mouse models of genomic lesions: utility as therapeutic ... · signaling (varney 2015) pten;...

ROSE ANN PADUAUMRS-1131, Hôpital Saint-Louis

Mouse Models of Genomic Lesions:Utility as Therapeutic Platforms

• 2 large studies: Papaemmanuil et al, Blood 2013; Haferlach et al, Leukemia 2014• 80% patients have at least 1 mutation, 6 genes mutated in > 10% patients • splicing factor and/or epigenetic regulators are mutated in most patients

• many recurrent mutations in MDS are very rare

Frequency of driver mutations in MDS (by subtype)

Boultwood 2015

GENEALTERATIONSfound in MDS

patients MDS disease entities

(Frequency)

ALTERATIONS STUDIED IN

MOUSE MODEL

TYPE OF MOUSE MODEL

MOUSE MODELS DISEASE ENTITIES

According to WHO Classification 2008

HEMATOLOGICAL FEATURES DESCRIBED

Spliceosome and polycombSF3B1 MDS, AML with MDS SF3B1 mutant Xenograft

KI

MDS-like

Mild, myeloid expansion

HSCs restricted to myeloid lineage with clonal evolution leading to

AML (Mian 2015)Increase in myeloid cells.

Deregulated expresion of target genes (Mmp9, Puma, Bcl2l1)

(Sathiaseelan ASH2015)SRSF2 Mutations in MDS,

AML with MDS SRSF2 mutant KI

KO

MDS

Embryonic lethal

Impaired Hematopoietic ddifferentiation (Kim 2015)

Fetal liver cells had increased apoptosis and decrease of

hematopoietic stem/progenitor cells (Komeno 2015)

U2AF1 Heterozygous mutations in 11%

MDS

U2AF1 mutant Tg Some features of MDS Altered hematopoieses and changes in pre-mRNA splicing in progenitors

(Shirai 2015)

EZH2 Inactivating mutations in MDS/MPN

EZH2 Deficiency KO MDS/MPNT-ALL and clonal expansion of

B cells

Thrombocytosis (Mochisuk-Kashio2015)

EZH1 Deletion EZH1 Deletion KO No Dysplasia or malignancy Abolished repopulating capacity of HSCs when combined with EZH2

loss (Mochisuk-Kashio 2015

New mice 1



(Frequency)


MOUSE MODEL

TYPE OF MOUSE MODEL




Interesting NewsTert BoneMarrow Failure

syndromes: DC, AA,MDS

TERT deficiency Conditional TertKO

Anemia Decreased erythroblasts, reduced HSCs, neutrophilia and increased

myelopoiesis (Raval 2015)

DNMT3A Mutated in MPN, MDS,AML

DNMT3A deficiency

Conditional KO MDS/MPN Cytopenia, hepatomegaly,expandedstem/progenitors (Guryanova 2015)

TIFABTRAF-

interacting protein with

forkhead-associated domain B

Haploinsufficiency in 5q- MDS

TIFAB deficiency KO BM failure Neutrophil dysplasia, cytopenia.Derepression of TLR4-TRAF6

signaling (Varney 2015)

PTEN Mutated in JMML (MDS/MPN)

PTEN deficiency KO in NF1 haplo-

insufficient background

Pediatric MDS/MPN (JMML-like)

Death at day20-35 days due to organ infiltration with

monocytes/macrophages Liu 2016)

RPS14 Haploinsufficiency 5q-MDS

RPS14 deficiency Conditional KO Anemia, megakaryocytedysplasia

Erythroid differentiaion defect, apoptosis, loss of HSC quiescence,

high levels of innate immune signaling (Schneider 2016)

New mice 2

Classification based on ApoptosisModel Apoptosis Classification of

Bethesda (BM blasts)

