TET2 in MPN and MDS: What is the role? - Imedex · TET2 in MPN and MDS: What is the role? ... Polycythemia Vera 13.8% 17% 10% 13% 7% 14/89 NA (357 MPN) ... The role of TET2 in MPN

15/05/2012

Lisbon_MDS/MPN_2012 1

TET2 in MPN and MDS:

What is the role?

Lisbon_MDS/MPN_2012

The role of TET2 in MPN and MDS

1- TET2 mutations in myeloid neoplasms

2- TET enzymes and 5-hydroxymethylcytosines

3- Function of TET2

4- TET2 mutations in the development of myeloid malignancies

5- Diagnostic and prognostic relevance of TET2 mutations

Lisbon_MDS/MPN_2012

15/05/2012





3- Function of TET2



Lisbon_MDS/MPN_2012

Discovery of TET2 mutations in myeloid malignancies

Common 4q24 deletion in four cases of hematopoietic malignancy: early stem cell involvement?

Viguié et al, Leukemia. 2005 Aug;19(8):1411-5.

Lisbon_MDS/MPN_2012

15/05/2012



Common 4q24 deletion in four cases of hematopoietic malignancy: early stem cell involvement?

Viguié et al, Leukemia. 2005 Aug;19(8):1411-5.

Lisbon_MDS/MPN_2012

p<0.001p=0.182

p=0.151

p=0.013

Early clonal dominance in some patients with JAK2 mutant MPNs

1.0

0.6

0 0

0.2

0.8

0.4

AK

2 V

617F

/ to

tal J

AK

2

JAK2 V617F clone (majority of PV patients)

JAK2 wild-type cells

0.0JA

CD34+CD38- CD34+CD38+ Erythroblastes

early progenitors

committedprogenitors

mature cells

Lisbon_MDS/MPN_2012

15/05/2012


p<0.001p=0.182

p=0.151

p=0.013

Early clonal dominance in some patients with JAK2 mutant MPNs

1.0

0.6

0 0

0.2

0.8

0.4

AK

2 V

617F

/ to

tal J

AK

2

JAK2 V617F clone (minority of MPN patients)

JAK2 V617F clone (majority of PV patients)

JAK2 wild-type cells

0.0JA

CD34+CD38- CD34+CD38+ Erythroblastes

early progenitors

committedprogenitors

mature cells New mutations ?

Lisbon_MDS/MPN_2012

500 kbRP11-1061M21 RP11-16G16

Cen TelRP11-356L5

Chromosome 4 q24

minimal 4q24 region

MDS


RP11-1061M21 RP11-16G16RP11-356L5

TET25' 3'

LOH 4q

Chromosome 4 q24

SNP-array

Patient #1 Patient #4

Acquired mutations in TET2

WT

Myeloidcells *

Q557Stop Del1237-39

#4

MDS AML

Delhommeau et al, Mutation in TET2 in myeloid cancers. N EnglJ Med 2009;360:2289-2301.

delLOH

TET25' 3'

106.11 Mb 106.35 Mb325 Kb

deletionArray-CGH

#5MPN

Lisbon_MDS/MPN_2012

15/05/2012


TET2 mutations in myeloid malignancies

DiseaseTET2

mutant total % reference

All myeloid malignancies 725 3247 22,3 A meta‐analysis of TET2 mutations shows a distinctmutations shows a distinct

distribution pattern in de novo acute myeloid leukemia and

chronic myelomonocytic leukemia.

Euba B, Vizmanos JL, García‐Granero M, Aranaz P, Hurtado C,

Migueliz I, Novo FJ, García‐Delgado M.

