Activation of a PML/TP53 axis underlies acute promyelocytic. leukemia cure

Transcription

1 Activation of a PML/TP53 ais underlies acute promyelocytic leukemia cure Julien Ablain, Kim Rice, Hassane Soilihi, Aurélien de Reynies, Saverio Minucci & Hugues de Thé a PLZF-RARA vs PLZF-RARA pathway name RA low & int & high RA int & high not low RA int & high source h 1h h 1h h 1h h 1h h 1h GO:37 immune system process 7.E%15 1.3E%17 1.E%.E% *** *** * *** * ** GO:955 immune response.5e% 7.1E%1.7E%.1 * ** * *** * * GO:95 defense response 1.3E%7.E%11.E%.1E% * ** NS *** NS * GO:1 membrane 1.E%7 1.1E%9 1.E%.9 ** NS NS ** NS NS GO:95 response to eternal stimulus 1.3E%5 1.E%9 1.7E%3 3.1E% NS ** NS *** NS NS GO:59 response to stimulus 5.E%5 1.5E%9.9E%.E% NS *** NS *** NS *** GO:95 inflammatory response 1.E%.E%9.1E% 1.E% NS ** NS *** NS * GO:5177 response to other organism 1.E%5.1E%.. * * NS * NS NS GO:911 response to wounding.e% 5.7E%.1E%3 3.5E% NS * NS *** NS NS GO:5 plasma membrane 9.7E%.E%..93 * NS NS *** NS NS GO:917 response to bacterium 3.E%.7E%7 1. * * NS NS NS NS GO:31 intrinsic to membrane 3.E%.3E%7 5.1E%.95 ** NS NS * NS NS GO:7 receptor activity.9e%7.1e%5.7e%.9 * NS NS * NS NS GO:399 myeloid cell differentiation 7.E%7 1.E%.5 9.E% ** NS NS NS NS NS GO:5135 interphase E%7 NS NS NS NS NS NS GO:517 regulation of cell cycle..5.e%.e%7 NS * NS NS NS NS GO:59 chromosome E%7 NS NS NS NS NS *** GO: cell cycle process e%7 NS * NS NS NS *** GO:7 DNA replication initiation E%7 NS NS NS NS NS ** GO:75 cell cycle checkpoint.71.1.e% 1.7E%7 NS * NS NS NS ** GO:95 response to stress 3.3E%.E%7 5.E%.3E% NS *** NS ** NS *** GO:1 DNA-dependent DNA replication E%9 NS * NS NS NS *** GO:79 cell cycle e%9 NS ** NS NS NS *** GO:97 response to DNA damage stimulus E%9 NS ** NS NS NS *** GO:59 DNA metabolic process E%1 NS ** NS NS * *** GO: DNA replication e%1 NS ** NS NS * *** NS:P>1 %5 *:1 % <P<1 %5 **:1 %11 <P<1 % ***:P<1 %11 b Untreated RA low RA high h % of cells 7 h Nature Medicine: doi:1.13/nm.31

2 c % of GFP positive cells RA: Ø low high d Fold induction ( h) Serpine1 Dusp5 Egr RA: low int high int high RA: low int high int high RA: low int high int high Fold induction ( h) Fold induction ( h) PLZF-RARA PLZF-RARA PLZF-RARA e % Annein-V positive cells Untreat. Do RA int RA high f Relative Nucleus Area (piels/mm)," 3,5" 5,",3" 15," 1,1" 5 P =. P = 3.1 " RA: untreat"ra"low"ra"high" Ø Figure S1: High-dose RA triggers cell cycle arrest with features of senescence, not apoptosis in mouse APLs. (a) GO terms associated with genes differentially regulated in the different eperimental settings. The p values are indicated. (b) Cell-cycle analysis of GFP-positive bone marrow cells from APL mice treated with low (1.5) or high (5) RA doses for h or 7 h. DNA content was assessed by propidium iodide staining. Percentages of cells in each cell-cycle phase are shown in a representative histogram. (c) Analysis of GFP-marked leukemia cells in RA-treated APL mice. Mouse APLs were stably transduced with the MLS.1SHC-GFP retroviral vector and GFP-positive APL bone marrow cells were sorted and re-transplanted into recipient mice. APL mice were treated with low (1.5) or high (5) RA doses for 7 h and GFP-positive cell percentages were assessed by flow cytometry. (d) Epression levels of Serpine1, Dusp5 and Egr1 in bone marrow cells of h RA-treated APL mice relative to untreated (mean of three independent mice, Nature Medicine: doi:1.13/nm.31

