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Targeting VEGF-mediated Tumor Angiogenesis in Cancer Therapy June 19-20, 2014 www.nyas.org/Angiogenesis2014 The New York Academy of Sciences, New York City

SCIENTIFIC Organizing Committee Gabriele Bergers, PhD, University of California, San Francisco Melanie Brickman Stynes, PhD, MSc, The New York Academy of Sciences Michele De Palma, PhD, EPFL School of Life Sciences, Lausanne Brooke Grindlinger, PhD, The New York Academy of Sciences Kerstin Hofmeyer, PhD, The New York Academy of Sciences Luisa Iruela-Arispe, PhD, University of California, Los Angeles Robert S. Kerbel, PhD, Sunnybrook Research Institute Gregory Plowman, MD, PhD, ImClone Systems / Eli Lilly and Company Gavin Thurston, PhD, Regeneron Pharmaceuticals

ACKNOWLEDGEMENT OF SUPPORT Silver Sponsor: Bronze Sponsor: Regeneron Pharmaceuticals, Inc. Grant Support: This program is supported by grants from AbbVie and Genentech.

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Welcome

T

he New York Academy of Sciences and the Scientific Organizing Committee are pleased to welcome you to our conference, Targeting VEGF-mediated Tumor Angiogenesis in Cancer Therapy. Since Dr. Judah Folkman’s seminal discoveries on the mechanism of angiogenesis in the 1970s, significant progress has been made in therapeutic development. A decade ago, the first antiangiogenic drug targeting the vascular endothelial growth factor (VEGF) signaling pathway received approval by the U.S. Food and Drug Administration (FDA). Several other anti-angiogenic drugs for the treatment of human cancers are also now on the market. While some individuals have experienced improvements in progression-free survival due to anti-angiogenic therapies that target VEGF, to date, these drugs are lagging behind in improving overall cancer patient survival in the clinic when compared to the promising results observed in animal disease models. The goal of this conference is to provide a neutral forum to discuss recent advances in our understanding of molecular and cellular mechanisms underlying normal and pathological angiogenesis and the mechanisms of action of anti-angiogenic therapies. Speakers will examine ways to improve VEGF-targeted cancer therapies by addressing urgent issues such as: (i) inherent / acquired resistance, (ii) immunomodulation, (iii) enhanced invasiveness, and (iv) lack of validated predictive and prognostic biomarkers. Presentations, including a Keynote Lecture by Harold F. Dvorak, MD, will explore VEGF-dependent and -alternative mechanisms of angiogenesis. “Point–Counterpoint” debates between leading experts will aim to resolve controversies regarding the function of vascular normalization, treatment-induced metastasis, and the validity of hypertension as a physiological biomarker. By bringing together international scientists and clinicians from diverse sectors, we hope to foster new collaborations and research to translate the potential of angiogenesis inhibition into clinical cancer therapy.

VEGF-mediated Tumor Angiogenesis 3

Welcome

To disseminate the knowledge and ideas exchanged by participants beyond the walls of the auditorium and past the event date, a Meeting Report will be prepared in collaboration with conference faculty for publication in Annals of the New York Academy of Sciences. Academy members are granted free access to the online version of Annals at www.nyas.org/annals. We ask you to take a moment to give us your feedback and help us further improve our scientific programming by completing the online survey for this event at www.nyas.org/Angio2014-eval, or by clicking on the survey link in the attendee follow-up email. We hope that this conference will meet your expectations, stimulate exciting discussions, and lead to fruitful new collaborations. Please do not hesitate to notify our staff of any questions, concerns, or suggestions.

Melanie Brickman Stynes, PhD, MSc Director, Life Sciences Conferences The New York Academy of Sciences

4 VEGF-mediated Tumor Angiogenesis

FACULTY DISCLOSURES

All faculty participating in this activity are required to disclose to the audience any significant financial interest and/or other relationship with the manufacturer(s) of any commercial product(s) and/or provider(s) of commercial services discussed in his/her presentation and/or the commercial contributor(s) of this activity. Judith Agudo, PhD None Evelyn Aranda, PhD N/A Carlos Bais, PhD Employee • Genentech, Inc. Laura E. Benjamin, PhD Employee • ImClone Systems Gabriele Bergers, PhD None Melanie Brickman Stynes, PhD, MSc None Oriol Casanovas, PhD None John S. Condeelis, PhD Research Support • Deciphera Speaker Bureau • Leica Consultant • Deciphera, MetaStat Shareholder • MetaStat

Dan Gabriel Duda, DMD, PhD* Consultant • Hexal, Sandoz Harold F. Dvorak, MD Research Support • ImClone Systems/Lilly, Pfizer Inc Lee M. Ellis, MD Consultant • Amgen, Genentech, Inc./Roche, ImClone Systems/Lilly Kyrre E. Emblem, PhD* Intellectual Property Rights • Nordic NeuroLabs AS Napoleone Ferrara, MD N/A Brooke Grindlinger, PhD Shareholder • General Electric Co. Axel Grothey, MD Research Support • Bayer, Bristol-Myers Squibb, Genentech Inc., ImClone Systems, Morphotek, Sanofi

Michele De Palma, PhD None

John V. Heymach, MD, PhD N/A

Elisabeth de Vries, MD, PhD Research Support • Roche

Kerstin Hofmeyer, PhD None

VEGF-mediated Tumor Angiogenesis 5

FACULTY DISCLOSURES

Herbert I. Hurwitz, MD Research Support • Amgen Genentech, Inc., Lilly, Pfizer Inc, Regeneron Pharmaceuticals, Roche, Sanofi Consultant • Amgen, Genentech Inc., Lilly, Regeneron Pharmaceuticals, Roche, Sanofi Luisa Iruela-Arispe, PhD None Jingquan Jia, MD, PhD N/A Robert S. Kerbel, PhD Research Support • Cerulean Consultant • Angiocrine Biosciences, Cerulean, Taiho Shareholder • Angiocrine Biosciences Honoraria • Genentech, Inc., Pfizer Inc, Regeneron Pharmaceuticals, Sanofi Feilim Mac Gabhann, PhD None

