OBJECTIVES/GOALS: The EWSR1-FLI1 gene fusion is implicated as a source of oncogenic activity in the majority of Ewing sarcoma (EwS) cases (>70%). Our studies will provide unparalleled insight into the transformative mechanics of EWS-FLI1 METHODS/STUDY POPULATION: EWS-FLI1 is an intrinsically disordered protein (IDP). IDPs do not form stable secondary or tertiary folds in biological conditions and ofter contain proline-rich regions and other multivalent binding regions. These regions interact with numerous partners resulting in highly dynamic and complex protein-protein interactions. Recent advances in proximity labelling techniques, such as the use of a biotin ligase fused to a protein of interest are exceptionally well suited to identifying IDP interactomes since they do not rely on the binding affinities but rather the distance between interacting proteins. Combining novel discovery proteomics with Nuclear Magnetic Resonance (NMR) approaches will provide unparalleled insight into the transformative mechanics of EWS-FLI1. RESULTS/ANTICIPATED RESULTS: Using the proximity labeling technique TurboID, which limits the number of false-positive interactions, we have shown that EWSR1 and EWS-FLI1 act within the spliceosome (responsible for mRNA splicing and processing). Using bioinformatics techniques, we show EWS-FLI1 interacts with peptidyl-prolyl isomerase (PPI) proteins, specifically PPIL1, through the EWS-FLI1 N-terminal region (EWS-LCD). We used NMR to provide insight into the interaction between PPIL1 and EWS-LCD, showing that EWS-LCD interacts with the catalytic region of PPIL1. We anticipate that PPIL1 isomerizes EWS-FLI1 to modulate its activity, and that EWS-FLI1 interacts with the spliceosome through formation of a biological condensate. However, future proximity labeling and NMR studies are needed to verify this activity. DISCUSSION/SIGNIFICANCE: Our studies will yield actionable insights regarding the protein-protein interfaces in EWS-FLI1 that can be targeted to attenuate the oncogenic activity of EWS-FLI1. More broadly, our results will be applicable toward understanding the etiology of other pediatric cancers and for guiding the development of novel targeted treatments.