AACR 2018 Proceedings: Abstracts 1-3027

By CTI Meeting Technology

American Association for Cancer Research 2018 Proceedings: Abstracts 1-3027

• Language: English
• Publisher: CTI Meeting Technology
• Release date: Apr 9, 2018
• ISBN: 9781370257430

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Proceedings of the AACR

Volume 59 | April 2018

Part A: Abstracts 1-3027

TABLE OF CONTENTS

TUMOR BIOLOGY:

Angiogenesis Inhibitors and Stimulators

Breast Cancer Metastasis

Dormancy and Aging: The Influential Microenvironment

Mechanisms Underlying Metastasis 1

Role of the Innate Immune System in Tumorigenesis

Targeting Cancer Stemness

The Mechanical Microenvironment in Tumorigenesis

Tumor Heterogeneity 1

EPIDEMIOLOGY:

Genetic Contributions to Cancer Epidemiology 1: GWAS and Pathway and Candidate Gene Studies

PREVENTION RESEARCH:

Chemoprevention of Cancer

BIOINFORMATICS AND SYSTEMS BIOLOGY:

Integrative Cancer Biology 1

MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

BCL-2 Family and Mitochondrial Apoptosis

Cancer Predisposition and Synthetic Lethality

Chromatin Structure and Function

Emerging Concepts

Functional Genomics

High-Throughput Sequencing 1

Metabolism and Cellular Functions 1

MicroRNA Regulation in Cancer Biology

Noncoding RNAs as Oncogenes and Tumor Suppressors

Oncogenes and Tumor Suppressor Genes

CLINICAL RESEARCH:

Biomarker Discovery 1

Clinical Risk Factors and Outcomes in Solid Tumors / Survivorship Research

Immune Response to Therapies 1

Modifiers of the Tumor Microenvironment 1

Molecular Diagnostics 1: Cytogenetics, Clinical Molecular Genetics, and Clinical Imaging

Radiation Oncology

CANCER CHEMISTRY:

Chemical and Structural Biology

IMMUNOLOGY:

Inflammation, Immunity, and Cancer

Response and Resistance to Immune Checkpoint Blockade

Vaccines 1

EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

Antibody-Drug Conjugates: Agents and Technology

Biomarker Identification and Novel Methods

Experimental Agents and Combinations for Hematologic Malignancies 1

Growth Factors and Protein Kinases as Targets

Modulators of Ionizing Radiation and Other Radiotherapeutics

New Therapy with New Mechanism of Action

Strategies to Reversing Drug Resistance

CLINICAL RESEARCH:

Pediatric Cancer: Poster Discussion

BIOINFORMATICS AND SYSTEMS BIOLOGY:

Computational Methods and Resources for Cancer Research

CANCER CHEMISTRY:

Molecules Addressing Challenges in Drug Discovery

CLINICAL RESEARCH:

Liquid Biopsy 1

ENDOCRINOLOGY:

Novel Roles of Steroid Hormone Receptors

EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

Novel Therapeutic Agents and Combinations

IMMUNOLOGY:

Engineering Immunotherapies for Anticancer Activity

MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

Cell Signaling Pathways

Epigenetic Mechanisms of Tumor Progression

MULTIDISCIPLINARY:

Radiation Science

TUMOR BIOLOGY:

Patient-Derived Models of Cancer: Present and Future

Building the Microenvironment through Crosstalk

MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

Targeting the Cell Cycle: Mechanism and Therapy-- Poster Discussion

TUMOR BIOLOGY:

Adaptation and Checkpoints in Tumorigenesis

Advances in the Generation and Analysis of Patient-Derived Xenografts

Biomarkers of Metastasis

Mechanisms Underlying Metastasis 2

Regulation of Stemness in Cancer

Translational Therapeutics in Cancer Models 1

Tumor Heterogeneity 2

EPIDEMIOLOGY:

Descriptive Epidemiology of Cancer

Genetic Contributions to Cancer Epidemiology 2: Next-Generation Sequencing and Familial Cancers

PREVENTION RESEARCH:

Molecular Mechanisms and Targets for Cancer Prevention

BIOINFORMATICS AND SYSTEMS BIOLOGY:

Application of Bioinformatics to Cancer Biology 1

Integrative Cancer Biology 2

MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

Autophagy

DNA Repair and Damage Response

Epigenetic Therapy

Genotype/Phenotype Correlations

High-Throughput Sequencing 2

Metabolic Pathways

Mitosis and Telomeres

Navigating the Cancer Chromatin Landscape

Novel Biomarkers and Drivers of the Cancer Transcriptome

Targeting the Cell Cycle: Mechanism and Therapy

CLINICAL RESEARCH:

Adoptive Cell Therapy 1

Biomarker Discovery 2

Liquid Biopsy 2

Molecular Diagnostics 2: Laboratory Correlates for Targeted Agents and Preclinical and Clinical Trials

Pediatric Cancer

Special Populations / Biostatistics in Clinical Trials

CANCER CHEMISTRY:

Target Based Drug Discovery

IMMUNOLOGY:

Immune Checkpoints 1

Immune Response to Therapies 2

Modifiers of the Tumor Microenvironment 2

Therapeutic Antibodies, Including Engineered Antibodies 1

ENDOCRINOLOGY:

Growth Factors and Nuclear Receptor Signaling

EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

Acquired Resistance Against Molecularly Targeted Therapies

Cell Cycle, Drug Resistance, and Combinations

Experimental Agents and Combinations for Hematologic Malignancies 2

Identification of Molecular Drug Targets

New Targets 1

Therapeutic Resistance: New Targets and New Inhibitors

TUMOR BIOLOGY:

Epithelial Cancer Stem Cell Biology

Epithelial-to-Mesenchymal Transition and EMT and TGFbeta in Metastasis

Novel Mechanisms Regulating Angiogenesis

Pediatrics 1: Genomics, Epigenetics, and Biomarkers

Surveying the Tumorigenic Microenvironment

The Metastatic Microenvironment

Translational Therapeutics in Cancer Models 2

Tumor Heterogeneity 3

EPIDEMIOLOGY:

Screening, Early Detection, and Risk Prediction

PREVENTION RESEARCH:

Biomarkers, Intervention, and Early Detection for Cancer Prevention

BIOINFORMATICS AND SYSTEMS BIOLOGY:

Application of Bioinformatics to Cancer Biology 2

New Software for Data Analysis

MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

CDK and CDK Inhibitors: Mechanism and Therapy

Ferroptosis, Metabolism, and Cancer Cell Death

Gene Discovery

Kinases and Phosphatases

Metabolic Regulation

Metabolism and Cellular Functions 2

Noncoding RNAs and Cancer

Nuclear Oncoproteins and Tumor Suppressor Genes

Receptors and Growth Factors

Transcription Factor Crosstalk and Aberrant Transcriptional Control

CLINICAL RESEARCH:

Adoptive Cell Therapy 2

Biomarker Discovery 3

Biomarkers of Therapeutic Response in Clinical Trials

Novel Immunotherapies and Laboratory Models in Pediatric Cancer

Prognostic Biomarkers

CANCER CHEMISTRY:

Antitumor Agents

Cancer Biology Insights Emerging from Proteomic Investigations

IMMUNOLOGY:

Immune Checkpoints 2

Immune Mechanisms Invoked by Therapies 1

Therapeutic Antibodies, Including Engineered Antibodies 2

EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

Design, Structure/Activity, and Modeling

Modulation of DNA Damage and Repair

Monitoring the Evolution of Therapeutic Resistance

New Agents and New Targets

New Targets 2

Novel Experimental Combinations

BIOINFORMATICS AND SYSTEMS BIOLOGY:

Systems and Computational Biology: Poster Discussion

CLINICAL RESEARCH:

Molecular Predictors of Response, Mediators of Resistance, Mechanisms of Action, Pharmacodynamic Markers, and Novel Disease Subsets

Use of Liquid Biopsies in Clinical Trials

EPIDEMIOLOGY:

Genetic and Molecular Epidemiology of Cancer Risk and Prognosis

EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

Identification of Biomarkers and Molecular Drug Targets

IMMUNOLOGY:

Defining New Immunotherapeutic Targets through Deep Molecular Characterization

MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

Drugging the Undruggable: Using Synthetic Lethality and Other Approaches to Develop New Treatment Strategies

Cancer Epigenetics and Epigenetic Therapy

Sampling the Cancer Genome and the Epigenome: Opportunities and Exquisite Vulnerabilities

MULTIDISCIPLINARY:

Cancer Survivorship and Disparities

TUMOR BIOLOGY:

Deciphering Cancer Mechanisms in Animal Models

Organ-Specific Metastasis

Sunday, April 15, 2018

TUMOR BIOLOGY:

Angiogenesis Inhibitors and Stimulators

#1

A kinome-wide CRISPR screen reveals BET inhibition-associated endothelial cell resistance to anti-angiogenic therapy.