Ian Miller, PhD N/A Gregory Plowman, MD, PhD N/A Thomas Powles, MBBS, MRCP, MD Research Support • Genentech, Inc., GlaxoSmithKline, Novartis, Pfizer Inc Speakers Bureau • GlaxoSmithKline, Novartis, Pfizer Inc Shahin Rafii, MD N/A Brian I. Rini, MD Research Support • GlaxoSmithKline, Pfizer Inc, Roche Consultant • GlaxoSmithKline, Pfizer Inc, Roche Alexander Scholz, PhD None Gavin Thurston, PhD Employee • Regeneron Pharmaceuticals

David F. McDermott, MD* Consultant • Bristol-Myers Squibb, Merck, Roche An * after the speaker’s name indicates that the speaker intends to discuss unlabeled use of a commercial product or an investigational use of a product not yet approved for this purpose. The speaker will disclose this information during his/her presentation. N/A = not available at time of printing. 6 VEGF-mediated Tumor Angiogenesis

AMENDMENT GRANT SUPPORT Funding for this conference was made possible (in part) by CA18665201 from the National Cancer Institute. The views expressed in written conference materials or publications and by speakers and moderators do not necessarily reflect the official policies of the Department of Health and Human Services; nor does mention by trade names, commercial practices, or organizations imply endorsement by the U.S. Government. DISCLOSURES Evelyn Aranda, PhD None

Jingquan Jia, MD, PhD None

Napoleone Ferrara, MD None

Ian Miller, PhD None

John V. Heymach, MD, PhD Research Support  AstraZeneca  GlaxoSmithKline Consultant  AstraZeneca  Eli Lilly  Genentech  GlaxoSmithKline  Pfizer

Gregory Plowman, MD, PhD Employee & Shareholder  Eli Lilly Shahin Rafii, MD Consultant  Angiocrine Bioscience, Inc

AGENDA June 19, 2014, Session I, 12:15 PM — Updated Abstract Instructive Angiocrine Contribution of Vascular Niche to Tumor Progression Shahin Rafii, MD, Weill Cornell Medical College, Ansary Stem Cell Institute, New York, New York, United States & Angiocrine Bioscience, New York, New York, United States Organ and tumor specific endothelial cells (ECs) are not just passive conduits to deliver oxygen and nutrients, but also establish an instructive vascular niche, which by elaboration of specific paracrine trophogens, (known as angiocrine factors), directly balances the rate of normal and malignant stem cell self-renewal and differentiation. For example, activation of the Akt-mTOR pathway in the sinusoidal ECs (SECs)

AMENDMENT stimulates expression of angiocrine factors, including Notch-ligands, Wnts, FGFs, and TGF-modulators, that induce expansion of authentic hematopoietic stem cells — While MAPkinase induces expression of angiocrine factors, that support differentiation of the stem cells into lineage-committed progenitors. Furthermore, after partial hepatectomy, SECs within the liver stimulate regeneration by angiocrine expression of Wnt2 and HGF. Pulmonary capillary ECs (PCECs) by deploying MMP14 and release of EGF-ligands sustain lung regeneration. Notably, transplantation of SECs or PCECs or tissue-specific endothelial progenitor cells (EPCs) into mice restores organ regeneration. These data establish the remarkable tissue-specific vascular heterogeneity in orchestrating organ regeneration. Indeed, we have recently shown that each organ is arborized with specialized capillary ECs endowed with a unique repertoire of angiocrine factors. Therefore, to capitalize on the potential of vascular cells for organ regeneration, we need to engineer tissue-specific ECs that can home and engraft in tissues promoting organ regeneration and repair. Most importantly, as ECs can be provoked to instigate pro-fibrotic changes, we need to manufacture tissue-specific ECs that drive organ regeneration without promoting maladaptive fibrosis. We have also recently shown that mouse/human tumor ECs activated by FGF4-FGFR1 deploy Notch-ligand Jagged1 (Jag1) to induce Notch2-Hey1 in neighboring B-Cell lymphoma cells (LCs) (Cao, Z. et al., Cancer Cell, 2014). This crosstalk enforces + + + aggressive CD44 IGF1R CSF1R LC phenotypes, including extra-nodal invasion and chemo-resistance. Inducible EC-selective-deletion of Fgfr1 + iΔEC/iΔEC or Jag1 in Eµ-Myc lymphoma model (Myc Fgfr1 , + iΔEC/iΔEC Myc Jag1 ), or impairing Notch2 signaling in mouse/human lymphoma xenografts, diminished lymphoma aggressiveness and prolonged mouse survival. Thus, targeting perfusion-independent functions of vascular niche by blocking the angiocrine FGF4FGFR1/Jagged1-Notch2 loop offers new opportunities to limit tumor aggressiveness and enhance chemo-sensitivity. June 19, 2014, Session II — Session Chair Shahin Rafii, MD, Weill Cornell Medical College & Angiocrine Bioscience June 20, 2014, Session IV, 9:00 AM — Updated Title Synergy of Chemotherapy and anti-VEGF Treatment? — and Updates from the 2014 ASCO Annual Meeting Lee M. Ellis, MD, The University of Texas MD Anderson Cancer Center

AGENDA Day 1 — Thursday, June 19, 2014 8:00 AM Breakfast and Registration 8:45 AM Opening Remarks Melanie Brickman Stynes, PhD, MSc, The New York Academy of Sciences Luisa Iruela-Arispe, PhD, University of California, Los Angeles 9:00 AM Keynote Lecture Heterogeneity of the Tumor Vasculature — Why Doesn’t Anti-VEGF/VEGF Receptor Therapy Work Better? Harold F. Dvorak, MD, Beth Israel Deaconess Medical Center Session I: Mechanisms of Tumor Angiogenesis Session Chair: Gabriele Bergers, PhD, University of California, San Francisco 9:45 AM

