Abstract B005: Emergent condensate behaviors of the EWS::FLI1 fusion protein in Ewing sarcoma

Abstract Fusion oncoproteins generated by chromosomal translocations are defining molecular drivers of several pediatric cancers, functioning in part by reprogramming gene expression and disrupting cellular differentiation. Ewing sarcoma, an aggressive cancer of bone and soft tissue, exemplifies this paradigm and is driven in the majority of cases by the oncogenic fusion protein EWS::FLI1, formed by joining the low-complexity domain (LCD) of the RNA-binding protein EWS to the DNA-binding domain (DBD) of the transcription factor FLI1. EWS::FLI1 alters transcriptional and RNA processing programs and assembles into aberrant biomolecular condensates, yet the molecular basis for these fusion-driven assemblies remains poorly understood. Here, we integrate NMR spectroscopy, mutagenesis, biophysical and cellular assays, microscopy, and all-atom molecular dynamics simulations to dissect how sequence features and domain interactions shape the condensate properties of EWS and EWS::FLI1. We find that the EWS LCD undergoes extensive self-association mediated by tyrosine-rich motifs that generate compact intramolecular conformations and robust multivalent networks driving phase separation. The RNA-binding domains of EWS engage the LCD through transient electrostatic and aromatic interactions, modulating condensate dynamics, whereas the FLI1 DBD of the fusion protein directly interacts with the EWS LCD via flexible “wings”. Incorporation of the FLI1 DBD markedly alters the material properties of condensates, producing emergent biophysical behaviors distinct from either parental protein. These altered condensate properties possibly contribute to the cellular dysregulation and toxicity associated with EWS::FLI1, in part through mis-localization and aberrant interactions with native EWS that disrupt normal nuclear organization and RNA processing. Together, these findings define a molecular framework in which weak, multivalent, and chemically specific interactions give rise to the emergent properties of the EWS::FLI1 fusion protein. By linking sequence-encoded features, interdomain crosstalk, and condensate material states, we propose a mechanistic description of how fusion with ETS-family DBDs reprograms the physical behavior of EWS assemblies. These emergent condensate properties provide a potential unifying description of how EWS::FLI1 perturbs transcriptional regulation, RNA metabolism, and nuclear organization in pediatric Ewing sarcoma. Citation Format: David S. Libich, Erich J. Sohn, Kandarp A. Sojitra, Aiola Stoja, Courtney N. Johnson, Jeetain Mittal, Alex A J. Bishop. Emergent condensate behaviors of the EWS::FLI1 fusion protein in Ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Fusion-Positive Cancer: From Discovery to Therapy; 2026 Jan 13-15; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(1_Suppl):Abstract nr B005.