biostudies-literatureUnknown44(38)Olmedo-Pelayo JDrug resistance is an ill-defined cause of dismal outcomes in cancer. Ewing sarcoma (EwS), a pediatric cancer characterized by high therapy failure rates, is driven by a single oncogenic event generating EWSR1::ETS gene fusions (primarily EWSR1::FLI1) in a silent genomic background. This provides a straightforward model to study the impact of gene fusions on drug responses. Here, we describe a novel mechanism of sensitivity to DNA topoisomerase 1 poisons in EwS. We discovered that EWS::FLI1 prevents the resolution of R-loops induced by these drugs via sequestering DHX9 helicase, ultimately resulting in R-loop accumulation, replication stress, and genome instability. In turn, excessive DHX9 or reduced EWS::FLI1 levels render EwS cells resistant to the active metabolite of irinotecan (SN-38)Oncogene3537-3552https://www.ebi.ac.uk/biostudies/studies/S-EPMC12436182biostudies-literatureEWS::FLI1-DHX9 interaction promotes Ewing sarcoma sensitivity to DNA topoisomerase 1 poisons by altering R-loop metabolism.PMC12436182Garcia-Dominguez DJde Alava EEhlers ACCarcaboso AMOhmura SDelgado-Bellido DAlonso JGrunewald TGPGranado-Calle EMackintosh CLlombart-Bosch AScotlandi KMachado ILobo-Selma LGomez-Herreros FJordan-Perez CMonteiro-Amaral ATOlmedo-Pelayo JfalseEWS::FLI1-DHX9 interaction promotes Ewing sarcoma sensitivity to DNA topoisomerase 1 poisons by altering R-loop metabolism.Drug resistance is an ill-defined cause of dismal outcomes in cancer. Ewing sarcoma (EwS), a pediatric cancer characterized by high therapy failure rates, is driven by a single oncogenic event generating EWSR1::ETS gene fusions (primarily EWSR1::FLI1) in a silent genomic background. This provides a straightforward model to study the impact of gene fusions on drug responses. Here, we describe a novel mechanism of sensitivity to DNA topoisomerase 1 poisons in EwS. We discovered that EWS::FLI1 prevents the resolution of R-loops induced by these drugs via sequestering DHX9 helicase, ultimately resulting in R-loop accumulation, replication stress, and genome instability. In turn, excessive DHX9 or reduced EWS::FLI1 levels render EwS cells resistant to the active metabolite of irinotecan (SN-38)2025-01-01T00:00:00Z2025 Oct2026-06-03T02:58:20.126Z2026-04-23T03:13:59.894ZS-EPMC124361824072166110.1038/s41388-025-03496-9