From the Isolation and Cloning of VEGF-A to FDA Approval of Multiple VEGF Inhibitors for Cancer and Intraocular Diseases Napoleone Ferrara, MD, University of California, San Diego

10:15 AM Effects of Extracellular Matrix Binding on the Biology of VEGF and VEGF-Therapy Luisa Iruela-Arispe, PhD, University of California, Los Angeles 10:45 AM Coffee Break 11:15 AM HOT TOPIC TALK (selected from submitted abstracts) The miR-126–VEGFR2 Axis Controls the Innate Response to Pathogen-associated Nucleic Acids Judith Agudo, PhD, Icahn School of Medicine at Mount Sinai 11:30 AM HOT TOPIC TALK (selected from submitted abstracts) Overcoming Resistance to VEGF-Blockade by Combinatorial Targeting of the Angiopoietin/Tie2 Axis Alexander Scholz, PhD, Stanford University School of Medicine 11:45 AM Interacting Signaling Pathways in Tumor Angiogenesis: VEGF, Delta/Notch, and Angiopoetin-1/Tie-2 Gavin Thurston, PhD, Regeneron Pharmaceuticals 12:15 PM Instructive Angiocrine Contribution of Vascular Niche to Tumor Progression Shahin Rafii, MD, Weill Cornell Medical College 12:45 PM Networking Lunch and Poster Viewing VEGF-mediated Tumor Angiogenesis 7

AGENDA Session II: ImmunOmodulation in Angiogenesis Session Chair to be announced 2:00 PM

HOT TOPIC TALK (selected from submitted abstracts) Mechanisms of Macrophage Promotion of Tumor Angiogenesis Evelyn Aranda, PhD, Albert Einstein College of Medicine

2:15 PM

Novel Immunotherapeutic Approaches for Cancer Therapy David F. McDermott, MD, Beth Israel Deaconess Medical Center

2:45 PM

Tackling Resistance to Anti-angiogenic Therapy: a Tumor-Immune Cell Dialogue Gabriele Bergers, PhD, University of California, San Francisco

3:15 PM

Coffee Break

3:45 PM

Overcoming Tumor Resistance to VEGFA Signaling Blockade by Co-targeting ANG2-TIE2 Signaling and Macrophages Michele De Palma, PhD, EPFL School of Life Sciences, Lausanne

4:15 PM

Imaging Immune Cells in the Context of Tumor Angiogenesis John S. Condeelis, PhD, Albert Einstein College of Medicine

Session III: Vascular Normalization in Angiogenesis 4:45 PM

Point – Counter Point Debate Does Vascular Normalization Improve Delivery of Cancer Therapeutics?



Moderator: Lee M. Ellis, MD, The University of Texas MD Anderson Cancer Center



Pro-Debater: Dan Gabriel Duda, DMD, PhD, Massachusetts General Hospital



Contra-Debater: Elisabeth G.E. de Vries, MD, PhD, University Medical Center Groningen

5:30 PM

Poster Session and Networking Reception

7:00 PM

Conclusion of Day 1

8 VEGF-mediated Tumor Angiogenesis

AGENDA Day 2 — Friday, June 20, 2014 8:00 AM

Breakfast and Registration

8:00 AM

Early Career Investigator Mentoring Workshop (concurrent)



Editor's Guide to Writing and Publishing Your Paper Brooke Grindlinger, PhD, The New York Academy of Sciences, Former Editor, The Journal of Clinical Investigation

Session IV: Challenges to Anti-angiogenic Therapy — Learning from Successes and Failures Session Chair: Gavin Thurston, PhD, Regeneron Pharmaceuticals 9:00 AM

Synergy of Chemotherapy and anti-VEGF Treatment (Mechanisms of Action of Targeting VEGF) Lee M. Ellis, MD, The University of Texas MD Anderson Cancer Center

9:30 AM

Optimal Strategies for Resistance Avoidance and Long-Term Treatment with Anti-angiogenic Therapy Axel Grothey, MD, Mayo Clinic College of Medicine

10:00 AM HOT TOPIC TALK (selected from submitted abstracts) Vessel Architectural Imaging Reveals Early Vascular Response to Anti-angiogenic Therapy Kyrre E. Emblem, PhD, Massachusetts General Hospital/ Harvard Medical School 10:15 AM HOT TOPIC TALK (selected from submitted abstracts) Population Pharmacodynamics: Mechanism-based Modeling of the Angiogenesis Receptor Kinome in Cancer Feilim Mac Gabhann, PhD, Johns Hopkins University 10:30 AM Coffee Break 11:00 AM Understanding VEGFR2 Targeting for Cancer Therapy Laura E. Benjamin, PhD, ImClone Systems 11:30 PM Anti-angiogenic Therapy: Impact on Invasion and Metastatic Disease Outcomes Robert S. Kerbel, PhD, Sunnybrook Research Institute

VEGF-mediated Tumor Angiogenesis 9

AGENDA 12:00 PM Point – Counter Point Debate Does Anti-angiogenic Therapy Induce Invasion and Metastasis?

