Resistance in the Clinical Setting Dr. Wilson H. Miller, Jr

1. Resistance in the Clinical Setting Dr. Wilson H. Miller, Jr

2. Potential Conflict of Interest

3. Resistance in the Clinical Setting • Wilson H. Miller Jr., M.D., Ph.D. • Segal Cancer Center • SMBD Jewish General Hospital • McGill University, Montreal, Quebec Canada

4. Mechanisms of Cellular Drug Resistance • Intrinsic Resistance Mechanisms • Host factors • Decreased intracellular drug accumulation (poor absorption, rapid metabolism, or excretion). • Inefficient delivery of a drug to its target (tumor cells). • Specific genetic and epigenetic drivers • Malignant cell growth is associated with tumor-specific activation of oncogenic pathways and inactivation of tumor suppressor genes. • •Specific drug targets may or may not be relevant to growth of a given tumor. • The wrong target cell? • Stem cell resistance

5. Mechanisms of Cellular Drug Resistance • Acquired Resistance Mechanisms • Decreased accumulation of drugs within cells • • Increased drug efflux. • • Reduced drug uptake. • Changes in drug-target interactions • Mutations in targeted oncogenes. • Changes in target gene expression. • Changes in signaling pathways that drive growth • Replacement of one TK pathway with another. • Interchangeable pro-angiogenic factors and pathways. • Multiple interdependent cell survival pathways. • Loss of checkpoints.

6. Three Main Mechanisms of Cellular Drug Resistance (1) Decrease in intracellular drug concentrations (2) Changes in drug-target interactions Mutation (3) Changes in signal transduction pathways Cell cycle arrest and repair

7. Decrease in Intracellular Drug Concentrations • Decrease of drug influx • Alterations of cell membrane structures. • Most chemotherapeutic drugs enter cells by passive diffusion. • Increase of drug efflux • Overexpression of transmembrane proteins (ABC superfamily of transporters). LRP/MVP Is the major component of the Vault protein Involved in cellular traffic

8. Examples of Chemotherapeutic Drugs with Increased Delivery to Tumors SarCNU Rationale • SarCNU is a novel chloroethylnitrosourea which demonstrates selective cytotoxicity against primary human gliomas in-vitro. • Selective uptake via the extraneuronal catecholamine uptake carrier allows increased concentration in tumor cells. • Preclinical toxicity studies confirm that SarCNU is less toxic than BCNU, the standard treatment of gliomas.

9. Examples of Chemotherapeutic Drugs with Increased Delivery to Tumors SarCNU • Phase I and pharmacokinetics study in advanced solid tumor malignancy. • 43 patients enrolled. • Myelosuppression and some pulmonary toxicity observed in patients.

10. Examples of Chemotherapeutic Drugs with Increased Delivery to Tumors Darinaparsin: Organic Arsenic • First in a new class of molecules. • Potentially safer and more active for cancer treatment than approved inorganic arsenic.

11. Darinaparsin (DAR) is more potent than As2O3at inducing apoptosis in a variety of leukemia and lymphoma cell lines. NB4 (APL) AsR2 (As-resistant APL) CCRF-CEM (NHL) IM9 (NHL) Diaz et al, 2009 Feb;23(2):431

12. DAR induces more cellular oxidative stress than As2O3. NB4 (APL) AsR2 (APL) NB4 (APL) AsR2 (APL) NB4 (APL) AsR2 (APL) Diaz et al, 2009 Feb;23(2):431

13. Increased ABCC1 exporter expression causes resistance toAs2O3 but not DAR in the arsenic-resistant cell line AsR2. 10.0 7.5 Arsenic (ppb) 5.0 2.5 0.0 control 1.0µM DAR 1.0µM As2O3 NB4 cells 12 10 ABCC1/GAPDH Relative quantity 8 6 AsR2 cells 4 2 0 NB4 AsR2

14. 7 6 5 4 As (ppb) 3 2 1 0 Control MK571 1.0 uM ATO 1.0 uM DAR MK571+1.0uM DAR MK571+ 1.0uM ATO ATO-resistant NB4-AR2 cells, are sensitized to ATO, but not DAR, by co-treatment with an ABCC1 inhibitor. A. B. Total intracellular As in AsR2 treated for 24hrs. Viable cell number in AsR2 treated for 24hrs. 1 7 . 5 1 5 . 0 1 2 . 5 cell number (x10 4 cell/ml) 1 0 . 0 ** 7 . 5 5 . 0 2 . 5 0 Control 2.0 uM DAR MK571 2.0 uM ATO MK571+ 2.0uM DAR MK571+ 2.0uM ATO

15. M e M e M e M e M e O H O H M e Butyric Acid M e M e M e M e M e M e Examples of Chemotherapeutic Drugs with Increased Delivery to Tumors Hybrid Molecules – Targeting the Oncogene with Two Therapeutic Agents O O Retinoic Acid O O RN1 O O Figure 2. Chemical structure of RN1 and it’s precursors.

