The photoreceptor program in Group 3 Medulloblastoma: Role of the TFs NRL and CRX
Celio Pouponnot Institut Curie UMR3347 CNRS / U1021 INSERM
Institut Curie - C. Pouponnot - 1 21 février 2018 Medulloblastoma (MB)
MB
cerebellum
Pediatric tumor of the cerebellum (Median age 7 yrs) Most frequent Malignant brain tumor of childhood Treatment : surgery, chemotherapy, radiotherapy WNT G4 è70-80% overall survival at 5 years SHH èImportant secondary effects G3
2 21 février 2018 Medulloblastoma (MB)
Ø Based on gene expression profil : 4 different molecular groups
MB subtype WNT HH 3 4 incidence 10% 25% 25% 40%
Overall survival Very good Intermediate Bad Intermediate (meta+++) (meta++)
MYC amplification MYCN & CDK6 Driver pathway WNT HH OTX2 amplification amplification
MYC & GABAergic & Expression Photoreceptor Neuronal & WNT signaling SHH signaling photoreceptor glutamatergic signature markers signature signature
• Cancers often express aberrant differentiation programs unrelated to the tissue of origin
•Although express as a result of cancer cell plasticity, they are not thought to actively participate to cancer progression.
•Group 3 MB expresses an aberrant photoreceptor differentiation of unknown significance. 3 3 21 février 2018 Group 3 are enriched in active enhancer/SE controling photoreceptor genes
4 21 février 2018 Group 3 are enriched in active enhancer/SE controling photoreceptor genes
Same enrichment observed in the analysis of enhancer-gene target in MB TF/SE interaction identifies two master regulators of the photoreceptor lineage: CRX and NRL
5 21 février 2018 NRL or CRX are involved in MB growth
CRX KD NRL KD
CRX or NRL KD increases survival of orthotpicallygrafted mice
Institut Curie 6 21 février 2018 NRL in Medulloblastoma MAF
NRL TA EHR DB LZ
Role of NRL in medulloblastoma
NRL belongs to the TF of the oncogenic MAF family (Multiple Myeloma)
NRL expression is restricted to the photoreceptors of the retina (not expressed in the cerebellum)
Role in photoreceptor terminal differentiation (KO NRL: lack of a subset of photoreceptors)
Mutations of NRL in human (retinitis pigmentosa)
è NRL has not yet been demonstrated to be involved in oncogenesis
Role of NRL in medulloblastoma?
Institut Curie 7 21 février 2018 NRL is not expressed during cerebellar development
Institut Curie 8 21 février 2018 Transcriptomic analysis of NRL KD in D458MED shNRL Mean shNRL Ctl rep1 rep2 rep3 NCOA1 ZNF385A
Up CYB5R1 PURB HNRNPU CRX FXR2 GNGT1 ARR3 RPS20 MRFAP1L1 NSF AIPL1 FAM57B NDUFS8 UNC119 NDRG3 ATP1A3 ELOVL2 SMUG1 STXBP1 PBX1 ABCA4 Down PDE6B CPE EPB41L2 PPP5C MEGF9 GNB3 PDC ALDOC ACTR1B RP1 GNGT2 RCVRN VTN SLC6A6 PDE6A RBP3 PLEKHB1 GUCA1A −27 0 4.8 Photoreceptor genes
