Introduction: We reported blocking of BET bromodomain binding proteins (BRDs) by use of an inhibitor (JQ1) and the associated strong downregulation 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 combined targeted treatment options.
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 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 strikingly observed a strong downregulation 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 a DNA-independent interaction of BRD4 with EWS/FLI1 and additional interaction with CDK9. Treatment of ES cells with a specific CDK9 inhibitor demonstrated a rapid downregulation of EWS-FLI1 expression and block of contact-dependent growth. Furthermore, CDK9 inhibition induced apoptosis in ES 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 ES with BRD and CDK9 inhibitors in a preclinical model was more effective than individual drug application alone.
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, Oxana Schmidt, Shudong Wang, Beat W. Schaefer, Stefan Burdach, Guenther H.S. Richter. Combined targeting of the Ewing sarcoma transcriptional program by blocking epigenetic readers and transcription initiation via EWS-FLI1 [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr B02.