kidney cancer and melanoma: progresses in clinical and translational research rome, november 23-24,...
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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 SigalottiCancer Bioimmunotherapy Unit,
Centro di Riferimento Oncologico, I.R.C.C.S, Aviano
EPIGENETIC ALTERATIONSEPIGENETIC ALTERATIONS
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 ?
Hypomethylation(gene expression)
Hypermethylation(gene silencing)
Morgan et al., Hum Mol Genet (2005)
GENETIC ALTERATIONS
IRREVERSIBLE
EPIGENETIC ALTERATIONS
DYNAMICDYNAMIC
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,….)
O
HOH
HH
HH
HO
N
N
N
NH2
O 1
43 5 2 6
5-AZA-2'-DEOXYCYTIDINE (5-AZA-CdR)
CH3 CH3 CH3CH3CH3
CH3 CH3 CH3 CH3 CH3
CH3 CH3 CH3CH3CH3
CH3 CH3 CH3 CH3 CH3
z5-AZA-CdR
DNAreplication
DNAmethylation
X
CH3 CH3 CH3CH3CH3
zCH3 CH3 CH3
DNMT XCH3 CH3 CH3 CH3 CH3
z
z
z
DNMT
CH3 CH3 CH3CH3CH3
zCH3 CH3 CH3
CH3 CH3 CH3 CH3 CH3
z
Hypomethylated DNA
DNMT
Epigenetically-regulatedimmune molecules
in cutaneous melanoma
CYTOTOXIC T CELLS AND/OR ANTIBODY-DEFINED TUMOR-ASSOCIATED ANTIGENS
Differentiation antigens Tumor over-expressed antigens Unique antigens Cancer Testis Antigens (CTA)
Different families of related antigens: MAGE, NY-ESO and SSX gene families and GAGE/PAGE/XAGE super-families ………..
CANCER TESTIS ANTIGENS (CTA)
ADVANTAGES OF CTA AS THERAPEUTIC TARGETS
CTA are not expressed in benign tissues with the exception
of testis and placenta
CTA are immunogenic in vivo inducing both CELLULAR
and/or HUMORAL immune responses
Distinct CTA can be expressed in malignant tissues of
different histological origin, providing common therapeutic
targets shared by human neoplasia regardless of their specific
histotype
Patient Metastasis#
Site ofmetastasis
MAGE-1 MAGE-2 MAGE-3 MAGE-4 GAGE 1-6 BAGE NY-ESO-1 PRAME Tyrosinase Melan-A/MART-1
Mel 320 I SC - + + - - - - + + +II SC - + + - - - - + + +III LN - + + - - - - + + +IV SC - + + - - - - + + +V SC - + + - - - - + + +
Mel 435 I LN +/- - +/- - + - - + + +II SC +/- - +/- - + - - + + +III SC +/- - +/- - + - - + + +IV M +/- - +/- - + - - + + +V SC +/- - +/- - + - - + + +
Mel 462 IA SC - + + - - - - + + +IB SC - + + - - - - + + +II SC - + + - - - - + + +
IIIA SC - + + - - - - + + +IIIB SC - + + - - - - + + +
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
0 CTA(11 %)
1 CTA(24 %)
2 CTA(21 %)
3 CTA(13 %)
4 CTA(13 %)
5 CTA(9 %)
6CTA(9 %)
7CTA(0 %)
Roeder et al., Arch Dermatol Res (2005)
Can epigenetic drugs help?
REGULATION OF CTA EXPRESSION IN HUMAN MELANOMA XENOGRAFTS BY 5-AZA-CdR
Xenografts
Mel 275 Mel 313 Mel 195
5-AZA-CdR - + - + - +
MAGE-1 - -
MAGE-2 - -
MAGE-3 -
MAGE-4 - - -
MAGE-A10 - -
GAGE 1-6 -
NY-ESO-1 - - -
PRAME -
PERSISTENCY OF 5-AZA-CdR-INDUCED CTA EXPRESSION IN HUMAN MELANOMA XENOGRAFTS
0,0E+00
3,0E-04
6,0E-04
9,0E-04
1,2E-03
5 10 20 30
NY
-ES
O-1
mo
l/-
ac
tin
mo
l
0
200
400
600
800
1000
1200
pre post2 post3 post4
ctrl
5-AZA-CdR
OD
405
IN VIVO GENERATION OF ANTI-NY-ESO-1 ANTIBODIES BY 5-AZA-CdR-TREATED MELANOMA CELLS
INTRATUMORHETEROGENEITY
LEVELS OF MAGE-A3 mRNA EXPRESSED BY DIFFERENT SINGLE CELL CLONES
FROM MEL 313 MELANOMA CELL LINE
Clone
1
3
5
7
1 2 3 4 5 6 7 8 9 10 11 12 13 14Mel 313cell line
MA
GE
-3 m
ol/
-act
in m
ol (
x10-3
)
CLONE 5
CLONE 14
ANALYSIS OF MAGE-A3 PROMOTER METHYLATION IN MEL 313 MELANOMA CLONES
0
4x10-3
8x10-3
1,2x10-2
1,6x10-2
0
1x10-3
2x10-3
3x10-3
UP-REGULATION OF MAGE-3 EXPRESSION IN SINGLE CELL CLONES
FROM MEL 313 MELANOMA CELL LINE BY 5-AZA-CdR
Clone 5 Clone 14
MA
GE
-3 m
ol/
-act
in m
ol
8,292
124,688
251,218218,05
-50
50
150
250
350
313#5 313#14
IFN
(pg
/ml)
B37
B37 5-AZA-CdR
RECOGNITION OF MEL313 MELANOMA CLONES BY A MAGE-3-SPECIFIC HLA-B37-RESTRICTED CTL CLONE
EFFECT OF 5-AZA-CdR ON LEVELS OF HLA CLASS I ANDCO-STIMULATORY MOLECULES IN MEL 275 MELANOMA CELLS
RECOGNITION OF HLA-A2-POSITIVE MEL 275 MELANOMA CELLS BY AN ANTI-GP100 CTL CLONE
0
10
20
30
25:1 12:1 6:1 3:1 1.5:1
Ctrl
5-AZA-CdR
% L
ysis
E:T ratio
5-AZA-CdR
-HLAClass I
-ICAM-1
ENHANCED AMOUNT OF IFN- RELEASING GP100-SPECIFIC HLA-A2- RESTRICTED CTL IN RESPONSE TO 5-AZA-CDR-TREATED
MEL 275 MELANOMA CELLS
DE NOVO EXPRESSION OF CTA IN AML AND MDS PATIENTS TREATED WITH A SINGLE COURSE OF 5-AZA-CdR
CTA-positive/
total samples
T0 2/33
T15 32/33
T30 10/15
T40 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
Epigenetic drugs
immunerecognition
cell cycle
apoptosis
angiogenesis
invasion &metastasis
CTA-basedVaccine(s)
SystemicAdministrationof 5-AZA-CdR
“Epigenetic”chemoimmunotherapyMethylation
MEDICAL ONCOLOGY AND IMMUNOTHERAPY DEPT. OF MEDICAL ONCOLOGYUNIVERSITY HOSPITAL OF SIENA
CANCER BIOIMMUNOTHERAPY UNITDEPT. 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