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Full Text (PDF) Published OnlineFirst March 6, 2018; DOI: 10.1158/2159-8290.CD-16-0861 RESEARCH ARTICLE Cross-Cohort Analysis Identifi es a TEAD4–MYCN Positive Feedback Loop as the Core Regulatory Element of High-Risk Neuroblastoma Presha Rajbhandari 1 , 2 , Gonzalo Lopez 1 , 3 , Claudia Capdevila 1 , Beatrice Salvatori 1 , Jiyang Yu 1 , 4 , Ruth Rodriguez-Barrueco5 , 6 , Daniel Martinez 3 , Mark Yarmarkovich 3 , Nina Weichert-Leahey 7 , Brian J. Abraham 8 , Mariano J. Alvarez 1 , Archana Iyer 1 , Jo Lynne Harenza 3 , Derek Oldridge 3 , Katleen De Preter9 , Jan Koster 10 , Shahab Asgharzadeh 11 , 12 , Robert C. Seeger 11 , 12 , Jun S. Wei 13 , Javed Khan13 , Jo Vandesompele 9 , Pieter Mestdagh 9 , Rogier Versteeg 10 , A. Thomas Look 7 , Richard A. Young8 , 14 , Antonio Iavarone 15 , Anna Lasorella 16 , Jose M. Silva 5 , John M. Maris 3 , 17 , 18 , and Andrea Califano1 , 19 , 20 , 21 ABSTRACT High-risk neuroblastomas show a paucity of recurrent somatic mutations at diag- nosis. As a result, the molecular basis for this aggressive phenotype remains elu- sive. Recent progress in regulatory network analysis helped us elucidate disease-driving mechanisms downstream of genomic alterations, including recurrent chromosomal alterations. Our analysis identi- fi ed three molecular subtypes of high-risk neuroblastomas, consistent with chromosomal alterations, and identifi ed subtype-specifi c master regulator proteins that were conserved across independent cohorts. A 10-protein transcriptional module—centered around a TEAD4–MYCN positive feedback loop—emerged as the regulatory driver of the high-risk subtype associated with MYCN amplifi cation. Silencing of either gene collapsed MYCN -amplifi ed MYCN( Amp ) neuroblastoma transcriptional hall- marks and abrogated viability in vitro and in vivo . Consistently, TEAD4 emerged as a robust prognostic marker of poor survival, with activity independent of the canonical Hippo pathway transcriptional coactivators YAP and TAZ. These results suggest novel therapeutic strategies for the large subset of MYCN-deregulated neuroblastomas. SIGNIFICANCE: Despite progress in understanding of neuroblastoma genetics, little progress has been made toward personalized treatment. Here, we present a framework to determine the downstream effectors of the genetic alterations sustaining neuroblastoma subtypes, which can be easily extended to other tumor types. We show the critical effect of disrupting a 10-protein module centered around a YAP/TAZ-independent TEAD4–MYCN positive feedback loop in MYCNAmp neuroblastomas, nominating TEAD4 as a novel candidate for therapeutic intervention. Cancer Discov; 8(5); 582–99. ©2018 AACR. 1 Department of Systems Biology, Columbia University, New York, New York. 12 Keck School of Medicine, University of Southern California, Los Angeles, 2 Department of Biological Sciences, Columbia University, New York, New York. California. 13 Genetics Branch, Oncogenomics Section, Center for Cancer 3 Division of Oncology and Center for Childhood Cancer Research, Chil- Research, NIH, Bethesda, Maryland. 14 Department of Biology, Massachu- dren’s Hospital of Philadelphia, Philadelphia, Pennsylvania. 4 Department setts Institute of Technology, Cambridge, Massachusetts. 15 Department of Computational Biology, St. Jude Children’s Research Hospital, Memphis, of Neurology and Pathology and Cell Biology, Institute for Cancer Genet- Tennessee. 5 Department of Pathology, Icahn School of Medicine at Mount ics, Columbia University, New York, New York. 16 Department of Pediatrics Sinai, New York, New York. 6 Departament de Patologia i Terapèutica Exper- and Pathology and Cell Biology, Institute for Cancer Genetics, Columbia imental, Facultat de Medicina i Ciències de la Salut, IDIBELL, Universitat University, New York, New York. 17 Department of Pediatrics, Perelman de Barcelona, L’Hospitalet de Llobregat, Barcelona, Spain. 7 Department School of Medicine, University of Pennsylvania, Philadelphia, Pennsylva- of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical nia. 18 Abramson Family Cancer Research Institute, Philadelphia, Pennsyl- School, Boston, Massachusetts. 8 Whitehead Institute for Biomedical vania. 19 Department of Biomedical Informatics, Columbia University, New Research, Cambridge, Massachusetts. 9 Center for Medical Genetics & York, New York. 20 Department of Biochemistry and Molecular Biophysics, Cancer Research Institute Ghent (CRIG), Ghent University, Gent, Belgium. Columbia University, New York, New York. 21 Herbert Irving Comprehensive 10 Department of Oncogenomics, Academic Medical Center, Amsterdam, Cancer Center and J.P. Sulzberger Columbia Genome Center, Columbia the Netherlands. 