Myeloproliferative neoplasms: recent advances in pathogenesis

Transcription

1 Myeloproliferative neoplasms: recent advances in pathogenesis Andy Chase University of Southampton and Wessex Regional Genetics Laboratory Salisbury, UK Uppsala 2010

2 Myeloproliferative neoplasms MDS/MN Myelodysplastic syndrome CML V ET MF CNL CEL Mastocytosis MN U CMML acml (BCR ABL neg) JMML MDS/MN U RA RARS RCMD RCMD RS RAEB MDS del(5q) MDS U

3 Myeloproliferative neoplasms MDS/MN Myelodysplastic syndrome CML V ET MF CNL CEL Mastocytosis MN U CMML acml (BCR ABL neg) JMML MDS/MN U RA RARS RCMD RCMD RS RAEB MDS del(5q) MDS U

4 athogenesis of MN & MDS/MN Tyrosine kinases and associated pathways Transcription factors SN array analysis and acquired UD Epigenetics

5 Cytogenetics of MN & MDS/MN ~ 99% normal karyotype or non specific changes eg +8 ~ 1% translocations creating tyrosine kinase fusion genes FGFR1 JAK2 DGFRA DGFRB 8p11 9p24 4q12 5q33

6 Tyrosine Imatinib kinase responsive fusions tyrosine MN kinase & MDS/MN fusions FI1L1 4q12 KIF5B 10p11 CDK5RA2 9q33 STRN 2p24 FLT3 13q12 LYN 8q12 SYK 9q22 DGFRA 4q12 N=6 STBN1 2p16 TM3 1q21 DE4DI 1q22 WDR48 3p22 GOLGA4 3p22 RKG2 4q21 HI1 7q11 KANK1 9p24 CCDC6 10q21 GIA1 11p13 ERC1 12p13 BIN2 12q13 SART3 12q23 ETV6 12p13 DGFRB 5q33 N=24 DTD1 20p11 GIT2 12q24 RABE1 17p13 ABL 9q34 JAK2 9p34 MYO18A 17q11 SECC1 17p11 NDE1 16p13 T53B1 15q22 NIN 14q24 TRI11 14q32 KIAA q32 CM1 8p21 BCR 22q11 FGFR1 8p11 LOC q13 N=10 ZNF198 13q12 LRRFI1 2q37 CE110 9q33 TRIM24 7q34 FGFR1O 6q27 FGFR1O2 12p11 CFS1 12q15 August 2010: 49 tyrosine kinase fusion genes

7 WHO classification of DGFRA, DGFB and FGFR1 fusions Myeloid and lymphoid neoplasms with eosinophilia and abnormalities of DGFRA, DGFRB or FGFR1 MN or MDS/MN with eosinophilia DGFRA and FGFR1 fusions can present as T lineage lymphoblastic lymphoma with eosinophilia all three can present as AML FI1L1 DGFRA is cryptic all other identified translocations are cytogenetically visible DGFRA and DGFRB fusions are imatinib sensitive

8 Function of fusion partner gene Drives expression rovides oligomerisation domain? Altered localisation Reduced degradation Increased STAT signalling

9 Altered localisation of fusion receptors cell surface Ub Ub CBL Internalisation: degradation/recycling Ub Ub CBL cell surface CBL X cytosol STAT5 signalling Toffalini 2009, Haematologica 94:

