CBL and EZH2 as new molecular markers in MPN

CBL and EZH2 as new molecular markers in MPN Andy Chase University of Southampton and Wessex Regional Genetics Laboratory Salisbury, UK Munich 2011

* Myeloproliferative neoplasms MDS/MPN Myelodysplastic syndrome CML PV ET PMF CNL CEL Mastocytosis MPN-U CMML acml (BCR-ABL neg) JMML MDS/MPN-U RA RARS RCMD RCMD-RS RAEB MDS del(5q) MDS-U

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

SNP 6.0 data analyzed by AsCNAR software Normal Amplification Deletion aupd Yamamoto et al., Am J Hum Genet 2007

aupd>20 Mb (n=148 MDS/MPN patients)

11q aupd is associated with CBL mutations Dunbar et. al. 2008, Cancer Res. 68:10349-10357 Grand FH et. al. 2009, Blood, 113:6182-6192 Sanada et. al. 2009, Nature, 460:904-908

* CBL is an E3 ubiquitin ligase ubiquitin conjugating enzyme E2 ub ubiquitin ligase CBL E3 ub ub ub ub P P KIT FLT3 etc. ub CBL E3 P P E1 ubiquitin activating enzyme multiple signals PI3K MAPK SRC proteasomal degradation

CBL tyrosine kinase binding (TKB) RING proline rich Y Y Y UBA N- -C GRB2, SRC, CRKL, PI3K. binds substrate binds E2 CBL is a multifunctional 120 kd protein: E3 ubiquitin ligase that targets substrates for proteasomal degradation acts as a scaffold for multiple signalling molecules eg PI3K, GRB2, SRC, CRKL Two mammalian homologs: CBLB and CBLC All have TKB, linker and RING domains CBLB, but not CBLC, has C-terminal signalling molecule binding domains A viral homolog, v-cbl, causes Casitas B-lineage lymphoma in mice v-cbl only retains the TKB domain

* CBL disease incidence CMML 5-22% (overall 13%) JMML 7-19% (overall 14%) acml 8% RARS-T 0/19 (0%) PMF 6% no mutations in PV or ET AML <1% higher in CBF subtypes MDS 2.5% higher in RAEB2, secondary MDS (CBLB and CBLC mutations reported with low incidence)

CBL and prognosis Kohlmann, 2010, J Clin Oncol: No association with survival in CMML Survival in CMML Survival in CMML 19 months 14 months P=0.05 CMML, acml, MF (mutated = 19, non-mutated = 87) Jankowska et al. Blood 2010 Grand F H et al. Blood 2009;113:6182-6192

* CBL: position of mutations binds substrate binds E2 GRB2, SRC, PI3K, CRKL WRGL Gelsi Boyer 2010 Kohlmann 2010 Exon 8 Exon 9 PTPQDHIKVTQEQYELYCEMGSTFQLCKICAENDKDVKIEPCGHLMCTSCLTSWQESEGQGCPFCRCEIKGTEPIV X X X X XX XX X X XX X X XXXXX X X X X XX X X X X X X X X X X X 4 x deletions / splice site mutations Adapted from Dunbar et al 2009

* CBL is an E3 ubiquitin ligase ubiquitin conjugating enzyme E2 ub ubiquitin ligase CBL E3 ub ub ub ub P P KIT FLT3 etc. ub CBL E3 P P E1 ubiquitin activating enzyme multiple signals PI3K MAPK SRC proteasomal degradation

* CBL is an E3 ubiquitin ligase ubiquitin conjugating enzyme ub E2 ub ubiquitin ligase CBL E3 X ub ub ub ub CBL E3 P P P P KIT FLT3 etc. E1 ubiquitin activating enzyme multiple signals PI3K MAPK SRC X proteasomal degradation

Transforming ability is associated with loss of E3 ubiquitin ligase activity Wt-CBL N454D S376F H398Y P417A R420Q Proliferation of 32D-FLT3 + CBL WP: FLT3 IP: FLT3 WB: HA-Ub WB: CBL

* Gain-of-function CBL mutants Sanada et al, 2009 (Nature) Mutant CBL NIH-3T3 Transformation Mutant CBL CBL -/- mouse background Transformation

* Juvenile Myelomonocytic Leukemia (JMML) Rare, aggressive, MDS/MPN of childhood Overall incidence of CBL mutation: 14% All CBL mutations germline 2/3 with developmental abnormalities: dev. delay, cardiomyopathy, cryptorchidism, growth delay High rate of spontaneous remission but with vasculopathies in later life Exon 8 Exon 9 PTPQDHIKVTQEQYELYCEMGSTFQLCKICAENDKDVKIEPCGHLMCTSCLTSWQESEGQGCPFCRCEIKGTEPIV X X X X XX XX X X XX X X XXXXX X X X X XX X X X X X X X X X X X CMML/aCML JMML Exon 8 Exon 9 Perez 2010 Loh 2009 Exon 8 4 x deletions / splice site mutations PTPQDHIKVTQEQYELYCEMGSTFQLCKICAENDKDVKIEPCGHLMCTSCLTSWQESEGQGCPFCRCEIKGTEPIV X XX X X X X X XXX XXX XXX XXXXXX Exon 9 5 x deletions / splice site mutations

