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An epigenetic approach to the treatment of acute leukemia and myelodysplastic syndromes

An epigenetic approach to the treatment of acute leukemia and myelodysplastic syndromes. Guillermo Garcia-Manero Assistant Professor of Medicine Department of Leukemia UTMDACC. Epigenetic Alterations. Histone residue Effect. H3 AC K9 + H4 AC K8 + H3 Ser10P + H3Met K4 +

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An epigenetic approach to the treatment of acute leukemia and myelodysplastic syndromes

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  1. An epigenetic approach to the treatment of acute leukemia and myelodysplastic syndromes Guillermo Garcia-Manero Assistant Professor of Medicine Department of Leukemia UTMDACC

  2. Epigenetic Alterations Histone residue Effect H3 AC K9 + H4 AC K8 + H3 Ser10P + H3Met K4 + H3 Met K9 – DNA methylation m m m m phosphorylation methylation acetylation Histone modifications

  3. DNA methylation Silencing of foreign DNA Genomic instability Spontaneous deamination Increased mutation rate Silencing of imprinted/ X inactivated genes Silencing of tumor suppressor genes

  4. + + + + + + + + + + + + p57KIP2 methylation in ALL A. COBRA CCRF-HSB2 CCRF-CEM BALL1 TALL1 SupT1 Molt3 PEER Jurkat M B.RT-PCR CCRF- CEM CCRF- HSB2 Jurkat BALL1 Molt3 SupT1 PEER TALL1 RT p57KIP2 GAPDH SupT1 AzaC PEER AzadC SupT1 PEER RT p57KIP2 decitabine GAPDH Kikuchi et al Oncogene 2002 Garcia-Manero et al. Blood 2003

  5. Global hypermethylator phenotype • Coordinated methylation of multiple CpG islands Non-neoplastic sequence Methylation defect ? M K-Ras + P53 - M M M Aberrantly methylated DNA Toyota M. PNAS 1999;96:8681-6 Toyota M. PNAS 2000;97:710-715

  6. DNA methylation in ALL Column: gene Row: patient Black boxes: methylated White boxes: unmethylated Garcia-Manero G. Clin Cancer Res 2002;8:2217

  7. Gene specific methylation in ALL Garcia-Manero G, et al Clinical Cancer Research, 2002;8:2217-2224

  8. Methylator phenotype Garcia-Manero G, et al Clinical Cancer Research, 2002;8:2217-2224

  9. Silencing of Molecular Pathways ATM DNA damage P53 P73 G1 Death P14/MDM2 CDKI P21 P27 P57 P16 P15 cyclin E CDK2 RP methylated cyclin D CDK4-6 S

  10. P57 methylation in ALL Initial presentation p57 Relapse p57 p21 p14 RT-PCR Garcia-Manero Blood 2003

  11. Silencing of a Cell Cycle Pathway ATM DNA damage P53 P73 G1 Death P14/MDM2 CDKI P21 P27 P57 P16 P15 cyclin E CDK2 RP methylated cyclin D CDK4-6 S

  12. Clinical implications 1-Upfront use of hypomethylating agents to restore normal methylation patterns 2-Incorporation into induction/consolidation or maintenance therapies 3-Early alloBMT 25% Minimal Residual Disease

  13. Cytosine Nucleoside Analogs

  14. Effect of decitabine on versican gene expression 0 0.8uM 0.4uM 0.2uM 0.1uM Versican GAPDH 0 0.8uM 0.4uM 0.2uM 0.1uM Versican GAPDH

  15. Decitabine Incorporates into DNA Hypomethylating at 1 microM Active in MDS Active in AML at low doses Active in CML at low and high doses Toxicity at high doses: Myelosuppression Azacytidine Incorporates into RNA Pro-drug for decitabine Hypomethylating at 10 microM Active in MDS Active in AML at high doses No reported single-agent activity in CML Toxicity at high doses: Myelosuppression, Mucositis, Serositis

  16. Low Dose Decitabine in MDS • 169 pts; median age 70 yrs • IPSS: int 1-48; int 2-50; high 71 • Decitabine 45 to 50 mg/m2 qd x 3 (135-150 mg/m2 per course) every 6 wks-8 courses • Response 49%, induction death 11 (7%) • Plts increase 42% after course 1, 63% after course 2 • Median response duration 9 mos; median survival 15 mos • Overall 30% cytogenetic response Wijermans.Blood 100 (Suppl 1): Abst 355, 2002

