Interaction between genetic ancestry and EWS-FLI induced transcription in Ewing sarcoma tumorigenesis

Abstract Ewing Sarcoma (ES) is a rare but deadly pediatric bone and soft tissue tumor, with little improvement in survival for decades despite knowing the driving fusion oncoprotein EWS-FLI1. EWS-FLI1 is notably toxic, acting as an aberrant transcription factor and leading to growth arrest and apoptosis in all but a few cell types. ES incidence in populations of European ancestry is nearly ten times that found in children with primarily African ancestry. To better understand how EWS-FLI1 transforms the transcriptome and the protective effect of the African genome, we leveraged this disparate genomic context to study ES tumorigenesis by introduction of EWS-FLI1 into cells derived from donors with a range of African ancestry followed by RNA sequencing (RNA-seq). Induced pluripotent stem cell (iPSC) lines with genome wide SNP genotyping data were obtained and local ancestry was assessed using RFMix to establish genetic ancestry. Two lines each of ~100% European and African ancestry and four lines with intermediate European/African admixture (45% to 90% African) were differentiated into neural crest cells (iNCC, a proposed cell-of-origin for ES) and transduced with a lentivirus expressing either a GFP reporter or a GFP-2A-EWS/FLI1 cassette. Interestingly, iNCC derived from European lines maintained a higher frequency of cells expressing GFP/EWS-FLI1 than the pure African lines, with admixed lines showing an intermediate frequency. RNA was extracted at 48- and 96-hour time points and sequenced for gene expression analysis. As expected, we identified pronounced changes to the transcriptional landscape in EWS-FLI1-induced cells compared to control with 13,578 differentially expressed genes (adjusted p-value < 0.05). More interestingly we identified 3,128 genes with ancestry-associated gene expression differences in response to EWS-FLI1 expression. Gene Set Enrichment Analysis (GSEA) using MSigDB 50 hallmark gene sets (v7.4) detected 24 significantly enriched gene sets with an inverse correlation with percent African ancestry, including those sets involved with oxidative phosphorylation, MYC v1 and v2, and MTORC1 signaling. To identify those expression changes that are key to ES tumorigenesis we are comparing the transcriptional profiles of our EWS-FLI1 expressing iNCC to ES tumors and ES cell lines as well as other models of ES derived from mesenchymal stem cells. Ongoing studies will include profiling of chromatin accessibility and EWS-FLI1 occupation for each ancestral iNCC line following EWS-FLI1 transduction to determine the early, ancestry-linked transitions that are permissive to oncogenic transformation. As EWS-FLI1 itself has proven elusive to direct targeting, studying its immediate downstream effects has the potential for establishing new druggable biologic pathways for treatment of ES. Citation Format: Rachel M. Moss, Kelsie L. Becklin, Lauren J. Mills, Branden S. Moriarity, Beau R. Webber, Logan G. Spector. Interaction between genetic ancestry and EWS-FLI induced transcription in Ewing sarcoma tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 702.