Improving Patient Outcomes by Targeting the VEGF/VEGFR Axis Wells Messersmith, MD, FACP Professor Director, Gastrointestinal Medical Oncology Program Co-Head, Division of Medical Oncology Program co-Leader, Developmental Therapeutics May 2013

Conflict of Interest: No employment, speaker’s bureaus, stock ownership, royalties, patents, etc Data Safety Monitoring Board for Morphotek Local PI of clinical trials by Genentech/Roche, GSK, AstraZeneca, EntreMed, Pfizer, Millenium, Bayer, Onconova, and NIH/CTEP. Slides: S. Kopetz (MDACC), Chris Lieu (Colorado)

Angiogenesis Outline/Objectives: Introduction Role of PlGF a. Angiogenesis b. Ongoing trials in GI cancers Role of PlGF Mechanisms of resistance

Angiogenesis Background Observation that tumor growth can be accompanied by increased vascularity was reported more than a century ago In 1939, Ide and colleagues first postulated the existence of a tumor-derived blood-vessel-growth stimulating factor In 1960s, experiments by Greenblatt and Shubik, and Ehrmann and Knoth, provided early evidence that tumor angiogenesis was mediated by diffusible factors produced by tumor cells In 1971, Folkman proposed that anti-angiogenesis might be an effective anticancer strategy C. Lieu, Colorado

VEGF VEGF family of growth factors and their receptor tyrosine kinases mediate proangiogenic effects Normal endothelial cells engaged in angiogenesis express numerous VEGF receptors, but produce very little detectable VEGF Tumor cells express VEGF receptors and also produce VEGF (autocrine mechanisms) Classic signaling is via VEGF-A binding to VEGFR-2, stimulating the PI3K/Akt as well as Raf/MEK/MAPK pathways

VEGF Signaling Kerbel R. N Engl J Med 2008;358:2039-2049

Large Molecule VEGF Inhibitors VEGF-A VEGF-R1 (Flt-1) Migration Invasion Survival VEGF-R3 (Flt-4) Lymphangiogenesis VEGF-R2 (KDR/Flk-1) Proliferation Permeability PlGF VEGF-B VEGF-C, VEGF-D Functions Y Bevacizumab Y Ramucirumab Aflibercept (VEGF Trap) PIGF = placental growth factor. Holash et al, 2002; Roy et al, 2006; Ghosh et al, 2000.

Proposed Mechanism of Anti-Angiogenics Inhibition of new vessel growth and vascular regression Vascular normalization Vascular constriction Direct effects on tumor cell function Offsetting effects of chemotherapy inducing VEGF levels

(selected anti-angiogenic agents) Phase III’s (selected anti-angiogenic agents) Colorectal Recently reported: Regorafenib (“CORRECT”) – positive Aflibercept (“VELOUR”) – positive Bevacizumab 2nd line (“TML”) – positive Brivinib (with cetux) – negative Ongoing Ramucirumab (VEGFR-2 mAb; “RAISE”): NCT1183780

Track Record of TKI’s in Colorectal Total > 10,000 Kopetz, MDACC

Track Record of TKI’s in Colorectal Agent FOLFOX Irinotecan FOLFIRI EGFRi + chemo Other Sunitinib Phase IIB Phase III Phase I/II Vandetanib Phase I Cediranib Phase II/III Vatalanib Phase IIIs Axitinib Bevacizumab Semaxinib 5-FU, phase III Motesanib Sorafenib Pazopanib Brivanib Cetux, Phase III Regorafenib Phase II Linifanib Positive Negative Ongoing

Track Record of TKI’s in Colorectal Kopetz, MDACC

(selected anti-angiogenic agents) Phase III’s (selected anti-angiogenic agents) Pancreas Cancer Gem +/- sorafenib “BYPAN” (NCT00541021) - negative Gem +/- bevacizumab (CALGB 80303) - negative Gem +/- Aflibercept (“VANILLA”) - negative Gem +/- axitinib (NCT00471146) – negative Gastro-esophageal Bevacizumab (“AVAGAST”) – negative (overall population) Ramucirumab (VEGFR-2) vs BSC – positive Pancreas Islet Cell / Carcinoid Sunitinib (NCT00428597) – positive Bev vs Interferon, SWOG 0518 (NCT00569127)

(selected anti-angiogenic agents) Phase III’s (selected anti-angiogenic agents) HCC 1st Line Sorafenib (“SHARP”) – positive Doxorubicin/Sorafenib vs S, CALGB 80202 (NCT00917384) Sorafenib vs brivanib (“BRISK FL”) – negative Sorafenib vs sunitinib (NCT00699374) – negative Sorafenib vs linifanib (ABT-869) (NCT01009593) 2nd Line Brivanib vs BSC (“BRISK PS”) (NCT 01217034) – terminated Ramucirumab vs BSC (“REACH”) (NCT01140347)

