Abstract 1466: Heterogeneity of the EWS-FLI1 transcriptome drives differences in the cellular phenotype of Ewing sarcoma cell lines

Abstract

Abstract Background: Ewing sarcoma (ES) is dependent on the oncogenic transcription factor EWS-FLI1 for oncogenesis and progression. EWS-FLI1 binds to chromatin to both induce and repress gene expression. These gene expression changes drive proliferation, disrupt the cell cycle, and block cellular differentiation. One approach to target EWS-FLI1 is to reverse the gene signature. However, we have observed an underappreciated but striking heterogeneity of the EWS-FLI1 transcriptome among cell lines. While many common downstream targets exist, at least an equal number of cell-line specific large magnitude gene expression changes are also present. In this study, we evaluate the phenotypic importance of these cell-line specific changes and evaluate potential mechanisms that EWS-FLI1 may use to drive divergent transcription. Methods: The EWS-FLI1 transcriptome was characterized by RNA sequencing, following siRNA silencing of EWS-FLI1 at two time points in 6 cell lines. Effects on specific genes was confirmed by qPCR and western blot analysis. Cellular migration was evaluated using scratch assays on the Incucyte Zoom. EWS-FLI1binding was evaluated using Cleavage Under Targets and Tagmentation assay (CUT&Tag) and the transcriptome of Ewing subpopulations was determined by single cell RNA sequencing. Results: Next generation RNA sequencing of highly optimized siRNA knockdowns of EWS-FLI1 in 6 ES cell lines showed striking differences in gene expression. Conditions were optimized to capture the same magnitude change in EWS-FLI1 expression at the shortest time point ever reported. Fewer common targets were identified across all cell lines than previously reported, perhaps due to the short time frame and focus on direct targets. Independently generated well-established lists of targets were enriched in the data. However, there were 184 genes that changed by a Log2FC &gt 3 (P &lt 0.0001) in only one cell line including important genes like WNT7B and CTLA4. Marked heterogeneity was observed in a previously described migratory transcriptional signature including the key effectors Zyxin and Integrin Alpha-5; an observation confirmed by unbiased pathway analysis and cellular migration assays. Importantly, increased cellular migration was only seen in one cell line with siRNA silencing and not with any EWS-FLI1 targeted small molecules. To characterize the source of heterogeneity, we evaluated EWS-FLI1 binding at Zyxin and Alpha-5 integrin and single cell RNA sequencing of the migratory sub-signature. CUT&Tag demonstrates that EWS-FLI1 is found bound at these genes even in non-migratory cell lines. Conclusion: Careful consideration of Ewing sarcoma model-specific gene expression changes is important to future therapeutic approaches as these unique changes influence cellular phenotype. Citation Format: Seneca S. Kinn-Gurzo, Susan M. Kitchen-Goosen, Maggie Chasse, Marie Adams, Rebecca Kaufman, Patrick J. Grohar. Heterogeneity of the EWS-FLI1 transcriptome drives differences in the cellular phenotype of Ewing sarcoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1466.