Abstract
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.
Highlights
Ewing sarcoma (EWS) represents the second-most common primary bone malignancy in children and young adults after osteosarcoma with an annual incidence of 2.9 cases per million population [1]
To investigate the dependency of the EWS fusion proteins on bromodomain and extraterminal (BET) bromodomain proteins in EWS, we knocked down BRD2, BRD3, or BRD4 by shRNA in CHLA-10 and RD-ES cells characterized by EWS/FLI1, and CADO-ES1 cells characterized by EWS/ ERG fusion
To test the protumorigenic and metastatic functions of the BET protein BRD4 —a well-characterized member of the BET family, the chorioallantoic membrane (CAM) model was used in which tumor growth and spontaneous metastasis in vivo can be measured in a short period of time [22]
Summary
Ewing sarcoma (EWS) represents the second-most common primary bone malignancy in children and young adults after osteosarcoma with an annual incidence of 2.9 cases per million population [1]. EWS is driven by characteristic chromosomal translocations that generate fusions between the EWS breakpoint region 1 (EWSR1) gene with members of the ETS family of transcription factors, most commonly to FLI1 [3] and with less frequency to ERG [4]. The EWS/ETS (EWS/FLI1 and EWS/ERG) oncogenic fusion proteins are often the only genetic alteration in these pediatric tumors functioning as an aberrant transcription factor [5, 6]. Similar to other transcription factors, direct pharmacological targeting of the EWS/ETS fusion proteins remains a formidable challenge, and proteins in this class are often considered undruggable due to a lack of small molecule recognizable pockets [8]. Identification and targeting of functionally relevant EWS/ETS-associated factors should be considered to mitigate its oncogenic function
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