Classification based on Apoptosis

Reference

BID-deficient Anti MPD/CMML(<15%) MPD/CMML Zinkel et al 2002

KRAS* ? MPD ? Braun et al 2003Chan et al 2003

NUP98-HOXD13 Pro MDS/AML (14%) MDS Lin et al 2005

NUP98-HD13+FLT3

? AML (>20%) ? Palmqvist et al 2006

NUP98-HD13+Meis1

? AML (65%) ? Pineault et al 2003

SALL4 Pro MDS-AML MDS Ma et al 2006

NRAS*/BCL-2 Pro MDS (15%) MDS Omidvar et al 2007

NRAS*/BCL-2 Anti AML (90%) AML Omidvar et al 2007

AML1+EVI 1 ? AML ? Watanabe-Ocochi et al 2008

EVI 1 Pro MDS MDS Delwell, perscomm

Presenter

Presentation Notes

So looking at other published animal model I propose that the classification correlates with apoptosis. So BID-deficient mice are anti-apoptotic and develop myloproliferative disease (MPD). These two KRAS-mediated MPDs have not been characterised. The Nup98/Hox13 fusion is pro-apoptotic with 14% bone marrow blasts and develop MDS. Sall4 is pro-apoptotic and develop AML.It is not known what the apoptosis status is of the AML. These next two are ours, which is controlled by where BCL-2 is localised. The last one here with EVI1 are pro-apoptotic and have an MDS phenotype. So it looks like in these models we have recapitulated the human disease in that in the MDS stage of the disease pro-apoptosis dominates and in the AML stage anti-apoptosis is the main feature. Of course in real life you expect a mixture at times as the disease is in transition.

Overall Aim

• To develop animal models as test platforms for therapeutics

• To develop reagents for improved patient treatment

• To identify biomarkers to predict response to treatment

The NRAS/BCL-2 mediated mouse model of High Risk MDS and AML post-MDS

ABNORMALCLONE

CLONALEXPANSION

LEUKEMICCLONE

EXPANSION

INITIATION

PROGRESSION

STEM CELL

Omidvar Cancer Research 2007

MRP8BCL-2

MMTVtTA/TetoBCL-2 HR-MDS

AML post-MDS

NRAS: coding for a small GTPase, involved in the regulation of proliferation, differentiation and

survival. Mutation of NRAS: frequent molecular abnormality in MDS and linked to AML.

Bcl2: regulator of apoptosis. Overexpression of Bcl2: observed in AML and MDS.

Presenter

Presentation Notes

Model of progression Starting with a stem cell an initiating event occurs giving rise to an abnormal clone, which has a selective advantage and clonally expands. Eventually other events occur giving rise to a leukemic clone, which also expands. Using a genetic approach we used transgenic mice bearing either mutant NRAS or BCL-2 as we have shown previously that mutations of NRAS are found in MDS patients and BCL-2 has been found to be upregulated in MDS patients as they progress. Furthermore 50% of AML patients have upregulation of BCL-2 which correlates with a poor prognosis. In our mouse models depending on where we express BCL-2 we get either MDS or AML-like disease

X BCL-2

0 20 40 60 80 100

0.0

0.2

0.4

0.6

0.8

1.0

Latency (days)

Prop

ortio

n Al

ive

FVB/NNRASBCL-2

NRAS/BCL-2

0 100 200 3000.0

0.2

0.4

0.6

0.8

1.0

Latency (days)

Prop

ortio

n Al

ive

+DOX(n=13)

-DOX(n=18)

AML

MDS

NRAS and BCL2 cooperate to give HR-MDS or AML post-MDS

90

180

HR-MDS

AML post-MDS

%blast

8±0.3

12±2

18±5

15±3

89±4

MMTV-LTR/ BCL-2

NRAS

Omidvar Cancer Res 2007

Presenter

Presentation Notes

When we cross the MMTVtTA/TBCL-2 mice with the MRP8mutant NRAS mice we get an MDS-like disease with around 15% bone marrow blasts with a lot of dysplasia, some liver invasion and a relatively indolent disease with a long latency period before they die. Note that when hBCL-2 is switched off the lethality of the disease can be rescued.When you cross the MRP8BCL-2 with mutant MRP8NRAS you get an AML-like disease with nearly 90% bone marrow blasts, massive invasion around the periportal tract of the liver, invaded spleens cells with the disappearance of the white pulp and expansion of the red pulp due to extramedullary hematoppiesis and the mice die within 3 months. We see some dysplasia, which indicates there was a preceding MDS. Note that the single RAS has normal differentiation with dysplasitc cells and clear livers and spleesn. The single Bcl-2 mice have increased bone blasts, but these are normal and the livers and spleen are also not invaded. Note the scales of the survival curves so MDS takes longer to die compared to the AML.

