Dynamic modelling of EWS::FLI1 fluctuations reveals molecular determinants of phenotypic tumor plasticity and prognosis in Ewing sarcoma [CUT&RUN]

The mechanisms underlying tumor cell plasticity driving drug resistance and disease progression remain poorly understood. In Ewing sarcoma (EwS), variations in EWS::FLI1 (EF) activity have been associated with epithelial-mesenchymal plasticity (EMP). Using degron technology, we titrated endogenous EF in an EwS cell line and linked phenotypic states to distinct EF thresholds. Strikingly, modest EF depletion promoted a pro-metastatic phenotype, which was diminished upon near-complete EF loss. Transcriptomic analysis revealed distinct gene clusters with heterogenous response patterns to varying EF dosage. Target genes most sensitive to subtle EF depletion contained GGAA microsatellites in EF-bound enhancers. Furthermore, we identified Krüppel-like zinc-finger transcription factors to be associated with EF-repressed EMP genes. EF rescue after partial depletion for a transient period identified persistently dysregulated genes associated with poor prognosis. This study underscores the therapeutic challenge of insufficient EF inhibition and provides a foundation for exploiting oncoprotein dynamics to uncover therapeutic vulnerabilities in fusion-driven cancers.