Chronic myelomonocytic leukemia. 13 eme Colloque Annuel du Cancéropôle Ile-de-France Februray 9t h, 2018

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1 Chronic myelomonocytic leukemia 13 eme Colloque Annuel du Cancéropôle Ile-de-France Februray 9t h, 2018

2 Chronic Myelomonocytic Leukemia (CMML) Typically, a 72-y man has a routine blood test that shows a monocytosis Monocytes > 1,000 / mm 3 & > 10% WBC If monocytosis persists for at least 3 months w/o other explanation, CMML according to the WHO The risk to die in the two following years is 50%

3 Relative survival (%) Simple definition Blood monocytes > 1000/µL CMML Severe prognostic (median survival, months) Months Srour SA et al, Brit J Haematol, 2016

4 WGS: three genomic signatures, of which 2 indicate an ageing-related process

5 TET2 ASXL1 DNMT3A EZH2 IDH2 SRSF2 U2AF1 SF3B1 ZRSR2 LUC7L2 CBL KRAS NRAs NF1 JAK2 RUNX1 PHF6 BCOR ETNK1 Paatients (%) A 60 characteristic, relatively % 75% 60% simple, 30 genomic 20 signature /pt 3 recurrent Epigenetic Splicing Signaling Others Merlevede et al, Nat Commun 2016

6 CMML clonal architecture

7 Subclonal mutations generate specific diseased cells / locations Systemic mastocytosis with associated CMML (SM-AHNMD) CD14+ cells TET2 S99fs TET2 E1874K SF3B1 K700E CARD4 S60N GPR137B T200M TMEM233 P32S PXYLP1 M78V PTCH1 A1118P SEMA4D R218X CFAP53 E413X Skin biopsy TET2 S99fs TET2 E1874K SF3B1 K700E CARD4 S60N GPR137B T200M TMEM233 P32S PXYLP1 M78V PTCH1 A1118P SEMA4D R218X CFAP53 E413X + KIT D816V MYH3 D862V

8 Genetic alterations in CMML A disease of ageing With a non specific but characteristic molecular signature (TET2, SRSF2, ASXL1, Ras path) Genetic alterations may account for 25% of the disease heterogeneity (RAS & proliferation, RUNX1 & thrombocytopenia, KIT & mastocytosis ) Itzykson R et al, Blood 2013 & J Clin Ooncol 2013 Merlevede J et al, Nature Commun 2016 & unpublished data

9 Current therapeutic approaches in CMML Active monitoring ESA Cytoreductive Hypomethylating Transplant Solary E, Itzykson R, Blood 2017

10 Serial whole exome sequencing - 3 HMA-treated patients, Responders

11 Variant frequency (%) A few months before treatment 20 0

12 Variant frequency (%) After the first DAC cycle 20 0

13 Variant frequency (%) After 10 DAC cycles clinical response 20 0

14 Variant frequency (%) after 23 cycles AML transformation 0

15 Eric Padron Serial analysis of an exceptional responder to AZA

16 Changes in gene expression (RNA Seq) are observed mostly in responders Change > 2 Up Down Total Untreated Treated, non-responders Treated, responders

17 Changes in DNA methylation (errbs) are observed only in responders

18 CMML response to hypomethylating agents? Dissociation between Improved phenotype DNA demethylation, Changes in gene expression & Persistence / accumulation of mutations Merlevede J, Droin N et al, Nature Commun 2016

19 At diagnosis Hematopoiesis imbalance mttet2, mtasxl1, mtsrsf2 TIF1g promoter, mir150 enhancer méthylation mtras

20 Responders to HMA Balanced hematopoiesis mttet2, mtasxl1, mtsrsf2 mtras

21 mir150 expression / rnu6b fold change CD16 MO1 (% of total monocytes) The response to demethylating agents includes An increase in mir-150 expression level A decrease in classical MO1 monocyte fraction below 94% Before AZA After AZA AZA Threshold 94% patient Responders Non-Responders CD Responders (N=7) Selimoglu-Buet D et al, submitted

22 TET2 S1691fs TET2 R1516X KDM6a R61X KRAS G12D CD34-positive cell reprogrammation

23 Clones selected for further analyses Age-matched control Co1 Co4 Co3 Co5 Age-matched control Co6 CMML patient A A1 A2 A3 A4 A5