Anti-hBCL-2

Anti-Actin

Omidvar Cancer Res 2007

Anti-BCL-2 Anti-RAS Merged+DAPI

BCL-2/NRAS

BCL-2 and mutant NRAS co-localize in the bone marrow

Sca-1+ Mac-1+.

Anti-Ras

Anti-Actin

RAS-GTP

hBCL-2:RAS-GTP

SpleenSca-1+ Mac-1+.

Presenter

Presentation Notes

Using the RAF1 pulldown assay, which brings down only active GTP-bound RAS we can see that RAS/BCL-2 is active in the expanded Sca1+ compartment, unlike the singles. In the Mac1 compartment where MRP8 drives expression we have active RAS in the single RAS and the double mice, but surprisingly also in the single BCL-2 mice. We hypothesize that this is because BCL-2 recruits endogenous mouse RAS to activate RAS. Below you can see that the hBCL-2 antibody sees the complex showing that BCL-2 physically binds the RAS-GTP complex. Using confocal microscopy you can see that RAS and BCL-2 co-localize.

Le Pogam Leuk Res 2013

RAS:BCL-2 complex localization correlates with apoptosis and prognosis in MDS/AML patients

ME:11%BM blast/APO-/High RAS:BCL-2 co-loc/Mito+ JM:4%BM blast/APO+/Low RAS:BCL-2 co-loc/PM+ME:11%BM blast/APO-/High RAS:BCL-2 co-loc/Mito+ JM:4%BM blast/APO+/Low RAS:BCL-2 co-loc/PM+

Model of Leukemic Progression

ABNORMALCLONE

CLONALEXPANSION

LEUKEMICCLONE

EXPANSION

INITIATION

PROGRESSION ?

Genomic Instability

NRAS

BCL-2 NRAS*

LEUKEMIC

Presenter

Presentation Notes

In order to ask what the mechanism is that may drive disease progression one of the mechanisms may be genomic instability.

gH2AX and Phosphorylated ATM on chromatin fibres of BM in NRAS/BCL2 AML post-MDS mice

gH2AX Phos ATM gH2AX+ Phos ATM

NRAS

FVBN

BCL-2NRAS+BCL-2

(N=4)

20-30

+ve fibers

30-50

0

10-18

%

Rassool Cancer Res 2007

Presenter

Presentation Notes

gH2AX and phosphorylated ATM are recruited at sites of DNA damage to initiate DNA repair. As you can see these are increased in the singles and the double transgenic AML mice.

Is the disease reversible?

Presenter

Presentation Notes

Est-ce que la maladie est réversible

Results of Preclinical Studies• Antioxidants

N-Acetyl Cysteine (Rassool Cancer Res 2007)

• Small molecules RACi (Rassool Cancer Res 2007)

BCL-2i (Beurlet Blood 2013)

• Immunotherapy all-trans retinoic acid (ATRA) – a vitamin A

derivative and DNA PML-RARA for APL(Padua Nat Med 2003; Robin Blood 2006; Furugaki Blood 2010;

Pokorna Mol Cell Probes 2013) or a non-specific DNA construct pVAX14(Le Pogam Oncotarget 2015; Patel Blood Cancer J 2015)