16. RN1 induces growth arrest in NB4 and R4 cell lines. NB4 R4 NB4 and R4 cells were treated with media, 10-5 M RA, butyrate, RA plus butyrate, or RN1. In NB4 cells, RA, RA plus butyrate, and RN1 significantly inhibited growth (P<0.001). In R4 cells, RN1 significantly inhibited growth (P<0.02).

17. Imatinib Treatment in CML Chronic Myeloid Leukemia (CML) • Characterized by the Philadelphia chromosome t(9;22). • Results in fusion of BCR and ABL genes. • Imatinib mesylate is the frontline therapy. • Imatinib is a selective inhibitor ofBcr-Abl, PDGF-R, Kit.

18. Imatinib Treatment in CML Models Multiple Resistance Mechanisms • Imatinib has revolutionized treatment for CML but resistance is a problem in a small percentage of patients. • Primary resistance • Insufficient inhibition of BCR-ABL • Low plasma levels of imatinib. • Activity of drug pumps. • Stem cells • Secondary resistance • Imatinib-resistant BCR-ABL kinase-domain mutations. • Overproduction of BCR-ABL (genomic amplification). • BCR-ABL-independent mechanisms (not well understood). • ? Activation of other kinases. • ? Other molecular events.

19. T315I** F382L F311L/I/V F317L G383D V299L L324Q L298V L248V L387F/M M343T E292V M388L E453G/K/A/V G250E/A/F A344V V289A/I S348L A397P Q252H/R E450G/Q/K A350V M472I Y253F/H G236E Q447R H396R/P K357R F486S S417Y E255K/V P-loop Activation loop E275K M351T/L V379I E459K/Q I418V S438C M244V D276G E355G/D T277A/N L364I D241G E279K F359V/C/D/I E281A M237I K285N BCR-ABL Mutations Associated with Imatinib Resistance Most mutated clones, except for T315I, may be eradicated with appropriate choice and combination among the second generation Abl TKIs (Dasatinib, Nilotinib, Bosutinib).

20. CML Stem Cells – Resistance to TKI’sPersistence of minimal residual disease • Possible mechanisms of stem cell resistance • High levels of ABC drug transporters. • Increased capacity for DNA repair. • Accumulation of mutations. • Quiescence. • Therapeutic Approaches for Stem Cell Resistance • Targeting the ABC transporters. • Targeting the different surface markers. • Targeting the pathways in stem cell renewal. • Targeting the quiescence.

21. Resistance in Signal Transduction Pathways – HER2 (ERBB2)

22. HER2 (ERBB2) Driven Breast Cancer • Overexpression of the Her2 (ErbB2) protein found in 18-20% of breast tumors. • Correlates with more aggressive tumors. • Current targeted therapies • Trastuzumab (Herceptin) – monoclonal Ab specifically targets Her2. • Treatment for early stage HER2+ breast cancer. • Resistance in vast majority of patients occur within 1 year. • HER2 mutations not commonly found. • Lapatinib-TKI inhibitor • Inhibits Her2 and EGFR.

23. Current Therapies to Overcome Trastuzumab Resistance • Lapatinib -TKI inhibitor • This combined inhibition can • overcome Herceptin resistance • in some cases. • LBH589 – Deacetylase inhibitor • Induces degradation of Her2, • ER and pAKT. • Phase Ib/IIa LBH589 in combo • with Trastuzumab for HER2+ • metastatic breast cancer. • Enhances Her2 inhibition in combo • with Trastuzumab or Lapatinib

24. Resistance in Signal Transduction Pathways: The importance of KRAS, BRAF and EGFR mutations in EGFR signaling in Colon Cancer • Ligand binding to EGFR • promotes heterodimerization, • activation and downstream • pathways; • Ras-Raf • MAPK • PI3K-Akt

25. The importance of KRAS status in Metastatic Colorectal Cancer • Ab against EGFR (Cetuximab and Panitumumab) inhibit downstream • pathways. • Mutated KRAS or BRAF leads to • constitutive activated pathway. • Mutated KRAS (~30% pts) • Mutated BRAF (~10% pts) • Cetuximab and Panitumumab • Only effective in KRAS and BRAF • wild type tumors.

