CELL-LINES: TRANSCRIPTION THERAPY BY RA

Transcription

1 PML/ PML/ RAR RAR CELL-LINES: TRANSCRIPTION THERAPY BY RA HDAC Co-repressors HDAC RA 1-6 M Coactivators HAT PML-INDUCED DIMERISATION: ENHANCED COREPRESSOR BINDING RARA AND REPRESSION BY HISTONE DEACETYLATION RARA TARGETS ACTIVATED Dose of RA? Arsenic? clinical remission differentiation

2 NBs ARE DISRUPTED IN APL PML PML + PML/RARA HUMAN APL CELLS Recruits proteins to facilitates PTM. Controls P53 activation PML/RARA dominant negative effects on PML: apoptosis resistance, proliferation? PLZF/RARA: PML NBs unaffected Immediately corrected by RA and arsenic treatment in vivo Daniel, Blood 1993, Koken EMBO J 1994, Weiss, Dyck Cell 1994

3 RA OR ARSENIC DEGRADE PML/RARA Zhu PNAS 1997

4 PML/RARA DEGRADATION PATHWAYS NLS RING B B Coiled-Coil DNA HORMONE As 2 O 3 K49 D522 RA AF2 Binding+Oxidation NB reformation SUMO conjugation RNF4-induced Poly-ubiquitination 19S association Dose-dependent DEGRADATION DEGRADATION Zhu PNAS 1997, 1999, Lallemand JEM 21, Nat Cell Biol 28 Jeanne Cancer Cell 21

5 As DIRECTLY CROSS-LINKS PML Zhang, Science, 21; Jeanne. Cancer Cell 21

6 As TRIGGERS PML OXIDATION (SUMO-INDEPENDENT ) Jeanne Cancer Cell 21

7 ROS- & ARSENIC -INDUCED DIMERISATION CONTROL MATRIX TRANSFERT & SUMOYLATION Zhang, Science, 21; Jeanne. Cancer Cell 21

8 A CC MOTIF CONTROLS ARSENIC BINDING, NB-FORMATION, SUMOYLATION & BIOLOGICAL RESPONSE B2 BOX: SIYCRGCSKPLCC SCALLDSSHSELKCDIS B2 KPLCCSCALLDSS Mutations in Arsenic-resistant patients Jeanne Cancer Cell, Zhang Science, 21 Goto Blood 211

9 WHAT DID WE LEARN FROM THE MOUSE? Differentiation does not underlies APL clearance PML/RARA loss required for APL cure through activation of a PML/P53 senescence axis The RA/Arsenic combination is synergistic in mice and patients Arsenic clear APL by inducing oxidative stress

10 UNCOUPLING RARA DEGRADATION AND TRANSACTIVATION ds Ablain, JEM 213

11 DIIIF TRANPLANTING APL WITH DIFFERENTIATED BLASTS! Never clonogenic 1 3 granulocytes are clonogenic in vivo TRANSCRIPTIONAL ACTIVATION: DIFFERENTIATION? PML/RARA DEGRADATION: LOSS OF SELF-RENEWAL Ablain, JEM 213

12 PMLXXXXXXXXXxxx GENETIC UNCOUPLING OF DIFFERENTIATION & APL CLEARANCE PML/RARA APLs Nasr et al. Nat Med 28

13 % survival RA INDUCES DOSE-DEPENDENT LOSS OF CLONOGENIC ACTIVITY Day 3 PML/RARA High None HIGH Low Intermediate 2 4 6

14 PLZF/RARA DNA GENETIC UNCOUPLING OF DIFFERENTIATION & APL CLEARANCE Nasr et al. Nat Med 28

15 NO LOSS OF SELF-RENEWAL AND CLONOGENIC ACTIVITY Day 3 PLZF/RARA 2 2 n

16 WHAT DID WE LEARN FROM THE MOUSE? Differentiation does not underlies APL clearance PML/RARA loss required for APL cure through activation of a PML/P53 axis The RA/Arsenic combination is synergistic in mice and patients Arsenic clear APL by inducing oxidative stress

17 ARRAYS TO EXPLORE THE BASIS FOR APL RESPONSE PML-RARA PLZF/R ARA As RA 1.5 RA 1 RA 1 RA1/A s RA 1 Differentiation ++ delayed APL clearance PML/RARA degradation + +/

18 TRANSCRIPTOMIC ANALYSIS a PML/RARA WT PML/RARA* PLZF/RARA RA1.5 RA1 RA1 RA1 RA1 RA1 RA1 Blast differentiation Loss of leukemiainitiating activity b RA1.5 & 1 & /- + PML/RARA WT PML/RARA WT PML/RARA* PLZF/RARA RA1 & 1 not 1.5 RA1 RA1 RA1 6h 12h 6h 12h 6h 12h 6h 12h 6h 12h Differentiation p<1 Genes induced by RA1-1, -7 p<1-7 NS NS p<1 but not RA1.5, -5 p<1 are -7 p<1 associated -7 p<1-7 NS p<1 with -7 a cell cycle arest signature. Cell P53 cycle arrest targets. NS NSP53 stabilisation: p<1 PML/RARA, not PLZF/RARA -7 NS p<1-7 NS NS NS NS c 6h 12h RA: Ø h 12h RA: Ø PML/RARA PLZF/RARA p53 p53 b-ac n b-ac n d p = SASP Julien Ablain, Nature Medicine 214 E2F NS

