P = 0.005).

Conclusion: In conclusion, irAEs indicate greater ORR, PFS and OS than non-irAEs in ICB treatment, and the relationship in anti-PD-1 therapy was stronger than in anti-CTLA-4 therapy. With appropriate management of irAEs, the clinical predictor, continued treatment may be possible to maximize its potential ICB benefits.

#4993

The Kynurenine Pathway as a possible resistance mechanism to immunotherapy in sarcomas.

Imane Nafia,1 Ariel Savina,2 Alban Bessede,1 Antoine ITALIANO3. 1 _Explicyte, Bordeaux, France;_ 2 _Institut Roche, Boulogne Billancourt, France;_ 3 _Institut Bergonie, Bordeaux, France_.

Background: Sarcomas are heterogenous malignant mesenchymal neoplasms known to be particularly resistant to immunotherapy, which thus calls the interest of the elucidation of related resistance mechanisms. We recently demonstrated an increase in Kynurenine Pathway (KP) activity in the plasma of patients undergoing Pembrolizumab and metronomic cyclophosphamide. The KP has already been described to favor tolerance and immune escape through the degradation of L-Tryptophan and production of a series of metabolites including L-Kynurenine. While Indoleamine 2,3 dioxygenase (IDO1), a first rate-limiting enzyme of the KP still represents an attractive therapeutic target, the exact functional role of the KP upon immunotherapy has not yet been clearly elucidated.

Methods: Using a preclinical syngeneic mouse model of sarcoma based on the inoculation of murine sarcoma MCA205 cell line, we investigated the modulation of the KP upon PDL1 blockade. Besides the evaluation of tumor growth and survival, intratumoral biopsy was performed and used for further gene expression analysis by RT-qPCR. More precisely, KP enzymes- and key cytokines-encoding genes were assessed. Using the same experimental setting on satellite mice, intratumoral microdialysis was performed on day 13 post-tumor inoculation and kynurenine contents were quantified within the tumoral microenvironment.

Results: PDL1 blockade, using a specific anti-PDL1 antibody (provided by Genentech), exhibited a clear anti-tumoral effect associated with an intratumoral inflammatory cytokines signature driven by Ifng, Tnfa and Il2. Interestingly, Ifng level was significantly and positively associated with response to anti-PDL1. As regards to KP enzymes, a slight upregulation of the tryptophan-degrading enzymes Ido1 and Ido2 was detected while no changes were observed for Tdo2. An increase of kynurenine-degrading enzymes, including Kmo (encoding for Kynurenine monoamine oxygenase) and Kynu (encoding for kynureninase), was also detected thereby suggesting that PDL1 blockade favors Kynurenine degradation as a possible compensatory mechanism. These results were confirmed by intratumoral microdialysis in the satellite study. Indeed, anti-PDL1 was able to decrease the Kynurenine to Tryptophan ratio(Kyn / Trp) while increasing intratumoral production of kynurenines catabolites.

In conclusion, this new set of data on a preclinical sarcoma model highlights for the first time that PDL1 blockade deeply modulates the Kynurenine Pathway with an effect not exclusively associated to the upper part of the pathway and thus warrants further investigation.

#4994

Differential gene expression analysis reveals pathways involved in anti-pd-1 therapy response prediction.

Yee Him Cheung,1 Jie Wu,1 Weihua Huang,2 Changhong Yin,2 Katherine Linder,3 Kaushal Parikh,4 Ke Tang,5 Christopher Kasbek,5 Jun Huang,5 Zeil Rosenberg,5 Michael Fanucchi,6 John Fallon,2 Nevenka Dimitrova7. 1 _Philips Research North America, Cambridge, MA;_ 2 _New York Medical College, Valhalla, NY;_ 3 _Baylor College of Medicine, Houston, TX;_ 4 _Mayo Clinic, Rochester, MN;_ 5 _Admera Health, South Plainfield, NJ;_ 6 _WMCHealth Cancer Institute, Valhalla, NY;_ 7 _Philips IntelliSpace Precision Medicine, Valhalla, NY_.

The goal of this study is to evaluate the potential use of gene expression profile of peripheral blood mononuclear cells (PBMCs) as a non-invasive biomarker for predicting the response of anti-PD-1 therapy in non-small cell lung cancer (NSCLC) subjects.

Peripheral blood of NSCLC stage 4 subjects treated with an anti-PD-1 monoclonal antibody was collected prior to treatment (week 0) and subsequent infusions. Total RNA was extracted, and libraries were prepared using TruSeq Stranded mRNA LT Sample Prep Kit and sequenced by Illumina NextSeq. The paired-end reads were mapped to human reference hg38 using STAR and gene expressions were calculated using RSEM. Preliminary analysis of differential gene expression (DE) was performed among the responders at respectively (T1) weeks 3-4, (T2) weeks 6-9 and (T3) weeks 12-16 after treatment, compared with the baseline at T0. Further DE analysis was performed between the baseline samples of responders and non-responders. Genes with FDR-adjusted p values less than 0.05 were selected for pathway enrichment analysis with DAVID.

Altogether 30 RNA-Seq samples of seven subjects were collected at multiple time points before and after treatment. According to standard RECIST 1.1 criteria, five subjects had a partial response to the therapy, whereas two subjects had stable disease or no response. In addition, liquid biopsy results were available for selected samples.

Among the responders, there are 203 DE genes for T1 with log2 fold change (LFC) of gene expression from -2.4 to 3.5, 859 DE genes for T2 with LFC from -3.4 to 3.3, and 395 DE genes for T3 with LFC from -3 to 6.8. Among the DE genes for the three time points, 155 genes are common to T1/T2, 250 common to T2/T3, and 104 common to T1/T2/T3. Pathway enrichment test using Gene Ontology Biological Process terms shows that the strongest DE pathways for initial response after treatment at T1 are regulation of immune response (FDR adjusted p = 2.5E-5), oxygen transport (p = 5.5E-3) and T cell costimulation (p = 3.9E-3). Based on the 104 common DE genes, oxygen transport (p = 0.029) is the only pathway consistently perturbed due to an increase in hemoglobin. When comparing the baseline of responders and non-responders, we identified a candidate gene signature, with some of the genes also reported in other studies. However, more samples are required to establish its statistical significance.

Systemic immune response after first dose of anti-PD1 treatment is reflected by DE genes in blood samples collected in weeks 3-4. Tumor regression might be indicated by the consistent increase in hemoglobin level. The results of these analyses show promise of developing a predictive signature for the response of anti-PD-1 immunotherapy in NSCLC subjects based on the gene expression profile of PBMCs. While these are preliminary results, we continue to recruit subjects, and will present the up-to-date results at the conference.

#4995

Mechanism of action of anti-PD-L1 antibody in a PD-L1-negative and immune desert-like tumor model.

Toshiki Iwai, Masamichi Sugimoto, Osamu Kondo. _Chugai Pharmaceutical, Kamakura, Japan_.

Purpose: Intratumoral PD-L1 plays an important role in preventing T cell-mediated cancer cell killing. Anti-PD-1/PD-L1 antibodies generally show higher clinical benefit specifically in patients with high intratumoral PD-L1 expression. However, anti-PD-L1 antibody, atezolizumab, provide clinical benefit even in the subgroup of cancer patients where PD-L1 is low or undetectable on tumor cells and tumor-infiltrating immune cells (TC0/IC0). In this study, we investigated the mechanism of action by which PD-L1-negative tumors responded to an anti-PD-L1 therapy by using a syngeneic mouse tumor model.

Experimental Design: We tested the antitumor effect of an anti-mouse PD-L1 monoclonal antibody (mAb) in vivo in murine models. Flow cytometry, tumor-stimulated IFNγ release assay, T cell repertoire analysis, RNA sequencing, protein immunoassays, and immunohistochemistry were performed on isolated tumors or on tumor-draining lymph nodes (dLN).

Results: The anti-PD-L1 mAb showed significant antitumor activity in 6 of the 17 tumor models tested. Model FM3A, one of these 6 sensitive models, showed little intratumoral PD-L1 expression and was classified as an immune desert-like tumor model, but PD-L1 was found to be highly expressed on CD103+CD11c+ cells in dLN in this model. Some of the CD44+CD62L−CD8α+ effector T cells in dLN expressed PD-L1 receptors, i.e. they were PD-1- and/or B7-1-positive cells. Using lymphocytes from dLN, we found that tumor cell-stimulated IFNγ production was significantly increased even in the control group, and it was further significantly increased in the anti-PD-L1 mAb group compared to that in the control group. We found that anti-PD-L1 mAb treatment firstly enhanced T cell priming and increased CXCR3+ activated T cells in dLN. FM3A tumors expressed CXCR3 ligands regardless of anti-PD-L1 mAb treatment. CXCR3 blockade significantly prevented activated CD8α+ T cells from increasing in tumors as a result of anti-PD-L1 mAb treatment but not in dLN and significantly attenuated tumor growth inhibition of anti-PD-L1 mAb.

Conclusions: We showed that an anti-PD-L1 mAb exerted antitumor activity in a TC0/IC0-like and immune desert-like tumor model through the blocking of PD-L1 in dLN. Tumor antigen presentation and T cell priming had already occurred in dLN at baseline prior to anti-PD-L1 mAb treatment in the FM3A model but PD-L1 on antigen-presenting cells prevented further T cell priming. We suggested that PD-L1 blockade in dLN appeared to trigger re-activation of T cells leading to antitumor activity. The results also suggested that the antitumor activity of anti-PD-L1 Ab requires both the enhancement of T cell priming by PD-L1 blockade as well as the trafficking of T cells to the tumor in response to the tumor via CXCR3 and its ligands system.

#4996

A real-time PCR-based approach to quantitatively assess tumor immune profiles and immune responses.

Marco A. De Velasco,1 Yurie Kura,1 Noriko Sato,1 Naomi Ando,1 Kazuko Sakai,1 Yasunori Mori,1 Barry R. Davies,2 Masahiro Nozawa,1 Kazuhiro Yoshimura,1 Kazuhiro Yoshikawa,3 Kazuto Nishio,1 Hirotsugu Uemura1. 1 _Kindai University Faculty of Medicine, Osaka-Sayama, Japan;_ 2 _Strategy, Oncology, IMED Biotech Unit, AstraZeneca, United Kingdom;_ 3 _Aichi Medical University, Japan_.

Exciting breakthroughs in tumor immunology have led to the discovery and development of several promising immunotherapeutic strategies for cancer patients. Thus far, immune checkpoint inhibitors have been the most encouraging for solid tumors, however, treatment responses are observed in only a subset of patients and these appear to be primarily dependent on a tumor's baseline immune profile. Additionally, tumors respond differently to immunotherapy and criteria to assess treatment responses are still being refined. We have identified a set of immune-related genes and developed a scoring system to profile the tumor's immune status and assess immunological responses. In this study, we used a qRT-PCR-based approach to assess this panel and determine baseline tumor immune-profiles in an immunocompetent mouse model of Pten-null prostate cancer. We also assessed and compared tumor immune responses following androgen withdrawal (via surgical castration) and anti-PD-L1 immune checkpoint blockade. A total of 96 genes were selected for this focused panel which consisted of cell-type, immuno-responsive and housekeeping control genes. Core modules were designated based on functional gene associations which included antigen presentation, tumor inflammation, effector cells, immunomodulatory cells, immunosuppressive signaling and immune checkpoints. Core modules were further subdivided in to appropriately relevant sub-modules. A score for each submodule was calculated and a weighed score was then assigned for each core module. Tumor immunophenotypes based on core and sub-core immune scores (IS) were corroborated with immunohistochemical and/or flow cytometric analyses. Mouse castration-naïve prostate tumors exhibited low scores indicating a '"cold tumor" phenotype with little variation between individual mice. Androgen withdrawal induced cancer cell death in these tumors that in turn promoted immune cell infiltration. Core and sub-core IS of individual mice identified varied signatures reminiscent of immune excluded and inflamed "hot tumor" phenotypes. Short-term treatment with anti-PD-L1 blockade (clone D265A, mouse/IgG1 kappa) elicited an increased immune response signature in tumors from surgically castrated mice, but not in tumors from intact mice. Notably, discernable differences were noted among individual responders supporting the notion that responses to immune checkpoint may indeed be dependent on baseline tumor immune-profiles. In summary, androgen withdrawal appears to promote a "hotter" immunological phenotype in this model of prostate cancer, that is increased further by PD-L1 inhibition. Moreover, this investigation provides proof of concept evidence for a qRT-PCR-based biomarker approach for immuno-oncology.

#4997

Responsiveness to immune checkpoint inhibitors in RET dependent cancers.

Aparna Hegde, Le Huang, Shuang Liu, Kenneth Hess, Maria Cabanillas, Mimi Hu, Naifa Busaidy, Steven Sherman, George Simon, George Blumenschein, Vassiliki A. Papadimitrakopoulou, David S. Hong, Funda Meric-Bernstam, John Heymach, Vivek Subbiah. _MD Anderson Cancer Center, Houston, TX_.

Background: The RET receptor tyrosine kinase can be oncogenically activated by gene fusions or point mutations. Multikinase inhibitors such as cabozantinib, lenvatinib, and vandetanib have demonstrated activity in RET dependent (RET+) malignancies. Recent development of selective RET inhibitors is poised to alter their treatment paradigm. However, the responsiveness of RET+ malignancies to immune checkpoint inhibition (ICI) is unknown. We compared the time to progression (TTP) on ICI with non-ICI therapy in patients with malignancies harboring activating RET mutations/fusions.

Methods: A retrospective chart review of all RET+ patients who were referred to the phase 1 clinical trials program at MD Anderson Cancer Center between September 2014 and October 2018 was conducted. Patients who had not discontinued treatment or had discontinued treatment for reasons besides disease progression were censored. Time to progression was estimated using Kaplan-Meier analysis.

Results: Out of 72 patients who had received systemic therapy for RET+ malignancies, 39 (54.2%) harbored RET mutations (RETm) and 33 (45.8%) harbored RET fusions (RETf). Nineteen patients (26.4%) received ICI and 53 (73.6%) received non-ICI therapy. Thirty four patients (47.2%) had medullary thyroid cancer, 29 (40.3%) had NSCLC and 9 (12.5%) had other types of cancer. PD-L1 expression was only available for 7 patients of which 5 (71.4%) were positive (≥ 1%). Patients had received a median of 1 (1-7) prior lines of therapy. Overall median TTP (mTTP) was 10.5 months (95% CI 6.7-30.1). Hazard ratio for ICI vs. non-ICI therapy was 1.73 (0.70, 4.26) p=0.25 in patients with RETf and 8.0 (2.27, 28.2) p=0.0025 in patients with RETm.

Conclusions: In patients with RET+ malignancies, mTTP was shorter with ICI compared to non-ICI therapy. The effect of type of therapy was dependent on the type of RET pathway aberration, with a statistically significant higher risk of progression in RETm malignancies treated with ICI compared to non-ICI therapy.

Time to Progression

---

RET | Therapy | N | mTTP (months) | Freedom from Progression at 6 months (%) | Freedom from Progression at 12 months (%)

Fusion | Non-ICI | 21 | 8.3 | 63 | 35

Fusion | ICI | 12 | 3.0 | 36 | 24

Mutation | Non-ICI | 32 | 31.9 | 82 | 73

Mutation | ICI | 7 | 5.6 | 43 | 0

#4998

Development of a personalized off-the-shelf whole-cell immunotherapy for breast cancer.

Vivekananda (Vivek) Sunkari,1 Sanne Graeve,1 George E. Peoples,2 Charles L. Wiseman,1 William V. Williams,1 Markus D. Lacher1. 1 _BriaCell Therapeutics Corp., Berkeley, CA;_ 2 _Cancer Insight, LLC., San Antonio, TX_.

BACKGROUND: Whole-cell cancer immunotherapies induce cancer-specific immune responses with the goal of long-term immune surveillance and remission. Non-replicating (irradiated) cancer cells are used to stimulate the immune system to recognize tumor-associated antigens and target tumor cells. Whole-cell immunotherapies have achieved regression of bulky, macroscopic tumors, but clinical trials have shown limited efficacy. SV-BR-1-GM is an HLA class I and II expressing, GM-CSF secreting breast cancer cell line. In a pilot clinical trial, an almost complete response of widely metastatic breast cancer was seen in a patient who allele-matched SV-BR-1-GM at HLA-DRB3. A follow-up Phase I/IIa clinical trial is ongoing in subjects with advanced breast cancer.

RESULTS: Extensive in vitro analysis demonstrated that SV-BR-1-GM cells not only have features of breast cancer cells but surprisingly also features of dendritic cells, the latter especially because of the expression of both HLA class I and class II complexes. SV-BR-1-GM cells "loaded" with a peptide known to bind to histocompatibility complexes containing HLA-DRβ3, as allele-encoded by SV-BR-1-GM, induced the activation of a CD4+ T cell clone specific for the peptide-DRβ3 complex, suggesting functionality of SV-BR-1-GM's HLA II machinery. To date, 24 subjects have been inoculated with the SV-BR-1-GM regimen in a Phase I/IIa trial with no adverse immediate hypersensitivity responses to low-dose inoculations with test cells (SV-BR-1 or SV-BR-1-GM). DTH response was evaluable in 18 patients with 72% developing DTH. The patient with the most pronounced DTH response, 01-002, also had a clinical response with regression of 20 of 20 lung metastases. Two other patients also had evidence of tumor regression. 6 patients were assessed for anti-SV-BR-1 antibodies. Whereas antibodies were found in sera of all patients, higher titers were measured in post-treatment compared to baseline samples. Patients who responded to the SV-BR-1-GM regimen with tumor regression matched SV-BR-1-GM at least at one HLA allele.

CONCLUSIONS AND OUTLOOK: SV-BR-1-GM cells may act as antigen-presenting cells directly activating HLA matching patient T cells. To include more patients predicted to derive clinical benefit from this whole-cell approach, SV-BR-1 cells are being engineered to, among others, overexpress exogenous HLA alleles. The goal is to develop a set of cell lines suitable for personalized off-the-shelf immunotherapy. The strategy will result in cell lines that match ~90% of the US population at 2 or more HLA alleles.

#4999

Characterization of myeloid derived suppressor cells after capecitabine and nivolumab combination treatment.

Qing Lin, Tienan Wang, Wenjun Yan, Ranran Wu, Lun Nie, Chensi Miao, Xing Wang, Feng Wang, Jie Zhang, Weilin Shi, Yanping Yuan, Tingting Shi, Ao Li, Wei Dong, Xu Chen, Jingying Sun, Qunsheng Ji. _WuXi AppTec co. Ltd., Shanghai, China_.

Capecitabine, a widely used oral chemotherapeutic agent, only functions in its active form, 5-FU. The metabolism of capecitabine to 5-FU frequently occurs in tumor tissues with high expression of thymidine phosphorylase, enzyme responsible for converting capecitabine. Extensive studies have demonstrated that capecitabine functions to inhibit growth of tumors, especially for metastatic breast and colorectal tumors, by impairing DNA synthesis and metabolism. However, effects of capecitabine on myeloid derived suppressor cell (MDSC) in the tumor microenvironment are poorly understood. In fact, conflicting results were reported that capecitabine either reduced CD11bhigh/Gr1high MDSC and enhanced T cell functions, or failed to alter the number and function of MDSC or the levels of GM-CSF/IL6 in blood. In this study, we established an optimized in-vitro MDSC suppressive assay and take the advantage of this system to investigate the function of capecitabine on MDSC-mediated T cell suppression. Myeloid cells in tumor environment acquire the expression of PD-L1, suggesting that blocking the PD1-PD-L1 axis might revert the function of MDSC. Therefore, we further explored the combination effect of capecitabine and nivolumab, a PD-1 antibody, on the function of MDSC in human PBMC cells.

#5000

**Cell cycle checkpoint kinase (CHK)1 inhibition induces immune modulation in recurrent** BRCA **wild-type high-grade serous ovarian cancer (HGSOC) patients.**

Erika J. Lampert,1 Min-Jung Lee,1 Jane B. Trepel,1 Akira Yuno,1 Ashley Cimino-Mathews,2 Joo-Sang Lee,1 Eytan Ruppin,1 Jayakumar Nair,1 Irene Ekwede,1 Jung-Min Lee1. 1 _Center for Cancer Research, National Cancer Institute, Bethesda, MD;_ 2 _Johns Hopkins Hospital, Baltimore, MD_.

Background: Data suggest cell cycle checkpoint inhibition with cyclin-dependent protein kinase 4 and 6 inhibitors may modulate the tumor immune microenvironment by augmenting T cell activation. It is unknown whether CHK1, which regulates the G2/M transition, may similarly engage in immune cross-talk. We hypothesize that CHK1 inhibition may increase tumor and peripheral immunogenicity via increased DNA damage and modulation of the innate and adaptive immune system in recurrent ovarian cancer.

Methods: We previously reported clinical efficacy of the CHK1 inhibitor (CHK1i) prexasertib in heavily pretreated BRCA wild-type (BRCAwt) HGSOC patients, yielding a 33% response rate. Blood samples were collected at baseline and 6-24 hours post-second dose on cycle 1 day 15 (C1D15) to assess immune subsets and γ-H2AX using multiparametric flow cytometry. Paired fresh core biopsies were collected at baseline and post-treatment on C1D15 for total RNA-seq. Archival tissue samples were also obtained.

Results: 23 of 24 patients had paired blood samples for correlative flow cytometry studies. There was a statistically significant increase in γ-H2AX from baseline to C1D15 in CD3+ and CD19+ lymphocytes (median fluorescence intensity 1.63 vs 1.74 p=0.034; 1.69 vs 1.76 p=0.040, respectively) suggesting increased DNA damage after CHK1i treatment. The percent of total CD14+ monocytes per mononuclear cells increased after treatment (31.6 vs 45.6% p=0.005), and an increase from baseline to C1D15 in HLA-DR on CD14+ monocytes, a marker of immunocompetence, was associated with improved PFS (3.5 months vs 9.25 months if decreased vs increased p=0.019). There was a significant decrease in CD4+ and CD8+ T cells on C1D15 compared to baseline (25 vs 19.6% p=0.008; 9.49 vs 7.63% p=0.005, respectively). However, GITR+ CD4+ and CD8+ T cells, suggesting activated effector T cells, increased on C1D15 (0.32 vs 0.82% p=0.0003; 0.23 vs 0.5% p<0.0001, respectively). Of the four The Cancer Genome Atlas molecular subtypes of HGSOC, immunoreactive was most common (7/18 [39%]) among 18 available pretreatment biopsies. Molecular subtype was not predictive of response and did not impact immunomodulatory effects seen. In 9 paired biopsies, there were no significant differences in expression levels of immunostimulatory (e.g. IFN-γ, IL-16) nor suppressive cytokines (e.g. IL10, TGF-β, CCL22). Immunohistochemical staining for PD-L1, Tregs, TILs, TAMs, and CD8 expression on archival tissue samples will be presented.

Conclusions: CHK1i treatment increases γ-H2AX levels in peripheral blood indicating prexasertib-induced DNA damage. The data suggest CHK1 inhibition elicits an anti-tumor innate immune response in the peripheral blood of heavily pretreated BRCAwt HGSOC patients. An increase in immunocompetent monocytes may be associated with clinical benefit and warrants further evaluation.

#5001

Open-label, non-randomized, exploratory pre-operative window-of-opportunity trial to investigate the pharmacokinetics and pharmacodynamics of the smac mimetic Debio 1143 in patients with resectable squamous cell carcinoma of the head and neck.

Carlos Gomez-Roca,1 Caroline Even,2 Christophe Le Tourneau,3 Neus Basté Rotllan,2 Jean-Pierre Delord,1 Jerome Sarini,1 Sebastien Vergez,1 Stephane Teman,2 Caroline Hoffmann,3 Philippe Rochaix,4 Bruno Gavillet,5 Elisabeth Rouits,5 Franck Brichory,5 Dalit Rechavi-Robinson,5 Vincent Bize,5 Sebastien Del Rizzo,5 Daniela Purcea,5 Silvano Brienza,5 Claudio Zanna,5 Gregoire Vuagniaux,5 Sergio Szyldergemajn5. 1 _Institut Universitaire du Cancer de Toulouse, Toulouse, France;_ 2 _Institut Gustave-Roussy, Paris, France;_ 3 _Institut Curie, Paris, France;_ 4 _Institut Universitaire du Cancer de Toulouse, France;_ 5 _Debiopharm International S.A., Lausanne, Switzerland_.

Background: Inhibitor of Apoptosis Proteins (IAPs) regulate apoptosis and modulate NFκB signaling, which in turn drives the expression of genes involved in immune and inflammatory responses. Debio 1143 is an orally available antagonist of IAPs with the potential to enhance tumor response, when combined with chemo/radiotherapy and/or immunotherapy, that is currently being tested in phase II clinical trials.

Methods: We investigated the pharmacokinetics and pharmacodynamic effects of Debio 1143 monotherapy (200 mg/day D1-15 +/-2 p.o.) in paired tumor samples collected at diagnosis and at time of surgical resection in a window of opportunity trial in patients with potentially resectable squamous cell carcinoma of the head and neck (SCCHN) (EudraCT Number: 2014-004655-31). Pharmacokinetic disposition of Debio 1143 was evaluated in tumor samples by quantitative mass spectrometry imaging (QMSI), and in plasma by LC-MS/MS. Immunohistochemistry for cellular IAP1 (cIAP1), cleaved caspase-3 apoptosis marker, Ki-67 proliferation marker, CD3+, CD4+ and CD8+ tumor-infiltrating lymphocytes (TILs), PD-1 and PD-L1 were performed in pre- and post-treatment specimens. Exploratory transcriptomic analyses were conducted to assess the effects of Debio 1143 on gene expression.

Results: Twelve patients were evaluated. Debio 1143 monotherapy was well tolerated. Tumor penetration was high, with Debio 1143 concentrations up to 55-fold those found in plasma at the time of the resection. Pharmacodynamic analysis of resected tumors showed a significant target engagement with degradation of cIAP1 (p < 0.05). There was no significant change in cleaved caspase-3 or proliferation measured by Ki67. Overall, the levels of CD8+ TILs, PD-1 and PD-L1 positive immune cells increased significantly (p < 0.05) compared to pre-treatment levels. Changes were observed in the expression of genes related to NFκB signaling.

Conclusions: This study demonstrates that Debio 1143 distributes widely into SCCHN tumors, engages its target cIAP1, and induces downstream effects that may modulate immunity in the tumor microenvironment supporting future combination therapy with immune-checkpoint agents in cancer patients.

#5002

Investigating the role of NF- κB signaling and immune checkpoint blockade therapy in melanoma.

Keith Wells, Jennifer Hintzsche, Carol M. Amato, Richard Tobin, Victoria Vorwald, Martin McCarter, Yiqun Shellman, Aik Choon Tan, William Robinson. _University of Colorado Denver, Aurora, CO_.

Immune checkpoint blockade (ICB) treatments, such as anti-PD1, improves survival in patients with metastatic melanoma. Despite durable response rates, 40-50% of patients show no benefit. Implicated in acquired resistance is the downregulation of antigen presentation, via dysfunction of the interferon-receptor pathway. However, little is known about intrinsic response mechanisms to ICB. The NF-κB-pathway is important for immune response as a key transcription factor for several antigen presentation proteins and cytokine/chemokine production. This study explored NF-κB signaling in patients with favorable, or unfavorable responses to anti-PD1 therapy. Fifty-two patients with metastatic melanoma were grouped into complete or partial responders (CR/PR; n = 6 and 15, respectively), and stable or progressive disease (SD/PD; n = 16 and 15, respectively). Consistent with other studies, tumor mutational burden assessed by whole exome sequencing (WES) was higher in the CR/PR group (p = 0.03). WES analysis also showed an enrichment of variants in NF-κB-pathway related genes within the CR/PR group (14/21, 67%) compared with SD/PD patients (6/31, 19%), p = 0.001. Notably, the hotspot variants G34E/R and G41E in NFKBIE, a negative regulator of NF-κB, were found exclusively in patients with complete or partial response to ICB (5/21, 24%). RNA-seq analysis of 19 tumors showed that the TNFα/NF-κB signaling pathway is upregulated in the CR/PR group, and highly expressed genes included CD83, HLA-DQB, and NFATC1. Therefore we hypothesized that when activated, particularly within the melanoma cells, this pathway contributes to an anti-tumor immune response. Using HEK293 cells, we demonstrated NFKBIEG34E increased NF-κΒ activity compared with the wildtype, upon TNFα stimulation. Consistently, flow cytometry also show an increase of targets of NF-κB transcription such as CD83, MHC class II, and PDL1. Similarly, the melanoma cell line A375, which harbors the NFKBIEG34E mutation, showed increases in protein expression when stimulated with TNFα and INFγ. Furthermore, progression free survival increased in patients that had tumors with an NF-κB-pathway variant compared with patients that were pathway wildtype (p = 0.03). Patients harboring tumors with NF-κB-pathway variants also had a greater response by RECIST criteria compared with wildtype. Taken together, our results suggest that an increase of pro-immunogenic NF-κB signaling in the tumor may contribute to the favorable response to ICB, highlighting a role for CD83 in the tumor microenvironment that merits further examination.

#5003

Vaccibody DNA vaccine platform VB10.NEO induces strong neo-antigen specific CD8+ T cell responses critical to cure established tumors in pre-clinical models.

Elisabeth Stubsrud, Stine Granum, Helene Zell-Flagstad, Audun Bersaas, Lise Madelene Skullerud, Monika Sekelja, Karoline Schjetne, Agnete Fredriksen. _Vaccibody, Oslo, Norway_.

BACKGROUND: Recent advances in the field of cancer immunotherapy have identified CD8+ T cell responses against tumor-specific neoantigens as a key driver of tumor regression and prolonged survival. VB10.NEO is a highly potent DNA plasmid vaccine with intrinsic adjuvant effect designed for efficient delivery of personalized tumor-specific neoantigens. VB10.NEO plasmid is translated in vivo and the secreted protein will covalently bind to endocytic receptors on APC by a targeting unit expressing CCL3 (MIP-1α) allowing efficient uptake and presentation of the neoantigens. In addition, CCL3 will attract immune cells by chemotaxis and induce maturation of APC locally. The objective of this study is to demonstrate the potential of VB10.NEO to induce tumor-specific T cell responses and control tumor growth.

METHODS: VB10.NEO was delivered i.m to study CD8+ and CD4+ neoantigen-specific T cell responses in four different pre-clinical mouse models. Immunogenicity was compared with delivery of neoantigen as traditional peptide-adjuvant immunization. The tumor protective effect of VB10.NEO in the presence or absence of anti-PD-1 therapy was investigated in the CT26 colon carcinoma model.

RESULTS: Vaccibody DNA Vaccine Platform VB10.NEO is flexible and can hold up to at least 40 neoepitopes. VB10.NEO vaccination induced strong neoantigen-specific T cell responses. Homologous boost vaccinations further augmented the response. T cell responses against predicted CD8+ T cell epitopes previously reported as non-immunogenic or activating only weak T cell responses using a conventional peptide-adjuvant or RNA immunization showed strong CD8+ T cell responses when delivered in the Vaccibody format, demonstrating a unique and strong priming of CD8+ T cells using VB10.NEO. The response was also accompanied by CD4+ T cell responses. In a therapeutic tumor setting VB10.NEO vaccinated mice (monotherapy) induced tumor protective responses. The effect was augmented when combining VB10.NEO with anti-PD-1 where complete regression of large established tumors was observed. All tumour-free mice rechallenged with a lethal tumor dose were protected indicating induction of long-lasting memory responses. Furthermore, the critical role of CD8+ T cells for the observed tumour protection was confirmed when depleting CD8+ T cells.

CONCLUSION: VB10.NEO immunotherapy induce strong CD8+ T cell responses critical for anti-tumor effect which demonstrate the unique characteristic of the Vaccibody platform to potentiate activation of CD8+ T cells. VB10.NEO in combination with anti-PD-1 further synergize the potent immune responses resulting in durable complete tumor regression in pre-clinical models supporting the scientific rational for the current ongoing clinical trial investigating VB10.NEO in combination with CPI in patients with advanced solid tumors.

#5004

A new pattern called Hyperprogression when using Immune checkpoint blockers in real world.

SEOREE KIM,1 Sang-Yeob KIM2. 1 _Division of Medical Oncology, Department of Internal Medicine, College of Medicine, The Catholic University of Korea, Seoul 06591, Korea., seoul, south Korea, Republic of Korea;_ 2 _Asan Institute for Life Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul 05505, Korea, seoul, south Korea, Republic of Korea_.

Introduction Although immune checkpoint blockades (ICBs) therapy can lead to favorable and durable results by reinvigorating the anti-tumor immune response in some patients, many other patients experience poor prognosis and even tumor overgrowth can be seen in real practice. So, we performed retrospectively hyperprogression-related studies on patients who underwent immunotherapy to find the predictable clinical factor. We also revealed the difference of immune composition in TME that are associated with HPD by multiplex IHC

Method We retrospectively evaluated the medical records of NSCLC patients (n=243) who treated with anti-PD-1/anti-PD-L1 therapy at 5 institutes of St. Mary's Hospitals between January 2014 to June 2018.

Results A total of 231 patients were included in the TGK analysis. Median age was 64.2 years with a majority being male (74.9%) and smokers (70.1%, n=162). Among them, 88.9% were heavy smokers with over 20 pack per years (n=144). Most of the ICB drugs were used in the second and above lines, but the proportion used in the fourth or higher heavy treated patients was 16%(n=37). The rate of over-growth was 33.3% (n=77) among these patients, the rate of hyperprogression was 32.4% (n=25). The proportion of patients harboring oncogenic driver mutation such as EGFR alteration was 18.6%. Univariate analyses indicated that HPD was significantly associated with oncogenic drive mutant status compared with non-HPD (18.6% [8 of 43] vs 9.04% [17 of 188]; P= 0.001). No significant differences were observed according to age, number of previous lines of therapy, or presence of more than 3 metastatic sites before ICBs. Multivariate analyses with a multivariate logistic regression model demonstrated that presence of less than 3 metastatic sites (odds ratio [OR], 4.769; 95% [CI], 1.384-16.433; P < .013) was an independent predictor for HPD. We also analyzed the association of NLR, PLR and CAR with HPD. Serologic markers post 6 weeks, i.e., at the time of first response evaluation, showed prognostic value for the most significant hyperprogression after ICB use. Kaplan-Meier OS estimates showed that there was a clear trend toward worse outcome for the patients with HPD (median OS, 5.6 months;95% CI, 4.5-10.3; P < 0.001) compared with the patients with non-HPD disease progression (median OS, 7.4 months; 95% CI, 15.1-19.6; P = 0.003) The multiplex IHC results showed that the percentage of T-cell marker expression of Tumor and Stroma in each slide was different between good responder and HPD.

Conclusion Even though the improved recognition of hyperprogression, the etiology of HPD remains unclear, apart from the pseudoprogression. However, we observed that some clinical factors and some serologic biomarker can be used to predict HPD. Furthermore, we undergo TMB and multiplex IHC to understand mechanism of hyperprogression.

#5005

Role of tumor neoantigen expression during immunotherapy of pancreatic cancer.

Adam L. Burrack, Jackson Raynor, Ellen J. Spartz, Margaret A. Olson, Ingunn M. Stromnes. _University of Minnesota, Minneapolis, MN_.

Pancreatic ductal adenocarcinoma (PDA) is a highly lethal malignancy with few effective therapies. Immune checkpoint blockade (ICB) has shown remarkable clinical responses in many advanced cancers. However, pancreatic cancer fails to respond to ICB for reason(s) not well understood. Therefore, we developed a new pancreas cancer animal model that we serendipitously discovered exhibits differential therapeutic responses following ICB. Specifically, primary tumor epithelial cells derived from C57Bl/6 KrasG12D/+;Trp53R172H/+;Ptf1a-Cre (KPC) mice were transduced with a luciferase-GFP to visualize tumor growth in vivo, single-cell cloned, and orthotopically implanted into the pancreas of B6 mice. Tumors rapidly grew and recruited numerous immune cell subsets. Unexpectedly, administration of 3 doses of either αPD-1 or αPD-L1 significantly reduced tumor size and prolonged animal survival (median overall survival 23 vs. 43 days, p=0.0023). In contrast, orthotopic implantation of parental or GFP+ KPC cells grew unabated following ICB, and clones derived from the same parental KPC cells transduced with ovalbumin failed to establish tumors. We mapped the immunodominant H-2Db luciferase immunotherapy-responsive epitope and generated fluorescently-labeled MHC class I tetramers to detect tumor-specific T cells. We found 20-40% of the tumor-infiltrating CD8+ T cells and 1-5% of the splenic CD8+ T cells bind tetramer at 3 weeks, resulting in a >3 log increase in cell number in tumor-bearing mice compared to naïve mice, which have on average 133 tetramer+ T cells. Tetramer+ T cells peaked in circulation 5 days following the first dose of anti-PD-L1, and persisted for at least 48 days. ICB significantly increased the frequency of intratumoral IFNγ+ tetramer+ T cells while decreasing the breadth of co-inhibitory receptor expression. We are currently employing single cell gene expression in combination with T cell receptor sequencing to identify molecular signature of immunotherapy responsive T cells. Our studies are reminiscent of the Goldilocks model of thymic TCR selection: tumor cells that express neoepitopes that elicit too strong of a T cell response will be eradicated, whereas as other neoepitopes, such as the one identified here, can render pancreatic tumor cells transiently susceptible to ICB. We also show that despite expression of an immunotherapy responsive epitope and sustained treatment with ICB, pancreatic cancer is not eradicated, highlighting the necessity to develop strategies to enhance pancreatic tumor-specific T cells in addition to ICB.

#5006

Comprehensive gene expression analysis of the tumor microenvironment in patients with advanced cancer treated with a personalized neoantigen vaccine, NEO-PV-01, in combination with anti-PD1.

Meghan E. Bushway,1 Ying Sonia Ting,1 Rana H. Besada,1 Tracey E. Sciuto,1 Jasmina Prabhakara,1 Julian Scherer,1 Kristen N. Balogh,1 April Lamb,1 Jennifer A. Kaplan,1 Lisa D. Cleary,1 Melissa A. Moles,1 Sarah E. Church,2 Yuqi Ren,2 Xing Ren,2 Richard B. Gaynor,1 Matthew J. Goldstein,1 Les H. Brail,1 Joel Greshock,1 Lakshmi Srinivasan1. 1 _Neon Therapeutics, Cambridge, MA;_ 2 _NanoString Technologies, Inc, Seattle, WA_.

Background: Neoantigens arise from DNA mutations in cancer cells and are important targets for T cell mediated anti-tumor immunity. NEO-PV-01 is a personal neoantigen vaccine of up to 20 peptides designed based on a patient's neoantigen and HLA profile that is directed at inducing tumor-specific T cell responses to neoantigens. Here we report comprehensive immune-related gene expression analysis of longitudinal tumor biopsies from patients with metastatic melanoma, bladder, and non-small cell lung cancer treated on our NT-001 trial with NEO-PV-01 + adjuvant in combination with nivolumab (NCT02897765) to correlate with clinical outcomes.

Methods: Tumor biopsies from all three tumor types were collected i) prior to treatment, ii) after 12 weeks of nivolumab monotherapy and iii) after completion of NEO-PV-01 vaccination. Targeted gene expression analysis on RNA extracted from FFPE blocks was performed using the NanoString™ nCounter platform. A custom set of 800 genes included markers for immune cell populations, cytolytic markers, immune activation and suppression, and the tumor microenvironment. Gene signatures of key immune features were calculated after normalization with housekeeping genes and used for subsequent analysis.

Results: Changes in the immune cell populations and the tumor microenvironment were detected after treatment with nivolumab and NEO-PV-01. Increases in various immune cell subsets, including T cells and B cells, as well as an increase in cytolytic phenotype were observed in tumor biopsies following treatment. Moreover, changes in the tumor microenvironment, consistent with the absence of tumor by histologic evaluation, were detected in many of the post-vaccination biopsies. In addition, these observations were consistent with data from peripheral blood that demonstrated durable de novo neoantigen-specific immune responses after vaccination. Additional exploratory analyses of the data demonstrate differential gene expression in patients' tumors that align with tumor responses to therapy.

Conclusion: Treatment with nivolumab and NEO-PV-01 leads to changes in the tumor microenvironment that are consistent with cell types and phenotypes that could contribute to an anti-tumor response. 

### Tumor Immune Microenvironment

#5007

Preclinical evaluation of JTX-8064, an anti-LILRB2 antagonist antibody, for reprogramming tumor-associated macrophages.

Heather Cohen, Yasmin Hashambhoy-Ramsay, Lauren R. Pepper, Jeffrey Y. Smith, Margaret Willer, Kevin Guay, Vikki Spaulding, Kristin O'Malley, Monica Gostissa, Abha Dhaneshwar, Edward C. Stack, Alessandro Mora, Donald R. Shaffer. _Jounce Therapeutics, Cambridge, MA_.

Introduction: Jounce has generated cell type-specific gene signatures as a means of probing The Cancer Genome Atlas and other large datasets to identify targets that may be important immune checkpoints. Using a tumor-associated macrophage (TAM) gene signature, we have found a strong correlation and coherence between TAMs and LILRB2 (leukocyte immunoglobulin like receptor B2; ILT4) across multiple tumors types. LILRB2 is a myeloid cell surface receptor containing four extracellular immunoglobulin domains, a transmembrane domain, and three cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Ligation of LILRB2 on myeloid cells, via its endogenous ligands (classical MHC I molecules [e.g. HLA-A, HLA-B] and non-classical MHC I molecules [e.g. HLA-G]), provides a negative signal that inhibits stimulation of an immune response. HLA-G is recognized as an important immunosuppressive molecule playing a role in maternal-fetal tolerance and being overexpressed in cancer - often associated with advanced disease stage and poor prognosis. As tumor-associated macrophages are known to suppress the anti-cancer immune response, these findings provide rationale for targeting LILRB2.

Methods and Results: We have generated a panel of monoclonal antibodies that bind specifically to LILRB2, but not other LILR family members, and can block binding of LILRB2 to MHC I molecules (i.e. HLA-A and HLA-G). In vitro differentiated monocyte-derived macrophages (MDMs) cultured for 24h in the presence of anti-LILRB2 antibodies and lipopolysaccharide (LPS) show polarization toward a more inflammatory phenotype - secreting higher levels of TNF-α and IL-6 with decreased amounts of IL-10 and CCL2 as compared to an isotype control antibody. NanoString mRNA analysis revealed that, in the absence of LPS or any additional stimuli, MDMs cultured with anti-LILRB2 antibodies showed gene changes consistent with inflammatory or M1-like polarization of macrophages. Anti-LILRB2 antibodies were also evaluated in human tumor histoculture and induced pharmacodynamic responses consistent with macrophage and T cell activation in a variety of tumor types. While mice do not express LILRB2 specifically, they do express a LILRB-like molecule known as Pirb. Mice that are deficient in Pirb display resistance to mouse colon 38 (MC-38) tumor growth suggesting this pathway functions as immune checkpoint in cancer.

Conclusions: Based on these preclinical data, JTX-8064, a high affinity LILRB2-specific humanized antagonist monoclonal antibody, is being developed as an immunotherapeutic to reprogram suppressive macrophages within the tumor microenvironment.

#5009

**Translational efficacy of oncolytic HSV-1 in glioblastoma using a human autologous** ex vivo **platform, CANscript™.**

Munisha Smalley,1 Vidushi Kapoor,1 Douglas Best,1 Carmela Passaro,2 Michal O. Nowicki,2 Suniti K. Saha,3 Komal Prasad,4 E. Antonio Chiocca,2 Sean E. Lawler,2 Aaron Goldman1. 1 _Mitra Biotech RxDx, Woburn, MA;_ 2 _Brigham and Women's Hospital, Boston, MA;_ 3 _IRIS Multispeciality Hospital, Ganguly Bagan, Kolkata, India;_ 4 _Narayana Health, Bangalore, India_.

Background: Oncolytic viruses (OV) have been a topic of great interest as therapeutic agents for indications such as glioblastoma multiforme (GBM), where current treatment options are poor and limited. Alongside engineering these viruses, finding useful pre-clinical models to elucidate the efficacy of the OV has been challenging. In particular, these viruses have been developed to overcome immune resistance, allowing for the reinvigoration of the immune contexture in the tumor microenvironment (TME). Current pre-clinical models, such as mouse models, are limited in their ability to recapitulate the TME fully. Thus, there is a need for more relevant pre-clinical models to study the efficacy of OV and downstream effects in the TME.

Methods: rQNestin34.5v.1 herpes simplex virus (rQNestin) has previously been investigated in both in vitro and in vivo studies of GBM. To assimilate the effects of rQNestin on the TME, a more humanized system was employed; CANscriptTM. CANscript is an ex vivo human tumor model, that recapitulates the native, patient-autologous TME, incorporating autologous patient-derived peripheral blood mononucleated cells. We treated GBM tissue (n=10) with rQNestin, capable of expressing GFP, and profiled GFP expression as a proxy for replication efficiency. We coupled this analysis with gene expression of immune-related pathways to gauge the modulation of the immune contexture by rQNestin. In addition, we performed multiplex cytokine analysis and multiplex immunohistochemistry to investigate the impact of rQNestin on the spatial context and activity of the immune compartment in the TME, ex vivo.

Results: Immunohistochemistry established that viral replication and tissue penetration was observed, ex vivo. Furthermore, RNA transcriptional profiling and cytokine analysis revealed that the oHSV-1 was capable of dynamically altering the tumor microenvironment, and dysregulating immune subsets within the tumor. Further stratification of tumor samples based on the pharmacodynamic profile identified subsets of patient tumors that induce adaptive immunity.

Concluding remarks: Here, we report that CANscript, an ex vivo human tumor model, can be used to evaluate the effects of OV in the TME - not only viral replication but also the direct effect on the immune compartment. CANscript will be an invaluable tool to investigate the response and resistance to OV. This platform has the potential to enable better pre-clinical modeling of OV development.

#5010

Potentiating immune checkpoint blockade therapeutic efficacy using a small molecule activator of integrin cell adhesion receptors.

Yared Hailemichael,1 Peter Vanderslice,2 Robert V. Market,2 Ronald J. Biediger,2 Darren G. Woodside,2 Upendra K. Marathi,3 Willem W. Overwijk1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _Texas Heart Institute, Houston, TX;_ 3 _7Hills Pharma, Houston, TX_.

Background: Immune checkpoint blockade (ICB) has therapeutic benefit in several human cancers, but in many patients, ICB - induced T cells do not infiltrate tumors, preventing clinical benefit. Intratumoral T cell accumulation requires firm adhesion mediated by the integrins very late antigen-4 (VLA-4) and lymphocyte function-associated antigen-1 (LFA-1) on activated T cells. The integrin class of cell adhesion receptors also play critical roles in multiple phases of tumor immune responses. Blocking the LFA-1 interaction with its ligand Intercellular adhesion molecule 1 (ICAM-1) abrogates CD8+ tumor infiltration after anti-CTLA4 therapy. Here, we evaluated the effect of an integrin activator, 7HP349, on promoting intratumoral T cell accumulation to potentiate CTLA-4 and PD-L1 checkpoint blockade anti-tumor activity.

Experimental Procedure: To evaluate the effect of 7HP349 in promoting ICB therapeutic activity, we combined 7HP349 with anti-CTLA-4 or anti-PD-L1 antibodies in preclinical murine models for melanoma and colon carcinoma.

Results: In cell adhesion assays, 7HP349 increased by >100-fold the number of VLA-4 or LFA-1 expressing T cells that bind to vascular cell adhesion molecule 1 (VCAM-1) and ICAM-1, respectively. 7HP349 facilitated chemo-attraction of T cells across matrices of VCAM-1 or ICAM-1, induced by the chemokine SDF-1α. In a PDL-1 negative B16/BL6 melanoma model, 7HP349 was dosed twice weekly at 1 mg/Kg intratumorally either alone as a single agent compared to vehicle control (13% vs 0%, p<0.01) or in combination with anti-CTLA-4 (44% vs 21%, p<0.01). After intraperitoneal administration, 7HP349 increased the complete response in combinations with anti-CTLA-4 (77% vs 27%, p<0.01). In addition, 7HP349 increased median survival in mice with CT-26 colon cancer when dosed either as a single agent versus control (21 vs 15 days, P<0.0012) or in combination with anti-PD-L1 (24 vs 21 days, P<0.02). 7HP349 may also increase the effectiveness of anti-CD137 without increasing liver toxicity. Levels of CD8 effector T cells (Teff) detected in tumor were significantly higher in mice treated with anti-CTLA-4 and 7HP349 than anti-CTLA-4 and vehicle. Some of these Teff showed specificity to p15E, an endogenous retroviral epitope expressed on B16. Mice treated with anti-CTLA-4 and 7HP349 showed increased depigmentation (vitiligo) suggesting immunity to melanocyte differentiation antigens.

Conclusion: 7HP349 described here represents a first-in-concept agent that positively regulate VLA-4 and LFA-1 function. Activation of integrin cell adhesion molecules with 7HP349 is a promising approach to enhance the anti-cancer activity of checkpoint blockade therapy with antibodies against CTLA-4 and PD-(L)1.

#5011

Targeting hypoxia-induced immune suppression to overcome immunotherapy resistance in prostate cancer.

Priyamvada Jayaprakash, Midan Ai, Arthur Liu, Pratha Budhani, Todd Bartkowiak, Jie Sheng, Casey Ager, Courtney Nicholas, Ashvin Jaiswal, Yanqiu Sun, Krishna Shah, Sadhana Balasubramanyam, Nan Li, Guocan Wang, Jing Ning, Anna Zal, Tomasz Zal, Michael Curran. _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Immune checkpoint blockade is effective in "hot" tumors like melanoma with pre-existing immune infiltrates; however, "cold" tumors like prostate cancer fail to respond. We found that prostate cancers harbor regions of hypoxia that resist T cell infiltration even in the context of anti-CTLA-4 (cytotoxic T lymphocyte associated protein-4) and anti-PD-1 (programmed cell death protein 1) blockade. These hypoxic zones serve as islands of immune privilege through the recruitment and suppressive polarization of immature myeloid cells into myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM). We found that targeted hypoxia ablation using TH-302, a hypoxia-activated prodrug, sensitized both transplantable and spontaneous models of prostate cancer to checkpoint blockade, coincident with enhanced T cell infiltration and effector function and loss of MDSC recruitment and suppressive function. Tumors treated with the combination of TH-302 and checkpoint blockade showed a reduced capacity to suppressively polarize new myeloid immigrants, implying a durable reconditioning of the tumor microenvironment (TME) into an immune-infiltrated, pro-inflammatory milieu. T cells infiltrating combination-treated tumors exhibited increased mitochondrial respiration, consistent with creation of a metabolically favorable milieu for T cell function. Based on these findings, we hypothesized that other approaches capable of metabolically rewiring the TME should promote anti-tumor immunity and sensitize checkpoint blockade-resistant tumors to immunotherapy. With this in mind, we performed a longitudinal study comparing a panel of different mitochondrial respiration inhibitors and a glutaminase inhibitor for their efficacy in reducing hypoxia, improving T cell infiltration and decreasing myeloid cell recruitment and suppressive polarization using immunofluorescence staining and confocal microscopy. Our preliminary data suggests that inhibitors targeting mitochondrial respiration, rather than those targeting glutamine metabolism synergize with checkpoint blockade and exhibit the highest efficacy in increasing T cell recruitment. We continue to characterize the dynamics of hypoxia reduction, duration of normalization following drug withdrawal, and impact on the immune microenvironment of these diverse approaches to metabolic reconditioning.

#5012

Targeting CD39 with a first-in-class inhibitory antibody prevents ATP processing and increases T-cell activation.

Alana G. Lerner, Maria Kovalenko, Megan Welch, Tracy dela Cruz, Jeff Jones, Clifford Wong, Bradley Spatola, Meghan Eberhardt, Andrew Wong, Wanchi Fung, Leanna Lagpacan, Vanessa Soros, John Corbin, Courtney Beers, Achim K. Moesta. _Tizona Therapeutics, South San Francisco, CA_.

The ATP/Adenosine pathway in the tumor microenvironment (TME) has emerged as an important immune-regulatory pathway. The ATPase CD39 is highly expressed in the TME, both on infiltrating immune cells and tumor cells across a broad set of cancer indications. CD39 processes pro-inflammatory extracellular ATP to ADP and AMP, which is then processed by CD73, to immunosuppressive adenosine. Inhibiting the enzymatic function of CD39 has the potential to shift the immunosuppressive milieu of the TME in a 2-pronged fashion: 1) Enhancement of immunostimulatory extracellular ATP released by damaged and/or dying tumor cells and 2) Inhibition of the generation and accumulation of suppressive adenosine within the TME, thereby unleashing an immune-mediated anti-tumor response.

Tizona has generated a novel first-in-class fully human anti-CD39 antibody, TTX-030, that inhibits CD39 ATPase enzymatic function with sub-nanomolar affinity and potency. TTX-030 is specific for CD39 and binds to CD39+ cancer cell lines and primary human leukocytes with high affinity. TTX-030 is capable of inhibiting CD39 at elevated ATP concentrations reported in the TME. Enzymatic inhibition by TTX-030 has demonstrated: preservation of pro-inflammatory extracellular ATP, reduction of adenosine accumulation, and inhibition of phosphate release by a variety of CD39-expressing cells, including tumor cells and immune cells. In vitro functional assays using stimulated PBMCs exposed to exogenous ATP demonstrated increased proliferation of both CD4+ and CD8+ T cells in the presence of TTX-030 and increased secretion of the pro-inflammatory cytokines IFN-γ, TNF-α, and IL-2. Treatment of mice with a mouse-specific CD39 inhibitory antibody in a syngeneic tumor model significantly decreased tumor growth.

In summary, TTX-030 is a selective and potent CD39 enzymatic inhibitor, capable of preventing adenosine-mediated immune suppression and increasing T-cell activation. Inhibition of CD39 with TTX-030 represents a unique therapeutic target aimed at modulating the immunosuppressive TME in cancer.

#5013

NG-641: An oncolytic T-SIGn virus targeting cancer-associated fibroblasts in the stromal microenvironment of human carcinomas.

Brian R. Champion, Matthieu Besneux, Marilena Patsalidou, Ana Silva, Manuela Zonca, Nalini Marino, Gianfranco di Genova, Sam Illingworth, Stefania Fedele, Lorna Slater, Fred Lilley, Darren Plumb, Katy West, Paul Cockle, Alice Brown. _PsiOxus Therapeutics Ltd, Abingdon, United Kingdom_.

NG-641 is a modified variant of enadenotucirev (EnAd), an Ad11p/Ad3 chimeric group B adenovirus, which retains all the functional properties of enadenotucirev while also expressing transgenes designed to target breakdown of the stromal barrier and immune suppression within the tumor microenvironment. Enadenotucirev has potent and selective anti-tumor activity against a range of epithelial cancer cells, with a blood stability profile that enables systemic dosing; it has now been administered intravenously to over 100 cancer patients. As an approach to immunogene therapy targeting stromal rich tumors, we have created a transgene-modified variant of EnAd expressing a bi-specific T-cell activator molecule (FAP-TAc) recognizing fibroblast activating protein (FAP) on cancer associated fibroblasts (CAFs) and CD3 on T-cells. Production of FAP-TAc by virus infected tumor cells should lead to T-cell mediated killing of CAFs and thus modification of the tumor microenvironment to drive effective anti-tumor immunity. This local production approach also bypasses potential delivery and safety issues associated with systemic dosing of such molecules. To enhance the activity of the FAP-TAc molecule, particularly in tumors with poor immune cell infiltration ("excluded" or "immune desert" phenotypes), NG-641 also encodes the transgenes CXCL9, CXCL10 and IFNα to recruit T-cells and enhance the overall immune response and cancer cell killing.

Initial studies with different viruses expressing FAP-TAc alone showed that FAP-TAc activity generated by NG-641 infection was essentially the same as that with viruses bearing only the FAP-TAc transgene. Following infection of tumor cells with NG-641, the virus selectively secretes functional FAP-TAc molecules, as determined in cocultures of FAP+ fibroblasts and T cells. Production of CXCL9, CXCL10 and IFNα was confirmed by ELISA assays, with functionality evaluated by reporter cell, FACS and cell migration assays. T cell-activation mediated by FAP-TAc lead to cytokine secretion and cytotoxicity towards the fibroblasts, both of which were enhanced by expression of the IFNα transgene. Studies with primary human tumor samples (containing tumor cells, CAFs and infiltrated immune cells) also demonstrated potent activation of the endogenous T-cells, indicating that virus produced FAP-TAc is a potent T-cell activator despite suppressive influences of the tumor microenvironment.

In conclusion, we have shown that CAFs can be effectively targeted for T-cell mediated destruction by NG-641, a tumor stroma targeting transgene-bearing oncolytic virus. This is associated with strong activation of endogenous T-cells to kill CAFs even in the presence of an immunosuppressive microenvironment. Systemic dosing of such a virus to patients with stromal rich tumors may provide an effective approach for driving productive anti-tumor immunity.

#5014

Profile of ARX001822, a highly potent, selective and orally bioavailable A2a antagonist effective in preventing adenosinergic suppression of T cell activation.

Peter M. Finan,1 Roy Pettipher,1 Jonathan White,2 Viral Patel,3 Ben Moulton,4 Soraya Porres,2 Karolina Gherbi,3 Elizabeth M. Rosethorne,5 Steven J. Charlton,3 Clive McCarthy1. 1 _Adorx Therapeutics, Edinburgh, United Kingdom;_ 2 _Charles River Laboratories, Harlow, United Kingdom;_ 3 _Excellerate Biosciences, Nottingham, United Kingdom;_ 4 _YProTech, Alderley Park, United Kingdom;_ 5 _University of Nottingham, Nottingham, United Kingdom_.

Adenosine is elevated in the tumour microenvironment and plays a critical role in suppressing T cell function through high affinity interaction with the A2a receptor. Genetic deficiency of A2a in mice is associated with enhanced cytotoxic responses and reduced tumour burden in syngeneic models. These effects are mimicked by small molecule A2a antagonists and some of these compounds are currently being evaluated in clinical trials for the treatment of solid tumours, particularly in combination with checkpoint inhibitors. However, the high levels of adenosine in the tumour microenvironment can dramatically reduce the effectiveness of competitive A2a antagonists. The challenge therefore is to identify highly potent and selective A2a antagonists which retain potency in the presence of high concentrations of adenosine and therefore have the potential to nullify the adenosinergic pathway within the tumour microenvironment ARX001822 binds A2a with high affinity (Ki=0.9 nM) and with greater than 660-fold selectivity over A1, A2b and A3. In a functional assay utilising CHO cells expressing recombinant A2a ARX001822 inhibited cAMP production in response to the selective A2a agonist CGS21680 in a competitive manner (KB= 0.3nM). Activation of A2a leads to the suppression of T cell-derived cytokine production and ARX001822 prevented this suppression even in the presence of high concentration of the adenosine receptor ligand NECA (IC50=38 nM). ARX001822 was also active in human whole blood, preventing NECA-mediated elevation of pCREB in CD8+ T cells and restoring production of interferon-γ with a potency 5-20 times higher than that of competitor molecules undergoing clinical evaluation in cancer. ARX001822 was orally bioavailable in rats and mice and was effective in inhibiting elevation of pCREB in mouse CD8+ T cells in an ex vivo pharmacodynamic assay. ARX001822 is a highly potent and selective A2a antagonist which is effective in preventing adenosinergic mediated suppression of cytokine production in the presence of high concentrations of adenosine receptor ligands and a full complement of plasma proteins. Knowledge of whole blood potency on both pCREB and interferon-γ modulation combined with the exposure required for activity in the pharmacodynamic model is helpful in estimating the clinical exposure required for A2a receptor blockade and downstream events related to modulating T cell function.

#5015

HERA-CD40L a hexavalent CD40 agonist induces T cell mediated anti-tumor immune response and shows superior activity in direct comparison to benchmark agonistic antibodies.

Christian Gieffers, Jaromir Sykora, Christian Merz, Mauricio Redondo Müller, David M. Richards, Julian Sefrin, katharina Billian-Frey, Karl Heinonen, Viola Marschall, Matthias Schröder, Harald Fricke, Meinolf Thiemann, Hill Oliver. _Apogenix AG, Heidelberg, Germany_.

CD40 ligand is a member of the TNF superfamily (TNF-SF) and a key regulator of the immune system. Its cognate receptor CD40 is expressed on antigen-presenting cells and on many tumor types, and has emerged as an attractive target for immunological cancer treatment.

Effective signaling for CD40/CD40L depends on the formation of a defined ligand/receptor complex triggered by interaction of a trimeric CD40L with three CD40 receptor chains allowing correct assembly of intracellular signaling complexes and respective signal transduction. Trimerization is a hallmark of the TNF-SF and has pivotal implications for the generation of respective TNFR-SF agonists in particular. However, ignoring the underlying trimeric structural concept bivalent antibodies are still the main agonistic biotherapeutic development candidates to address TNFR-SF members including CD40. Such bivalent antibodies are inherently associated with limited agonistic activity that requires Fc/FcγR interactions or potentially show increased toxicity caused by super-clustering of endogenous ligand receptor pairs.

To overcome the inadequacies of antibodies, we have developed HERA-CD40L composed of three receptor binding domains in a single chain arrangement, linked to an Fc-silenced human IgG1 thereby generating a hexavalent molecule. HERA-CD40L mimics the natural ligand, induces potent agonistic activity and, importantly, does not require FcγR mediated crosslinking. Comparison of HERA-CD40L to anti-CD40 benchmark antibodies (including CP-870,893) revealed superiority for HERA-CD40L in all assays tested. (i) In contrast to antibodies, HERA-CD40L showed strong activation of NFkB signaling upon treatment of B cells. (ii) HERA-CD40L treatment, but not clinical benchmark antibodies, converts immature phagocytic macrophages into mature/professional APCs and promoted differentiation towards the M1 spectrum macrophages. (iii) Furthermore, HERA-CD40L treated PBMCs stimulate potent allogeneic anti-tumor T cell response that was not detectable for CD40-antibodies.

In vivo, a murine surrogate of HERA-CD40L stimulated clonal expansion of OT-I specific murine CD8+ T cells without affecting non-specific immune cells. In the syngeneic CT26wt mouse model mHERA-CD40L treatment converts cold into hot tumors by increasing infiltration of CD8+ and CD4+ T cells. In addition mHERA-CD40L showed single agent anti-tumor activity in the CD40-negative syngeneic MC38-CEA mouse model, suggesting an involvement of the immune system in controlling tumor growth.

In summary HERA-CD40L is a potent agonist able to establish single agent anti-tumor immune responses. Comparison to bivalent benchmark antibodies showed superior biological activity of HERA-CD40L and qualifies this molecule as an ideal candidate for combinatorial cancer treatments.

#5016

STACT-TREX1: A systemically-administered STING pathway agonist targets tumor-resident myeloid cells and induces adaptive anti-tumor immunity in multiple preclinical models.

Anastasia M. Makarova, Alexandre Iannello, Chris S. Rae, Beverly King, Marina Besprozvannaya, John Faulhaber, Justin Skoble, Christopher D. Thanos, Laura Hix Glickman. _Actym Therapeutics, Inc., Berkeley, CA_.

Systemic delivery of STING agonists to tumor-resident myeloid cells will be required to elicit optimal type I interferon-mediated anti-tumor immunity in a metastatic disease setting. To this end, we have generated a microbial-based immunotherapy (STACT- Salmonella Typhimurium (Attenuated) Checkpoint Therapy) that utilizes a highly attenuated, clinically developed strain that is specifically enriched in tumors after intravenous administration. The strain has been modified to reduce pro-inflammatory TLR signaling in order to limit its immunosuppressive microbial profile and significantly improve its therapeutic index. An inhibitory microRNA to TREX1 was designed and introduced into the STACT strain. TREX1 is a 3′ exonuclease immune checkpoint that degrades cytosolic DNA, thereby preventing it from binding cGAS and activating the STING pathway. Mutations in human TREX1 cause a type I interferonopathy known as Aicardi-Goutières syndrome and autoimmune chilblain lupus. Systemic delivery of small-molecule inhibitors targeting TREX1 are intractable due to its ubiquitous expression in healthy tissue. We have engineered our systemically administered therapy to specifically deliver RNAi against TREX1 to the myeloid compartment of the tumor microenvironment, rather than to the inflammatory TNFα- and IL-6-producing stromal compartment. This enables targeted cGAS/STING induction of type I interferon, thereby promoting optimal T cell priming against tumor neoantigens. STACT-TREX1 was evaluated for therapeutic efficacy in several tumor models, including CT26 and MC38 colon carcinoma models, and B16.F10 melanoma. A high degree of tumor-specific colonization of STACT-TREX1 therapy was observed after a single intravenous tail vein administration, with 100,000-fold enrichment detected in tumor tissue relative to spleen and liver. The therapy was well tolerated, with very low TNFα and IL-6 serum cytokines detected. In multiple flank tumor models, potent tumor growth inhibition and complete tumor regressions were observed as a monotherapy. Efficacy was CD8-dependent, and cured mice were protected from tumor re-challenge. Importantly, immune correlates post-treatment demonstrated a significant shift away from the recruitment of microbe-induced innate myeloid populations, and towards type I interferon-induced adaptive immunity. This novel therapeutic approach will allow for systemic delivery of a STING pathway agonist that induces type I interferon specifically in tumor-resident myeloid cells, inducing potent adaptive anti-tumor immunity in solid tumors that are refractory to existing immunotherapies.

#5017

**MEDI1191, a novel IL-12 mRNA therapy for intratumoral injection to promote T** H **1 transformation of the patient tumor microenvironment.**

Nadia Luheshi,1 Susannah Hewitt,2 Fabien Garcon,1 Shannon Burke,1 Amanda Watkins,1 Kristen Arnold,2 John Zielinski,2 Philip Martin,3 Michael Sulikowski,1 Christopher Bagnall,1 Jean-Martin Lapointe,1 Gordon Moody,3 Han Si,3 Christopher Morehouse,3 Robert W. Wilkinson,1 Ronald Herbst,3 Joshua Frederick2. 1 _MedImmune Ltd., Cambridge, United Kingdom;_ 2 _Moderna Inc., Cambridge, MA;_ 3 _MedImmune, Gaithersburg, MD_.

Patients who respond to PD-L1 / PD-1 immune checkpoint blockade tend to have an inflamed, TH1 polarised tumor microenvironment (TME), characterised by expression of interferon-γ (IFNγ) and PD-L1. Novel therapies that induce TH1 transformation of the patient TME therefore have the potential to enhance anti-tumor immunity. As a central mediator of TH1 immune responses, interleukin 12 (IL-12) directly induces IFNγ release from activated NK, NKT and T cells, and is known to play a key role in driving anti-tumor responses. However systemic recombinant IL-12 was poorly tolerated in early clinical trials. We therefore designed MEDI1191 as a novel IL-12-based therapy designed for injection directly into tumors, composed of a lipid nanoparticle (LNP)-formulated mRNA encoding human IL-12.

We previously reported that intratumoral (IT) mouse (m) IL-12 mRNA, the surrogate for MEDI1191, promotes cytotoxic T cell-dependent anti-tumor immunity and enhances responses to PD-L1 blockade in pre-clinical models. Here, we demonstrate that IFNγ is also required for the anti-tumor activity of mIL-12 mRNA. A single dose of mIL-12 mRNA significantly increased expression of IFNγ and TH1 genes in MC38 tumor-bearing mice. Treatment with an IFNγ neutralising antibody blocked mIL-12 mRNA anti-tumor activity in this model. In addition, we report here that MC38 tumor rejection in response mIL-12 mRNA / anti-PD-L1 combination therapy correlates with increased cytotoxic T cell infiltration into tumors, and expansion of tumor-reactive T cells in the periphery.

We next investigated the pharmocodynamic activity of MEDI1191 in patient tumor-derived models. A single IT dose of MEDI1191 induced human IL-12p70 expression in mice bearing four different patient-derived xenograft tumors. Furthermore, in an ex vivo patient tumor slice culture assay, MEDI1191 induced dose-dependent IL-12 release, IFNγ expression and upregulation of TH1-signature gene expression. IL-12 protein secretion was induced in slices of all patient tumors tested. However, the magnitude of the IFNγ response to MEDI1191 varied between patient tumors. Quantification of the tumoral T cell and NK cell numbers within the patient tumor samples revealed a positive correlation between MEDI1191-induced IFNγ release and baseline tumor NK infiltrate.

These preclinical data demonstrate the potential for MEDI1191 to induce IFNγ-dependent TH1 transformation of the TME, and support the development of MEDI1191 as a potential treatment for patients with solid tumors, alone and in combination with inhibitors of the PD-1/PD-L1 T cell checkpoint.

#5018

Activation of dendritic cells by immunostimulatory CD40L/4-1BBL-encoding oncolytic virotherapy in melanoma.

Jessica Wenthe,1 Tanja Lövgren,1 Mantas Šilanskas,1 Emma Eriksson,1 Angelica Loskog2. 1 _Uppsala University, Uppsala, Sweden;_ 2 _Uppsala University, Lokon Pharma AB, Uppsala, Sweden_.

Checkpoint blockade antibody therapy has revolutionized the treatment of metastatic melanoma. However, up to 50% of patients establish treatment resistance, which is frequently connected to a non-T cell inflamed phenotype. Poor T cell activation and infiltration is suggested to be caused by a lack of functional dendritic cells (DCs) that are crucial for T cell priming. Indeed, in progressing melanoma patients, immature DCs are often increased in the tumor lesion and draining lymph node. Herein, we investigated the effect of an immunostimulatory oncolytic adenovirus LOAd703 in a humanized melanoma-DC model. LOAd703 (serotype Ad5/35) carries two immunostimulatory transgenes; trimerized membrane-bound (TMZ)-CD40 ligand and 4-1BBL. Viral replication is restricted to tumor cells but transgene expression is controlled by a CMV promoter, which allows expression also in healthy cells of the surrounding tumor microenvironment (TME).

LOAd703 efficiently infected and killed the human melanoma cell line 526-mel as evaluated by MTS viability assay. Infected 526-mel cells expressed both TMZ-CD40L and 4-1BBL as verified by flow cytometry. To investigate LOAd703's effect on DC maturation, immature DCs were differentiated from CD14+ monocytes using GM-CSF and IL-4 and infected with LOAd703 or stimulated with MEGACD40L®, which is a highly active CD40L oligomer. Flow cytometry analysis revealed that LOAd703-infected DCs upregulated the expression of maturation markers and co-stimulatory molecules in a similar manner as high doses of MEGACD40L®. These included CD83, CD80, CD86, CCR7, ICAM-1 and MHC molecules. Interestingly, CD70 that is required for CD27 stimuli of T cells and lowers T cell receptor signaling threshold, was highly upregulated on the DCs using LOAd703, but not MEGACD40L®. Tumor cells are often immunosuppressive which may reduce the capacity of LOAd703 to activate DCs. To model this, immature DCs were co-cultured with LOAd703-infected 526-mel cells, which is also better mimicking the events in the TME. Co-cultured DCs upregulated the same markers as before, but this upregulation was twofold higher for CD83 and CD80. In agreement with the previous observation that CD40L signaling alone did not induce CD70 expression, CD70 expression on DCs was lower in the co-culture, indicating that a direct infection of the virus and potentially signaling through TLRs together with CD40L signaling is required to induce CD70 on DCs or that it may be inhibited by immunosuppressive factors released by the tumor. LOAd703-matured DCs also upregulated PD-L1 and thus it would be of interest to combine LOAd703 therapy with PD-L1/PD-1 blockade.

In conclusion, LOAd703 killed human melanoma cells and induced the expression of TMZ-CD40L and 4-1BBL in infected cells. Moreover, LOAd703 infection activated DCs to express costimulatory molecules, as well as CCR7, which is essential for a systemic immune response.

#5019

Allosteric inhibition of SHP2 suppresses CSF1R signaling and selectively reduces viability of M2 tumor associated macrophages contributing to anti-tumor immunity.

Elsa Quintana,1 Chris J. Schulze,1 Tiffany J. Choy,1 Darienne R. Myers,1 Kasia Mordec,1 Dylan Daniel,2 Mark A. Goldsmith,1 Jan A. Smith1. 1 _Revolution Medicines, Redwood City, CA;_ 2 _MI Bioresearch, Ann Arbor, MI_.

The protein-tyrosine phosphatase SHP2 promotes oncogenic RAS/MAPK pathway activation in different tumor types. In immune cells, SHP2 binds to phosphorylated ITIM and ITAM domains on regulatory receptors, including PD-1. We have shown that RMC-4550, a SHP2 allosteric inhibitor, attenuates tumor growth in syngeneic mouse tumor models with effects equivalent to, or greater than those of checkpoint inhibitors. Consistent with anti-tumor immunity as the mechanism, tumor growth inhibition (TGI) was not observed in immunocompromised mice. TGI was associated with changes in the tumor immune microenvironment, in both the adaptive and innate arms. Similar to checkpoint blockade, the frequency of CD8+T cell infiltrates was increased upon treatment with RMC-4550. Polarized macrophage populations were significantly shifted in favor of antitumor immunity, with marked increases in M1 and decreases in M2 cells, effects not seen with checkpoint blockade. The objective of the present study was to determine if the effect on tumor-associated macrophage (TAM) polarization is secondary to a release of the biochemical brakes on adaptive immunity and/or a result of a direct effect of SHP2 on TAMs.

In vivo administration of RMC-4550 in the CT26 syngeneic model significantly decreased M2, and increased M1, TAM after CD8+T cell depletion or IFNγ blockade, similar to the response in control animals. In vitro, SHP2 inhibition attenuated CSF1R signaling in murine bone marrow-derived macrophages and selectively induced apoptotic cell death in M2, but not M1, polarized macrophages. In addition, RMC-4550 treatment in vitro reduced the suppressive potential of human M-MDSCs. Collectively these observations suggest that SHP2 inhibition directly impacts the survival and function of suppressive monocytic immune cells. Given the effect of SHP2 inhibition on the CSF1R signaling pathway, we assessed whether the anti-tumor activity of RMC-4550 was similar to that of CSF1R blockade. In contrast to RMC-4550, anti-CSF1R antibody did not induce a significant delay in CT26 tumor growth. These results confirm that RMC-4550 has pleiotropic effects on the immune system including modulation of both adaptive and innate mechanisms. In summary, we propose that SHP2 plays a central role in inducing immune suppression in the tumor microenvironment by both inhibiting T cells and supporting the viability of pro-tumorigenic macrophages. Thus, SHP2 inhibition represents a novel investigational strategy with dual activity: direct inhibition of cancer cell growth in certain tumors as well as promotion of an anti-tumor immune response by direct transformation of the tumor immune microenvironment. Tumors that are intrinsically dependent upon SHP2 and exhibit a myeloid-rich microenvironment could be particularly susceptible to this dual-mechanism therapeutic strategy.

#5020

Therapeutic potential of an immune modulatory CCL22-based vaccine.

Inés Lecoq,1 Katharina L. Kopp,2 Rikke Christensen,2 Ayako W. Pedersen,2 Evelina Martinenaite,1 Mai-Britt Zocca,2 Mads H. Andersen1. 1 _Center for Cancer Immune Therapy, Copenhagen, Denmark;_ 2 _IO Biotech, Copenhagen, Denmark_.

CCL22 is a macrophage-derived chemokine that exerts immunosuppressive functions by the recruitment of regulatory T cells (Tregs) through the CCL22/CCR4 axis. It is expressed by cells in the tumor microenvironment (TME) in many different cancer tissues including breast, lung, and pancreatic cancer and is thought to promote the suppression of anti-cancer immunity. Recently, we described that CCL22-specific T-cells from cancer patients can kill CCL22-expressing tumor cells, and activation of CCL22-specific T cells may directly influence the CCL22 concentration in the TME, suggesting that CCL22 may be an interesting target for an immune modulatory vaccine.

Thus, in the present study we have targeted CCL22-expressing cells by activation of CCL22-specific effector T cells by peptide vaccination in tumor models. We present here that CCL22-targeting peptide vaccination leads to expansion of predominantly CD8+, IFNγ-secreting CCL22-specific T cells in both BALB/c and C57BL/6 mice. Furthermore, CCL22 vaccination results in delayed tumor growth and enhanced survival in tumor harboring hosts in CT26 and Pan02 models. Preliminary assessment of gene expression in the tumors reveals that CCL22 vaccination leads to a reduction of CCL22 expression, alongside other immune suppressive molecules, such as IDO1 in the TME. Characterization of vaccine-mediated changes of cellular composition of the TME is currently ongoing.

In conclusion, our findings provide a rationale for a novel cancer immunotherapy to target the immune suppressive TME.

#5021

**LNAplus** TM **antisense oligonucleotides targeting CD39 and CD73 have potent anti-tumor activity by modulating immunosuppressive tumor microenvironment.**

Julia Festag,1 Tamara Thelemann,1 Abhishek S. Kashyap,2 Richard Klar,1 Sandra Kallert,2 Melanie Buchi,2 Lisa Hinterwimmer,1 Monika Schell,1 Stefanie Raith,1 Sven Michel,1 Alfred Zippelius,2 Frank Jaschinski1. 1 _Secarna Pharmaceuticals GmbH & Co. KG, Planegg/ Martinsried, Germany; _2 _Cancer Immunology, University Hospital Basel, Basel, Switzerland_.

The ectonucleotidases CD39 and CD73 have emerged as promising therapeutic targets to enhance antitumor immune responses, as they act in concert to convert extracellular immune-stimulating ATP to immunosuppressive adenosine. CD39 and CD73 are expressed on different immune cells as well as on cancer cells and support the tumor in escaping immune recognition and destruction. Accordingly, in order to enhance immunity against tumors it would be favorable to increase extracellular ATP- and to reduce adenosine concentrations in the tumor microenvironment. As an alternative approach to small molecule inhibitors or antibodies, we developed antisense oligonucleotides (ASOs) using the OligofyerTM tool to suppress the expression of CD39 and CD73. ASOs were synthesized as Gapmers with flanking locked nucleic acids (LNA) to increase stability and affinity to the target RNA. Knockdown efficacy of ASOs on mRNA and protein level was investigated in cancer cell lines and primary human T cells. CD39 and CD73 activity was evaluated by measuring levels of ATP, AMP and adenosine in cell supernatants. As functional readout we analyzed T cell proliferation and viability in presence of extracellular ATP. The impact of ASO-mediated target knockdown in vivo on anti-tumor immune responses was analyzed in a syngeneic mouse tumor model. Unformulated CD39- and CD73-specific ASOs achieved potent target knockdown on mRNA and protein level in tumor cell lines and primary human T cells in vitro. Furthermore, degradation of extracellular ATP or AMP was significantly blocked by CD39- or CD73-specific ASOs while formation of adenosine was suppressed. Supplementation of cell culture medium with ATP impaired proliferation and viability if T cells expressed CD39 or CD73. Notably, ASO-mediated knockdown of CD39 or CD73 reversed this inhibitory effect of ATP. Systemic treatment of mice with CD39- and CD73-specific ASOs resulted in a knockdown of CD39 and CD73 mRNA expression in spleen and liver. Treatment of tumor-bearing mice with CD39-specific ASO led to dose dependent reduction of CD39 protein expression e.g. in Tregs, tumor-associated macrophages and myeloid-derived suppressor cells. Strikingly, the frequency of intratumoral Tregs was considerably diminished in CD39 ASO-treated animals. Moreover, tumor growth was strongly reduced by CD39-specific ASO. Anti-tumor efficacy was improved when combining CD39 ASO with anti-PD-1 treatment.

Taken together, targeting of CD39 and CD73 by ASOs represents a very promising state-of-the art therapeutic approach to improve immunity against tumors.

#5022

iGRAMMy: Cloud-based characterization of microbial landscape in colorectal cancers.

Li C. Xia,1 Dongmei Ai,2 Man Guo,2 Hanlee Ji1. 1 _Stanford University, Stanford, CA;_ 2 _Beijing Univ of Sci and Tech, Beijing, China_.

Many studies have identified pervasive tumor-infiltrating bacteria in colon, gastric, kidney, and other tumors. Infiltrating bacteria have functional significance, affecting the immune system state, affecting a tumor's ability to metastasize, and modifying patient's response to therapies. Only a few tools can infer infiltrating bacteria based on the RNA-and DNA-Seq data of tumor samples. These tools are not optimized for cloud-based computation, where large public datasets such as The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC) now reside. Here, we present a novel cloud-based bioinformatics tool - iGRAMMy, that accurately, efficiently, and robustly estimate microbial relative abundance in sequenced tumor and matched normal samples. We developed iGRAMMy by integrating read filtering, remapping, and relative abundance estimation steps of whole genome (WGS-seq) and/or transcriptome (RNA-seq) based microbiome analysis. We validated iGRAMMy by comparing estimated abundance to positive and negative controls of a published dataset as golden standard. We applied iGRAMMy to study all colorectal cancer samples within the TCGA cohort and reported the microbial landscape of these tumors. We identified a significantly higher abundance of overall bacterial infiltration in tumor as compared to normal samples. We found species like Bacteroides fragilis, Bacteroides dorei and Fusobacterium nucleatum were significantly higher in tumors, which were corroborated by previous reports. We uniquely identified Bacteroides intestinalis was also significantly more abundant in tumor samples. We released iGRAMMy and the entire analysis as a workflow pipeline on cancer genomics cloud, where researchers can easily replicate the study and use the tool for their own data.

#5023

Immunomodulation of the multi-tyrosine kinase inhibitor TAS-115 in a mouse model of prostate cancer.

Marco A. De Velasco,1 Yurie Kura,1 Noriko Sato,1 Naomi Ando,1 Kazuko Sakai,1 Kazuhiro Yoshimura,1 Masahiro Nozawa,1 Kazuhiro Yoshikawa,2 Kazuto Nishio,1 Hirotsugu Uemura1. 1 _Kindai University Faculty of Medicine, Osaka-Sayama, Japan;_ 2 _Aichi Medical University, Japan_.

Increasingly, immunotherapeutic agents are making their way into the clinic for the management of cancer, however, not all patients respond to single agent treatments. Susceptibility to immunotherapy is highly dependent on a tumor's immune composition. Agents that are able modulate the tumor's microenvironment (TME) to improve the balance between cytotoxic T cells and immunosuppressive cells such as myeloid-derived suppressor cells (MDSCs) M2-polarized macrophages and T regulatory cells (Tregs) are highly sought. We previously evaluated the preclinical activity of the multikinase inhibitor TAS-115 on mouse model of prostate cancer and showed that its antitumor effect was due in large part to its influence on the TME. In this study we examine the consequences of TAS-115 administration alone or in combination with androgen withdrawal via surgical castration or anti-androgen therapy with apalutamide, and focus on its influence on antitumor immunity. Pten/Trp53-double knockout mice were treated with TAS-115 alone or in combination with surgical castration or apalutamide for 4 weeks. Overall, TAS-115 reduced prostate tumor burden by 10.5%, however, 37.5% (3/5) of mice experienced no response (7.5%), while reductions of 14% and 27% were observed in 25% (2/8), 37.5% (3/5) of the remaining mice, respectively. A similar trend was noted in mice treated with the TAS/apalutamide combination but TAS plus surgical castration. Changes in cancer cell proliferation and apoptosis were insignificant with TAS-115 treatment, however, 2 of 4 TAS-115-responsive mice used for immunohistochemical (IHC) analysis showed a ~3-fold increased cleaved caspase-3 expression over non responsive mice. All TAS-115-treated mice showed reduced levels of phosphorylated STAT-3. Flow cytometric analysis of dissociated tumor samples revealed a reduction of tumor associated macrophages (TAMs) particularly when TAS-115 was given in combination with castration or apalutamide. Further analysis by IHC and qRT-PCR showed a M2-M1 polarization switch after TAS-115 therapy accompanied with decreased accumulation of monocytic MDSCs. An increase in tumor infiltrating neutrophils was also observed in mice receiving TAS-115. Although overall T cell infiltration was not improved TAS-115, there was an enhanced of activated CD8+ T cells, which was especially evident in responders. However, Treg infiltration was also increased after the administration of TAS-115. Overall our findings suggest that TAS-115 induced the reduction of immunosuppressive MDSCs and TAMs and may have induced an M2-M1 switch to provide a less immunosuppressive tumor microenvironment resulting in improved T cell mediated responses and provides evidence to support further investigation into using molecular targeting agents such TAS-115 as immune-modulators in combination with other immunotherapies to enhance or restore cytotoxic T cell activity.

#5024

Treatment with synthetic RIG-I agonist triphosphate RNA leads to local and systemic anti-tumor effects in a mouse melanoma tumor model.

Mike W. Helms,1 Eric Parmantier,2 Kerstin Jahn-Hofmann,1 Felix Gnerlich,1 Lars König,3 Christiane Metz-Weidmann,1 Monika Braun,1 Gabriele Dietert,1 Kaj Grandien,1 Joachim Theilhaber,4 Hui Cao,4 Tim Wagenaar,4 Max Schnurr,3 Stefan Endres,3 Dmitri Wiederschain,4 Sabine Scheidler,1 Simon Rothenfusser,3 Bodo Brunner1. 1 _Sanofi, Frankfort, Germany;_ 2 _Sanofi, VITRY, France;_ 3 _Ludwigs-Maximilian Universtät München, Munich, Germany;_ 4 _Sanofi, Cambridge, MA_.

RIG-I is a highly important cytosolic pattern recognition receptor (PRR) involved in sensing RNA virus infection and inducing interferon (IFN) production. RIG-I's natural ligand, triphosphate RNA (ppp-RNA), is proposed to be a valuable addition to the growing arsenal of cancer immunotherapy treatment options. This study validates the use of intratumoral treatment with synthetic RIG-I agonist ppp-RNA for the therapy of human cancer, with melanoma as potential entry indication amenable to intratumoral treatment. Firstly, we demonstrate that RIG-I expression is closely correlated to cellular and cytokine immune activation in a wide variety of tumor types. Secondly, cellular models of human melanoma confirm susceptibility of cancer cells to ppp-RNA treatment, revealing unexpected heterogeneity between cell lines in their selectivity for RNA features, including sequence, secondary structures and presence of triphosphate. Cellular RNA treatment responses (type I IFN, FasR, MHC-I, cytotoxicity) were demonstrated to be RIG-I dependent using RIG-I KO cells. Thirdly, we show that ppp-RNA treatment of a mouse melanoma tumor model, leads to significant local and systemic anti-tumor effects and survival benefits, associated with a type I IFN response, tumor cell apoptosis and innate and adaptive immune cell activation. For the first time, we demonstrate systemic presence of tumor antigen specific CTLs following treatment with RIG-I agonist. Overall our study demonstrates that ppp-RNA or analogs thereof have the potential to play an important role for cancer treatment in the next wave of immunotherapy. However, potential challenges in the generation and formulation of potent RIG-I agonists remain to be solved.

#5025

Immune modulatory activity of MP0250, a tri-specific VEGF and HGF neutralizing DARPin drug candidate.

Ulrike Fiedler, Laurent Juglair, Nicolo Rigamonti, Daniel C. Snell, Niina Veitonmaeki, Michael T. Stumpp, Andreas Harstrick, Victor Levitsky, Sandra Dietschy, Keith M. Dawson, Jutta Haunschild. _Molecular Partners AG, Schlieren-Zurich, Switzerland_.

The VEGF/VEGFR and HGF/cMet pathways are implicated in tumor survival, growth, angiogenesis, invasion and metastasis. We developed MP0250, a multi-specific DARPin protein that binds to and neutralizes VEGF-A and HGF. The phase I clinical trial has been concluded and showed that MP0250 was well tolerated, had sustained exposure and produced signs of anti-tumor activity. Our hypothesis is that MP0250 potentiates anti-tumor drugs and reverts adaptive therapy resistance by reshaping the tumor microenvironment. This concept is currently being tested in phase II clinical trials in multiple myeloma in combination with bortezomib and in NSCLC in combination with osimertinib. Here we show that the effects of MP0250 on the tumor microenvironment can also have immune modulatory activity and can restore anti-tumor immunity. Signaling by VEGFR and cMET lead to multiple immune-suppressive effects including T-cell exclusion and recruiting of immune-suppressive cell into tumors. Therefore, we studied the immune modulatory activity of MP0250 in combination with an anti-PD1 antibody in syngeneic mouse models: one colorectal cancer mouse model and two different mouse-derived isograft models (MDI) that express high and intermediate levels of HGF. We showed that MP0250 monotherapy produced strong anti-tumor activity in all tested models. In combination with the anti-PD1 antibody, the anti-tumor activity was enhanced in the MC38 model and the MDI model expressing high levels of HGF. Immune histochemical analysis showed that MP0250 reduced the number of tumor blood vessels and promoted blood vessel normalization which in turn should allow better T-cell infiltration into tumors. Indeed this was observed when MP0250 was combined with anti-PD1; the number of CD4 positive T-cells in tumors was increased compared to the single-agent therapy groups. Moreover, flow cytometry analysis showed that MP0250 significantly reduced the number of tumor infiltrated neutrophils, which is in line with recent findings that HGF/cMET signaling mediates the recruitment of neutrophils into tumors and generates an immune-excluded tumor microenvironment. Taken together we have shown that MP0250 has immune modulatory activity and potentiates the activity of anti-PD1 therapy in mice. This paves the way for a clinical combination of MP0250 with anti-PD1 or other immune activating therapies in patients.

#5026

The association of genomic features and immunosuppression in gastric cancer.

Shogo Kumagai,1 Yosuke Togashi,1 Kohei Shitara,1 Takahiro Kinoshita,1 Katsuya Tsuchihara,2 Hiroyoshi Nishikawa1. 1 _National Cancer Center Hospital East, Kashiwa-shi, Japan;_ 2 _National Cancer Center Hospital East, Kashiwa-City, Japan_.

While the clinical efficacy of anti-PD-1 antibody against gastric cancer has been shown, the clinical response is not satisfied. More detailed analyses of immune status in gastric cancer therefore are required. Here, we show that a sub-population of gastric cancer with a specific driver gene mutation develops immunosuppressive tumor microenvironment (TME), not only inhibiting migrations of CD8+ T cells, but also recruiting regulatory T cells (Tregs). The immunological phenotypes of TME were examined with multi-color flow-cytometry and RNA sequencing of surgically resected samples from 22 gastric cancer patients. Gastric cancers were clustered into two groups according to immunologic gene signatures: high- and low-immunogenic tumors. The low-immunogenic group was further divided into two groups with the ratio of effector Tregs to CD4+ T cells ≥20% or < 20%. Among the low-immunogenic group with high effector Tregs to CD4+ T cells ratio (n=5), two patients harbored a specific driver gene mutation. In order to elucidate the detail mechanism by which the specific driver gene mutation in gastric cancer inhibited migrations of CD8+ T cells and recruited Tregs, we evaluated comprehensive gene expressions using the specific driver gene mutation-overexpressed cell lines. Lower expression of chemokines crucial for CD8+ T-cell recruitment (CXCL10) was detected in the specific driver gene mutation-overexpressed cell lines than in the specific driver gene wild-type-overexpressed cell lines. In addition, Gene Set Enrichment Analysis (GSEA) demonstrated enrichment of gene sets associated with fatty acid synthesis in the specific driver gene mutation-overexpressed cell lines compared with the specific driver gene wild-type-overexpressed cell lines. Indeed, abundant fatty acids were detected in TME of driver gene mutation-overexpressed tumors and were dominantly utilized by Tregs, contributing the compelling survival of Tregs in the TME than effector T cells. These results suggested that gastric cancer cells with the specific driver gene mutation reduce the production of chemokines recruiting CD8+ T cells, produce a favorable TME for Tregs. We then developed mouse models and explored TILs and confirmed that the specific driver gene mutation-overexpressed tumors have lower CD8+ T cells and higher Tregs than wild-type-overexpressed tumors. Efficacy of anti PD-1 antibody was ameliorated in the specific driver gene mutation-overexpressed tumors compared with wild-type-overexpressed tumors. Our findings propose that certain types of cancer cells with the specific driver gene mutation inhibit immigration of CD8+ T cells, recruit Tregs, developing an immune suppressive TME associated with the resistance to anti PD-1 antibody. Further studies are required to develop new therapeutic methods of anti PD-1 antibody in combination with inhibitors which block down-streams of the specific driver gene mutation signaling.

#5027

Sequencing the tumor microenvironment and myeloid derived suppressor cells to understand response to immunotherapy in primary HER2 positive breast cancer.

Evanthia T. Roussos Torres, Christine Rafie, Emily Davis, Luciane T. Kagohara, Elana J. Fertig, Elizabeth M. Jaffee. _Johns Hopkins University, Baltimore, MD_.

The purpose of this study is to identify how the epigenetic modulator entinostat reprograms myeloid derived suppressor cells (MDSCs) to prime the tumor microenvironment (TME) and modulate immunotherapy response. Early trials investigating single agent ICIs, anti-CTLA-4 and anti-PD-1/PD-L1, resulted in overall response rates of 5-20% in patients with metastatic HER2 positive breast cancer but little is understood about use of these therapies in patients with early stage/ primary disease. Suboptimal and inconsistent immune responsiveness is likely the result of multiple suppressive signals within the TME that provide a formidable barrier to T cell infiltration leading to an opportunity for TME modulation to potentially improve response. We and others have shown that immunosuppressive factors can be overcome by adding the class 1 histone deacetylase inhibitor, entinostat, to ICIs to reprogram MDSCs and upregulate T cell attracting signals within the TME. We hypothesize that gene expression signatures of reprogrammed MDSCs are predictive of response with subsequent immunotherapy treatment. To test this hypothesis, we used the neu-N syngeneic mouse model of HER2 positive breast cancer. We investigated differences in survival and tumor burden in response to combinations of entinostat with anti-PD-1 and anti-CTLA-4. Having previously identified MDSCs as important targets of entinostat that modulate response to ICIs, we used methods we developed to study MDSC function. Given the broad effects of entinostat on global gene expression including that of MDSCs, we used single cell RNA-seq (scRNA-seq) on primary tumors and isolated intratumoral MDSCs to determine what is driving changes in MDSC function in primary tumors. We plan to use data obtained from scRNA-seq to develop signatures of response and identify changes in responders vs. non-responders to help develop biomarkers of response in primary disease. Our results thus far show that treatment with entinostat + ICIs improves survival, and decreases tumor burden in primary tumors. Ex-vivo studies of MDSCs demonstrate changes in immunosuppressive capabilities of MDSCs and show differences in infiltration and function of granulocytic vs. monocytic MDSCs. We expect that evaluation of the ongoing sequencing data will identify more specific immune targets within primary TME and help us understand the importance of targeting MDSCs in early disease. We predict this work will identify additional candidate targets for therapeutic testing to convert breast cancers into immune responsive tumors, and that this will facilitate improved response to ICIs that will ultimately translate into improved survival of patients with breast cancer.

#5028

Characterization of the tumor immune microenvironment in treatment-naïve EGFR-mutant NSCLC uncovers a low IFN-gamma suppressive immune phenotype.

Xiuning Le, Marcelo V. Negrao, Alexandre Reuben, Won-Chul Lee, Edwin Parra, Jun Li, Tatiana Karpinets, Carmen Behrens, Boris Sepesi, Ara Vaporiciyan, Jack Roth, Cara Haymaker, Emily Roarty, Jianhua Zhang, Chantale Bernatchez, Jianjun Zhang, Ignacio Wistuba, Don Gibbons, John Heymach. _MD Anderson Cancer Center, Houston, TX_.

Background: Although immune checkpoint blockade has been successfully utilized in treating patients with non-small cell lung cancer (NSCLC), the benefit of immunotherapy for patients with advanced EGFR-mutant (EGFRm) NSCLC has been limited. Data from IMpower150 trial's subgroup analysis suggests modulation of the tumor immune microenvironment (TME) with antiangiogenic agents and chemotherapy might enhance response to anti-PD-1/PD-L1 blockade in EGFRm lung cancers. We aim to gain a deep understanding of EGFRm NSCLC TME as the first step to make EGFRm amenable to immune therapy.

Methods: We queried two cohorts of resected stage I-III lung adenocarcinomas: PROSPECT (94 tumors) and Immune Genomic Profiling of NSCLC (ICON, 76 tumors). In the PROSPECT cohort (14 EGFRm vs 80 WT), we compared 10 immune markers by IHC (PD-L1, PD-1, CD3, CD4, CD8, CD45RO, CD57, CD68, FoxP3 and Granzyme B) and 44 immune regulator genes' expression levels and pathway signatures from microarray data. We subsequently validated the observation of differentially expressed genes in the ICON cohort (15 EGFRm vs 61 WT) using IF, RNAseq and WES.

Results: In the PROSPECT cohort, lower PD-L1 (2.2 vs 10.4 H-score, p<0.01) and lower GzmB (212 vs. 358 counts/mm2, p=0.02) were observed in the EGFRm compared to WT tumors. This is validated in the ICON cohort, showing lower PD-L1 expression (0.18% vs 7.28% p=0.05) and lower TMB (1.8 vs 9.0 mut/Mb, p<0.01) in EGFRm compared to WT tumors. IFN-gamma gene expression (3.89 vs 4.71 p=0.03 in PROSPECT) and signatures were low in the EGFRm tumors, suggesting a suppressive tumor microenvironment. Different than some prior reports, we found CD8+ T cells were not significantly different in EGFRm vs. WT groups. Among known immune regulators, TGFbeta was higher in the EGFRm tumors in the PROSPECT cohort, but not in the ICON cohort. Other known immune regulators, including CTLA4, LAG3, TIM3, TIGIT, IL6 and VEGFA were not differentially expressed.

Conclusion: Our analysis showed that EGFR-mutant NSCLCs demonstrate a PD-L1 low, GzmB low, IFN-gamma low immune phenotype, suggesting a suppressive tumor microenvironment. We found that the CD8+ T cells were present in the tumor, but likely suppressed functionally by the negative regulators in the TME. These results direct future analysis of suppressive immune cell populations (CD4+ subpopulation, macrophages and dendritic cells) in EGFRm lung cancers, and represent an initial step for rationale combination of immune therapy to modulate the suppressed TME, which might lead to enhanced treatment efficacy to benefit patients with EGFR-mutant lung cancers.