L k L h 2012 A 2

MPN 130 1121 11,6

MDS 182 835 21,8

MDS/MPN (347 CMML) 191 371 51,5

AML 222 920 24,1

de novo AML 176 753 23,4 Leuk Lymphoma. 2012 Apr 2.de novo AML 176 753 23,4

secondary AML 46 167 27,5

[1] Saint-Martin et al,.Blood 2009;114:1628 – 1632.[2] Szpurka et al, Leuk Res 2010;34:969– 973.[3] Nibourel et al, Blood 2010;116:1132 – 1135.[4] Kohlmann et al, J Clin Oncol 2010;28:3858 – 3865.[5] Colaizzo et al, J Th romb Haemost 2010;8:1142– 1144.[6] Rocquain et al, BMC Cancer 2010;10:401.[7] Teff eri et al, Leukemia 2010;24:1128 – 1138.

[8] Abdel-Wahab et al, Cancer Res 2010;70:447 – 452.[9] Smith et al, Blood 2010;116:3923 – 3932.[10] Ko et al, Nature 2010;468:839 – 843.[11] Pollyea et al, Hematol Oncol 2011;29:157 – 160.[12] Bacher et al, Ann Hematol 2010;89:643 – 652.[13] Metzeler et al, J Clin Oncol 2011;29:1373 – 1381.

Lisbon_MDS/MPN_2012

MPN Frequency of TET2 mutations

Frequency of TET2 mutations (details)

patients with TET2 mutation/total number of patients

Reference

Polycythemia Vera 13.8% 17%10%13%7%

14/89NA (357 MPN)

13/1001/14

[1][2] [3][4]

Essential Thrombocythemia 8.5% 5%4%

3/57NA (357 MPN)

[1] [2]

TET2 mutations in myeloproliferative neoplasms

11%8%

( )9/841/12

[ ][3][4]

Primary Myelofibrosis 20.5% 17%8%25%43%

10/60NA (357 MPN)

4/163/7

[1] [2][3][4]

Post-PV/ET Myelofibrosis 23.5% 14%33%40%

3/211/34/10

[1][3][4]

Post-MPN AML 22.7% 17%17%32%13%

2/123/186/192/16

[1][4][5] [6]

[1] Tefferi A et al, Leukemia.2009;23:905-911.[2] Abdel-Wahab et al, Blood 2009;114:144-147.[3] Delhommeau et al, N Engl J Med 2009;360:2289-2301.

13%25%

2/1616/63

[6][7]

Systemic Mastocytosis 29% 29% 12/42 [8]

Chronic myelogenousleukemia, chronic phase

0% 0%0%

0/140/71

[9][10]

Chronic myelogenous leukemia, non-chronic phase

13.3% 15%10%

6/402/20

[9][10]

[4] Saint-Martin et al, Blood 2009;114:1628-1632.[5] Couronne et al, Leukemia 2010;24:201-203.[6] Beer et al, Blood 2010;115:2891-2900.[7] Abdel-Wahab, Cancer Res 2010;70:447-452.

[8] Tefferi et al, Leukemia 2009;23:900-904.[9] Makishima et al, Blood 2011;117:e198-206.[10] Roche-Lestienne et al, Leukemia 2011

15/05/2012


Large deletions

conserved dom.TET2

conserved dom. loss or truncation

of the TET2 protein

box 1 box 2

TET2 mutations in myeloid malignancies

Nonsense mutations

Frameshifts

Missense mutations

Small in frame deletion

Large deletions

MPN

Allmyeloid

alteration of conserved residues

Double-stranded -helix 2OG-Fe(II)-dependent dioxygenase domain

Cysteine-rich domain

Fe(II)-binding

2-Oxoglutarate-binding

Mal.

Adapted from Mohr et al, exp hem, 2010Lisbon_MDS/MPN_2012




3- Function of TET2



Lisbon_MDS/MPN_2012

15/05/2012


TET oncogene family

Mohr et al, exp hem, 2010

Lisbon_MDS/MPN_2012

TET protein function

Tet1 2OG-Fe(II)-dependent

dioxygenase

5-methylcytosine 5-hydroxymethylcytosine

Tahiliani et al. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by mll partner tet1. Science 2009;324:930-935.

