1. Receptor tyrosine kinases (RTKs) drive key cancer pathways and can be exploited as therapeutic targets, as shown by drugs like imatinib that inhibit mutated kinases in cancers. 2. RTK inhibitors have shown efficacy against cancers dependent on single kinases, but resistance often emerges through secondary mutations or bypass pathways. 3. Effective combination therapies are needed to overcome resistance, such as combining RTK inhibitors with other drugs that block downstream or bypass pathways.

1. Receptor tyrosine kinases as therapeutic targets for cancer John Heymach, M.D., Ph.D. Depts. of Thoracic/Head and Neck Medical Oncology and Cancer Biology University of Texas M.D. Anderson Cancer Center March 13, 2009

2. Key points Even though cancer cells are complex, they may be addicted to one or a few key pathways (“oncogene addiction”), often driven by an RTK. 2. Some RTKs are validated or very promising as therapeutic targets. Particularly for tumors with “oncogene addiction”. 3. We are testing RTK inhibitors in the clinic. Some work well for some diseases. 4. Resistance to RTK inhibitor may arise through several different mechanisms. Understanding these mechanisms of resistance is important for designing better combination therapies.

3. Cancer cells are very complex. Genetically unstable. Large number of mutations Multiple deregulated pathways.  Conventional wisdom was: “Targeting any one pathway is not likely to be successful.”

4. Hahn and Weinberg 2002

5. Efficacy of imatinib, an inhibitor of BCR-ABL, in CML (Druker et al, NEJM, 2001) Out of 54 patients treated with imatinib who had failed interferon alfa, at 300 mg or higher bid 53 complete hematological responses 29 cytological responses

6. Efficacy of imatinib, an inhibitor of BCR-ABL, in CML (Druker et al, NEJM, 2001) Out of 54 patients treated with imatinib who had failed interferon alfa, at 300 mg or higher bid 53 complete hematological responses 29 cytological responses  Conventional wisdom: “Perhaps hematological malignancies can be treated by targeting a single pathway. But solid tumors are different.”

7. 18 FDG-PET of patient with GIST treated initially with SU5416 and later with imatinib mesylate . Heymach et al, CCR, 2004 Pre- and post- treatment with SU5416 Pre- and post- treatment with imatinib

8. Efficacy of imatinib, an inhibitor of c-KIT, in gastrointestinal stromal tumors (Demetri et al, NEJM, 2002) Chemotherapy ineffective Families with inherited GIST found to have activating KIT mutations Imatinib tested in 147 patients 81% with partial response or prolonged stable disease  Selected solid tumors can be treated by targeting a single pathway

9. The Normal Cell Growth factors DNA Apoptosis Senescence Anti-Growth signals Limited nutrients/ microenvironment

10. The Cancer Cell Growth factors DNA Apoptosis Senescence Anti-Growth signals Contact inhibition/ microenvironment

11. The Hallmarks of Cancer Self-sufficiency in growth signals Insensitivity to anti-growth signals Evading apoptosis Limitless replicative potential Sustained angiogenesis Tissue invasion and metastasis (Genetic instability)  RTKs may be involved in all, or almost all, of these traits. Hanahan and Weinberg, Cell 2000

12. Hallmarks of cancer Luo et al, Cell 2009

13. “ Oncogene addiction” Despite the multitude of genetic and epigenetic alterations in a cancer, a given tumor is likely to be driven by only a few select changes- i.e. gain of an oncogene or loss of a tumor suppressor. Maintenance often depends on the continued activity of certain oncogenes. Myc K-RAS, HRAS BCR-ABL EGFR Her2 Others being studied: BRAF, MDM2, PI3K pathway Luo et al, Cell 2009

14. RTK inhibitors Small molecule inhibitors: bind to ATP pocket and prevent receptor phosphorylation (ATP donates phosphate) Examples: EGFR inhibitors: gefitinib and erlotinib Kit and BCR inhibitor: imatinib VEGFR inhibitors: SU5416, SU11248 ***Because of structural similarity of different tyrosine kinase domains, small molecule inhibitors typically inhibit several different RTKs Antibodies: typically bind to extracellular domain of receptor or ligand and prevent receptor activation Example: EGFR inhibitor: cetuximab (Erbitux) VEGF: bevacizumab (Avastin)

15. EGFR complexed with Erlotinib Stamos and Eigenbrot, 2002 http://www.ncbi.nlm.nih.gov

16. Structure of wild-type and mutated EGFR tyrosine kinase domain Key structures C and N lobes (ATP cleft in between) P lobe: phosphate binding A loop: activation Gazdar et al, Trends Mol Med. 2004 Oct;10(10):481-6.

