Abstract 129: Understanding the molecular drivers that regulate Ewing sarcoma cell fate

Abstract

Abstract Like other translocation-positive sarcomas, Ewing sarcoma (ES) harbors a pathognomonic fusion protein (FP) capable of widespread epigenetic reprogramming that can lead cells to acquire a high-grade, undifferentiated phenotypic state. To better understand how this might occur, we employed a CRISPR knockout (CRISPR-KO) model of A673, targeting exon 4 of the N-terminus EWSR1 portion of the FP and generated a clonal pool (EWSR1 CRISPR KO). We used single-cell RNA and ATAC sequencing (scMultiome) to identify the transcriptional and chromatin changes after the FP knockout (FP KO). We identified several clusters of ES cell heterogeneity. Since the EWSR1-KO is a pooled population, we used known FP-regulated genes to delineate cells bearing the FP KO from wild-type (FP WT) cells. We observed robust expression of FP-activated genes, PRKCB, and LIPI, in many clusters suggesting regular FP function (FP WT). Conversely, FP-repressed genes, LOX, and IGFBP3 were elevated in a single cluster containing only EWSR1-KO cells. This suggests the FP was successfully knocked out in this cluster (FP KO). That FP KO cluster also lost transcriptional resemblance to ES. A granular identification with cell lines confirmed that all clusters except FP KO were identified as A673 cells. However, the FP KO resembled cell lines with a fibroblastic phenotype. We analyzed enriched motifs and observed no significant difference between the FP KO and FP WT for EWSR1::FLI1. However, the FP KO cells were enriched for motifs in the Fos/Jun family and Smad2/3, which have roles in EMT and mesenchymal differentiation. The FP-KO cells significantly expressed the Fos/Jun family of proteins. Next, we identified peak-gene associations within a ± 500 kb window around the TSS. When we analyzed peaks linked to genes expressed in FP-WT, we found enrichment of the Fos/Jun protein family (p < 1e-34), Ets family, and GGAA-microsatellites. However, the FP-KO were strongly enriched for Fos/Jun protein family (p < 1e-540) with no change in Ets family motif. Knocking out the FP in the A673 cell line significantly shifted the ES lineage. We observed peaks linked to genes positively associated or regulated by the FP were enriched for Fos/Jun family, Ets family, and GGAA-microsatellites. There may be a cooperative interaction between these TFs that affects the genes linked to the FP. The fact that FP loss enriched the Fos/Jun family motifs, but not Ets family or GGAA-microsatellites, suggests that FP loss activates FP-repressed genes and enables chromatin remodeling to open previously inaccessible DNA binding sites. Citation Format: Danh Truong, David McCall, Clement Agyemang, Sandhya Krishnan, Alexander Lazar, Joseph Ludwig. Understanding the molecular drivers that regulate Ewing sarcoma cell fate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 129.