Abstract 4035: Elucidating the DNA binding dynamics of EWSR1::FLI1 using biochemical methods

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Abstract Importance: This work signifies a breakthrough in our ability to study Ewing sarcoma by developing a protocol to purify functional WT EWSR1::FLI1. This development changes the level of detail to which we can study EWSR1::FLI1 and potentially other difficult to purify fusion oncoproteins. Objective: Ewing sarcoma is a pediatric bone and soft tissue sarcoma that arises in adolescents and young adults. The mutation that causes >85% of Ewing sarcoma cases is caused by the fusion oncoprotein, EWSR1::FLI1. Efforts to purify recombinantly expressed EWSR1::FLI1 have proven challenging for decades due to aggregation caused by the low complexity domain (LCD) of EWSR1. Our goal was to purify recombinantly expressed and functional EWSR1::FLI1 to quantitatively understand the nature of binding dynamics to various DNA sequences. Methods: We successfully expressed and purified EWSR1::FLI1 using a newly developed protocol. We performed electrophoretic mobility shift assays (EMSAs) to test binding of EWSR1::FLI1 on naked- and nucleosome-wrapped DNA. Binding constants were determined with DNA containing either a canonical high affinity (HA) ETS DNA binding sequence or a 2X GGAA microsatellite repeat. Results: Our laboratory has successfully developed a protocol to consistently achieve highly pure and functional EWSR1::FLI1. EWSR1::FLI1 has approximately 7-fold tighter binding to a 2X GGAA repeat, than to HA ETS sequence. For comparison, the minimal FLI DNA binding domain proteins (ETS85 and ETS102) bind to HA ETS sequences and GGAA microsatellites at similar affinities. These experiments suggest that the EWSR1 LCD alters the binding dynamics of the ETS DBD domain and that EWSR1::FLI1 preferentially binds to GGAA microsatellites. We tested the pioneer function of EWSR1::FLI1 by performing EMSAs with nucleosome-wrapped DNA. EWSR1::FLI1 bound to nucleosome-wrapped DNA containing GGAA repeats with only 3-fold less affinity than naked DNA. For comparison, when ETS85 and ETS102 bound nucleosome-wrapped DNA, this affinity was ∼30-fold less, showing potentially weak pioneering function of the FLI DBD. Conclusions& Relevance to Pediatric Care: These studies allow us to understand the physical nature of fusion oncoproteins such as EWSR1::FLI1 and will be the foundation for therapeutics that could be used to treat Ewing sarcoma patients. Citation Format: Jack Tokarsky, Blanche Chen, Michael G. Poirier, Emily Theisen. Elucidating the DNA binding dynamics of EWSR1::FLI1 using biochemical methods [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 4035.