Institut Curie 9 21 février 2018 Transcriptomic analysis of NRL KD in D458MED shNRL Mean shNRL Mean shNRL Ctl shNRL Ctl rep1 rep2 rep3 rep1 rep2 rep3 NCOA1 SMARCD3 ZNF385A LOC645513 RREB1
Up CYB5R1 PURB AMOTL2 HNRNPU TBC1D8 CRX JAG2
FXR2 Up CREBL2 GNGT1 DOK6 ARR3 CPLX3 RPS20 ERLIN1 MRFAP1L1 AK1 NSF FGD4 AIPL1 DDX31 FAM57B NDUFS8 IMPG2 UNC119 FLJ27352 NDRG3 SSX2IP ATP1A3 ARL4D ELOVL2 RNF5 SMUG1 TMEM57 STXBP1 GTPBP8 PBX1 KCTD21 ABCA4 Down PDE6B MXRA7 CKB CPE ICK EPB41L2 Down SPATA20 PPP5C NLK MEGF9 GNB3 ADCK1 PDC GNB3 ALDOC USH2A ACTR1B MAK RP1 ARL6 GNGT2 RD3 RCVRN KIAA1107 VTN NRN1 SLC6A6 TESK2 PDE6A FSTL5 RBP3 CNTN1 PLEKHB1 GUCA1A NRL −27 0 4.8 −7.1 0 3.4 Photoreceptor genes Group3 genes NRL regulates the photoreceptor differentation program and some of the group 3 specific genes
Institut Curie 10 21 février 2018 In vitro KD of NRL
Ø Growth curves/Viability assay
D458Med: DAOY : endogenous NRL expression no endogenous NRL expression
5 12,0 12,0 shCtl 8,0 8,0 shNRL #1 4,0 4,0 Count x10 Count shNRL #2 0,0 0,0 Cell 0 2 4 6 8 0 2 4 6 8 Day Day
PDX culture 1 PDX culture 2
3,0E+04 2,0E+05 NI 2,0E+04 shCtl 1,0E+05 1,0E+04 shNRL #1 Luminescence
0,0E+00 0,0E+00 0 2 4 6 0 2 4 6 shNRL #2 Day Day
NRL is required for cell viability/growth
11 Institut Curie 11 21 février 2018 NRL protects MB cells from apoptosis
Ø Apoptosis : cleaved caspase 3 (FACS)
60 *** 40 *** 20 NRL KD 0 induces apoptosis in MB cell % apoptotic cells apoptotic % shCtl shCtl lines and in PDXs shNRL #1 shNRL #2 shNRL #1 shNRL #2 shNRL D458Med DAOY
PDX culture 1 PDX culture 2 100 ** 100 ** ** 80 80
60 * 60
40 40
20 20
% apoptotic cells apoptotic % 0 0 NI #1 NI shCtl shCtl shNRL #1 shNRL shNRL #2 shNRL shNRL #2 shNRL shNRL
12 Institut Curie 12 21 février 2018 Transcriptomic analysis of NRL KD in D458MED
List of NRL correlated genes in human MB samples
Institut Curie 13 21 février 2018 Transcriptomic analysis of NRL KD in D458MED
List of NRL correlated genes in human MB Top 100 genes samples
Institut Curie 14 21 février 2018 Transcriptomic analysis of NRL KD in D458MED
shNRL Mean shNRL Ctl rep1 rep2 rep3 RALGPS2 ARHGAP28 PALMD Up LOC100130155 ATP5A1 CRX BCL2L1 GNGT1 ENOSF1 IMPG2 SSX2IP BCMO1 ARL4D PCK2 AIPL1 List of NRL correlated Genes significantly EML1 CALCOCO2 genes in human MB Top 100 genes deregulated ABCA4 PDE6B samples following NRL KD C14orf129 KCNH6 CTBS HSBP1L1
Down MNAT1 GNB3 USH2A MAK PHYHD1 PDC SLC1A7 CERS4 ARL6 RD3 RP1 DBP EPS8 GNGT2 RCVRN VTN GPR160 NRL
−6.2 0 4.6 NRL correlated genes
A Subset of the top 100 genes the expression of which is correlated to that of NRL are deregulated in KD NRL cells.
