Abstract B012: The effect of a novel therapy on DNA methylation in Ewing Sarcoma

Abstract Ewing sarcoma (EwS) is a rare and aggressive disease affecting the bone and soft tissue of children and young adults. A FET::ETS fusion protein, typically EWSR1-FLI1 is pathognomonic for this disease. EwS is genomically quiet with widespread dysregulation driven by aberrant fusion activity leading to elevated endogenous levels of replication stress and epigenetic drift. While five-year survival rates are moderate for localized primary disease (∼70%), metastasized, relapsed, and refractory disease have much lower rates of survival (15-35%) and are associated with increased epigenetic heterogeneity. Treatment options are currently limited to systemic therapies which have not seen major advancement since the development of interval compression of chemotherapy and often lead to secondary complications for these patients. Our lab has identified a novel combination of replication stress response inhibitors (RSRi), DDK (DDKi) and WEE1 (WEE1i), which sensitize EwS cells to their own elevated levels of replication stress by removing repair strategies and forcing mitotic catastrophe and apoptosis. In one in vivo study, cell line derived xenografts in NSG mice were treated with DDKi (simurosertib) and WEE1i (adavosertib) in the following design: 1) vehicle control (n=5), 2) Irinotecan + Temozolomide (n=9), 3) Irinotecan (full dose) + DDKi + WEE1i (n=7), 4) Irinotecan (half dose) + DDKi + WEE1i (n=9), for a total of 30 samples. We have shown that this RSRi combination is able to effectively limit tumor growth over time even in the context of decreased chemotherapeutic dose. As increased replication stress can cause epigenetic drift, changes in methylation associated with treatment condition were investigated by reduced representation bisulfite sequencing. Differential methylation analysis was performed on individual CpG sites, CpG structures (islands, shores) and transcriptional elements (promoters, enhancers, transcription start sites). In this context, key pathways were found to be differentially methylated between treatment conditions, including those involved in cell signaling, motility, metabolism, and neuronal processes. Overall, this targeted therapy presents a promising therapeutic option for this pediatric patient population. Citation Format: Emily Isenhart, Ajay Gupta, Joyce Ohm. The effect of a novel therapy on DNA methylation in Ewing Sarcoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Methylation, Clonal Hematopoiesis, and Cancer; 2025 Feb 1-4; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2025;85(3 Suppl):Abstract nr B012.