Abstract 2624: Aneuploidy induction mechanism in Ewing sarcoma

Ewing sarcoma (ES) is the second most prevalent sarcoma that develops in the bones of adolescents and children. A major aberration in Ewing sarcoma is the chromosomal translocation between chromosomes t(11;22), resulting in the loss of one allele of EWSR1 and acquisition of EWSR1-FLI1 fusion gene. Trisomy 8 (∼50%) is the major aneuploidy in ES tumors and is associated with low survival rate in patients. This study aims to elucidate the molecular mechanism underlying the aneuploidy induction in ES. To study the role of loss of one EWSR1 allele and gain of the EWSR1-FLI1 fusion gene inducing aneuploidy, we established a conditional DLD-1 cell line that enables knockdown of EWS derived from one EWSR1 allele using Auxin-induced Degron system and expression of EWSR1-FLI1 using a Tet-on system. The DLD-1 cell line was employed in this study because it is a near diploid cell line that has been utilized to investigate aneuploidy induction by the gene of interest. Treatment of the cell with Auxin (AUX) degrades EWSR1 protein expressed from one allele of EWSR1, while treatment with Doxycycline (DOX) induces the expression of EWS-FLI1. Simultaneous degradation of EWSR1 and EWSR1-FLI1 expression is achieved by treating the cell line with both AUX and DOX. To investigate the effect of loss of one EWSR1 allele and gain of EWSR1-FLI1 fusion gene, cells were treated with AUX (EWSR1 knockdown derived from one allele), DOX (EWSR1-FLI1expression), and AUX/DOX (EWSR1 knockdown and EWSR1-FLI1 expression), for 2 to 8 days. The results showed that all three-drug treated samples led to an increase in cell size. Further, Fluorescence-activated Cell Sorting analysis (FACS) revealed that the majority of the small cells were diploid (2N), whereas the majority of large cells were aneuploid (>3N). While Ewing sarcoma cells are described as small round cells, there was a variation observed in Ewing sarcoma cell sizes, A673 and SKES1, but not in the normal MSC cells.Next, we investigated whether the treatment of AUX and DOX induces trisomy 8 using FISH assay. Both AUX and DOX treated cells displayed a higher incidence of trisomy 8 compared to the control group. Furthermore, the AUX/DOX treated cells displayed a significantly higher incidence of trisomy 8 compared to AUX, DOX treated, and control cells, suggesting that the expression of EWSR1-FLI1 fusion protein and EWSR1 knockdown have a cumulative effect on trisomy 8 induction. While trisomy 8 has been reported in various cancers with a poor prognosis, the induction mechanism remains unclear. To the best of our knowledge, this is the first observation describing the collaborative activities between the EWSR1-FLI1 fusion protein and EWSR1 knockdown in inducing trisomy 8. We are currently investigating the molecular function of trisomy 8 induction in this cell line and in Ewing sarcoma cell lines. Citation Format: Priyani H. Hapugaswatta, Mizuki Azuma. Aneuploidy induction mechanism in Ewing sarcoma [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 2624.