Dendriticcells

T cells

ES cells

Lisbon_MDS/MPN_2012

15/05/2012


TET protein function

Tahiliani et al. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by mll partner tet1. Science 2009;324:930-935. Lisbon_MDS/MPN_2012

Decarboxylase

Cycle of cytosine / 5mC / 5hmC

Mercher et al, Médecine Sciences 2011; 12;27 : 1064-1066

Tahiliani et al. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 2009 ; 324 : 930-5.

Gu et al. The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes.Nature 2011 ; 477 : 606-10.

Dawlaty et al. Tet1 is dispensable for maintaining pluripotency and its loss is compatible with embryonic and postnatal development. Cell Stem Cell 2011 ; 9 : 166-75.Molecular biology. Demystifying DNA demethylation. Science 2011 ; 333 : 1229-30.Guo et al. Nabel & Kohli. : Hydroxylation of 5-methylcytosine by tet1 promotes active DNA demethylation in the adult brain. Cell 2011;145:423-434.

Guo et al. Emerging roles of TET proteins and 5-hydroxymethylcytosines in active DNA demethylation and beyond. Cell Cycle 2011 ; 10 : 2662-8.

15/05/2012





3- Function of TET2



Lisbon_MDS/MPN_2012

Expression of TET2 in human hematopoietic cells

3

4

5

6

hematopoietic differentiation of hES cells

TET2 m

RN

Are

ssio

n

1

10

leukemia cell linesprimary

cells

Delhommeau et al, N Engl J Med

0

1

2

3

0 1 2 4 6 8 9 12 17 21 26 28Days in hematopoietic

induced differentiation culture

norm

aliz

ed

expr

0,01

0,1

1

MO7E HL60UT7 K562 UKE1U937HELCD34+ Granulocytes

2009;360:2289-2301.

Lisbon_MDS/MPN_2012

Langemeijer et al, Nat Genet 2009;41:838-842.

15/05/2012


TET2 knock-down in human leukemic cell lines

Lisbon_MDS/MPN_2012

Dot blot

Pronier et al, Blood 2011;118:2551-2555.

TET2 knock-down in human CD34+ cells

0,200,400,600,801,001,201,401,601,802,002,20

CD34+

2,2

1,2

TET2

mRNAshRNATET2

shRNAscr

0,00mRNA

Pronier et al, Blood 2011;118:2551-2555.

Lisbon_MDS/MPN_2012

15/05/2012


TET2

wt

5-hmC tib d

m m wt wt mcontrols1:1

1:2

Quantification of 5hmC in primary cells according to TET2 status

MPN Pronier et al, Blood 2011;118:2551-2555.

Methylene blue (MB)

antibody 1:4

1:8

1:1

1:2

1:4

1:8

Ko et al, Nature 2010;468:839 – 843.

Other myeloid

malignancies

Lisbon_MDS/MPN_2012

TET2

CXXC??

Polycomb

ComplexTET2

CXXC??

TET2 function : hydroxylation of 5-methylcytosine-> DNA demethylation in hematopoietic stem cells?

5-hmC

5-mC 5-mU

TET AID

CpG CpGCpG

transcriptional repression

CpG CpG

TET2

CXXC??A

BERDNMTR

CXXC??

5-mC

5-hmC

H3K4me3

H3K4me1 R

A Activator

Repressor

Cytosine

transcriptional activationCpG CpG

TET2CXXC??

BERDNMTs

Consequences on HSC biology?

Lisbon_MDS/MPN_2012Pronier & Delhommeau Curr Hematol Malig Rep. 2012 Mar;7(1):57-64.

15/05/2012


Early progenitors (CD34+/CD38-)


Early progenitors (CD34+/CD38-)lymphoid → myeloid

Lisbon_MDS/MPN_2012


Impairs terminal erythroid differentiation in vitro


shRNA scramble shRNA TET2


Lisbon_MDS/MPN_2012

15/05/2012



Impairs terminal erythroid differentiation in vitro


shRNA scramble shRNA TET2


Disturbs in vitro granulo-monocytic differentiationshRNA scramble shRNA TET2

Lisbon_MDS/MPN_2012

37,4% 52,4%

CD

14

CD15 CD15Pronier et al, Blood 2011;118:2551-2555.

increase in

Tet2 knock-down in mouse hematopoietic cells

increase in monocytic

differentiation

Ko et al, Nature 2010;468:839 – 843.

