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

Abstract

Abstract 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<0.05). To determine the effect on DNA damage repair, DSB were quantified in DDX3- knockdown cells and controls, treated with or without RK-33, at various time points after irradiation. At early time points, treatment with RK-33 had no impact on the number of ES and control cells (MHH-ES, TC71, MSD-10) with >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<0.01). The time needed for full recovery of radiation-induced DSB in untreated cells was less than 6 hr, while RK-33 pre-treated cells failed to recover even after 24 hr. Irradiated DDX3-knockdown cells demonstrated impaired DNA damage repair as well. To expand our investigation into the mechanism by which DDX3 regulates DNA damage repair, we studied the subcellular localization of the enzyme in response to radiation. We found that DDX3 translocates into the nucleus and colocalizes with γH2AX in irradiated cells, suggesting a dual role, direct and indirect, in DNA damage repair. In vivo, we found that animals treated with subtherapeutic external beam radiation concurrently with RK-33 had a substantial increase in tumor response. Our results suggest that DDX3 plays a crucial role in regulating DNA damage repair dynamics in ES, both indirectly by affecting the translation of involved proteins, and directly by localizing to sites of DSB. Therefore, inhibiting DDX3 using RK-33 may represent a novel approach to radiosensitization of cancer cells. 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.