© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. or or I.J.-L. ( Paris, France. Translational Research, Institut Curie, Paris, France. France. Frankfurt am Main, Germany. Paris, France. Denis Diderot, CNRS UMR 8550, PSL, Paris, France. Institut de Biologie de l’École Normale Supérieure (IBENS), PSL Research University, Paris, France. Group for Pediatric Biology, Institute of , München (LMU), Munich, Ludwig-Maximilians-Universität Germany. 7 U1163, Laboratory of Embryology and Genetics of Congenital Paris Malformations, Descartes - Sorbonne Paris Cité University, Imagine Institute, Paris, France. Research Center, Paris, France. Research University, INSERM, U830, Equipe Labellisée Ligue contre le Cancer, Paris, France. National de la Recherche Scientifique (CNRS), Unité Mixte de Recherche (UMR) 8104, University Paris Descartes UMR-S1016, Paris, France. 1 tem sys a nervous have sympathetic peripheral childhood the of in tumor a cancer neuroblastoma, a of die who people six in one Nearly treatment aspect modules. tumors evocative of by of type deconvoluted CRC CRC lines: We and the (NCCs). nervous Neuroblastoma Hermann Rohrer Sandrine Grossetête-Lalami Angel M Carcaboso Birgit Geoerger Virginie Raynal Valentina Boeva by transcriptional circuitries Heterogeneity of neuroblastoma cell identity defined Nature Ge Nature Received 19 December 2016; accepted 28 June 2017; published online 24 July 2017; Gustave Gustave Roussy, Vectorology and Anticancer Therapies, UMR 8203, CNRS, Université Paris-Sud, Université Paris-Saclay, Villejuif, France. Institut Curie, Paris Sciences et Lettres (PSL) Research University, INSERM, U900, Mines-ParisTech, Paris, France.

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13 8 been shown to act as major drivers of neuroblastoma oncogenesis. In shown to been oncogenesis. as act of major drivers neuroblastoma landscape of (PDXs), landscape xenografts five six of patient-derived which had DBH and TH of expression and SH-EP cells a exhibiting S without substrate-adherent phenotype phenotype), (N DBH and TH enzymes biosynthetic noradrenergic consistent is expressing and processes result neurite-like displaying cells This SH-SY5Y with respectively. II, and I groups in included and SH-EP, SH-SY5Y its subclones, were whereas line, heterogeneous cell SK-N-SH phenotypically the included These II. and I groups Four analysis. between position this an intermediate occupied lines in cell neuroblastoma lines hNCC the resembled closely II Group comprising the GIMEN, SH-EP and GICAN neuroblastoma cell lines. ( ( lines hNCC both in or lines cell in at two neuroblastoma least identified of super-enhancers number changes. Principal component analysis (PCA) based on scores by ROSE the algorithm were defined Table 1 identity cell about information integrative provide analysis, motif sequence and further defined by super-enhancer mapping of H3K27 acetylation (H3K27ac) be can which CRCs, of neuroblastoma. program expression gene the this CRCs work, we the have transcriptional determined , Valérie Combaret , Elise Diaz Fig. 1 Fig. 6 We examined a panel of 25 neuroblastoma cell lines ( doi:10.1038/ng.392 , , Alban Lermine a ) and two primary human NCC (hNCC) lines 3 4 ): group I, with 18 neuroblastoma cell lines, and group II, II, group and lines, cell neuroblastoma 18 with I, group ): , , Simon Durand Institut Institut Curie Genomics of Excellence (ICGex) Platform, Institut Curie 10 9 3 , LPS-ENS, LPS-ENS, Université Pierre et Marie Curie (UPMC), Université , 10 18 , Bertrand Ducos

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© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. ( super- Most identity. cell enhancers of group sympathetic II overlapped of with super-enhancers of hNCC hallmark lines a be to appear differentiation TF network controlling normal sympathetic neuron and specification cell lines consistent with previous super-enhancer data on a few neuroblastoma ( PHOX2A strongest super-enhancers comprise a set of TF loci including for each group ( ( PCA the in included when I group with MYCN overall. population cell SK-N-AS the to normalized were RT-qPCR,by data and evaluated was Expression line. cell SK-N-AS ( ( bold. in shown are II I and group of SEs in enriched are motifs binding whose TFs lines. cell neuroblastoma in (bottom) member family JUN or a FOS with or (top) PHOX2B with a CRC in participate to ( Methods). Online in described are 1 (batches batch from were cells SK-N-SH lines. cell neuroblastoma of a panel in control, at binding H3K27ac for profiles ChIP-seq for I ( groups in scores H3K27ac median highest the with SEs 100 the for plot wild-type ALKwt, scores. log (SE) super-enhancer 1 Figure s r e t t e l  without or with I, group in lines n Fig. 1 Fig. Fig. 1b Fig. h d a (0–80 = 31), for the expression values of 22 TF genes identified in CRCs of cell lines. ( lines. cell of CRCs in identified genes TF 22 of values expression the for = 31),

Our analysis found found analysis Our We then sorted super-enhancers according to median H3K27ac signal PC2 –0.0 –0.06 –0.04 0.00 0.02 ) Module 2 PHOX2B c amplification ( amplification –0.024

), consistent with the results of ), results consistent the analysis. PCA with the , 14 GATA3 Super-enhancer landscape identifies various CRCs and identities in neuroblastoma cell lines. ( lines. cell neuroblastoma in identities and CRCs various identifies landscape Super-enhancer d Group , , HAND2 MNA MNA Cell line PDX , , 1 1 e PHOX2B HAND1 5 and and . . PHOX2B, HAND2 and GATA3 participate in a complex

–0.020 ALKw ALKmut I

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KLF7 SK−N−SH GIMEN , , MAF ALK hNCC -amp ALKw ALKmut ISL1 GICAN SH−EP GLIS3 GATA2 PHOX2A 2 , SK−N−AS Supplementary Table2 Supplementary SH−SY5Y MYCN F c NR3C1 IRF1 t and BHLHE41 IRF2 MAFF –0.012 IRF3 hNCC hNCC ALKw rep1 GLIS3 rep2 MEF2D FLI1 IRF1 Supplementary Supplementary Table 3 PRRX , ,

super-enhancers for 10 out of 18 cell cell 18 of out 10 for super-enhancers IRF2 RUNX GATA3 IRF3 t RUNX

