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Kidney cancer and melanoma: progresses in clinical and translational research

Kidney cancer and melanoma: progresses in clinical and translational research. ROME, November 23-24, 2007. EPIGENETIC IMMUNOTHERAPY OF HUMAN MELANOMA: MOLECULAR BASES AND PERSPECTIVE CLINICAL APPLICATIONS. Luca Sigalotti Cancer Bioimmunotherapy Unit,

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Kidney cancer and melanoma: progresses in clinical and translational research

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  1. Kidney cancer and melanoma: progresses in clinical and translational research ROME, November 23-24, 2007 EPIGENETIC IMMUNOTHERAPY OF HUMAN MELANOMA: MOLECULAR BASES AND PERSPECTIVE CLINICAL APPLICATIONS Luca Sigalotti Cancer Bioimmunotherapy Unit, Centro di Riferimento Oncologico, I.R.C.C.S, Aviano

  2. EPIGENETIC ALTERATIONS

  3. EPIGENETICS Heritable changes in the expression of single genes or patterns of genes not based on modifications of the DNA sequence Methylation in C5 of cytosine within CpG dinucleotides Histone modifications Changes in chromatin structure TO TRANSCRIBE OR NOT TO TRANSCRIBE ?

  4. Morgan et al., Hum Mol Genet (2005) Hypomethylation (gene expression) Hypermethylation (gene silencing)

  5. GENETIC ALTERATIONS EPIGENETIC ALTERATIONS DYNAMIC IRREVERSIBLE

  6. EPIGENETIC MODIFICATIONS ARE REVERSIBLE PHARMACOLOGICALLY Inhibitors of DNMT (e.g., 5-AZA-cytidine, 5-AZA-2’- deoxycytidine, Zebularine) Inhibitors of HDAC (e.g., TSA, depsipeptide, SAHA,….)

  7. 5-AZA-2'-DEOXYCYTIDINE (5-AZA-CdR)

  8. CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 z z z z z z z CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 DNMT DNMT DNMT DNA replication 5-AZA-CdR DNA methylation X X Hypomethylated DNA

  9. Epigenetically-regulated immune molecules in cutaneous melanoma

  10. CYTOTOXIC T CELLS AND/OR ANTIBODY-DEFINED TUMOR-ASSOCIATED ANTIGENS

  11. CANCER TESTIS ANTIGENS (CTA) Different families of related antigens: MAGE, NY-ESO and SSX gene families and GAGE/PAGE/XAGE super-families ………..

  12. ADVANTAGES OF CTA AS THERAPEUTIC TARGETS

  13. EXPRESSION OF MAA IN SEQUENTIAL AND CONCOMITANT MELANOMA METASTASES

  14. CTA EXPRESSION IN MELANOMA STEM CELLS

  15. However…….

  16. CO-EXPRESSION OF 7 CTA IN 53 METASTATIC MELANOMAS 7CTA (0 %) 6CTA (9 %) 0 CTA (11 %) 5 CTA (9 %) 1 CTA (24 %) 4 CTA (13 %) 3 CTA (13 %) 2 CTA (21 %)

  17. Roeder et al., Arch Dermatol Res (2005)

  18. Can epigenetic drugs help?

  19. REGULATION OF CTA EXPRESSION IN HUMAN MELANOMA XENOGRAFTS BY 5-AZA-CdR

  20. NY-ESO-1 mol/b-actin mol PERSISTENCY OF 5-AZA-CdR-INDUCED CTA EXPRESSION IN HUMAN MELANOMA XENOGRAFTS

  21. IN VIVO GENERATION OF ANTI-NY-ESO-1 ANTIBODIES BY 5-AZA-CdR-TREATED MELANOMA CELLS OD405

  22. INTRATUMOR HETEROGENEITY

  23. LEVELS OF MAGE-A3 mRNA EXPRESSED BY DIFFERENT SINGLE CELL CLONES FROM MEL 313 MELANOMA CELL LINE 7 5 MAGE-3 mol/b-actin mol (x10-3) 3 1 Mel 313 cell line 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Clone

  24. CLONE 5 CLONE 14 ANALYSIS OF MAGE-A3 PROMOTER METHYLATION IN MEL 313 MELANOMA CLONES

  25. 1,6x10-2 1,2x10-2 8x10-3 4x10-3 0 UP-REGULATION OF MAGE-3 EXPRESSION IN SINGLE CELL CLONES FROM MEL 313 MELANOMA CELL LINE BY 5-AZA-CdR 3x10-3 MAGE-3 mol/b-actin mol 2x10-3 1x10-3 0 Clone 5 Clone 14

  26. RECOGNITION OF MEL313 MELANOMA CLONES BY A MAGE-3-SPECIFIC HLA-B37-RESTRICTED CTL CLONE

  27. EFFECT OF 5-AZA-CdR ON LEVELS OF HLA CLASS I AND CO-STIMULATORY MOLECULES IN MEL 275 MELANOMA CELLS

  28. 30 Ctrl 5-AZA-CdR 20 % Lysis 10 0 25:1 12:1 6:1 3:1 1.5:1 E:T ratio RECOGNITION OF HLA-A2-POSITIVE MEL 275 MELANOMA CELLS BY AN ANTI-GP100 CTL CLONE

  29. ENHANCED AMOUNT OF IFN-g RELEASING GP100-SPECIFIC HLA-A2- RESTRICTED CTL IN RESPONSE TO 5-AZA-CDR-TREATED MEL 275 MELANOMA CELLS 5-AZA-CdR a-HLA Class I a-ICAM-1

  30. DE NOVO EXPRESSION OF CTA IN AML AND MDS PATIENTS TREATED WITH A SINGLE COURSE OF 5-AZA-CdR T: indicates time (days) from the beginning of Decitabine treatment

  31. apoptosis cell cycle angiogenesis invasion & metastasis immune recognition PHARMACOLOGIC DNA HYPOMETHYLATION AS A “POSITIVE” REGULATOR OF THE BIOLOGY OF CANCER CELLS Epigenetic drugs

  32. Systemic Administration of 5-AZA-CdR Methylation “Epigenetic” chemoimmunotherapy CTA-based Vaccine(s)

  33. Maresa Altomonte Lucia Anzalone Edi Bolzanaro Lorelai Brasoveanu Luana Calabrò Ilaria Cattarossi Francesca Colizzi Enzo Cortini Sandra Coral Alessia Covre Riccardo Danielli Chiara De Nardo Anna Maria Di Giacomo Elisabetta Fratta Ester Fonsatti Massimo Guidoboni Annunziata Gloghini Elda Lamaj Antonia Anna Lettini Samuele Massarut Giampaolo Nardi Hugues Nicolay Laura Pezzani Cristina Santantonio Luca Sigalotti Daniela Marconi MEDICAL ONCOLOGY AND IMMUNOTHERAPY DEPT. OF MEDICAL ONCOLOGYUNIVERSITY HOSPITAL OF SIENA CANCER BIOIMMUNOTHERAPY UNITDEPT. OF MEDICAL ONCOLOGY, CRO AVIANO

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