Abstract 3879: Threshold dynamics of EWS::FLI1 reveal drivers of tumor plasticity and prognosis in Ewing sarcoma

Abstract Ewing sarcoma (EwS) is a highly aggressive pediatric cancer driven by the EWS::FLI1 (EF) fusion oncoprotein. Emerging evidence suggests that variations in EF expression levels may contribute to tumor cell plasticity, promoting treatment resistance and relapse. Both tumor intrinsic and extrinsic influences such as microenvironmental and therapy related factors, may influence EF thresholds. However, the phenotypic consequences of EF fluctuations at different amplitudes and intervals remain poorly understood. To address this, we engineered a EF threshold modulation system in A673 cells by tagging endogenous EF with a fluorescent mNeonGreen (mNG) and a degron tag (dTAG). Using mNG fluorescence as a quantitative proxy for EF levels, we established a stable and highly reproducible EF gradient through treatment with graded concentrations of the dTAGV-1 ligand, ranging from 3 hours to 4 weeks. Phenotypic assays revealed that partial EF depletion (35-65%), but not near-complete loss (90-95%), significantly enhanced migratory and invasive behaviors in vitro, while minor reductions (15-35%) were sufficient to impair anchorage-independent growth. In vivo studies corroborated these findings, as partially EF-depleted cells upon EF restoration exhibited increased extravasation in zebrafish models and enhanced lung metastasis in immunocompromised mice. To elucidate the molecular basis underpinning EF threshold-dependent phenotypes, we integrated acute transcriptional changes from nascent RNA sequencing (SLAM-seq) with EF chromatin occupancy (CUT&RUN) data following 3 hours of dTAGV-1 gradient treatment. A 15% reduction in EF resulted in immediate downregulation of EF-activated genes enriched for GGAA microsatellites in enhancers and upregulation of EF-repressed genes associated with monomeric ETS binding sites in EF-bound promoter regions. Interestingly, the largest group of EF repressed genes, associated with epithelial-to-mesenchymal transition (EMT), showed graded upregulation in response to increasing dTAGV-1 concentrations. For these genes, EF binding was predominantly localized to promoter regions enriched for Krüppel-related zinc finger protein binding sites, implicating novel EF coregulators in mediating the migratory phenotype. Strikingly, transient EF depletion (3 weeks) followed by rapid EF restoration led to sustained upregulation of EMT-related genes and migratory behavior for up to 2 weeks after dTAGV-1 ligand washout. Supporting these observations, approximately 20% of these genes correlated with poor prognosis in a cohort of 196 EwS patients. Our findings underscore the importance of EF expression dynamics in driving EwS cell plasticity and highlight therapeutic challenges posed by insufficient direct EF inhibition. These insights provide a foundation for exploiting oncogene dynamics to uncover therapeutic vulnerabilities in single oncogene driven cancers. Citation Format: Veveeyan Suresh, Christoph Hafemeister, Andri Konstantinou, Florencia Cidre-Aranaz, Sarah Grissenberger, Caterina Sturtzel, Karla Queiroz, Dorota Kurek, Andrea Wenninger-Weinzierl, Martha Zylka, Ana Sastre, Javier Alonso, Martin Distel, Anna Obenauf, Thomas Grünewald, Florian Halbritter, Heinrich Kovar, Valerie Fock. Threshold dynamics of EWS::FLI1 reveal drivers of tumor plasticity and prognosis 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 3879.