Juvenile Myelomonocytic Leukemia ( JMML)

Leukemia Disease Model and Drug Screening

Dr. Baoqiang Guo

B.guo@mmu.ac.uk
 Learning Outcomes
• Define Juvenile Myelomonocytic Leukemia ( JMML)
• Describe and recognise JMML
• Typical patient presentation
• Typical peripheral blood and bone marrow picture
• Genetic analysis and sensitivity to GM-CSF

• Discuss how to develop a JMML iPSCs disease model

 What is JMML?

JMML is a chronic myeloproliferative

disorder that typically affects young Normal
Blood
children: more than 95% of cases are
film
diagnosed before age 4

Phenotype/cell stem origin JMML

arises from hematopoietic stem cells
(HSCs) (Cooper et al., 2000). Clonal
proliferations of myeloid, monocyte- JMML
macrophages, erythroid, and Blood
film
sometimes lymphoid progenitor cells
are seen.
 Noonan syndrome and JMML

1. Noonan syndrome (NS) is an autosomal dominant developmental disorder with an estimated

incidence of 1 in 2500.

2. This syndrome is caused by germline gain-of –function mutation in genes encoding components
of Ras/MAPK signalling pathway.

3. Mutations in the oncogene PTPN11 are the most frequent in this syndrome ( 50% of NS cases) .

4. Approximately 10% of NS patients present with JMML.

5. Somatic PTPN11 mutations have been identified in 35% of the de novo non-syndromic JMML (Sporatic
JMML).
 The character of JMML

1. PTPN11 mutation in NS and JMML indicated high phosphatase activity and their
hematopoietic cells indicated high sensitivity to GM-CSF.

2. Syndromic and non-syndromic JMML patients are resistant to any chemotherapy, the
only treatment choice is allo-BMT with high relapse rate.
Why do we target PTPN11 mutations?

iPS cell line

PTPN11 gene encode a protein Shp2

The consequence of PTPN11 oncogenic mutations

GF, cytokines, and extracellular Oncogenic Shp2

iPS cell line
matrix

Shp2 inhibitor:
Normal development SHP099 and TNO155
Multiple signalling pathways are possibly
involved in transformation of NS to JMML
 How to define the pathogenesis of NS transformation to
JMML ?
Why iPS?
iPS cells
Why not directly patients or cell line?

1. No cell line available. Directed differentiation

2. Ethical reason.
3. We can not have enough patients in certain
HSCs/HPCs
years. Disease iPS cells bank.
4. Using iPS differentiation to catch the
transformation point.
5. Repeatable MS/iTraq

Proteomics
NS-
JMML
Non- New proteins-new
de novo
JMML
JMML signalling pathway
NS

New targets
iPS cells
The Nobel Prize for Induced Pluripotent Stem Cells (iPSCs)
in Physiology or Medicine 2012
• Mature adult cells can be reprogrammed
to become pluripotent iPSCs

• These ground breaking discoveries have

completely changed our view of the
development and cellular specialisation

Reprogramming of human cells:

1. Drug toxicity
2. Disease modelling and drug screening
3. Replacement therapy.
Differentiation of iPS cells to three germ layers for
translational application
 How to do disease modeling and drug screening

1. The disease in a dish

2. iPS cells from a patient represent an early stage of disease

3. The establishment of in vitro differentiation models of specific cell-type for

dissecting pathogenic events responsible for disease initiation and progression.
Sequence analysis of our two iPSCs with PTPN11 mutation
Directed differentiation of iPS cells into HSCs and HPCs

1. EB ( Embryoid bodies) formation methods.

2. Co-culture with Stromal cell line feeder cells (OP9).
3. Feeder-free monolayer differentiation system.
From iPS cells to myeloid progenitors with Embryo body formation method

iPS cells grow on

iMEF-feeders

Matrigel- 48 to 72 hours
coated

Ultralow
attachment
Embroid body formation- a crucial step for iPSCs
differentiation to HSCs
2-3days on Matrigel or rhLN521
Scraper
iPSCs-on iMEF Collagenase B TryPLE Cluster
20Mins 1-3Mins 20-50 cells

