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Heterogeneity of neuroblastoma cell identity defined by transcriptional circuitries

Abstract

Neuroblastoma is a tumor of the peripheral sympathetic nervous system1, derived from multipotent neural crest cells (NCCs). To define core regulatory circuitries (CRCs) controlling the gene expression program of neuroblastoma, we established and analyzed the neuroblastoma super-enhancer landscape. We discovered three types of identity in neuroblastoma cell lines: a sympathetic noradrenergic identity, defined by a CRC module including the PHOX2B, HAND2 and GATA3 transcription factors (TFs); an NCC-like identity, driven by a CRC module containing AP-1 TFs; and a mixed type, further deconvoluted at the single-cell level. Treatment of the mixed type with chemotherapeutic agents resulted in enrichment of NCC-like cells. The noradrenergic module was validated by ChIP-seq. Functional studies demonstrated dependency of neuroblastoma with noradrenergic identity on PHOX2B, evocative of lineage addiction. Most neuroblastoma primary tumors express TFs from the noradrenergic and NCC-like modules. Our data demonstrate a previously unknown aspect of tumor heterogeneity relevant for neuroblastoma treatment strategies.

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Figure 1: Super-enhancer landscape identifies various CRCs and identities in neuroblastoma cell lines.
Figure 2: Different identity of neuroblastoma primary tumors and impact of chemotherapy on cell identity.
Figure 3: PHOX2B, HAND2 and GATA3 are master TFs defining the super-enhancer (SE) landscape of noradrenergic neuroblastoma.
Figure 4: PHOX2B is critical for the growth of noradrenergic neuroblastoma cells.

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Acknowledgements

We are grateful to M. Ponzoni (IRCCS Istituto Giannina Gaslini), M. Schwab (German Cancer Research Center), J. Couturier (Institut Curie) and R. Versteeg (University of Amsterdam) for providing neuroblastoma cell lines. We thank F. Tirode and C. Kamoun for help with RNA-seq analysis and alignment of NGS data, respectively; O. Blanchard for help in cell culture experiments; and M. Caly for PHOX2B immunohistochemistry. We are grateful to the animal facilities team, the Experimental Pathology Department and the Plateforme Génomique of Institut Curie. We thank N. Clément, T. Adam-de-Beaumais and B. Mallon for their help in the identification of neuroblastoma diagnosis–relapse pairs and V. Bernard for pairs validation. We thank V. Saint-André for scientific discussion, J. Maliash-Planchon and the Unité de Génétique Somatique for preparation of patient samples. We thank D. Figarella-Branger (BB-033-00097, CRB AP-HM, CRB TBM, AC-2013-1786), M. Clapisson (CRB Centre Léon Bérard, AC-2008-101), O. Minckes, C. Blanc-Fournier and N. Rousseau (CHU, Tumorothèque de Caen Basse Normandie) for providing tumor patient samples. This work was supported by grants from Institut Curie, INSERM, the Ligue Nationale contre le Cancer (Equipe Labellisée), the Société Française de Lutte contre les Cancers et les Leucémies de l'Enfant et l'Adolescent, the Institut National du Cancer (PRT-K14-061 and PHRC IC 2007-2009) and by the following associations: Association Hubert Gouin “Enfance and Cancer,” Les Bagouz à Manon, les amis de Claire, Courir pour Mathieu, Dans les pas du Géant and Olivier Chape. The MAPPYACTS protocol is supported by the Institut National du Cancer (PHRC-K14-175), the Fondation ARC (MAPY201501241), the Société Française de Lutte contre les Cancers et les Leucémies de l'Enfant et l'Adolescent (Fondation Enfants et Santé), the Fondation AREMIG and the Association Thibault BRIET. High-throughput sequencing was performed by the ICGex NGS platform of the Institut Curie, supported by the grants ANR-10-EQPX-03 (Equipex) and ANR-10-INBS-09-08 (France Génomique Consortium) from the Agence Nationale de la Recherche (Investissements d'Avenir program); by the Canceropole Ile-de-France; and by the SiRIC-Curie program -SiRIC Grant INCa-DGOS- 4654. Biomark analysis was done using the High Throughput qPCR-HD-Genomic Paris Centre platform supported by grants from Région Ile-de-France (21016711). G.S. is supported by the Annenberg Foundation (11-385). V.B. is supported by the ATIP-Avenir Program, the ARC Foundation (ARC-RAC16002KSA-R15093KS), Worldwide Cancer Research (WCR16-1294 R16100KK) and the “Who Am I?” Laboratory of Excellence ANR-11-LABX-0071, funded by the French Government through its Investissement d′Avenir program, operated by the French National Research Agency (ANR) (ANR-11-IDEX-0005-02). H.R. is supported by the Mayent-Rothschild program from Institut Curie and the Wilhelm-Sander-Stiftung. The laboratory of T.G.P.G. is supported by LMU Munich's Institutional Strategy LMUexcellent within the framework of the German Excellence Initiative, the Mehr LEBEN für krebskranke Kinder—Bettina-Bräu-Stiftung, the Walter Schulz Foundation, the Wilhelm-Sander-Stiftung (2016.167.1) and the German Cancer Aid (DKH-111886 and DKH-70112257).