Michael Y. He, Michael M. Halford, Marc G. Achen, Steven A. Stacker. Peter MacCallum Cancer Centre, Melbourne, Australia.

Angiogenesis inhibition is a useful strategy for treating cancer. However, the efficacy of anti-angiogenic therapy (AAT) in clinical oncology has been limited largely by highly variable patient response and the inevitable occurrence of resistance. The modest patient benefits have underscored a pressing need for qualified biomarkers and better knowledge of resistance mechanisms. Blood endothelial cells (ECs) are one of the main targets of AAT in the tumor microenvironment. Hence, understanding the EC response to AAT will provide insight into tumor response. To identify molecular modifiers of the EC response to AAT (in this study, bevacizumab, a humanized neutralizing anti-VEGF-A monoclonal antibody), we developed a high-throughput genetic screening platform. This involved a three-dimensional microcarrier-based culture system, CRISPR-Cas9-driven gene loss-of-function (LOF) and VEGF-A-dependent serum-free culture conditions for response modifiers. A pooled kinome-wide CRISPR-Cas9-based screen of 763 genes (with four single guide RNAs/sgRNAs targeting each gene) identified 18 candidate genes that upon LOF (represented by sgRNAs) were significantly enriched or depleted in the bevacizumab versus control treatment arm (P ≤ 0.005, FDR ≤ 0.3). Further candidate evaluation using siRNAs validated six genes whose knockdown conferred EC resistance or sensitization to bevacizumab (P < 0.05). Of these, knockdown of BRD2 or BRD3, which encode the epigenetic reader bromodomain-containing protein 2 or 3 (BRD2 or BRD3), respectively, conferred EC resistance to bevacizumab. The bromodomain and extraterminal domain (BET) inhibitors JQ1 and I-BET762, which selectively target the BET family of proteins (BRD2, BRD3, BRD4 and BRDT), reproduced the results of BRD2 or BRD3 LOF, with a more prominent phenotype (P < 0.05). Drug dose-response assessment indicated an anti-angiogenic effect of BET inhibitors regardless of the presence of bevacizumab. This inhibitory effect was unexpectedly attenuated when cells were co-treated with bevacizumab under VEGF-A-dependent conditions. Experiments to investigate the mechanistic basis for this phenotype of resistance are ongoing and include differential gene expression analysis using RNA-Seq and in vivo evaluation. Applying a non-biased approach to identify molecular modifiers of the EC response to AAT, we demonstrate in this study that BET inhibition is unexpectedly associated with resistance to bevacizumab, despite BET inhibition alone having an anti-angiogenic effect. These observations prompt further evaluation of epigenetic regulation in tumor angiogenesis, particularly in the context of interaction between BET inhibition and VEGF blockade. Clinically, these findings may facilitate development of potential predictive and/or response biomarkers and strategies to overcome resistance to AAT and/or BET inhibitors.

#2

CXCR2+ tumor cells mediate vascular mimicry driving anti-angiogenic therapy (AAT) resistance in glioblastoma (GBM).

Kartik P. Angara, Thaiz F. Borin, Mohammad H. Rashid, Roxan Ara, Bhagelu R. Achyut, Ali S. Arbab. Augusta University, Augusta, GA.

Glioblastoma (GBM) is a hypervascular and hypoxic neoplasia of the central nervous system with an extremely high rate of mortality. Owing to its hypervascularity, anti-angiogenic therapies (AAT) have been used as an adjuvant to the traditional surgical resection, chemotherapy, and radiation to normalize blood vessels and control abnormal vasculature. The benefits of AAT have been transient, and evidence of relapse exemplified in the progressive tumor growth following AAT reflects development of resistance and alternative neovascularization mechanisms in these resilient tumors to counter the AAT therapy insult. Vascular mimicry (VM) is the uncanny ability of tumor cells to acquire endothelial-like properties and lay down vascular patterned networks reminiscent of host endothelial blood vessels. The VM channels serve as an irrigation system for the tumors to meet with the increasing metabolic and nutrient demands of the tumor. In our current studies to understand the tumor-inherent mechanisms of AAT resistance, we identified a crucial pro-migratory and pro-angiogenic chemokine, CXCL8 or IL-8, to be highly upregulated in the GBM tumors treated with AAT. AAT-treated groups had significantly higher populations of CXCR2+ stem and endothelial-like subpopulations and these cells lined the VM-like vascular structures carrying functional RBCs in the tumors. These stem cell-like and endothelial-like populations were decreased following treatment with HET0016 or SB225002. Furthermore, knocking down CXCR2 led to smaller tumor size in the animals and improperly developed vascular structures without CXCR2+ GBM cells lining them. Also, HET0016 and SB225002 disrupt the tube-forming capability of U251 GBM cells in an in vitro Matrigel angiogenesis assay. This confirms our hypothesis that CXCR2+ GBM cells initiate VM and contribute to AAT resistance in GBM. Our present study suggests that tumor cell autonomous IL-8/CXCR2 pathway contributes to VM-mediated AAT resistance in GBM and that HET0016 and SB225002 have a great potential to target therapeutic resistance and can be combined with other chemotherapeutic agents in preclinical and clinical trials.

#3

Metronomic chemotherapy with cyclophosphamide (Cy) and the repositioned drug losartan (Los) for the treatment of M-234p triple negative murine mammary adenocarcinoma.

Julian Guercetti,¹ Leandro E. Mainetti,¹ M.Carolina Grillo,¹ Antonela Del Giúdice,¹ Maria V. Baglioni,¹ Juan M. Cáceres,¹ Ainelén Arboatti,² Daniel Francés,² Viviana R. Rozados,¹ Maria J. Rico,¹ O.Graciela Scharovsky¹. ¹Facultad de Ciencias Médicas, UNR, Rosario, Argentina; ²Facultad de Ciencias Bioquímicas y Farmacéuticas, UNR, Rosario, Argentina.

Metronomic chemotherapy (MCT) refers to the chronic, equally spaced, delivery of low doses of chemotherapeutic drugs, without extended interruptions. Drug repositioning in oncology refers to the use of drugs formulated for other indications that showed antitumor potential. Los is an antagonist of angiotensin II receptor used to treat hypertension. We aim to study the combined effect of metronomic Cy+Los on M-234p tumor. Female BALB/c mice were orthotopically challenged with M-234p (day 0) and distributed, on day 8, into 4 groups (n=5-6/group) treated as follows, GI: Control, without treatment; GII: Cy 25mg/kg/day in the drinking water; GIII: Los 200mg/kg/day in the drinking water; GIV: Treated as GII+GIII. Mice were weighted, and tumor volume measured 3 times/week. When tumors were exponentially growing, mice were euthanized, tumors excised, and blood samples taken for immunohistochemistry, western blot and flow cytometry. GIV showed tumor growth inhibition compared to GI, GII and GIII and, as a consequence, higher survival (P<0.005). Also, GIV showed 60% (3/5) of complete tumor regressions, without relapses. No weight losses or sings of toxicity were observed. IHC analysis showed a lower N° of Ki67+ cells in GIV (P<0.05). Moreover, a significant increase of apoptosis in GIV vs Control was shown by TUNEL assay (P<0.05). No differences in circulating CD4+, CD8+ and Treg cells were shown by flow cytometry but, a marginally significant increase in Th17 cells in GII and GIV was seen. To share some light on the effect of the therapy on the cancer associated fibroblasts, the protein αSMA was measured by western blot; the expression of this marker was significantly lower in GIV vs GI (P<0.05). To evaluate the anti-metastatic effect of the therapy, mice were inoculated i.v. with 5x10⁵ M-234p cells. On day 3, mice were distributed in the same experimental groups. When the first mouse showed signs of metastatic disease, all of them were euthanized, lungs excised and stained to highlight metastasis. The N° of lung metastasis was significantly lower in GII and GIV. No weight losses or any other sing of toxicity were observed. These results clearly show that metronomic chemotherapy with a combination of Cyclophosphamide and Losartan, administered as an intervention strategy, can inhibit, not only the growth of a triple negative mammary adenocarcinoma, causing permanent regressions, but also its metastasis, while being devoid of toxicity. Those effects would be achieved, at least partially, by inhibition of tumor proliferation, increase of apoptosis and modification of tumor microenvironment through tumor fibroblasts. The characteristics and the efficacy of the therapeutic schedule herein utilized suggest its implementation at the clinical setting in the near future.

#4

Antitumor activity of DLX1008, a single chain antibody fragment binding to VEGF-A, in in vivo preclinical models of Kaposi sarcoma and glioblastoma.

Anthony B. Eason,¹ Sang-Hoon Sin,¹ Emese Szabó,² Douglas J. Phillips,³ Miriam Droste,³ Abdijapar Shamshiev,³ Dirk P. Dittmer,¹ Michael Weller². ¹The University of North Carolina at Chapel Hill, Chapel Hill, NC; ²University Hospital Zurich and University of Zurich, Zurich, Switzerland; ³Cell Medica Switzerland AG, Schlieren, Switzerland.