* RASopathies Chan et al, 2009b, Loh, 2011

CBL - summary CBL mutations are associated with 11q UPD and are nearly always homozygous Highest incidence in MDS/MPN and PMF Most mutations in linker and RING domain (exons 8 and 9) and result in loss of ubiquination activity CBL mutations have both tumour suppressive and gain-offunction properties CBL mutations in JMML are constitutional with a high rate of spontaneous remission and can be associated with developmental abnormalities

aupd>20 Mb (n=148 MDS/MPN patients)

7q36.1 Chr. 7 12 aupd samples 52 Mb, >400 genes Tyrosine kinases excluded Sequenced 15 candidate genes C7orf33 CUL1 EZH2 7q UPD SNP 6.0 acgh

7q aupd is associated with EZH2 mutations Premature STOP 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

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 gain-of-function EZH2 mutation (Tyr 641) identified in lymphomas of germinal-centre origin (Morin et al., Nat Genet 2010)

EZH2 function EZH2 is a polycomb gene that modifies chromatin - methylates lysine 27 of histone H3 (H3K27me) - associated with transcriptional repression EZH2, SUZ12 and EED are the core components of the polycomb repressive complex PRC2 PRC2 EED SUZ12 EZH2 RBBP4/7 SIRT1 JARID2 PHF1 Histone H3K27 methylation is opposed by H3K4 methylation catalysed by MLL PRC2 targets: developmental, cell cycle and differentiation

* 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

* EZH2 disease incidence CMML 6-13% (overall 10%) acml 13% MDS/MPN-U 10% PMF 4-13% (overall 6%) PV/ET - MF 0-5 (overall 4%) PV 1/30 (3%) ET 1/30 (3%) CML-BC 0/40 AML <1% -7/7q- 0/54 MDS 1.5-23% (overall 8%)

Location of mutations within EZH2 Nonsense and premature stop codon mutations Missense mutations N- -C E D I D II CXC SET 18% 77% 62% 75% 88% 1 100 200 300 400 500 600 700 751 EED SUZ12 Methyltransferase activity

% survival % survival EZH2 mutation is associated with a poor prognosis unmutated MDS/MPN (n=115) mutated MDS/MPN (n=19) mutation negative (n=182) heterozygous mutation (n=22) homozygous mutation (n=10) P=0.0006 P=0.089 (het vs hom) months after diagnosis months after diagnosis Survival in CMML Alive at 3 years: 33.3 vs 69.9% Alive at 3 years: 33.9 vs 49.7 vs 89.8% Grossmann, 2011, Leukemia, 25:877

* EZH2 mutation is associated with a poor prognosis P<.001 EZH2 mut EZH2 WT P=.032 EZH2 mut EZH2 WT Guglielmelli et al. EHA 2011 EZH2 mutations in 22/370 (6%) PMF Median survival 31.6 months vs 137 mos Survival in MDS Bejar, 2011, NEJM HR for death = 2.13

* Mutation of other H3K27me proteins in MDS/MPN 1-2% 8% of CMML 1% EED SUZ12 UTX 12% EZH2 me me K27 me EED: 1/87 MDS/MPN (not with 11q UPD) SUZ12: 4 cases with mutation (1-2%) 1 of 2 cases with 17q aupd and 2 of 2 cases with focal 17q11.2 deletions (one with an NF1 mutation) Jankowska, Blood, 2011: UTX mutations in (4/52) 8% of CMML

EZH2 - a paradox? Loss-of-function mutations in MDS/MPN But overexpression in AML and solid tumours Inactivating mutations of both EZH2 and UTX in CMML

Polycomb Group Genes and Cancer +/- +/+ +++ -/- Polycomb Stem cell loss Tumour Normal Stem cells Tumour Sauvageau, 2008, PLoS Biology

Summary - Molecular pathogenesis of MPN & MDS/MPN Epigenetic regulation TET2 IDH1/2 ASXL1 EZH2 SUZ12 EED DNMT3 UTX Notch pathway NOTCH NCSTRN Splicing machinery SRSF2 U2AF35 PRPF40B ZRSR2 SF3B1 SF1 Unknown? Tyrosine kinase signalling TK fusions CBL RAS JAK2 KIT NF1 SH2B3 PTPN11 Transcription factors RUNX1 WT1 CEBPA

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)