1. Robert S. Kerbel, PhD Canada Research Chair in Tumor Biology, Angiogenesis and Antiangiogenic Therapy Sunnybrook Health Sciences Centre University of Toronto Acquired (“Evasive”) Resistance to Antiangiogenic Drugs and Tumor Flare-Up 2 nd Quebec Conference on Therapeutic Resistance in Cancer Montreal, November 5, 2010

2. Potential Conflict of Interest Dr. Robert S. Kerbel –Consultant (2004-present) Adnexus SAB member GSK MolMed Oxigene Taiho Pharmaceuticals Japan YM Biosciences

3. Bioessays 13: 31-36, 1991 Inhibition of tumor angiogenesis as a strategy to circumvent acquired resistance to anti-cancer therapeutic agents Kerbel RS

4. Nature 390: 404-407, 1997 Antiangiogenic therapy of experimental cancer Does not induce acquired drug resistance Boehm T, Folkman J, Browder T, O'Reilly MS Nature 390: 335-336, 1997 A cancer therapy resistant to resistance Kerbel RS ?

5. Recent Clinical Trial Results Raising Concerns About Antiangiogenic Therapy 1.Many phase III trials with disappointing results failure of oral TKIs alone or when combined with chemotherapy failure or small PFS benefit only when bevacizumab combined with chemotherapy; no OS benefit breastovarian gastric prostate and failure of 1 st adjuvant phase III trials breast prostate colon ‘CO8’ in CRC ‘AVANT’ in CRC

6. Llovet JM et al., N Engl J Med 359: 378-390, 2008 Sorafenib in advanced hepatocellular carcinoma

7. Yu et al (RS Kerbel) Science 295: 1526-1528, 2002 Effect of p53 status on tumor response to antiangiogenic therapy Conclusions: 1.Antiangiogenic therapy selectively enriches for p53 mutant cells  resistance 2. p53 mutant cells reside in more hypoxic regions distal to blood vessels

8. Some Proposed Modes of Acquired Resistance to VEGF Pathway–Targeting Antiangiogenic Drugs Selection of variants having enhanced ability to survive under hypoxic conditions Target one pathway (eg, VEGF) and a compensatory, alternate pathway takes over (eg, bFGF, IL-8, ……) “Evasive resistance” Rapid vascular remodeling (maturation) of remaining tumor vasculature, or “vascular co-option”

9. VEGF is a dominant player in tumor angiogenesis – but there are lots of other backups, eg, PlGF (VEGFR-1) Angiopoietins (Tie2) Dll4 (notch receptors) Sex hormones bFGF HGF/SF (c-met receptor) IL-8 PDGF SDF-1 (CXCR4 receptor) (FGFRs)

10. Cancer Cell 8: 299-309, 2005 Drug resistance by evasion of antiangiogenic targeting of VEGF signaling in late-stage pancreatic islet tumors Casanovas O, Hicklin DJ, Bergers G, Hanahan D.

11. “Anti-angiogenic therapy using brivanib….in a mouse model of pancreatic neuroendocrine cancer (PNET), results in sustained vascular blockade, without evidence for evasive/acquired resistance …” Elizabeth Allen, Ian B. Walters, I. Celeste Rivera, and Douglas Hanahan  Tumors acquiring resistance to DC101 (a VEGFR-2 Mab) respond to VEGFR-2/bFGFR antagonist (brivanib)  Also, rapid development of resistance previously detected using DC101 not observed with brivanib TKI  Efficacy of the drug (brivanib) appears even more effective when used in the first-line treatment setting Poster presented at the EORTC-NCI-AACR Molecular Targets meeting in Boston, 2009

12. D Huang et al, Cancer Res 70: 1063-1071, 2010 Interleukin-8 mediates resistance to antiangiogenic agent sunitinib in renal cell carcinoma.

13. antiangiogenic therapy H1F-1   tumor response; elevated hypoxia bFGF  Hypoxia/HIF-1–Mediated Upregulation of Multiple Proangiogenic Growth Factors HGF  SDF-1 

14. Tang TC, et al (RS Kerbel). Development of a resistance-like phenotype to sorafenib by human hepatocellular carcinoma cells is reversible and can be delayed by metronomic UFT chemotherapy. Neoplasia, in press, Nov. 2010 Response to Sorafenib, and Subsequent Relapse of Human HCC Tumors Transplanted Into the Liver sorafenibib Days Therapy initiated ~2 wks after transplantation

15. Implications for ‘Sequential / Salvage’ Therapy with VEGF Pathway Inhibitors to Treat Progressive Disease sunitinib responserelapse sorafenib responserelapse sunitinib bevacizumab responserelapse sunitinib