  17. Low-Dose Decitabine in MDS Wijermans.Blood 100 (Suppl 1): Abst 355, 2002

  18. Phase I low dose study of 5-aza-2’-deoxycytidine • 48 patients (2 patients registered twice: total 50) • Median age 60 (2-84) • AML 35 (73%) • Untreated 5 • CR <12 months 28 • CR>12 months 2 • MDS 7 (14%) • ALL 1(2%) • CML 5 (10%) • Median number previous tx 2 (0-11) Issa, Garcia-Manero. Blood 2004

  19. Dose response: low dose 5-aza-2’-deoxycytidine

  20. Results: Phase I low dose 5-aza-2’-deoxycytidine N(%)

  21. 1960 1970 1980 1990 2000 2003 2004 Chronology of Azacitidine Development Event Synthesis (Czech scientists) Initial Laboratory Work Initial Phase I Trials and PK Work as Cytotoxic Development as a Cytoxic (Upjohn) Submission to the FDA as CT Agent (Upjohn) NCI/CALGB Phase II- Studies 8421 and 8921 (Silverman/Holland) NCI/CALGB Phase III- 9221 (1992) Publication of CALGB 9221 (Silverman) Pharmion Resubmission Pharmion Confirmatory Trial Vidaza Approval Silverman LR, et al. Leukemia 1993. 7(S1): 21-29. Silverman LR, et al. Mol and Cell Diff 1994: 505.

  22. Continue untilEndpoint + No S t r a t i f y R a n d o m i z e Exit Criteria 1) Supportive Care* RA RARS RAEB RAEB-T CMML + Yes Aza C(dose as per arm #2) A S S E S S • Response • Continue Rx • No Response • - Off Study 2) Aza C75mg/m2/d x 7 days q28 x 4 QOL QOL QOL M M M 0 29 57 113 Day CALGB 9221A Randomized Phase III Controlled Trial of SubcutaneousAzacitidine in Myelodysplastic Syndromes * Minimum duration of supportive care = 4 months unless transform to AML; death or pltsš 20 x 109/L at week 8 or later QOL – Quality of Life Assessment M = Bone Marrow Aza C – Azacitidine S.C. Silverman L, et al. J Clin Oncol 2002. 18:2414-26. Kornblith AB, et al. J Clin Oncol 2002. 18:2427-39

  23. SC AZA Crossover • No. Evaluated 92 99 49 CR 0 (0%) 7 (7%)* 5 (10%) PR 0 (0%) 15 (16%) ** 2 ( 4%) Improved 5 (5%) 38 (37%) ** 16 (36%) Total 5 (5%) 60 (60%) ** 23 (47%) P - value * < 0.01 **<0.001 Analysis of Response Silverman L, et al. Randomized Controlled Trial of Azacitidine in Patients with MDS: A Study of the CALGB J Clin Oncol 2002. 18:2414-26. Reprinted with permission from the American Society of Clinical Oncology.

  24. 1.0 + + + + + + + + 0.8 + Azacitidine Supportive Care + + 0.6 + + + + + + Probability of Remaining Event-Free + + + + + 0.4 + + + + + + + + + + + + + + + + + + + + + + + + 0.2 + + + + + + P=.001 0.0 0 6 12 18 24 30 36 42 48 54 Months p=0.007 Time to AML Transformation Silverman L, et al. Randomized Controlled Trial of Azacitidine in Patients with MDS: A Study of the CALGB J Clin Oncol 2002. 18:2414-26. Reprinted with permission from the American Society of Clinical Oncology.

  25. 1.0 Azacitidine Supportive Care 0.8 Median 20 months 14 months 0.6 Probability of Remaining Event-Free 0.4 + + + + + + + + + + + 0.2 + + + + + + + + + + + + + + + + + + + + + 0.0 0 6 12 18 24 30 36 42 48 54 Months p = 0.10 Overall Survival Silverman L, et al. Randomized Controlled Trial of Azacitidine in Patients with MDS: A Study of the CALGB J Clin Oncol 2002. 18:2414-26. Reprinted with permission from the American Society of Clinical Oncology.

  26. Azacitidine Supportive Care 1.0 1.0 0.8 0.8 Probability of Survival 0.6 0.6 Induction Azacitidine Crossed before 6 months Did not cross before 6 months 0.4 0.4 Probability of Survival 0 6 12 18 24 30 36 42 48 54 0.2 0.2 Months p=0.1 0.0 0.0 Months from 6-month Landmark 0 5 10 15 20 25 30 35 40 45 50 p=0.03 Survival: Landmark Analysis Silverman L, et al. Randomized Controlled Trial of Azacitidine in Patients with MDS: A Study of the CALGB J Clin Oncol 2002. 18:2414-26. Reprinted with permission from the American Society of Clinical Oncology.

