Juvenile and ChronicMyelo-MonocyticLeukemia

red blood cell

Haematopoietic stem cell

Lympho-myeloid progenitor cell

lymphoid progenitor cellMEP

BFU-E

CFU-E

CFU-MK

CFU-GM

CFU-M CFU-G

platelet granulocyte T cell B cell NK

erythro

MGK

CFU-BCFU-T

monocyte

MDS/MPN of the WHO classification

Clonal diseases of the HSC

Excluded in 2009 : PDGFBR rearrangement and eosinophilia

1. Chronic myelomonocytic leukemia

2. Juvenile myelomonocytic leukemia

3. Atypical chronic myeloid leukemia,

4. Refractory anemia with ring sideroblasts and thrombocytosis

5. MDS/MPN unclassifiable

No BCR-ABL or Ph1Bone marrow blast cells < 20%

JMML

An agressive myeloid malignancy of chilhood

MPN / MDS in the WHO classification

- Infant or young child (0-6), male > female, with fever and pallor, - Circulating WBC count > 10G/L; Peripheral monocytosis > 1 G/L- A few circulating myeloid precursor cells

- Sometimes : skin rash, cough, bloody stools

- Splenomegaly, hepatomegaly

- Lack of BCR-ABL- Less than 20% blast cells in the bone marrow

JMML

An agressive myeloid malignancy of chilhood

Cytogenetic abnormality (monosomy 7, 25%)

Hypersensitivity of myeloid progenitor cells to GM-CSF (not to IL-3 or G-CSF)

Increased HbF for age

Treatment : ABMT (EFS 52%)

AML4 / AML5 or spontaneous improvement

Genetic syndromes that predispose to JMML

Neurofibromatosis, type I (NF1) : prone to JMML in the first decade

- Autosomal dominant disorder (or spontaneous)- At least 6 café au lait macules, - At least 2 neurofibromas or 1 plexiform neurofibroma,- Lisch nodules (iris hamartomas), axillary or inguinal freckling, and/or optic gliomas

- NF1 encodes neurofibromin protein, a GTPase activating protein for Ras- Enhances the hydrolysis of the active, GTP-bound conformation of Ras

- In the bone marrow of children with JMML: loss of the wild type allele

- Mouse models of nf1 conditional deletion reproduce the disease

Genetic syndromes that predispose to JMML

Noonan syndrome (NS)

- Autosomal dominant (or spontaneous) disorder- Facial dysmorphism, short stature, webbed neck, cardiac anomalies,- Varying levels of impaired cognition,- Self-resolving myeloproliferative disorder in infancy that resembles JMML

- Germine mutations in PTPN11 (former SHP2) in 50% of cases- Encodes a non-receptor protein tyrosine phosphatase (also SHP2)- That connects tyrosine kinase receptors to Ras

- Other Noonan : germline mutations in SOS1, a RAS-GEF (Guanine nucleotide-exchange factor) KRAS

Gene mutations in JMML

SHP2Yp

Grb2

SOS1

Ras-GDP

Ras-GTP

NF1

CBL

Raf

Mutually exclusive mutations in

PTPN11 35%NF1 25%N/KRAS 20%CBL 10%

Other in SOS1, FLT3, ASXL1

No TET2 mutation

AKT STAT5

ERK

MEK

CMML

a disease of the lederly

defined by only one positive criteria

MPN / MDS in the WHO classification

Clonal disease of HSC with monocytosis

- Persistant monocytosis (> 1 G/L)

- Lack of Ph1 or Bcr-Abl

- Blood and bone marrow blast cells < 20%

- Cell dysplasia, at least one cell line

Molecular abnormalities identified

in human CMML

1 – Cytogenetic abnormalities : 15-40%

2 – Uniparental disomy : ~50 %

3 – Mutations in

Epigenetic genes : TET2 ; ASXL1 ; AML1/RUNX1; IDH1/2; EZH2; UTX

Signalling : N/KRAS; CBL; FLT3-ITD, JAK2; NOTCH2 /NCST / MALM

Splicing : SRSF2; ZRSR2; J2AF35; SF3B1

4 – Deregulated expression of

TIF1, miRNA, CJUN, CFOS

IDH/TET2 pathway: 72% mutations

Molecular abnormalities identified

in human CMML (GFM 175 patients)

