Control of melanoma dedifferentiation program and immune escape by NFATc2

Andrea Anichini Human Tumors Immunobiology Unit

Fondazione IRCCS Istituto Nazionale dei Tumori, Milan

Background 1. Melanoma dedifferentiation

Consequences

+ - Intrinsic and acquired resistance to

MAPK inhibition

+ -

Promotion of immune escape and of tumor infiltration by myeloid cells

+ -

Upregulation of stemness (CD271) and dedifferentiation (AXL) markers

+ - Shift from a proliferative

to invasive gene programs

MITF + -

MITF-regulated genes, pigmentation and MDAs + -

Definition

Intrinsic regulators of melanoma dedifferentiation

ZEB1, TWIST1 In cooperation with oncogenic BRAF

Caramel, Cancer Cell 2013

Extrinsic regulators of melanoma dedifferentiation

IFN-g, NGF Furuta, J. Invest Dermatol. 2014

Drug exposure, hypoxia, and nutrient starvation

Ravindran Menon, Oncogene 2014

TNF-a

CD3

MITF b catenin

Loss of MITF in-vivo in the absence of a T cell infiltrate

Two types of melanomas with distinct MITF profiles

SOX10 / MITF AP-1 / TEADS

Master regulators of distinct transcriptional states

Proliferative gene network

Invasive gene network

MITF+ melanomas Tissue section Cell line

Pt#14

Pt#8

MITF- melanomas Tissue section Cell line

Pt#95

Pt#5

Hoek, K. S. et al. In vivo switching of human melanoma cells between proliferative and invasive states. Cancer Res. 68, 650–656 (2008).

Sensi M. et al. Human cutaneous melanomas lacking MITF and melanocyte differentiation antigens express a functional Axl receptor kinase. J Invest Dermatol. 131:2448-57, 2011.

2010

Background 2. NFAT family of Transcription Factors in normal and neoplastic cells

NFATc2 NFATc11

10

100

MF

INFATc2 NFATc1

50m 50m

Me#29318

BRAFV600E

p53WT

Me#15392

BRAFV600E

p53WT

Me#3046/2

BRAFV600E

p53n.d.

NFATc1

NFATc2

N N C C N N C C N N C C

+ - + - + - + - + - + - FBS

PARP

Tubulin

120 kDa

55 kDa

140 kDa

89 kDa

Perotti et al. J. Invest. Dermatol. 2012

Background 3. NFATc2 is a potential therapeutic target in melanoma

No

rmal

ized

d

ensi

ty v

alu

e

Hypothesis: NFATc2 may be an intrinsic regulator of melanoma dedifferentiation

NFATc2 expression correlates with a melanoma dedifferentiated profile

+ -

Outline of main findings

NFATc2

MITF and MITF-regulated genes

+ -

CD271 + -

MART-1 18 kDa

NFATc2

42 kDa β-actin

MITF

120 kDa

56 kDa

52 kDa GPNMB

PGC-1α 90 kDa

NFATc2 120 kDa

55 kDa α-Tubulin

27 kDa Rab27a

MITF 56 kDa

52 kDa

125 kDa

75 kDa

Protein level: NFATc2 expression more frequent in melanomas lacking MITF and proteins coded for by MITF target genes

NFATc2 is preferentially expressed in melanoma cells with a dedifferentiated profile

Inverse correlation between NFATc2 and Melanocyte Differentiation Antigens (MDA)

Perotti et al. Oncogene 2015

qPCR: Inverse correlation between NFATc2 and MITF

Protein level Inverse correlation between

NFATc2 and MITF in 24 cell lines

Inverse correlation between CD271 and Melanocyte Differentiation Antigen expression in melanoma cell lines

NFATc2 is preferentially expressed in melanoma cells with a dedifferentiated profile