#5029

OT-101/Chemotherapy- A novel mechanism of action (MOA) in glioblastoma immunization therapy.

Larn Hwang, David Nam, Fatih Uckun, Vuong Trieu. _Oncotelic Inc, Agoura Hills, CA_.

Background: Escalating Intratumoral heterogeneity (IH) resulting in xenogenization which is countered by overexpression of TGF-β2. OT-101 is a TGF-β2-specific Phosphorothioate Antisense. Here we report the results of a randomized Phase 2b clinical trial (NCT00431561) comparing the safety and efficacy of OT-101 versus temozolomide (TMZ) in adult patients with WHO grade III/IV high-grade glioma. We took advantage of the known xenogenization activity of alkylating agent such TMZ to explore the impact of xenogenization on survival on treatment with OT-101.

Methods: This is a phase 2b, multi-national, multi-center, open-label, active-controlled, randomized parallel group dose-finding study to evaluate the efficacy and safety of OT-101 in adult patients with recurrent high-grade glioma, administered intratumorally as continuous high-flow microperfusion over a 7-day period every other week.

Results: A total of 134 patients, 89 patients in the OT-101 test group and 45 patients in the temozolomide (TMZ) group. OT-101 was superior to TMZ in doubling the number of pts in the good survival group, 64 of 97 pts (66%); whereas, only 14 of 45 pts (31%) treated with TMZ did (p=0.0001).

mOS | OT-101 | TMZ

---|---|---

|

Subseq. TMZ | Subseq. TMZ (or other alkylating agent)

Prev. TMZ | No | Yes | No | Yes

No | 4.2mos (N=14) | 26.2mos (N=30) | 5.6mos (N=8) | 13.2mos (N=9)

Yes | 4.8mos (N=19) | 29.5mos (N=34) | 6.6mos (N=14) | 36.6 mos (N=14)

There were two cases of neutropenia in the 90 pts treated with OT-101 versus 8, 10, and 8 pts with leukopenia, neutropenia, and thrombocytopenia, respectively, among 45 pts treated with TMZ (2% vs. 56%, p<0.0001).

Conclusions: Improved survival was achieved by repeated exposure to TMZ/alkylating agent. This was further enhanced by OT-101 and consistent with the proposed MOA of OT-101/Chemo as reactivation of immunity during TGF-β suppression and subsequent xenogenization by TMZ.

#5030

Microtubule destabilization by plinabulin generates anti-tumor immune response through repolarization of intratumoral macrophages.

Elham Pishali Bejestani,1 G. Kenneth Lloyd,2 Melanie Buchi,1 Reto Ritschard,1 Petra Herzig,1 Ramon Mohanlal,2 Lan Huang,2 James R. Tonra,2 Alfred Zippelius,1 Abhishek Kashyap1. 1 _University Hospital Basel, Basel, Switzerland;_ 2 _Beyondspring Pharmaceuticals, New York, NY_.

The M2 phenotype is dominant in tumor associated macrophages (TAMs) and correlates with poor prognosis. As M1 polarized macrophages possess anti-tumor functions, in vivo reprogramming of macrophages is a promising strategy for cancer treatment. We recently demonstrated that microtubule destabilizing chemotherapeutic agents such as plinabulin induce potent dendritic cell maturation and thereby augment anti-tumor immune responses; yet the effects of plinabulin on macrophage polarization remain unknown. Here we demonstrate that plinabulin, which is currently being investigated for both its anti-tumor effect and prevention of chemotherapy-induced neutropenia in phase II/III clinical trials,induces M1 polarization of ex vivo cultured human monocyte-derived M2 macrophages. This was characterized by a reduction in the cell surface expression of M2 markers CD163 and CD206 and an increase in the expression of co-stimulatory M1 markers CD80 and CD86. Phenotypically, plinabulin-polarized human M1 macrophages secreted increasing levels of iNOS and inflammatory cytokines IL-1β, IL-6 and IL-12 while IL-10 and IL-4 secretion decreased. This suggests a functional skewing from immunosuppressive M2 to pro-inflammatory M1 macrophages upon exposure to plinabulin. Similarly, plinabulin induced the M1 polarization of murine FACS sorted F4/80+ intratumoral M2-TAMs, characterized by increased CD80 and decreased CD206 expression. Moreover, systemic treatment (i.p.) of tumor bearing mice with plinabulin led to a significant reduction in the number of tumor-infiltrating TAMs with a concomitant M1 polarization of the remaining TAMs. Indeed, plinabulin treatment of mice bearing subcutaneous MC38 colon cancer or orthotopic EMT6 breast cancer tumors significantly delayed tumor growth. This efficacy of plinabulin remained unaltered in Rag2-/- mice lacking T cells, suggesting that macrophages are required for its anti-tumor activity. These results identify targeting of TAMs by plinabulin as a promising therapeutic strategy. Testing of plinabulin in combination with other macrophage targeting drugs are currently being explored to investigate the dual effect in reprogramming the tumor immune microenvironment.

#5031

Regulatory T cell ablation accelerates early stage breast cancer progression.

Wei Du, Leandro Martinez, Valentina Robila, Nicholas Clark, Michael Idowu, Paula Bos, Paula Bos. _Virginia Commonwealth University, Richmond, VA_.

Breast cancer is the most common malignancy in women worldwide. Early detection of pre-invasive breast tumor has dramatically improved due to the routine screening for women. Ductal carcinoma in situ is a non-obligate precursor of breast cancer, and it only progresses to invasive breast cancer in about 40% of patients. We have previously shown that transient ablation of regulatory T (Treg) cells in established breast tumors results in significant reduction of primary and lung metastatic tumor growth. In this study, we aim to investigate the specific role of Treg cells at the pre-malignant stages. Using a spontaneous, stage-wise MMTV-driven polyoma middle T (PyMT) model of murine mammary carcinogenesis, crossed to Foxp3DTR knock-in mice where injection of diphtheria toxin leads to a complete ablation of Treg cells for around 7 days, we found that ablation of Treg cells at the pre-invasive tumor stage resulted in a significant increase in the number of mammary glands developing tumors, as well as tumor growth compared to control mice. Histological examination of tissues revealed that the area of the incipient tumors was larger in Treg cell-ablated mice, and presented a more advanced tumor stage. In addition, Treg cell ablation resulted in a pronounced mammary inflammatory infiltrate, including an increase in tumor-associated macrophages and a soluble cytokine network conducive to tumor progression. Interestingly, Treg cell ablation increased the percentage of cancer stem/progenitor cells in the mammary compartment. Overall, our study demonstrates that unlike their role in established cancers, Treg cells ablation promotes breast cancer progression at early stages.

#5032

High ERbeta expression correlates with the tumor suppressor 15-PGDH and PGD2 synthase in a female cohort of colorectal cancer patients.

Geriolda Topi, Pujarini Dash, Shakti R. Satapathy, Syrina Mehrabi, Roy Ehrnström, Marie-Louise Lydrup, Anita Sjölander. _Lund Univ., Malmö, Sweden_.

Background: The estrogen receptor β (ERβ) is the predominant ER in the colon mucosa. We have previously reported that high expression of ERβ is independently associated with a better prognosis in female patient with colorectal cancer (CRC). β-catenin and cyclooxygenase-2 (COX-2) are well known to be involved in CRC tumorigenesis. We have shown that a high level of CysLT2R, a low level of CysLT1R and high mast cell density are correlated with good prognosis in CRC patients. β-catenin and some inflammatory mediators, such are cyclooxygenase-2 (COX-2), cysteinyl leukotriene receptor 1 and 2 (CysLT1R, CysLT2R), prostaglandin D2 (PGD2), 15-PGDH activity and mast cells have been associated with CRC. Aim: To investigate the association between ERβ and other tumor modulators in CRC patients, as well as the underlying mechanism in colon cancer cell lines.

Material and methods: A tissue microarray of primary CRCs from 320 female patients was stained for the expression of ERβ, COX-2, 15-PGDH, CysLT1R, CysLT2R, ß-catenin, PGD synthase and mast cells. The Immunohistochemistry technique was used to evaluate the staining intensity, which was assigned an Immuno-Reactive Score (IRS). The zebrafish xenograft model was used to study colon cancer cells metastasis. ERβ -041 (an ERβ agonist) was injected into the zebrafish perivitelline space to allow for xamination of its effect on colon cancer cells. After 48 h, the tail, considered the distant metastatic site, was investigated for the presence of the metastasized cells. The effect of ERβ on colon cancer cells (SW-480 and HCT-116) after treatment with ERβ-041 was studied by wound healing and trans-well migration assay, as well as by survival assay.

Results: Patients with high ERβ expression had significantly higher levels of membrane ß-catenin, CysLT2R, 15-PGDH and PGD synthase, which all are known to have an anti-tumor effect. These patients had significantly lower levels of CysLT1R, COX-2 and nuclear ß-catenin expression, which are known to have pro-tumorigenic effects. In addition, patients with high ERβ expression had higher numbers of mast cells, which are a major source of PGD2 synthase and PGD2. In the zebrafish xenograft model, the ERB-041 treated cells showed no distant metastasis, unlike the untreated cells. ERβ stimulation of colon cancer cells produced a significant decrease in migration and survival compared to that of the untreated control cells after 48 h post-stimulation.

Conclusion: High ERβ expression was significantly correlated with anti-tumorigenic inflammatory mediators and lower nuclear ß-catenin in patients with CRC. These results strengthen our hypothesis regarding the beneficial role of ERβ in CRC patients. Our results raise the possibility of cross-talk between ERβ, CysLT1R and CysLT2R signalling.

#5033

The role of the epigenetic regulator SATB2 in colon cancer progression.

Donal J. Brennan,1 Kirsha Naicker,1 Sudipto Das,2 Bruce Moran,1 Rut Klinger,1 Fredrik Ponten,3 Stephen Hewitt,4 Karin Jirström,5 Annette T. Byrne,2 Jacintha O'Sullivan,6 William M. Gallagher,1 Darran P. O'Connor2. 1 _University College Dublin, Dublin, Ireland;_ 2 _Royal College of Surgeons in Ireland, Dublin, Ireland;_ 3 _Uppsala University, Uppsala, Sweden;_ 4 _National Cancer Institute, Bethesda, MD;_ 5 _Lund University, Lund, Sweden;_ 6 _Trinity College Dublin, Dublin, Ireland_.

SATB2 is a nuclear matrix-associated transcription factor that orchestrates gene expression by regulating higher-order chromatin structure. Using antibody-based screening of 48 normal human tissues and 20 cancer types, we identified SATB2 as a protein almost exclusively expressed in gastrointestinal tissue. We confirmed this observation at the mRNA level in 10,533 human tissue samples. Differential expression of SATB2 was observed in colorectal cancer, with loss of expression occurring along the adenoma-carcinoma sequence. Additionally, SATB2 expression was markedly decreased in metastatic SW620 colon cancer cells and siRNA knockdown of SATB2 expression in parental SW480 cells increased their growth and migratory capacity. Ectopic expression of SATB2 in the metastatic variant reversed the observed phenotype. Using tissue microarray and automated image analysis of SATB2 expression in colorectal cancers (n=309), SATB2 was demonstrated by multivariate Cox regression analysis to be an independent predictor of disease-specific survival (HR=0.52, 95% CI 0.32-0.83, p=0.006). SATB2 mRNA levels were examined in a second cohort (n=290) and again, SATB2 was demonstrated to be an independent predictor of disease-specific survival (HR=0.40, 95% CI 0.18-0.92, p=0.031). Interestingly, in colorectal cancer patients, SATB2 levels significantly correlated with CD3+ T-cell infiltrates in the tumours (p=0.006) and inversely correlated with COX2 expression (p=0.019). In two independent colorectal cancer cohorts (n=467), SATB2-low tumours were found to be significantly enriched in the CMS1 (immune-related) subtype (p<0.001). Gene set enrichment analysis revealed that SATB2 low tumours demonstrated altered immune signalling with significant increases in IFNγ (p=0.001), IL6 (p=0.001), IL8 (p<0.001) TFGβ (p<0.001), which was mirrored by in vitro manipulation of SATB2 expression in colon cancer cells. Furthermore, in a longitudinal cohort of patients with ulcerative colitis, we observed a significant correlation between loss of SATB2 expression and occurrence of future cancers (p=0.013). We therefore postulate that SATB2 acts as a master regulator of the inflammatory response in the gut and loss of expression is significantly associated with the progression of colorectal cancer.

#5034

**Response to RRx-001 in SCLC is positively correlated with a reduction of CD206 expression on HLA-DR** low/- **monocytes.**

Min-Jung Lee, Akira Yuno, Sunmin Lee, Jane B. Trepel. _National Institutes of Health (NIH) National Cancer Institute, Bethesda, MD_.

Background: In tumors such as small cell lung cancer (SCLC), myeloid-derived suppressor cells and M2-polarized macrophages promote tumor growth, angiogenesis and metastasis. In a Phase II study called QUADRUPLE THREAT (NCT02489903), where RRx-001, a macrophage repolarizing agent, was combined with a platinum doublet (etoposide + cisplatin/carboplatin or EP) in previously platinum treated SCLC, we correlated expression of the M2 marker, CD206, on HLA-DRlow/-monocytes, a phenotype that correlates with a poor prognosis, with response to RRx-001 in 14 patients with SCLC. CD206 expression on HLA-DRlow/-monocytes was associated with response to chemotherapy, and overall survival.

Methods: Patients were administered 4 mg RRx-001 once weekly until progression at which point EP was started. The EP chemotherapy consisted of etoposide 100 mg/m2IV on days 1-3 of a 21-day cycle and either cisplatin 80 mg/m2IV on day 1 or carboplatin AUC 5-6 IV on day 1. Study treatment with RRx-001 and chemotherapy continued until progression or intolerable toxicity. Peripheral blood was collected in cell preparation tubes with sodium citrate from 14 patients for exploratory studies during screening and after therapy on Days 1, 8 and 15. Peripheral blood mononuclear cells (PBMCs) were isolated from blood at the National Institutes of Health by centrifugation and multiparameter flow cytometric analysis was performed.

Results: A strong correlation was present between decreased CD206 expression on HLA-DRlow/-monocytes (between baseline, Day 8 and Day 15) and the objective response rate (Pearson's coefficient 0.61). A decrease in expression of CD206 on HLA-DRlow/-monocytes during treatment was also associated with improved OS of 12.5 months (HR 0.785) and, conversely increased CD206 expression on HLA-DRlow/-monocytes during treatment was associated with poorer OS (6.8 months; HR 1.27). Spearman correlations were used to quantify the relationship between the change in CD206 expression on HLA-DRlow/-monocytes at baseline, Day 8 and Day 15 of treatment with RRx-001 and outcome. Spearman, Pearson and Kendall correlation estimates were consistent. Kaplan-Meier survival analysis was performed for correlation between OS and the change in CD206 expression on HLA-DRlow/-monocytes between baseline, Day 8 and Day 15. All data were analyzed using SAS.

Conclusions: During treatment with RRx-001, reduced expression of the protumorigenic M2 marker CD206 on peripheral monocytes positively correlated with increased response and survival, suggestive of a systemic increase in the ratio of M1/M2 macrophages that may, in turn, attenuate the metastatic process.

#5035

Blockade of EphB4-ephrin-B2 interaction remodels the tumor immune microenvironment in head and neck cancers.

Shilpa Bhatia,1 Ayman Oweida,1 Shelby Lennon,1 Laurel Darragh,1 Sanjana Bukkapatnam,1 Benjamin Van Court,1 David Raben,1 Natalie Serkova,1 Antonio Jimeno,1 Eric Clambey,1 Elena Pasquale,2 Sana Karam1. 1 _University of Colorado Denver, Aurora, CO;_ 2 _Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA_.

Introduction: Identifying targets in the tumor microenvironment (TME) that act as barriers to an effective anti-tumor immune response has become an area of intense investigation. In the current study, we established EphB4-ephrin-B2 signaling as a key pathway that regulates both innate and adaptive arms of the immune system. Eph receptor tyrosine kinases and their membrane-bound ephrin ligands have been implicated in human malignancies and in immune cell development, migration, and activation in inflammatory models. However, direct evidence that supports the role of Eph-ephrin interaction in cancer-related immune response is lacking. We hypothesized that EphB4-ephrin-B2 interaction regulates TME by sustaining immunosuppressive cells-Tregs and TAMs thus negatively impacting the functional ability of CD8 T cells.

Materials and methods: We used orthotopic models of head and neck squamous cell carcinoma to determine the role of EphB4-ephrin-B2 interaction in tumor immune microenvironment. Mice were treated with control agent or an EphB4-ephrin-B2 blocker in the absence or presence of radiation (RT). Tumor immune cell infiltrates were analyzed using mass cytometry and flow cytometry applications. ELISA or multiplex cytokine array were utilized to determine circulating cytokine/chemokine levels in plasma.

Results: We observed that inhibition of EphB4-ephrin-B2 signaling in vivo significantly reduced tumor growth and decreased intratumoral Tregs, TAMs, and increased the activation of Teffector cells, without affecting CD4 T cell numbers. This was correlated with decreased Treg proliferation and activation when EphB4-ephrin-B2 signaling is inhibited. Since RT remains the mainstay in treatment of head and neck squamous cell cancer (HNSCC) patients, we combined EphB4-ephrin-B2 inhibitor with RT in our tumor model and observed further increase in CD8 and CD4 T cell infiltrates and activation status, and a significant decline in circulating TGF-β1 levels and an increase in CXCL10 levels compared to the control group. A significant reduction of TAMs, favoring a polarization towards an anti-tumoral M1 phenotype, was also observed in EphB4-ephrin-B2 inhibitor+RT group. We also compared the efficacy of combining EphB4-ephrin-B2 inhibitor with RT to anti-PDL1+RT in an in vivo model known to develop resistance to anti-PDL1+RT therapy. Our data demonstrated that combining EphB4-ephrin-B2 inhibitor with RT was equally effective to that of anti-PDL1+RT in terms of anti-tumor growth response.

Conclusions: Our study provides the first insight into a novel role for EphB4-ephrin-B2 interaction in modulating tumor immune microenvironment in HNSCC. Our findings present a potential alternative in the form of EphB4-ephrin-B2 targeted therapeutics that can be tested in clinical trials in combination with RT for HNSCC patients.

#5036

Induction of highly efficacious anti-tumor activity and modulation of tumor microenvironment: Cell-free off the shelf therapeutic modality.

Archana Thakur,1 SriVidya Yarlagadda,2 Kyungmin Ji,2 Dana L. Schalk,1 Johnson Ung,,1 Edwin T. Bliemeister,1 Amro Aboukameel,2 Eli Casarez,1 Bonnie F. Sloane,2 Lawrence G. Lum1. 1 _University of Virginia Cancer Center, Charlottesville, VA;_ 2 _Wayne State University, Detroit, MI_.

Adoptive transfer of Bispecific antibody Armed activated T cells (BATs) show promising anti-tumor activity in clinical trials in solid tumors. The cytotoxic activity of BATs occur upon engagement with tumor cells via the bispecific antibody bridge which stimulates BATs to release not only the lytic and cytotoxic molecules (perforin/granzyme) but also cytokines, chemokines and other signaling molecules extracellularly. We hypothesized that the release of extracellular soluble factors by this complex interaction of T cells, bispecific antibody, and tumor cells may serve as a potent anti-tumor and immune activating conditioned media (CM). In a 3D tumorsphere model, tumor+BATs-CM (n=10) showed potent cytotoxicity (p<0.001) against multiple breast (MDA-MB-231, BT-20, SK-BR-3 and MCF-7) and other cancer cell lines (p<0.001) compared to control tumor-CM or BATs-CM. Tumor+BATs-CM (n=6) was able to reduce the proportion of CD44hi/CD24lo cancer stem like cells to 0.7% compared to 4.9% in control CM. The addition of tumor+BATs-CM decreased the proportions of T regulatory cells (5% to 1.1%; p<0.02) and myeloid derived suppressor cells (3.8% to 1.2%; p<0.03), but increased activation and proliferation of effector T cells in 3D cultures compared to control CM (n=3). Size based CM factionation showed that most activity is retained in the <50kDa but >10kDa fraction. Multiplex analysis showed high levels of IL-2, IL-15, IFN-γ, TNF-α, GM-CSF, granszyme B (GZB), IL-13, MIP-1β, IP-10, MIG and RANTES. These factors are likely responsible for the cytolytic and immune activating effects. Phospho-specific signaling protein arrays showed enhanced JAK1/STAT-1/STAT-5A, Rac/cdc42/STIM-1) pathways in tumor+BATs-CM (n=3). Exosomal microRNA (miR) in tumor+BAT-CM showed higher expression of several miRs that are associated with T cell function and activation compared to control CM (n=2). Simulations using cocktails of multiple cytokines were done to test anti-tumor activity, IFN-γ/TNF-α/GZB showed potent cytotoxicity directed at breast (58-78%) and pancreatic cancer (50-72%) cell lines compared to 45-65%, 20-27%, 18-25% with IFN-γ, TNF-α and GZB individually, respectively. In a xenograft breast cancer model, IV and intra-tumoral injections of 10x concentrated tumor+BAT-CM (3x/week for 4 weeks;150μl CM/injection) was able to inhibit tumor growth significantly (p<0.01) compared to the control CM treated mice (n=10 mice/group). Therapeutic advantages of CM include: 1) a ready off-the-shelf product; 2) a decrease in regulatory and manufacturing costs. In summary, BATs-Tumor complex derived CM provides a clinically controllable cell-free platform to target various tumor types with diverse anti-cancer immune activating mediators regardless of the heterogeneous nature of the tumor cells and mutational burden as a novel and potent off-the-shelf therapeutic modality. 

## EPIDEMIOLOGY

### Influences on Cancer Risk and Outcomes

#5037

Erythrocyte levels of cadmium and lead and risk of B-cell non-Hodgkin lymphoma and multiple myeloma.

Emily L. Deubler,1 Susan M. Gapstur,1 W. Ryan Diver,1 Mia M. Gaudet,1 James M. Hodge,1 Victoria L. Stevens,1 Marjorie L. McCullough,1 Laura G. Haines,2 Keith E. Levine,2 Lauren R. Teras1. 1 _American Cancer Society, Atlanta, GA;_ 2 _RTI International, Durham, NC_.

Heavy metals such as cadmium and lead are ubiquitous in the environment and have been classified as carcinogens by the International Agency for Research on Cancer. While research is limited, associations of heavy metals with risk of lymphoid malignancies is plausible given that other environmental factors have been associated with these cancers. To further explore associations of cadmium and lead exposure with risk of lymphoid malignancies, we conducted a nested case-control study within the Cancer Prevention Study-II (CPS-II) Nutrition cohort, a U.S. prospective cohort of men and women. Incident B-cell non-Hodgkin lymphomas (B-NHL) and multiple myelomas (MM) were identified from 32,704 CPS-II participants who were cancer-free at time of blood collection between 1998-2001. Cases included the following histologic subtypes: chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), multiple myeloma (MM), and other B-cell lymphoma. Each case was matched to two eligible controls that were selected among those who were cancer-free at the time of the case's diagnosis. The matching factors included race, age, gender, and blood draw date. Cadmium and lead levels were measured in stored erythrocytes from the blood draw. Conditional logistic regression models were used to estimate relative risks (RR) and 95% confidence intervals (CI) for lymphoid malignancy overall and stratified by NHL subtype. The final analytic cohort consisted of 375 B-NHL or MM cases (95 DBLCL, 90 CLL/SLL, 62 FL, 76 MM, and 52 other B-cell lymphoma), and 750 matched controls. The cohort was 56% male, and an average of 69.9 years of age at the time of blood draw. There was a significant, positive association between erythrocyte levels of lead and risk of lymphoid malignancy overall (RR: 1.17, 95% CI: 1.03 - 1.33 per 1 standard deviation (SD; 17.6 µg/L) increase in lead level). This association appeared to be driven by FL (per SD of lead RR: 1.76, 95% CI: 1.21 - 2.57). No associations between lead and DLBCL, CLL/SLL, MM, or other B-cell lymphoma were observed. Results appeared similar when additionally controlling for alcohol use and smoking status. There was no association between cadmium and lymphoid malignancies overall; however, cadmium was inversely associated with risk of multiple myeloma (Q4 vs Q1 RR: 0.30, 95% CI: 0.12 - 0.75). Preliminary results from this nested case-control analysis suggests that a positive association between erythrocyte lead levels and risk of lymphoid malignancies (particularly follicular lymphoma) may exist. Reasons for an inverse association between cadmium and risk of multiple myeloma are unclear but might be due to chance. Further research is needed to fully explore and confirm these findings.

#5038

Chrysotile asbestos fibers in tissue adjacent to laryngeal squamous cell carcinoma in cases with a history of occupational asbestos exposure.

Stephanie K. Wronkiewicz,1 Victor Roggli,2 Damaris Kuhnell,3 Rondi A. Butler,4 Karl T. Kelsey,5 Scott M. Langevin3. 1 _Univ. of Cincinnati College of Arts and Sciences, Cincinnati, OH;_ 2 _Duke University, Durham, NC;_ 3 _Univ. of Cincinnati College of Medicine, Cincinnati, OH;_ 4 _Brown University College of Medicine, Providence, RI;_ 5 _Brown University School of Public Health, Providence, RI_.

Asbestos describes a group of naturally occurring fibrous silicate mineral compounds. Its use dates back thousands of years but became widespread in the late 19th century through the late 20th century due to its favorable industrial properties, including its strength, flexibility and thermal properties, with peak use in the United States occurring in the 1970s. Asbestos has been associated with a number of chronic respiratory diseases, including malignancy, and was first linked to mesothelioma and lung cancer in the early to mid part of the 20th century. Despite the known health risks, asbestos is not universally banned and is still in use in many countries (including the United States), with an estimated 125 million occupationally-exposed individuals worldwide. In addition to mesothelioma and lung cancer, asbestos has also been implicated as a risk factor for squamous cancers involving the upper airway, in particular laryngeal and pharyngeal carcinoma, as indicated by recent meta-analyses; however, evidence for the latter associations is based solely on epidemiologic data from observational studies. The primary objective of this work was to attempt to strengthen the evidence via empirical demonstration of persistent asbestos fibers embedded in the tissue surrounding the tumor, thus providing a more definitive biological link between exposure and disease. Six HPV-negative laryngeal and pharyngeal squamous cell carcinoma cases were selected from a large population-based case-control study of head and neck cancer, plus one case with no occupational exposure history as a control. Briefly, detailed occupational history was available for 1,056 head and neck cancer cases enrolled through major teaching hospitals located in Boston, Massachusetts. Occupational data was reviewed to identify those with an occupational history of asbestos exposure. Tissue cores were obtained from adjacent non-neoplastic tissue in tumor blocks from the initial primary tumor resection from a subset of 6 cases with a history of occupational-asbestos exposure and 1 case with no reported occupational exposure (control). A count of asbestos fibers >5um was performed on the cores from each case at Duke University using a scanning electron microscope equipped with an energy dispersive x-ray analyzer (EDXA). Despite the low volume of input tissue, chrysotile asbestos fibers were identified in 3/6 of evaluated cases with a history of occupational asbestos exposure; all 3 cases had tumors originating in the larynx. This is the first study to demonstrate the presence of asbestos fibers in the larynx of cases with laryngeal squamous cell carcinoma, adding an additional line of physical evidence implicating asbestos as an etiologic factor for laryngeal squamous cell carcinoma.

#5039

Medical radiation exposure and risk of retinoblastoma: A report from the Children's Oncology Group.

Omar Shakeel,1 Nelson Pace,2 Philip J. Lupo,1 Michael E. Scheurer,1 Arupa Ganguly,3 Greta R. Bunin4. 1 _Baylor College of Medicine, Houston, TX;_ 2 _Exponent Inc., Oakland, CA;_ 3 _University of Pennsylvania, Philadelphia, PA;_ 4 _Children's Hospital of Philadelphia (Retired), Philadelphia, PA_.

Retinoblastoma, which results from mutations in the RB1 gene, is the most common intraocular malignancy in childhood. Approximately 10% of cases are familial, and the remaining cases (both bilateral and unilateral) are considered sporadic. Although little is known about the causes of sporadic cases, there is some evidence that parental exposure to medical radiation is associated with sporadic bilateral retinoblastoma in offspring. These findings, however, have not been confirmed. Further, the relation between medical radiation exposure and sporadic unilateral retinoblastoma has not been investigated. Therefore, we evaluated the role of medical radiation exposure on sporadic bilateral and unilateral retinoblastoma. Eligible patients were diagnosed with sporadic bilateral or unilateral retinoblastoma from 1998 to 2011 and treated at one of nine participating institutions. Controls were recruited from the friends and relatives of cases. Telephone interviews were conducted with parents to obtain information on demographic factors and exposures, including medical procedures prior to conception of the index child. Gonadal radiation doses were estimated utilizing PCXMC and ImPACT software. Logistic regression models were used to evaluate the associations between parental medical radiation exposure and retinoblastoma. In maternal analyses, there were 298 bilateral cases, 184 unilateral cases, and 404 controls. In paternal analyses, there were 268 bilateral cases, 155 unilateral cases, and 358 controls. We found that compared to mothers without medical radiation exposure, mothers who reported a lower gastrointestinal (GI) series were more likely to have a child who developed bilateral retinoblastoma (odds ratio [OR]=6.9, 95% confidence interval [CI]: 2.9-16.4). A similar association was observed for unilateral retinoblastoma though the confidence interval included the null (OR=2.8, 95% CI: 0.8-9.7). When evaluating gonadal dose, increasing maternal exposure was associated with bilateral retinoblastoma (P for trend=0.03). Further, compared to unexposed mothers, mothers in the highest dose category were more likely to have a child who developed sporadic bilateral retinoblastoma (OR=2.3, 95% CI: 1.4-4.1). Notably, the same trend was not observed for unilateral retinoblastoma. Transversely, increasing paternal gonadal dose was associated with unilateral retinoblastoma (P for trend=0.03) but not bilateral retinoblastoma. These results are in contrast to previously hypothesized patterns (i.e., that maternal exposure would be associated with unilateral retinoblastoma, whereas paternal exposure would be associated with bilateral retinoblastoma due to a de novo mutation in RB1 of paternal origin). Our findings could point to a more complex etiologic framework for this important pediatric malignancy.

#5040

Involvement of PI3K/Akt pathway in cadmium triggered aggressive prostate cancer.

Priyanka Kulkarni,1 Pritha Dasgupta,1 Nadeem S. Bhat,2 Yutaka Hashimoto,1 Sharanjot Saini,1 Altaf A. Dar,3 Varahram Shahryari,1 Marisa Shiina,1 Soichiro Yamamura,1 Yuichiro Tanaka,1 Rajvir Dahiya,1 Shahana Majid1. 1 _UCSF VA Medical Ctr., San Francisco, CA;_ 2 _University of Miami, Miami, CA;_ 3 _Califonia Pacific Medical Center, San Francisco, CA_.

Cadmium (Cd) has been implicated in cancer development and classified as a type I carcinogen by the International Agency for Cancer Research. The etiology of prostate cancer development is associated with multitude of causative risk factors including exposure to cadmium. However, the molecular mechanisms associated with Cd-induced prostate cancer remain elusive. This study provides evidence that PI3K/Akt signaling is a major molecular pathway involved in Cd induced malignant transformation of normal prostate epithelial cells. Functionally, Cd exposure induced aggressive behavior as indicated by increased proliferation, migration and invasion in Cd-RWPE1 cells compared to parental RWPE1. PI3K/Akt pathway is constitutively activated in prostate cancer driving the most aggressive forms of cancer and metastasis. Consistent with these findings, the RT2 PCR array analysis of 84 genes involved in the PI3K/Akt pathway revealed induction of gene expression in catalytic units (P110α, Akt, mTOR, NFKB1, RAF etc.) with a concomitant reduction in expression of regulatory units (PIK3R1, PIK3R2, PTEN etc.) of the PI3K/Akt pathway in Cd-RWPE1 cells compared to parental RWPE1. This was confirmed by individual quantitative real-time PCR analysis using TaqMan gene expression assay probes. Effect of Cd on the translation of the PI3K/Akt pathway genes was examined by immunoblot assays. Gene Set Enrichment Analysis (GSEA) for differentially expressed genes in Cd-RWPE1 showed 5 overlapping pathways that were enriched in Cd-treated cells (Cd-RWPE1) and negatively correlated with parental RWPE1. The overlapping pathways include KEGG Apoptosis pathway (ES=0.56, NES=1), KEGG ERBB pathway (ES=0.25, NES=1), KEGG MAPK pathway (ES=0.48, NES=1), KEGG Pathways in cancer (ES=0.33, NES=1) and KEGG Prostate Cancer pathway (ES=0.35, NES=1). Interestingly, all these pathways are implicated in prostate cancer progression and metastasis. We randomly selected up- and down-regulated genes from the PI3K/Akt pathway in PCR array and performed profile analysis in a prostate adenocarcinoma data set (n=534) from the TCGA/GDC data base. We observed upregulation of the oncogenes along with downregulation of tumor suppressors in prostate cancer compared to normal prostate samples. Taken together, these data reveal that Cd exposure induced aggressive malignant characteristics in normal prostate epithelial cells via modulation of the PI3K/Akt signaling pathway. Understanding the molecular mechanisms involved in the malignant transformation of normal prostate epithelial cells may help develop optimal therapeutic strategies for advanced prostate cancer.

#5041

Determination of carcinogenic risk by immunosuppression and genotoxicity indexes.

Olga Ostash, Nina Balenko, Lyudmila Grigorenko, Igor Chernichenko, Yuriy Dumanskyi, Olga Litvichenko, Larisa Sovertkova. _State Institution "O.M. Marzeiev Institute for Public Health of the National Academy of Medical Sciences of Ukraine", Kyiv, Ukraine_.

We studied the immunological effects of the exposure of endogenic nitrosamines and their precursors jointly with the electromagnetic field of industrial frequency (50 Hz). The experiment was performed in 160 white outbred female rats aged 6-8 weeks divided into 4 groups: 1- intact control; 2 — introduction of the precursors of nitrosamines (sodium nitrite — 100 mg/kg, tetracycline — 20 mg/kg); 3 — irradiation with magnetic field of 50 Hz, 90 µT; 4 — introduction of the precursors of nitrosamines and irradiation with magnetic field of 50 Hz. The precurcors were introduced once a day, 5 days a week during 3 month. Magnetic field irradiation was carried out for 7 hours daily. Endogenic synthesis of nitrosamines was controlled by means of their determination in liver and kidneys. Genotoxic changes were assessed with the help of micronuclear test. The more profound changes in the immune system under joint effect of endogenically formed nitrosamines, their precursors and magnetic field of industrial frequency were established in comparison with the isolated action of these factors. These changes were manifested in stable suppression of T- and B-cell chains, expansion of their spectrum due to the activation of unspecific factors and development of mildly expressed reactions of autosensibilization and sensibilization, hypersensitivity of delayed type.The T-cell chain of the immunity was earliest sensitive one to the effect of studied factors, suppression of the immunity was revealed in a month and was stable for 3 months during the whole experiment. The correlation, previously established by us, between the levels of early suppression of T-lymphocytes and genotoxic effect, both isolated and joint factors' effect was confirmed. The maximum decrease of the level of suppression was noted under joint effect of the factors and complied with the maximum frequency of micronuclei in a bone marrow. The less expressed suppression was observed under isolated effect of the factors and accompanied by the lower level of the frequency of micronuclei. These results demonstrate the possibility of the use of the complex of early parameters of immunosupression and genotoxicity as the early criteria for the prognostication of the hazard of the effect of the industrial frequency magnetic field and chemical carcinogens of nitrosamine class. The increase of the suppression of T-cell chain of the immunity and genotoxic effect was established under joint exposure of the levels of endogenic nitrosamines and magnetic fields, in comparison with isolated effect, which indicates a potential oncogenic hazard of joint effect of chemical carcinogenic nitrosamines and magnetic field.

#5042

Folate and other B-vitamins and the risk of breast cancer in younger women.

Serena C. Houghton,1 A Heather Eliassen,2 Shumin Zhang,3 Jacob Selhub,4 Bernard A. Rosner,5 Walter C. Willett,5 Susan E. Hankinson1. 1 _University of Massachusetts Amherst, Amherst, MA;_ 2 _Brigham and Women's Hospital and Harvard Medical School, Amherst, MA;_ 3 _Brigham and Women's Hospital and Harvard Medical School, Boston, MA;_ 4 _Tufts University, Boston, MA;_ 5 _Harvard T.H. Chan School of Public Health, Boston, MA_.

Purpose: Prior epidemiologic cohort studies have generally found no associations of folate and other B-vitamins with breast cancer overall, but differences by menopause status have been suggested. However, prior studies have been limited by small numbers to examine the relationship between different plasma measures and related polymorphisms and breast cancer risk among younger women. Therefore, we examined the association of plasma B-vitamins and metabolites, and related genetic variants, with risk of breast cancer among predominantly premenopausal women.

Methods: We conducted a nested case-control study within the Nurses' Health Study II. From blood samples collected in 1996-1999 and follow-up through 2007, plasma measures were available for 610 cases and 1,207 controls. Unconditional multivariable logistic regression was used to estimate relative risks (RR) of breast cancer and 95% confidence intervals (CIs). We examined whether associations varied by methylenetetrahydrofolate reductase (MTHFR) and dihydrofolate reductase polymorphisms, breast cancer risk factors, or tumor characteristics.

Results: Plasma vitamin B12 was associated with a 71% increase in risk of breast cancer comparing the highest versus lowest quintile (95%CI=1.22-2.39, p-trend=0.02). Plasma folate (comparable RR=1.20, 95%CI=0.85-1.68), pyridoxal 5'-phosphate (RR=1.19, 95%CI=0.85-1.65), cysteine (RR=1.15, 95%CI=0.81-1.63), homocysteine (RR=0.90, 95%CI=0.65-1.25), and cysteinylglycine (RR=0.92, 95%CI=0.65-1.31) were not associated with overall breast cancer risk. Folate was significantly positively associated with invasive (RR highest tertile vs. lowest=1.48, 95%CI=1.09-2.01) and estrogen receptor-positive/progesterone receptor-positive breast cancer (RR T3 vs. T1= 1.59, 95%CI= 1.12-2.27) and the invasive association was suggestively stronger for bloods collected post-fortification (p-heterogeneity=0.19). Several nutrient/breast cancer associations varied across subgroups defined by age, smoking, alcohol, multivitamin use and MTHFR status (p-interaction<0.05).

Conclusions: Overall, plasma B-vitamins and metabolites were not consistently associated with lower breast cancer risk. For increasing plasma folate and B12 levels higher risk of invasive breast cancer was observed. Additionally, there may be associations in subgroups defined by related genetic variants, breast cancer risk factors, and tumor factors. Future studies in younger women are needed to confirm findings.

#5043

Pre-diagnostic blood levels of organochlorines and risk of non-Hodgkin lymphoma in three population-based cohorts in China and Singapore.

Bryan A. Bassig,1 Xiao-Ou Shu,2 Andreas Sjodin,3 Woon-Puay Koh,4 Yu-Tang Gao,5 Jennifer Adams-Haduch,6 Mark Davis,3 Renwei Wang,6 Yong-Bing Xiang,5 Mark Purdue,1 Bu-Tian Ji,1 Gong Yang,2 Richard Jones,3 H. Dean Hosgood,7 Wei Jie Seow,4 Wei Hu,1 Wei Zheng,2 Jian-Min Yuan,8 Qing Lan,1 Nathaniel Rothman1. 1 _NCI-DCEG, Bethesda, MD;_ 2 _Vanderbilt University, Nashville, TN;_ 3 _Centers for Disease Control and Prevention, Atlanta, GA;_ 4 _Saw Swee Hock School of Public Health, Singapore, Singapore;_ 5 _Shanghai Cancer Institute, Shanghai, China;_ 6 _University of Pittsburgh Cancer Institute, Pittsburgh, PA;_ 7 _Albert Einstein College of Medicine, New York, NY;_ 8 _University of Pittsburgh, Pittsburgh, PA_.

Background: Organochlorines (OCs) are environmentally persistent compounds that have been extensively used as pesticides and for other industrial applications. Residues of OCs have been detected at hazardous waste sites and in various environmental media worldwide and serum levels of OCs continue to be detectable in the general population. Specific OCs have been associated with non-Hodgkin lymphoma (NHL) risk with varying degrees of evidence. These associations have not been evaluated in Asia, where the exposure patterns of substantially high levels of certain OC pesticides and lower levels of polychlorinated biphenyls (PCBs) are different from Western populations. China accounted for nearly 20% of the worldwide production of dichlorodiphenyltrichloroethane (DDT) through 1983 when it was restricted, and historical usage of hexachlorocyclohexane (HCH) in China has been among the highest in the world.

Methods: We evaluated the risk of NHL in relation to pre-diagnostic blood levels of five OC pesticides/metabolites (hexachlorobenzene, β-HCH, oxychlordane, trans-nonachlor, and p,p'-DDE, the primary metabolite of DDT) and four PCB congeners (118, 138-158, 153, 180) in a case-control study of 167 NHL cases and 167 controls matched on age, sex, and blood collection date. The study was nested within three population-based cohorts of Chinese men and women in Shanghai and Singapore. Associations between lipid-adjusted OC concentrations and NHL risk were analyzed using conditional logistic regression.

Results: Median levels of p,p'-DDE and β-HCH were up to 12 and 65 times higher, respectively, in Shanghai (blood collected between 1986-2000) compared to reported levels in population-based cohorts in the United States (CLUE; Nurse's Health Study) and Norway (Janus) with blood collected between 1972-1990. Median levels of p,p'-DDE and β-HCH were more comparable in Singapore (blood collected between 2000-2004) relative to the Western cohorts (1-2 fold concentration differences). Levels of β-HCH were associated with increased risk of overall NHL (3rd vs. 1st tertile OR=1.78, 95%CI=0.98-3.23; ptrend =0.049) in the pooled analysis of three cohorts. No significant associations were observed for other OCs and NHL risk, including for p,p'-DDE. Results for β-HCH and p,p'-DDE were consistent across cohorts.

Discussion: Associations between β-HCH and NHL risk have not been consistent in studies of Western populations. Our findings provide the first evidence suggesting associations between blood levels of β-HCH and NHL risk in a population in Asia that experienced far higher exposures. Although there is limited evidence that DDT (IARC Group 2A) is associated with NHL based on studies in Western populations, our findings among Asians, who had higher p,p'-DDE levels than reported in the general population in the West, do not support an association with environmental exposure.

#5044

Prostate adenocarcinoma incidence and risk factors in Veterans with well controlled HIV infection.

Kathryn E. Royse,1 Jose M. Garcia,2 Donna L. White,1 Jennifer R. Kramer,1 Yongquan Dong,1 Suchismita Raychaudhury,1 Peter A. Richardson,1 Christine Hartman,1 Elizabeth Y. Chiao1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _University of Washington School of Medicine, Seattle, WA_.

(a) Although prostate cancer is projected to be one of the most frequently diagnosed cancers in HIV-infected men overall, little is known about its risk in the sub-group with well-controlled HIV-infection.

(b) We performed a retrospective cohort study to determine age-adjusted incidence of prostate adenocarcinoma in HIV-positive male veterans utilizing Veterans Administration (VA) healthcare between 10/01/1999 and 12/31/2016. HIV infection and prostate adenocarcinoma diagnosis as well as related clinical, sociodemographic, and lifestyle risk factors were obtained using extant VA administrative healthcare databases, the VA cancer registry, and augmented prostate cancer diagnosis with direct electronic medical record (EMR) review. We defined well controlled HIV as >60% of time with an undetectable HIV viral load and limited our analyses to those with a minimum of 90 days between HIV and prostate diagnosis, death, their last recorded health care encounter, or study end. We employed time-varying Cox proportional hazard regression models and used backward elimination to identify risk factors associated with incident prostate adenocarcinoma; effects are reported as Hazard ratios (HR) and 95% confidence intervals (CI).

(c) During an average 10.26 years of follow-up, we identified 587 incident prostate adenocarcinomas among our cohort of 19,079 HIV positive men with well-controlled infection (age-adjusted incidence rate [IR] = 83.31 per 100,000-person years, 95% CI: 76.83-90.33); with a significant increasing trend over time. We identified several factors associated with significant increased risk of incident prostate adenocarcinoma, after adjusting for HIV medication use, in our well-controlled HIV-positive cohort including substance abuse (HR=1.68, 95% CI: 1.27-2.24, p=0.0003), age at HIV diagnosis (HR=1.73, 95% CI: 1.25-2.39, p=0.0010), and black race (HR=2.07, 95% CI: 1.58-2.72, p<.0001). History of alcohol abuse (HR=0.54, 95% CI: 0.39-0.75, p=0.0002), longer time with well-controlled infection (HR=0.63, 95% CI: 0.59-0.67, p<.0001), and receiving integrase inhibitors (HR=0.64, 95% CI: 0.45-0.92, p=0.041) were associated with reduced risk. Several risk factors included in the multivariable model, such as PSA testing, testosterone levels, and maximum BMI, were not found to be significantly associated with or protective for prostate cancer.

(d) Further research is needed to confirm our findings and to better identify sub-groups of well-controlled HIV-positive men at greatest increased prostate cancer risk.

#5045

Pineal gland volume and risk of prostate cancer.

Latifa A. Bazzi,1 Lara Sigurdardottir,2 Sigurdur Sigurdsson,3 Unnur Valdimarsdottir,4 Johanna Torfadottir,5 Thor Aspelund,4 Lenore Launer,6 Tamara Harris,6 Vilmundur Gudnason,3 Lorelei Mucci,7 Sarah Markt8. 1 _University of Michigan School of Public Health, Ann Arbor, MI;_ 2 _Icelandic Cancer Society, University of Iceland, Reykjavik, Iceland;_ 3 _Icelandic Heart Association, Reykjavik, Iceland;_ 4 _Icelandic Heart Association, University of Iceland, Reykjavik, Iceland;_ 5 _University of Iceland, Reykjavik, Iceland;_ 6 _National Institute on Aging, Bethesda, MD;_ 7 _Harvard T.H. Chan School of Public Health, Boston, MA;_ 8 _Case Western Reserve University, Cleveland, OH_.

Background: The parenchyma of the pineal gland, an endocrine gland in the brain, produces the circadian hormone melatonin. Previously, we found low levels of melatonin were associated with an increased risk for advanced prostate cancer. This study used data from men in the Age, Gene/Environment Susceptibility-Reykjavik (AGES-Reykjavik) Study to evaluate the relationship between pineal volume, melatonin levels, and prostate cancer risk.

Methods: Participants were enrolled in the AGES-Reykjavik Study from 2002 to 2006 and underwent detailed clinical assessments at baseline that included biospecimen collection, MRI of the brain, medical history, and health questionnaires on dietary and lifestyle factors. We included 802 men who had information on pineal size, where parenchyma, calcification, and cyst volume were estimated individually and manually from the MRIs. Cox proportional hazards regression was used to calculate hazard ratios and 95% confidence intervals for risk of prostate cancer during follow-up through 2014, comparing parenchyma volume tertiles.

Results: There was a positive association between pineal parenchyma volume and urinary levels of melatonin. During follow-up, 135 men were diagnosed with prostate cancer, 30 of which were advanced prostate cancer. Men with volumes in the upper tertile had no statistically significant increased risk of prostate cancer compared to men with volumes in the lowest tertile (HR: 1.0, 95% CI: 0.7, 1.5), and men with volumes in the middle tertile had only a borderline statistically significant decreased risk of prostate cancer compared to men with volumes in the lowest tertile (HR: 0.7, 95% CI: 0.4, 1.0). Compared to men without pineal cysts, there was a borderline statistically significant association between men with pineal cysts and decreased risk of prostate cancer (HR: 0.7, 95% CI: 0.5, 1.0). Compared to men without pineal calcifications, there was no statistically significant association between men with pineal calcifications and increased risk of prostate cancer (HR: 1.1, 95% CI: 0.7, 1.6).

Conclusions: Pineal parenchyma volume was associated with melatonin levels, but there was no statistically significant association between parenchyma volume alone and prostate cancer risk. Further studies are needed to investigate the relationship between pineal parenchyma volume, presence of calcifications and cysts, melatonin, and prostate cancer.

#5046

Gallbladder disease and the risk of hepatocellular carcinoma: US case-control study.

Kenda Al-Assi,1 Rikita Hatia,1 Vijayashri Rallapalli,1 Yehia I. Abugabal,1 Ahmed Abdelhakeem,1 Reham Abdel-Wahab,1 Kanwal Raghav,1 Prasun K. Jalal,2 Ahmed Kaseb,1 Asif Rashid,1 Donghui Li,1 Manal Hassan1. 1 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Baylor College of Medicine, Houston, TX_.

BACKGROUND:

Hepatocellular carcinoma (HCC) is the most common primary cancer of the liver with a five-year survival rate of less than 5 percent. Gallbladder disease (GBD) is defined as the presence of cholelithiasis or cholecystitis. Both cholelithiasis and cholecystitis have been reported as risk factors in the development of gastrointestinal cancers, such as biliary tract and pancreatic cancers. Moreover, patients with GBD tend to develop chronic liver diseases, which may progress to cirrhosis. However, the role of GBD in the development of non-cirrhotic related HCC has not been elucidated. We aimed to investigate the role of GBD and the risk of HCC development in the absence of cirrhosis.

METHODS:

We conducted a case-control study at The University of Texas M.D. Anderson Cancer Center. Cases were defined as pathologically confirmed HCC. Controls were healthy individuals without prior history of cancer, who were spouses of other cancer patients seen at MD Anderson. All subjects were USA residents. Participants were personally interviewed for prior history of GBD and for several HCC risk factors (environmental, behavioral, chronic medical conditions, and family history of cancer). Blood samples of all participants were tested for markers of hepatitis B and C viruses. We reviewed the pathology and radiology records of HCC patients to assess presence and absence of cirrhosis. Multivariable logistic regression analysis was conducted to estimate the adjusted odds ratio (AOR) and 95% confidence interval (CI) for the association between GBD and HCC in the absence of cirrhosis and with adjustment for potential confounders.

RESULTS:

Between 2000 and 2017, a total of 1333 cases and 1104 controls were enrolled in the study. After review, 347 HCC case patients showed no evidence of cirrhosis and were eligible for this analysis. The prevalence of GBD based on the participants' recall during personal interview was 22.7% in the cases. Upon further assessment by review of medical records, the prevalence of GBD was 40.3% in the cases. Individuals with GBD were two times more likely to develop HCC than individuals with no history of GBD (AOR = 2.0; 95% CI: 1.4 – 2.8). The estimated AOR did not meaningfully change when we rely on the prevalence of GBD recalled by cases (22.7%) versus prevalence of GBD obtained from medical records (40.3%). The association between GBD and HCC continue to be significant in the absence of viral hepatitis, through a restricted analysis among non-viral non-cirrhotic population after controlling for age, sex race, alcohol use, cigarette smoking, diabetes mellitus, and family history of cancer.

CONCLUSIONS:

We conclude that GBD is a significant risk factor for HCC development in absence of cirrhosis. Future research aiming at investigating the underlying mechanism of GBD-induced HCC in absence of cirrhosis should be warranted. In addition, patients with GBD should be screened for evidence of cirrhosis.

#5047

Change in age of diagnosis of oropharyngeal cancer in the United States, 1975-2015.

Brittany J. Cline,1 Matthew C. Simpson,1 Aleksandr R. Bukatko,1 Eric Adjei Boakye,2 Kahee A. Mohammed,1 Nosayaba Osazuwa-Peters1. 1 _St. Louis University School of Medicine, Saint Louis, MO;_ 2 _Southern Illinois University School of Medicine, Springfield, IL_.

Introduction: Human papillomavirus (HPV) is the most common etiology of oropharyngeal squamous cell carcinoma (OPSCC), causing 70-90% of cases, and has continued to increase in incidence. While HPV-associated OPSCC has been associated with younger age at diagnosis compared to other head and neck cancers, research studies suggest that age of diagnosis might have increased in the last decade. There is currently no long-term, population-based estimate of age of diagnosis of oropharyngeal cancer, or differences in age of diagnosis based on race and gender. This study aimed to describe the change if any in the age of diagnosis of OPSCC in the last four decades.

Methods: The mean age at diagnosis of OPSCC patients from the Surveillance, Epidemiology, and End Results (SEER) 9 database diagnosed from 1975-2015 was computed for each year (n = 30,320). An independent samples t-test compared mean age at diagnosis by sex (female, male), and analysis of variance compared mean age at diagnosis by race/ethnicity (Hispanic, non-Hispanic (NH) white, NH black, NH other). Joinpoint regression estimated yearly increases/decreases in mean age of diagnosis by sex and race/ethnicity through annual percent changes (APC), which were summarized with average annual percent changes (AAPC).

Results: OPSCC patients were predominantly NH white (80.0%) and male (76.8%) with a mean age at diagnosis of 60.3 years (standard deviation = 10.9 years). Females had a significantly higher mean age at diagnosis than males (62.4 versus 59.7, p < 0.01). Hispanic (mean = 59.2) and NH black patients (mean = 57.3) had a significantly lower mean age at diagnosis than NH white patients (mean = 60.8). Overall, mean age at diagnosis remained stable from 1975-1996 (APC = 0.00, p > 0.05), significantly decreased from 1996-2002 (APC=-0.86, p < 0.01), and significantly increased from 2002-2015 (APC = 0.35, p < 0.01). However, the overall AAPC showed a stable trend in age of diagnosis from 1975-2015 (AAPC = -0.02, p > 0.05). The AAPCs in age of diagnosis for gender (males and females) and race/ethnicity also both remained stable from 1975-2015 (p > 0.05), except for NH blacks, with an estimated 0.13% yearly increase in age of diagnosis from 1975-2015 (AAPC = 0.13, p < 0.01).

Conclusions: The overall age at diagnosis for OPSCC has remained stable in the United States between 1975 and 2015. The younger age at diagnosis among NH blacks and Hispanics is important in prevention, early detection, and surveillance of OPSCC in the United States

#5048

Determination of haptoglobin, hemoglobin genotypes and malaria incidence in Nigerian breast cancer patients.

Titilope M. Dokunmu,1 Patience Obi,1 Oluwakemi A. Rotimi,1 Omolara A. Fatiregun,2 Sulaiman O. Agodirin,3 Solomon O. Rotimi1. 1 _Covenant University, Ota, Nigeria;_ 2 _Lagos State University Teaching Hospital, Lagos, Nigeria;_ 3 _University of Ilorin, Ilorin, Nigeria_.

Breast cancer is the second leading cause of cancer morbidity and mortality globally. Cancer chemotherapy commonly result in hemolysis, which impacts patient overall health. There is a need to determine genetic factors associated with hemolysis in breast cancer patients. Haptoglobin (Hp), a polymorphic protein plays important role in hemoglobin clearance and disease predisposition, but has been reported to have no prognostic factor in breast cancer. However, understanding selection pressure that drives certain gene mutations in specific populations and how it confers protection or susceptibility to diseases is crucial. In Nigeria, breast cancer, malaria infection and sickle cell disease are prevalent and associated with hemolysis, but little is known of their association in breast cancer patients. This study aims to determine relationship between haptoglobin, hemoglobin genotypes and submicroscopic malaria co-morbidity in clinically diagnosed breast cancer and healthy Nigerian women. DNA was extracted from blood using standard methods. Haptoglobin 2 and hemoglobin genotypes were detected by RFLP-PCR, while Plasmodium falciparum infection was detected by primer specific amplification of plasmodium cytochrome oxidase III gene (cox III) in 75 clinically diagnosed breast cancer (BC) and 287 healthy women (control; HC). Proportions were determined and compared in the two groups and test of association was carried out with significance level set at P <0.05. In BC groups, 3 (4.1%) of 72 Hp 2-2 phenotypes was detected compared to a significantly higher occurrence of 48 (16.7%) of 287 in HC group (p <0.05). Conversely, malaria infection was detected in 68 (94.4%) BC versus 255 (88.9%) in HC group. A similar proportion had Hp deletions (2 in BC and 8 in HC group). There was a low prevalence of hemoglobin S genotype in the entire population and relative risk for Hp 2-2 polymorphism in hemoglobin genotypes was not significantly different. In conclusion, this study reports in breast cancer and healthy women an inverse correlation of haptoglobin (Hp2-2) genotype with malaria incidence in southwest Nigeria. The results imply a possible protection against hemolysis and can play significant role in determining choice of cancer therapy for good patient treatment outcomes.

#5049

Associations of aspirin, non-aspirin NSAIDs, statins, and metformin with risk of biliary tract cancer: A Swedish population-based cohort study.

Lorena Marcano-Bonilla,1 Cathy Schleck,1 William Harmsen,1 Omid Sadr-Azodi,2 Terry Therneau,1 Lewis Roland Roberts,1 Nele Brusselaers2. 1 _Mayo Clinic, Rochester, MN;_ 2 _Karolinska Institutet, Stockholm, Sweden_.

Objectives: Biliary tract cancers (BTCs) comprise the second most common type of hepatobiliary cancer. Given the increase in global BTC incidence, with associated morbidity and mortality, together with the limited therapeutic options, there is increasing interest in strategies for disease prevention. We performed a population-based cohort study to determine the association between low dose aspirin, non-aspirin NSAIDs, statins, metformin, other risk factors and the risk of biliary tract cancer (BTC), while assessing confounding by sex.

Methods: We conducted a nationwide Swedish population-based cohort study using the Swedish Prescribed Drug Registry, which virtually completely enumerates use of prescribed medications nationwide since 2005. BTC diagnosis (intrahepatic cholangiocarcinoma [iCCA], extrahepatic cholangiocarcinoma [eCCA] or gallbladder cancer [GBC]) was ascertained from the Swedish Cancer Registry. Age-scaled Cox models, with exposure as time-varying covariates, were used to calculate hazard ratios (HRs), separately for men and women.

Results: In the 5.7 million person cohort, the risk of iCCA was significantly lower in men using statins (HR 0.62,95%CI 0.39-1.00,p=0.05), with a non-significant reduction in women. Statin use was associated with a significantly decreased risk of eCCA in both women (HR 0.60,0.38-0.94,p=0.03) and men (HR 0.47,0.28-0.80,p=0.01). Low dose aspirin (HR 0.76,0.60-0.97,p=0.03) was associated with a lower risk of GBC only in women, while statins (HR 0.72,0.55-0.93,p=0.01) showed a significantly decreased risk of GBC in women and a non-significant reduction in men. For all BTC subtypes, combined use of low dose aspirin and statins did not confer additional risk reductions beyond those achieved by statins alone. Male and female users of non-aspirin NSAIDs appeared to be at increased risk of BTC and its subtypes. Metformin did not significantly affect risk of BTC.

Conclusion: We performed the largest population-based cohort study evaluating risk and protective factors for BTC. Our results provide strong evidence in favor of the chemopreventive roles of low dose aspirin and statins in a subtype-and sex-specific manner. Individual risk factors contribute to development of BTC subtypes in different magnitudes. If randomized controlled clinical trials validate our findings and provide favorable evidence of the efficacy and safety of these drugs in the prevention of BTCs, our results could potentially be practice-changing, leading to improvements in the outcomes in this population.

#5050

Testosterone supplementation in relation to prostate cancer in a US commercial insurance claims database.

Michael B. Cook,1 Daniel C. Beachler,2 Lauren E. Parlett,3 Philip T. Cochetti,4 William D. Finkle,5 Stephan Lanes,6 Robert N. Hoover1. 1 _National Cancer Institute, Rockville, MD;_ 2 _HealthCore Inc, Wilmington, DE;_ 3 _HealthCore Inc., Alexandria, VA;_ 4 _University of Pennsylvania, Philadelphia, PA;_ 5 _Consolidated Research, Inc., Beverly Hills, CA;_ 6 _HealthCore Inc., Wilmington, DE_.

Background: Testosterone supplementation (TS) has dramatically increased in the United States (US). We conducted a study of TS and prostate cancer risk using a large US commercial insurance research database.

Methods: From the HealthCore Integrated Research Database (HIRDSM), we selected men aged 30 years or greater who were new users of TS during 2007-2015. We selected two male comparison groups: 1) unexposed (matched 10:1); 2) new users of a phosphodiesterase type 5 inhibitor (PDE5i). Incident prostate cancer was defined as diagnosis of prostate cancer within four-weeks following prostate biopsy. Propensity scores and inverse probability of treatment weights were used in Poisson regression models to estimate adjusted incidence rates, incidence rate ratios (IRRs) and 95% confidence intervals (CI) using doubly robust estimation. Subgroup analyses included stratification by prostate cancer screening, hypogonadism, and follow-up time.

Results: The adjusted prostate cancer IRR was 0.77 (95%CI: 0.68, 0.86) when comparing TS with the unexposed group and 0.85 (95%CI: 0.79, 0.91) in comparison with the PDE5i group. Inverse associations between TS and prostate cancer were observed in a majority of subgroup analyses, although in both comparisons estimates generally attenuated with increasing time following initial exposure. Amongst TS users, duration of exposure was not associated with prostate cancer.

Conclusion: In this study, men who received TS did not have a higher rate of prostate cancer compared with the unexposed or PDE5i comparison groups. The inverse association between TS and prostate cancer could be the result of residual confounding, contraindication bias, or undefined biologic effect.

#5051

Associations between autoimmune conditions and gastric cancer risk among elderly US adults.

Minkyo Song, M. Constanza Camargo, Andriy Derkach, Eric A. Engels, Charles S. Rabkin. _National Cancer Institute, Bethesda, MD_.

Background: The associations of multiple autoimmune conditions with gastric cancer may reflect their co-occurrence with autoimmune gastritis and its clinical manifestation pernicious anemia (PA), known risk factors for this malignancy. We analyzed diagnoses in US elderly aged >65 years to investigate the spectrum of these associations and the extent of mediation by PA.

Methods: We conducted a population-based nested case-control study using the Surveillance, Epidemiology and End Results (SEER)-Medicare linked database. 35,499 gastric cancer cases first diagnosed in SEER during 1992-2013 were compared to 200,000 cancer-free controls matched by age, sex and year of selection from a 5% random sample of Medicare beneficiaries. Autoimmune conditions were identified from inpatient Medicare claims. Logistic regression models estimated associations between autoimmune conditions and gastric cancer, as well as their direct and indirect effects on cancer risk.

Results: Among 40 autoimmune conditions evaluated, four were associated with increased gastric cancer risk (odds ratio, 95% confidence interval): autoimmune hepatitis (3.32, 1.30-8.50), PA (1.94, 1.81-2.08), uveitis (1.33, 1.03-1.70) and pure red cell aplasia (1.30, 1.09-1.54). Amyotrophic lateral sclerosis was associated with decreased risk (0.23, 0.06-0.96), based on 2 gastric cancer cases vs. 47 controls with this diagnosis. Within the control population, PA was positively associated with eight other autoimmune conditions: aplastic anemia (4.45, 2.47-8.35), Crohn's disease (3.61, 2.48-5.25), pure red cell aplasia (2.83, 2.03-3.95), polymyositis/dermatomyositis (2.82, 1.34-5.95), celiac disease (2.51, 1.24-5.08), Addison disease (2.17, 1.32-3.56), rheumatoid arthritis (1.75, 1.53-2.01) and ulcerative colitis (1.57, 1.07-2.31). Mediation analysis indicated that PA may account for 20% (p=6.52E-09) of pure red cell aplasia's effect on gastric cancer risk. PA failed to show significant mediation of the effects of autoimmune hepatitis, uveitis and amyotrophic lateral sclerosis.

Conclusions: Our study confirmed previously reported associations of PA and several other autoimmune conditions with gastric cancer risk. The association that we found between pure red cell aplasia and gastric cancer was partially mediated by PA. We also identified an intriguing set of autoimmune conditions associated with PA, many of which have been previously linked to gastric cancer; their lack of association with cancer in our data may be due to low population prevalence and insufficient statistical power. The relatively small fraction of mediation by PA does not preclude a more substantial role of subclinical autoimmune gastritis in gastric cancer etiology. Alternatively, autoimmunity may contribute through a different mechanism, perhaps via aggravation of mucosal infection by Helicobacter pylori.

#5052

Immune profiles in the San Francisco Adult Glioma Study using Immunomethylomics.

John K. Wiencke,1 Annette Molinaro,1 Warrier Gayathri,1 Jennifer Clarke,1 Jennie Taylor,1 Devin Koestler,2 Joe Wiemels,3 Helen Hanson,1 Lee Sean,1 Terri Rice,1 Lucie McCoy,1 Lucas Salas,4 Wrensch Margaret,1 Brock Christensen,4 Karl T. Kelsey5. 1 _University of California, San Francisco, San Francisco, CA;_ 2 _University of Kansas, Kansas City, KS;_ 3 _University of Southern California, Los Angeles, CA;_ 4 _Dartmouth University, Lebanon, NH;_ 5 _Brown University, Providence, RI_.

Glioma patients demonstrate abnormalities in peripheral blood leukocytes that have been associated with survival time. Here we assessed immune cell profiles in archival blood samples obtained 5-25 years ago using a novel epigenetic approach called immunomethylomics. We first validated the approach to measure the proportions of CD4 T, CD8 T cells, B cells, NK cells, neutrophils, and monocytes in archival blood using the new 850,000 feature EPIC Illumina methylation bead array. The immunomethylomic assay was shown to match the performance of multiparametric flow cytometry and thus is a highly accurate method for immune profiling. We next measured cell profiles in blood from 312 molecularly defined AGS subjects. In this cohort we enriched for patients with triple negative tumor subtypes (IDH wildtype, 1p19q negative, TERT non-mutant). Elevations in the neutrophil lymphocyte ratio (NLR) significantly increased with grade, whereas the proportions of T and B cells decreased with increasing grade. Using an established cut point of > 4.0 for the NLR revealed that this immune parameter was associated with shorter survival times in glioblastoma (median overall survival (mOS) 12.6 vs. 16.9 months) and non-glioblastoma (mOS 16.7 vs. 36 months) patients. Ongoing analyses of the cohort will be presented to evaluate the effects of age, gender, surgery, chemoradiation and tumor grade on the survival results. Because DNA based immune cell profiles do not require intact cells nor preserved proteins, they provide a powerful tool to glean potentially important immunologic information from historic stored samples that would otherwise be useless using current cytometry-based approaches.

#5053

Epstein-Barr virus prevalence in classical Hodgkin lymphoma tumors is explained by histologic subtype, not race/ethnicity in a multiethnic US population.

Rachel Bolanos,1 Amie Hwang,1 Chun Chao,2 Christopher Flowers,3 Sheeja Pullarkat,4 Jose Aparicio,1 Sophia Wang,5 Karen Mann,3 Leon Bernal-Mizrachi,3 Joo Song,5 Christian Steidl,6 Christine Lee,4 Wendy Cozen,1 Iran Siddiqi1. 1 _University of Southern California, Los Angeles, CA;_ 2 _Southern California Kaiser, Pasadena, CA;_ 3 _Emory University, Atlanta, GA;_ 4 _University of California Los Angeles, Los Angeles, CA;_ 5 _City of Hope, Duarte, CA;_ 6 _University of British Columbia and British Columbia Cancer Agency, Vancouver, British Columbia, Canada_.

Epstein-Barr virus (EBV) is present in a varying proportion of classical Hodgkin lymphoma (cHL) tumors reportedly associated with age, sex, race/ethnicity and histologic subtype. As part of a study to examine the tumor microenvironment and survival in a multiethnic set of US cHL cases, we examined the distribution of EBV expression in tumor blocks from a subset of 269 of the available cases. We confirmed cHL diagnosis and histological subtype in H&E sections of FFPE tumor blocks of excisions and core biopsies from cases provided by Southern California Kaiser, Winship Cancer Center at Emory University, City of Hope National Medical Center, Grady Memorial Hosiptal, University of California Los Angeles and University of Southern California hospitals diagnosed from 1996-2016. Immunostain results were available for most cases. Tissue microarrays were constructed with two 2 mm cores from each case with 29 cases on each array. EBER was assessed using in situ hybridization and scored as negative, positive in HRS cells or positive in the surrounding normal infiltrate. Demographic and clinical information, including the biopsy anatomic site, subtype, race/ethnicity (Hispanic, African American, Asian, non-Hispanic white), age at diagnosis and gender. Multiple logistic regression was conducted to assess the relationship between age, race/ethnicity, gender, subtype and EBV tumor status. Race/ethnicity data was available at this time for 237 cases. Of these, 186 were nodular sclerosis (NS), 53 were mixed cellularity (MC), 8 were lymphocyte depleted (LD), 3 were lymphocyte rich (LR), and 10 were not otherwise specified (NOS). Fifty-seven cases were African American, 48 were non-Hispanic white, 115 were Hispanic, 16 were Asian and one was other race. 106 were female (42%). Age at diagnosis ranged from 5-84 years, with 118 (50%) in the adolescent/young adult (AYA) range (15-35). EBV prevalence in HRS cells by subtype was 0% for LD, 47% for MC, 33% for LR, 23% for NS and 50% for NOS. When restricted to the two most common subtypes, NS and MC, histologic subtype alone was a statistically significant predictor of EBV tumor status (p=.0008) and when adjusting for age, sex, site and race/ethnicity (p=.0011). In NS cases, EBV HRS cell positivity was highest in non-Hispanic whites (31%), followed by Hispanics (23%) and African Americans (18%). Among MC cases, it was very high among African Americans (80%) compared to Hispanics and non-Hispanic whites (33-39%). The patterns were similar when restricted to the AYA age group. 6.5%, 4% and 20% of EBV-negative NS, MC and NOS cases, respectively, had EBV present in non-malignant lymphocytes but not in HRS cells. Histologic subtype was the strongest predictor of EBV HRS cell positivity in this set of multiethnic cHL patients. Unlike previous reports, EBV-positive tumors were not more common among non-whites, except for African American MC cases.

#5054

Are co-occurring structural birth defects associated with risk of acute lymphoblastic leukemia among children with Down syndrome.

Jeremy M. Schraw,1 Tiffany M. Chambers,1 John P. Woodhouse,1 Peter H. Langlois,2 Mark A. Canfield,2 Angela E. Scheuerle,3 Michael E. Scheurer,1 Sharon E. Plon,1 Karen R. Rabin,1 Philip J. Lupo1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _Texas Department of State Health Services, Austin, TX;_ 3 _University of Texas Southwestern Medical Center, Dallas, TX_.

Background: Children with Down syndrome (DS) have a 15- to 20-fold increased risk of developing acute leukemia (ALL). While children with DS typically also present with multiple co-occurring major and minor structural birth defects, very little is known about whether the number and type of these co-occurring birth defects in children with DS are associated with risk of ALL.

Methods: The Genetic Overlap Between Anomalies and Cancer in Kids (GOBACK) Study included linking data from population-based birth defects and cancer registries in Texas for the years 1999-2013. We performed a case-control analysis of ALL risk in participants diagnosed with DS. We evaluated the risk of ALL according to the presence of major birth defects in eight organ systems, as well as by the number of birth defects.

Results: We identified 7,684 children with DS (controls) and 81 children with DS-ALL (cases) from among 5.7 million live births. There was a high burden of co-occurring birth defects in both the DS and DS-ALL groups, with 97% compared to 98% being diagnosed with at least one co-occurring birth defect (p = 0.39), respectively, and 68% compared to 71% being diagnosed with at least one major birth defect (p = 0.33). Similar to what has been reported among the general population of ALL patients, children with DS and ALL had a significantly higher mean birthweight (3088 vs 2891 g, p <0.001) than children with DS overall, and were born to older parents (mean maternal age 33.7 vs. 31.7 yrs, p = 0.01; mean paternal age 36.6 vs. 33.8 yrs, p = 0.02). Although there were trends towards increased prevalences of major birth defects overall and in most organ systems among the DS-ALL group, none reached statistical significance. Similarly, we identified a non-significantly greater mean number of total birth defects in the DS-ALL group (p = 0.2). Neither number of total birth defects nor number of major birth defects were associated with ALL in multivariable Cox regression models.

Conclusions: In this population-based assessment, we did not find strong evidence that co-occurring structural birth defects were related to ALL risk among children with DS. However, the small numbers of children with DS-ALL make it difficult to draw definitive conclusions.

#5055

**Earlier birth cohort, lower BMI, and increased FEV** 1 **are risk factors for cancer in adults with cystic fibrosis.**

John B. Doyle,1 Rita M. Knotts,2 Claire L. Keating,1 Emily A. DiMango,1 Julian A. Abrams1. 1 _Columbia University Irving Medical Center, New York, NY;_ 2 _New York University Langone Health, New York, NY_.

Introduction: Adults with cystic fibrosis (CF) have a higher risk of multiple cancer types compared to the general population, and this risk appears to be particularly increased in transplant recipients. However, other risk factors for malignancy have not been well-described. This study assessed patient factors associated with cancer in adults with CF.

Methods: This was a retrospective study of prospectively collected data from the CF Foundation Patient Registry from 1992 - 2015. All adults cancer-free at 18 years of age were followed until either their most recent year in the registry or the year of their cancer diagnosis. Age, sex, CFTR mutation class, history of transplant, birth cohort, and BMI and FEV1 five years prior to cancer diagnosis or most recent registry entry were assessed as predictors of cancer using multivariable logistic regression. Analyses were repeated stratified by history of transplant. Data on cancer type was unavailable in the registry.

Results: Of 26,453 adults with CF, 525 (2.0%) had cancer diagnosed by histology at a mean age of 40.5 (SD=12.8) years. Of cancer patients, 183 (35.1%) were transplant recipients and 302 (64.7%) had a severe CFTR mutation (Class I-III). Among all CF adults, factors independently associated with cancer included lower BMI, higher FEV1, history of transplant, and birth cohort (Table). Among non-transplanted patients, cancer was associated with higher FEV1, unknown mutation class, and birth cohort. Among transplanted patients, cancer was only associated with birth cohort.

Conclusion: A history of transplant, lower BMI, higher FEV1, and earlier birth cohorts are associated with cancer in adults with CF. More recent birth cohorts have lower risks of cancer even after adjustment for age, possibly related to major improvements in CF care. Future studies are warranted to identify risk factors associated with specific cancer types and to better refine cancer screening recommendations in this patient population.

Factors associated with cancer in CF (models include associated variables on univariate analyses)

---

|  | All CF patients (n=12,019) | Non-transplanted CF patients (10,790) | Transplanted CF patients (n=2,402)

|  | Adjusted odds ratio | 95% confidence interval | Adjusted odds ratio | 95% confidence interval | Adjusted odds ratio | 95% confidence interval

|

Age | 0.99 | 0.98-1.01 | 0.98 | 0.96-1.00 | 1.00 | 0.98-1.02

|

Sex (male) | 0.98 | 0.76-1.27 | 0.80 | 0.59-1.07 | 1.16 | 0.83-1.62

|

BMI | 0.95 | 0.91-0.99 | |  | |

|

FEV1 percent predicted | 1.01 | 1.00-1.02 | 1.01 | 1.00-1.02 | |

|

History of transplant | 2.86 | 2.07-3.96 | N/A | N/A | N/A | N/A

Birth cohort | <1970 | 1.00 | Referent | 1.00 | Referent | 1.00 | Referent

1970-1974 | 0.37 | 0.25-0.55 | 0.34 | 0.21-0.53 | 0.50 | 0.30-0.84

1975-1979 | 0.27 | 0.17-0.41 | 0.18 | 0.11-0.31 | 0.38 | 0.21-0.69

1980-1984 | 0.08 | 0.05-0.14 | 0.07 | 0.04-0.14 | 0.24 | 0.12-0.47

1985-1989 | 0.06 | 0.03-0.11 | 0.04 | 0.02-0.09 | 0.12 | 0.05-0.31

1990-1994 | 0.01 | 0.00-0.05 | 0.01 | 0.00-0.04 | 0.10 | 0.03-0.33

>1994 | - | |

- | |

0.08 | 0.01-0.68

CFTR Mutation class | Class I-III | 1.00 | Referent | 1.00 | Referent | 1.00 | Referent

Class IV-V | 0.88 | 0.59-1.32 | 0.78 | 0.50-1.21 | 0.76 | 0.39-1.49

Unknown | 0.17 | 0.54-1.09 | 0.66 | 0.44-1.00 | 1.27 | 0.83-1.93

#5056

Exercise and inflammation on the risk of cancer.

Catherine Handy Marshall,1 Zeina Dardari,1 Miguel Cainzos-Achirica,2 Martin B. Mortensen,3 Khurram Nasir,4 Mouaz H. Al-Mallah,5 Michael D. Miedema,6 Ron Blankstein,7 Roger S. Blumenthal,1 Kala Visvanathan,1 Michael J. Blaha1. 1 _Johns Hopkins School of Medicine, Baltimore, MD;_ 2 _Bellvitge University Hospital and Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Spain;_ 3 _Aarhus University, Denmark;_ 4 _Yale School of Medicine, New Haven, CT;_ 5 _King Saud bin Abdulaziz University for Health Sciences, Saudi Arabia;_ 6 _Minneapolis Heart Institute, Minneapolis, MN;_ 7 _Brigham and Women's Hospital, Boston, MA_.

Introduction: High systemic inflammation and low levels of exercise are associated with increased risk of cancer. We hypothesized that in the setting of inflammation, exercise mitigates cancer risk.

Methods: To address this, we identified 6,388 participants, free of cancer and cardiovascular disease, enrolled in the Multi-Ethnic Study of Atherosclerosis. Regular moderate/vigorous intentional exercise was categorized as present or absent based on self-report from validated questions. A composite inflammatory score was created with a point given for C-reactive protein, IL-6, fibrinogen, and GlycA above normal, with composite scores > 2 points categorized as high. Cancer incidence was ascertained based on ICD codes abstracted from hospitalization or cancer registry data. Cox proportional hazard models were used to calculate subsequent risk of incident cancer. Models were adjusted for baseline age, gender, race, pack years smoking, education, income, health insurance status, body mass index, high-density lipoprotein levels, healthy diet adherence, statin and aspirin use.

Results: The mean age was 62 years (±10.2 years), 53% female, 39% white, 26% black, 22% Hispanic, and 12% Chinese-American. Compared to the reference group (individuals reporting no exercise and with low levels of inflammation), in multi-variable adjusted models, individuals with high levels of inflammation and no exercise were at highest risk of cancer (hazard ratio = 1.17; 95% confidence interval 1.00 – 1.37), while those with low levels of inflammation who exercised were at the lowest risk of cancer (HR 0.80, 95% CI 0.68-0.95). Those who had high levels of inflammation, but regular exercise had no increased risk of cancer (HR 0.94, 95% CI 0.68-1.30). There was no significant interaction between exercise and inflammation. When considering individual inflammatory markers, there was a similar pattern (Table).

Conclusion: Intentional moderate/vigorous exercise may lower the risk of cancer in individuals with high versus low levels of chronic inflammation.

Cox proportional hazard models for incident cancer by exercise and measures of inflammation, adjuste

---

|

no exercise  | any exercise

|

HR | 95% CI | HR | 95% CI

Low composite score (0 - 1) | Ref | |  | 0.80 | 0.68 | 0.95

High composite score (>2) | 1.17 | 1.00 | 1.37 | 0.94 | 0.68 | 1.30

P for interaction = 0.25 | |  | |  | |

Low CRP (<2 mg/L) | Ref | |  | 0.80 | 0.68 | 0.95

High CRP (>2 mg/L) | 1.23 | 1.05 | 1.43 | 0.98 | 0.71 | 1.35

P for interaction = 0.94 | |  | |  | |

Low IL-6 (< 1.8 pg/mL) | Ref | |  | 0.80 | 0.68 | 0.95

High IL-6 (>1.8 pg/mL) | 1.07 | 0.91 | 1.26 | 0.86 | 0.62 | 1.19

P for interaction = 0.18 | |  | |  | |

Low GlycA (<400 µmol/L) | Ref | |  | 0.80 | 0.68 | 0.95

High GlycA (>400 µmol/L) | 1.19 | 1.02 | 1.39 | 0.96 | 0.69 | 1.32

P for interaction = 0.73 | |  | |  | |

Low fibrinogen (<450 mg/dL) | Ref | |  | 0.80 | 0.68 | 0.95

High fibrinogen (>450 mg/dL) | 0.97 | 0.75 | 1.25 | 0.77 | 0.51 | 1.18

P for interaction = 1.00 | |  | |  | |

#5057

Single cell RNA sequencing reveals altered natural killer-like, effector CD8+ T lymphocytes in smokers.

Suzanne N. Martos, Michelle R. Campbell, Marie A. Iannone, Gary S. Pittman, Ma Wan, Douglas A. Bell. _NIH-NIEHS, Research Triangle Park, NC_.

Tobacco smoke exposure is a risk factor for many human diseases and the global disease burden attributed to smoking remains substantial. In addition to DNA damage, smoking-induced epigenetic changes may contribute to etiology of complex smoking-associated diseases. Smoking alters the epigenome and transcriptome of human blood leukocytes. However, interpretation of bulk genomic approaches is limited because changes could indicate altered distribution of cell (sub)populations or changes in expression within (sub)populations. To characterize smoking-related gene expression changes in primary immune cells, we performed single cell RNA sequencing on human peripheral blood mononuclear cells (PBMCs) from smokers (n=4) and nonsmokers (n=4). Transcriptomes of 45,965 cells (78,183 reads per cell) revealed an altered population of Natural Killer (NK)-like T lymphocytes in smokers. Compared to NK cells, the NK-like T cell cluster had elevated expression of CD8A, CD8B, CD3D, CD3E, CD3G, CD6, and CD2, indicative of CD8+ T lymphocytes. Relatively rare in nonsmokers (2.2%), the transcriptionally unique subset of CD8+ T cells comprised 8.7% of PBMCs in smokers. Among CD8+ T cell subtypes, the increase in NK-like CD8+ T cells (Mann-Whitney p = 0.03) corresponded with a decrease in Naïve CD8+ T cells (p = 0.03). We did not observe changes in the frequencies of two additional CD8+ T cell clusters or in the overall frequency of CD8+ T cells. Mass cytometry of a 26-antibody leukocyte panel confirmed no differences in the frequencies of total CD8+ T (CD3+/CD8+/CD56-), total NKT (CD3+/CD56+) or CD8+ NKT (CD3+/CD8+/CD56+) cells between smokers and nonsmokers. This suggests smoking is associated with an increased number of CD8+ T cells that share characteristics with NK cells, but are not NKT cells. Consistent with an NK-like phenotype, altered effector CD8+ T cells had elevated expression of genes reported to be upregulated in T cells reprogrammed to NK-like cells. Compared to other effector CD8+ T cells, altered effector CD8+ T cells had reduced IL7R and increased FCGR3A (CD16), IFNG (interferon gamma), GZMB (granzyme B), and PRF1 (perforin) expression. In mice, granzyme B and perforin expressing CD8+ T cells contribute to the development of atherosclerotic plaques. Our data highlights a potential link between smoking-induced functional changes in human CD8+ T cells and atherosclerosis and /or immune surveillance in cancer.

#5058

The association between smoking status at diagnosis and breast cancer specific mortality in Ireland: A population based study.

Maeve Kiely,1 Joesph McDevitt,2 Linda Sharp3. 1 _National Cancer Institute, NIH, Bethesda, MD;_ 2 _National Cancer Registry Ireland, Cork, Ireland;_ 3 _Institute of Health & Society, Newcastle University, United Kingdom_.

Background: Breast cancer is the most commonly diagnosed cancer worldwide and is a leading cause of breast cancer mortality in females. In recent years, 5 year survival rates have greatly improved for breast cancer however a clear disparity exists between survival in countries of high income compared to low and middle income. Disparity in survival also exists within individual countries. The identification of modifiable lifestyle factors can be an impactful and cost effective way to improve breast cancer outcomes worldwide, particularly in low income counties. An association between active smoking at diagnosis and breast cancer specific mortality has been reported by a number of studies to date. However, there is a paucity of studies undertaken in European populations. This large study was conducted to investigate whether smoking status at diagnosis is an independent prognostic factor for breast cancer specific mortality in the Irish female population.

Methods: All data was derived from the National Cancer Registry Ireland database which records all incident cancers in the Republic of Ireland. Completeness of case ascertainment at 5 years after diagnosis is estimated to be at 98%. Multivariable Cox proportional Hazards models were used to compare breast cancer specific mortality in current smokers, ex-smokers and never smokers. Subgroup analyses by breast cancer molecular subtype was also conducted.

Results: Data for women with known smoking status at time of diagnosis included 37,711 invasive breast cancer cases that accrued 263,626 women-years. Current smokers had a significantly increased risk of breast cancer specific mortality compared to never smokers (multi-variable HR, 1.3; 95% CI, 1.19, 1.41). Ex-smokers did not have a significantly different hazard ratio compared to never smokers (HR, 1.02; 95% CI, 0.92, 1.12). In subgroup analyses, risk of dying from breast cancer was increased most for current smokers with HER2+ disease compared to never smokers in the 5 years post diagnosis (HR, 1.53; 95% CI, 1.11, 2.12). This was followed by luminal A disease (HR, 1.39: 95% CI, 1.17, 1.65).

Conclusion. Breast cancer patients who are current smokers at diagnosis have a statistically significant increased risk of breast cancer specific mortality. This risk differs depending on molecular subtype of disease.

#5059

Early life risk factors and childhood cancer risk.

Shaina L. Stacy,1 Jeanine M. Buchanich,1 Zhen-qiang Ma,2 Christina Mair,1 Linda Robertson,1 Ravi K. Sharma,1 Evelyn O. Talbott,1 Jian-Min Yuan1. 1 _University of Pittsburgh, Pittsburgh, PA;_ 2 _Pennsylvania Department of Health, Harrisburg, PA_.

Prenatal exposures, including maternal, delivery, and newborn characteristics, may affect childhood cancer risk. Our objective was to examine whether such birth certificate-derived factors are associated with increased risk of subsequent cancer development, including leukemia and central nervous system (CNS) tumors, in children ages zero to 13. Pennsylvania state birth and cancer registry files obtained from the PA Department of Health were linked to create a cohort of children born from 2003 to 2015 who developed cancer from 2003 to 2016. After exclusions, 1,834,796 infants were included with a total of 13,785,309 person-years of observation. Among these, there were 2,355 children diagnosed with any cancer, 748 with leukemia, and 545 with CNS tumors. Older maternal age increased the hazard of developing any childhood cancer, leukemia, and CNS tumors. Children born to severely obese mothers (body mass index ≥40 kg/m2) were at over 50% higher hazard of leukemia (P=0.01) after adjustment for age and other significant risk factors; however, maternal obesity did not significantly increase the risk of childhood CNS tumors. Among the infant characteristics, having a fetal growth ratio 30% higher than expected was significantly associated with increased hazard of any childhood cancer, leukemia, or CNS tumors (hazard ratios 2.19, 1.79, and 1.77, respectively). Other fetal and newborn factors associated with higher risk of total childhood cancer included fetal intolerance of labor, lower gestational age, and having a low Apgar score at birth. This analysis demonstrated significant and independent impacts of maternal obesity and fetal growth rate on childhood cancer development, particularly leukemia, after accounting for multiple risk factors. If confirmed, weight control during pregnancy would be an effective means of childhood cancer prevention.

#5060

Effect of aspirin on gut microbiome in a pilot randomized double-blind trial.

Anna E. Prizment,1 Jeremiah Menk,2 Christopher Staley,2 Sithara Vivek,2 Guillaume Onyeaghala,2 Bharat Thyagarajan,2 Ryan Demmer,2 Dan Knights,2 Kathie Meyer,3 Aasma Shaukat,2 Alexander Khorutz,2 Michael J. Sadowsky,2 Robert J. Straka,2 Timothy Church2. 1 _Univ. of Minnesota, Masonic Cancer Center, Minneapolis, MN;_ 2 _Univ. of Minnesota, Minneapolis, MN;_ 3 _University of North Carolina, Chapel Hill, NC_.

Background: Aspirin use is associated with decreased risk of colorectal cancer (CRC), possibly by modulating the gut microbiome. We conducted a pilot double-blind randomized trial to evaluate the effect of aspirin on the gut microbiome. (ClinicalTrials.gov NCT02761486).

Methods: Fifty healthy individuals 50-75 years old were randomized (3:2) to receive a daily standard dose of aspirin (325 mg, N=30) or placebo (lactose, N=20) for 6 weeks followed by a 6-week washout. Stool specimens were collected at baseline, 3, 6, 9, and 12 weeks, and analyzed using 16S ribosomal RNA gene sequencing (V4 region) and Mothur software [ver. 1.35.1]. Fifteen genera associated with CRC were pre-specified from a published meta-analysis of the gut microbiome and CRC [Shah, 2017]. Among these 15 genera, eight genera, which were present in >10% of subjects at baseline in our study, were examined. Additionally, we examined four taxa previously associated with aspirin in a cross-sectional study of non-steroidal anti-inflammatory drugs and the gut microbiome [Rogers, 2016]. Mixed-effects logistic regression was used to estimate associations between aspirin use and changes in the relative abundance of taxa from pre- to post-treatment (baseline to week 6) via an interaction term (treatment*time). Log of odds ratio (β estimate) and P-values for the interaction term comparing aspirin to placebo for week 6 versus baseline are presented.

Results and conclusions. Out of the 8 pre-specified genera found to be associated with CRC in the meta-analysis, the interaction term was significant for 3 genera: Parabacteroides (β =-0.43, P<0.0001), Dorea (β =-1.56, P=0.02), and Akkermansia (β =0.30, P=0.009). These results suggest that aspirin decreases the relative abundance of two bacteria increased in CRC cases compared to non-cases - Parabacteroides and Dorea., while it increases the relative abundance of Akkermansia, which has been associated with anti-cancer immune response and improved survival of CRC patients. The following taxa that were previously associated with aspirin [Rogers, 2016] were also associated with aspirin in the present trial: family Ruminococcaceae (β= 0.33, P<0.0001), genera Bacteroides (β= -0.39; P<0.0001) and Prevotella (β= 0.51; P<0.0001). Our study suggests that aspirin changes the relative abundances of several gut bacteria previously shown to be associated with CRC.

#5061

Adherence to guidelines for cancer survivors and health-related quality of life among Korean breast cancer survivors.

Dahye Koh,1 Sihan Song,1 Sang-Eun Moon,1 Jihyoung Cho,2 Young Bum Yoo,3 Se Kyung Lee,4 Jung Eun Lee1. 1 _Seoul National University, Seoul, Republic of Korea;_ 2 _Keimyung University School of Medicine, Daegu, Republic of Korea;_ 3 _Konkuk University Medical Center, Seoul, Republic of Korea;_ 4 _Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea_.

Backgrounds: Breast cancer is the most frequent cancer affecting women worldwide and the second most common cancer among Korean women; nevertheless, the survival rate of patients with breast cancer has continuously increased. This improvement in survival rate of breast cancer indicates the significance of healthcare for better prognosis and quality of life. We aimed to determine the association between adherence to American Cancer Society (ACS) guidelines for cancer survivors and health-related quality of life (HRQoL) among Korean breast cancer survivors.

Methods: We included a total of 302 breast cancer survivors (aged 54.18±9.11 years) who had been diagnosed with stage I to III primary breast cancer according to the American Joint Committee on Cancer (AJCC) and had breast cancer surgery at least 6 months before enrollment. We collected dietary information using food frequency questionnaire (FFQ) and other lifestyle factors using a structured questionnaire. We obtained clinical information from medical records at each hospital. We assessed HRQoL levels using the 36-Item Short Form Health Survey (SF-36). We calculated adherence scores based on "Nutrition and Physical Activity Guidelines for Cancer Survivors" released by the ACS: (1) achieving and maintaining a healthy body weight; (2) engaging in regular physical activity; and (3) following the ACS guidelines for cancer prevention; achieving a dietary pattern that is high in vegetables, fruits, and whole grains and low in processed and red meat. We examined the least square means (LS-means) and 95% confidence intervals (CIs) of HRQoL according to the quintiles of adherence scores using the generalized linear models (GLM).

Results: The adherence scores ranged from three to 12. We observed that increasing adherence scores were associated with increasing levels of physical functioning (PF) and vitality (VT) in all breast cancer survivors of our study; LS-means (95% CIs) of the lowest and the highest quintiles of adherence scores were 69.61 (63.92-75.30) and 78.35 (72.02-84.67), respectively (p for trend=0.04) for PF and 55.97 (49.65-62.30) and 64.23 (57.19-71.26), respectively (p for trend=0.05) for VT. Among participants with stage II or III, we found that high adherence to ACS guidelines was related to higher levels of HRQoL components of each physical component summary (PCS) and mental component summary (MCS). However, we did not observe positive associations among participants with stage I.

Conclusions: Increasing adherence to ACS guidelines was associated with increasing levels of physical functioning and vitality of HRQoL among Korean breast cancer survivors. Positive associations between adherence scores and HRQoL levels were limited to breast cancer survivors with stage II or III. Further prospective studies are needed to evaluate a temporal relationship between adherence to guidelines and HRQoL levels.

#5062

Periprostatic adipose tissue and prostate cancer recurrence after radiation therapy.

Emma H. Allott,1 Lauren E. Howard,2 Taofik Oyekunle,2 Amanda M. De Hoedt,3 Joseph K. Salama,3 Stephen J. Freedland4. 1 _Queens University Belfast, Belfast, United Kingdom;_ 2 _Duke University, Durham, NC;_ 3 _Veterans Affairs Medical Center, Durham, NC;_ 4 _Cedars-Sinai Medical Center, Los Angeles, CA_.

Introduction: Obesity is associated with higher prostate cancer mortality, but mechanisms are not completely understood. Obesity is a heterogeneous phenotype, and fat distribution varies even between individuals of similar body mass index (BMI). Accumulation of visceral adipose tissue, a metabolically active fat type, has been more strongly linked with adverse cancer outcomes than overall obesity measured by BMI. We hypothesized that excess periprostatic adipose tissue, a type of visceral fat enveloping the prostate, would be more strongly related to poor prostate cancer outcomes than overall obesity.

Methods: We quantified abdominal and pelvic adipose tissue distribution using CT scans from prostate cancer patients treated with radiation at the Durham NC Veterans Administration. Visceral fat area (VFA) was measured at L4/L5 and periprostatic fat (PPAT) area at the pubic symphysis by thresholding on Hounsfield Units -190 to -30 to identify adipose tissue. Pre-radiotherapy height and weight was abstracted from medical records and used to calculate BMI. BMI was categorized as ≥30 vs. <30 kg/m2, and adipose tissue measures categorized as ≥ vs. < median. Recurrence was defined as PSA of 2 ng/ml over the nadir, according to the Phoenix definition. Cox proportional hazards analysis was used to examine associations between overall, abdominal and pelvic obesity and risk of recurrence, adjusting for age, race, year, biopsy Gleason, biopsy PSA, clinical stage, and receipt of androgen deprivation therapy (ADT). We performed stratified analysis by receipt of ADT.

Results: Of 407 prostate cancer patients treated with radiation from 2005 - 2011, 84 (21%) experienced recurrence during a median follow-up of 9 years. Overall obesity defined as BMI ≥30 kg/m2 was not associated with risk of recurrence overall or stratified by ADT. Neither was any measure of abdominal/pelvic obesity related to risk of recurrence overall. However, there was a suggestion that associations between abdominal/pelvic obesity and risk of recurrence varied by ADT receipt. Higher VFA was suggestively associated with increased risk of recurrence in men who received radiation only (HR 1.79; 95% CI 0.87-3.66), but inversely associated with risk of recurrence in men treated with radiation + ADT (HR 0.49; 95% CI 0.24-1.03; p-heterogeneity=0.002). Similarly, higher PPAT area was weakly associated with increased risk of recurrence in men treated with radiation only (HR 1.33; 95% CI 0.71-2.48) but inversely associated with recurrence risk in men who received radiation + ADT (HR 0.46; 95% CI 0.23-0.93; p-heterogeneity=0.002).

Conclusions: Visceral obesity appeared more strongly linked to prostate cancer recurrence than overall obesity. If confirmed, our findings suggest that visceral and pelvic obesity, two metabolically active adipose tissue depots, may have varying effects on prostate cancer progression dependent on the hormonal environment of the individual.

## PREVENTION RESEARCH

### Cancer Chemoprevention and Interception: From Mechanism to Translation

#5063

Mediation by differential DNA methylation of known associations between single nucleotide polymorphisms and bladder cancer risk.

Kristina M. Jordahl,1 Amanda I. Phipps,1 Timothy W. Randolph,2 Lesley F. Tinker,2 Rami Nassir,3 Lifang Hou,4 Garnet L. Anderson,1 Karl T. Kelsey,5 Emily White,1 Parveen Bhatti6. 1 _University of Washington/Fred Hutchinson Cancer Research Center, Seattle, WA;_ 2 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 3 _University of California, Davis, Davis, CA;_ 4 _Northwestern University Feinberg School of Medicine, Evanston, IL;_ 5 _Brown University, Providence, RI;_ 6 _BC Cancer Research Centre, Vancouver, British Columbia, Canada_.

Though bladder cancer has been the subject of multiple well-powered genome-wide association studies (GWAS), the mechanisms underlying the relationship between associated single nucleotide polymorphisms (SNPs) and bladder cancer risk remain largely unknown. The study focused on rs798766, rs401681, rs2294008, and rs8102137, which have been associated with bladder cancer and are also cis-acting methylation quantitative loci (mQTL). We assess whether the effects of these SNPs on bladder cancer are mediated through proximal methylation changes in pre-diagnostic blood at mQTL-associated CpG sites in a study of 440 matched case-control pairs nested within the prospective Women's Health Initiative (WHI) based on the subset of cases identified during follow-up (n = 412) and of controls (n = 424) with complete covariate information.

Mediation analyses were conducted with each of the four mQTL as the exposure, all corresponding CpG sites as the mediators, and incident bladder cancer as the outcome. We used a regression-based approach appropriate for dichotomous outcomes and multiple mediators. The relevant linear and logistic regression models were adjusted for matching variables and potential confounders, including race/ethnicity, smoking status, and pack-years of smoking. Effect estimates and confidence intervals (CIs) for the natural direct effect (NDE) and natural indirect effect (NIE) were estimated using a bootstrapping approach. To improve understanding of mechanisms underlying observed mediated effects, we conducted exploratory mediation analyses for individual SNP-CpG pairs, stratified by smoking status.

