Abstract 4170: The RNA helicase, DDX3, modulates DNA damage repair in Ewing sarcoma

Introduction: Ewing sarcoma (ES) is a primary bone sarcoma occurring in adolescents and young adults, accounting for 15% of childhood/adolescent cancer. We previously reported that an RNA helicase, DDX3, regulates the translation of a variety of proteins in ES cells, including those modulating DNA damage repair. Therefore, we hypothesized that inhibiting DDX3, using a small molecule called RK-33, would radio-sensitize ES cells by preventing DNA damage repair. Methods: ES cell lines (MHH-ES and TC71) were transfected with DDX3- targeted shRNA or a control, scramble shRNA. RT-PCR and western blotting confirmed knockdown of DDX3. RK-33 cytotoxicity was evaluated using cell viability assay. DNA double strand breaks (DSB) were quantified by immunofluorescent staining for γH2AX, and subcellular localization of DDX3 was determined by immunofluorescence. ES patient derived xenografts (PDX) were subcutaneously implanted in 4 cohorts of NSG mice. Results: Expression of DDX3 mRNA and protein was decreased in DDX3-knockdown cells compared with controls. To validate that DDX3 is inhibited by RK-33, we compared the cytotoxicity of this agent in control cells and in the DDX3 knockdown cell lines. Parental ES and control cells (MHH-ES, TC71, MSD-10) were more sensitive to RK-33, with IC50 values that were significantly lower than their corresponding DDX3-knockdown lines (M2C7, M1F6; 4 μM, 2.7 μM and 2.6 μM vs 42 μM and 11.6 μM, respectively, p 10 γH2AX foci, but after 24 hours, the average number of irradiated cells with >10 foci was significantly higher in RK-33-treated cells than in cells treated with vehicle alone (p Citation Format: Marwa Afifi, Breelyn A. Wilky, Catherine Kim, Venu Raman, David Loeb. The RNA helicase, DDX3, modulates DNA damage repair in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4170.