11 Division of Hematology/Oncology, Saban Research University, New York, New York. Institute, The Children’s Hospital Los Angeles, Los Angeles, California. 582 | CANCER DISCOVERY MAY 2018 www.aacrjournals.org Downloaded from cancerdiscovery.aacrjournals.org on October 1, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst March 6, 2018; DOI: 10.1158/2159-8290.CD-16-0861 INTRODUCTION of high-risk neuroblastoma, all newly diagnosed patients are empirically treated with intensive cytotoxic chemoradio- Neuroblastoma is a malignancy arising from the developing therapy to achieve disease remission. Thus, further elucida- sympathetic nervous system that typically affects very young tion of the molecular mechanisms responsible for high-risk children. About half of the cases present with widespread neuroblastoma pathogenicity is required for guiding novel, metastatic disease and/or extraordinarily focal amplifi cation more effective and less toxic precision-oncology strategies. of the MYCN gene, often with >100 copies. These high-risk Following on recent results from the assembly and inter- patients have poor 5-year survival probability (<40%), despite rogation of regulatory network models (interactomes) in intensive multimodal treatment regimens ( 1 ). Diagnostically, human cancers, using systems biology approaches ( 7 ), we neuroblastomas show relatively few recurrent somatic point focused on the discovery of more universal tumor dependen- mutations ( 2–5 ). In contrast, it has been known for decades cies in high-risk neuroblastomas. This approach has been that high-risk neuroblastomas harbor complex and recurrent highly successful in elucidating small regulatory modules, somatic structural copy-number alterations (CNA), affecting dubbed tumor checkpoints, comprising master regulator large chromosomal regions, as well as focal amplifi cation of (MR) proteins that mechanistically regulate the transcrip- the MYCN oncogene ( 6 ). Identifi cation of the causal driver tional state of the tumor. MR proteins have been shown to genes associated within these CNAs remains a challenge. elicit either essentiality or synthetic lethality in several can- Despite signifi cant understanding of the genetic landscape cers ( 8–16 ). Critically, aberrant activity of tumor checkpoint Note: Supplementary data for this article are available at Cancer Discovery Children’s Hospital of Philadelphia, Colket Translational Research Build- Online (http://cancerdiscovery.aacrjournals.org/). ing, 3501 Civic Center Boulevard , Philadelphia, PA 19104-4318. E-mail: P. Rajbhandari and G. Lopez contributed equally to this article. [email protected] Corresponding Authors: Andrea Califano, Herbert Irving Comprehen- doi: 10.1158/2159-8290.CD-16-0861 sive Cancer Center, Columbia University, 1130 St. Nicholas Avenue, New © 2018 American Association for Cancer Research. York, NY 10032. E-mail: [email protected] ; and John M. Maris, MAY 2018 CANCER DISCOVERY | 583 Downloaded from cancerdiscovery.aacrjournals.org on October 1, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst March 6, 2018; DOI: 10.1158/2159-8290.CD-16-0861 RESEARCH ARTICLE Rajbhandari et al. MRs arises from one or more genetic alterations in their subtype showed a highly undifferentiated phenotype and upstream pathways (14, 17), thus providing a rationale for high mitosis–karyorrhexis index (MK; Supplementary Fig. the heterogeneous mutational landscape of tumors with S1I and S1J, respectively), potentially resulting from down- highly similar transcriptional states (15). MR proteins have regulation of differentiation programs and upregulation been shown to represent critical molecular tumor vulner- of cell proliferation and cell growth pathways, respectively abilities that can be effectively targeted pharmacologically (Fig. 1E and F). Although the majority of MYCNA-subtype in several human malignancies (10, 11, 16, 18, 19), thus samples harbored MYCN amplifications resulting in MYCN providing novel therapeutic opportunities. Here, we sought overexpression, a few samples appeared to compensate to interrogate regulatory networks of high-risk neuroblasto- for low MYCN mRNA levels by significantc-MYC (MYC) mas to identify and validate MR proteins representing highly overexpression. Consistent with previous findings, MYCN/ conserved, tumor-specific vulnerabilities for specific subtypes MYC deregulation in neuroblastoma may occur via tran- of this malignancy. scriptional deregulation mechanisms, independent of their genomic amplification status (22, 23). All samples in this subtype showed high MYCN/MYC-specific signature activ- RESULTS ity (ref. 24; Supplementary Fig. S1M and S1N). Although Our approach represents the integration of computational samples in other high-risk subtypes presented broad 2p inference and experimental validation, both
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