Institut Curie 15 21 février 2018 NRL and BCL-XL expression are correlated
Fattet dataset Wnt HH 3 4 XL - NRL Fattet dataset BCL-XL GSE12992 XL expression of BCLof -1.5 0 2.8 - Log2 expression Log2
corr.score: 0.719 / rank: 31/18901 genes) BCL Log2 expression of Wnt HH 3 4 NRL
Kool dataset Wnt HH 3 4 XL - NRL Kool dataset BCL-XL GSE10327 XL expression of BCLof -1.5 0 2.4 - corr.score: 0.349 / rank: 925/18901 genes expression Log2 Log2 expression of BCL NRL Wnt HH 3 4
Northcott dataset #1 Wnt HH 3 4 XL - NRL Northcott dataset #1 BCL-XL GSE21140 XL expression - of BCLof -1.5 0 3.1 Log2 expression expression Log2 corr.score: 0.622 / rank 131/22370 genes BCL Log2 expression of NRL Wnt HH 3 4
Northcott dataset #2 HH 3 4 XL - Northcott dataset #2 NRL BCL-XL GSE37385 XL expression - of BCLof -1.3 0 3.7 Log2 expression Log2 corr.score: 0.516 / rank: 82/15896 genes BCL Log2 expression of Wnt HH 3 4 NRL
Institut Curie 16 21 février 2018 BCL-XL is a NRL direct target gene
-1694 -1681
MARE consensus TGCTGACTCAGCA ChIP known MAF hRhodopsin TGCCGATTCAGCC hInsulin TGCAGCCTCAGCC target genes 20,0 hBCL-XL TGCTGCCTCAGCC
10,0 +1 BCL-XL promoter Fold induction Fold 0,0 Ctl Ctl Ctl Ctl NRL Western-Blot NRL NRL NRL promoter 3’UTR promoter 3’UTR DAOY D458MED PDX culture 1 shNRL shNRL D458Med DAOY Empty vector NRL shCtl #1 #2 shCtl #1 #2 NRL BCL-XL β-actin
NRL binds to the BCL-XL promoter and induces its expression
Institut Curie 17 21 février 2018 IS BCL-XL a potential therapeutic target in MB?
shBCL-XL 100
- Ctl #1 #2 cells 80 60 BCL-XL 40
apoptotic 20
β-actin % 0 - Ctl #1 #2 shBCL-XL
EC shbclxl D458D458MED shBclxL PDX2 shCtrl (1/8) 100 Ctl (0/7) shCtrl (0/7) 100 Ctl (1/8) shBCL-XL #1 (0/8) shBclxL#3 (0/8) shBclxL#3 (4/8) (%) ) shBCL-XL #1 (3/8) ) (%)
% shBclxL#5 (7/8) ( % shBCL-XL #2 (1/8) shBclxL#5 (1/8)
shBCL-XL #2 (6/8) ( l
l a a v i v 50 i
50 v v Survival r r Survival u u S S
0 0 0 20 40 60 0 20 40 60 DayDay DayDay
KD of BCL-XL induces apoptosis in MB and increases survival
18 Institut Curie 18 21 février 2018 BCL2 proteins, as potential therapeutic targets
Ø BCL inhibition using pharmacological agents : TW37
è Target the BH3-binding groove BH3 domain of Bcl Bcl-2 anti-apoptotic in Bcl-2 anti-apoptotic proteins pro-apoptotic protein protein
In vivo experiments
Inhibition of Tumor regression tumor formation
Institut Curie 19 21 février 2018 19 TW37 induces MB regression
In vivo experiments : tumor regression
TW37 TW37
100 0 10 12 16 18
5 Ctl (n=12) Ctl TW37 Ctl TW37 10 Start p<0.005
Number of of Number 1 TW37 (n=11) photons/s x10
0,1 D9 D19 7 12 17 22 before treatment after treatment Day
1
0,8 0,6 Inhibitor of BCL anti-apoptotic 0,4 Survival 0,2 family induces tumor regression p<0.0001 0 0 20 40 60 80 100
Stop TW37 treatment
Institut Curie 20 21 février 2018 Model
NRL and CRX are master regulators of the Group 3 MB photoreceptor program
Photoreceptor transcriptional program is a dependency in Group 3 MB
NRL control cell cycle by inducing CCND2 expression
NRL protects MB from apoptosis by inducing BCL-XL expression
BCL-XL as a potential therapeutic target in Group 3 MB