Moderator: Gregory Plowman, MD, PhD, ImClone Systems / Eli Lilly and Company



Pro-Debater: Oriol Casanovas, PhD, Catalan Institute of Oncology - IDIBELL



Contra-Debater: Napoleone Ferrara, MD, University of California, San Diego

12:45 PM Networking Lunch and Poster Viewing Session V: BIOMARKERS FOR ANTI-ANGIOGENIC CANCER TREATMENT Session Chair: Dan Gabriel Duda, DMD, PhD, Massachusetts General Hospital 2:00 PM

Genetic and Circulating Markers Associated with Response to Anti-VEGF Therapy John V. Heymach, MD, PhD, The University of Texas MD Anderson Cancer Center

2:30 PM

Novel Biomarkers that Are of Prognostic Value and Can Predict Response or Resistance to Anti-angiogenic Therapy Thomas Powles, MBBS, MRCP, MD, Barts Cancer Institute

3:00 PM

HOT TOPIC TALK (selected from submitted abstracts) Direct Evidence of Targeted Inhibition by Anti-angiogenic Agents from a Wound-healing Model in Patients with Advance Solid Tumors Jingquan Jia, MD, PhD, Duke University Medical Center

3:15 PM

HOT TOPIC TALK (selected from submitted abstracts) Metabolic PET Imaging Approaches as Early Biomarkers of Cardiac Toxicity Following Treatment with the Tyrosine Kinase Inhibitor Sunitinib Ian Miller, PhD, Royal College of Surgeons in Ireland

3:30 PM

Coffee Break

10 VEGF-mediated Tumor Angiogenesis

AGENDA 4:00 PM

Identification and Analysis of in vivo VEGF Downstream Markers Link VEGF Pathway Activity with Efficacy of Anti-VEGF Therapies Carlos Bais, PhD, Genentech, Inc.

4:30 PM

Point – Counter Point Debate Is Hypertension a Valid Biomarker for Anti-angiogenic Tumor Therapy?



Moderator: John V. Heymach, MD, PhD, The University of Texas MD Anderson Cancer Center



Pro-Debater: Brian Rini, MD, Cleveland Clinic



Contra-Debater: Herbert I. Hurwitz, MD, Duke Department of Medicine

5:15 PM

Closing Remarks

5:30 PM

Adjourn

The New York Academy of Sciences requests that you do not take photographs or make audio or video recordings of the conference presentations, or present unpublished data on any open-access websites, unless specific permission is obtained from the speaker. VEGF-mediated Tumor Angiogenesis 11

ABSTRACTS Speaker abstracts are listed in order of presentation.

Day 1 — Thursday, June 19, 2014 Keynote Lecture Heterogeneity of the Tumor Vasculature — Why Doesn’t Anti-VEGF/ VEGF Receptor Therapy Work Better? Harold F. Dvorak, MD, The Center for Vascular Biology Research and the Department of Pathology, Beth Israel Deaconess Medical Center, Boston, Massachusetts, United States; Harvard Medical School, Boston, Massachusetts, United States The microvasculature that supplies human cancers and mouse tumors is similar, and similarly heterogeneous, and includes at least six distinct blood vessel types. Some of these vessel types are present in all tumors thus far examined, whereas others are present in only some cancers and not in others. We have used an adenovirus expressing VEGF-A164 (Ad-VEGF-A164) to replicate each of these vessel types in immunodeficient nude mice and have found that anti-VEGF/VEGF receptor (VEGFR) therapy targets some vessel types, particularly early-formed mother vessels, but not other vessels that form later. Even so, the mechanism(s) by which anti-VEGF/VEGFR therapy damages Ad-VEGF-A164-induced surrogates and tumor vessels is not entirely clear. Our findings in the Ad-VEGF-A164 model are consistent with those of others demonstrating that similar types of therapy are effective in regressing rapidly growing mouse tumors but are less effective in treating many human cancers, as well as more slowly growing mouse tumors that have already acquired an established vasculature. If anti-vascular cancer therapy is to become more effective, new targets in addition to VEGF/VEGFR may be needed.

Session I: Mechanisms of Tumor Angiogenesis From Isolation and Cloning of VEGF-A to FDA Approval of Multiple VEGF Inhibitors for Cancer and Intraocular Diseases Napoleone Ferrara, MD, University of California, San Diego, La Jolla, California, United States Angiogenesis, the development of new blood vessels, is a fundamental pathophysiological process. Vascular endothelial growth factor (VEGF)-A was isolated and cloned in 1989 and during the following decade was shown to be a key regulator of blood vessel growth in embryonic development and in a variety of other physiological processes. Several VEGF inhibitors were reported to block tumor growth in numerous preclinical models, consistent with an important role of VEGF-A in tumor angiogenesis. Bevacizumab, a humanized antiVEGF-A monoclonal antibody, and several other VEGF pathway inhibitors have been approved for the treatment of advanced tumors. Furthermore, blocking

12 VEGF-mediated Tumor Angiogenesis

ABSTRACTS VEGF-A prevented vision loss and had a dramatic impact on the progression of neovascular age-related macular degeneration as well as other intraocular neovascular disorders. We have recently been studying the mechanisms of resistance to anti-VEGF therapies in various tumor models. These studies indicate that multiple proangiogenic mechanisms may be implicated. We identified factors produced by myeloid cells and by fibroblasts. In recent studies, we identified Interleukin 17, a key product of Th17 Helper T cells as a factor mediating angiogenic escape and resistance to VEGF inhibitors. Efforts are ongoing to determine the translational and clinical significance of such findings.