10 Tyrosine Imatinib responsive tyrosine kinase fusions kinase fusions in atypical MNs STBN1 2p16 TM3 1q21 DE4DI 1q22 WDR48 3p22 GOLGA4 3p22 RKG2 4q21 HI1 7q11 CCDC6 10q21 GIA1 11p13 ERC1 12p13 BIN2 12q13 FLT3 13q12 SART3 12q23 LYN 8q12 ETV6 12p13 DGFRB 5q33 N=24 FI1L1 4q12 SYK 9q22 DTD1 20p11 GIT2 12q24 KIF5B 10p11 RABE1 17p13 DGFRA 4q12 ABL 9q34 JAK2 9p34 MYO18A 17q11 SECC1 17p11 NDE1 16p13 T53B1 15q22 NIN 12q24 TRI11 14q32 CCDC88C 14q32 CDK5RA2 9q33 N=6 CM1 8p21 STRN 2p24 Endocytic, ER, golgi Centrosomal BCR 22q11 FGFR1 8p11 LOC q13 N=10 ZNF198 13q12 LRRFI1 2q37 CE110 9q33 TRIM24 7q34 FGFR1O 6q27 FGFR1O2 12p11 CFS1 12q15 August 2010: 49 tyrosine kinase fusion genes

11 Tyrosine kinase and associated signalling in MN & MDS/MN TK fusion genes FLT3 ITD, KD ML W515 KIT D816V LNK JAK2 JAK2 RAS GD RAS GT JAK2 V617F, exon12 LNK (SH2B2) NRAS, KRAS NF1 TN11 cytosol NF1 TN11 CBL CBL

12 Tyrosine kinase and associated signalling in MN & MDS/MN TK fusion genes FLT3 ITD, KD ML W515 KIT D816V LNK JAK2 JAK2 RAS GD RAS GT JAK2 V617F, exon12 LNK (SH2B2) NRAS, KRAS NF1 TN11 cytosol NF1 TN11 CBL CBL

13 Tyrosine kinase and associated signalling in MN & MDS/MN TK fusion genes FLT3 ITD, KD ML W515 KIT D816V LNK JAK2 JAK2 RAS GD RAS GT JAK2 V617F, exon12 LNK (SH2B2) NRAS, KRAS NF1 TN11 cytosol NF1 TN11 CBL CBL

14 Tyrosine kinase and associated signalling in MN & MDS/MN TK fusion genes FLT3 ITD, KD ML W515 KIT D816V LNK JAK2 JAK2 RAS GD RAS GT JAK2 V617F, exon12 LNK (SH2B2) NRAS, KRAS NF1 TN11 cytosol NF1 TN11 CBL CBL

15 Tyrosine kinase and associated signalling in MN & MDS/MN TK fusion genes FLT3 ITD, KD ML W515 KIT D816V LNK JAK2 JAK2 RAS GD RAS GT JAK2 V617F, exon12 LNK (SH2B2) NRAS, KRAS NF1 TN11 cytosol NF1 TN11 CBL CBL

16 Cryptic fusion genes are often associated with genomic copy number changes 1. Small deletions eg. FI1L1-DGFRA in CEL 2. Episomal amplification eg. NU214-ABL in T-ALL Cools et al Cryptic translocations eg. EML1-ABL in T-ALL BCR/ABL Keersmaecker et al., 2005 Graux et al. 2004

17 Cryptic tyrosine kinase fusions CGH arrays designed to target all tyrosine kinases and 400 additional cancer/haemopoiesis related genes Result: No evidence of cryptic tyrosine kinase fusions deletions of RUNX1 and CEBA RUNX1 CEBA

18 RUNX1, CEBA, WT1, NM1 in MDS/MN RUNX1, CEBA, WT1, NM1 sequenced in MDS/MN RUNX1 CEBA NM1 WT1 acml (n=59) % CMML (n=87) % MDS/MN U (n=11) %

19 RUNX1, CEBA, WT1, NM1 in MDS/MN RUNX1, CEBA, WT1, NM1 sequenced in MDS/MN RUNX1 CEBA NM1 WT1 acml (n=59) % CMML (n=87) % MDS/MN U (n=11) % Ernst, 2010, Haematologica, 95(9), Gelsi Boyer, 2008, BMC Cancer, 8:299 Kuo, 2009, Leukemia, 23: 1426 Kohlmann, 2010, J Clin Oncol, 28:3858