Institut Curie 21 21 février 2018 Team Signaling and Cancer progression Alexandra
Chloé Bertrand Morgane Alain
Magalie Sabine
Charlène Celio
Collaborations: O. Ayrault/ F.Do z / O. Delattre (Institut Curie) Paul Northcott (St Jude) R.J. Weschler-Reya (Burhnam Institute) Charles Lin (Baylor College of Medecine) C. Haberler (Inst. Of Neurology) FranckInstitutInstitut Bourdeaut Curie Curie --nomnom dede(Institut l’émetteur l'émetteur - -Curie)TitreTitre dede lala présentationprésentation 22 21 février 2018 Group 3 MB is enriched in photoreceptor genes
Kool et al., 2008 and Cho et al. 2011
23 21 février 2018 NRL and CRX control photoreceptor genes
Genes with high H3K27ac in NRL and CRX high G3 tumors
Correlation of H3K27ac and expression
Correlation with presence of NRL/CRX co-motifs
Enrichment of photoreceptor genes
24 21 février 2018 BCL2 proteins, as potential therapeutic targets
Ø BCL inhibition using pharmacological agents : TW37
In vivo experiments
Inhibition of Tumor regression tumor formation
Start TW37 Stop Orthotopic treatment treatment grafting Tumor growth & Tumor growth Mice survival
D0 D10 D18
TUMORS DEVELOPED 25 Institut Curie 25 21 février 2018 NRL and CRX control photoreceptor genes
NRL and CRX control the expression of photoreceptor genes in a subset a G3 tumors
26 21 février 2018 NRL controls BCL-XL expression
RT-qPCR BCL-XL mRNA 3,0 Western-Blot * 2,0 DAOY D458MED PDX culture 1 shNRL shNRL 1,0 Empty NRL shCtl #1 #2 shCtl #1 #2 Relative Relative ** ** * vector
expression ** 0,0 ** ** NRL NRL shCtl shCtl shCtl
Empty vector BCL-XL shNRL #2 shNRL shNRL #1 shNRL shNRL #1 shNRL shNRL #2 shNRL shNRL #1 shNRL shNRL #2 shNRL β-actin DAOY D458Med PDX PDX culture1 culture 2 p<0,05
BCL-XL is a direct NRL target gene
Institut Curie 27 21 février 2018 NRL and CRX expression in G3 and G4 subtypes
Institut Curie 28 21 février 2018 BCL-XL is a NRL target gene
ChIP
20,0 -1694 -1681
MARE consensus TGCTGACTCAGCA 10,0 known MAF hRhodopsin TGCCGATTCAGCC TGCAGCCTCAGCC target genes hInsulin hBCL-XL TGCTGCCTCAGCC induction Fold 0,0 Ctl Ctl Ctl Ctl NRL NRL NRL NRL +1 BCL-XL promoter promoter 3’UTR promoter 3’UTR
D458Med DAOY
NRL binds to the BCL-XL promoter in MB BCL-XL is direct target of NRL in group3 MB
29 Institut Curie 29 21 février 2018 NRL and MYC expression in G3 and G4 subtypes
Institut Curie 30 21 février 2018 NRL controls the cell cycle in MB
DAOY ONS-76 D458MED HDMB03 80,0 80,0 100,0 *** *** Ctl 60,0 80,0 shNRL #1 60,0 60,0 shNRL #2 60,0 *** 40,0 cells 40,0 40,0 ***
% 40,0 20,0 20,0 20,0 20,0
0,0 0,0 0,0 0,0 S S S S G2+M G2+M G2+M G0/G1 subG1 G0/G1 subG1 G0/G1 subG1 G2+M G0/G1 subG1
NRL KD leads to a decrease in S phase and an increase in subG1
Institut Curie 31 21 février 2018 NRL controls CyclinD2 expression
6,0 CCND2 4,0 ChIP 2,0
0,0 Fold induction IP : Ctl NRL Ctl NRL Ctl NRL Ctl NRL promoter 3’UTR promoter 3’UTR D458MED DAOY
DAOY D458MED PDX D458MED 4,0 2,0 1,2 1 2 shNRL 3,0 * 0,8 Ctl #1 #2 2,0 1,0 0,4 NRL expression 1,0 * Relative CCND2 * ** ** ** 0,0 0,0 0,0 ** CCND2 vect NRL Ctl #1 #2 Ctl #1 #2 Ctl #1 #2 β-actin shNRL shNRL shNRL