1 FLI1 b MYCN TBX18 hNCC rep. hNCC rep. SK-N-FI CLB-CAR CLB-MA CLB-GA 1 MEF2D

FOSL1 Median normalized H3K27ac score 120,000 160,000

2 PRRX1 40,000 80,000 FOSL2 RUNX1

and the the and RUNX2 TBX18 2 1 amplification, and for three three for and amplification,

and and FOSL1 Fig. 1 Fig. 2 0 ALK AL FOSL2 KLF7 CIC, ER K TBX2 HAND2 −1 −0.8 −0.6 −0.4 −0.2 0 0.2 0.4 0.6 0.8 1 and and TCF4 ALK 4 0 2 F GATA2 a ). All PDXs clustered clustered PDXs All ). ALK PHOX2B GATA3 ). These findings are findings These ). ). h PHOX2A ; ALKmut, mutant mutant ; ALKmut, ) Pearson correlation matrix, based on RNA-seq data in neuroblastoma cell lines and PDXs PDXs and lines cell neuroblastoma in data RNA-seq on based matrix, correlation ) Pearson SE rank NFIB 6 0 PHOX2B INSM2 e [0–80] oncogene locus oncogene Cell ID i ). ). In group I, the HAND1 8 0 Group Module 9 0 10 MYT1 Module Module ZNF423 0 SEs, respectively. ( respectively. SEs, 36 L 1.00 HAND2 1 HIF1 I 7 Overlap withhNCCSEs No overlapwithhNCCSEs 100

37 A 2 1

4 PHOX2 8 18

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46 160,000 200,000 240,000 120,000 b 42 ) and II ( II ) and

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able at the transcript and protein levels in group II and hNCC lines lines hNCC and II group in levels protein and transcript the at able ( tumors primary and lines cell neuroblastoma most in expressed highly are TFs Both PHOX2A expression PHOX2B regulates development, ous system ( PHOX2A and ing motif common to several proteins, including PHOX2B est score. For group I, this analysis identified a TAATYYAATTA bind DNA sequence motifs enriched in the super-enhancers with the high intermediate the in and (SK-N-SH). group (SH-EP) II group (SH-SY5Y), I group in 4 Tables with ated associ super-enhancers indicate not did scores super-enhancer of analysis supervised Furthermore, PCA. the in identified were status PDXs ( 32

28 4.0

To TFs for driver groups I detect and II, we i-cisTarget used to find 33 0 38 2 0 35 i f ) Single-cell analysis showing heterogeneity of cell identity in the the in identity cell of heterogeneity showing analysis ) Single-cell ) Immunoblot analysis of PHOX2B, with vinculin as a loading a loading as vinculin with PHOX2B, of analysis ) Immunoblot BCL6

MYCN 2 c Supplementary Fig. 3 MY

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NB–69 CLB-MA CLB–GA SJNB1 CLB-PE Nature Ge Nature SK–N–FI SH–SY5Y GIMEN

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3 SK-N-BE(2)-C 1 3 3

1 SK-N-FI 8 - - - - - .

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. CRCs of the six PDXs were highly similar to those of the group I cell I cell group of the those to similar highly PDXs were six of the CRCs expressed in NCCs and/or mesenchymal derivatives. The are set latter the of TFs Most JUN. or FOS and PHOX2B with ciated in position the PCA had a CRC that TFs of several included sets asso ( member family JUN or FOS a or PHOX2B with either CRC a in be to predicted TFs for Fig. 10 ( II group to specific JUN and FOSL2 FOSL1, and I, II. group of landscape super-enhancer the influences of activity neuroblastoma group I the while super-enhancers AP-1 TF ture hNCCs FOS and JUN family members, both of which are expressed in imma ( lines hNCC the and II group in motif AP-1 in enrichment showed ( cells GICAN or SH-EP at enhancer 6 Fig. Supplementary ( lines cell neuroblastoma other all in expressed was but 1). (batch experiment ChIP-seq the in used ones the than noradrenergic more were 2) (batch experiment this 2 ( reads. MNA, cases. NCC-like and noradrenergic fully between continuum (Pearson tumors of set whole the in correlate negatively modules 2) (module NCC-like ( tumors. primary neuroblastoma 498 of a set in lines cell of CRCs in identified genes 2 Figure Nature Ge Nature ( lines Supplementary Figs. 8 Figs. Supplementary 2 A CRC calling algorithm ) and three in-house pairs. FC, fold change. ( change. fold FC, pairs. in-house three ) and Fig. 1 Fig. ), ), consistent with our i-cisTarget results. We therefore searched c

) Identity of tumor pairs at diagnosis and relapse shown by expression profiling. The series includes seven pairs from data set GSE65303 (ref. (ref. GSE65303 set data from pairs seven includes series The profiling. expression by shown relapse and diagnosis at pairs tumor of ) Identity Different identity of neuroblastoma primary tumors and impact of on cell identity. ( identity. cell on chemotherapy of impact and tumors primary neuroblastoma of identity Different a n 1 g Module PHOX2B etics 1 ). Because super-enhancer strength correlated linearly linearly correlated strength super-enhancer Because ). . These results suggest . that results suggest These in PHOX2B the participates 1

ADVANCE ONLINE PUBLICATION ONLINE ADVANCE and no PHOX2B Fig. 1 Fig. ). There was a corresponding lack of super- of lack corresponding a was There ). and and Supplementary Fig. 7 Fig. Supplementary GATA3 Module HAND2 9 , c 1 HAND1 9 g 9

TH Module 2 log FC identified PHOX2B as specific to group ). Cell lines showing an intermediate intermediate an showing lines Cell ). −1 −2 ). AP-1 is a heterodimer composed of composed heterodimer a is AP-1 ). PHOX2B 0 1 2 2 KLF7 and

PHOX2A GATA3

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d NR3C1 ) Treatment of SK-N-SH cells with chemotherapy favors cells with an NCC-like identity. Cells used in used Cells identity. NCC-like an with cells favors chemotherapy with cells SK-N-SH ) of Treatment IRF1