17. Epidermal Growth Factor Receptors R K R K Signal transduction cascade Ligand Ligand binding Dimerization Phosphorylation

18. EGFR Inhibitors Plasma membrane R K R K Signal transduction cascade Ligand Antibodies: Erbitux (cetuximab), ABX-EGF, ICR62, Omnitarg(pertuzumab) TK inhibitors: Iressa (gefitinib), Tarceva (erlotinib), EKB-569, CI-1033, PKI166

19. EGFR Inhibitor Specificity Plasma membrane R K R K Gefitinib (Iressa) Erlotinib (Tarceva) R K R K R K R K erbB1 erbB2 erbB3 erbB4 R K R K EKB-569 CI-4033

20. EGFR and Tumor Specificity Plasma membrane R K R K R K R K R K R K erbB1 erbB2 erbB3 erbB4 R K R K Gastric, Ovarian H&N NSCLC Prostate Breast: B1,B2,B4

21. Kinase dendrogram for selected RTKIs Adapted from Fabian, M, Nature Biotechnology VOL 23, March 2005

22. Mechanisms of resistance to RTK inhibitors 1. Target not critical for a given tumor (primary resistance) 2. Incomplete receptor inhibition Inhibitor not sufficiently potent Secondary mutations that prevent binding of inhibitor (i.e. T790M mutation in lung cancer prevents gefitnib or erlotinib from inhibiting EGFR) 3. Target bypass: the tumor circumvents the inhibited pathway by activation of other pathways (i.e. MET)

23. Potential mechanisms of resistance to targeted agents R R Ligand R R Ligand Pathway X Effect (I.e. tumor cell survival) Pathway X Effect (I.e. tumor cell survival) 1. Incomplete target inhibition Effective target inhibition X (partial) inhibitor

24. Potential mechanisms of resistance to targeted agents R R Ligand R R Ligand Pathway X Effect (I.e. tumor cell survival) R R Ligand Pathway X Effect (I.e. tumor cell survival) Pathway X Effect (I.e. tumor cell survival) R2 R2 Ligand 2 1. Incomplete target inhibition 2. “Target bypass” Effective target inhibition X X Pathway Y A B

25. Approaches to combating resistance R R Ligand R R Ligand Pathway X Effect (I.e. tumor cell survival) Pathway X Effect (I.e. tumor cell survival) R2 R2 Ligand 2 1. Incomplete target inhibition 2. “Target bypass” X Pathway Y A B Add additional inhibitor or try more potent inhibitor for same target

26. Approaches to combating resistance R R Ligand R R Ligand Pathway X Effect (I.e. tumor cell survival) Pathway X Effect (I.e. tumor cell survival) R2 R2 Ligand 2 1. Incomplete target inhibition 2. “Target bypass” X Pathway Y A B Inhibit downstream or bypass pathways

27. EGFR Tyrosine Kinase Inhibitor Therapy in NSCLC

28. Leading causes of cancer death in the U.S. Jemal et al Ca Can J Clin 2003, 53: 5-26 29,000 Pancreatic 31,000 Prostate 42,000 Breast 57,000 Colorectal 155,000 Lung deaths Tumor type

29. Median survival in advanced NSCLC (SEER data) 1970s-1990s Breathnach et al (2001) JCO 19(6): 1734 6.9 months 7.3 months

30. Probably yes. All recent randomized studies have similar results No clear efficacy benefit for nonplatinum combinations or triplet combinations Are we approaching the ceiling for improved benefit from combination chemotherapy regimens? Schiller JH et al. N Engl J Med . 2002;346:92-98. Cisplatin/Paclitaxel Cisplatin/Gemcitabine Cisplatin/Docetaxel Carboplatin/Paclitaxel 1.0 0.8 0.6 0.4 0.2 0.0 0 5 10 15 20 25 30 Months Stage IIIB/IV Patient Survival, % ECOG 1594

31. NCI Canada BR.21 Advanced NSCLC Tarceva vs Placebo Phase III Trial Previously Treated Advanced NSCLC N = 638 Stratified by : Center PS, 0/1 vs 2/3 Response to prior Rx (CR/PR:SD:PD) Prior regimens, (1 vs 2) Prior platinum, (Yes vs no) Shepherd, et al. PASCO 2004 Erlotinib 150 mg PO QD R A N D O M I Z E D Placebo PO QD 2:1 randomization

32. Overall Survival *Adjusted for stratification factors at randomization, and HER1/EGFR status. HR = hazard ratio. TARCEVA ™ (erlotinib) PI. TARCEVA (n=488) Placebo (n=243) Median survival (mo) 1 - year survival (%) Months Survival distribution function 1.00 0 5 10 15 20 25 30 0.75 0.50 0.25 0 TARCEVA Placebo 6.7 4.7 31.2 P <0.001 * HR=0.73 (95% CI, 0.61-0.86) 21.5