Background:

Angiogenesis mediated by vascular endothelial growth factor (VEGF) is a hallmark of several cancers. Efficacy of VEGF inhibitors could be increased by improving affinity or tumor penetration, for example by reducing the size of the drug. Recently, the exceptionally stable anti-VEGF single chain variable fragment (scFv) brolucizumab (RTH258, ESBA1008, Novartis) completed phase 3 clinical trials in ophthalmology and was shown to be more efficacious than the larger aflibercept. The same molecule, named DLX1008, is currently in pre-clinical development in gliobastoma and Kaposi sarcoma (KS) by Cell Medica. KS is the most angiogenic cancer known to date. Carcinogenesis is induced by KS-associated herpesvirus (KSHV) and compromised of endothelial cells expressing high levels of VEGFR. KS is unique because angiogenesis (growth of non-tumor endothelial cells) as well as proliferation of the primary tumor cells are dependent on VEGF. No VEGF antagonist has yet been demonstrated to unequivocally improve overall survival in either KS or glioblastoma, which may be due to limited tumor penetration.

Methods:

Binding affinity to VEGF-A was determined by kinetic exclusion assay (KinExA).

For in vivo studies, we used the L1T2 model of KS, whereby KSHV-positive human cells (L1T2/ ATCC® VR-1802™) are implanted subcutaneously onto NGS mice, and the U87 model of glioblastoma, where human glioblastoma cells (U87/ ATCC® HTB-14™) are implanted subcutaneously or intracranially onto SWISS nude mice. Tumor growth was followed by caliper measurements and pathology evaluated by histochemistry or imaging.

Results:

DLX1008 binds to VEGF-A165 with a KD of 1.05 pM. Bevacizumab binds with a KD of 32.4 pM. Hence, the affinity of DLX1008 to human VEGF is around 30-fold tighter than that of bevacizumab.

DLX1008 reduced growth of L1T2 cells in vivo (p ≤ 0.007 by mixed effects model, n = 10) compared to vehicle control at 15 mg/kg once daily 5x/week and (p≤0.001, n = 10) in a second biological repeat. Imaging showed that the tumor died from the inside out in response to DLX1008. DLX1008 reduced the growth of U87 cells subcutaneously implanted in vivo (p ≤ 0.0021 by ANOVA, n = 10) and improved median survival of mice intracranially implanted with U87 cells (p ≤ 0.00026) compared to control scFv at 50 mg/kg twice daily. Staining of intracranial U87 tumors showed decreased CD31-positive blood vessel density as well as decreased p-VEGFR1 immunoreactivity in vascular structures and glioma cells.

Conclusions:

The high affinity, highly stable scFv DLX1008 shows highly reproducible anti-tumor activity in in vivo preclinical models of KS and glioblastoma. Efficacy was attributable to reduced angiogenesis. Its small size offers advantages in tissue penetration and distribution as compared to bevacizumab. DLX1008 is an excellent antibody to advance into clinical trials.

#5

The histone acetyltransferase inhibitor CPTH6 impairs tumor angiogenesis acting on both endothelial and cancer cells.

Marta Di Martile,¹ Marianna Desideri,¹ Simonetta Buglioni,¹ Carla Azzurra Amoreo,¹ Daniela Trisciuoglio,² Donatella Del Bufalo¹. ¹Regina Elena National Cancer Institute, Rome, Italy; ²National Research Council, Rome, Italy.

Protein acetylation is typically catalyzed by enzymes with histone acetyltransferase (HATs) or histone deacetylase (HDACs) activity. To date, it has been identified the involvement of protein acetylation in different tumorigenic signaling events, including angiogenesis. The biological process of neo-vasculature formation from pre-existing blood vessels is widely considered to be an essential process to sustain tumor growth as well as to provide a route for tumor cell metastatization. In this context, the role exerted by HDACs in tumor angiogenesis is well known, whilst the role of HATs is largely unknown. Among molecules with HAT inhibitory activity, the thiazole derivative CPTH6 has been characterized by our group for its antitumor activity in different tumor models, including non-small cell lung cancer (NSCLC). In this study, we assessed the effect of CPTH6 on angiogenesis-related properties of both endothelial and NSCLC cells. The human umbilical vein endothelial cell (HUVEC) and H1299 NSCLC cell lines were used. HUVEC and H1299 morphogenesis was analyzed by plating cells on matrigel and evaluating their ability to organize capillary-like structures. The effect of CPTH6 on protein acetylation was assessed by WB analysis. Transwell supports were employed to evaluate HUVEC migration and invasion. Human Angiogenesis Antibody Array was used to test the conditioned media derived from CPTH6-treated H1299 cells. C57/BL6 and nude mice were used to perform matrigel plug assay and to evaluate tumor growth, respectively. IHC analysis of CD31 was employed to evaluate the number of intra-tumor vessels in tumor xenografts. The HAT inhibitor CPTH6 affected some endothelial cell functions in vitro. In particular, CPTH6 impaired HUVEC invasion, migration and differentiation abilities at doses that did not alter proliferation. Although CPTH6 did not affect histone H3 acetylation, it slightly reduced α-tubulin acetylation in HUVEC. In addition, CPTH6 decreased the neovascularization in vivo, as evidenced by the impairment of the VEGF-induced vascularization of matrigel plugs. Interestingly, CPTH6 affected also the angiogenesis-related properties of cancer cells. In particular, this compound reduced the ability of H1299 to organize capillary like structures and, conditioned media derived from CPTH6-treated H1299 cells, impaired HUVEC morphogenesis. Accordingly, CPTH6 reduced the secretion of some pro-angiogenic factors (VEGF, EGF, ANG, TIE-2, TNF-α) and, at the same time, increased the release of anti-angiogenic ones (Endostatin, PLG). Finally, in H1299-tumor xenografts, CPTH6 decreased significantly the number of intra-tumor vessels, even though it did not impair tumor growth. Overall, this study adds information to the role of HATs in tumor angiogenesis, and proposes HAT inhibition as an attractive target for antiangiogenic therapy of NSCLC.

#6

Role of Notch signaling in bevacizumab-induced vascular normalization in glioblastoma.

Norihiko Saito, Kazuya Aoki, Nozomi Hirai, Satoshi Fujita, Haruo Nakayama, Morito Hayashi, Takatoshi Sakurai, Satoshi Iwabuchi. Toho University Ohashi Medical Center, Tokyo, Japan.

Tumor angiogenesis occurs in the setting of a defective vasculature, which is associated with increased vascular permeability and enhanced tumor permeability. Bevacizumab is used to treat malignant glioma and was found to reduce microvascular density and prune abnormal tumor microvessels. Using electron microscopic observation of two autopsy cases, we investigated the effects of blood vessel normalization in glioblastomas treated with bevacizumab. Notch-1 and SMA immunostaining were used to compare initial surgical specimens with postmortem specimens obtained after bevacizumab treatment. Postmortem samples showed marked proliferation of SMA-positive cells (pericytes) in tumor vessels and marked proliferation of Notch-1–positive cells around vessels. Electron microscopic images confirmed the presence of pericytes surrounding the vascular endothelium. These findings suggest that bevacizumab treatment promotes vascular normalization by recruiting mature pericytes. Next, we investigated the effects of bevacizumab on VEGF inhibition in glioma stem cells. Bevacizumab treatment attenuated activation of VEGFR2 and increased Notch signaling expression. VEGF inhibition by bevacizumab treatment attenuated proliferation and self-renewal of glioma stem cells and induced endothelial and pericyte differentiation. In tumor angiogenesis, vascular endothelial growth factor induces sprouting angiogenesis and recruitment of vascular endothelial cells such as tip cells, stalk cells, and phalanx cells. Fully mature phalanx cells are in close contact with pericytes. Our results suggest that bevacizumab treatment induces glioma stem cells to differentiate to endothelium and pericytes. These mechanisms might be important in normalizing tumor vasculature after bevacizumab treatment and could be useful in improving the effectiveness of current glioma therapy.

#7

Platinum resistance in epithelial ovarian cancer is dependent on a PDGFR alpha-VEGF-A signalling mechanism that activates downstream angiogenesis pathways.

Aya El Helali,¹ Nuala McCabe,¹ Christopher Steele,² Lara Dura Perez,² Christina L. O'Neill,³ Naomi Dickson,² Niamh McGivern,¹ Caolan Harkin,¹ Andrena McCavigan,² Reinhold J. Medina,³ Laura A. Knight,² Stephen McQuaid,⁴ Jacqueline A. James,⁴ Caroline O. Michie,⁵ Charlie Gourley,⁵ W Glenn McCluggage,⁶ Denis P. Harkin,² Richard H. Wilson,¹ Alan W. Stitt,³ Richard D. Kennedy¹. ¹Centre for Cancer Research and Cell Biology, Belfast, United Kingdom; ²Almac Diagnostics, Belfast, United Kingdom; ³Centre for Experimental Medicine, Belfast, United Kingdom; ⁴Northern Ireland Molecular Pathology Laboratory, Belfast, United Kingdom; ⁵Nicola Murray Ovarian Cancer Research Centre, Edinburgh, United Kingdom; ⁶Department of Pathology, Royal Group of Hospitals Trust, Belfast, United Kingdom.

