Abstract A016 EWSR1 in Ewing Sarcoma Cells Exhibits Enhanced Recruitment to Sites of Active Transcription Compared to That Observed in Non-Ewing Sarcoma Cells

Abstract The initiating genetic event in several pediatric tumor-types, including Ewing sarcoma (EWS), involve translocations affecting the EWSR1 locus. The EWSR1 gene encodes EWSR1, a transcriptional regulator. In EWS cells expressing the fusion oncoprotein EWSR1::FLI1, studies have shown disrupted function of EWSR1 function and that this contributes to the deregulated gene expression observed in these tumors. Recently, we reported the nucleoplasmic organization of endogenous EWSR1 in EWS cells. In brief, we determined that at a resolution of ∼120 nm, EWSR1 is present in two states, a distributed state which is present throughout the nucleoplasm and as foci. Both EWSR1 states localize with nascent RNA, while foci also significantly colocalize with phosphorylated RNA polymerase II (p-RNA pol II). In this study, we have examined EWSR1’s localization at a higher resolution (40 nm) in EWS and non-EWS cells. For this study, we used two EWS cell lines, A673 and TC-32, and a non-EWSR1 driven sarcoma cell line, HT-1080 that expresses EWSR1 and FLI1. Using gene editing, we generated EWSR1 reporter cell lines that express either mNeonGreen or the FLAG peptide fused to the N-terminus of EWSR1. In A673 cells, we also generated isogenic cell lines that express an 11 amino acid peptide (HiBiT) fused to either EWSR1 or EWSR1::FLI1. Following validation of successfully modified clones, we used fluorescence or immunofluorescence (IF) and super-resolution confocal and stimulated emission depletion microscopy to assess protein localization. We observed that EWSR1’s overall organization in A673, TC-32 and HT-1080 cells is similar with over 95% of nuclear localization and its existence in two states, distributed and foci. At a resolution of 40 nm or less, we found that EWSR1 foci in EWS and non-EWS cells are composed of 3 to 6 distinct IF signals. Furthermore, in all three cell lines, we observed that the distributed EWSR1 signal exists as a fibro-granular network that colocalizes with nascent RNA. While these features proved similar between the three cell lines, we detected some differences in EWSR1’s organization in EWS and non-EWS cells. Specifically, in the HT-1080 cells, EWSR1 foci are smaller (∼80 nm) than that seen in EWS cells (A673: ∼247 nm, TC-32: ∼578 nm). Also, EWS cells exhibit minimal localization of EWSR1 to nucleoli, but in HT-1080 cells, we observed EWSR1 in one or more nucleoli of ∼30% of cells. In addition, we observed significantly less colocalization of EWSR1 foci with p-RNA pol II in the HT-1080 cells (Pearson’s correlation coefficient (PCC) = 0.47) compared to EWS cells (PCC, A673 = 0.85; TC-32 = 0.87). One reason for these differences could be the interaction of EWSR1 with EWSR1::FLI1 affecting EWSR1’s localization. To begin to evaluate this hypothesis, we examined the nuclear localizations of EWSR1::FLI1 and EWSR1 and observed colocalization of these proteins (PCC=0.45). Collectively, our study of endogenous EWSR1 shows its localization in EWS cells differs from that of non-EWS cells, which could affect the regulation of gene expression in EWS. Citation Format: Soumya Sundara Rajan, Tamara L. Jones, Vernon J. Ebegboni, Langston Lim, Michael J. Kruhlak, Natasha J. Caplen. EWSR1 in Ewing sarcoma cells exhibits enhanced recruitment to sites of active transcription compared to that observed in non-Ewing sarcoma cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pediatric Cancer Research; 2024 Sep 5-8; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl):Abstract nr A016.