Our results suggest that substantial proportions of the modest effects of rs401681 (NIE = 1.05; NIE CI = 0.89-1.25; NIE percent = 98.5%) and rs2294008 (NIE = 1.10; NIE CI = 0.90-1.33; NIE percent = 77.6%) on bladder cancer risk are mediated through differential DNA methylation at nearby mQTL-associated CpG sites. There was little evidence supporting mediation for associations of rs8102137 and rs798766 with bladder cancer risk. Exploratory analyses suggest that, in smokers, the effect of rs2294008 is primarily mediated by methylation changes at CpG sites involving genes that bind to and modulate the α7 subunit of nicotinic acetylcholine receptors (α7-nAChRs).

Overall, this study suggests compelling mechanisms by which known bladder cancer risk variants are associated with susceptibility to bladder cancer. While results need confirmation, the study demonstrates the value of combining SNP and DNA methylation data.

#5064

Sirt6 is a novel tumor suppressor in human urothelial cancer and activated by methysticin.

DongJun Fu, Noriko N. Yokoyama, Mattew Tippin, Xiaolin Zi. _UC Irvine, Orange, CA_.

Sirtuin 6 (SIRT6) plays an important role in longevity, metabolism, DNA-repair, and inflammatory response. In human urothelial cancer, a high level of SIRT6 expression predicts a better survival for patients. We found that overexpression of SIRT6 in human muscle-invasive bladder cancer cell line J82 reduced cell migration and invasion, but didn't reduce the in vitro cell growth. Kava is a traditional psychotropic beverage made from the rhizomes of Piper methysticum G. Forst. Previous epidemiological studies have shown an inverse relationship between cancer incidence and kava consumption in South Pacific Island Nations. We have shown that Methysticin, a major kavalactone in the Kava plant, inhibits the growth, migration and invasion of bladder cancer lines. However, these anti-cancer activities of Methysticin are attenuated in cells with low expression of SIRT6, expression of enzymatically mutant SIRT6 or SIRT6 knockout, which suggested that the anti-cancer activities of Methysticin are at least in part dependent on SIRT6 expression. Molecular modelling indicates that Methysticin docks into the site next to a loop near the acetylated peptide substrate binding site of SIRT6. The Cellular Thermal Shift Assay (CETSA, an in vivo assay) demonstrated that Methysticin significantly decreased heat-induced protein degradation of SIRT6. Treatment of J82 cells with Methysticin also resulted in a dose-dependent reduction of H3K18 acetylation. Taken together, these results suggest that Methysticin acts a novel SIRT6 activator for its anti-bladder activity. Further studies have demonstrated that Methysticin activates the TBK1/IRF3/STING pathway by increasing the protein expression of TBK1 and the phosphorylation of TBK1 and STING, which suggest that Methysticin may be able to enhance anti-tumor immunity. Therefore, our results support further investigation of Methylation for bladder cancer prevention.

#5065

**Pharmacological modulation of inflammation and p53 signaling synergize to prevents muscle invasive bladder cancer** in-vivo **.**

Venkateshwar Madka,1 Yuting Zhang,1 Nandini Kumar,1 Gopal Pathuri,1 Nicole Stratton,1 Stanley Lightfoot,1 Adam S. Asch,1 Vernon E. Steele,2 Altaf Mohammed,2 Chinthalapally V. Rao1. 1 _Univ. of Oklahoma Health Sciences Ctr., Oklahoma City, OK;_ 2 _National Cancer Institute, Rockville, MD_.

Bladder cancer (BC) is the second most common genitourinary cancer and a leading cause of death globally. Muscle invasive BC (MIBC) has high mortality (>85% patients) leading to death within 2 years of diagnosis, if untreated. Although new treatment options were approved recently, it is still very challenging and most expensive to manage. Preventing BC is highly desirable to reduce recurrence, mortality and improve quality of life. Inactivation of p53 signaling and chronic inflammation are frequent hallmarks of MIBC. In spite of the promising chemopreventive effects of non-steroidal anti-inflammatory agents (NSAIDs), their clinical translation is hampered due to side-effects associated with their chronic intake and higher doses. Here we investigated a combinatorial approach to modulate inflammation with NSAIDs (licofelone or NO-Naproxen) and p53 signaling pathways using CP-31398 (CP) for preventing MIBC bladder in-vivo. Transgenic UPII-SV40T mice developing spontaneous MIBC were generated and fed control or experimental diets containing the licofelone (150ppm), NO-naproxen (300 ppm), CP (150ppm) alone or in combination starting at early tumor stage (6 weeks age). After 34 weeks of agent administration, mice were euthanized and urinary bladders were evaluated. Control diet fed transgenic mice developed high grade, muscle invasive, urothelial transitional cell carcinoma (TCC) leading to 3-5 fold increase in bladder weights (140.2±9.8 mg Vs 27.3±0.8 mg; p<0.0001) and (34.2±0.8 mg vs 14.8±0.53 mg; p<0.0001) in males and females compared with wild type mice. These tumors had dysregulated cell cycle and inflammation markers similar to human tumors. Treatment with licofelone, NO-Naproxen or CP alone led to significant suppression of bladder tumor. While NSAIDs had significant inhibitory effect in males (65% - 78%; p<0.0001) and females (31% - 34%; p<0.01-p<0.0001) compared to control group, CP had strongest tumor growth suppressive effect (80%; p<0.0001 and 36%; p<0.0001) in both genders. CP had no effect on invasion in male mice while in females ~50% inhibition was observed, while moderate effect of licofelone (12% - 42% inhibition; p<0.005) and NO-Naproxen (38% - 42% inhibition; p<0.001) was observed. Importantly, the combination of two agents led to a synergistic effect leading to significant inhibition of both tumor growth (~80% in males; p<0.0001 and 55% females; p<0.0001) and invasion ~62% inhibition (p<0.0001) in both genders. Molecular analysis of urothelial tumors showed inhibitory effect on proliferation and inflammatory markers (PCNA, Cyclins, p53, p21, COX2, and IL1β). Our results suggest that safer dose combination of targeted agents may yield better chemopreventive effects than higher dose of individual agents. Specifically, NSAID plus CP may be a promising combination for preventing MIBC. (Supported in part by NCI-PREVENT program - NCI-CN-53300)

#5066

Chemoprevention of breast cancer by targeting glucose metabolism with HJC0152.

Hyejin Kim,1 Jiabin Dong,1 Lili Wang,1 Jimin Xu,2 Dan Zhang,3 Ruping Yan,4 Hao Zou,4 Haiying Chen,2 Xi Liu,1 Yun Zhu,1 Yu Xue,2 Jia Zhou,2 Qiang Shen1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _University of Texas Medical Branch, Galveston, TX;_ 3 _Shandong University of Traditional Chinese Medicine, Jinan, China;_ 4 _Kunming Medical University, Kunming, China_.

Most mammalian cells use glucose as the primary fuel source, which is metabolized via glycolysis to pyruvate and further transferred into mitochondria for generating ATP through the Krebs cycle under normal condition. However, metabolism is characteristically reprogrammed in cancer cells or highly proliferative cells with a preferential ATP production through generating lactate by lactate dehydrogenase (LDH/LDHA), referred to as the Warburg effect or metabolic reprogramming toward anaerobic glycolysis. Efficient control of energy metabolism is the key to maintaining metabolic homeostasis, while disturbance in energy balance provokes diseases such as obesity, diabetes and cancer. However, the mechanisms underlying efficient energy metabolic homeostasis and breast cancer development are poorly understood. The transcription factor Signal Transducer and Activator of Transcription 3 (STAT3) is activated downstream of many cytokines and growth factor receptors. STAT3-targeting genes and their functions vary depending on the cellular context, primarily in correlation with cell survival and proliferation. Recently, reports show that active STAT3 with phosphorylation at Y705 residue plays an important role in regulating energy metabolism via transcriptional induction of its well-recognized transcriptional target HIF-1α. HJC0152, a novel small-molecule glucose metabolism modulator, was proprietarily developed using a combination approach of structure-based drug design strategies and molecular modeling techniques in our attempt to develop orally bioavailable non-peptide STAT3 inhibitors for anticancer use. A panel of mammary epithelial cells and breast cancer cells treated with HJC0152 exhibited suppressed cell growth and induced apoptosis in vitro. Intriguingly, HJC0152 reduces glucose uptake and Glut1 protein level. In addition, HJC0152 treated BC cell line showed decreased protein level of glycolytic enzymes including HK2, PFKL, ALDOA, PDHK, PKM2, LDHA and important metabolism regulator HIF-1α in a time-dependent manner. Furthermore, HJC0152 has significant in vivo efficacy in reducing/preventing mammary tumor development in transgenic mouse models of estrogen receptor (ER)-negative breast cancer. These results provide a rationale to develop HJC0152 as a promising drug candidate and preventive therapy for breast cancer and other cancers with aberrant glucose metabolism. In addition, HJC0152 can serve as a molecular probing tool for elucidating the key factors driving the development of breast cancer in the context of metabolic dysregulation and diseases. This work was supported by grant R01CA226001 from the NIH/NCI.

#5067

**Identification of key drivers of cancer stemness and progression regulated by vitamin D compounds in ductal carcinoma** in situ **breast cancer.**

Naing Lin Shan, Min Ji Bak, Li Cai, Roman Wernyj, Davit Sargsyan, David Cheng, Audrey Minden, Ah-Ng Tony Kong, Nanjoo Suh. _Rutgers University, Piscataway, NJ_.

Ductal carcinoma in situ (DCIS), the pre-invasive form of breast cancer, counts for one out of every five new breast cancer diagnoses. About one-third to half of the DCIS patients will progress to invasive breast cancer within 10 years of initial diagnosis. The disappearance of myoepithelial layer and disruption of the basement membrane are the hallmarks of transition from DCIS to invasive ductal carcinoma (IDC). Our laboratory has previously shown that vitamin D compounds decreased the MCF10DCIS.com xenograft tumors and inhibited the invasive transition from DCIS to IDC. We have also demonstrated that vitamin D compounds inhibited mammosphere formation by targeting breast cancer stem-like population and putative stem cell markers in MCF10DCIS cells. However, the key molecular mechanisms that govern the transition from DCIS to IDC are yet to be elucidated. In this study, we have performed RNA sequencing analysis of mammospheres treated with vitamin D compounds to identify potential genes that regulate breast cancer stemness and potentially the progression of DCIS to IDC. The mammosphere culture system has been shown to enrich stemness in tumor cells grown in low-attachment tissue culture plates. Upon treatment of mammospheres with vitamin D compounds, we identified down-regulation of genes that are involved in maintenance of breast cancer stem-like cells (e.g. GDF15, XBP1 and ALDH1A3), genes that mediate epithelial-mesenchymal transition, invasion and metastasis (e.g. LCNZ and S100A4), genes that confer breast cancer chemo-resistance (e.g. NGFR, PPP1R1B1, and AGR2) and genes that serve as biomarkers of basal breast cancer (e.g. NGFR). We also observed the up-regulation of genes that are associated with basal-like phenotype (e.g. KRT6A and KRT5), genes that regulate breast tumorigenesis (e.g. EMP1) and genes that regulate breast cancer stem cell self-renewal (e.g. DICER1). Our data set encompasses over 20,000 genes, providing us with unbiased insight to understand the overall gene expression profiling in DCIS breast cancer stem cells treated with vitamin D compounds. Thus, this study helps us identify genes that are key drivers of breast cancer stemness and progression and those regulated by vitamin D compounds. Further mechanistic studies of these significant genes will elucidate the natural history of DCIS progression to IDC, and provide us with target genes to prevent breast cancer progression. (This project is funded by Busch Biomedical Grant, Rutgers University)

#5068

Genomic regulation of the cell cycle as a function of ARID1A occupancy in breast epithelial cells.

Sham Jdeed, Balint L. Balint, Iván P. Uray. _University of Debrecen, Debrecen, Hungary_.

The idea of chemoprevention introduced a new concept both in clinical practice and experimental approaches. While single agent ER-independent chemopreventive therapy based on RXR-selective retinoids and other agents has produced promising results, even minor toxicity remains an obstacle to their clinical use. We performed unbiased high throughput screening to identify synergistic growth suppressive combinations of agents in normal mammary epithelial cells. A RXR-selective agonist and a beta-adrenergic inhibitor, as a candidate combination, at low concentrations demonstrated marked cancer preventive activity in a mouse model of Her2-induced ER-negative breast cancer. To elucidate the molecular mechanisms of this chemopreventive activity proteomic analyses of the candidate drug combination ensued. These revealed increased levels and phosphorylation of the DNA damage sensor ATM, along with elevated levels of the ARID1A subunit of the SWI/SNF chromatin remodeler in normal breast epithelial cells, but not in in situ breast carcinoma cells. Thus, we hypothesized that the maintenance of genomic integrity through ARID1A is a critical factor in the effectiveness of cancer preventive agents and helps reduce the odds of transformation. Genomic regions of increased accessibility are mapped by high throughput sequencing enabling multidimensional assays of the regulatory chromatin regions. Enhancers identified through chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) for H3K27 acetyl complemented with histones H3K4 will be discussed. We propose that ARID1A may mediate antiproliferative effects through genome-wide pausing of transcription of cell cycle regulatory genes. These results will help establish a role for the crosstalk of rexinoid and adrenergic signaling in the maintenance of a conserved chromatin remodeling mechanism and thus, premalignancy in breast epithelial cells.

#5069

Breast cancer prevention by triterpenoids from allspice.

Jie Gao,1 Kenza Mamouni,2 Georgios Kallifatidis,2 Siva Panda,3 Muthusamy Thangaraju,2 Bal L. Lokeshwar2. 1 _Department of Clinical and Diagnostic Sciences at University of Alabama, Birmingham, AL;_ 2 _Georgia Cancer Center, Augusta University, Augusta, GA;_ 3 _Department of Chemistry and Physics, Augusta university, GA_.

Breast cancer ranks second as a lethal cancer in women. Although survival following initial diagnosis is ~ 100% in first five years, cancer progression and mortality is imminent in subsequent years. The slow progression to the lethal form of breast cancer has prompted development of multiple avenues to delay the progression, metastasis and mortality using potent prevention strategies, including the use of nutraceuticals. Oleanolic acid (OA) and ursolic acid (UA) are two triterpenoids found in edible plant parts-fruits and seeds with potent cancer preventive, and selective cytotoxic activities against multiple cancers including breast cancer. We conducted cytotoxic assays of the combination of OA and UA. We found the combination has enhanced efficacy as compared to OA or UA alone. The combination of OA and UA and UA alone caused cell death by increased autophagy but not apoptosis in both MCF7 and MB231 human breast cancer cells. Further analysis revealed increased autopagosomes and autophagic flux, inhibition of either process reduced cytotoxicity, indicating cytotoxic autophagy is the primary mechanism of their action. Therefore, a combination of OA and UA with conventional therapies could enhance their therapeutic efficacy while limiting systemic toxicities of existing therapies.

#5070

Sulforaphane is a novel inhibitor of breast cancer-induced osteolytic bone resorption.

Subrata K. Pore, Joseph D. Latoche, Carolyn J. Anderson, Juraj Adamik, Deborah L. Galson, Kurt R. Weiss, Boeun Lee, Rebecca J. Watters, Prashant N. Kumta, Shivendra V. Singh. _Univ. of Pittsburgh, Pittsburgh, PA_.

Bone is the preferred site for metastatic spread in women with advanced breast cancer, and skeletal complications is associated with high morbidity and mortality. Nearly 70% of luminal-type breast cancer patients with metastases to bone experience skeletal complications due to enhanced osteoclastogenesis (osteoclast activation) that increases bone resorption, pain, pathological fractures, spinal cord compression, and hypercalcemia. The present study was designed to determine the effect of a cruciferous vegetable component (Sulforaphane; SFN) on breast cancer-induced osteoclastogenesis. Osteoclast differentiation in mouse bone marrow monocytes (BMM) was inhibited significantly upon the addition of 10% conditioned-media (CM) from SFN-treated breast cancer cells belonging to different subtypes, including MDA-MB-231, MCF-7, and SK-BR-3, in comparison with corresponding control. PCR-based gene expression profiling identified a common set of genes downregulated by SFN treatment compared to vehicle-treated control in MDA-MB-231, MCF-7, and SK-BR-3 cells, including osteoclastogenesis promoting transcription factors (RUNX2, NF-κB, and SOX-9) and certain soluble molecules (MMP9, TNFα, and Cathepsin K). Many of these gene expression changes were confirmed in MDA-MB-231, MCF-7, and SK-BR-3 cells by RT-PCR or western blotting. To determine the in vivo efficacy, SFN was administrated orally (1 mg per mouse; three times/week) to athymic mice intracardially injected with MDA-MB-231-Luc cells. SFN administration significantly inhibited the multiplicity of bone metastases and increased the bone volume relative to total volume. SFN-mediated prevention of osteoclastogenesis was associated with a significant decrease in TRAP-positive osteoclasts in bones and the levels of Cathepsin K, IL-8, and RANKL in serum. Altogether, this study demonstrates, for the first time to the best of our knowledge, that SFN is a potent inhibitor of breast cancer-induced osteoclastogenesis and bone resorption in vitro and in vivo. This study was partly funded by a pilot project grant from the UPMC Hillman Cancer (NCI grant P30 CA047904; Robert L. Ferris- Principal Investigator) and RO1 CA142604 and CA129347 awarded by the National Cancer Institute (Shivendra Singh- Principal Investigator).

#5071

Palbociclib inhibits the stemness of mammary epithelial cells in premalignant tissues of MMTV-erbB2 transgenic mice.

Amanda B. Parris,1 Yongxuan Liu,2 Zhikun Ma,1 Erin W. Howard,1 Xiaoshan Feng,2 Xiaohe Yang1. 1 _North Carolina Central University, Kannapolis, NC;_ 2 _First Affiliated Hospital of Henan University of Science and Technology, Luoyang, China_.

Palbociclib is the first CDK4/6-Cyclin D inhibitor approved by the FDA for ER+/Her2(erbB2)-breast cancer treatment. With the success of the drug in hormone-positive breast cancer, it is encouraging to explore more options of Palbociclib as an anticancer agent. erbB2 is a receptor tyrosine kinase (RTK) frequently overexpressed in breast cancer. The molecular hallmarks of erbB2+ breast cancer include the upregulation of cyclin D. The aim of this study was to explore the potential of Palbociclib as a preventive agent using MMTV-erbB2 transgenic mice, with a focus on the stemness of mammary epithelial cells (MECs) in the premalignant tissues. We first tested the in vivo effect of Palbociclib on tumor cells on MMTV-erbB2 mammary tumors using syngeneic tumor graft models. The animals bearing grafted tumors were treated with saline (control), low (75 mg/kg/day) and high (150 mg/kg/day) Palbociclib via oral gavage every 3 days for 29 days. We found that Palbociclib significantly inhibited tumor growth in a concentration dependent manner. To test the effect of Palbociclib on MEC stemness in premalignant mammary tissues, the animals at 9 wks of age were treated with saline, low and high (0, 75, 150 mg/kg/day) doses of Palbociclib every 3 days for 4 wks. We found the efficiency of both primary and secondary mammosphere formation of MECs from drug treated mice was significantly inhibited, suggesting the inhibition of mammary stemness in the premalignant tissues. Consistently, results from 3D cultures showed that colony numbers of the cells from drug treated mice were also lower than the control. Using CD24 and CD49f as markers, flow cytometry was performed to analyze the effect of Palbociclib on MEC subpopulations, including luminal, basal, and stromal populations. We showed that the percentage of luminal cells and putative mammary reconstitution units (MRUs) in drug treated mice was significantly inhibited. We also found that the percentage of CD61+/CD49+ cells, which are enriched with luminal progenitor cells and the origin of tumor initiation cells of this model, in Palbociclib treated tissues were also inhibited. The data suggest that Palbociclib induces MEC reprogramming in the premalignant tissues. Moreover, we found that the protein levels of pRb, E2F1, cyclin D1, c-myc, and cdc2 in cell cycle regulation, and pAKT, pERK, pmTOR, and p4EBP1 in the RTK pathway, pER, and DVL2 and LRP6 in the Wnt pathway were significantly downregulated in Palbociclib treated tissues, which was more evident in the high dose group. Taken together, our data indicate that Palbociclib inhibits MEC proliferation and stemness in the premalignant mammary tissues. Downregulation of cell cycle regulators and the signaling in ER, RTK, and Wnt pathway plays a critical role in this process. Our data support further investigation of Palbociclib in the prevention of ER+/ErbB2+ breast cancer.

#5072

Targeting the mTOR/TORC Pathway for the prevention of er-negative and triple-negative breast cancer.

Abhijit Mazumdar, Jamal Hill, Yun Zhang, Lakshmi Reddy Bollu, Alejandro Contreras, Michelle Savage, Shizuko Sei, Altaf Mohammed, Powel Brown. _UT MD Anderson Cancer Ctr., Houston, TX_.

Background: Women with "triple-negative breast cancer" (TNBC), are currently treated with chemotherapy, and have a very poor prognosis. TNBC tumors also frequently have p53 and BRCA1 gene mutations. Dysregulation of PI3K-mTOR pathway has been commonly associated with ER-negative breast cancers with poor prognosis. The mTOR inhibitor everolimus is used to treat ER-positive tumors that have become resistant to anti-estrogen therapy. We hypothesized that targeting mTOR may prevent development of ER-negative and BRCA1-mutant breast cancers and asked whether the mTOR inhibitor everolimus exhibited tumor preventive efficacy in several mouse models of breast cancer.

Methods: p53-null mammary gland donor mice were transplanted into cleared fat pads of p53 wild-type mice. BRCA/p53-deficient mice: We produced BRCA1 mice by breeding males and females. All mice were separated into two groups 1) Control and 2) everolimus. MMTV-erbB2 and p53 null mammary gland mice were treated with everolimus (5mg/kg, by oral gavage). BRACA1 mice were given 2mg/kg and 5mg/kg 2X a week of everolimus. All of these mice spontaneously developed mammary tumors within 12 months. Mice were observed daily, toxicity and the percentage of tumor free mice were recorded. Tumor incidence and time to tumor formation was visualized using Kaplan- Meier curves and analyzed using the generalized Wilcoxon test.

Results: In MMTV-erbB2 mice everolimus reduced tumor incidence and was associated with an increase in median survival time from 240 days to 410 days. At 365 days, when all mice in control group died, only half of the mice treated with everolimus had developed tumors (p=0.0001). Everolimus also reduced tumor incidence in p53 null mammary gland mice. At 420 days, 50% of the control mice developed mammary tumors compare to only 7 % of the everolimus treated mice (p=0.04). Everolimus also reduced tumor incidence in BRCA1 mice. At 262 days, when 13 (out of 18) (72%) of the control mice developed mammary tumors compare to only 7 (out of 18) (38%) in everolimus treated group developed tumors (p=0.02). Long term treatment (>150 days) of everolimus was associated with mild toxicity that includes slight weight loss (<10%) and skin changes (matted hair, skin erythema) in MMTV-erbB2 and p53-null mammary gland models. We have not observed any visible toxicity in BRCA1 mice yet. Results of mammary tissue biomarkers will be presented.

Conclusions: The mTOR inhibitor everolimus prevented mammary tumorigenesis in all three mouse models. Our results suggest that everolimus can be an effective cancer preventive drug and that further studies with reduced everolimus dose alone or in combination with other targeted therapies are warranted. In the future, clinical trials of the everolimus should be considered for the prevention of breast cancer in high-risk patients. Supported by NCI PREVENT Cancer Preclinical Drug Development Program (HHSN-2612015000-18I (PB)).

#5073

mPGES-1 **deficiency impairs self-renewal properties of colon cancer stem cells .**

Masako Nakanishi, Daniel W. Rosenberg. _Univ. of Connecticut Health Ctr., Farmington, CT_.

Dysregulation of prostaglandin E2 (PGE2) signaling is a hallmark of many cancers, including colorectal cancer (CRC). Direct suppression of inducible PGE2 synthesis by genetic deletion of Ptges1 (mPGES-1) affords dramatic cancer protection to the colon. However, the precise mechanisms by which this effect occurs remain incompletely understood. Recent evidence points to a fundamental role of PGE2 signaling in the expansion of cancer stem cells. In the present study, using an organoid system established from Apc mutant mice, we have evaluated the influence of PGE2 and related prostanoids on the viability of cancer stem cells in the colon. To establish cancer organoids, colon tumors were harvested from 16 week-old ApcΔ14/+ mice with or without mPGES-1 (D14:WT and D14:KO). Colon tumor multiplicity in D14:KO mice is approximately half the number of tumors in D14:WT mice. Tumor tissues were digested with collagenase and dispase, and cultured in Matrigel with complete organoid media. Organoid growth was scored daily over a one-week time period, and passaged. Tumor organoids generated from D14:WT and D14:KO mice showed no obvious differences in their growth characteristics nor morphological features during the first week of ex-vivo growth. After the first passage, however, the growth rate of D14:KO organoids was significantly reduced (175% vs. 80%, WT and KO, respectively), and by the second passage, D14:KO organoids failed to maintain cell viability. Immunohistochemical analysis of the organoids showed strong nuclear localization of ß-catenin and loss of Apc expression regardless of mPGES-1 genotype, indicating intact Wnt-driven cancer growth mechanisms that were independent of inducible PGE2 signaling. These observations indicate that alternative mechanisms are contributing to the compromised stem cell expansion observed in the D14:KO organoids. Since mPGES-1 is expressed within the tumor stroma, we postulated that the inactivation of mPGES-1 would cause a marked microenvironmental change. To test this possibility, we used GC-MS/MS to measure a panel of prostanoids within the tumor tissue. Targeted lipidomic analysis showed significant metabolite redirection of tissue prostanoids, with a significant increase (2-fold) in PGD2 in the D14:KO mice. Interestingly, administration of PGD2 was recently shown to reduce the growth and regeneration potential of stem cells within a hair follicle organ culture system. Given new evidence that PGD2 may have a direct influence on 'stem cell-ness' in other organ systems, we propose that the colon tumor microenvironment, deficient in inducible PGE2, may undergo permanent molecular changes that directly influence the growth characteristics and viability of cancer stem cells as reflected in our organoid culture system. New data is presented to define the long-lasting influence of eicosanoid metabolic changes on cancer-derived stem cell viability and proliferative capacity.

#5074

Optimizing erlotinib plus sulindac dosing regimens in a preclinical model of FAP.

Ahmetmursel Ulusan,1 Praveen Rajendran,1 Wan-Mohaiza Dashwood,1 Altaf Mohammed,2 Shizuko Sei,2 Powel H. Brown,3 Eduardo Vilar-Sanchez,3 Roderick H. Dashwood1. 1 _Texas A &M College of Medicine, Houston, TX; _2 _National Cancer Institute (NCI), Rockville, MD;_ 3 _University of Texas MD Anderson Cancer Center, Houston, TX_.

Introduction Colorectal cancer (CRC) involves sporadic cases and hereditary syndromes, such as familial adenomatous polyposis (FAP). In FAP patients, surgical intervention often is coupled to prevention strategies using nonsteroidal anti-inflammatory drugs. However, none of the current therapeutic options is fully effective. A recent trial in FAP patients combined standard of care Sulindac (SUL) treatment with daily Tarceva/erlotinib (ERL), and had good efficacy in reducing adenomatous polyp burden. Toxicity concerns, however, raised questions over the combination strategy for long-term prevention (Samadder et al. 2016). Using the Apc‐mutant polyposis in rat colon (Pirc) model as a mimic of human FAP, we sought to optimize the dosing regimens for SUL+ERL for an improved safety profile, while retaining efficacy in the GI tract.

Methods In a short-term pharmacodynamic biomarker study, rats (n=7) were fed AIN control diet or AIN diet containing 250 ppm SUL, either alone or in combination with ERL, given by oral gavage at 6 or 12 mg/kg (daily), 21 mg/kg (twice weekly), or 42 mg/kg (once weekly). Colon and small intestine (SI) polyps were resected at 0.5, 1, 2, 3, 7, 10, and 14 days after dosing, and assessed by immunoblotting (IB) or RT-qPCR for changes in pErk/Erk, pAkt/Akt, and other biomarkers. In a follow-up efficacy study, rats (n=10) were given SUL, ERL, or ERL+SUL using dosing regimens that were dictated by recovery times for pErk in the biomarker study. Colon and SI polyps were recorded at necropsy for location, incidence, multiplicity, and volume. Tumors and adjacent normal tissues were taken for histopathology and molecular analyses.

Results The biomarker study revealed that pErk was inhibited in Pirc colon polyps for up to 10 days after discontinuing ERL treatment, with full recovery on or around day 14. Nuclear β-catenin, c-Myc, Mmp-7 and Cyclin D1 also were attenuated by ERL+SUL in colon polyps. In the efficacy study, results accrued via endoscopy at 3, 4 and 5 months were remarkably consistent with data obtained during final necropsy, at 6 months. Compared to AIN controls, multiple ERL+SUL groups exhibited significant suppression of polyps in the colon (78-98% inhibition, p<0.001) and SI (91-100% inhibition, p<0.001). Molecular analyses revealed that pErk was inhibited in adenomatous polyps, along with downregulation of β-catenin targets (c-Myc, Mmp-7). No overt toxicity was detected, other than mild skin changes in groups given ERL.

Conclusions These studies have the potential to define safe and effective dosing strategies for SUL+ERL, improving efficacy against colon and SI polyps, while circumventing toxicity and resistance. Outcomes from the current work, plus an ongoing one-year toxicity/resistance trial in Pirc, should be directly translatable to the clinical management of FAP patients exhibiting similar pathology and phenotype. Supported by NCI Contract Number HHSN261201500018I, Task Order HHSN26100004.

#5075

**Naproxen blocks spontaneous lung adenoma progression to adenocarcinoma in Kras-** G12V **mice.**

Gaurav Kumar,1 Venkateshwar Madka,1 Craig Logsdon,2 Anil Singh,1 Nicole Stratton,1 Stanley Lightfoot,1 Altaf Mohammed,3 Chinthalapally V. Rao1. 1 _Univ. of Oklahoma Health Sciences Ctr., Oklahoma City, OK;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 3 _National Cancer Institute, Rockville, MD_.

The aim of the present study was to investigate the effects of a non-steroidal anti-inflammatory drug (NSAID), naproxen, on a spontaneous mouse model of lung adenocarcinoma. Lung cancer is the most frequently diagnosed cancer and the primary cause of cancer-related deaths worldwide. Inflammation plays an important role in the development and progression of lung and several other cancers. The association between NSAID use and lung cancer risk suggests a beneficial effect on patients' lung cancer progression. Preventing lung cancer can help to significantly reduce cancer-related mortality. Compared with other NSAIDs, naproxen is relatively safer in terms of cardiovascular risk. Eight-week-old transgenic KrasG12V male and female mice (n=20) or littermate wild-type (n=5) mice were fed (AIN76A) diets containing naproxen (0 ppm or 400 ppm) in modified AIN76-A diet for 28 weeks. At 36 weeks of age, mice were euthanized. Lungs were collected and evaluated for lung tumor incidence and multiplicity, and were saved in formalin for histopathological identification of adenoma and adenocarcinoma. By 12 weeks of age, KrasG12V mice developed visible lung tumors that enlarged in size and progressed to adenocarcinoma by 24-36 weeks of age. Lung tumor incidence was observed in 100% of KrasG12V mice. Dietary administration of naproxen did not show any overt-toxicities. No significant changes were observed in body weight gain or any major organ's (liver, kidney, and pancreas) gross morphology or weight in mice fed with naproxen compared with mice fed control diet. Due to reduced tumor burden, lungs of the naproxen-fed mice weighed less (230.6± 12.2 mg, p=0.019) (Mean±SEM) than those of the control group (384.0±68.1 mg). Importantly, mice fed control diet developed 19.8±0.96 total lung tumors (2.5±0.3 adenoma, 17.3±0.7 adenocarcinoma). The results suggest that naproxen inhibits total lung tumor formation by ~52% (9.4±0.85; p<0001) and adenocarcinoma by ~64% (6.1±0.6; p<0001), compared with control diet. However, we observed no significant difference in the number of adenomas in mice fed with control diet and mice fed naproxen diet. These data suggest that naproxen delays the progression of lung adenoma to adenocarcinoma. Biomarker analysis of lung tumors from mice exposed to naproxen showed significantly reduced tumor cell proliferation (PCNA, Cyclin D1) and increased apoptosis (p21, Caspase-3). These results support a chemopreventive role of naproxen in spontaneous-induced lung adenocarcinoma formation. (Supported by the Kerley-Cade Chair Endowment & partly by the NCI-PREVENT program HHSN261201500038i)

#5076

You can teach an old drug new tricks: Repurposing drugs to prevent mesothelioma.

Bethan G. Rogoyski,1 Ankur Karmokar,1 Lynne Howells,1 Farhat Khanim,2 Dean Fennell,1 Anne Thomas,1 Karen Brown1. 1 _University of Leicester, Leciester, United Kingdom;_ 2 _University of Birmingham, Birmingham, United Kingdom_.

Global rates of mesothelioma are rising. Yet surgical and therapeutic treatment options are lacking, resulting in unacceptably low survival rates. We propose that prevention-based strategies, utilising repurposed drugs could significantly reduce patient mortality.

An initial panel of 100 repurposed agents at biologically achievable doses was screened in three mesothelioma cell-lines using viability assays. Apoptosis and cell-cycle assays were used to corroborate initial findings and identify treatments that consistently reduced mesothelioma viability in vitro. Primary patient tissue and a subsequent PDX model developed in NOD/SCID mice, have provided a basis for ex vivo explant experiments to augment initial findings in a more translational model, as well as providing some insight into mechanism of the top two agents. Tumour growth response to the top candidate drug in vivo using the PDX model was shown to be equally promising. A second in vivo study testing two top candidates in combination is now underway, as well as proteomic assessment of both in vitro and in vivo samples to further comprehend underlying mechanism of action.

Niclosamide and zinc acetate comprise the two top candidate drugs having reduced mesothelioma cell line viability to 27.4% and 59.3% of that of untreated cells, further reducing viability to 6.45% in combination - results congruent with V-FITC and FACS analysis which both show cell cycle alterations and apoptosis in response to treatment. BAP-1 being one of few genes consistently associated with mesothelioma aetiology, a CRISPR-knockout BAP-1 cell-line was generated to model early-stage disease and showed similar response to treatment as the mesothelioma cell-lines. FACS analysis of treated ex vivo tissue also showed up to a 27% increase in apoptosis in response to combined treatments. In vivo response to daily dietary Niclosamide supplementation produced a statistically significant decrease in PDX mouse tumour volume compared with a control diet.

Current reliance on disuse of asbestos has resulted in a huge unmet need for therapeutic options for the treatment and prevention of mesothelioma. Survival rates remain static at 12 months, despite a rise in diagnoses predicted over the next decade. However, the latency period between asbestos exposure and mesothelioma diagnosis provides an unparalleled opportunity for a chemopreventive strategy. We show that two repurposed drugs hold significant potential in reducing mesothelioma progression. Not only do zinc and niclosamide reliably reduce mesothelioma viability across various diseases models and stages, they have the additional benefit of long-term safety and side-effect profiles. This method of repurposing drugs not only increases patient tolerability to treatment - an important consideration in chemoprevention - but also reduces any temporal or financial investment of translating these drugs to clinical application.

#5077

Proteomic profiling reveals chemopreventive targets in esophageal adenocarcinoma.

Katherine M. Weh,1 Connor L. Howard,1 Amy B. Howell,2 Jennifer L. Clarke,3 Laura A. Kresty1. 1 _University of Michigan, Ann Arbor, MI;_ 2 _Rutgers University, Chatsworth, NJ;_ 3 _University of Nebraska, Lincoln, NE_.

Esophageal adenocarcinoma (EAC) is characterized by rising incidence rates and high mortality due to late stage diagnosis and a lack of efficacious options for prevention and treatment. While reasons for the rapid increase in EAC are being unraveled, persistent, symptomatic reflux of gastric and duodenal contents, known as gastroesophageal reflux disease is considered the strongest risk factor. Our laboratory utilizes a rat surgical model of reflux-induced EAC to evaluate mechanisms by which cranberry proanthocyanidins (C-PAC) delivered in the drinking water inhibit reflux-induced EAC. Findings show that C-PAC (690ug/rat/day) inhibits EAC formation by 83% at 40 weeks of study and mechanisms of inhibition are under investigation. We utilized nano LC-MS/MS proteomic profiling to identify proteins that were dysregulated due to reflux-induced EAC and reversed with C-PAC treatment. Proteomic profiling revealed that C-PAC treatment restored 63 proteins dysregulated in the context of reflux. EIF3F, PSMD2 and ACTR2 were the top proteins up-regulated by reflux; whereas, top markers restored by C-PAC included GSTT2, OGN and PCMT1. Metacore integrated software showed top pathways up-regulated by reflux and down-regulated C-PAC included Translation_regulation of translation initiation (EIF-linked), Immune response and Regulation of telomere length. Conversely, C-PAC treatment upregulated the glutathione metabolism pathway which is consistent with GSTT2 restoration. Disease enrichment analysis revealed multiple gastrointestinal tract diseases linked to reflux, which C-PAC down regulated supporting that proteomic profiling may inform other potential disease targets for C-PAC. Finally, beyond the 63 reflux-driven proteins C-PAC reversed, we considered proteins, pathways and processes which could not be reversed by C-PAC (n=269). As an example, Process networks upregulated by reflux (not reversed by C-PAC) included Translation initiation (RPL-linked), Elongation, mRNA processing, G2-M and S-phase of the cell cycle. Thus, proteomics profiling may inform the development of complementary preventive combinations for improved cancer inhibitory efficacy, especially for a cancer like EAC, with high mutational burden. Western blot analysis of several markers in the rat esophagus including GSTT2 and COX2 support the data obtained through proteomic profiling. Future directions include interrogating specific pathways that are highly modulated by reflux-induced EAC and restored by C-PAC including DNA damage, repair and extracellular matrix and adhesion. Last, proteomic profiling has also allowed for identification of post-translational modifications which may provide insight into regulation of key dysregulated proteins.

#5078

Modulation of glucose metabolism and lactate efflux in tumors by bitter melon juice in its efficacy against pancreatic cancer.

Deepanshi Dhar, Rama Kant, Komal Raina, Michael F. Wempe, Natalie J. Serkova, Chapla Agarwal, Rajesh Agarwal. _Univ. of Colorado Denver -AMC, Aurora, CO_.

Pancreatic cancer (PanC) is currently ranked as the fourth leading cause of cancer-related deaths across the United States. The dismal statistics for the year 2018 estimates about 55,440 new incidences and 44,330 PanC-associated fatalities in men and women combined, with a 5-year survival rate of less than 10%. PanCs possess a very intricately designed metabolic profile favoring excess aerobic glycolysis in addition to altered glutamine metabolism, contributing to tumor cell proliferation and PanC progression. Targeting metabolic phenotype in PanC with natural/diet-derived agents has immense scope for better non-toxic intervention, improved overall survival and quality of life; the major concerns with the use of frontline PanC chemotherapeutics. Herein, we utilized bitter melon juice (BMJ), a natural agent, which we recently reported to possess anticancer potential via targeting PanC stem cell and bulk cell populations and exerting a role in elevating drug sensitivity of gemcitabine resistant PanC cells. The established role of BMJ in activating master metabolic regulator APMK in PanC cells served as a basis for pursuing deeper investigation into the underlying metabolic alterations leading to BMJ efficacy in PanC. We investigated the comparative metabolic profiles of PanC cells with differential KRAS mutational status (PANC1 - mutated KRAS and BxPC3 -; wild type KRAS), on BMJ exposure. Specifically, we employed Nuclear magnetic resonance (NMR) metabolomics and in vivo imaging platforms to assess whether BMJ modulates PanC cell metabolome, together with establishing the molecular metabolomic targets to better understand the relevance of altered metabolism in PanC management by BMJ. Multinuclear NMR metabolomics was performed post 72 hours of BMJ treatment followed by PLS-DA (partial least square discriminant analysis) assessments on the quantitative metabolic data sets to visualize the treatment group clustering; altered glucose uptake, lactate export and energy state were identified as the key components responsible for cell death induction. We next employed PANC1 xenograft model for assessing in vivo BMJ efficacy against PanC. Positron Emission Tomography ([18FDG]-PET) and Magnetic Resonance Imaging (MRI) on PANC1 tumor-bearing animals reiterated the in vitro results, with BMJ-associated significant changes in tumor volumes, tumor cellularity and glucose uptake. Additional studies in BMJ-treated PanC cells and xenografts displayed a strong decrease in the expression of glucose and lactate transporters GLUT1 and MCT4, respectively, supporting their role in metabolic changes by BMJ. Collectively, these results highlight BMJ-induced modification in PanC metabolomics phenotype and establish primarily lactate efflux and glucose metabolism, specifically GLUT1 and MCT4 transporters, as the potential metabolic targets underlying BMJ efficacy in PanC.

#5079

**Targeting cholecystokinin-2 receptor (CCK2R) for pancreatic ductal adenocarcinoma prevention in p48** Cre/+ **-LSL-Kras** G12D/+ **mice model.**

Altaf Mohammed,1 Naveena B. Janakiram,2 Chen Suen,1 Nicole Stratton,2 Stanley Lightfoot,2 Anil Singh,2 Gopal Pathuri,2 Rebekah Ritchie,2 Venkateshwar Madka,2 Chinthalapally V. Rao2. 1 _National Cancer Institute, Rockville, MD;_ 2 _Univ. of Oklahoma Health Sciences Ctr., Oklahoma City, OK_.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer that is difficult to treat. Hence, developing strategies that delay/inhibit/prevent the progression of pancreatic intraepithelial neoplasms (PanINs) to PDAC are of utmost importance. Gastrin signaling mediated through gastrin/cholecystokinin-2 receptor (CCK2R) and its down-stream signaling molecules are overexpressed in both human and KrasG12D mouse PDACs. Particularly, CCK2R is highly expressed in PDAC stellate cells and is involved in fibrosis and PDAC progression. Thus, targeting CCK2R for pancreatic chemoprevention is a valid approach for PDAC prevention. Two CCK2R antagonists, netazepide (YF476) and JNJ-26070109, were tested for their effect on PanINs and their progression to PDAC in p48Cre/+-LSL-KrasG12D/+ mice. Six-week-old p48Cre/+-LSL-KrasG12D/+ (22-24/group) mice were fed (AIN-76A) diets containing 0, 250 or 500 ppm netazepide or JNJ-26070109 for 38 weeks. After sacrifice at 44 weeks of age, pancreata were collected, weighed, and evaluated histopathologically for PanINs and PDAC. To understand the molecular mechanisms, levels of proliferation and apoptosis (PCNA, p21, and caspase3) were analyzed by Immunohistochemistry (IHC). Results demonstrated that control diet fed mice showed 69% and 33% incidence of PDAC in male and female mice. Dietary administration of low dose and high dose JNJ-26070109 inhibited the incidence of PDAC by 88% (p<0.003) and 71% (p<0.004) in male mice and by 100% and 24 % (not significant, p>0.05) in female mice respectively. Dietary administration of low dose and high dose netazepide inhibited the incidence of PDAC by 74% (p<0.018) and 69% (p<0.02) in male mice and by 45% and 33 % (not significant, p>0.05) in female mice, respectively. Both agents reduced PanIN 3 (carcinoma in-situ) multiplicity compared to untreated mice in both genders, with JNJ-26070109 showing greater inhibitory effect compared to netazepide. Both agents showed a decrease in caspase-3, p21, and PCNA expressions. Transcriptome analysis showed downregulation of Cldn1, Sstr1, Apod, Gkn1, Siglech, Cyp2c44, Bnc1, Fmo2, 623169, Kcne4, Slc27a6, Cma1, Rho GTPase activating protein 18 and Gpr85 genes in JNJ-26070109-treated mice compared to untreated mice. Netazepide-treated mouse pancreas specifically showed down regulation of Riks, Zpbp, Ntf3, Lrrn4, Aass, Skint3, Kcnb1, Dgkb, Ddx60, and Aspn genes compared to untreated mice pancreas. In summary, targeting the CCK2R pathway by netazepide and JNJ-26070109 showed significant chemopreventive effects in suppression of PDAC and PanIN3. Overall, JNJ-26070109 showed better chemopreventive efficacy on PDAC incidence and PanIN 3 multiplicity as compared to netazepide. However, caution is recommended while selecting the doses as the agents appeared to exhibit gender-specific effects. [Supported by NCICN25001-26}

#5080

Decursinol intercepts LNCaP human prostate cancer xenograft growth bypassing androgen receptor-prostate specific antigen axis.

Sangyub Kim, Chongtao Qin, Deepkamal N. Karelia, Arati Sharma, Cheng Jiang, Junxuan Lu. _Penn State College of Medicine, Hershey, PA_.

The slow-growing nature of most prostate cancers (PCa) provides multiple windows of opportunity to block or delay disease progression to reduce patient suffering and mortality. This is especially true following the failure of radical prostatectomy (RP) and radiation therapy (RT) of localized PCa with biochemical recurrence detectable by a rise in plasma/serum prostate specific antigen (PSA) and prior to androgen deprivation therapy (ADT). Costly aside, ADT is not curative and causes many adverse effects which negatively affect the quality of life of the patients. Currently there is no standard of care regimen for post-RP/RT patients with rising PSA to stump the trajectory. Many patients seek food or supplement-based interventions with a hope to delay ADT. The root of Korean Angelica gigas Nakai (AGN) contains decursin (D) and its isomer decursinol angelate (DA) as the major pyranocoumarins. We have previously shown that daily gavage of AGN and D/DA suppressed the growth of androgen receptor (AR)+ and AR- xenograft tumors and TRAMP neuroendocrine carcinomas. We also showed that D/DA rapidly converted to decursinol (DOH) in rodents and humans, and DOH inhibited LNCaP-AR xenograft growth, therefore the likely in vivo active compound. To assess merit of DOH to intercept recurrent PCa, we tested in NSG SCID mice bearing s.c. inoculated human LNCaP PCa xenograft (a) its efficacy to inhibit tumor growth and b) serum PSA as an efficacy metric. A day prior to start of daily DOH gavage (120 mg/kg body weight) for the tumor bearing mice, blood was drawn through tail vein for baseline PSA. After 4 weeks, the mice were bled for post-PSA and their tumors/prostate were dissected and weighed. Gavage delivered DOH did not affect body weight, but significantly reduced the tumor volume and weight without decreasing the weight of the prostate, distinct from ADT drugs. The post-PSA showed a significant and tight linear correlation with the tumor weight for mice in the vehicle group (r2=0.94, n=6). Five of 7 DOH-treated mice showed post-PSA/tumor weight plot on the same regression line but two non-responders displayed exaggerated post-PSA/weight ratio. The much slower PSA rise in the DOH-responders (2.9 fold, n=5) than vehicle group (7.8 fold, n=6, p=0.031) directly reflected a reduction of tumor burden by DOH treatment. Histological (H&E) analysis showed that DOH responder tumors had greater necrosis and less hemorrhage than non-responders and control tumors. Immunohistochemistry staining for AR and PSA did not detect any reduction of either protein in the responder tumors which contained reduced mitotic marker phospho-histone 3. Our preclinical modeling therefore suggests serum PSA as a reliable efficacy metric for DOH-mediated PCa interception in post-PR/RT patients, involving mitosis inhibition and necrosis without affecting the cancer AR/PSA axis.

#5081

Leelamine is a novel inhibitor of fatty acid synthesis in prostate cancer.

Krishna B. Singh, Shivendra V. Singh. _University of Pittsburgh, Pittsburgh, PA_.

Increased beta-oxidation of fatty acids to meet energy demand is a rather unique characteristic of a subset of human prostate cancers. A safe and effective intervention for inhibition of fatty acid synthesis is still a clinically unmet need. We have shown previously that leelamine (LLM), a phytochemical derived from pine tree bark, suppresses expression and activity of full-length androgen receptor (AR) and its splice variants in vitro and in vivo in preclinical models of prostate cancer. Because AR is implicated in regulation of fatty acid metabolism, the present study was undertaken to determine the effect of LLM on this metabolic pathway. Treatment of a castration-resistant (22Rv1) as well as an androgen-responsive (LNCaP) human prostate cancer cell line with LLM (2.5 and 5 µmol/L) resulted in downregulation of key fatty acid synthesis enzyme proteins including ATP citrate lyase (ACLY), acetyl-CoA carboxylase 1 (ACC1), fatty acid synthase (FASN), and sterol regulatory element-binding protein 1 (SREBP1). LLM treatment also decreased intracellular levels of total free fatty acids and neutral lipid droplets in LNCaP and 22Rv1 cells. Consistent with the in vitro results, we also observed a significant decrease in ACLY and SREBP1 protein expression and number of neutral lipid droplets in vivo in tumor tissue sections of 22Rv1 xenografts after intraperitoneal administration of LLM (9.1 mg/kg bw/ day, 5 times/week) compared to controls. Studies are in progress to determine if overexpression of AR and/or SREBP1 confers protection against fatty acid synthesis inhibition by LLM. In conclusion, it is reasonable to postulate that suppression of AR-SREBP1 regulated fatty acid metabolism is an important mechanism in prostate cancer inhibition by LLM. This study was supported by the grant RO1 CA101753 awarded by the National Cancer Institute.

#5082

SIRT1 functions as a double-edged sword in prostate cancer.

Shih-Bo Huang,1 Dinesh Thapa,2 Roble G. Bedolla,1 Amanda R. Muñoz,1 Xiaoyu Yang,1 Paul Rivas,1 Robert L. Reddick,1 Hiroshi Miyamoto,3 Rita Ghosh,1 Addanki Pratap Kumar1. 1 _University of Texas Health Science Center at San Antonio, San Antonio, TX;_ 2 _Dana-Farber Cancer Institute, Boston, MA;_ 3 _University of Rochester Medical Center, Rochester, NY_.

SIRT1 is a NAD+ dependent deacetylase known to regulate a plethora of biological processes through posttranslational regulation of proteins including those that function as tumor promoters and suppressors. In order to define the role of SIRT1 in prostate pathogenesis, we used 2 mouse models: (i) PTEN knockout (PTENKO) mouse model by pharmacological activation of SIRT1 with resveratrol (RES) a known activator of SIRT1, & (ii) orthotopic implantation model with genetic silencing of SIRT1 (SIRT1 shRNA). We also used genetic and pharmacologic inhibition of SIRT1 in cell culture models to understand the mechanism. We tested whether SIRT1 modulation is beneficial when targeted early (before the establishment of prostatic lesions) or late (after the establishment of prostatic lesions) in the PTENKO model. RES intervention was initiated in 4-5-week (early intervention) and 10-15-week-old (late intervention) PTENKO mice. Analyses of samples collected longitudinally during progression revealed that early intervention with RES reduced incidence of high-grade prostate intraepithelial neoplastic lesions (HGPIN) when given for 14 weeks with no significant difference at 7 or at 11 weeks. On the other hand, late intervention after the establishment of HGPIN lesions with RES had no beneficial effect. Importantly, longer treatment duration (28 weeks) resulted in significantly increased incidence of invasive prostate carcinoma. Furthermore, RES had no significant effect on the development of orthotopic prostate tumors following implantation of LNCaP cells in nude mice. In contrast, orthotopic implantation of SIRT1 stably silenced LNCaP cells showed significant impairment in tumor development. Immunohistochemical evaluation showed nuclear localization of SIRT1 in human prostate tumors and was associated with increased risk of biochemical recurrence. Mechanistic investigations revealed (i) suppression of AR signaling in hormone-sensitive LNCaP but not in castration-resistant 22Rv1 cells; (ii) RNAseq coupled with gene ontology enrichment analysis using SIRT1 silenced cells under conditions of androgen stimulation and inhibition identified genes involved in cell cycle checkpoint and senescence as top pathways affected by SIRT1 loss of function. In silico analysis shows that the identified SIRT1-regulated targets are associated with disease aggressiveness and poor disease-free survival. Taken together these data demonstrate that (i) SIRT1 plays a contextual role during prostate pathogenesis by functioning as tumor suppressor during early stage and as a tumor promoter during late stage; (ii) SIRT1 inhibition suppresses tumor development and (iii) RES is a better preventive than therapeutic agent. Therefore, our findings offer promising avenues to develop SIRT1-regulated pathways as novel therapeutic targets to inhibit prostate cancer recurrence. Supported by CPRIT Training Grant RP 170345 (SH) and CPRIT RP 150166 (APK)

#5083

BSCHL-2046, a lead chalcone derivative from a newly synthesized library, is a potent neddylation inhibitor against the growth of prostate cancer cell lines.

Matthew A. Tippin, Dong Jun Fu, Liankun Song, Victor Pham, Xaolin Zi. _University of California, Irvine, Irvine, CA_.

Members of the chalcone family of molecules and their derivatives have been shown to inhibit various forms of cancer cells. In particular, Flavokawain A has been shown to inhibit prostate cancer cell lines. In order to capitalize on the bioactive nature of chalcone derivatives, we created a library of chalcone derivatives based on the scaffold of Flavokawain A but with an additional anti-cancer moiety. Here we have demonstrated that BSCHL-2046, the most potent member of the library, inhibits cell growth of prostate cancer cell lines including LNCaP, C4-2B, DU145, 22Rv1, and PC3 with IC50's ranging from 0.44µM-7.5µM. BSCHL-2046 inhibited Ubc12 neddylation in an in vitro assay at an IC50 of 1.5µM and demonstrated complete inhibition at a concentration of 6µM. BSCHL-2046 also reduced Nedd-8 conjugations to both Cullin-1 and Ubc12 in vivo in DU145 cells. The inhibition of neddylation by BSCHL-2046 resulted in an increased expression of p27 and CDT-1. Using molecular modeling, BSCHL-2046 was predicted to bind directly to the Cullin-RING E3 ligase complex. The Cellular Thermal Shift Assay (CETSA, an in vivo assay) demonstrated that BSCHL-2046 significantly decreased heat-induced protein degradation of E3 subunits, supporting BSCHL-2046 directly binding to the complex. Furthermore, BSCHL-2046 at a concentration of 500nM and 1.25µM displayed a 75% to complete inhibition of colony formation of C4-2B cells in soft agar. Taken together, these results demonstrate BSCHL-2046 and its structural analogs are potent neddylation inhibitors that deserve further investigation for the prevention and treatment of prostate cancer.

#5084

Epigenetic modifications involving reactivation of RECK inhibiting MMP-9 and MMP-2 in prostate cancer.

Eswar Shankar, Omair Iqbal, Natarajan Bhaskaran, Gauri Deb, Gregory T. MacLennan, Pingfu Fu, Sanjay Gupta. _Case Western Reserve Univ., Cleveland, OH_.

Metastasis is responsible for more than 90% of prostate cancer-associated mortality in the United States. One of the distinctive reason for metastasis has been the imbalance of matrix metalloproteinases (MMPs) as a result of reduced expression of RECK (reversion-inducing-cysteine-rich protein with Kazal motifs) a novel tumor suppressor. RECK has been shown to be a potent inhibitor of MMP-2 and MMP-9. Loss of RECK has been frequently identified in clinical prostate cancer specimens and prostate cancer cell lines, therefore mechanistic understanding of its loss and approaches to restore its expression would facilitate inhibition of metastasis. Green tea polyphenols (GTP) and its major constituent, epigallocatechin-3-gallate (EGCG) has been shown to suppress prostate cancer metastasis, however the mechanism has not been fully elucidated. We determined whether treatment with GTP has ability to restore induction of RECK and play a key role in suppressing invasiveness in prostate cancer. Human prostate cancer LNCaP tumor were implanted in the ventral prostate of athymic nude mice for 2 weeks followed by per-oral intake of GTP at 7.5 and 15.0 mg/kg body weight freshly prepared in 100µl PBS. Other groups were treated with DNA methyltransferase inhibitor-5-aza-2'-deoxycytidine (AZA), histone deacetylase inhibitor-trichostatin A (TSA) and histone methyltransferase inhibitor-3-Deazaneplanocin A (DZNep) individually at 0.1 mg/kg body weight intraperitoneally at alternate days/week; and combination of AZA+TSA and DZNep+TSA at similar doses and times. Treatment of mice with GTP resulted in marked decrease in tumor growth and its local invasion in dose dependent manner, compared to treatment with epigenetic inhibitors and their combination after 8 weeks of intervention. GTP treatment significantly reduced serum levels of MMP-2, MMP-9 and VEGF, compared to treatment with epigenetic inhibitors alone. Combination of AZA+TSA exhibited similar effect which was equivalent to the lower dose of GTP treatment. Furthermore, GTP treatment significantly reduced EZH2 and H3K27me3 and class I HDAC protein levels in tumors. GTP partially reversed RECK hypermethylation and significantly enhanced its expression in the tumor tissue. Similar findings were noted in cell culture where treatment of PC-3 and LNCaP cells with 20µM EGCG and 10µg/mL GTP for 72 h significantly induced RECK expression at mRNA and protein levels along with decrease invasiveness in these cells. Our findings suggest that induction of RECK is a key epigenetic event reactivated by GTP/EGCG that results in suppression of MMP-2/MMP-9, matrix degradation and angiogenesis to delay prostate cancer invasion and its subsequent progression.

#5085

Molecular analysis of chemopreventive effects of grape antioxidants resveratrol and quercetin in transgenic adenocarcinoma of the mouse prostate.

Chandra K. Singh, Mary A. Ndiaye, Gagan Chhabra, Charlotte A. Mintie, Nihal Ahmad. _University of Wisconsin-Madison, Madison, WI_.

Prostate cancer (PCa) is one of the most frequently diagnosed cancers of the male population and current treatments are insufficient to fully manage this neoplasm. Therefore, identification of novel mechanism-based approaches are needed for PCa management. Earlier, we demonstrated that a combination of the grape antioxidants resveratrol and quercetin impart superior anti-proliferative responses in multiple human PCa cell lines, as well as a significant anti-tumor response in transgenic adenocarcinoma of the mouse prostate (TRAMP) (Cancer Res 75(15 Suppl):2801). The rationale of the study was based on the fact that i) both resveratrol and quercetin are naturally present in several plants ii) quercetin improves bioavailability of resveratrol by inhibiting its sulfation, and iii) separately, both agents have shown potential for management of PCa in previously published studies. This study extended our previous work and determined the mechanisms of chemopreventive effects of resveratrol-quercetin combination employing a mouse PCa RT² Profiler PCR array that profiles 84 key PCa-related genes. For this, we employed tumor tissues generated in a chemoprevention protocol where TRAMP mice were given AIN76A diet supplemented with resveratrol (600 mg/kg), quercetin (60 mg/kg), or a combination of both. PCR array analysis found significant modulation (≥2-fold) in 14, 15, and 10 genes in the quercetin, resveratrol, and combination groups, respectively. To explore the involved gene networks using Ingenuity Pathway Analysis (IPA), we selected total 22 genes with ≥2-fold change in any one group and ≥1.5-fold change in other group(s). IPA analysis identified that resveratrol-quercetin modulated genes supported the cumulative actions of increased apoptosis, as well as inhibition of cell viability/proliferation, hyperplasia, vasculogenesis, and angiogenesis. Further, IPA predicted inhibition of major PCa promoting upstream signaling molecules viz. Pi3k, Vegf, Csf2, Ca2+, and Pth. This PCR array also identified decreased levels of Igf1, Egfr, Egr3, and Il6, which are known to support PCa progression, as well as found increased levels of Nkx3-1, which is a tumor suppressor in PCa. Furthermore, IPA exploration identified a gene network where decreased Igf1 emerged as a central regulatory player, interacting with most of the resveratrol-quercetin modulated genes. Additionally, employing IHC, immunoblot, and RT-qPCR analyses, we found marked decrease in the levels of cell proliferation markers Ki67 and PCNA, oxidative stress biomarker 4-HNE, EMT marker vimentin, and prosurvival marker Bcl2. These results suggest that this natural combination of grape polyphenols may be useful as a chemopreventive regimen for PCa. Further detailed studies including clinical trials are needed to determine the translational significance of our findings.

#5086

Acetyl-L-carnitine (ALCAR) inhibits angiogenesis, migration and macrophage recruitment in prostatic cancer cells.

Adriana Albini,1 Antonino Bruno,2 Denisa Baci,3 Matteo Gallazzi,3 Matilde Tramacere3. 1 _University of Milano-Bicocca, Monza, Italy;_ 2 _Science and Technology Pole (PST), MultiMedica, Milano, Italy;_ 3 _Science and Technology Pole (PST), MultiMedica, Milan, Italy_.

Many efforts have been addressed on the identification of novel active compounds from natural sources, endowed with anti-proliferative, anti-oxidant, chemopreventive properties, and their ability to target the tumour microenvironment (TME). Through metabolomics approaches we previously found that in serum samples from prostate cancer (PCa) patients, three carnitine family members were significantly decreased, suggesting a potential protective role of carnitine against PCa. Acetyl-L-carnitine(ALCAR) is an aceticacid ester of carnitine with high bioavailability and is involved in the transportof fatty acids across the inner mitochondrial membrane. We have showed that ALCAR was able reduce angiogenesis in vitro, acting on the VEGF/VEGFR2 and CXCR4/CXCL12 axes. We also found that ALCAR inhibits inflammatory angiogenesis in vivo, by reducing endothelial cells and macrophage recruitment in the matrigel plugs. Here we investigated the ability of ALCAR to interfere with key functional steps of prostate carcinogenesis and identified the molecular mechanisms involved. The effects of ALCAR on PCa cells were investigated in vitro by functional assays (adhesion, migration and invasion assays), molecular and biochemical approaches (RT-PCR, FACS, Byoplex and western blot). We found that ALCAR reduces apoptosis on PCa cells (PC3, Du-145, LNcap) and inhibits crucial tumorigenic steps such as adhesion, migration and invasion. We than confirmed the results from functional assays at molecular levels, and we found that ALCAR blocks CXCR4/CXCL12 axes, a key regulator of malignant migratory/aggressive phenotype. In accordance with our published paper, we confirmed the anti-angiogenic and ant-inflammatory properties of ALCAR, that we found to lower VEGF and CXCL8 production in PCa cell lines. Furthermore, we found a significantly reduced expression of inflammatory-related chemokines/cytokines involved macrophage recruitment such as CCL2, IL-6 and TNFα in PCa cells. Our results highlight the angio/chemopreventive and anti-inflammatory properties of ALCAR and allow the identification of multiple and overlapping mechanisms of action through which ALCAR inhibits PCa progression / metastasis. Our findings provide the rational for the employment of ALCAR, as a possible supplement for approaches of chemoprevention in subjects at high risk to develop cancer.

#5087

Carvedilol prevents skin cancer independently of its antioxidant property.

Mengbing Chen, Sherry Liang, Bradley Andresen, Ying Huang. _Western Univ. of Health Sciences, Pomona, CA_.

Carvedilol is an FDA approved β-blocker traditionally prescribed for cardiovascular diseases. Our previous work demonstrated that carvedilol has preventive activity against ultraviolet radiation (UVR)-induced skin damage, inflammation, and carcinogenesis. However, the photoprotective mechanism for carvedilol remains unknown. Interestingly, not all β-blockers have cancer preventive activity, suggesting that targeting the β-adrenergic receptors may not be responsible for carvedilol's anticancer activity. Since carvedilol has been shown as a potent antioxidant and such property is not shared by majority of other β-blockers (e.g., metoprolol), we hypothesized that the antioxidant property may be, in part, responsible for carvedilol's protective activity against photocarcinogenesis. The nontumorous mouse epidermal JB6 P+ cells were subjected to various doses of UVR exposure and H2O2 treatment to identify the optimal doses to examine protective activity. UVR and H2O2 dose-dependently reduced cell viability. 25mJ/cm2 UVR and 100 μM H2O2 reduced cell viability detected by Trypan blue exclusion assay to 58.1% and 60.6%, respectively. 25mJ/cm2 UVR and 300 μM H2O2 increased the formation of intracellular fluorescent 2',7'-dichlorofluorescein (DCF) fluorescence, a marker of reactive oxidative species (ROS) detected by flow cytometry, to 5- and 4-fold compared to non-treated control, respectively. The cells were treated before or after UVR and H2O2 exposure with metoprolol, carvedilol or 4-hydroxycarbazole (4-OHC). 4-OHC is an intermediate compound for carvedilol synthesis that possesses an identical UV absorption profile as carvedilol. Additionally, the carbazole moiety is likely responsible for the antioxidant activity of carvedilol. Carvedilol, but not metoprolol and 4-OHC, was able to inhibit EGF-induced JB6 transformation in soft agar. Carvedilol, but not metoprolol and 4-OHC, also attenuated UVR and H2O2 induced cell death. Carvedilol, but not 4-OHC, inhibited UVR-induced NF-κB and AP-1 promoter activity. In addition, unlike carvedilol, 4-OHC failed to prevent chronic UVR induced skin carcinogenesis in SKH-1 mice. However, 4-OHC and carvedilol similarly attenuated UVR- and H2O2-induced ROS formation in JB6 P+ cells, while metoprolol had no effect. These results indicate that the photoprotective effects of carvedilol are primarily due to an unknown mechanism that is independent of β-adrenergic receptors and reactive oxygen species scavenging.

#5088

Silibinin inhibits UVB-induced promotion and progression of microscopic basal cell carcinoma formation via targeting bone morphogenic protein 2.

Cynthia M. Rigby, Gagan Deep, Anil K. Jain, David J. Orlicky, Chapla Agarwal, Komal Raina, Rajesh Agarwal. _Univ. of Colorado Denver-AMC, Aurora, CO_.

Non-melanoma skin cancers (NMSCs) account for about half of all malignancies diagnosed annually in the United States. Around 80% of NMSCs are basal cell carcinoma (BCC) and 20% are squamous cell carcinoma (SCC). Whereas the efficacy of several chemopreventive agents has been examined and reported against both BCC and SCC, a majority of these studies have focused on the test agent's activity in a long-term setting to determine the amount of tumors formed. Notably, the studies evaluating the efficacy of chemopreventive agents during early stage/s of BCC development are lacking. Accordingly, utilizing the well-established patched (Ptch)+/- mouse model of UVB-induced BCC formation, we excised skin samples from UVB exposed mice prior to tumor formation to study the promotion/progression of BCC and to determine the target/s of silibinin, a well-known skin cancer (SCC) chemopreventive agent, in tumor growth inhibition. Ptch+/- and Ptch+/+ mice were irradiated with UVB 240mJ/cm2 3 times per week with or without topically applied silibinin 5 times per week. As early as one month, we found that UVB exposure significantly increased the number of mast cells in Ptch+/- mice by about 48% (P<0.05), which was completely inhibited (to control levels) by silibinin topical treatments. In Ptch+/+ mice, which do not develop BCC tumors, we did not observe any increase in mast cells following UVB exposure, suggesting this could be a specific pathway in the development of BCC. To decipher the molecular mechanism of these findings, we performed a PCR profiler array analysis of several genes involved in signal transduction pathways which showed strong differences between Ptch+/+ and Ptch+/- mice that were unexposed, UVB irradiated, and silibinin treated. Most notably, following UVB exposure for 1 month, in Ptch+/- mice the expression of Bone morphogenetic protein 2 (BMP-2), Hairy/enhancer-of-split related with YRPW motif 1 (Hey1), and Inhibitor of DNA binding 1 (Id1) was significantly upregulated when compared to Ptch+/+ mice. Additional studies focusing on BMP-2 found that silibinin strongly inhibits UVB-induced expression of BMP-2 in Ptch+/- mouse skin. Consistent with these results, we also found that silibinin strongly attenuates UVB-induced BMP-2 expression and DNA damage in Ptch+/- mouse skin ex vivo. Regarding BCC formation, silibinin treatment inhibited UVB-induced microscopic BCC formation in Ptch+/- mice; microscopic tumor number and size were reduced by 73% and 84%, respectively. Together, our results suggest a possible role of BMP-2 in early stages of BCC development and that silibinin plausibly acts through BMP-2 to inhibit microscopic BCC formation. In conclusion, our previous studies in SCC models and current findings in BCC model suggest that silibinin could be a multi-target agent capable of being a chemopreventive agent for both types of NMSCs.

#5089

Physicochemical properties of exogenous molecules correlated with their biological efficacy as protectors against inflammation and carcinogenesis.

René Victor V. Bensasson. _UMR 7245 - CNRS/MNHN, Paris, France_.

A large number of molecules referred to here as M, several of which are extracted from plants, belong to different chemical classes and exhibit chemoprotective activity against inflammation and carcinogenesis in rodents and in vitro cell culture. The aim of this article is to review and analyse our investigations on QSAR linear correlations observed between physicochemical parameters of the tested molecules M and their biological efficacies measured in vivo and/or in vitro. The physico-chemical parameters characterize the energy required to oxidise M into M•+, E(M•+/M) and are determined theoretically using mainly semiempirical (AM1) and density functional theory (DFT) quantum mechanical methods. The biological efficacies of the molecules M are related to (i) quenching inflammation, a critical root of tumour progression, (ii) inhibiting angiogenesis, required for invasive tumour growth, (iii) inducing a cancer-protective enzyme NAD(P)H-quinone oxidoreductase, (iv) inhibiting the action of topoisomerases involved in DNA replication and transcription, known as targets for anticancer drugs (v) inhibiting the abnormal proliferation of cells in the forestomach of mice, (vi) inhibiting the onset and progression of aberrant crypt foci in the rat colon. Our in silico theoretical evaluations of the physico-chemical parameters of M correlated linearly with biological activities should orient the rational design of new congeners with greater potency and should reduce the expensive use of in vivo and in vitro bioassays.

#5090

Epitope mapping of mouse TERT for vaccine development.

Yurong Song,1 Jason Marshall,1 Travis Kerr,1 Ligia Pinto,1 Shizuko Sei,2 Robert Shoemaker2. 1 _Leidos Biomedical Research, Inc., Frederick, MD;_ 2 _National Cancer Institute, Bethesda, MD_.

Telomerase reverse transcriptase (TERT) is one of two essential components of telomerase, an enzyme complex that generates and maintains telomeres. Telomerase is expressed mainly in embryonic and adult stem cells. Telomere biology has recently been implicated in the pathogenesis of a variety of diseases, and mutations in telomerase components result in a predisposition to solid malignancies. More recent findings show that TERT is not only expressed in late stage primary tumor cells and metastatic cells, but also expressed in incipient cancer stem cells and/or tumor-initiating cells, indicating that TERT has essential roles at every stage of tumorigenesis. However, the clinical benefit of a TERT (hTERT)-targeting vaccine, given as a single agent or in combination with chemotherapeutic agents, has been limited in patients with advanced cancer, possibly due to insufficient epitope coverage as well as tumor-induced immune suppression. Thus, targeting TERT in tumor-initiating cells in early stage lesions may be more effective in preventing cancer development and progression. To develop cancer preventive TERT vaccines, novel immunogenic epitopes must be first identified and evaluated in a relevant preclinical model of tumorigenesis. The goal of this project is to identify the epitopes of mouse TERT with high immunogenicity using MHC I-peptide binding assay by flow cytometry analysis. The in vitro peptide-induced MHC Class I stabilization assay was carried out using RMA/S cell line, which was derived from a Rauscher leukemia virus-induced C57BL/6 T cell lymphoma and is deficient in transporter associated with antigen processing (TAP). Results of screening the overlapping peptide library will be presented.

Funded by NCI Contract No. HHSN261200800001E

#5091

**Inhibition of mutagenicity of 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine ( PhIP) by aqueous extract (crude) and organic extract (pulegone) of** Calamintha nepeta. **.**

Yanlingxue Wan, Ryan Hayes, Joshua Li, Kristin Ferrer, Padma T. Uppula, Brian Yuen Yau Wong. _Andrews Univ., Berrien Springs, MI_.

Inhibition of mutagenicity of 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) by aqueous extract (crude) and organic extract (pulegone) of Calamintha nepeta.

Calamintha nepeta (CN) is an aromatic mint used as a traditional medicinal herb in Europe and the Mediterranean for its broad range of biological activities; these include anti-inflammatory, antioxidant, antimicrobial, anti-ulcer, insecticidal, and cancer-preventive properties. Previous studies have revealed the apoptotic and cancer-preventing attributes of its major organic fractions: pulegone (PUL, 2760 ppm), D-limonene (385 ppm), and menthone (590 ppm). PUL (6.4 - 800 mg/plate) was not mutagenic in Salmonella typhimurium TA97, TA98, TA100, TA1535, or TA1537 regardless of S9 metabolic activation. When cooking at high temperatures, creatine, sugars, and amino acids begin to produce 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), which yielded positive results in the Ames mutagenicity tests. PhIP is one of the most abundant heterocyclic amines (HCAs) in cooked meat and the U.S. Department of Health and Human Services' National Toxicology Program has denounced it as "reasonably anticipated to be a human carcinogen". Thus, cancer of the prostate, breast, lung, esophagus, stomach, and colorectal may be related to high intake and high exposure to PhIP. This study assessed and compared the effects of total aqueous and organic extracts of CN on PhIP-induced mutagenesis using TA98 as the bacterial tester strain and rat liver 9000 x g supernatant (S9) as the metabolic activation system. Our results showed that both the crude aqueous extract and organic essential oil extract (PUL) of the herb significantly inhibited the mutagenicity of PhIP (both 0.1 and 1 µg/plate), bioactivated by S9 (800 mg/plate), in a dose-response fashion (p < 0.05). The percent inhibition of revertant formation for these two extracts against 0.1 µg/plate of PhIP were: 32%, 74%, 79%, 84%, and 90% regarding the total aqueous extract (0.1, 0.5, 1.0, 1.5, and 3 mg/plate) and 28%, 36%, 64%, 75%, and 97% for the PUL essential oil of the organic extract (1.0, 10, 50, 100, and 200 µg/plate). Both the aqueous and organic extracts revealed the same trend of significance (p < 0.05) concerning percent inhibition of revertant formation against PhIP (1 µg/plate) in a dose-response fashion. The organic fraction (PUL) maintained a stronger antimutagenic effect in comparison to the total aqueous effect. These results suggest that both aqueous crude extract and organic PUL extract of CN contain antimutagenic phytochemicals concerning PhIP-induced mutagenicity. Further study of their modulatory effects on metabolism and the DNA binding of PhIP is warranted to reveal the potential antimutagenicity mechanism and their chemopreventive properties against PhIP and potentially against other HCAs.

#5092

Participant satisfaction with cancer chemoprevention clinical trials: The Mayo Clinic Cancer Prevention Network (CPN) experience.

David Zahrieh,1 Ryan P. McMurray,1 Nathan R. Foster,1 Paul J. Limburg,2 Sumithra J. Mandrekar1. 1 _Mayo Clinic, Rochester, MN;_ 2 _Mayo Clinic College of Medicine, Rochester, MN_.

INTRODUCTION Participating in a clinical trial is a personal choice and an individual experience. Several barriers to recruitment of high risk men and women in chemoprevention trials have been documented; however, to our knowledge, there are no existing reports on participant satisfaction in such trials. Therefore, our aim was to describe and evaluate participants' perception of their experience with participating in chemoprevention trials conducted by CPN by summarizing the 5-item "Was It Worth It" (WIWI) tool for each trial and across the trials, and according to race/ethnicity, sex, age, site, and organ of primary interest.

METHODS Because participants who participate in chemoprevention trials tend to be at increased cancer-risk, but otherwise healthy, the WIWI tool can help answer simple questions about participants' assessment of whether or not participation in such a trial was worthwhile. The WIWI tool was consistently administered at the end of the intervention period in each trial conducted by CPN; since 2003, 7 trials have completed the study intervention period and are included in the results reported here.

RESULTS The scientific focus of the 7 trials targeted 5 organs: breast, colorectum, esophageal, hepatobiliary, and lung. Of the 485 participants registered to these 7 trials, 419 (86.4%) were non-Hispanic white, 19 (3.9%) non-Hispanic black, 32 (6.6%) Hispanic, 14 (2.9%) other race/ethnicity, and 1 (0.2%) unknown race/ethnicity; there were 120 (24.7%) females. In total, 446 (92.0%) participants completed the WIWI questionnaire at the end of their respective intervention period. Across the trials, 93% [95% CI] [90.6%, 95.4%] of the participants indicated that participation was worthwhile and 92% [89.5%, 94.5%] reported that they would participate again. Additionally, 414 (92.8%) participants indicated that they would recommend participating in the current research study to others, while 10 (2.3%) indicated that they would not and 22 (4.9%) were uncertain. Furthermore, several participants provided actionable feedback to the question "If there was one thing that could have been done to improve your experience in this research study, what would it be?" Participant feedback on overall changes in quality of life and on their expectations with participation in the trial was positive but more nuanced, varying according to trial and participant composition.

CONCLUSION In this dataset of chemoprevention trial participants enrolled through the CPN consortium, highly favorable feedback was evident with respect to the overall study experience, suggesting that the recruitment and retention methods employed are facilitating success. These data can be used to help advance the NCI accrual quality improvement program in developing/enhancing strategies to improve accrual, retention, and adherence as well as enhance participant diversity in subsequent trials.

## BIOINFORMATICS AND SYSTEMS BIOLOGY

### Methods and Tools for Cancer Analysis

#5093

Clinical-grade NGS analytic platform with explicit negative calling to improve variant reporting across different laboratory protocols: Accuracy study.

Edward P. Tang, Charlie C. Kim, Kristine C. Mechem, Glenda G. Anderson. _Farsight Genome Systems, Sunnyvale, CA_.

Introduction: Research-grade bioinformatic pipelines share a common workflow with a logical shortcut that infers where no variant was called, no variant exists. The complexity of NGS protocols makes this inference clinically unsound. Incomplete enrichment, or a sample with deletions in the region of interest can result in false negative errors. Clinical-grade NGS analytics should explicitly evaluate each region of clinical interest to distinguish regions with no informative variants from those with too little evidence to make a call. An ideal platform would produce reliable results from sequencers and library prep in clinical use without protocol-specific tuning. This study evaluates the performance of a novel Clinical-grade Analytic Platform designed to meet these challenges.

Study Design: 189 samples from the NA12878 cell line, processed by 13 different laboratories, using 18 different protocols and 11 different instruments were sourced as raw sequencing data from public repositories. Each sample file was run through the Clinical-grade Analytic Platform to produce a cVCF result, a detailed accounting of variants present, regions with no variant present and regions with insufficient coverage across 14,626 exons in the 1043-gene PanCancer TestPanel. High Confidence TRUTH, obtained from the GIAB consortium, established that NA12878 DNA contains 598 variants and 14,230 exons with no variant across the 1043 genes. The cVCFs were compared to TRUTH to quantify performance.

Results: The Clinical-Grade NGS Analytic Platform ran all 189 samples without protocol or laboratory-specific tuning, with an overall sensitivity of 95% and specificity of 99%. This performance includes samples with average read depth ranging from 18x to 10,000x, although regions with local read depths above 30x performed better. Hybridization capture-based methods generally outperformed amplicon-based methods at all read depths. Full presentation will discuss performance stratified by instrument, library prep method, local read depth, and variant type.

Conclusion: The Clinical-grade Analytic Platform, Farsight Correo, showed strong performance across a broad range of read depths, instruments and library prep methods. The platform's unique ability to distinguish between "No Variant Present" and "Insufficient Coverage" contributed significantly to this performance. Robust, cross-protocol performance is clinically valuable. It can enable clinical laboratories to expand or change NGS protocols without replacing analytics. It can enable companion diagnostic programs to include multiple sequencing protocols and partners. And finally, explicit negative variant calling can help AI and machine learning mirror the underlying biology; integrating variant presence and absence into new pathway-based models to significantly advance genomics-guided medicine.

#5094

SCOPE: A normalization and copy number estimation method for single-cell DNA sequencing.

Rujin Wang, Danyu Lin, Yuchao Jiang. _University of North Carolina at Chapel Hill, Chapel Hill, NC_.

Whole genome single-cell DNA sequencing (scDNA-seq) enables characterization of copy number profiles at the cellular level. This circumvents the averaging effects associated with bulk-tissue sequencing and has increased resolution yet decreased ambiguity in deconvolving cancer subclones and elucidating cancer evolutionary history. ScDNA-seq data is, however, sparse, noisy, and highly variable even within a homogeneous cell population, due to the biases and artifacts that are introduced during the library preparation and sequencing procedure. Here, we propose SCOPE, a normalization and copy number estimation method for scDNA-seq data. The distinguishing features of SCOPE include: (i) utilization of cell-specific Gini coefficients for quality controls and for identification of normal/diploid cells, which are further used as negative control samples in a Poisson latent factor model for normalization; (ii) modeling of GC content bias using an expectation-maximization algorithm embedded in the Poisson generalized linear models, which accounts for the different copy number states along the genome; (iii) a cross-sample iterative segmentation procedure to identify breakpoints that are shared across cells from the same genetic background. We evaluate performance of SCOPE on real scDNA-seq data sets from cancer genomic studies. Compared to existing methods, SCOPE more accurately estimates subclonal copy number aberrations and is shown to have higher correlation with array-based copy number profiles of purified bulk samples from the same patient. We further demonstrate SCOPE on three recently released data sets using the 10X Genomics single-cell CNV pipeline and show that it can reliably recover 1% of the cancer cells from a background of normal and recover the cancer subclonal compositions of 10,000 breast cancer cells.

#5095

Statistical modeling of transcriptional regulatory states in single-cell RNA-Seq data of tumor and infiltrated immune cells.

Changlin Wan, Wennan Chang, Xiaoyu Lu, Yifan Sun, Kaman So, Sha Cao, Xiongbin Lu, Chi Zhang. _Indiana University, Indianapolis, IN_.

Single-cell RNA-seq of tumor tissue has greatly strengthened our understanding of tumor development as well as the heterogeneous tissue micro-environment. Many models are dedicated to detect deferentially expressed genes among single cells of different conditions, or to discover (novel) sub-cell types via various clustering methods. However, current statistical models often neglect the zero/low expressions, and use one single modality to fit the rest of the non-zero ones. We believe that zero and low expressions are observed partly due to limited experimental resolution; on the other hand, occasionally, low expressions are falsely observed as non-zero due to the existence of incompletely degraded mRNA. In addition, the severe heterogeneity and fast dynamics of single cells makes it invalid to fit their expressions in one single modality. Here, we propose a left truncated mixture Gaussian (LTMG) distribution to uncover the multimodality in single cells' gene expression while properly handling the zero and low expressions. LTMG is motivated by the kinetic relationships among the mRNA abundance, transcriptional regulatory signals (TRSs), and mRNA metabolism in a cell. Specifically, observations of a gene's expression are fitted as mixture Gaussian distributions which assumes zero and low expressions as left censored data. By treating each mixing component as one state of the transcriptional regulators, we combined our LTMG model and our in-house bi-clustering algorithm to infer modules of genes co-regulated by certain (unknown) transcriptional regulatory signal, which is only shared by a subset of all the single cells. Application of the method to three high quality single-cell RNA-seq data sets of T cells extracted from liver, colon and lung cancer micro-environments, identified varied transcriptional regulations specific to subclasses of T cells.

#5096

Exploring novel prognostic genetic markers using elastic-net regularization and association rule mining in HER2-negative metastatic gastric cancer patients.

Sejung Park,1 Woo Sun Kwon,2 Inhye Jeong,3 Hyun Jeong Kim,2 Jong Soo Hong,4 Jaeyoung Kim,5 Jingmin Che,6 Chung Mo Nam,7 Hyun Cheol Chung,8 Sun Young Rha8. 1 _Songdang Institute for Cancer Research, Department of Biostatistics and Computing, Yonsei University College of Medicine, Seoul, Republic of Korea;_ 2 _Songdang Institute for Cancer Research, Yonsei University College of Medicine, Seoul, Republic of Korea;_ 3 _Songdang Institute for Cancer Research, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Republic of Korea;_ 4 _Department of Biomedical Systems Informatics, Department of Biostatistics and Computing, Yonsei University College of Medicine, Seoul, Republic of Korea;_ 5 _Bio-Center, Daewoong Pharmaceutical Co.,Ltd., Yongin, Republic of Korea;_ 6 _Development of Medical Research, Bio-Age Inc, Seoul, Republic of Korea;_ 7 _Department of Preventive Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea;_ 8 _Songdang Institute for Cancer Research, Brain Korea 21 PLUS Project for Medical Science, Division of Medical Oncology, Department of Internal Medicine, Yonsei Cancer Center, Yonsei University Health System, Seoul, Republic of Korea_.

Trastuzumab is the standard of care and has been shown to prolong survival of HER2-positive gastric cancer (GC) patients. However, HER2-positive is present only in about 20% of GC patients, there is a need to explore prognostic genetic markers for 80% of HER2-negative GC patients. The aim of this study was to identify the prognostic markers by elastic-net regularization and association rule mining and evaluate prognosis with selected genetic markers in HER2-negative metastatic GC (mGC) patients. A total of 143 HER2-negative mGC patients who have been treated with first-line chemotherapy in Yonsei Cancer Center, Korea between 2011 and 2018 were retrospectively assessed. Patients were categorized into two groups (responder or non-responder) based on the median progression-free survival (PFS: 7.8 months (95% confidence interval (CI), 6.3 - 8.9)) of the first-line chemotherapy. Targeted sequencing data of patients was obtained from CancerSCANTM and CANCER MASTER, and were analyzed with common variants of two panels. We used combined tools of elastic-net with 1,000 times of re-sampling bootstrap method and association rule mining to filter out and select critical genetic markers distinguishing responder or non-responder groups. To assess prognosis significance of selected genetic markers, we developed prognostic score based on the sum of sub-scores. If the right-hand-side of the rules signifies a responder, the patient is given a value of 0 when applying to the rule and 1 when they do not. PFS were determined by the Kaplan-Meier method and compared using the log-rank test. The 85 candidate genetic markers were selected by elastic-net with 60% of variable inclusion probability. Among the 85 candidate genetic markers, 12 genetic markers involved in 26 rules with lift greater than 1.5 were related to the responder group from association rule mining. The selected 12 genetic markers are included as follows: EGFR/KRAS normal copy number, APOBEC3B deletion, FGFR2/MLH1/PHLPP2/PARP4/SH2B3/BARD1/ALK/GNAS wild type, and NCOA3 mutation. The prognostic scores generated by selected genetic markers were 3, 4 and 5 respectively, and patients with higher scores had significantly poor prognosis than those with lower scores. Median PFS of each score group were 9.8 months (95% CI, 8.8 - 11.6), 7.5 months (95% CI, 5.7 - 8.6), and 5.2 months (95% CI, 1.2 - 6.7) for patients with consecutive scores of 3, 4 and 5 (P=0.017). The combined tool of elastic-net and association rule mining effectively found prognostic genetic markers and can be applied to develop the prognostic scoring system. The results of novel combined tool identified 12 multiple prognostic genetic markers and demonstrated significant survival differences in prognostic scores among HER2-negative mGC patients, although further studies are required.

#5097

Clinical trial outcomes for drug combination therapies can be predicted by modeling independent action using cancer cell line screens.

Alexander Ling,1 R. Stephanie Huang2. 1 _UChicago/UMN, Saint Paul, MN;_ 2 _University of Minnesota, Saint Paul, MN_.

Background:

In many cancer settings, it is evident that combination drug therapies show increased efficacy compared to monotherapies. However, the development of new combination therapies is slowed by the fact that it is infeasible to experimentally evaluate the vast number of possible drug combinations. In an effort to overcome this problem, we developed a computational method which uses monotherapy screening data in cell lines to estimate the efficacy of combinations of two or more chemotherapy drugs. When validated against published phase 3 clinical cancer trial results, our method is capable of predicting clinical trial success or failure with a high degree of sensitivity and specificity.

Methods and Results:

We processed existing high-throughput in vitro drug screening datasets (i.e. GDSC and CTRPv2) to obtain fitted dose response curves for more than 600,000 drug-cell line pairs, including data for 695 unique compounds and 1347 unique cell lines. We then used this dataset to develop a method of estimating the efficacy of combinations of two or more drugs. While previous efforts to predict drug combination efficacy have largely focused on dose-agnostic estimates of drug synergy, we modeled dose-dependent estimates of independent drug action, where each patient treated with a drug combination only derives benefit from the single drug in the combination to which they are most sensitive. For method validation, we systematically searched clinicaltrials.gov to identify completed, phase 3 cancer clinical trials that evaluated drug combinations for which we could estimate efficacy and which had at least 50 patients per trial arm. Of the >9,000 clinical trials considered, 37 trials (11 successful and 26 unsuccessful) met our inclusion criteria.

Using clinically determined plasma concentrations for each drug, we then predicted relative efficacy for each of the experimental drug combinations versus the control group therapies in these trials. These predictions were clustered into two groups (predicted high benefit vs low benefit) using k-means clustering, and these clusters were compared to whether or not the clinical trials were considered a success by the original study investigators. Our method correctly classified 84% of the trials, with a sensitivity of 73% and a specificity of 88%. Furthermore, efficacy predictions for combinations containing erlotinib were dependent on EGFR mutation status, suggesting that this method may be useful for identifying molecular subtypes which are sensitive to a given combination therapy.

Conclusion:

Clinically meaningful predictions of drug combination efficacy can be generated from single-agent, in vitro drug screens under the assumption of independent drug action. This enables efficacy predictions to be made for >150,000 2-drug combinations and millions of 3- and 4-drug combinations using the existing GDSC and CTRPv2 datasets.

#5098

In silico **analysis of the membrane targeting mechanism of the pleckstrin homology domains within the oncogenic Grb2-associated-binding protein family.**

Antonio Lopez, Ezza Awan, Areeba Zaheer, Shaneen Singh. _City Univ. of New York Brooklyn College, Brooklyn, NY_.

Members of the metazoan Grb2-associated-binding (GAB) family of proteins are crucial regulatory elements of receptor tyrosine kinase (RTK) signaling pathways. These scaffold proteins link and amplify plasma membrane receptor signaling to downstream targets by staging protein-protein interactions. Aberrant GAB expression is associated with improper growth and development, and several members of the GAB family have been implicated as oncogenes. GAB1, GAB2, and GAB3 are overexpressed in various cancers including lung, breast, colorectal, nervous system, and ovarian cancers, along with certain leukemias and melanomas. Recent studies suggest a direct relationship between GAB expression levels and tumorigenesis, where induced or silenced expression respectively accelerated or inhibited tumor proliferation. Silencing of GAB1 in colorectal cancers and GAB2 in glioblastoma via miRNA, and GAB3 in gliomas via siRNA inhibits tumor proliferation indicating that GAB proteins can be potentially effective targets in cancer therapy. The most conserved structural moiety in all members of the GAB family is an N-terminally located pleckstrin homology (PH domain) that directs subcellular localization and is crucial for effective protein function. Given the challenges facing RNA-based cancer therapies, more practical therapeutic avenues may be found in disrupting or inhibiting proper PH domain function; however, the mechanism of membrane targeting of all GABs remains to be elucidated. To this end, we have performed a comprehensive computational analysis of the six known GAB family members - chordate GAB1, GAB2, GAB3, and GAB4; Caenorhabditis elegans Suppressor of Clear (SOC1); Drosophila melanogaster Daughter of Sevenless (DOS) and their respective homologs to establish intra-familiar relationships via sequence analysis. Furthermore, in silico homology-based and ab initio modeling techniques were employed to propose suitable structural models of the PH domains and confirm the structural conservation of the fold. The refined theoretical models were docked against phosphoinositide head-groups to establish binding affinities and the mechanism of GAB PH domain localization. Our results suggest that non-specific electrostatics may be a key component for membrane targeting of GAB PH domains acting in concert with interaction through specific conserved residues and reveal an alternative binding pocket in addition to the canonical binding site. Our study is the first step in rational design of potential therapies based on targeting GAB function via the PH domain.

#5099

Evaluation of two next-generation sequencing platforms for genomic analysis in circulating tumor cells.

Chi Shan Candy Lam, Wei Dai, Josephine Mun Yee Ko, Maria Lung. _University of Hong Kong, Hong Kong, Hong Kong_.

Background: Circulating tumor cells (CTCs) are released from primary or metastatic tumors into the cancer patient's bloodstream and may establish secondary tumors in distant body sites. Their frequencies are thought to have great prognostic impact and their mutation signatures may yield pointers in treatment decisions. However, CTCs are extremely rare in the blood and are challenging to sequence. Current next-generation-sequencing (NGS) technologies and analysis procedures must be carefully strategized to maximize sensitivity, while maintaining specificity and controlling false discovery rates.

Aim of the study: This study aims to establish a robust and sensitive NGS bench work and analysis pipeline for genetic analysis of CTCs with good positive predictive value (PPV) and low false discovery rate (FDR), which can ultimately be translated into clinical application. We will compare and evaluate the performance of two NGS platforms for CTC analysis in the Hong Kong cancer patients.

Methods: This study will streamline the sequencing and analyses of two NGS approaches, i.e. amplicon-based or hybridization capture-based, for their application for CTC analyses. Firstly, a cancer cell line with known mutations will be spiked into normal blood cells at different percentages and sequenced with both approaches independently. The amplicon-based platform covers 60 cancer relevant and actionable genes, while the customized capture kit for the hybridization-based platform includes 245 cancer relevant genes curated in our group. Both platform's sensitivity, specificity, PPV and FDR will be evaluated. Secondly, CTCs from several cancer types with high incidence in Asia will be enriched by size, enumerated for comparative analysis, and sequenced. Sequencing results will be used to refine the analysis pipelines for both platforms. Results of sequencing analyses, including potential druggable targets and prognostic markers identified, will be determined.

Results: We successfully detected over 80% of known mutations at as low as 1.25% CTC purity, and with FDRs below 10% at 2.5% CTC purity by the hybridization-based method. We detected damaging missense, stopgain, or splicing mutations with the amplification-based platform in all 19 samples sequenced so far. More than 80% of blood samples from cancer patients had at least 5 CTCs detected. Preliminary comparison of the sequencing results of one high CTC purity sample showed that more than 50% of variants detected by this platform was also identified by hybridization-based sequencing.

Conclusion: Our study sheds light on studying the genomic profiles of the rare CTCs for clinical application and identifying the potential actionable targets in Asian cancer patients.

Acknowledgements: This study is supported by the HKU Platform Technology Fund and the Hong Kong Research Grants Council Area of Excellence grant AoE/M-06/08 to MLL.

#5100

Optimization of amplicon design for polymerase chain reaction.

Bolong He, Hao Qin, Fugen Li, Hanyan Yang. _3DMed, Shanghai, China_.

Amplicon design is critical important in DNA sequencing technology. Previously the amplicon designer uses exhaustive trial and error method, relying on the individual experience to compare the cost of different amplicon combinations. However, optimization of amplicon design has remained challenging due to the lack of a systematic method. As the magnitude of target DNA region and amplicon combination increases, the complexity of manual calculation and judgment will grow exponentially. In this study, our aim was to design a mathematical model to minimize the cost of amplicon combination. For given amplicon design problem, a linear integer programming model is constructed according to the optimization target and the restrictions. Let's denote our target regions by R_1⋯ R_M, and amplicon by A_1,⋯, A_N, and conflict value c_ij for the amplicon pair A_i and A_j. The model's restriction is to find a set of amplicons that could cover the target regions as much as possible, and for each R_i we have at most two A_j which will cover it simultaneously. The model's optimization target is to minimize the total conflict value. The model is implemented by Python programming and uses Gurobi optimization software. In a project to design amplicon for DNA sequencing, the target regions consist of about 90,000 bits, and there are about 35,000 amplicon available. The above optimization method running on the personal computer with 8GB and 2.2GHz Intel i5-5200 CPU, take about 24 hours and produce total conflict value of 1374. The best trial and error practice took more than one week and produced total conflict value of 4527. Optimization of amplicon design for polymerase chain reaction proves to be more precise and efficient than the exhaustive trial and error method. Using optimization method will help standardize the product design for DNA sequencing.

#5101

CAB-CAR-T: The prioritization of cell surface protein targets for conditionally active biologics to treat all solid tumors.

Hiba A. Shaban, Laurence Jadin, James Onuffer, Farzad Haerizadeh, Alissa Kerner, Gregory Schreiber, Gregory Frost, Scooter Willis. _F1 Oncology, West Palm Beach, FL_.

Introduction

Proteins on the surface of cancer cells represent viable targets for conditionally active biologic (CAB) CAR-T therapy that only work in the tumor microenvironment. By modeling properties of cell surface proteins from TCGA datasets we seek to identify optimal targets across all TCGA cancer cohorts that when used in CAB-CAR-T therapies will provide the greatest number of treatment options for patients across all cancer malignancies.

Method

Cell surface proteins(n=1086) were identified and meta data specific to each gene was organized from a variety of public databases for data modeling. Various approaches based on ideal CAB-CAR-T properties are used to rank the cell surface proteins as therapeutic targets. The 31 TCGA cohorts representing the most comprehensive collection of genomic profiled tumor samples and outcomes of all cancers is used to rank cell surface proteins for the percentage of patients in each cohort who would be eligible for treatment based on predetermined mRNA cutoffs.

Various approaches were used to filter the ranked list based on ideal CAB-CAR-T properties: known antibodies that can be used for initial CAB-CAR-T development, non-receptor as a static protein structure, highly expressed in CCLE indicating mRNA expression is a feature of cancer cell lines and low expression in critical tissues like Heart, Lung, Liver, Muscle etc. Different ranked lists of cell surface proteins were used to determine the number of CAB-CAR-T products required to treat 90% of patients in TCGA cohorts. A patient with the highest mRNA expression above the mean plus one standard deviation as determine across all TCGA samples is assigned to that specific protein biomarker as eligible for treatment and removed from the list of patients still to be treated. A bootstrap p-value for the ranked lists was determined by calculating the minimum number of randomly selected cell surface proteins that would give 90% coverage of the TCGA cohort.

Results

It was shown that it is reasonable to find ranked list of genes with high mRNA expression in TCGA and minimum expression in off-target critical tissue that 5-7 CAB-CAR-T products could be used to treat 90% of TCGA patients. To achieve 100% treatment coverage each additional CAB-CAR-T product added to the list had minimum inclusion of additional patients for treatment.

