Download 5-AZA-CdR

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 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
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-CdR)
NH 2
N
4
3
5
2
1 6
O
N
HO
O
H
H
H
H
OH
H
N
CH3
CH3
CH3
CH3 CH3
CH3
CH3
CH3
CH3 CH3
CH3
CH3
DNA
replication
CH3
CH3 CH3
DNA
methylation
CH3
X
DNMT
z
CH3
z
CH3
CH3 CH3
CH3
CH3 CH3
CH3
CH3 CH3
CH3
CH3 CH3
CH3
CH3
z
CH3 CH3
CH3
z
CH3
CH3
CH3 CH3
z
DNMT
CH3
CH3
z
CH3
5-AZA-CdR
CH3 CH3
z
DNMT
X
CH3
CH3
Hypomethylated DNA
CH3
Epigenetically-regulated
immune 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)
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
 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
EXPRESSION OF MAA IN SEQUENTIAL
AND CONCOMITANT MELANOMA METASTASES
Patient
Metastasis
#
Site of
metastasis
MAGE-1
MAGE-2
MAGE-3
MAGE-4
GAGE 1-6
BAGE
NY-ESO-1
PRAME
Tyrosinase
Melan-A/
MART-1
Mel 320
I
II
III
IV
V
SC
SC
LN
SC
SC
-
+
+
+
+
+
+
+
+
+
+
-
-
-
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Mel 435
I
II
III
IV
V
LN
SC
SC
M
SC
+/+/+/+/+/-
-
+/+/+/+/+/-
-
+
+
+
+
+
-
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Mel 462
IA
IB
II
IIIA
IIIB
SC
SC
SC
SC
SC
-
+
+
+
+
+
+
+
+
+
+
-
-
-
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
CTA EXPRESSION IN MELANOMA STEM CELLS
However…….
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 %)
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
+
-
-
-
-
-
-
-
+
NY-ESO-1 mol/b-actin mol
PERSISTENCY OF 5-AZA-CdR-INDUCED CTA
EXPRESSION IN HUMAN MELANOMA XENOGRAFTS
1,2E-03
9,0E-04
6,0E-04
3,0E-04
0,0E+00
5
10
20
30
IN VIVO GENERATION OF ANTI-NY-ESO-1 ANTIBODIES
BY 5-AZA-CdR-TREATED MELANOMA CELLS
1200
OD405
1000
800
ctrl
600
5-AZA-CdR
400
200
0
pre
post2
post3
post4
INTRATUMOR
HETEROGENEITY
MAGE-3 mol/b-actin mol (x10-3)
LEVELS OF MAGE-A3 mRNA 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-A3 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-CdR
1,6x10-2
3x10-3
1,2x10-2
2x10-3
8x10-3
1x10-3
0
4x10-3
Clone 5
0
Clone 14
RECOGNITION OF MEL313 MELANOMA CLONES BY
A MAGE-3-SPECIFIC HLA-B37-RESTRICTED CTL CLONE
350
251,218
IFN (pg/ml)
250
218,05
124,688
150
50
B 37
8,292
B 37 5A ZA -CdR
-50
313#5
313#14
EFFECT OF 5-AZA-CdR ON LEVELS OF HLA CLASS I AND
CO-STIMULATORY MOLECULES IN MEL 275 MELANOMA CELLS
RECOGNITION OF HLA-A2-POSITIVE MEL 275 MELANOMA
CELLS BY AN ANTI-GP100 CTL CLONE
30
Ctrl
5-AZA-CdR
% Lysis
20
10
0
25:1
12:1
6:1
E:T ratio
3:1
1.5:1
ENHANCED AMOUNT OF IFN-g RELEASING GP100-SPECIFIC HLA-A2RESTRICTED CTL IN RESPONSE TO 5-AZA-CDR-TREATED
MEL 275 MELANOMA CELLS
5-AZA-CdR
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-CdR
T0
CTA-positive/
total samples
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
apoptosis
cell cycle
angiogenesis
Epigenetic drugs
invasion &
metastasis
immune
recognition
Systemic
Administration
of 5-AZA-CdR
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
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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
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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