NRL binds to the CCND2 promoter and regulates its expression
Institut Curie 32 21 février 2018 Role of BCL-XL in NRL-mediated apoptosis
50 40 cells Empty vector BclxL 30 shNRL shNRL 20 shCtl #1 #2 shCtl #1 #2 10
apoptotic 0 %
NRL NI NI shCtl shCtl Bcl-xL shNRL#1 shNRL#2 shNRL#1 shNRL#2
β-actin Empty vector BCL-XL
BCL-XL overexpression rescues NRL-induced apoptosis
è BCL-XL is a key mediator of NRL-mediated protection against apoptosis
Institut Curie 33 21 février 2018 The retina: the window of the brain
Ganglion cell Layer Inner plexiform layer
Inner nuclear layer Outer plexiform layer Outer nuclear layer Inner segment Outer segment Pigment epithelium
Choroïd
The retina is a laminar structure composed of different cell type
GCL: Ganglion cells INL: Amacrine, Horizontal, Bipolar and Müller cells (Glia) ONL: Photoreceptors
CRX and NRL are master regulator of a subtype of photoreceptor
Institut Curie 34 21 février 2018 Maf transcription factors and cancer v-maf was initially identified as an oncogene transduced in the genome of avian retrovirus AS42 that induces musculo-aponeurotic fibrosarcoma Nishizawa et al., PNAS 1989
Musculo- Aponeurotic Fibrosarcoma
LTR Δgag v-Maf Δenv LTR AS42 avian oncogenic retrovirus Maf transcription factors belong to the AP1 superfamily
JUN MAF
FOS CREB
ATF Maf transcription factors belong to the AP1 superfamily
(Musculo-Aponeurotic Fibrosarcoma) Transactivation Leucine zipper Domain EHR domain GSK3 p38 p38 S14 S49 T53 T57 S61 S65 T134 S336 c-Maf Basic domain MafA Large MAF MafB
NRL
MafF / MafG / MafK (Small Maf)
Eychène, Rocques & Pouponnot, Nat. Rev. Cancer, 2008 b-Zip Maf well established oncogenes
MAF
Maf are well established ONCOGENES : (No data on NRL)
in vitro: Maf transformed CEF (Nishizawa et al., Oncogene 2003; Pouponnot et al., Oncogene 2006)
in animal models: Transgenic mice developed T and B cell lymphomas (Morito et al., Cancer Res 2006; 2011)
in human: Translocations in Multiple Myeloma (8-10%) and overexpression (50%) (Chesi et al., Blood 1998; Hanamura et al., Jpn J. Cancer Res. 2001;Boersma-Vreugdenhil et al., Br. J. Haematol. 2004)
Overexpression sufficient / no activating mutation found in human
Eychène, Rocques & Pouponnot, Nat. Rev. Cancer, 2008 Maf oncogenes
MAF Maf are well established oncogenes :
BUT
Oncogenic role uncoupled from their physiological function
-do not control major cellular functions such as cell cycle, apoptosis, migration…
-Terminal differentiation transcriptional control of tissue-specific genes that control a specific aspect of a given tissue.
work on this paradox
ECTOPIC EXPRESSION exert oncogenic activity in tissues where they are not normally expressed
Eychène, Rocques & Pouponnot, Nat. Rev. Cancer, 2008 NRL in Medulloblastoma MAF
NRL TA EHR DB LZ
Role of NRL in medulloblastoma
NRL expression is restricted to the photoreceptors of the retina (not expressed in the cerebellum)
Role in photoreceptor terminal differentiation (KO NRL: lack of a subset of photoreceptors)
Mutations of NRL in human (retinitis pigmentosa)
Fulfill he criterias of MAF-mediated transformation
Role of NRL in medulloblastoma?