16. HJ Burstein et al. (KD Miller), J Clin Oncol 26: 1810-1816, 2008 Phase II study of sunitinib malate, an oral multitargeted tyrosine kinase inhibitor, in patients with metastatic breast cancer previously treated with an anthracycline and a taxane

17. Ebos et al., "Multiple circulating proangiogenic factors induced by sunitinib malate are tumor-independent and correlate with antitumor efficacy." Proc Nat'l Acad Sci, USA 104:17069-74, 2007

18. Sunitinib treatment of normal mice also increases circulating G-CSF, SDF-1, SCF and OPN in a dose- dependent fashion VEGF SDF-1  SCF sTIE-2 OPN EPO sVEGFR-2IL-6 G-CSF PDGF-AB Ebos et al PNAS 104: 17069-74, 2007

19. Circulating Bone Marrow-Derived Pro-angiogenic Cell Populations RS Kerbel "Tumor Angiogenesis" New Engl J Med 358: 2039-2049, 2008

20. Possible consequences of the host-dependent multi-cytokine ‘surge’ induced by antiangiogenic TKIs 1.contribute to tumor flare up/rebound? 2.contribute to drug resistance? 3.contribute to tumor growth/malignant progression?

21. Tumor Flare-up or 'Rebound' After Cessation of Antiangiogenic TKI Therapy Acta Oncol. 48: 927-31, 2009 The reverse side of the victory: flare up of symptoms after discontinuation of sunitinib or sorafenib in renal cell cancer patients. A report of three cases. Desar IM, Mulder SF, Stillebroer AB, van Spronsen DJ, van der Graaf WT, Mulders PF, van Herpen CM Acta Oncol. 48: 621-624, 2009 Flare-up: an often unreported phenomenon nevertheless familiar to oncologists prescribing tyrosine kinase inhibitors. Wolter P, Beuselinck B, Pans S, Schoffski P

22. Cancer Cell: Vol 15(3):220-239 March 3, 2009

23. Protocol for initial examination of the possibility of anti- angiogenic drug-induced acceleration of metastatic disease e.g. daily treatment of normal mice with sunitinib for 7 days e.g. daily treatment of normal mice with vehicle control for 7 days inject luciferase tagged human breast cancer cells 1 day evaluate metastatic burden and survival days 5 –30 inject luciferase tagged human breast cancer cells 1 day evaluate metastatic burden and survival days 5 –30

24. Impact of Sunitinib Pretreatment (or Post-treatment) on Progression of Micrometastases Days Post Tumor Implantation 7 14 21 30 Group A 1 2 3 4 5 6 7 Group D 1 2 3 4 5 Group E 1 2 3 4 5 Group B 1 2 3 4 5 6 7 Group C 1 2 3 4 5 6 7 27 Ebos et al. Cancer Cell, 15: 232-239, 2009

25. Impact on Survival Times

26. Impact of Long Term Sunitinib Treatment on Established Primary Tumor Growth Ebos et al., Cancer Cell 15: 232-239, 2009

27. Short-term adjuvant sunitinib treatment increases spontaneous metastasis after removal of primary human breast cancer xenograft 5 Before 30 Group A Group B Days After Tumor Resection Photons/sec Ebos et al., Cancer Cell 15: 232-239, 2009 (survival times also decreased)

28. Ebos JM et al., Cancer Cell 15: 232-239, 2009 Accelerated metastasis after short-term treatment with a potent inhibitor of tumor angiogenesis. “Our results may be pertinent to the consideration of several prominent issues in cancer therapeutics including…… use of antiangiogenic drugs in the adjuvant setting……”

29. 0.00.51.01.52.02.53.0 0 20 40 60 80 100 DFS (years) FOLFOX +BEV Phase III Adjuvant Bevacizumab+Chemo NASBP ‘CO8’ Trial in Stage II & III CRC HR FOLFOX BEV only

30. “While we originally hoped the significant survival benefit of Avastin seen in metastatic disease in colorectal cancer would be translated to the early setting, it is becoming increasingly clear that the effects of Avastin are different in the metastatic and early disease settings for patients with colon cancer.’’ said Hal Barron, M.D., Head of Global Product Development and Chief Medical Officer at Roche.

31. SKOV-3-13 10 days after intraperitoneal transplantation of luciferase positive SKOV-3 cells Impact of Pazopanib and Metronomic Topotecan Chemotherapy in a Model of Advanced Ovarian Cancer SKOV-3-13 Non-tumor SKOV-3-6 SKOV-3-11 K. Hashimoto et al, Mol Cancer Ther 9: 996-1006, 2010

32. days after start of treatment % survival Impact of chronic daily metronomic oral topotecan chemotherapy plus pazopanib K. Hashimoto et al.

33. Acknowledgments John Ebos Christina Lee Georg Bjarnason Kae Hashimoto Jamie Christensen (Pfizer)