33. Analysis of tumor from patients with major response to EGFR inhibitors Approximately 10-20% of patients had dramatic tumor shrinkage after treatment (major response) In majority of tumors from patients with major response, mutations in EGFR tyrosine kinase domain observed (exon 19 deletion, point mutation) Mutation led to constitutive activation of EGFR and increased sensitivity to EGFR inhibition. Tumors with amplification of EGFR may also have increased sensitivity Patients who never smoked, or were of Asian origin, had a high frequency of EGFR mutations. (Paez et al, Science, 2003; Lynch et al, NEJM, 2003)

34. Never smokers: overall survival- erlotinib vs Placebo 1.0 0.8 0.6 0.4 0.2 0 0 5 10 15 20 25 Survival rate Erlotinib Placebo Months on study Herbst et al., ASCO 2004

35. Secondary mutations in EGFR (T790M) lead to acquired resistance to EGFR TKIs Kobayashi et al, NEJM 2005

36. Secondary mutations in EGFR (T790M) lead to acquired resistance to EGFR TKIs Kobayashi et al, NEJM 2005 T790M mutation

37. Amplifications of MET receptor lead to resistance in previously EGFR-addicted NSCLC cells. Engelman et al, Science, 2007

38. Pivotal role of VEGF in tumor angiogenesis 2. VEGF increases endothelial protease expression 1. Tumor secretes VEGF 4.VEGF induces mobilization and differentiation of BM- derived CEPs 3. Endothelial cell migration proliferation, and capillary tube formation promoted by VEGF Bone marrow

39. Different approaches to inhibition of VEGF signaling Ligand sequestration: MAbs, soluble receptors (i.e. bevacizumab) GRB2 SOS ras PLC  p85 Inhibition of tyrosine phosphorylation and downstream signaling inhibition Transcription factor inhibition Tyrosine kinase inhibition: TKIs TKI  tyrosine kinase inhibitor Receptor blocking: MAbs

40. SU11248: Multitargeted Receptor Tyrosine Kinase Inhibitor VEGFR-1 VEGFR-2 VEGFR-3 Fms PDGFR  PDGFR  CSF1R KIT FLT3 Split Kinase Domain RTKs Mendel DB, et al. Clin Cancer Res 2003;9:327–37 *Receptor phosphorylation FLT3 (WT) 0.25 KIT 0.01 VEGFR-2 0.009 *Cellular IC 50 (µM) PDGFR  0.008 EGFR 8.9 MET 12.0 VEGFR-2 0.009 PDGFR  0.008 FGFR1 0.83 EGFR >10 Enzymatic K i (µM) VEGFR-3 0.017 N H O N H F N H O N OH COOH HOOC H

41. LSC analysis of PDGFR-beta and VEGFR-2 phosphorylation after treatment of GIST patients with SU11248 for 10-14 days Patients (n=20) PD SD PR PD = progressive disease; SD = stable disease; PR = partial response % change in phosphorylation post-treatment Davis et al, ECCO 2005 - 

42. Lessons from tumor-based biomarkers of activity For SU5416/SU6668, lack of clinical activity likely due, at least in part, to incomplete target inhibition. It is critical to not only identify important targets, but also to determine the duration and degree of target inhibition needed for anti-tumor activity. 2. SU11248-treated GIST patients with clinical benefit had a: greater post-treatment induction in TEC apoptosis (9.55 vs 1.78) and TC apoptosis than patients with progressive disease greater degree (but not complete) inhibition of PDGFR- β and VEGFR phosphorylation. There is likely to be room for improvement with more potent inhibitors or combinations.

43. Key points (again) Even though cancer cells are complex, they may be addicted to one or a few key pathways (“oncogene addiction”), often driven by an RTK. CML: addicted to BCR-ABL Gastointestinal stromal cancer: Addicted to C-KIT Some NSCLC: EGFR mutations. 2. Some RTKs are validated or very promising as therapeutic targets. Particularly for tumors with “oncogene addiction”. BCR-ABL (CML) KIT (GIST) PDGFR (GIST) EGFR (lung cancer) VEGFR (multiple diseases) RET (medullary thyroid) 3. We are testing RTK inhibitors in the clinic. Some work well for some diseases. Imatinib: GIST, CML Erlotinib: NSCLC Sunitinib: GIST, RCC 4. Resistance to RTK inhibitor may arise through several different mechanisms. Understanding these mechanisms of resistance is important for designing better combination therapies. Secondary mutations or other changes that prevent the inhibitor from binding. Activation of a “bypass” pathway.