Abstract 7053: Proteomics and integrative pathway analysis to understand the mechanism of action of mithramycin and etoposide combination induced cell death in Ewing sarcoma cells

Abstract Ewing’s sarcoma (ES) is a highly aggressive cancer that primarily originates in the bones or soft tissues and is predominantly diagnosed in adolescents and young adults. The EWS-FLI1 fusion protein is considered a hallmark of ES, being expressed in approximately 85% of cases. Prognosis for advanced ES remains poor, with survival rates around 30%, and current chemotherapy regimens are associated with severe long-term side effects. Mithramycin has emerged as a promising inhibitor of EWS-FLI1; however, its clinical application is limited by dose-dependent toxicities. We hypothesize that combining mithramycin with standard chemotherapy could enhance therapeutic outcomes through synergistic effects, potentially allowing for reduced doses of both agents to maximize efficacy while minimizing toxicity. To investigate this, we evaluated the combined impact of mithramycin and etoposide on ES cell lines. Using TC205 cells, we further explored the underlying mechanisms through proteomics and integrative pathway analysis (IPA). In this study, we assessed the dose- and time-dependent effects of mithramycin and etoposide in ES cell lines (CHLA10 and TC205) and non-cancerous cardiomyocytes (H9C2) to optimize treatment conditions. In vivo assays demonstrated a significant reduction in tumor growth with combination therapy compared to single-agent treatment. Using TC205 cells, we assessed the impact of mithramycin and etoposide both as monotherapies and in combination. Cells were plated in triplicate for each group (control, mithramycin, etoposide, and mithramycin + etoposide) and treated with optimized doses. After 48 hours, cells were harvested, and protein samples were subjected to proteomics and IPA to elucidate the mechanisms of action of both mono- and combination therapies. The combination of mithramycin and etoposide significantly inhibited cell viability compared to individual treatments. Proteomics analysis identified 517 altered proteins (t-test with BH correction), with 142 upregulated and 375 downregulated. IPA revealed significant changes in markers associated with disease and physiological function networks, including homologous recombination, cytostasis of sarcoma cell lines, cell movement of sarcoma cell lines, and osteoclastogenesis of limb bones. More than 70 disease and function networks and over 200 canonical pathways were significantly affected. Key pathways included eukaryotic translation initiation, cell cycle checkpoints, neutrophil degranulation, and cell cycle control of chromosomal replication. Overall, these findings reveal that the combination of mithramycin and etoposide alters critical networks and pathways, enhancing therapeutic efficacy in ES cell lines. Citation Format: Christoffer B. Lambring, Khadiza Zaman, Elin Stone, Natalie Gierat, Ameya Bhargava, Laszlo Prokai, Riyaz Basha. Proteomics and integrative pathway analysis to understand the mechanism of action of mithramycin and etoposide combination induced cell death in Ewing sarcoma cells [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 7053.