Abstract A27: BET bromodomain proteins in Ewing sarcoma regulate a specific transcriptional program, tumorigenicity and apoptosis by interacting with EWS-FLI1 and the positive transcription elongation factor
Authors: Hensel Tim , Giorgi Chiara , Becker-Dettling Fiona , Calzada-Wack Julia , Neff Frauke , Schmidt Oxana , Wang Shudong , Schäfer Beat W. , Burdach Stefan , Richter Günther HS 
Published: December 2017

Introduction: Ewing sarcomas (ES) are highly malignant bone or soft tissue tumors that are genetically defined by balanced chromosomal EWS/ETS translocations. Previously, we reported the use of an inhibitor (JQ1) blocking BET bromodomain binding proteins (BRDs) and the associated strong down-regulation of the predominant EWS-ETS protein EWS-FLI1. Here we analyzed the mechanistic effects of this treatment by EWS-FLI1 interaction studies and the evaluation of possible down-stream players in order to shed further light into this intricate network and to develop refined treatment regimens for ES patients.

Experimental procedures: Function of BRDs was analyzed by application of specific inhibitors (JQ1, I-BET151), RNA interference (RNAi) with the generation of stable and inducible knockdowns or knockouts by the generation of BRD4 CRISPR/Cas9 cell lines. To analyses 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.

Summary: By use of JQ1 we strikingly observed a strong down-regulation of the predominant EWS-ETS protein EWS-FLI1 in a dose dependent manner, which was confirmed by treatment with I-BET151. Microarray analysis revealed JQ1 treatment to block the typical ES associated expression program. The effect on this expression program was partially mimicked by RNAi with BRD3 or BRD4 expression but not by BRD2 blockade. Furthermore, knockout studies of BRD4 by CRISPR/Cas9 as well as knockdowns of individual BRD2, 3 or 4 did not recapitulate proliferation restrictions as observed for JQ1, hinting towards an interdependency for all 3 proteins.

Subsequent functional studies on the protein level demonstrated an interaction of EWS-FLI1 with BRD4 as well as phosphorylated CDK9 as an essential partner of the positive transcription elongation factor (p-TEFb). Additional analysis revealed a particular regulation of the X-linked inhibitor of apoptosis protein (XIAP) and CASP8 and FADD-like regulator (CFLAR), as a result of CDK9 activity. Treatment of ES cells with a specific CDK9 inhibitor further demonstrated a rapid down regulation of EWS-FLI1 expression and block of contact dependent growth. Furthermore, CDK9i induced apoptosis in ES as depicted by cleavage of Caspase 8 or PARP, which we similarly observed after JQ1 treatment.

Conclusion: Here we demonstrate a possible interdependency of BET proteins regulating the ES specific expression profile and a possible substitution effect observed after knockdown or knockout of individual BRD proteins. We further demonstrate that ES are also susceptible to treatment with inhibitors targeting the p-TEFb complex that resemble features observed after JQ1 treatment. Most interestingly, we observed EWS-FLI1 to interact with BRD4 and CDK9 to presumably regulate an ES specific expression profile and suppress activation of apoptotic mechanisms. Hence treatment of ES with BRD inhibitors in combination with a CDK9i seems a new treatment option that could significantly improve the particular need to specifically block the pathognomonic EWS-ETS transcriptional program and malignant phenotype of ES.

Citation Format: Tim Hensel, Chiara Giorgi, Fiona Becker-Dettling, Julia Calzada-Wack, Frauke Neff, Oxana Schmidt, Shudong Wang, Beat W. Schäfer, Stefan Burdach, Günther HS Richter. BET bromodomain proteins in Ewing sarcoma regulate a specific transcriptional program, tumorigenicity and apoptosis by interacting with EWS-FLI1 and the positive transcription elongation factor [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr A27.