Intron retention (IR) is increasingly recognized as a feature of long noncoding RNAs (lncRNAs), yet the mechanisms that shape IR in lncRNAs and the functional consequences of this process remain largely unexplored. To investigate how IR contributes to lncRNA regulation, we performed a genome-wide screen to identify factors controlling IR in the lncRNA PURPL. This approach uncovered a prominent role for U2AF2, which promotes retention of a specific intron in PURPL through a weak polypyrimidine tract. IR of this intron drives nuclear enrichment of PURPL and enhances cell proliferation, revealing biological relevance. Transcriptome-wide analyses showed that although U2AF2 broadly supports canonical splicing consistent with its well-established function in promoting splicing, it also facilitates IR within a distinct subset of RNAs, including the nuclear speckle-associated lncRNA MALAT1. Loss of U2AF2 disrupts MALAT1 speckle localization and using MALAT1 knockout cells reconstituted with wild-type or intron deleted variants, we identified a single intron critical for MALAT1's speckle localization. Deletion of this intron from endogenous MALAT1 impaired speckle localization and reduced cell migration, phenocopying the loss of MALAT1. Together, these findings reveal IR as a key regulatory mechanism governing lncRNA localization and function and uncover an unexpected role for U2AF2 in promoting IR within specific lncRNA contexts.

HNRNPH1 is a regulator of alternative splicing, but few studies have defined the splicing events it mediates. Here, we used short- and long-read RNA sequencing to interrogate the transcriptome-wide effects of HNRNPH1 depletion and its regulation of specific splicing events. Differential alternative splicing analysis revealed effects on the transcriptome that involved all splice event categories. We confirmed HNRNPH1's regulation of a splicing event involving TCF3-exons 18a and 18b that encode distinct TCF3 transcription factor isoforms. Extending this finding, we present evidence that in neuroblastoma, HNRNPH1 is a MYCN target, potentially explaining the higher levels of HNRNPH1 and TCF3-exon 18a transcript variants in this tumor type. Analysis of two skipped exon events determined that HNRNPH1 regulates the splicing of exons encoding part of the EIF4G1 translation initiation factor's N-terminus and an exon included in the 5'UTR of specific transcript variants encoding the mitotic kinase AURKA. Using reporter constructs, we show this AURKA 5'UTR exon enhances expression, suggesting HNRNPH1 could contribute to regulating AURKA protein levels. Our findings highlight HNRNPH1's roles in regulating the expression of proteins with diverse cellular functions.

Background: Ewing sarcoma (EWS) is a rare cancer of the bone and soft tissue, most prevalent in children and young adults. The treatment of EWS has progressed relatively little in over 30 years. Survival rates for patients, particularly those with metastatic and/or relapsed disease remain poor, highlighting the urgent need for innovative treatment options. Methods: Here, we have explored the therapeutic potential of the oncolytic Maraba virus strain MG1 using various in vitro models of EWS, including established cell lines, doxorubicin-resistant derivatives, spheroid cultures and primary patient-derived Ewing sarcoma cell cultures. We examined the direct oncolytic activity of MG1 and its ability to stimulate the immune-mediated killing of EWS by human healthy donor peripheral blood mononuclear cells. Results: We show that MG1 undergoes productive replication and exerts direct oncolysis of established EWS cell lines, doxorubicin-resistant EWS cell lines and patient-derived Ewing sarcoma cell cultures more recently established from tumours. In contrast, primary mesenchymal stem cells (the likely cell of origin of EWS) were resistant to MG1, with IFN-I being a major determinant of tumour cell selectivity. MG1-treated PBMC produced IFN-I and killed EWS cells in vitro, in a natural killer (NK) cell-dependent manner. Conclusions: The ability of MG1 to kill EWS cells directly and stimulate NK cell cytotoxicity against this tumour suggests that MG1 may provide therapeutic benefit for EWS patients where the efficacy of conventional treatments is currently limited.

Chromosomal instability (CIN), a major hallmark of cancer, can be driven by defects in the integrity of centromere or kinetochore structure. Coordinated control of phosphorylation and dephosphorylation activities during cell division is critical to ensure chromosomal stability. Overexpression of the centromeric histone H3 variant CENP-A is observed in many cancers, and its mislocalization to noncentromeric regions promotes CIN. We identified protein phosphatase 1 (PP1) nuclear targeting subunit (PNUTS) as a top candidate in a genome-wide siRNA screen for gene depletions that lead to increased nuclear CENP-A levels. Here, we define a role for PNUTS in preventing CENP-A mislocalization and CIN. Depletion of PNUTS resulted in high nuclear CENP-A levels throughout the cell cycle in a PP1-dependent manner. Consistent with these results, mislocalization of CENP-A and its interacting partner CENP-C were observed on mitotic chromosomes from PNUTS-depleted cells. Defects in kinetochore integrity and CIN phenotypes were also observed in PNUTS-depleted cells. Mechanistically, we show that depletion of the histone H3.3 chaperone DAXX suppresses the mislocalization of CENP-A and micronuclei incidence in PNUTS-depleted cells. In summary, our studies highlight the importance of phospho-regulation mediated by PNUTS in preventing CENP-A mislocalization and CIN.

