Abstract PR13: Targeting p-TEFb as well as the EWS-ETS-specific transcriptional program in Ewing sarcoma synergistically blocks tumor growth
Authors: Hensel Tim , Giorgi Chiara , Schmidt Oxana , Yen Hsi-Yu , Steiger Katja , Fulda Simone , Niggli Felix K. , Weigert Wilko , Wang Shudong , Schäfer Beat W. , Burdach Stefan , Richter Günther H.S. 
Published: February 2019

Abstract
Introduction: Ewing sarcomas (EwS) are highly malignant bone or soft tissue tumors. Genetically, EwS are defined by balanced chromosomal EWSR1/ETS translocations, which give rise to chimeric proteins (EWSR1-ETS) that generate an oncogenic transcriptional program associated with altered epigenetic marks throughout the genome. By use of inhibitors blocking BET bromodomain binding proteins (BRDs) we strikingly observed a strong dose-dependent inhibition of the predominant EWSR1-ETS protein EWS-FLI1. Here we analyzed in depth the mechanistic effects of epigenetic EWSR1-FLI1 inhibition and possible contribution of transcriptional elongation to the EwS-specific phenotype and malignancy.
Experimental Procedures: Function of BRDs as well as transcriptional elongation was analyzed by application of specific BRD (JQ1, I-BET151) or CDK9 inhibitors (CDK9i), RNA interference (RNAi) with the generation of stable and inducible knockdowns of BRDs in EwS cell lines. To analyze the resulting changes Co-IP, ChIP-qPCR, RT-PCR, Western blotting, cell cycle analysis, proliferation and invasion assays, whole-transcriptome analysis via microarrays as well as xenograft mouse models were utilized.
Results: By use of JQ1 or iBET we observed a strong downregulation of the predominant EWSR1-ETS protein EWSR1-FLI1 and its associated expression program. The effect on this expression program was partially mimicked by RNAi of BRD3 or BRD4 but not by BRD2. However, long-term treatment with JQ1 resulted in the development of resistance. Interestingly, coimmunoprecipitation experiments revealed a DNA-independent interaction of BRD4 with EWSR1-FLI1 and further interaction with CDK9. CDK9 together with CCNTs are part of the positive transcription elongation factor b (P-TEFb) activating productive elongation of mRNA transcripts. Treatment of EwS cells with specific CDK9i again demonstrated a rapid downregulation of EWSR1-FLI1 expression and block of contact-dependent growth. Microarray analysis following CDK9 inhibition uncovered a transcriptional program only partially related to JQ1 inhibition. However, CDK9 inhibition induced apoptosis in EwS as depicted by downregulation of XIAP and CFLAR and consequently cleavage of Caspase 8, PARP and increased CASP3 activity, similar to JQ1. Combined treatment of EwS with BRD and CDK9 inhibitors in vitro and in a preclinical mouse model in vivo was overall more effective than individual drug application.
Conclusion: Translocation-driven tumors such as EwS are very susceptible to combined treatment with epigenetic inhibitors. Here we demonstrate that treatment with inhibitors targeting the p-TEFb complex could interrupt communication between EWSR1-FLI1, BRD4, and CDK9, further impeding EWSR1-ETS transcriptional activity and its associated pathognomonic expression program. Hence, treatment of EwS with BRD inhibitors in combination with a CDK9i seems a new treatment option that could significantly block the pathognomonic EWSR1-ETS transcriptional program and malignant phenotype of ES.
Citation Format: Tim Hensel, Chiara Giorgi, Oxana Schmidt, Hsi-Yu Yen, Katja Steiger, Simone Fulda, Felix K. Niggli, Wilko Weigert, Shudong Wang, Beat W. Schäfer, Stefan Burdach, Günther H.S. Richter. Targeting p-TEFb as well as the EWS-ETS-specific transcriptional program in Ewing sarcoma synergistically blocks tumor growth [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr PR13.