24 Summary of semi-solid medium cultures

25 The size of MK colonies by CMML clones is increased

26 MK dysplasia D14 D24

27 Cells generated in liquid medium D0 D14 D20

28 Functinal heterogeneity over genetic heterogeneity

29 CRISPR/Cas9 introduction of SRSF2 P95H mutant allele

30 ipscs in vitro differentiation phenotype A1 CD14+ Common genetic background A2 CD33+ CD41+ KDM6A R61X TET2 S1691FS TET2 R1516X A4 CD123+ CD235a+ Distinct phenotype Variable epigenome Summary CD34 + cells KDM6A R61X TET2 S1691FS TET2 R1516X PATIENT CLONAL EVOLUTION KDM6A R61X TET2 S1691FS TET2 R1516X KRAS G12D A5 A3 CD33+ in vitro differentiation phenotype CD123+ CD14+ CD235a+ CD41+ Common genetic Background KDM6A R61X TET2 S1691FS TET2 R1516X KRAS G12D Distinct phenotype Variable epigenome CD14+ CRISPR/Cas9 SRSF2 P95H CD33+ CD41+ A3 P95H CD123+ CD235a+

31 Multi-level heterogeneity of chronic myelomonocytic leukemia cells illustrated by patient-derived induced pluripotent stem cells

32 A role for mature cells of the clone? IL-6 Mature cells produce IL-6 that affects leukemic progenitor cell fate Cancer Cell, 2011 Targeting IL-6 prevents CML development in mice Cancer Cell 2015 BCR-ABL LSC BCR-ABL Progenitor

33 wt/wt wt/exc exc/exc wt/wt wt/exc exc/exc MIF mrna expression (relative to HPRT) Plasmatic MIF (ng/ml) Plasmatic MIF (ng/ml) TET2 down-regulation increases the production of MIF Cell lines tet2 +/- or tet2 -/- mice Kasumi MO7E UT7 TF1-a * * * * N=3 SCR shrna TET2 shrna K427 *** **** ANO model **** **** KDa 12.5 Kasumi MO7E UT7 TF1-a SCR sh SCR sh SCR sh SCR sh MIF Actin

34 TET2 down-regulation increases MIF gene expression HDAC1 HDAC2 Healthy donor classical monocytes TET2 - MIF gene A A A A CMML TET2 mut classical monocytes RNA + HAT Pol II TFIID MIF gene A A Droin N et al, submitted

35 TET2 mutated monocytes overproduced MIF, which promotes monocyte production MIF CMML HSPC Progenitor

36 CD123 + plasmocytoid cell islands HES CD123 Described initially in the 80 s In diverse myeloid malignancies More frequent in CMML Plasmocytoid shape Strong expression of CD123 Mrinal Patnaik, Mayo-clinic

37 25% CMML accumulate pdcs in the bone marrow

38 pdcs are part of the leukemic clone

39 pdc amplification is associated with RAS path mutations

40 RAS path mutations in CMML Hypersensitivity of Myeloid progenitors to GM-CSF pdc progenitors to FLT-3L

41 AML transformation (%) pdcs excess correlates with Treg and an increased risk of leukemic transformation P = 0.01 pdc rich pdc poor Months)

42 mttet2 early clonal dominace TIF1g promoter, mir150 enhancer méthylation Hyper signal (RAS) MDSC Defective apoptosis, MIF pdcs

43 General conclusion CMML genetic abnormalities well-identified: Stepwise linear regression models showed that mutations account for 25% of variability of clinical phenotype The respective parts of epigenetics, mature cells of the clone, micro/macro environment still poorly understood Genetics Epigenetics Mature cells Environment Therapeutic strategies to explore: signaling & apoptosis, immune cell modulation, cytokine inhibition Deinninger M, Tyner JW, Solary E, Nature Rev Cancer 2017

44 INSERM U1170 Villejuif Sequencing, epigenetics Nathalie Droin Monocyte subsets, mir-150 Dorothée Selimoglu-Buet IPS clones Allan Beke, Lucie Laplane Plasmocytoid dendritic cells Nolwenn Lucas, Matthieu Duchmann Monocyte apoptosis Franck Debeurme Clonal analyses Raphael Itzykson, Jane Merlevede Margot Morabito, Jeffie Lafosse Philippe Rameau

45 Collaborations P. Fenaux R Itzykson T. Braun L Adès S de Botton C Willekens B Quesnel C Berthon E Jourdan & others & patients O Vagner-Ballon, Créteil F Delhommeau, Saint Antoine L Delva, Dijon M. Stratton, Cambridge M Patnaik, Rochester V Santini, Firenze M Figueroa, Miami E Padron, Tampa S Ogawa, Kyoto M Deininger, Salt Lake City Jeff Tyner, Portland