Combination Flavopiridol and SN38 drives toxicity and loss of DNA damage repair in Ewing Sarcoma

Abstract

EWS-Fli1 is an oncogenic transcription factor that causes Ewing sarcoma. EWS-Fli1 is a fusion protein comprised of an N-terminal low complexity domain from the RNA-binding protein EWSR1 and the C-terminal DNA-binding domain of the ETS transcription factor Fli1. EWS-Fli1 drives expression of oncogenes and decreases expression of tumor suppressor proteins. EWS-Fli1 acts as a transcriptional regulator by recruiting RNA Pol II to promoter regions. Ewing sarcoma is particularly susceptible to DNA damaging chemotherapy compared to other cancers. However, the underlying reason for this sensitivity to DNA damage has not been elucidated. Given the role of EWS-Fli1 in disrupting transcription, we hypothesized that EWS-Fli1 is concurrently disrupting the DNA damage response. During transcription, RNA Pol II is phosphorylated by CDK9 to escape initiation into elongation. We tested the ability of transcription inhibition to affect the DNA damage response by combining treatment a CDK9 inhibitor, flavopiridol, and the DNA damage agent, SN38. This combination drastically diminished cell viability in Ewing cell lines. This sensitivity was not found in non-Ewing cell lines U2OS and HEK293T/17. We also find in Ewing cells that the combination treatment decreases the DNA damage markers gamma-H2A.X and phospho-CHK1 compared to non-Ewing's cells. Together, these results suggest Ewing sarcoma cells are sensitized to DNA damage due in part to transcriptional dysregulation and suggest combination CDK9 inhibition with DNA damage treatment could provide a therapeutic approach for Ewing Sarcoma.