Presenter

Presentation Notes

ONLY published resulys So eventually keep ABT but not AZA

NRAS* BCL-2

Treatment of Mice with N-Acetyl cysteine

6 weeksNAC

Offspring tested for DNA damage and

misrepair

Controluntreated

X

Presenter

Presentation Notes

So the experimental design in order to ask if NAC will reduce genomic instability is shown here. We breed the mice with NAC in the water and when the pups are born we maintain the parents in NAC. AT 6 weeeks of age we then take the cells from the mice and test for DNA damage and misrepair. Ici cette expérience a été réalisée pour déterminer si NAC peut réduir l'instabilité génomique. Les souris sont alimentées en présence de NAC dans l'eau de boisson et quand les souriceaux naissent nous maintenons NAC dans l’eau de boisson des parents. – A 6 semaines nous prélevons les cellules des souris et nous analysons les dommages et les défauts de réparations de l'ADN.

FVBN

Foci 2±2 10±2 18±4

NRAS NRAS/BCL2

Immunostaining for gH2AX in BM MNC Nuclei fromN Acetyl cysteine-treated NRAS/BCL2 Mice

Untreated

NACTreatment

in vivo

N=3

N=3

Foci 4±31±1 10±2

100 nuclei examined /experiment


Presenter

Presentation Notes

You can see here foci stained with gH2AX, which is recruited at the sites for repair after DNA damage. There is an increase of staining in the single NRAS and the doubles, which are reduced after NAC treatment, showing that NAC is reducing damage.

MB1 22

MB1 22

NRAS/BCL2 + NAC

NRAS + NAC

MB 1 13NRAS/BCL2

0

5

10

15

20

25

FVB/N RAS RAS/BCL2

Mouse spleen cells

Mis

repa

ir Fr

eque

ncy

%

M B1 16NRAS

N=3

NHEJ Misrepair Frequencies in NRAS/BCL2 Mice Following NAC Treatment in vivo

White Colony PCR products around breakpoint

NACuntreated


Presenter

Presentation Notes

Here are the results. As you can see with NAC the huge deletions seen here in the doubles are repaired. The same result is obtained with the single NRAS. So NAC is also reducing the misrepair. .

BCL-2 inhibition with ABT-737 prolongs survival in an NRAS/BCL-2 mouse model of myeloid leukemia by targeting leukemia initiating cells

0

,2

,4

,6

,8

1

0 25 50 75 100 125 150 175 200Days

ABT(n=23)

Control(n=27)

Cum

ulat

ive

Surv

ival

0

,2

,4

,6

,8

1

0 25 50 75 100 125 150 175 200

ABT-737 (n=35)

Control (n=60)

DaysC

umul

ativ

e su

rviv

al

MDS from onset of disease (3-6 mths) AML from birth (3-4 weeks)

Beurlet Blood 2013; Gorombei, in preparation

ABT-737 75 mg/kg IP 3x per week for 5 weeks

P = 0.0005 P < 0.0001

ABT-737

RAS:BCL-2 Complex Co-localizes from the Mitochondria to Plasma Membrane

Beurlet Blood 2013

Genes regulated after ABT-737 treatment of MDS and AMLDifferential exon specific gene expression – Sca1 + splenocytes

AML

ADHESION RHO

CELL CYCLE

APOPTOSIS

MICROENVIRONMENT ICOS

TRAFFICKING RAB

IMMUNE TLRs

GENOMIC INSTABILITY

STEM CELL

EPIGENETICS ADA

MDSCELL CYCLE

APOPTOSIS

MICROENVIRONMENT ICOSIMMUNE TLRs

STEM CELL ALDH

EPIGENETICS ADA

MDS –399 genes

AML –997 genes

Secondary transplants of spleen cells showing that ABT-737 targets leukemia initiating cells

AML: p<0.005

days (post transplant)

MDS untreated(n=4)

MDS + ABT-737Post day (n=3)

MDS: p<0.05

AML (MRP8BCL-2/NRAS-TBone Marrow (LP) Bone Marrow (HP) Liver Spleen % BM Blast

52

56

66Gr-1

Mac-1

FVB/N-T NRAS-T

BCL-2-T BCL-2/NRAS-T

R1 R2

Bone Marrow

R1 R2

R1 R2R1 R2

Gr-1

Mac-1

Gr-1

Mac1

HR-MDS + ABT-737Post day 33 (n=4)

DNA Vaccination Scheme

Stimulates both CTL and Antibody production

Padua & Chomienne Discovery Med, 2004

Gene coding for a tumour antigen

MHC I

MHC II

Secreted antigen

Intracellularprotein

Golgiapparatus

Proteosomes

APC

CTLPerforingranzyme

B Cell

Modulate immune response

Such as Tregs?

