Myriam AlcalayGenomica Funzionale

Dpt.o Oncologia Sperimentale, Istituto Europeo di OncologiaDpt.o Medicina, Chirurgia e Odontoiatria, Università degli Studi di Milano

Leucemogenesi e proteina difusione AML1/ETO

Verona, 21 maggio 2009

t(8;21) Acute Myeloid Leukemias

(DNA-directed Transcription Factor)

ETO (Co-repressor of Transcription)

AML1

Normal Leukemic

Down-regulation of genes involved in myeloid differentiation Up-regulation of genes involved in HSC maintenance

AML1/ETO-mediatedtranscriptional regulation

Structure of AML1/ETO

Genomic analysis

Strategy to:

1) correlate AML1/ETO binding pattern with effects ontranscription

2) characterize AML1/ETO binding regions with an unbiasedapproach

A. Gardini et al., PLoS Genet. 2008 Nov;4(11):e1000275.

Transcription in the Genomic Era

Expression ArrayAnalysis ofover 47,000transcripts

Genomic Tiling ArrayAnalysis of protein binding patterns throughout thewhole genome= array-based chromatin immunoprecipitation(ChIP-chip)

The model system: U937 cell line

VD3+TGFβ

VD3+TGFβ

U937-A1E

U937 differentiate into monocytes upon treatment with VitD3 + TGFβ.

AML fusion proteins transfected into U937 differentiation block

U937 stably transfected with AML1/ETO under the control of ametallothionein promoter (mt)ZnSO4

U937-Mt

Expression array

HGU133v2.0Plus

1316regulated genes (FC>1,5)

55% decreased

45% increased

ControlRNA

AML1/ETORNA

ChIP-chip platform 1

NimbleGen Human HG17 Promoter Array set-4kb to +1kb of 24,434 annotated genes

700,000 probes

Gene A Gene B

AML1/ETO peaks in in the promoters of 2,513 unique genes

Expression-binding correlation

1316regulated

genes

2513occupied

promoters

358 “direct” targets

70%30%

DOWN UP

(247) (111)

Not all AML1/ETO-dependent transcriptional regulation is associated topromoter binding (27.2%)

Not all AML1/ETO binding events result in transcriptional regulation (14.2%)

AML1/ETO binds to the promoters of upregulated genes

ChIP-chip platform 2

Chr.19 Tiling Array

350,000 overlapping probes

Gene A Gene B

408 high-stringency AML1/ETO peaks on chromosome 19

AML1/ETO binding on chr.19

Topography of AML1/ETO peaks

AML1/ETO binding regions show enrichment of specificsequences that are consensus binding sites for 4 knowntranscription factors: AML1, Ets1, AP.1 and the E-protein HEB.

Sequence analysis

E proteins interact with ETO andAML1/ETO

Heb is an interactor of AML1/ETO in U937

HEB is an interactor of AML1/ETO

AML1 – control cells

AML1 – A1E expressing cells

AML1/ETO

Peaks of endogenous AML1protein largely overlap toAML1/ETO peaks.Only 9% of AML1 peaksdisappear after expression ofAML1/ETO.

HEB – control cells

HEB – A1E expressing cells

AML1/ETO

AML1/ETO causes a majorrearrangement in HEB bindingprofile.

Wild-type

AML1/ETO

Gardini et al., PLoS Genetics, 2008

AML1 and HEB binding on chr.19

Role of HEB in AML

1- Heb is normallyexpressed only inLin- cells (whichsubpopulation?)

2- HEB is expressedspecifically in cellsderiving from a mousemodel of AML1/ETO-dependent leukemia

Growth curve of AML1/ETO expressing U937 cells

0

200000

400000

600000

800000

1000000

1200000

0 24 48 72 96

time (hours)

No.

of

cells

3- In the absence of HEB,AML1/ETO expression inducesapoptosis or cell cycle arrest

HEB siRNA

control

In the absence of HEB, cells cannot sustain AML1/ETO expression

-AML1/ETO binding is not restricted to promoters, nearly half ofrecruitment is within the gene body

-AML1/ETO does not function primarily by displacing native AML1

-AML1/ETO brings HEB on its target regions and dramaticallysubverts HEB positioning in the genome

-Indirect transcriptional regulation may derive from delocalization ofHEB and/or other TF for their target promoters

Other transcription factors (PU.1, AP1,...)?

