EWS/ETS-Driven Ewing Sarcoma Requires BET Bromodomain Proteins.

The EWS/ETS fusion transcription factors drive Ewing sarcoma (EWS) by orchestrating an oncogenic transcription program. Therapeutic targeting of EWS/ETS has been unsuccessful; however, identifying mediators of the EWS/ETS function could offer new therapeutic options. Here, we describe the dependency of EWS/ETS-driven transcription upon chromatin reader BET bromdomain proteins and investigate the potential of BET inhibitors in treating EWS. EWS/FLI1 and EWS/ERG were found in a transcriptional complex with BRD4, and knockdown of BRD2/3/4 significantly impaired the oncogenic phenotype of EWS cells. RNA-seq analysis following BRD4 knockdown or inhibition with JQ1 revealed an attenuated EWS/ETS transcriptional signature. In contrast to previous reports, JQ1 reduced proliferation and induced apoptosis through MYC-independent mechanisms without affecting EWS/ETS protein levels; this was confirmed by depleting BET proteins using PROTAC-BET degrader (BETd). Polycomb repressive complex 2 (PRC2)-associated factor PHF19 was downregulated by JQ1/BETd or BRD4 knockdown in multiple EWS lines. EWS/FLI1 bound a distal regulatory element of PHF19, and EWS/FLI1 knockdown resulted in downregulation of PHF19 expression. Deletion of PHF19 via CRISPR-Cas9 resulted in a decreased tumorigenic phenotype, a transcriptional signature that overlapped with JQ1 treatment, and increased sensitivity to JQ1. PHF19 expression was also associated with worse prognosis in patients with EWS. In vivo, JQ1 demonstrated antitumor efficacy in multiple mouse xenograft models of EWS. Together these results indicate that EWS/ETS requires BET epigenetic reader proteins for its transcriptional program and can be mitigated by BET inhibitors. This study provides a clear rationale for the clinical utility of BET inhibitors in treating EWS.Significance: These findings reveal the dependency of EWS/ETS transcription factors on BET epigenetic reader proteins and demonstrate the potential of BET inhibitors for the treatment of EWS. Cancer Res; 78(16); 4760-73. ©2018 AACR.

[1]  T. Kirchner,et al.  Targeting the undruggable: exploiting neomorphic features of fusion oncoproteins in childhood sarcomas for innovative therapies , 2019, Cancer and Metastasis Reviews.

[2]  F. Robert,et al.  The Mediator Complex: At the Nexus of RNA Polymerase II Transcription. , 2017, Trends in cell biology.

[3]  M. Rivera,et al.  Cancer-Specific Retargeting of BAF Complexes by a Prion-like Domain , 2017, Cell.

[4]  M. Bulyk,et al.  Polycomb-like proteins link the PRC2 complex to CpG islands , 2017, Nature.

[5]  S. Lessnick,et al.  Recent advances in targeted therapy for Ewing sarcoma , 2016, F1000Research.

[6]  Yan Guo,et al.  BET bromodomain inhibitors suppress EWS-FLI1-dependent transcription and the IGF1 autocrine mechanism in Ewing sarcoma , 2016, Oncotarget.

[7]  Liguo Wang,et al.  BET bromodomain-mediated interaction between ERG and BRD4 promotes prostate cancer cell invasion , 2016, Oncotarget.

[8]  J. Bradner,et al.  Targeting the epigenetic readers in Ewing Sarcoma inhibits the oncogenic transcription factor EWS/Fli1 , 2016, Oncotarget.

[9]  B. Schäfer,et al.  Targeting the EWS-ETS transcriptional program by BET bromodomain inhibition in Ewing sarcoma , 2015, Oncotarget.

[10]  J. Alonso,et al.  EWS/FLI1 Target Genes and Therapeutic Opportunities in Ewing Sarcoma , 2015, Front. Oncol..

[11]  Lei Zheng,et al.  MicroRNA-195-5p acts as an anti-oncogene by targeting PHF19 in hepatocellular carcinoma. , 2015, Oncology reports.

[12]  Darryl J Pappin,et al.  BET Bromodomain Inhibition Suppresses the Function of Hematopoietic Transcription Factors in Acute Myeloid Leukemia. , 2015, Molecular cell.

[13]  Nathan C. Sheffield,et al.  Epigenome Mapping Reveals Distinct Modes of Gene Regulation and Widespread Enhancer Reprogramming by the Oncogenic Fusion Protein EWS-FLI1 , 2015, Cell reports.

[14]  Yan Liu,et al.  Targeting transcriptional addictions in small cell lung cancer with a covalent CDK7 inhibitor. , 2014, Cancer cell.

[15]  W. Huber,et al.  Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 , 2014, Genome Biology.

[16]  Shawn M. Gillespie,et al.  EWS-FLI1 utilizes divergent chromatin remodeling mechanisms to directly activate or repress enhancer elements in Ewing sarcoma. , 2014, Cancer cell.

