Purpose: Ewing sarcoma (EWS) is the second most frequent malignant bone and soft-tissue tumor in children and young adults. More than 85% of EWS expressed the oncogenic transcription factor EWS-FLI1 fusion protein. With current therapies, about 75% of patients with localized disease survive, but patients with metastatic or recurrent disease have a dismal outcome with survival rates less than 20%. Although significant advances have been made in understanding the molecular basis of EWS, little has changed in the treatment of EWS patients over the past four decades. Efforts have been focused on the development of therapeutics targeting the EWS-FLI1 oncoprotein; however, no effective drugs have been developed. Therefore, our goal was to accelerate promising and new combination therapies to EWS patients through drug repurposing and rediscovery, and to expand the treatment options and improve treatment outcomes for patients with this rare and life-threatening disease.

Methods: We first employed an integrated gene expression signatures-based bioinformatics approach to identify FDA-approved drugs that are predicted to reverse the EWS disease gene signatures and/or mimic the silencing of the EWS-FLI1 oncogene. We then evaluated the drug hits from the in silico approaches in three benign sarcoma cell lines (Hs 919.T., Hs 822.T., Hs 863.T.) and a panel of six genetically diverse EWS cell lines (A673, TC71, SK-ES-1, RD-ES, CHLA-258 and COG-E-352) by using CellTiter-Blue® Cell Viability Assay. The cytotoxic activities and drug interaction of the most potent drugs in various combinations were further assessed in vitro by using 8x8 matrix dose response assay. The combination index (CI) was calculated by the CompuSyn software according to the Chou and Talalay's method. Cellular apoptosis and DNA damage response after drug treatments in EWS cells were analyzed by Western blot.

Results: Twenty-six FDA-approved drugs were identified through in silico prediction approaches (FDR < 0.05). Among them, MS-275, elesclomol, daunorubicin and vindesine are the most potent and selective drugs in killing EWS cells (72 h, IC50 0.004 - 3.65 µM), as compare to the benign sarcoma cells (IC50s in EWS cells were at least 10 times lower than the counterparts in benign controls). The combination of MS-275 (Class I HDAC inhibitor) and elesclomol (a selective mitochondrial reactive oxygen species inducer) was the most robust and synergistic treatment in inhibiting EWS cell growth, with 57%-82% of the combinations showing synergy (CI < 1) in the matrix dose response test across all tested EWS cell lines with either EWS-FLI1 or EWS-ERG fusion. Western blot data indicated that MS-275 and elesclomol induced caspase-dependent apoptosis in EWS cells in both time- and dose-dependent manner. Both MS-275 and elesclomol treatment led to DNA double-strand breaks (DSBs), as evidenced by the phosphorylation of γH2AX. MS-275 treatment inhibited homologous recombination (HR) pathway by downregulating BRCA1 and RAD51, while MS-275 regulated non-homologous end-joining (NHEJ) pathway in a cell type-dependent manner.

Conclusions: Our studies revealed that the combination of MS-275 and elesclomol were synergistic and that MS-275 sensitized EWS cells to elesclomol through targeting DNA DSBs repair. This combination therapy may provide an alternative treatment strategy for children and young adults diagnosed with Ewing sarcoma.

Citation Format: Yan Ma, Bin Chen, Scott J. Weir, Atul Butte, Andrew K. Godwin. In silico and in vitro drug screening identifies new therapeutic approaches targeting DNA double-strand breaks repair in Ewing sarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B20.