Introduction:

40-45% of patients with High Grade Serous Ovarian Cancer (HGSOC) will eventually relapse with platinum resistant disease. Tothill et al and TCGA are two independent gene expression datasets which have demonstrated the presence of a mesenchymal molecular subgroup, characterised by upregulation of angiogenesis regulating genes. Angiogenesis is known to be an integral pathological feature of HGSOC and anti-angiogenics have dominated the field of drug development in EOC. However, despite this, anti-angiogenic agents have failed to demonstrate a significant impact on overall survival (OS) benefit. In this study, we asked if platinum resistance could be associated with an improved response to anti-angiogenic agents and what the underlying biological rationale for this could be.

Methods:

A meta-analysis of 14 phase II and III clinical trials in EOC were used to investigate the association between platinum resistance and response to anti-angiogenic agents. In addition, we analysed gene expression in 12 matched pre- and post-chemotherapy EOC samples. Novel isogenic cisplatin-resistant HGSOC cell lines were established to study the development of an angiogenic phenotype. Further studies were performed in novel ascites-derived primary cell lines from HGSOC patients with known outcomes following platinum-based chemotherapy.

Result:

In the clinical trial meta-analysis, an OS benefit for antiangiogenics was observed in platinum-resistant disease (p=0.029), whilst platinum-sensitive EOC only derived progression free survival (PFS) (p=<0.0001) benefit and not OS (p=0.125). In the 12 matched pairs of patient samples, post-platinum samples had a higher micro-vessel density (MVD) relative to their paired treatment-naïve sample (p= 0.0001). Additionally, an in vivo angiogenesis matrigel plug assay demonstrated that cisplatin-resistant EOC cell lines were associated with an increase in MVD (p=<0.0001). MVD was reduced in the platinum-resistant cells following treatment with bevacizumab (p=0.001). Ascites-derived primary cells established from platinum-resistant patients demonstrated overexpression of VEGF-A, consistent with stimulating angiogenesis. Gene expression analysis of pre- and post-platinum paired samples identified that PDGFRα (p=0.007) and PDGFRβ (p=0.005) were differentially expressed in the post-platinum therapy samples. In vitro validation in the platinum-resistant cell lines demonstrated that VEGF-A expression was regulated by PDGFRα.

Discussion:

We have demonstrated that previous platinum therapy for EOC is associated with an increase in tumor PDGFα and VEGF-A expression, correlating with a response to anti-angiogenic therapies. This data suggests that platinum therapy resistance may inform the selection of EOC patients for novel antiangiogenic therapies in future clinical trials.

#9

Efficacy and functional study of tetraarsenic oxide as an anticancer drug in cervical cancer cell lines and cervical cancer patient-derived xenograft mouse.

Jeong-Won Lee, Jae Ryoung Hwang, Young-Jae Cho, Ji-Hye Kim, Ji Yoon Ryu, E-Sun Baik. Samsung Medical Center, Seoul, Republic of Korea.

Background: Arsenic compound has been used as a medicine in China and has been studied to treat acute promyelocytic leukemia, hepatocellular carcinoma, and melanoma. Tetraarsenic oxide (TAO; As4O6) is an inorganic arsenic compound that has been demonstrated to inhibit angiogenesis and cell growth in cervical cancer cells. In this study, we demonstrated anti-cancer effect of TAO in cervical cancer patient-derived xenograft (PDX) mouse model and the functional mechanism related with cervical cancer cell death.

Materials & Methods: We used SiHa, Caski, and HeLa cells for cervical cancer cells and HUVEC cells to study functional mechanism of TAO in an in vitro. MTT assay was performed to assess cell death and MMP-2-specific ELISA assay was used to detect MMP-2 expression. To establish the patient-derived xenograft (PDX) mouse model of cervical cancer, surgical patient tumor specimens were reduced to small pieces (less than 2-3 mm), implanted into the subrenal capsules of the left kidneys of BALB/C nude mice, and propagated by serial transplantation. The PDX model used in this study was generated with a tumor that was histologically defined as a FIGO stage Ib1 invasive squamous cell carcinoma. The patient was a 46-year-old woman who received primary debulking surgery followed by radiation therapy for 28 times.

Results: We measured IC50 of TAO in cervical cancer and HUVEC cells. IC50 of TAO in SiHa, Caski, and HUVEC was around 3 uM and it was about 0.6 uM in HeLa cells. Although TAO inhibited MMP-2 expression, an important protein for angiogenesis, in both cervical cancer cell lines and HUVEC cells, the functional mechanism was different in between cervical cancer cells and HUVEC cells. TAO inhibited Akt activation in SiHa, Caski, and HeLa. However, TAO had no effect on activation of Akt in HUVEC cells. TAO inhibited expression of VEGF receptor 2 in HUVEC cells but not in cervical cancer cells. We found that TAO inhibited autophagy determined by p62 expression level in cervical cancer cells but not in HUVEC cells. Cervical cancer PDX mouse study demonstrated that TAO inhibited tumor growth.

Conclusions: TAO inhibited cell growth of cervical cancer cells and HUVEC. The functional signaling pathway of TAO-induced cell death might different in between cervical cancer cells and endothelial cell, HUVEC. TAO inhibited tumor growth in cervical cancer PDX mouse probably through inhibition of cancer cell growth and inhibition of angiogenesis via inhibition of endothelial cell growth. These results suggest that TAO may be considered as a novel therapeutic compound for the treatment of cervical cancer.

#10

Evaluation of metronomic chemotherapy regimens in preclinical orthotopically implanted colon cancer models, and in patient-derived xenografts.

Jose Lopez,¹ Paloma Valenzuela,¹ Valerie Gallegos,¹ Karla Parra,¹ Valeria Rolih,¹ Diana Gonzalez Garcia,¹ Joel Martinez,¹ Urban Emmenegger,² Guido Bocci,³ Robert S. Kerbel,² Giulio Francia¹. ¹UT El Paso, El Paso, TX; ²University of Toronto, Toronto, Ontario, Canada; ³University of Pisa, Pisa, Italy.

Recent advances for the treatment of late stage colon cancer include a metronomic capecitabine plus bevacizumab maintenance regimen as reported for the CAIRO3 phase 3 clinical trial (Simkens et al, Lancet: 2015; 385: 1843-52) . We previously reported a phase 2 trial evaluating metronomic cyclophosphamide (CTX) plus UFT (a 5-fluorouracil prodrug) and celecoxib in gastrointestinal cancer patients (Allegrini et al, Angiogenesis: 2012; 15, 275-286). We also reported the preclinical evaluation of metronomic oral topotecan in mouse models of colon cancer (Hackl et al, Gut: 2013; 62, 259-71). Here we report our studies using orthotopic (intracecal) implantation of human HT29/luc/hCG colon cancer cells in SCID mice. Disease progression was noninvasively assessed using the transfected human chorionic gonadotropin (hCG), whose levels in the mouse urine correlate with tumor burden - and this was coupled with luciferase (luc) bioimaging of the mice. These implanted tumor models were used together with patient derived xenografts (PDX), to evaluate metronomic regimens of etoposide (80mg/kg/day, p.o.), gemcitabine (given i.p.; at either 120mg/kg every 3 days, or at the much lower 1mg/kg/day), or CTX given as a monotherapy (20mg/kg/day given via the drinking water) or combined with neutralizing antibodies against VEGF or EGFR. Our results indicate; 1) effective anti-tumor activity of daily etoposide, and of 2) daily gemcitabine (as well as when the gemcitabine was given every 3 days), and that 3) orthotopic HT29/luc/hCG colon tumors are weakly responsive to CTX or CTX plus targeted agents compared to the same tumors implanted subcutaneously or intraperitoneally. The effectiveness of metronomic etoposide was confirmed in a colon PDX model in NSG mice (n=5 mice/group; p<.05) - obtained from Jackson labs. Notably ovarian cancer and breast cancer PDX models did not respond to etoposide therapy (n=5/group), indicating the response may be tumor specific. Collectively, these results highlight the importance of advanced disease models to confirm sometime overly optimistic experimental therapeutic results from subcutaneously or intraperitoneally implanted tumor models. They also demonstrate the promising anti-tumor activity of metronomic etoposide and gemcitabine regimens for colon cancer, and they lead us to advocate the concerted use of both implanted xenografts and PDX for the testing of new therapeutic strategies for the treatment of colon cancer.

#11

Antitumor activity of lenvatinib in the renal cell carcinoma cell line RENCA model resistant to a VEGF specific inhibitor.

Kenji Ichikawa, Saori Watanabe Miyano, Yukinori Minoshima, Junji Matsui. Tsukuba Research Laboratories, TSUKUBA-SHI, IBARAKI, Japan.