No specific mutationHigh frequency of TET2/IDH pathway mutations (> 70%)Combinations of mutations in signaling, epigenetic, splicingMore mutations to identify (NGS)

Molecular abnormalities identified

in human CMML

Yoshida Nature 2011

Molecular abnormalities identified

in human CMML

CML

BCR-ABL

TE

JAK2* hZ

PV

JAK2* HZ

MF

JAK2*other

CMML

TET2* - IDH1/2*other

TET2 rare JAK2 rare

Increasing complexityfrom myeloproliferative neoplasms

to myeloproliferative / myelodysplastic diseases

AML1 ASXL1 CBL JAK2 KRAS NRAS

29 MUTATIONS IN 17 PATIENTS

# CLONES

TET2

52 MUTATIONS IN 46 PATIENTS

Clonal expansion occurs in theCD34+/CD38- compartment

CMML : clonal amplification at the stem cell level

Stem cells

Progenitors

Maturecells

Normal MPN CMML

LMMP?

?

TIF1 involved in zebrafish erythropoiesis (severe anemia in « moonshine » mutant) human erythropoiesis (ex vivo differentiation of CD34+ cells)

TIF1 knock-out embryonic lethal in mice

TIF1 (Transcription Intermediary Factor 1 )

1127 AA1127 AARINGRING B1B1 B2B2 CoiledCoiled--coilcoil TSSTSS PHDPHD BromoBromo

RBCC ou TRIMRBCC ou TRIMTIF Signature TIF Signature SequenceSequence

FeaturesFeatures of of chromatinchromatinremodelingremodeling proteinprotein

MeMe33H3K4H3K4AcetylatedAcetylated

lysineslysines

TIF1- belongs to a group of four proteins (TIF1 ) - modulates the TGF- signaling pathway- is a transcriptional co-regulator (TAL1/PU1)

Ageing Tif1 / mice developa CMML-like disease

Peripheral blood

00

11

22

33

44 CtrlCtrl//

11--1313 1414--2626 2727--3939 ≥≥4040

Mon

ocyt

es (k

/mm

Mon

ocyt

es (k

/mm

33 ))

WeeksWeeks

n=11n=11n=23n=23

n=12n=12n=23n=23

n=12n=12n=20n=20

n=9n=9n=9n=9

**

****

Gr1Gr1--FITCFITC

Mac

1M

ac1 --

Ale

xa64

7A

lexa

647

CtrlCtrl //

44%44% 1%1%3%3% 51%51%

CtrlCtrl //

1 cm1 cm

Spleen

-139GGGAGGAYGT TYGTGYGTA YGTGYGYGTGT YGTAAT YGTTT TT TTTTAAA YGYGYGA YGYG

-139GGGAGGACGTCCGTGCGTACGTGCGCGTGCCGCAACCGCCCTCCTTCAAACGCGCGACGCG unconverted

non methylatedpartially methylatedtotally methylated

Control TIF1low

TIF1norm

alexpression (Subset# 1) (Part of subset# 2)

TIF1 gene promoter is methylatedin 35-50% of CMML

35%of patients

Months

Cum

ulat

ive

Pro

babi

lity

of S

urvi

val

0 6 12 18 24 30

0.0

0.2

0.4

0.6

0.8

1.0

17 15 11 7 2 0

18 14 11 9 3 1

low TIF1ghigh TIF1g

Low TIF1

Normal TIF1

1.0

0.8

0.6

0.4

0.2

0.0

0 6 12 18 24 30Months

Cum

ulat

ive

prob

ablil

ity

of s

urvi

val

TIF1 expression leveldoes not predict decitabine efficacy

0

50

100

150

200

250

300

mRN

Aex

pres

sion

(rel

ativ

e)