3 measured by Q-PCR). (e) Proportion of Annein-V-positive bone marrow cells in APL mice treated for 1 h as indicated. Doorubicin (Do) was used as a positive control. (f) Quantification of nucleus size in GFP-marked leukemia cells following treatment of APL mice with RA low (1.5) and RA high (5) after 7 h (n = 1). a 1. Trp53 Fold induction (1 h) Untreat. RA int RA high b Trp53 ctrl#1 ctrl# sh#1 sh# c Relative colony numbers 1 1 P =.5 P =. Untreated RA 1 7 M ctrl sh Trp53 #1 sh Trp53 # d 7 d 1 1 d 3 P =. n = P = 5.1 n Time to death (d) Time to death of secondary recipients (d) ctrl #1 sh Trp53 #1 ctrl #1 + RA int sh Trp53 #1 + RA int treatment f Survival benefit (RA int vs untreated) P =. Trp53 +/+ Trp53 /! e 7 d d P =.5 n = P =.1 n Time to death (d) Time to death of secondary recipients (d) ctrl # sh Trp53 # ctrl # + RA int sh Trp53 # + RA int treatment g RA 1 M: Trp53 +/+ Trp53 /! + + Figure S: A key role for Trp53 activation in RA-induced loss of APL-initiating cell self-renewal in vivo. (a) Epression levels of Trp53 in bone marrow cells from APL mice treated with RA int (1) or RA high (1) for 1 h relative to untreated (untreated: n =, RA int: n =, RA high: n =, measured by epression arrays). (b) Nature Medicine: doi:1.13/nm.31

4 Western blot analysis of Trp53 in -transformed mouse hematopoietic progenitors after retroviral transduction of vectors epressing scrambled shrnas (ctrl #1 and ctrl #) or shrnas directed against Trp53 (sh #1 and sh #). (c) Relative numbers of colonies (sh #1 or #/ctrl colony ratios) formed by transformed mouse hematopoietic progenitors after retroviral transduction of vectors epressing scrambled shrna (ctrl) or shrnas against Trp53 (sh #1 and sh #), and grown in methylcellulose for 7 d in the presence or absence of 1 7 M RA. Data represent the mean ± SD of three independent eperiments. (d) & (e) Left panels: survival of mice inoculated with APL cells transduced with the vectors described in (b), and treated for 7 d with RA int (1). Right panels: survival of secondary recipients of bone marrow cells from the mice described above but that had been treated for 3 d with RA int (1). (f) Quantification of the survival benefit (treated/control survival ratios) of 7 d RA int (1) treatment of mice injected with Trp53 +/+ or Trp53 / APL cells (n=). (g) Western blot analysis of in Trp53 +/+ or Trp53 / APL cells treated e vivo by 1 M RA for 1 d. Nature Medicine: doi:1.13/nm.31

5 a Pml d Survival benefit (RA int vs untreated) ctrl sh#1 sh# P = Pml +/+ Pml /! b P = P =. Relative colony numbers 1 1 Untreated ctrl sh Pml #1 sh Pml # e Pml +/+ RA 1 RA 7 M Pml /! RA 1 M: + + c Bone marrow of APL mice Pml /! Pml +/+ CD11b Pml /! Pml+/+ Gr1 Untreated RA int d 3 RA int d Figure S3: Loss of Pml impedes RA-triggered APL clearance. (a) Western blot analysis of Pml in -transformed mouse hematopoietic progenitors after retroviral transduction of vectors epressing scrambled shrna (ctrl) or shrnas directed against Pml (sh #1 and sh #). (b) Relative numbers of colonies (sh #1 or #/ctrl colony ratios) formed by transformed mouse hematopoietic progenitors after retroviral transduction of vectors epressing scrambled shrna (ctrl) or shrnas against Pml (sh #1 and sh #), and grown in methylcellulose for 7 d in the presence or absence of 1 7 M RA. Data represent the mean ± SD of three independent eperiments. (c) MGG staining and FACS analysis of bone marrow cells of mice injected with Pml +/+ or Pml / APL cells and treated with RA int (1) for 3 or d. (d) Quantification of the survival benefit (treated/control survival ratios) of 7 d RA int (1) treatment of mice injected with Pml +/+ or Pml / APL cells (n=). (e) Western blot analysis of in Pml +/+ or Pml / APL cells treated e vivo by 1 M RA for 1 d. Nature Medicine: doi:1.13/nm.31