Effects of Extracellular Matrix Binding on the Biology of VEGF and VEGF-Therapy M. Luisa Iruela-Arispe, PhD, Christiana Ruhrberg, PhD, Carmen M. Warren, PhD, Tom Chen, PhD, Department of Molecular, Cell and Developmental Biology, UCLA, Los Angeles, California, United States Vascular Endothelial Growth Factor (VEGF) is essential for the formation, growth, and survival of blood vessels during both developmental and pathological conditions. The Vegf gene is alternatively spliced into multiple isoforms with varying affinities to the extracellular matrix (ECM), dictated by the presence of exon 7, commonly referred to as the heparin binding domain. Compared to soluble VEGF, ECM-associated VEGF provides distinct signaling outcomes, resulting in differences in vascular patterning. While the importance of matrix interactions to VEGF signaling has been well documented, much less is known about the specific components that bind to VEGF in vivo. Surprisingly, endothelial specific knockout of Ext1, an enzyme necessary for heparin sulfate biosynthesis, had no effect on blood vessel development or function. Here we present work on the identification of VEGF binding partners both in vitro and in vivo. Using surface plasmon resonance, we observed tight association of VEGF with various ECM molecules in vitro. We extended this work with mass spectrometry experiments to determine proteins that interact with VEGF in vivo. Using this unbiased approach, we discovered ECM components that have not yet been described as VEGF interactors. Finally, we explored the functional consequences of VEGF binding to the ECM and showed that this association significantly increases the halflife of VEGF by roughly 100-fold. Overall, our findings demonstrate that VEGF is capable of binding to a vast array of ECM molecules and that these associations can alter the bioavailability of VEGF.

VEGF-mediated Tumor Angiogenesis 13

ABSTRACTS The miR-126–VEGFR2 Axis Controls the Innate Response to Pathogen-associated Nucleic Acids Judith Agudo1, Albert Ruzo1, Navpreet Tung1, Hélène Salmon1, Marylène Leboeuf1, Daigo Hashimoto1, Christian Becker1, Lee-Ann Garrett-Sinha2, Alessia Baccarini1, Miriam Merad1, and Brian D. Brown1 1Icahn School of Medicine at Mount Sinai, New York, New York, United States, 2State University of New York at Buffalo, Buffalo, New York, United States

MicroRNA-126 (miR-126) is a microRNA predominately expressed by endothelial cells and controls angiogenesis. Unexpectedly, we found miR-126 is also required for the innate response to pathogen-associated nucleic acids, and that miR126-deficient mice had increased susceptibility to pseudotyped-HIV infection. miRNA profiling and deep-sequencing indicated that miR-126 was highly and specifically expressed by plasmacytoid dendritic cells (pDCs), the major type I interferon producing cells. In the absence of miR-126, pDCs were unable to become properly activated or secrete high levels of interferons in response to stimulation, and underwent premature apoptosis, indicating that miR-126 controlled the survival and function of pDCs. Molecular analysis revealed that miR-126 regulated the expression of a number of innate response genes. Interestingly, in pDCs miR-126 also controlled the expression of VEGF-receptor 2 (VEGFR2), a receptor described to be predominately restricted to endothelial cells. However, gene expression analysis of more than 200 different immune cell populations indicated that VEGFR2 is also highly and exclusively expressed on pDCs amongst hematopoietic cells. Deletion of the gene encoding VEGFR2 specifically in DCs resulted in impaired pDC development and reduced type I interferon production; indicating that VEGFR2 signaling is also important for pDC function, and suggesting that miR126 regulates pDCs, at least in part, through its control of VEGFR2 expression. These studies indicate that the miR-126–VEGFR2 axis is an important regulator of the innate response to pathogen infection, which operates through multiscale control of pDCs, and assigns an important new function to the clinically targeted receptor VEGFR2 and its ligand VEGF-A.

14 VEGF-mediated Tumor Angiogenesis

ABSTRACTS Overcoming Resistance to VEGF-Blockade by Combinatorial Targeting of the Angiopoietin/Tie2 axis Alexander Scholz, PhD1,2, Sebastian Cremer, MD1, Patrick N. Harter, MD1, Michel Mittelbronn, MD1, Paul van Slyke, PhD3, Daniel J. Dumont, PhD3, Karl H. Plate, MD1 and Yvonne Reiss, PhD1 1University Cancer Center & Institute of Neurology, Goethe University Medical School, Frankfurt, Germany, 2Laboratory of Immunology and Vascular Biology, Stanford University, Palo Alto, United States, 3Sunnybrook Health Center, Toronto,

Canada Glioblastoma multiforme (GBM) is the most common and the most malignant form of brain tumor with a median overall survival of just about 14 months. Until now, standard treatment consists of combined radio- and chemotherapy. More recently, antiangiogenic therapy targeting VEGF has been approved for the treatment of recurrent GBM. Even though anti-VEGF therapy has prolonged symptom-free survival, its role in first-line treatment is less clear. Furthermore, several pre-clinical studies suggest that resistance to anti-angiogenic therapy could be mediated by infiltration of proangiogenic myeloid cells. To define new therapeutic options for anti-angiogenic therapy in GBM, we addressed the role of the Angiopoietin(Ang)/Tie signaling pathway. We found Ang-2 to be upregulated in vessels of malignant human brain tumors while being absent in normal brain. Furthermore, Ang-2 expression correlated with WHO grade and the amount of infiltrating myeloid cells. In a mouse model with endothelial cell-specific overexpression of Ang-2, we observed significantly increased numbers of myeloid cells in an orthotopic, syngeneic glioblastoma model, thus corroborating our findings in the human system. Interfering with Ang-2/Tie2 signaling by either blockade of Ang-2 or overactivation of the receptor lead to increased overall survival, vessel normalization, and reduced infiltration of myeloid cells in the Gl261 glioma model. Combining anti-Ang-2 and anti-VEGF treatment showed synergistic effects with regard to survival and myeloid cell infiltration. Thus, targeting Ang-2 and VEGF in glioblastoma represents a promising approach for future therapy by targeting two major components of solid tumors, angiogenesis, and inflammation.