19 % survival % survival % GFP posi ve cells in the BM BM of APL mice Spleen weight (g) P53 REQUIRED IN APL CLEARANCE a 1 b Untreated RA1 day 3 c Day 6 ctrl sh p53 ctrl +RA1 p=1-5 sh p53 +RA1 p53 -/- p53 +/ ,2 p53 +/ p53 P53 extinction retard RA response, but -/- not differentiation! 2 µm,6.6 Survival benefit reflects LIC clearance P,4.4 and requires P53,8.8,2.2 untreated RA1 d3 RA1 d6 d days p53 +/ days p53 -/- e 12 1 Day 3 p53 +/ Day 3 p53 -/ untreated RA1 treatment Time to death (day) Time to death of secondary recipients (day) untreated RA1 Julien Ablain, Nature Medicine 214

20 NOT APOPTOSIS, SENESCENCE-LIKE

21 PML NUCLEAR BODIES Disrupted by PML/RARA Restored by therapy Apoptosis, Senescence, Stem Cell quiescence, P53 control?

22 12h) RA1 Spleen weight (g) % survival Fold induc on (12h) untreated Spleen weight (g) % survival % survival % survival RA1 untreated Spleen weight (g) % survival % survival a RARA 53 Bone marrow 6h PML & NB-REFORMATION DRIVE P53 ACTIVATION & LIC CLEARANCE DAPI Bone marrow 6h e marrow 6h e pml +/+ DAPI pml 5 µm b 1.2 1,2 1. 1,8.8,6.6,4.4,2.2 pml +/+ pml -/- 1. PML/RARA 1 p53,6.6 b-ac n PML/RARA.2 p53 p53 (longer expo) RA: Ø h 12h PML/RARA RA: Ø p53 b-ac n pml a b e 5 µm pml +/+ pml +/+ pml -/- pml -/- RA: Ø p=3.1-4 p=.5 p=3.1-4 p=.5 untreated RA1 d3 RA1 d6 6h 1.2 1,2,8.8,4.4,2 b-ac n 12h f pml 6 f b 6h 1.2 1,2 1. 1,8.8,6.6,4.4,2.2 12h untreated RA1 d3 RA1 d6 pml +/+ pml -/- untreated RA1 d3 RA1 d6 f c p=3.1-4 g p=.5 cell death 7.3E-6 signal Pathway transduction name p-value 1.1E-5 tissue remodeling 2.1E-5 cell death 7.3E-6 protein secretion 3.5E-5 signal transduction regulation of cell proliferation1.1e-5 1.6E-4 tissue remodeling p53 response 2.1E-5 2.E-3 protein secretion 3.5E-5 regulation of cell proliferation 1.6E-4 6 p53 response 2.E-3 g 5 c Pathway name c Pathway name 12 cell death signal transduction 7 day treat. 1 tissue 7 remodeling day treat. protein secretion regulation 8 of cell proliferation p53 response day treat Time to death (day) Time 2 to death 4 (day) 6 d pml +/+ pml -/ p53 S/P A Day Gene 3 RA1 RA1 RA1 RA1 Day Symbol 1 Nr4a X X 1 Vdr X X X 8 Gs X Tgm P2rx X 6 Bcl2l X 6 Cd X X Lif X X 4 Intu Serpine X X X 2 Ddit X X Klk1b Ndrg X X Gm Lrp X Mmp Time to Il1a death of secondary recipients (day) X X X Asb Cxcl X X Time to Dgke death of 1.535secondary recipients (day pml +/+ : untreated RA1 pml -/- : 1.5<FI<2 2<FI<3 untreated 3<FI<5 5<FI RA1 PML-RARA WT p-value d h Day Time to death of secondary recipients (day pml +/+ : untreated RA1 pml -/- : untreated RA1 p-value 7.3E-6 1.1E-5 2.1E-5 3.5E-5 1.6E-4 2.E-3 Nr4a1 (Nur77) Serpine1 (Pai-1) pml +/+ Time : to death untreated (day) RA1 pml -/- : untreated RA1 RA: h PML-RARA* PLZF-RARA Nr4a1 (Nur77) Serpine1 (Pai-1) pml +/+ pml -/- p53 S/P A Gene RA1 RA1 pml +/+ RA1 RA1 h Symbol pml -/- Nr4a X p53 S/P X A Vdr Gene X X X RA1 RA1 RA1 RA1 Gs2 Symbol X Nr4a1 Tgm X X Vdr P2rx X X X Gs2 Bcl2l X Cd X X Tgm Lif X X P2rx X Intu Bcl2l X Serpine X X X Julien Ablain, Nature Medicine 214