6 a Pathway'name GO:313()(regulation(of(tissue(remodeling GO:517()(positive(regulation(of(secretion GO:91()(regulation(of(apoptosis GO:191()(regulation(of(cell(death GO:571()(positive(regulation(of(protein(secretion GO:19()(cell(death GO:15()(death GO:715()(signal(transduction GO:99()(regulation(of(signal(transduction GO:57()(regulation(of(protein(secretion GO:1()(regulation(of(cell(communication GO:915()(apoptosis GO:151()(programmed(cell(death GO:19()(positive(regulation(of(cell(death GO:771()(tissue(remodeling GO:93()(secretion GO:51()(positive(regulation(of(protein(transport GO:51()(regulation(of(secretion GO:71()(cell(surface(receptor(linked(signaling(pathway GO:93()(protein(secretion GO:53()(negative(regulation(of(cellular(process GO:775()(multicellular(organismal(development GO:117()(regulation(of(cytokine(production GO:99()(negative(regulation(of(signal(transduction GO:71()(multicellular(organismal(homeostasis GO:()(induction(of(apoptosis(by(etracellular(signals GO:11()(cytokine(production GO:3373()(response(to(vitamin GO:715()(cell(communication GO:59()(response(to(stimulus GO:3()(negative(regulation(of(apoptosis GO:39()(negative(regulation(of(programmed(cell(death GO:5()(negative(regulation(of(cell(proliferation GO:95()(response(to(eternal(stimulus GO:5()(negative(regulation(of(cell(death GO:97()(endocytosis GO:35()(positive(regulation(of(apoptosis GO:737()(regulation(of(ERK1(and(ERK(cascade GO:73()(cellular(ion(homeostasis GO:3()(positive(regulation(of(programmed(cell(death GO:397()(response(to(cytokine(stimulus GO:917()(induction(of(apoptosis GO:35()(developmental(process GO:17()(regulation(of(cell(proliferation GO:15()(induction(of(programmed(cell(death GO:553()(bone(resorption GO:59()(homeostatic(process GO:5171()(cellular(response(to(stimulus GO:513()(regulation(of(protein(transport GO:5715()(positive(regulation(of(cytokine(secretion p53(response p,value 5.7E)7 1.E) 1.E).E).5E) 7.3E) 7.5E) 1.1E)5 1.E)5 1.E)5 1.E)5 1.E)5 1.7E)5.E)5.1E)5 3.1E)5 3.E)5 3.3E)5 3.E)5 3.5E)5 3.E)5 3.7E)5 3.7E)5 3.E)5.1E)5 5.E)5.5E)5.7E)5 7.E)5.E)5.E)5.7E)5 9.E)5 1.E) 1.1E) 1.3E) 1.E) 1.E) 1.E) 1.5E) 1.5E) 1.5E) 1.5E) 1.E) 1.E) 1.E) 1.7E) 1.7E) 1.7E) 1.7E).E)3 b RA: Nra1 Vdr Gs Tgm Pr7 Bcll15 Cd3 Lif Intu Serpine1 Ddit3 Klk1b1 Ndrg Gm191 Lrp1 Mmp5 Il1a Asb Ccl Dgke Validation h - 1 h Pml /! Pml +/+ Pml /! Trp53 I H I H I H I H target Pml +/+ Log induction Figure S: Pml-dependent transcriptional changes. (a) GO terms associated with genes deregulated in bone marrow cells of RA-treated Pml +/+ APL mice but unaffected in those of their Pml / counterparts. Genes differentially regulated between Pml +/+ and Pml / APL cells in untreated mice were ecluded. (b) Heatmap showing epression levels of the genes described in Figure 5b in bone marrow cells of RA int- (I: 1) and RA high (H: 1)-treated Pml +/+ or Pml / APL mice relative to untreated mice. Genes reported to be transcriptionally regulated by Trp53 or demonstrating Trp53 binding in non-apl cell lines (L. Attardi, personal communication) are indicated. Nature Medicine: doi:1.13/nm.31