26. Response to Cetuximab According to the Presence or Absence of KRAS Mutation in the Overall 114 Patients Lievre, A. et al. J Clin Oncol; 26:374-379 2008

27. (A) Progression-free survival (B) overall survival according to the presence or absence of KRAS mutation PFS 32 vs. 9 weeks P = 0.0000001 OS 14.3 vs. 10.1 months P = 0.0017 Lievre, A. et al. J Clin Oncol; 26:374-379 2008

28. Signal Transduction Pathways: The importance of KRAS, BRAF and EGFR mutations in EGFR signaling in lung adenocarcinoma

29. Oncogene mutations in the EGFR pathway in lung adenocarcinoma • About 50% of lung adenocarcinoma harbor somatic mutations of six genes that encode proteins in the EGFR signaling pathway: • KRAS mutations • EGFR mutations • Her-2 mutations • Her-4 mutations • BRAF mutations • Phosphatidylinositol 3-kinase (PI3K) mutations

30. KRAS mutations in lungadenocarcinoma • KRAS mutation in 30% of lung adenocarcinoma. • Association with smoking. Poor prognostic factor in resected tumors. • Lack of sensitivity of KRAS mutated tumors to geftinib or erlotinib (EGFR inhibitors).

31. Activating Mutations in the EGFR Correlate with EGFR-TKI Sensitivity

32. EGFR mutations in lung adenocarcinoma associated with sensitivity but additional mutations can mediate resistance Sharma, Nat Rev Cancer, 2007

33. Resistance in Angiogenic Targeted Therapy

34. Current Angiogenic Inhibitors in Clinical Use and Clinical Trials • Bevacizumab (Avastin) • Sunitinib (Sutent) • Sorafenib (Nexavar) • Cederanib (Recentin - AZD- 2171) • VEGF-Trap Many others in development

35. Modes of Resistance to Anti-Angiogenic Therapy Upregulation of pro-angiogenicsignaling pathways • FGF, ephrin and angiopoietin families. Recruitment of BM derived cells • Endothelial and pericyte progenitors are incorporated as • components of new vessels to build new blood vessels • Pro-angiogenicmonocytes fuel the tumors with cytokines, • growth factors and proteases. • Increased pericyte coverage protects tumor blood vessels • Helps tumor endothelium to survive and function.

36. Overcoming Resistance to Anti-Angiogenic Therapy • The combination of antiangiogenesis agents with cytotoxic • chemotherapy has increased the activity of chemotherapy in • breast, colon, lung cancer and in melanoma. • Data on toxicity of targeted agents in older individuals are • limited: the risk of thrombosis with avastin and of serious • cutaneous reactions with cetuximab appears to increase with age.

37. Conclusions • Overcoming Resistance • Targeted therapy has been very successful in situations where a single or few targets are responsible to maintain the disease (CML, HER2 positive breast cancer). • Inhibiting a single target in a complex signaling pathway is unlikely to provide sufficient therapeutic activity for the treatment of most genetically unstable human cancers. • -Multiple activating signals and cross talk. • -Signals transmitted via multiple pathways. • The combination of 2 or more targeting agents seems to be more effective and safer, at least in the case of inhibition of the signal transduction cascade.

38. Conclusions: More work needed • Need to continue to characterize mechanisms of action, mechanisms of resistance, signaling pathways. • Continued research to improve our understanding of the heterogeneity and complexity of the tumor microenvironment. • Continue to identify mutations in targeted oncogenes and targets in the downstream pathways. • The use of technological advances in genomics, proteomics and biomarker development to better predict tumor types and patient subsets that may be particularly responsive to treatment.

39. The Importance of Pharmacodynamic Markers B I O M A R K E R S Gene expression • Data processing • Data integration • Pathway linkage • Analysis • Data coherence Preclinical Enzyme activity Samples Tumor cell markers Clinical Metabolomics Experiments Analysis Informatics Discovery

40. Translational research should be part of the solution The complexity of resistance in patients demonstrates the need for • Developing new models of • Multi-disciplinary and multi-institutional collaborations • Academic and industrial partnerships • Designing biomarker-driven clinical trials to • Collect clinical samples • Identify biomarkers predicting resistance • Study mechanism of resistance identified in patients (vs. in cell lines) • Develop new or improved molecules The Quebec – Clinical Research Organization in Cancer was designed to answer these challenges.