Abstract EWS/FLI1 is a unique chimeric fusion protein found in approximately 85% of Ewing sarcoma (ES) tumors. Blocking this fusion protein in conjunction with administration of anticancer agents could be an effective strategy for treating ES. Baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5), also known as survivin, is overexpressed in several malignancies, including ES. Survivin is associated with aggressive growth of cancer and resistance to standard chemotherapy and thus serves as a prognostic marker for ES and other cancers. EWS/FLI1 and Specificity protein1 (Sp1) jointly mediate the expression of cyclins to facilitate cell growth. Mithramycin-A (Mit-A) has been identified as a potent inhibitor of EWS-FLI1 and its downstream targets. Preclinical research from our laboratory demonstrated that clotam, a small molecule, exhibits anticancer activity by targeting Sp1 and survivin. The objective of our investigation is to test the efficacy of Mit-A or clotam to induce the antiproliferative activity of standard chemotherapeutic agents, vincristine (Vin) or etoposide (Eto), in ES cell lines. TC71, TC32, CHLA32, CHLA10 and TC205 cells were treated with increasing concentrations of Mit-A/clotam/Vin/Eto. Cellular growth inhibition was evaluated at 48 h post-treatment using CellTiter-Glo kit (Promega). In order to determine the combination response, cell growth inhibition of each chemotherapeutic drug was assessed in the presence or absence of Mit-A or clotam. Further studies were performed to investigate the effect of individual agents and combinations on apoptosis and cell cycle phase distribution. Levels of cleaved poly (ADP-ribose) polymerase (c-PARP) were determined by Western blot analysis and the apoptotic cell population quantitated by flow cytometry analysis of Annexin-V stained cells. Cell cycle phase distribution was also measured by flow cytometry using propidium iodide. Results showed a dose-dependent antiproliferative effect with all agents. The combination of Mit-A or clotam with Vin or Eto showed greater effect on apoptosis and cell cycle arrest compared to the effects of Vin or Eto alone. Overall, these results indicate the potential clinical advantage of combination treatment involving Mit-A or clotam for inhibiting the growth of ES cells. Molecular profiling analysis is under way to elucidate the candidate pathways associated with beneficial effect of our combination treatments and with other chemotherapeutic agents. Citation Format: Anish Ray, Sagar Shelake, Umesh Sankpal, Lina Albeer, Holly Lout, Abigail Hunter, Kathryn Dunlap, Rajasekhar Maram, W. Paul Bowman, Riyaz M. Basha. Combination treatment strategies to enhance the effect of standard chemotherapeutic agents in Ewing sarcoma cells [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 3188.