Conclusions

By modeling various properties of cell surface proteins to establish future development of CAB-CAR-T products it is reasonable to expect 90% patient coverage with 10 distinct therapies. Further modeling will be performed to exam combination therapies where tumor heterogeneity is an important criteria for the ranked list to have efficacy, with a goal of maximizing complete responses (CRs) and minimize the chance of relapse in the future.

#5102

Deep learning method for the classification of CNV based on the next generation target sequencing.

Jidong Lang, Geng Tian. _Geneis (Beijing) Co.Ltd, Beijing, China_.

Background: Copy number variations (CNVs) are of great importance to many cancers. Recently, the next-generation sequencing has made detecting CNV by sequencing possible. Traditional CNV determination methods involve expensive experiments, which is costly. Recently, several algorithms were proposed the detection of CNV, but none of them could utilize only the coverage information at a specific gene to make the determination of CNV at the gene.

Methods: We collected 1132 samples with 51 mesenchymal-epithelial transition factor (MET) CNV samples and 1081 samples with no CNVs. We split the exons of MET to multiple 50-bp windows with the stride of 40-bp. At each window the supporting reads were counted, and reads numbers at each exons were piled up, generating a matrix. The training set containing 38 CNV-positive matrices and 38 CNV-negative matrices were extracted from all the matrices, leaving the others as the test set. The deep learning network, convolutional neural network (CNN) were applied to distinguish between the CNV-positive matrices and the CNV-negative matrices. For comparison, logistic regression model was also applied for the classification task.

Results: The training set were split into 5 pieces containing 15 samples each and 5-fold cross validation were run for the CNN or the logistic regression model. After running 10 5-fold cross validation, we got an accuracy of 85.73 ± 9.24% for CNN and the average accuracy of 87.07 ± 8.99% for the logistic regression model. We chose 3 best models in both CNN and logistic regression models for the determination of the test set. The CNN models gained the accuracy of 71.84 ± 2.19% while the logistic regression models gained the accuracy of 69.70 ± 2.56%. Furthermore, the Area Under Curve (AUC) of the CNN models were 0.6335 ± 0.0124, higher than that of the logistic regression models which was 0.5666 ± 0.0115.

Conclusions: We proposed a method to determine the samples with CNV by using the convolutional neural network with only the coverage information within the specific region. It provides the possibility of CNV detection for cancer patients using only the next generation sequencing data.

#5103

The dark cancer kinome - untapped opportunities for the development of novel drugs.

Derek J. Essegian, Rimpi Khurana, Vasileios Stathias, Valery Chavez, Jaime R. Merchan, Stephan Schürer. _Univ. of Miami Miller School of Medicine, Miami, FL_.

Kinases are firmly established drug targets in cancer. There are currently 44 FDA approved kinase drug and hundreds of compounds are in clinical development. However, less than 10% of the Kinome is currently targeted and a large proportion is considered understudied by the NIH Illuminating the Druggable Genome Program (https://druggablegenome.net/). No small molecule inhibitors are known for these "dark" proteins, yet many may be opportune novel cancer targets.We developed a computational pipeline to identify and prioritize understudied kinases as cancer drug targets. We analyzed the complete set of tumors in The Cancer Genome Atlas (TCGA). For 33 different cancers we performed differential expression analysis and identified 39 dark kinases that exhibit significant upregulation in at least four types. Using co-expression analysis we built functional networks prioritizing drug targets. To identify small molecules that reverse their expression levels, we leveraged transcriptional response signatures obtained from dozens of human cancer cell lines exposed to tens of thousands of small molecules from the Library of Integrated Network-based Cellular Signatures (LINCS). To identify small molecules that directly bind to and inhibit dark kinases, we have have combined an advanced AI (artificial intelligence) model trained on activity data from across the Kinome with structure-based simulations.Using the computational pipeline, we identified the dark Ca2+/Calmodulin dependent kinase PNCK as the most differentially overexpressed kinase in kidney cancer patients. Our analyses have demonstrated statistically significant correlation between PNCK mRNA levels and various clinical and pathological outcomes, including histologic grade, clinical staging and overall survival. We have confirmed high levels of PNCK expression in 5 renal cell carcinoma cell lines (Caki-1, ACHN, 786-O, A704 and A498). Knockdown and overexpression studies have suggested PNCK and the CaMK pathway may contribute to cellular proliferation and cell cycle progression. We have applied our AI-based screening pipeline to a library of >20 million commercially available compounds and confirmed three PNCK inhibiting chemotypes. In summary, using a novel computational pipeline, we have identified and experimentally validated PNCK as a prospective novel drug target in an understudied pathway that is highly upregulated in kidney cancer. We identified first in class small molecules that target this previously dark kinase as prospective starting points for optimization into a clinical candidate.

#5104

Pan-cancer classification on gene expression data by neural network.

Kijin Yu, Bong-Hyun Kim, Peter Chang Whan Lee. _University of Ulsan College of Medicine, Seoul, Republic of Korea_.

Cancer classification based on molecular signatures such as gene expression profiles has been provided insights on causes of the disease or provided guidance for selecting patient's treatment regimen. We developed a cancer classifier that can identify 13 different types of cancers or a normal tissue based on bulk RNA-seq data as well as single cell RNA-seq data. Training was performed with 6,483 samples from 13 cancer tissues and 640 samples from tumor-adjacent normal tissues in The Cancer Genome Atlas based on 300 most significant genes for the binary classification of each cancer. Then, we compared Neural network, Support vector machine, Naïve Bayes and Random Forest methods. Neural network performed consistently better than other methods, even it took shorter time to train. We further applied our approach to bulked single cell data that is generated from single cell RNA-seq and found our model successfully classified cancer types and normal samples. Gene set enrichment analysis comparing cancer types found genes in cell cycle-related pathways, which highlights the differences between cancer types might be in their abilities to grow in different conditions. We also found genes that expressed in multiple cancers that can be novel biomarkers and therapeutic targets.

#5105

A plug-and-play infrastructure for scalable bioinformatics operations.

Juan S. Medina-Martínez,1 Juan E. Arango-Ossa,1 Gunes Gundem,1 Max F. Levine,1 Minal Patel,1 Noushin R. Farnoud,1 Venkata D. Yellapantula,1 Gao Teng,1 Joseph G. Mccarter,1 Elsa Bernard,1 Franck Rapaport,2 Dominik Glodzik,1 Ross L. Levine,1 Andrew Kung,1 Elli Papaemmanuil1. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _Rockefeller University, New York, NY_.

Genome profiling represents a critical pillar for clinical, translational, and basic research studies. Hospitals, core facilities, and research enterprises invest significant resources to generate genomic data sets. Yet, data management and analysis is frequently manual, which demands significant operator time and often results in siloed resources rendering them as single-use assets. Centralization of the genomic capital in a framework that enables automated processing, metadata integration and continuous interrogation maximizes return for investment and serves as the critical catalyst for research innovation, clinical translation and reproducible research. We developed Isabl, a plug-and-play infrastructure for scalable bioinformatics operations. Isabl provides solutions for databasing, assets management, tracking, automated and reproducible data processing. Dynamic reporting and meta-analysis across data assets is enabled.

Isabl is built on four main components. First, an individual-centric and extensible relational database with tracking support for samples (temporal, spatial, aliquot), experimental data (assays, platforms, sequencing runs), cohorts (clinical trials, research projects) and versioned bioinformatics applications (assembly aware, tools, results). Second, the database is exposed through a fully featured RESTful API that enables horizontal integration with information systems such as sequencing cores LIMS, variant visualization platforms like cBioPortal, and where applicable, clinical and biospecimen institutional databases. Third, a Software Development Kit (SDK) built for Next Generation Sequencing assets management. The SDK enables automated execution of data import and language-agnostic bioinformatics applications (alignment, variant calling, post-processing) with support for cohort and individual level reporting features. Furthermore, the SDK facilitates dynamic retrieval of results using vertical and horizontal queries (individual and cohort level, respectively). Lastly, Isabl comes with a Single Page Web Application that fosters user interaction with multidisciplinary teams (i.e. researchers, project coordinators, engineers, clinicians) facilitating tracking of analyses, results visualization, and dynamic query processing.

Isabl is currently supporting the Memorial Sloan Kettering Genome Pediatrics Precision Medicine Initiative, a prototype platform that delivers integrated, real-time automated reporting of clinical targeted gene re-sequencing, research whole genome and transcriptome profiling data; as well as linked data from pre-clinical models (i.e. PDX) and single cells studies. As an open-source tool, Isabl democratizes access to a purpose built, automated, scalable and fully integrable bioinformatics architecture. Isabl will be available at https://github.com/isabl-io.

#5106

The GenePattern Notebook environment for reproducible cancer research .

Michael M. Reich,1 Thorin T. Tabor,1 Ted Liefeld,1 Barbara Hill,2 Helga Thorvaldsdottir,2 Jill P. Mesirov1. 1 _University of California, San Diego, La Jolla, CA;_ 2 _Broad Institute of MIT and Harvard, Cambridge, MA_.

As the availability of genetic and genomic data and analysis tools from large-scale cancer initiatives continues to increase, the need has become more urgent for a software environment that supports the entire "idea to dissemination" cycle of an integrative cancer genomics analysis. Such a system would need to provide access to a large number of analysis tools without the need for programming, be sufficiently flexible to accommodate the practices of non-programming biologists as well as experienced bioinformaticians, and would provide a way for researchers to encapsulate their work into a single executable document including not only the analytical workflow but also the associated descriptive text, graphics, and supporting research. To address these needs, we have developed GenePattern Notebook, based on the GenePattern environment for integrative genomics and the Jupyter Notebook system. GenePattern Notebook unites the phases of in silico research - experiment design, analysis, and publication - into a single interface.

GenePattern Notebook presents a familiar lab notebook format that allows researchers to build a record of their work by creating cells containing text, graphics, and executable analyses. Researchers add, delete, and modify cells as the research evolves, supporting the initial research phases of prototyping and collaborative analysis. When an analysis is ready for publication, the same document that was used in the design and analysis phases becomes a research narrative that interleaves text, graphics, data, and executable analyses. The online notebook format allows researchers to explain the analytical and scientific considerations of each step in any level of detail, promoting reproducibility and adoption.

GenePattern Notebook features are designed to help nonprogramming users. We have developed additional cell types allowing users to select analyses, specify inputs, navigate results, send result files to new analyses, and create richly formatted text, all without the need for programming.

The GenePattern Notebook Environment is also a platform for open science and reproducible research. Authors can elect to publish a notebook, making it visible to all users, who can then create a copy and use it to reproduce the author's results or adapt it to their own work. The repository provides links to public notebooks that can be used in a publication as permalinks. Researchers can invite collaborators to work together on a notebook prior to publication.

A free, cloud-based GenePattern Notebook workspace is available at http://www.genepattern-notebook.org, where researchers can develop, share, and publish notebook documents. We have provided a collection of template notebooks that walk users through various genomic and machine learning analyses, and are collaborating with research laboratories to create integrative cancer genomics notebooks.

#5107

Putative biomarkers for tumor sample purity prediction based on gene expression data.

Yuanyuan Li. _National Institute of Environmental Health Sciences, Research Triangle Park, NC_.

Tumor purity is the percent of cancer cells present in a sample of tumor tissue. The non-cancerous cells (immune cells, fibroblasts, etc.) have an important role in tumor biology. We applied a supervised machine learning method, XGBoost, to data from 33 TCGA tumor types to predict tumor purity using RNA-seq gene expression data. Across the 33 tumor types, the median correlation between observed and predicted tumor-purity ranged from 0.75 to 0.87 with small root mean square errors, suggesting that tumor purity can be accurately predicted using expression data. We further confirmed that a ten-gene set (CSF2RB, RHOH, C1S, CCDC69, CCL22, CYTIP, POU2AF1, FGR, CCL21, and IL7R) whose expression levels alone were predictive of tumor purity regardless of tumor type. We tested the predicative power of the ten-gene set on an independent dataset that was not from TCGA and showed that the expression levels of the ten genes can accurately predict the cancer cell proportions in those independent samples. The correlation coefficient between the predicted and experimentally obtained tumor purity was 0.88. Our analyses suggested that the ten-gene set may serve as a biomarker for tumor purity prediction using gene expression data.

#5108

Somatic small variant and copy number alteration calling with the Genome Analysis Toolkit.

Lee Lichtenstein, Jonn Smith, David Benjamin, Aaron Chevalier, Kristian Cibulskis, Samuel K. Lee, Eric Banks. _Broad Institute of MIT and Harvard, Cambridge, MA_.

Somatic small mutations, SNVs or Indels, and copy number alterations are the two categories of mutations with the largest impact on cancer tumors. The Broad Institute has released somatic variant calling workflows for small mutations (M2) and copy number alterations (ModelSegments) based on the Genome Analysis Toolkit (GATK). The suite of workflows can call variants in capture or whole-genome sequencing data and will include functional annotations (Funcotator), such as protein change (for small variants) and impacted gene (for all variants). Common artifacts in sequencing data, such as those arising from oxidative DNA damage, FFPE/deamination, or mapping errors, are corrected automatically. Evaluation of the workflows is standardized and repeatable, which allows tracking of performance across versions, both detection performance (e.g. sensitivity, precision), as well as runtime performance (e.g. CPU and RAM usage). A matched normal is not required for a given tumor sample, since the workflows can leverage pre-processed panels of normals (PoNs). The workflows are freely available, are portable (i.e. can be run on local, on-prem, or cloud compute), are optimized for cost reduction, and can be tuned to optimally leverage available compute.The measured sensitivity of M2 was at least 0.93 for small somatic nucleotide variants (SNVs) and 0.83 for small insertions/deletions (Indels) on DREAM1, DREAM2, and DREAM3 challenges, and on a titrated mixture of germline samples (>=100x depth, AF = 0.2). The measured precision of M2 ranged from 0.91 to 0.98 on DREAM1, DREAM2, and DREAM3 for both SNVs and Indels. The false positive rate (FPR) of M2 was between 0.03 and 0.21 FP/Mb for SNVs, and between 0.0 and 0.1 FP/Mb for indels, on twelve paired, replicate normal-normal samples. The cost of the M2 workflow is about USD$1.15 for a pair of 35x WGS matched tumor-normal samples, using Google Cloud Compute, and required about 32 hours of CPU time on a single core with 3GB RAM.

The measured sensitivity of ModelSegments was at least 0.91 for deletions and amplifications across three cohorts of TCGA whole-exome samples (Stomach adenocarcinoma N=39, Thyroid carcinoma N=50, and Lung adenocarcinoma N=60). The measured specificity for the same set of cohorts was at least 0.96 for both deletions and amplifications. All results reported here were using the corresponding SNP Array results as a truth set.

GATK MS cost was approximately USD$0.65 on a 30x WGS pair using Google Cloud Compute and required about 6 hours of CPU time with a single core. The RAM usage was varied automatically in the workflow to minimize cost, but was in the range of 2-13GB.

#5109

National cyberinfrastructure and bioinformatic analysis support available to the cancer research community.

Bhavya Papudeshi, Carrie Ganote, Sheri Sanders, Thomas G. Doak. _Indiana University, Bloomington, IN_.

National Center for Genomic Analysis Support (NCGAS) is an NSF-funded center whose goal is to help biologists optimize their bioinformatic pipelines to best address their research questions. To help reach these goals, NCGAS provides a range of services based on the researchers needs. We currently host gateways, Trinity Galaxy and Genepattern, with built-in workflows to support cancer researchers who have little background in command line work. Both these gateways are projects funded by the Information Technologies in Cancer Research (ITCR) program of NIH, NCI, to aid in the genomic and transcriptomic characterization of cancers. NSF-funded researchers comfortable in command line can gain access to IU cyberinfrastructure (HIPAA compliant), to run or develop bioinformatic workflows. Bioinformatic packages are already installed system wide, with reference databases, and new programs are always added upon user's request. NCGAS is also an ambassador of Extreme Science and Engineering Discovery Environment (XSEDE) resources, providing access to other HIPAA compliant national cyberinfrastructure (Texas Advanced Computing Center, Pittsburgh Supercomputing Center, etc.), available to the entire research community. XSEDE resources include the Jetstream cloud, based at IU and TACC, allowing researchers to use preconfigured virtual machines (VMs) already setup, create custom VM environments for their own work, and to share with classes and collaborators. Data can be transferred and managed between all of these resources securely with already available Globus endpoints which allows transfer for terabytes of data quickly. Additionally, NCGAS hosts workshops to help researchers get started. These training events can be scheduled online or at your local university upon request. NCGAS currently supports projects working on transcriptome, genome-level assembly, phylogenetics, metagenomics, and meta-transcriptomics analysis through providing a range of compute resources that best serve the research project.

#5110

An online tool for summarizing and searching human cancer-genomic publications.

Kevin Williams, Myron Zhang, Anayancy Ramos, Andrew Johnson, Stefanos Stravoravdis, Megan Heenehan, Garrett M. Dancik. _Eastern Connecticut State Univ., Willimantic, CT_.

PubMed catalogs over 33,000 articles published with the keywords "cancer" and "gene" in the past year alone. The large volume of cancer genomic publications necessitates the development of text-mining tools to help cancer researchers navigate and summarize articles efficiently. Here we present a Cancer Publication Portal (CPP) that allows a researcher to search and summarize cancer genomic literature based on a gene of interest. CPP integrates data from several sources, including PubTator, which uses robust text-mining tools to identify associations between articles and biological concepts; the Medical Subject Headings (MeSH) database; the HUGO Gene Nomenclature Committee human gene name database; PubMed, a database of biomedical literature citations; and the National Cancer Institute (NCI) Thesaurus. To begin a search, a user selects a gene of interest. CPP then summarizes the relevant cancer-related publications mentioning this gene through tabular and graphical summaries showing the frequency of articles stratified by references to any (1) cancer type, (2) pharmacological agent, (3) genomic mutation, and (4) additional human gene. Additionally, CPP catalogs and summarizes articles based on mentions of >30 additional cancer-related terms, based on an analysis of terms that are significantly more likely to occur in cancer-related abstracts. CPP allows users to quickly obtain statistics, e.g., to find the frequency of articles mentioning EGFR and Erlotinib across cancer types. Interactive summaries allow users to narrow in on a topic of interest by applying additional filters, in which case the summaries are updated and the process can be repeated. At any point, the user can browse through the current set of article abstracts and citation information to get more information about an article. Finally, we introduce a Citation Analysis module for helping users identify "important" articles. This module identifies articles which cite articles in a provided list. The module then generates an edge list that can be imported into network analysis tools such as Gephi for visualization and analysis. CPP currently includes information for ~1.1 million cancer-related publications associated with >19,000 human genes. The underlying data is stored in an MySQL database while R/Shiny is used to implement the web interface. CPP is available from the following link: https://gdancik.github.io/bioinformatics/CPP/

#5111

HuPharm: An interactive data analysis platform for preclinical studies.

Binchen Mao, Sheng Guol, Qixiang Li. _Crown Bioscience Inc., Taicang, China_.

Background: Preclinical studies, the first stage of the drug development process, must always have proper planning, execution, analysis and reporting in order to preserve scientific integrity. The decision to advance to human clinical trials depends heavily on results of these studies. For To facilitateing in vivo pharmacology data analysis and interpretation, we developed a web platform (HuPharm) that automates statistical pipelines and report generation. Its intuitive and interactive interface will greatly help researchers, who more often than not are non-statisticians, on data validation, exploratory data analysis and statistical analysis.

Methods: We use RStudio's shiny package to build an interactive web application (HuPharm), and we use knitr and rmarkdown packages to generate a highly informative data analysis report. HuPharm is deployed on the web using Shinyapps.io developed by RStudio. Based on the distribution of input data, HuPharm will automatically performs parametric tests such as ANOVA and Welch's t-test or non-parametric tests such as Kruskal-Wallis test and Mann-Whitney U test. Additionally, HuPharm is capable of conducting survival analysis, including both non-parametric Kaplan-Meier analysis and Ccox regression. Aside from classical tests, we have also implemented a number of state-of-the-art analytics on HuPharm, such as multilevel/hierarchical linear models, frailty analysis and joint modeling of longitudinal and survival data. Last by not the least, power analysis module in HuPharm can help researchers with their study design by power and sample size calculation.

Results: Based upon user inputs, HuPharm will dynamically generate a series of tables and figures to facilitate preclinical data analysis, such as tumor volume summary statistic table and plots, tumor growth curves, omnibus group comparison plots, all pairwise group comparison table and post hoc analysis results table. In addition, HuPharm can generate one nicely formatted study report including all those aforementioned figures and tables in both html and pdf format. Powered by shiny server's interactive design, users can easily adjust the parameters to explore their data in a highly flexible and responsive way.

Conclusions: HuPharm is a convenient and powerful tool to aid researchers in the field of preclinical trials studies for their study design and data analysis. It will help to add rigor and improve the quality of preclinical studies.

#5112

LinkedOmics: Analyzing multi-omics data within and across 32 cancer types.

Suhas Vasaikar,1 Peter Straub,2 Jing Wang,1 Bing Zhang1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _Vanderbilt University School of Medicine, Nashville, TN_.

Background: The Cancer Genome Atlas (TCGA) project has performed molecular profiling of human tumors using genomic, epigenomic, transcriptomic, and proteomic platforms, and each tumor is comprehensively characterized by around 100,000 molecular attributes in addition to typical clinical attributes. To make these data directly available to the entire cancer research community, several data portals have been developed. However, none of the existing data portals allow systematic exploration and interpretation of the complex relationships between the vast amount of clinical and molecular attributes.

Methods: We developed LinkedOmics (http://www.linkedomics.org), a web platform that focuses on the discovery and interpretation of associations between clinical and molecular attributes. LinkedOmics includes three data analysis modules. The LinkFinder module allows flexible exploration of associations between a molecular or clinical attribute of interest and all other attributes, providing the opportunity to analyze and visualize associations between billions of attribute pairs for each cancer cohort. The LinkCompare module enables easy comparison of the associations identified by LinkFinder, which is particularly useful in multi-omics and pan-cancer analyses. The LinkInterpreter module transforms identified associations into biological understanding through pathway and network analysis. All modules provide user-friendly data visualization.

Results: The current version of LinkedOmics contains multi-omics data and clinical data for 32 cancer types and a total of 11,158 patients from the TCGA project. Currently we have ~50 PDX (patient derived xenograft) samples data. It is also the first multi-omics database that integrates mass spectrometry (MS)-based global proteomics data generated by the Clinical Proteomic Tumor Analysis Consortium (CPTAC) on selected TCGA tumor samples, as well as CPTAC Colon, Endometrial, Kidney and Breast cancer. In total, LinkedOmics has more than a billion data points. We used several case studies to demonstrate the utility of LinkedOmics in revealing functional impact of somatic mutation or copy number alteration on mRNA or protein expression, in deriving multi-omics based protein signature for poor prognosis, in performing pan-cancer analysis to identify survival-associated gene expression signature, and in connecting novel pan-cancer poor prognosis markers to tumor invasiveness and aggressiveness.

Conclusions: LinkedOmics provides a unique platform for biologists and clinicians to access, analyze and compare cancer multi-omics data within and across tumor types. With 5 case studies we demonstrated the power of LinkedOmics in cancer research. Although the current version of LinkedOmics includes only TCGA and CPTAC data, it can be easily extended to support other cohort-based or user provided multi-omics studies.

#5113

A flexible pipeline for precision medicine biomarker detection and prediction of treatment response.

V Keith Hughitt,1 Sayeh Gorjifard,1 Aleksandra M. Michalowski,1 John K. Simmons,2 Ryan Dale,3 Eric C. Polley,4 Jonathan J. Keats,5 Beverly A. Mock1. 1 _NCI, Bethesda, MD;_ 2 _Personal Genome Diagnostics, Baltimore, MD;_ 3 _NIH, Bethesda, MD;_ 4 _Mayo Clinic, Rochester, MN;_ 5 _TGen, Phoenix, AZ_.

Recent years have seen an explosion in the availability of paired molecular profiling and drug screen data, providing an unprecedented opportunity for the development of targeted therapies based on an individual's genetic background. Despite a number of recent successes in diseases ranging from cystic fibrosis to cancer, significant hurdles remain in our ability to accurately predict treatments based on molecular profiling data. In particular, few such tools exist that allow the integration of heterogeneous data types (e.g. genomic, transcriptomic, and somatic mutations), along with high-throughput drug screen data to make predictions about treatment efficacy. Here, we describe a generalized open-source pipeline developed for the analysis of precision medicine data, Pharmacogenomics Prediction Pipeline, or "P3". The modular design of P3 enables the inclusion of arbitrary input data types and the selection from multiple alternative machine learning algorithms, while automated statistical and visualization reporting steps incorporated throughout the pipeline assist in parameter tuning and early detection of problematic data components. Molecular profiling data is further enriched by the incorporation of external biological information in the form of pathway and gene set annotations such and Gene Ontology (GO) and The Molecular Signatures Database (MSigDB). To demonstrate the use of P3 for preclinical biomarker prediction, we applied P3 to an unpublished multiple myeloma dataset consisting of exome, RNA-Seq, CNV, and drug screen data for 1,912 compounds across 47 tumor cell lines. Specifically, P3 was used to predict molecular features associated with response to treatment for all drugs where a differential response to treatment was observed across patients. Furthermore, molecular profiling and drug screen data for 267 drugs and over a thousand cell lines spanning multiple cancer types from the Genomics of Drug Sensitivity in Cancer (GDSC) project were analyzed using P3, providing insights into shared mechanisms of drug sensitivity and resistance across different cancer and treatment types.

#5114

Deep phenotype extraction to facilitate cancer research: Extending DeepPhe to ovarian cancer.

Alicia Beeghly-Fadiel,1 Jeremy L. Warner,1 Sean Finan,2 James Masanz,2 Harry Hochheiser,3 Guergana Savova2. 1 _Vanderbilt Univiversity Medical Center, Nashville, TN;_ 2 _Boston Children's Hospital, Boston, MA;_ 3 _University of Pittsburgh, PIttsburgh, PA_.

Introduction: To facilitate research on personalized cancer medicine, we developed the DeepPhe system for deep phenotype extraction of computable longitudinal summaries from electronic medical records (EMR). DeepPhe provides an advanced natural language processing (NLP) pipeline capable of extracting cancer details from clinical notes, storage and export of the resulting details, and a visualization tool with interactive graphical capabilities at both the cohort and individual patient levels. Our initial software release included training and evaluation of breast cancer specific attributes (Savova et al. Cancer Research 2017). As part of our iterative process of testing and improving DeepPhe, we have recently expanded to ovarian cancer.

Methods: We used DeepPhe to extract ovarian cancer characteristics from EMR, including tumor location(s), laterality, temporality, and clinical and pathologic components of stage of disease at diagnosis. Tool performance was quantified by F1 scores, defined as the harmonic mean between precision and recall, with F1=1.00 indicating perfect performance, compared to the gold standard of information from manual data abstraction.

Results: Among a test set of 26 primary epithelial ovarian cancer cases with 1,675 annotated notes from UPMC, the overall F1 was 0.9327. F1 scores for attributes of tumor location, laterality, and temporality were 0.8621, 0.5000, and 1.000, respectively. Performance was perfect for all clinical stage of disease components (T, N, and M), while pathologic components scored 0.7879, 0.7692, and 0.9200, respectively. Evaluation of 16 additional UPMC cases is currently underway and ovarian cancer specific attributes are being expanded to include degree of surgical cytoreduction (debulking) and presence of residual disease. Generalizability will be further assessed by evaluating DeepPhe's performance on EMR for 427 ovarian cancer cases from the Vanderbilt University Medical Center.

Conclusions: Our results demonstrate that DeepPhe can be adapted to additional cancer types without losing good-to-excellent performance characteristics. While some concepts, such as laterality, remain challenging for extraction, current results are considered adequate for semi-automated abstraction approaches. Further refinement and expansion to additional clinical and tumor characteristics, including genomics, tumor biomarkers, treatments, and patient outcomes are currently underway. Open-source and freely available for research use, DeepPhe tools (https://github.com/DeepPhe/DeepPhe-Release) can facilitate large-scale extraction of detailed information from EMR systems for research on cancer.

#5115

Enhancing data quality for clinical studies of investigational cancer immunotherapy drug candidates by a new data analytics tool.

Chengsen Xue, Joanne Cuomo, Melissa L. Baptiste, Kai Chen, Danielle L. Kurkowski, Thomas W. Mc Closkey. _ICON Laboratory Services, Farmingdale, NY_.

Cancer immunotherapy is rapidly becoming a treatment of choice in fighting deadly cancers, especially late stage or metastatic cancers. Humanized antibodies such as alemtuzumab and nivolumab alter the dynamics of regulatory T cells, cytotoxic T cells, B cells, NK cells and dendritic cells. All of these cells can be analyzed for a wide array of intracellular and surface biomarkers with flow cytometric technology. The biggest challenges with the use of flow cytometry in the clinical trial setting are the assessment of the amount of data produced and the lack of replicable standards for each set of biomarkers as it relates to the particular characteristics and functions of leukocytes.

Previously, we reported a new method to improve data quality in clinical research using big data analytics and data visualization (AACR 2018 Bioinformatics and System Biology, Abstract #2605). By statistical analysis and pattern recognition, we can simultaneously monitor unlimited data points related to numerous biomarkers. Here we describe widening the application of the new analytic method for use in the field of flow cytometry and cancer immunotherapy.

A flow cytometer measures multiple fluorescence signals at once, and antibodies directed against different cellular proteins can be conjugated with a variety of fluorochromes. When an antibody, such as anti-PD-L1 Alexa Fluor 647, has a quality issue, caused either by the manufacturing process or a technical error within the clinical laboratory, an error signal can be detected by running data analytic. Through consistently monitoring each reagent used, we are working toward improving the reliability of the process itself and the quality of data.

Human-machine interaction and automation are some of the most profound changes in the clinical research setting. The entire process relies heavily on the high volume of sample processing and data generated by advanced machine systems, and intermittent human intervention between each cycle of data collection, analysis, reporting and finalization. Every step in flow cytometric analyses, such as antibody quality and data validation, as well as improvement in staff training and administration of proficiency testing may be improved by this new data analytic tool. In the near future clinical research combining with artificial intelligence and machine learning will be more efficient. This will substantially reduce the time, cost and failure rate of research and development in creating new drugs.

#5116

Benchmarking the HLA typing performance of three HLA assay and two software.

Rui Meng,1 Tao Zhu,2 Yu Gong,3 Yanan Chen,4 Hua Dong,4 Ye Yizhou,4 Fugen Li,4 Ping Liu,5 Minya Yao6. 1 _Huazhong University of Science and Technology, Wuhan, China;_ 2 _Zhejiang Cancer Hospital, Hangzhou, China;_ 3 _Second Affiliated Hospital, Zhejiang University College of Medicine, Hangzhou, China;_ 4 _3DMed, Shanghai, China;_ 5 _The Second Xiangya Hospital of Central South University, Changsha, China;_ 6 _The First Affiliated Hospital of Zhejiang University, School of Medicine, Hangzhou, China_.

Background:The adaptive immune response intrinsically depends on hyper variable human leukocyte antigen (HLA) genes. Correct HLA typing is crucial for successful donor-patient matching in organ transplantation and recently is also an emerging biomarker for immune checkpoint inhibitor. With the great progress on next generation sequencing (NGS) technology, sequencing accuracy and throughput have increased while the cost for data have decreased faster than Moore's Law, thus HLA tying by NGS has been widely used from laboratory to clinic. Method:We tested three NGS assay for HLA typing and two recently published HLA typing software from 24 samples. Three assays involve two target capture methods (WES and HLA target sequencing) and one amplification sequencing method. Two software involve Xhla and HLAHD, respectively. First, we compared 3 assay performance based on PE150 read length and xHLA software by both 4 alleles and 8 alleles. Second, we compared the performance of two widespread HLA typing software xHLA and HLA-HD according to different assay type, read depth, read length and speed. Results:First of all, for PE150 datasets on three assay, the accuracy rate of HLAHD is all 100%, while xhla in WES is 99%, amplicom 98.6%, target HLA sequencing 100%. On reads length evaluation, the accuracy of HLAHD decreases gradually from PE150 to PE100 and PE75, and the capture method is more sensitive to reads length than the amplification assay, while Xhla is very robust on read length. On coverage evaluation, when the amplicom sequencing data was down-sampled 500X to 10X, the accuracy of HLAHHD and XHLA is both 100% in 50X and above and decrease gradually when less than 50X. In terms of time, with the increase in sequencing depth, running time for both software increase. HLAHD prediction from fastq file is longer than that from bam file, especially when the panel data is relatively large, the time will increase exponentially. Conclusions:Our benchmarking dataset show that current HLA assay and computational methods for HLA typing generally provide satisfactory results, while different assay type matched with different software, read depth and read length do affect the performance.

#5117

Comprehensive structural analysis of cancer genomes by genome mapping.

Ernest Lam. _Bionano Genomics Inc., San Diego, CA_.

Tumors are often comprised of heterogeneous populations of cells, with certain cancer-driving mutations at low allele fractions in early stages of cancer development. Effective detection of such variants is critical for diagnosis and targeted treatment. However, typical short sequence reads are limited in their ability to span across repetitive regions of the genome and to facilitate structural variant (SV) analysis. Based on specific labeling and mapping of ultra-high molecular weight (UHMW) DNA, we developed a single-molecule platform that has the potential to detect disease-relevant SVs and give a high-resolution view of tumor heterogeneity.

We developed a DNA isolation and sample preparation workflow that preserves the DNA integrity and conserves structural variation information from blood, cells and preserved tissue. Single molecules are labeled at specific motifs and analyzed in massively parallel nanochannels. The single-molecule maps are used in a bioinformatics pipeline that effectively detects structural variants at low allele fractions. It includes single-molecule based SV calling and fractional copy number analysis. Preliminary analyses using simulated and well-characterized cancer samples showed high sensitivity for variants of different types at as low as 5% allele fractions. The candidate variants are then annotated and further prioritized based on control data and publicly available annotations. The data are imported into a graphical user interface tool that includes new visualization tools (such as Circos diagrams) for real-time interactive visualization and curation.

Bionano offers sample preparation, DNA imaging and genomic data analysis technologies combined into one streamlined workflow that enables high-throughput genome mapping on the Bionano Saphyr system. Together, these components allows for efficient analysis of any genome of interest.

#5118

TIDE-E: an online evaluator of tumor immune-suppressive function of gene sets.

Jingxin Fu,1 Wubing Zhang,2 Peng Jiang,1 Xiaole S. Liu1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Tongji University, Shanghai, China_.

Recent immunotherapy studies have generated a diverse collection of datasets, covering different aspects of cancer immune evasion. However, to the best of our knowledge, there are no public tools that can systematically rank genes regarding their function on tumor immune evasion and comprehensively evaluate biomarkers across multiple clinical cohorts. Therefore, we presented the TIDE-E (evaluator) website for users to evaluate their gene sets across a large panel of data cohorts (http://tide.dfci.harvard.edu). The website integrated two categories of data resources, human clinical datasets from public immunotherapy studies, TCGA, PRECOG, and METABRIC databases, as well as functional genomics datasets from CRISPR screens in preclinical models. For a user-defined gene set, the web application can test its performance as immunotherapy response biomarkers across multiple clinical studies, and rank the gene members according to their relevance in promoting tumor immune evasion.

#5119

Customizable all-in-one cancer panels to detect copy number variation, DNA rearrangement and mutations by targeted NGS.

Arjun Vadapalli,1 Ashutosh Ashutosh,2 Heather Tao,1 Linus Forsmark,1 Christian Le Cocq,1 Akansha Khare,1 Bahram Arezi,1 Gilbert Amparo,1 Michael Ruvolo,1 Carlos Pabon,1 Jayati Ghosh,1 Jimmy Jin,1 Tracy Liu,1 Douglas Roberts1. 1 _Agilent technologies, Santa Clara, CA;_ 2 _Agilent Technologies, Santa Clara, CA_.

Background: The current standard of genomic profiling of cancer tissues relies upon multiple separate technologies to aid in tumor characterization. Here we describe a customizable NGS design and analysis workflow for cancer samples that can detect single nucleotide polymorphisms (SNPs), insertions and deletions (INDEL), somatic copy number alterations (SCNAs), and translocations (TLs) in a single assay.

Methods: We have created a library of strategically placed target-enrichment probes to provide for specific detection of SNP/INDEL, SCNAs, and translocation in genes identified as important in several cancer subtypes. The probes are optimized with the SureSelect XTHS protocol, which enables extremely sensitive detection of rare exonic mutation events within a heterogeneous sample. SCNA detection is further enhanced with additional probes targeting high minor allele frequency SNPs within the target gene boundary. Translocation detection is enabled by targeting intronic regions of fusion gene candidates. The data analysis is performed by the software SureCall that calls SNPs across all regions covered by probes. SCNA detection utilizes either a matched normal or standard control DNA and aberrant regions are identified by jointly segmenting the read depth ratios and SNP allele frequencies. The clonal structure is discerned using two Bayesian cancer models in a hierarchical manner. Translocation are identified by analyzing split reads across putative fusion breakpoints that are supported by both high- and low-confidence reads that pass quality filters.

Results: We demonstrate rare allele detection in cancer cells down to 1%. We also show detection of 3 copies of MET at 20% tumor fraction and 16 copies of ERBB2 at 2% tumor fraction. For translocations, DNA level gene fusions were detected for ALK and ROS fusion genes at <=3% tumor fraction. In 39 ERBB2 FISH-positive FFPE samples, ERBB2 CNV detected by AIO panel was 97.4% concordant with FISH (scored according to standard ASCO/CAP convention). Using FISH as the gold standard, the false negative rate was 2.6% and false positive rate was 2.4%. In 43 ALK FISH-positive FFPE samples, ALK fusion was detected using a small AIO lung panel which includes probes spanning introns 18/19 of the ALK gene and is 90.7% concordant with FISH (Agilent SureFISH ALK BA). Using FISH as the gold standard, the false negative rate was 9.3% and false positive rate was 2.3%.

Conclusions: This work represents an important advancement in the development of a single assay to detect copy number variation, DNA rearrangement and mutations.

#5120

Managing human tissues, bio-fluids, and clinical trial data at Fidelis Research leveraging a cloud-based system.

Aditi Gade,1 Arun Apte,2 Shonali Paul,1 Iliyan P. Hristov3. 1 _CloudLIMS Software Solutions, Indore, India;_ 2 _CloudLIMS.com, Wilmington, DE;_ 3 _Fidelis Research AD, Sofia, Bulgaria_.

Introduction: Founded in 2012, Fidelis Research provides high-quality human tissues and bio-fluids specimens along with de-identified patient disease information to pharmaceutical, biotechnology, and pre-clinical contract research companies to facilitate their research. Fidelis has collection sites in South East Europe - Bulgaria, Romania, Croatia, Serbia, and Turkey. Through these sites, the company identifies patients, obtains consents, and then collects various types of samples. Additionally, the company has also established Clinical Research Units that conducts clinical trials. The primary goal of Fidelis Research is to provide its customers with integrated services ranging from pre-clinical to market authorization stage of product development. To achieve this, a Laboratory Information Management System (LIMS) was indispensable.

Description: At any given point, Fidelis works on multiple collection projects for different clients, collecting samples from several subjects and sites. The samples are shipped to the biobank facilities. This requires excellent data management to ensure that the right data is associated with the right samples, thus facilitating their shipping to clients. The main challenges the company faced before implementing CloudLIMS' solution had to do mainly with the amount of time spent on manually entering data, the difficulties with ensuring the accuracy of data and QC, risks of overlapping or deleting data by multiple users, inconsistency in data presentation and usability for reporting and billing purposes.

Results: After deploying the cloud-based LIMS, Fidelis is now able to efficiently manage and store human tissues and bio-fluids specimens collected along with the donor information such as age, sex, diagnosis, date of surgery, medical history, etc. With a cloud-based system, sharing specimens' data internally and externally has become much easier and hassle-free. Fidelis' clients have benefited from more consistently presented data, accurate reports, and faster turnaround times. Fidelis has integrated its label generation and printing with CloudLIMS, benefiting the company and its clients.

Conclusion: Cloud-based LIMS facilitates Fidelis Research in providing high-quality, ethically-sourced human biospecimens for the research and development needs of pharmaceutical, and biotechnology companies. Additionally, the LIMS also helps Fidelis Research to comply with the EU Directives, FDA regulations, and the applicable national laws and ensure data security. The improvements achieved in the company's operations range from shorter turnaround times for processing samples storage, shipping, data management and billing, to facilitating auxiliary services such as label generation and printing.

#5121

Discovery and screening of protein biomarkers with the FirePlex Technology Platform.

Amy Perea, Russell Neuner, Bianca Heinrich, Graeme Doran, Wayne Austin, Conor Rafferty, Matt Camilleri, Michael Tackett, Long To, Elnaz Atabakhsh, James Murray, Daniel Pregibon. _Abcam, Inc., Cambridge, MA_.

In patients and animal models, molecular biomarkers are used as indicators of normal and pathogenic processes. In drug discovery and screening pipelines, molecular biomarkers are used to assess the mechanism of action, efficacy, and toxicity of lead compounds. To address the need for rapid and sensitive biomarker quantitation, we have developed the FirePlex® Technology Platform. Utilizing patented hydrogel particles and a three-region encoding design, FirePlex assays allow for true, in-well multiplexing, providing flexible and customizable quantification of miRNA and protein analytes. To facilitate biomarker discovery studies, we offer our standard-throughput, flow cytometry-based assays which enable quantitation of up to 75 miRNA or protein analytes per sample, from only 20 µl of input. These assays demonstrate 5 logs of dynamic range and offer highly sensitive quantitation of analytes in serum, plasma, cell culture supernatant, urine, and CSF without the need for sample processing or RNA isolation. For drug discovery and screening studies, we offer our high-throughput FirePlex (FirePlex®-HT) Immunoassays for quantitation of up to 10 protein analytes per sample from only 6.25 µl biofluid input, in 384-well plate format. FirePlex-HT assays provide 3-4 logs dynamic range, demonstrate 1-100 pg/ml sensitivity, and have been validated in serum, plasma, and cell culture supernatant. The two-step workflow, no-wash assay format, and readout on high-content imagers limit hands-on time and are amenable to automation, thus making FirePlex-HT ideally suited for high-throughput screening studies. In addition, here we introduce our high-throughput miRNA assays for screening miRNA biomarkers in 96-well or 38-4well plate format, with readout on high-content imagers. Here we present data from miRNA and protein profiling studies using the FirePlex platform, and introduce the simplified workflow of the FirePlex-HT® immunoassays with data demonstrating the performance for quantifying key cytokines in multiplex, in biological samples. Together, this novel combination of multiplexed, high-sensitivity assays and bioinformatics tools enables rapid quantitation of protein biomarker signatures in biofluid specimens.

## MOLECULAR AND CELLULAR BIOLOGY / GENETICS

### Advanced Omics

#5122

Comprehensive detection of germline and somatic structural mutation in cancer genomes by Bionano Genomics optical mapping.

Andy Pang. _Bionano Genomics Inc., San Diego, CA_.

In cancer genetics, the ability to identify constitutive and low-allelic fraction structural variants (SVs) is crucial. Conventional karyotype and cytogenetics approaches are manually intensive. Microarrays and short-read sequencing cannot detect calls in segmental duplications and repeats, often miss balanced variants, and have trouble finding low-frequency mutations.

We describe the use of Bionano Genomics's Saphyr platform to comprehensively identify SVs for studying cancer genomes. DNA >100 kbp is extracted, labelled at specific motifs, and linearized through NanoChannel arrays for visualization. Molecule images are digitized and de novo assembled, creating chromosomal arm scale genome maps. Somatic mutations can be identified by running the variant annotation pipeline that compares the cancer sample's assembly SVs against >600,000 SVs in Bionano's control sample SV database, and against a matched control sample's SVs, if avaliable. Also, two new Bionano pipelines leverage these long molecules to identify additional somatic SVs: the copy number variation (CNV) and the molecule mapping pipelines. By examining the coverage-depth of molecules alignment to the public reference, the pipeline can identify megabases long CNVs. Similarly, clusters of split-molecule alignments can reliably find translocations and other rearrangements.

We applied this suite of discovery tools to identify SVs in a well-studied melanoma cell line COLO829. We collected data from the tumor and the matched blood cell line, constructed contiguous assemblies (N50 >50 Mbp), and called >6,000 SVs in each genome. Then, we classified 51 as somatic by comparing the tumor and the blood control. The two new pipelines further increased sensitivity to rearrangements, for example they captured a BRAF duplication, and other chromosome-arm CNVs. We apply these thorough approaches to multiple well-studied cancer lines to identify novel SVs missed by previous studies. In conclusion, with one comprehensive platform, Saphyr can discover a broad range of traditionally refractory but relevant SVs, and further improves our understanding of cancer.

#5123

Characterization of genetic mutation spectra and identification of gene amplification and fusion variants in cell-free nucleic acid from cultured cancer cell media and liquid biopsy specimens using Oncomine™ Pan-Cancer Cell-Free Assay.

Ru Cao,1 Kris Lea,1 Madhu Jasti,1 Jeff Schageman,1 Khalid Hanif,1 Yanchun Li,1 Jian Gu,1 Varun Bagai,1 Priyanka Kshatriya,1 Harriet Wikman,2 Sonja Loges,2 Kelli Bramlett1. 1 _ThermoFisher Scientific, Austin, TX;_ 2 _University Medical Center Hamburg-Eppendorf, Hamburg, Germany_.

Currently the standard practice in tumor biomarker research still relies on invasive tumor biopsy from local sites following by molecular testing or NGS assay. However, this only provides a very limited characterization of tumor composition. Recent studies in non-invasive biomarker research have demonstrated the potential advantages of using cell-free nucleic acids isolated from blood plasma to study genetic heterogeneity of tumor population and dissect the complex cancer clonal architecture. However, it has been challenging to apply to practical research due to low sample yield and sensitivity of detection approaches. Moreover, presence of both cfDNA and cfRNA requires methods capable of interrogating both types of analytes to maximize the utility of each plasma sample and characterize the comprehensive spectrum of mutations including single nucleotide variants, gene amplifications, and structural variants.

Recently developed Oncomine™ Pan-Cancer Cell-free Assay employs an amplification-based approach from Ion Torrent NGS technology and achieves exceptional sensitivity and specificity with input amount as little as 20 ng. It includes the most comprehensive genetic content to simultaneously interrogate both cfDNA and cfRNA. Multiple libraries were pooled together for templating on Ion Chef™ and sequenced on Ion GeneStudio S5 systems.

In this study, a panel of cancer cell lines harboring multiple variant types were selected and cultured for extended time after apoptosis. We were able to extract cell-free nucleic acids that were released into the cell media and mimic the circulating DNA profile of liquid biopsy samples using in vitro model. Using Oncomine™ Pan-Cancer Cell-free Assay, we successfully detected all the expected variants in these cancer cell lines including gene amplification (MET, ERBB2, CDK4) and fusion variants (ALK fusion and MET exon skipping). Subsequently, we applied this assay to a set of longitudinal liquid biopsy samples collected from a human subject with NSCLC during the course of 15 months. The results showed that a well-known TP53 mutation R248Q was consistently detected in the longitudinal samples with varying allelic frequencies. Interestingly,

additional gene amplifications including MET, CDK4 and FGFR3 were identified at late time points. Furthermore, these observations were confirmed by digital PCR and concordant with FISH analyses in solid tumor.

Overall, this study demonstrates that Pan-Cancer assay provides a unique and complete NGS solution for comprehensive genetic mutation assessment using in vitro and in vivo liquid biopsy models.

#5124

A machine learning based approach to identify biomarkers of environmental toxicant exposures relevant to liver cancer disparities in rural Illinois.

Brandi Smith,1 Zeynep Madak-Erdogan2. 1 _University of Illinois at Urbana-Illinois, Champaign, IL;_ 2 _University of Illinois at Urbana-Illinois, Urbana, IL_.

Although there have been nationwide reductions in recent cancer death, liver cancer mortality remains to be a problem in rural Illinois. Researchers anticipate that exposure to environmental toxicants in rural Illinois, may be possible causes of increased liver cancer mortality in females. The purpose of our study is to identify potential biomarkers, which are related to early liver toxicity through machine learning. We accessed gene expression data from the open source Toxicogenomics Project-Genomics Assisted Toxicity Evaluation System (TG-GATES) database from rats that had been exposed to over 170 toxicants relevant to liver cancer. We performed a differential gene expression analysis to identify significant features or genes, which are related to hepatotoxicity endpoints, specifically necrosis. Then we built our predictive model using supervised machine learning (ML) through feature selection and classification models. We performed differential gene expression analysis and feature selection to decrease the dimensionality of the gene set. We then used classification modeling to classify genes related to necrosis. Lastly, we tested our predictive model, on an independent data set. Our model predicted specific genes that were highly related to liver toxicant exposure.

#5125

A novel method for isolating high-quality UHMW DNA from 10 mg of freshly frozen or liquid-preserved animal and human tissue including solid tumors.

Yang Zhang,1 James Broach2. 1 _Bionano Genomics Inc., San Diego, CA;_ 2 _Penn State College of Medicine, Hershey, PA_.

High-quality UHMW DNA is key to success in optical mapping of long genomic DNA using Bionano Genomics Saphyr® system for de novo genome assembly and structural variation detection. Ideally, ~100 mg of tissue is frozen immediately upon collection using liquid nitrogen or dry ice, and stored at -80°C before DNA isolation. Practically, tissue amounts may be limited and collection often occurs at sites where low temperature preservation for storage and shipping may be unavailable.

Here we present a novel method to isolate UHMW DNA from 10 mg of either freshly frozen or room temperature preserved animal and human tissue for subsequent enzymatic labeling using direct labeling and staining (DLS) to generate Bionano maps. With a TissueRuptor, 10 mg tissue is homogenized followed by a three-step purification process before embedding the purified nuclei in agarose gel plugs. This process takes less than 3 hours for a batch of 6 samples. Using this method, we have successfully isolated high quality UHMW DNA from various tissue types of rat and human including solid tumors.

The method is very attractive compared to other protocols because: 1) tissue preservation, shipping and handling can be done at room temperature, 2) uses very small amount of tissue - possible application for rare samples and human tissue biopsy tests, and 3) the length and quality of the resulting single molecules generate high quality optical maps for genome finishing and SV calling.

#5126

Genetic analysis of metachronous pancreatic cancers.

Hirano Tomonori,1 Nobuyuki Kakiuchi,1 Yasuhide Takeuchi,1 Yoshikage Inoue,1 Tomomi Nishimura,1 Yoichi Fujii,1 Akira Yokoyama,1 Hideki Makishima,1 Toshihiko Masui,1 Shinji Uemoto,1 Sachiko Minamiguchi,1 Hironori Haga,1 Kenichi Chiba,2 Hiroko Tanaka,2 Yuichi Shiraishi,2 Satoru Miyano,2 Norimitsu Uza,1 Yuzo Kodama,3 Hiroshi Seno,1 Tsutomu Chiba,4 Seishi Ogawa1. 1 _Kyoto University, Kyoto, Japan;_ 2 _The University of Tokyo, Tokyo, Japan;_ 3 _Kobe University Graduate School of Medicine, Kobe, Japan;_ 4 _Kansai Electric Power Hospital, Osaka, Japan_.

[Introduction] Early detection of pancreatic cancer is a key to curable surgery, although many are diagnosed in advanced stage. However, even in those cases in which cancer was detected early enough for curative resection, metachronous pancreatic cancer may occur in residual pancreas, whose pathogenesis, including its relationship with primary cancer, has been poorly understood. In this study, we aimed to reveal the origin of metachronous pancreatic cancers using an unbiased detection of somatic mutations in primary and metachronous cancers as well as adjacent precursor lesions.

[Methods] Samples were obtained from longitudinal sampling from above lesions using laser microdissection from formalin-fixed paraffin-embedded surgical specimens. DNA was extracted and analyzed for somatic mutations of each lesion by whole-exome sequencing with matched normal DNA. On the basis of shared and private mutations across different samples, we interrogated history of clonal evolution of these lesions.

[Results] Four patients were enrolled who underwent curative surgery for early pancreatic cancer and subsequently for metachronous tumors in the residual pancreas. Pathology from surgery for primary cancer were margin-negative in all patients. The median interval between the initial and second surgery was 29.8 months (22.8 - 38.3 months). The number of precursor lesions analyzed was from 0 to 5 per patient. The median number of somatic mutations per sample was 78 (range: 41-92) in cancers and 20 (14-42) in precursor lesions. None of the patients have known pathogenic germline mutations. With a median of 78 and 20 mutations per sample, all samples had one or more driver mutations. In each case, all driver mutations that were detected in cancers were shared between primary and metachronous cancer. It indicated that metachronous cancer branched off from primary cancers at later stage of carcinogenesis. And negative margin at initial surgery suggested that metachronous cancer was formed by dissemination or metastasizing rather than Intraductal progression. By contrast, none of the mutations other than a hotspot KRAS mutations were shared between precursor lesions and cancers, suggesting multiple independent clonal growth of precancerous cells in the cancer bearing pancreas.

[Conclusions] Our study shows that metachronous pancreatic cancers are derived from the same origin of initial cancer. Therefore, even in the case of early pancreatic cancer careful checkup for recurrence in the residual pancreas is important.

#5127

Association of inactivating calcium sensing receptor exon 7 SNPs with hypercalcemia-related disease phenotypes.

Ky'Era Actkins,1 Heather Beasley,1 Annika Faucon,2 Lea Davis,2 Amos Sakwe1. 1 _Meharry Medical College, Nashville, TN;_ 2 _Vanderbilt University Medical Center, Nashville, TN_.

The calcium sensing receptor (CaSR) is a ubiquitously expressed G-protein coupled receptor known to be a major calcium sensor in most tissues and organ systems. It is known to regulate systemic calcium homeostasis by sensing slight increases in systemic calcium and by influencing the secretion of parathyroid hormone by parathyroid chief cells. Unfortunately, the CaSR is known to be invariably mutated and inactivating mutations in particular, are associated with the development of cancer-induced hypercalcemia (CIH). Among the several inactivating CASR polymorphisms described thus far, the rs1801725 (A986S SNP) is common among Caucasian subjects while the rs1801726 (Q1011E SNP) is common among African American subjects. However, whether these mutations contribute to other hypercalcemia-related disease phenotypes remains unclear. In this study, we carried out a phenome-wide association study (PheWAS) using the Synthetic Derivative (SD), a database at VUMC containing de-identified electronic health records (EHRs) linked to patient DNA samples in the BioVU biorepository. Using 39,893 genotyped individuals in the Multi-Ethnic Genotyping Array (MEGA), we observed that SNP rs1801725 was significantly associated with disorders of bone and cartilage (p = 5.93e-6, OR = 1.28) as well as with secondary malignancy of bone (p = 1.46e-5, OR = 1.21); whereas SNP rs1801726 was significantly associated with osteoporosis related phenotypes (p = 5.93e-6, OR = 1.28). Given that breast cancer and other solid tumors frequently metastasize to skeletal tissues, these data suggests that the inactivating rs1801725 and rs1801726 CaSR variants differentially influence hypercalcemia-related comorbidities and cancer metastasis to bone. We will further verify these effects using the associated clinical data and perform genetic risk analyses for the expression of these SNPs and the associated disease phenotypes.

#5128

Polyethnic-1000: Advancing cancer genomics by studying ethnically diverse, underserved patient populations in New York.

Fieke E. Froeling,1 Nicolas Robine,2 Benjamin Hubert,2 Michael Zody,2 Dayna Oschwald,2 Harold E. Varmus,3 Charles L. Sawyers,4 David A. Tuveson,1 On behalf of the NYGC P1000 Consortium. 1 _Cold Spring Harbor Laboratory, Cold Spring Harbor, NY;_ 2 _New York Genome Center, New York City, NY;_ 3 _Weill Cornell Medicine, New York City, NY;_ 4 _Memorial Sloan Kettering Cancer Center, New York City, NY_.

Background and aims Recent advances in DNA sequencing technologies have revolutionized approaches to the prevention, risk assessment, early detection, diagnosis, and treatment of cancers. However, many ethnic groups, especially non-European populations, have been significantly under-represented in cancer research, including clinical trials, and have not received equal benefits in clinical practice. As a result, our current knowledge about tumor biology, cancer risk, and response to treatment has primarily been derived from patients of European descent. These inequities limit our understanding of the many types of cancer and may exacerbate health disparities in the United States. This multi-institutional study, named Polyethnic-1000, aims to address both the scientific and social issues by creating a dynamic research platform within the ethnically diverse greater New York City area.

Methods and results Polyethnic-1000 is a collaborative effort organized by the New York Genome Center (NYGC), involving staff and patients at academic centers and partnering hospitals in the New York City region. The genomics and informatics capabilities of the NYGC will be used to determine how inherited and somatically acquired genetic variations affect the behavior of cancers occurring in ethnically diverse populations. In a first, retrospective stage we are establishing the necessary infrastructure and workflow from sample acquisition to whole-exome and RNA sequencing, data analysis and data sharing within the consortium. Then we will start a prospective study enabling the formation of cohorts of interest for particular cancer types and particular ethnicities, with uniform consent allowing germline and somatic sequencing with broad data sharing of the somatic variants identified.

Conclusion By establishing a collaborative network, Polyethnic-1000 will deepen our understanding of the contributions that ethnicities make to the incidence and biology of cancers, potentially improving outcomes for patients who currently lack access to the most recent advances in medical science.

#5129

Actionable activating oncogenic HER2 transmembrane and juxtamembrane domain mutations.

Kanika Bajaj. _Genentech, South San Francisco, CA_.

Deregulated HER2 is a target of many approved cancer drugs. We analyzed 111,176 patient tumors and identified recurrent mutations in HER2 transmembrane domain (TMD) and juxtamembrane domain (JMD) including G660D, R678Q, E693K and Q709L. Using a saturation mutagenesis screen and testing of patient-derived mutations we found several activating TMD and JMD mutations. Structural modeling and analysis showed that the TMD/JMD mutations function by improving the active dimer interface or stabilizing an activating conformation. Further, we found that HER2 G660D employed asymmetric kinase dimerization for activation and signaling. Importantly, anti-HER2 antibodies and small molecule kinase inhibitors blocked the activity of TMD/JMD mutants. Consistent with this, a G660D germline mutant lung cancer patient showed remarkable clinical response to HER2 blockade.

#5130

Single-cell mutational profiling of paired AML samples at diagnosis, remission and relapse: Implications for therapeutic resistance and MRD detection.

Alexey Aleshin,1 Robert Durruthy-Durruthy,2 Ryan Corces,1 Michaela Liedtke,1 Dennis Eastburn,2 Ravindra Majeti1. 1 _Stanford University, Menlo Park, CA;_ 2 _Mission Bio, Inc., South San Francisco, CA_.

Acute myeloid leukemia (AML) is a molecularly heterogeneous disorder of the bone marrow with poor long-term clinical outcomes due to a high risk of relapse after initial therapy. Relapse is thought to occur from minimal residual disease (MRD) consisting of leukemic blasts present below the limit of morphologic detection. Genomic sequencing studies have suggested complex subclonal architecture of AML, however bulk sequencing techniques may not fully resolve individual subclones present at diagnosis, remission and relapse. Here, we use a high-throughput single cell sequencing technique to delineate the subclonal structure of AML and identify clones present at diagnosis and at time of remission associated with disease relapse.Matched diagnosis, remission, and relapse samples were examined for 20 de novo AML cases including 15 relapsed and 5 non-relapsed controls. Mutational bulk sequencing was performed by NGS panel sequencing and exome sequencing was available in select cases. Single cell processing was performed using the Tapestri platform (Mission Bio, inc). Briefly, individual cells were isolated using a microfluidic approach, followed by barcoding and genomic DNA amplification for individual cancer cells confined to droplets. Barcodes were then used to reassemble the genetic profiles of cells from next generation sequencing data. We applied this approach to individual AML samples, for accurate single nucleotide variant (SNV) and indel calling for up to 300 multiplexed loci from up to 10,000 cells in a single run.Targeted single-cell sequencing was able to recapitulate all mutations identified by bulk sequencing. Additionally, single cell analysis allowed for resolution of subclonal architecture and tumor phylogenetic evolution beyond what was predicted from bulk sequencing alone. We identified rare subclonal populations associated with relapse at time of diagnosis and remission. Additionally, single-cell sequencing unambiguously resolved pre-leukemic/clonal hematopoiesis-associated clones, which persisted following treatment. Analysis of paired samples at diagnosis and relapse identified subclonal populations below detection of standard bulk sequencing methods. These included rare single-cell populations associated with treatment resistance including independent subclones with IDH1 and IDH2 mutations, co-occurring RAS and FLT3 mutations, as well as persistent low level clones associated with MRD positivity and disease relapse. Our results suggest a greater degree of subclonal heterogeneity in de novo AML samples than inferred from bulk sequencing methods alone and shows the utility of single-cell sequencing for treatment monitoring, MRD identification, and understanding of disease resistance mechanisms.

#5131

Breast cancer with insertion or deletion exhibits the immunogenic phenotype.

Peng Yuan,1 Xue Wang,2 Yuzi Zhang,3 Jing Zhao,3 Shangli Cai,3 Yongmei Yin4. 1 _National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China; _2 _Cancer Hospital Chinese Academy of Medical Sciences, Beijing, China;_ 3 _3D Medicines Inc., Shanghai, China;_ 4 _The First Affiliated Hospital of Nanjing Medical University, Nanjing, China_.

Background: Although immunotherapy has been proven to be effective in a wide range of malignant tumors, breast cancer remains to be one of poorly immunogenic tumors and only a small subset of patients with breast cancer achieved benefits from immunotherapy. Therefore, identification of better predictive biomarkers to guide patient selection is highly desirable. It has been reported that insertion or deletion (indels) could create a novel open reading frame and generate more neoantigen which may mediate response to immunotherpay. However, the pattern of indels mutations in breast cancer is still unclear.

Methods: Whole-exome sequencing data, RNA-Seq data and clinical data of 1096 breast tumors from The Cancer Genome Altas (TCGA) database were used to analyze the pattern of indels in breast cancer. Next generation sequencing (NGS) data of 81 breast tumors from clinical dataset were also used to validate the indels mutation pattern in different molecular subtype.

Results: 81.7% (895/1096) of breast tumors in TCGA dataset harbored at least one indels mutation. Hormonal receptor (HR) negative tumors were associated with higher burden of indels mutations than HR positive tumors in both TCGA dataset (P = 0.05) and NGS-clinical dataset (P = 0.003). Indels were significantly correlated with higher TMB and neoantigen level in TCGA cohort (P < 0.0001). In addition, tumors with at least eight indels mutations (cut off as 80% percentile) exhibited even higher TMB (P < 0.0001) and neoantigen (P < 0.0001) level. Among 45 immune related genes, the mRNA expression of 22 genes were significantly higher in tumors with indels mutations, such as LAG3, IL18, IL6, CTLA4 and PDCD1. Indels group also showed a high levels of genome instability in terms of HRD-LOH (P = 0.004), NtAI (P = 0.000), wGII (P = 0.001) and LST (P =0.014).

Conclusions: Breast tumors with indels mutations exhibited the immunogenic phenotype. Further studies are warranted to investigate the potential value of indels as a predictive biomarker for immunotherapy in breast cancer.

#5132

Assessing tumor mutational burden and profiling variants from FFPE samples using a PCR-based next-generation sequencing assay.

Ruchi Chaudhary,1 Charles Scafe,1 Dinesh Cyanam,2 Vinay Mittal,2 Warren Tom,1 Janice Au-Young,1 Seth Sadis,2 Fiona Hyland1. 1 _Thermo Fisher Scientific, South San Francisco, CA;_ 2 _Thermo Fisher Scientific, Ann Arbor, MI_.

Introduction: High Tumor Mutational Burden (TMB) has shown association with benefit from immune checkpoint blockade therapies. While TMB as computed from whole exome sequencing (WES) is still a gold standard, the high input material (tumor and germline DNA) requirement and complex bioinformatics refrains exploring this biomarker in individual labs. Herein, we develop a PCR-based targeted panel for computing TMB and detecting important variants from FFPE research samples.

Methods: A targeted panel was designed that covered 1.7 Mb of genomic region from 409 key cancer genes. First an UDG treatment is applied to repair samples with FFPE fixation error. Utilizing Ion AmpliSeq chemistry, the workflow requires very little (only 20 ng) input DNA. The assay enables a 3-day turn-around time from sample to the final report. The workflow enables < 60 minutes of hands-on time for automated library preparation and templating on a batch of 4 samples. Sequencing is performed on high throughput semiconductor sequencing platform to achieve sufficient depth (~1200x coverage) and accuracy. The analysis pipeline accompanies variant caller parameters optimized for high accuracy in variant detection. The workflow is tumor sample only, therefore, TMB (nonsynonymous somatic mutations/Mb) is calculated by removing germline variants using population databases. Eight NSCLC FFPE tumors were analyzed. Samples were also tested without UDG repair. Count of somatic C:G>T:A mutations below 15% allelic frequency was called deamination.

Results: In-silico analyses using 466 lung adenocarcinoma, 375 skin cutaneous melanoma, and 274 colon adenocarcinoma samples from TCGA MC3 dataset displayed high correlation between WES TMB and predicted panel TMB (r2 = 0.90 on lung, r2 = 0.96 on melanoma, and r2 = 0.98 on colon). Deamination reduced by average 85.67% (SD 16.87) on FFPE samples after applying UDG repair. Mean TMB of first six samples was 6.97 (SD 2.00), and TMB of last two samples was 43.58 and 87.14. Among two high TMB samples, first had gain-of-function and loss-of-function mutations in BRAF and NF1 genes and had 59.4% C:G>A:T somatic mutations relating to molecular smoking signature. On the second high TMB sample, the assay detected gain-of-function and loss-of-function mutations in FGFR3, NOTCH1, KRAS, HNF1A, and CREBBP genes and estimated 64.7% C:G>T:A somatic mutations at CpG sites consistent with deamination of 5-methylcytosine.

Conclusions: We have developed a workflow on the Ion Torrent sequencing platform with Ion AmpliSeq chemistry to estimate TMB from FFPE research samples. This solution will advance research in immuno-oncology.

#5133

Identification of distinct gene expression profiles in African Americans and Caucasian men with prostate cancer.

Joakin Mori, Raven Williams, Hui-Xian Lin, Alahni Becks, Jason White, Balasubramanyam Karanam, Clayton Yates, Honghe Wang. _Tuskegee University, Tuskegee, AL_.

Introduction African Americans (AA) are disproportionately affected by prostate cancer (PCa) compared to European Americans (EA). Genetic and epigenetic differences have been implicated in this racial disparity. Present PCa diagnostic and treatment regimens have focused mainly on gene fusion product involving the androgen driven transmembrane protease serine 2 (TMPRSS2) and the transcription factor, ETS related gene (ERG). However, TMPRSS2:ERG (T2E) fusion product is common mainly to EA (50-80% of patients) and has lower prevalence amongst other ethnicities including AAs (10-30%), Japanese and Chinese patients. In addition, T2E negative PCa subtype has been associated with increased risk of developing castration resistant PCa (CRPC). This study aims at classifying PCa by molecular and pathologic subtypes that drive disease aggressiveness in patients of AA and EA ancestry.

Methodology Prostate cancer tissue slides obtained from EA and AA patients were stained for ERG expression. Laser-capture micro-dissected RNAs from formalin-fixed paraffin-embedded tissues were used for RNA-seq. DESeq2 was used as a statistical procedure to call differentially expressed genes (DEGs) in different samples. Data set from Powell was also analyzed for DEGs between ERG positive and ERG negative samples. Leading-Edge Analysis (GSEA v2.2.4) was performed to determine the representation of DEGs across enriched gene sets.

Results 32.4% of EA were ERG positive compared to 29% of AA that stained positive. More EA samples (26.4%) had high intensity staining (2≥ on a 1 - 3 scale) compared to AA samples (25.8%). Over 11% of EA samples had high (4) distribution of ERG positive staining compared 6.5% in AA samples. Analysis of the Powell dataset (GSE41696) revealed 301 DEGs and gene set enrichment analysis identified immune system process, regulation of cellular component and homeostatic process as most biological processes affected by DEGs. Other processes significantly affected included: cell motility, cell activation and regulation of locomotion. At least 15 genes including: CCL5, TLR4, PIK3CB, CD86, KIT, CD40, ITGB3, PDGFRA, LEF1, HIF1A, CLU, PLA2G6, GLI3, PIK3CA and TNNB1, were present in at least 10 enriched gene sets. The DEGs across enriched gene sets showed gene signatures involved in Wnt signaling, PI3K, chemokines and receptor tyrosine kinases.

Conclusion Specific sets of genes are differentially enriched between EGR-/AR- and ERG+/AR+. These findings suggest that PCas have a distinct gene expression profile in the absence of the T2E fusion, which may act as a driver of disease aggressiveness and racial health disparities in PCa. Identifying these differences in gene-signature expression and signaling transduction pathways will allow stratification of men across races and identify, early on, those individuals who are likely to develop the most aggressive disease.

#5134

Landscape of genomic alterations across solid tumors based on a comprehensive clinical sequencing analysis of 5355 Chinese cancer patients.

Ming Yao,1 Minghui Wang,2 Mingwu Chen,3 Tao Shou,4 Jingyu Cao,5 Hui Chen,1 Aodi Wang,1 Lijuan Chen,1 Jinwei Hu,1 Shuirong Zhang,1 Kai Wang1. 1 _OrigiMed, Shanghai, China;_ 2 _Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China;_ 3 _First Affiliated Hospital of Guangxi Medical University, Shanghai, China;_ 4 _First People's Hospital of Yunnan Province, Shanghai, China;_ 5 _The Affiliated Hospital of Qingdao University, Qingdao, China_.

Background: Precision oncology highlights the individualization of diagnosis, prognosis and treatment based on molecular stratification of cancer patients (pts). Genetic patterns in many types of cancer vary among geographical regions, leading to variation in evidence-based recommendations for clinical management of pts. Genomic alterations (GAs) of solid tumors from Chinese pts have not been fully studied across all cancer types.

Methods: Tumor tissues and matched blood from 5355 Chinese pts were collected and sequenced by a next generation sequencing based 450-gene panel assay. About 60% of the samples were FFPE tissues. In our study, 59% were males and 41% were females with a median age of 58 years old (range 1-92). The major cancer types were NSCLC (36%), CRC (8%), HCC (7%), gastric cancer (5%), pancreatic cancer (5%), and also cholangiocarcinoma, esophageal carcinoma, SCLC, gallbladder carcinoma, ovarian cancer, breast cancer, head and neck cancer, sarcoma, renal carcinoma, uterine neoplasms and others. 1% were pediatric patients . Single base substitutions, short and long insertions/deletions (indels), copy number alterations (CNA), fusions/rearrangements, TMB and MSI status were assessed by NGS.

Results: On average, our study identified 7 mutations per patient. Gene rearrangements and long indels (50bp - 2k) were detected in 15% and 5% of the cancers. In total, 58% of pts harbored GAs in druggable genes defined as targets of FDA approved drugs. Highest mutation frequencies were found in TP53 (60%), EGFR (23%), KRAS (18%), CDKN2A (13%), TERT (12%), PIK3CA (11%), APC (11%), LRP1B (10%), ARID1A (9%), and RB1 (8%) across all tumor types. The frequency of other druggable genes included BRCA1/2 (5%), BRAF (3%), HER2 amplifications (3%), MET amplifications (2%), ALK fusions (2%), RET fusions (0.7%), ROS1 fusions (0.7%), FGFR fusions (0.7%), NTRK fusion (0.6%). Frequency of GAs in druggable genes varied among different tumor types. For instance, EGFR mutations occurred in 49% of NSCLC, 7% of SCLC, 2% of gastric cancer, and 2% of CRC. NTRK fusions enriched in sarcoma (4%), gastric cancer (2%) and CRC (1%). The median TMB of the whole cohort was 4.6 muts/Mb and 6% had TMB higher than 20 muts/Mb. Top 3 tumors with highest median TMB were SCLC, CRC and gastric cancer, while pancreatic cancer and sarcoma had low TMB. As a pan-cancer biomarker for immunotherapy, pts with MSI-H accounted for 1.3% of solid tumors, including 9% of uterine neoplasms, 8% of CRC, 6% of gastric cancer and 2% of ICC.

Conclusions: In summary, we have constructed a large-scale data set including somatic mutations, CNAs, fusions/rearrangements, TMB and MSI status from 5355 Chinese pts with more than 19 tumor types, which provided an extensive understanding of disease-specific indicators, outcomes and therapeutic strategies for both targeted and immune therapies in Chinese pts.

#5135

Genome-wide CRISPR-based screening reveals ABCG2 as a novel drug resistance gene in pancreatic cancer.

Ryne C. Ramaker, Andrew A. Hardigan, Emily R. Gordon, Richard M. Myers, Sara J. Cooper. _HudsonAlpha Institute for Biotechnology, Huntsville, AL_.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly cancers and incidence is increasing due to the increase in risk factors such as diabetes and obesity. One reason for the high mortality rate associated with this cancer is the high rate of resistance to commonly used chemotherapies. Our previous work showed a link between patient survival and expression of drug resistance genes, thus we undertook genome-wide CRISPR knockout and activation screens to identify genes conferring resistance to cytotoxic chemotherapies in two pancreatic cancer cell lines. Our screen revealed hundreds of genes with potential roles in drug resistance, many of which have expression levels that correlate with patient survival. We have validated our top candidates by stably expressing individual guide RNAs in each of three pancreatic cell lines, Panc-1, BxPC-3 and MiaPaCa-2 and screening for viability following treatment with three chemotherapeutic agents, oxaliplatin, gemcitabine, and irinotecan. ABCG2, an ABC transporter, was revealed as our top hit, conferring resistance to multiple drugs in multiple cell lines. Our results would suggest that repression of ABCG2 might improve sensitivity to cytotoxic chemotherapies. We test this hypothesis using an ABCG2 inhibitor and demonstrate the effect of this inhibitor on cell viability, drug sensitivity and transcriptomic profiles in the context of cell lines with variable ABCG2 expression.

#5136

Adapting long read sequencing technologies for targeted and single cell applications.

Shruti Iyer, Sara Goodwin, W. Richard McCombie. _Cold Spring Harbor Laboratory, Woodbury, NY_.

Structural variations (SV), a hallmark of genomic instability in cancer, include insertions, deletions, duplications, inversions, or translocations that can either activate oncogenes or inactivate tumor suppressor genes. These variants contribute to the complexity of the cancer genome but have not been successfully resolved yet. While short-read sequencing has aided cancer genomics, it has performed poorly in SV detection, with false positive and false negative rates of 50% or more. Long-read sequencing generates reads that are >10 kb long and has helped identify thousands of genomic features pertinent to cancer that were previously missed by short-read sequencing. Long reads can span SV with a single continuous read, giving a clearer idea of the variation, its position, and size. Currently, long-read sequencing methods are greatly limited by the DNA preparation step, with fragment lengths and yield often compromised by standard extraction methods. The potential to generate reads at the megabase level relies greatly on library preps using large amounts (several μg) of DNA input, which prevents its application to samples like patient tumors that are often limited in DNA. Our overarching goal is to develop a targeted approach that generates large contiguous reads from moderate to low input DNA masses. To this end, we use different extraction, amplification, fragmentation, and enrichment strategies to efficiently target and resolve complex regions of the genome that are often associated with disease conditions. Development of a targeted long-read sequencing strategy using low DNA input would give us the potential to explore the landscape of genetic variation across several individuals and heterogeneous cell populations. We are currently employing and adapting CRISPR-based targeting techniques to enrich for specific regions of the genome from intact cells and/or high-molecular-weight DNA.

#5137

Comprehensive analysis of POLE and POLD1 mutation in 9322 Chinese cancer patients.

Hua Dong,1 Yuezong Bai,1 Xinkai Cao,1 Yanyan Wang,1 Lin Shi,1 Fugen Li,1 Yizhou Ye,2 Shuyang Sun3. 1 _3DMed, Shanghai, China;_ 2 _3DMED INC, Shanghai, China;_ 3 _Shanghai Ninth People's Hospital, Shanghai, China_.

POLE and POLD1 proofreading mutations can cause ultra-hypermutant-phenotype in cancer, but the distribution of POLE and POLD1 mutation frequency in Chinese population are not clear compared to microsatellite instability (MSI) and tumor mutation burden(TMB). Herein, we examined mutation profiles of POLE and POLD1 in 9322 Chinese cancer patients by two target sequencing panels including POLE and POLD1 gene. Tissue samples from 5866 patients were sequencing with a lab validated CP381 gene panel while plasma samples from 3155 patients were sequencing with a lab validated CT150 gene panel in a CAP certified lab 3DMed. Wet lab experiments and dry lab bioinformatics analysis were done by standard FFPE and ctDNA sample processing and analysis pipeline. POLE and POLD1 pathogenic mutation was defined by published research evidence. Of the 9322 cancer patients, 23 cases showed POLE and POLD1 pathogenic mutations (20 POLE and 3 POLD1), with the total mutation frequency 0.25% (0.35% for tumor sample and 0.006% for plasma sample). However, except for two Intrahepatic cholangiocarcinoma, one melanoma and one endometrial cancer patients, all the left mutation were from colon and rector cancer (CRC), account for a total mutation frequency 1.4% in CRC (19/1335). Although Endometrial cancer is reported with high POLE/POLD1 mutation, there were only 49 endometrial cancer patients in our cohort with mutation frequency 2%(1/49). POLE/POLD1 pathogenic mutations are micro satellite stable (MSS) in 20 cases while the left 3 were MSI-H. Eight cases have co-occurrence with MMR pathway gene loss of function mutation (any one of MSH2/MSH6/MLH1/PMS2), we infer those MMR gene mutations are not driver mutation but passenger mutation induced by POLE and POLD1 proofreading mutations as those eight cases are mostly MSS. Two patients with POLE pathogenic mutation with immune checkpoint inhibitor treatment were followed up for further response evaluation. POLE-and POLD1 pathogenic mutation are very rare in Chinese cancer patients, enrich in CRC type with 2.9% mutation frequency. POLE/POLD1 pathogenic mutations are usually exclusive to MSI-H patients, thus could expand the patient population in colon cancer which have potential to benefit from immune-therapy.

#5138

Inhibition of kPNB1 and NUPR1 binding increase the anti-cancer drug sensitivity.

Chan Hee Park,1 Seung Wook Ham,1 HyeKyoung Shin,2 Kyung Soo Oh1. 1 _Chungang University, Seoul, Republic of Korea;_ 2 _Yonsei University College of Medicine, Seoul, Republic of Korea_.

Karyopherin beta 1 (KPNB1, known as importin beta 1) is carrier protein, it is known as transporter of protein from cytoplasm to nucleus through nuclear pore complexes (NPCs) using KPNAs as an adapters. The nuclear protein-1 (NUPR1, known as p8/Com-1) is identified as a transcription factor and it is showed multifunctional mechanisms related to cellular stress response such as serum-starvation, oxidative stress and drug stimulation. In this study, we investigated protein-protein interaction between NUPR1 and KPNB1, it might be a new target for cancer therapy.For the evaluation of binding affinity between protein and inhibition effect by compound 1, we processed the single molecule binding assay. Cytotoxicity of combination treatment with doxorubicin and compound 1 were validated several cancer cell lines such as MDA-MB-231, PC-3, SK-OV-3, and SiHa. Gene expression patterns were analyzed RNA-Seq and validated with qRT-PCR and western-blotting. KPNB1 bind to NUPR1 as a high affinity compare to KPNB1 single treatment assay and there was no difference according to concentration of NUPR1. KPNB1 and NUPR1 protein-protein interaction was inhibited by compound 1 according to concentration of compound 1 showed 71%, 44%, and 31% of inhibition rate with 50nM, 10nM, 2.5nM of compound 1, respectively. In doxorubicin single treatment, NUPR1 move to nucleus more than 90% of cells compare to non-treated MDA-MB-231 cells. Otherwise, NUPR1 was exist both nucleus and cytoplasm more than 90% cells, which is indicate that translocation of NUPR1 to the nucleus was blocked by compound 1 treatment. And combination treatment showed synergistic effect of cytotoxicity in various cancer cells such as such as MDA-MB-231, SiHa, PC-3, and SK-OV-3 (1.4 ~ 6 fold). Additionally, NUPR1 related genes were showed change of gene expression in compound 1 combination compared to doxorubicin single treatment. In these results suggest that translocation mechanism of NUPR1 into nucleus bind to KPNB1 and this binding was inhibited by compound 1 which is new candidate drug combination method for cancer therapy.

#5139

Development of novel copy number variation (CNV) reference materials in genome in a Bottle background.

Kunal Banjara, Lu Zheng, Samyuktha Dasari, Kara Norman. _Thermo Fisher Scientific, Fremont, CA_.

Introduction:

Advancements in tumor characterization by solid and liquid biopsy are instrumental in the evolution of personalized medicine. Recent improvements in assay sensitivity for profiling DNA variants such as single nucleotide polymorphisms (SNP) and indels have facilitated expansion of both solid and liquid biopsy applications. However, measurement of copy number variations (CNV) is a relatively new application, and few reference materials exist to aid in assay development and optimization. In this study, we demonstrate for the first time the development of 17 whole gene CNV reference standards in a background of the highly characterized NIST Genome in a Bottle GM24385 genomic DNA.

Method:

DNA molecules containing full genes of MET, ERBB2, ERBB3, PIK3CA, EGFR, BRAF, FGFR1, FGFR2, FGFR4, KIT, KRAS, MYC-N, PDGFRA, MDM2, CD274, MYC-L and MYC were each spiked separately into background GM24385 genomic DNA to generate 2.8 copies of each gene. Copy number for each of the 17 CNV samples was determined using Bio-Rad® Droplet DigitalTM PCR (ddPCRTM). To assess linearity over a range of copy numbers, MET and ERBB2 CNV ladder reference materials with copy gains at 6 different levels 3, 6, 9, 12 and 15 copies were also generated. To mimic circulating tumor DNA, each CNV sample was fragmented and size selected to recover a population containing ~170 bp size fragments. Copy number was also verified post size selection using ddPCRTM and the Ion S5TM XL system. Multiplexed triple CNV controls with MET, EFGR and ERBB2 were also developed at 3, 6, 9, 12 and 15 copies and analyzed by ddPCRTM.

Results:

Copy number for all 17 genes measured at 2.80 ± 0.28 copies by ddPCR. When tested in a ctDNA copy number ladder format, MET and ERBB2 showed good linearity of R2=0.99 for observed versus expected # of copies. Good correlation between ddPCR and NGS was also observed, with a slope of 1.004 for MET and 0.969 for ERBB2 across increasing copy numbers and standard deviation (SD) of ± 10%. Notably, SD increased at copy numbers higher than 12. The average size of fragmented ctDNA CNV reference standards were observed to be ~170 bp on the Agilent 2100 Bioanalyzer system. Finally, the multiplexed triple CNV control also performed as expected on ddPCR with SD of ± 10%.

Conclusion:

A novel method for producing CNV and ctDNA CNV controls has been developed that enables preparation of any copy gain level with a known gDNA background. Simpler QC materials mimicking patient samples will enable simpler CNV test method development and analytical validation, which will be critical for laboratories to introduce CNV solid and liquid biopsy testing into the field.

#5140

Discovery of biomarkers predicting response to radiation and anti PD1 therapy in head and neck squamous cell cancer.

Joon Sang Lee,1 Michele Sanicola-Nadel,1 Alexei Protopopov,1 Emma Wang,1 Joachim Theilhaber,1 Tun Tun Lin,1 Michael Korrer,2 Sohini Roy,2 Young Jun Kim,2 Jack Pollard1. 1 _Sanofi, Cambridge, MA;_ 2 _Vanderbilt, Nashville, TN_.

Introduction: Treatment of head and neck squamous cell cancer (HNSCC) frequently includes surgery, radiation, chemotherapy, targeted agents, and increasingly combination of the former with immune checkpoint inhibition. An understanding of the mechanisms mediating response to these therapies as well as biomarkers that can better tailor therapy on an individualized basis and reduce treatment-related toxicity would benefit patients.

Methods: To uncover these mechanisms and biomarkers, we analyzed whole transcriptome RNA-Seq data of two different cohorts of head and neck squamous cell cancer patients: pre and post-treatment samples from radiation therapy and pre-treatment samples from anti-PD1 therapy. We evaluated the abundance of eight immune and two stromal cell populations and pathway activity changes by using Gene Set Variation Analysis (GSVA).

Results and Conclusions: Following radiation genes involved in epithelial to mesenchymal transition, extracellular matrix remodeling, and wound healing are upregulated. Additionally, there is evidence of tumor microenvironment remodeling post-radiation consistent with increased fibroblast abundance and decreased immune cell infiltration. Furthermore, innately resistant tumors to anti-PD1 also display similar signatures of gene regulation, suggesting a common mechanism mediating response to both types of treatment. Notably, these signatures are strongly correlated to activation of the transforming growth factor (TGF-β) signaling pathway and suggest that attenuating its signaling may improve anti-PD-1 response and also response to radiation treatment in head and neck squamous cell cancer.

#5141

T-cell receptor repertoire analysis by next-generation sequencing peripheral blood mononuclear cells from multiple myeloma or smoldering multiple myeloma patients.

Yong Zhang, Svetlana Petrovskaya, Emma C. Scott, Luis Santana-Quintero, Tigran Ghazanchyan, Amy Rosenburg, V. Ashutosh Rao, Gideon M. Blumenthal, Marc Theoret, Richard Pazdur, Julia A. Beaver, Dickran Kazandjian. _FDA, Silver Spring, MD_.

Multiple myeloma (MM) is a hematological malignancy mostly assessed by bone marrow involvement, so it is attractive to develop a method using peripheral blood. We hypothesized that peripheral blood mononuclear cells (PBMCs) may replace tedious and invasive bone marrow biopsies as a biomarker. In this study, we aimed to investigate if T-cell receptor (TCR) repertoire has clinical significance in MM or Smoldering MM (SMM) diagnosis or prognosis. Using baseline genomic DNA (PBMC) from 24 patients with SMM and 31 patients with MM, we performed next-generation sequencing on the Illumina NextSeq 500 platform by immunoSEQ TCRB Kits from Adaptive Biotechnology to quantitatively assay the complementarity determining region 3 (CDR3) of human T-cell receptor β (TCRβ) gene. To evaluate T-cell diversity and clonality, we performed pipeline analysis by ImmunoAnalyzer from Adaptive Technology, and downloaded sequencing data with manual analysis by JMP13 software. Compared to TCRB data from 280 normal control (Age > 40) PBMCs (Nat Genet. 2017, 49(5):659-665), we found less TCR templates from both MM and SMM baseline based on size of TCRB repertoire, and less TCR rearrangements or unique clonotypes based on structure of TCRB repertoires. Only MM baseline showed higher maximum clonotype frequency. No difference of T-cell clonality was found from MM and SMM. Furthermore, we performed the same sequencing on 18 SMM patients at different treatment time points including chemotherapy after cycle 8, 20, and 32. Our results showed patient samples after cycle 8 had less TCR templates and TCR rearrangements compared to those from the other three time points although no significant difference were found in maximum clonotype frequency or T-cell clonality. TCR overlap analysis will be further performed to track potential clonotype changes. Thus, we identified TCR repertoire analysis may have potential clinical significance as biomarker assay for MM and SMM patients.

#5142

New genomic alterations are frequently detected in recurrent nasopharyngeal carcinoma compared with the primary tumors.

William C. Cho,1 Yi-Ting Yang,2 Kein T. Tan,2 Victor W. Ma,1 Wah Cheuk,1 Roger K. Ngan,3 Shu-Jen Chen2. 1 _Queen Elizabeth Hospital, Kowloon, Hong Kong;_ 2 _ACT Genomics, Co. Ltd., Taipei, Taiwan;_ 3 _The University of Hong Kong, Gleneagles Hong Kong Hospital, Hong Kong_.

Introduction: Nasopharyngeal carcinoma (NPC) is a common cancer in Southern China and Southeast Asia. The clinical outcomes of patients with early stage disease have improved significantly in recent years, but a substantial number of patients would develop recurrent disease and it is the major cause of mortality. Understanding the genetic mechanism of relapse may help develop novel treatment modalities and strategies for this group of patients. This study aims to compare and identify the genetic alterations in primary and recurrent NPC by next-generation sequencing.

Materials and Methods: The archived paired samples of 7 primary and 8 matched relapsed tumor tissues from seven NPC patients (one with 2 relapsed points) who developed recurrence after treatment were retrieved. Using deep targeted next-generation sequencing, we evaluated a panel of 440 genes in these 15 NPC samples. Genomic alterations found in cancer samples at the first diagnosis (primary tumors) were compared with those found in the relapsed tissues (progression after treatment).

Results: Overall, we found that all patients showed a number of mutations in both the primary tumors and relapsed tissues. The most frequently mutated genes were MUC16 (85.7%), ADAMTS9 (80%), MITF (73.3%), ADAMTSL1 (73.3%), PTPRD (66.7%), TEK (66.7%), FOXP1 (53.3%) and IRS2 (53.3%). We observed that MCL1 (42.9%), SOX2 (42.9%) and PIK3C2G (28.6%) mutations were only found in the primary tumor. Comparing to the primary tumors, all patients acquired new mutations in the relapsed tissues. The seven patients have each acquired 6, 13, 14, 20, 29, 31 and 35 new genomic alterations respectively. PAX5 (42.9%), PIK3CA (42.9%), CREBBP (28.6%), DPYD (28.6%), FANCG (28.6%), BIRC2 (28.6%), BIRC3 (28.6%) and PTEN (28.6%) were the most frequently detected gene mutations newly acquired in the relapsed tissues.

Conclusion: This study demonstrated that mutation events might be present at diagnosis or arise during cancer treatment. The finding that all primary tumors harboring a number of mutations could explain the subsequent relapse and might therefore warrant more precise therapies. Tumor relapse is associated with the acquisition of additional gene mutations, particularly copy number variant deletion in PAX5 gene and single nucleotide variant missense in PIK3CA gene. Our results revealed substantial discordances between the primary tumors and recurrences, stressing the need for analysis of the relapsed tissues. Further analysis of these mutations and the study of their downstream protein may be useful for the development of novel treatment for this group of patients.

#5143

Transcriptomic analysis of primary retinoblastoma with affymetrix HTA2 chip using a detection score reveals a central core of mRNA shared by all the samples.

Diana E. Alvarez Suarez,1 Hugo Tovar,2 Enrique Hernández,2 Manuela Orjuela,3 Lourdes Cabrera,4 Claudia Hernández,5 Daphne García,5 Adriana Hernández,6 Verónica Ponce6. 1 _CINVESTAV-Centro de Investigación Y estudios Avanzados del Instituto Politécnico Nacional., ciudad de México, Mexico;_ 2 _INMEGEN,, Ciudad de México, Mexico;_ 3 _Columbia University, New York, NY;_ 4 _Hospital Infantil de México, Ciudad de México, Mexico;_ 5 _Hospital de Pediatría, CMN SXXI, Ciudad de México, Mexico;_ 6 _Hospital de Pediatría, CMN SXXI, IMSS, ciudad de México, Mexico_.

Introduction: Retinoblastoma (Rb) is an intraocular malignant tumour of early childhood, considered the most robust clinical model of genetic predisposition to develop cancer. Previous transcriptomic studies in Rb are based on differential analysis of expression data, ignoring the detection scores that can be obtained from high throughput technologies. The detection score allows to obtain profiles of present/absent mRNAs across samples without the need of a control sample. In this study, 8 primary cultured tumors and Rb cell line Y79 were analyzed using the detection scores, with the aim to discover what is similar and what is different among them in terms of mRNA detection.

Methodology: Tumors were obtained from enucleated eyes of Rb patients treated at Hospital de Pediatría, CMN SXXI, Instituto Mexicano Del Seguro Social (IMSS) and Hospital Infantil de México Federico Gómez, Secretaría de Salud in Mexico City. Tumor tissues were cultured for a week and total RNA extracted. We used Affymetrix Human Transcriptome Array 2.0 able to detect many isoforms. Pre-processing included normalization with RMA (Average Robust Multiarray) and DABG (Detection Above Background) to calculate the detection score for each exon as present or absent. We carried out subsequent analysis to determine the detection score of each transcript and each gene in two corresponding datasets. For each of these detection datasets, hierarchical clustering (HC) was performed producing two color detection maps for present and absent elements.

Results: We obtained detection scores for 21,986 genes and 59,629 annotated transcripts (isoforms). In the detection map of genes obtained with HC, we discovered a central core of 3,391 mRNAs cluster shared by all the samples, while in the detection map of transcripts we discovered a 12,799 mRNAs cluster shared by all the samples. Absent mRNAs cluster were also shared across the samples with 9,005 in the genes and 18,651 in the transcripts detection maps. Notably, variability accounted for a cluster of 9,590 and 28,179 across the genes and transcripts detection maps, respectively. Furthermore, we determined the Rb transcriptomic size by counting the mRNAs in each sample and calculating the average across the datasets with 8,238 and 28,396 mRNA detected, 13,747 and 31,232 not detected corresponding to gene or transcript detection maps. When Y79 was introduced in the datasets, these numbers change considerably, and the corresponding detection maps show mRNA clusters specific for the cell line, but not present in the primary tissues.

Conclusion: Rb samples shared a central mRNA cluster of detected elements and a cluster of not detected elements, despite tumor heterogeneity. This systems level analysis brings a coherent and easy way for biological interpretation of high throughput data.

#5144

The potential biomarkers and rational combination of immunotherapy in microsatellite stable colorectal cancer.

Xuman Lu,1 Zhe Ji,2 Jing Zhao,3 Yuzi Zhang,3 Mengli Huang,3 Shangli Cai,3 Jun Huang1. 1 _The Sixth Affiliate Hospital of Sun Yat-sen University, Guangzhou, China;_ 2 _Northwestern University, Chicago, IL;_ 3 _3D Medicines Inc., Shanghai, China_.

Purpose Immune checkpoint blockades (ICBs) have been reported to be used in early stage colorectal cancer (CRC) as neoadjuvant therapy. However, it seems that microsatellite stable (MSS) CRC patients obtained no significant clinical benefit in studies with small sample size. We aimed to explore the potential biomarkers and rational immunotherapy to optimize response to ICBs in MSS CRC.

Procedures Whole exome sequencing and RNAseq of 612 CRC patients from The Cancer Genome Atlas were analyzed. T cell dysfunction/exclusion signatures and Tumor infiltrating lymphocytes were calculated from TIDE model and TIMER algorithm, respectively. Mann Whitney U test and Kruskal test were used to compare the difference among two or more than two groups. P value was adjusted by BH method for multiple comparisons.

Results There were 109 stage I, 228 stage II, 184 stage III and 91 stage IV in 612 CRC patients. The MSI-H prevalence, tumor mutation burden, PD-L1 mRNA and CD8 T cell infiltration decreased with the increase in pathologic stage (all P < 0.01). In MSS CRC, mRNA expression of immune checkpoint related genes including PD-L1, CTLA4 and LAG3 were significantly higher in stage I-III than stage IV (adjusted P < 0.01, < 0.01 and 0.04, respectively). For immune activation genes (CD8A, CXCL9, CXCL10, GBP1, GZMB, GZMA, IFNG, FAS, STAT1, TAP1), their mRNA expression levels increased with the decrease of stage (All adjusted P < 0.01). Besides, stage I-III MSS CRC had lower T cell exclusion signature score than stage IV (adjusted P = 0.02). No similar results were observed in MSI-H. Compared with MSI-H, MSS had higher M2 macrophage signature score (P < 0.001), but no association were observed between M2 macrophage signature and stage.

Conclusions Early stage CRC with MSS had higher levels of positive predictive features for ICBs treatment, which suggested stronger immune responsiveness. Combination of M2 macrophage inhibitor and ICBs has the potential to turn "cold tumor" into "hot tumor" and acts as a promising therapeutic regimen, especially for early stage CRC. More clinical trials are needed to confirm this hypothesis.

#5145

A new discovering motif on SOS1 PR domain clues Grb2-SOS1 association.

Tsung-Jen Liao,1 Hyunbum Jang,2 Ruth Nussinov,2 David Fushman1. 1 _University of Maryland, College Park, MD;_ 2 _National Institute of Health, Frederick, MD_.

Grb2 is an adaptor protein that recruits Ras-specific guanine nucleotide exchange factor (GEF), such as Son of sevenless 1 (SOS1), to the plasma membrane. SOS1 exchanges GDP by GTP, activating Ras. Grb2 consists of an SH2 domain in the middle and flanks nSH3/cSH3 domains at both ends. Both nSH3/cSH3 domains bind to SOS1's C-terminal proline rich (PR) domain and have the preference for interacting with the PXXPXR motif. Our NMR data suggest that the arginine residue, followed by prolines in the PXXPXR motif, is responsible for the SH3-SOS1 association, yielding large chemical shift perturbations (CSPs) on the binding interface of SH3. The SOS1's PR motif, PVPPPVPPRRRP, exhibits the strongest binding affinity to both SH3 domains. Using comprehensive techniques involving molecular dynamics (MD) simulation and NMR measurement, we uncover a new SOS1's PR motifs, PKLPPKTYKREH, which shows strong binding to cSH3 but exhibits very weak affinity to nSH3. Unlike the arginine residue in the PXXPXR motif, the lysine residue in the PXXPPKXXKR motif induces weak interaction with cSH3. However, high affinity interaction is mainly derived from the hydrophobic interaction between the PXXP motif and cSH3, indicating the different binding modes of interaction. Our results suggest that Grb2 nSH3/cSH3 bind to SOS1 at distinct locations and provide atomic detailed models of Grb2-SOS1 association. Recent Grb2 targeted therapy reveals that peptide inhibitors, which interrupt Grb2-SOS1 interaction, effectively prevent the migration of breast cancer cell. The mechanism of Grb2-SOS1 interaction may enlighten the promising drug treatment development.

#5146

ATP-binding cassette transporter, ABCG2, activation is responsible for chemo-resistant, SIK3 low expressed ovarian cancer.