Abstract Ewing sarcoma (EWS) is an aggressive bone and soft tissue tumor that affects children and young adults. Ewing sarcomas harbor few mutations beyond the chromosomal translocation that initiates disease, and the mechanistic basis for the metastasis of these tumors remains poorly understood. The epigenome of EWS cells reflects the regulatory state of genes associated with the DNA binding activity of the fusion oncoproteins EWSR1::FLI1 or EWSR1::ERG. In this study, we examined the EWSR1::FLI1/ERG’s repression of transcription factor genes, particularly those encoding transcriptional regulators of cell differentiation. To examine EWSR1::FLI1/ERG’s regulation of gene expression, we assayed DNA binding using ChIP-seq or CUT&RUN, RNA using RNA-sequencing (RNA-seq) or qRT-PCR, and protein using immunoblotting or immunofluorescence. We depleted the expression of EWSR1::FLI1/ERG proteins using RNAi and overexpressed ETS1 using a full-length cDNA or CRISPR activation.RNA-seq analysis of control and EWSR1::FLI1-silenced TC-32, TC-71, and A673 EWS cells identified 67 genes encoding proteins with DNA binding activity that exhibited significant increases in expression following depletion of EWSR1::FLI1. Comparison of the expression of these 67 transcription factor genes in EWS cell lines (n=41) and tumors (n=79) showed 37 expressed at a wider range in tumors relative to cell lines (p <0.01). This more variable expression in EWS tumors suggests that one or more of these transcription factors could exert a phenotypic effect. Focusing on one of the EWSR1::FLI1-repressed target genes, ETS1, we detected EWSR1::FLI1 binding and an H3K27me3 repressive mark at this locus. Depletion of EWSR1::FLI1 results in ETS1’s binding of promoter regions. Analysis of EWS cells in which we profiled ETS1 binding and assessed the effects of ectopically expressed ETS1 defined 265 genes as positively regulated by ETS1 of which 103 also exhibited an increase in expression following depletion of EWSR1::FLI1. Of these 103 genes, we focused on ETS1’s regulation of TNS3, as assessment of multiple EWS tumor expression profiles, showed positive correlation of ETS1 and TNS3 expression. TNS3 encodes TENSIN3, a focal adhesion protein that regulates cytoskeletal reorganization and contributes to cell migration by connecting the cytoplasmic tail of integrins to the actin cytoskeleton. EWS cell lines (SK-N-MC (EWSR1::FLI1) and ES-5838 (EWSR1::ERG), in which we activated ETS1 expression (CRISPRa) exhibited a migratory phenotype and increased TNS3 expression. Critically, the activated ETS1 EWS cell lines show TNS3 accumulation at leading cell edges, with F-actin cytoskeletal reorganization, a phenotype associated with cell migration. Our study demonstrates that EWS cells expressing ETS1 exhibit a more migratory phenotype, which, has the potential to promote the dissemination of cells and, thus, metastasis. Citation Format: Vernon Justice Ebegboni, Tamara L. Jones, Tayvia Brownmiller, Erica Pehrsson, Patrick Zhao, Soumya Sundara Rajan, Natasha J. Caplen. ETS1, a target gene of the EWSR1::FLI1 fusion oncoprotein, regulates the expression of the focal adhesion protein TENSIN3 [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 2855.