VEGF-mediated Tumor Angiogenesis 15

ABSTRACTS Interacting Signaling Pathways in Tumor Angiogenesis: VEGF, Delta/Notch, and Angiopoetin/Tie-2 Gavin Thurston, PhD, Regeneron Pharmaceuticals, Tarrytown, New York, United States Blocking angiogenesis in cancer is recognized as a useful approach for the treatment of many solid tumors. Although angiogenesis is a complex process that requires the coordinated interplay of a variety of signaling pathways, much of the focus to date has been on targeting the VEGF pathway, which is an important upstream activator of tumor angiogenesis. However, not all preclinical and clinical tumors are equally responsive to VEGF inhibition. Therefore, more recent regimens are exploring VEGF blockers in combination with other anti-angiogenic agents or with cytotoxic chemotherapy. Additional angiogenic targets include Delta-like ligand 4 (Dll4) and angiopoietin-2 (Ang2), which are both expressed by endothelial cells in tumors and act as ligands for Notch or Tie2 receptors in the vasculature, respectively. In preclinical tumor models, combined blockade of VEGF plus these other angiogenic pathways can produce enhanced anti-vascular effects, such as greater decreases in tumor perfusion, compared to single agent treatments. These enhanced anti-vascular effects are associated with more potent tumor growth inhibition. Full benefit of combination anti-angiogenic therapy in the clinic will require better mechanistic understanding of which tumors vessels are resistant to individual treatments, as well as the development of biomarkers to identify resistant and susceptible tumor vessels.

Instructive Angiocrine Contribution of Vascular Niche to Tumor Progression Shahin Rafii, MD, Weill Cornell Medical College, New York, New York, United States Abstract not available at time of printing.

16 VEGF-mediated Tumor Angiogenesis

ABSTRACTS Session II: Immunemodulation in Angiogenesis Mechanisms of Macrophage Promotion of Tumor Angiogenesis Evelyn Aranda, PhD, Bin-Zhi Qian, and Jeffrey W. Pollard, PhD, Albert Einstein College of Medicine, Bronx, New York, United States Tumor-associated macrophages (TAM) play a major role in promoting malignancy in a wide variety of cancers. Our previous results in a mouse model of breast cancer caused by the expression of the Polyoma Middle T oncoprotein (PyMT) in the mammary epithelium, have demonstrated that the ablation of the macrophage colony stimulating factor-1 (CSF-1) resulted in a reduced rate of progression of tumors to malignancy and an inhibition of metastases. This was associated with a dramatic reduction in TAMs and inhibition of the angiogenic switch. Vascular endothelial growth factor (VEGF) and Wnts have been shown to be regulators of normal and pathological angiogenesis but little is known about the mechanisms that macrophages use to increase angiogenesis. Here we aimed to find the role of macrophages in tumoral blood vessel formation. Our results show that sorted TAMs from PyMT mice increase in vitro endothelial cell tube formation and hemoglobin content in Matrigel plugs. Furthermore, a significant reduction of hemoglobin content and infiltration of CD34-positive cells was found in plugs implanted in macrophage-deficient Csf1op null mice when compared to wild-type mice. Targeted gene ablation of VEGF in macrophages shows less angiogenesis in plugs while ablation of Porcupine (Porcn), a protein involved in the secretion of Wnts, increases the levels of VEGF in macrophages, tube formation, and infiltration of cells in Matrigel. These data show that macrophages are essential players in the formation of blood vessels and suggest a crosstalk of VEGF and Wnts in the regulation of tumoral angiogenesis.

Novel Immunotherapeutic Approaches for Cancer Therapy David F. McDermott, MD, Beth Israel Deaconess Medical Center, Boston, Massachusetts, United States An improved understanding of the molecular mechanisms governing the host response to tumors has led to the identification of signaling pathways involved in limiting the anti-cancer immune response. One of the most critical pathways responsible for mediating tumor-induced immune suppression, or “immune checkpoint”, is the programmed death-1 (PD-1) pathway. Many human solid tumors express programmed death-1 ligand 1 (PD-L1), and this is often, though not always, associated with a worse prognosis. Tumor-infiltrating lymphocytes (TILs) from patients with cancer typically express PD-1 and have impaired anti-tumor functionality. Proof-of-concept has come from several preclinical studies in which blockade of PD-1 or PD-L1 has enhanced T-cell function and tumor-cell lysis. Three monoclonal antibodies against PD-1, and one against VEGF-mediated Tumor Angiogenesis 17

ABSTRACTS PD-L1, have reported phase 1 data. All four agents have shown encouraging preliminary activity with good safety profiles, and additional data are awaited. Other factors in the tumor microenvironment have been shown to suppress tumor immune responses in preclinical models. For example, there is evidence that VEGF decreases dendritic cell (DC) function, and thus by extension impacts antigen presentation and T-cell activation. Concurrently restoring DC and T-cell functionality may improve the anti-tumor T-cell response and lead to improved clinical activity. Trials evaluating the combination of “immune checkpoint inhibitors” and angiogenesis inhibitors are ongoing. This talk will summarize the emerging data in this field and discuss the potential for collaboration between tumor immunotherapists and cancer “micro-environmentalists”.

Tackling Resistance to Anti-angiogenic Therapy: a Tumor-Immune Cell Dialogue Lee B. Rivera1, Kan Lu1, David Meyronet3, Judy Varner4, and Gabriele Bergers1,2 1Helen Diller Family Comprehensive Cancer Center, UCSF, San Francisco, California, United States, 2Brain Tumor Research Center, UCSF, San Francisco, California, United States, 3Hospices Civils de Lyon, Lyon, France, 4Moores

Cancer Center, UCSD, San Diego, California, United States Bevacizumab (Avastin; Genentech/Roche), a humanized monoclonal antibody directed against VEGF-A, and several receptor tyrosine kinase inhibitors blocking the VEGF/VEGFR pathway (e.g., Sunitinib, sorafenib) have been FDA-approved for use in various tumor types in the US. Despite encouraging beneficial effects, patients inevitably develop resistance and frequently fail to demonstrate significantly better overall survival. Emerging evidence suggests that tumors bypass anti-angiogenic therapy while VEGF signaling remains inhibited through a variety of mechanisms by either re-establishing neovascularization, or by altering their behavior to propagate and progress without the need to satisfactorily reinitiate angiogenesis. Leveraging from the results obtained during the course of anti-angiogenic therapy in pancreatic and brain tumor mouse models and patient tumor samples, we found that relapsing tumors exposed cellular signals associated with resistance that were rather transiently regulated in response to contextual signals that tumor cells received from the immune cell compartment and responded to during the course of treatment. These data also revealed that the nature of tumor cells determines which resistance pathways will be facilitated by immune cells. Further, we observed that various innate immune populations exhibit an oscillatory behavior by which they substitute for each other to promote resistance by using the same central signaling pathway. These studies support the concept of reprogramming innate immune cell populations, rather than targeting them, as a sufficient and feasible approach to abrogate angiogenesis and restore T cell-mediated antitumor immunity.

