Abstract 2621: Mitotic activities of Ewing sarcoma proteins

Ewing sarcoma (ES) is a pediatric bone tumor in critical need of targeted chemotherapies with fewer side effects. ES’s driving hallmark, the t(11:22) chromosomal translocation, generates two aberrations of the EWS protein: the loss of an EWS allele and expression of EWS/FLI1 fusion protein, denoted as EWS aberrations. Identifying drug targetable EWS aberration-induced changes to tumor biology and drug sensitivity is required to optimize current treatment strategies and develop new targeted therapies. To accomplish this, we established a DLD-1 cell line that lacks the t(11:22) chromosomal translocation and EWS aberrations, while enabling conditional EWS/FLI1 expression with doxycycline (Tet-On system), single-allele EWS knockdown with auxin (auxin-inducible degron system), or a combination of both (EWS/FLI1-EWS KD). This allows us to dissect the contribution of each EWS aberration to phenotypic changes in the cell. Since EWS is a known regulator of tubulin acetylation, we investigated whether tubulin acetylation levels changed under EWS aberration conditions. Both immunocytochemistry and western blotting revealed EWS/FLI1 expressing and EWS/FLI1-EWSKD cells displayed lower acetylated tubulin signal intensity after one and seven days, while EWS knockdown cells displayed higher acetylated tubulin signal intensity. As tubulin acetylation prevents microtubule damage, we probed cells with an antibody that visualizes microtubule damage and repair sites. EWS/FLI1 expressing and EWS/FLI1-EWS KD cells exhibit a higher signal intensity, covering a broader area compared to the control cells. Our data suggests that the combination of EWS/FLI1-EWS KD increases microtubule damage sites by reducing microtubule acetylation. When the EWS/FLI1-EWS KD cells were treated with nocodazole, the rate of cell death increased compared to control nocodazole-treated cells, indicating the cells had been sensitized to microtubule disruption. Currently, we are investigating how EWS aberrations change the regulation of microtubule acetylation and how this mechanism affects sensitivity to microtubule disruptors. Understanding this mechanism could identify new drug targets and treatment strategies in ES to improve drug efficacy and reduce secondary side effects for patients. Citation Format: Evan Schulz, Mizuki Azuma. Mitotic activities of Ewing sarcoma proteins [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 2621.