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IRF3 GLIS3 MEF2D

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f RUNX1 and and RUNX2

6 TBX18 amplification; FPKM, fragments per kilobase of transcript per million mapped mapped million per transcript of kilobase per fragments FPKM, amplification; - - FOSL1 FOSL2 All cell lines with with lines cell All Both types of identity are in observed several heterogeneous cell lines. members but lacking PHOX2B and noradrenergic marker expression. ized by expression of a distinct module including FOS and JUN family the TH enzymes and/or DBH, or (ii) an identity,NCC-like character of GATA3 and HAND2 PHOX2B, expression and including module either (i) a sympathetic noradrenergic identity, characterized by a CRC ity in neuroblastoma cell lines and suggest that individual cells assume ( population same the 2 within 1 or module of module TFs expressing cells comprise and heterogeneous were lines cell SK-N-SH and AS 12 Fig. ( II group or I group in TFs several of coexpression lated at the level. Immunoblot analysis confirmed the anticorre were modules These FOSL2. and FOSL1 included which PHOX2B, ule 1, included GATA3which and HAND2, 2, and module I groups and II ( distinguishing TF two main modules identified analysis This modules. TF connected fully define further ( expression gene with Fig. 1 Fig. Taken together, data these demonstrate a novel of type heterogene −1 −0.8 −0.6 −0.4 −0.2 0 0.2 0.4 0.6 0.8 1 i b ). Furthermore, single-cell analysis showed that the SK-N- the that showed analysis single-cell Furthermore, ). , , R ) Mean expression values of the noradrenergic (module 1) and and 1) (module noradrenergic the of values expression ) Mean Supplementary Fig. 13 Fig. Supplementary = −0.49, one-sided permutation test test permutation one-sided = −0.49, d b Average expression (log FPKM) Average expression (log FPKM) of module 2 of module 2 3.5 4.0 3.0 4.5 2.0 2.5 2.5 1.0 1.5 2.0 3.0 MYCN hNC rep Supplementary Fig. 11 Fig. Supplementary 2 C MNA tumors Non-MNA tumors amplification except one (CHP-212) had a had (CHP-212) one except amplification hNC rep a Average expression(logFPKM) SH-EP Average expression(logFPKM 2 ) Pearson correlation matrix for the 22 TF 22 the for matrix correlation ) Pearson 6 5 1 C SK-N-SH cisplatin and and of module of module Supplementary Table 6 Supplementary 4 1 7 1 SK-N-SH P Ctrl < 10 doxorubicin ), we used the latter to latter the used we ), SK-N-S 6 1 SH-SY5 SK-N-SH −10 ) SK-N-SH Ctrl s r e t t e l 8 H Supplementary Supplementary ) and define a define ) and Y Fig. 1 Fig. 2 8 h ): ): mod ).  - - - - © 2017 Nature America, Inc., part of Springer Nature. All rights reserved. two modules to explore primary tumor identity. All but two tumors tumors two but All identity. tumor primary explore to modules two the of expression average the used we Next, neuroblastomas. pri mary in module CRC noradrenergic PHOX2B–HAND2–GATA3 define a further and lines cell in observations our confirm therefore results These TF genes. module of NCC-like that the with correlated of expression that indicated ( genes TF module NCC-like the of expression in intercorrelations positive detected We 1. also module correla between positive tions strong were observed WeCRCs line tumors. cell these for in calculated identified module each of TFs the of primary sion of set large a from tumors data expression studied we tumors, primary neuroblastoma of characteristic also are lines cell lastoma without lines cell in represented were identities three all whereas identity, noradrenergic ( GATA3. SEs. and HAND2 PHOX2B, neuroblastoma by top 500 to occupied simultaneously are peaks H3K27ac ( motifs. at binding TF GATA3 binding and HAND2 PHOX2B, of identification 3 Figure s r e t t e l  ( module noradrenergic the of expression high showed

To explore whether the different identity classes seen in neurob in seen classes identity different the whether Toexplore d b e a Percentage of super-enhancers Bits 100 0 1 2 2 60 80 20 40

0 0 (0–80) (0–60) (0–180) (0–100) PHOX2B, HAND2 and GATA3 are master TFs defining the super-enhancer (SE) landscape of noradrenergic neuroblastoma. ( neuroblastoma. noradrenergic of landscape (SE) super-enhancer the defining TFs master are GATA3 and HAND2 PHOX2B, ( 1 PHOX2B n = 498; GEO GEO 498; = 2 To 10 PHOX2B 3 0 p 4 ( MYCN b 5 ) and and ) 100−200 6 PHOX2B To , , GSE4971 HAND2 p 7 ALK amplification ( amplification SE SE SE PHOX2B 8 PHOX2B ( H3K27ac PHOX2B HAND2 GATA3 200−300 9 c ) SEs. ( SEs. ) To and and 10 1 p ). Correlations between expres between Correlations ). Fig. 2 Fig. 11 , , HAND2 GATA3 12 PHOX2B HAND2 GATA3 d 300−400 ) CRC of activating TFs that define a noradrenergic module. ( module. noradrenergic a define that TFs activating of CRC ) Fig. 1 Fig. 13 To a ). Our analysis further further analysis Our ). p 14 expression levels in in levels expression and and 15 g ). 400−500 16 To GATA3 Binding by 17 p None GATA3 PHOX2B HAND2 PHOX2B–GATA3 HAND2–GATA3 HAND2–PHOX2B HAND2–PHOX2B–GATA3 18 PHOX2B HAND2 GATA3 i. 2 Fig. is anti- is Bits 0 1 2 b b , 1 c ). ). - - - - ) Tracks for ChIP-seq profiles for PHOX2B, HAND2, GATA3 and H3K27ac H3K27ac and GATA3 HAND2, PHOX2B, for profiles ChIP-seq for Tracks )

8 7 6 5 4 3 2 c more resistant to the three agents used ( used agents three the to resistant more were cells SH-EP lines. cell SH-SY5Y noradrenergic and SH-EP like NCC- the on chemotherapy of effect the investigated we response, treatment and identity cell of heterogeneity between link possible a ( stages disease two the between observed were pairs sample in diagnosis–relapse ten evaluated NCC multipotent avian from its differentiation with neuron consistent peripheral is of module promotion this from genes of downregulation 10 of in majority the was observed ( lines cell to the Similarly lines. for II cell group identity, as described NCC-like full with cases to rare may correspond expression module NCC-like and high low with noradrenergic two cases The remaining tumors. in primary identity of cell heterogeneity suggesting module, NCC-like of the values to low high from observed was A continuum Supplementary Fig. 14 Fig. Supplementary (0–80) (0–60) (0–180) (0–100) Next, expression of the NCC-like and noradrenergic modules was modules and noradrenergic NCC-like of the expression Next, −10 , two-sided Wilcoxon signed-rank test). A role for for role A test). signed-rank Wilcoxon two-sided , f HAND2 ) Summary of densities of 2,078 binding sites corresponding corresponding sites binding 2,078 of densities of Summary ) f

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© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. geting both types of cells during treatment. during cells of types both geting after Together,treatment. of the data importance tar these underline noradrenergic to NCC-like from and chemotherapy, under identity NCC-like to adrenergic from switch a on rely may This identity. cell in NCC-like cells supports the concept of plasticity in the reversion of The observation that tumors identity. at relapse are NCC-like not systematically an enriched to noradrenergic induce a also from may transdifferentiation treatment that possibility the However, exclude cannot resistance. we drug better with correlates thus identity like ( respectively 2, and 1 modules of expression increased or decreased the in resulted platin or cis Treatment doxorubicin with line cell SK-N-SH of parental the (−Dox). controls untreated two or doxycycline with treated xenografts two in staining hematoxylin and Welch’s unpaired tailed s.e.m.; (mean water drinking the to added was (−Dox) only sucrose or (Dox) mm ~170 of a volume reached tumors When 1783. shPHOX2B with transduced cells CLB-GA xenograft subcutaneous (mean 8 day at counted was cells living of number the and 0), (day ng/ml 100 10 (100%). doxycycline without cells shCTL-infected to relative doxycyline, without or with cultured 1783, shPHOX2B or 1437 shPHOX2B shCTL, ( group). per replicates 1 or ng/ml (100 doxycycline of presence or absence the in cells CLB-GA shRNA-treated of kinetics ( induction. doxycycline h after 72 at ( 1437) shPHOX2B targeting vectors shRNA 2 different ( targeting shRNA an with infected or (control) noninfected cells neuroblastoma CLB-GA in control) loading as vinculin (with immunoblot h by 72 at confirmed was treatment ( cells. neuroblastoma 4 Figure Nature Ge Nature binds directly PHOX2B tumors. and lines cell in observed were sion b e a c ) PHOX2B immunoblot of SH-SY5Y neuroblastoma cells infected with with infected cells neuroblastoma SH-SY5Y of immunoblot ) PHOX2B 3 PHOX2B Strong correlations among among correlations Strong