Anti-vascular endothelial growth factor (VEGF) therapies have been in clinical use to treat patients with multiple types of cancers, especially in renal cell carcinoma (RCC) and hepatocellular carcinoma (HCC). However, benefits of anti-VEGF therapies are still limited because of the intrinsic/acquired resistance to the treatment. Lenvatinib mesilate (lenvatinib) is an oral multiple receptor tyrosine kinase (RTK) inhibitor that suppresses the kinase activities of VEGF receptors (VEGFR1-3), in addition to other proangiogenic and oncogenic pathway-related RTKs including fibroblast growth factor receptors (FGFR1-4), the platelet-derived growth factor receptor (PDGFR) α, KIT, and RET. Lenvatinib has been approved as a monotherapy for the treatment of radioiodine-refractory differentiated thyroid cancer and in combination with everolimus for the treatment of patients with RCC treated with one prior anti-VEGF therapy in the US and EU. In this study, we established an anti-VEGF treatment resistant preclinical mouse tumor model. We also examined whether lenvatinib, which targeted multiple receptor tyrosine kinases, showed anti-tumor activity in this model. To establish model resistant to anti-VEGF treatment, an expression vector encoding VEGFR-Fc protein (VEGF decoy) was introduced into a mouse RCC cell line RENCA. First, we confirmed the RENCAVEGFR-Fc transfectants secreted VEGF decoy, which selectively interacted with recombinant VEGF protein but not with ligands to other RTKs in in vitro. Tumor growth in vivo of RENCAVEGFR-Fc was significantly slower than that of RENCAMock. RENCAMock transfectants took 16 days to reach a mean tumor volume (TV) of 500 mm³ whereas RENCAVEGFR-Fc transfectants required 72 days to reach same TV. Furthermore, 72 days after inoculation of RENCAVEGFR-Fc transfectants, we resected tumor tissues and confirmed the expression of VEGF decoy protein. Tumor microvessel density was decreased in resected RENCAVEGFR-Fc tumor compared to the same size of RENCAMock tumor. From the above results, we characterized RENCAVEGFR-Fc tumor was resistant to VEGF decoy. Finally, anti-tumor activity of lenvatinib was challenged against RENCAMock or RENCAVEGFR-Fc tumors. When tumor volume reached approximately 300mm³, 10 mg/kg of lenvatinib was orally administered once daily for 14 days. Treatment of lenvatinib led to almost complete inhibition of tumor growth in both RENCAMock and RENCAVEGFR-Fc models. In conclusion, we developed a mouse RENCAVEGFR-Fc model resistant to anti-VEGF treatment, and lenvatinib showed anti-tumor activity in the resistant model as with RENCAMock model. Additional preclinical studies will be investigated to elucidate the signaling pathways associated with resistance to anti-VEGF treatment, in which lenvatinib may inhibit such as FGFR1-4, PDGFRα, KIT and RET.

#12

Antitumor activity of lenvatinib in combination with everolimus or an anti-PD1 antibody in preclinical RCC models.

Yusuke Adachi, Takayuki Kimura, Masahiro Matsuki, Junji Matsui, Yasuhiro Funahashi. Eisai Co., Ltd., Tsukuba-shi, Ibaraki, Japan.

Lenvatinib mesilate (lenvatinib) is an oral multiple receptor tyrosine kinase (RTK) inhibitor that selectively inhibits the kinase activities of VEGFR1-3, in addition to other proangiogenic and oncogenic pathway-related RTKs including FGFR1-4; PDGFRα; KIT; and RET. Lenvatinib plus everolimus for advanced renal cell carcinoma (RCC) after one prior anti-VEGF therapy was approved in the US and EU in 2016. Currently, Phase3 clinical study of lenvatinib in combination with everolimus or pembrolizumab for metastatic RCC patients is in progress. However, the detailed mechanisms underlying the efficacy of these combination treatments remain to be elucidated. In this study, we investigated the antitumor activities of lenvatinib in combination with everolimus or anti-PD1 mAb in preclinical RCC models. We examined antitumor activities in four human RCC (A-498, VMRC-RCW, Caki-1, and ACHN) xenograft models orally treated with lenvatinib, everolimus, and their combination. Expression of Ki67 and phosphorylation of S6 in RCC xenograft models were analyzed by immunofluorescence (IF) staining. Inhibitory effects of lenvatinib and everolimus on the phosphorylation of S6K (T389 and T421/S424), and S6 (S235/S236) were analyzed by western blot in vitro. We also examined antitumor activity of combination treatment of lenvatinib and anti-PD-1 mAb in RAG murine RCC model. The combination of lenvatinib with everolimus showed greater antitumor activity than that of either mono-treatment in all of 4 RCC xenograft models we examined and led to tumor regression in three out of 4 models (A-498, Caki-1, and ACHN). IF imaging showed expression of Ki67 was further suppressed with the combination treatment compared to lenvatinib and everolimus mono-treatments. Phospho-S6 (p-S6) signal were localized in perivascular regions of control group. Lenvatinib mono-treatment decreased p-S6 in the region apart from vessels, but some staining of p-S6 in perivascular regions were remained. The combination treatment further suppressed p-S6 staining regardless of the regions. In RAG syngeneic model, the combination treatment of lenvatinib at 10 mg/kg and anti-PD-1 mAb showed tumor shrinkage and tumors were regressed to nonpalpable sizes in 3 of 6 mice, although lenvatinib and anti-PD-1 mAb mono-treatments only slowed growths of RAG tumor. Our results indicate that combination of lenvatinib and everolimus enhances the inhibitory effects of mTOR signaling and tumor proliferation and showed greater antitumor activities. Combination of lenvatinib and anti-PD-1 mAb showed greater antitumor activity including tumor shrinkage, which was not demonstrated by each monotherapy in the murine RCC syngeneic model. These preclinical results provide one of the mechanisms of combinational effect of lenvatinib and everolimus, or PD-1 blockade in RCC.

#13

Anti-angiogenic action of leukotriene-C4induced 15-hydroxyprostaglandin dehydrogenase in colon cancer cells is a TNF-α dependent phenomenon.

Shakti R. Satapathy, Anita Sjölander. Lund University, Malmo, Sweden.

Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide. Cyclooxygenase-2 (COX-2), which plays a key role in the biosynthesis of prostaglandin E2 (PGE2), is often up-regulated in CRC and in other types of cancer. PGE2 induces angiogenesis and tumor cell survival, proliferation and migration. The tumor suppressor 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is a key enzyme in PGE2 catabolism, converting it into its inactive metabolite 15-keto-PGE2 and is often down-regulated in cancer. Interesting enough, CRC patients expressing high levels of Cysteinyl leukotriene receptor 2 (CysLTR2) have a good prognosis and therefore, we investigated a potential link between CysLTR2-signaling and the tumor suppressor 15-PGDH in colon cancer cells. TNF-α is considered as the main regulators of COX-2 and mPGES-1 that contribute to the increased synthesis of PGE2, which is inhibited by overexpressed 15-PGDH. Level of the pro-tumorigenic PGE2 are increased in CRC, previously attributed to increased production via TNF-α mediated COX-2 up-regulation but more recently attributed to decreased catabolism due to down-regulation of 15-PGDH. Elevation of 15-PGDH expression by leukotriene C4 (LTC4), a CysLTR2 ligand, exhibited anti-tumor activity in colon cancer cells with significant phosphorylation of β-catenin and down-regulation of anti-apoptotic marker Bcl-2 with concurrent activation of CASPASE-3 expression. We also observed a dramatic down-regulation of TNF-α on mRNA level and NF-κβ on both mRNA as well as protein level with LTC4 induced 15-PGDH in a TNF-α dependent manner. Moreover, TNF-α regulated anti-angiogenic action of 15-PGDH in HT-29 and Caco-2 colon cancer cells by depleting the mRNA level of MMP-2 and MMP-9 and protein level of VEGFR-1. Furthermore, our preliminary observation also suggested disrupted tube formation in HUVEC with LTC4 induced 15-PGDH which is mediated by TNF-α. Hence, restoration of 15-PGDH expression through CysLTR2-signaling promotes the anti-angiogenic action against colon cancer cells, indicating an anti-tumor as well as the anti-metastatic efficacy of CysLTR2-signaling.

#14

Preclinical evaluation of novel HIF-1α/P300 binding inhibitors.

Jonathan D. Strope,¹ Emily M. Harris,¹ Shaunna L. Beedie,¹ Cindy H. Chau,¹ Kristina M. Cook,² Christopher J. Schofield,³ Kirk R. Gustafson,⁴ William D. Figg¹. ¹National Cancer Institute, Bethesda, MD; ²The University of Sydney, Sydney, Australia; ³University of Oxford, Oxford, United Kingdom; ⁴National Cancer Institute, Frederick, MD.