18 VEGF-mediated Tumor Angiogenesis

ABSTRACTS Overcoming Tumor Resistance to VEGFA Signaling Blockade by Co-targeting ANG2-TIE2 Signaling and Macrophages Michele De Palma, PhD, The Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology Lausanne (EPFL), Lausanne, Switzerland Angiopoietin-2 (ANG2/ANGPT2) is a context-dependent TIE2 receptor agonist that enables angiogenesis in concert with vascular-endothelial growth factor (VEGF)-A. Indeed, ANG2 neutralization improves tumor angiogenesis and growth inhibition by VEGFA or VEGF receptor-2 (VEGFR2) blockade in several tumor models. However, it is unclear whether ANG2 may also sustain VEGFA-independent tumor angiogenesis. To address this question, we employed two tumor models — late-stage RIP1-Tag2 pancreatic neuroendocrine tumors (PNETs) and orthotopic MMTV-PyMT mammary carcinomas — which are transiently responsive but rapidly become resistant to VEGFR2 blockade. We found that ANG2 expression was higher in the PNETs than mammary tumors and was further increased by VEGFR2 inhibition. Combined ANG2/VEGFR2 blockade suppressed revascularization and progression in the vast majority of the PNETs, whereas it only had minor additive effects in the mammary tumors. Notably, a minority of the PNETs revascularized after ANG2/VEGFR2 blockade, thus showing adaptive resistance to the double anti-angiogenic treatment. We found increased tumor-associated macrophage (TAM) numbers in association with the resistant tumors, and therefore explored the utility of pharmacologically depleting TAMs in combination with the anti-angiogenic treatment. The results of these studies will be presented at the meeting.

Imaging Immune Cells in the Context of Tumor Angiogenesis John S. Condeelis, PhD, Albert Einstein College of Medicine, New York, New York, United States Imaging at single cell resolution in vivo has been used to discover that carcinoma cells form migratory streams and intravasate when associated with macrophages and linear collagen-I fibers. The tumor cell-macrophage tropism results from EGF/ CSF1-paracrine chemotaxis causing the formation of cell pairs and streams that move unidirectionally to blood vessels [Roussos et al., Nat. Rev. Cancer, 2011]. This emergent behavior is cell autonomous and can be reconstituted in vitro using the three purified cell types and collagen-I fibers [Sharma et al., IntraVital, 2012]. Once the three cell types touch each other they spontaneously assemble a cell complex involving the direct contact between a tumor cell, endothelial cell, and macrophage [Roh-Johnson et al., Oncogene, 2013]. This triad is called the “Tumor MicroEnvironment of Metastasis (TMEM)” and its presence in human breast tumors has been used as a prognostic to predict distant metastatic recurrence [Robinson et al., Clin. Cancer Res., 2009; Rohan et al., JNCI, 2014 in Press].

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ABSTRACTS TMEM functions as a dedicated site of tumor cell transendothelial migration for intravasation and extravasation [Roh-Johnson et al., Oncogene, 2013]. The gene expression profile of the tumor cells involved in TMEM function has been determined and has supplied prognostic markers for predicting tumor cell dissemination in breast tumors [Robinson et al., Clin. Cancer Res., 2009; Rohan et al., JNCI, 2014 in Press; Agarwal et al., Breast Cancer Res., 2012]. The micro-pharmacology of the signaling between cells within TMEM suggest that TMEM is similar in function to an angiogenic sprout suggesting that re-tasking of drugs directed toward inhibition of angiogenesis, but at much lower concentrations, may block tumor cell dissemination.

Session III: Vascular Normalization in Angiogenesis Point – Counter Point Debate Does Vascular Normalization Improve Delivery of Cancer Therapeutics? Pro-Debater: Dan G. Duda, DMD, PhD, Kyrre E. Emblem, PhD, Marek Ancukiewicz, PhD, Christine Lu-Emerson, MD, Elizabeth R. Gerstner, MD, Tracy T. Batchelor, MD, A. Greg Sorensen, MD, and Rakesh K. Jain, PhD; Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, United States Anti-angiogenic therapy has shown clear activity and improved survival benefit for certain tumor types. However, an incomplete understanding of the mechanisms of action of anti-angiogenic agents has hindered optimization and broader application of this new therapeutic modality. In particular, the impact of anti-angiogenic therapy on tumor blood flow and drug and oxygen delivery (i.e., the role of vessel pruning versus normalization) remains controversial. This controversy has become critical as multiple phase III trials of anti-VEGF agents combined with cytotoxics failed to show overall survival benefit in newly diagnosed glioblastoma (nGBM) patients and several other cancers. Using MRI techniques and blood biomarkers in prospective phase II clinical trials of cediranib with chemoradiation vs. chemoradiation alone in nGBM patients, we shed light on mechanisms of nGBM response to cediranib, a pan-VEGF receptor tyrosine kinase inhibitor. We demonstrate that improved perfusion occurs only in a subset of patients in cediranib-containing regimens, and is associated with improved overall survival in these nGBM patients. Moreover, an increase in perfusion is associated with improved tumor oxygenation status as well as with pharmacodynamic biomarkers, such as changes in plasma placenta growth factor and sVEGFR2. In conclusion, tumor perfusion changes after anti-angiogenic therapy may distinguish patients who benefit from those who do not early in the course of this expensive and potentially toxic form of therapy, and these results may provide new insights into the selection of glioblastoma patients most likely to benefit from anti-VEGF treatments. Contra-Debater: Elisabeth G.E. de Vries, MD, PhD, The University Medical Center Groningen, the Netherlands Abstract not available at time of printing. 20 VEGF-mediated Tumor Angiogenesis