5 Cell index Tumor volume (mm ) Vinculi cells were plated in 24-well plates with or without doxycycline at doxycycline without or with plates 24-well in plated were cells 1,000 1,500 2,000 2,500 3,000 3,500 –1 50 0 1 2 3 4 ± 0 0 n 0 n s.d.; s.d.;

= 8 mice per group). *** group). per = 8 mice PHOX2B is critical for the growth of noradrenergic noradrenergic of growth the for critical is PHOX2B 9 8 7 6 5 4 3 2 1 0 –Dox Control n cells n etics 100 1 Time (h) = 6 replicates). ns, not significant. ( significant. not ns, = 6 replicates). Dox µ g/m Treatment time(d) l S *

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≤ x 0.05, two- 0.05, shPHOX2B l shPHOX2 –Dox *** 1783 expres

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x B l - - - lines with noradrenergic identity. noradrenergic with cell lines neuroblastoma of landscape super-enhancer the defining TFs, GATA3master are and HAND2 PHOX2B, that demonstrate results ( regions regulatory active within regions long that showed HAND2, PHOX2B and GATA3 bind ~400-bp- the same ( GATA3 and roblastoma were by super-enhancers co-occupied PHOX2B, HAND2 with TF binding sites. More than 90% of the strong and recurrent neu according to average super-enhancer score and evaluated intersection least two neuroblastoma cell lines. We ranked at super-enhancer regionsin predicted regions super-enhancer 4,336 of TFs these by pancy ( another’ssuper-enhancers one to bind and regulated GATA3super-enhancer are and HAND2 PHOX2B, that showing module, noradrenergic the of existence cal Fig. 16 Supplementary super-enhancers and also in the the in peaks H3K27ac the to and GATA3 ( lastoma binding for cell motifs line and PHOX2B,identified HAND2 neurob CLB-GA the in TFs these for analysis ChIP-seq performed development system nervous sympathetic during HAND2 protein toma treatment. toma neuroblas for strategy valuable a be could identities both targeting whether determine to help will experiments Further heterogeneity. cells of both identities, showing a previously unknown aspect of tumor comprise tumors primary identity. Most or NCC-like noradrenergic sympathetic to corresponding identities, tumor different predicate networks TF Distinct neuroblastoma. of landscape epigenomic and to PHOX2B tity therefore key lineage as TFs well toappearas these addicted to be mice in proliferation GATA3HAND2and with neuroblastline in sympathetic controlling GATA3on knockdown knockdown in several cell lines, which is consistent with previous data and which ( in detectable but low is line, expression protein cell and RNA PHOX2B CLB-PE the of identity noradrenergic the with ( identity noradrenergic a maintain to cells the for sufficient was expression residual the that suggested data RT-qPCR and RNA-seq to sufficient However, lines. was cell SH-SY5Y and CLB-GA the of identity decrease the affect PHOX2B whether evaluated then We growth impaired also tumor cells CLB-GA in PHOX2B of expression Decreased ( growth cell neuroblastoma ( protein PHOX2B of tem in noradrenergic CLB-GA and SH-SY5Y cells. Inducible decrease anti- inducible a doxycycline- generated we growth, on neuroblastoma knockdown suggested previously been has proliferation cell on neuroblastoma knockdown proliferation neuroblast decreased to leads structures autonomic understood. poorly remains neuroblastoma to position in mutations frameshift genes and super-enhancer-marked among It has been demonstrated that cancer dependencies can be found found be can dependencies cancer that demonstrated been Ithas Our work provides fundamental insights into the transcriptomic transcriptomic the into insights fundamental provides work Our We a observed reduction of proliferation upon Supplementary Fig. 6 Fig. Supplementary Supplementary Fig. 19 Fig. Supplementary 2 8 2 . To further document the consequence of of consequence the document further To . 9 Fig. Fig. 3 . PHOX2B i. 3 Fig. 2 n vivo in 3 , , and PHOX2B, HAND2 and GATA3 cross-regulate a ). ). Binding regions for all three TFs corresponded 1 e Fig. 4a Fig. 6 1 ). Additionally, positional binding analysis analysis binding positional Additionally, ). . Neuroblastoma cells of noradrenergic iden noradrenergic of cells Neuroblastoma . 1 7 ). ). These results therefore the confirm biologi ( short hairpin RNA sys hairpin short (shRNA) expression 5 , whereas conditional knockout of of knockout conditional whereas , ( i. 4e Fig. Supplementary Fig. 20 Fig. Supplementary ). 25 Fig. 3 Fig. Fig. 4c Fig. , Phox2b , b 2 PHOX2B 6 PHOX2B ) resulted in significant inhibition of of inhibition significant in resulted ) MYCN , its role in sporadic neuroblastoma neuroblastoma sporadic in role its , , d f ). This observation is consistent consistent is observation This ). and and ). We next investigated the occu the We investigated ). next , d -deficient mice completely lack lack completely mice -deficient and and super-enhancer ( are associated with predis with associated are , , 19 GATA3 upeetr Fg 18 Fig. Supplementary Supplementary Fig. 17 Fig. Supplementary , 2 2 7 4 . An effect of of effect An . . Although missense missense Although . Fig. 3 Fig. HAND2 , , ). These results areresults These ). HAND2 s r e t t e l 1 f 6 ). Together,). our . We therefore therefore We . Fig. 3b, and and and PHOX2B PHOX2B PHOX2B Phox2b GATA3 Fig. 1 Fig. c ALK and ). ). ). ).  ------f