Keng-Fu Hsu,1 Yu-Ling Liang,1 , Chin-Han Wu,1 Neng-Yao Shih2. 1 _National Cheng Kung Univ. Hospital, Tainan, Taiwan;_ 2 _National Health Research Institutes, Tainan, Taiwan_.

Background: Epithelial ovarian cancer (EOC) has a high tumor-associated mortality rate among the gynecological cancers because of chemo-resistant development.

Materials and Method: The human EOC cell lines, SKOV3, OVRCA4, were obtained from the American Type Culture Collection (ATCC). For determine ATP-binding cassette transporters and SIK3 expression, Western blot and Quantitative real-time polymerase chain reaction (qRT-PCR) were used. We used a lentiviral small-hairpin RNA (shRNA) system to knock down SIK3 in the ovarian cancer cell lines. Microarray data derived from SKOV3, OVRCA4 cells expressing sh-luc, sh-SIK3 (01), and sh-SIK3 (63) were analyzed by MetaCore and Ingenuity Pathways Analysis (IPA). Top 10 up-and down-regulated genes and other metastasis-related genes and 5 most highly enriched networks were identified. For determining cell viability when cells treated with cisplatin and Taxol, WST-1 assay was used. Immunohistochemistry and quantification of ABCG2 and SIK3 were performed in EOC tissues. Statistical analysis of OS and PFS were performed using SPSS statistical software (Version 22.0; IBM Corp, Armonk, NY). Survival curves were generated using the Kaplan-Meier method, and differences in survival were assessed using the log-rank test.

Results: Knockdown SIK3 expression upregulates ABCG2 expression, activity as well as resistance to chemotherapeutic agents. The ABCG2 and SIK3 expression levels in 204 EOC patient were mutually reversed. In all stages of the disease, high expression of SIK3 (SIK3-H) was associated with a significant better OS, PFS, compared with low expression of SIK3 (SIK3-L) (127.0 vs 46.0 months, 66.0 months vs 26.0 months respectively). Patients with SIK3-L and ABCG2-H carried the worst prognosis.

Conclusion: (1) High expression of SIK3 indicates a better prognosis in primary ovarian cancer and serous type disease, especially in advanced serous disease. (2) Ovarian cancer patients with SIK3-L and ABCG2-H expression carried the worst prognosis due to chemoresistance

#5147

Clinical benefit of molecularly-guided treatment strategies on progression free survival in patients with advanced gastrointestinal malignancies.

Blake Buzard, Tyler Gonser, Melissa Hinrichsen, Anu Amallraja, Kirstin Williams, Pradip De, Brian Leyland-Jones, Rachel Elsey, Tobias Meissner, Casey Williams. _Avera Cancer Institute, Sioux Falls, SD_.

Introduction: Despite many advances, the treatment for most patients with advanced solid tumors in the abdomen continues to be a clinical challenge. The purpose of our study was to evaluate the clinical benefit of molecularly-guided treatment strategies on progression free survival in patients with advanced gastrointestinal malignancies.

Materials and Methods: We performed a retrospective chart review of 112 patients with advanced (>90% stage IV) gastrointestinal cancer (n=60 colon, n=21 pancreas, n=10 cholangio, n=8 esophageal, n=13 others) that underwent targeted DNA sequencing of 343 genes and assessed mutations, treatments and outcomes. We compared evidence-based standard of care therapy to molecularly-guided treatment on progression free survival time pre- and post-sequencing. We further assessed KRAS mutational status on patient outcomes, as well as potential correlation between time to molecularly-guided treatment and patient outcomes.

Results: Out of 112 patients sequenced and presented to our molecular tumor board, 50% received molecularly-guided treatment. Most frequent molecularly-guided regimens included the addition of either a MEK and/or an mTOR inhibitor. The MEK inhibitor Trametinib was recommended for 58/112 patients, due to activating mutations in the RAS-MAPK pathway (46 of all patients were KRAS mutated), and 27/58 patients received Trametinib. The mTOR inhibitor Everolimus was recommended for 32/112 patients due to PI3K-mTOR pathway activating mutations, with 18/32 patients receiving Everolimus. Doublet therapy of Trametinib and Everolimus was given to n=13 patients, with n=5 patients receiving Everolimus only and n=14 patients receiving Trametinib only as targeted therapy. Comparing patients for whom either Trametinib and/or Everolimus was given after recommendation with patients who did not receive the recommendation, we did not find a difference in outcome. However, overall progression free survival (PFS) increased for patients receiving molecularly-guided treatment after sequencing compared to patients who received standard treatment, by an average PFS of 9 months. Patients who did not receive molecularly-guided therapy until later in their disease course had poorer outcomes compared to those who received it earlier in their treatment.

Conclusions: Molecularly-guided therapy is an emerging treatment strategy for patients with gastrointestinal cancers. Our work showed that progression free survival was increased in patients who received molecularly-guided therapy compared to standard treatment. More research is needed to assess any direct correlations between most genetic mutations and clinical outcomes.

#5148

Immunohistochemical Analysis of High-Risk Prostate Cancer before and after Treatment with Neoadjuvant Intense Androgen Deprivation Therapy.

Nicole V. Carrabba,1 Scott Wilkinson,1 Stephanie Harmon,1 Baris Turkbey,1 Huihui Ye,2 Peter Pinto,1 David VanderWeele,1 Fatima Karzai,1 William Dahut,1 Peter Choyke,1 Adam Sowalsky1. 1 _NIH, Bethesda, MD;_ 2 _Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA_.

Background

Prostate cancer is the second most commonly-diagnosed and second leading cause of cancer deaths in men in the United States. The balance of intervention and avoiding unnecessary treatment poses a challenge in prostate cancer. Predicting the risk of disease progression is difficult due to frequent intratumoral heterogeneity and inadequate tissue sampling in tumor classification. Our hypothesis proposes that resistance or response to novel therapies can be predicted through biopsy examination of tissue obtained prior to treatment, when these biopsies are representative of major high-risk regions.

Methods

The basis for this correlative study is a clinical trial in which men with intermediate and high-risk features were treated with enzalutamide + GnRH agonist for 6 months prior to radical prostatectomy. We obtained samples of tissue before treatment by MRI/US-fusion biopsy, corresponding to areas of pathologic response or resistance. After mapping residual tumor burden in these RP specimens and the corresponding regions to the pre-treatment biopsies, we examined several potential mechanisms that mediated resistance or sensitivity by performing quantitative biopsy and whole mount immunohistochemistry (IHC) on an autostainer. These include AR, PTEN, GR and PD-L1 among others. Using Definiens Tissue Software, we quantified IHC staining intensity to measure morphological characteristics in pre-and posttreatment specimens.

Results

Initial results highlight the ability to successfully map pre-treatment biopsy to post-treatment RP through combinatorial imaging. Residual tumor shows a diversity of oncogenic mechanisms and pathways of resistance by immunohistochemical staining across a panel of IHC markers in prostate cancer representing oncogenes and tumor suppressors. Result show that in some patients, mechanisms remained consistent pre and post-treatment, and many patients display intratumoral heterogeneity having multiple foci with different morphologies. At the Annual Meeting, we will report the final quantitative results across the cohort.

Conclusions

Our ability to understand the impact of intense neoadjuvant ADT is crucial for applying precision approaches to future combination therapies with the goal of evoking more durable responses. This research will ultimately identify predictors of response or resistance to this novel therapy for high risk prostate cancer patients. Clinicians will be able to better tailor treatment, preventing unnecessary treatment and comorbidity associated with this treatment in patients who are predicted to not respond. Furthermore, identification of recurrent resistance mechanisms may allow for precision adjuvant therapy in select nonresponders. 

### Cellular and Stromal Interactions of Tumor Cells

#5149

**Subclonal sociability: Interactions of** HER2 **and** PIK3CA **mutant cells in breast cancer.**

Ian Waters,1 Swathi Karthikeyan,1 Lauren Dennison,1 David Chu,2 Ben Ho Park3. 1 _Johns Hopkins School of Medicine, Baltimore, MD;_ 2 _University of California San Francisco, San Francisco, CA;_ 3 _Vanderbilt University Medical Center, Nashville, TN_.

Background: Tumor heterogeneity is a common characteristic of many cancers and plays an important role in their development and eventual metastasis. The traditional clonal evolution model describes cancer cells as descendants of a single progenitor that has accumulated a stepwise series of mutations. However, recent sequencing studies have revealed the presence of genetically distinct subclones in numerous tumors. PIK3CA is the most commonly mutated gene in breast cancer while HER2 is overexpressed in ~20% of breast cancers. Activating missense mutations in the gene that encodes HER2 have also been discovered, and our lab has previously shown the effects of these mutations and their synergy with PIK3CA mutations in a double-mutant cell. This project explores the interactions between cells with either a HER2 or a PIK3CA mutation when they are present as genetically distinct subclones in co-cultures.

Methods: Isogenic MCF-10A cells containing mutations in PIK3CA, HER2, or no mutation were co-cultured in growth factor-deprived conditions. In these conditions, PIK3CA mutant cells are known to be able to grow, while HER2 mutant and wild-type (WT) cells arrest. The cellular makeup of the co-cultures was determined using droplet digital PCR (ddPCR) with probes against the respective mutations. Cells were tagged with stable expression of either zsGreen (PIK3CA mutants) or tdTomato (HER2 and WT) and observed under fluorescent microscopy. Further imaging was performed using the IncuCyte live cell imaging system (Essen Bioscience), which allowed for periodic imaging of cells in culture. Co-cultures were treated with Neratinib or Alpelisib (BYL719), which are HER1/2/4 and PIK3CA inhibitors, respectively. Cells in each condition were counted and ddPCR was performed to determine allelic fractions of the co-cultures.

Results: When cultured together, PIK3CA mutant cells can induce proliferation in HER2 mutant cells, but not WT cells. Over time, HER2 mutant cells grow to make up >50% of the co-cultures. Imaging and transwell assay data (not included on poster) implicate cell-cell contact rather than paracrine factors as the primary driving factor of this phenotype. HER2 and PIK3CA inhibitors can effectively target the co-cultures and inhibiting the growth of the PIK3CA cells appears to halt the growth of the HER2 mutants as well.

Conclusions: PIK3CA mutant cells can induce proliferation of quiescent HER2 mutant cells in growth-factor deprived conditions. This effect appears to the cell-cell contact mediated rather than driven by secreted factors. Co-cultures that contain PIK3CA and HER2 mutant cells have therapeutic vulnerabilities that can be exploited using drugs that are currently approved or in clinical trials. These results have important implications in understanding early tumor development and suggest that therapeutic approaches that target subclonal populations may be effective in certain tumors.

#5150

Adipose-derived stem cell disruption of gap junction intercellular communication in obesity-associated endometrial cancer.

Li-Ling Lin,1 Srikanth Polusani,1 Guangcun Huang,1 Chun-Lin Lin,1 Chiou-Miin Wang,1 Nicholas Lucio,1 Mikhail Kolonin,2 Alexes Daquinag,2 Pawel Osmulski,1 Bruce Nicholson,1 Edward Kost,1 Tim Huang,1 Nameer B. Kirma1. 1 _UT Health Science Center at San Antonio, San Antonio, TX;_ 2 _UT Health Houston, Houston, TX_.

Endometrial cancer is the most common gynecologic cancer. It is divided into two major types, a less aggressive endometrioid type and a more aggressive serous type. Recent clinical observations underscore obesity as a major risk factor for endometrial cancer. In this study, we hypothesized that obesity can result in long term epigenetic modifications in the endometrial compartment, leading to the promotion of endometrial cancer. We examined the role of adipose-derived stem cells (ASCs), which are adipocyte progenitors, in the endometrial tumor microenvironment. By staining for ASCs in a tumor tissue microarray, we observed ASC infiltration that was specific to samples from obese patients, correlating with body mass index (R2=0.77, p<0.001). Using a co-culture system to simulate ASC microenvironmental effects and transcriptomic analysis, we showed that exposure of immortalized endometrial epithelial cells (EECs) to ASC secreted factors led to the repression of cell-cell communication pathways, most notably gap junctions and related factors tight junction proteins TJP and PKC genes. This gene repression was associated with induction of DNA Methylation in the promoter of the major GJ gene GJA1 (encoding connexin 43, Cx43) in the ASC-exposed EECs. Importantly, we further demonstrated this DNA hypermethylation in the promoters of the GJA1, TJP2 and PKC genes in primary endometrial tumors from obese patients compared to non-obese patients. We assessed the effects of epigenetic regulation on gap junction intercellular communication (GJIC) and cell-cell interactions using cellular calcein dye transfer assays and atomic force microscopy (for nano-scale assessment of cell-cell adhesion) by treating endometrial cancer cells with a demethylating agent (DAC). DAC treatment resulted in increased level of cell-cell adhesiveness and communication via gap junction coupling. Specific reactivation of GJA1 (Cx43) by expression vector in endometrial cancer cells led to decreased cellular motility. Because we found that PAI-1 is a major adipokine in the ASC secretome, inhibition of PAI-1 in ASC-exposed EECs led to a cell population expression profile with lower GJ expression, based on single-cell PCR studies. Collectively, the data demonstrate multi-scale regulation of cellular communication via paracrine actions and direct cell-cell coupling via gap junctions by epigenetic silencing influenced by ASCs. This leads to disruption of cellular homeostasis and enhanced motility, promoting endometrial cancer in obese patients.

#5151

Deciphering the molecular mechanisms driving infiltrative histopathological type of colorectal cancer liver metastases.

Miran Rada, Anthoula Lazaris, Stephanie Petrillo, Abdellatif Amri, Peter Metrakos. _McGill University, Montreal, Quebec, Canada_.

Colorectal carcinoma (CRC) remains the second leading cause of cancer death in the western world. Over 50% of CRC patients develop liver metastases (LM) and 90% will succumb to their disease. Liver resection of the LMs provides the only possibility of cure, but only 20% of colorectal cancer liver metastases (CRCLM) patients are resectable. The combination of angiogenic inhibitors (AI: anti-VEGF) with chemotherapy is the current form of treatment. Unfortunately, 65-70% of the patients continue on chemotherapy until resistance develops and then are treated with second, third and some times a fourth line of treatment, with an expected median overall survival of 24-28 months. We have no way of identifying those CRCLM patients that would respond/benefit to the addition of anti-angiogenic therapies (e.g. Bevacizumab). Recently we have identified two CRCLM histologic growth patterns (HGP) that predict treatment response and survival: 1) Desmoplastic (DHGP), a desmoplastic ring separating cancer cells from the liver parenchyma and lesions grow by angiogenesis; 2) Replacement or infiltrative (RHGP), tumor cells infiltrate the parenchymal cells in the liver as the lesions grow by co-opting the sinusoidal blood vessels between the liver cell plates. We showed that CRCLM patients with predominantly desmoplastic HGP metastasis receiving AIs plus chemotherapy have more than double the 5-year overall survival compared to patients with replacement HGP who have received the same treatment. In addition, our clinical data revealed that Angiogenic Inhibitors could negatively affect outcomes in patients with replacement HGPs. These non-angiogenic lesions do not respond to angiogenic inhibitors. To further our understanding of the molecular differences between the two HGPs we demonstrated by knocking out ARPC3 (Actin-related protein 2/3 complex subunit 3, involved in actin polymerization) in the human colon cancer cell line, HT-29, that cancer cell motility is a crucial process that regulates histological growth pattern in CRCLM. HT29 CRC cells injected directly into the mouse liver grow into replacement HGPs, while HT29s silenced for ARPC3 grow into desmoplastic HGPs lesions. However, the molecular mechanisms that regulate ARPC3 in CRCLM remain unknown. To further dissect the molecular mechanisms differentiating desmoplastic from replacement HGPs, we performed RNA-seq analysis of CRCLM lesions from chemonaïve patients. Our data revealed that both TGFβ1 and RUNX1 were upregulated in RHGP comparing to DHGP lesions. This has been further validated by immunoblotting and immunohistochemistry. Consistently, RUNX1 has been reported as a downstream of TGFβ1 and transcriptional factor for ARPC3. Collectively, our data suggests that TGFβ1 and RUNX1 contribute to the formation of infiltrative type of colorectal cancer liver metastases possibly through upregulation of ARPC3.

#5152

EMT cells increase the metastatic potential of neighboring carcinoma cells via non-canonical activation of GLI signaling through secretion of VEGF-C.

Deguang Kong,1 Deepika Neelakantan,1 Hengbo Zhou,1 Michael T. Lewis,2 Heide L. Ford1. 1 _University of Colorado Denver Anschutz Medical Campus, Aurora, CO;_ 2 _Baylor College of Medicine, Houston, TX_.

Breast cancer is a devastating disease that claims around 50 million lives per year worldwide, and almost all of these deaths result from metastatic disease rather than from primary tumor burden. Recent studies clearly demonstrate that there is significant intratumor heterogeneity in breast cancer, and that this heterogeneity contributes to malignant progression. Our laboratories previously demonstrated that breast cancer cells that had undergone an oncogenic epithelial-to-mesenchymal transition (EMT) could increase metastasis of neighboring cells via activation of non-canonical GLI transcription factor activity in a paracrine manner that is dependent on Six1 expression in the EMT cells. However, the mechanism by which these Six1-expressing EMT cells activate GLI signaling thereby imparting aggressive properties on neighboring cells remained unknown. Herein we describe the novel discovery that VEGF-C, which is transcriptionally upregulated by Six1, mediates paracrine non-canonical activation of GLI and resultant enhancement of metastatic properties in cells that do not express Six1. Our data demonstrate that VEGF-C is upregulated in HMLER-Snail1 cells, MCF7-Six1 cells and in Met-1 cells endogenously expressing Six1, in a manner that depends on Six1 expression, and that VEGF-C is secreted into the conditioned media (CM) of these cells. Inhibition of VEGF-C in the HMLER-Snail1, MCF7-Six1, and the Met-1 models abrogates paracrine GLI activation and attenuates non-cell autonomous induction of proliferation, migration, and invasion in vitro. In vivo, we show that cells expressing Six1 can enhance the growth and metastasis of those not expressing Six1, and studies will be discussed that examine whether VEGF-C inhibition in Six1 expressing cells disrupts the crosstalk and inhibits non-cell autonomous induction of metastasis by Six1. Finally, by interrogating the TCGA dataset we find that VEGF-C and GLI1 expression significantly positively correlate in human breast cancer encompassing all molecular subtypes. Taken together, these data suggest that VEGF-C secretion may be a novel and conserved paracrine means by which EMT cells activate GLI in neighboring tumor cells that do not express these EMT-inducing transcription factors (TF), ultimately enhancing overall metastasis of heterogenous breast tumors.

#5153

Measuring T-cell avidity and enrichment using an acoustic force based technology.

Ali Raja,1 Wouter Scheper,2 Elena Merino,1 Rens Braster,1 Gerrit Sitter,1 Felix Oswald,1 Ton Schumacher,2 Andrea Candelli,1 Leif Anderson1. 1 _Lumicks, Boston, MA;_ 2 _University of Amsterdam, Amsterdam, Netherlands_.

The key driver for effective immunotherapies is the overall strength binding of the immune cell and the target cell (e.g. tumor cells). The overall strength is known as 'avidity', a parameter reflecting interaction efficiency. One of the main obstacles has been the generation of immunotherapies promoting effective and long-lasting immune responses, due to the lack of tools measuring this parameter. Here, we have made used of acoustic forces as a novel method to isolate immune cells based on their avidity to specific targets, such as (tumor) cells. The force that cell-target need to experience to be separated is called 'rupture force'. In this study, we were able to identify the rupture forces of tumor specific and non-specific T cells and separate these different populations applying controllable acoustic forces. T cells engineered with a melanoma antigen recognizing T-cell receptor needed 6 times more relative force than the control, non-specific T cells, to be separate from their melanoma cells. These findings indicate that the specific melanoma T cells bind with higher efficiency and higher avidity than the non-specific ones, and that they can be separated with this method. Furthermore, 1.4 to 3.6-fold enrichment of high avidity T cells from a mixed specific: non-specific (1:10) T cell population was obtained after their isolation using acoustic forces and confirmed using FACS measurements. Therefore, we demonstrate here that acoustic forces can be use as a tool to measure avidity.

#5154

Developing Micro-fluidic Chip to understand Altruistic stem cell reprogramming.

Bidisha Pal,1 Sorra Sandhya,2 Joyeeta Talukdar,3 Lekhika Pathak,2 Hong Li,4 Bikul Das1. 1 _The Thoreau Lab for Global Health, Lowell, MA;_ 2 _KaviKrishna Lab, Assam, India;_ 3 _Kavikrishna Lab, Assam, India;_ 4 _Forsyth Institute, Cambridge, MA_.

Background:Tumor microenvironment (TME) is a complex network of cancer and stromal cell interactions in specialized niches. Cancer stem cells (CSCs) and mesenchymal stem cells (MSCs) co-exist as significant components of TME that contribute to tumor progression. However, it is not clear whether MSCs compete or co-operate with CSCs for niche occupancy. In principle, stem cell competition eliminates the competing neighbors (e.g. p53 mutant cancer cells) while stem cell altruism enhances group-fitness in the niche. We found that the conditioned media of oral cancer CSCs, ABCG2+ cells, induced stem cell altruism in CD271+ MSCs (1). This indirect demonstration suggests the potential role of stem cell altruism in TME mediated tumor progression. Here we hypothesize that CSC mediated stem cell altruism of MSCs can be studied using an organ-on-chip microfluidic device. Methods:Naïve CD271+ MSCs were maintained in vitro by using an established culture method that we previously reported (2). Development of organ-on-chip microfluidic platform involved coating of 96 wells with polydimethylsiloxane (PDMS) and squalene to enhance cellular adhesion and delay stem cell differentiation. Two wells were linked by microfluidic channels to facilitate cell-to-cell interaction. We used 1000 ABCG2+ CSCs obtained from SCC-25 cell line (3) and equal numbers of naïve CD271+ MSCs. CSCs co-cultured with MSC in a Boyden-chamber assay was used as control population. After two weeks of culture, MSCs were evaluated for potential reprogramming to altruistic stem cell (ASC) phenotype as previously described. Results:We observed that MSCs collected from organ-on-chip exhibit stem cell altruism as indicated by loss of p53 and gain of HIF-2alpha expression as measured by In-Cell western assay (1). These MSCs also exhibited secretion of GSH, another characteristic of ASCs. Subsequently, these cells activated p53/MDM2 oscillation, and underwent apoptosis/differentiation, another important characteristic of ASCs. While, MSCs cultured in Boyden chamber assay, showed rapid proliferation and loss of CD271+ expression, and gain of markers associated with carcinoma-associated-fibroblasts including SMA and cytokeratin 8. Conclusion:Overall, our findings suggest that microfluidics-based organ-on-chip device can be used to understand stem cell altruism in TME. 1. PMID:22689594.2. PMID: 288841133. Talukdar J et al In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 920.

#5155

**Increased longevity of continuous bone marrow cultures and radioresistance of bone marrow stromal cells from SOD1** 93A **ALS (Amyotrophic Lateral Sclerosis) mice.**

Andrew Henderson,1 Michael W. Epperly,1 Renee Fisher,1 Donna Shields,1 Lora Rigatti,2 Christopher Donnelly,2 Simon Watkins,2 Joel S. Greenberger1. 1 _UPMC Hillman Cancer Center, Pittsburgh, PA;_ 2 _University of Pittsburgh, Pittsburgh, PA_.

Introduction: The SOD1G93A mouse model of ALS, demonstrates hind limb paralysis beginning at 90 - 100 days of age with stage 4 paralysis at 125 days of age and progressive neuromuscular loss.

Materials and Methods: To determine whether deficiency of functional SOD1 influenced parameters of hematopoiesis, long-term bone marrow cultures were established from ALS and control mice. Bone marrow stromal cell lines derived from LTBMCs were tested for clonogenic radiation survival. We tested the effect of bone marrow transplant after total body irradiation on delay of paralysis.

Results: SOD1G93A marrow cultures demonstrated significant increase in production of hematopoietic progenitor cells (p < 0.0001) and overall longevity of production of hematopoietic cells (p = 0.0354), and bone marrow stromal cell lines were significantly radioresistant (D0 = 1.33 ± 0.09, and ñ = 8.57 ± 1.8) compared to control C57BL/6J mice (D0 = 1.59 ± 0.11, p = 0.117; and ñ = 3.4 ± 0.4, p= 0.0466). Total body irradiation and bone marrow transplantation with GFP+ donor marrow demonstrated a significant increase in paralysis free interval from 129.2 ± 3.0 to 240.7 ± 21.1 days (p = 0.0010), normalization of blood/brain barrier permeability, and increase in M2 marrow origin microglial cells in proximity to degenerating anterior horn cell/motor neurons. Isolated brain and spinal cord irradiation did not prolong the paralysis free interval (129.0 ± 2.7 days, p = 0.7748).

Conclusions: The results of hematopoiesis in LTBMCs in the absence of functional SOD1 showed improved LTBMC longevity and radioresistance of marrow stromal cells both unexpected pleiotropic effects of the SOD1G93A genotype. Marrow transplant after TBI prolonged the paralysis free interval in ALS mice.

#5156

In vitro application of tumor-treating fields to suppress tunneling nanotubes in mesothelioma.

Akshat Sarkari,1 Michal Munster,2 Einav Zeevi,2 Moshe Giladi,2 Emil Lou1. 1 _Univ. of Minnesota, Minneapolis, MN;_ 2 _Novocure, LTD, Haifa, Israel_.

The purpose of this study was to evaluate the effects of tumor-treating fields (TTFields) on tunneling nanotubes (TNTs) in malignant mesothelioma. TTFields have emerged as a therapeutic modality for the treatment of glioblastoma, through application of alternating electric fields, which exert dipole alignment and dielectrophoretic force. The dielectrophoretic force is intensified in cellular compartments that are non-uniform in size and shape such as the mitotic furrow. TNTs are filamentous actin protrusions that are conduits for intercellular communication and transport of vital cell signals that stimulate cell growth, invasion, and resistance to treatment. TNTs are highly prevalent in malignant pleural mesothelioma. We hypothesized that by creating dielectrophoretic force, TTFields may disrupt or prevent the formation of mesothelioma TNTs and thus sensitize these cells to the cytotoxic effect of chemotherapy and TTFields. TTFields (1.1-1.6 V/cm; 150-200 kHz) were applied using the inovitro system to VAMT and MSTO malignant mesothelioma cell lines. TNT index (average # of TNTs/cell) was determined at 24, 48, and 72 hours of TTFields application. Cell viability assessment using evaluation of cell count and colony formation assays were performed to determine effects of TTFields either alone or in combination with varying concentrations of cisplatin (range 0-10 µM) or pemetrexed (0-512 nM). In separate experiments, we examined the effects of cell cycle inhibition on TNTs using the compound AZD 5438 (Tocris Biosciences). Application of continuous TTFields suppressed TNT formation by 60% over a 72-hour period. This suppression was achieved by the 24-hour timepoint, and maintained over the 72 hours. Assessment of cell viability showed high rate of cell death at the stated frequencies and intensities. The addition of cisplatin or pemetrexed to TTFields application significantly decreased the cell count. The combination of TTFields and low concentration cisplatin (0.1-1 µM) resulted in enhanced treatment efficacy as evaluated using the colony formation assay. Complete inhibition of the cell cycle paradoxically stimulated a significantly higher number of TNTs. Here, we show that TTFields application suppresses TNTs formation in malignant mesothelioma cell lines. Treatment with TTFields leads to enhanced treatment efficacy when combined with standard of care chemotherapeutic drugs cisplatin and pemetrexed. Additional optimization of the applied TTFields intensities and frequencies may further improve treatment efficacy. The sharp rise in TNTs seen following cell cycle inhibition is a potential cellular stress response to cancer-directed treatment. Suppression of TNT formation, and thus TNT-mediated intercellular communication networks in tumors, is a potential mechanism of TTF efficacy in treatment of mesothelioma, glioblastoma, and other invasive cancers.

#5157

Comparison of exosomes shed by breast cancer cell lines with varying metastatic potential.

Kelly A. Martin, Nicholas R. Hum, Aimy Sebastian, Gabriela G. Loots. _Lawrence Livermore National Laboratory, Livermore, CA_.

Exosomes are endosomal secreted vesicles containing a variety of genetic and non-genetic material that can be transferred between cells (DNA, RNA, protein, lipid, etc.). Cancer derived exosomes have been implicated in a wide range of cancer mechanisms such as promotion of tumor growth, tumor angiogenesis, immune evasion, drug resistance, and metastasis. In addition to functional significance, cancer exosomes may possess novel biomarkers with potential uses for non-invasive liquid biopsies for cancer detection and monitoring of disease progression. Here we examined differences that may exist in extracellular vesicles or exosomes (EVs) shed by cancer cell lines with various metastatic potential (MDA-MB-231, highly metastatic; MCF-7, weakly metastatic; MCF-10a, normal breast epithelial cells). Morphological analysis of EVs was performed using fluorescent Nanosight Tracking Analysis and revealed decreased mean diameter of EVs derived from MCF-7 cells compared to the other two cell lines, there was also increased exosome particle yield in this cell line. Small RNAs from EVs cargo were also analyzed via sequencing (exoRNA-Seq). The majority of sequenced RNA aligned to coding regions of the human genome (~60%) across all cell lines. Non-coding RNA classification also showed little variability across cell lines examined regardless of metastatic potential, with ~38% of RNA reads corresponding to non-coding RNAs. Pairwise comparison of these cell lines demonstrated that each line packaged unique cargos into their shed exosomes. The non-coding regions corresponded to a variety of small RNAs (miRNA, snoRNA, snRNA, miscRNA, rRNA) but included other RNA features such as pseudogenes and antisense RNAs. We found 304 genes significantly up-regulated and 150 genes significantly down-regulated when comparing metastatic cancer exosomes (MCF-7 and MDA-MB-231) to normal, non-tumorigenic exosomes (MCF-10A). Among examined microRNAs we found several miRNAs associated with metastatic behavior that have previously been implicated in cancer biology, which will be prioritized for validation. Also, 21 RNAs were upregulated at high levels in exosomes shed by metastatic cells, and their expression level was directly proportional with the metastatic character, suggesting that these RNAs may be tested for their potential as biomarkers. Future research will expand upon these newly identified genetic signatures of metastatic exosomal cargo and further validate their influence in driving metastasis.

This study received funding from DOD grant BC151687. This work was conducted under the auspices of the USDOE by LLNL (DE-AC52-07NA27344). IM number: LLNL-POST-758941

#5158

Hypoxia alters the release and size distribution of extracellular vesicles in pancreatic cancer cells to support their adaptive survival.

Mary C. Patton, Haseeb Zubair, Mohammad Aslam Khan, Seema Singh, Ajay P. Singh. _University of South Alabama, Mobile, AL_.

Pancreatic tumors are characterized by poor vasculature and fibrous stromal tissue networksthat create an extensive hypoxic tumor microenvironment. Extracellular vesicles (EVs) play important roles in pancreatic tumor pathobiology by supporting inter-cellular communications. They arebroadly classified into three subtypes: exosomes (Exo; 30-150 nm), microvesicles (MVs; 100 nm-1 µm) and apoptotic bodies (Abs; 1-5 µm), according to their cellular origin. Here, we studied the effect of hypoxic stress on the release kinetics and size distribution of EVs in pancreatic cancer cells. Further, we also investigated the role of different EV subtypes in adaptive survival of pancreatic cancer cells under hypoxia.Our data demonstrated that under hypoxic conditions, pancreatic cancer cells (MiaPaCa and AsPC1) shed greater amount of EVs with most noticeable changes recorded for the small size EVs. Moreover, all EVs (small, moderate, large) showed a shift towards reduced size depending upon the extent of hypoxia. When examined for subtype-specific markers, we observed mixed profiles. Thrombospondin (marker for Abs) and Arf6 (marker for MVs) were exclusively detected in large and moderate size fractions, respectively, under both normoxia and hypoxia. However, CD9 (marker for Exo) was detected in both small and moderate size EVs under hypoxia. Furthermore, in release kinetics studies we observed cyclic increases in accumulation of EV subtypes under hypoxic conditions. In addition, our data demonstrated that EVs from hypoxic cancer cells promoted survival of cancer cells under hypoxia,with small EVs being the most active. Altogether, our findings establish that hypoxia alters shedding of EVs in supporting adaptive survival of pancreatic cancer cells; associated differences could possibly be exploited for effective cancer management.

#5159

Isoform specific expression of cadherin10 is associated with progression and drug resistance in non-small cell lung cancer.

Rajib Ghosh,1 Phattrakorn Powan,1 Liying W. Rojanasakul,2 Yon Rojanasakul1. 1 _West Virginia University, Morgantown, WV;_ 2 _National Institute for Occupational Safety and Health, Morgantown, WV_.

Lung cancer is the leading cause of cancer related death worldwide accounting for 1.69 million of total 8.8 million cancer deaths in 2015 alone. Nearly 85% of all lung cancer cases are classified as non-small cell lung cancer (NSCLC) with a 5-year survival rate of 16%, despite of all advanced targeted treatment modalities. Like any other cancer, cadherin proteins play a critical role in lung cancer progression, metastasis and drug resistance. Classical type I and type II cadherin proteins are calcium-dependent transmembrane glycoproteins with a cytoplasmic tail that mediates downstream signaling and a large extracellular N-terminal head that engages in homophilic and heterophilic adhesion. While alterations of type I cadherins (N-cadherin and E-cadherin) are well studied, functions as well as alterations of type II cadherins, such as cadherin-8, -9, -10, -12, and -18, remain largely unknown. Recent whole genome sequencing of patient samples towards Precision Medicine initiatives revealed 2,687 donors affected by 9,151 cadherin10 (CDH10) mutations alone across the 63 cancer projects worldwide. The majority of mutations in CDH10 are missense substitutions largely distributed throughout the entire gene length, without any specific hotspot. CDH10 copy number amplification is also reported in a significantly large number of lung cancer patients. Alternative splicing of this gene potentially results in multiple transcript variants. To resolve the conundrum of Loss-of-heterozygosity (LOH) and copy number amplification, we decided to look further in to expression and function of CDH10 using array of lung cancer cell lines and human lung cancer patient samples. Here we report, for the first time, that full-length CDH10 expression as well as the tumor suppressive function are compromised in the course of NSCLC development, while aberrant smaller isoforms appear in the cell lines and tumors that drive the oncogenic progression. Expression of such smaller isoforms were further altered when the cells were treated with commonly used chemotherapy drugs, such as Cisplatin, Doxorubicin and Paclitaxel indicating a direct relationship between drug resistance and appearance of these smaller isoforms. We found that the downregulation of full-length CDH10 using siRNA results in an increase in proliferation and invasion, indicating a potential anti-oncogenic function of full-length CDH10 protein. Therefore, we found that mutations and alternative splicing potentially downregulates full-length CDH10 expression along with its tumor suppressive function. However, appearance of smaller aberrant isoforms accounts for tumorigenic progression and drug resistance.

#5160

Extracellular vesicles from scirrhous gastric cancer cells induce premetastatic niche formation for peritoneal metastasis.

Tomohisa Okuno, Masakazu Yashiro, Shuhei Kushiyama, Sadaaki Nishimura, Shingo Togano, Kenji Kuroda, Tatsuro Tamura, Takahiro Toyokawa, Hiroaki Tanaka, Kazuya Muguruma, Kosei Hirakawa, Masaichi Ohira. _Osaka City Univ. Grad. School of Medicine, Osaka, Japan_.

Background: Human scirrhous gastric carcinoma (SGC) is also known as diffusely infiltrating carcinoma, type 4 on Borrmann's criteria, and linitis plastica-type carcinoma. SGC is characterized by frequent peritoneal metastasis, high proliferation and infiltration activity with extensive fibrosis in the tumor stroma. We previously reported that a soluble factor(s) from SGC cells changed the peritoneum to pre-metastatic niche such as round shape of mesothelial cells and peritoneal fibrosis, which is a favorable environment for cancer cells to metastasize. Extracellular vesicles (EVs; Exosomes) might be one of soluble factor(s) which might change the peritoneal component to pre-metastatic niche. In this study, we investigated the effect of EVs from SGC cells on the pre-metastatic niche formation of the peritoneum.

Methods: Four SGC cell lines, OCUM-2M, OCUM-2MD3, OCUM-12, KATO-III, 2 non-SGC cell lines, MKN74, MKN45, and peritoneal mesothelial cells (PM cells) were used. The effect of EVs from gastric cancer cells on the peritoneum was examined in vitro and in vivo, as follows. PKH26-labeled EVs from OCUM-2MD3 cells were intravenously injected into nude mice for 3 weeks, then we examined the distribution of PKH26-labeled EVs by a fluorescence microscope. Next, we injected EVs intraperitoneally into nude mice, and investigated the morphology of peritoneum. Effect of EVs on the gene expression of PM cells was examined by RT-PCR. Clinical significance of EVs markers, CD9 and CD63, was evaluated by immunohistochemistry using 63 human gastric cancer specimens.

Results: PKH26-labeled EVs frequently distributed to peritoneum and the liver. The morphology of PM cells changed from round shape to spindle shape by EVs addiction in vitro and in vivo. mRNA expression level of ZEB and N-cadherin of PM cells was significantly increased following the addiction of EVs. The high expression of EVs

markers, CD9 and CD63, was significantly correlated with frequent peritoneal metastasis.

Conclusion: EVs from SGC cells might frequently distribute to mesothelial cells of peritoneum. EVs from SGC cells might play an important role for the formation of a pre-metastatic niche on peritoneum by the morphologic change of peritoneal mesothelial cells.

#5161

**Probing leukemia vulnerabilities** in vivo **using domain-focused CRISPR screening.**

Yiliang Wei, Christopher Vakoc. _Cold Spring Harbor Laboratory, Cold Spring Harbor, NY_.

The cancer microenvironment plays an important role in the disease emergence, development, recurrence, and treatment resistance. For human myeloid malignancies, the bone marrow microenvironment contains a complex structure with a number of different cell types. The sophisticated interaction network between myeloid leukemia and the microenvironment adds great challenges to elucidating the pathogenesis of the disease, and discovering novel therapeutic mechanisms. Over the last few years, CRISPR-Cas9 genome editing technology has risen as a powerful tool for identifying essential genes for cancer growth. Our lab has further developed an advanced CRISPR-Cas9 screening approach that uses pooled sgRNAs targeting functional protein domains to significantly improve screening efficiency. In contrast to the massive amount of in vitroCRISPR-Cas9 screening studies, very few studies have focused on developing an in vivoCRISPR-Cas9 screening approach. Compared to in vitroscreening, a huge advantage of in vivoscreening is that it allows cancer cells in direct contact with the native microenvironment, which would lead to the identification of essential signalings for cancer development in vivo. In this study, we developed an effective and efficient in vivo domain-targeting CRISPR-Cas9 screening approach with a human acute myeloid leukemia (AML) xenograft model. We designed and constructed a number of domain-targeting sgRNA libraries covering different cellular functions, including G-protein coupled receptors, ion channels, solute carriers, membrane associated proteins, kinases, phosphatases, glycosylation pathway proteins, proteases, transcription factors, and chromatin regulators. We performed the CRISPR-Cas9 screens both in vivo and in vitro at the same time to identify in vivo specific essential genes for AML. With the focused-size libraries, we are able to achieve high screening quality and to nominate high confident in vivo specific targets. Among them, we re-captured the in vivo specific essentialities of the CXCR4 and CD47 genes, which have been well studied for their critical connections with bone marrow microenvironment. In addition, we identified and validated several in vivo specific targets associate with glycosylation pathway, integrin linked pathways, and chromatin regulators. Our preliminary results have demonstrated this approach can effectively identify essential genes for cancer microenvironment, which in vitroscreening is incapable of. We aim to use this approach to further identify in vivo specific targets in other pathways; and to understand their mechanism connecting to the cancer microenvironment; and to identify small molecules with therapeutic potency that could disrupt these connections.

#5162

Role of IFN-gamma-activation of distinct tumor and stromal cell populations in colorectal carcinoma pathogenesis.

Nathalie Britzen-Laurent,1 Wei Guo,2 Victoria Langer,1 Svetlana Khoziainova,2 Thomas Weisenburger,1 Thomas H. Winkler,1 Julia Straube,1 Maximilian J. Waldner,1 Christoph Becker,1 Elisabeth Naschberger,1 Lisa Skottke,1 Tripal Philipp,1 Sergei Grivennikov,2 Michael Stürzl1. 1 _Univ. of Erlangen-Nuremberg, Erlangen, Germany;_ 2 _Fox Chase Cancer Center, Philadelphia, PA_.

Tumor and stromal cell plasticity induced by different tumor microenvironments (TMEs) has a significant impact on tumorigenesis of colorectal carcinoma (CRC). Specifically the role of the immunological TME has been investigated intensively. The prognosis of CRC is influenced by the density and localization of infiltrating T cells. Moreover, the presence of an mRNA expression profile indicative of a type 1 adaptive immune response (high IFN-γ and IFN-induced gene expression, cytotoxic T cell (CTL) signature) represents a positive prognostic factor for patients with CRC. IFN-γ is a major mediator of the Th1 immune response, which is produced by NK, NKT, Th1 cells and CTL. The role of IFN-γ in anti-tumor immune response has been mainly attributed to the immune-modulatory activity of the cytokine, such as the recruitment and activation of cytotoxic T-cells or monocytes. However, the expression of the IFN-γ receptor is ubiquitous and studies of IFN-γ-induced genes expression in CRC revealed that many different cell types are stimulated by the cytokine within a tumor, including tumor cells and stromal cells such as macrophages/monocytes and endothelial cells. In vitro, IFN-γ shows anti-tumorigenic activities in CRC cell lines, activates monocytes and exerts potent anti-angiogenic effects on endothelial cells. Here we investigated the cell-type-specific impact of the response to IFN-γ on CRC development using mouse strains with conditional knock-out of the IFN-γ receptor in intestinal epithelial cells (Villin-Cre), T-cells (CD4-Cre), myeloid cells (CD11b-Cre) and endothelial cells (Tie2-Cre + BMT). Tumor growth was chemically induced by injection of azoxymethane combined with three cycles of treatment with dextran-sodium sulfate. This model recapitulates inflammation-induced carcinogenesis. Our results revealed increased tumor numbers and load when the IFN-γ response was blocked in epithelial cells, despite an initial attenuation of inflammation. Tumors that developed in Ifngr2-Villin-Cre mice showed an attenuated IFN-γ response, and a decrease of CD8+ T cell infiltration, cell death and hypoxia. Abrogation of the IFN-γ-response in endothelial cells also fostered tumor growth, which could be attributed to an increase of angiogenesis. Surprisingly, only a modest effect was seen in the T-cell-specific knock-out, in comparison to the myeloid-specific knock-out, which was associated with a strong increase of tumor numbers and load. The latter indicated that the anti-tumorigenic activity mediated by IFN-γ relies to a larger extent on the innate than on the adaptive immune response. Our study documents that the anti-tumorigenic activity of IFN-γ is based on direct effects on epithelial tumor cells and on the vasculature, as well as on the involvement of the innate immune response.

#5163

Angiocrine induction of colorectal cancer cell HER3 activation and cell survival is independent of CRC mutational status.

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

INTRODUCTION: Colorectal cancer (CRC) is the second-leading cause of cancer-related death in the United States. A better understanding of the regulation of CRC cell survival pathways is necessary for developing new therapeutic strategies for patients with metastatic CRC (mCRC). We have previously found that endothelial cells (ECs) from the liver, the most common site of CRC metastases, secrete soluble factors in their conditioned medium (CM) that, in turn, increase cell growth and chemoresistance of CRC cells. We also determined that EC-induced CRC cell survival is mediated, in part, by HER3/ERRB3 signaling.

OBJECTIVES: In the current study we sought to determine if alterations in driver mutations mediate the role of liver ECs in activating HER3 signaling and promoting cell survival in CRC cells.

METHODS: Primary ECs from non-malignant liver parenchyma were isolated as previously described (Wang et al., Mol Cancer Res., 2018). CRC cell lines with distinct molecular profiles (MSI status, and wildtype or mutant KRAS, BRAF, and PIK3CA) were incubated with either their own CM (control) or CM from ECs. The effects of CM on CRC cell growth and response to 5-fluorouracil (5-FU) was determined by MTT and colony formation assays. The effect of angiocrine signaling on chemoresistance was also determined by fluorescence-activated cell sorting (FACS) for apoptosis after treating CRC cells with 5-FU, either in control CM or EC CM. HER3 blockade was accomplished by 1) siRNA knockdown or 2) a HER3 antibody.

RESULTS: CM from liver ECs significantly increased cell growth and chemoresistance in CRC cells regardless of the gene alteration profiles in in vitro assays. We confirmed that EC CM activated the HER3-AKT signaling pathway in CRC cells. Furthermore, we found that HER3 blockade attenuated EC CM-induced AKT activation and cell survival in CRC cells independent of the cell mutational profile.

CONCLUSIONS: Our results demonstrate that HER3 mediates liver EC-induced cell growth and chemoresistance in CRC cells independent of the driver mutational profiles of the cells. The genetic background of CRC cells is not a mediator of EC CM-induced HER3 activation and cell survival. These findings suggest that effects of HER3 targeting of tumor cells in patients with mCRC should be independent of MSI status, or RAS, BRAF, or PIK3CA mutational status.

#5164

Signaling crosstalk within prostate tumor microenvironment mediates castrate resistant disease progression.

Manisha Tripathi,1 Srinivas Nandana,1 Jen-Ming Huang,2 Manabu Kato,2 Rajeev Mishra,2 Leland Chung,2 Li Xin,3 Neil Bhowmick2. 1 _Texas Tech University Health Sciences Center, Lubbock, TX;_ 2 _Cedars Sinai Medical Center, Los Angeles, CA;_ 3 _Baylor College of Medicine, Houston, TX_.

Juxtacrine signaling with the stromal compartment is important to the development of castrate resistance prostate cancer. We found that Notch signaling is upregulated in prostate cancer associated fibroblasts (CAFs) compared with normal tissue associated fibroblasts (NAFs). Further, inhibition of Notch signaling in CAFs resulted in decreased Wnt signaling and decrease in expression of Notch pathway genes. Interestingly, androgen receptor antagonism resulted in increased Notch signaling and the upregulation of downstream Wnts. Further, generation of chimeric tissue recombinant xenografts with human RV1 prostate cancer epithelia with mouse prostate stromal cells with activated Notch signaling that are derived from NICD transgenic mice resulted in increased tumor size compared with wild type control stromal recombinants. Conversely, recombinants derived from prostatic fibroblasts of RBPJ-knockout mice that have a loss of Notch signaling demonstrated diminished tumorigenesis, compared with xenografts with the wild type fibroblasts. In trying to determine the mediator of Wnt-associated paracrine prostate epithelial expansion, we found downstream YAP activation in the RV1 cells. In a reciprocal epithelial-to-fibroblast communication we found that the RV1 cells treated with conditioned media from NICD fibroblasts showed increased IL-6 expression. Conversely, conditioned media from prostate cancer cells caused increased Notch target gene expression in CAFs, which was blocked upon addition of IL-6 neutralizing antibody. Taken together our results suggest that there is a stromal-derived feed-back - feed-forward signaling loop involving Notch and Wnt ligands that activates YAP in the epithelia for paracrine IL-6-dependent Notch activity back in the stromal fibroblasts. Critically, disruption of this cross-talk in the fibroblasts can potentially limit the gain of stem features and castrate resistance of the adjacent epithelia in halting lethal progression of prostate cancer.

#5165

Secreted CCDC80 from hepatic stellate cells promote metastasis of hepatocellular carcinoma.

Kwang-Suck Kim,1 SaeHwan Lee,2 Nayoung Jun,1 Hyog Young Kwon1. 1 _Soonchunhyang Institute of Medi-bio Science, Soonchunhyang University, Cheonan, Republic of Korea;_ 2 _2Liver Clinic, Soonchunhyang University Cheonan Hospital, Cheonan, Republic of Korea_.

Liver cirrhosis is the most important risk factor for hepatocellular carcinoma (HCC) and hepatic stellate cell (HSC) has pivotal role in developing cirrhosis by producing various extracellular matrix (ECM). HSC are essential mediators of tumor microenvironment of HCC through remodeling of ECM and angiogenesis. We found that CCDC80 expression is upregulated in activated HSCs from RNA-sequencing with human fetal stellate cell. However, the role of CCDC80 was not well known in hepatocarcinogenesis. First, we validated mRNA expression of CCDC80 was increased in activated HSC lines and protein expression was also increased in cultured media. Functional analysis of knockdown and over expression CCDC80 was investigated in both HCC and HSC cell lines, and HCC cell lines were analyzed for proliferation (MTT assay), migration (using transwell), invasion (using Matrigel-coated transwell), and wound healing assay with condition media of CCDC80 knockdown HSCs. Expression of epithelial-mesenchymal transition (EMT) markers in HCC was evaluated with conditioned media. Knockdown of CCDC80 resulted in significantly reduced invasion, migration and wound healing behaviors of HCC cell lines. Overexpression of CCDC80 was increased invasion and migration. Expression of E-cadherin was increased and snail and vimentin expression were decreased in HCC cell lines cultured with conditioned media of CCDC80 knockdown HSCs. The associations between CCDC80 and EMT markers were validated in CCDC80 transfected HSC cell line compared with control. The mouse xenograft model also has shown the lower tumor growth, tumor weight and metastatic tumor nodule number in CCDC80 knockdown HSC with HCC co-injection compared with normal HSC and HCC co-injected mouse. Taken together, secreted CCDC80 from HSCs affected the aggressiveness and EMT of HCC cell lines. CCDC80 might be a novel biomarker and therapeutic target for patients with HCC.

#5166

Extracellular matrix protein tenascin C increases phagocytosis mediated by CD47 loss-of-function in glioblastoma.

Ding Ma,1 Senquan Liu,2 Bachchu Lal,1 John Laterra,1 Mary Ann Wilson,1 Shuli Xia1. 1 _Kennedy Krieger Research Inst., Baltimore, MD;_ 2 _Johns Hopkins School of Medicine, Baltimore, MD_.

Glioblastomas (GBMs) are highly infiltrated by myeloid-derived innate immune cells that contribute to an immunosuppressive nature of the brain tumor microenvironment (TME). CD47 has been shown to mediate immune evasion as the CD47-SIRPα axis prevents phagocytosis of tumor cells by macrophages and other myeloid cells. In this study, we established CD47 homozygous deletion (CD47-/-) in human and mouse GBM cells by genome editing to investigate the impact of eliminating the "don't eat me" signal on tumor growth and tumor-TME interactions. CD47 knockout (KO) did not significantly alter tumor cell proliferation in vitro, but significantly increased phagocytosis of tumor cells by macrophages in co-cultures. Compared with CD47 wild type xenografts, orthotopic xenografts derived from CD47-/- tumor cells grew much slower with enhanced tumor cell phagocytosis and increased recruitment of M2-like tumor associated microglia/macrophages (TAMs). CD47 KO increased tumor-associated extracellular matrix protein tenascin C (TNC) in xenografts, which was further examined in vitro. CD47 loss-of-function upregulated TNC expression in tumor cells via a Notch pathway mediated mechanism. ShRNA-based TNC knockdown in tumor cells enhanced the growth of CD47-/- xenografts in vivo, and decrease the number of TAMs. TNC knockdown inhibited phagocytosis of CD47-/- tumor cells in co-cultures. Furthermore, TNC stimulated release of pro-inflammatory factors including TNF-α via a toll-like receptor 4 (TLR4) and STAT3-dependent mechanism in human macrophage cells. These results reveal a vital immunomodulation role of TNC in brain tumor biology and demonstrate the prominence of the TME extracellular matrix in affecting the anti-tumor function of brain innate immune cells.

#5167

Cell autonomous and non-autonomous actions of CCL22 in oral carcinogenesis.

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

Oral cancer, a subtype of head and neck cancer, has been the 4th leading cause of male cancer death in Taiwan for the past decade. Smoking, alcohol consumption, and betel quid chewing are major risk factors for oral cancer in Taiwan. The crosstalk between immunity and cancer cells can both inhibit and enhance tumor growth and is now classified as a cancer hallmark. The accumulation of regulatory T cells (Tregs) in several tumor types potentially plays a critical role in tumor evasion of immune surveillance. Oral cancer is also characterized by an increase in the number of infiltrating Tregs; however, the significance of the increase on the patient prognosis remains controversial. We found that the expression of CCL22, a chemokine for Treg recruitment, was not only positively with poor prognosis in oral cancer patients but also significantly correlated with that of FOXP3, a master regulator for Treg development and functions, in the same patients. The correlation is also validated in TCGA database analysis of head and neck cancer patients. The depletion of CCL22 expression reduced oral cancer cell proliferation, migration and invasion, suggesting an oncogenic role of CCL22 in oral cancer. Since CCR4 is the receptor for CCL22, we studied the in vitro and in vivo impact of deregulated CCL22-CCR4 signaling axis in oral carcinogenesis. We first confirmed the surface expression of CCR4 in oral cancer cells by flow cytometry. Ectopic CCL22 overexpression increased oral cancer cell migration and invasion without the effect on proliferation. Through injection of CCL22-altered oral cancer cells in both syngeneic and athymic backgrounds, the cell autonomous and non-autonomous actions of CCL22 and the mechanism responsible for the CCL22 deregulation in oral cancer will be discussed. This study will not only further the role of CCL22-CCR4 signaling axis in modulating the crosstalk between oral cancer and its microenvironment but also provide a novel therapeutic strategy for treating oral cancer.

#5168

The role of activated beta-catenin/Wnt signaling in the progression of castration resistance of prostate cancer in bone.

Justin Roberts, Estefania LaBanca, Peter Shepherd, Jun Yang, Michael Starbuck, Nora Navone. _MD Anderson Cancer Center, Houston, TX_.

Bone metastases typically develop in patients with advanced prostate cancer (PCa). These metastases are osteoblastic (bone-forming) and constitute the main cause of morbidity and mortality. Androgen deprivation is commonly used to treat bone metastases of PCa, but responses to such therapy are short, and eventually, the disease progresses to a castration-resistant form. Bone is the primary site of castration-resistant PCa (CRPC) progression. Further development of therapies for bone metastases of PCa requires an understanding of the mechanisms underlying the growth of CRPC in bone. Clinical and laboratory-based investigations have implicated a role for PCa cell – bone cell interaction in the growth of PCa and have demonstrated that osteoblasts produce factors that favor the growth and survival of PCa cells. Collectively, these observations strongly suggest that PCa cells interact with osteoblasts and that this interaction contributes to the growth of PCa in bone. Recently, a study showed 18% of patients with metastatic CRPC harbored alterations in the Wnt canonical pathway, including hotspot mutations of β-catenin (β-cat), the molecular node of the Wnt canonical pathway, that leads to pathway activation. This report agrees with previous publications of β-cat nuclear accumulation, an indirect measure of Wnt-canonical pathway activation, in 20 to 40% of advanced PCas. We previously reported that a patient derived xenograft (PDX) MDA PCa 118b (118b) developed in our lab harbors a β-cat mutation (D32G) and has an activated Wnt canonical pathway. 118b induces an osteoblastic reaction in the bone of mice in vivo. In vitro, 118b induces osteoblast proliferation in co-culture. In contrast, the PCa cell line, PC3, induces osteolytic lesions in vivo and in vitro inhibits osteoblast proliferation in co-culture. Strikingly, expression of β-cat mutated at D32G in PC3 cells reverses this inhibitory effect of PC3 cells resulting in increased proliferation of osteoblasts when grown in co-culture. We therefore hypothesize β-cat/Wnt signaling in PCa cells mediates the PCa-bone interaction that favors PCa growth in bone. Our future studies are focusing on the importance of β-cat mutations recently identified in patients with metastatic CRPC and determining whether these mutations also result in an osteoblastic phenotype. Additionally, we are screening our collection of PCa PDX lines for increased β-cat expression through immunohistochemistry and will sequence DNA from PDX lines that demonstrate increased nuclear β-cat expression to test for activating mutations in β-cat. Our aim is to gain insight into the mechanism of the paracrine interaction between PCa cells and bone that supports the growth of PCa bone lesions with the ultimate goal of developing therapies to block these interactions and reducing the morbidity and mortality associated with metastatic disease.

#5169

Pancreatic tumorigenesis evokes mechanisms of tissue injury and repair.

Kathleen E. DelGiorno,1 Chi-Yeh Chung,1 Raj Giraddi,1 Eugene Ke,1 H. Carlo Maurer,2 Maya Ridinger-Saison,1 Wahida H. Ali,1 Crystal Tsui,1 Cynthia Ramos,1 Razia Naeem,1 Makoto Ohmoto,3 Linjing Fang,1 Gidsela Luna,1 Conor Fitzpatrick,1 Caz O'Connor,1 Uri Manor,1 Ichiro Matsumoto,3 Kenneth P. Olive,2 Geoffrey M. Wahl1. 1 _The Salk Institute, La Jolla, CA;_ 2 _Columbia University, New York, NY;_ 3 _Monell Chemical Senses Center, Philadelphia, PA_.

Despite numerous advances in our understanding of pancreatic ductal adenocarcinoma (PDA) genetics and biology, this disease is expected to become the second leading cause of cancer-related deaths in the U.S. by 2020. These statistics largely reflect the fact that by the time PDA is detected, it has already spread, making the study of early events in tumorigenesis invaluable. Harold Dvorak is credited with suggesting that tumors behave as wounds that do not heal, specifically that they are able to induce the stroma required for their maintenance and growth. Decades of research have provided an array of molecular mechanisms supporting this hypothesis. When injured, the pancreas undergoes acinar to ductal metaplasia (ADM) where digestive enzyme-producing acinar cells transdifferentiate to ductal cells; a process thought to allow for tissue healing and repair. Though a number of insightful studies have been conducted to determine the underlying mechanisms of this process, it is still incompletely understood. Using a number of high-resolution imaging techniques and lineage tracing models, we have found that chronic pancreatic injury is sufficient to induce formation of a number of differentiated cell types during ADM, including tuft cells, which are absent from the normal pancreas and may function in tissue repair.

Tuft cells are solitary chemosensory cells found throughout the hollow organs of the respiratory and digestive tracts. Their expression of taste, neuronal, and inflammatory cell signaling factors is thought to enable monitoring of intraluminal homeostasis and local response via effectors. Previous studies demonstrate that, in mice, tuft cells are absent from the normal pancreas, but transdifferentiate from the acinar cell epithelium in response to oncogenic Kras expression. Interestingly, while they increase during the genesis of pancreatic intraepithelial neoplasia (PanIN), they are not detected in PDA. Tuft cell formation is also characteristic of human pancreatitis and PanIN. These data suggest a conserved, transient, but currently undefined role for tuft cells in early tumorigenesis. Here, we employ novel mouse models to elucidate this role and to identify consequences of tuft cell ablation. These studies suggest that an important function of tuft cells involves production of immune-modulatory factors in response to injury and oncogenesis. Consistent with this, we show that pancreas-specific Pou2f3 ablation eliminates tuft cell formation and enhances disease progression. Collectively, these data suggest that neoplastic lesions that form in response to oncogenic mutation evoke the cellular heterogeneity that occurs during ADM in response to tissue injury. We conclude that tuft cells and, by inference, the associated metaplastic and neoplastic lesions, play a protective role early in pancreatic injury and tumorigenesis.

#5170

Targeting FGFR signaling to disrupt cellular cross-talk in pancreatic cancer.

Abigail Coetzee, Edward Carter, James Heward, Faraz Mardakheh, Richard Grose, Hemant Kocher. _Barts Cancer Institute, Queen Mary University of London, London, United Kingdom_.

Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis with a 5 year survival rate of less than 5%. PDAC tumors consist of a desmoplastic stroma, which limits the effectiveness of chemotherapy. Pancreatic stellate cells (PSCs), which form a key part of this stroma, become activated in response to tumor development. Activated PSCs enter a cross-talk with cancer cells to induce tumor cell proliferation and invasion, leading to metastatic spread. Nuclear fibroblast growth factor receptor 1 (nFGFR1) has been found in PSCs at the invasive edge of PDAC tumors. Inhibition of FGFR1 prevents its nuclear translocation in PSCs, which results in decreased invasion in 3D in vitro PDAC models. Nuclear translocation of FGFR1 in PSCs appears to be a vital mechanism that triggers the transcription of key proteins involved in PDAC invasion.

We have used a powerful combination of chromatin immunoprecipitation (ChIP-seq) and sub-cellular mass spectrometry to determine the transcriptional targets of nFGFR1 and consequent sub-cellular protein flux. These techniques have allowed us to dissect the functional consequences of FGFR1 knockdown or inhibition in the PSCs. Candidate drivers of invasion are being validated in state-of-the-art 3D in vitro PDAC models. We have extended these functional studies to combination therapy with the clinical agent gemcitabine (targeting cancer cells) and all-trans retinoic acid (ATRA, modulating PSCs), providing translational relevance for our findings. We are embarking on validating this novel strategy using in vivo co-culture xenograft models with specific reference to FGFR1. Effectively disrupting the cross-talk between the tumor and stroma, either alone or in combination with other therapies, could translate to improved therapeutic responses in PDAC patients in the clinic.

#5171

GRP78 as a potential modulator of cell adhesion markers in metastatic prostate cancer and multiple myeloma.

Christopher Cultrara,1 David Sabatino,1 Jenny Zilberberg2. 1 _Seton Hall Univeristy, South Orange, NJ;_ 2 _Hackensack University Medical Center, Hackensack, NJ_.

Background: Glucose regulated protein 78 (GRP78) is a resident chaperone of the endoplasmic reticulum and a master regulator of the unfolded protein response under physiological and pathological cell stress conditions. GRP78 is overexpressed in many cancers, regulating a variety of signaling pathways associated with tumor initiation, proliferation, adhesion and invasion which contributes to metastatic spread. GRP78 can also regulate cell survival and apoptotic pathways to alter responsiveness to anticancer drugs. Tumors that reside in or metastasize to the bone and bone marrow (BM) space can develop pro-survival signals through their direct adhesive interactions with stromal elements of this niche thereby resisting the cytotoxic effects of drug treatment. In this study, we report a direct correlation between GRP78 and the adhesion molecule N-cadherin (N-cad), known to play a critical role in the adhesive interactions of multiple myeloma and metastatic prostate cancer with the bone microenvironment.

Methods: N-cad expression levels (transcription and protein) were evaluated upon siRNA mediated silencing of GRP78 in the MM.1S multiple myeloma and the PC3 metastatic prostate cancer cell lines. Furthermore, we evaluated the effects of GRP78 knockdown (KD) on epithelial-mesenchymal (EMT) transition markers, morphological changes and adhesion of PC3 cells.

Results: We demonstrate that GRP78 KD: 1) induced the concomitant downregulation of N-cad in both PC3 and MM.1S cells, 2) resulted in significant decreases of both N-cad and E-cad protein levels in PC3 cells, 3) induced TGF-β1 and Snail-2 expression, potentially accounting for the observed downregulation of E-cad. Our data also suggests that N-cad regulation via GRP78 KD supersedes the effects of TGF-β1; i.e., N-cad was downregulated in spite of the fact that TGF-β1 expression was significantly increased upon GRP78 KD.

Conclusion: This work implicates GRP78 as a modulator of cell adhesion markers in MM and PCa. Our results may have clinical implications underscoring GRP78 as a potential therapeutic target to reduce the adhesive nature of metastatic tumors to the bone niche.

#5172

Deposition of platelets in sinusoids and its contribution to hepatocarcinogenesis.

Hiroki Tanaka,1 Kie Horioka,1 Masahiro Yamamoto,2 Koji Sawada,1 Takumu Hasebe,1 Kosuke Yamazaki,3 Katsuhiro Ogawa1. 1 _Asahikawa Medical University, Asahikawa, Japan;_ 2 _Yamagata University, Yamagata, Japan;_ 3 _Japanese Red Cross Hokkaido College of Nursing, Kitami, Japan_.

Platelets can activate hepatocyte proliferation not only via releasing the platelet factors, but also promoting for liver sinusoidal endothelial cells (LSEC) and Kupffer cells to produce the factors such as IL-6 and TGF-α that are indispensable for hepatocyte proliferation. We previously reported that in the transgenic (TG) mice with liver specific human BRAFV600E mutation, which corresponds to the mouse BrafV637E that is highly prevalent in mouse DEN-induced hepatic tumors, exhibit increased liver weight five times larger than that of normal mice. Furthermore, hepatocytes in these mice overexpress thrombopoietin (TPO) in association with megakaryocytosis/thrombocytosis, and large numbers of platelets were deposited in the liver sinusoids. In the present study, we investigated whether platelets are deposited in liver sinusoids in hepatic tumors in human and mice. CD61 immunohistochemical staining revealed the increased numbers of platelets were deposited in the sinusoids in foci, adenomas and hepatocellular carcinomas (HCC) induced by neonatal treatment with DEN compared to surrounding normal hepatic tissues. Moreover, increased numbers of platelets were deposited in sinusoids of dysplastic nodules and well-differentiated HCC compared to normal liver tissue and cirrhotic nodules in human. In addition, we evaluated the correlation between the tumor diameters and peripheral platelets numbers in the patients who were primarily diagnosed as HCC in our institute. The result indicated that the numbers of peripheral platelets are positively correlated with the tumor diameters. These results indicate that platelets contribute to development of HCC via interacting sinusoidal cells.

#5173

Development of a novel tunable synthetic 3D hydrogel platform for the study of tumor and stromal cell interactions.

Hsu-Kun Wang, Vi Chu, Nick Asbrock. _MilliporeSigma, CA_.

Numerous cancer cell models exist used to investigate disease mechanisms and to screen potential cancer therapeutics. Roughly 90% of promising preclinical drugs fail to result in efficacious human treatments. Traditional two-dimensional (2D) tissue culture models lack realistic complexity, while animal models are expensive, time consuming, and too frequently fail to reflect human tumor biology. Recently, three-dimensional (3D) cell culture models are a new method to generate new drug candidates before moving to expensive and time-consuming animal models. We have developed a biochemically defined hydrogel platform formed by mixing various polymers with chemical crosslinkers with enhanced functionality. The hydrogel system employs one of two types of backbone polymers: a synthetic non-degradable polyvinyl alcohol (PVA) or an enzymatically-degradable dextran. Both polymers are functionalized either with fast or slow thiol-reactive groups. Crosslinkers consist of either PEG non-cell-degradable or a CD cell-degradable crosslinkers containing peptide sequences that create cleavage sites for matrix metalloproteases (MMPs) that allow cell migration. The technology provides mechanical and biochemical cues to investigate both morphological and physiological properties of cells in a 3D environment. The hydrogel allows precise control over hydrogel stiffness, gelation speed, cell migration and allows cell recovery for downstream applications. Here, we demonstrate the utility of this hydrogel platform to grow epithelial, fibroblast and tumor cells in 3D cell cultures with high cell viabilities and functionalities. Furthermore, we have constructed a 3D tumor/stromal cell co-culture using the hydrogel to model the dynamic tumor cell microenvironment. This technology will allow the creation of more accurate 3D cancer cell models for basic research and drug discovery applications.

#5174

Expression, regulation and role of PXDN, a collagen IV crosslinker, in normal development and cancer of the mammary gland.

Anna K. Sigurdardottir, Eirikur Briem, Zuzana Budkova, Thorarinn Gudjonsson, Gunnhildur Á. Traustadottir. _University of Iceland/Stem Cell Research Unit, Reykjavik, Iceland_.

Branching morphogenesis is an integral process in the development of the breast gland, which is instigated through the activity of stem cells in the terminal duct lobular unit (TDLU). An important mechanism during branching is epithelial to mesenchymal transition (EMT) a process that is often hijacked in fibrosis and cancer formation. D492 is a breast epithelial progenitor cell line that generates TDLU-like structures in 3D culture and in co-culture with endothelial cells undergoes EMT that has given rise to the D492M cell line. We have shown that D492 and D492M differ greatly in terms of gene and microRNA expression.

One of the most downregulated miRNAs in D492M compared to D492 is miR-203a that has previously been shown to target EMT transcription factors such as SNAI1 and SNAI2. When we overexpressed miR-203a in D492M we observed that the most downregulated gene was peroxidasin (PXDN), which we subsequently confirmed as a novel target for miR-203a.

PXDN is involved in collagen IV crosslinking in the basement membrane and has been linked to various diseases, in particular fibrosis and cancer. Our preliminary data shows that PXDN is expressed in epithelial cells and fibroblasts in the breast, as well as in fibrotic foci within the stroma. An emerging topic in mammary gland research is tissue stiffness, which has been shown to play an important role in both development and cancer progression. In response to tissue injury or disease, fibroblasts become activated into myofibroblasts that contribute to increased tissue rigidity. In this relation, PXDN formation and excretion from myofibroblasts in the kidney has been found to contribute to the development of renal fibrosis. We aim to investigate the role of PXDN in mammary gland development and disease and the molecular mechanisms behind the regulation of PXDN.

#5175

Expression profile and role of LOXL3 in astrocytomas.

Talita de Souza Laurentinho, Roseli da Silva Soares, Suely Kazue Marie, Sueli Mieko Oba-Shinjo. _School of Medicine of University of Sao Paulo, Sao Paulo, Brazil_.

Astrocytomas are the most common primary central nervous system tumors in adults. The WHO classification based on histologial characteristics according consider four malignant grades, glioblastoma (GBM) being the most malignant (grade IV). Molecular classification of GBM is based on the integration of somatic mutations, DNA methylation and specific transcript/protein expression and consider four subtypes: proneural, classic, mesenchymal and neural. Lysyl oxidase family is composed of five members (LOX, LOXL1, LOXL2, LOXL3 and LOXL4) which are enzymes responsible for catalyzing lysine-derived cross-links of collagen and elastin. The aim of the present work was to analyze the gene expression of LOXL3 in astrocytomas of different grades and different GBM molecular subtypes, the impact of LOXL3 expression level on overall survival in GBM cases of our cohort. These data were also analyzed in the TCGA database to validate our findings. LOXL3 expression increased with astrocytoma malignant grades, and proved to be a prognostic factor, as GBM patients with lower LOXL3 expression presented longer survival time than those with higher expression (p<0.041). Analogous data was obtained in the TCGA database, when considering temozolamide-treated GBM patients (p=0.049). Additionally, LOXL3 expression was higher in mesenchymal GBM subtype than in proneural and classic GBM subtypes in TCGA cohort (p<0.001). To further understand LOXL3 functional role in gliomagenesis, LOXL3 was silenced with two distinct siRNA sequences in U87MG, a mesenchymal subtype of GBM cell line. LOXL3 down regulation was confirmed at gene and protein expression levels. Functional assays with silenced LOXL3 demonstrated a decrease in the cell proliferation (p<0.05), and an increase in the apoptosis associated to temozolamide treatment (p<0.05). Immunofluorescence analysis in U87MG cells showed LOXL3 colocalized with mitochondria. Interestingly, such colocalization decreased when LOXL3 was downregulated, associated to a clear morphological alteration in mitochondria shape. A rough increase in mitochondria volume was observed, suggesting fused mitochondria. In addition, such condition was associated to a 23% decrease in mitochondrial DNA copy number. Altogether, our findings suggest that LOXL3 is upregulated in GBM, with impact in prognosis and might play a role in mitochondria dynamics and/or mitophagy, modulating apoptosis. Transcriptome analysis, currently in progress, will further clarify the associated players in this signaling pathway.

### Changes in Chromatin Structure

#5176

Topological characterization of chromosome territories 9 and 22 and BCR-ABL1 genes in bone marrow CD34+ cells.

Eunice Fabián-Morales, Yameli Lizeth Rodríguez Torres, David G. Vallejo Escamilla, Rodrigo González-Barrios, Alfredo De La Torre Luján, Alejandro López-Saavedra, Clementina Castro-Hernández, Luis A Herrera Montalvo. _Instituto Nacional de Cancerología, Mexico City, Mexico_.

During interphase, chromosomes occupy a distinct, limited space within the nucleus, referred to as chromosome territory (CT). It has been postulated that the non-random CT's spatial organization within the cell nucleus contributes to determining the outcome of chromosomal translocations. Comparative analysis of the spatial arrangement of translocation partners and their frequency of translocation suggest that translocations occur preferentially among proximally positioned genome regions. The consistence appearance of t(9;22) in pluripotent hematopoietic stem cells, apart from being etiological factor for chronic myeloid leukemia (CML), is a clear example of what is considered a non-random chromosome aberration.

In order to highlight the importance of spatial proximity and topological features in determining the t(9;22) formation, we evaluated these elements using three-dimensional fluorescence in situ hybridization and 3D Structure Illumination Microscopy. This analysis was carried out in CD34+ cells from 5 CML patients in complete response, in Mobilized Bone Marrow (MBM) from 5 healthy donors, and also in peripheral blood mononuclear cells (PBMC). 3D reconstructions from at least 25 cells per sample were analyzed. We determined the nucleus' and CTs' volume, the distance between BCR and ABL (BA) genes and their CTs, and the distances between them and the nuclear center.

We found that nuclear volumes were significantly larger in CD34 + cells from MBM and CML patients compared to PBMC controls. Likewise, the absolute CTs volume analysis shows a major volume of CT9 compared to CT22. In order to avoid the bias of size differences due to nuclear volumes, we normalized the CTs' volumes and expressed them as a percentage of the nuclear volume in each studied population, however it does not show a significant variation. After normalizing the distance between the CTs a

To determine the distance between BA genes and their CTs we calculated the nearest distance between the mass center of CT9 and CT22. Shorter distances were observed between BA from MBM and CML patients in complete response compared to PBMC samples. After normalizing the distance between the CTs and BA genes against the radius of the nucleus. We observed that the location of CT22 tended towards the center, whereas that of CT9 towards the nuclear periphery. Meanwhile BA genes were located closer to the nuclear center than the respective CTs' geometrical center.

Given that CTs are discrete chromatin domains, histone modifications might have a significant role in dictating the topological CTs characteristics. Therefore, we have mapped some relevant histone modifications like H3K9ac and H3K27me3 across CT9 and 22 using 3D immuno-FISH. The enrichment of H3K27me3 seems to be greater in CT22 from samples of CML patients in complete response. These are preliminary results and we pretend increase the sample size to delineate conclusive results.

#5177

Spatial co-fragmentation pattern of cell-free DNA recapitulates in vivo chromatin organization and identifies tissue-of-origin.

Yaping Liu,1 Tzu-Yu Liu,1 David Weinberg,1 Chris J. De La Torre,2 Catherine L. Tan,2 Anthony D. Schmitt,2 Siddarth Selvaraj,2 Vy Tran,3 Louise C. Laurent,3 François-Clément Bidard,4 Imran S. Haque1. 1 _Freenome Inc, South San Francisco, CA;_ 2 _Arima Genomics, San Diego, CA;_ 3 _University of California San Diego, San Diego, CA;_ 4 _Institut Curie, Paris, France_.

Three-dimensional chromatin organization varies across different cell types and is essential for gene regulation. Functional genomic elements that reside kilobases to megabases away can be brought into spatial proximity by chromatin folding. In fixed cells, DNA fluorescence in situ hybridization and super-resolution microscopy can measure the distances between loci at ~10-100nm resolution, while chromosome conformation capture followed by next-generation sequencing (Hi-C) is able to profile genome-wide chromatin organization at kilobase-pair level resolution by measuring contact probabilities between pairs of loci. These methods provide a static snapshot of genome compaction and organization in different cellular states. However, assessing in vivo genome-wide chromatin organization changes non-invasively and longitudinally in patients is challenging due to the limitations of current technologies. Recently, circulating cell-free DNA (cfDNA) in blood has been shown as a promising biomarker to capture the genetic and local epigenetic changes within patients.

Here, we inferred in vivo chromatin organization in blood cells from co-fragmentation patterns of cfDNA by using fragment lengths estimated from paired-end whole genome sequencing (WGS). We performed cfDNA WGS on 100 healthy, 34 colorectal cancer, 48 lung cancer, and 19 melanoma patients. The inferred chromatin organization is highly concordant with Hi-C performed on white blood cells and not explained by technical biases, sequence composition, or other epigenetic factors. Further, we developed methods to identify the tissue-of-origin of cfDNA based on its co-fragmentation pattern and Hi-C signal in reference cell types, which confirmed that most cfDNA in healthy individuals is derived from hematopoietic cells. In cancer patients, we observed an increased contribution to cfDNA from cancer cells that was quantitatively correlated with estimated tumor fraction in cfDNA and qualitatively matched tumor type. We also verified the results using publicly available cfDNA WGS data from different healthy and cancer patients. These results are consistent with previous studies that directly measured DNA methylation or that inferred nucleosome positions from WGS on cfDNA. However, our method has distinct advantages including using only low-coverage WGS, not requiring bisulfite treatment, and providing a more robust and quantitative estimation of cell type contributions. Collectively, our results demonstrate the potential of using cfDNA WGS to non-invasively assess the in vivo three-dimensional chromatin organization and determine tissue-of-origin in different physiological and pathological conditions, which may be useful for detecting, monitoring and treating different diseases.

#5178

Genomic and transcriptomic alterations of chromatin factors in head and neck cancer.

Hui Cheng,1 Riyue Bao,2 Carter Van Waes,1 Vassiliki Saloura1. 1 _National Insts. of Health, Bethesda, MD;_ 2 _University of Chicago, Chicago, IL_.

Chromatin factors play central roles in the transcriptional regulation and maintenance of genomic stability and have been implicated in cancer initiation and progression in multiple cancer types. No study has systematically investigated the genomic and expression alterations of chromatin factors in head and neck squamous cell carcinoma (HNSCC). To this purpose, we utilized the molecular profiles from over 510 HNSCCs in the Cancer Genome Atlas (TCGA), and thoroughly characterized the mutational, copy number and transcriptional alterations of 457 chromatin factors and histones. Over 75% of all HNSCC samples have genomic alterations in protein methyltransferases and protein demethylases. Protein lysine methyltransferases MLL2 and NSD1 are the most frequently mutated chromatin factors in this disease. Actin Like 6A (ACTL6A), a component of the SWI/SNF chromatin remodeling complex, SUMO specific peptidase 5 (SENP5) and Nuclear receptor binding SET domain protein 3 (NSD3) are the most frequently copy-number amplified and overexpressed chromatin factors. Interestingly, hierarchical clustering of gene expression of a set of chromatin factors identified a molecular subtype with significantly better overall survival (OS), regardless of HPV-status, stage and NSD1 mutation status, a factor recently reported to render better OS in HNSCC. Systematic characterization of the deregulation of chromatin factors may elucidate novel epigenetic mechanisms and provide novel targets for cancer therapy in HNSCC. Supported by NIDCD intramural projects ZIA-DC-000016, 73 and 74.

#5179

**Chromatin dynamics** in vivo **reveal the complexity of cell fate transitions in pancreatic cancer initiation.**

Rohit Chandwani,1 Steven Leach,2 Richard Koche3. 1 _Weill Cornell Medicine, New York, NY;_ 2 _Dartmouth College, Lebanon, NH;_ 3 _Memorial Sloan Kettering Cancer Center, New York, NY_.

Pancreatic cancer initiation features a hallmark preneoplastic transdifferentiation event wherein the acinar cell acquires a ductal phenotype. Despite these transitions in cell fate, little is known about the chromatin specification of pancreatic cell types and the epigenetic dysregulation of normal acinar cells in tumor initiation. To address these questions, we employed a lineage-traced autochthonous mouse model to examine systematically perturbed acinar cells; in addition, we sorted pancreatic progenitor, islet, and ductal cell populations. We observe in our system that Kras activation alone does not disturb acinar cell chromatin nor the histologic appearance of the pancreas. By contrast, caerulein alters chromatin significantly in metaplasia and even in regeneration, with putative enhancers derepressed despite normal histology. In the context of Kras activation and caerulein administration, we find a broad and stable reorganization of chromatin, reflecting cooperativity between oncogenic stress and an inflammatory insult. The chromatin of the reprogrammed acinar cell bears few, if any, ductal features and instead reflects a largely novel cell fate. Analysis of these acinar-derived cells reveals the induction of specific proliferative and progenitor master transcription factors, opening of their binding sites in chromatin, and activation of associated transcriptional programs. Together, our findings suggest that (1) loss of acinar cell identity is resistant to oncogenic stress and is susceptible to inflammation; (2) the acquired acinar cell fate reflects neither true metaplasia nor transdifferentiation nor dedifferentiation events, and (3) acinar cell regeneration is incomplete. Our data thus demonstrate that histologic appearance does not adequately reflect cellular identity, highlighting the complexity of cell fate decisions in the preneoplastic pancreas and revealing potential master regulators of acinar cell identity.

#5180

Enhanced and controlled chromatin extraction for chromatin-based epigenetic assays in FFPE tissues.

Jian Zhong,1 Zhenqing Ye,1 Chad Clark,1 Samuel Lenz,1 Justin Nguyen,2 Huihuang Yan,1 Keith Robertson,1 Gianrico Farrugia,1 Zhiguo Zhang,3 Tamas Ordog,1 Jeong-Heon Lee1. 1 _Mayo Clinic, Rochester, MN;_ 2 _Mayo Clinic, Jacksonville, FL;_ 3 _Columbia University, New York, NY_.

Background: Epigenetic dysregulation is involved in the etiology and progression of various human diseases. Formalin-fixed paraffin-embedded (FFPE) samples represent the gold standard for archiving pathology samples, and thus FFPE samples are a major resource of samples in clinical research. However, chromatin-based epigenetic assays in the clinical settings are limited to fresh or frozen samples and are hampered by low chromatin yield in FFPE samples due to the lack of reliable and efficient chromatin preparation methods. Here, we introduce a new chromatin extraction method from FFPE tissues (Chrom-EX PE) for chromatin-based epigenetic assays.

Results: During rehydration of FFPE tissues, applying tissue-level cross-link reversal to the deparaffinized tissue at 65°C dramatically increased chromatin yield in the soluble fraction. We found the resulting chromatin to be compatible with ChIP-qPCR and ChIP-seq analysis of histone marks (H3K4me3, H3K27me3, and H3K27Ac) and RNA polymerase II. The chromatin prepared by Chrom-EX PE showed a gradual fragmentation pattern with varying incubation temperature. At temperatures below 37°C, the majority of soluble chromatin was over 1 kb in size. The soluble chromatin prepared at 45-60°C showed a typical nucleosomal pattern shown in cell lines and frozen tissues. The majority of the chromatin prepared at 65°C is close to mononucleosomal size. These observations indicate that chromatin preparation from FFPE samples can be controlled for downstream chromatin-based epigenetic assays.

Conclusions: Our method achieves efficient extraction of high-quality chromatin from FFPE samples suitable for targeted and genome-wide epigenetic assays while minimizing degradation and damages. This approach provides a new platform for developing chromatin-based assays applicable to archived FFPE samples to circumvent the over-fixed nature of FFPE tissues.

This work was supported, in part, by National Institutes of Health grant P01DK068055 and the Mayo Clinic Center for Individualized Medicine Epigenomics Program.

#5181

Global alteration of CTCF binding in the cancer genome.

Celestia Fang,1 Zhenjia Wang,2 Carlos A. Martinez,1 Panagiotis Ntziachristos,1 Chongzhi Zang2. 1 _Northwestern University, Chicago, IL;_ 2 _University of Virginia, Charlottesville, VA_.

The goal of this study is to understand the functions of altered CTCF binding in the cancer genome and its role in tumorigenesis. CTCF is a zinc finger protein that binds to DNA and functions as a transcriptional factor (TF) or a chromatin insulator. Disruption of individual CTCF bindings have been reported in several systems that associate with DNA methylation changes and result in aberrant chromatin interaction and differential expression of genes associated with the topological associated domains (TADs). We systematically analyzed over 500 CTCF ChIP-seq datasets across different human tissues and identified cancer-specific aberrant (gained) and disrupted (lost) CTCF binding sites in several cancer types. Through integrative analysis with other genomic data including RNA-seq, hi-C and DNA methylation profiles, we found that CTCF binding alterations correlate with differential chromatin interaction in the TADs, but most altered binding sites do not have DNA methylation changes. Instead, lost CTCF bindings at gene promoter regions associate with transcriptional repression, while gained bindings primarily occur in the distal enhancer regions bound by oncogenic transcription factors inducing gene expression. We validated these findings in T-cell acute lymphoblastic leukemia (T-ALL) using patient derived samples and identified NOTCH1 among oncogenic TFs bound at enhancers co-occupied with T-ALL-specific gained CTCF binding. These findings indicate that alteration of CTCF binding is an epigenomic signature of cancer cells, and our approach demonstrates a framework of integrating existing multi-omics data for mechanistic studies of cancer gene regulation.

#5182

Chromatin reader machinery as target for overcoming resistance to DNA-demethylating epi-drug decitabine.

Khushboo Agrawal, Petr Vojta, Rastislav Slavkosky, Ivo Frydrych, Petr Dzubak, Marian Hajduch. _Palacky University, Olomouc, Czech Republic_.

Based on the current understanding of the epigenetic landscape, cancer methylome is highly disrupted, which makes DNA methylation an excellent target for anti-cancer cures. To date, azacytidine and its congener, decitabine (DAC) are the most successful DNA demethylating epigenetic drugs. However, scientists are yet to find the reversal of clinical resistance to these drugs. Yet another challenge remains to be the decreased efficacy of these promising therapies in the cure of solid tumors. We used a soild tumor, HCT116 colorectal cancer cell line and developed resistance against DAC. DAC-resistant HCT116 cells were used to study the epigenetic cross-talk between DNA methylation and chromatin modifications. The screening of parental and DAC-resistant HCT116 cells against inhibitors of epigenetic writers-erasers-readers unveiled increased sensitivity of resistant cells to BET inhibitor (inhibitor of reader enzymes), (+)-JQ1. BET inhibitor further mediated augmented response on cell cycle phases of resistant cells, increased anti-proliferative effects in xenograft models of resistant cells, and synergystic effects in combination with DAC in HCT116 parental cells, both in vitro and in vivo. We then sequenced the transcriptome of DAC-sensitive and -resistant HCT116 cells, and (+)-JQ1 treated-(DAC-resistant) cells, using RNA-seq. The RNA-seq data revealed the overexpression of critical oncogenes, and their binding inactivation of key tumor suppressor genes (TSGs) in resistant cells. The most significant and biologically relevant transcriptional changes were further validated by qRT-PCR, and in addition their methylation status was determined by bisulphite sequencing. We discovered that the expressions of down-regulated TSGs were driven by promoter methylation, exposing these tumor-suppressive signatures as biomarkers which might differentiate between DAC-resistance and sensitivity, whereas, overexpression of oncogenes was independent of promoter methylation. Interestingly, the expressions of up-regulated oncogenes which define cell identity, mainly those involved in signaling of inflammatory pathways (including some bromodomain-specific genes) were reversed on treatment with (+)-JQ1. Further, siRNA-mediated genetic inhibition of bromodomains in resistant cells phenocopied therapeutic inhibition by (+)-JQ1. These data unveil the chromatin "reader proteins", as regulators of dysregulated oncogenic expressions in DAC-resistant cells. The present study provides novel insights into the epigenomic landscape of DAC-resistant colorectal cancer cells, and put forward, the alternative therapeutic regimen for DAC-resistant patients.

This study was supported by Czech Ministry of Education, Youth and Sports (LO1304, LM2015091, LM2015064), Technology Agency of the Czech Republic (TE01020028) and Internal Grant Agency of Palacky University (IGA_LF_2016_019). 

### Deregulation of Histone Modifications

#5183

ONC201 induces acetylation of histone binding within the p21 (CDKN1A) gene promoter.

Yiqun Zhang, Lanlan Zhou, Shengliang Zhang, Marie D. Ralff, Shuai Zhao, Philip H. Abbosh, Rahmat Sidker, Wafik S. El-Deiry. _Fox Chase Cancer Center, Philadelphia, PA_.

ONC201/TIC10 is a promising anticancer agent that upregulates cytotoxic TRAIL pathway signaling in cancer cells. Preliminary clinical data indicates ONC201 induces clinical benefit in a subset of patients with histone H3 K27M glioma, among other tumors. Since H3 K27M mutation reduces levels of H3K27 di- and tri-methylation and in turn alters the expression of genes by epigenetic modulation, we investigated ONC201 effects on epigenetic regulation in cancer. We treated the colorectal cancer cell line HCT116 with the histone deacetylase inhibitor entinostat or ONC201. Chromatin immunoprecipitation (ChIP) was performed with anti-histone H3 (acetyl K9) antibody using lysates from drug-treated tumor cells. Immunoprecipitated DNA was probed by PCR using primers across the promoter of p21 (WAF1; CDKN1A). The results indicate that both ONC201 and entinostat induce the acetylation of histone H3 binding within the p21 promoter. We further observed that ONC201 plus entinostat have synergistic effects in this p21 promoter acetylation activity. Activation of p21 gene expression by enhancing histone acetylation may be relevant in cancer suppression by ONC201.

#5184

N-CoR2 epigenetically regulates adhesion-dependent branching morphogenesis and its loss correlates with malignant progression of the mammary gland.

Shenq-Shyang Huang,1 Valerie M. Weaver,2 Kelvin K.C. Tsai3. 1 _National Tsing Hua University, Hsinchu, Taiwan;_ 2 _University of California San Francisco, San Francisco, CA;_ 3 _Taipei Medical University, Taipei, Taiwan_.

Branching morphogenesis depends upon extracellular matrix remodeling and dynamic cell-cell and cell-extracellular matrix interactions, although how this developmental program is coordinated remains unclear. Expression profiling of mammary tissue at different stages of branching morphogenesis combined with histological and functional studies identified the histone deacetylase (HDAC) regulator, nuclear receptor co-repressor 2 (N-CoR2), as a key orchestrator of adhesion-dependent branching morphogenesis in the mammary gland. Molecular studies revealed that N-CoR2 actively represses fibronectin (FN) and thrombospondin-1 (THBS-1) promoter accessibility in an HDAC-dependent manner. Consistently, histological analysis revealed that N-CoR2 is abundantly expressed in the luminal epithelial cells localized along the outer rim of invading terminal end buds (TEB) and that its expression is lowest at sites of TEB branching bifurcation. In addition, reducing N-CoR2 expression drove the scattering of human mammary acini in vitro and promoted promiscuous branching of humanized mouse mammary tissue in vivo, suggesting that downregulation of N-CoR2 is required for branching morphogenesis in mammary gland. Furthermore, downregulation of N-CoR2 in breast cancer cells increased the expression of both FN and THBS-1 and reduced the expression of E-cadherin and enhanced the migratory capability of breast cancer cells. Intriguingly, immunohistochemical and clinical correlative analysis indicated that loss of N-CoR2 correlates with perturbed cell-cell and enhanced cell-ECM adhesion and acquisition of an invasive phenotype and local lymph node metastasis in a subset of breast carcinomas. These findings suggest N-CoR2 may epigenetically regulate adhesion-dependent branching morphogenesis in the mammary gland and that its loss could promote malignant progression of some breast cancers.

#5185

Squamous cell cancers and chronic obstructive pulmonary disease share polycomb repressive cComplex 2 dysregulation.

Aria L. Byrd, Christine F. Brainson. _University of Kentucky, Lexington, KY_.

Non-small cell lung cancers often share the aberrant differentiation patterns observed in the bronchiolar epithelium of chronic obstructive pulmonary disease (COPD), including squamous and mucinous phenotypes. Additionally, smokers with COPD are 3.5 times more likely to develop squamous lung cancer than smokers with pathologically normal lung epithelium. The Polycomb Repressive Complex 2 (PRC2), which contains the methyltransferase EZH2, mediates proper cell differentiation via tri-methylation of histone H3 at lysine 27 (H3K27me3), leading to epigenetic silencing of genomic regions. Our previous data demonstrate that squamous lung cancers often have low H3K27me3. Therefore, we hypothesized that loss of proper PRC2-mediated gene repression changes lung epithelial cell fate and drives squamous and mucinous phenotypes in COPD as well. To examine this hypothesis, we stained lung tissue from healthy patients and patients with COPD for H3K27me3 and observed that areas of squamous and mucinous metaplasia had a significantly decreased abundance of nuclear H3K27me3 stain. To demonstrate that this effect was due to loss of EZH2 activity, EZH2 transcripts were knocked down with short hairpins in human bronchiolar epithelial cells. When grown in air-liquid-interface cultures, shEZH2 cells had significantly more goblet cells and significantly fewer club cells than control cultures. We also tested this hypothesis in genetically engineered mice in which we could conditionally delete Ezh2. To examine growth, differentiation and self-renewal potentials of different lung epithelial cells, we FACS-isolated distal lung bronchioalveolar stem cells (BASCs) and cultured them as organoids. We observed a 2-fold decrease in organoid number of Ezh2 null vs wild type bronchiolar cells and a significant difference in organoid diameter between Ezh2 wild type, heterozygous and null bronchiolar cells. Organoids with squamous morphology were only seen in the Ezh2 null genotype. Lastly, we performed Gene Set Enrichment Analysis and found that genes up-regulated in human COPD patients relative to healthy smokers were also up-regulated in murine EZH2 knock-out adenocarcinoma cells. Together, these data suggest that loss of proper PRC2-mediated gene silencing is a shared epigenetic state in COPD and squamous lung cancers. Our laboratory is actively investigating the mechanisms through which EZH2 activity is lost in bronchiolar epithelium and plan to use our knowledge in attempt to 'renormalize' dysregulated epithelium to a more normal epigenetic state. Funded by American Cancer Society IRG-85-001-25, K22 CA20103 and Molecular Mechanisms of Toxicity Training Grant T32ES07266

#5186

Discovery of a novel histone deacetylase 6 inhibitor that kills drug-resistant breast cancer.

Catríona Dowling,1 Eugene Dillion,2 Michael Hemann,3 Gerard Cagney,2 Anthony Letai,4 Triona Ni Chonghaile1. 1 _Royal College of Surgeons in Ireland, Dublin, Ireland;_ 2 _University College Dublin, Dublin, Ireland;_ 3 _Massachusetts Institute of Technology, Boston, MA;_ 4 _Dana-Farber Cancer Institute, Boston, MA_.

Cytotoxic chemotherapy is the standard of care for patients with triple negative breast cancer (TNBC). Most patients with advanced TNBC progress after chemotherapy and die from metastatic disease. Currently, no established targeted therapeutics or biomarkers of outcome/response have been clinically approved in the context of TNBC. The aggressive nature of TNBC is reflected by an increased likelihood of distant recurrence and death within five years following primary intervention and a shorter survival once diagnosed with metastatic disease. To identify novel therapeutics for the treatment of chemoresistant TNBC, we performed a high-throughput screen to identify small molecules that are cancer selective but can kill drug-resistant TNBC cells. We screened a total of 30,000 compounds in duplicate across the TNBC cell line MDA-MB-321 in which the pro-death proteins BAX/BAK were knocked down, and the non-transformed "normal" MCF10A cell line. There was a hit rate of 0.3% in the screen and 85 compounds were retested in the validation cherry pick screen. From this screen 18 compounds were further validated with low-throughput assessment for mitochondrial independent killing and selectivity for cancer cells. To identify the mechanism of action of the lead compound we used a genetic approach to generate an RNAi signature for the compound, which indicated the lead compound was most likely that of histone deacetylase inhibitor (HDAC). Using an in vitro HDAC inhibitor screen, we identified that the compound specifically inhibited HDAC6. Using a series of cell based assays, we determined that treatment of cells with the compound BAS-2 resulted in the same phenotypical response (inhibition of migration, invasion and 3D spheroid formation) as HDAC6 knockout. To identify substrates of HDAC6 in cells we analyzed the proteome for increased acetylation by mass spectrometry following HDAC6 knockout or inhibition with the specific inhibitor, BAS-2. Remarkably, we identified a series of new substrates for HDAC6 that are common in both the HDAC6 knockdown and BAS-2 treated cells. In conclusion, we have identified a novel HDAC6 specific inhibitor that selectively kills cancer cells independent of mitochondrial apoptosis, alters acetylation of over a 50 common proteins and inhibits spheroid formation of TNBC.

#5187

Effect of histone deacetylase inhibitor on PD-L1 expression in lung cancer cells.

Umamaheswari Natarajan,1 Thiagarajan Venkatesan,2 R Vijayaraghavan,1 Shila Samuel,1 Appu Rathinavelu2. 1 _VRR Inst. of Biomedical Science, Kattuppakkam, Chennai, India;_ 2 _Nova Southeastern University, Fort Lauderdale, FL_.

Programmed Cell Death Ligand-1 (PD-L1) is frequently expressed on tumor cells and tumor-infiltrating immune cells. The expression of PD-L1 is indicative of immunosuppression and cause for poor prognosis in several cancers. The effects of PD-L1 are mediated through its binding to the Programmed Cell Death-1 (PD-1) receptor, which is a co-inhibitory surface molecule that is expressed on lymphoid and non-lymphoid derived cells. After PD-L1 binding the PD-1 receptor mediates the signals intracellularly to suppress peripheral T-cell responses. Therefore, determining the mechanisms of up-regulation or down-regulation of the PD-L1 is very crucial for the purpose of devising strategies to reactivate the T-cells that can lead to enhanced tumor attack. However, the cellular mechanisms that determine the levels of PD-L1 are not fully understood at this time. Hence, we analyzed different cancer cell lines for the expression levels of PD-L1 and confirmed the highest-level of expression in HCC827 lung cancer cells. In addition, H460, H1975 (Lung Cancer), SJSA1 (Osteosarcoma) and U-87 MG (Glioblastoma) cells were found to express PD-L1 in high levels. Relatively, lower level expression of PD-L1 was detected in H226 (Squamous Cell Carcinoma) PANC-1 (Pancreatic Cancer), CFPAC-1 (Pancreatic, Ductal Adenocarcinoma) and LNCaP (Prostate Cancer) cells compared to HCC827. Since, alterations in gene expressions are often regulated by epigenetic modifications, we conducted experiments to determine whether the expression of PD-L1 can also be altered by the HDAC inhibitor SAHA (Suberoylanilide hydroxamic acid). In our experiments, SAHA (7.5 μM) was able to reduce the expression of PD-L1 in a time and dose dependent manner and produced more than 50% decrease in the protein levels within 24 hrs. while the level of PD-L1 was significantly reduced, concomitant decreases in EGFR, phospho-EGFR levels were also observed in HCC827 cells following SAHA treatment. Furthermore, SAHA was able to increase the levels of acetyl-H2B, acetyl-H3 and acetyl-H4, which confirmed the impact of HDAC inhibition on the acetylation status of histones. Since de-acetylation mediated unwinding of the DNA is typically responsible for the increased expression of genes such as p21/CDKN1A, it is suspected that the decrease in the PD-L1 levels may be due to reduced transcription of the CD274 gene that is coding for PD-L1. However, it is not clear whether the decrease in PD-L1 expression observed is because of p21 mediated negative control on the transcription process or due to some other mechanism. Additional studies are required to fully understand the actual mechanisms that may be involved in the regulation of PD-L1 expression by HDACs and their inhibitors. (This research was supported by the Royal Dames of Cancer Research Inc., Fort Lauderdale, Florida).