Normal control-iPS derived NS-iPS-derived embroid NS/JMML-leukemia –iPS

embroid body (EB) body (EB) derived embroid body (EB)
Normal Control, NS and NS/JMML -derived
Hematopoietic stem cells (HSCs)

Control Noonan syndrom JMML leukemia

Normal Control, NS and NS/JMML -derived
Hematopoietic stem cells (HSCs)
Control Noonan syndrom JMML leukemia
Dysregulated hematopoiesis was found in NS/JMML by EB
colony forming assay (CFU-assay) and Cytospin-Giemsa
staining EB14-Giemsa staining

CFU-GM BFU-E/CFU-E

Con
Con

B
A

NS
NS

NS/JMML
NS/JMML

Colony forming assay showing dysregulated hematopoiesis A: showing that huge colonies were found in 15-
day colonies of Day 14 differentiated EB cells derived from NS/JMML-iPS cells. B: Giemsa staining of day-14 EB
cells differentiated from iPS cell generated from control, NS and NS/JMML patients.
Dysregulated haematopoiesis could be observed for drastic
increase in myeloid phenotype CD33+CD34+

CD33CD34

P e r c e n ta g e o f C D 3 3 + C D 3 4 + c e lls
40

**
30

20

ns
10

0
C o n tro l NS N S /J M M L
Dysregulated haematopoiesis could be observed for drastic
increase in myeloid phenotype CD34+CD45+ as well
Dysregulated haematopoiesis was also found in CD34+CD18+
primitive cells

CD34+CD18+
Hemogenic endothelial cells-CD34+CD31+
Common significantly expressed proteins in JMMLs : WTs, NOONANs : WTs and JMMLs : NOONANs
Log2 Ratio outside the range of -0.832 to 0.782 AND p-value < 0.05 in at least 3 out of 4 ratios in each
category.

147 proteins were observed to change significantly

between the JMML samples and WT samples in at
least 3 out of 4 JMML : WT ratios.
P02751, FN1
P11413, G6PD
75 proteins were observed to change significantly P04083, ANXA1
between the NOONAN samples and WT samples Q14195, DPYSL3
in at least 3 out of 4 NOONAN : WT ratios. P13533, MYH6

18 proteins were observed to change

significantly between the JMML
samples and NOONAN samples in at
least 3 out of 4 JMML : NOONAN ratios.
P23219, PTGS1
O00534, VWA5A
P07996, THBS1

P13727, PRG2
Q93084, ATP2A3
P16401, HIST1H1B
P05107, ITGB2
P31146, CORO1A
P29350, PTPN6
P52566, ARHGDIB
P26447, S100A4 Q04828, AKR1C1
Q02539, HIST1H1A BioVenn
iTRAQ proteomics analysis of CD33 samples

Protein expression ratios for JMMLs against NOONANs that

have p-value < 0.05 for all ratios

40 proteins with expression ratios that have p-value < 0.05 in:

117- JMML K1 R1 : 115- NOONAN VPA7 R1

117- JMML K1 R1 : 116- NOONAN VPA7 R2
118- JMML K1 R2 : 115- NOONAN VPA7 R1
118- JMML K1 R2 : 116- NOONAN VPA7 R2
Gene Ontology, GO BP analysis for signalling pathway
1500 N o A d d itio n C XC L12

N u m b e r o f c e lls m ig ra tin g
*
*
GO BP Terms 1000

leukocyte migration

regulation of multicellular organismal development

*
defense response 500

cell migration

leukocyte chemotaxis
cell motility 0
NS N S /J M M L
localization of cell
movement of cell or subcellular component

inflammatory response

ion transport

C C L 3 g e n e e x p r e s s io n ( fo ld c h a n g e )
9
peptide cross−linking **
Log2FC 8
locomotion
7
- 1 regulation of cell adhesion
1 6