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. provided the in-house pairs of diagnosis–relapse samples with the help of E.L., G.P. sequencing experiments. andG.S. participated in supervised the study design and lentiviral andparticles provided help with lentiviral infections. S.B. coordinated analysis. E.D. and B.D. generated the Biomark data. M.F.O. and T.G.P.G. generated and A.M.C. provided neuroblastoma PDXs. I.M. performed the reproducibility lines. A.L. performed alignment of RNA-seq and ChIP-seq data. E.D.-D., B.G., D.S. H.C.E. and S.T. provided hNCC cell lines and V.C. provided neuroblastoma cell contributed of chemotherapeutic agents. C.P.-E. performed the PHOX2B in the study design. A.P. generated and analyzed the doxycycline-inducible anti- C.L.-B. performed V.B. coordinated bioinformatics analysis and I.J.-L. coordinated the whole study. V.B. and I.J.-L. conceived the study, analyzed the data and wrote the manuscript. and the German Cancer Aid (DKH-111886 and DKH-70112257). Stiftung, the Walter Schulz Foundation, the Wilhelm-Sander-Stiftung (2016.167.1) Excellence Initiative, the Mehr LEBEN für krebskranke Kinder—Bettina-Bräu- Munich’s Institutional Strategy LMUexcellent within the framework of the German the Wilhelm-Sander-Stiftung. The laboratory of T.G.P.G. is supported by LMU 02). is H.R. supported by the Mayent-Rothschild program from Institut Curie and operated by the French National Research Agency (ANR) (ANR-11-IDEX-0005- funded by the French Government through its Investissement d R16100KK) and the “Who Am Laboratory ofI?” Excellence ANR-11-LABX-0071, (ARC-RAC16002KSA-R15093KS), Worldwide Cancer Research (WCR16-1294 (11-385). V.B. is supported by the ATIP-Avenir Program, the ARC Foundation Région Ile-de-France G.S. (21016711). is supported by the Annenberg Foundation Throughput qPCR-HD-Genomic Paris Centre platform supported by grants from -SiRIC Grant 4654. BiomarkINCa-DGOS- analysis was done using the High program); by the Canceropole Ile-de-France; and by the SiRIC-Curie program Consortium) from the Agence Nationale de la Recherche (Investissements d’Avenir grants ANR-10-EQPX-03 (Equipex) and ANR-10-INBS-09-08 (France Génomique performed by the ICGex NGS platform of the Institut Curie, supported by the AREMIG and the Association Thibault BRIET. High-throughput sequencing was de Leucémies l’Enfant et l’Adolescent (Fondation Enfants et Santé), the Fondation ARC Françaisethe Société (MAPY201501241), de Lutte contre les Cancers et les is supported by the Institut National du Cancer (PHRC-K14-175), the Fondation Mathieu, Dans les pas du Géant and Olivier Chape. The MAPPYACTS protocol Gouin “Enfance and Cancer,” Les Bagouz à Manon, les amis de Claire, pour Courir PHRC IC and2007-2009) by the following associations: Association Hubert de l’Enfant et l’Adolescent, the Institut National du Cancer (PRT-K14-061 and Françaisethe Société de Labellisée), Lutte contre les Cancers et les Leucémies grants from Institut Curie, INSERM, the Ligue Nationale contre le Cancer (Equipe Normandie) for providing tumor patient samples. This work was supported by Minckes, C. Blanc-Fournier and N. Rousseau (CHU, Tumorothèque de Caen Basse TBM, AC-2013-1786), M. Clapisson (CRB Centre Léon Bérard, AC-2008-101), O. patient samples. We thank D. Figarella-Branger CRB (BB-033-00097, AP-HM, CRB J. Maliash-Planchon and the Unité de Génétique Somatique for preparation of V. Bernard for pairs validation. We thank V. Saint-André for discussion, scientific their help in the identification of neuroblastoma diagnosis–relapse pairs and of Institut Curie. We thank N. Clément, T. Adam-de-Beaumais and B. Mallon for team, Pathologythe Experimental Department and the Plateforme Génomique for PHOX2B immunohistochemistry. We are grateful to the animal facilities O.data, respectively; Blanchard for help in cell culture experiments; and M. Caly F. Tirode and C. Kamoun for help with RNA-seq analysis and alignment of NGS (University of Amsterdam) for providing neuroblastoma cell lines. We thank (German Cancer Research Center), J. Couturier (Institut Curie) and R. Versteeg We are grateful to M. Ponzoni (IRCCS Istituto Giannina Gaslini), M. Schwab online version of the pape Note: Any Supplementary Information and Source Data files are available in the pape the the in available are references, and codes accession statements of including Methods, and data availability any associated Me s r e t t e l  AUTHOR CO A

c kn thod o w shRNA cell lines. S.D. performed the analysis single-cell and study r . ledgme in vitro s N TRIBUTIO in vitro experiments. V.R. performed all sequencing experiments. n ts r . experiments and ChIP experiments and participated N S in vivo experiments and online online ′ Avenir program, version of version

7. 9. 8. 6. 5. 4. 3. 2. 1. claims jurisdictional in published maps and affiliations. institutional reprints/index.htm at online available is information permissions and Reprints The authors declare no competing interests.financial manuscript.final I.J.-L., V.B. and O.D. provided support.financial All authors read and approved the nervous development and TFs. I.J.-L. and O.D. provided laboratory infrastructure. and infrastructure data storage. andH.R. T.D. provided in sympathetic expertise and B.G. S.G.-L. participated in RNA-seq analysis. E.B. provided computational 29. 28. 25. 24. 23. 22. 21. 20. 19. 18. 17. 16. 15. 14. 13. 12. 11. 10. 27. 26. COMPETI

