Identification of EWS-FLI1 Regions Necessary for Ewing Sarcoma Proliferation

Ewing sarcoma is pediatric bone malignancy defined by a translocation between EWS and ETS family transcription factor. EWS-FLI1 (EF) is the most common translocation and codes for a novel transcription factor that combines the N-terminus of EWS, which contains LC domain comprised of tyrosine rich peptide repeats, and C-terminal portion of FLI1, which contains the ETS DNA binding domain. EF is a key transcriptional regulator known to both activate and repress genes. While understanding of the molecular mechanism by which EF controls transcription have become clearer, targeted therapeutic interventions against EF or its transcriptional program have yet to make clinical impact. This is due in part to a poor understanding of how the N-terminus of EWS is contributing to the oncogenic program and a disparate range of reported effects after EF depletion on Ewing sarcoma cells. This report shows the adaptation of two inducible degron systems, Small Molecule Assisted Shut-Off (SMASh) and Auxin-inducible degron (AID), into the endogenous locus of EF in a series of Ewing sarcoma cell lines to define the phenotype of EF depletion. Across multiple cell line and degradation mechanisms, EF depletion in Ewing sarcoma cells leads to decreased cell proliferation through G1/S arrest that can be rescued through re-expression of EF. Having established a baseline for EF depleted cells, I developed a proliferation-based assay to test the functionality of mutant EF constructs on their ability to drive proliferation in the setting of endogenous EF depletion. I tested a series of EF truncations, with single or multiple exon deletions in the EWS portion of the translocation. Expression of EF constructs with loss of any single exon was tolerated and allowed for continued proliferation, but loss of at least exons 1-4 on the N-terminus and loss of exons 5-7 from the C-terminus resulted in non-functional EF constructs. Given that there appear to be redundant elements within the N-terminus of EF, I tested truncated and mutant version of a minimal rescue construct containing only exon 1-5 of the N-terminus of EF. Mutation of only 3 tyrosines to serine within the minimal construct was sufficient to prevent proliferation in Ewing cell lines.