Gene Ontology Biological Process (GO BP) enrichment analysis was conducted 2

using the DAVID Bioinformatics software to identify significantly enriched GO BP 1

terms. The statistically significant GO BP FAT category terms were then 0

NS N S /J M M L
illustrated in a GOChord plot. The colors in which genes are presented reflect
their log 2-fold change as per legend.
Examples with very big significant protein level fold changes in
CD33+ cells from differentiated 14-day of control, NS and
NS/JMML –iPSCs
ITGβ2 Protein S100-A4

Fold change of Protein S100-

30.0
12.0
25.0
Fold change of ITGβ2

10.0 20.0

A4 leve
protein level

8.0 15.0
6.0 10.0
4.0 5.0
2.0 0.0
Con NS NS/JMML
0.0
Con NS NS/JMML

Galectin-10 1.2
CRBP-1

Fold change of CRBP-1

Fold change of Galectin-

14.0 1
10 protein level

protein level
12.0 0.8
10.0
0.6
8.0
6.0 0.4
4.0 0.2
2.0
0
0.0
Con NS NS/JMML
Con NS NS/JMML
 Validation of key proteins found with MS-iTraq

Con
70 **
** **

(p e rc e n ta g e o f C D 3 3 + c e lls )
60

N u m b e r o f IT G  2 c e lls
50
SSC-A

40
NS
30

20

10 Con NS NS/JMML
S100A4
0
Actin
NS/JMML C o n tro l NS N S /J M M L

CD18/ITGβ2
Curaxin selectively inhibited the proliferation of NS/JMML-iPS-
derived progenitor cells
DMSO Nutlin JQ1 Curaxin C+J C+N

Con

NS

NS/JMML

P8 p7 p6 p5 p4 p3 p2 p1 p0
Day 8. CellTrace Violet
Curaxin (CBL0137) preferentially inhibits the colony-forming ability of
NS/JMML-iPS-derived Cells in comparison with NS-iPS-derived cells

ns
100 ns

C o lo n y n u m b e r ( % c o n tr o l)
NS

N S /J M M L
80 ns

60
**
40
*
20

0
1 lin 1 7 1
JQ ut JQ 3 JQ
N + L 01 +
li n B 7
ut C 13
N L0
B
C
Curaxin induces primitive cells of bone marrow of patients with
sporadic JMML
All population CD34+
100 ns
N u m b e r o f c e lls in e a c h p o p u la tio n

100 C o n tro l
C o n tro l
C o n tro l + C B L 0 1 3 7
C o n tro l + C B L 0 1 3 7 ns

P e r c e n a tg e C D 3 4 + c e lls
JM M L
JM M L 80
80 JM M L + C BL0137 **
JM M L + C BL0137 *
( p e r c e n ta g e )

60
60 ** **

40 40

20 20
ns ns
ns ns ns ns

0 0
L a te a p o p to tic A p o p to tic L iv e L a te a p o p to tic A p o p to tic L iv e

CD33+ CD31+
100 C o n tro l ns
100 C o n tro l
C o n tro l + C B L 0 1 3 7
ns ns
C o n tro l + C B L 0 1 3 7
P e r c e n ta g e C D 3 3 + c e lls

P e r c e n ta g e C D 3 1 + c e lls
JM M L JM M L
80 ** 80
JM M L + C BL0137 JM M L + C BL0137

60 ** 60

40
40

20
*
20 ns ns
ns ns
ns ns
0
0 L a te a p o p to tic A p o p to tic L iv e
L a te a p o p to tic A p o p to tic L iv e

Cells were treated in liquid culture for 3 days, and the number of cells undergoing
apoptosis in different cell populations assessed by measuring annexin V and 7-AAD
 Conclusions

1. NS/JMML-iPSCs disease model recapitulate JMML patients

character

2. Curaxin is potential therapeutic drug for the treatment of

JMML
 Learning Outcomes
• Define Juvenile Myelomonocytic Leukemia ( JMML)
• Describe and recognise JMML
• Typical patient presentation
• Typical peripheral blood and bone marrow picture
• Discuss how to develop a JMML iPSCs disease model
Thank you for
attending the lesson