#5188

Epigenetic changes mediated by polycomb repressive complex 2 are associated with cisplatin acquired resistance in testicular germ cell tumor.

Ratnakar Singh, Zeeshan Fazal, Emmanuel Bikorimana, Andrea Corbet, Jennifer Rodriguez, Alyssa Steege, Sarah J. Freemantle, Michael J. Spinella. _University of Illinois Urbana Champaign, Champaign, IL_.

Testicular germ cell tumors (TGCTs) represent the most common carcinoma affecting young men. TGCTs serve as a paradigm of chemo-sensitive tumors as more than 80% of metastatic disease can be cured with cisplatin-based chemotherapy. However, therapy resistance can occur, which results in poor patient outcomes. The mechanisms of cisplatin resistant in these tumors remain largely undefined. To study the mechanism of cisplatin acquired resistance we generated several independent, acquired resistant clones of parental NT2/D1, 833K and 2102EP testicular cancer cells employing an in vitro protocol designed to mimic the standard-of-care cisplatin regimen used to treat TGCT patients. We further characterized 10 of these clonal lines and confirmed stable cisplatin resistance ranging from 5- to 30-fold. After confirming the cisplatin resistance, we performed RNA seq transcriptomic profiling, differential gene expression analysis, DAVID and GSEA. Remarkably, RNA-seq based transcriptional profiling revealed highly significant alterations shared between the majority of the resistant cells regardless of their parental cell of origin. This includes apparent amplification of chromosomal region 17q21-25 in 8 of 10 lines. Unexpectedly, 7 of 10 lines had a highly significant enrichment of genes regulated by H3K27 methylation and polycomb repressive complex 2 (PRC2). Transcription factor prediction analysis of genes differentially expressed between resistant and parental cells revealed that polycomb complex members were among the most highly ranked predicted transcription factors responsible for the gene expression changes. These include EZH2, SUZ12, JARID2 and BMI1. We will present our ongoing studies investigating the role of PRC2 and H3K27 methylation in mediating cisplatin resistance in these cells and in patient samples. Together our data suggests that repression of H3K27 methylation may be a mechanism of cisplatin acquired resistance in TGCTs and that restoration of PRC2 function could be a viable approach to overcome treatment failure.

#5189

Activity of polycomb repressive complex 2 determines sensitivity to epigenetic therapy.

Fan Chen, Christine F. Brainson. _University of Kentucky, Lexington, KY_.

EZH2 is a histone methyltransferase that tri-methylates histone H3 at lysine 27 (H3K27me) in the context of the Polycomb Repressive Complex 2, and is highly correlated with poor prognosis of many cancers. However, recent data suggest a de-coupling of EZH2 expression from its enzymatic activity, and many poor prognosis tumors also have low H3K27me3. Therefore, we hypothesize that deletion of Ezh2 would drive more aggressive tumors which may have distinct epigenetic vulnerabilities. To test this hypothesis, we generated the LSL:KrasG12D/+; p53 flox/flox (LSL: lox-stop-lox) mice (Kras/p53 mice) with either zero, one or two floxed alleles of Ezh2 and induced lung tumors with intranasal adeno-Cre virus administration. Supporting our hypothesis, we observed mice with two floxed alleles of Ezh2 (Ezh2 null) had higher tumor burden, higher tumor grade and lower survival than Ezh2 wild-type or heterozygous Kras/p53 mice. Ezh2 null tumors also had a higher propensity to metastasize than Ezh2 wild-type tumors. Consistent with these results, Ezh2 null Kras/p53 tumors and cell lines had increased expression of genes involved in epithelial-mesenchymal transition (EMT), KRAS-driven tumorigenesis, metastasis, and cell cycle regulation. In order to examine the therapeutic vulnerabilities of Ezh2 depleted lung cancers, we built a panel of 2-dimensional cell lines and 3-dimensional organoid cultures of the Kras/p53 tumors that were Ezh2 null, heterozygous and wild-type. We reasoned that using the H3K27me3 demethylase inhibitor might be able to stabilize the residual H3K27me3 in Ezh2 null cells and decrease their aggressive properties. Intriguingly, while we observed no difference in sensitivity to H3K27me3 demethylase inhibitor in the various Ezh2 genotypes of 2-dimensional cultures, both 3-dimensional cultures and in vivo allografts demonstrated Ezh2 null tumors but not Ezh2 WT or heterozygous are sensitive to the H3K27me3 demethylase inhibitor. These data are interesting because they suggest that assays of 3-dimensional organoids may more faithfully recapitulate responses to epigenetic drugs than standard cell lines, and that aggressive tumors with low H3K27me3 might be selectively killed by demethylase inhibitor. We are now expanding our studies to include other targeting agents, including inhibitors of the kinase WEE1 and inhibitor of the chromatin reader BRD4.

#5190

EZH2 epigenetically silences anti-tumor immunity in melanoma via dual histone and DNA methylation.

Jessamy Tiffen, Dilini Gunatilake, Stuart Gallagher, Peter Hersey. _The Centenary Institute, Australia_.

Despite major advances in immuno- and targeted therapies for metastatic melanoma, not all patients respond to such treatments and the development of therapy resistance remain key obstacles. Novel targets are required and the epigenetic modifier EZH2 may represent such a solution.

EZH2 methylates histones causing chromatin compaction and silencing of gene expression. Additionally, EZH2 has been shown to interact with DNA methyltransferases (DNMTs). Aberrant EZH2 in cancer results in the silencing of hundreds of tumor suppressor genes that would normally constrain cancer growth, and 26% of patients in the Australian Melanoma Genome Project (AMGP) display such abnormal EZH2.

Using ChIP-seq, microarrays and DNA methylation sequencing, we identified a subset of EZH2 target genes that are silenced by both histone and DNA methylation in melanoma. Interestingly many of these genes play a role in the anti-tumor immune response, including RASSF5 and ITGB2 that facilitate lymphocyte infiltration into tumors and correlate with better survival outcomes in melanoma patients when their expression is high. We show that such genes can be significantly up-regulated by combined treatment with inhibitors of both EZH2 and DNMT in melanoma cells in vitro.

Our studies suggest that aberrant EZH2 in melanoma drives aggressive disease via suppression of genes that would normally trigger the anti-tumor immune response. The progression of several preclinical, small molecule inhibitors of EZH2 and DNMTs remains promising.

#5191

Functional role of EZH2 in neuroendocrine prostate cancer.

Md Imtiaz Khalil, Shu Yang, Anthony Blankenship, Zachary Connelly, Xiuping Yu. _Louisiana State University Health Sciences Center-Shreveport, Shreveport, LA_.

Background: Neuroendocrine prostate cancer (NEPC) is the most aggressive malignant variant of prostate cancer (PCa). Since there is no effective treatment available for NEPC to date, devising a therapeutic intervention requires identifying the mechanisms of how prostate cancer cells gain neuroendocrine phenotype. Evidence suggest that neuroendocrine differentiation is associated with elevated expression of Enhancer of Zeste Homolog 2 (EZH2). EZH2 is the catalytic subunit of Polycomb repressive complex 2 (PRC2) that functions to regulate histone H3 methylation and causes transcriptional repression of target genes. We hypothesize that the elevated expression of EZH2 in NEPC may render the tumors cells an undifferentiated state which enables the lineage switch and trans-differentiation into neuroendocrine malignancies.

Method and Results: First, we extracted microarray data to analyze the expression of polycomb genes during PCa progression, using Gene Expression Omnibus (GEO) database from NCBI. The expressions of Polycomb genes (EZH2, EED, SUZ2, CBX1) were up-regulated in metastatic castrate-resistant prostate cancer and the increased EZH2 expression was in concert with elevated expressions of NEPC marker (CHGA, FOXA2, SOX2) and decreased expression of FOXA1, a prostate differentiation marker. Additionally, we conducted IHC staining in sections derived from benign prostate hyperplasia, low- and high- grade prostate adenocarcinoma, patient-derived NEPC xenograft tumors (LuCaP), and mouse models of NEPC (12T10 LADY and TRAMP). EZH2 and FOXA2 were overexpressed in all NEPC tumors while FOXA1 was abundant in prostate adenocarcinoma but decreased in NEPC. Also, we cultured prostate adenocarcinoma, LNCaP cells in androgen-depleted media and analyzed the gene expression of major polycomb genes and NEPC markers by qPCR. The expressions of Polycomb genes (BMI1, RING1a, CBX1) were induced by androgen depletion, concurrent with the induction of NEPC gene, CD56. Furthermore, we stably knocked down EZH2 in DU145 and PC3 cell lines (both have NEPC features) using shRNA and measured both the RNA and protein level of EZH2, PCa and NEPC markers using qPCR and western blot. We found that the levels of AR, FOXA1 and E-cadherin are upregulated in both DU145 and PC3 cells when EZH2 was knocked down.

Conclusion: Our data support that EZH2 is functionally involved in the development of NEPC.

Future direction: We will overexpress EZH2 in LNCaP cells to study if elevated EZH2 expression promotes NEPC. We will also perform RNA-seq and ChIP-seq to study how the altered expression of EZH2 affects the global transcriptome and identify the downstream target genes of EZH2 in PCa. Additionally, we will conduct in vivo animal experiments to determine EZH2's role in NEPC using NEPC PDX models and transgenic mouse models. Findings from this research would provide detailed insights into developing therapeutics against EZH2 mediated NEPC. (Funding source: R03 and FWCC).

#5192

EBV-associated histone modifications regulate DNA damage repair pathway and associate with cisplatin resistance in nasopharyngeal epithelial malignancy.

Merrin Man Long Leong, Arthur Kwok Leung Cheung, Wei Dai, Sai Wah Tsao, Maria Li Lung. _The University of Hong Kong, Hong Kong_.

Introduction: Epstein-Barr virus (EBV) infection is associated with various cancers. Some studies have confirmed EBV can induce hypermethylation to suppress the expression of several key cancer-related genes in gastric cancer. Previously, we showed that nasopharyngeal carcinoma (NPC), an EBV-associated malignancy, is characterized by low mutation rate, but harbors extensive hypermethylation compared with other cancer types. Also, the de novo methylated regions in NPC extensively overlap with the histone bivalent marks (H3K4me3 and H3K27me3) derived from human embryonic stem cells . This suggests an important role of EBV in regulating histone modifications in NPC development. Hence, further studies focusing on the EBV-associated histone modifications are necessary.

Aim: This study aims to elucidate the role of EBV in regulating a transcription-activation histone mark (H3K4me3) and a suppressive mark (H3K27me3) in nasopharyngeal epithelial (NPE) cells.

Methodologies: Two pairs of EBV+/- NPE cell lines were used for chromatin immunoprecipitation sequencing (ChIP-Seq) to identify the genes regulated by EBV-associated histone modifications. ChIP-QPCR and RT-QPCR were utilized to validate the ChIP-Seq results. The candidate genes and pathways identified from the ChIP-Seq results were validated by in vitro and in vivo functional studies.

Results: A panel of 16 DNA damage repair family members were identified with significant reduction of H3K4me3 in the EBV+ NPE cells. One of the candidate genes, MLH1, was validated with down-regulation in the EBV+ NPC cell lines and NPC specimens, and the expression level was demonstrated to associate with cisplatin resistance. Also, the base excision repair (BER) pathway was significantly enriched with reduction of H3K4me3 after EBV infection. Down-regulation of the 7 members from BER was validated in the EBV+ NPE cell lines and NPC specimens. The defective DNA damage repair in the EBV+ NPE cell lines was further validated by the comet assay. Furthermore, after EBV infection, gain of H3K27me3 in MLH1 and the 7 members from BER pathway was identified by ChIP-QPCR. Interestingly, our results from bisulfite sequencing reveals that the promoter regions of the candidate genes are hypomethylated in both EBV +/- NPE cells.

Conclusion: EBV modulates histone bivalent marks independent of the promoter DNA methylation status to regulate DNA damage repair in the host cells.

Acknowledgements: This work was supported by the Research Grants Council of the Hong Kong Special Administrative Region, People's Republic of China: Grant number AoE/M-06/08 to MLL and Seed Funding Programme for Basic Research of HKU 201308159003 to AKLC.

#5193

Conditional knockout of histone chaperone FACT in mice: Consequences for normal tissues.

Masaki A. Ito, Poorva Sandlesh, Imon Goswami, Laura A. Prendergast, Katerina Gurova. _Roswell Park Comprehensive Cancer Center, Buffalo, NY_.

Histone chaperone FAcilitates Chromatin Transcription (FACT) is, a complex of SSRP1 and SPT16, involved in chromatin remodeling during transcription, replication, and DNA repair. FACT is selectively expressed during early stages of development and later exclusively present in the stem cell niches in adult normal tissues. It is expressed in undifferentiated aggressive tumors with poor prognosis.¹ FACT inhibition is not tolerated by most tumor, unlike normal cells. This vulnerability of tumor cells upon FACT inactivation suggests evaluating FACT inhibition as an antitumor therapeutic approach.¹ Since FACT knockout (KO) is embryonically lethal (E3.5),² study of FACT's function in different non-tumor cells of mammalian organisms was not possible and was mostly studied in single cell organisms and in tumor cell lines. Therefore, to assess the safety of FACT inhibition as an anticancer approach, by understanding its role in normal physiology, we generated a conditional KO of Ssrp1 mouse model using CreERT2-LoxP system.³ We harvested stem cells of different lineages to better understand the role of FACT in normal mouse physiology. Using the hematopoietic stem cells (HSCs) from the bone marrow (BM), we performed BM colony formation assay. We found that FACT inactivation significantly decreased the number of colonies of HSCs. Furthermore, inhibiting FACT significantly increased caspase 3 expression in mouse adipose stem cells (ASCs), resulting in apoptosis and decreased survival. These results prompted us to comprehensively evaluate roles of FACT in stem cell development and survival. To this end, fibroblasts in a conditional knockout mouse were reprogrammed by retroviral vector encoding four pluripotency transcription factors (Oct3/4, Klf4, Sox2 and c-Myc). We have found successful generation of induced pluripotent stem (iPS) cells 14 days after reprogramming. In conclusion, FACT associates with development and/or survival of mouse HSCs and ASCs. Further evaluation using iPS cells is expected to delineate roles of FACT in various stages of stem cell development and thus normal physiology. 1. Garcia H, et al. Cell Rep, 2013 4(1) 2. Cao S, et al. Mol Cel Bio. 2003 23(15) 3. Sandlesh P et al. PLOS ONE. 2018 13(6)

#5194

Epigenomic and transcriptional profiling of CRC cell lines with distinct response patterns to the BET inhibitor BI 894999.

Fabio Savarese, Daniel Gerlach, Larissa Koller, Susy Straubinger, Paula Elena Träxler, Onur Kaya, Norbert Schweifer, Ulrike Tontsch-Grunt, Norbert Kraut. _Boehringer Ingelheim RCV GmbH & Co. KG, Vienna, Austria_.

The bromodomain and extra-terminal (BET) protein BRD4 is a "reader" of epigenetic information and binds to acetylated chromatin. BRD4 acts as key regulator of transcriptional elongation by activating P-TEFb at distinct genes. It is via this specific activation of potentially oncogenic transcripts, e.g. MYC, that BRD4 is thought to contribute to tumorigenesis. Inhibition, via so-called BET inhibitors, has indeed proven to be a novel and attractive treatment option tested in ongoing clinical trials for both hematological as well as solid cancers. While BET inhibition shows good efficacy in pre-clinical models of hematological indications, solid tumors show much more heterogeneous responses to monotherapy. Consequently, biomarkers predicting response to treatment with BET inhibitors are of high interest. Colorectal cancer (CRC) is a heterogeneous disease in which novel treatment options are urgently needed. Our pre-clinical studies show that the BET inhibitor BI 894999 is highly active in a fraction of cell lines tested. H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) and transcriptional profiling of sensitive and resistant cell lines identified a small set of H3K27ac peaks associated with sensitivity to BET inhibition. Much of the transcriptional response in CRC cell lines however was cell line specific, leading to only few commonly regulated genes within sensitive versus resistant cell lines. Regarding pharmacodynamic biomarkers in CRC, we could confirm modulation of HEXIM1 after treatment in both sensitive and resistant cell lines. In agreement with previous results (Cohen et al, Nature Comm. 2017), we identified AP1 binding sites in BRD4-bound regulatory regions, which indicates that BRD4 may regulate expression of these genes cooperatively with the transcription factor AP1. Furthermore, the transcription factor TCF4 showed enrichment, suggesting possible interactions between BRD4 and the WNT pathway in a subset of CRC cell lines. The AXIN1, a core WNT pathway component, e.g. is down-regulated in 5/9 cell lines, which however does not track with overall BI 894999 sensitivity. These results, further supporting heterogeneous responses to single-agent BETi treatment, will be discussed. These studies support the further evaluation of BI 894999 as a therapeutic option for colorectal cancer therapy.

#5195

Efficacy of histone deacetylase inhibitors in a 3D cell culture model.

Macarena Irigoyen, Gonzalo Castillo. _Bioensis, LLC., Bothell, WA_.

Epigenetic dysregulation, changes in gene expression not due to changes in the DNA sequence, is the underlying cause of many diseases including cancer. Identification of enzymatic chromatin-complex modulators that can restore chromatin homeostasis is critical to developing better therapeutic agents for both, hematologic and solid tumors. 3D cell culture models have been developed to overcome the limitations of monolayer in vitro assays, to include the complexity of the tumor microenvironment known to modulate epigenetic factors, such as cell-cell interaction and allowing for formations of metabolic gradients, factors known to play a role in epigenetic drug efficacy and resistance. In this study, we evaluated the efficacy of class I and II Inhibitors of histone deacetylases, SAHA and Trichostatin A, in a 3D cell culture in vitro assay across several human tumor carcinoma cell lines. The cell lines were grown in 3D cell cultures, and efficacy of the drugs was evaluated at 72 hours and ten days. All the cell lines grown as spheroids proved to be highly sensitive to SAHA and Trichostation A. AGS, a stomach adenocarcinoma cell line, showed the highest sensitivity to SAHA with an IC50 of 270 nM. Trichostatin A, on the other hand, displayed higher sensitivity to the ovarian carcinoma cell line, SKOV3, with an IC50 of 190 nM. In all cases, maximum potency was seen at the 10-day incubations with IC50s below 1 µM for all the cell lines in the panel. Drug-drug combinations with the epigenetic drugs were also studied, the results are discussed.

#5196

Vorinostat exhibits anticancer effects through modulation of nuclear receptors and tumor suppressor genes in sub-types of triple negative breast cancer cells.

Fatemeh NouriEmamzadeh,1 Beverly Word,1 Ebony Cotton,1 Kai Littlejohn,1 Gustavo Miranda-Carboni,2 Beverly Lyn-Cook1. 1 _NCTR, Jefferson, AR;_ 2 _University of Tennessee Health Science Center, Memphis, TN_.

Aberrantly expression of histone deacetylases (HDAC) play a critical role in tumorigenesis. The expression of individual HDACs has been shown to correlate to decreases in both, disease-free and overall survival, thus targeting HDACs is emerging as a new therapy for cancer. Histone acetylation is associated with elevated transcription, while deacetylation is associated with gene repression. Emerging evidence suggests their role in triple negative breast cancer (TNBC) progression. TNBC is an aggressive type of cancer that has poor prognosis and molecular studies have shown several subtypes within TNBC. These cancers lack targeted treatments and several options used such as, PARP inhibitors, angiogenesis inhibitors, androgen receptor inhibitors and others have shown limited efficacy, however histone deacetylase inhibitors are showing promising results in clinical trials. Vorinostat, a histone deacetylase inhibitor, also known as suberanilohydroxane acid (SAHA) was approved by the FDA for treatment of cutaneous T cell lymphoma. In this study, vorinostat was investigated in subtypes of TNBC cell lines, MB231(MSL) and two basal-like 2 TNBC cell lines, HCC70 and HCC1806. Cells were treated with vorinostat for 24 hrs. Vorinostat significantly up-regulated the tumor suppressor gene NR4A1 by 20 fold, NR4A3 by 10 fold and AR5A by 5 fold. RORα gene had the highest up-regulation, 60 fold, whereas PPARγ 4.5 fold. Estrogen related receptor A, ESRRA was also up-regulated in HCC70 cells. A decrease in expression was noted in DDX5, which is known to be elevated in basal-like tumors. A different profile was noted in the MB231 TNBC cells. Vorinostat also decreased growth and cell invasion, which shows its functional effects on these aggressive TNBC cell lines These changes suggest that vorinostat, which is currently in clinical trials for TNBC may be exerting its effect through modulation of orphan nuclear receptors and reactivation of tumor suppressor genes through epigenetic mechanisms. Further studies are needed to determine other signaling pathways modulated by this HDAC inhibitor.

#5197

Histone deacetylase inhibitors promote breast cancer metastasis by elevating NEDD9 expression.

Zong-long Hu, Ya-fang Wang, Yi Su, Yan-yan Shen, Yan-fen Fang, Jian Ding, Yi Chen. _Shanghai Institute of Materia Medica, Shanghai, China_.

The approved pan-histone deacetylase (HDAC) inhibitors have brought impressive therapeutic benefits for patients with a few subtypes of hematologic malignancies. Favorable outcomes in breast cancer have not been observed, although multiple HDAC inhibitors have been therapeutically validated in preclinical breast cancer models. Here, we report that pan-HDAC inhibitors provoke breast cancer metastasis in vitro and in vivo, which will severely impede their clinical benefits. Given the role of HDAC inhibitors in the epigenetic control of transcription, we utilized RNA sequencing (RNA-seq) analysis to analyze global gene-expression changes following LBH589 treatment of MCF-7 cells. Pathway enrichment analysis of the 1591 identified genes based on GO analysis showed that 146 GO were significantly altered. Interestingly, most of the changes were related to cytoskeletal, cell junction, and biological adhesion pathways. Genes in the above mentioned GO were intersected to obtain seven genes by Venn analysis. NEDD9 was among the most significantly upregulated transcripts by LBH589, for which was suggested as a metastasis marker in the last decade. We substantiated this result by detecting the mRNA and protein levels of NEDD9 in HDAC inhibitors treated cells. As with the RNA-seq results, a notable upregulation of NEDD9 mRNA and protein were induced by HDAC inhibitors in breast cancer in vitro and in vivo. Mechanistically, pan-HDAC inhibitors enhance acetylation of H3K9 at the NEDD9 gene promoter to upregulate NEDD9 expression via inhibition of HDAC4, and activate FAK phosphorylation. Notably, we demonstrated that FAK inhibitor can reverse pan-HDAC inhibitor-induced breast cancer metastasis, which may improve HDAC inhibitors efficacy for breast cancer. In summary, we demonstrated an NEDD9-FAK signaling pathway, which was induced by pan-HDAC inhibitors, leading to breast cancer metastasis, and thereby caused limitation of the clinical benefits of HDAC inhibitors on breast cancer. We propose that HDAC4 has an upstream regulatory role in NEDD9 expression, leading to FAK phosphorylation and subsequent stimulation of breast cancer metastasis through epigenetic regulation. Our results suggest a need for increased isoform selective HDAC inhibitor research and development.

#5198

5AZA and decitabine exhibit different molecular effects on non-Hodgkin lymphoma cells: Involvement of DNMT3a.

Raffaele Frazzi,1 Tonia De Simone,1 Olga Serra,2 Annamaria Buschini,2 Laura Canovi,1 Tiziana De Luca,1 Francesco Merli1. 1 _Azienda Unità Sanitaria Locale IRCCS di Reggio Emilia, Reggio Emilia, Italy;_ 2 _University of Parma, Parma, Italy_.

Introductory sentence. Demethylating drugs represent a powerful tool for epigenetic modulation of chromatin structure in cancer cells. 5-azacytidine (5-AZA) and 5-aza-2'-deoxycytidine (decitabine) are promising therapeutic solutions also in mature B-cell neoplasms, although the molecular mechanisms of action have yet to be elucidated. Here we describe their effects on non-Hodgkin lymphoma cells and demonstrate how hypomethylation is not homogeneous across the genome. Experimental procedures. Toledo (GC-derived) and NU-DUL-1 (ABC-like) diffuse large-B cell lymphoma (DLBCL; non-Hodgkin) cell lines. Peripheral blood mononuclear cells (PBMCs) isolation. Epitect methyl II methylation qPCR and global chromatin methylation quantitative assays. Immunoblottings. Proliferation assays. CpG islands bioinformatics analysis. Comet assay and phosphorylation of gamma-H2AX histone assay. Gene silencing through siRNAs. New data. A panel of 9 CpG islands within a set of genes that we previously identified has been investigated as a potential target for hypomethylating activity of 5AZA and decitabine. KLF4, DAPK1 and SPG20 promoters (located on chr. n°9 the first two and n°3 the latter) are not affected by a single dose of 5AZA after 48h or 7 days of incubation (the CpG islands within these promoters are fully unmethylated in healthy PBMCs). However, global chromatin demethylation is clearly visible either after 48h or 7 days in both cell lines. By comparison, decitabine demethylating effects return to the initial values after 7 days. MZB1 promoter results demethylated by both treatments, while MGMT is demethylated only in NU-DUL-1 by decitabine but not 5AZA. These data suggest that hypomethylating agents act selectively on discrete regions of the genome. 5AZA and decitabine markedly down-regulate DNMT1 in a dose-dependent fashion and activate PARP. Despite DNMT1 downregulation, KLF4, DAPK1 and SPG20 promoters result unaffected. DNMT1 silencing in our cells do not cause any change in the promoter methylation of the selected targets. The de novo methyltransferase DNMT3a is expressed only by NU-DUL-1 and its silencing leads to a partial MZB1 demethylation and to the significant decrease in global chromatin methylation whereas DNMT1 silencing does not. In order to assess an involvement of the genotoxic damage, Comet assays were performed up to 24 hours of incubation with the drugs. We demonstrate that 5AZA does not induce any significant genotoxic damage while decitabine causes a tail formation starting at 6h after drug administration. Conclusions. Our data show that 5AZA and decitabine exhibit different mechanisms of action on lymphoma cells. Different DLBCL-derived cell lines display different changes when exposed to single doses of hypomethylating drugs, underlying the fact that cell of origin may play a role during the response. DNMT3a contributes to chromatin methylation in lymphoma cells.

#5199

A novel role for BAP1 in development and tumor suppression.

Dawn A. Owens,1 Jeffim N. Kuznetsov,1 Stefan Kurtenbach,1 Tristan Agüero,2 Mary Lou King,3 J. William Harbour1. 1 _Bascom Palmer Eye Institute, Sylvester Comprehensive Cancer Center and Interdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, Miami, FL;_ 2 _National University of Tucumán, Tucuman, Argentina;_ 3 _Sylvester Comprehensive Cancer Center and Interdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, Miami, FL_.

Uveal melanoma (UM) is the second most common form of melanoma and one of the most deadly types of cancer. Recent discoveries in our laboratory and others have led to a deeper understanding of the driver mutations UM. The initiating mutation that is present in virtually all UM occurs in one of four genes in the Gaq signaling pathway (the "Gaq" mutations) and result in sustained activation of proliferative signals. A second mutation occurs later in one of three genes – BAP1, SF3B1 or EIF1AX (the "BSE" mutations), which determines metastatic risk – BAP1 (high risk), SF3B1 (intermediate risk) or EIF1AX (low risk). Surprisingly, we recently discovered that the Gaq mutation and the subsequent BSE mutation occur very close in molecular time. This work suggests that Gaq mutations may cause epigenetic changes that predispose to BSE mutations. We have shown that BAP1-mutant UMs exhibit a de-differentiation and stem-cell like phenotype, suggesting that BAP1 may normally regulate differentiation programs during embryonic development. Consistent with this possibility, BAP1 loss in mouse is embryonic lethal. To study the role of BAP1 in early vertebrate development, therefore, we chose Xenopus laevis (African clawed frog), a widely used model for studying gene function during development. We found that targeted depletion of the Xenopus BAP1 protein (xBap1) causes a striking developmental phenotype affecting multiple germ layers, and that this phenotype is associated with disruption of super enhancers around differentiation genes that are normally maintained by BAP1. Taken together, these studies suggest that loss of BAP1 promotes tumor progression and metastasis, at least in part, by disrupting tightly controlled differentiation programs that govern cell identity. These findings may nominate new therapeutic targets in BAP1-mutant cancers.

### Mechanisms and Consequences of Transcriptional Deregulation

#5200

High CD73 expression, regulated by estrogen signaling, associates with cancer aggressiveness in estrogen receptor (+) breast cancer.

Eriko Katsuta, Vivek Anand, Li Yan, Subhamoy Dasgupta, Kazuaki Takabe. _Roswell Park Comprehensive Cancer Center, Buffalo, NY_.

Background: CD73, a cell surface enzyme, catalyzes the generation of adenosine from ATP and ADP in the tumor microenvironment along with CD39. Accumulated extracellular adenosine functions as immune-suppressor, and also binds to adenosine receptors which promotes angiogenesis and cell proliferation that results in accelerate cancer progression. However, the clinical significance and molecular function of CD73 expression in breast cancer remains unclear.

Methods: Utilizing breast cancer cohorts of TCGA and GEO, clinical significance and underlying mechanisms were investigated. Molecular experiments were carried out in MCF7 cells, ER(+) breast cancer cell line.

Results: In TCGA cohort, CD73 expression level was significantly lower in ER(+) breast cancers compared to ER(-) tumors. Higher CD73 expression was associated with worse overall survival in ER(+) tumors (p=0.003), but not in ER(-) tumors. Gene Set Enrichment Analysis revealed that estrogen response gene sets (Early; p=0.043, Late; p=0.021) were significantly enriched in CD73 low expressing ER(+) tumors, suggesting estrogen signaling may repress CD73 expression. To test this hypothesis, we analyzed the expression of CD73 and CD39 in MCF7 cells treated with estrogen, tamoxifen. Our data revealed that estrogen treatment suppressed CD73 and CD39 expression, whereas tamoxifen treatment enhanced expression of the genes. These findings suggest that CD73 and CD39 gene expression is suppressed by estrogen signaling, whereas binding of ER antagonists such as tamoxifen can remove the repressive effect on gene expression. On the other hand, epithelial-mesenchymal transition (EMT) (p<0.001) and angiogenesis (p<0.001) gene sets were significantly enriched in CD73 high expressing ER(+) tumors. CIBERSORT demonstrated that CD73 high expressing ER(+) tumors have less infiltrating CD8(+) T cells, memory B cells and plasma cells, implying that CD73 high expressing tumors have immune suppressive environment, which is in agreement with the notion that CD73 high tumors are immunosuppressive. Finally, we found that CD73 expression was significantly elevated post-chemotherapy compared to tumors prior to the treatment (p=0.007), and CD73 high expression patients showed worse relapse-free survival in neoadjuvant chemotherapy patients cohort (p=0.003).

Conclusion: Molecular studies revealed that CD73 expression is regulated by estrogen signaling. Increased expression of CD73 significantly correlates with worse outcomes in ER(+) breast cancer patients. This may be due to upregulated pro-metastatic gene signatures such as EMT and angiogenesis as well as less infiltration of anti-cancer immune cells by adenosine generated by CD73 in the tumor microenvironment. Our data reveals an intriguing mechanism which may be responsible for recurrence and metastasis of ER(+) breast cancer.

#5201

Using Taqman assays to verify eQTL links arising from GWAS studies.

Stephen M. Jackson, Harita Veereshlingham, Kamini Varma. _Thermo Fisher Scientific, South San Francisco, CA_.

Tremendous progress has been made using genome-wide association studies (GWAS) to link genetic variation with phenotypes and pathologies. In spite of these success, it has been estimated that as many as 90% of SNPs identified in GWAS studies map to non-coding regions, complicating the mechanistic interpretation of the results. It is thought that these non-coding SNPs fall into regulatory regions and influence the expression of a gene or genes. The study of expression quantitative trait loci (eQTLs) provides a method for understanding the link between genetic variants and altered gene expression, and could potentially provide new insights connecting GWAS results to molecular mechanisms. To illustrate how eQTLs can be found and verified, we generated transcriptomic information from various tumor samples using Applied Biosystems' Clariom D microarrays. We also generated SNP genotyping data from the same samples using Applied Biosystems' Axiom UK Biobank microarrays. To find putative eQTLs, we compared the SNPs genotypes and the gene expression levels in these samples to the GTEx database. Potential eQTLs in this set of samples were verified using Applied Biosystems' Taqman SNP Genotyping and Taqman Gene Expression assays. We therefore illustrate a workflow where candidate eQTLs can be confirmed and screened in larger cohorts using the more economical qPCR reagents available from Applied Biosystems.

#5202

**Molecular mechanism of transcriptional regulation of** REV7 **, a DNA damage response gene.**

Yoshiki Murakumo, Yuko Shimada, Takuya Kato, Yasutaka Sakurai. _Kitasato Univ. School of Medicine, Sagamihara, Japan_.

REV7 is involved in several biological response after DNA damage, including the double strand break repair and translesion DNA synthesis. It has bee reported that high levels of REV7 expression are observed in several malignant tumors and are associated with resistance to chemotherapeutic agents such as cisplatin. In normal tissues, REV7 expression is extremely high in germ cells compared with the other normal cells, suggesting the cell type-specific expression of REV7. However, the mechanism of regulation of REV7 expression has not been understood. To assess the mechanism of regulation of REV7 expression, we analyzed the promotor region of the human REV7 locus to identify transcriptional regulators controlling REV7 expression. The transcriptional start point of the REV7 gene was determined by 5'-RACE using human testis cDNA. About 3 kb of the upstream region of the REV7 locus was isolated by genomic PCR and the promoter activity was analyzed by the luciferase reporter assay. Using several deletion mutants, we found that there are two transcriptional activation regions in the upstream of the REV7 locus. Bioinformatic analyses revealed several motifs of transcription factor binding sites in these regions, and we focused on one molecule as a candidate for the transcription factor controlling REV7 expression. We will present and discuss our further analyses.

#5203

Segregating activities of the estrogen receptor in gene regulation.

Mei Yang, Zhao Zhang, Kexin Xu. _University of Texas Health Science Center at San Antonio, San Antonio, TX_.

Upon stimulation with its ligand estradiol (E2), the estrogen receptor (ER) obviously can induce both transcriptional activation and repression in breast cancer cells. Although the mechanism of gene activation has been extensively scrutinized, how ER targets are turned off is generally overlooked. Some theories have been proposed to explain the trans-suppression process, such as engagement of corepressors, physiological squelch of coactivators, or decommission of RNA Polymerase II, etc. However, none of these models really elucidate the intrinsic and external factors that segregate ER activities in transcriptional regulation. In this study, we identified a functional domain of ER, which is specifically required for E2-mediated gene repression. While the DNA-binding domain (DBD) was essential for the overall response to E2, deletion of AF2 fragment at C-terminus of ER specifically reverted the E2-repressed transcriptional program into E2-activated one. We further demonstrated that RNAs transcribed from these E2-repressed enhancers (eRNAs) act as the exterior determinants of ER activity in gene repression. In addition to stabilizing promoter-enhancer looping structure, these eRNAs recruit ER to particular chromatin loci, and facilitate the hierarchical formation of a functional complex responsible for transcriptional repression. Thus, our findings suggest that by targeting distinct functional domains of ER, we may be able to reprogram expression of a specific subset of ER targets, which play critical roles in conferring malignant phenotypes of breast cancer cells. Meanwhile, as a distinctive class of cis-acting elements besides chromatin regulatory regions, eRNAs help to refine ER-dependent transcriptional program that contributes to the development and progression of breast cancer.

#5204

ICAM-1 and IL-17E upregulate FOXO1 to suppress the expression of oncogenic PKC-ι in human melanoma.

Wishrawana Sarathi Ratnayake, Christopher Apostolatos, Sloan Breedy, André Apostolatos, Mildred Acevedo-Duncan. _Univ. of South Florida, Tampa, FL_.

Throughout the span of the last 15 years, the number of cases of melanoma has been on a steady increase. Approximately, 92,000 new melanoma cases will be diagnosed in the United States in 2018. Our previous studies demonstrated that protein kinase C-iota (PKC-ι) is crusial for melanoma progression, which promotes the activation of nuclear factor (NF)-κB through a PI3K/AKT manner, thereby supporting survival and progression. In addition, PKC-ι induced the metastasis of melanoma cells by stimulating ephithilial-mesenchmal transition. Results also showed that PKC-ἱ specific inhibitors diminished the expression of both PKC-ι and phospo-PKC-ι, suggesting that PKC-ι plays a role in regulating its own expression in melanoma cells in-vitro {Ratnayake, W.S., et al. Cell Adhes. Migr. (2018) https://doi.org/10.1080/19336918.2018.1471323}. In this study, we report the transcriptional regulation mechanisms of PRKCI (PKC-ι gene) expression in melanoma. PROMO and Genomatix Matinspector methods were used to identify c-Jun, interferon-stimulated gene factor 3 (ISGF3), paired box gene 3 (PAX3), early growth response protein 1 (EGR1) and forkhead box protein O1 (FOXO1), which bind on or near the promoter sequence of the PRKCI and they were analyzed for their roles in PKC-ι expression. siRNAs were used to silence selected transcription factors, and the results revealed that, silencing of FOXO1 and c-Jun significantly altered the expression of PRKCI. In addtion, we knocked down NF-κB in order to demonstrate changes on activities of c-Jun and FOXO1. Both phosphorylated and total PKC-ι levels increased upon FOXO1 silencing and decreased upon c-Jun silencing, suggesting that c-Jun turns is an upregulator, while FOXO1 acts as a downregulator of PRKCI expression. Multiplex ELISA assays were used to analyze multiple pathways and the expression of cytokines with respect to crosstalk between signaling pathways affected by specific inhibiton PKC-ι. Western blot data for all siRNA treatments of PKC-ι, NF-κB, FOXO1 and c-Jun were supported by real-time qPCR. Taken together, results suggested that the regulation of PKC-ι expression was strongly associated with FOXO1 and c-Jun through the trascriptional deactivation/activation. In addition, we observed a significant decrease in the mRNA levels of both interleukin (IL)-6 and IL-8, with a significant increase in the levels of IL-17E and intercellular adhesion molecule 1 (ICAM-1) upon the knockdown of expression of PKC-ι in both cell lines. This suggested that PKC-ι expression was affected by said cytokines in an autocrine modus. Overall, the findings suggest that PKC-ι inhibition suppresses its own expression, diminishing oncogenic signaling such as NF-κB, PI3K/AKT and JNK/c-Jun while upregulating anti-tumor signaling such as ICAM-1/FOXO1, thus rendering it an effective novel biomarker for use in the design of novel targeted therapeutics for melanoma metastasis.

#5205

**EWS-FLI1 activates** HOXD13 **expression through enhancer reprogramming.**

April Ann Apfelbaum, Laurie Svoboda, Brian Magnuson, Mats Ljungman, Elizabeth Lawlor. _University of Michigan, Ann Arbor, MI_.

Ewing sarcomas (ES) are bone tumors of mesenchymal stem cell (MSC) origin. ES is defined by EWS-FLI1 fusions, which initiate tumorigenesis via enhancer reprogramming. Posterior homeobox D (HOXD) genes, in particular HOXD13, are over-expressed by ES, where they function as obligate, cooperative oncogenes. These genes play crucial roles in limb development and spatiotemporal control of HOXD13 expression depends on long-range enhancers that flank the HOXD locus. We hypothesize that HOXD13 is aberrantly activated in the ES cell of origin as a result of EWS-FLI1-dependent hijacking of developmental enhancers.

We interrogated ChIP-seq and RNA-seq data to identify EWS-FLI1 binding sites and HOXD13 activation states in ES cells. These analyses revealed an EWS-FLI1 binding site within the developmentally conserved posterior HOXD enhancer region, which we will refer to as Desert Enhancer 268 (DE268). These sites were also marked with the active enhancer marks, H3K27Ac and H3K4me1. EWS-FLI1 knockdown was associated with loss of EWS-FLI1 binding, loss of H3K27Ac and H3K4me1, and loss of HOXD13 expression. Findings were independently validated by ChIP-qPCR and RT-qPCR. Through BruUV-seq we also identified that DE268 has eRNA expression that is lost upon EWS-FLI1 knockdown. Thus, HOXD13 expression in ES cells is maintained by EWS-FLI1-mediated enhancer activation. To address whether hijacking of HOXD13 enhancers is an initiating event in tumorigenesis, we evaluated enhancer profiles in MSCs ± EWS-FLI1. Whereas DE268 was not active in parent MSC, EWS-FLI1 binding and enhancer activation were evident in EWS-FLI1+ MSC. However, although EWS-FLI1 reproducibly binds and activates DE268, expression of HOXD13 is limited to discrete conditions. Together, these results suggest that EWS-FLI1 is necessary for enhancer activation, but that the fusion is alone is insufficient for induction of HOXD13 expression. Ongoing studies will utilize CRISPRi technologies to determine the contribution of DE268 on HOXD13 gene expression.

#5206

Role of super-enhancers in HPV+ head and neck squamous cell carcinoma.

Fernando T. Zamuner,1 Ilya Vorontsov,2 Emily Flam,1 Vera Mukhina,2 Ludmila Danilova,3 Tingting Ou,1 Elena Stavrovskaya,4 Theresa Guo,1 Eric Windsor,5 Dylan Z. Kelley,1 Michael Parfenov,6 Michael Considine,3 Elana J. Fertig,3 Alexander Favorov,3 Daria A. Gaykalova1. 1 _Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins Medical Institutions, Baltimore, MD;_ 2 _Laboratory of Systems Biology and Computational Genetics, Vavilov Institute of General Genetics, Russian Academy of Sciences, Russian Federation;_ 3 _Division of Oncology Biostatistics and Bioinformatics, Department of Oncology, Johns Hopkins Medical Institutions, Baltimore, MD;_ 4 _Department of Bioengineering and Bioinformatics, Moscow State University, Russian Federation;_ 5 _Department of Biotechnology, Maryland Holistics LLC, Silver Spring, MD;_ 6 _Department of Genetics, Harvard Medical School, Boston, MA_.

Head and neck squamous cell carcinomas (HNSCC) are the sixth leading cause of cancer worldwide with different incidences, mortalities, and prognosis for different subsites. Infection by Human Papilloma Viral (HPV) can cause the development of HPV+ HNSCC, the most rapidly growing population of cancer patients. The molecular biology of HPV+ HNSCC is related to abnormal transcriptional regulation. Super-enhancers (SEs) were recently identified as critical regulators of gene expression during cell differentiation and disease development. SEs are long clusters of enhancers that recruit master transcription factors (TFs) and coactivators to regulate the expression of target genes. We have recently developed a new whole-genome analytical pipeline to optimize ChIP-Seq procedures on primary surgical samples, and this protocol helped us to define the whole-genome distribution of H3K27ac, the hallmark of SEs, in HPV+ HNSCC samples. To detect SEs, we used a novel LILY algorithm that corrects the ChIP-Seq signal for copy number variation and CpG density. The analysis revealed that HPV+ HNSCC-specific SEs regulate genes critical for head and neck cancer development, such as EGFR, TP63, JAG1, IRF6, SMAD3, MAP4K2, SNAI1, TNFAIP3, ANO2, and TNFRSF1A. These genes are located in the vicinity of HNSCC-specific SEs, and the expression of those genes was altered by JQ1, the inhibitor of BRD4, the main component of SE machinery, supporting the role of SE in their regulation. The following Cistrome analysis allowed us to elucidate the TF composition of the SE machinery. Thus, we have found out that P63, P53, SOX2, FOSL1, SMAD3, and SNAI2 are TFs that are the most overrepresented in the HPV+ HNSCC-specific SEs. These comprehensive analyses have revealed novel insight into the HPV+ HNSCC biology and paved the basis for the implications for epigenetic therapeutics for this tumor types, and potentially other virus-related malignancies.

#5207

Functions of nucleolin in its interactions with the MYC promoter G-quadruplex.

Guanhui Wu, Luying Chen, Clement Lin, Buket Onel, Danzhou Yang. _Purdue University, West Lafayette, IN_.

DNA G-quadruplexes (G4s) are four-stranded non-canonical secondary structures formed in guanine-rich DNA sequences with functional significance and are found to be specifically enriched in the transcriptional regulatory regions of cancer-related genes. In particular, c-MYC, one of the most commonly deregulated genes in human cancers, has a DNA G4 motif in its proximal promoter which functions as a transcription inhibitor and can be targeted by G-quadruplex-interactive compounds, and is thus considered as an attractive target for cancer therapeutics. Nucleolin is overexpressed in cancer cells and is shown to be an important transcriptional regulator. Nucleolin has been found to specifically bind the c-MYC promoter G-quadruplex and to be involved in the regulation of MYC transcription, however, little is known about how the c-MYC promoter G-quadruplex interacts with and is regulated by nucleolin. By using AcGFP-tagged nucleolin, we observed nucleolin is mainly present in the cell nucleolus and nucleoplasm. We show that the nucleolin protein interacts to the MYC promoter region in vivo using chromatin immunoprecipitation assay coupled with next-generation sequencing analysis (ChIP-seq) in cancer cells. To show the association of nucleolin with G4s in vivo, we treated MCF7 cells with a G4-stabilizing ligand and monitored cellular localization of nucleolin and G4 sites using an immunofluorescent staining assay. Within 24 hr after treatment, we observed a large increase of endogenous G4 structures as well as a rapid cellular translocation of nucleolin from the cell nucleolus to the cell nucleoplasm. Importantly, the nucleolin foci were significantly colocalized with the G4 foci, suggesting the direct interaction of nucleolin and G4s in vivo. In the meantime, both MYC mRNA and protein levels were significantly reduced, indicating the nucleolin/MYC G4 interaction represses MYC transcription. To understand the interactions of nucleolin with the MYC G4 structure, we prepared various nucleolin protein constructs and studied its interactions with the MycG4 structures in vitro using NMR, EMSA, fluorescence, CD, AUC, and UV-crosslinking methods. We found that nucleolin specifically recognizes the c-MYC promoter G-quadruplex and forms a 1:1 complex with MycG4. Importantly, MycG4 maintains its G4 structure in the nucleolin-MycG4 complex. Additionally, CD melting of the MYC G4-nucleolin complex showed that MYC G4 is stabilized by nucleolin by more than 10 Celsius. In a conclusion, our in vitro and in vivo results provide important insights into the nucleolin protein interactions with the MYC promoter G4 structure and the G4-associated biological functions of nucleolin in cancer cells.

#5208

Genome-wide AR enhancer activity in prostate cancer.

Dogancan Ozturan,1 Flora Huang,2 Tunc Morova,2 Mohammadali Saffarzadeh,2 Nathan A. Lack2. 1 _Koc University, Istanbul, Turkey;_ 2 _Vancouver Prostate Centre, Vancouver, British Columbia, Canada_.

Prostate cancer (PCa) is one of the most commonly diagnosed forms of cancer with one out of every nine North American men developing the disease in their lifetime. Despite robust early detection methods, new therapeutics, and advancements in surgical operations, PCa is still second leading cause of cancer death in men. An increasing number of studies have shown that non-coding mutations plays a critical role in the development and progression of PCa. These somatic mutations can disrupt regulatory elements such as enhancers and alter the transcriptional landscape of the cancer. Discovering and interpreting the non-coding elements is crucial to understand the nature of this disease.

At all stages of PCa, androgen receptor (AR) signaling is essential to the cancer. Recent work from our laboratory demonstrated that there is a significant increase in somatic mutations at AR binding sites (ARBS). Given the importance of AR-mediated transcription, as well as the recurrent nature of these mutations, we proposed that these somatic mutations could play an important role in PCa development. To stratify the mutations, we conducted a massively multi-parallel enhancer assay to identify those ARBS sites with enhancer activity. With STARR-seq we found that 8% of the ARBS had strong inducible AR enhancer activity (341/4139). Interestingly, the majority of these sites did not contain a canonical androgen response element. With these results we incorporated high-throughput chromosome conformation capture (HiC) to formally define the genes regulated by each of these AR enhancers. Based on these results we have selected and characterized several non-coding ARBS mutations. We believe that the importance of the regulatory role of these regions will play a tremendous role in understanding of the prostate cancer and its progression.

#5209

Efficacious targeting of TERT oncogene rearrangement with BET bromodomain inhibitor and proteasome inhibitor combination therapy.

Jingwei Chen,1 Christopher Nelson,2 Peiyan Liu,1 Andrew E. Tee,1 Bernard Atmadibrata,1 Karen MacKenzie,2 Jamie Fletcher,1 Toby Trahair,1 Patsie Polly,3 Roger Reddel,2 Hilda Pickett,2 Tao Liu1. 1 _Children's Cancer Institute Australia, Sydney, Australia;_ 2 _Children's Medical Research Institute, Sydney, Australia;_ 3 _Department of Pathology, UNSW Medicine, UNSW, Sydney, Australia_.

Background: TERT oncogene rearrangement with transcriptional super-enhancers leads to TERT over-expression in one third of high-risk neuroblastoma patients, and this subtype of neuroblastoma patients shows very poor prognosis. Transcriptional super-enhancers of MYC oncogene are controlled by the BET bromodomain protein BRD4. However, factors important for super-enhancer activity in TERT-rearranged neuroblastoma are unknown.

Aims: To examine the role of BRD4 in regulating TERT over-expression and cell proliferation in TERT-rearranged neuroblastoma cells, and to identify the best combination therapy with BRD4 inhibitors and other anticancer agents against TERT-rearranged neuroblastoma.

Methods and Results: Knocking down BRD4 with BRD4 siRNAs decreased TERT mRNA and protein expression as well as telomerase activity in TERT-rearranged neuroblastoma cell lines. Alamar blue assays showed that transfection with TERT siRNAs or BRD4 siRNAs considerably reduced cell proliferation. After screening of the Food and Drug Administration-approved oncology drug library, we identified the proteasome inhibitor, carfilzomib, as the compound exerting the best synergistic anticancer effects, in combination with the BRD4 inhibitor OTX-015, against TERT-rearranged neuroblastoma cells. Immunoblot and telomerase activity assays showed that OTX-015 and carfilzomib synergistically decreased TERT protein expression and telomerase activity. Beta-galactosidase and Annexin-V staining of TERT-rearranged neuroblastoma cells revealed that OTX-015 and carfilzomib did not induce senescence but synergistically caused apoptosis, and that transfection with a TERT-over-expression construct rescued the tumour cells from the combination treatment. Importantly, in mice xenografted with TERT-rearranged neuroblastoma cells, combination therapy considerably suppressed tumour progression. Immunoblot analysis of mouse tumour tissues revealed that OTX-015 and carfilzomib dramatically reduced TERT protein expression. Immunohistochemistry analysis showed that OTX-015 and carfilzomib co-operatively reduced the proportion of neuroblastoma cells positively stained with PCNA, indicating that the combination treatment synergistically repressed tumour proliferation.

Conclusions: The BET bromodomain protein BRD4 is essential for TERT oncogene expression and cell proliferation in TERT-rearranged neuroblastoma cells. Combination therapy with OTX-015 and carfilzomib considerably reduces TERT expression, induces TERT-rearranged neuroblastoma cell death and tumour growth inhibition, and is a very promising novel strategy for the therapy of TERT-rearranged neuroblastoma.

#5210

Darolutamide impairs prostate cancer growth by altering chromatin conformation and transcriptional activity of genes involved in cell proliferation and survival.

Simon J. Baumgart, Ekaterina Nevedomskaya, Ralf Lesche, Bernard Haendler. _Bayer AG, Berlin, Germany_.

Androgen signalling is essential for early and late stage prostate cancer (PCa), as demonstrated by the efficacy of androgen receptor (AR) antagonists. The AR gene and its regulatory element are frequently amplified in castration-resistant tumors for which improved treatments are highly needed. Recent findings underscore that gene regulatory elements play an important role in tumor development due to their impact on chromatin conformation and gene regulation. Here we describe the genome-wide effects of darolutamide, a novel AR antagonist which just completed a pivotal clinical phase III trial, by analyzing AR occupancy and its impact on chromatin looping, histone H3K27 acetylation and downstream gene regulation. Genome-wide RNA-seq and ChIP-seq were used to analyze the androgen-sensitive cell lines VCaP, LAPC4 and LNCaP treated with R1881, or with R1881 and darolutamide. Binding of AR and of the pioneer factor FOXA1, as well as histone H3K27 acetylation were determined. Chromatin conformation was detected using the HiChIP method with an AR-specific antibody. Differential gene expression was analyzed by DESeq2 and Gene Set Enrichment Analyses. ChIP-seq peak identification was done by MACS2. HiChIP analysis was performed with the hichipper pipeline. Darolutamide efficiently blocked AR signalling at the level of transcriptional regulation and altered chromatin landscape interaction. Analysis of transcriptomic data showed a strong reduction of the androgen response, as well as down-regulation of proliferation and cell cycle genes by darolutamide. Concordantly, AR occupancy was decreased genome-wide compared to androgen stimulation. Importantly, no binding cluster was identified which showed increased AR occupancy after darolutamide treatment, as reported for other AR antagonists. Interestingly, we observed in darolutamide-treated samples a parallel reduction of FOXA1 occupancy at AR sites, which argues for a co-dependence between this pioneer factor and the AR. Also, we found that H3K27 acetylation, a histone mark linked to active regions, was markedly down-regulated at specific enhancer and gene regions following darolutamide treatment, further pointing to reduced activity of enhancer regions recognized by the AR. Finally, determination of chromatin looping mediated by the AR between enhancers and gene promoters showed a dense network at genes involved in prostate cancer survival. The loss of AR binding at these regions was in line with the significantly reduced transcriptional response observed following darolutamide treatment. In conclusion, we found that darolutamide strongly prevented genome-wide AR binding in multiple hormone-sensitive cellular PCa models, importantly at genes involved in cell proliferation and survival. We furthermore highlight the cross-talk between the pioneer factor FOXA1 and the AR.

#5211

**Super enhancer-associated master transcription factor** SOX4 **suppresses anti-tumor immunity in NAFLD-associated hepatocellular carcinoma.**

Ka Wing Cheung,1 Feng Wu,1 Wenshu Tang,1 Weiqin Yang,1 Jingying Zhou,1 Patrick Tan,2 Yuk Lap Kevin Yip,3 Ka Fai To,3 Sze Lok Alfred Cheng3. 1 _The Chinese University of Hong Kong, Kowloon, Hong Kong;_ 2 _Duke-NUS Graduate Medical School, Singapore;_ 3 _The Chinese University of Hong Kong, Hong Kong_.

Hepatocellular carcinoma (HCC) has become a prominent global health threat due to its occurrence and lethality. Increasing prevalence of non-alcoholic fatty liver disease (NAFLD) have been found culpable for HCC initiation, via disruption of the liver microenvironment. Super enhancers (SEs) and master transcription factors (TFs) translate microenvironmental changes into chromatin remodelling and activation, which subsequently disrupt the transcriptome profile and initiate oncogenesis. This project aims at profiling the SE status and unveiling the master regulators involved in diet-induced HCC progression. Nanoscale chromatin immunoprecipitation sequencing (nano ChIP-seq) against histone mark H3K27ac in 10 pairs of primary human NAFLD-HCC tumors and adjacent non-tumor tissues revealed potential tissue-specific SEs (averaged 541 and 512 per HCC tumor and non-tumor tissues, respectively). Global mRNA profile was detected by RNA sequencing (RNA-seq) to support the enhancer-target gene transcription axes in the enriched metabolic and immune response pathways. A non-alcoholic steatohepatitis-induced HCC (NASH-HCC) mouse model was established by subcutaneous injection of a diabetogenic agent streptozotocin (STZ) (200 μg) two days after birth followed by a 19-week high-fat high-carbohydrate (HFHC) diet from the age of 4 weeks. Another carcinogen diethylnitrosamine-induced HCC (DEN-HCC) mouse model was established by intraperitoneal injection of DEN (25 mg/kg) followed by a 22-week HFHC diet from the age of 6 weeks. Tumor-enabling SEs were profiled in primary human NAFLD-HCC tissues and a network of master TFs were identified using integrated bioinformatic analysis. Intriguingly, tumor-enabling SEs target critical genes involved in PDGFB signalling and NAFLD pathogenesis, which are significantly correlated with high master TF SOX4 expression. Notable overexpression of Sox4 and Pdgfb was also observed in liver tumors in two HCC mouse models, significantly correlating with the CD11b+ CD11c+ IL10+ IDO+ tolerogenic dendritic cell population. Integrated analysis of ChIP-seq and RNA-seq in primary human tissues revealed a super enhancer-associated master regulatory network in NAFLD-HCC development. Our findings suggest that epigenetic regulation of the SOX4 and PDGFB may contribute to an immunosuppressive liver tumor environment, providing insights for novel therapeutic strategy against this rapidly-increasing dreadful disease. This project is supported by Collaborative Research Fund C4017-14G and the Focused Innovations Scheme 1907309.

#5212

**Ablation of SMAD4 repression on** Elf3 **promotes the progression of lung cancer** in vivo **.**

Jian Liu, Tianyuan Wang, San-pin Wu, Jian-liang Li, Francesco J. Demayo. _NIEHS, Research Triangel Park, NC_.

Lung cancer is the leading cause of cancer death among both men and women in United States. ELF3, a transcription factor, overexpresses in human and mouse lung cancer and plays an oncogenic role in lung cancer cells1,2. Understanding the regulatory mechanisms for ELF3 may help to develop related target therapies. Ablation of Smad4 and Pten in mice results in an increase of Elf3 expression and the development of adeno-squamous carcinoma1. Results from in vivo ChIP-Seq analysis demonstrate SMAD4 occupancy near the Elf3 promoter in the pulmonary epithelium1. Therefore, we hypothesize that this SMAD4 occupancy is required to repress Elf3 expression and subsequently suppresses lung cancer progression. To test this hypothesis, we deleted this SMAD4 binding region in vivo and characterized the phenotypic consequences.

CRISPR/Cas9 was used to ablate the SMAD4 ChIP-Seq binding peak on Elf3 promoter region in mice. This resulted in mice with 12 and 17 nucleotide deletions in the Elf3 promoter region. ChIP-qPCR analysis showed that SMAD4 binding was decreased while H3K27ac bindings were increased in the Elf3 promoter region. The reduction in SMAD4 binding and the epigenetic changes in the Elf3 promoter region resulted in an increased expression of ELF3 in these lungs of these mice. Histological analysis of these mouse lungs showed the presence of pulmonary hyperplasia and the development of lung tumors. In order to identify potential interacting factors with SMAD4 in the Elf3 promoter, motif analysis of the deleted SMAD4 binding peak identified GATA2, ETS1 and OXT2 as potential interacting factors.

In summary, small disruption of Elf3 promoter region bound by SMAD4 leads to the increase of ELF3 expression with the enhanced binding of H3K27ac and the decrease of SMAD4 binding and promotes lung hyperplasia and lung cancer development. SMAD4 may interact with GATA2, ETS2 and OXT2 in the regulation of Elf3 expression.

Key references

1. Liu, J. et al. ErbB2 Pathway Activation upon Smad4 Loss Promotes Lung Tumor Growth and Metastasis. Cell Rep, doi:10.1016/j.celrep.2015.02.014 (2015).

2. Wang, H. et al. Overexpression of ELF3 facilitates cell growth and metastasis through PI3K/Akt and ERK signaling pathways in non-small cell lung cancer. Int J Biochem Cell Biol 94, 98-106, doi:10.1016/j.biocel.2017.12.002 (2018).

#5213

A modified L-penetratin peptide targeting e2f-1, 2 and 3 is effective in combination with cisplatin or pemetrexed against prostate and lung cancer cell lines.

Gulam M. Rather,1 Michael Anyanwu,1 John E. Kerrigan,2 Kathleen W. Scotto,1 Olga Garbuzenko,3 Tamara Minko,3 Zoltan Szekely,3 Joseph R. Bertino1. 1 _Rutgers Cancer Institute of New Jersey, New Brunswick, NJ;_ 2 _Rutgers School of Arts and Science, Rutgers, The State University of New Jersey, Piscataway, NJ;_ 3 _Ernest Mario School of Pharmacy, Rutgers: The State University of New Jersey, Piscataway, NJ_.

Overexpression of the "activating" transcription factors, E2F1,2 and-3a induces genes involved in DNA synthesis and leads to cellular proliferation, tumor growth, and invasion. Therefore, inhibiting the overexpression of one or more activating E2Fs is a recognized target in cancer therapeutics. Dysregulation/overexpression of E2Fs is also controlled by deletion or mutations in the retinoblastoma protein in tumors. In our previous studies we showed that a novel penetratin conjugated 7-mer peptide (PEP) inhibited transcription of the E2F1, 2 and 3a by bounding tightly to E2F promoters. The PEP was cytotoxic at low micro molar concentrations to several malignant cell lines. As the PEP was unstable in vivo, the PEP was encapsulated in PEGylated liposomes (PL-PEP). Treatment of xenografts of the pRB negative small cell lung cancer H-69 and castrate resistant prostate cancer DU145 tumors propagated in mice with the PL- PEP, caused tumor regression. To increase stability and potency of the PEP, L-Arg in the PEP was replaced by D-Arg. Molecular simulation studies showed that the D-Arg PEP secondary structure is more stable than the L- Arg peptide structure in water. In vitro studies showed that the D-Arg PEP was more potent and more resistant to degradation by serum proteases than the L- form. As E2F is important for DNA repair, and for transcription of thymidylate synthase, we tested the effects of the PEP in combination with cisplatin, a DNA damaging drug and pemetrexed, a potent thymidylate synthase inhibitor. Combination studies of the D-ARG pep with cisplatin (in DU145, PC3, LnCap and 4T1) cells and with pemetrexed (in non-small cell lung cancer, H2009, H441, H1975 and H2228) resulted in synergistic cytotoxicity as determined by Chou-Talalay analysis.

#5214

Targeting the estrogen receptor pathway in luminal breast cancer through inhibition of p300/CBP.

Aaron Waddell, Iqbal Mahmud, Guimei Tian, Daiqing Liao. _University of Florida, Gainesville, FL_.

Luminal breast cancer represents approximately two thirds of all breast cancer cases and is characterized by the expression of hormone receptors, such as the estrogen receptor alpha (ER). ERα is a member of the steroid nuclear receptor family and is involved in a hormonal signaling pathway that drives tumor growth through upregulation of genes involved in cell cycle progression, such as MYC and CCND1. Antagonists of ER function are the standard of care treatment for ER+ luminal breast cancer. However, resistance to ER antagonists is common in advanced breast cancer cases. Therefore, there is an urgent need to investigate new therapeutics that can be utilized to treat tumors that are resistant to traditional therapies. p300/CBP are two paralogous acetyltransferases that catalyze histone 3, lysine 18 and 27 acetylation (H3K18ac and H3K27ac) at promoters and enhancers to promote gene expression. ER mediated transcription is critically reliant upon the recruitment of co-activators, including p300/CBP. However, the mechanistic role of p300/CBP catalytic activity in globally regulating ER mediated transcription remains poorly understood. Inhibition of p300/CBP, as a critical co-activator of ER, potentially represents an applicable strategy for inhibiting ER function in luminal breast tumors. Importantly, the catalytic histone acetyltransferase (HAT) domain of p300/CBP is under intense investigation as a target of small molecule therapeutics in cancer. We analyzed publicly available data and report that p300/CBP are upregulated at the mRNA level in breast cancer, including the luminal subtypes, and that their expression negatively correlates with patient survival. We also found that pharmacologic inhibition of p300/CBP HAT activity in ER+ cell lines potently suppresses ER mediated transcription, downregulates ER, c-Myc and Cyclin D1 protein levels and prevents estrogen induced growth in vitro. Studies are underway to understand how p300/CBP regulates ER signaling using genetic, pharmacologic, and biochemical methods to assess the therapeutic effects of pharmacologic p300/CBP inhibition on ER+ breast cancer. (Supported by Bankhead-Coley Research Program, and James and Esther King Biomedical Research Program, Florida Department of Health)

#5215

Targeting of HOX-PBX binding in glioblastoma multiforme as a novel therapeutic treatment.

Richard Morgan,1 Monika Primon,1 Steven Shnyder,1 Susan Short,2 Balveen Kaur,3 Bangxing Hong,3 Izhar Bagwan,4 William Rogers,4 Hardev S. Pandha4. 1 _University of Bradford, Bradford, United Kingdom;_ 2 _University of Leeds, Leeds, United Kingdom;_ 3 _University of Texas, Houston, TX;_ 4 _University of Surrey, United Kingdom_.

The HOX genes encode a family of transcription factors that play an essential role in embryonic patterning. They are also aberrantly expressed in numerous cancers, including glioblastoma (GBM). Three Amino Acid Loop Extension Homeobox (TALE) proteins act as important co-factors for HOX proteins, modulating their binding affinities to genomic targets. TALE proteins include the Pre-B-cell leukemia homeobox (PBX) proteins 1-4, which bind anterior HOX proteins, facilitating their nuclear entry and limiting their degradation. HTL00-1 is a 2nd generation hexapeptide drug that inhibits HOX-PBX dimer formation, and has been shown to induce rapid apoptosis in cancer cells, but not normal cells, through the rapid upregulation of genes including cFos, DUSP1, and EGR1. We have found that both HOX and TALE genes are markedly dysregulated in primary GBM tumors as well as in murine (GL261), adult (LN18, U87-MG, U251-MG) and pediatric (KNS42, SF188) GBM cell lines, all of which are sensitive to HTL-001. These genes were even more highly expressed in experimentally induced GBM cancer stem cells (CSCs) compared with parental lines, with a corresponding increase in sensitivity to HTL-001. We also investigated the in vivo activity of HTL-001 in Black 6 mice carrying GL-261 subcutaneous and orthotropic tumors with twice weekly intraperitoneal delivery. HTL-001 was shown to cross the blood brain barrier using Alexa660 labelled peptide, and significant anti-tumor activity was observed in both subcutaneous and orthotropic models with increased survival (p<0.02 and p<0.0078, respectively). Resected tumors from HTL-001 treated mice showed marked evidence of apoptosis, tumor vasculature disruption and focal necrosis compared to untreated tumors. Taken together, our findings indicate that HOX-PBX inhibition is a potential therapeutic target for adult and pediatric GBM patients.

#5216

**Identification of** Sox2 **regulatory factors in small cell lung cancer.**

Hannah Wollenzien,1 Mira Yousef,2 Ellen Voigt,2 Michael S. Kareta2. 1 _University of South Dakota, Vermillion, SD;_ 2 _Sanford Research, Sioux Falls, SD_.

Sox2 is one of the core pluripotency genes that has also been implicated to be oncogenic in a multitude of cancers. We have shown that Sox2 is required for the formation of SCLC using mouse models and human cell culture. SCLC predominantly arises from in the lung from pulmonary neuroendocrine cells, which are formed from Sox2-expressing progenitors that are then silenced upon terminal differentiation. However, upon transformation Sox2 becomes highly upregulated in SCLC. One means of repressing Sox2 is the Rb tumor suppressor, which binds to the Sox2 locus and represses transcription by the recruitment of many epigenetically silencing factors. However, there are still some gaps in our knowledge of the repression of Sox2 by Rb, such as which factors are recruiting Rb to Sox2. We describe a novel technique of Cre-mediated Protein-DNA Immunoprecipitation (CrePI) to identify proteins at the Sox2 locus in an unbiased manner to further elucidate the mechanism of Rb repression of Sox2.

#5217

RB loss reprograms AR and E2F1 signaling in models of prostate cancer progression.

Amy C. Mandigo, Chris McNair, Matthew J. Schiewer, Karen E. Knudsen. _Thomas Jefferson University, Philadelphia, PA_.

Prostatic adenocarcinoma (PCa) is the most frequently diagnosed non-cutaneous malignancy, and the third leading cause of cancer death in males in the United States. A crucial component to the development and progression of PCa is the activity of the androgen receptor (AR). As such, targeting the AR-signaling axis through androgen deprivation therapy (ADT) is the first line of therapy for PCa. However, cells invariably become resistant to this therapy and men relapse with the incurable form of the disease termed, castration-resistant prostate cancer (CRPC). In addition to AR, another principal component aiding in the progression of disease is the retinoblastoma tumor suppressor protein (RB). RB functions to repress tumor development by negatively regulating the activity of the E2F family of transcription factors, preventing cell cycle progression. RB is lost in roughly 30% of CRPC tumors and is sufficient to induce a CRPC phenotype in hormone-sensitive cells under ADT conditions. Analyses into the molecular significance of RB loss on disease progression identified a potential cooperation between AR and the RB-E2F1 signaling axis. Biological assessment performed in isogenic RB knockdown (i.e. hormone-sensitive and CRPC models) identified distinct functional consequences of RB loss depending on AR status and disease state. Transcriptome analysis identified divergently regulated gene signatures between disease stages in the presence of AR activation, which were not seen under ADT conditions, implicating a unique role of AR in transcriptional regulation with the loss of RB. Data to be discussed will also include further comparison of the E2F1 and AR cistromes in the absence of RB to identify the mechanism by which the AR-RB-E2F1 signaling axis function in promoting the progression of disease.

#5218

Progesterone receptor-mediated repression of STAT5A-target genes implicated in breast cancer development.

Sean Holloran, Gloria Trinca, Christy Hagan. _University of Kansas Medical Center, Kansas City, KS_.

Recent clinical data suggest that progesterone and the progesterone receptor (PR) promote the development of breast cancer. PR itself is a transcription factor, and likely cooperates with other transcription factors to alter tumorigenesis in the mammary gland. ChIP-seq data from T47D breast cancer cells treated with R5020 (synthetic progesterone) showed that PR binding sites were enriched in signal transducers and activators of transcription 5A (STAT5A) gamma interferon activated sites (GAS), the genomic binding sites for STAT5A. This suggests possible adjacent or overlapping binding sites for STAT5A and PR. STAT5A is activated in a similar manner to PR: phosphorylation leads to dimerization and translocation to the nucleus, DNA binding and regulation of target genes involved in mammary gland development, inflammation, and proliferation. The clinical data surrounding the role for STAT5A in breast cancer risk and progression are mixed, and suggest that STAT5A activity in breast cancer is very context dependent, varying based on hormone receptor status. PR and STAT5A are both required for normal mammary gland development and, interestingly, when knocked out arrest at the same stage of mammary gland ductal branching. These data imply putative crosstalk between PR and STAT5A, perhaps through an interaction between these two transcription factors. Co-IP experiments in T47D cells showed an interaction between PR and STAT5A that is regulated by the addition of PR ligand and/or prolactin (STAT5A activator in the breast/mammary gland). To determine the implication of this interaction on STAT5A transcriptional activity we performed a luciferase assay. We transfected a STAT5A target gene (CISH) linked to a luciferase reporter into T47D cells stably expressing shRNA for PR and non-silencing (NS) control. Interestingly, when both ligands for STAT5A and PR were present there was repression of STAT5A transcription of the luciferase reporter compared to when STAT5A ligand was present alone. This repression was lost in cells expressing shRNA for PR. This PR dependent repression was verified in the context of chromatin looking at select STAT5A-target gene (CISH, EREG, and PTHrP), expression via qPCR; prolactin-activated expression of these genes was potently repressed in response to PR ligand treatment. Of note, one of the STAT5A-target genes shown to be repressed by PR, PTHrP (parathyroid hormone related protein), has been linked to breast cancer progression, and specifically, immune cell recruitment to mammary gland tumors. Studies are ongoing now to determine how PR-mediated repression of PTHrP may affect immune cell recruitment to mammary gland tumors models in mice. Understanding the crosstalk between PR and STAT5A, and what transcriptional programs are jointly regulated/repressed by these two transcription factors, could lead to the discovery of novel, PR-based therapies in breast cancer.

#5219

Kaiso as a novel therapeutic target in castration-resistant prostate cancer (CRPC).

Hui-Xian Lin,1 Honghe Wang,1 Jason White,1 Balasubramanyam Karanam,1 Anghesom Ghebremedhin,1 Benjamin Adu Addai,1 William E. Grizzle,2 Clayton Yates1. 1 _Tuskegee University, Auburn, AL;_ 2 _University of Alabama at Birmingham, Birmingham, AL_.

Androgen receptor (AR) is a critical driver in the progression of prostate cancer (PCa). Androgen deprivation therapy(ADT) has been a standard treatment of PCa. However, the PCa develops to castration resistant prostate cancer (CRPC) in almost 53% of patients after 18-32 month's therapy. Metastatic CRPC has poor prognosis and mean survival time is fewer than 2 years. Kaiso belongs to a BTB/POZ zinc finger protein family and is known as a transcriptional repressor. Kaiso expression and localization have been reported to correlate with the prognosis and metastatic potential in several human malignancies. Previous works from our lab have demonstrated that transcription factor Kaiso was upregulated in the progression of PCa. Therefore, our objective is to explore the interrelationship between Kaiso and AR, further to clarify Kaiso as a potential therapeutic target in PCa progression and CRPC.

To investigate molecular mechanisms underlying how aberrant expression of Kaiso contributes to CRPC, the androgen sensitive human prostate cancer LNCaP cells were treated with 10µM anti-androgen Enzalutamide (MDV3100). Kaiso expression levels were increased as detected by RT-PCR and immunofluorescence. To systematically investigate Kaiso targets in PCas, we performed Kaiso ChIP-Seq assay using prostate cancer cell lines LNCaP, C4-2B and PC3. The Kaiso ChIP-Seq peaks indicate androgen receptor motif enrichment in AR expressing LNCaP and C4-2B cells but not in AR-negative PC3 cells, indicating Kaiso could cooperate with AR as co-regulator and potentially control transcription of a subset of its target genes. The interaction of them was confirmed by co-immunoprecipitation of AR using Kaiso antibody in LNCaP cells. To identify Kaiso target genes that interacted with AR pathway, Kaiso knockdown and overexpression stable cell lines were established in LNCaP cells and C42B cells. RT2 Profiler PCR array of AR pathway were performed using LNCaP scramble (LNCaP-Scr) cells and shKaiso cells. The results showed multiple genes were upregulated and downregulated, such as FOLH1, PTEN, RAC3 and IGF1. More importantly, LNCaP Kaiso overexpression cells were more resistant to MDV3100 treatment compared to resistance observed in LNCaP-Scr cells. There were significant negative correlations between Kaiso expression levels and castration-sensitivity.

Our results suggest that Kaiso is a novel AR-interacting protein. Kaiso regulates the genes in AR pathway. Targeting Kaiso presents a potential therapeutic strategy for disrupting AR signaling in CRPC and may prevent CRPC development.

These studies were supported by grants U54MD007585, (NIH/RCMI) [CY], U54CA118623-01 (NIH/NCI) [CY]

#5220

Transcription regulatory networks associated with luminal master regulator expression and breast cancer survival.

Jung S. Byun,1 Sandeep Singhal,2 Samson Park,3 Dae IK Yi,3 Ambar Caban,2 Nasreen Vohra,4 Eliseo J. Perez-Stable,1 Anna Napoles,1 Kevin L. Gardner2. 1 _NIMHD, Bethesda, MD;_ 2 _Columbia University, New York, NY;_ 3 _NCI, Bethesda, MD;_ 4 _East Carolina University, Greenville, NC_.

The disparity in breast cancer burden of African Americans compared with European Americans is one of the most revealing instances in oncology associated with ethnicity. Recent studies demonstrated that there were highly intratumoral genetic heterogeneity and increased the frequency of basal subtypes among African Americans than European Americans. Because significant race-based disparities in breast cancer patients with hormone-receptor positive still persistent even after reflecting socio-economic status, there may be substantial roles playing by intrinsic biological factors contributing to the disparities. Transcriptional regulation governed by estrogen receptor plays critical roles in major changes in chromatin landscape that facilitate the assembly of other transcriptional complexes affecting mammary tumor initiation and proliferation. We investigated the estrogen receptor and its essential pioneer transcription factors how their expressions are associated with racial disparity with their prognostic significance. Co-expression analysis of estrogen receptor and its pioneer transcription factors suggest that ER-positive patients of women of European ancestry show much more favorable survival than women of African ancestry. Differential comparison of protein expression integrated with network-level gene expression analysis reveals significant differences in the predictive ability of the luminal master regulators based on race and survival. Moreover, we identified genes in the downstream of these master regulators that are highly correlated with race and survival. The comparative analysis of the predictive value of race-based cutoffs suggests that these master regulators of luminal differentiation have reduced transcriptional activity in the downstream regulatory network pathways in African ancestry. Functional characterization of the luminal master regulators and their downstream pathways may provide a better understanding of the intrinsic mechanisms that drive racial disparities in breast cancer survival.

#5221

Snail promotes neurite outgrowth in prostate cancer cells.

Gabrielle J. Edwards. _Clark Atlanta University, Atlanta, GA_.

Neurite Outgrowth is a process where developing neurons produce new tentacle-like extensions as they grow in response to guidance cues. The projection can be an axon or a dendrite (nerve fibers). Nerve growth factors, or neurotrophins which are important for survival or growth, are one family of such stimuli that regulate neurite growth. Studies have also proposed that cancer cells stimulate their own innervations. Therefore, the concept of neoneurogenesis includes the development of nerve endings (axons)towards the tumor. Cancer cells are able to secrete neurite outgrowth-promoting molecules and axon guidance molecules that would stimulate and initiate the growth of these new axons to particular areas of the tumor. Neurite outgrowth involves reciprocal signaling interactions between tumor cells and nerves where invading tumor cells have acquired the ability to respond to pro-invasive signals within the peripheral nerve environment. Neurite Outgrowth could serve as a possible mechanism leading to invasion of cancer cells into the nerve sheath and subsequent metastasis. Snail1 or Snail is a zinc-finger protein that can down-regulate cell adhesion proteins such as E-cadherin by binding several E-boxes located in the promotor region, thereby inducing the epithelial mesenchymal transition (EMT) process to promote tumor migration and metastasis. Snail has also been shown to promote neuroendocrine differentiation. The hypothesis is that Snail can promote neurite outgrowth. For this study we utilized various prostate cancer cell lines: C42 NS, E006AA-hT (controls); C42 Snail shRNA, E006AA-hT (stable Snail knockdowns); LNCAP Neo (empty vector control) and LNCAP Snail (stably over-expressing Snail). Conditioned media collected from these cells was cultured with nerve cells (NS20Y) for 48 hours followed by a quantitative neurite outgrowth assay. Nerve growth factor (NGF) was utilized as a positive control. We also included Lanreotide, an inhibitor of neuroendocrine differentiation. After 48 hours, the nerve cells were incubated with Fix/Stain solution containing Cell Viability, and Cell Membrane stain, and nerve extensions quantified by analysis with a plate reader. Our results showed that C42 NS and E006AA-hT NS (Snail-high) conditioned media led to a higher neurite outgrowth compared to the Snail knocked down cells. We also observed that neurite outgrowth was blocked by Lanreotide inhibitor. In conclusion, Snail promotes neurite outgrowth via secretion of soluble factors, which can be inhibited by Lanreotide, a drug currently being used to treat patients with neuroendocrine tumors. Therefore, targeting cancer cell interaction with nerve cells may be helpful in halting prostate cancer progression/metastasis.

GRANT SUPPORT: NIH 1P20MD002285, NIH/NCRR/RCMI G12RR003062-22 and NIH/NIGMS/RISE 5R25GM060414

#5222

Repression of neuronal genes protects the pancreas from certain injuries and aberrant plasticity.

Julie K. Bray, Ola Elgamal, Lais Da Silva, Dhruvit Sutaria, Xiuli Liu, Kristianna Fredenburg, Thomas D. Schmittgen. _University of Florida, Gainesville, FL_.

Among the major cancer types, pancreatic ductal adenocarcinoma has one of the lowest survival rates with little improvement over the past 3 decades. To address this dire clinical need, this study explores the unique and underexploited neuronal property of the pancreas that correlates with greater injury and pancreatic cancer development. The pancreas has a remarkable innate plasticity, and a shift toward neuroendocrine transdifferentiation has been shown to correlate with poor survival and tumor progression in numerous cancers, such as lung and prostate cancers. Recent reports show a subpopulation of cells within pancreatic tumors that express neuroendocrine-related genes correlate with poor outcome and chemoresistance. We hypothesize one regulator of this ductal-neuroendocrine lineage plasticity may be REST, a transcriptional repressor most known for orchestrating neuronal development. Tissue microarray of human pancreatic cancer shows patients negative for REST had a lower overall survival than patients positive for REST. To investigate the role of REST in the pancreas, we developed a novel transgenic mouse model with REST conditionally knocked out of the pancreas. REST KO resulted in an increase of neuroendocrine markers and an increase of zymogen granules, resulting in a lighter, cloudy appearance of the pancreas. When pancreatic injury was introduced by injections of cholecystokinin analog caerulein to induce acute pancreatitis, the REST KO mice show greater serum amylase levels and higher pancreatic edema indicating greater pancreatitis. Furthermore, an increase in neuronal-related genes was observed within REST KO pancreas relative to control. We then investigated the role of REST during cancer-promoting injuries such as chronic pancreatitis. Mice were administered a 10-week series of caerulein injections, and histology was evaluated. As expected, REST KO mice experienced greater inflammation, injury, and pre-neoplastic lesions. Further directions include investigating REST KO during later stages of pancreatic cancer injury by crossing REST KO mice with mutant KRAS mice, as oncogenic KRAS is a leading driver of pancreatic cancer. We expect REST KO to increase neuroendocrine properties that will accelerate KRAS-induced pancreatic cancer development. In conclusion, we hypothesize that loss of REST increases neuroendocrine-like properties of the pancreas that leads to a greater injury, suggesting that REST plays a protective effect against pancreatic damage and pancreatic cancer development. Furthermore, our novel REST KO mice may prove to be a useful model to investigate how neuroendocrine properties may promote pancreatic cancer development and drug resistance. Understanding neuronal influences during pancreatic cancer development could have multiple implications for cancer patients and warrants further investigation.

#5223

Epigenetic silencing mediated by non-phosphorylated STAT5B prevents differentiation in acute myeloid leukemia.

Jakub Szybinski,1 Daniel Sasca,1 Jan Heidelberger,2 Karolin Klumb,1 Viral Shah,1 Anna Dolnik,3 Matthias Theobald,1 Lars Bullinger,3 Petra Beli,2 Thomas Kindler1. 1 _University Medical Center of Mainz, Mainz, Germany;_ 2 _Institute of Molecular Biology, Mainz, Germany;_ 3 _Charité, University Medical Center, Berlin, Germany_.

STAT5 proteins are transcription factors involved in the regulation of proliferation, apoptosis and differentiation. Upon ligand-mediated activation, phosphorylated STAT5A (pSTAT5A) and pSTAT5B form homo- or heterodimers, translocate to the nucleus and activate transcription. Recent data indicate an important role of un-phosphorylated STAT5 (uSTAT5) in the regulation of differentiation. For example, the presence of uStat5 shifts the balance toward the maintenance of leukemic stem cells, whereas genetic depletion of Stat5induced differentiation. Further, uSTAT5 has been shown to act as a repressor of megakaryocytic differentiation via restricting the access of ERG to its target genes. To further investigate the function of uSTAT5 in mammalian AML, we established several cell line models expressing doxycycline-inducible shRNA directed against either STAT5A or STAT5B. In uSTAT5-expressing AML cells targeting of STAT5A or STAT5B suppressed cell growth and induced differentiation. These effects were significantly stronger upon knockdown of STAT5B compared to STAT5A. Global RNA sequencing demonstrated enrichment of differentiation programs upon downregulation of uSTAT5B. To further explore the biological function of uSTAT5, we performed SILAC-based global proteomics after pulldown of either STAT5A or STAT5B. While uSTAT5A was found to be associated with proteins involved in RNA processing, uSTAT5B primarily co-precipitated with chromatin- and histone-binding proteins, like the transcriptional repressor ETV6 or the histone H3K4 demethylase KDM5C. To dissect the specific roles of pSTAT5B and uSTAT5B, we performed co-immunoprecipitation assay upon treatment of AML cells with GM-CSF or vehicle control. As expected, GM-CSF treatment caused strong phosphorylation of STAT5A/B, however, uSTAT5B-binding of ETV6 was almost completely abolished, indicating a specific association only with uSTAT5B. As depletion of uSTAT5B induced differentiation and uSTAT5B interacts with KDM5C and ETV6, we hypothesized that uSTAT5B prevents transcription of genes involved in differentiation. To address this question, we performed H3K4me3 ChIP-seq analysis in THP-1 cells upon knockdown of STAT5B. Downregulation of uSTAT5B caused strong accumulation of H3K4me3 at promoter regions of previously silenced genes. Finally, we wanted to investigate whether targeting of KDM5C can reverse disturbed differentiation in AML cells. Treatment with the KDM5 inhibitor CPI-455 significantly inhibited proliferation and induced apoptosis, but only in AML samples expressing uSTAT5B. In summary, our data provide evidence that uSTAT5B specifically blocks differentiation in AML cells via its interaction with KDM5C followed by repression of H3K4me3 at distinct promoter regions. Targeting of uSTAT5B or its interacting partners might represent an interesting novel strategy in AML therapy.

#5224

Conversion of human glioblastoma cells into neurons by neuronal transcription factors.

Xin Wang, Zifei Pei, Aasma Hossain, Tania T. Barnatan, Yuting Bai, Gong Chen. _Pennsylvania State University, University Park, PA_.

Background: Glioblastoma is one of the most severe primary cancer types in the central nervous system. Because of genomic and epigenetic heterogeneity, GBM is infiltrative and resistant to conventional treatments such as radiation, chemotherapy and molecular targeting drugs. Glioblastoma often arises from astrocytes, which can be directly converted into neurons according to our earlier work, so we hypothesize that glioblastoma cells might also be converted into non-proliferating neurons. This trans-differentiation therapy might provide a unique approach for glioblastoma treatment.

Methods: NeuroD1 was chosen as one of the candidate factors in this study because we have shown its critical roles in astrocyte-to-neuron conversion. Neurog2 and Ascl1 were also tested to understand possible different conversion mechanisms. Single transcription factor or GFP was overexpressed via retrovirus in human glioblastoma cells. Twelve hours after virus infection, culture medium was changed into differentiation medium containing neurotropic factors for neuronal maturation. Immuostainng and other tests were conducted at different days post infection.

Results: Retrovirus yielded high infection efficiency in fast-proliferating glioblastoma cells. All three factors tested were capable of converting glioblastoma cells into neuron-like cells efficiently. Besides morphological change, robust pan-neuronal markers were expressed during the conversion, such as immature neuronal markers DCX, Tuj1 and mature neuronal makers MAP2, NeuN. Majority of the converted cells from glioma were immunopositive for glutamatergic neuron marker vGluT1 and hippocampal neuron marker Prox1. Reactive astroglial marker GFAP decreased after conversion, but cancer marker EGFR and IL13Ra2 remained during conversion. Cell proliferation was inhibited during conversion indicated by Ki67 and BrdU. Robust synaptic puncta along dendrites were found in glioma-converted cells, indicated by SV2 immunostaining. Patch-clamp recordings revealed that most of the converted neurons could fire multiple action potentials or single action potential.

Conclusion: Our data suggest that several neuronal transcription factors are capable to convert human glioblastoma cells into neuron-like cells efficiently. The converted cells obtained a variety of neuron-specific markers with functional synaptic networks and active electrophysiological properties. This neuronal conversion was also confirmed by reduction of reactive astroglial marker GFAP. Although some cancer markers remain in the converted neurons, glioblastoma cells stopped proliferating once being converted. In summary, our study suggests that converting human glioblastoma cells into neurons could be a potential therapeutic approach for glioblastoma treatment to at least control cancer cell proliferation and inhibit tumor progression.

#5225

Androgen deprivation upregulates SPINK1 expression and potentiates cellular plasticity in prostate cancer.

Ritika Tiwari,1 Nishat Manzar,1 Vipul Bhatia,1 Anjali Yadav,1 Shannon Carskadon,2 Nilesh Gupta,2 Amina Zoubeidi,3 Nallasivam Palanisamy,2 Bushra Ateeq1. 1 _Indian Institute of Technology Kanpur, Kanpur, India;_ 2 _Henry Ford Health System, Detroit, MI;_ 3 _University of British Columbia, Vancouver, British Columbia, Canada_.

The Serine Peptidase Inhibitor, Kazal type 1 (SPINK1) overexpression represents the second-largest prostate cancer (PCa) subtype associated with increased risk of recurrence and poor prognosis. Regardless of molecular subtype, androgen-deprivation therapy (ADT) remains the mainstay treatment for locally advanced and metastatic PCa patients. However, majority of the treated individuals eventually progress to castration-resistant stage and a subset of these patients develop ADT-induced neuroendocrine prostate cancer. Despite evidences of detrimental effects of ADT on PCa, possible role of androgen signaling in SPINK1-mediated prostate oncogenesis remains unexplored. Here, we show that androgen receptor (AR) functions as a direct transcriptional repressor of SPINK1, and blocking AR signaling relieves its repression, leading to upregulation of SPINK1. In agreement, we observe an inverse association between SPINK1 levels and AR expression across multiple patient cohorts, and in neuroendocrine differentiated LNCaP cells. We show that AR and its corepressor, the RE1-silencing transcription factor (REST), occupy SPINK1 promoter and inhibits its transcription. On the other hand, in the absence of AR, lineage reprogramming factor SOX2 in turn binds to SPINK1 promoter leading to its positive transcriptional regulation in androgen-deprived conditions with concomitant increase in neuroendocrine markers. Additionally, stable knockdown of SPINK1 results in reduced epithelial-mesenchymal transition, decreased stemness and drug resistance. Collectively, our findings provide a plausible explanation to the paradoxical clinical outcomes of ADT, arising due to increase in SPINK1 levels. Finally, we emphasize the need to take a well-informed decision prior to ADT and develop alternative therapeutic strategies for castrate-resistant PCa patients.

#5226

Non-canonical functions of TERT in glioblastoma pathobiology.

Shayesteh R. Ferdosi,1 Saumya Bollam,1 Sen Peng,1 Brett Taylor,1 Vijay Gokhale,2 Laurence Hurley,2 Michael Berens,1 Harshil Dhruv1. 1 _Translational Genomics Research Institute, Phoenix, AZ;_ 2 _University of Arizona Cancer Center, Tucson, AZ_.

Mutations in the promotor region of TERT are the most common non-coding mutations across cancers, especially in glioblastoma (GBM), which has a TERT mutation frequency rate over 80%. In the absence of any effective molecular targeting therapy for GBM, elucidating oncogenic signaling of TERT could open new avenues in GBM treatment. Canonically, mutations of TERT, which result in TERT upregulation, maintain telomere length in the nucleus and promote indefinite proliferation of cancer cells. However, a non-canonical function of TERT in the mitochondria has recently been suggested. We screened GBM cell models against a novel small molecule inhibitor (RG1534, Reglagene Inc.) that interferes with the functionality of a mutated hTERT promoter. RG1534 selectively suppresses glioma cell viability without affecting non-transformed normal human astrocytes. More interestingly, RG1534 treatment leads to rapid apoptosis induction in glioma cell lines that does not correlate with the time course of the telomere shortening effect. We further validated this rapid apoptosis behavior in glioma cell lines using siRNA and CRISPR/Cas-9 mediated hTERT knockdown. We also measured the protein expression of TERT in subcellular fractions of glioma cell lines and demonstrated the presence of higher TERT expression in mitochondrial extract compared to the nuclear extract. Finally, using MitoSOX dye we demonstrated significant increase of ROS generation in glioma cells treated with RG1534 as compared to control. In summary, our results demonstrate that non-canonical functions of TERT may play critical role in glioma pathobiology and require more detail investigation.

#5227

Chromatin folding disruptions in human cancers.

Kadir C. Akdemir. _MD Anderson Cancer Center, Houston, TX_.

Genomic material within the nucleus is folded into successive layers in order to package and organize the long string of linear DNA. This hierarchical level of folding is closely associated with transcriptional regulation and DNA replication. Genes within the same folding domain demonstrate similar expression and histone-modification profiles. Therefore, boundaries separating different domains have important roles in reinforcing the stability of these domain-wide features. Indeed, domain boundary disruptions in human genetic disorders or human cancers lead to misregulation of certain genes, due to de novo enhancer exposure to promoters. However, the frequency of boundary disruptions in human cancers, and whether there are recurrently affected boundaries in specific cancer types, remains unclear. Here, to understand effects and distributions of somatic structural variations (SVs) across TADs, we utilized 288,457 high-confidence somatic structural variations from 2658 tumor-normal pair high-coverage whole genome sequencing datasets across various cancer types. We comprehensively profiled structural variations effects on the domain boundaries and the regulation of genes in human cancers. Our findings demonstrate that the impact of TAD disruptions are substantially varied across tumor types and these events are reflective of overall SV burden and type. Notably, most of the TAD disruptions could result in appreciable changes in nearby gene expression in cancer cells. In addition, we demonstrated that SVs can lead to fusion of discrete TADs. Notably, complex rearrangements drastically change chromatin folding maps in the cancer genomes.

#5228

**Acute depletion of CTCF disrupts enhancer-promoter regulation of** MYC **in B-cell acute lymphoblastic leukemia.**

Chunliang Li, Yang Zhang. _St. Jude Children's Research Hospital, Memphis, TN_.

Acute lymphoblastic leukemia (ALL), including B-ALL and T-ALL, is the most common childhood cancer and the leading cause of cancer death in children and young adults. Comprehensively genomic sequencing strategies have successfully sub-classified ALL based on genetic alterations including chromosomal rearrangement and aberrant gene mutations, of which frequent coding mutations of CTCF were discovered. However, the function of CTCF in ALL was not clearly defined yet. In a cohort of 264 human pediatric B-ALL patients, particularly in those carrying CTCF loss-of-function mutations, we identified a positive correlation of mRNA expression between CTCF and MYC. Then we integrated the miniAID-mClover3 cassette to the both alleles of endogenous human CTCF locus, which allows acute auxin-mediated degradation of the CTCF protein in a human pediatric B-ALL cell line. By employing combination strategies including whole transcriptome analysis and Capture-C, we discovered that only small number of genes were differentially expressed in CTCF depleted cells, among which MYC and it's downstream genes, not other B-ALL associated oncogenes, were specifically enriched. Mechanically, chromatin interaction capture assay reveals that acute depletion of CTCF primarily disrupted direct interaction between MYC promoter and its distal enhancer residing ~1.8 Mb downstream, subsequently down-regulating target genes in human B-ALL cancer cells. Thus, CTCF/MYC regulation axis is essential in B-ALL development.

#5229

The mutually exclusive and diverse NuRD chromatin remodeling complex in cancer progression.

Adone Mohd-Sarip,1 Diana Zatreanu,2 Jeroen Demmers3. 1 _Queen's University Belfast, CCRCB, Belfast, United Kingdom;_ 2 _Institute of Cancer Research, London, United Kingdom;_ 3 _Erasmus MC, Rotterdam, Netherlands_.

Introduction: ATP-dependent chromatin remodelers are frequently mutated in cancers. Similar to the better studied SWI/SNF remodelers, the sequencing of cancer genomes has uncovered frequent mutations in genes encoding the Nucleosome remodeling and histone deacetylase (NuRD) subunits. Upon closer inspection, NuRD complex is highly amplified in breast cancer, deleted in prostate cancer, yet mutated in lung cancer. These observations suggest that (in)activation of NuRD might contribute to oncogenesis in a cell-type specific manner. Prompted by these findings, and to gain insights into the functions of the NuRD complex, we first need to understand the composition of this complex(es).

Material & Methods: We carried out Immunoprecipitation (IP) with antibodies raised against NuRD members DOC1 and CHD4, followed by mass spectrometry (MS) with HeLa nuclear extracts. This was followed by 'reverse' IP-MS i.e. using antibodies raised against these novel NuRD-associated factors (AFs) on HeLa nuclear extracts and data mining for the NuRD-AFs (cBioPortal). Re-expression and knockdowns of DOC1, CHD4 and NuRD-AFs were executed in selected breast, prostate and lung carcinoma cell lines. High resolution MNase nucleosomal mapping followed by qPCR was performed at specific target loci.

Results & Discussions: Proteomics data suggest that ZFHX3 (cancer determinant), ZBTB2 (developmental regulator) and SMAD4 (BMP/TGF-beta signalling pathway) as NuRD-AFs. Interestingly, DOC1 IP reveals interaction only with SMAD4 while CHD4 immunoprecipitation uncovered interaction only with ZFHX3. Besides, ZBTB2 associated with both DOC1 and CHD4-NuRD. It is worth noting that these novel NuRD-AFs were found to be frequently altered in specific cancers e.g. prostate, non-small cell lung and breast carcinomas. Re-expression and knockdowns of NuRD-AFs affected epithelial-mesenchymal transitions (EMT) and chromatin dynamics during cancer progression.

Conclusion: Remodeling of chromatin is an essential step towards gene expression, therefore in order to study the critical role of NuRD-AFs during carcinogenesis, a higher resolution picture of the dynamic changes in chromatin and nucleosomal structure is pertinent. These mapping experiments revealed nucleosome occupancy as well as (in)accessibility of chromatin during carcinogenesis, in particular at specific target loci. Chromatin dynamics uncovered in specific carcinoma cell lines thereby generating a comprehensive picture of the various chromatin states during carcinogenesis and expanding our understanding of chromatin function. The impact will be protein/gene expression as well as chromatin (in)accessibility and structure could be used in (early) diagnostics and prediction of carcinogenesis or biomarker discovery, in combination with already available diagnostics/biomarkers; towards a more targeted and personalised therapy for cancer patients.