Angiogenesis Outline/Objectives: Introduction Role of PlGF a. Angiogenesis b. Ongoing trials in GI cancers Role of PlGF Mechanisms of resistance

Role of Placenta Growth Factor Angiogenic protein in VEGF family involved in cancer, inflammation, pre-eclampsia, cardiovascular disease Unlike VEGF, PlGF is undetectable in healthy tissues; upregulated in tumors Contributes to angiogenic and inflammatory “switch” Produced by malignant cells, endothelial cells, smooth-muscle cells, pericytes, cancer-associated fibroblasts, tumor-associated macrophages, and other stromal cells Serum PlGF is prognostic in colorectal cancer (Wei, 2009) Also prognostic in gastric cancer (Chen, 2004) and HCC (Ho, 2007)

Mechanism of Placenta Growth Factor Loges et al, Clin Cas Res 2009. ©American Association for Cancer Research

Insights on PlGF Four isoforms of PlGF in humans PlGF transmits its own signal via VEGFR-1 (results is phosphorylation of different residues than VEGF-A) PlGF augments VEGF-A signaling by displacing it from soluble VEGFR-1 (a negative regulatory mechanism), thereby increasing availability of VEGF-A PlGF also causes transphosphorylation of VEGFR-2 PlGF upregulates the expression of proangiogenic molecules such as VEGF, FGF-2, and MMP-9 PlGF knock-out mice are healthy (unless challenged with inflammation, cancer, etc)

Role of Placenta Growth Factor Loges et al, Clin Cas Res 2009. ©American Association for Cancer Research

PlGF in bevacizumab resistance Colorectal cell lines that are chronically exposed to anti-VEGF-A therapy with bevacizumab show upregulation of compensatory pathways, including PlGF These “bev-adapted” cell lines are also more metastatic Fan et al, British J Cancer. ©Cancer Research UK

PlGF and GI Toxicity In chemically induced models of intestinal injury: Up to 6 fold increase in PlGF in the injured colon (minimal VEGF increase) In PlGF knockout mice (-/-), Substantial intestinal injury Healing angiogenesis response is absent Can be rescued by PlGF Increased weight loss and chronic intestinal injury Wild-type PlGF Knock-Out (severe injury) S. Kopetz, MDACC Hindryckx et al Lab Invest, ‘10, Fischer, Nat Rev Onc, ’08 ©Nature Publishing Group

Angiogenesis Outline/Objectives: Introduction Role of PlGF a. Angiogenesis b. Ongoing trials in GI cancers Role of PlGF Mechanisms of resistance

Resistance Intrinsic Resistance Tumor cells can use existing blood vessels in vasculature-rich organs (lungs) Absence of VEGF or VEGF receptors in metastatic tumors Acquired Resistance Induced pro-angiogenic factor substitution (FGF, PlGF, etc) Recruitment of bone marrow-derived cells to restore neovascularization

Resistance Pathways Numerous compensatory factors and cell types Ellis et al. Clin Cancer Res 2008;14:6371-6375 Numerous compensatory factors and cell types

Preclinical Models of Resistance Preclinical trials in a mouse model of pancreatic neuroendocrine (islet cell) cancer, Rip1–Tag2 Rip1–Tag2 mice were treated with a monoclonal antibody (DC101) that specifically blocked VEGFR signaling (in particular VEGFR2) Initial response denoted by tumor stasis and reductions in tumor vascularity Casanovas et al. Cancer Cell 2005:8;299-309

Preclinical Models of Resistance Response phase was transitory (10–14 days) and was followed by tumor regrowth Dense tumor vasculature restored Casanovas et al. Cancer Cell 2005:8;299-309

Preclinical Models of Resistance The relapsing tumors were found to express higher levels of the mRNAs for the pro-angiogenic factors fibroblast growth factor 1 and 2 Tumor-derived cells subjected to hypoxic conditions similarly upregulated most of the genes Casanovas et al. Cancer Cell 2005:8;299-309

RECIST Measurements for Representative Patient Clinical Evidence RECIST Measurements for Representative Patient Prior to Progression “Angiogenic Activity” Hypothesis: resistance to angiogenesis inhibition should precede clinical progression Kopetz; Lieu

PlGF and bFGF are Increased Prior to Progression p<0.01 compared to baseline ULN = upper limits of normal for healthy controls Kopetz. GI ASCO. 2009

Conclusion Inhibition of angiogenesis results is modest but meaningful benefit for multiple tumor types, including colorectal cancer. Dearth of biomarkers for who benefits; who is harmed; how resistance occurs VEGF-A has been the main focus, but the role other angiogenic signaling proteins (such as PlGF) under active study and drug development Overcoming resistance via multiple bypassing pathways may be challenging

Angiogenesis Overview Thank You! Wells.Messersmith@ucdenver.edu 31