20 Acquired uniparental disomy (aud) FLT3 ITD: ch 13 isodisomy Griffiths et al, Leukemia 2005 V617F Mitotic recombination V617F V617F CEBA: ch 19 isodisomy Fitzgibbon et al, Cancer Res, 2005 WT1: ch 11 isodisomy Fitzgibbon et al, Cancer Res, 2005 RUNX1: ch 21 isodisomy Fitzgibbon et al, Cancer Res, 2005 NF1: ch 17 isodisomy Stephens, et al Blood, 2006 Flotho et al., Oncogene, 2007

21 Mechanism of progression to homozygosity: acquired uniparental disomy (aud)) arising from mitotic recombination * * Courtesy of Manoj Raghavan

22 SN 6.0 data analyzed by AsCNAR software Normal Amplification Deletion aud Yamamoto et al., Am J Hum Genet 2007

23 aud>20 Mb (n=148 MDS/MN patients)

24 aud>20 Mb (n=148 patients) ML TET2 CBL FLT3

25 7q aud analysis 12/148 (8%) MDS/MN with 7q aud Sequenced 15 candidate genes - no mutations 52 Mb, > 400 genes

26 Chr. 7 7q36.1 C7orf33 CUL1 EZH2 7q UD SN 6.0 acgh

27 7q aud is associated with EZH2 mutations remature STO codon Missense mutation Nonsense mutation C>T R207X C>G C576W del A K685fsX Ernst et al, 2010, Nature Genetics, 42:722. Nikoloski et al, 2010, Nature Genetics, 42: 665

28 EZH2 mutations are acquired atient #03 atient #30 T-cells Granulocytes C>T (Q328X) del G (A656fsX18)

29 EZH2 function EZH2 is a polycomb gene that modifies chromatin tri methylates lysine 27 of histone H3 (H3K27me3) associated with transcriptional repression EZH2, SUZ12 and EED are the core components of the polycomb repressive complex RC2 RC2 EED SUZ12 EZH2 SET Histone H3K27 methylation is opposed by H3K4 methylation catalysed by MLL

30 EZH2 and chromatin modification RC2 EED SUZ12 EZH2 SET Transcription

31 EZH2 and chromatin modification RC2 EED SUZ12 EZH2 SET RC2 Transcription

32 EZH2 and chromatin modification RC2 EED SUZ12 EZH2 SET RC2 H3K27me3 Transcription

33 EZH2 and chromatin modification RC2 EED SUZ12 EZH2 SET RC2 H3K27me3 RC1 Transcription

34 EZH2 and chromatin modification RC2 EED SUZ12 EZH2 SET RC2 H3K27me3 RC1 H2AK119ub Transcription

35 EZH2 and chromatin modification RC2 EED SUZ12 EZH2 SET RC2 H3K27me3 RC1 H2AK119ub DNMT Transcription

36 EZH2 and chromatin modification RC2 EED SUZ12 EZH2 SET RC2 H3K27me3 RC1 H2AK119ub DNMT Transcription

37 EZH2 and chromatin modification RC2 EED SUZ12 EZH2 SET DNMT Transcription

38 H3K27 and H3K4 methylation marks developmental regulators: bivalent model Stem cell H3K27me3 H3K4me3 Activation of tissue specific and lineage specific genes Repression of self renewal and non lineage specific genes Mature cells

39 H3K27 and H3K4 methylation marks developmental regulators: bivalent model Stem cell H3K27me3 H3K4me3 X Activation of tissue specific and lineage specific genes Repression of self renewal and non lineage specific genes Mature cells

40 EZH2 and cancer EZH2 can act as an oncogene by overexpression Upregulated in advanced aggressive cancers e.g. prostate cancer, breast cancer and AML EZH2 can transform normal cells in vitro and in vivo EZH2 inhibition can inhibit the growth of AML and breast cancer cells A single recurrent EZH2 mutation (Tyr 641) identified in lymphomas of germinal-centre origin (Morin et al., Nat Genet 2010)

