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Preclinical studies with OSU-HDAC42 A new orally bioavailable HDAC inhibitor Pierluigi Porcu, M.D.

Preclinical studies with OSU-HDAC42 A new orally bioavailable HDAC inhibitor Pierluigi Porcu, M.D. Division of Hematology/Oncology Comprehensive Cancer Center The Ohio State University. Histone Deacetylase Inhibitor OSU-HDAC42.

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Preclinical studies with OSU-HDAC42 A new orally bioavailable HDAC inhibitor Pierluigi Porcu, M.D.

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  1. Preclinical studies with OSU-HDAC42 A new orally bioavailable HDAC inhibitor Pierluigi Porcu, M.D. Division of Hematology/Oncology Comprehensive Cancer Center The Ohio State University

  2. Histone Deacetylase Inhibitor OSU-HDAC42 Preclinical testing under the Rapid Access to Intervention Development (RAID) Program at the National Cancer Institute Class I-II HDACi IC50 = 20-30nM N-hydroxy-4-(3-methyl-2-phenyl-butyrylamino)benzamide Courtesy of Ching-Shih Chen, PhD, PharmD, College of Pharmacy, The Ohio State University

  3. OSU-HDAC42 SAHA Anti-Proliferative Effects of OSU-HDAC42 and SAHA in Prostate Cancer Cell Lines PC-3 LNCaP DU-145 Relative % Viable Concentration (µM) IC50 (µM): OSU-HDAC42 0.48 0.39 0.43 SAHA 3.10 2.20 1.62 Kulp SK, Chen CS, Wang DS, Chen CY, Chen CS. Clin Cancer Res. 2006 12: 5199-206.

  4. Differential Effects of HDAC Inhibitorson Akt Dephosphorylation

  5. Effect of OSU-HDAC42 on Apoptosis in CLL Patient Cells Relative % Live (annexin-negative)

  6. M 0.5mM 1mM 3mM 5mM 10mM M 0.25mM 0.5mM 1mM 2mM 5mM Flavopiridol HDAC42 Cytotoxicity of flavopiridol and HDAC42 in fresh Sezary cells Courtesy of J.C. Byrd

  7. Untreated 0.92 uM No ZVAD 150uM ZVAD OSU-HDAC42-induced Apoptosis in CLL cells is Caspase-dependent

  8. TNF-Related Apoptosis Inducing LigandTRAIL • TRAIL selectively induces apoptosis in many cancer cells • CLL cells have shown to be resistant to TRAIL • Class I but not Class II HDACi sensitize CLL cells to TRAIL (Inoue et al. Cancer Res. 2006; 66: 6785-92) • Thus, Class I HDAC inhibitors in particular may show promise as a combination therapy with TRAIL

  9. OSU-HDAC42 + TRAIL Relative % Live (annexin-negative)

  10. C-FLIP in OSU-HDAC42 Treated CLL Cells (24 hr) Pt. 1 Pt. 2 Pt. 3 0.038 DP 5 uM SAHA OSU-HDAC42 - .46 uM .92 uM - .46 uM .92 uM - c-FLIP 55 kDa GAPDH 37 kDa C-FLIP:GAPDH 1.6 1.8 1.6 1.0 1.0 0.7 1.2 0.9 0.8

  11. Conclusions • OSU-HDAC42 effectively induces caspase-dependent apoptosis in CLL cells at 48 hr • HDAC42 induces apoptosis in fresh malignant T-cells at concentrations similar to those active in CLL cells • OSU-HDAC42 sensitizes CLL patient cells to TRAIL but this sensitization is not due to FLIP degradation

  12. HDAC inhibition, Virus Latencyand Immunity in Lymphoma

  13. Lymphoma Immunity Infection Infections, Immunity and Lymphoma Cytokine polymorphisms HLA Haplotype Chemokine polymorphisms KIR Haplotype

  14. CMV and T-cell lymphoma • Association between CMV and T-cell Lymphoma • Herne KL, Talpur R, Breuer-McHam J, Champlin C, and Duvic M. Cytomegalovirus seropositivity is significantly associated with mycosis fungoides and Sézary syndrome. Blood 101: 2132-2135, 2003 • Gupta RK, Ramble J, Tong CY, Whittaker S, MacMahon E. Cytomegalovirus seroprevalence is not higher in patients with mycosis fungoides/Sezary syndrome. Blood 107: 1241-1242, 2006 • Reactivation of CMV in T-cell Lymphoma • Porcu P, Baiocchi RA et al. OSU-0303, A Phase I trial of subcutaneous (SQ) alemtuzumab and CHOP in T-cell lymphoproliferative disorders. ASCO 2006 • Six patients ineligible for positive EBV viral load at registration