### Regulation of Gene Expression in Cancer

#5230

Modulation of SF3B1 causes global intron retention and downregulation of the B-cell receptor pathway in chronic lymphocytic leukemia.

Qimei Han,1 Jianbo Wang,2 Austin Y. Shull,1 Fang Shi,3 Libin Deng,4 Jeong-Hyeon Choi,1 Eun-Jeong Park,1 Shuo Tu,4 Lirong Pei,1 Farrukh T. Awan,5 Roni Bollag,1 Locke J. Bryan,1 Hong-bo Xin,4 Chandraiah Lagisetti,6 Thomas R. Webb,6 Victor Jin,2 Huidong Shi1. 1 _Augusta University, Augusta, GA;_ 2 _University of Texas Health Science Center, San Antonio, TX;_ 3 _Georgia Institute of Technology, Atlanta, GA;_ 4 _Nanchang University, Nanchang, China;_ 5 _The Ohio State University, Columbus, OH;_ 6 _SRI International, Menlo Park, CA_.

Splicing factor SF3B1 is frequently mutated in chronic lymphocytic leukemia (CLL) patients and has been suggested as a potential therapeutic target. In this study, we demonstrated that SF3B1 modulator sudemycin D6 (SD6) effectively inhibits cell growth and induces apoptosis in CLL cells. RNA sequencing analysis revealed significant increases in global intron retention in SD6-treated CLL cells. Pathway analysis of the genes associated with increased intron retention suggested that B-cell receptor (BCR) and PI3K signaling pathways were among the most important pathways being affected by SD6. The increases in intron retention were inversely correlated with decreases in mRNA and protein levels of the affected BCR/PI3K pathway molecules including BLNK, BTK, AKT, PLCγ2, and PI3Kδ. SD6 treatment also induced a time-dependent exon-skipping event in MCL-1 mRNA and resulted in significant down-regulation of another anti-apoptotic gene TRAF1, thus collectively contributing to the SD6-induced apoptosis. Furthermore, SD6 treatment can overcome the pro-survival and pro-growth signals and synergize with established CLL therapies ibrutinib, idelalisib, and venetoclax to induce apoptosis in primary CLL cells co-cultured with bone marrow stromal cells and T-cell-derived cytokines. Finally, in vivo treatment with SD6 at 10mg/kg/day for 7 days significantly inhibited the growth of xenograft tumors that were established by subcutaneous inoculation of 5×106 MEC1 CLL cells into NOD mice. Collectively, these results provide a strong rationale for the future clinical development of spliceosome modulators and potential combination therapies for the treatment of CLL.

#5231

Regulatory mechanism of carboxylesterase 2 expression and its role in human colorectal cancer.

Momoko Ishimine,1 Hyeon-Cheol Lee,1 Hirofumi Nakaoka,2 Hajime Orita,1 Toshiyuki Kobayashi,1 Ituro Inoue,2 Koichi Sato,1 Takehiko Yokomizo1. 1 _Juntendo University School of Medicine, Tokyo, Japan;_ 2 _National Institute of Genetics, Shizuoka, Japan_.

Irinotecan (camptothecin-11 (CPT-11)) is an anticancer drug that has been used in the treatment of a wide spectrum of cancer, and irinotecan-containing regimens such as FOLFIRI and FOLFOXIRI are standard chemotherapy regimens for advanced colorectal cancer. Irinotecan is a prodrug that is converted to its active metabolite 7-ethyl-10-hydroxycamptothesin (SN-38) by carboxylesterases CES1 and CES2. CES2 has a far higher hydrolytic efficiency against irinotecan than CES1. Since CES2 expression is higher than other CES isozymes in colon tissue, it might contribute to local (i.e., intratumoral) activation of irinotecan. Recent studies have shown that CES2 expression is regulated by p53 in human colon carcinoma cell lines. TP53 is a tumor suppressor gene and the frequency of its mutations in colorectal cancer is approximately 50%. Therefore, TP53 mutation may cause downregulation of CES2 expression and affect the sensitivity of colorectal cancer to irinotecan. In our previous study, we showed that both CES2 expression and carboxylesterase activity were significantly lower in most colorectal cancer specimens than in adjacent normal tissue. Nevertheless, there was no clear relationship between CES2 expression and TP53 gene status. We also found that p21 expression was significantly reduced in the tumors compared with adjacent normal tissue independently of their TP53 mutational status. Notably, there was a strong positive correlation between p21 and CES2 expression even in the tumors with nonfunctional p53. Recent studies have reported that colorectal tumors developed in proximal colon (right side) and distal colon (left side) exhibit different molecular characteristics and histology. Patients with left-sided colorectal tumors respond well to conventional adjuvant chemotherapies, and the prognosis of these patients is better than that of patients with right-sided colorectal tumors. To test whether location of tumor affects CES2 expression, we compared the CES2 expression levels of tumors that arose from proximal colon with those from distal colon. We also tested the potency and the selectivity of a recently reported CES2 inhibitor. Our results provide important insights into the regulatory mechanism of CES2 expression and its role in colorectal cancer.

#5232

PES1 is highly expressed and its biological function in gastrointestinal tumor.

Qin Feng,1 Shenyi Lian,1 Ping Wang,1 Yue Wang,1 Zhongwu Li,1 Chengchao Shou,1 Tingting Ren,2 Dongmei Lin1. 1 _Peking University Cancer Hospital and Institute, Beijing, China;_ 2 _Peking University People's Hospital, Beijing, China_.

Nucleolus, one of the most important organelles in cell nuclear of eukaryotic cells, is made of rRNA, rDNA and nucleoproteins. Its main function is rRNA synthetic. PES1 is part of nucleolar protein, and was identified in the mutational study of the embryonic development of Zebrafish. Its main biological functions include forming nucleolus, promoting cell proliferation and regulating cell cycle. A few researches indicate the close relationship between PES1 and tumorigenesis.

The purpose of this study is to explore the role of PES1 in tumorigenesis and the possible mechanism. Three specific monoclonal antibodies against PES1 were made through hybridoma technique. Using these antibodies, it is found that PES1 had significantly higher expression in gastric and colon carcinoma than in tumor-adjacent and -distant tissues. Immunohistochemical assay on gastric carcinoma specimens and colon carcinoma specimens indicated that PES1 had significantly higher expression in carcinoma than in tumor-adjacent tissues. Moreover, we demonstrated the localization of PES1 was changed from nucleolus to nucleoplasm significantly under the stimulation of chemotherapeutic drugs and UV. The phenomenon of PES1 translocation in nucleus was weaken in drug-resistant cancer cell lines. These results suggest that the translocation of PES1 in nucleus might be related to DNA damage repair and drug resistance mechanism of cancer cells.

In summary, PES1 expression is significantly higher in gastric and colon carcinoma tissue. And dynamic changes of PES1 localization in the cell nucleus is an important biological function and is worth further research.

#5233

MEF2C **dysregulation is associated with immature T cell immunophenotype, absence of biallelic deletion of TCR-gamma and inferior survival.**

Anita Chopra, Jay Singh, Deepak Verma, Rajive Kumar, Sameer Bakhshi, Jayanth Kumar, Rachna Seth, Atul Sharma. _All India Institute of Medical Sciences, New Delhi, India_.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy affecting both children & adults. Unlike B-ALL, the prognostic markers are not clearly defined in T-ALL. In this study, we evaluated the correlation of MEF2C gene expression in T-ALL with immunophenotype & absence of biallelic deletion of TCR-gamma (ABD) & tested its potential significance as prognostic marker.

A total of 106 T-ALL patients, including 75 children & 31 adults (males 93; females 13) were included. Immunophenotyping was done in all cases at diagnosis on bone marrow/peripheral blood samples using CD3, CD7, CD4, CD2, CD45, CD5, CD8, CD1a, CD13, CD33, CD117, HLA-DR, CD34, CD65 & CD11b antibodies. The patients were categorized into immature (pre & pro T-ALL; n=48), cortical (n=47) & mature (n=11) T-ALL based on immunophenotypic features. MEF2C gene expression was quantified by RQ-PCR. ABD was done in 90 patients in which DNA was available by quantitative DNA PCR (Q-PCR) for TCR-gamma rearrangements using the protocol described before1. Minimal residual disease in bone marrow samples at the end of induction therapy was also measured. Kruskal-Wallis test was performed to determine the correlation of MEF2C expression with immunophenotypes & TCR-gamma chain status. Kaplan Meier survival analysis was done to evaluate the significance of MEF2C dysregulation on event free survival & overall survival.

We found MEF2C gene to be significantly overexpressed (p=0.013) in patients with immature T-ALL as compared to cortical & mature T-ALL cases. It was significantly overexpressed in early T-cell precursor (ETP-ALL) cases (n=15) versus non ETP-ALL (p=0.03). By qPCR, 29 of 90 patients (32.22%) were classified as ABD, 44 (48.89%) as non-ABD & 17 (18.89%) as indeterminate. MEF2C expression was higher in ABD patients (p=0.011). Minimal residual disease was found more frequently in MEF2C dysregulated patients (p=0.033). On Kaplan Meier survival analysis, event free survival (EFS) was not significantly different between two groups (p=0.33). Overall survival (OS) was worse in MEF2C dysregulated patients as compared to others (p=0.017). ETP-immunophenotype was associated with poorer EFS (p=0.013). ABD did not correlate with EFS & OS.

To conclude, MEF2C dysregulation was associated with immature T-immunophenotype, ABD & inferior survival at diagnosis. MEF2C gene expression can be used as potential candidate to risk stratify T-ALL cases.

Reference 1. Gutierrez A, Dahlberg SE et al. Absence of biallelic TCRgamma deletion predicts early treatment failure in pediatric T-cell acute lymphoblastic leukemia. J Clin Oncol. 2010;28:3816-23.

#5234

Epigenetic control of heparanase expression through CRISPR/dCas9.

Guihua Zeng,1 Fu-Sen Liang,2 Lina Cui1. 1 _University of Florida, Gainesville, FL;_ 2 _Case Western Reserve University, Cleveland, OH_.

Heparanase is the only known enzyme that cleaves heparan sulfate (HS) and hence participates in the degradation and remodeling of the extracellular matrix (ECM). Pathological expression of heparanase at abnormal levels can contribute to the development of various diseases. In our research, directed by clustered regularly interspaced short palindromic repeats (CRISPR) system, the promoter region of heparanase was successfully and specifically targeted with screened and combined single guide RNAs (sgRNAs). Linking dCas9 with different histone modification enzymes (eg. P300, hHDAC3, LSD1), the expression of heparanase was successfully up- or down-regulated through acetylation, deacetylation and demethylation. This approach allows us to tune heparanase expression in various cell lines on demand, enabling the study of heparanase activity in real time.

#5235

PIN1 and FOXP3 regulate SKP2 expression.

Wei-Hsiung Yang, Chiung-Min Wang, William H. Yang. _Mercer Univ. School of Medicine, Savannah, GA_.

S-phase kinase-associated protein 2 (SKP2), a component of the E3 ubiquitin ligase SKP1-CUL1-Fbox complex, acts as an oncogene/oncoprotein as evidence suggests that overexpression of SKP2 results in cell cycle dysregulation and tumorigenesis as well as associates with poor prognosis in numerous cancers. The unique peptidyl-propyl isomerase PIN1, which isomerizes the cis-trans peptide bond between pSer/pThr and proline, regulates its target proteins' cell localization, stability, and function. However, whether SKP2 is the target protein of PIN1 remains unknown. Herein, we demonstrate that SKP2 expression is regulated by PIN1 and FOXP3. First, we observed that knock-down PIN1 using siRNAs reduced SKP2 protein level in several cancer cell lines (MCF7, PC3, DU145, HepG2, and JEG3 cells). Secondly, we found that overexpression of PIN1 increased SKP2 protein level. Furthermore, PIN1 dose-dependently enhanced SKP2 promoter activity in gene reporter assay. Since FOXP3 is a known transcriptional repressor for the oncogene SKP2, we investigated whether PIN1 affects FOXP3-mediated SKP2 expression. Interestingly, we observed that PIN1 dose-dependently counteracted FOXP3-mediated SKP2 promoter activity. Finally, we demonstrated that mutations on PIN1 (W34A) and FOXP3 (Y342F) abolished PIN1-mediated and FOXP3-mediated SKP2 expression, respectively. Taken together, our results suggest that PIN1 functions as a novel regulator of SKP2 and with FOXP3 may be involved in tumor development and progression.

#5236

A qualitative change in transcriptome during MDCKII 3D epithelial morphogenesis is linked to intracellular trafficking.

Tianfang Wang,1 Shaying Zhao,1 Shi-Yuan Cheng2. 1 _University of Georgia, Athens, GA;_ 2 _Northwestern University, Evanston, IL_.

The majority of human cancers are derived from epithelial tissues. Understanding epithelial morphogenesis is hence important in cancer research. Madin-Darby canine kidney II (MDCKII) cells, cultured in 3D and 2D conditions, are used extensively as a model for studying cell polarity, epithelial morphogenesis and carcinogenesis. Like other cell differentiation systems, gene expression plays a key role in MDCKII epithelial morphogenesis.

To better understand the change in the transcriptome, we performed a time course RNA-seq analysis of MDCKII 3D cytogenesis, along with fully polarized 2D cells for comparison. Surprisingly, our study reveals that the change is not linear, but rather clearly biphasic. Specifically, about 3,000 genes are significantly up- or down-regulated between 24 hour and day 3 after seeding, when the lumen is forming, compared to < 120 such genes during other time intervals. Because previous studies have shown that Rab11-coordinated intracellular trafficking plays an essential role in MDCKII lumen formation, we hypothesize this qualitative change in the transcriptome is linked to intracellular trafficking. To test this hypothesis, we used knockdown clones of AVL9 and DENND5A, both interacting with Rab11 and participating in trafficking. Knockdown cells have altered cell polarity and defective trafficking, but not as significant changes in transcriptome as wild type cells, supporting our hypothesis. We then focused on β-catenin to better understand the mechanism. Our study reveals that in wild type cells, following the first cell division, β-catenin is depleted from the nucleus and enriched in cell-cell junction. This results in MYC transcriptional silencing (supported by ATAC-seq analysis), which in turn initiated down-regulation of numerous MYC target genes. These observations are however not observed in knockdown cells.

Our study supports the qualitative change ("switch on-off") model, rather than the quantitative (gradual) change model, in transcriptome remodeling during epithelial cell differentiation. Moreover, our work supports that intracellular trafficking likely initiates this quantitative change in transcriptome.

#5237

Development of a gene expression database of renal cell carcinoma cases by NGS-combined HiCEP to identify tumor markers.

Makoto Kawaguchi,1 Hirotaka Matsuo,1 Ryoko Araki,2 Seiko Shimizu,1 Mikiya Takao,1 Akiyoshi Nakayama,1 Yosuke Kitamura,1 Masumi Abe,2 Keiichi Ito,1 Nariyoshi Shinomiya1. 1 _National Defense Medical College, Tokorozawa, Saitama, Japan;_ 2 _National Institute of Radiological Sciences, Chiba, Japan_.

Objectives: High coverage expression profiling (HiCEP) is an AFLP-based comprehensive gene expression analysis technique invented in Japan. HiCEP has two unique characteristics. First, it can detect low amounts of mRNA with high sensitivity and reliability. Second, HiCEP enables highly quantitative and reproducible mRNA expression analyses. However, it requires complicated processes, including TA cloning of isolated transcripts, to obtain sequence information on detected peaks. We performed next-generation sequencing (NGS)-combined HiCEP and tried to establish a gene expression database of renal cell carcinoma (RCC) cases to identify effective tumor markers.

Materials and methods: We collected cancerous and macroscopically non-cancerous regions from 83 RCC cases. Of these, six cases with clear cell RCC were analyzed by HiCEP. Total RNA was extracted from the cancerous and non-cancerous tissues of six clear cell RCC cases, and transcribed to cDNA. The cDNA was synthesized and subjected to digestion with the restriction enzymes MspI or MseI, followed by adapter ligation. Selective PCR by 256 kinds of primer pairs was used to amplify the HiCEP fragments, and products with fluorescently-labeled primer were then analyzed by capillary electrophoresis. HiCEP fragments were sequenced using a next-generation sequencer (ion PGM, Thermo Fisher Scientific). We compared the expression levels of HiCEP peaks in cancerous tissues with those in non-cancerous tissues.

Results: We detected several HiCEP peaks in cancerous tissues that showed five times higher expression than in normal tissues. We determined the sequences of the HiCEP fragments by NGS, and developed the first ever cancerous tissue HiCEP fragment database.

Conclusion: We successfully established an RCC gene expression database by NGS-combined HiCEP. We are now performing replication analyses and further analyses of blood samples from the same RCC cases to be able to identify diagnostic and prognostic markers of RCC.

#5238

Clinical significance of GET4 expression in colorectal cancer.

Kensuke Koike,1 Takaaki Masuda,1 Kuniaki Sato,1 Yuushi Motomura,1 Jyunnichi Takahashi,1 Dai Shimizu,1 Shotaro Kuramitsu,1 Atsushi Fujii,1 Akihiro Kitagawa,1 Miwa Noda,1 Yusuke tsuruda,1 Hajime Ootu,1 Yousuke Kuroda,1 Hidetoshi Eguchi,1 Takashi Nakagawa,2 Koushi Mimori1. 1 _Kyushu University Beppu Hospital, Beppu-shi, Ooita-ken, Japan;_ 2 _Kyushu University Hospital, Fukuoka-shi, Fukuoka-ken, Japan_.

Background: Colorectal cancer (CRC) is one of the most common cancers worldwide and the second leading cause of cancer-related deaths in developed countries, with the majority of deaths being attributed to distant metastasis. Therefore, it is necessary to identify a novel biomarker for cancer progression and metastasis, one that could also be a useful therapeutic target in CRC. Amplification of chromosome 7p is frequent in CRC, and it has been considered to harbor driver genes that promote tumorigenesis or tumor progression by the gain of function. We aimed to identify novel candidate driver genes on chromosome 7p and to clarify the clinical significance of their expression in CRC.

Material and Methods: 1. We selected the candidate genes that satisfied the following criteria using CRC data from The Cancer Genome Atlas (TCGA). 1) The DNA copy number and mRNA expression are positively correlated with each other, 2) overexpressed in the tumor tissues compared to the normal tissues. 2. The mRNA expression of the candidate genes was measured in 147 surgically-resected CRC tissues and the paired normal tissues in our hospital by quantitative RT-PCR. The differences of mRNA expression between CRC tissues and normal tissues were analyzed by Mann Whitney U-test. 3. Survival analysis between high and low expression group of the candidate genes was performed by the Kaplan-Meier method. Correlation between the mRNA expression of the candidate genes and the clinicopathological factors were analyzed by Fisher's exact test. 4. Gene Set Enrichment Analysis (GSEA) was performed in CRC data from TCGA to clarify the correlation between the candidate genes and gene sets that are associated with tumorigenesis or tumor progression.

Results: The Golgi to ER traffic protein 4 (GET4) was identified as a candidate driver gene. GET4 is known to be one factor of the BCL2-associated athanogene 6 (BAG6) chaperone complex and function as a regulator for the nucleo-cytoplasmic transport of BAG6. The BAG6 complex is implicated in diverse cellular processes including apoptosis, co-chaperone, and DNA damage response. The expression of GET4 was significantly higher in CRC tissues than in normal colon tissues (p=0.03), and it correlated with depth invasion (p=0.02), and lymphatic invasion (p=0.01). The high GET4 expression group had a significantly poorer prognosis than the low expression group (p=0.02). On multivariate analysis, distant metastasis was an independent prognostic factor affecting OS (p<0.05) with hazard ratios (95% CI) of 66.6 (5.46-1649.3) among clinicopathological factors. GSEA showed that GET4 expression was negatively correlated with the pathway associated with the Rb-related protein p130, which belongs to the RB gene family proved to be a tumor suppressor.

Conclusions: GET4 could be a promising driver gene of CRC on chromosome 7p possibly through regulating the location of BAG6. GET4 may be a therapeutic target as well as a poor prognostic biomarker in CRC.

#5239

Cisplatin disrupts MDM2-DAXX-HAUSP complex, p53 accumulation, cell cycle regulation, and apoptosis in acute leukemia cells.

Sanjay Kumar, Paul Tchounwou. _Jackson State Univ., Jackson, MS_.

Cisplatin (CDDP) is an antitumor drug successfully used in the treatment of various types of human malignancies. It enhances toxicity of arsenic trioxide (ATO) in head and neck squamous cell carcinoma cell, small cell lung cancer, ovarian cancer cells, and oral squamous cell carcinoma cells (, and chronic myelogenous leukemia cells. CDDP induces cytotoxicity, DNA damage, oxidative stress, and apoptosis in acute promyelocytes leukemia (APL) cells. However, the molecular mechanisms of CDDP-induced P53 activation, cell cycle arrest, and apoptosis in APL cells remain poorly understood. We hypothesized that CDDP inhibits proliferation of APL cells through MDM2-DAXX-HAUSP complex disruption, p53 activation leading to cell cycle arrest and apoptosis. To test hypothesis, we used three APL cell lines and mice model of APL and investigated CDDP effects on cells growth, complex disruption, cell cycle progression, and apoptosis by applying western blotting, IP, gene knockdown techniques, immune/histochemistry, and confocal imaging. We found that CDDP - induced stress signal transmitted by protein kinase (ATM, ATR) and its downstream targets (CHK1 & CHK2) phosphorylation leading to down-regulation of complex molecules expression and accumulation of p53. CDDP-induced p53 expression modulated cell cycle progression and apoptosis in APL cells, and stimulated the formation of more promyelocytes with dense granules through reduction of MDM2 expression in bone marrow cells. CDDP also activated p53 in APL mice liver tissues. This novel mechanism of action of CDDP in APL cells may help in the design of new anti-leukemic drugs for treatment of APL patients.

Acknowledgements: This research was supported by National Institutes of Health NIMHD [grant # G12MD007581], through the RCMI-Center for Environmental Health at Jackson State University.

#5240

Long-term treatment with IL-4, IL-6, and/or IL-17 increases the expression of the NADPH oxidase DUOX2 in human colon cancer cells in vitro.

Agnes N. Juhasz, Mariam Konate Monnard, Guojian Jiang, YongZhong Wu, Abdalla Amr Abdelmaksoud, Jinqiu Chen, Xialong Luo, Noemi Kedei, Smitha Antony, Jennifer L. Meitzler, Jiamo Lu, Iris M. Dahan, Krishnendu Roy, James H. Doroshow. _National Cancer Inst., Bethesda, MD_.

Colorectal cancer (CRC) develops through a multistep process that is enhanced by chronic inflammation and includes complex interactions between the colonic epithelium and pro-inflammatory cytokines capable of stimulating cancer initiation and progression due, in part, to enhanced production of reactive oxygen species (ROS). Expression of colonic DUOX2, a H2O2 generator, and NOX1, a superoxide producer, is increased in patients with inflammatory bowel disease and adenomatous polyps. For these experiments, we investigated the effect of prolonged treatment with pro-inflammatory cytokines known to be associated with CRC (IL-4, IL-6, and IL-17A alone and in combination) on NADPH oxidase (NOX) expression in human colon cancer lines, and early passage, two-dimensional cultures of conditionally-reprogrammed patient-derived colon cancer cells (PDCs). HT-29 colon cancer cells were treated with IL-4, IL-6, IL-17A, IL-4+IL-17A, and IL-6+IL-17A at a concentration of 25ng/ml each for 4, 6, 8, 15, and 30 days. We measured the effect of these cytokines on expression of NOX family members NOX1, DUOX2, and DUOXA2 by quantitative RT-PCR, and by Western analysis. The production of ROS by NOX proteins was measured with chemiluminescence (superoxide) and Amplex Red assays (H2O2), respectively. DUOX2 protein, which is absent at baseline in HT-29 cells, was easily detectable after 4 days, and increased as a function of the duration of cytokine exposure and for cytokine combinations. In addition to HT-29 cells, similar results were obtained using Ls513 and DLD-1 colon cancer lines. Six PDC lines were also studied: T 280-R, F725, F1126, CK7962, CK7354 and 027-R1. Modest increases in NOX1 expression were observed for 2 lines (T 280-R, CK7962) following 6 days of treatment with IL-6+IL-17A or IL-4; however, NOX1 was highly expressed in 4 cell lines following exposure to the combination of IL-4+IL-17A. Moreover, DUOX2 mRNA and protein were highly expressed after IL-6+IL-17A treatment for 6 days in the T 280-R and F725 cell lines, and very highly expressed after IL-4+IL-17A treatment in all 6 PDCs. Enhanced NOX1 and/or DUOX2 expression in CRC cells was accompanied by concomitant enhancement of NOX-specific ROS. RNA-Seq analysis of cytokine-modulated NOX expression in HT-29 cells and a NOX1 shRNA inhibited subline of HT-29 cells, suggests that both NF-κB and Stat signaling pathways are involved in the control of DUOX2 expression in both established and early passage human CRC cells. These experiments suggest that multiple pro-inflammatory cytokines known to be present in the CRC microenvironment may contribute to the development of a hyperoxidant milieu through the up-regulation of the two NOX species, NOX1 and DUOX2, expressed in the colon.

#5241

Whole transcriptome TempO-Seq profiling of focal areas of H&E stained FFPE: Differentiation of normal colon and cancer phenotypes between donors.

Christy Trejo,1 Elliot Imler,1 Milos Babic,1 Peter Shepard,1 Raymond Nagle,2 Joanne Yeakley,1 Bruce Seligmann1. 1 _BioSpyder Technologies, Inc., Carlsbad, CA;_ 2 _University of Arizona, Tucson, AZ_.

Profiling gene expression from small amounts of FFPE using methods that require extraction of RNA from the tissue, such as RNA-seq, have been problematic. We report use of a modification of the whole transcriptome TempO-Seq® assay (Yeakley et al, PLOSone, 2017; DOI:10.1371/journal.pone.0178302) to profile 2 mm2 areas of 5 μm thick FFPE sections by scraping the tissue and assaying lysates directly without extracting the RNA. Duration of fixation and the amount of sample down to a 2 mm2 area do not impact the performance of the assay. Good results can be achieved using FFPE blocks that are up to 30 years old. As a consequence of the ability to assay areas as small as 2 mm2 from H&E stained tissue, we were able to scrape and profile areas of colorectal cancer vs adjacent normal and obtain replicates within donors, and then compare between donors. The assay shows excellent reproducibility, with R2 > 0.95 between biological replicates (separate samples taken from the same patient and lysed/processed separately). While the normal tissues from the patients exhibited a range of expression variability, they all clustered very tightly when analyzed via principal component analysis. The cancer tissue profile from each patient was not only substantially different from the normal tissue profile of that patient, but substantially different from all profiles within the normal tissue cluster of all patients. Furthermore, the cancer tissue profiles were separated into distinct subgroups. Changes in regulatory pathways leading to oncogenesis could be directly distinguished between patients. These results demonstrate the ability to use whole transcriptome TempO-Seq profiling to investigate molecular subtypes of colon cancer using archival FFPE samples. We are expanding the cohort of patients being profiled to investigate these signatures further, and to draw out signatures which allow direct subtype classification purely from expression data.

#5242

To investigate the molecular mechanism and therapeutic roles of PSF-1 in leukemia.

Han-Yun Hsieh, Wei-Zhen Jia, Nobuyuki Takakura. _Osaka University, Osaka, Japan_.

Recently, DNA replication protein PSF-1 gets more and more attention on the research field, PSF-1 not only plays the critical role in normal cell DNA replication but also involves in cancer cell proliferation and anti-apoptosis. PSF1 highly expressed in many types of cancers, such as non-small lung cancer, breast cancer, and colon carcinoma. However, the role and mechanism of PSF1 in leukemia are still uncleared. Compared to the human normal peripheral blood mononuclear cells (PBMCs), the expression level of PSF1 was significantly increased in several types of leukemia, including acute and chronic type leukemia. We also found that PSF1 participate in leukemic cell proliferation and anti-apoptosis. Since PSF1 presents in many types of leukemia, PSF-1 might act as a potential therapeutic target of pan-leukemia. However, in order to enhance the therapeutic effect of leukemia, against its recurrence and metastasis, leukemic stem cells (LSCs) become the novel therapeutic research target. Previous studies indicated that PSF-1 is essential for stem cell proliferation. Based on the study, we selected PSF-1 as the candidate. In this research, we would like to investigate the role of PSF-1 in leukemia and its cancer stem cell niche. Further to establish the new therapeutic method of LSC-specific clinical treatment.

#5243

CRISPR knockout: How to get high biallelic knockout.

Yongqi Li, Li Min, Ellen Liu, Yiran Wang. _Origene Technologies Inc, Rockville, MD_.

Although homology directed repair (HDR)-mediated gene knockout / knockin is well established, it cannot necessarily be applied in some cell types and organisms with low HDR frequencies. Herein we describe an improved technology for genome-wide gene knockout / knockin using CRISPR-Cas9 targeted genome editing: CRISPR KN2.0. CRISPR KN2.0 technology is specifically designed to provide an easy-to-use and get around 50% biallelic knockout. This gene knockout / knockin technology is designed to knock out any DNA locus and knock in a functional cassette containing Green Fluorescence Protein (GFP) and puromycin resistant gene to facilitate screening process. Both of these cassettes are expressed under EF1A promoter after genomic integration. CRISPR KN 2.0 has been used to successfully knock out many genes / knocked in functional cassette in HeLa, HEK293T and MIA PaCa-2 (a human pancreatic carcinoma cell line) cells. Studies carried out in house and by some colleagues show that CRISPR KN 2.0 is highly efficient and render improved knockout/knockin rate (over 50% positively integrated colonies after puromycin selection).

#5244

BET inhibition decreases the expression of DNA repair enzymes in pancreatic cancer.

Aubrey Lynn Miller, Samuel C. Fehling, Patrick L. Garcia, Karina J. Yoon. _Univ. of Alabama at Birmingham, Birmingham, AL_.

Pancreatic ductal adenocarcinoma (PDAC), the most common type of pancreatic cancer (PC), is now the third leading cause of cancer related deaths in the US. This is a highly aggressive disease in that 80% of patients present with locally advanced or metastatic disease and their only treatment option is systemic chemotherapy. Ultimately patient tumors develop resistance to therapy and resulting in a median survival of ~6 months. Therefore, there is an imperative need to identify therapies that provide a more durable response for this patient population.

The family of bromodomain and extraterminal domain (BET) proteins has recently become a target of interest for the treatment of PDAC. The BET proteins (BRD2, BRD3, BRD4, and BRDT) function to regulate transcription by recruiting positive transcriptional activators to the promoters of genes. Our lab has shown that inhibition of BET bromodomain function using the BET inhibitor JQ1 suppresses PDAC tumor growth in vivo and inhibits cell viability in vitro. Importantly, PDAC cells and tumors exposed to JQ1 show evidence of DNA damage. We hypothesized that because of the role BET proteins are known to play in regulating gene expression, that the observed JQ1-induced DNA damage may result from JQ1 inhibiting the expression of DNA repair genes whose expression are dependent on BET protein transcriptional complexes. qRT-PCR and immunoblot analysis demonstrated that treatment of the pancreatic cancer cell line BxPC3 with JQ1 inhibited the expression of two double strand break repair proteins, Ku80 and RAD51, both of which have been shown to be overexpressed in cancer including PDAC. Although it has been established that JQ1 co-occupies 99% of the genomic loci that BRD4 is known to bind to, specific gene products whose expression is dependent on BRD4 in PDAC have not been thoroughly investigated.

The goal of the current study was to determine if the expression of DNA repair genes Ku80 and RAD51 were specific gene targets of BRD4 transcriptional complexes. We exposed BxPC3 cells to JQ1 for 48 hours and assessed changes in the association of BRD4 with the promoter loci of Ku80 and RAD51 using chromatin immunoprecipitation (ChIP) assays. qRT-PCR of the ChIP demonstrated that treatment with JQ1 significantly (p<0.05) decreased the association of BRD4 with the promoters of both genes. To further confirm that these gene products were dependent on BRD4 for their expression, we down-regulated BRD4 using shRNA and evaluated the effect on the expression of Ku80 and RAD51. Immunoblot revealed that by reducing the expression of BRD4 by 53% in BxPC3 cells, the expression of Ku80 and RAD51 were also decreased by 35% and 93% respectively. We conclude that the expression of Ku80 and RAD51 are dependent on BRD4 transcriptional complexes and are gene targets that contribute to the anti-tumor efficacy of JQ1 in PDAC.

#5245

Integrative analysis of KIF4A, 9, 18A, and 23 and their clinical significance in low-grade glioma and glioblastoma multiforme.

SangYeon Cho, DongWoon Kim. _Medical School of Chungnam National University, Daejeon, Republic of Korea_.

To determine the prognostic significance of kinesin superfamily gene (KIF) expression in patients with brain cancer, including low-grade glioma (LGG) and glioblastoma multiforme (GBM), we comprehensively analyzed KIFs in 515 LGG and 595 GBM patients. Among KIFs, KIF4A, 9, 18A, and 23 showed significant clinical implications in both LGG and GBM. The mRNA and protein expression levels of KIF4A, 9, 18A, and 23 were significantly increased in LGG and GBM compared with those in the normal control groups. The mRNA expression levels of KIF4A, 9, 18A, and 23 in LGG were significantly increased in the high-histologic-grade group compared with those with a low histologic grade. Genomic analysis showed that the percent of mRNA upregulation of KIF4, 9, 18A, and 23 was higher than that of other gene alterations, including gene amplification, deep deletion, and missense mutation. In addition, LGG patients with KIF4A, 18A, and 23 gene alterations were significantly associated with a poor prognosis. In survival analysis, the group with high expression of KIF4A, 9, 18A, and 23 mRNA was significantly associated with a poor prognosis in both LGG and GBM patients. Gene Set Enrichment Analysis (GSEA) revealed that high mRNA expression of KIF4A, 18A, and 23 in LGG and GBM patients showed significant positive correlations with the cell cycle, E2F targets, G2M checkpoint, Myc target, and mitotic spindle. By contrast, high mRNA expression of KIF9 in both LGG and GBM patients was significantly negatively correlated with the cell cycle, G2M checkpoint, and mitotic spindle pathway. However, it was significantly positively correlated with EMT and angiogenesis. This study has extended our knowledge of KIF4A, 9, 18A, and 23 in LGG and GBM and shed light on their clinical relevance, which should help to improve the treatment and prognosis of LGG and GBM.

#5246

A paradox: Expression of the myelin and lymphocyte protein 2 (MAL2) is down-regulated in human hepatocholangio and renal carcinomas in contrast to its up-regulation in other cancers.

Alfonso Lopez-Coral,1 Joeffrey J. Chahine,2 Bhaskar V. Kallakury,2 Pamela L. Tuma1. 1 _The Catholic University of America, Washington, DC;_ 2 _MedStar Georgetown University Hospital, Washington, DC_.

Recent work has found that Myelin and Lymphocyte Protein 2 (MAL2), a protein that functions in polarized protein sorting, is frequently overexpressed in many epithelial-derived human cancers, and has been linked to poor prognosis in patients with colorectal and pancreatic cancers. Yet, its role in tumorigenesis is unknown. Our recent overexpression/knockdown studies in polarized hepatic WIF-B cells, Hep3B hepatocellular carcinoma cells and hepatoma-derived Clone9 cells all indicate that MAL2 is a tumor suppressor. How can MAL2's function as a tumor suppressor be reconciled with its upregulated expression and link to bad prognosis in human cancers? MAL2 resides on chromosome region 8q24, a region frequently amplified in multiple epithelial-derived human cancers along with c-Myc, a well-known oncogenic driver whose overexpression dysregulates gene expression leading to downstream decreased expression of MAL2 via inactivation of the Miz1 transcription factor. Our prediction is that lower grade lesions from human epithelial-derived cancers display high MAL2 expression that will diminish as cancers progress into higher grade lesions and metastases with a concomitant increase in c-Myc expression. We tested this hypothesis with a cohort, of 23 cholangiocarcinoma (CC), 18 hepatocellular (HCC) and 20 renal cell (RCC) human carcinoma cases whose formalin fixed paraffin embedded tissue sections containing benign and later-stage tumor lesions were immunostained using the Dako Autostainers Link 48 along with Mal2 rabbit polyclonal (ABCAM), Miz-1 monoclonal ZBTB17(NOVUS), c-Myc monoclonal Y69 (ABCAM) and Ki-67 monoclonal Mib-1(Dako) antibodies. Nuclear and/or cytoplasmic immunoreactivity was scored based on the intensity and percentage of positive cells in both the benign epithelium and adjacent tumor component in each case. In all three carcinoma types, both MAL2 and Miz-1 expression was high in the benign tissue. In the tumor component MAL2 and Miz-1 expression was respectively lost in 15/23(65%) and 22/23(96%) CC, 13/18(72%) and 17/18(94%) HCC, 13/20(65%) and 16/20(80%) RCC cases. In contrast only 2/20 (10%) and 1/20(5%) RCC cases showed an upregulated expression of MAL2 and Miz-1 respectively compared to the benign component, with all three remaining carcinoma cases showing no up-or-down regulation in protein expression. However, no enhanced c-Myc expression was detected in the tumor lesions despite the higher proliferative index as indicated by Ki-67 labeling. These results indicate a concordant down-regulation of both MAL2 and Miz-1 in CC, HCC, and RCC supporting a tumor suppressor role in these cancers. We are currently extending these studies to other epithelial-derived cancer tissue types and also are more closely examining MAL2, Miz1 and c-Myc expression across graded lesions to better test our hypothesis.

#5247

Overexpression of methylenetetrahydrofolate dehydrogenase 1 like (MTHFD1L) in bladder cancer: A potential therapeutic target.

Marie-Lisa Eich, Maria D. Rodriguez Pena, Darshan S. Chandrashekar, Jennifer B. Gordetsky, George J. Netto, Sooryanarayana Varambally. _University of Alabama at Birmingham, Birmingham, AL_.

Introduction: Methylenetetrahydrofolate dehydrogenase 1 (MTHFD1L) is a mitochondrial enzyme involved in the folate cycle by catalyzing the reaction of formyl-tetrahydrofolate to format and tetrahydrofolate (THF). THF is crucial for de novo purine and thymidylate synthesis and is also involved in the regeneration of methionine. Cancer cells rely on purines derived from the de novo pathway for the nucleotides whereas normal cells favor the salvage pathway. Thus, MTHFD1L could be a valuable therapeutic target for anticancer drug design. This study aimed to examine MTHFD1L expression in bladder cancer (BLCA).

Material and Methods: Applying the publicly available cancer transcriptome database UALCAN, we searched for overexpressed genes in bladder cancer associated with survival probability. These in silico analyses indicated a high expression of MTHFD1L in bladder cancer. Furthermore, MTHFD1L expression was analyzed in different molecular subtypes, stage and race groups. Survival probability in MTHFD1L low-and high-expressing tumors was also assessed using UALCAN. Additionally, by searching the publicly available data base Human Protein Atlas we analyzed MTHFD1L immunostaining patterns in bladder cancer.

Results: We analyzed data for 19 normal bladder tissues and 408 primary bladder cancers from the TCGA dataset. MTHFD1L was significantly (p<0.0001) overexpressed in tumorous tissue compared to normal. This overexpression was observed independent of stage, race and age. Furthermore, we found a higher number of MTHFD1L mRNA transcripts in tumors belonging to the basal/squamous and in the neuronal TCGA subtype. Kaplan-Meier survival analysis indicated a poorer survival in patients with high MTHFD1L expression (p=0.05). Evaluation of immunohistochemistry staining pattern in bladder cancer using the Human Protein Atlas database showed a differential expression among human bladder cancer tissues. We are performing in vitro studies to evaluate the role of MTHFD1L in bladder cancer initiation and progression.

Conclusions: Our preliminary findings on MTHFD1L suggest that its overexpression is associated with poor patient survival in bladder cancer. These results suggest that MTHFD1L could be important in the biology of BLCA and a potential target for therapy. Future studies will investigate the functional significance of MTHFD1L and its regulation in bladder cancer.

#5248

Expression of somatic alleles in TCGA data reveals allelic imbalance with implications for treatment and survival.

Shannon T. Bailey, Jim Lund, Jeffrey R. Gulcher. _WuXi NextCODE, Cambridge, MA_.

Next-generation technologies, including whole-genome sequencing of DNA and RNA, have facilitated the identification of different cancer subclasses and genes responsible for tumor growth and progression, and guide the application of targeted therapies for personalized medicine. In this study, we employ a novel, combined approach for assessing cancer mutations.

As standard practice, our group inspects individual DNA sequencing reads underlying each somatic mutation call to assess quality and confirm calls made by variant calling algorithms. This approach provides confidence in identifying likely candidate driver genes while eliminating calls unsupported by aligned reads. We have recently expanded this method to include the assessment of RNA variants as well. In this approach, called DNA mutations are inspected in sequencing reads from both DNA and RNA.

In analysis of 850 tumors from the TCGA breast cancer dataset, we observed inconsistent levels of mutation expression i.e., RNA imbalance, where only less than half of the mutations identified have similar variant call ratios in both RNA and DNA. We observe three types of RNA allelic imbalance: higher RNA mutation call ratios compared to that in DNA, higher DNA mutation call ratios compared to that in RNA, and RNA expression where the DNA mutation is absent. We also observe sites where a mutation is present in DNA, but the RNA is not expressed. Notably, we found a patient with a E545K mutation in PIK3CA DNA that was not expressed in the RNA, suggesting that treatment with PI3 kinase inhibitors will be ineffective for this patient.

When analyzing the most commonly mutated genes in this cohort, we found that the mutation expression patterns are not random but specific to individual genes. For example, GATA3 mutations are almost always expressed with a higher RNA call ratio, BRCA1 and LRP6 mutations are rarely expressed, and PIK3CA mutations are almost always expressed with the same call ratio. We also noticed differences in survival patterns with the different categories of RNA expression for certain genes. We found that patients with higher RNA call ratios in GATA3 have better overall survival compared with patients that have lower RNA call ratios, which is consistent with reported better survival outcome for patients with GATA3 mutations. We also observed that patients with lower RNA mutation call ratios in TP53 had better survival, and those with higher MAP3K1 call ratios had better overall survival.

As DNA and RNA are commonly assayed in cancer studies such as TCGA, we present a novel, combined approach for better identifying mutations of interest. We believe this approach provides an improved methodology for assessing mutations in patients and providing insight into patient outcome. These results also suggest caution when evaluating DNA alone and the necessity for confirming the expression of mutations in RNA.

#5249

The transcriptional regulation of CCND1 by miR-15a-5p in the nucleus of colorectal carcinoma cell lines.

Zhenzhen Liu, Xue Bai, Chunyan Li. _Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China_.

Although miRNAs are important post-transcriptional regulators of gene expression in cytoplasm, more and more evidences demonstrate that miRNAs also have nuclear functions. The majority of miRNAs are present in both the cytoplasm and the nucleus. The canonical function of translational repression in cytoplasm is well studied, but the functions of nuclear miRNAs are currently not well understood. From previous studies, we found that miR-15a-5p (miR-15a for short) can be detected in the nucleus of the colon cancer cell line. By transfected miR-15a labelled with biotin, we validated that miR-15a could bind to DNA (especially dsDNA) in the nucleus of colorectal carcinoma cell line HCT116. To find the targets of miR-15a in the nucleus, we scanned the promoter region of the potential targets which were predicted by Targetscan, miRWalk, and miRDB. The promoter of CCND1 has one region that is complementary to miR-15a. By integrating the RNA-seq data and miRNA-Seq data from NCI60 cell line panel and TCGA (The Cancer Genome Atlas), we found that CCND1 was negatively regulated by miR-15a in colorectal cancer. To confirm that miR-15a regulates CCND1's transcription in the nucleus, we constructed the promoter of CCND1 and a luciferase reporter into one plasmid, then site-specific mutation was introduced into the potential binding site of miR-15a in the promoter of CCND1. The luciferase assay demonstrated that the mutation in the binding site decreased the expression of CCND1, which validate that miR-15a may regulate the expression of CCND1 by binding to the promoter of CCND1 in the nucleus. The next step, we will further study the function of nuclear miR-15a distinct from the canonical paradigm in carcinogenesis.

#5250

Notch signaling in ER+ breast cancer in response to endocrine therapy agents.

Ciera S. Singleton, Lucio Miele, Judy S. Crabtree. _Louisiana State University Health Sciences Center-New Orleans, New Orleans, LA_.

Breast cancer is the most frequent cancer in women in the world, and more than 70% of breast cancer cases are estrogen receptor α positive (ER+). Standard treatment for ER+ breast cancers is endocrine therapy that consists of selective estrogen receptor modulators (SERMs) such as tamoxifen in pre-menopausal patients; aromatase inhibitors (AIs); or selective estrogen receptor down-regulators (SERDs); fulvestrant, and treatment is dependent on the menopausal state of the patient. Many luminal tumors acquire resistance to therapy and recur, leading to disease progression and mortality. Resistance may result from the activation of quiescent signaling pathways that drive cancer stem cell (CSC) activity, such as the Notch signaling pathway. Previous studies demonstrate that activation of Notch via the NOTCH1 receptor recruits ERα in the absence of estrogen to facilitate transcription of estrogen responsive genes. However, NOTCH4 is often upregulated in resistant ER+ breast cancers, driving endocrine therapy resistance via breast CSC activity. We hypothesize that NOTCH4 promotes the transcription of estrogen-responsive genes in the absence of estrogen in a selective manner that is unlike NOTCH1. Understanding how NOTCH4 regulates the transcription of estrogen responsive genes upon treatment with endocrine therapy may suggest the need for combination therapy for ER+ breast cancer that includes a Notch targeted therapy. We have measured mRNA expression of classic estrogen responsive genes in ER+ breast cancer cell lines that either overexpress NOTCH1 or NOTCH4. Our data demonstrate differential expression of estrogen-responsive genes between NOTCH1 and NOTCH4 in the presence of tamoxifen. We have also measured Notch contribution to breast cancer stem cell activity through mammosphere formation assays using ER+ breast cancer cell lines in the presence of fulvestrant and Notch signaling inhibitors. The expression of classical estrogen responsive genes from mammospheres was altered in the presence of fulvestrant, Notch signaling inhibitors, or combinations of the two treatments. Further, combination treatment led to the disruption of mammosphere formation suggesting that combination therapy in the clinic may be suitable for the prevention of endocrine resistance by inhibition of BCSC activity through NOTCH4.

#5251

STAT1 and miR155 expression in human prostate cancer tissue.

Ines Benedetti,1 Lia Barrios Garcia,1 Juan Rebollo2. 1 _Universidad de Cartagena, Cartagena, Colombia;_ 2 _Universidad del Sinú, Cartagena, Colombia_.

Background: Transcription factors (TFs) and microRNAs (miRNAs) form a gene regulatory network at transcriptional and posttranscriptional level. Increasing evidence shows that miRNAs regulates molecular pathways in cancer by targeting various oncogenes and tumor suppressor genes. However, the mutual regulations between TFs and miRNAs in cancer cells have not been well characterized. Signal transducer and activator of transcription 1 (STAT1) has been reported as a tumor suppressor, but recent evidence suggests that may also play a tumor-promoting role. An STAT1-miR155 feedback loop has been identified in normal tissues. We aimed to explore the STAT1 and miR155 expression in prostate cancer by a bioinformatics analysis combined with immunohistochemical evaluation of STAT1 in normal and prostate cancer tissues.

Design: The expression of miR155 was explored in the dbDEMC2 database of Differentially Expressed miRNAs in Human Cancers. Potential targets of this miRNA were searched in the microRNA-target interactions database (miRTarBase), and STAT1 was selected. The protein expression level of STAT1 was analyzed in normal and tumor tissues, from the TCGA databases, by GEPIA bioinformatics tools; and by immunohistochemistry in a prostate tissue microarray (TMA), constructed from prostatectomy samples of patients with localized prostate cancer (PCa), including representative areas from PCa and benign prostate tissue (BPT). Immunohistochemical expression of STAT1 was evaluated by two pathologists, an H-score was obtained for each spot by microscopic assessment of the percentage of epithelial cells with positive staining. Differences in STAT1 expression were assessed by comparing H-Scores between BPT and PCa tissues using Mann-Whitney test, a p value less than 0.05 was considered statistically significant. The Ethical Review Board of the academic institution approved this study.

Results: The analysis of miR155 and STAT1 expression in the Genome Cancer Atlas Database showed that both are overexpressed in a variety of tumors including prostate cancer. STAT1 expression at protein level was evaluated in 143 tissue cores representing PCa, and 78 tissue cores representing BPT. Mean STAT1 expression in cores with PCa, was significantly higher than mean of STAT1 expression in cores with BPT (p less than 0.0001). There was a higher mean staining score in PCa (210, 95% CI: 196,1-227,8), compared to BPT (105,5, 95% CI: 87,1-123,9). STAT1 staining was present predominantly in the cytoplasm, also in the membrane and nucleus, of prostate cancer tumor cells.

Conclusions: Although miR155 and STAT1 are supposed to be involved in a negative auto-regulatory feedback mechanism, they were both found overexpressed in most of the prostate cancer tissues, which could be explained by the loss of the regulatory feedback loop equilibrium in the tumor environment. Additional studies to detect the in situ expression of miR155 are in progress to validate these results.

#5252

High expression of PLK1, polo-like kinase 1, is significantly associated with DNA repair deficiency, inactivated TP53, and worse prognosis in breast cancer.

Takashi Takeshita, Mariko Asaoka, Eriko katsuta, Yan Li, Kazuaki Takabe. _RoswellPark Comprehensive Cancer Center, Buffalo, NY_.

Backgrounds: Polo-like kinase 1 (PLK1), the representative member of the PLK family, plays a pivotal role both in mitosis, especially in G2/M phase, and in the regulation of DNA damage repair. It is suggested that overexpression of PLK1 in cancer cells may disturb the behavior of G2-M DNA damage checkpoint for cancer cell survival. Therefore it was of interest whether PLK1 expression has any clinical significance in breast cancer (BC), which is known to have less DNA damage and mutations compared from other cancers.

Methods: A total of 1025 women with BC from The Cancer Genome Atlas (TCGA) and a total of 237 women with BC from neoadjuvant (NAC) cohort are enrolled. We developed computer-based analysis to verify the clinical significance of PLK1 mRNA expression in these large BC cohorts.

Results: In TCGA cohort, PLK1 high expressing tumors were statistically significantly associated with more aggressive clinical factor. Gene Set Enrichment Analysis shows that PLK1 high expressing tumors were significantly associated with cell cycle related gene sets. In addition, PLK1 high expressing tumors were significantly associated with deficiency of homologous recombination (HR), which is one of the important mechanisms of DNA repair, and high expression of TP53, which is an intermediary to the HR of PLK1. All these results suggest that PLK1 high expressing tumors associate with worse cancer biology. Recently tumor immune microenvironment (TIM) attracted many researchers' interest for its association with better response to NAC and better survival. In analysis of TIM, we found that PLK1 high expressing tumors were significantly correlated with anti-cancer related tumor infiltrating lymphocytes (TILs) and higher immune cytolytic activity CYT. Thus, PLK1 high expressing tumors associate with TIM that is against cancer. Since both higher HR deficiency (HRD) and anti-cancer related TILs are predictors of high sensitivity to chemotherapy, response of PLK1 high expressing tumors to NAC were analyzed, which did not show significant association. On the other hand, survival analysis demonstreated that PLK1 high expressing tumors were statistically significantly associated with poor prognosis in the whole and the hormone receptor-positive/human epidermal growth factor receptor 2-negative group. Our result suggest that although PLK1 high expressing tumors attract TILs and assoaciate with HRD, those anti-tumor effects are overweighed by its association with aggressive cancer biology that translate to worse survival particularly in the subtype known to have less mutations.

Conclusions: We have demonstrated the clinical significance of PLK1 mRNA expression in BC. High PLK1 expression was associated with more aggressive clinical factors, cell cycle related genes, higher HRD, inactivated TP53, high levels of TILs, and worse prognosis in BC.

#5253

Regulation of estrogen related receptor α by ING4 and its potential role in breast cancer pathogenesis.

Aymen A. Shatnawi,1 Vincent Giguere2. 1 _University of Charleston, Charleston, WV;_ 2 _McGill University, Montreal, Quebec, Canada_.

Breast cancer is one of the most frequently diagnosed tumors in the United States that affects one in eight women during their lifetime. It is second only to lung cancer as cause of cancer death in women. The orphan members of the nuclear receptor superfamily, estrogen-related receptors ERRα and γ (ERRs) isoforms are considered the master regulators of energy metabolism and their transcriptional pathways are highly associated with the cancer phenotype. Realizing the important function of co-regulators on ERRs transcriptional activity and their impact on cancer development and progression, we have identified the tumor growth family member 4 (ING4), a protein that has been implicated in several solid tumors and physically interacts with ERRα in breast cancer cellular models and modulates its transcriptional activity. Examination of the Oncomine database revealed that both ING4 and ERRα gene expression were significantly higher in invasive ductal and lobular breast carcinomas compared to normal ones, and their increased expression is inversely correlated with the overall patient survival rate in The Cancer Genome Atlas (TCGA) database. Knocking down ING4 in a breast cancer cell line using siRNA technology significantly decreased the expression of ERRα, and overexpression of ING4 upregulates the endogenous expression of ERRα. Well-defined ERRα target genes were also regulated by ING4 in the same manner as ERRα. Also, depleting ING4 expression significantly increased breast cancer cell migration in in vitro scratch assays, while both ING4 and ERRα knockdown decreased breast cancer cell proliferation. Collectively, these findings provide evidence for positive regulation of ERRα by ING4. Understanding the mechanism by which ING4/ERRα interaction regulates cellular pathways in breast cancer, underscores the impact of such regulation on biological and pharmacological actions. Thus, they can be used for the development of improved novel pharmaceuticals that target ERRα/ING4 complex or its downstream pathways.

#5254

CARMIL3 promotes tumor progression in triple negative breast cancer: A possible novel biomarker.

Jeeyeon Lee, Dong Won Baek, Soo Jung Lee, Jae-Hwan Jeong, In Hee Lee, Jin Hyang Jung, Ho Yong Park, Yee Soo Chae, Jieun Kang. _Kyungpook National University Hospital, Daegu, Republic of Korea_.

Purpose: CARMIL3 (LRRC16B, capping protein regulator and myosin 1 linker 3) was previously identified as a novel oncofetal-like gene. It influences cell proliferation, cell cycle control and transformation, possibly mediated by cyclin B1. This study aimed to investigate the function of CARMIL3 in triple negative breast cancer (TNBC) cells and tissues.

Methods: CARMIL3 mRNA was measured using a qRT-PCR in breast cancer cells (MDA-MB-231, MCF7, SK-BR3 and T-47D) and tissues from 20 patients with TNBC. The effects of CARMIL3 on cell proliferation, migration, and invasion were determined using MTT, wound healing, and Matrigel Transwell assays.

Results: Results showed that, the CARMIL3 mRNA expressions were significantly increased in breast cancer cell lines, especially in triple negative breast cancer cell lines (MDA-MB-231 and MDA-MB-468) when compared to normal breast epithelial cells (MCF10A). Downregulation of CARMIL3 inhibited the cell proliferation, migration and invasion of MDA-MB-231. Furthermore, high CARMIL3 mRNA expression was found in 20 TNBC specimens than normal controls (p<0.0001).

Conclusions: In conclusion, CARMIL3 expression in TNBC affects cancer progression, which suggests that CARMIL3 can be a new biomarker for patients with TNBC.

#5255

Breast cancer subtype classification using a multi-gene expression profile.

Sara Ravaioli,1 Francesca Pirini,1 Andrea Rocca,1 Maurizio Puccetti,2 Massimiliano Bonafè,1 Giovanni Martinelli,1 Sara Bravaccini1. 1 _Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy;_ 2 _AUSL Imola, Imola, Italy_.

Background: Immunohistochemistry (IHC) is the method conventionally used in clinical practice to define breast cancer (BC) subtypes. This technique is subjective and semi-quantitative. Molecular approaches like multigene panels are increasingly used in clinical practice for their reliability and accuracy. This preliminary study was performed to assess the applicability of the NanoString Breast Cancer 360™ panel, which analyze 770 transcripts of genes involved in a number of signatures useful to define the molecular subtype. We evaluated the correlation between the gene expression levels with the IHC values of conventional biomarkers on 12 BC formalin-fixed paraffin-embedded (FFPE) samples.

Methods: Immunostaining was performed by using Ventana Benchmark Ultra system. Anti-ER, -PR, -Ki67 and -HER2 Ventana antibodies were used. Tumors were classified according to the most recent St. Gallen classification. RNA was isolated from FFPE tumor with AllPrep DNA/RNA FFPE Kit and quantified by Nanodrop. The Breast Cancer 360™ panel assay was performed according to manufacturer's instructions. The normalized count for each gene analyzed using nSolver analysis software was compared with the respective IHC biomarker status by Mann-Whitney test to assess the correlation between the two methods. The mean mRNA count was compared between luminal and triple negative BC by unpaired T test. All statistical analyses were performed by Graphpad Prism software 5.

Results: The 12 BC samples were classified in 3 luminal A, 3 luminal B (HER2+) and 6 triple negative tumors by IHC. The expression level of ESR1, PGR, MKI67 and ERBB2 genes showed a statistically significant correlation with the corresponding protein expression (p=0.0022, p=0.0054, p=0.0025 and p=0.0091, respectively).

A significant difference in the expression levels of ERBB2 gene between HER2+ and HER2- BC was observed (mean mRNA counts 16848 vs 1307, p=0,035). Furthermore, ESR1 expression was significantly higher in ER+ than in ER- BC (mean mRNA counts 9105 vs 195, p=0,021). We also evaluated the differential expression between luminal and triple negative BC for AR, EGFR, HIF1A, MKI67, MYC, NOTCH1, RAD51 genes. The expression levels for all these genes were significantly higher in triple negative cases than luminal BC, except for AR that was higher in luminal tumors (p<0.05).

Conclusions: In this study Breast Cancer 360™ panel showed high concordance with IHC data. We are aware that our preliminary results require validation in a larger case series to establish the real value of this multi-gene expression profile in BC. However, our results highlight the potential of this molecular approach to properly identify tumor subtypes, allowing a better management of BC patients.

#5256

Molecular signatures associated with obesity-related tumor growth in breast cancer women.

Diddier Prada, Cristian Arriaga-Canon, Jose Diaz-Chavez, Carlo Cortes, Marco Andonegui-Elguera, Pedro Perez-Collado, Rodolfo Muniz, David Cantu-de-Leon, Paula Cabrera-Galeana, Enrique Bargallo, Luis Herrera, Fernando Penaloza. _Instituto Nacional de Cancerologia, Mexico City, Mexico_.

Obesity has been widely associated with chronic diseases, including cancer. Obesity increases the risk of developing several types of cancer, both in women and men, especially breast cancer in postmenopausal women. However, the role of obesity after tumors is established and its potential mechanisms are unknown. Our study aims to determine the molecular signatures associated with obesity-related tumor growth in a cohort of breast cancer patients from a country with an obesity epidemic. We evaluated a cohort of patients with breast cancer (n=151) at diagnosis, including patients in all clinical stages and 78% of them showed overweight or obesity. We determined the role of body mass index (BMI) on clinical characteristics and, using RNA-seq analyses in biopsy samples at diagnosis, we determined potential genes associated. We found a positive correlation between BMI and tumor size at diagnosis (r = 0.147, p-value = 0.006) and age (r=0.116, p-value=0.03). Lower BMI was observed when the number of positive nodes increased (p-value = 0.015). In a random subset from these patients (n=12) and using robust linear models, we determined that 10 genes (GPRC5B, SLITRK6, KRT23, VTCN1, TNFRSF12, PTP4A2, BACE2, EPH8, CYP21A1P, and TMEM52) were positively associated with BMI. CSTA and IGKV1-16 showed a negative association with BMI. Gene ontology analyses revealed that VTCN1 and PTP4A3 were linked to increased proliferation and tumor progression. CSTA, a protease inhibitor, was also associated with poor prognosis. Our study suggested a deleterious effect of obesity on breast cancer patients, which could be caused by changes in cell expression on key genes in tumor cells.

#5257

Activation of the oncogene ERG by the Ras/ERK and PI3K/AKT pathways.

Brady G. Strittmatter, Peter Hollenhorst. _Indiana University- Bloomington, Bloomington, IN_.

The TMPRSS2-ERG re-arrangement occurs in ~50% of prostate cancers and results in androgen driven aberrant expression of the transcription factor ERG. ERG acts as an oncogene in the prostate and activates a transcriptional program which results in cell migration, epithelial to mesenchymal transition, and tumorigenesis. Activation of both the Ras/ERK and PI3K/AKT signaling pathways has been demonstrated to be necessary for transcription of ERG target genes and is required for ERG mediated phenotypes in the prostate, however, the exact mechanism of this requirement is not known. Recently, our lab demonstrated that ERK1/2 phosphorylation of ERG abrogates ERG's interaction with the co-repressor EZH2 and the PRC2 and is necessary for ERG-mediated transcription of genes involved in cell migration. It has been demonstrated that ERG also requires activation of the PI3K/AKT pathway for ERG-mediated tumorigenesis in the prostate, however how the Ras/ERK and PI3K/AKT pathways cooperate in transcription of ERG target genes and ERG mediated phenotypes is not well understood. Here we use genomic and transcriptomic approaches to demonstrate that Ras/ERK and PI3K/AKT pathway requirements for ERG function differ between distinct ERG-mediated mediated gene expression programs which correlate with distinct phenotypic outputs. Overall, our results demonstrate how the Ras/ERK and PI3K/AKT pathways contribute to ERG mediated tumorigenesis in the prostate and point the way towards new therapeutic targets. 

### Tracking Metabolic Profiles and Subtype-specific Metabolic Interventions

#5258

1 **H MRS analysis of pancreas metabolites altered by cachexia.**

Raj Kumar Sharma, Santosh K. Bharti, Paul T. Winnard Jr, Yelena Mironchik, Marie-France Penet, Zaver M. Bhujwalla. _The Johns Hopkins University School of Medicine, Baltimore, MD_.

Cachexia is a multifactorial syndrome characterized by skeletal muscle weight loss and reduced physical activity. The extreme weight loss due to cachexia results in a particularly poor quality of life causing profound weakness, listlessness, and inability to function [1, 2]. Severe weight loss decreases the tolerance to treatments and anticancer therapies, and leads to the reduced survival of patients. In pancreatic cancer, the syndrome affects nearly 80% of patients [3]. Cancers can stimulate cachexia through the dysfunction of multiple organs [4]. Metabolic abnormalities may interfere with the regular functioning of these organs to increase the severity of the disease. Here we determined metabolic changes in the pancreas with cachexia-inducing and non-cachexia inducing tumor growth using high-resolution quantitative 1H magnetic resonance spectroscopy (MRS) of pancreas tissue obtained from normal, non-cachectic (Panc1) and cachectic (Pa04C) mice bearing pancreatic ductal adenocarcinoma (PDAC) xenografts.

Cancer cells were inoculated in the right flank of six to eight week old male severe combined immunodeficient mice. The pancreas was removed from mice following euthanization once tumors were ~300 mm3, snap frozen and stored at -80°C prior to dual phase extraction. 1H MRS was performed on the water phase. All 1H MR spectra with water suppression were acquired on a 750 MHz MR spectrometer using a single pulse sequence. All data processing analyses and quantification were performed with TOPSPIN 3.5 software. All statistical analysis were performed with MetaboAnalyst software [5].

Multivariate analyses performed to analyze the differences in the metabolic profiles among the groups (Control n = 9, Pa04C = 10 and Panc1 = 8) revealed differences in the overall metabolic pattern in the pancreas from normal, cachectic and non-cachectic groups. A significant decrease of leucine, isoleucine, valine, BCAA, glutamate, choline, pyruvate, glycine, niacinamide, fumarate and increase of alanine, pyruvate, and NAD was detected in cachectic mouse pancreas compared to control pancreas. A significant increase in alanine, phenylalanine, pyruvate, phosphocholine and decrease in glycine, NAD, niacinamide was observed in cachectic mouse pancreas compared to non-cachectic mouse pancreas. Pathway impact analysis using MetaboAnalyst web software indicated alterations in branch chain amino acid pathways and glutamate, glutamine metabolism. These results provide new insights into changes in pancreas metabolism with cachexia, and support investigating metabolic targets and biomarkers to reduce cachexia-associated morbidity.

Supported by NIH R01CA193365 and R35CA209960.

Ref: 1. Fearon K et al, The Lancet Oncology. 2011; 2. Penet MF, Bhujwalla ZM. Cancer journal. 2015; 3. Winnard PT, Jr. et al, Cancer research. 2016; 4. Argiles JM et al, Nature reviews Cancer. 2014; 5. Xia J, Wishart DS. Nature protocols. 2011.

#5259

Fatty acid oxidation represents a metabolic vulnerability in glioblastoma in nutrient-deprived conditions.

Shiva Kant, Antony Dayalan, Pravin Kesarwani, Prakash Chinnaiyan. _Beaumont Health, Royal Oak, MI_.

Glioblastoma represents an aggressive, primary brain tumor with limited treatment options. We performed an integrative, cross-platform analysis coupling global metabolomics and gene expression profiling in >100 patient-derived gliomas to begin to understand the diverse metabolic programs driving the aggressive phenotype of this malignancy. Alterations in fatty acid β-oxidation (FAO) emerged as a key metabolic node differentiating glioblastoma from low-grade astrocytoma, as demonstrated by an accumulation of acylcarnitines. Metabolic heterogeneity was observed within glioblastoma that could further define tumors as FAO 'high' and 'low'. Integrative analyses identified these metabolic subtypes to be enriched with mesenchymal (MES) and proneural (PN) glioblastoma subtypes, respectively. These findings were metabolomically and functionally recapitulated in molecular subtype-specific preclinical models. Analysis of gene expression profiles from these lines uncovered an orchestrated transcriptional program designed to promote fatty acid uptake, activation, and mitochondrial oxidation. Studies designed to determine the biologic consequence of enhanced FAO in glioblastoma only identified a role in glucose-deprived conditions, where it served as a vital, alternate source for ATP synthesis. Based on these findings, we tested the hypothesis that dual targeting of glycolysis and FAO would elicit energetic stress-mediated cell death in glioblastoma. Accordingly, the glycolysis inhibitor 2DG and the FAO inhibitor etomoxir only demonstrated anti-proliferative activity in MES glioblastoma cell lines when used as single agents in vitro, while the combination resulted in robust, necroptosis-mediated cell death. Synergistic anti-tumor activity was observed following combined glycolysis and FAO inhibition when extended in vivo in an orthotopic model. Collectively, our findings suggest FAO provides metabolic plasticity in MES glioblastoma, allowing these cells to adapt to nutrient-deprived microenvironments. Combinatorial strategies designed to inhibit glycolysis and FAO represents an attractive therapeutic approach in glioblastoma.

#5260

Different cell metabolism by FAP-α overexpression in triple negative breast cancer cells.

Noriko Mori, Balaji Krishnamachary, Jiefu Jin, James Barnett, Yelena Mironchik, Zaver M. Bhujwalla. _Division of Cancer Imaging Research, The Russell H. Morgan Department of Radiology and Radiological Science, The Johns Hopkins University School of Medicine, Baltimore, MD_.

Fibroblast activation protein-α (FAP-α) is a cell surface serine protease that is attracting increasing interest because of its role in immune suppression and cancer metastasis. To understand the role of FAP-α in cancer cell metabolism we engineered triple negative MDA-MB-231 cells to stably overexpress FAP-α and compared metabolic profiles of parental and FAP-α overexpressing cells using 1H MR spectroscopy. We observed a significant increase of aspartate, glycerophosphocholine, lactate, myoinositol and phosphatidylcholine with FAP-α overexpression. These results identify a previously unknown role of FAP-α in modifying cancer cell metabolism that may provide new insights in its functional roles in cancer progression.

MDA-231luc (parent cells), derived from MDA-MB-231, were purchased from Sibtech Inc. MDA-231luc-FAPα (231-FAPα) cells were engineered using the gene for human FAP that was subcloned into lentiviral vector pMA3211. Immunoblot analysis with FAP-α antibody confirmed the overexpression of FAP-α protein in 231-FAPα cells. Approximately 3.5x107 cells were used for dual-phase cell extraction as previously described [1]. 1H MR spectra were recorded on a Bruker Biospin Avance-III 750 MHz NMR spectrometer and analyzed using TOPSPIN 3.5 software. Integrals of the metabolites of interest were determined and normalized to the number of cells and internal standard. Metabolite levels were quantified as arbitrary units (A.U.) from three experimental samples from each cell line. Statistical significance was evaluated using the unpaired one tailed Student t test.

We identified 18 metabolites from the aqueous phase (8 amino acids, 3 choline metabolites and 7 others) and 10 signals from the lipid phase (7 Fatty acids (FA), Cholesterol, Phosphatidylethanolamine (PtdE), -N(CH3)3: Phosphatidylcholine (PtdCho) & Sphingomyelin (SM)) using 1H MR spectra. We found that 4 metabolites (aspartate, glycerophosphocholine (GPC), lactate and myo-inositol) from the aqueous phase and 2 signals (FA: -CH2-CH2-CH=, and -N(CH3)3) from the lipid phase were significantly higher in 231-FAPα cells. It is likely that PtdCho level was significantly higher in 231-FAPα cells since the majority of -N(CH3)3 signals are from PtdCho. All the FA signals tended to be higher in 231-FAPα cells compared to parental cells. Since the average cell numbers at the time of cell collection were similar, cell density differences did not contribute to differences in metabolite levels. We are currently investigating the molecular mechanisms underlying these differences. The significant differences in water-soluble and lipid-soluble extracts provide new insights into the metabolic modulatory role of FAP-α.

[1] Krishnamachary B, et al. Cancer Res. 2009;15;69(8):3464-71

This work was supported by NIH R35CA209960.

#5261

Choline kinase downregulation decreases prostate cancer associated fibroblast viability.

Jesus Pacheco-Torres, Tariq Shah, Flonne Wildes, Balaji Krishnamachary, Zaver M. Bhujwalla. _The Johns Hopkins University School of Medicine, Baltimore, MD_.

Introduction

We previously observed an increase of prostate CAFs (PCAFs) in prostate cancers that metastasize, consistent with the known role of fibroblasts in inducing growth, confer castration-resistance and increasing metastatic potential. While aberrant choline metabolism has been identified in prostate cancer cells, choline metabolism in fibroblasts and PCAFs is unexplored and may identify a treatment strategy through downregulation of enzymes such as choline kinase alpha (Chk-α), the enzyme that converts choline to phosphocholine (PC). Chk-α is typically increased in cancer cells. Here we characterized the metabolic profile of normal prostate fibroblasts and PCAFs as well as determined the effect of Chk-α downregulation with small interfering RNA (siRNA).

Methods

Experiments were performed using the human prostate myofibroblasts (WPMY-1, derived from stromal cells from the peripheral zone of the histologically normal adult prostate) and PCAFs (obtained from an adenocarcinoma of the prostate gland). Cell extracts were obtained using a dual-phase extraction method. High-resolution 1H MR spectra were recorded on a Bruker Biospin Avance-III 750 MHz NMR (Bruker Biospin) spectrometer operating at a 1H frequency of 750.21 MHz using a 5-mm broad band inverse (BBI) probe head equipped with z-gradient accessories. 1H MR spectra were manually phased and automated baseline corrected using TOPSPIN 3.2 software. Integrals of the metabolites of interest were determined and normalized to the TSP reference and the number of cells. Metabolites were estimated from at least three experimental samples. Statistical significance was evaluated using the Student's t-test. To target Chk-α, cells were transiently transfected with small interfering RNA (siRNA) against Chk-α at a concentration of 100 nM for 48h using standard protocol. Total RNA was isolated, complementary cDNA synthesized and quantitative real-time PCR performed using IQ SYBR Green supermix and gene specific primers. Viability studies were performed using CCK8 assay as previously reported.

Results and Discussion

Metabolic profiles were significantly different between WPMY-1 and PCAFs, with significant differences in choline, phosphocholine, glutamate and alanine, among others. siRNA directed against Chk-α reduced mRNA levels in both WPMY-1 and PCAFs. We observed a significant decrease of cell viability in PCAFs but not in normal prostate fibroblasts. These data support investigating Chk-α as a target to eliminate CAFs in tumors. Increased glutamate observed in PCAFs also support targeting enzymes and transporters in glutamine/glutamate metabolism as potential therapeutic strategies against PCAFs. Future studies with CAFs from different cancers should further validate the metabolic differences between normal fibroblasts and CAFs identified in this study.

Supported by NIH R35CA209960. JPT is supported by Martin-Escudero Foundation.

#5262

Lipidomic profiling of neuroblastoma cells after treatment with novel withalongolide A 4, 19, 27-triacetate (WGA-TA).

Chitra Subramanian,1 Rajendiran Thekkelnaycke,1 Tanu Soni,1 Valerie P. Opipari,1 Barbara N. Timmermann,2 Mark S. Cohen1. 1 _Univ. of Michigan, Ann Arbor, MI;_ 2 _Univ. of Kansas, Kansas, KS_.

Introduction: Neuroblastoma (NB) is a pediatric solid tumor that affects approximately 700 children every year in the United States. Despite advances in treatment, the cure rate for high risk advanced NB having N-myc amplification has a poor response of 40-50%. Standard of care treatment for high risk NB patient consists of surgery, intensive chemotherapy and radiotherapy. Recently, we and others have shown that withanolide treatment of NB induce apoptosis of NB cells, through several mechanism including lipid oxidation, down regulation of N-myc and suppression of Akt/mTOR/NF-κB activation. Therefore, we hypothesized that mass spectrometric analysis of untargeted lipidomic profiling after treatment of NB cells with withanolide WGA-TA will identify novel prognostic and diagnostic biomarkers of NB.

Methods: Validated neuroblastoma cell lines IMR32 (N-myc amplified) and SH-EP (N-myc non-amplified) were grown in 2D culture in appropriate growth medium. Lipids were extracted from cells using modified Bligh and Dyer method. Lipidomic profiling was carried out using LC MS/MS. Principal component analysis (PCA) was used to identify differentially regulated metabolites.

Results: Principal component analysis of NB cell lines and WGA-TA treatment showed complete separation of samples as well as internal controls. Initial analysis of lipid profiling of IMR32 and SH-EP cells showed differential regulation of several lipids. N-myc non-amplified SH-EP cells have upregulation of phospholipids such as phosphocholine (PC), phosphor ethanol amine (PE), sphingomyelin (SM) and free fatty acid, whereas glycerol lipid diacyl glycerol (DG) is down regulated in SH-EP cells compared to IMR32 cells. Treatment of IMR32 cells with WGA-TA led to up regulation of phospho lipids such as PC, phosphoserine (PS), SM, Lyso PE, free fatty acids just like N-myc non-amplified cells. But, triglycerides (TG) were up regulated after treatment with WGA-TA in both IMR32 and SH-EP cells.

Conclusions: WGA-TA treatment of N-myc amplified cells led to upregulation of phospholipids similar to that of N-myc non-amplified cells indicating that CDP-Choline pathway lipids might play an important role in NB. Further analysis is needed to unravel the link between N-myc and lipid metabolism in NB.

#5263

1 **H and** 13 **C MRS-based metabolic markers of IDH1 mutant glioma response to temozolomide therapy.**

Elavarasan Subramani, Chloe Najac, Georgios Batsios, Pavithra Viswanath, Marina Radoul, Anne Marie Gillespie, Russell O. Pieper, Sabrina M. Ronen. _University of California San Francisco, San Francisco, CA_.

Low-grade gliomas, driven by mutations in the isocitrate dehydrogenase 1 (IDH1) gene, are less aggressive than primary glioblastoma (GBM), but nonetheless always recur and ultimately lead to patient death. To improve patient survival, one therapeutic strategy is treatment with the alkylating chemotherapeutic agent Temozolomide (TMZ), previously reserved for the treatment of primary GBM. Though the treatment of IDH1 mutant patients with TMZ improves survival, early detection of treatment-associated effects remains challenging. The goal of this study was, therefore, to determine the value of magnetic resonance spectroscopy (MRS)-based biomarkers for detection of response to treatment. To this end, we examined the global metabolic alterations that occurred following TMZ treatment in a genetically engineered IDH1 mutant immortalized Normal Human Astrocyte (NHAIDHmut)-based cell model using 1H MRS combined with chemometrics and 13C MRS combined with labeling of cells with [1-13C]-glucose and [3-13C]-glutamine. Cells were treated either with the IC50 value of TMZ (100 μM; N=5), or with DMSO (0.2%; N=5) for 72 hours. Then, metabolites were extracted from cells using the dual-phase extraction method. 1H-MRS and proton-decoupled 13C-MRS spectra were acquired using a 500 MHz Bruker Avance spectrometer. 1H MRS data was subjected to multivariate analysis using SIMCA. Specific metabolites were also quantified using Mnova7, integrals normalized to TSP and to cell number and statistical significance of differences determined using unpaired Student's t-test. Pathway enrichment analysis of dysregulated metabolites was performed using MetPA. principal component analysis score plot showed separation of TMZ-treated from DMSO-treated control cells. Further, improved separation between the groups was obtained by orthogonal-partial least squares discriminant analysis. Most significant metabolites contributing to class separation were identified using correlation ≥0.6 and variable importance in projection score ≥1. Glutamine, glutamate, 2-hydroxyglutarate (2-HG), pyruvate, succinate, glucose, phosphocholine, isoleucine, valine, lysine, phenylalanine, NAD(P)+ and AMP/ADP/ATP were observed to be significantly higher in TMZ-treated cells as compared to controls. Based on pathway impact score, tricarboxylic acid (TCA) cycle was identified to be the significantly altered pathway following treatment Further, there was a significant increases in glucose- and glutamine-derived glutamate and 2-HG, indicating an increase in flux to the TCA cycle following treatment. These findings demonstrate that IDH1 mutant glioma show a clear metabolomic fingerprint in response to TMZ therapy, which, combined with complementary hyperpolarized 13C MRS approaches, may help assess early response to TMZ therapy in mutant IDH1 glioma.

#5264

Metabolic reprogramming with β-alanine to overcome chemotherapy resistance in pancreatic cancer.

Danny Yakoub, Smitha T. Totiger, Alexandra Moran, Sujit Suwal, Julio Pimentel, Lauren O'Donell, Jamie Walls, Nagaraj Nagathihalli, Nipun Merchant. _University of Miami-Miller School of Medicine, Miami, FL_.

Background: Increased aerobic glycolysis (Warburg effect) leading to acidic microenvironment confers chemoresistance to weakly basic Gemcitabine, the standard treatment in Pancreatic ductal adenocarcinoma (PDAC). Metabolomic perturbations in two PDAC cell lines having differential sensitivity to Gemcitabine was investigated by profiling over 40 metabolites using 1H-NMR spectroscopy. Notably, a significant decrease in the concentration of β-alanine (βA) was observed in Gem resistant compared to Gem sensitive cell line, suggesting βA may have a role in chemotherapy response. We aimed to evaluate the role of βA supplementation in improving Gem efficacy in Gem resistant cell lines

Methods: PDAC Panc-1 cell line extracts treated with Gem with and without βA supplementation were analysed via 1H-NMR spectroscopy. Aerobic glycolysis was quantified by measuring extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) using the Seahorse XF analyser platform. Global mRNA profiling was done by Next Generation Sequencing. Proliferation, migration and cell cycle analysis was measured by Xcelligence real time cell analysis system and flow cytometry, respectively. PANC-1 and BxPC-3 tumor xenografts treated with Gemcitabine with and without βA for 4 weeks and the tumor volumes and weights were measured.

Results: βA supplementation in Gem resistant cells results in: (1) decreased basal glycolytic rate, proton leak and ATP production; (2) increased pyruvate, decreased lactic acid and NAD+ concentrations with decreased LDH-A transcription; also increased carnosine levels with a more basic pH in the conditioned media; (3) enhanced sensitivity to Gem treatment, with a significant reduction in proliferation, cell migration, and increased apoptosis and relative hENt-1 mRNA levels and (5) xenograft tumor volumes and weights were significantly reduced upon βA supplementation.

Conclusion: βA supplementation reprograms cell metabolism by normalizing energy production and reducing microenvironment acidification, thus enhancing chemo-sensitivity in Gem resistant PDAC cells and xenografts. βA can be potentially used as a co-therapeutic in PDAC.

#5265

Spleen metabolism altered by human pancreatic cancer xenografts.

Santosh Kumar Bharti, Paul T. Winnard, Raj Kumar Sharma, Yelena Mironchik, Marie-France Penet, Zaver M. Bhujwalla. _The Johns Hopkins University School of Medicine, Baltimore, MD_.

Cancer-induced cachexia accounts for approximately 20% of all cancer deaths [1] and affects nearly 80% of pancreatic cancer patients [2, 3]. Cachectic patients experience a wide range of symptoms affecting several organ functions such as muscle, liver, brain, and heart that decrease quality of life and worsen prognosis. A major characteristic of cachexia is the accelerated skeletal muscle and fat storage wasting causing nutrient mobilization both directly as lipid and amino acids, and indirectly as glucose derived from the exploitation of liver gluconeogenesis that reaches the tumor through the bloodstream [4]. Here, for the first time, we have performed high-resolution quantitative 1H magnetic resonance spectroscopy (MRS) of spleen tissue obtained from normal mice and mice bearing PDAC that are cachectic (Pa04C) and non-cachectic (Panc1).

The Panc1 (non-cachectic), and Pa04C (cachectic) PDAC cell lines were used [5]. Eight-week-old male severe combined immunodeficient mice were inoculated in the right flank with cancer cells (5×106). Mice were euthanized once tumors were ~ 300 mm3. Control, cachectic and non-cachectic groups consisted of 9, 10 and 9 mice per group respectively. Spleen water soluble metabolites were extracted, freeze dried, reconstituted in D2O PBS and transferred to an NMR tube for spectral acquisition with a 750 MHz (17.6T) MR spectrometer [6].

As anticipated, mice with cachexia-inducing Pa04C tumors showed significant weight loss with time. We observed, for the first time, that spleens from cachectic Pa04C tumor bearing mice showed a profound weight loss compared to normal mice and mice with Panc1 tumors. However, an increase of liver and spleen size has been previously reported in terminal cachectic human patients from colorectal cancer measured by CT scan 2-11 months prior to death [7]. A significant decrease in almost all amino acids was observed in cachectic (Pa04C) mouse spleens compared to normal and non-cachectic (Panc1) mouse spleens. Differences in choline metabolites, creatine, glutamine, glutamate, glutathione and aspartate were observed in cachectic mouse spleens compared to non-cachectic mouse spleens and spleens from healthy control mice. The significant decrease of amino acids in the cachectic spleens may reflect increased utilization of amino acids by the tumor or other organs during the cachexia muscle/protein wasting [4]. These results provide new insights into changes in spleen metabolism during cachexia, and support investigating metabolic targets to reduce cachexia associated morbidity.

Supported by NIH R01CA193365 and R35CA209960.

Reference: 1. Argiles et al. Nat. Rev. Cancer 2014, 14 (11), 754; 2. Fearon et al. HPB (Oxford) 2010, 12 (5), 323; 3. Ozola et al. Pancreatology 2015, 15 (1), 19; 4. Porporato et al. Oncogenesis 2016, 5, e200; 5. Winnard et al. Can. Res. 2016, 76(6): 1441; 6. Penet et al. Clin. Can. Res. 2015, 21 (2), 386; 7. Lieffers et al. Am J Clin Nutr. 2009, 89 (4), 1173

#5266

Kinetic measurements of intracellular ATP levels in co-culture models using live-cell analysis.

Cicely L. Schramm, Michael L. Bowe, Laura A. Skerlos, Grigory S. Filonov, Yong X. Chen, Daniel M. Appledorn. _Sartorius, Ann Arbor, MI_.

Differential metabolic requirements of tumor cells have gained recognition as attractive therapeutic targets. For example, clinical trials are currently underway to test the efficacy of the glutaminase-1 (GLS1) inhibitor CB-839 in a variety of indications, including triple-negative breast cancer (TNBC). Standard approaches to monitoring drug induced metabolic perturbations are limited to endpoint assays that lack cell-specific data in complex co-culture models and provide limited kinetic information. Here we describe a live-cell approach to image and analyze intracellular ATP levels of tumor cells in mono- or co-culture over time using the IncuCyte® CytoATP Sensor. A panel of breast cancer cell lines stably expressing a genetically encoded, fluorescent ATP sensor or a control (non-ATP binding) sensor were generated and cultured alone or with a monolayer of stromal cells. The effect of CB-839 treatment on tumor cell ATP levels was monitored over days and analyzed using the IncuCyte® S3 for Metabolism, which is equipped with a specialized optical module and image acquisition mode. In concordance with previous results, TNBC cell lines were more sensitive to GLS1 inhibition than their receptor-positive counterparts. Across all TNBC lines tested, CB-839 treatment resulted in a reduction in intracellular ATP that was sustained for the duration of the 3-day time course. In contrast, most receptor-positive cell lines displayed little to no effect of GLS1 inhibition. In some cases, a transient reduction followed by recovery within 48 hours was observed, highlighting the value of kinetic analysis. Co-culture with CCD-1086SK fibroblasts attenuated the effect of CB-839 in TNBC cell lines. This was not due to drug buffering, as the reduction in ATP induced by CB-839 treatment of MCF7 cells was unaffected by co-culture with stromal cells. Visualization tools included in the IncuCyte® ATP Analysis Software Module provide a quick, qualitative assessment of data across the assay plate and a view into the heterogeneity of responses to treatment. Measurements of proliferation, cell death, and mitochondrial membrane potential provided additional data supporting the observations generated via ATP readout. Overall, these data highlight the value of live-cell, kinetic analysis of metabolic and cell health measurements.

#5267

Unbiased metabolic profiling of atypical teratoid/rhabdoid tumors predicts sensitivity to the glutamine metabolic inhibitor 6-diazo-5-oxo-L-norleucine.

Sabrina Z. Wang,1 Brad Poore,1 Jesse Alt,1 Antoinette Price,1 Sariah Allen,2 Brent Orr,2 Rana Rais,1 Barbara Slusher,1 Charles Eberhart,1 Eric H. Raabe,1 Jeffrey H. Rubens1. 1 _Johns Hopkins School of Medicine, Baltimore, MD;_ 2 _St. Jude Children's Research Hospital, Memphis, TN_.

Atypical teratoid rhabdoid tumors (AT/RT) are deadly tumors of infancy in need of new, targeted therapies. Molecular analyses have revealed 3 epigenetically distinct subgroups of AT/RT. High MYC-expressing tumors are a particularly aggressive subgroup, with a 5-year median survival of 18.5%. MYC is difficult to directly target given large, flat, featureless protein-protein binding sites. We performed the first unbiased metabolic profile of AT/RT cell models by high-performance liquid chromatography-mass spectrometry. This study revealed that high MYC-expressing AT/RT have a unique metabolic profile (Partial Least Squares-Discriminant Analysis). Pathway analysis highlights a dependence on glutamine metabolism in high-MYC expressing AT/RT. High MYC-expressing cell lines grow poorly in glutamine-free media compared to low-MYC cell lines (MTS assay, glutamine-free media vs normal media). Due to this dependence on glutamine metabolism, we hypothesized that high-MYC expressing AT/RT would be especially sensitive to glutamine metabolic inhibitors. We show that the glutamine analogue, 6-diazo-5-oxo-L-norleucine (DON) slows high-MYC expressing AT/RT cell growth, induces high rates of apoptosis, and improves survival in orthotopic mouse models of high-MYC expressing AT/RT (p =0.0027 by log-rank test). In contrast, low-MYC expressing models are relatively insensitive to DON. In uniformly labeled glutamine metabolic analyses of AT/RT cells, DON treatment led to depletion of glutathione levels by preventing the production of glutamate. DON treatment synergized with carboplatin to kill AT/RT cells in vitro and extend the life of mice bearing AT/RT orthotopic xenografts (p< 0.001 by log rank test). We aim to translate these findings into a new clinical trial to improve survival in this deadly disease.

#5268

Outcome prediction of metastatic colorectal cancer patients undergoing liver resection by analyzing serum metabolomics.

Alfredo Budillon, Susan Costantini, Angela Sorice, Francesca Capone, Silvia Marchese, Elena Di Gennaro, Carlo Vitagliano, Fabiana Tatangelo, Alfonso De Stefano, Franco Bianco, Paolo Delrio, Francesco Izzo, Antonio Avallone. _Istituto Nazionale Tumori Fondazione G. Pascale - IRCCS, Napoli, Italy_.

In metastatic colorectal cancer (mCRC) patients (pts), treatment strategies integrating liver resection with more effective therapies offer better 5-year survival rates than palliative chemotherapy alone. However, resectability is established only on clinical-morphovolumetric criteria, liver resection is a complex and costly procedure and relapse occurs in almost 2/3 of pts after potentially curative resection. Therefore, prompt identification of pts at higher risk of recurrence is critical to avoid not-beneficial, expensive procedures. Aberrant metabolism is an emerging hallmark of cancer and recent observations suggest that specific metabolic changes can be used to stratify pts for prognosis and drug-response. We evaluated by 600MHz NMR spectroscopy the metabolomics profiling on sera from 30 mCRC pts, enrolled in the Obelics trial (NCT01718873), which investigated different schedules of bevacizumab in combination with oxaliplatin plus fluoropyrimidines regimens, and subdivided on the basis of outcome, in good (R) vs bad responders (NR) according to PFS: 12 months or longer (R, n = 12) and shorter than 12 months (NR, n = 18). We compared the samples of the two mCRC pts groups, collected at response evaluation when resectability was established in case of appropriate tumor reduction and PCA, sPLS-DA and loading plots evidenced metabolites with statistically different levels between the two sub-groups. ROC curves were performed to identify the cutoff levels of these significant metabolites to be correlated with patient survival. In this way we demonstrated that low levels of 3-hydroxybutyrate and of hydroxyproline as well as high levels of histidine correlated with both poor progression free survival (PFS) and overall survival (OS). Notably, either 3-hydroxybutyrate or histidine are better predictor of both PFS and OS compared to pathologic response on resected metatastases. Lipidomics analysis confirmed clear differences between R and NR pts indicating statistically significant increase of lipids in NR pts, with both higher triglycerides and phospholipids correlating with poor PFS and OS. This latter effects, may reflect, at least in part a non specific inflammatory response; indeed a significant increase of pro-inflammatory cytokines was also demonstrated in NR pts sera by cytokinomics using multiplex ELISA approach. Finally, basal serum metabolomics analysis in both NR and R pts demonstrated that on-treatment evaluation is more informative than pre-treatment evaluation to stratify patients for outcome. Overall, these data suggest that NMR-based metabolomics is a potent and affordable method that could play a role in the prediction of mCRC outcome.

#5269

Discovery and validation of plasma acylcarnitines for the early diagnosis of hepatocellular carcinoma.

Eric Yi-Liang Shen,1 Abellona U,2 Simon Taylor-Robinson,2 Mark Thursz,2 Elaine Holmes,2 Jeremy Nicholson3. 1 _Chang Gung Memorial Hospital, Taoyuan, Taiwan;_ 2 _Imperial College London, London, United Kingdom;_ 3 _Murdoch University, Perth, Australia_.

Introduction:

Hepatocellular carcinoma (HCC) is one of the commonest and the leading cause of cancer death globally. The main cause of this high mortality rate is that patients with HCC are usually diagnosed at late stages since they have only subtle clinical presentations at early stages. This late diagnosis is a result of the requirement of high-end imaging modalities, the use of invasive procedures, and the lack of reliable tumour biomarkers. Metabolomics is the study of metabolites in an organism in response to the changes in health status. The purpose of this study was to identify and validate plasma acylcarnitines as potential diagnostic biomarkers of HCC by using techniques in the field of metabolomics.

Methods:

228 EDTA-plasma samples of healthy controls, cirrhotic patients, or treatment-naïve patients with HCC, collected from Imperial College Healthcare Tissue Bank, were used as a discovery and training set. Patient factors, including basic patient characteristics, aetiologies of HCC, and Child-Pugh scores, and data relating to tumours, such as the multiplicity, the sizes in diameter, the status of macro-vascular invasion, were collected. A targeted assay was adapted to measure concentrations of carnitine and acylcarnitines with different chain length using the TQ-S tandem quadrupole mass spectrometer. Non-parametric univariate analyses were applied to evaluate the statistical difference between HCC and cirrhosis. Logistic regression was preferred to develop diagnostic models. A Gambian cohort of 165 plasma samples was then used as an external validation.

Results:

Among sixteen measured carnitine and acylcarnitines, three acylcarnitines, namely acetylcarnitine, succinylcarnitine, and hexanoylcarnitine, statistically increased in HCC patients comparing to cirrhotic patients. A linear model incorporating these three acylcarnitines and alpha-fetoprotein was then developed. The area under the ROC curve of this multivariable regression model was 0.782 (95% C.I., 0.695-0.870) in the training set. When applied to the validation cohort, the sensitivity and specificity of this model at identifying HCC patients from cirrhosis were 82.2% and 67.8%, respectively.

Conclusion:

This report provides a comprehensive profile of the changes of carnitine and acylcarnitines among HCC, cirrhotic patients and healthy controls, and determines the value of acylcarnitines as diagnostic biomarkers for HCC.

#5270

A potential role of branched chain amino acid metabolism in breast cancer cell invasiveness.

Maxwell B. Colonna, Arthur S. Edison, Shaying Zhao. _University of Georgia, Athens, GA_.

Carcinomas have two broadly distinct modes of invasion: collective and individual. Collective invasion is characterized as groups of cells that migrate while retaining cell-cell contacts. Individual invasion occurs through mechanisms such as the acquiring of mesenchymal-like features by cancer cells. While these different modes of invasion have been well studied at the genomic and transcriptional levels, their metabolic alterations remain much less understood. To address this deficiency, we performed untargeted NMR metabolomics, in conjunction with RNA-seq, on two variants of MCF7, a widely used breast cancer cell line. We report herein several findings. First, the two MCF7 variants, one parental (referred to as MCF-7 hereafter) and another having undergone many passages (referred to as Augusta hereafter), differ morphologically, with MCF-7 being more epithelial and Augusta being more mesenchymal-like. Importantly, fibroblast co-culture experiments indicate that MCF-7 cells resemble collective invasion, while Augusta cells resemble individual invasion. Furthermore, both scratch-assay and gene set enrichment analysis of RNA-seq data indicate that Augusta cells are more invasive. Second, our NMR metabolomics analysis reveals that branched chain amino acids (BCAAs) represent a significant metabolic difference between MCF-7 and Augusta cells. Partial least squares discriminant analysis of the metabolomics data identified BCAAs as essential distinguishing features between the two cell lines. Specifically, compared to MCF-7 cells, BCAAs are significantly depleted inside Augusta cells, consistent with the upregulation of several key BCAA degradation genes. We hypothesize that Augusta cells can more efficiently utilize BCAAs, and are performing 13C-labeled BCAA flux analysis to test this hypothesis. Previous studies have shown that an aberrantly expressed BCAA metabolism gene sustains growth and enhances invasiveness of breast cancer cells. However, increased catabolism of BCAAs has not been reported in literature on this subject. Our simple cell line model allows for a deeper molecular understanding of BCAA metabolism in breast cancer cell invasion.

#5271

Molecular imaging of pyruvate kinase M2 (PKM2) with [18F]DASA-23 detects temozolomide- and tumor treating fields (TTFields)-induced changes in glycolysis in glioblastoma.

Chirag B. Patel, Corinne Beinat, Yuanyang Xie, Edwin Chang, Sanjiv S. Gambhir. _Stanford University School of Medicine, Palo Alto, CA_.

Introduction: Pyruvate kinase M2 (PKM2) is an enzyme that catalyzes the final step in glycolysis, a key step in tumor metabolism and growth, making PKM2 an important marker of cancer glycolytic reprogramming. The 1-((2-fluoro-6-[18F]fluorophenyl)sulfonyl)-4-((4-methoxyphenyl)sulfonyl)piperazine ([18F]DASA-23) radiotracer has been developed to measure the expression of PKM2. Glioblastoma (GBM) is traditionally treated with surgical resection, temozolomide (TMZ) chemotherapy, and radiation therapy. Tumor treating fields (TTFields), the application of alternating electric fields (100-500 kHz, 1-4 V/cm) to tumors, is emerging as the fourth therapeutic modality in GBM. There is an important need to assess early on whether a patient's GBM is responding to a given standard-of-care or experimental therapy. In this study we evaluated the ability of [18F]DASA-23 to detect changes in metabolism in response to standard-of-care (TMZ) and emerging (TTFields) therapies, in cell culture and an orthotopic murine model of human GBM.

Methods: Human U87-MG GBM cells were subjected to either 200 kHz TTFields or the IC50 of TMZ for three or six days (n≥3/condition), followed by evaluation of 30-minute cellular uptake of [18F]DASA-23. In parallel experiments, immunofluorescence for PKM2 was performed to confirm the [18F]DASA-23 uptake results. Finally, U87-MG human GBM cells were orthotopically implanted in nude mice (N=5), and evaluated with 7T small animal magnetic resonance imaging (MRI) and [18F]DASA-23 microPET/CT before and one week post-initiation of vehicle or TMZ therapy.

Results: There was a significant interaction between the treatment (vehicle, TMZ, or TTFields) and treatment duration (3 or 6 days) on PKM2 expression as measured by cellular uptake of [18F]DASA-23 (p=0.005, 2-way ANOVA). Immunofluorescence for PKM2 in TTFields-exposed and unexposed U87-MG cells revealed reduced cell count and less intense PKM2 staining due to TTFields. In the orthotopic mouse model, the tumor/normal brain (T/N) [18F]DASA-23 uptake ratio (unitless) at one week post-treatment was divided by the pre-treatment T/N ratio. The resulting ratio of ratios was significantly smaller in the TMZ cohort (1.0±0.4) compared to in the vehicle cohort (2.2±0.2), p=0.004.