A key component of tumor progression and metastasis is the hypoxic response. The hypoxic response regulates angiogenesis, tumor invasion, and metabolism. The Hypoxia Inducible Factor (HIF) is the transcriptional system responsible for the hypoxic response. The inhibition of the hypoxic response via inhibition of the HIF-1 pathway by disrupting its association with the transcriptional coactivator p300 presents a potential therapeutic target for multiple cancers where HIF is upregulated. To that end, we describe the preclinical development of previously described novel marine pyrroloiminoquinone alkaloids found using a HIF-1α/p300 assay in a high throughput screen of extracts from the National Cancer Institute’s Natural Products Repository. Pyrroloiminoquinone alkaloids are a novel class of HIF-1α inhibitors, which interrupt the protein−protein interaction between HIF-1α and p300 and consequently reduce HIF-related transcription. The 2 lead candidates, discorhabdin L and H were assessed in inhibition of angiogenesis in an in vitro HUVEC assay and ex vivo rat aortic ring assay. The compounds were also evaluated for in vivo efficacy in prostate cancer cell tumor xenografts. Results show that development of these compounds for clinical use is warranted and may prevent the progression of multiple tumor types.

#15

PA28γ contributes to tumor angiogenesis through regulation of IL-6 and CCL2 in oral squamous cell carcinoma.

Xin Zeng, Jing Li, Jiajia Liu, Ning Ji, Hui Feng, Qianming Chen. State Key Laboratory of Oral Diseases,West China Hospital of Stomatology, Sichuan University, Chengdu,Sichuan, China.

Background: PA28γ has been shown to promote tumor development and progression. Although PA28γ has been suggested to play a role in tumor angiogenesis, direct evidence for the molecular mechanisms underlying this phenomenon have not been investigated.

Methods: Oral squamous cell carcinoma (OSCC) stable cell lines with varying levels of PA28γ expression were constructed. EA.hy926 human endothelial cells were co-cultured with these lines to evaluate the influence of PA28γ on EA.hy926 cell proliferation, migration, invasion, and tube formation. Using a subcutaneous xenotransplantation tumor model of under-expressing PA28γ HSC-3 cells, we explored effects on tumor angiogenesis in nude mice. We performed screens of the different angiogenesis-related genes and cytokines being expressed using PCR and cytokine chips to identify candidates with the potential to affect tumor angiogenesis. Then, candidates were verified using Western blot, and neutralization tests immunohistochemistry. Finally human OSCC clinical cohort was used to validate.

Results: PA28γ could enhance the ability of OSCC cells to interact with endothelial cells, thereby recruit endothelial cells and induce angiogenesis. Ablation of PA28γ inhibits tumor-induced angiogenesis in xenografts mice model in vivo. In vivo zebrafish model experiments showed that depleted PA28γ remarkably blocked the formation of intersegmental vessels in zebrafish embryos. Furthermore, Angiogenesis-related PCR, cytokine chips and Western blot analysis indicated that PA28γ through regulate IL-6 and CCL2 expression, thereby subsequent activation of the Stat3/VEGF and PI3K/MMP-1 axis to promote angiogenesis. Neutralization or block results IL-6 and CCL2 has an essential role in PA28γ-controlled promotes of angiogenesis. In addition, clinical OSCC cohort validate that PA28γ positively correlates with IL-6 or CCL2 expression.

Conclusion: Collectively, our data show that PA28γ contributes to the tumor angiogenesis, by regulating IL-6 and CCL2. PA28γ thus represents a novel therapeutic target for treating PA28γ-positive OSCC cancers.

#16

A wide-spectrum analysis of angiogenesis-related factors in ovarian cancer reveals a signature potentially for prognosis and therapeutic targeting.

Chih-Lin Kuo. National Yang-Ming University, Taipei, Taiwan.

Ovarian cancer is the first leading cause of death from woman gynecological malignancies worldwide. It is typically diagnosed at a late stage with a poor 5-year survival rate less than 30%. In the progress of ovarian cancer, angiogenesis-related factors not only induce neovascularization for supporting tumor growth but also accelerate ascites production, which helps the peritoneal metastasis and is associated with poor disease prognosis. Therefore, angiogenesis-related factors are thought to be particularly crucial in the persistence of ovarian cancer and have become key targets in clinical research. Although the expression of some angiogenesis-related factors in ovarian cancer has been sporadically reported, analysis of the profile of angiogenesis-related factors in a whole spectrum manner is needed to evaluate their cruciality and relationship. In our study, totally 104 angiogenesis-related factors were used to analyze their transcriptional changes along with ovarian cancer progression in the data of 601 patients obtained from TCGA datasets (AgilentG4502A_07_2 and AgilentG4502A_07_3). 33 candidates with significantly differential expression (p < 0.05) between normal and any stage were initially revealed. To narrow down the candidates, these factors were ranked by the significance of expressional changes between normal and all various stages and 13 factors with the most significant difference (p < 0.0001) were selected and their fold changes were further shown in a heat-map manner; these included MMP9, VEGFA, CXCL10, TIMP2, AGRP, MMP8, IGFBP-1, ENG, TEK, FGF7, CSF3, ANGPTL5, ANGPTL7. Moreover, Ingenuity Pathway Analysis (IPA) and DAVID database analysis were applied to analyze their functional categories in angiogenesis. In conclusion, we developed a signature of angiogenesis-related factors for patients with ovarian cancer. With appropriate validation, it can be applied as a prognostic index or used to select certain candidates for developing targeted therapies.

#17

Inhibition of LRG1 normalizes tumor vessels and improves efficacy of cancer therapeutics.

John Greenwood,¹ Marie N. O'Connor,¹ David Kallenberg,¹ Rene-Filip Jackstadt,² Angharad Watson,³ Julia Ohme,³ Laura Dowsett,¹ Jestin George,¹ Xiaomeng Wang,¹ Ann Ager,³ Owen J. Sansom,² Stephen E. Moss¹. ¹University College London, London, United Kingdom; ²Cancer Research UK Beatson Institute, Glasgow, United Kingdom; ³Cardiff University, Cardiff, United Kingdom.

Introduction: The aim of this study was to determine whether deletion of the gene coding for the secreted glycoprotein, leucine-rich alpha-2-glycoprotein 1 (LRG1), or blockade of its action through function-blocking antibody treatment, improves tumor vascular function.

Experimental Procedures: The role of LRG1 was investigated in subcutaneous B16/F0 and LL2 mouse tumor models and in genetically engineered mouse models of intestinal (ApcMin) and pancreatic (KPC) cancers. Tumors were evaluated in wild type (WT) or Lrg1-/- mice or in WT mice treated with 15C4, a LRG1 blocking antibody. Tumor growth and survival were monitored and post-mortem analysis of vascular density, structure and function were undertaken. The effect of blocking LRG1 function on the efficacy of cisplatin or adoptive T cell therapy in B16/F0 tumor-bearing mice was determined.

Results: In Lrg1-/- mice or following functional blockade of LRG1 in WT animals there was a significant reduction in B16/F0 and LL2 tumor growth and improved survival in the ApcMin and KPC tumor-bearing mice. Vascular density was reduced in the B16/F0 and the KPC tumors but not in those of ApcMin. Most notably, we found that loss of LRG1 results in improved pericyte-endothelial cell association in the B16/F0 and ApcMin tumors. In the B16/F0 tumors we also observed an increase in the proportion of perfused vessels, and a reduction in vessel permeability and tumour hypoxia, consistent with our hypothesis that LRG1 is a vascular disrupting factor. Normalizing tumor vasculature to enhance vessel patency, reduce hypoxia and vascular leakage, and improve delivery of therapeutics has become a major objective. We therefore evaluated the effect of inhibiting LRG1 activity with the 15C4 antibody on the efficacy of cisplatin or adoptive CD8+ T cell therapy on B16/F0 tumor growth. Co-therapy revealed a highly significant reduction in tumor growth compared with monotherapy alone.

Conclusions: These data show that LRG1 subverts physiological angiogenesis by promoting dysfunctional vessel growth, and that therapeutic targeting of LRG1 reduces tumor neovascular growth and normalizes vascular function. We propose, therefore, that LRG1 is a potential therapeutic target in cancer, and that its inhibition may aid the delivery and efficacy of tumour therapeutics.

Funded by grants from the Medical Research Council UK, The Wellcome Trust, Rosetrees Trust, UCL Business and Moorfields Eye Hospital Special Trustees.

#18

MMP9 inhibition in mouse models of breast cancer: Therapeutic synergy with vinorelbine-based chemotherapy.

Francesca Reggiani,¹ Loredana Vecchi,¹ Valentina Labanca,¹ Patrizia Mancuso,¹ Amanda Mikels-Vigdal,² Francesco Bertolini¹. ¹European Inst. of Oncology, Milan, Italy; ²Gilead Sciences, Inc., Foster City, CA.

The combination of different therapeutic approaches, such as simultaneous targeting of tumor cells and stroma, may be a superior strategy to enhance anti-tumor responses in metastatic breast cancer (BC). Recently, the pro-tumorigenic activity of adipose tissue was further examined and suggested MMP9 promotes BC invasion, angiogenesis, and lung metastasis in mice (Reggiani et al., Cancer Res 2017). In the present work, MMP9 inhibition was combined with distinct therapeutic agents.