Induction and maintenance of memory?

pVAX14 enhances the survival in HR-MDS Mice and induces stable disease

pVAX14

3.5kb

Le Pogam Oncotarget 2015

pVAX14 enhances the normalizes Signaling proteins

Le Pogam Oncotarget 2015

D1 D40D20DiagnosisD0

DNA Booster

Sampling

Presenter

Presentation Notes

Modifications of MEK1 in spleen cells of HR- MDS and normal FVB/N mice. Representative NanoPro traces showing MEK 1 and Actin of spleen extracts from HR-MDS treated with either HBSS vehicle or pVAX14 using the protocol illustrated in 4A. Boxed are Peak 1 representing a phosphorylated isoform and peak 2 a dephosphorylated isoform. Quantitations are shown in histogram (n=2 in triplicate). Actin was used as a housekeeping protein to control for proteins loading of mice treated with control vehicle HBSS and or with pVAX14. Measured on day 30. Conclusion: Flipper induces dephosphorylation of one of the MEK1 isoforms

Immune responses: pVAX14 targets progenitors, Increasesmemory T-cells, IFNg and MYD88 expression in HR-MDS Mice

IFNg Elispot

Le Pogam, Gorombei, in preparationLe Pogam Oncotarget 2015

Presenter

Presentation Notes

BM cells from the mice of high leukemic burden were used as targets of CD3+ enriched cells isolated from the spleen of MDS mice on day 82 of the protocol illustrated in Figure 4A; cells enriched for CD3+ from control vehicle (HBSS) or pVAX14 treated mice using CD3 magnetic beads. The ratio of CD3+ cells to MDS BM for culture in methocult (Stem cell technologies, Vancouver, Canada) was 10:1. Myeloid colonies were counted after 7days of incubation at 370C and 5% CO2. There is a significant difference between the E:T 100:1, with the lowest colonies coming from the mouse with the highest platelet count. Increased interferon gamma (IFNg) producing cells upon treatment with pVAX1 in high risk MDS mice using the protocol shown harvested on day 30. There are clearly low and high IFNg producers following pVAX14 treatment. P<0.025 Conclusion: pVAX14 induces IFNg and targets progenitors pVAX14 Increased Memory T-cells of HR-MDS mice on day 30. pVAX14 Vs Control vehicle (HBSS): p<0.01, Mann Whitney test. I ncreased MYD88 expression upon treatment with pVAX14 assayed after treatment (post day 50). pVAX14 MYD88 expression levels were significantly different from Vehicle (HBSS) (p≤0.03). Conclusion: pVAX14 induces IFNg and MYD88 expression of HR-MDS mice.

These cells shed exogenous or endogenous peptides

At the same time TAA are also present from apoptotic cells or

induction of apoptosis

Activation of lymphocytes CD4+ and CD8+ T cells cytotoxic T lymphocytes Secretion of IFN gamma Production memory T cells Production of IgG and

specific antibodies

Hypothesis: Increase of pre-existing immune response.