è NRL has not yet been demonstrated to be involved in oncogenesis NRL overexpression in MB
Wnt HH 3 4 Fattet dataset NRL GSE12992 NRL -1.5 0 2.8 expression Wnt HH 3 4
Wnt HH 3 4 300 Kool dataset NRL GSE10327 NRL
-1.5 0 2.4 expression Wnt HH 3 4 200
Wnt HH 3 4 Northcott dataset #1 GSE21140 NRL 100
-1.5 0 3.1 RelativeNRL expression NRL expression Wnt HH 3 4 0 HH 3 4 Northcott dataset 76
- 1 2 #2 NRL D458 NRL GSE37385 DAOY MB PDX ONS -1.3 0 3.7 expression Wnt HH 3 4
NRL is overexpressed in the metastatic MB group 3 How NRL is expressed in group 3 MB
OTX2
AGGGGATTAG
NRL
-850
During normal retina development , OTX2 is able to regulate NRL expression (Montana et al., J Biol Chem (2011); Kautzmann et al., J Biol Chem (2011)
OTX2 is amplified in group 3 MB
Does OTX2 regulates NRL expression in MB? How NRL is expressed in group3 MB
ChIP shOTX2 Ctl #1 #2
60,0 OTX2 3'UTR 40,0 Promoter β-actin 20,0 Fold induction 0,0 NRL IP : Ctl NRL Ctl NRL D458MED DAOY β-actin
OTX2 is bound to the NRL promoter in the D458 MB cell line OTX2 KD decreases NRL expression
OTX2 regulates NRL expression in MB OTX2 expression OTX2 expression OTX2 expression Fattet Northcott Wnt Wnt dataset dataset HH HH #1 3 3 4 4 is not restricted
OTX2 expression OTX2 expression Northcott Kool Wnt Wnt dataset to group3 group3 to MB dataset HH HH #2 3 3 4 4 Epigenetic modifications of the NRL locus in MB
WNT vs G3 SHH vs G3 Others vs G3 G4 vs G3 0.036 4.9e−06 2.7e−06 0.00024
0.8
0.6
Methylation 0.4
Wnt HH 3 4 medulloblastoma
G3 specific enhancer are The NRL locus is hypomethylated in G3 %MB nucleated at the NRL locus
Epigenetic modifications and OTX2 expression drive NRL expression in Group 3
Collaboration with Dr. P Northcott (St Jude) C. Lin & F.Bourdeaut& Dr. O. Delattre (Institut Curie) Model
OTX2, epigenetic deregulation
NRL
Other genes : Photoreceptor differentiation Photoreceptor genes BCL-XL, CCND2
? Medulloblastoma
First demonstration of oncogenic activity of the MAF family member, NRL
NRL protects MB from apoptosis by inducing BCL-XL expression
High potential of anti-BCL therapy in MB
Photoreceptor differentiation program is instrumental to group3 MB growth through NRL Genes with high H3K27ac in NRL/CRX high G3 tumors are enriched in photoreceptor genes
Institut Curie - nom de l'émetteur - Titre de la présentation 47 21 février 2018 Team: Raf and Maf signaling in oncogenesis and development Alain Eychène & Celio Pouponnot Staff scientists:
Alain Eychène DR1 INSERM Celio Pouponnot CR1 CNRS Sabine Druillennec CR1 INSERM Laure Lecoin IR2 CNRS Magalie Larcher IE2 Curie
Non permanent:
Alexandra Garancher PhD Coralie Dorard PhD Céline Barbotin PhD Morgane Morabito PhD
Collaborations F. Bo u rd e a u t Curie U830 O. Ayrault Curie UMR3347/U1021 O. Delattre Curie U830 F. D o z Curie Pédiatrie S.Puget Necker G. Fortin CNRS Neuro PSI GIF J. Champagnat CNRS Neuro PSIGIF
Institut Curie - nom de l'émetteur - Titre de la présentation 49 21 février 2018 Institut Curie - nom de l'émetteur - Titre de la présentation 50 21 février 2018 Institut Curie - nom de l'émetteur - Titre de la présentation 51 21 février 2018 Institut Curie - nom de l'émetteur - Titre de la présentation 52 21 février 2018 Institut Curie - nom de l'émetteur - Titre de la présentation 53 21 février 2018