[17]  Li Ding,et al.  Genomic landscape of Ewing sarcoma defines an aggressive subtype with co-association of STAG2 and TP53 mutations. , 2014, Cancer discovery.

[18]  A. McKenna,et al.  The genomic landscape of pediatric Ewing sarcoma. , 2014, Cancer discovery.

[19]  Zhaohui S. Qin,et al.  Therapeutic Targeting of BET Bromodomain Proteins in Castration-Resistant Prostate Cancer , 2014, Nature.

[20]  Rahul Jandial,et al.  Altered Expression of Polycomb Group Genes in Glioblastoma Multiforme , 2013, PloS one.

[21]  A. Martínez-Torteya,et al.  SurvExpress: An Online Biomarker Validation Tool and Database for Cancer Gene Expression Data Using Survival Analysis , 2013, PloS one.

[22]  D. Zheng,et al.  An H3K36 methylation-engaging Tudor motif of polycomb-like proteins mediates PRC2 complex targeting. , 2013, Molecular cell.

[23]  Zhaohui S. Qin,et al.  Characterization of the EZH2-MMSET histone methyltransferase regulatory axis in cancer. , 2013, Molecular cell.

[24]  Yang Shi,et al.  Phf19 links methylated Lys36 of histone H3 to regulation of Polycomb activity , 2012, Nature Structural &Molecular Biology.

[25]  Stefan Knapp,et al.  The bromodomain interaction module , 2012, FEBS letters.

[26]  Arul M Chinnaiyan,et al.  PARP-1 inhibition as a targeted strategy to treat Ewing's sarcoma. , 2012, Cancer research.

[27]  H. Hakonarson,et al.  Evaluating the role of the FUS/TLS-related gene EWSR1 in amyotrophic lateral sclerosis. , 2012, Human molecular genetics.

[28]  S. Robson,et al.  Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia , 2011, Nature.

[29]  U. Dirksen,et al.  Risk of recurrence and survival after relapse in patients with Ewing sarcoma , 2011, Pediatric blood & cancer.

[30]  P. Sandy,et al.  Targeting MYC dependence in cancer by inhibiting BET bromodomains , 2011, Proceedings of the National Academy of Sciences.

[31]  R. Young,et al.  BET Bromodomain Inhibition as a Therapeutic Strategy to Target c-Myc , 2011, Cell.

[32]  William B. Smith,et al.  Selective inhibition of BET bromodomains , 2010, Nature.

[33]  Angela N Koehler,et al.  A complex task? Direct modulation of transcription factors with small molecules. , 2010, Current opinion in chemical biology.

[34]  Derek Y. Chiang,et al.  The landscape of somatic copy-number alteration across human cancers , 2010, Nature.

[35]  Stephen C. Haroldsen,et al.  Microsatellites as EWS/FLI response elements in Ewing's sarcoma , 2008, Proceedings of the National Academy of Sciences.

[36]  M. Suvà,et al.  EWS-FLI-1 expression triggers a Ewing's sarcoma initiation program in primary human mesenchymal stem cells. , 2008, Cancer research.

[37]  Shwu‐Yuan Wu,et al.  The Double Bromodomain-containing Chromatin Adaptor Brd4 and Transcriptional Regulation* , 2007, Journal of Biological Chemistry.

[38]  Vladimir N Uversky,et al.  Multiple aromatic side chains within a disordered structure are critical for transcription and transforming activity of EWS family oncoproteins , 2007, Proceedings of the National Academy of Sciences.

[39]  J. Tchinda,et al.  Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer. , 2006, Science.

[40]  Pablo Tamayo,et al.  Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[41]  Qiang Zhou,et al.  Recruitment of P-TEFb for stimulation of transcriptional elongation by the bromodomain protein Brd4. , 2005, Molecular cell.

[42]  J. Brady,et al.  The bromodomain protein Brd4 is a positive regulatory component of P-TEFb and stimulates RNA polymerase II-dependent transcription. , 2005, Molecular cell.

[43]  S. Burdach,et al.  DNA Microarrays Reveal Relationship of Ewing Family Tumors to Both Endothelial and Fetal Neural Crest-Derived Cells and Define Novel Targets , 2004, Cancer Research.

[44]  M. Schemper,et al.  EWS/FLI-1 antagonists induce growth inhibition of Ewing tumor cells in vitro. , 1996, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[45]  P. Sorensen,et al.  A second Ewing's sarcoma translocation, t(21;22), fuses the EWS gene to another ETS–family transcription factor, ERG , 1994, Nature Genetics.

[46]  G. Thomas,et al.  Gene fusion with an ETS DNA-binding domain caused by chromosome translocation in human tumours , 1992, Nature.

[47]  A. Stathis,et al.  BET Proteins as Targets for Anticancer Treatment. , 2018, Cancer discovery.