Leucemogenesi e proteina di fusione AML1/ETO

Transcription

1 Verona, 21 maggio 2009 Leucemogenesi e proteina di fusione AML1/ETO Myriam Alcalay Genomica Funzionale Dpt.o Oncologia Sperimentale, Istituto Europeo di Oncologia Dpt.o Medicina, Chirurgia e Odontoiatria, Università degli Studi di Milano

2 t(8;21) Acute Myeloid Leukemias AML1 (DNA-directed Transcription Factor) ETO (Co-repressor of Transcription) Normal Leukemic Down-regulation of genes involved in myeloid differentiation Up-regulation of genes involved in HSC maintenance

3 AML1/ETO-mediated transcriptional regulation

4 Structure of AML1/ETO

5 Genomic analysis Strategy to: 1) correlate AML1/ETO binding pattern with effects on transcription 2) characterize AML1/ETO binding regions with an unbiased approach A. Gardini et al., PLoS Genet Nov;4(11):e

6 Transcription in the Genomic Era Expression Array Analysis of over 47,000 transcripts Genomic Tiling Array Analysis of protein binding patterns throughout the whole genome = array-based chromatin immunoprecipitation (ChIP-chip)

7 The model system: U937 cell line ZnSO 4 U937 stably transfected with AML1/ETO under the control of a metallothionein promoter (mt) U937 differentiate into monocytes upon treatment with VitD3 + TGFβ. AML fusion proteins transfected into U937 differentiation block U937-Mt U937-A1E VD3+ TGFβ VD3+ TGFβ

8 Expression array HGU133v2.0Plus AML1/ETO RNA Control RNA 1316 regulated genes (FC>1,5) 55% decreased 45% increased

9 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

10 Expression-binding correlation 1316 regulated genes 2513 occupied promoters 358 direct targets 70% 30% DOWN UP (247) (111) Not all AML1/ETO-dependent transcriptional regulation is associated to promoter binding (27.2%) Not all AML1/ETO binding events result in transcriptional regulation (14.2%) AML1/ETO binds to the promoters of upregulated genes

11 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

12 AML1/ETO binding on chr.19 Topography of AML1/ETO peaks

13 Sequence analysis AML1/ETO binding regions show enrichment of specific sequences that are consensus binding sites for 4 known transcription factors: AML1, Ets1, AP.1 and the E-protein HEB.

14 HEB is an interactor of AML1/ETO E proteins interact with ETO and AML1/ETO Heb is an interactor of AML1/ETO in U937

15 AML1 and HEB binding on chr.19 AML1 control cells AML1 A1E expressing cells AML1/ETO Peaks of endogenous AML1 protein largely overlap to AML1/ETO peaks. Only 9% of AML1 peaks disappear after expression of AML1/ETO. HEB control cells HEB A1E expressing cells AML1/ETO causes a major rearrangement in HEB binding profile. AML1/ETO Wild-type AML1/ETO Gardini et al., PLoS Genetics, 2008

16 Role of HEB in AML 1- Heb is normally expressed only in Lin- cells (which subpopulation?) 2- HEB is expressed specifically in cells deriving from a mouse model of AML1/ETOdependent leukemia No. of cells Growth curve of AML1/ETO expressing U937 cells time (hours) control HEB sirna 3- In the absence of HEB, AML1/ETO expression induces apoptosis or cell cycle arrest In the absence of HEB, cells cannot sustain AML1/ETO expression

17 -AML1/ETO binding is not restricted to promoters, nearly half of recruitment is within the gene body -AML1/ETO does not function primarily by displacing native AML1 -AML1/ETO brings HEB on its target regions and dramatically subverts HEB positioning in the genome -Indirect transcriptional regulation may derive from delocalization of HEB and/or other TF for their target promoters Other transcription factors (PU.1, AP1,...)?

18 Control of LSC functions

19 AML fusion proteins regulate genes involved in self-renewal of HSC Stem cells Progenitors Mature cells Sel-renewing stem cells Bmi-1 p18 Actively cycling cells p21 GO stem cell Unfrequently dividing cells p16 p53 Post-mitotic cells Senescence Apoptosis

20 p21 p21 cell-cycle inhibitor implicated in HSC maintenance p21 X HSC G0G0 HSC HSC HSC G0 PROG PROG PROG HSC quiescence is essential for self-renewal; in the absence of cell-cycle inhibitor p21, there is rapid exhaustion of the HSC compartment upon proliferative stimuli (serial transplantation, myelosuppressive chemotherapy) Cheng, Science 2000 p21 is essential for HSC self-renewal

21 AML fusion proteins up-regulate p21 expression

22 p21 is essential for leukemogenesis WT Lin- A1E or PR Leukemia Leukemia 3-8 ms later transplantation 2-11 ms later transplantation p21 -/- Lin- P/R Leukemia No Leukemia p21 -/- Lin- A1E No Leukemia

23 WHY?? - Why is p21 essential for AML oncogene-dependent transformation? - Which negative effect is exerted by AML fusion proteins on HSC that is compensated by p21? An impairment of HSC functions (e.g. diminished self-renewal and functional exhaustion) was recently described in mice deficient in several genomic-maintenance pathways, due to accumulation of genomic damage (Nature 2007) Does AML fusion protein expression and/or p21 loss induce accumulation of DNA damage?

24 AML fusion proteins induce DNA damage Murine Lin- cells Human CD34+ cells DAPI/γH2AX/GFP DAPI/γH2AX

25 p21 prevents accumulation of DNA damage In the absence of p21, there is accumulation of DNA damage after AML fusion protein expression

26 LSC proliferate more in the absence of p21 p21 cell cycle restriction in LSC

27 50-fold decrease of LSC in a p21-/- background p21 prevents exhaustion of LSC

28 Expression of AML fusion proteins induces DNA damage and upregulation of p21 Up-regulation of p21 in LSC induces cell cycle restriction, which allows for DNA repair. The net result is the maintenance of a pool of LSC carrying a moderate degree of damaged DNA. In the absence of p21, fusion protein expression induces HSC proliferation and accumulation of DNA damage. Upon transformation, uncontrolled proliferation leads to further accumulation of DNA damage, functional exhaustion of LSC and cell death

29 Biological implication Unique checkpoint regulation in SCs Progenitors, other cells (More differentiated) Pr Activation of a p53-dependent checkpoint Apoptosis/Senescence Clearance of the damaged DNA Oncogene expression DNA damage SC Activation of a p21-dependent checkpoint Cell cycle restriction Accumulation of DNA damage

30 Therapeutic implication Chemotherapy Initial tumor Residual non-cycling cancer stem cells Relapse Quiescent LSCs might be responsible for tumor relapse after chemotherapy Inhibition of DNA-repair mechanisms may be incompatible with AML oncogene expression

31 Thank HEB you! Alessandro Gardini Natalia Meani Silvia Licciulli Gaia Scafetta Marco Saia Microarray Unit: Simone Minardi Elisa Venturini PG Pelicci Andrea Viale Francesca De Franco Annette Orleth Valeria Cambiaghi Matteo Cesaroni Lucilla Luzi