Control of LSC functions

AML fusion proteins regulate genes involved in self-renewal of HSC

GO stem cell

Sel-renewingstem cells

Stem cells Progenitors Mature cells

Actively cycling cells

Unfrequentlydividingcells

GO stem cell

Sel-renewingstem cells

Stem cells Progenitors Mature cells

Actively cycling cells

Unfrequentlydividingcells

GO stem cell

Sel-renewingstem cells

Stem cells Progenitors Mature cells

Actively cycling cells

Unfrequentlydividingcells

Post-mitoticcells

Senescence Apoptosis

Bmi-1

p21

p16 p53

p18

p21

p21 cell-cycle inhibitor implicated in HSC maintenance

G0G0p21X

HSC

HSC HSC HSC

PROG PROG PROG

G0

HSC quiescence is essential forself-renewal; in the absence ofcell-cycle inhibitor p21, there israpid exhaustion of the HSCcompartment upon proliferativestimuli (serial transplantation,myelosuppressive chemotherapy)

Cheng, Science 2000

p21 is essential for HSC self-renewal

AML fusion proteins up-regulate p21 expression

Leukemia

3-8 ms later

WT Lin- A1E or PRLeukemia

transplantation

No Leukemiap21-/- Lin- A1E

transplantation

No Leukemia

2-11 ms later

Leukemiap21-/- Lin- P/R

p21 is essential for leukemogenesis

An impairment of HSC functions (e.g. diminished self-renewal andfunctional exhaustion) was recently described in mice deficient inseveral genomic-maintenance pathways, due to accumulation of

genomic damage (Nature 2007)

- Why is p21 essential for AML oncogene-dependent transformation?

- Which “negative effect” is exerted by AML fusion proteins on HSC thatis compensated by p21?

Does AML fusion protein expression and/or p21 lossinduce accumulation of DNA damage?

WHY??

AML fusion proteins induce DNA damage

Murine Lin- cells

DAPI/γH2AX/GFP

Human CD34+ cells

DAPI/γH2AX

In the absence of p21, there is accumulation of DNAdamage after AML fusion protein expression

p21 prevents accumulation of DNA damage

LSC proliferate more in the absence of p21

p21 cell cycle restriction in LSC

50-fold decrease of LSC in a p21-/- background

p21 prevents exhaustion of LSC

In the absence of p21, fusionprotein expression induces HSCproliferation and accumulation ofDNA damage.

Expression of AMLfusion proteins inducesDNA damage and up-regulation of p21

Upon transformation, uncontrolledproliferation leads to furtheraccumulation of DNA damage, functionalexhaustion of LSC and cell death

Up-regulation of p21 inLSC induces cell cyclerestriction, which allowsfor DNA repair.

The net result is themaintenance of a pool ofLSC carrying a moderatedegree of damaged DNA.

SC

Oncogeneexpression

Progenitors, other cells(More differentiated)

Activation of ap21-dependent

checkpoint

DNA damage

Cell cycle restrictionAccumulation

of DNA damage

PrActivation of ap53-dependent

checkpoint

Apoptosis/SenescenceClearance of

the damaged DNA

Unique checkpoint regulation in SCs

Biological implication

Therapeutic implication

Initial tumorResidual non-cycling

cancer stem cells Relapse

Chemotherapy

Quiescent LSCs might be responsible for tumorrelapse after chemotherapy

Inhibition of DNA-repair mechanisms may beincompatible with AML oncogene expression

Microarray Unit:Simone MinardiElisa Venturini

Alessandro Gardini

Natalia MeaniSilvia LicciulliGaia ScafettaMarco Saia

PG Pelicci

Andrea Viale

Francesca De FrancoAnnette OrlethValeria Cambiaghi

Matteo CesaroniLucilla Luzi

HEBThank you!