Epigenetics and Cancer + Nilofer Azad, MD Assistant Professor, Gastrointestinal Oncology/Phase I Program Sidney Kimmel

1. Epigenetics and Cancer + Nilofer Azad, MD Assistant Professor, Gastrointestinal Oncology/Phase I Program Sidney Kimmel Comprehensive Cancer Center October 19, 2010

2. Simplified Model of Epigenetic Regulation of Gene Expression CMAJ 2006;174(3):341-8

3. How do genes get turned on and off? Protein Complex Protein Complex M M M DNA of Gene X Promoter Coding section Non-coding section Coding section Histone Histone Gene is transcribed = ON Gene blocked from being transcribed = OFF

4. DNA Methyltransferase inhibitors • Two currently FDA approved agents • 5-azacytidine(Vidaza) • 5-aza-2'-deoxycytidine (decitabine, Dacogen)

5. 5-azacitidine • FDA approved in 2004 for myelodysplasia • Dose: 75 mg/m2 SQ daily x 7 d / 28 d cycle • Mechanism of action: Incorporated into DNA → suicide inhibitor of DNMTInduces global hypomethylation • Time to clinical response: Average = 4 months

6. Histone deacetylase inhibitors • Three currently FDA approved agents • Vorinostat (Pan-HDACi)(SAHA, Zolinza) Oral agentApproved for cutaneous T-cell lymphoma • Depsipeptide (Pan-HDACi)(Istodax) Intravenous agent Approved for cutaneous T-cell lymphoma • Valproic acid (weak inhibitor) anti-seizure

7. Lung Cancer

8. Rationale for double epigenetic blockage in lung cancer • Epigenetic gene silencing mediated by DNA methylation and histonedeaceylation is a key contributor to lung carcinogenesis • Preclinical studies suggest that combining DMNTi with HDACi synergistically enhances expression of silenced tumor suppressor genes • Clinical studies combining DMNTi and HDACi have shown remarkable clinical activity in MDS/AML • Hypothesis: similar effect in NSCLC

9. MS275 SNDX-275 5 5 - - Aza AC Day 1 8 15 Day 1 8 15 22 29 36 22 29 36 Trial Schema • 5AC Dosing = 40 mg/m2 SQ daily on days 1-6 and 8-10 • SNDX-275 dosing = 7 mg PO (fixed dose) days 3 and 10 • Cycle length = 28 days

10. Phase I Toxicity Data

11. Updated Response Data 28 Evaluable Patients • 1 Complete Response – On treatment for 14 months • 1 Partial Response Responded for 8 months – then new SCLC Still no progression of his NSCLC 9 months off epigenetic therapy • 8 Stable Disease One on treatment for 18 months; Five treated for 4 months One treated for 3 months then stopped due to schedule One still being treated (on cycle 12 now) • 17 Progressive Disease • 8 Not evaluable (finished less than 1 cycle) • 5 Actively being treated

12. Overall Survival Median OS: 8.2 months

13. Images of Patient with Complete Response 56 year old woman with stage I lung cancer that was resected and treated with adjuvant chemotherapy. She progressed after salvage chemotherapy with radiation at relapse. She had a response after 2 cycles, continued improvement after 4; 14 cycles were given. She had 3 prior therapies for advanced disease.

14. Images of patient withPartial Response 58 year old male treated with 3 prior therapies; Chemotherapy refractory disease. He completed 8 cycles.

15. Images of patient with partial response: liver metastases Pre-treatment Cycle 2 Cycle 4 Cycle 8

16. Hypotheses for biology of the complete responder • Fewer number of previous therapies… • Higher serum level of 5-azacitidine… • Epigenetics… • Responding patient was a previously resected stage I NSCLC patient • Analysis of her tumor and mediastinal lymph nodes found a methylation pattern that predicted she was at high risk for early recurrence

17. Gene DNA Hypermethylation Markers Are Better for Prognosis than Standard Staging p16 and H-cadherin 1.00 P<0.0001 Negative (U) n=79 0.75 Stage 1 Proportion Disease-Free 0.50 OR = 25 fold Positive (M) N=11 0.25 Stage 3 Molecular Re-staging 0.00 0 1 2 3 4 5 Brock et al, 2008 Years After Surgery

18. Epigenetic Therapy Study Design: Treatment Schema 5-Azacitidine 40 mg/m2 SQ Day 1-5, 8-10 Entinostat 7mg PO Day 3 and 10 Every 28 days, for 6 cycles Intended Accrual: 172 patients R A N D O M I Z E 2 Stage IA or IB NSCLC s/p surgery with curative intent Standard Care Intended Accrual: 86 patients 1 Within 4-8 weeks of completing surgery

19. Colon Cancer

20. Colorectal Cancer is Common 2009 Estimated U.S. Cancer Deaths Men294,120 Women271,530 Available at: http://www.cancer.org. Lung and bronchus 31% Prostate 10% Colon and rectum 8% Pancreas 6% Leukemia 4% Liver/bile duct 4% Esophagus 4% Urinary bladder 3% Non-Hodgkin Lymphoma 3% Kidney 3% All other sites 24% 26% Lung and bronchus 15% Breast 9% Colon and rectum 6% Pancreas 6% Ovary 4% Non-Hodgkin lymphoma 3% Leukemia 3% Uterine corpus 2% Brain/other nervous system 2% Liver/bile duct 25% All other sites Colorectal cancer represents 2nd leading cause of death Available at: http://www.cancer.org.

