Abstract A004 The ETS1-driven expression of TNS3 in Ewing sarcoma cells enhances the formation of the focal adhesions that promote cell movement

Abstract The mechanisms that regulate Ewing sarcoma (EWS) cell dissemination are poorly characterized. We have reported that though cell line studies show the EWSR1::FLI1/ERG fusion oncoproteins represses ETS1 expression, there is inter-tumoral variations in its mRNA levels and higher expression of ETS1 in tumors correlate with reduced overall patient survival. ETS1 encodes a transcription factor that regulates the movement of several early progenitor cell sub-types, and activation of ETS1 in EWS cells enhanced their movement and migration. This suggests that ETS1 expression could promote cell dissemination. We also showed that ETS1 expression in EWS cells upregulated the expression of the focal adhesion (FA) protein TENSIN3 (TNS3). Critically, depletion of TNS3 reverted the enhanced formation of FAs induced by ETS1 and partially rescued the increased migratory phenotype. In this study, we investigated the ETS1-induced changes in the phenotype of EWS cells by assessing TNS3 function in the context of other FA proteins. We generated epigenetic and transcriptomic profiles of EWS cells using ChIP-seq, CUT&RUN-seq, or RNA-seq in unperturbed, siRNA-treated or CRISPRa-EWS cells. We used dCas9-VP64 (control) or ETS1-activated (ETS1a) SK-N-MC cells and performed immunofluorescence and super resolution microscopy employing antibodies against TNS3, vinculin (VCL), paxillin (PXN), pan-talin (TLN), and focal adhesion kinase (FAK). Previously, we observed TNS3 and the FA scaffold protein VCL at the membrane projections of ETS1a EWS cells, however, we did not determine whether this reflected a change in VCL expression and/or protein redistribution. Epigenetic analyses showed EWSR1::FLI1/ERG, and ETS1 binding (following depletion of EWSR1::FLI1) at VCL and its expression increased 6- and 3-fold following EWSR1::FLI1 and EWSR1::ERG depletion, respectively. In contrast, we observed only a slight increase in VCL expression (RNA or protein) in ETS1a EWS cells. This suggests that our visualization of VCL in the ETS1a EWS cells reflected a change in its localization, not expression. Consistent with this finding, in the ETS1a EWS cells, we observed an increase in VCL’s non-nuclear intensity relative to control cells. This observation reflected VCL’s recruitment to the plasma membrane, a phenotype that TNS3-depletion reversed. To further study whether TNS3 is the predominate determinant of FA formation in ETS1a EWS cells, we examined other FA components, specifically PAX, TLN1, TLN2, and FAK. We observed no evidence of EWSR1::FLI1/ERG or ETS1 directly regulating these genes. Nevertheless, PXN, TLN, and FAK, all showed an increase in both total and non-nuclear intensities following ETS1 activation, suggesting that ETS1 indirectly activates the expression of other FA proteins. Nevertheless, depletion of TNS3 decreased the non-nuclear intensities of these FA proteins, confirming the importance of TNS3 in the formation FAs in the ETS1-modfied lines. We thus conclude that if expressed in an EWS cell, ETS1’s regulation of TNS3 that will enhance FA formation and cell movement. Citation Format: Vernon J. Ebegboni, Tamara J. Jones, Shaoli Das, Erica C. Pehrsson, Soumya Sundara Rajan, Natasha J. Caplen. The ETS1-driven expression of TNS3 in Ewing sarcoma cells enhances the formation of the focal adhesions that promote cell movement [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 A004.