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© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. histones or iDeal ChIP-seq kit for transcription factors (Diagenode) using using (Diagenode) factors transcription for kit ChIP-seq iDeal or histones kit for ChIP-seq iDeal the using was performed (ChIP) immunoprecipitation and analysis. ChIP-seq guardians or parents law. national from to according obtained was consent informed Written 3272. reference 3, France de Ile Personnes des Protection de Comité and 0811728; reference 1, France de Ile Personnes des Protection de Comité L12–171; and L07–95 IV,references Sud-Est Personnes des Protection de Comité mittees com ethics the of decision the by authorized was study This Board. Review Curie’s Institutional Institut the by approved was profiles samples, neuroblastoma expression of of study including patients, from material Analysis biological 1. of pair of relapse the from derived was PDX MAP-GR-B25-NB-1 The MAPPYACTS protocol. the through obtained were samples relapse The 4; stage all Relapse; and Patient samples. identifier (ClinicalTrials.gov protocol NB-1 and MAP-GR-B25-NB-1 PDXs were obtained through the MAPPYACTS in accordance with European MAP-IC-A23-NB-1, MAP-GR-A99-legislation. performed were experiments All 135/11). protocol Barcelona, de Universitat at Animal local de the Etico Éxperimentación (Comité care and committee use animal by approved were Déu de Joan Sant Recerca de Institut at studies of mice SCID female Animal at of engraftment. weeks 3–6 mice nude Swiss or female weeks using 10–11 obtained were models HSJD-NB-011 and 17) under the number The MAP-IC-A23-NB-1 2015032614359689v7. (IC-pPDX- approvedModels, by PDX the experimental ethic committee Pediatric 26 (CEEA26—Gustave of Roussy) Development project the within maintained and developed are models PDX These PDXs. MAP- MAP-GR-B25 for and used GR-A99-NB-1 were mice NSG female whereas engraftment, at weeks 6–8 (refs. IGR-N835 NB8, All used. PDXs but had MAP-IC-A23-NB-1 and MAP-GR-B25-NB-1 HSJD-NB-011) MAP-GR-A99-NB-1, (IGR-N835, 4 stage or (IGR-NB8) 3 stage A23-NB-1), models. PDX Gibco). from were which FGF2, and EGF except by Sigma-Aldrich supplied products 2 (all factor growth fibroblast pg/ml 200 and insulin ng/ml 1 factor, growth epidermal pg/ml 100 glucagon, pg/ml 10 10 hydrocortisone, FCS (Eurobio), 100 12% with supplemented (Gibco) DMEM:F12 Glutamax in grown were cells Briefly, material. of for embryonic use human the Agency Biomedical French lines were grown as described 100 and (Eurobio) FCS or 20% 15% 10%, with lines, cell for other Healthcare) (GE or DMEM in NB69, in IMDM (Gibco) for (according NB-EBc1 to the provided conditions), SH-EP, lines, and cell GICAN CLB for Healthcare) (GE RPMI in atmosphere at 5% CO 37 °C with were cultured lines cell Neuroblastoma cell lines. Cells were checked routinely by PCR for the absence of mycoplasma. Control-FREEC using genomic copy number profile calculated from the input ChIP-seq data obtained by of was comparison performed the line authentication Cell cells. in adrenergic enriched was 2 Batch agents. chemotherapeutic the of evaluation the for used was 2) (batch batch second A analysis. single-cell and ChIP-seq the for Group, Oncology respectively. One batch of SK-N-SH (batch cells 1) was used Children’s the from and Cultures Cell Authenticated of Collection European the from obtained were lines cell NB-EBc1 and NB69 The Ponzoni. M. from gift kind a was GICAN line and Versteeg, R. from obtained were TR-14 and by M. provided Schwab kindly and derived were J. lines Couturier. were cell LinesLAN-1 lines GIMEN,and N206,SH-EP cell The SJNB1, V.SJNB6, CLB Combaret. by SJNB8, (ATCC). SJNB12 Collection the Culture Type from obtained American were SK-N-SH and SK-NF-I SK-N-DZ, SK-N-BE(2)C, study have been previously described lines. cell hNCC and Neuroblastoma ONLIN doi: H3K27ac, for Abcam) polyclonal, (rabbit ab4729 antibodies: following the 10.1038/ng.3921 E E M E Neuroblastoma PDXs were obtained from stage L2 (MAP-IC- L2 stage from obtained were PDXs Neuroblastoma THOD Three diagnosis–relapse pairs of tumors (Pair1/2/3-Diagnosis µ µ µ g/ml penicillin–streptomycin, g/ml 10 penicillin–streptomycin, mM HEPES, 100 ng/ml g/ml penicillin–streptomycin (Gibco). Primary hNCC hNCC Primary (Gibco). penicillin–streptomycin g/ml g/ml transferrin, 400 pg/ml 3,3,5-thio-iodo-thyronine, 3,3,5-thio-iodo-thyronine, pg/ml 400 transferrin, g/ml 3 34 1 S H3K27ac, PHOX2B, HAND2 H3K27ac, and GATA3 chromatin with SNP array profile and STR profiling for ATCC for profiling STR and profile array SNP with Supplementary Table2 Supplementary , 3 5 ) were obtained using female Swiss nude mice of mice nude Swiss female using obtained were ) 3 2 under bioethical approval PFS14-011 from the 3 0 . CHP-212, IMR-32, SH-SY5Y, SK-N-AS, NCT0261396 Neuroblastoma cell lines used in this this in used lines cell Neuroblastoma 3 3 . . None were related to the lines cell MYCN ) were studied in this work. work. this in studied were ) amplification. PDXs amplification. IGR- 2 ). 2 in a humidified in a humidified -

using using the TruSeq preparation ChIP library kit according to the manufacturer’s histones. for kit ChIP-seq iDeal the using lines cell for above described as performed were ChIP and chromatin of fragmentation cells, of Lysis wheel. rotating a on agitation with min 8 for formaldehyde 1% adding by performed was chromatin of Cross-linking PBS. in resuspended then and in listed are Primers DNA. input the to pair primer each for compared and Biosystems) (Applied Mix Master Green powerSYBR using regions genomic specific on antibody for each by qPCR validated was efficiency ChIP sequencing, Before precipitated with beads and with the magnetic Ipurepurified kit (Diagenode). temperature and 4 reverse cross-linked h at 65 °C with proteinase K. DNA was ChIP, at for 3 min 30 room h After at eluted for 4 was TFs. only °C chromatin antibodies with precleared were beads magnetic A–coated Protein GATA3. 1 with °C 65 at h with at 4 wheel °C on a overnight 4 rotating performed was K. ChIP proteinase cross-linked reverse and input quantify to for kept used was chromatin TFs of 1% of equivalent The TFs. for cells million 3.75 and for H3K27ac cells of 1 million chromatin with performed was bp. ChIP ~300 and lysates were sonicated to obtain sheared chromatin to an average length of buffer, lysis of addition the by isolated was Chromatin temperature. room at min 5 for concentration) (final mM 125 with quenching by followed min 10 for formaldehyde 1% with cross-linked were cells Tentively. million GATA3, respec and HAND2 PHOX2B, for Biotechnology Cruz Santa from polyclonal) (goat sc-22206X and sc-9409 monoclonal), (mouse sc-376997X sample except for the CLB-GA cell line, for which the experiment was was experiment the which for line, cell CLB-GA the for except sample respectively). regions, 1,819 and (1,901 cells SH-EP and GIMEN in detected was of number super-enhancers The highest 385). (s.d. was 1,252 line cell per identified super-enhancers of number average the 25 lines, For cell ROSE. neuroblastoma by determined was super-enhancers from enhancers typical distinguishing of score the threshold The score. to super-enhancer according HMCan) by bias content GC and number copy for corrected (already values density H3K27ac normalized of sum the to ing were excluded. Then each region received a super-enhancer score correspond tance of 12.5 kb, and regions ( ROSE lines. cell lastoma neurob these for profiles number copy known cell matched each profiles of These line. profiles number copy obtain to files) *.bam input, parameters; (LILY package). signal peak and length peak between values correlation of basis the on discarded also were values) score HMCan low (i.e., signal low with Peaks regions. amplification to correspond may they as peaks, 100 first the discounting experiment, each of peaks highest 5,000 the in median value density the of basis the on profiles rescales script LILY The the below). in (see included package script a with samples between normalized then profiles were Density (*.bed). peaks called further regions enrichment large and narrow and (*.wig) bias number copy and content GC the for corrected files pro density ChIP included output HMCan analysis. the from excluded were blacklist ENCODE hg19 the ‘false’. from duplicates, Regions removing 0.1; threshold, final 20; iterations, of number bp; 200 distance, merging 0.05; old, 50 400 bin bp; bp; 25 length, large small bin kb; length, length, fragment maximum bp; 250 length, fragment median bp; 100 length, ment (ref. v1.30 HMCan using were called regions (peaks) in regions. Enriched signal genomic amplification detect to order in kept were reads duplicate discarded; were 20) < (Q quality (ref. v2.1.0 Bowtie2 using hg19/GRCh37 genome nt). 100 reads, single mode; run (rapid instrument (KAPA Kit) Quantification Library qPCR and on by sequenced the Illumina HiSeq2500 quantified were libraries DNA resulting the cycles, 14 of step tion amplifica a final After bp). (100–600 ChIP H3K27ac for the only performed was selection Size adapters. Illumina indexed TruSeq to ligation and tailing dA repair, end of steps consecutive to subjected was DNA Briefly, protocol. µ Illumina sequencing libraries were prepared from ChIP and input DNA input and ChIP from prepared were libraries sequencing Illumina pestle a with powder to reduced were tumors frozen samples, PDX For ChIP-seq experiments for H3K27ac were performed once for every every for once performed were H3K27ac for experiments ChIP-seq To call enhancers and super-enhancers, we used LILY, a version modified of Control-FREEC The analysis. ChIP-seq g of antibody for H3K27ac, 2 2 H3K27ac, for antibody of g 12 , 3 9 . First, large H3K27ac peaks were stitched together, using a default dis Supplementary Table 7 Supplementary ChIP-seq reads were mapped to the human reference reference human the to mapped were reads ChIP-seq 3 1 3 algorithm was applied to input samples (default (default samples input to applied was algorithm 7 ) with the following parameters: minimum frag minimum parameters: following the with ) µ g for HAND2 and 5 5 and HAND2 for g . ± 2.5 kb from the transcription start site) 3 7 3 . The regions were sorted sorted were regions The . 6 ). Reads of low mapping mapping low of Reads ). µ Nature Ge Nature g for PHOX2B and and PHOX2B for g P value thresh value n etics 3 8 ------

