Kidney cancer and melanoma: progresses in clinical and translational research ROME, November 23-24, 2007 EPIGENETIC IMMUNOTHERAPY OF HUMAN MELANOMA: MOLECULAR.

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