21. Stage I-II Colorectal Cancer Staging Adenoma Pre-cancer lesion Stage I localized, not through “muscularis” (muscle wall in the colon) Stage II through muscularis, but no lymph nodes Stage III cancer in nodes, but not other organs Stage IV metastatic (liver, lung, etc) Stage III IV

22. Disease Stage at Time of Diagnosis Stage I 15% Stage II 20%–30% Stage III 30%–40% Stage IV 20%–25% Hamilton IM, Grem JL. Current Cancer Therapeutics. 3rd ed. 1998;157.

23. Risk of recurrence after primary resection in Stage II and III Colon Cancer 85% recur within 3 years 85% Sargent, D. et al. J Clin Oncol; 27:872-877 2009

24. Metastatic Disease

25. History of Treatment for Colorectal Cancer • ~1960: 5-FU is a cornerstone of first-line therapy; bolus/infusion • ~1985: Addition of LV (biomodulator) to 5-FU bolus regimens • 1998: Irinotecan as single agent approved as second-line • 2000: Irinotecan approved as first-line in CRC (bolus IFL) • 2001: Capecitabine approved as first-line in CRC in selected pts • 2002: Oxaliplatin approved as second-line agent (FOLFOX) • 2004: Oxaliplatin approved as first-line agent in infusional regimen • 2004: Approval of Cetuximab (Erbitux) & Bevacizumab (Avastin) • 2006: Approval of Panitumumab (Vectibix) • 2008: KRAS mutations predict lack of benefit of EGFR mAb’s

26. No active drug ~4-6 mo 5-FU/LV 12-14 mo IFL ~ 15-16 mo FOLFOX4 ~ 20 mo IFL + bevacizumab 20.3 mo FOLFOX/FOLFIRI 21.5 mo FOLFOX/FOLFIRI + biologics ? 0 6 12 18 24 Median OS (mo) Incremental Survival Advantage in First-LineMetastatic Colorectal Cancer Are we hitting a wall with current drugs?

27. Therapy for Advanced Colorectal Cancer: Response rates and survival First LineSecond LineThird Line - FOLFOX or - FOLFOX or - Irinotecan + - CAPOX or - FOLIRI or Cetuximab - FOLFIRI - Irinotecan alone - Cetuximab +/- Bevacizumab - Irinotecan/Cetuximab - Panitumumab +/- Bevacizumab Response Rates in Randomized Trials: 50-60% 15% 10% Survival Benefit in Randomized Trials: Yes Yes Yes

28. Epigenetics in CRC • Many genes have silenced expression due to epigenetic changes • Targeting epigenetically abnormal tumors may be more effective than targeting abnormal mutations in genes • CRC may be uniquely appropriate for this strategy • A subset of colon cancer have more gene promoter methylation Ahuja et al.

29. Combination Epigenetic Therapy • First study of epigenetic therapy in CRC • Primary Objective: • To determine the preliminary efficacy via tumor shrinkage rate of the combination of 5-azacitadine and entinostat in patients with metastatic colorectal cancer • Secondary Objective: • To see what is happening in the tumor itself and circulating cells in blood before and after treatment with these drugs

30. Study Schema 28 days C1d1 C1d3 entinostat C1d10 entinostat C2d1 C2d3 entinostat C2d10 entinostat C3d1 5-aza days 1-5 and 8-10 q cycle 5-aza days 1-5 and 8-10 q cycle = plasma sampling for research purposes = tumor sampling for research purposes

31. Ongoing and Upcoming Studies • Lung Cancer • New schedule • Adjuvant treatment of early stage disease • Breast • Same schedule in triple negative and hormone resistant metastatic cancer

32. Conclusions • Despite progress, colon cancer is a still leading source of death • Epigenetic therapy offers a novel way to approach treating cancer, based on the abnormal gene expression seen in cancers compared to normal cells • We are presently enrolling a trial of patients with late-stage colon cancer an treating them with epigenetic agents, 5-azacitidine and entinostat

33. Breast Cancer

34. Epigenetics and breast cancer • Multiple genes are methylated and thus silenced in breast cancer1 • ER, RAR beta, cyclin D, Twist, RASSF1A, and HIN-1 1 Pu RT. Mod Pathol 2003;16(11):1095-101.

35. Zebularine inhibits growth of MDA-MB-231 cell lines alone or in combination Billam M. BCRT 2010

36. Clinical studies: Vorinostat in MBC • Phase 2

37. SKCCC J0785/TBCRC 008A Multi-Institutional Randomized Phase II Study Evaluating Response and Surrogate Biomarkers to Carboplatin and nab-Paclitaxel (CP) with or without Vorinostat (SAHA) in HER2- Negative Breast Cancer Principal Investigator: Vered Stearns, MD Fellow: Roisin Connolly, MB.BCh

38. Eligible patients with locally advanced or metastatic breast cancer (up to 60) Cohort B (up to 30)Hormone-resistant5-AZA + entinostat Cohort A (up to 30)Triple-negative5-AZA + entinostat Disease Progression at Any Time Cohort A or Cohort B 5-AZA + etinostat + hormonal therapy MD discretion Event Monitoring Study schema

39. Conclusions • Epigenetics is a new way to look at cancer biology and therapy • Ongoing trials in major tumor types in the metastatic setting • Plans to move therapy into earlier stage disease may be even more successful

40. Acknowledgements • First and foremost, our patients • SU2C researchers • Research support staff at all our institutions