MITF + -

+ - NFATc2

42k Da

120k Da NFATc2

CD271

β-actin

75k Da

CD271 expression in NFATc2+ cell lines

Quantitative assessment of western blot data

CD271 and NFATc2 expression by flow cytometry in 24 cell lines

Targeting of NFATc2 promotes melanoma differentiation

NFATc2

MART-1

GPNMB

120 kDa

56 kDa

52 kDa

95 kDa

18 kDa

75 kDa

125 kDa

90 kDa

42 kDa

MITF

gp100

PGC-1α

β- actin

MITF

Me71

75 kDa CD271

β-actin 42 kDa

Me71

siRNA

42 kDa

90 kDa

125 kDa

75 kDa

Me71

β-actin

PGC-1α

GPNMB

120 kDa

42 kDa

52 kDa

95 kDa

18 kDa

56 kDa

Me71

NFATc2

MART-1

β-actin

MITF

gp100

shRNA

β-actin

PGC-1α

GPNMB

42 kDa

90 kDa

125 kDa

75 kDa

Me71

NFATc2

MART-1

β-actin

MITF

gp100

120 kDa

42 kDa

52 kDa

95 kDa

18 kDa

56 kDa

Me71

NFATc2 inhibitor

Me71 Me71

Ctrl

shRNA_14

Ctrl

shRNA_15

NFATc2

shRNA_86a

NFATc2

shRNA_86b

NFATc2

shRNA_87a

Me71

NFATc2 shRNA_86a NFATc2 shRNA_87a Ctrl shRNA_15

gp100

NFATc2

MITF

GPNMB RAB27a PGC-1a Gp100 Tyrosinase MART-1 Melanin production Pigmentation

- - - - - - - - + + + + + + + +

+ CD271 -

Targeting of NFATc2 rescues melanoma differentiation

Genes modulated by NFATc2 silencing

Gen

es m

od

ula

ted

b

y TN

Fa

Up Down

Up

Down

46 49

41 14

TNF-a

T cells

TNF-a

TNF-a

TNF-RI/II

NFATc2+ melanomas express surface-bound TNF-a

NFATc2 profile

+ + + + + + - - -

mTNF-α

Events

TNFR1 TNFR2 TNF-α

NFATc2+ cell line (Me71)

Surface

staining Intracellular

staining

Surface

staining Surface

staining

Events

mTNF-α TNFR1 TNFR2 TNF-α

NFATc2- cell line (Me45)

Surface

staining Intracellular

staining

Surface

staining

Surface

staining

mRNA expression Protein expression

mTNFa correlates with TNFR1/2 NFATc2 correlates with TNFR1/2

Targeting of NFATc2 suppresses endogenous TNF-a

Ctrl siRNA

NFATc2

siRNA-1

mTNF-α

Ctrl siRNA

NFATc2

siRNA-2

Events

TNF-α

Events

Surface staining intracellular staining

Ctrl siRNA

NFATc2

siRNA-1

Ctrl siRNA

NFATc2

siRNA-2 Me71

Events

TNF-α

Events

Ctrl

shRNA_15

NFATc2

shRNA_86a

mTNF-α

Me71

Ctrl

shRNA_15

NFATc2

shRNA_87a

Ctrl

shRNA_15

NFATc2

shRNA_86a

Ctrl

shRNA_15

NFATc2

shRNA_87a

Endogenous TNF-a is under NFATc2 control

MITF

Ctrl 0.06 0.12

β-actin 42 kDa

MART-1 18 kDa

56k Da

52 kDa

Ab anti-TNF-α

(μg/ml)

NFATc2+ MITFlo mTNF-a+

cell line

MART-1

- 0.06 0.12 - 0.06 0.12

Ab anti-TNF-α

(μg/ml)

Ab anti-TNF-α

(μg/ml)

rTNF-α

42 kDa

18 kDa

β-actin

- - - + + +

Me71

rTNF-α

75 kDa CD271

β-actin 42 kDa

Me71

Role of endogenous TNF-a in melanoma dedifferentiation

NFATc2-TNFa axis

NFATc2

MITF

TNF-a

TNF-a

TNF-RI/II

?

C-Myc Brn2

Upstream regulator analysis of genes modulated by NFATc2 silencing

NFATc2

expression

Brn2 45 kDa

56 kDa

52 kDa MITF

Vinculin 110 kDa

-/+ + ++ +/- - - + + + ++ - +

45 kDa Brn2

56 kDa

52 kDa MITF

110 kDa Vinculin

45 kDa Brn2

56 kDa

52 kDa MITF

110 kDa Vinculin

Me71 Me98

Preferential expression of Brn2 in MITF- NFATc2+ melanomas

Targeting of Brn2 upregulates MITF

c-myc 65 kDa

57 kDa

42 kDa β-actin

120 kDa NFATc2

Me71

45 kDa Brn2

c-myc 65 kDa

57 kDa

42 kDa β-actin

120 kDa NFATc2

Me98

Brn2 45 kDa

65 kDa

57 kDa c-myc

42 kDa β-actin

120 kDa NFATc2

Me71

45 kDa Brn2

120 kDa NFATc2

42 kDa β-actin

65 kDa

57 kDa c-myc

Me71

45 kDa Brn2

Targeting of NFATc2 suppresses c-Myc and Brn2

MART-1 18 kDa

β-actin 42 kDa

MITF 56k Da

52 kDa

Ctrl 0.06 0.12

Ab anti-TNF-α

(μg/ml)