DNA plasmids injected in humans are expressed

in myocytes, derm cell, in antigen presenting cells (APC)

DNA sequences (CpG sequences)activate TLRs

Mechanism of Action of pVAX14DNA Vaccination Scheme

Modified from Forni, Nature Reviews, Genetics

Antibodies

CTLs

Fenaux & GFM

High risk MDSPatients Treated with 6 cycles of

5-azacitidine and have stable

disease

Follow-upImmunomonitoring

Exploratory studies

2mg DNA

1mg DNA

4mg DNA (+ ATRA)

27

Phase I/II Protocol and monitoring on MDS withongoing reference treatment of 5-azacytidine

• Patricia Krief• Marika Pla• Satyananda Patel – Past Post doc• Petra Gorombei- Past PhD student• Laura Guerenne – Past PhD student• Stephanie Beurlet - Past PhD student• Carole le Pogam - Past PhD student • Pierre Fenaux – Clinical Lead• Lionel Aides

UMRS-1131 (Department of Hematology)Christine Chomienne

CollaboratorsIUH, University Paris Diderot,

Hôpital St-LouisCytology:

Maria-Elena NogueraPathology:Anne Janin

Christophe LeboeufImaging:

Niclas Setterblad

Nuclear Medicine:Hôpital Lariboisière ,

Laure SardaUniversitè Paris Diderot

Pascal Merlet

Institut CochinMichaela Fontenay

Cardiff UniversityNader Omidvar

Robert West

King’s College LondonMohamed Saeed

Ghulam Mufti

AbbottSteve Elimore

GenosplicePierre de la Grange

Protein SimpleAndrea Tu

Carsten Loeuk

University of MarylandFeyruz Rassool

Johns HopkinsSteve Baylin

University of California, San FranciscoScott KoganMike Bishop

StanfordIrv Weismann

Alice FanDean Felsher

University of Texas MD Anderson

Marina KonoplevaMichael Andreeff



(Frequency)


MOUSE MODEL

TYPE OF MOUSE MODEL




Gene silencing and altered tumor suppressor genesNPM1 t (3,5) translocation

NPM/MLF1MDS, AML with MDS

<1% (Arber 2003;Yoneda-Kato

1996; Grisendi 2005)NPM1 MutationsMDS/AML(Bains

2011)(4.4%)

NPM1 NPM1+/- Some features of MDS,MDS/MPN like and Myeloid leukemia (Sportoletti 2008)

Dyserythropoiesis , Dysmegakariopoiesis (Grisendi

2005)Leukocytosis with organ infiltration

(Sportoletti 2008)

RPS14 Haplo insufficiency5q- syndrome(Ebert

2008; Eisenmann2009)

LSCE**Cd74-

Nid67region

KO MDS with isolated del(5q)5q- syndrome like (Barlow

2009)

Macrocytic Anemia , Mild thrombocytopenia and mild

neutropenia , DyserythropoiesisDysmegakariopoiesis, Pro-

apoptotic profile

SPARC Haplo insufficiency5q-

syndrome(Eisenmann2009)

SPARC KO MDS with isolated del(5q) Some features of 5q-

syndrome (Lehmann 2007)

Thrombocytopenia, No DysplasiaImpaired ability to form BFU-E

APC Haplo insufficiency5q- syndrome

(Eisenmann 2009)(95%)

APC Heterozygote KO

MDS/MPN CMML like(Lane 2010; Wang

2010)

Macrocytic anemia, Leukocytosis, Monocytosis, Splenomegaly

BAP1 Heterozygous frameshift mutation

RMCD (Dey 2012)

BAP-1 Conditional KOCreERT2

MDS/MPNCMML like (Dey 2012)

Anemia, thrombocytopenia, leukocytosis with monocytosis,

splenomegaly, dyserythropoiesis, dysmyelopoiesis Beurlet

Hematologica 2013

Animal Models

Altered stem cell and niche

Dicer 1 No Dicer TgInducible Osterix

MDSRCMD like (Raaijmakers

2010)

Neutropenia +/-Anemia+/- Thromcytopenia Dysgranulopoiesis Dysmegariopoiesis

Pro-apoptotic profile ,2% AML transformation

Tet2 Mutations (loss of function) CMML

20%(Teferri 2009), 22%(Delhommeau

2009)MDS 6%(Teferri ,

19% (Delhommeau)

MPN 23%(Teferri2009b)

MPD 12% (Delhommeau)

Tet2 KOTet2LacZ/LacZ

Conditional KOMix1Cre

MDS/MPNCMML like (Quivoron

2011) (Incomplete penetrance)

