The combination of EWSR1-FLI1 and loss of one EWSR1 allele leads to the induction of trisomy 8

Ewing sarcoma is a pediatric cancer that develops in skeletal elements. The majority of Ewing sarcoma patients carry the aberrant EWSR1-FLI1 fusion gene. Despite trisomy 8 being an additional common aberration associated with a poor prognosis for patients, its induction mechanism remains unknown. When the EWSR1-FLI1 gene is formed, the cell loses one wildtype EWSR1 allele. To elucidate the induction mechanism of trisomy 8, we generated a cell line that allows for the conditional induction of EWSR1-FLI1 expression and EWSR1 knockdown (derived from a single EWSR1 allele. Specifically, the conditional cell line was generated by integrating the Tet-on EWSR1-FLI1 construct into the AAVS locus and adding a miniAID tag at the 5’ end of the EWSR1 locus using auxin-degron system. A combination of the EWSR1-FLI1 expression and degradation of one allele-derived EWSR1 induced a high incidence of trisomy 8 within eight days, enhancing colony formation. Mechanistically, trisomy 8 is induced by the haploinsufficiency of EWSR1, and the remaining EWSR1 proteins are likely inhibited by interaction with EWSR1-FLI1. Our data showed that the knockout of EWSR1 alone was sufficient to increase the incidence of trisomy 8. Expression of wild-type EWSR1 in EWSR1 knockout cells rescued the high incidence of trisomy 8. In contrast, the EWSR1:R565A mutant, which lacks the ability to interact with Aurora B kinase, failed to rescue this phenotype. We propose that the combination of EWSR1-FLI1 expression and loss of EWSR1 contributes to the induction of trisomy 8 through the compromised EWSR1-Aurora B pathway.