65 kDa

57 kDa c-myc

Me71 Me98

65 kDa

57 kDa c-myc

45 kDa Brn2

56 kDa

52 kDa MITF

β-actin 42 kDa

rTNF-α

MART-1 18 kDa

NFATc2 120 kDa

MITF 56 kDa

52 kDa

β-actin 42 kDa

65 kDa

57kDa c-myc

42 kDa β-actin

45 kDa Brn2

56 kDa

52 kDa MITF

65 kDa

57 kDa c-myc

Me71

Endogenous TNF-a regulates c-Myc and Brn2

NFATc2 TNF-a c-Myc Brn2 MITF

Targeting of c-Myc suppresses Brn2 and rescues MITF expression

65 kDa

57 kDa c-myc

Brn2 45 kDa

56 kDa

52 kDa MITF

18 kDa MART-1

42 kDa β- actin

c-myc 65 kDa

57 kDa

56 kDa

52 kDa MITF

45 kDa Brn2

18 kDa MART-1

42 kDa β- actin

Me71 Me98

18 kDa MART-1

c-myc 65 kDa

57 kDa

45 kDa Brn2

56 kDa

52 kDa MITF

42 kDa β- actin

65 kDa

57 kDa

c-myc

45 kDa Brn2

56 kDa

52 kDa MITF

42 kDa β-actin

18 kDa MART-1

Me71 Me98

NFATc2 TNF-a c-Myc Brn2 MITF

NFATc2 TNF-a

TNF-a

TNF-RI/II

Brn2 C-Myc MITF

GPNMB RAB27a PGC-1a Gp100 Tyrosinase MART-1 Melanin production Pigmentation T cell recognition CD271

- - - - - -

+

- - -

GPNMB RAB27a PGC-1a Gp100 Tyrosinase MART-1 Melanin production Pigmentation T cell recognition CD271 -

+ + + + + + + + +

Oncogene, 2015

The final model

NFATc2 and MITF expression HLA-A2, b2M expression and

CTL-mediated lysis MDA expression

NFATc2HI MITFLo melanoma cell lines are poorly recognized by MDA-specific CTLs

CTL lines to

MART-127-35

MART-127-35

gp100209-217

*** *** ***

*** ***

*** *** ***

*** ***

***

*** *** ***

**

*

***

***

* **

Ctrl shRNA_15

NFATc2 shRNA_86a

NFATc2 shRNA_87a

Ctrl shRNA_15

NFATc2 shRNA_86a

NFATc2 shRNA_87a

Ctrl shRNA_15

NFATc2 shRNA_86a

NFATc2 shRNA_87a

Targeting of NFATc2 improves CTL-mediated melanoma recognition

a

Eve

nts

HLA-A2

Ctrl

shRNA_15

NFATc2

shRNA_86a

Ctrl

shRNA_15

NFATc2

shRNA_87a

CTL lines to

MART-127-35

MART-127-35

Gp100209-217

b

Me 71 Ctrl shRNA_15

Me 71 Ctrl shRNA_15

Me 71 Ctrl shRNA_15

NFATc2

NFATc2 vs MITF in-vivo

MITF NFATc2

Conclusions

Acknowledgements

Human Tumors Immunobiology Unit

Roberta Mortarini Valentina Perotti Elena Tassi Giulia Grazia

Paola Baldassari Ilaria Bersani Alessandra Molla Claudia Vegetti Gabriella Nicolini

Surgical Oncology Unit Andrea Maurichi Mario Santinami

Dept. of Pathology Gabrina Tragni

Functional Genomic Core Facility Loris de Cecco Edo Marchesi Silvana Canevari

Grant support

-Expression of NFATc2 promotes a MITF- CD271+ dedifferentiated profile in melanoma cells. -NFATc2 promotes expression of mTNF-a that in turn negatively regulates MITF and differentiation through a Myc-Brn2 axis. -Melanoma differentiation can be rescued by targeting NFATc2, Myc and Brn2 and by neutralization of endogenous TNF-a. -Targeting of NFATc2 improves recognition of melanoma cells by MDA-specific CTLs.