  15. Cytomegalovirus (CMV) reactivation in T-cell lymphomas before systemic therapy Grady T., et al. Abstr. 722, ASH 2005 • 52 CTCL: stage IA-B (30 pts), IIA-B (18 pts), and III (4) • Prior therapy: untreated (6 pts), topical steroids (24 pts), topical targretin (11 pts), topical alylating agents (7), PUVA (21 pts), electron beam radiation (8) • CMV seropositive = 31/52 (60%) • Elevated whole blood CMV viral loads = 15/31 (50%) • Hybrid Capture Assay (Digene). Median cutoff ratio 2.87 (>1.0 = positive), range 1.2 - 68.4 • Significant correlation between lower (<200/mm3) CD8+ T-cell counts and higher (patient/cutoff ratio >2) CMV titers (p<.003) • Six of 12 (50%) tested tissue samples (skin, LN, bone marrow) from the 15 patients with elevated CMV viral loads were positive for CMV IE-RNA

  16. 12 EBV and CMV seropositive patients 14 mos 8 mos 16 mos 5 mos 12 mos Patients 16 mos 19 mos 11 mos 8 mos 21 mos 4 mos 7 mos = CMV = EBV CMV and EBV reactivation in T-cell lymphoma off immunosuppressive therapy

  17. Role of HDAC/HAT in the Control ofLytic Viral replication • Switch from latency to/from lytic viral infection is controlled by HDAC/HAT-mediated chromatin remodeling in multiple experimental systems • EBV EBNA-3C recruits HDAC to induce latency • CMV IE-1 antagonizes histone deacetylation to induce lytic cycle • HIV-1 HDAC-1, HDAC-6, p300, Tat regulate viral replication • HTLV-1 p300 induces, HDCA-1 inhibits, viral replication • HHV-8 (KSHV) LANA interacts with HDACs and suppresses ORF50 • HDACi (VPA) therapy leads to greater depletion of the viral reservoir in HIV-infected patients with undetectable viremia on HAART (Lancet 366: 549, 2005) • Activation of viral replication sensitizes EBV-positive tumor cells to the pro-apoptotic effect of chemotherapy and antiviral therapy

  18. Histone deacetylase inhibitors induce EBV early lytic gene (vTK) activation in LCL lines Depsipeptide

  19. HDACi (DP) Induce EBV vTK and BGLF4 and Enhance AZT-GCV-induced apoptosis Antiviral Tx: AZT + GCV (mg/ml) Media 10 mg/mL 50 mg/mL 100 mg/mL Media 2% 4% 12% 17% DP (10 nM) 11% 22% 43% 45% Annexin V

  20. Selective activity of Depsipeptide against Latency III EBV-positive cell lines Xenotransplanted 10 x 106 cells NK-cell depleted SCID mice received Depsipeptide 5 mg/m2 i.p. on days 2, 4, 7 and 10 Roychowdhury S, Baiocchi RA et al. J. Natl. Cancer Inst. 96: 1447-1457, 2004

  21. Modulation of normal NK cell receptors DMSO HDAC42 (50nM) HDAC42 (250nM) 5.42 12.77 5.70 22.89 7.03 43.83 NKG2D 6.67 104.06 6.54 44.05 7.03 74.22 KIR

  22. Modulation of HLA with HDAC42 in LCL DMSO HDAC42 (250nM) HDAC42 (50nM) 5.26 375.27 9.13 196.27 2.92 602.52 Class I 5.78 444.39 6.06 441.52 5.44 495.29 Class II

  23. Effect of HDACi (DP) on PBMC IFNg Production Patients treated with Depsipeptide (13 mg/m2) on Phase II trial (Shah MH et al.) Ex vivo PBMC plated in IL-12 (10ng/ml) + IL-18 (100ng/ml) Incubated at 37oC 16hr; CD56+ events collected PRE Tx 4 hr Post Tx 20 hr Post Tx Pre Tx Cycle 2 1% 4% 2% 2.5% Intracellular flow cytometry for IFNg

  24. Lymphoma Immunity Infection Infections, Immunity and Lymphoma

  25. Conclusions • HDACi therapy may influence CMV and EBV reactivation in seropositive patients and require monitoring of viral loads • The viral status of the lymphoma may influence the response to HDACi and may be a relevant tumor marker • Treatment with HDACi may induce or upregulate viral antigens that can serve as targets of therapy • Treatment with HDACi may influence innate and adaptive immunity

  26. Acknowledgments Ohio State University Comprehensive Cancer Center Clara D. Bloomfield, MD Michael A. Caligiuri, MD John C. Byrd, MD Michael R. Grever, MD Carlo Croce, MD OSU T-cell Lymphoma Group Terrence Grady, DO, PhD Robert A. Baiocchi, MD PhD Lapo Alinari, MD Mark Bechtel, MD Carl Morrison, MD Cynthia Magro, MD Work Funded by NCI K23 Grant CA-2435632, Berlex to P. Porcu and Amgen Research Fellowship Grant to T. Grady

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