  27. 1.0 Aza/Low Supp Care/Low 0.8 Aza/High Supp Care/High 0.6 Probability of Survival 0.4 0.2 0.0 50 40 30 20 10 0 Months Survival: FAB Classification Silverman L, et al. Randomized Controlled Trial of Azacitidine in Patients with MDS: A Study of the CALGB J Clin Oncol 2002. 18:2414-26. Reprinted with permission from the American Society of Clinical Oncology.

  28. 80 70 Physical Function*p = .018 60 50 Fatigue **p = .0031 40 EORTC 30 20 Dyspnea**p = .0003 10 0 0 250 107 Supportive Care Crossover AZA Time (days) Quality of Life Impact: EORTC Fatigue, Dyspnea & Physical Functioning of Crossover Patients on Supportive Care for 4 months Prior to Crossover (N=30) * Higher Scores = Better Functioning ** Lower Scores = Symptom Improvement Kornblith et al., Impact of Azacytidine on QOL of Patients with MDS Treated in a Randomized Phase III Trial: a CALGB StudyJ Clin Oncol 2002; 20: 2441-52. Reprinted with permission from the American Society of Clinical Oncology.

  29. MDS Treatment Selection Low Risk High Risk (Low, Int-1) (Int-2, High) Clinical Trial Clinical Trial Growth Factors AZA ATG Chemotherapy AZA SCT CC5013 SCT Clinical Trial Clinical Trial Growth Factors AZA ATG Chemotherapy AZA CC5013 Young (Potentially SCT Eligible) Old

  30. Model of epigenetic therapy DNMT INHIBITORS Other ? HDACS Other histone modifiers?

  31. Therapeutic De-Silencing Unmethylated Expressed DNA Methylation Methyl-Binding Protein Recruitment Histone Deacetylase Recruitment Histone Methylase Recruitment Methylated Silenced

  32. Phase I study of SAHA(a histone deacetylase inhibitor) • Overall response rate by disease • AML: 3/14 (21%) 2 CRp x 4-6 weeks 1 CR > 6 months • MDS: 0/2 • ALL: 0/2 • CML: 0/1 • CLL: 0/3

  33. Pre-SAHA (Day 1) 2 hr post-SAHA (Day 14) Pre-SAHA (Day 22) SAHA induces histone acetylation in vivo Cohort 1, 100 mg SAHA, TID Bone Marrow Cells Peripheral Blood Cells

  34. Combination of 5-aza-2’-deoxycytidine and valproic acid: Induction of apoptosis Yang H, Garcia-Manero. Leuk Res, in press

  35. Effect on histone acetylation

  36. Results of a phase I/II study of 5-aza-2’-deoxycytidine and valproic acid (N=40)

  37. Response expectations

  38. Valproic acid levels (N=43) (µg/ml) (mg/L) P=0.02 P=0.04 Higher VPA levels in responders vs non (free: 36 vs 21; bound: 127 vs 104 p=0.02)

  39. Histone Acetylation (N=38) Total # Acetylated 37% P=0.002 Number 11% 0% VPA dose: Pt #13 Pt #31 Ac-H3 Ac-H4 B-actin C+ DAY 0 1 5 10 21 C- C+ 0 1 5 10 21

  40. LINE methylation (N=30) Method: bisulfite pyrosequencing

  41. P21 mRNA induction (N=30) Relative expression ratio (2-∆∆CT) Real time TaqMan results

  42. Ongoing studies • Decitabine low dose randomized study in MDS • Phase II studies of decitabine in CML • Phase I/II study of combination of decitabine with valproic acid • Phase I study of SAHA • Phase I study of MGCD0103 • Low-dose study of 5-azacytidine • Phase I/II of 5-azacytidine, valproic acid and ATRA • Phase II study of 5-azacytidine post alloSCT

  43. Dept of Leukemia Hagop Kantarjian Jean-Pierre Issa Jerry Daniel Jason Williamson LanLan Shen Yutaka Kondo Hui Yang Koyu Hoshino Blanca Sanchez-Gonzalez Andreia Canalli Sherry Pierce Jan Davis Jackie Fiorentino Hematopathology Carlos Bueso-Ramos Pediatrics Sima Jeha (St Judes) Biostatistics Gary Rosner Terry Smith Sapporo Medical School Minoru Toyota Funding NIH/NCI Physician-Scientist MDACC ASCO CDA Ladies Leukemia League Peggy Howard Fund Leukemia SPORE SuperGen/MGI Pharmion Acknowledgements

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