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. performed using the Position Analysis tool of the RSAT package the of tool Analysis Position the using performed ( FOSL2) and FOSL1 1, (module modules two defined expression their of correlation the of basis the on method) McQuitty ( TFs 37 in resulted This II. or I group of lines cell of 50% than more in FOSL2) or FOSL1 JUNB, (JUN, TFs PHOX2B TF or AP-1 homeobox as the CRC same the in COLTRON to occur 4 Figs. ( super-enhancers neuroblastoma of motifs AP-1 and obox home ( in enrichment significant analysis a discovered our analysis enrichment motif in As super-enhancer a with asso not were ciated that TFs 17 excluded we predictions, COLTRON the From of 50% ( I over group in from lines present cell TFs kept We sample. given a of CRC be a to in predicted involved was TF given a super-enhancer whether see to (ii) cliques ranked with and parsed files then We study.the our samples, in lines cell 2 neuroblastoma than more in 31 detected was of region set our in sion expres gene with correlated score super-enhancer (i) properties: following COLTRON by detected were respectively. hNCCs, and II and I overlapping of the 100 peaks top regions in groups H3K27ac super-enhancers i-cisTarget the applied we hNCCs, of super-enhancers and II) and I groups of lines (cell enhancers analysis of groups super-enhancer scores between I and II. about fold changes and SH, SK-N-AS, SJNB12, CHP-212). SY5Y), group II (GICAN, SH-EP, GIMEN) and the intermediate group (SK-N- SJNB8, IMR-32, N206, LAN-1, SK-N-DZ, SK-N-BE(2)C, SK-N-FI, TR14, SH- CLB-MA, CLB-CAR, CLB-BER-Lud, CLB-PE, NB69, NB-EBc1, SJNB1, SJNB6, the first principal components suggested their separation into group I (CLB-GA, enhancer regions to the first two principal components. Analysis of samples in super-enhancers. 2 hNCC lines) was performed on log super- of predictions regions. enhancer positive false and events cell-line-specific remove to two neuroblastoma cell lines or hNCC samples regions). This (5,975 was done active genes. samples. hNCC both in 4,791 super-enhancers 1,639 to genes to assigned assigned and were detected we Similarly, super-enhancers neuroblastoma total, In ( genes of number a to assigned be can enhancer super- each and regions, super-enhancer several have can gene each Of note, 0.361, = (threshold study this in corresponding to an analyzed adjusted samples of set full the in super-enhancer and score expression gene between those correlation selected highest the with super-enhancer—we a by regulated possibly therefore and lines cell information human (TADs) in eight domains using associating hg19) of topologically 78, locations about release (RefSeq genes RefSeq the to assigned ( neuroblas putative as super-enhancers toma annotated were sample one than more in of detected number super-enhancers overlapping median with the regions 4,336 of Overall, one-half super-enhancers. threshold a as using subregions, into subpeaks several with excluded regions long separated we and one, into regions enhancer lines cell super- 25 neighboring the several of To kb. stitching 12 in avoid than shorter regions predicted regions super-enhancer the super- active 2. as replicate annotated in were enhancers 93% 1, replicate in super- and ( super-enhancers 500 calling calculation super-enhancer score of enhancer samples reproducibility replicate the these used We document to replicates. biological two in performed Nature Ge Nature GATA3, nt; 5 HAND2, nt). 8 PHOX2B, nt; 5 sizes: cleotide Motif discovery in ChIP-seq peaks of GATA3, HAND2 and PHOX2B was was GATA3, of PHOX2B and HAND2 peaks ChIP-seq in discovery Motif lines hNCC and samples PDX lines, cell neuroblastoma the in CRCs super- neuroblastoma in enriched motifs binding TF known detect To PCA for 33 samples (25 neuroblastoma cell lines, 6 neuroblastoma PDXs and in at least active regions super-enhancer we only kept analysis, For further superimposed we super-enhancers, neuroblastoma of list a Togenerate and and 4 0 . Among all genes located in the same TAD same a as the super-enhancer— in located genes all . Among 8 n ), among these 52 TFs, we selected those that were predicted by predicted were that those selected we TFs, 52 these among ), etics Supplementary Table3 Supplementary Supplementary Fig. 22 Fig. Supplementary 4 P 1 n values for the two-sided Wilcoxon test for differential method to the list of 2,227, 1,850 and 1,640 valley valley 1,640 and 1,850 2,227, of list the to method n upeetr Tbe 3 Table Supplementary = 18) or group II ( II group or 18) = = 7, includes PHOX2B; module 2, 2, module PHOX2B; includes = 7, P 1 value (‘FDR’) of 0.05, 9 upeetr Fg 21 Fig. Supplementary on the basis of super-enhancers with the the with super-enhancers of basis the on Fig. 1 Fig. Supplementary Table 3 2 values of super-enhancer scores of 5,975 g ). Clustering of the 37 genes (hclust, (hclust, genes 37 the of Clustering ). shows contributions of the super- the of contributions shows ). n = 3). This resulted in 69 TFs. TFs. 69 in resulted This 3). = upeetr Fg 10 Fig. Supplementary Supplementary Table 3 Table Supplementary ). Super-enhancers were were Super-enhancers ). Supplementary Fig. Supplementary 11 includes information ). Among the top top the Among ). Supplementary Supplementary n = 15, includes includes = 15, 4 2 (oligonu ). ). ). ). ). ). - - - - -