A monoclonal antibody that inhibits mouse MMP9 was administered to tumor-bearing (4T1) immune-competent BALB/c mice, alone or in combination with low-dose chemotherapy (vinorelbine, V or cyclophosphamide, CTX) or checkpoint inhibitors (anti-PD1 or anti-PDL1 monoclonal antibodies). MMP9 inhibition was also evaluated in diet-induced obese BC mice (Balb/c or FVB background).

The anti-tumor response of different combination treatments was assessed by tumor growth measurement, lung metastasis assessment, tumor angiogenesis evaluation through immunofluorescence, and immune system modulation by flow-cytometry.

The combination of V and MMP9 inhibition was superior to either agent alone. Tumor growth was reduced in mice administered with combined V and MMP9 inhibition compared to single treatment or control mice, administered with isotype control and saline (41% V+anti-MMP9 vs control, p=0.001). Lung metastases were significantly impaired (37% V+anti-MMP9 vs control, p=0.009). MMP9 inhibition exhibited the greatest reduction in intratumor angiogenesis among therapeutic agents tested (59% vs control, p=0.004). The combination of MMP9 inhibition with V compared to control, reduced B (1%, p=0.008), T cells (2.5%, p=0.01), and macrophages (35%, p=0.05) in the tumor.

Inhibiting MMP9 in diet-induced obese BALB/c mice was more effective in reducing tumor growth and metastatic progression, when compared to normal-weight mice (BC volume: 46%, p=0.01; lung metastasis: 53%, p=0.01). These data suggest that the increase of MMP9 release, observed in adipose progenitors when exposed to BC cells (Reggiani et al., Cancer Res 2017), was a mediator of the obesity-dependent pro-tumorigenic activity. Of note, when MMP9 inhibition was initiated in early phases of tumor growth in ErbB2+ BC FVB mice, the reduction of local tumor growth was more pronounced (started at 15 days vs 45 days: 18%, p=0.01; 15 days vs isotype control: 11%, p=0.01; 45 days vs isotype control: 64%, p=0.3 ns).

MMP9 inhibition is efficacious in mouse models of BC with concomitant obesity. In human BC patients, response to chemotherapy is reportedly lower, and the risk of relapse is higher in obese sub-population (Ewertz et al., JCO 2011). The additive effects of MMP9 inhibition with V may warrant further studies to characterize underlying molecular mechanisms in BC patients with high body mass index (BMI).

#19

Trastuzumab resistance accompanies vasculogenic mimicry in HER2-positive breast cancer cells.

Masafumi Shimoda, Ami Hori, Shinzaburo Noguchi. Osaka University Graduate School of Medicine, Suita, Japan.

Trastuzumab (Tzm) is a drug that targets HER2/ERBB2/Neu, and is essential for the treatment of HER2-positive breast cancer. However, resistance to the drug is a major obstacle in controlling the progression of this devastating disease. We hypothesized that Tzm load might induce a phenotypic change in HER2-positive breast cancer cells, enabling them to escape and survive Tzm activity. We conducted comprehensive immunophenotyping to detect the phenotypic changes in HER2-positive breast cancer cells loaded with Tzm, and compared the immunophenotype of Tzm-loaded cells with that of control cells based on the criteria determined a priori. Out of 242 cell surface antigens, 9 antigens were significantly upregulated and 3 were significantly downregulated. Surprisingly, all the antigens were related to endothelial and stem cell phenotypes, suggesting that Tzm load induced vasculogenic mimicry (VM). Moreover, we found VM markers like COX2, MMP2, MMP14, phospho-SMAD2/3, VEGFA, HIF1A, and TWIST1 to be upregulated in Tzm-loaded cells. However, Tzm-loaded cells did not exhibit tube formation on Matrigel, a hallmark of VM. Since the Tzm-loaded cells were still sensitive to Tzm, we used three Tzm-resistant cell lines to investigate if Tzm resistance accompanied VM. All Tzm-resistant cell lines exhibited tube formation on Matrigel. Importantly, several growth factors including EGF, FGF2, IGF1, and VEGF promoted VM in these cells, suggesting that single molecular-targeted drugs did not effectively inhibit VM as other growth factors could quickly compensate the VM pathways. We then examined if eribulin, a tubulin-binding chemotherapeutic drug that promotes vascular remodeling, inhibited VM in Tzm-resistant cells. Eribulin inhibited tube formation in Tzm-resistant cells at a clinically relevant concentration. In conclusion, Tzm load induces an incomplete vasculogenic phenotype in HER2-positive breast cancer cells. The cells exhibit VM after eventually acquiring Tzm resistance. Since VM drives metastasis, its regulation in Tzm-resistant HER2-positive breast cancer appears to be a promising approach in suppressing the progression of HER2-positive breast cancer.

#20

Inhibition of mutant EGFR in NSCLC promotes endothelin-1-mediated NSCLC disease progression and angiogenesis.

Stephen L. Ollosi,¹ Margaret Soucheray,² Jeffrey Becker,² Ines Pulido,³ Annika Dalheim,² Fatima Al-Shahrour,² Wei Qui,² Michael Nishimura,⁴ Julian Carretero,³ Simon Kaja,² Takeshi Shimamura². ¹Loyola University, Lemont, IL; ²Loyola University, IL; ³University of Valencia, Spain; ⁴Loyola University, Maywood, IL.

Despite recent advances in the treatment of NSCLC targeting of EGFR kinase domain mutations with tyrosine kinase inhibitors (TKIs), work needs to be done to reduce morbidity and improve survival for NSCLC patients. In NSCLC, tumor angiogenesis has been identified as important therapeutic target in combination with EGFR TKIs. However, only small advancements have been made for the use of angiogenesis inhibitors in NSCLC and it remains elusive why the inhibition of VEGF-mediated neovascularization is not therapeutically efficacious. We present evidence that a subpopulation of NSCLC cells with the EGFR TKI-induced epithelial to mesenchymal transition (EMT) contributes to the attenuation of response to anti-VEGF/VEGFR therapy. One of the hallmarks of cancer is heterogeneity and we have previously demonstrated that tumor heterogeneity within NSCLC cells lines harboring EGFR kinase domain mutations gives rise to divergent resistance mechanisms in response to treatment. In vivo admix models are instructive in studying intratumoral heterogeneity and in elucidating therapeutic responses. While NSCLC cell with acquired EGFR TKI resistance and EMT phenotype did not exhibit growth advantage in vivo, a 50% epithelial EGFR TKI sensitive and 50% mesenchymal EGFR TKI resistant admix provided significant growth advantage in vivo assessed by caliper measurement. Moreover, the admix tumors are resistant to EGFR TKI treatment. Interestingly, short-term in vitro co-culture of epithelial and mesenchymal cells did not provide a proliferative/growth advantage. The preliminary result led us to hypothesize that the epithelial-mesenchymal admix helps to create a tumor-host niche that is suitable for EGFR TKI resistance. To this end, we utilized the Luminex multiplex assay system to quantify secreted growth factors, cytokines, and chemokines. We have discovered that epithelial EGFR TKI sensitive cells secrete a significant amount of VEGF-A and cells with acquired/transient EGFR TKI resistance with an EMT phenotype secrete substantial amount of endothelin-1 (EDN1). Using an in vitro tube formation assay, we showed that secreted VEGF-A and EDN1 in admix conditions work synergistically to promote angiogenesis. Ectopic overexpression of EDN1 in EGFR-mutated HCC827 cells and predominantly secreting VEGF-A conferred significant resistance to gefitinib in vivo compared to control HCC827 cells. Importantly, the increased HUVEC cell tube formation in conditioned media from admix cells rich in both VEGF-A and EDN1 was significantly attenuated by the dual inhibition of VEGFR and EDNRA. Notably, the exposure of HCC4006 cells to 100nM gefitinib for 72 hours alone promoted transition from predominately VEGF-A secretion to EDN1 secretion. This suggests that the therapeutic efficacy of combining EGFR inhibitor with VEGFR inhibitors in the treatment of EGFR mutation positive NSCLC should be significantly tempered.

Breast Cancer Metastasis

#21

p66ShcA is a contextual breast cancer metastasis promoter or suppressor depending on the tumor microenvironment.

Jesse Hudson,¹ Kyle Lewis,¹ Julien Senécal,² Alexander Kiepas,² Sébastien Tabariès,² Valérie Sabourin,¹ Ryuhjin Ahn,¹ Rachel La Selva,¹ Peter Siegel,² Giuseppina Ursini-Siegel¹. ¹McGill University Lady Davis Institute for Medical Research, Montreal, Quebec, Canada; ²McGill University Goodman Cancer Research Centre, Montreal, Quebec, Canada.