Lisbon_MDS/MPN_2012

15/05/2012


increase in



differentiation

Ko et al, Nature 2010;468:839 – 843.

Lisbon_MDS/MPN_2012

Figueroa et al, Cancer Cell 2010;18:553-567.

increase in Lin-

Sca+C-kit+ %

increase in



differentiation

Ko et al, Nature 2010;468:839 – 843.

Lisbon_MDS/MPN_2012

Figueroa et al, Cancer Cell 2010;18:553-567. Moran-Crusio, Cancer Cell 2011;20:11-24.

increase in Lin-

Sca+C-kit+ %

increased replating

ability

15/05/2012


Tet2 gene disruption models in the mouse

Quivoron C et al.Tet2 inactivation results in pleiotropic hematopoietic abnormalities in mouse and is a recurrent event during human lymphomagenesis. Cancer Cell 2011;20:25-38.2011;20:25 38.

Moran-Crusio et al. Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation. Cancer Cell 2011;20:11-24.

Li et al. Deletion of Tet2 in mice leads to dysregulated hematopoietic stem cells and subsequent development of myeloid malignancies. Blood 2011; 118(17):4509-18.

Ko et al. Ten-eleven-translocation 2 (tet2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice. Proc Natl Acad Sci U S A 2011;108:14566-14571.

Shide et al.TET2 is essential for survival and hematopoietic stem cell homeostasis. Leukemia. 2012 Apr 3. doi: 10.1038/leu.2012.94.

Lisbon_MDS/MPN_2012


increasedincreased replating

ability

Moran-Crusio, Cancer Cell 2011;20:11-24.

increased LSK fraction

Lisbon_MDS/MPN_2012

15/05/2012



Competiti e

Lisbon_MDS/MPN_2012Moran-Crusio, Cancer Cell 2011;20:11-24.

Competitive repopulation assays


Lisbon_MDS/MPN_2012

Quivoron et al, Cancer Cell 2011;20:25-38.

Myeloid malignancies with features reminiscent of myelomonocytic leukemia

+ Aberrant lymphoid maturation and lymphoid malignancies

15/05/2012





3- Function of TET2



Lisbon_MDS/MPN_2012

- SRC NOD-SCID

TET2 mutations are present in hematopoietic stem cells in myeloid malignancies

- lymphoid/myeloid progenitors

- committed progenitors CD

19

CD

56

CD15 CD15

HSClympho-myeloid progenitor cell

lymphoid progenitor cell

MEP

BFU E

CFU-GMCD15 CD15

CD

19

CD

56

- mature myeloid cells

cellBFU-E

CFU-E

CFU-MK

CFU-MCFU-G

RBCs plateletsGranulo-

cytes

T cell

B cellNK

Erythro-blast Mega-

karyocyte

Mono-cytes

Lisbon_MDS/MPN_2012

Viguié et al, Leukemia. 2005 Aug;19(8):1411-5.Delhommeau et al, N Engl J Med 2009;360:2289-2301.

15/05/2012


Single cell level analysis

Are TET2 mutations early events in the development of myeloid malignancies?

Patients with TET2 and JAK2 mutations

38

CD

19

BFU-E BFU-EB/Myeloid/NK

clone

Single cell level analysis

- early progenitors (CD34+CD38-) in B/M/NK long term cultures

- committed progenitors (CD34+CD38+) in methylcellulose assay

CD

3

CD34

CD

56

CD15

JAK2

TET2

Pat

ien

t #0

5

LOH

*

* *LOH

- Genotyping of individual colonies

HSC

Common early TET2 mutations

MDSmutation

MPNmutation

Multi-step model

AML

Increased apoptosis

Early progenitors

Committed progenitors

effectsof common

earlymutations

Effects of specific

Increasedproliferation

Selective advantageclonality


mutation

Normal hematopoiesis

Abnormaldifferentiation

MDS

Mature cells

mutationsNormaldifferentiation

MPN AML

Blockeddifferentiation

Lisbon_MDS/MPN_2012

15/05/2012


-> TET2 mutations frequently precede JAK2 mutations

Lisbon_MDS/MPN_2012

But the opposite order of events also occurs….