Conclusion: Together, these data suggest the potential for non-invasive assessment of GBM's response to various therapies using [18F]DASA-23, a radiotracer that measures PKM2 expression. Clinical studies are currently being completed to evaluate the diagnostic ability of [18F]DASA-23 in patients with intracranial malignancies and future studies will evaluate the ability of [18F]DASA-23 to predict responders vs. non-responders to therapy in recurrent GBM.

#5272

Sensitive bioluminescent assays for monitoring changes in tumor and immune cell metabolism.

Donna Marie Leippe, Natasha Karassina, Michael P. Valley, Gediminas Vidugiris, James J. Cali, Jolanta Vidugiriene. _Promega Corporation, Madison, WI_.

The tumor microenvironment is complex, containing diverse cell populations with individual nutrient requirements. Tumor and immune cells must respond to the dysregulated and changing conditions of the tumor microenvironment, as well as compete for fuels, to remain functional and viable. As metabolism is critical for proper cell function, e.g. for effective immune cell activity, understanding the metabolic needs of these cells is essential. We have developed a series of bioluminescent metabolite detection assays for monitoring cell metabolism. The assays share a common NAD(P)H bioluminescent detection technology that can be coupled with specific dehydrogenases to monitor key metabolic pathways such as: glycolysis with glucose and lactate detection; glutamine metabolism with glutamine and glutamate measurements; and lipolysis and lipogenesis with glycerol and triglyceride measurements. The assays were developed to have high sensitivity permitting detection of low levels of metabolites (femtomole detection limit), an important feature when samples contain only a few cells or small volumes. The assays can be used in a non-lytic format by analyzing metabolites in samples of collected cell culture medium. In this way changes over time can be monitored and the cells remain intact for additional measurements, such as multiplexing with viability or cytotoxicity assays. We have used these assays to study the metabolic activity of tumor cell lines and T cells. The induction of aerobic glycolysis is an early step in the activation of T cells. Here we present an example of detecting T cell activation in vitro by monitoring glycolysis and quantitating the increase in lactate secretion over time. Activation was observed only in the presence of both glucose and glutamine, confirming the requirements for both fuels and biochemical pathways for activation. In addition to understanding fuel requirements of cells, we also used the assays to follow changes of metabolic pathways in cancer cell lines treated with small molecule compounds. Treatment with such compounds could provide a means of manipulating tumor nutrient usage and metabolite secretion in the tumor microenvironment. In our experiments, metabolite levels were found to be altered by effectors of glycolysis, oxidative phosphorylation and glutamine metabolism. Multiplexing with viability and cytotoxicity assays provided information about cell health during the experiments.

#5273

Multi-omics approaches reveal potential role for corticosterone in prostate cancer.

Iqbal Mahmud,1 Bongyong Lee,2 Ranjan Perera,2 Timothy J. Garrett1. 1 _Univ. of Florida, Gainesville, FL;_ 2 _Johns Hopkins University School of Medicine, Baltimore, MD_.

Prostate cancer (PCa) is the most commonly diagnosed cancer in American men. In 2018, 164,690 new cancer cases and 29,430 cancer deaths are projected to occur in the United States. Currently, there is no efficient diagnostic and safe treatment option for the management of PCa. Using Ultra-High Performance Liquid Chromatography coupled High Resolution Mass Spectrometry (UHPLC-HRMS), we globally profiled more than 1400 metabolites across 79 urine samples related to PCa (19 control, 23 benign prostate hyperplasia (BPH), 11 prostatitis, and 26 PCa samples). Among all, corticosterone was identified as a top differential metabolite. Corticosterone levels were markedly elevated in PCa urine samples and modest but significant elevation of the corticosterone in BPH and prostatitis urine samples compared to controls, with an excellent area under the receiver operating characteristic curve of 0.95, 0.94, and 0.93, respectively. In addition, the expression of corticosterone biosynthetic gene, Steroid 11-beta-hydroxylase (CYP11B1), was elevated in PCa tissues, cell lines, and urine samples, while Corticosteroid 11-beta-dehydrogenase isozyme 2 (HSD11B2), which metabolize corticosterone, were markedly reduced in prostate tumors. With the understanding that androgen signaling is the key factor for prostate cancer, we investigated the role of androgen in regulating CYP11B1 and HSD11B2. We analyzed several androgen treated PCa cell lines derived global gene expression datasets and revealed that treatment with androgen increase in CYP11B1 expression and a concomitant decrease in HSD11B2 expression levels. Interestingly, we uncover that genetic knockdown of Androgen Receptor (AR) diminished CYP11B1 expression and alternatively, increased HSD11B2. Moreover, AR based chromatin occupancy (ChIP-seq) data also suggest direct binding of AR to the promoter of CYP11B1 in multiple PCa cells. The role of corticosterone in PCa is previously unknown. Our multi-omics approaches thus indicate that corticosterone might be potential prognostic marker for PCa. The tumor biology and mechanism through which corticosterone associates with PCa is being investigated in our laboratory.

#5274

Multi-omics integration analysis robustly predicts high grade patient survival and identified altered fatty acid metabolism in high grade bladder cancer.

Venkata Rao Vantaku,1 Jianrong Dong,1 Chandrashekar R Ambati,1 Dimuthu Perera,1 Sri Ramya Donepudi,1 Chandra Sekhar Amara,1 Vasanta Putluri,1 Ravi Shiva Shankar,1 Matthew J Robertson,1 Danthasinghe Waduge Badrajee Piyarathna,1 Mariana Villanueva,1 Friedrich-Carl von Rundstedt,2 Balasubramanyam Karanam,3 Leomar Y Ballester,4 Martha K. Terris,5 Seth P. Lerner,1 Apolo B Andrea,6 Hugo Villanueva,1 Andrew Sikora,1 Yair Lotan,7 Arun Sreekumar,1 Cristian Coarfa,1 Nagireddy Putluri1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _Universitätsklinikum Jena, Germany;_ 3 _Tuskegee University, Augusta, GA;_ 4 _University of Texas Health Science Center, Houston, TX;_ 5 _Augusta University, GA;_ 6 _National Institutes of Health, Bethesda, MD;_ 7 _UTSouthwestern, Dallas, TX_.

Cancer metabolism varies depending on tumor grade. Currently, there is an absence of integration of multi-omics data to predict Bladder cancer (BLCA) survival. We aimed to identify a metabolic signature in high-grade BLCA by integration of unbiased metabolomics, lipidomics and transcriptomics to predict patient survival and to discover novel therapeutic targets. In the current study, using a robust mass spectrometry platform, we conducted global unbiased metabolic and lipid profiling analysis. We identified 518 differential metabolites and 19 lipids of various classes using the NIST MS metabolomics compendium and lipidblast MS/MS libraries, respectively, between low-grade and high-grade BLCA then mapped them to associated genes. Pathway analysis revealed a unique set of biochemical pathways highly deregulated in high-grade BLCA. Integromics analysis identified a molecular gene signature associated with patient poor survival. Importantly, low expression of fatty acid associated enzymes in high-grade tumors was associated with low fatty acid β- oxidation and acyl carnitines in high-grade BLCA and confirmed using tissue microarray. Our key finding is that impaired fatty acid β-oxidation (FAO) by downregulation of fatty acid associate enzymes suggests a crucial role in the progression of low grade to high grade BLCA. Using a metabolic-centered multi-omics based integrative analysis provided a system-level perspective of BLCA that would facilitate the development of novel therapeutics for high-grade BLCA.

#5275

Cancer specific caloric restriction using novel small molecule improves the therapeutic regime for triple negative breast cancer.

Dhanir Tailor, Angel Resendez, Vineet Kumar, Catherine Going, Sharon Pitteri, Sanjay Malhotra. _Stanford School of Medicine, Palo Alto, CA_.

Hyperglycemic and hyper insulin condition are the signature symptoms for type two diabetes which is the most favorable condition for the development of cancer. Women having a type two diabetes has 20-27% high risk to develop breast cancer including triple negative breast cancer (TNBC). Microenvironment including hyperglycemic, hyper insulin condition leads to the therapy resistance. According to 'Warburg's effect' cancer cells consume a high amount of glucose in comparison to normal cells via aerobic glycolysis process. To take care of this condition currently, several clinical studies are going on for the combination therapy of type 2 diabetes and chemotherapy drugs together including erlotinib and metformin. The major drawback of this kind of type 2 diabetes drugs is that they won't affect cancer cells under hyperglycemic and hyper insulin conditions. These drugs including metformin work via AMP-activated protein kinase (AMPK) activation to switch aerobic glycolysis to oxidative phosphorylation. We have developed a novel AMPK activator (SU-18) who selectively induces an oxidative phosphorylation in TNBC cells and glycolysis in normal cells. We found that SU-18 induces G2/M phase arrest, mitochondrial stress and apoptosis in TNBC cells as same as metformin but at 1000 fold less concentration. It selectively reduces the glucose consumption, lactate and ATP production in TNBC cells where normal cells act opposite to this. SU-18 activity is through AMPK activation which got abolished in presence of AMPK inhibitor (Compound c). During mouse xenograft studies, it inhibits the tumor growth and progression. It also improves the blood glucose level in hyperglycemic mice. Comparative studies with metformin suggest that SU-18 works irrespective of hyperglycemia and insulin conditions, whereas metformin, is ineffective in this condition. FDG-PET scan analysis also supported to our in vitro glucose consumption data that treatment with SU-18 reduces the FDG accumulation in tumor cells. This study advocates the clinical candidature of SU-18 for TNBC patients.

#5276

Enhancement of cisplatin activity against triple negative breast cancer cells by atorvastatin.

Diandra Zipinotti dos Santos,1 Isabella dos Santos Guimarães,2 Nayara Gusmão Tessarollo,1 Taciane Barbosa Henriques Barbosa Henriques,2 Paulo Cilas Lyra Junior,1 Marcele L L. de Souza,1 Maria C. Gomes,3 Ian Victor Silva,1 Leticia B. A. Rangel2. 1 _Federal Univ. of Espirito Santo, Vila Velha, Brazil;_ 2 _Federal Univ. of Espirito Santo, Vitoria, Brazil;_ 3 _Federal Univ. of Espirito Santo, Brazil_.

Breast cancer (BC) is the second most common kind of cancer in the world and the most common among women. Its frequency rate is becoming alarming, being this way a major challenge to global health. It is worth mentioning that the lack of effective therapeutic options, in particular for certain subtypes, such as triple-negative tumor, still present a challenge for clinicians who often have to resort to highly unspecific cytotoxic therapies. Unfortunately, most patients relapse after a period of remission and eventually tumors becomes refractory to frontline therapy. This makes it desirable to identify drugs which synergize with chemotherapeutics and potentially reduce the dose of them that is necessary to treat patients. In the current financial and economical context, drug repositioning is gaining increasing interest as an alternative strategy for drug development. Cancer cells frequently exhibit specific alterations in their metabolic activity. Thus, targeting lipid metabolism in cancer cells could have therapeutic benefits and it does appear as promising auxiliary strategies in the fight against cancer. The present work aimed to investigate the effect of atorvastatin (ATV) on paclitaxel (PTX), cisplatin (CDDP) and olaparib (OLAP) cytotoxic on human BC cell lines MDA-MB-231 and MCF-7, and the mechanisms by which this interaction occurs. ATV decreased cellular metabolic viability (CMV; MTT assay) in a dose-time-dependent manner, especially in MDA-MB-231 being the maximum effect observed in with ATV 100µM (- 90%; p<0.001). In addition, ATV 5µM potentiates PTX and CDDP activities in MDA-MB-231, but not in MCF-7. Moreover, ATV had synergistic inhibitory effects with OLAP decreasing cellular metabolic viability in MDA-MB-231 (- 90%; p<0.001). . Cell cycle distribution analyzed by flow cytometry indicated that coadministration of ATV and CDDP in MDAM-MB-231 result in cell cycle arrest at G0/G1 phase as compared with that of the control and individual drug-treated cells. Altogether, our data suggest that association of ATV with conventional antineoplastic drugs seems to be beneficial, especially when associated with CISP for the MDA-MB-231 lineage. In any event, our study opens a new avenue in the fight against BC while possibly introducing a low cost substance in the portfolio of anticancer drugs and offering a promising strategy to increase the efficacy of them in breast cancer.

#5277

Glutamine antagonists synergize with L-asparaginase in MYCN-driven rhabdomyosarcoma.

Micah J. Maxwell,1 Brad A. Poore,2 Allison Hanaford,1 Jesse Alt,1 Rana Rais,1 Barbara S. Slusher,1 Charles G. Eberhart,1 Eric H. Raabe1. 1 _The Johns Hopkins University School of Medicine, Baltimore, MD;_ 2 _The University of Pittsburgh School of Medicine, Pittsburgh, PA_.

Rhabdomyosarcoma is the most common soft tissue sarcoma in children. Chromosomal translocations producing fusion transcription factors (PAX-FOXO1) are associated with poor prognosis. A subset of fusion-positive rhabdomyosarcomas have downstream activation of the transcription factor MYCN, a known oncogenic driver. Attempts to inhibit directly either PAX-FOXO1 or MYCN have been unsuccessful. MYCN is known to drive tumor cell reliance on glutamine metabolism. 6-diazo-5-oxo-L-norleucine (DON) is a well-characterized glutamine analog that irreversibly inhibits glutamine-utilizing enzymes. DON was well tolerated in a phase I pediatric clinical trial, but it has never been systematically tested in rhabdomyosarcoma. We show that high MYCN confers sensitivity to DON therapy in vitro. We have also developed a targeted glutamine antagonist compound, JHU083, which is similarly effective against MYCN-driven rhabdomyosarcoma cell lines. Furthermore, DON administered by intraperitoneal injection twice weekly significantly reduces flank tumor volume in a murine xenograft MYCN-driven rhabdomyosarcoma model (mean tumor volume 757 mm3 vs. 2167 mm3 in control animals, p-value 0.0000197 by t-test). In stable isotope resolved metabolomics experiments, DON primarily prevents asparagine synthesis, depleting intracellular asparagine levels by at least 50% in MYCN-driven cell lines (p-value = 0.0003). Finally, DON combined with L-asparaginase synergistically inhibits growth of MYCN-driven rhabdomyosarcoma cell lines (CI << 1 by the Chou-Talalay method, indicating strong synergy; p-value = 0.0001). We conclude that DON depletes cellular pools of asparagine, and combination therapy with DON and L-asparaginase synergistically inhibits the growth of MYCN-driven rhabdomyosarcoma. These studies provide the preclinical justification for potential clinical trials for the use DON or DON prodrugs in combination with L-asparaginase as new therapeutic options for patients with MYCN-driven rhabdomyosarcoma.

#5278

Separate and combined effects of metabolic reprogramming interventions, autophagy inhibition, and carboplatin on murine triple-negative breast cancer cells.

Alyssa J. Cozzo, Michael F. Coleman, Ciara H. O'Flanagan, Jane B. Pearce, Magdalena A. Rainey, Stephen D. Hursting. _UNC-Chapel Hill, Chapel Hill, NC_.

Triple-negative breast cancers (TNBC) are an aggressive BC subset currently lacking targeted therapies. TNBCs exhibit aberrant fuel metabolism, including increased flux through glycolysis and the pentose phosphate pathway, increased lactate production (Warburg metabolism), and elevated dependence on glutamine (glutaminolysis). While dietary interventions such as caloric restriction, intermittent fasting, or ketogenic diets have demonstrated benefits (e.g, reduced tumor incidence, progression, and sensitization to some chemotherapeutic agents) in preclinical BC models, translation to humans has proven challenging, and concerns remain regarding tolerance and long-term safety of extreme diets in cancer patients. We hypothesized that targeted disruption of select metabolic pathways could recapitulate the beneficial effects of the aforementioned dietary interventions and improve the efficacy of carboplatin, part of first-line chemotherapy for primary and metastatic TNBCs. Moreover, we posited that blocking autophagy would further enhance the anticancer effects of metabolic reprogramming interventions (MRIs) alone and in combination with carboplatin. To test these hypotheses, we are using a high-throughput screening approach employing several MRIs with or without the addition of chloroquine and/or carboplatin in vitro. Serum starvation (an in vitro model of caloric restriction), rapamycin, BMS-754807 (a dual IGF-1R/insulin receptor), and nicotinamide significantly reduced cell viability (assessed by MTT assay) and clonogenicity (assessed by colony formation assay) of M-Wnt and M6 cells, both well-characterized TNBC cell lines derived from mammary tumors of MMTV-Wnt-1 and C3-TAg transgenic mice, respectively. The growth suppressive effects of these MRIs were further enhanced when combined with autophagy inhibition via chloroquine (CQ). Treatment of M-Wnt and M6 cells with CQ (20-80 µM) also increased in vitro cytotoxicity of carboplatin. Ongoing analyses and planned in vivo studies will confirm whether co-treatment with MRIs and/or CQ are an effective strategy for increasing the efficacy of carboplatin in TNBC cells.

#5279

**Metabolic profiling defines a new characterization of acute myeloid leukemia and identifies** NPM1 **-mutated cases as a distinct subgroup.**

Giorgia Simonetti,1 Antonella Padella,2 Eugenio Fonzi,3 Martina Pazzaglia,3 Margherita Perricone,3 Maria Chiara Fontana,3 Samantha Bruno,3 Maria Teresa Bochicchio,1 Eugenia Franchini,3 Jacopo Nanni,3 Giovanni Marconi,3 Italo F. do Valle,4 Rossella De Tommaso,3 Anna Ferrari,1 Enrica Imbrogno,1 Claudio Cerchione,1 Cristina Papayannidis,3 Emanuela Ottaviani,3 Daniel Remondini,4 Giovanni Martinelli1. 1 _Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) Srl - IRCCS, Meldola (FC), Italy;_ 2 _Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Institute of Hematology "L. e A. Seràgnoli", Bologna, Italy;_ 3 _Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Institute of Hematology "L. e A. Seràgnoli", Italy;_ 4 _Department of Physics and Astronomy, University of Bologna, Italy_.

Genomic and functional alterations of enzymatic activity drive cancer metabolic reprogramming along with mutations of tumor suppressors and oncogenes. IDH1/2 lesions represent a paradigmatic example in acute myeloid leukemia (AML). The study aimed to stratify AML patients based on their metabolic landscape and to map potential connections between their metabolic and genomic profile. The genomic landscape of 166 AML patients was obtained by whole exome sequencing. Variants were called by MuTect and Varscan2. Six additional cases were analyzed by targeted sequencing (SOPHiA GENETICS). Metabolites were quantified by mass spectrometry of bone marrow cells (35 CD34+ and 15 CD33+ AML from the above-mentioned cohort) and compared with CD34+ cord blood and CD33+ healthy blood cells (n=21 each, Metabolon) by Welch's t-test. In AML, 17% of somatic variants targeted metabolism-related genes: 38% were enzymes (according to Recon2), including 3% of electron transport chain genes encoded by mitochondrial DNA (COX1-3, ND1-6), 3% were AML-related genes with a known involvement in cell metabolism (e.g. IDH1/2, MYC, KRAS) and 59% were metabolic regulators, defined by gene ontology annotation. Ninety-one% of patients carried at least one mutation in a metabolism-related gene, with 43% of variants rated as damaging. The most represented pathways were lipid, carbohydrate, nucleotide (AK9, H6PD), amino acids (IDO2) and glucose metabolism (PKM, HK3).

Principal component analysis of metabolic data showed a distinct profile between AML and healthy cells, with a predictive accuracy of 86% and 95% for CD34\+ and CD33\+ cells, respectively. Conversely, few differences were observed between CD34\+ and CD33\+ AML. Unsupervised hierarchical clustering clearly defined 3 AML clusters (C1-3). Moreover, 3 subgroups could be identified in C3 without ambiguous assignments. C1 was enriched for NPM1-mutated (mut) cases (83%, 33%, 27% in C1, C2 and C3, respectively, p=0.03). NPM1-mutated AML were distinguished by a 12-metabolites signature. They showed increased levels of spermidine, cytidine 2′ or 3′-monophosphate (P), thymidine 3′-monoP, uridine-2',3'-cyclic monoP and decrease of inosine 5'-monoP, suggesting altered polyamine, pyrimidine and purine metabolism. Moreover, NPM1-mut AML had reduced levels of intermediates involved in acyl carnitine, lysophospholipid, phosphatidylethanolamine and sphingolipid metabolism. Overall, mutations of metabolism-related genes are common in AML. We defined a metabolic-based classification of AML and identified a new metabolic signature based on 12 metabolites that distinguish NPM1-mut AML from wild-type cases and healthy CD34+/CD33\+ cells. Major alterations in the nucleotide and polyamine pathways suggest novel potential therapeutic approaches.

Supported by: EHA research fellowship award, AIRC, FP7-NGS-PTL, Fondazione del Monte.

#5280

Branched chain amino acids represent indispensible metabolic substrates in glioblastoma.

Antony H. Prabhu, Shiva Kant, Pravin Kesarwani, Prakash Chinnaiyan. _Beaumont Health, Royal Oak, MI_.

Glioblastoma (GBM) is an aggressive brain tumor with limited treatment options. Considerable advances have been made in understanding the genetic alterations driving these tumors, however, the metabolic alterations and mechanisms underlying these aggressive phenotypes remain unclear. We performed global metabolomic profiling of patient derived low-grade astrocytoma (LGA, n= 28) and GBM (n=80). Hierarchical clustering (HC) identified amino acid (AA) metabolism as a dominant metabolic node in GBM compared to LGA. Considerable inter-tumoral heterogeneity of AA metabolism was observed in GBM, with HC identifying two clearly distinct metabolic subtypes consisting of increased and decreased concentrations of AA and their metabolic intermediates. VIP analysis identified the accumulation of branched chain amino acids (BCAA)- leucine, valine and isoleucine as the top ranked metabolites differentiating these subtypes. Based on this, GBMs were classified as BCAA-high and BCAA-low. BCAA-high GBM was enriched with mesenchymal and classical subtypes while BCAA-low GBM was enriched with proneural and neural subtypes. Integrative analysis coupling these metabolic signatures with gene expression profiling performed on matched tissue demonstrated upregulation of BCAA metabolism-associated genes in BCAA-high GBM, along with global transcriptional programs designed to activate cell cycle progression. When extended into molecular subtype specific preclinical models, we demonstrated that BCAA were indispensible for survival of mesenchymal GBM cells, which was independent of the presence or absence of other non-essential and essential amino acids. Further, these cells were able to maintain long-term survival in a dormant state when grown in media devoid of all essential and non-essential AA other than BCAA and glutamine. Knockdown of key enzymes in BCAA metabolism led to robust cytotoxicity in this model, underscoring the importance of BCAA in GBM survival. Ongoing studies are designed to determine the biologic consequence and intermediary metabolism of BCAA in GBM. Collectively these results identify BCAA metabolism as a therapeutic target in GBM.

#5281

Identification of compensatory pathway for glutamate production.

Ryoichi Asaka, Tu Nguyen, Brian J. Krisch, Karim Nabi, Addison Quinones, Jessica Tan, Marjorie J. Antonio, Ting Li, Felipe Camelo, Kiet Nguyen, Sunag Udupa, Edward Gabrielson, Anne Le. _Johns Hopkins University, Baltimore, MD_.

Measurement and Methods: In this study, we sought to expand our knowledge of glutamine metabolism beyond glutaminolysis. Using mass spectroscopy-based stable isotope-resolved metabolomics (SIRM) with 13C515N2-labeled-glutamine, we precisely identified the metabolites produced from glutamine both in vitro and in vivo. Tumors were harvested 2 hours post first glutamine injection. Metabolites were then extracted from tumors, and analyzed using Agilent 6520 Q-TOF mass spectrometer and 1H-NMR. Metabolite intensities were later normalized to protein concentration following analysis.

Results: Our results showed that total glutamate levels were lower in BPTES-NP treated tumors as compared to vehicle control ones. Interestingly, we found an increase in (m+5) labeled glutamate (mass of the parent ions (m) and 5 more mass units due to 13C415N1-glutamate or 13C5-glutamate labelling) in BPTES-NP treated tumors as compared to the vehicle control tumors. Moreover, we found that (m+5) glutamate is a product of (m+7) glutamine being converted to (m+5) alpha-ketoglutaramate (KGM) via glutamine-pyruvate transaminase and further on into alpha-ketoglutarate (aKG) by omega-amidase, which can finally produce the identified (m+5) glutamate through glutamate dehydrogenase. Further analysis using 1H-NMR detailed a significant increase in overall KGM intermediate intensity in treatment groups compared to the control groups, confirming the upregulation of compensatory pathway (glutamine-KGM- aKG-glutamate) to produce glutamate upon treatment of GLS1 inhibitor.

Conclusion: These results explain the reasons behind the limited clinical outcomes for single therapy with a GLS1 inhibitor, and provide potential therapeutic targets: glutamine-pyruvate transaminase, for combination treatments with GLS1 inhibitors to prevent the compensation for glutamate production amid GLS1 inhibition.

#5282

Transition to and from a cancer stem-like state is marked by rapid metabolic shifts that are observable in real time using fluorescence lifetime imaging microscopy (FLIM).

Tong Xu, Cosimo Arnesano, Alireza Delfarah, Zhifei Luo, Kevin Delijani, Emmanuelle Hodara, Peggy Farnham, Nicholas A. Graham, Scott E. Fraser, Amir Goldkorn. _University of Southern California, Los Angeles, CA_.

Introduction: Highly tumorigenic, drug resistant cancer stem-like cells play a key role in therapy resistance and disease progression. We and others have previously shown that cancer cells can transition cyclically in and out of this aggressive phenotype in a manner that is rapid and spontaneous and does not require new mutations, drug selection, or ectopic gene expression. Phenotypic plasticity has been linked to altered cellular metabolic states, so we explored whether cancer cells exhibit distinct metabolic shifts as they switch to and from a cancer stem-like phenotype.

Materials and Methods: Bladder cancer cells (J82 cell line) were fractionated by Hoechst staining and FACS into a side population (SP) marked by high tumorigenicity and drug resistance, and a non-side population (NSP) lacking these characteristics. SP and NSP cells were profiled by Liquid Chromatography-Mass Spectrometry (LC-MS) metabolomics after labeling with [U-13C]-glucose or [U-13C]-glutamine, as well as by RNAseq. The results were analyzed by Metabolite Set Enrichment Analysis (MSEA) and Gene Set Enrichment Analysis (GSEA), respectively. Fluorescence Lifetime Imaging Microscopy (FLIM), which measures bound vs. free cellular NADH, was used to image SP and NSP cells separately or in co-culture with or without cisplatin.

Results: SP cells exhibited LC-MS MSEA profiles marked by upregulated TCA cycle activity and increased glutamine usage relative to NSP cells. Consistent with this finding, SP cells also exhibited RNAseq GSEA profiles marked by upregulated oxidative phosphorylation relative to NSP cells. Distinct FLIM metabolic signatures were successfully assigned to SP and NSP cells and used to image these subpopulations over time. When grown separately or in co-culture, SP cells' FLIM signature gradually shifted to an NSP signature. Conversely, NSP cells' FLIM signature gradually shifted an SP signature. When treated with cisplatin, SP cells survived and retained an unchanged SP FLIM signature. In contrast, fewer NSP cells survived cisplatin treatment, and NSP cells that did survive shifted towards an SP FLIM signature.

Conclusion: Spontaneous rapid transition to and from a cancer stem-like state involves a metabolic shift to and from oxidative phosphorylation and the TCA cycle. These distinct metabolic profiles can be tracked directly and noninvasively by FLIM, revealing that cancer cells spontaneously switch between the metabolic states over time but preferentially shift toward the cancer stem-like state when exposed to cisplatin. Thus, metabolic plasticity can be characterized, tracked and exploited therapeutically to disrupt cancer resistance and dissemination.

#5283

Prognostic value of resectable hepatocellular carcinoma: Clinico-pathologic findings.

Jin Young Kim, Mi Hwa Heo, So Jin Shin. _Keimyung Univ. Dongsan Medical Ctr., Daegu, Republic of Korea_.

We investigated the prognostic factors of resectable hepatocellular carcinoma (HCC). Preoperative peripheral blood factors, fluorine-18 fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) and pathologic parameters were investigated for progression free survival (PFS). From February 2008 to December 2012, a retrospective analysis was conducted on 65 patients who had received liver resection. Peripheral blood parameters were collected within 7 days based on operation date. The 18F-FDG uptake was calculated based on the maximum standardized uptake value of the tumor (SUVmax), the liver mean SUV and the tumor-to-normal SUV ratio (TNR). Immuno-histochemical (IHC) stain results were reviewed by a pathologist through specimen. A total of 65 patients were included (46 males, 19 females; average age, 57.5 ± 8.9 years). The median PFS of HCC was 52.9 months. SUVmax was correlated with PFS and the optimal cut off value of SUVmax was 3.25 (p = 0.005). Median PFS of low SUVmax was 76.4 months and high SUVmax was 8.5 months. The optimal cut-off value of TNR was 1.77 for PFS. The median PFS of HCC in patients with low TNR (< 1.77) and high TNR (≥ 1.77) was 82.3 and 7.0 months, respectively (p = 0.002). AST to platelet ratio index (APRI) could be a predictable marker for recurrence of HCC (p = 0.034). The median PFS of high PNI and low PNI were 64.9 and 10.9 months, respectively (p = 0.004). The IHC expression of glucose uptake transporter 2 was correlated with SUVmax. Preoperative SUVmax, TNR, APRI and PNI could predict recurrence after curative resection of HCC.

#5284

Hypoxia promote self-renewal in pancreatic cancer by modulating L-2-Hydroxyglutarate level.

Vineet K. Gupta, Nikita Sharma, Kousik Kesh, Roey Hadad, Vikas Dudeja, Ashok Saluja, Sulagna Banerjee. _University of Miami, Miami, FL_.

Pancreatic cancer cells undergo extensive metabolic reprogramming to fulfil the demands of metabolic nutrients to fuel their rapid proliferation. This results in abnormal accumulation of various metabolites which causes both metabolic and nonmetabolic dysregulation and potential transformation to malignancy and therefore termed as "oncometabolites". Hypoxia is a major hallmark of pancreatic tumors which associated with increase in self-renewal. The present study investigates the role of Hypoxia mediated altered oncometabolite in regulating self-renewal in pancreatic cancer. GC-mass spectrometric analysis of pancreatic cancer cells under hypoxia showed significant accumulation of 2-hydroxyglutarate (2-HG) both in cells and culture supernatant. Further analysis of hypoxic cells showed selective accumulation L- enantiomer form. Self-renewal requires critical balance between stemness and differentiation. Present study showed for the first time that L-2-HG regulates self-renewal by increasing expression of genes associated with stemness (Sox-2, CD133) and by decreasing expression of differentiation genes (PdX-1, HB9, NKX6.1). Further analysis showed that L-2-HG mediated epigenetic changes regulates these self-renewal gens in pancreatic cancer. Our results indicated that hypoxia mediated metabolic changes results in accumulation of L-2-HG which upregulate self-renewal by shifting critical balance of gene expression towards stemness in pancreatic cancer which contribute to chemoresistance and metastasis in Pancreatic cancer.

### Tumor Suppressors, Carcinogenesis, and Tumor Cell Resistance

#5285

The role of HIV-1 TAT interactive protein 2 in modulating tumor response to hypoxia.

Qingyu Stephanie Zhou,1 Xiaofang Guo,1 Minghua Li,1 Hua Pan2. 1 _Univ. of South Florida College of Pharmacy, Tampa, FL;_ 2 _Univ. of South Florida College of Medicine, Tampa, FL_.

While there is ample evidence that HIV-1 TAT interactive protein 2 (HTATIP2) is a tumor suppressor exerting its pro-apoptotic and anti-metastatic activities by regulating the expression of a subset of pro-apoptotic and anti-apoptotic genes and metastasis suppressor genes, it remains unclear if HTATIP2 has a role in tumor response to hypoxia. The objective of this study was to define the role of HTATIP2 in mediating certain molecular and cellular changes in response to hypoxia in tumors. We employed a series of isogenic A549 human lung adenocarcinoma sublines that were genetically deficient for HTATIP2 and/or HIF-1α to examine the potential modulatory effect of HTATIP2 on oncogenic signaling pathways mediated by HIF-1α and HIF-2α, and established the HTATIP2-deficient A549 xenograft model to explore the impact of deletion of HTATIP2 on tumor response to hypoxia. Results from the human HIF-regulated cDNA plate array study revealed that under hypoxic condition (0.5% O2), several genes promoting tumor metastasis and resistance to apoptosis and anticancer treatment, including HIF1α, CYSD, MMP1, MXI1, NDRG2, PAI1 and NDRG1, were upregulated to a greater extent in HTATIP2 knockdown A549 cells than in the control A549 cells. In contrast, genes involved in angiogenesis, glucose metabolism and protection of apoptosis under hypoxia, such as PDGF, PDK1, CA9, and SAG, were upregulated to a greater extent in control A549 cells than in HTATIP2-knockdown A549 cells. Results from the in vivo study demonstrated that the mean growth rate of HTATIP2-deficient A549 xenograft tumors was significantly increased as compared with that of parental A549 xenograft tumors irrespective of the presence or absence of sorafenib treatment. Moreover, the data of photoacoustic imaging (PAI) of tumor hypoxia revealed that the parental A549 tumors were better oxygenated than the counterpart HTATIP2-deficient A549 tumors. After the 10-day sorafenib treatment, the oxygenated regions in sorafenib-treated parental A549 tumors were significantly reduced as compared with the vehicle-treated parental A549 tumors, whereas no significant change in the oxygenated regions was found between vehicle- and sorafenib-treated HTATIP2-deficient A549 tumors. The overall finding of our study suggests that downregulation of HTATIP2 affords growth advantage to tumor cells by fine-tuning tumor adaptation to hypoxia, leading to tumor progression and aggression.

#5286

Enhancing the anti-cancer efficacy of transcriptional super-enhancer inhibitors in neuroblastoma cells.

Andrew E. Tee, Olivia C. Ciampa, Chelsea Mayoh, Matthew Wong, Tao Liu. _Children's Cancer Institute Australia, Sydney, Australia_.

Patients with neuroblastoma due to MYCN oncogene amplification and consequent N-Myc protein overexpression show poor prognosis. The CDK7/super-enhancer inhibitor THZ1 blocks MYCN gene transcription and reduces neuroblastoma cell proliferation. We recently performed a primary drug screen on a library of Approved Oncology Drugs obtained from the NIH and discovered that the receptor tyrosine kinase (RTK) inhibitors Ponatinib, Lapatinib and Nilotinib in combination with THZ1 exerted synergistic anti-cancer effects against BE(2)-C neuroblastoma cells. When MYCN-amplified neuroblastoma cells were treated with a range of doses of THZ1, and combinations of THZ1 and the RTK inhibitors, it synergistically induced neuroblastoma cell death whilst having minimal cytotoxic effects in non-malignant fibroblast. Affymetrix microarray differential gene expression analysis in neuroblastoma cells after treatment with THZ1 and RTK inhibitor combinations identified KRCC1 and PNUTS as the genes most synergistically reduced by the combination therapy. RT-PCR and immunoblot analyses confirmed that THZ1 and RTK inhibitors synergistically downregulated PNUTS mRNA and protein expression and reduced N-Myc protein but not N-Myc mRNA expression. Subsequently, knocking down of PNUTS gene expression resulted in decreased N-Myc protein but not mRNA expression and decreased cell proliferation in MYCN-amplified neuroblastoma cells. This study has therefore identified the combination therapy of THZ1 and RTK inhibitors is a novel and effective therapeutic strategy against MYCN-amplified neuroblastoma by reducing PNUTS and N-Myc expression.

#5287

Prostate-derived Etsfactor modulates AR-mediated gene expression and promotes luminal differentiation.

Fengtian Wang, Hari K. Koul. _LSU Health Shreveport, Shreveport, LA_.

Introduction: Transcription regulation, governed by the presence of transcription factors and the accessibility of regulatory elements such as enhancers is crucial in restricting lineage plasticity. Deregulated expression of differentiation-related transcription factors leads to cancer progression and poor prognosis. Prostate-derived ETS factor (PDEF) is one of the cell identity-related transcription factors characterized by the abundance of H3K27ac near its genomic locus. Highly expressed in prostate luminal cells, PDEF has been shown to promote the transcription of PSA in collaboration with AR. Our previous studies showed loss of PDEF is associated with higher Gleason score and tumor metastasis through MMP9 and Twist1. However, the role of PDEF, as a transcription factor, remains poorly understood. In this study, we evaluated the effects of PDEF on modulation of AR cistrome and luminal/epithelial differentiation.

Methods: TCGA data were extracted from cBioportal and analyzed using R. Gene set enrichment analysis (GSEA) was performed using default settings and gene sets were downloaded from MySigDB. Genomic coverage analysis was performed using SeqPlots. ChIP-seq analysis was performed using GALAXY. PC3 cells were grown in DMEM/F12 medium and retroviral expression was used to generate cells with stable PDEF expression. ChIP experiments were performed using Chromatrap ChIP-seq kit followed by standard qPCR with SYBR-Green. Gene track was visualized with IGV.

Results: Despite the oncogenic role of AR signaling, it plays a pivotal part in prostate luminal differentiation. We observed that prostate cancer progression is associated with loss of PDEF accompanied by a transcription switch from canonical AR targets to non-canonical in TCGA database. Moreover, AR genomic coverage in prostate luminal cell-specific enhancer regions is decreased in tumor tissues compared with the paired adjacent normal tissue. Stable-expression of PDEF in PC3 cells restored the expression of canonical AR targets as well as the enhancer pattern similar to LNCaP cells. PDEF ChIP-seq analysis of multiple cell lines revealed that PDEF co-localize with H3K4me1. ChIP-qPCR confirmed that PDEF co-localized with AR at the PSA enhancer region. We hypothesize that PDEF is critical for maintaining prostate luminal specific enhancers. Expressing PDEF in LNCaP-abl cells also increased the transcription of canonical AR targets and inhibited the transcription of non-canonical AR targets. Independent of AR, we discovered that PDEF binds to the promoter region of CK18. Moreover, a positive correlation was observed between PDEF and CK18 in cell culture and prostate cancer samples in the TCGA database.

Conclusion: These results show that PDEF plays an important role in the maintenance of canonical AR signature and luminal cell differentiation in prostate cancer in-part by rewiring the transcriptional program.

#5288

Evaluating the potential to repurpose statins for ovarian cancer therapy.

Paul T. Kroeger, Ronny Drapkin. _University of Pennsylvania, Philadelphia, PA_.

The recent exciting results using primary PARP inhibitor maintenance after upfront treatment for advanced ovarian cancer in BRCA1/2 mutation carriers highlight the importance of identifying druggable vulnerabilities in this disease. The fact that not all ovarian cancer patients will benefit from PARP inhibitors or experience durable responses, places great emphasis on the need to identify additional therapies. Re-purposing existing FDA-approved drugs offers an opportunity to accelerate progress in this area. Statins are drugs primarily prescribed for hypercholesterolemia and have been intensely researched for decades. Some of this work has demonstrated, through epidemiologic approaches and limited experimental studies, that statins have anticancer properties. We hypothesized that statins can be repositioned as part of a therapeutic regimen for HGSOC. Here, we demonstrate that certain ovarian cancer cell lines are exquisitely sensitive to treatment with statins. Our ongoing studies reveal that HGSOC cells that are sensitive to stain treatment manifest an amplification of chromosome 1q21. Within this genomic region are the S100 proteins, previously identified as potential biomarkers in several cancer types, as well as the anti-apoptotic protein MCL1. We performed a Reverse Phase Protein Array (RPPA) analysis on higher dose/short exposure, and low dose/longer exposure statin-treated cells. In both conditions, MCL1 levels were altered in statin-sensitive cell lines, while there was no such change in non-sensitive lines. After identifying MCL1 as a potential marker of response, Western blot analysis demonstrated a dose-dependent decrease in protein level of MCL1 with increasing statin concentration. Additionally, a short pro-apoptotic splice variant of MCL1 was produced upon statin treatment, but only in the sensitive lines. Mechanistically, we find that phospho-YAP (inactive) is upregulated after statin treatment. This data corroborated previous research that suggested that multiple different statins decrease nuclear YAP. Given its role as an oncogene, the apparent ability of statins to deactivate YAP and downregulate MCL1 steers statins in a promising direction for their repositioning for future therapeutic use.

#5289

Gain-of-function mutant p53 predisposes head and neck keratinocytes and squamous cell carcinoma cells to replicative stress and genomic instability through minichromosome maintenance complex component 5.

Mei Zhao, Tianxiao Wang, Chen Zhen, Jing Wang, Curtis R. Pickering, Junjie Chen, Jeffrey N. Myers, Ge Zhou. _MD. Anderson Cancer Center, Houston, TX_.

As the guardian of genome, p53 plays important roles in maintaining the genome integrity, and TP53 mutations thereby often lead to genomic instability in cancers. In addition to the loss-of-function, many TP53 mutations can lead to the gain-of-functions (GOF) that often promote tumor development and tumor progression. As an effort to identify the mechanisms involved in mutant p53 GOF activities in head and neck squamous cell carcinoma (HNSCC), we performed an unbiased proteomic screen to identify the mutant p53 G245D interactome in HNSCC cells by immunoprecipitation, and quantitative proteomics using stable isotope labeling with amino acids in cell culture and liquid chromatography coupled with tandem mass spectrometry. Our results identified that MCM5, a member of replication licensing heterohexameric origin recognition complex minichromosome maintenance 2-7 (MCM2-7), interacts with mutant p53s, which suggests a possible role of mutant p53 in regulation of DNA replication. Consistent with this, we show that overexpression of mutant p53 in p53-depelted oral keratinocytes and HNSCC cells predisposes cells susceptibility to replication stress by inhibiting the dormant origin firing, leading to increased chromosomal instability (CIN) under replication stress. Moreover, by overexpressing and downregulating MCM5, we demonstrate that MCM5 modulates GOF mutant p53-mediated susceptibility to replication stress and CIN. Given the importance of MCM2-7 in replication licensing, initiation, and elongation, our discovery of the relationship between mutant p53 and MCM5 suggests that the MCM2-7 complex is one of the key downstream effectors, through which GOF mutant p53s regulate DNA replication and genomic instability in HNSCC.

#5290

**Loss-of-** Irf5 **in mammary epithelial cells drives spontaneous mammary tumorigenesis by regulating EMT.**

Su Song, Dan Li, Kyle Kampta, Betsy Barnes. _Feinstein Institute, Manhasset, NY_.

Interferon regulatory factor 5 (IRF5) is a member of the IRF family of transcription factors. In human breast cancer (BC), IRF5 expression is lost in ~ 60% of ductal carcinoma in situs (DCISs) and ~90% of invasive ductal carcinomas (IDCs). BCs expressing the lower quartile of IRF5 transcript expression were found to significantly correlate with poor prognosis for recurrence-free survival. To examine how loss-of-Irf5 promotes breast tumor initiation and progression, we generated Irf5 knockout (Irf5-/-) mice on a BALB/C background that are susceptible to spontaneous mammary tumorigenesis. At 1 year-old, female nulliparous Irf5-/- mice (n=21) had a >3-fold increase in the incidence of spontaneous mammary tumorigenesis as compared to Irf5+/+ littermate mice (WT, n=25). As early as 6 months old, we detected dramatic differences in the presence of atypical ductal hyperplasia, and at 9 months old, ~50% of Irf5-/- mice had ADH while only 17% of WT littermates had detectable ADH (n=12/group). Immunofluorescence (IF) staining showed expansion of the luminal epithelial compartment in Irf5-/- mice with ADH and/or mammary tumors. Further, proliferation of Irf5-/- luminal epithelial cells was significantly increased over WT mice (15 week-old, n=3/group, 2-way ANOVA, Genotype factor F (1, 4) = 0.04493), supporting a loss of cell growth control in this compartment. To identify the molecular mechanism(s) driving enhanced mammary tumorigenesis, we performed RNA-seq on FACS-sorted luminal and basal epithelial cells from 9 month-old Irf5-/- and Irf5+/+ littermate mice (n=3 mice/group). Gene Set Enrichment Analysis (GSEA) identified signatures that were either positively or negatively regulated in Irf5-/- luminal and basal epithelial cells as compared to WT littermates. We found that epithelial to mesenchymal transition (EMT)-transcription factors were significantly enriched in both Irf5-/- luminal (p=0.046) and basal (p=0.004) epithelial cells. A diverse array of signaling pathways and molecules that participate in EMT induction were also found enriched in Irf5-/- cells, including Wnt/β-catenin (Luminal p=0.021; basal p=0.294), Sonic hedgehog (Shh) (luminal p=0.849), Gli1 (Irf5-/- vs. WT, luminal 3.56 vs. 1.35; basal 10.04 vs. 8.88) and PI3K signaling (luminal p=0.941; basal p=0.970). Findings suggest that loss-of-Irf5 promotes EMT through one or more of these pathways, thus contributing to the striking increase in spontaneous mammary tumorigenesis that occurs in Irf5-/- mice. Results from this study are expected to identify mechanisms and pathways that may be therapeutically targeted in patients with IRF5-negative BC.

#5291

Netrin-1 expression and targeting in multiple myeloma.

David Fahed,1 Kamel Chettab,1 Patrick Mehlen,2 Doriane Mathe,3 Anne Tourette,3 Morgane Denis,3 Alexandra Traverse-Glehen,4 Lionel Karlin,4 Charles Dumontet4. 1 _Anticancer Antibodies Team, CRCL, INSERM U1052, CNRS UMR5286, CLB, UCBL, Lyon, France;_ 2 _Dependence Receptors, Cancer and Development Team, CRCL, INSERM U1052, CNRS UMR5286, CLB, UCBL, Lyon, France;_ 3 _Antineo, Lyon, France;_ 4 _Hospices Civils de Lyon, Lyon, France_.

Netrin-1 was first described as an axonal chemoattractant in peripheral nervous system leading to axon outgrowth. Later on, after well characterizing its receptors, deleted in colorectal cancer (DCC) and uncoordinated-5 (UNC5), and their role in apoptosis, they were classified as dependence receptors. Dependence receptors belong to a family of receptors distinguished from other classical receptors by their capability of inducing cell death in the absence of their respective ligands. Their importance in tumorigenesis suggest that this family of receptors along with their ligands could constitute a novel target in cancer therapy. Recent studies showed upregulation of netrin-1 and its receptors in lung cancer tumors after treatment with conventional chemotherapies. The aim of our study was to analyze the expression of netrin 1 and its receptors UNC5B and DCC in multiple myeloma and to determine the impact of a monoclonal antibody targeting netrin-1 on myeloma cell lines in vitro and in vivo. This antibody, NP137, produced by NetrisPharma, is now in early phase clinical trials. In the first place, we performed a screening of various human myeloma cell lines, including AMO-1, LP-1, MM1S, RPMI8226, U266 and OPM-2, for netrin-1 and its different receptors using RT-qPCR and western blotting. We also analyzed the expression of netrin 1 and its receptors in fresh bone marrow aspirates by flow cytometry and in bone marrow biopsies using immunohistochemistry obtained from patients with multiple myeloma. Our results showed that myeloma cell lines expressed netrin-1 and its receptors DCC and UNC5B as determined both by RTqPCR and by western blotting. This expression was also analyzed in patient samples with 16 bone marrow biopsies using immunohistochemistry and in 10 fresh bone marrow aspirates by flow cytometry. Netrin-1 was identified by IHC in 6/16 samples while DCC and UNC5B were identified in all and in 13 samples, respectively. In fresh samples, both netrin-1 and its receptors were found to be more highly expressed than in normal lymphocytes. The exposure of myeloma cell lines to anti-netrin-1 humanized antibody in vitro did not induce a significant decrease in cell viability in vitro, whereas treatment of SCID mice with established RPMI8226 myeloma tumors lead to a significant reduction of tumor size in comparison to controls, suggesting a possible targeting of netrin 1 in this disease. Combination of NP137 with conventional anti-myeloma agents will be presented. In conclusion, these results suggest that netrin 1 signaling could constitute a novel original target in multiple myeloma.

#5292

Cell-autonomous suppression of colon carcinogenesis by type-2 cGMP-dependent protein kinase.

Bianca N. Islam, Yali Hou, Sarah K. Sharman, Darren D. Browning. _Medical College of Georgia at Augusta University, Augusta, GA_.

A growing body of evidence demonstrates that cGMP signaling has anti-tumor effects in the colon, and could harbor novel targets for colon cancer prevention. The tumor suppressive components that function downstream of cGMP remain largely understudied. In the present work we have characterized the expression of cGMP-dependent protein kinases (PKG1, PKG2) in normal and cancerous tissue from humans. PKG1 was highly expressed in both normal and tumor tissue, where it localized to supportive cells in the lamina propria and stroma (respectively). In contrast, PKG2 expression was notably decreased in tumors compared to matched normal tissue, where it was identified in the epithelium by IHC. Neither PKG isoform was detected at the RNA or protein level in colon cancer cell lines. To interrogate a potential tumor-suppressor role of PKG2 in the colon epithelium we bred PKG2 knockout (KO) mice into a C57BL/6J background. These animals exhibited increased intestinal turnover as reflected by crypt hyperplasia (Ki67) and elevated luminal apoptosis (cleaved caspase 3). When subjected to AOM/DSS treatment, the PKG2 KO mice developed almost twice the number of polyps per mouse as wild type siblings (1.75-fold, p=0.0037). Mouse colon organoids were used to confirm that PKG2 was the only isoform expressed in the epithelium, where it was observed in both proliferating and differentiating epithelial compartments. Colon organoids derived from PKG2 KO mice exhibited more rapid proliferation, and a reduced ability to differentiate compared to wild type controls. Taken together our results show that PKG2 is the central target of cGMP in the colon epithelium, where it suppresses carcinogenesis by controlling proliferation in a cell autonomous manner.

#5293

Celf2 suppresses non-small cell lung carcinoma growth by inhibiting the prex2-pten interaction.

Yiu To Yeung,1 Suyu Fan,2 Bingbing Lu,3 Xiang Li,3 Ann M. Bode,1 Zigang Dong1. 1 _The Hormel Institute, Austin, MN;_ 2 _The China-US (Henan) Hormel Cancer Institute, Zhengzhou, China;_ 3 _Zhengzhou University, Zhengzhou, China_.

The phosphoinositide 3-kinase (PI3-K)/Akt signaling pathway is important in the regulation of cell proliferation through its production of phosphatidylinositol 3,4,5-triphosphate (PIP3). Activation of this pathway is frequently observed in human cancers, including non-small cell lung cancer. The PI3- K/Akt pathway is negatively regulated by the dual-specificity phosphatase and tensin homolog (PTEN) protein. PTEN acts as a direct antagonist of PI3-K by dephosphorylating PIP3. Studies showed that PTEN phosphatase activity is inhibited by PREX2, a guanine nucleotide exchanger factor (GEF). Multiple studies revealed that CELF2, an RNA binding protein, cooperates synergistically with PTEN as a tumor suppressor in many cancers. However, the underlying mechanism as to how CELF2 enhances PTEN activity remains unclear. Here, we report that CELF2 interacts with PREX2 and reduces the association of PREX2 with PTEN. Consistent with this observation, PTEN phosphatase activity is upregulated with CELF2 overexpression and overexpression of CELF2 represses both Akt phosphorylation and cell proliferation only in the presence of PTEN. CELF2 gene delivery significantly inhibited PDX tumor growth. We analyzed 87 paired clinical lung adenocarcinoma samples and results showed that CELF2 protein expression is down-regulated in tumor tissues and associated with poor prognosis. Analysis of TCGA datasets showed that CELF2 expression is also associated with shorter patient survival time in many cancers. Overall, our work suggests that CELF2 plays a novel role in PI3-K signaling by antagonizing the oncogenic effect of PREX2.

#5294

The PanNET-related histone H3.3 chaperone Daxx regulates lineage specification and tissue homeostasis in the pancreas.

Teresa Sposito,1 Marie-Sophie Nguyen Tu,2 Guy A. Rutter,2 Marco Novelli,1 Chrissie Thirlwell,1 Paolo Salomoni1. 1 _UCL, London, United Kingdom;_ 2 _Imperial College, London, United Kingdom_.

Progressive age-dependent replacement of canonical H3.1/2 with H3.3 in post-mitotic cells is a crucial mechanism for maintaining genome integrity at heterochromatic sites. The histone chaperone DAXX cooperates with the chromatin remodeller ATRX for the deposition of the non-canonical histone H3.3 at heterochromatin, while it prevents the expression of endogenous retroviral elements in another complex involving the SETDB1 methyltransferases. Loss of function mutations affecting ATRX/DAXX and MEN-1 have been recently identified as a subgroup of pancreatic neuroendocrine tumours (PanNETs) leading to poor prognosis and more aggressive phenotype compared to the ATRX/DAXX and MEN-1 wild-type counterparts. We developed a mouse model for conditional Daxx inactivation in mouse islets to understand the impact of DAXX loss on pancreas homeostasis and PanNETs development. We found that loss of Daxx in the endocrine pancreas, using the Insulin-1Cre driver, leads to aberrant glucose metabolism with the development of hyperglycemia and consequently insulin resistance in mice. We have also shown that loss of DAXX is linked to increased expression of α-cell lineage genes upon glucose stimulation of isolated islets in vitro, resembling the recently reported phenotype of PanNETs harboring ATRX/DAXX or MEN1 mutations. Nonetheless, loss of Daxx was not sufficient to lead to PanNET development. We are currently investigating the chromatin and transcriptional perturbations caused by Daxx loss and how these could be linked to the aberration of cell lineage specification and tissue dysfunction in the pancreas. More generally, this work will provide new insight into how epigenetic alterations contribute to misregulation of developmental programmes and cancer development in the pancreas.

#5295

Targeting Rb phosphatase activity to circumvent CDK4/6 inhibitor resistance.

Kathleen H. DiSalvo, Brian C. Velez, Rebecca L. Kravtsov, Nancy A. Krucher. _Pace Univ., Pleasantville, NY_.

The Retinoblastoma (Rb) tumor suppressor protein plays a role in the control of proliferation, apoptosis, differentiation, invasion and the maintenance of genome stability. Its activity is regulated by phosphorylation on several serine/threonine sites, which is thought to inactivate its tumor suppressive function. Cyclin dependent kinases (CDKs) are responsible for most of the Rb phosphorylation that occurs in cells. It is well established that alterations in the Rb pathway (consisting of CDK4/6, D-type cyclins, the CDK inhibitor p16INK4a, and Rb itself) that lead to excessive phosphorylation of Rb have been observed in almost all cancer types. Alterations that lead to increased CDK activity toward Rb are more common than mutations to the Rb gene itself, a finding that prompted investigations into the development of clinical treatments that inhibit the phosphorylation of Rb by CDKs. The CDK4/6 inhibitors palbociclib and abemaciclib were recently clinically approved for use in breast cancer. However, acquired resistance to CDK4/6 inhibition has been shown to limit clinical efficiency. Targeting Rb phosphorylation by CDK4/6 inhibition has been shown to activate alternate pro-survival pathways such as the AKT pathway. In our studies we developed a method of siRNA-mediated activation of Rb phosphatase that targets Rb phosphorylation in attempt to circumvent the development of resistance. Phosphatase Nuclear Targeting Subunit (PNUTS) associates with and regulates PP1 activity toward Rb. In several cancer cell types, such as HCT116 colon cancer, MIA PaCa-2 pancreatic cancer and MCF7 breast cancer, PNUTS depletion causes dephosphorylation of Rb without AKT activation. Furthermore, in Panc1 pancreatic cancer cells, activation of AKT is observed after 1-hour palbociclib (500nM) treatment, whereas PNUTS depletion does not induce AKT. In MD-MBA-231 breast cancer cells, 1-hour treatment of palbociclib or abemaciclib (20nM) induces the AKT pathway as well as the STAT pathways, however PNUTS depletion in these cells does not induce AKT activation. It has been shown that CDK4/6 inhibitors activate AKT by causing loss of Rb interaction with SIN1, a component the mTORC2 complex. Our studies reveal that PNUTS depletion causes dephosphorylation of Rb, without disrupting Rb-mediated inhibition of mTORC2. Thus, the stimulation of Rb phosphatase activity may target Rb phosphorylation without the development of resistance.

#5296

DNAJA1 promotes cancer progression by stabilizing mutant p53.

Tomoo Iwakuma. _Univ. of Kansas Medical Ctr., Kansas City, KS_.

The tumor suppressor p53 is mutated in approximately 50% of human cancers. Majority of them are missense mutations, which results in accumulation of dysfunctional p53 proteins in tumors. Increasing evidence suggests that accumulation of mutant p53 is crucial for its oncogenic gain-of-function activities, such as proliferation, metastasis, and chemotherapy resistance. Previous work from our lab has revealed that DNAJA1, a HSP40 family member, specifically binds to and stabilizes conformational/misfolded mutant p53. DNAJA1 downregulation induces ubiquitination and proteasomal degradation of conformational p53 mutants, but does not affect the levels of wild-type p53 or DNA contact mutants. Based on these observations, we hypothesize that DNAJA1 promotes cancer progression in a manner dependent on conformational/misfolded mutant p53. To test this hypothesis, we examined the effects of DNAJA1 deletion/knockdown on malignant properties of head and neck squamous cell carcinoma (HNSCC) cells. Genetic deletion/knockdown of DNAJA1 resulted in reduced proliferation, colony formation, migration, and orthotopic tumor growth of HNSCC cells expressing conformational mutant p53, but not DNA contact mutant p53. These cellular phenotypes induced by DNAJA1 knockdown/knockout were substantially rescued by overexpression of DNAJA1 or mutant p53. We also conducted RNA sequencing analyses using HN31 cells (control, DNAJA1 knockdown or mutant p53 knockdown). Notably, 77.2% of downregulated genes by p53 knockdown were overlapped with genes downregulated by DNAJA1 knockdown. Moreover, genes involved in cancer-related pathways, including adhesion and actin cytoskeleton organization, were commonly downregulated. These results strongly suggest that DNAJA1 promotes HNSCC progression mostly dependent on conformational mutant p53. Our results may also suggest that DNAJA1 could be a novel therapeutic target for cancers carrying conformational/misfolded mutant p53.

#5297

Targeting androgen receptor by tumor suppressive maspin in prostate cancer.

Sijie Tang,1 Xueqi Lian,2 Jiajia Jiang,2 Huiyin Chen,2 Jiaqian Guo,2 Can Huang,2 Xiaohua Li,3 Luo Gu1. 1 _Nanjing Medical University, Affiliated AoYang Hospital of Jiangsu University, Nanjing, China;_ 2 _Affiliated AoYang Hospital of Jiangsu University, Suzhou, China;_ 3 _Nanjing Medical University, Affiliated AoYang Hospital of Jiangsu University, National Center for Gene Testing Technology Application & Demonstration (Anhui), The Laboratory of Clinical Genomics, Hefei KingMed Diagnostics Group, China_.

Androgen deprivation therapy remains the foundation of treatment for high-risk locally or systemically advanced prostate cancer, and also gains more treatment efficacy from combination with androgen receptor (AR) pathway inhibitors. Thus, new generation of AR inhibitor should be desirable for investigation of therapeutic improvement on prostate oncology. Tumor suppressive maspin was a endogenous HDAC 1 inhibitor and involved in regulation of multiple genes expression, which was associated with better outcomes of cancer chemotherapy. In current study, overexpression of maspin in prostate cancer cells using ectopic recombinant gene expression technology was found to decrease AR level, and knocked down of maspin expression by siRNA technique enhanced AR level in turns. Meanwhile, the maspin-mediated AR repression consequentially down regulated the transcription of AR-targeted genes (e.g. NKX3.1, TMPRSS2). Chromatin immunoprecipitation assay showed that maspin bound with natural rather than artificial AR transcription promoter. Bioinformatic analysis of microarray data extracted from NCBI GEO database (https://www.ncbi.nlm.nih.gov/geo/ ) also proved the inverse correlation of downregulation of maspin expression with upregulation of AR level in advanced prostate cancer. Treatment with protease inhibitor MG132 didn't rescue the loss of AR expression mediated by increased maspin through either ectopic recombinant transgenic expression or induction by treatment of MS-275 or 5'-Aza. Finally, MS-275-induced maspin expression increased the level of p-H2A.X and PARP cleavage, and augmented the treatment efficacy of AR antagonist

MDV3100 on down regulation of PSA mRNA. Taken together, our data demonstrated that AR was a novel target of maspin. Maspin-mediated AR transcription repression promoted the therapeutic efficacy of AR inhibitor MDV3100 in prostate cancer, thus it could be speculated to benefit the AR-related prostate cancer management.

#5298

Finding new molecules to bypass the tumor suppressor gene TP53 and restore cell cycle checkpoint control.

Henry Moore, Jessica Blackburn. _University of Kentucky, Lexington, KY_.

The tumor suppressor gene, tumor protein 53 (TP53), has been shown to be mutated in more than half of all human cancer cases. p53 controls the G1/S phase checkpoint of the mitotic cell cycle, protecting the fidelity of DNA and inducing apoptosis in cells with irreparable DNA damage. Mutations in TP53, which are often missense mutations that interfere with p53 DNA binding activity or protein folding, result in protein inactivation and loss of checkpoint control, allowing cells with potentially oncogenic mutations to survive and proliferate. TP53 mutations have been shown to enhance tumor progression, increase metastatic potential, and mediate chemotherapy resistance. Because of the importance of p53 in cancer, many studies have aimed to identify small molecules that can "reactivate" mutated p53 protein in cell culture, although this has met with little success thus far. The aim of this research is to identify the extent to which the effects of non-functioning p53 can be completely bypassed, and cell cycle checkpoint restored, using already-established FDA approved compounds in a zebrafish model of p53 loss.

Zebrafish were genetically engineered to have their entire TP53 locus removed using TALEN (Transcription activator-like effector nuclease), rendering them unable to produce the p53 protein. p53 deficient and wild-type zebrafish larvae were then subjected to gamma-irradiation to induce DNA damage. An apoptotic cell staining assay was then performed to allow for the qualitative and quantitative analysis of apoptosis levels throughout the larvae. As expected, TP53 null fish had significantly less apoptotic cells than control after DNA damage. We are in the process of testing 770 FDA approved compounds for their effects in inducing an apoptotic response in p53 null larvae after gamma-irradiation. Positive hits will be carried forward to human cancer cells with TP53 mutation, and the mechanism of action of the drug will be determined using biochemical approaches. These results could identify novel pathways that work in concert with p53 and will allow us to harness new mechanisms of DNA damage induced apoptosis to kill cancer cells. Repurposing FDA-approved drugs can also rapidly translate to clinical benefit in a wide number of cancers driven by TP53 mutation.

#5299

Role of short chain free fatty acid receptors in colorectal cancer.

Amal AlGhamdi,1 Saeed AlMahri,1 Maaged AlAkiel,2 Sameer Mohammad,1 Mohammad Aziz1. 1 _King Abdullah International Medical Research Center, Riyadh, Saudi Arabia;_ 2 _King Saud Bin Abdul Aziz University for Health Sciences, Riyadh, Saudi Arabia_.

One of the well established risk factors for colorectal cancer is dietary profile. A well recognized negative association of dietary fiber with colorectal cancer warrants further understanding of the underlying mechanism. Bacterial fermentation of dietary fibre results in formation of short chain free fatty acids in the gut. There are two receptors for these fatty acids one of which has been implicated in colorectal cancer. Free fatty acid receptor 2 has been associated with development of colorectal adenoma in a mouse model. We analyzed the differential expression of FFAR2 gene in colorectal cancer patients. Further, we generated genetically engineered colorectal cancer cell line with reduced level of expression of these receptors. These engineered cells were found to have enhanced rate of cell proliferation. In order to understand the mechanism of increased proliferation, we explored the involevement of glucose uptake and cAMP pathways. While there was an increased level of glucose uptake in engineered cells, there was no evidence of involvement of Protein kinase A mediated cAMP pathway. Further studies are needed to fully establish the connection between dietary fibre, gut microbiota and colorectal cancer.

#5300

LIN9 as a potential negative regulator of the glioblastoma stem cell state.

Farid Farkouh, Milan G. Chheda, Arijita Jash. _Washington University School of Medicine in St. Louis, St. Louis, MO_.

Introduction: Glioblastoma (GBM) is the most lethal primary brain tumor. The GBM stem cells are treatment-resistant cells. We performed an RNAi screen to identify genetic drivers of the GSC state. LIN9 suppression scored strongly in the opposite direction: LIN9 suppression led to a significant increase in neurosphere size and number. In other cellular contexts, LIN9 interacts with pRB and cooperates to promote differentiation. It also inhibits DNA synthesis and oncogenic transformation. The purpose of this study is to better understand the role of LIN9 in GBM.

Methods: Lentivirus for suppression and overexpression of LIN9 was produced via transfection of 293T cells. We transduced patient-derived 0308 GSCs. qRT-PCR was used to determine the expression of various genes.

Results: LIN9 suppression in 0308 GSCs results in increased sphere formation and increased cell proliferation but not increased cell size. Additionally, LIN9 suppression results in upregulation of Sox2 in human astrocytes. In contrast, LIN9 overexpression in 0308 cells results in decreased sphere formation and decreased proliferation. Further ongoing experiments will determine if suppression and overexpression of LIN9 have an effect on the expression of stem cell markers (ex. SOX2, Nestin, OLIG2) and differentiation markers (ex. GFAP, MAPK2, GAL4). We expect LIN9 suppression will result in an increase in the expression of stem cell markers and a decrease in expression of differentiation markers. We also expect the opposite results for LIN9 overexpression.

Conclusions: LIN9 is a potentially important negative regulator of the glioblastoma stem cell state.

#5301

The role of ID4 in regulating SAT1 gene expression in prostate cancer phenotype.

Majid Al-Zahrani. _Clark Atlanta University, Atlanta, GA_.

Introduction: Inhibitor of DNA binding 4 (ID4), a bHLH protein and a transcriptional regulator, is a tumor suppressor gene in prostate cancer. ID4 is expressed in prostate cancer cells (LNCaP), which are androgen sensitive, but epigenetically silenced in a more aggressive DU145 cells. The epigenetic silencing of ID4 is associated with increasing tumor grade and castration resistant prostate cancer (CRPC). ID4 regulates the expression of some other genes such as SAT1, which is a rate limiting enzyme found in the X chromosome involved in the catabolic pathway of polyamine metabolism by catalyzing the N(1)-acetylation of spermidine and spermine. Recently, SAT1 was also shown to mediate histone H3 acetylation suggesting its role in regulating gene expression. Next generation sequencing (NGS) data showed a significant low expression of SAT1 when ID4 is up regulated and vise-versa, which led to a hypothesis that ID4 might regulate the expression of SAT1 gene in prostate cancer cells.

Experimental procedures: The NGS (exon sequencing) was performed on prostate cancer cells either over-expressing ID4 (DU145(+)ID4) or cells in which ID4 was genetically silenced (LNCaP(-)ID4). Moreover, immunohistochemistry (IHC) analysis was performed on mice prostates tissues (WT and ID4-/- mice) and DU145(+)ID4 tumor xenografts tissues to analyze SAT1 protein expression in the presence and the absence of ID4.

Results: The NGS and IHC data analysis suggested that Spermidine/spermine N(1)-acetyltransferase (SAT1) is up-regulated in cells lacking ID4 such as (LNCaP(-)ID4) but down-regulated in cells which express ID4 (DU145(+)ID4). These results prompted us to investigate the effect of silencing SAT1 on ID4 expression and on cancer phenotype.

Conclusion: The expression of ID4 gene in prostate cancer cell lines LNCaP, and DU145(+)ID4 cells showed low expression of SAT1 gene compared with cells lacking ID4 gene as in LNCaP(-)ID4 and DU145 cells.

Acknowledgment: These studies were supported by the NIH/NIMHD/P20 Grant

#P20MD002285.

#5302

Interplay of Parkin, a 'tumor suppressor' in metabolic deregulation of cancer.

Zafar I. Bhat, Khalid Imtiyaz, M. Moshahid Rizvi. _Jamia Millia Islamia, New Delhi, India_.

Cancer is a disease of uncontrolled proliferation which initially arises as a result of dysregulation in some gene candidates responsible for maintaining a normal cellular state. Some genes are suppressed with a concomitant increase in the expression of certain dormant ones. The former group constitutes the tumor suppressors while as the later- the oncogenes. Parkin a PARK 2 gene product is a potent tumor suppressor, has been well reported to be down regulated in many cancer types. Besides its tumor suppressor role, it regulates the redox state of the cell and recently it was reported to be an important factor in TP53 (a master tumor suppressor protein) mediated tumor suppression provides a clue about the role of Parkin in metabolic deregulation- an emerging hallmark of cancer! To this end, here we present that how Parkin is regulated under different environmental conditions and how it governs metabolic plasticity- glycolytic phenotype and hence cellular migration.

To conclude, the results show that Parkin differentially regulates metabolism by modulating certain potent marker candidates responsible for Warburg effect and associated carcinogenesis.

[MMA Rizvi is the corresponding author for this work.]

#5303

Mechanism of the novel HDAC1-YAP epigenetic regulatory mechanism in Hippo pathway of human cancers.

Nai-Kuan Wang,1 Ming-Yuan Chao,2 Yu-Hsuan Huang,1 Jiun-I Lai2. 1 _National Yang Ming University Hospital, Taipei, Taiwan;_ 2 _National Yang Ming University, Taipei, Taiwan_.

The Hippo pathway is an evolutionarily conserved signaling pathway that regulates multiple biological functions, including organ size, cell proliferation, contact inhibition, and is also significantly implicated in cancer. In multiple cancer types, dysregulation of the Hippo pathway leads to heightened activity of the oncoproteins YAP and TAZ that contributes to oncogenesis. In mammalian cells, YAP is regulated by a phosphorylated cascade mediated by the kinases MST1/2 and LATS1/2. This cascade, known as the canonical Hippo pathway, receives upstream cues such as cell density, mechanical stress and other factors and ultimately results in phosphorylation of YAP/TAZ and its cytosolic degradation. Pertubation of the Hippo pathway leads to YAP intranuclear translocation and downstream activation of target genes, inducing proliferative signals and thus initiating oncogenesis. YAP/TAZ upregulation have been described in cancer, and YAP driven animal cancer models have been established. Much effort has been towards studying downstream effects of YAP activation in cancer, but the role of upstream epigenetic regulation of YAP is unclear. We asked whether if histone deacetylation has a role in YAP expression. Histone deacetylases (HDACs) are essential in controlling transcription, and HDAC inhibitors are approved in several cancer types. We discovered that HDAC inhibitors decrease YAP expression significantly without inhibiting TAZ levels, in multiple cancer cell lines. Interestingly, the downregulation occurs at the transcriptional level, suggesting that this inhibition is independent of canonical Hippo pathway. HDAC inhibition mediated downregulation of YAP leads to decreased nuclear translocation of YAP. We then show that the transcription inhibition of YAP expression is mediated by HDAC1, a member of the class I HDACs. This is of particular interest since HDAC1, normally involved in transcriptional silencing, conversely maintains YAP expression in human cancers. To clarify the exact mechanism, transcriptional profiling by RNA-seq and miR-seq were then performed on samples from cells treated with HDAC inhibitors or siRNA targeting HDAC1. Overlap of the consensus differentially expressed genes identified transcription factors that play a role in this pathway. Analysis of human RNA-seq data in cancers with HDAC inhibition validated our findings in human gene expression databases.In summary, we have discovered a novel pathway showing that inhibition of HDAC1 downregulates YAP expression, further inhibiting its intranuclear translocation and related downstream signaling. Interestingly, inhibition of HDAC1 conversely suppresses YAP expression. Our study uncovers a novel link between HDAC1 and the Hippo pathway and paves rationale for HDAC inhibitors, of which several drugs are approved, for a potential role of YAP driven cancers.

#5304

Gem-interacting protein (Gmip) is potentially a new lung tumor suppressor.

Yousef G. Amaar. _VA Medical Ctr., Loma Linda, CA_.

Introduction: We have identified a novel RASSF1C-PIWIL1-piRNA pathway that appears to promote lung cell proliferation and migration. PIWI-like proteins interact with PIWI interacting RNAs (piRNAs) to form complexes that regulate gene expression at the transcriptional and post-transcriptional levels leading to stimulation of stem cell renewal and proliferation. We previously have shown that RASSF1C regulates the expression of the PIWIL1-piRNA gene axis, suggesting the hypothesis that the RASSF1C-PIWI-piRNA pathway may promote lung cancer stem cell development and progression, in part, by epigenetically modulating the expression of growth promoting and growth inhibiting genes. To validate this hypothesis, we used a non-small cell lung cancer cell model to identify candidate genes targeted by the RASSF1C-PIWIL1-piRNA pathway through a gene methylation mechanism.

Methods: We have previously conducted a study to assess the impact of over-expressing RASSF1C and knocking down RASSF1C and P1WIL1 expression on global gene DNA methylation in the NSCLC cell line H1299 using the Reduced Representation Bisulfite Sequencing method. Candidate Differentially Methylated Regions (DMR) were identified by comparing DNA methylation profiles of experimental and control cells. Statistically significant candidate genes residing in hypo- and hyper-methylated regions in lung cancer cells were identified.

Results: We found that over-expression of RASSF1C and knocking down RASSF1C and PIWIL1 modulated DNA methylation of genomic regions. Among the candidate target genes identified is Gmip, a RhoA-specific GTPase-activating protein. We found over-expression of RASSF1C increases and knock-down of RASSF1C or PIWIL1 decreases intragenic methylation of Gmip. Consistent with this, RT-PCR analysis shows that Gmip mRNA levels are reduced in lung cancer cells over-expressing RASSF1C while Gmip mRNA levels are increased in cells with RASSF1C or PIWIL1 knocked down. Further, Kaplan-Meier analysis of survival of lung adenocarcinoma patients in The Cancer Genome Atlas shows that high expression of Gmip is associated with significantly higher patient survival, suggesting that Gmip may function as a tumor suppressor.

Conclusion: The RASSF1C-PIWI-piRNA pathway may drive epigenome regulation and genesis of lung cancer stem cells through modulation of novel genes such as Gmip. Interestingly, Gmip is among 3781 mRNAs that are predicted to be targeted by the PIWIL1-piRNA complex in mouse male germ cells. Our findings are the first to suggest that Gmip is a human lung tumor suppressor. Since virtually nothing is known about Gmip function in human cancer, its function as a tumor suppressor in lung cancer needs to be studied and characterized.

#5305

**Reexpression of** LSAMP **, a gene frequently deleted in African American prostate cancers, suppresses tumor growth and β-catenin activity.**

Kevin Babcock, Taduru Sreenath, Charles P. Xavier, Inger L. Rosner, Shiv Srivastava, Albert Dobi, Shyh-Han Tan. _Center for Prostate Disease Research, Uniformed Services University of the Health Sciences and the Walter Reed National Military Medical Center, Bethesda, MD_.

Introduction: African American (AA) men have the highest prostate cancer incidence and mortality rates in the US. The biological contribution to this disparity, however, is not well understood. LSAMP inactivation has been implicated in several cancers, and was recently identified in prostate cancer. A higher frequency of LSAMP inactivation has been observed in AA prostate cancer, and this aberration has been associated with a significantly greater risk of disease progression. In the characterization of LSAMP in prostate cancer cells lines, we found the copy number to be variable and the expression to be low or undetectable. LNCaP, MDA PCa 2B, and DU 145 prostate cancer cell lines were stably transduced to express LSAMP in an inducible or constitutive manner. LSAMP expression in these cell lines resulted in reduced cell proliferation, and induced a reversion to indolent cell-cell, and cell-extra-cellular-matrix adhesion characteristics, consistent with its tumor suppressive role. LSAMP expression also resulted in the down-regulation of receptor tyrosine kinases EPHA3, FGFR2, and FGFR4, and reduced activation of their downstream ERK and AKT pathways. Several Integrins were also up-regulated upon LSAMP expression. Additionally, β-catenin localization was altered, suggesting a potential reduction in transcriptional activity. We assessed the tumor suppressive function of LSAMP further, using in vitro assays and in vivo mouse models.

Methods: LSAMP expressing and control DU 145 cells were used to investigate the tumor suppressive function of LSAMP in vivo. Athymic nude mice were injected either subcutaneously, to determine effect of LSAMP expression on prostate tumor growth rates, or intravenously, to determine effect of LSAMP expression on tumor formation. We performed the TOPflash/FOPflash luciferase reporter assay to determine whether LSAMP expression modulates transcriptional activity of β-catenin in vitro.

Results: LSAMP expression resulted in a significant inhibition of tumor growth in the subcutaneous xenograft model. In the intravenous xenograft model, LSAMP expression resulted in a reduced incidence of distant metastases. Consistent with the negative modulation of signal transduction, and β-catenin localization previously observed, LSAMP expression resulted in a reduction of β-catenin transcriptional activity in vitro.

Conclusion: These studies provide in vivo evidence of the suppressive function of LSAMP in prostate tumors, corroborating previous in vitro and clinical findings. LSAMP expression reduced tumor growth rates, and incidence of distant metastases. LSAMP expression also reduced β-catenin transcriptional activity in vitro. These findings provide further support for a biological mechanism underlying the aggressive prostate cancer phenotype observed with LSAMP inactivation.

#5306

Targeting p53 deficiency with oncolytic viruses in myeloma and lymphoma.

Sophie Vantyghem,1 Anne Lok,1 Geraldine Descamps,1 Benoit Tessoulin,1 David Chiron,1 Berthe-Marie Imbert,2 Philippe Moreau,3 Steven Le Gouill,3 Martine Amiot,1 Agnes Moreau-Aubry,1 Catherine Pellat-Deceunynck1. 1 _CRCNA Inserm CNRS, Nantes, France;_ 2 _CRTI, Inserm, CHU Nantes, Nantes, France;_ 3 _CHU Nantes, Nantes, France_.

TP53 is the most frequently deleted and/or mutated gene in cancers and the deletion/mutations are associated with resistance to therapy in numerous cancers including Multiple Myeloma and Mantle Cell Lymphoma. In Multiple Myeloma and Mantle Cell Lymphoma patients and in B-cell malignancies, TP53 deletion and TP53 mutations are frequently associated. Treatments for these diseases have improved in the past decade but patients with p53 deficiency (deletion and mutation) have a reduced response to all treatments. Although the role of p53 loss in tumor emergence was recently shown to be related to its loss of DNA repair coordination, resistance to therapy is assumed to be related to the inability of the p53 defective protein to transactivate apoptotic genes such as BBC3 (Puma), PMAIP1 (Noxa) and BAX. However, since p53 coordinates cellular regulation in response to numerous stresses, including viral infection, its loss of function should induce tumor-specific vulnerabilities such as viral infection. We thus we assessed whether p53 mutant cells would be highly permissive to two oncolytic viruses i.e., Measles Virus (RNA negative virus) and TVEC (double strand DNA). We assessed the sensitivity of myeloma and lymphoma cells to the two viruses in 35 cell lines and 40 primary samples characterized for p53 status. In cell lines, we showed that viral load (measured by N gene and DNA polymerase expression, respectively) and cell death were associated with TP53 status for both viruses: TP53abn cell lines were preferentially infected and killed by Measles Virus or TVEC when compared to the TP53wt cell lines (p=0.046 and p=0.026, respectively). Resistance to infection was not related to IFN production. Interestingly, coculture of tumor cells with HS-5 stromal cells did not prevent any infection or death. Measles Virus infection occurred exclusively via CD46 (CD150 was mostly absent on myeloma and lymphoma cells), and we demonstrated using p53 activation/silencing that p53 repressed CD46 expression directly (ChIP assay) and indirectly (miRNA192). Receptors mediating TVEC infection of myeloma/lymphoma cells and mechanisms supporting p53-related TVEC infection are under investigation. In patients' samples (mononuclear cells from bone marrow or peripheral blood), we showed that both viruses preferentially targeted myeloma/lymphoma cells and that p53 deficient tumor cells (assessed by FISH, DNA sequencing and lack of DR5 increase upon exposure to nutlin3a) were more infected/killed than p53 competent tumor cells. In summary, myeloma and lymphoma cells were highly sensitive to Measles Virus or TVEC and resistance to infection was abrogated in p53 deficient cells, arguing for a clinical trial in refractory patients with p53 deficiency.

#5307

Androgen Receptor promotes localization of E-cadherin to cell-cell junctions through Abi1 signaling.

Baylee Porter,1 Disharee Nath,1 Susan A. Krum,2 Gennady Bratslavsky,1 Vladimir A. Kuznetsov,1 Leszek Kotula1. 1 _SUNY Upstate Medical Univ., Syracuse, NY;_ 2 _University of Tennesse Health Science Center, Memphis, TN_.

According to American Cancer Society, prostate cancer is a slow progressing disease that affects nearly 50 percent of males over the age of 60. Every year, 165,000 new cases are diagnosed and 30,000 deaths are reported. Current therapies such as Androgen Depravation Therapy (ADT) and Anti-Androgen treatments such as Enzalutamide increase quality of life and extend survival but do not provide a definitive treatment and are ineffective in highly metastatic prostate cancer. Castrate Resistant Prostate Cancer (CRPC) is an untreatable highly metastatic form of prostate cancer and once diagnosed the survival rate is 1-2 years. Hence, the understanding of molecular mechanisms of CRPC is of critical importance. We previously linked prostate cancer to Abi1 gene. Abl interactor 1 (Abi1) is a scaffolding protein and an integral member of the WAVE Regulatory Complex (WRC). WRC is actin nucleation promoting factor that acts upstream of Arp2/3 to enhance the speed of actin polymerization and is critical for various cellular functions including cell-cell junction formation. Our previous research using a prostate cancer mouse models shows that loss of Abi1 expression leads to down-regulation of cellular adhesion proteins such as E-cadherin. Studies using our CRISPR KO RWPE cell line indicate that loss of Abi1 disrupts cell-cell junction formation via decreased levels of E-cadherin. Analysis of patient data demonstrated loss of Abi1 in a subset of CRPC patients. Castrate resistant tumor growth is androgen independent therefore anti-androgen therapy is ineffective. In search of a link between the androgen signaling and the role of Abi1 in prostate cancer we found that Abi1 gene contains androgen receptor (AR) binding sites. Consistent with the idea that Abi1 is an AR responsive gene, expression levels of Abi1 are increased following AR stimulation with the synthetic androgen, R1881, of the hormone sensitive cell line LNCaP. We also observed increased levels of E-cadherin at the cell-cell junctions. In contrast, cells lacking Abi1 do not form proper cell junctions neither respond to AR stimulation in the same way as wild type cells. We propose that Abi1 is a critical part of androgen signaling by promoting cell-cell junctions and tissue integrity. Supported by NCI R01 CA161018 (LK).

#5308

BRMS1L, a metastatic suppressor gene, is transcriptionally regulated by p53.

Takashi Tokino, Ryota Koyama, Masashi Idogawa, Yasushi Sasaki. _Sapporo Medical Univ., Sapporo, Japan_.

The tumor suppressor p53 plays a pivotal role in the cell fate determination in response to diverse upstream signals. p53 regulates a number of genes that are involved in cell cycle arrest, apoptosis, senescence, and maintenance of genomic stability. Recent studies revealed that p53 is also important for the regulation of cell invasion and migration. Our group has previously revealed that the p53 transactivates anti-invasion genes such as CLCA2, ICAM2, and LIMA1. However, the role of the p53 downstream pathway in cancer invasion, migration, and metastasis remains unclear. In this study, microarray analysis showed that breast cancer metastasis-suppressor 1- like (BRMS1L) is upregulated by p53. We identified two responsive elements of p53 family proteins in the first intron and upstream of BRMS1L. These response elements are well conserved among mammals. Functional analysis showed that ectopic expression of BRMS1L inhibited cancer cell invasion and migration; knockdown of BRMS1L by siRNA induced the opposite effect. Importantly, clinical databases revealed that reduced BRMS1L expression correlated with poor prognosis in patients with breast and brain cancer. Together, these results strongly demonstrate that BRMS1L is one of the mediators downstream of the p53 pathway, and that BRMS1L inhibits cancer cell invasion and migration, which are essential steps in cancer metastasis. Collectively, our results suggest that BRMS1L is involved in cancer cell invasion and migration, and could be a therapeutic target for cancer.

#5309

Inhibition of cancer stem-like cells in esophageal squamous cell carcinoma with SOCS-1 gene therapy.

Ayumi Yamamoto, Eri Munekage, Satoshi Serada, Shigehiro Tsujii, Kosuke Hiramatsu, Minoru Fujimoto, Kazuhiro Hanazaki, Tetsuji Naka. _Kochi University, Japan_.

INTRODUCTION: Cancer stem-like cells are thought to be one of the causes of cancer initiation and progression. They are associated with the resistance to radiotherapy and chemotherapy for various types of tumor including esophageal squamous cell carcinoma (ESCC). Therefore, cancer stem-like cells are promising therapeutic targets of cancer. Suppressor of cytokine signaling-1 (SOCS-1) was identified as a negative regulator of various cytokine signaling. We have previously demonstrated that overexpression of SOCS-1 showed a potent anti-tumor effect on ESCC through inhibiting of the JAK/STAT signaling pathway. This study aimed to evaluate the anti-tumor effect of SOCS-1 gene therapy using adenoviral vector (AdSOCS-1) against cancer stem-like cells in ESCC.

METHODS: ESCC cell lines were separated into aldehyde dehydrogenase (ALDH) positive and ALDH negative cells by ALDEFLOUR, and then sphere formation assays were performed. The inhibitory effects of AdSOCS-1 and AdLacZ on the sphere formation were evaluated using TE8, TE14 ESCC cell lines. The inhibition of sphere formation was also evaluated using ESCC cells obtained from ESCC PDX mice (ESCC8). Next, the expression of transcription factors associated with cancer stem-like cells in ESCC was evaluated by RT-PCR. The inhibitory effects on tumor formation by AdSOCS-1 were evaluated by tumorigenicity assay using TE8 xenograft mice.

RESULTS: In the sphere formation assay, ALDH positive TE8 cells and TE14 cells represented strong sphere formation capacity. Compared with AdLacZ infected ALDH positive cells, sphere formation of AdSOCS-1 infected ALDH positive cells was significantly inhibited. The inhibition of sphere formation was also confirmed in ESCC cells obtained from ESCC8 PDX mouse tumor tissue. Interestingly, the expression level of Sox2, a critical stem cell factor regulated by STAT3, was also decreased by AdSOCS-1. Furthermore, AdSOCS-1 decreased the tumorigenic activity of the ALDH positive ESCC tumor cells. CONCLUSIONS: A potent anti-tumor effect of SOCS-1 gene therapy on ESCC may be partially explained by inhibiting the expression of a STAT3 regulated transcription factor critical for cancer stem-like cells.

#5310

**Novel, single-tube, multiplex rhPCR technology for detection of low-frequency variants in** BRCA1 **and** BRCA2 **.**

Nicole Sponer,1 Yu Wang,2 Junzhou Wang,1 Shengyao Chen,1 Kevin Lai,1 Scott Rose,2 Patrick Lau1. 1 _Integrated DNA Technologies, Redwood City, CA;_ 2 _Integrated DNA Technologies, Coralville, IA_.

Targeted next-generation sequencing (NGS) technologies serve as critical tools for cancer diagnostics. For example, individuals with pathological BRCA1 and BRCA2 (BRCA1/2) mutations have a dramatically increased risk for certain cancers, including breast and ovarian cancer. Those mutations are either germline or somatically acquired, and play an important role in cancer risk assessment, prevention of malignancy, and management of disease. A variety of methodologies are currently available to screen for BRCA1/2 mutations. Amplicon-based sequencing technologies are prevalent due to their easy workflow but have specific challenges due to inherent limitations of PCR, such as difficulties with amplification of overlapping amplicons for contiguous coverage in a single-tube or with detection of somatic low-frequency variants due to high polymerase error rates. We developed a robust, highly multiplex rhPCR workflow to screen for BRCA1/2 mutations across their entire coding regions in a single-tube. This method allows for detection of low-frequency variants through use of unique molecular identifiers (UMIs), which correct for PCR and sequencing errors and dramatically increase specificity. This method supports a wide DNA input range (1 to 50 ng) from cell-free, formalin-fixed paraffin-embedded (FFPE), or small biopsy samples. Our rhAmpSeq™ chemistry uses a two-enzyme system coupled with uniquely designed RNA-DNA hybrid primers, which enable the amplification of overlapping, multiplexed amplicons. Thus, we eliminate the need to split precious DNA samples into multiple tubes and prevent UMI hopping. Here, we demonstrate the performance of our novel, highly specific amplicon-based technology for detection of low-frequency variants of BRCA1/2 and compare our technology to current industry standards.

#5311

Establishment of neurofibroma cells and dedifferentiated fat (DFAT) cells from tumor tissues from patients diagnosed with NF1 (Neurofibromatosis type 1).

Yoshimi Arima,1 Hiroyuki Nobusue,1 Shigeki Sakai,2 Kazuo Kishi,2 Toshiki Takenouchi,2 Kenjiro Kosaki,2 Hideyuki Saya1. 1 _Keio University, School of Medicine, Institute for Advanced Medical Research, Tokyo, Japan;_ 2 _Keio University, School of Medicine, Tokyo, Japan_.

Background: The NF1 tumor suppressor gene encodes neurofibromin and is a functional Ras GTPase-activating protein (RasGAP) that negatively regulates the Ras signaling pathway by accelerating the conversion of activated Ras-GTP to inactive Ras-GDP. NF1 gene germline mutations cause various Neurofibromatosis type 1 (NF1) symptoms, including neurofibroma development. We are developing in vitro models that recapitulate the pathological and clinical properties of neurofibromas with the aim of developing therapeutic strategies to treat patients with NF1 gene-deficient tumors.

Methods: Neurofibroma cells and dedifferentiated fat (DFAT) cells were established from NF1 patient tumors. Tissue samples were obtained during tumor resection at our hospital from patients who met NIH clinical diagnostic criteria for NF1. Whole-blood specimens were also obtained for gene analysis. All patients provided written informed consent. The institutional review board at our university approved this aspect of our study. Tumor tissues were dissociated in DMEM containing collagenase. The neurofibroma cells at the bottom of the tube and the floating stromal adipocytes were collected separately after centrifugation. To establish DFAT cells, the stromal adipocytes were placed in a culture flask filled with 20% FBS-DMEM, and then the flask was inverted and incubated at 37 °C in a humidified atmosphere of 5% CO2. The stromal adipocytes floated up through the medium and adhered to the ceiling of the flask. After 1 week, the cells were firmly attached to the ceiling and had dedifferentiated. The DFAT cells as well as the neurofibroma cells can be passaged. The DFAT cells exhibited multipotent differentiation abilities into a variety of cell types.

Results: We established neurofibroma cells and DFAT cells from NF1-associated neurofibromas. We performed flow cytometry analysis and found that the cells derived from NF1 patients expressed SOX10, S100, and CD90, all of which are expressed in Schwann cells. We identified the NF1 mutations in patients by next-generation sequencing. Peripheral blood specimens from patients 1 and 2 were positive for c.1466A>G, p.Tyr489Cys and c.3213_3214delAA, p.Ser1072Hisfs*16 mutations of NF1, respectively. We also identified NF1 mutations in the cells that we had established from tumors. In the tumor specimen of patient 1, we identified an additional somatic mutation, c.6772C>T, p.Arg2258X of NF1 gene.

Conclusions: We established NF1 gene-deficient neurofibroma cells and NF1 gene-deficient DFAT cells from the tumor tissues from NF1 patients with NF1 gene mutations. These cells may well be useful in studying the pathophysiology of NF1 gene-deficient tumors as well as cell-based drug screening to facilitate the development of new treatments.

#5312

Chd5 regulates a ribosome biogenesis switch controlling neural cell fate specification.

Alea A. Mills,1 Dong-Woo Hwang2. 1 _Cold Spring Harbor Laboratory, Cold Spring Harbor, NY;_ 2 _Stony Brook University, Stony Brook, NY_.

Cell fate specification of neural stem/progenitor cells (NSCs) is an intricate process that determines neural cell identity during development while deregulation of cell identity can pave the way for brain malignancies. While transcriptional mechanisms undoubtedly affect this process, translational mechanisms are much less understood. We identified the chromatin remodeler Chromodomain Helicase DNA binding protein 5 (CHD5) as a tumor suppressor encoded at human 1p36—a region of the genome frequently deleted in a variety of cancers including glioma. Here we show that deficiency of Chd5 causes transcriptional de-repression of multiple ribosomal subunit genes, increases protein synthesis, and expands the activated stem cell pool leading to perturbation of NSC fate. Chd5 deficient NSCs have reduced levels of the transcriptional repressive histone mark H3K27me3 during early cell fate specification, and this culminates in the generation of excessive numbers of astrocytes at the expense of neurons at later stages of differentiation. Exogenous Chd5 rescues these cell fate defects while simultaneously re-establishing H3K27me3, and inhibition of the H3K27me3-specific demethylase Utx restores appropriate cell fate specification in NSCs lacking Chd5. Our findings define a Chd5-Utx-H3K27me3 axis pivotal in ribosome biogenesis and translation during neurogenesis, highlighting the critical role of chromatin dynamics and translational control in homeostasis and malignancy in the brain.

#5313

**Frequent downregulation of** SALL3 **by genetic and epigenetic alterations is involved in progression and chemoresistance of triple negative breast cancers.**

Yosuke Matsushita,1 Masato Komatsu,1 Kazuma Kiyotani,1 Tetsuro Yoshimaru,1 Hiromu Suzuki,2 Yasuo Miyoshi,3 Mitsunori Sasa,4 Toyomasa Katagiri1. 1 _Tokushima University, Tokushima, Japan;_ 2 _Sapporo Medical University, Sapporo, Japan;_ 3 _Hyogo College of Medicine, Nishinomiya, Japan;_ 4 _Tokushima Breast Care Clinic, Tokushima, Japan_.

Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer and worse disease-specific outcomes compared with other subtypes. Because of the lack of estrogen and progesterone receptors and HER2 gene amplification, cytotoxic agents remain the mainstay of treatment for TNBC patients. Although TNBC is enriched for germline and somatic BRCA mutation, a phenotype termed BRCAness, PARP inhibitors as monotherapy failed to improve the outcome of TNBC patients. Therefore, we aimed to uncover molecular characterization in TNBC cases by whole-exome sequencing analysis of genomic DNAs from 36 Japanese patients with TNBC compared with their corresponding normal. We identified 36 genes that were recurrently mutated (>10% of cases) in TNBC cases, including TP53 and PIK3CA as described in the previous next generation sequencing analysis of TNBC cases. Remarkably, we identified a number of epigenetic-related genes involved in histone modification and DNA methylation, which were mutated in 20 out of 36 (55.6%) cases. Among them, we focused on spalt like transcription factor 3 (SALL3) gene which shows recurrent somatic mutations, and are most frequently downregulated in TNBC cases. Ectopic overexpression of the wildtype SALL3, but not the somatically mutated SALL3 into BT549 breast cancer cells, which expresses low level of SALL3 gene, caused the significant suppression of cell growth. Importantly, promoter regions of SALL3 gene was frequently hypermethylated and transcriptionally silenced in only TNBC cases, but not in other types of breast cancer by TCGA data analysis. Moreover, the expression of SALL3 was restored after treatments of 5-aza-2'-deoxycitidine in TNBC cell line, HCC1937. Notably, siRNA-mediated knockdown of SALL3 expression in BT20 cells enhanced chemoresistance against paclitaxel and docetaxel, respectively. Moreover, low expression of SALL3 was associated with significantly shorter relapse free survival. Notably, because SALL3 retains eight C2H2-type zinc finger domains which are extremely conserved in mammalian transcription factors, we hypothesized that SALL3 transcriptionally regulates genes which are involved in progression and chemoresistance of TNBC. We identified the several candidates of SALL3-taget genes by gene expression and chromatin immunoprecipitation analyses. Our findings provide the evidence of a pathophysiological role for SALL3 as a tumor suppressor which is possibly associated with progression and chemoresistance of TNBC.

#5314

Smad4 loss upregulates chemokine expression and promotes tumor inflammation and growth in head and neck cancer.

Jude Masannat, Xuefeng Wang, Biwei Cao, FeiFei Song, Ritu Chaudhary, Janis de la Iglasia, Ciaara Gorgoglione, Robbert Slebos, Christine Chung. _Moffitt Cancer Center, Tampa, FL_.

Background: Head and neck squamous cell carcinoma (HNSCC) is the 6th most common cancer worldwide. Of the patients diagnosed with HNSCC, previous findings show that SMAD4 loss is more common in recurrent HNSCC patients compared with newly diagnosed cases and is strongly associated with poor patient survival and metastases. SMAD4 is a tumor suppressor gene and is a central mediator of the transforming growth factor β (TGF-β) signaling pathway and acts as a co-SMAD which binds receptor-regulated SMADs such as SMAD2 and 3. SMAD2/3/4 complex is important for regulating downstream gene transcription that is involved in tumor invasion and metastasis.

Purpose: Since the TGF-β pathway is highly dysregulated in HNSCC, we investigate in this study how SMAD4 downregulation alters TGF-β pathway signaling and the tumor microenvironment.

Results: Using isogenic HNSCC cell lines, we visualized using immunofluorescence staining that levels of activated phospho-SMAD2 in the nucleus are correlated with expression of SMAD4. Genome-wide screening using Gene Set Enrichment Analysis (GSEA) further revealed changes in gene expression during SMAD4 knockdown in HNSCC cells. From GSEA results, the 20 most significantly altered Gene Ontology (GO) pathways were determined. It was shown that majority of these GO pathways are associated with regulating neutrophil migration and chemotaxis with an upregulation of CXC chemokine gene expression, specifically with CXCL1. In contrast, anti-tumor chemokines, such as CXCL9 and CXCL11, were downregulated during loss of SMAD4 expression. Using an orthotopic mouse model we show that in vivo, decreased expression of SMAD4 promotes tumor take and growth.

Conclusion: Collectively, these results suggest that HNSCC tumors with SMAD4 loss may have poor prognosis attributed to immunosuppressive functions of chemokine expression that acts on tumor-associated neutrophils. 