Cont

rol

Subs

et#

1

Subs

et#

2

Wild-type

Mutated

Months

19 15 11 7 219 17 14 12 5

0 6 12 18 24 300 6 12 18 24 30

Cum

ulat

ive

Prob

abili

tyof

Sur

viva

l

0.0

0.2

0.4

0.6

0.8

1.0

Mutated

Wild-type

TET2

13 10 9 7 325 22 16 12 4

Months

Cum

ulat

ive

Pro

babi

lity

of S

urvi

val

0 6 12 18 24 30

0.0

0.2

0.4

0.6

0.8

1.0

27 24 18 15 6 0

10 7 6 3 1 1

AML1 WTAML1 MUT

Mutated

Wild-type

0 6 12 18 24 30

10 7 6 3 1 Mutated28 24 18 15 6 Wild-type

AML1ASXL1

Gene mutations in TET2, ASXL1, AML1 do not affect survival

Months

Cum

ulat

ive

Pro

babi

lity

of S

urvi

val

0 6 12 18 24 300.

00.

20.

40.

60.

81.

0

17 16 13 12 5 0

18 13 9 4 1

low MYBhigh MYB

Months

Cum

ulat

ive

Pro

babi

lity

of S

urvi

val

0 6 12 18 24 30

0.0

0.2

0.4

0.6

0.8

1.0

16 15 14 10 2 0

19 14 8 6 3 1

low CJUNhigh CJUN

P = 0.06

Cum

ulat

ive

prob

ablil

ity

of s

urvi

val 1.0

0.8

0.6

0.4

0.2

0.0

Low

High

CJUN 1.0

0.8

0.6

0.4

0.2

0.0

CMYB

Low

HighP = 0.01

0 6 12 18 24 30 0 6 12 18 24 30Months Months

CJUN level correlates with responseCMYB level correlates with survival

Chronic myelomonocytic leukemia

Persistant monocytosis (> 1000/µL)

Why do these patients die?

The best recognizedprognostic factor is blast cell count

Diagnostic feature

Peripheral blood monocytes

Left shift and myelocytes

Peripheral blood blasts

Bone marrow blasts

Dysplasia

t(9;22) or BCR-ABL

PDGFRA/B

CMML1

> 1 G/L

< 10%

< 5%

< 10%

0, one, more

No

No

CMML2

> 1 G/L

> 10%

5-19%

10-19%

0, one, more

No

No

Forward scatter

Side

scat

ter

Healthy donor CMML patient

CD14

Cel

lnum

ber

CD14

Cells counted as « monocytes »in the peripheral bloodinclude 2 populations

TET2 mutation(s) in the two cell populations

NoneR1214W; L1420FSQ652XMutation splice donor site exon 6 + L1855FSR544X ; E1492FSNoneE320X ; R1359HK450X ; I1163FSH1219YF1104FS , D1384VNoneT1883FSNoneQ1501FS ; G1861RQ591XNoneL615FS ; K1422FSK1005FS ; S1324FSS354FS ; L615FS

UPN

4754717279818596

100136153107171174180194207211216

-défensinesHNP1-3

CD14-/CD24+

Granuleux immatures

CD14+/CD24-

Monocytes

Lymphocyte

ExosomesIL-13

STAT3STAT6

Macrophage

M-CSF

The immunosuppressive propertiesof the CMML immature granulocytes

Take home messages

1 – JMML / CMML are distinct diseases

2 – JMML is a RAS pathway disease with hypersensitivity to GM-CSF

3 – CMML is a TET2 disease with many different mutations

4 – Epigenetic changes in addition to mutations (TIF1, miRNA)

5 – Immunosuppressive dysplastic granulocytes

To further explore the disease pathogenesis : eric.solary@igr.fr