Kidney cancer and melanoma progresses in clinical and

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

EPIGENETIC ALTERATIONS

EPIGENETICS Heritable changes in the expression of single genes or patterns of genes not based on modifications of the DNA sequence Methylation in C 5 of cytosine within Cp. G dinucleotides Histone modifications Changes in chromatin structure TO TRANSCRIBE OR NOT TO TRANSCRIBE ?

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

GENETIC ALTERATIONS EPIGENETIC ALTERATIONS IRREVERSIBLE DYNAMIC

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, …. )

5 -AZA-2'-DEOXYCYTIDINE (5 -AZA-Cd. R)

CH 3 CH 3 CH 3 DNA replication CH 3 CH 3 z CH 3 CH 3 X z CH 3 Hypomethylated DNA CH 3 z DNMT z CH 3 z DNA methylation DNMT CH 3 z CH 3 5 -AZA-Cd. R CH 3 CH 3 CH 3 CH 3 z DNMT X CH 3

Epigenetically-regulated immune molecules in cutaneous melanoma

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

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

ADVANTAGES OF CTA AS THERAPEUTIC TARGETS

EXPRESSION OF MAA IN SEQUENTIAL AND CONCOMITANT MELANOMA METASTASES

CTA EXPRESSION IN MELANOMA STEM CELLS

However…….

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

Roeder et al. , Arch Dermatol Res (2005)

Can epigenetic drugs help?

REGULATION OF CTA EXPRESSION IN HUMAN MELANOMA XENOGRAFTS BY 5 -AZA-Cd. R

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

OD 405 IN VIVO GENERATION OF ANTI-NY-ESO-1 ANTIBODIES BY 5 -AZA-Cd. R-TREATED MELANOMA CELLS

INTRATUMOR HETEROGENEITY

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

ANALYSIS OF MAGE-A 3 PROMOTER METHYLATION IN MEL 313 MELANOMA CLONES CLONE 5 CLONE 14

MAGE-3 mol/b-actin mol UP-REGULATION OF MAGE-3 EXPRESSION IN SINGLE CELL CLONES FROM MEL 313 MELANOMA CELL LINE BY 5 -AZA-Cd. R 1, 6 x 10 -2 3 x 10 -3 1, 2 x 10 -2 2 x 10 -3 8 x 10 -3 1 x 10 -3 0 4 x 10 -3 Clone 5 0 Clone 14

RECOGNITION OF MEL 313 MELANOMA CLONES BY A MAGE-3 -SPECIFIC HLA-B 37 -RESTRICTED CTL CLONE

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

RECOGNITION OF HLA-A 2 -POSITIVE MEL 275 MELANOMA CELLS BY AN ANTI-GP 100 CTL CLONE 30 Ctrl 5 -AZA-Cd. R % Lysis 20 10 0 25: 1 12: 1 6: 1 E: T ratio 3: 1 1. 5: 1

ENHANCED AMOUNT OF IFN-g RELEASING GP 100 -SPECIFIC HLA-A 2 RESTRICTED CTL IN RESPONSE TO 5 -AZA-CDR-TREATED MEL 275 MELANOMA CELLS 5 -AZA-Cd. R a-HLA Class I a-ICAM-1

DE NOVO EXPRESSION OF CTA IN AML AND MDS PATIENTS TREATED WITH A SINGLE COURSE OF 5 -AZA-Cd. R T 0 CTA-positive/ total samples 2/33 T 15 32/33 T 30 10/15 T 40 3/3 T: indicates time (days) from the beginning of Decitabine treatment

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

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

MEDICAL ONCOLOGY AND IMMUNOTHERAPY DEPT. OF MEDICAL ONCOLOGY UNIVERSITY HOSPITAL OF SIENA CANCER BIOIMMUNOTHERAPY UNIT DEPT. OF MEDICAL ONCOLOGY, CRO AVIANO • • • • 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