Leukocytosis MonocytosisAnemia Thrombocytopenia

Splenomegaly

Conditional KOVavCre

MDS/MPNCMML like (Moran-

Crusio 2011)

Leukocytosis MonocytosisMyeloid dysplasia

Splenomegaly

RAS signaling molecules

FLT3 FLT3-ITDMDS, CMML

5%(Stover 2005)

FLT3-ITD KIEndogenous

MDS/MPNCMML like(Lee 2007)

Leukocytosis MonocytosisSplenomegaly

PDGFRβ Translocation t(5;12)

CMML with eosinophilia

Rare(Greipp 2004)

TEL-PDGFRβ BMTLTR-MCSV*

MDS/MPNCMML like(Stover 2005)

Leukocytosis with Neutrophilia and Monocytosis Hepatosplenomegaly

NF1 Loss and mutationPediatric

MDS/MPN14%(Side 1997)

NF1 Inducible KO Mx1CreXNF1flox

MDS/MPNJMML like(LeDT 2004)

Leukocytosis Monocytosis Splenomegaly

PTPN11 MutationsJMML

34%(Tartaglia2003)

Ptpn11D61Y KIEndogenous

MDS/MPNJMML like(Chan 2009)

Anemia Leukocytosis MonocytosisSplenomegaly Hypersensivity to

GM-CSF

Animal Models

Beurlet Hematologica

2013

RAS signaling moleculesNRAS Point mutation

MDS, CMML20 - 40%

(Padua 1998; Paquette 1998; Bowen 2005)

NRASG12D rT/BMTLTR- LK *

AML with maturation or MDS/MPNAtypical CML

like(Mackenzie 1999)

Leukocytosis Monocytosis+/- high count of myeloblasts

Pro-apoptotic profile of lymphocytes and myelomonocytic cells

NRASG12D rT/BMTLTR- MCSV*

AML (monocytic/monoblas

tic) (Parikh 2006)Or MDS/MPN

CMML like

High BM blasts with monocytic/monoblastic differentiation

Leukocytosis with Monocytosis Hepatosplenomegaly

NRASG12D TgMRP8 promoter

Cooperation with BCL-2

MDS(Omidvar 2007)RAEB-1 like

or AML post-MDS(Omidvar 2007)

Thrombocytopenia Dysmyelopoeisis, dysplasia, propapoptotic profile

BM blasts about 15%Thrombocytopenia Neutropenia, some

dysplasia, anti-apoptotic profileBM blasts >60%

LSL-NRASG12D Conditional KIMix1Cre

MPD(Li 2011) Leukocytosis, splenomegaly

LSL-NRASG12D Conditional KIMix1Cre

+MOL4070LTR

AML-like(Li 2011) Myeloblasts M4/M5 subtype

KRAS Point mutationMDS,CMML, JMML

20- 25%(Braun 2004;Chan

2004)

LSL-KRASG12D Conditional KIMix1Cre

MDS/MPNJMML or CMML

like(Braun 2006; Van Meter 2007 )

Anemia Dyserythropoiesis Leukocytosis Monocytosis

KRASG12D rT/BMTLTR-MCSV*

MDS/MPNCMML like(Parikh

2007)

Leukocytosis with MonocytosisHepatosplenomegaly

Beurlet Hematologica 2013

Animal Models

Nuclear and Transcription FactorsEVI1

Evi1-MDS1Inv(3), t(3,12), t(3;21)

MDS, MDS/AML, t-MDS/AML(Rare) (Mitani 1994;Mochizuki

2000;Suzukawa 1994)Overexpression

(Common)(Russell 1994)

Evi1 rT/BMTLTR-MSCV*

MDSRCMD like(Laricchia-

Robbio 2006)

Pancytopenia Dyserythropoiesis Dysmegakariopoiesis, Pro-apoptotic profile

RUNX1/AML1 MutationsMDS subgroup MDS/AML

(5 to 20%)(Dicker 2010)t-MDS/AML

(40%)(Harada 2009)