Schaub et al, Blood 2010;115:2003-2007.

Lisbon_MDS/MPN_2012

15/05/2012


But the opposite order of events also occurs….


….. and complex multiclonal diseases may exist

Schaub et al, Blood 2010;115:2003-2007.

Lisbon_MDS/MPN_2012

TET2 mutation

HSCAML

MPN transformation

MPNmutation

Progression / Transformation

Early progenitors

Committed progenitors

effectsof common

earlymutations

Effects of specific


mutationSelective advantageclonality

MPN

Transformation

Normal hematopoiesis

Mature cells

mutations

AML

Blockeddifferentiation

Lisbon_MDS/MPN_2012

15/05/2012


PV h

Clonal evolution at the time of progression to AML

Saint-Martin et al, Blood 2009;114:1628-1632.

WT 17%

V617F 66%

TET2 550X 17%

JAK2 WT/VF TET2550X/WT

JAK2 WT TET2 WTJAK2 WT/VF TET2 WT

PV phase

JAK2V617F -> TET2550X

Lisbon_MDS/MPN_2012

PV h A t L k i

Clonal evolution at the time of progression to AML

Saint-Martin et al, Blood 2009;114:1628-1632.

WT 17%

V617F 66%

TET2 550X 17%

JAK2 V617F TET2 550X

9pUPD JAK2 VF/VF

16%

TET2 857 fs 77%

9pUPD JAK2 wt/wt

6%

JAK2 WT/VF TET2550X/857fs

JAK2 WT/VF TET2550X/WT

JAK2 WT TET2550X/857fs

JAK2 WT TET2 WTJAK2 WT/VF TET2 WT

PV phase Acute Leukemia

V617F TET2550X TET2857f

JAK2 VF/VF TET2550X/857fs

JAK2V617F -> TET2550X-> TET2857fs

Lisbon_MDS/MPN_2012

15/05/2012


Molecular analysis of post MPN-AML

Beer PA et al, Blood 2010;115:2891-2900.

Additionnal mutations in NRAS, RUNX1, TP53, CBL, TET2, FLT3

Lisbon_MDS/MPN_2012

Molecular analysis of post MPN-AML

Abdel-Wahab, Cancer Res 2010;70:447-452.

Lisbon_MDS/MPN_2012

15/05/2012





3- Function of TET2



Lisbon_MDS/MPN_2012

Diagnostic and prognostic relevance of TET2 mutations in myeloid malignancies

Many myeloid malignancies withTET2 mutationsMany other acquired genetic alterations in myeloid neoplasms→ role of TET2 mutations?

- MPN

Polycythemia vera Primary myelofibrosis

Lisbon_MDS/MPN_2012

Tefferi et al, Leukemia 2009;23:905-911.

15/05/2012



- MPN

Brecqueville et al, Genes

Primary myelofibrosis

q ,Chromosomes Cancer 2012 DOI 10.1002/gcc.

PV ET PMF Post-ET MF

Post-PV MF

MPN/MDSBlast phase

Lisbon_MDS/MPN_2012

PV ET PMF ET MF PV MF MDSBlast phase

ASXL1SUZ12

TET2DNMT3A

IDH2

JAK2MPLCBLNF1

SF3B1

Some patients with polycythemia vera are treated with pegylated interferon -2a (IFNa)

High rate of hematologic response

High rate of molecular response

20

40

60

80

10

0

% V

61

7F

20

40

60

80

10

0

% V

61

7F

P<.001

%V617F

%V617F

0 5 10 15 20

0

Months

0 6 12 18

0

Months

Kiladjian et al, Blood. 2006 Sep 15;108(6):2037-40.