41 EZH2 mutation screening Disease WHO subgroup Cases analysed No. of mutations MDS MDS/MN CMML Atypical CML MDS/MN-U Classic MN V ET 30 0 MF Atypical MN CEL/HES SM 30 0 AML -7/7q CML-BC 40 0 %

42 Location of mutations within EZH2 Nonsense and premature stop codon mutations Missense mutations E D I D II CXC SET 18% 77% 62% 75% 88% EED Methyltransferase activity

43 EZH2 mutations abrogate histone methyltransferase activity Co expression of RC2 components in Sf9 insect cells Immunoprecipitation and histone methyl transferase assay Confirmation of RC2 components in coomassiestained gel Ezh2 Suz12 Eed WT Y731D WT?SET C576W Y646C R690C Histone methylation reaction H3K27me3 (3H) Coomassie stained histones

44 EZH2 mutation and homozygosity are associated with a poor prognosis unmutated MDS/MN (n=115) mutated MDS/MN (n=19) mutation negative (n=182) heterozygous mutation (n=22) homozygous mutation (n=10) % survival = % survival =0.089 (het vs hom) months after diagnosis months after diagnosis

45 EZH2 mutation is an early event Y733X het Y733X het Y733X het atient #21 Nov 2004 MF Feb 2006 MF June 2007 AML R690H het R690H hom R690H hom atient #27 June 2006 ET Aug 2007 MF Oct 2008 AML

46 olycomb Group Genes and Cancer / +/ +/+ +++ Stem cell loss Tumour Normal Stem cells Tumour Sauvageau, 2008, LoS Biology

47 TET2 Inactivating mutations 6 kb coding region (25 fragments) Missense variants of unknown pathogenicity TET2 variants identified: MN 10% MDS 25% MDS/MN 40% AML 20% (? mainly secondary AML) TET2 often but not always precedes V617F

48 Chromosome 20 ASXL1 Mutations in exon 12 resulting in truncation and loss of C terminal HD finger Enhances RAR by cooperation with SRC 1 (Cho et al, 2006, J Biol Chem) Represses RAR by coooperating with H1 to modulate LSD1 activity (Lee et al, 2010, J Biol Chem) Gelsi Boyer, 2009, BJH, 145:788 Classic MN 8% CMML 43% MDS 20% 1 AML 10% (include 1934dupG) With BA1 forms H2A deubiquitinating complex R DUB (Scheuermann et al, 2010, Nature) ASXL1: c.1934dupg;p.gly646trpfsx12 Abdel Wahab et al, 2010, Leukemia

49 Epigenetic modifiers in MDS/MN EZH2 methylation of histone H3K27 TET2 demethylation of methylated cytosine ASXL1 epigenetic repression and activation (1934dupG excluded) CMML (N=99) EZH2 ASXL acml (N=69) EZH ASXL TET2 34 TET2 No evidence of association or mutual exclusion

50 Summary EZH2 Mutations associated with 7q aud (but not with 7/7q in AML) Most commonly MDS/MN, myelofibrosis and MDS Both an increase and decrease in H3K27 methylation may lead to malignancy

51 Summary Molecular pathogenesis of MN & MDS/MN Epigenetic regulation ASXL1 unknown TET2 WT1 EZH2 Transcription factors CEBA RUNX1 CBL TK fusions TN11 NRAS, KRAS JAK2 V617F NF1 Tyrosine kinase signalling LNK KIT D816V

52 Acknowledgements Wessex Regional Genetics Laboratory (Salisbury, UK) Collaborators Katerina Zoi (Athens, Greece) Andreas Hochhaus (Jena, Germany) Andreas Reiter (Mannheim, Germany) Hans Drexler (Braunschweig, Germany) Andrew Duncombe (Southampton, UK) Francisco Cervantes (Barcelona, Spain) David Oscier (Bournemouth, UK) Jacqueline Boultwood (Oxford, UK)