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
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 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,...)?
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
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??
In the absence of p21, there is accumulation of DNAdamage after AML fusion protein expression
p21 prevents accumulation of DNA damage
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