Abstract
Background: Previously, we used inhibitors blocking BET bromodomain binding proteins (BRDs) in Ewing sarcoma (EwS) and observed that long term treatment resulted in the development of resistance. Here, we analyze the possible interaction of BRD4 with cyclin-dependent kinase (CDK) 9. Methods: Co-immunoprecipitation experiments (CoIP) to characterize BRD4 interaction and functional consequences of inhibiting transcriptional elongation were assessed using drugs targeting of BRD4 or CDK9, either alone or in combination. Results: CoIP revealed an interaction of BRD4 with EWS-FLI1 and CDK9 in EwS. Treatment of EwS cells with CDKI-73, a specific CDK9 inhibitor (CDK9i), induced a rapid downregulation of EWS-FLI1 expression and block of contact-dependent growth. CDKI-73 induced apoptosis in EwS, as depicted by cleavage of Caspase 7 (CASP7), PARP and increased CASP3 activity, similar to JQ1. Microarray analysis following CDKI-73 treatment uncovered a transcriptional program that was only partially comparable to BRD inhibition. Strikingly, combined treatment of EwS with BRD- and CDK9-inhibitors re-sensitized cells, and was overall more effective than individual drugs not only in vitro but also in a preclinical mouse model in vivo. Conclusion: Treatment with BRD inhibitors in combination with CDK9i offers a new treatment option that significantly blocks the pathognomonic EWS-ETS transcriptional program and malignant phenotype of EwS.
Highlights
Ewing sarcoma (EwS) is a highly malignant bone and soft tissue neoplasia arising predominantly in the pelvis and long bones in children and young adults with early metastasis to lung and bone [1].EwS is defined by specific balanced chromosomal EWSR1/ETS translocations that give rise to oncogenic chimeric proteins, the most common being EWS-FLI1 as a consequence of the t(11;22)(q24;q12)translocation [2,3,4]
We demonstrated that EwS are susceptible to treatment with epigenetic inhibitors, such as JQ1, blocking BET bromodomain activity and the associated pathognomonic EWS-ETS
We confirmed that the treatment of EwS cell lines with normal concentrations of
Summary
Ewing sarcoma (EwS) is a highly malignant bone and soft tissue neoplasia arising predominantly in the pelvis and long bones in children and young adults with early metastasis to lung and bone [1].EwS is defined by specific balanced chromosomal EWSR1/ETS translocations that give rise to oncogenic chimeric proteins, the most common being EWS-FLI1 as a consequence of the t(11;22)(q24;q12)translocation [2,3,4]. Specific inhibitors of BET proteins such as I-BET151 or JQ1 result in the displacement of BRDs from chromatin and inhibition of transcription at key genes such as BCL2, MYC, and CDK6 [12], and induce growth arrest and differentiation of cancer cells [15,16]. We used inhibitors blocking BET bromodomain binding proteins (BRDs) in Ewing sarcoma (EwS) and observed that long term treatment resulted in the development of resistance. Treatment of EwS cells with CDKI-73, a specific CDK9 inhibitor (CDK9i), induced a rapid downregulation of EWS-FLI1 expression and block of contact-dependent growth. Combined treatment of EwS with BRD- and CDK9-inhibitors re-sensitized cells, and was overall more effective than individual drugs in vitro and in a preclinical mouse model in vivo
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