ABSTRACTS Day 2 — Friday, June 20, 2014 Editor's Guide to Writing and Publishing Your Paper Brooke Grindlinger, PhD, The New York Academy of Sciences, New York, New York, United States Publishing is critical to the scientific profession, yet training and guidance on the topic is very limited. Brooke Grindlinger, PhD, former Science Editor at The Journal of Clinical Investigation, will discuss what makes a good paper, strategies for selecting the appropriate journal, the review process, and how to navigate resubmissions. Session IV: Challenges to Anti-angiogenic Therapy – Learning from Successes and Failures

Synergy of Chemotherapy and Anti-VEGF Treatment (Mechanisms of Action of Targeting VEGF) Lee M. Ellis, MD, The University of Texas MD Anderson Cancer Center, Houston, Texas, United States Targeting VEGF has been a successful anti-neoplastic approach in numerous cancers. However, the magnitude of benefit has been less than expected. A better understanding of the mechanism(s) of action of this class of drugs would help provide insight on how to improve efficacy, and biomarker identification, but despite valiant attempts, we still do not fully understand how these drugs work. The mechanisms of action of VEGF targeted therapies are diverse and probably cannot be attributed primarily to any single effect. This is more complicated by the fact that multi-kinase inhibitors affect numerous pathways in addition to VEGF signaling. With rare exception, the addition of VEGF-Receptor (VEGFR) targeted tyrosine kinase inhibitors (TKIs) to chemotherapy has not led to meaningful improvements in survival in patients with solid malignancies. In fact, in recent metaanalysis of VEGFR TKIs and chemotherapy (Funakoshi et al., Cancer Treatment Reviews, 2014), the relative risk of a fatal adverse event was increased by nearly 50%. Antibodies to VEGF (Bevacizumab) and more recently VEGFR2 (Ramucirumab) have led to improvements in survival in some, but not all clinical trials where these combinations have been studied. The same holds true for VEGF-Trap (Aflibercept). This presentation will discuss various mechanisms of action of VEGF targeted therapies.

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ABSTRACTS Optimal Strategies for Resistance Avoidance and Long-Term Treatment with Anti-angiogenic Therapy Axel Grothey, MD, Mayo Clinic College of Medicine, Rochester, Minnesota, United States Anti-angiogenic therapy, in particular, anti-VEGF (vascular endothelial growth factor) therapy has become a component of standard treatment algorithms in various malignancies over the last decade. In contrast to cytotoxic chemotherapy or agents targeting tumor cells directly, anti-VEGF agents presumably exert their antitumor effect by modulating the tumor-host interaction or tumor microenvironment and affecting the host-provided tumor vasculature. The cellular targets of antiangiogenic therapy are genetically stable since they are routinely not derived from tumor cells. Thus, mechanisms of resistance to anti-angiogenic agents could differ from the ones associated with directly tumor-targeting agents. In fact, it has been questioned if resistance to anti-angiogenic agents exists at all. Recent data of a phase III trial in metastatic colorectal cancer confirmed that continuing the antiVEGF-A antibody bevacizumab beyond tumor progression from first- to second-line therapy is associated with improved survival, thereby challenging the established concept of tumor resistance to anti-angiogenic therapy. A large body of evidence has been generated over the last years demonstrating that a prolonged duration of anti-VEGF therapy is important for optimizing outcomes. Research on mechanisms of secondary resistance has suggested that upregulation of alternate pro-angiogenic factors beyond VEGF could play an important role. A longitudinal analysis of these factors could provide valuable insights regarding treatment approaches to counteract these mechanisms of resistance. It is conceivable that additional anti-angiogenic agents will have to be added to protracted and continued anti-VEGF therapy over time to maximize the duration of tumor control.

Vessel Architectural Imaging Reveals Early Vascular Response to Anti-angiogenic Therapy Kyrre E. Emblem, PhD1,2, Elizabeth R. Gerstner, MD2, Tracy T. Batchelor, MD2, Dan G. Duda, DMD, PhD2, Marco C. Pinho, MD2,3, Bruce Rosen, MD2, PhD, A. Greg Sorensen, MD2,4, Patrick Y. Wen5, MD, and Rakesh K. Jain, PhD2 1Oslo University Hospital, Oslo Norway, 2Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, United States, 3University of Texas Southwestern Medical Center, Dallas, Texas, United States, 4Siemens Healthcare Health Services, Malvern, Pennsylvania, United States, 5Dana-Farber

Cancer Center and Harvard Medical School, Boston, Massachusetts, United States Randomized phase III trials of bevacizumab with standard chemoradiation in glioblastoma (RTOG 0825 and AVAglio) indicate that first-line anti-VEGF treatment does not extend survival in unselected patient populations. Because anti-angiogenic drugs reduce vessel permeability, conventional MRI alone may not be adequate for monitoring treatment responses. Here, we address the lack of appropriate imaging biomarkers in clinical trials in brain tumors by going beyond conventional 22 VEGF-mediated Tumor Angiogenesis

ABSTRACTS MRI. Our initial experience in 70 glioblastoma patients enrolled in two phase II trials (NCT00305656, NCT00662506) of cediranib, an oral pan-VEGFR inhibitor, withand without chemoradiation, shows the promise for Vessel Architectural Imaging (VAI) [Emblem et al., Nature Medicine, 2013]. VAI combines perfusion MRI from small (