Abstract 3864: Combined targeting of the EWS/ETS transcriptional program by blocking epigenetic readers and transcription initiation in Ewing sarcoma
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: July 2017

Introduction: Previously, we reported blocking of BET bromodomain binding proteins (BRDs) by use of an inhibitor (JQ1) and the associated strong down-regulation of the predominant EWS-ETS protein EWS-FLI1 in Ewing sarcoma (ES). Here we analyzed in depth the mechanistic effects of this treatment by EWS-FLI1 interaction studies and the evaluation of possible combined targeting.

Experimental procedures: Function of BRDs was analysed 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 analyse 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 strikingly observed a strong down-regulation of the predominant EWS-ETS protein EWS-FLI1 and subsequent microarray analysis revealed JQ1 treatment to block the typical ES associated expression program. The effect on this expression program was partially mimicked by RNAi for BRD3 or BRD4 but not by BRD2. However, knockout studies of BRD4 by CRISPR/Cas9 as well as knockdowns of individual BRD2, 3 or 4 did not recapitulate JQ1-mediated proliferation restrictions and blockade and tumor development in xenograft mice as observed for JQ1. However, co-immunoprecipitation experiments revealed an DNA independent interaction of BRD4 with EWS/FLI1 and further interaction with CdK9. Treatment of ES cells with a specific CDK9 inhibitor demonstrated a rapid down regulation of EWS-FLI1 expression and block of contact dependent growth. Furthermore, CDK9 inhibition induced apoptosis in ES as depicted by down regulation of XIAP and CFLAR and consequently cleavage of Caspase 8, PARP and increased CASP3 activity, similar to JQ1. Combined treatment of ES with BRD and CdK9 inhibitors was more effective than individual drug application.

Conclusion: Translocation driven tumors such as ES 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 EWS-FLI1, BRD4 and CDK9 further impeding EWS-ETS transcriptional activity and its associated pathognomonic expression program.

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. Combined targeting of the EWS/ETS transcriptional program by blocking epigenetic readers and transcription initiation in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3864. doi:10.1158/1538-7445.AM2017-3864