Introduction: Src homology and collagen A (ShcA) adaptor proteins are essential during breast cancer progression. However, the role of the largest isoform, p66ShcA, is conflicting and still poorly understood. Under high levels of stress p66ShcA is phosphorylated on serine36, within its CH2 domain, allowing it to translocate to the mitochondria and induce the formation of reactive oxygen species (ROS) to promote apoptosis. Previously, we provided the first in vivo evidence that p66ShcA can influence both pro and anti-tumorigenic functions in ErbB2+ luminal breast cancer. Stable overexpression of p66ShcA reduced tumor outgrowth while simultaneously elevating the expression of mesenchymal genes to promote tumor plasticity. In this study, we evaluated the role of p66ShcA in metastatic dissemination to the lung in a model of basal breast cancer, which typically is associated with poor outcome. Hypothesis: p66ShcA regulates basal breast cancer metastasis to the lung. Methods: Screening a panel of basal breast cancer cells in vivo selected to the lung, liver and bone, we found p66ShcA enriched in lung and liver metastatic variant cell lines relative to parental breast cancer cells and those in vivo selected through the mammary fatpad. Using CRISPR/Cas technology we genetically deleted p66ShcA and rescued with p66ShcA-WT or a p66ShcA-S36A mutant. Lung metastatic basal breast cancer cells were injected into the fourth gland of the mammary fatpad. Tumors were measured using calipers 3 times/week following first palpation (>50mm3) followed by surgical resection to monitor the lung metastatic burden. In addition, we performed tail vein injections to monitor lung metastatic burden following direct entry into the circulation. Results: Loss of p66ShcA significantly reduced the metastatic burden to the lung following surgical tumor resection and this reduction was partially rescued by stable overexpression of wild type (WT), but not S36A mutated p66ShcA from the primary site. These effects were not due to altered anti-oxidant expression levels, changes in oxidative DNA Damage or microvessel density. Intriguingly, we found that WT-rescue of p66ShcA significantly elevated the migratory speed of breast cancer cells in vitro and corroborates our in vivo metastatic burden data. However, this is in stark contrast to our tail vein data, where WT-rescue of p66ShcA significantly inhibited lung metastasis. Conclusion: p66ShcA is required for efficient metastasis to the lung in a mitrochondrial-ROS-dependent fashion from the primary site. Our data suggest that cues from the tumor microenvironement of the mammary fatpad are essential for successful colonization and outgrowth at oxygen rich sites, such as the lung, as breast cancer cells with elevated expression of p66ShcA directly entering the circulation suppressed lung metastatic burden.

#22

The Y537S ESR1 mutation is a dominant driver of distant ER-positive breast cancer metastasis.

Guowei Gu,¹ Lin Tian,¹ Meng Gao,² Yassine Rechoum,¹ Luca Gelsomino,¹ Derek Dustin,¹ Arnoldo Corona-Rodriguez,¹ Amanda R. Beyer,¹ Anna Tsimelzon,¹ Xiang Zhang,¹ Sebastiano Ando',³ Suzanne Fuqua¹. ¹Baylor College of Medicine, Houston, TX; ²MD Anderson Cancer Center, Houston, TX; ³University of Calabria, Cosenza, Italy.

Background: Estrogen receptor (ESR1) mutations occur at a high frequency in metastatic breast tumors in patients treated with hormonal therapy in the metastatic setting. We do not know if these mutations are involved in metastasis. Experimental design and methods: We generated ESR1 Y537S homozygous mutations using CRISPR Casp-9 technology. Treatment synergy was evaluated using Compusyn. Athymic mice were used in tumor xenograft studies. ChIP-Seq and transcriptome analyses were performed. Results: We generated CRISPR ESR1 Y537S mutation homozygous knock-in clones and lentiviral stable pools in MCF-7 cells. Transcriptome profiling revealed elevated expression of Hallmark pathways, including epithelial mesenchymal transition (EMT) and estrogen-regulated gene expression. Mutant cell growth was resistant to tamoxifen, but responsive to fulvestrant treatment. Synergistic treatment effects were observed with fulvestrant and the mTOR inhibitor everolimus or the CDK4/6 inhibitor palbociclib. CRISPR Y537S mutant knock-in cells grown in the mammary fat-pad of athymic mice spontaneously metastasized to distant organs including the lung, intestine, and kidneys. In the presence of estrogen, there was no difference in the frequency of distant macrometastases between parental wild-type ER and CRISPR Y537S mutant ER mice. However, in the absence of estrogen, mimicking aromatase inhibitor treatment, 80% of CRISPR Y537S mutant ER mice displayed overt distant macrometastases, but none were observed in parental wild-type ER mice (p=0.04). Distant tumors retained ER expression and hormone sensitivity. Comparison of residual tamoxifen-treated metastatic tumors with tumors grown at the primary mammary fat-pad site using immunoblot analysis demonstrated significant reduction in estrogen-regulated gene expression, but no effect on the expression of biomarkers associated with EMT, suggesting a disconnect between EMT and distant metastasis in mutant cells. EMT genes were also identified as direct binding site targets in Y537S mutant cells compared with wide-type ER using ChIPSeq. We discovered that expression of the Y537S mutant was dominant, driving the growth of distant metastatic tumors when co-expressed with wild-type ER cells. A Y537S ER mutant-specific gene expression signature predicted poor disease-free survival of ER-positive patients using the METABRIC database, and lung-specific metastasis-free survival in a Memorial Sloan Kettering dataset. Conclusion: The Y537S ER mutation is a driver of distant metastasis in ER-positive breast cancer cells. A Y537S ER mutant-specific gene expression signature predicted poor disease-free, and distant lung metastasis in ER-positive patients. Mutation status is a potential new predictive factor for hormone therapy of metastatic breast cancer patients on maintenance hormonal therapy.

#23

Chemotherapy and tau interplay facilitates breast-to-brain metastasis.

Behnaz Saatian, Krutika Deshpande, Alex Julian, Brooke Naomi Nakamura, Ling Shao, Josh Neman. University of Southern California, Los Angeles, CA.

Breast cancer represents the second most common cause of brain metastasis with the rate of 10-16% after lung cancer. The overall survival in brain metastatic patients is very short ranging from 2 to 25 months. Considering the high incidence of brain metastasis and the limitation of available therapies, developing preventive strategies and early detection methods are required to improve the survival rate in the patients. Many primary and brain metastatic breast cancer patients represent cognitive deficits. Besides, chemo-treated breast cancer patients show memory problems before brain metastases diagnosis. Although the cause of cognitive impairments after chemotherapy have not been explored, a recent study revealed that chemotherapy promotes breast cancer metastasis. Correspondingly, our mRNA expression analysis showed downregulation of the tight junction markers in chemo-treated choroid plexus cells (CPs), suggesting that chemotherapy is likely to cause an increase in the blood-cerebral spinal fluid barrier (BCSFB) permeability. Chemotherapy also induces upregulation of tau expression in CP cells. Tau is known as a pathological hallmark of Alzheimer’s disease (AD) and neurodegenerative disorders. The abnormally phosphorylated and aggregated form of tau play critical role in neurodegenerative diseases as of AD. Tau expression, both at the mRNA and protein level, is significantly elevated in breast to brain metastases (BBMs). Therefore, we hypothesized that chemotherapy upregulates tau expression in breast cancer cells leading to the BCSFB and blood-brain barrier (BBB) permeability, and consequently, breast to brain metastasis and neurodegeneration. To test our hypothesis, the effect of chemotherapy on the BCSFB and BBB permeability were investigated using the trans endothelial electrical resistance (TEER) and migration assays in the in vitro models. Our results show an increase in abnormal tau expression in chemo-treated primary breast cancer cells. BBM-derived tau is abnormal and forms paired helical filaments (PHFs), similar to Alzheimer’s. We conclude that breast to brain metastasis is facilitated through an interplay between chemotherapy, and tau expression and release from breast cancer cells. Additionally, we suggest that tau plays a key role in neurodegeneration defects in brain metastatic patients. This study opens a new path towards prevention and early detection of breast cancer metastasis to the brain, cognitive decline and neurodegeneration in metastatic patients.

#24

Extracellular HMGA1 promotes tumor invasion and metastasis in breast cancer.

Olga Méndez,¹ Vicente Peg,² Candida Salvans,¹ Mireia Pujals,¹ Yolanda Fernández,³ Ibane Abasolo,³ Jose Perez,⁴ Ana Matres,¹ Josep Tabernero,⁴ Javier Cortés,⁴ Joaquín Arribas,¹ Josep Villanueva¹. ¹Vall D'Hebron Inst. of Oncology, Barcelona, Spain; ²Pathology department. Vall d´Hebron Institute de Recerca, Barcelona, Spain; ³Vall D'Hebron Institut de Recerca, Barcelona, Spain; ⁴Department of medical oncology. Vall D'Hebron University Hospital, Barcelona, Spain.

The recent in-depth study of the cancer secretome suggests that a fraction of the intracellular proteome might be playing alternative roles in the extracellular space during tumorigenesis through its unconventional secretion. A proteomic study aimed at evaluating the role of unconventional protein secretion in the context of tumor invasion led us to