Lisbon_MDS/MPN_2012

15/05/2012


c.4577delC, p.Gln1526Serfsc.1849G>T, p.Val617Phe

TET2 exon 11JAK2 exon 14

Some patients have both JAK2 and TET2 mutationsKiladjian et al. Leukemia 2010;24:1519-23.

Prior IFNa therapy

Month 24

Month 36

Wild type JAK2Wild type TET2

Good JAK2molecularresponse

Poor TET2molecularresponse

Lisbon_MDS/MPN_2012


Many myeloid malignancies withTET2 mutationsMany other acquired genetic alterations in myeloid neoplasms→ role of TET2 mutations?

- MDS

Mutated TET2

Non mutated TET2 MutatedTET2 n=55

Non mutatedTET2

p=0.37

320 MDS + 35 CMML patients

Lisbon_MDS/MPN_2012

Kosmider et al, Blood. 2009;114:3285-3291

TET2 n=55

Overall survival (Months)

100 2000

Smith et al, Blood. 2010;116(19):3923-3932

15/05/2012



- CMML

Kosmider et al,n=18

n=25

n=21

CMML 1 and 2 CMML 1

rviv

al

Kosmider et al, haematologica. 2009; 94(12):1676-1681

n=10O

vera

llsu

CMML (treated with decitabine)

Lisbon_MDS/MPN_2012

Itzykson et al, Blood. 2011;118(14):3824-3831

Diagnostic and prognostic relevance of TET2 mutations

- AMLTET2 mutations : adverse prognostic factorMetzeler et al J Clin Oncol 2011 29:1373-1381Chou et al, Blood. 2011;118(14): 3803-3810Weissmann et al, Leukemia doi:10.1038/leu.2011.326

Lisbon_MDS/MPN_2012

15/05/2012



- AML

Lisbon_MDS/MPN_2012

Patel et al, N Engl J Med 2012;366:1079-89.


- AML

Lisbon_MDS/MPN_2012

Patel et al, N Engl J Med 2012;366:1079-89.

15/05/2012


Conclusion

Mutations in TET2 → low 5-hmC content in primary cells from patients with myeloid malignancies

Function of TET2 not fully elucidated : differences between the types of mutations ? Link with other enzymes / mutations (IDH1/2)? Consequences on DNA methylation?

5-mC

5-hmC

DNA methylation?

Dang et al, Nature 2009;462:739-744

Figueroa et al, Cancer Cell 2010;18:553-567.

Ko et al, Nature 2010;468:839 – 843.

TET2 mutations in other cancers: lymphoid malignancies: role in hematopoietic stem cell biology?

Prognostic value to be integrated in a comprehensive genetic analysis of myeloid malignancies

Lisbon_MDS/MPN_2012

REMERCIEMENTS

Aline Massé

Isabelle Plo

Barbara da Costa Reis Monte Mor Elodie Pronier Hayat Mokrani

Inserm U1009

Raphaël Itzykson

Eric SolaryWilliam Vainchenker

Hôpital Saint-Antoine Hôpital Saint-Louis Pitié Salpétrière

Support, and grants

Hôpital Saint-AntoineNicole Casadevall, Carole Almire, Tounes Ledra, Dorota Jiezorowska,Christophe marzac, Irène Teyssandier

Jean SoulierJ.Jacques KiladjianBruno CassinatInserm UMR S 1008Frédéric PendinoInserm U985Olivier Bernard

Sabrina Dupont

Pitié SalpétrièreCécile Saint MartinChristine Belanné-ChantelotUniversity of ChicagoLucy A; Godley

Documents

TET2 in MPN and MDS: What is the role? - Imedex · TET2 in MPN and MDS: What is the role? ... Polycythemia Vera 13.8% 17% 10% 13% 7% 14/89 NA (357 MPN) ... The role of TET2 in MPN