Mutant AML1 D171N

Mutant AML1 S971fs

rT/BMTLTR-Retrovirus*(unspecified)

AML with MDS related changes, MDS RAEB-2

(Watanabe-Okochi 2008)

Multilineage Dysplasia, Leukocytosis Hepatosplenomegaly, High blasts count in BM

Multilineage Dysplasia, PancytopeniaBM blasts<20%

NUP98 TranslocationsMDS, MDS/AML, t-MDS/AML

1 - 2 % (Lam 2001)

NUP98-HOX TgVAV promoter

MDSRCMD like sometimes AML (Choi 2008; Slape

2008)

Thrombocytopenia, Anemia, Neutropenia Multilineage dysplasiaPro-apoptotic profile

CBFβ Translocation Inv 16t-MDS/AML

1,5% (Leroy 2002)

CBFβ-MYH11 Conditional KIMx1Cre promoter

AML (Kuo 2008) AML with monocytic differentiationMultilineage differentiation impairment

MLL Translocationst-MDS, t-MDS/AML

12% (Block 2002; Rowley 1997; Taki1997)

AmplificationMDS Mostly RAEB, t-MDS,

AML with MDSrc<1% (Anderson 2001)

MLL-AF9 KIEndogenous promoter

AML (Johnson 2003) No MDS featuresAML with Mac-1+/c-KIT+ phenotype

MLL-AF9 rT/BMTLTR-MSCV*

AML (CHEN 2008) No MDS featuresAML with Mac-1+/c-KIT+ phenotype

MLL-ENL Conditional KI endogenous promoter

MPD (Takacova 2012) Accelerated to AML with caffeine, an inhibitor of DNA damage response kinases ATM and ATR with

increase in Mac-1/c-KIT/GR-1 phenotype

C/EBPα MutationsMDS, CMML Rare, AML

5 to 14% (Fuchs 2008; Gombart2002)

C/EBPα mutant p30

KIEndogenous Promoter

AML(Kirstetter 2008) No MDS featuresAML with Mac-1+/c-KIT+ phenotype

SALL4 OverexpressionAML and MDS

60% (4B isoform)(Ma 2006)

SALL4B isoform TgCMV promoter

MDSRCMD like(Ma 2006)

NeutropeniaMultilineage dysplasia

50% AML transformationPro-apoptotic profile in young mice

Animal Models

Beurlet Hematologica 2013

RAS and BCL-2 co-localization in MDS/AML patients correlates with WHO classification

Patient BM blasts (% )

RAS:BCL-2

Col-loc

Mito PM Apoptosis NRAS Genotype WHO Classification Karyotpye

JM 4 Low Neg Pos APO+ NRAS12/13*** CMML I 46, XX, del 5q, del 11q [20]

EC 5 High Neg Pos APO+ Wildtype RAEB I 46, XX, del 11q [20]

MS 5 Low Pos Pos APO+ Wildtype CMML I 46, XY [20]

AB 10 Low Neg Pos APO+ Wildtype RAEB II 46, XY [20]

JP 11 Low Neg Pos APO+ Wildtype RAEB II 46, XY [20]

ME 11 High Pos Neg APO- Wildtype RAEB I 46, XX [20]

CX 12 Low Neg Pos APO+ Wildtype RAEB II 46, XY, del 5q [20]

LJ 14 High Pos Neg APO- Wildtype RAEB II 46, XY [20]

EH 18 High Pos Neg APO- ND RAEB II 46, XX [20]

CB 26 High Eqv Pos APO+ Wildtype AML post-MDS 46, XX [20]

BA 32 High Pos Pos APO- Wildtype AML post-MDS 46, XY, del 5q [20]

GA** 35 High Neg Pos APO+ NRASV12 AML post-MDS 46, XX [20]

RA 38 High Pos Neg APO- Wildtype AML post-MDS 46, XY [20]

Le Pogam Leuk Res 2013

mouse models of genomic lesions: utility as therapeutic ... · signaling (varney 2015) pten;...

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