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Authors and Affiliations

Authors

Contributions

V.B. and I.J.-L. conceived the study, analyzed the data and wrote the manuscript. V.B. coordinated bioinformatics analysis and I.J.-L. coordinated the whole study. C.L.-B. performed in vitro experiments and ChIP experiments and participated in the study design. A.P. generated and analyzed the doxycycline-inducible anti-PHOX2B shRNA cell lines. S.D. performed the single-cell analysis and study of chemotherapeutic agents. C.P.-E. performed the in vivo experiments and contributed in vitro experiments. V.R. performed all sequencing experiments. H.C.E. and S.T. provided hNCC cell lines and V.C. provided neuroblastoma cell lines. A.L. performed alignment of RNA-seq and ChIP-seq data. E.D.-D., B.G., D.S. and A.M.C. provided neuroblastoma PDXs. I.M. performed the reproducibility analysis. E.D. and B.D. generated the Biomark data. M.F.O. and T.G.P.G. generated lentiviral particles and provided help with lentiviral infections. S.B. coordinated and supervised sequencing experiments. G.S. participated in the study design and provided the in-house pairs of diagnosis–relapse samples with the help of E.L., G.P. and B.G. S.G.-L. participated in RNA-seq analysis. E.B. provided computational infrastructure and data storage. H.R. and T.D. provided expertise in sympathetic nervous development and TFs. I.J.-L. and O.D. provided laboratory infrastructure. I.J.-L., V.B. and O.D. provided financial support. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Valentina Boeva or Isabelle Janoueix-Lerosey.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–22, Supplementary Tables 1, 2 and 7 and Supplementary Note. (PDF 3758 kb)

Life Sciences Reporting Summary (PDF 163 kb)

Supplementary Table 3

Characteristics of neuroblastoma and hNCC super-enhancers. Group I: all neuroblastoma cell lines with the exception of SH-EP, GIMEN, GICAN, SK-N-AS, SJNB12, SK-N-SH and CHP-212. Group II: SH-EP, GIMEN and GICAN. (XLSX 1059 kb)

Supplementary Table 4

Supervised analysis of super-enhancer (SE) scores according to MYCN status. (XLSX 22 kb)

Supplementary Table 5

Supervised analysis of super-enhancer (SE) scores according to ALK status. (XLSX 20 kb)

Supplementary Table 6

Raw Ct values measured for housekeeping genes (GAPDH, ACTG1, ACTB, RPL15) and TFs of modules 1 and 2 for single cells of the SK-N-AS, SH-EP, SH-SY5Y and SK-N-SH cell lines using the Fluidigm Biomark HD. (XLSX 58 kb)

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Boeva, V., Louis-Brennetot, C., Peltier, A. et al. Heterogeneity of neuroblastoma cell identity defined by transcriptional circuitries. Nat Genet 49, 1408–1413 (2017). https://doi.org/10.1038/ng.3921

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