Mission Mission shRNA and library cloned into the pLKO-Tet-On system all-in-one RNAs sh1783 and sh1437 ( systems. shRNA Doxycycline-inducible 2 d. every RNAs were using extracted NucleoSpin RNA kit (Macherey-Nagel). (7.5 followingResazurin-based, the manufacturer’s instructions (Sigma-Aldrich). the usingmeasured then was viability Cell h. 48 ence in the untreated cells. Cells were treated with chemotherapeutic agents for bicin. Seeding densities for each cell lines were optimized to reach 80% conflu plated in 96-well plates 2 d before the addition of cisplatin, etoposide or doxoru Treatment of cell lines with chemotherapy. the in vided analysis. expression gene single-cell and immunoblotting read alignment, and RNA-sequencing transcriptome regions. 2,078 the for density rescaled average the We plotted then was rescaled to have a maximum value of 1 corresponding to the neuroblastoma super-enhancers. ChIP-seq density peak for each TF maximum.for each region strongest 500 the within were located of them 2,078 line. cell for CLB-GA the genome human the throughout regions such 14,693 We on obtained TFs. three centered regions all of peaks overlapped 2,400-bp that those kept and all sites binding ChIP-seq PHOX2B extracted first we positions, maximum #1027281; Qiagen) using RNAimax transfection reagent (Thermo Fisher Fisher (Thermo reagent transfection RNAimax using Qiagen) #1027281; siRNA control #SI04212446; Hs_GATA3_8 and #SI04202681 Hs_GATA3_7 with 20 nM siRNA (Hs_HAND2_3 #SI00131915, Hs_HAND2_6 #SI03046736, assays. growth and siRNA v5). 1607151 160531 5524-20 number (authorization l de Nationale, l’Education de Approval in research). mals for from cancer Ministère study was this received French Competent the Authority, and UKCCCR (86/609/EEC), (guidelines for the welfare and Community use of ani European the of recommendations and as calculated mm 3,000 around of volume a reached tumors when killed were Mice day. every caliper a with monitored was growth Tumor groups. water) drinking in sucrose 5% and mg/l) (2 (doxycycline treatment the or water) drinking in mm of 170 around volume a reached tumors When Biosciences). (BD Matrigel and PBS of mix in the of flanks 6-week-old NSG mice (Charles River Laboratories) in an equal against shRNA with duced mice. and experiments Xenotransplantation Coulter). a using ViabilityVi-cell(Beckman cells XR Cell Analyzer and atgroup) for day CLB-GA 8 ( The numbers of living cells were counted at days 4, 7, 10 and 14 (triplicates per plates 24-well in the presence or of absence 100 doxycycline ng/ml or 1 counting, 2 × 10 plicates per group) or no doxycycline. Medium was refreshed after 48 h. For cell plate in 200 trodes. 10 instrument (ACEA Biosciences) monitoring impedance across gold microelec assays. Proliferation (ref. 11 at day violet crystal with stained and dishes 6-well 10 × 6 assays, formation colony For 1 or ng/ml (100 doxycycline of addition the after h 72 by immunoblot assessed was efficacy knockdown PHOX2B respectively. and cells, CLB-GA SH-SY5Y for experiments, culture all during maintained and infection puromycin with 1 or ng/ml Selection 400 at (Invitrogen) Polybrene. without h 48 for particles viral with described previously as infected were cells (Addgene). Lentiviral particles were produced in HEK293T cells, and CLB-GA SK-N-SH cells were plated in 6-well plates and then treated with cisplatin cisplatin with treated then and plates 6-well in plated were cells SK-N-SH peak PHOX2B the around profiles density ChIP-seq average Tocalculate µ b M) or doxorubicin (100 nM) for 7 d. Medium and drugs were changed were drugs and Medium d. 7 for nM) (100 doxorubicin or M) the smallest. Experiments were conducted in accordance with the the with accordance in conducted were Experiments smallest. the 4 infected CLB-GA or SH-SY5Y cells were seeded per well of a 96-well µ Supplementary Note Supplementary l medium containing doxycycline at 100 ng/ml or 1 V 4 = = infected CLB-GA or 10 3 a , mice were randomly assigned to the control (5% sucrose sucrose (5% control to the assigned randomly were , mice /2 × × /2 Cells were counted in real time with an xCELLigence xCELLigence an with time real in counted were Cells b × (( × Supplementary TableSupplementary 7

PHOX2B HAND2 ′ µ a Enseignement Supérieur et de la Recherche Recherche la de et Supérieur Enseignement g/ml, respectively, was performed 24 h after after h 24 performed was respectively, g/ml, + + n . = 5 or 6 technical replicates) for SH-SY5Y replicates) = 5 or 6 technical b 4 and and transduced cells were plated at day 0 in in 0 day at plated were cells transduced (sh1783) were injected subcutaneously subcutaneously injected were (sh1783) )/2), with with )/2), Details of these experiments are pro are experiments these of Details 5 infected SH-SY5Y cells were plated in GATA3 SH-EP and SH-SY5Y cell lines were

4 PHOX2B- . SH-SY5Y cells were incubated incubated were cells SH-SY5Y . 10 × 10 × 10 a in vitroin knockdown was performed performed was knockdown being the largest diameter diameter largest the being ) ) were from Sigma selected specific short hairpin hairpin short specific 6 Toxicology Assay Kit, CLB-GA cells trans cells CLB-GA doi: 4 4 10.1038/ng.3921 ). µ g/ml (quintu µ µ g/ml). g/ml). g/ml. g/ml. 4 3 3 ------

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. 30. code able through the European avail Genome-Phenome are Archive (EGA) under samples accession patient for data RNA-seq as well RNA-seq and ChIP-seq number accession Expression under Gene in (GEO) available Omnibus are PDXs and tumors lines, cell the for data Data availability. at available is data ChIP-seq cancer availability. Code Summary Reproducibility A compared. statistically are that groups the equal between variation require not does and distribution normal the follow to data the need ( tumors of set the in modules NCC-like and ergic 10 and ( and score gene expression super-enhancer between The number of permutations was 10 test. permutation one-sided a implemented we correlation), Pearson no esis, Statistical analysis. the in PHOX2B immunohistochemistry. ( Cell Coulter) XR (Beckman Vi-cell Analyzer Viability a using counted was cells living of number The Scientific). doi:

10.1038/ng.3921 Schleiermacher, G. yrdzto aayi idcts rdmnn rarneet o ery replicating early ofregions rearrangementsneuroblastoma.in predominant indicates analysishybridization EGAS0000100250 Supplementary Note Supplementary 6 in the test for correlation between gene expression of the noradren the of expression gene between correlation for test the in Raw data for cell line ChIP-seq and RNA-seq and processed Code for the pipeline for super-enhancer detection from from detection super-enhancer for pipeline the for Code To calculate t al. et 5 . obnd 4clr aytpn ad comparative genomic karyotyping 24-color Combined and for this paper is available. is paper this for . P values for Pearson correlation (null hypoth Details of are Details experiments these provided 4 http://boevalab.com when calculating CancerGenet. Cytogenet. n = 5 or 6 technical replicates). technical 6 or 5 = GSE9068 Fig. Fig. Supplementary Fig. 11 Fig. Supplementary P 3 values for correlation 2 Rw aa o PDX for data Raw . /LILY ). This test does not not does test This ).

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