Abstract 7054: Mithramycin and chemotherapy combination induces apoptosis in Ewing sarcoma cells by upregulating reactive oxygen species and associated pathways

Abstract Ewing’s Sarcoma (ES) is a rare and aggressive bone cancer that primarily affects children and adolescents. The disease is characterized by the presence of the EWS-FLI1 fusion protein, a transcription factor found in 85% of ES tumors that drives oncogenesis and tumor progression. Advanced ES has a grim prognosis with survival rates below 30%. Standard chemotherapy often results in significant long-term morbidities due to side-effects. Mithramycin has shown promise in inhibiting EWS-FLI1, however, its clinical application has not been established due to dose-dependent toxicities. We hypothesize that combining mithramycin with chemotherapy could offer a potential strategy to achieve synergistic effects, enabling lower dosages of both drugs to enhance efficacy while minimizing toxicity. We tested the combination effect of Mithramycin and Etoposide using ES cell lines and determined the response on inducing higher apoptosis through altering reactive oxygen species (ROS). Cardiomyocytes were used to test systemic toxicity against non-cancerous cells. Ewing Sarcoma cells (CHLA 10 and TC205) and cardiomyocytes (H9C2) were cultured and treated with Mithramycin, Etoposide, and their combination using optimized concentrations determined for each drug. Cell viability, Caspase 3/7 activity and ROS production were measured using respective luminescence-based assays. Flow cytometry with Annexin V/7AAD staining evaluated apoptosis levels, and protein expression of apoptosis-related markers, such as c-PARP, was analyzed using a capillary-based Simple Western system. Statistical analyses were performed using GraphPad Prism to evaluate significance. The combination of Mithramycin and Etoposide significantly inhibited ES cell viability compared to either agent alone. Apoptotic markers were markedly elevated in the combination-treated cells and associated with an upregulation of Caspase 3/7 activity and ROS production. Flow cytometry analysis confirmed an elevated apoptotic response, with a higher percentage of Annexin V-positive cells in the combination group. Additionally, Western blot analysis revealed increased c-PARP expression, consistent with enhanced apoptosis. In H9C2 cardiomyocytes, no treatment exhibited significant toxicity at equivalent concentrations, indicating the potential for no cardiac side effects. These results demonstrate the synergistic effects of Mithramycin and Etoposide in targeting cancer cells while sparing healthy tissue. Protein samples were subjected to proteomics and Integrative Pathway Analyses are currently being conducted to precisely elucidate the involvement of ROS-associated markers in understanding the mechanism of action and in vivo efficacy of this combination. Investigating additional biomarkers of response and resistance may provide further insights for personalized therapy. Citation Format: Christoffer B. Lambring. Mithramycin and chemotherapy combination induces apoptosis in Ewing sarcoma cells by upregulating reactive oxygen species and associated pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7054.