23 PML PML PML PML PML PML PML.5,1.1 A NEW MODEL FOR RA-RESPONSE? Day 3 untreated RA1 RA1/As /+ pml -/- pml +/+ pml -/- h Transcrip onal repression RA Blast differen a on RARa PML RARa RA i PML PML PML tra on NB disrup on PML NB reforma on Partner recruitment P53 E2F Loss of self-renewal RA-RESISTANCE OF PLZF/RARA APL

24 WHAT DID WE LEARN FROM THE MOUSE? Differentiation does not underlies APL clearance PML/RARA loss required for APL cure through activation of a PML/P53 senescence axis The RA/Arsenic combination cures APL in mice and patients Arsenic clear APL by inducing oxidative stress

25 RA / ARSENIC ANTAGONIZE FOR DIFFERENTIATION!

26 RA/ARSENIC CLEARS APL (3 DAYS) Control ATRA As2O3 ATRA+As2O3 Lallemand, JEM 1999, Rego PNAS 2, Nasr, Nat Med 28

27 RA/ARSENIC SYNERGY IN MICE Lallemand JEM 1999

28 SYNERGISTIC DEGRADATION? NLS RING B B Coiled-Coil DNA HORMONE As 2 O 3 K49 D522 RA AF2 Binding+Oxidation NB reformation SUMO conjugation RNF4-induced Poly-ubiquitination 19S association Dose-dependent DEGRADATION DEGRADATION Zhu PNAS 1997, 1999, Lallemand JEM 21, Nat Cell Biol 28 Jeanne Cancer Cell 21

29 ML ML RA1/As PML PML RA1 untreated Rela ve expression (12h) Spleen weight (g) T of second Rela ve d ARSENIC TARGETS NORMAL PML PROTEIN 25.5 b-ac n 2 untreated RA1 ENHANCING NB RE-FORMATION ctrl pml-i pml-i K16R pml -/- APL Bone marrow 6h DAPI 5 µm pml e Nr4a1 (Nur77) pml +/+ pml -/- h ,5 Pai-1 (Serpine1) RA/arsenic, transplantation 2 at Day 3 Transcrip onal repression RARa RARa pml +/+ pml -/- pml -/- : RA RA1 Gene Symbol PML f,7.7 pml +/+,6.6,5.5,4.4,3.3,2.2,1.1 Day 3 untreated RA 1.5<FI<2 2<FI<3 3<FI<5 5<FI pml +/+ pml +/+ pml -/- RA int RA int/as RA int RA int/as Nr4a Vdr Cd Lif Serpine Ddit Ndrg Il1a Time to death of secondary recipients (day) RA1 Julien Ablain, Nature Medicine 214 p=. B differ

30 *+, # *+, # *+, # *+, # *+, #! " # $%$ &D" : %=32"=28 &. / >?-/ I &! " # '%' &D " : %=32"=28 &. / >?-/ I & A NEW MECHANISM FOR RA/ARSENIC SYNERGY: DUAL TARGETING OF PML (" =>" C%#32"I 28& *+, &)2O2)I & *+, & 82S3"8"$1%& >@6&. /. ² #. /. ² # *+, # *+, # >3" EF7*6&(38#" +A : G=>=>" ) *&6$) $897" 56& V%6"%#28& *+, &"I I 1#9"$1%& / I & / I & *+, # *"3=%23& 32#3: 9=( 2%=& +, - """"". /" >+: " #C6*" Julien Ablain, Nature Medicine 214

31 APL PATIENTS? Patients get definitively off treatment. Both drugs can be oral! Shen, Hu, PNAS 24, 29 Lu Blood 22 LoCoco NEJM 213, Zhu JCO 213

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33 A MIRACLE OR A MODEL? Transcriptional activation unessential PML/RARA degradation required, but insufficient Nuclear body reformation and P53 activation underlie APL cure. RARA drives, but PML cures: Consistent with mutations in resistant APLs (PML/RARA, PML or P53 ) The mighty mouse: APL cell lines p53 mutants!

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35 NB are druggable: PML induction by IFN NB biogenesis by Oxidative stress Trigger degradation of subsets of proteins? Trigger P53 activation and senescence?

36 NUCLEAR BODY PHARMACOLOGY IFN/ARSENIC to drive partner sequestration or degradation.. Loss of self-renewal APL, ATL, CML, others? Protein degradation (viral, PolyQ, others?) The APL miracle may be prolonged

37 Lck-TAX- OR HTLVI-DRIVEN ATL PML-DEPENDENT SENESCENCE? DEGRADATION PML-DEPENDENT? El Sabban, Blood 22, El Hajj, J. Exp. Med. 21

38 Slow regression Prolonged complete remissions

39 Jun ZHU Valérie LALLEMAND Rihab NASR Marie-Claude GUILLEMIN Omar FERHI Juliane HAFTERMEYER Julien ABLAIN Zhu CHEN Marion JEANNE Laurent PERES PP PANDOLFI Caroline BERTHIER Ali BAZARBACHI Hassane SOIHILI Saverio MINUCCI Kim RICE