Abstract A major challenge in osteosarcoma (OS) immunotherapy is overcoming the immunosuppressive tumor microenvironment (TME), to restore immune recognition and destruction of tumor cells. One of the key immune cell subsets known to contribute to the immunosuppressive TME are M2-polarized tumor-associated macrophages (TAMs). Our work aims to identify novel oncolytic virus (OV) combination immunotherapies to induce direct lysis of OS tumor cells, activate immune-mediated destruction of OS, and remodel the immunosuppressive TME. The direct lytic activity of oncolytic herpes simplex virus (oHSV; HSV1716) treatment was assessed using LIVE/DEAD flow cytometry and viral replication was measured using standard plaque assay. NK cell degranulation and killing assays were performed by co-culture of OS cell lines with peripheral blood mononuclear cells (PBMC) treated ± oHSV; NK cell degranulation and target cell death was assessed by flow cytometry. TAMs were generated in vitro by co-culture of PBMC with OS cell lines or primary bone marrow-derived mesenchymal stem cells (MSCs) for 7 days. Phagocytosis assays were performed by co-culture of TAMs with OS cells using fluorescent cell tracker stains and flow cytometry. Multicellular spheroids were generated by co-culture of firefly luciferase-expressing OS cell lines with CD14+ monocytes and bone marrow derived MSCs for 7 days in low adhesion plates, viability of tumor cells after treatment was assessed by addition of d-luciferin and quantification of luminescence. Treatment of four OS cell lines with oHSV for 72 hours resulted in <20% cell death by direct lysis. However, after treatment with oHSV-GFP, >95% of OS cells were GFP+ and viral titer increased up to 105-fold when compared to viral input. This highlighted high levels of infection and replication of oHSV within OS cell lines, despite limited oncolysis. Moreover, oHSV treatment of PBMC significantly enhanced the immune-mediated killing of OS cell lines, and this was NK cell dependent. Monolayer co-culture of PBMC with OS cell lines or MSC generated TAMs with significantly increased pro-tumor M2-like phenotype (CD14+CD206+CD163+), when compared with PBMC cultured alone, and TAMs suppressed NK cell degranulation against OS cell lines. Treatment of TAMS with oHSV encoding GM-CSF reduced the proportion of M2 TAMs. Moreover, treatment of OS target cells with anti-EGFR monoclonal antibody (mAb) and co-culture with in vitro generated TAMs significantly increased their phagocytic activity. Multicellular spheroids incorporating MSCs and CD14+ monocytes displayed a high degree of resistance against immune-mediated killing, when compared with OS cell line alone as monolayer or spheroid cultures. However, combination of OV with anti-EGFR mAb significantly increased immune-mediated killing against multicellular spheroids. oHSV combination strategies with mAb may be a promising treatment strategy against OS, to exert direct oncolysis, stimulate immune-mediated destruction, and remodel the immunosuppressive TME. Citation Format: Tyler K. Barr, Victoria A. Jennings, Alison Taylor, Jessica Murby, Natasha J. Caplen, Javed Khan, Richard Baugh, Heather E. Owston, Dennis McGonagle, Fiona Errington-Mais, Graham P. Cook. Oncolytic HSV1716-GMCSF combination strategies to remodel the immunosuppressive osteosarcoma tumor-microenvironment and promote anti-tumor immunity [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 B049.

Abstract Intron retention (IR) is a form of alternative splicing in which an intron that should be spliced out from a precursor transcript, is retained in the mature RNA after the splicing is completed. Although there is emerging evidence of widespread IR in protein-coding genes and long noncoding RNAs (lncRNAs), the underlying molecular mechanisms remain largely unclear. Here, we report the discovery of novel transcripts from the p53-induced lncRNA PURPL, in which intron 2 is retained. To determine the molecular mechanism(s) of IR in PURPL, we conducted a CRISPR-based screen in 3 different cell lines. For this purpose, we utilized a genome-wide guide RNA library expressing a reporter minigene containing the sequence of PURPL intron 2 and its flanking exons. Considering the Percent Intron Retention as readout, we unexpectedly identified proteins of the basal splicing machinery as potential promoting factors of PURPL IR, including the U2-Auxiliary Factor 2 (U2AF2) splicing factor which was one of the top hits of the screen. We next analyzed ENCODE eCLIP-seq datasets to identify RNA binding proteins that could regulate intron 2 and decided to focus on U2AF2 which showed the highest number of binding sites and strongest eCLIP signals within PURPL intron 2 sequence. We validated the effect of U2AF2 by knocking it down, confirming that U2AF2 is a positive regulator of PURPL intron 2 retention as opposed to its canonical function. We also identified the RNA Binding Protein SON as a splicing regulator that acts as an antagonist of U2AF2 in regulating PURPL intron 2 retention. To determine the global impact of U2AF2 knockdown, we performed Iso-Seq and RNA-seq upon U2AF2 depletion. We found that although U2AF2 predominantly acts to promote the splicing of introns in most cellular transcripts, it promotes intron retention in a subset of transcripts. One of these targets is MALAT1, a lncRNA known to play role in splicing by interacting with splicing factors in nuclear speckles. Interestingly, U2AF2 depletion results in MALAT1 exclusion from nuclear speckles. We are currently in the process of characterizing the effect of U2AF2 in the function of the two lncRNAs. These data provide mechanistic insights on PURPL and MALAT1 splicing and function and reveal a previously unrecognized non-canonical function of U2AF2 in promoting intron retention. Citation Format: Ioannis Grammatikakis, Amit Behera, Corrine Corrina R Hartford, Erica C Pehrsson, XiaoLing Li, Yongmei Zhao, Biraj Shrethsa, Tayvia Brownmiller, Natasha J Caplen, Kannanganattu V Prasanth, Thomas Gonatopoulos-Pournatzis, Ashish Lal. Molecular mechanisms of intron retention in Long Non-Coding RNAs [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A013.

Disruption of DNA damage repair via impaired homologous recombination is characteristic of Ewing sarcoma (EWS) cells. We hypothesize that this disruption results in increased reliance on nonhomologous end joining to repair DNA damage. In this study, we investigated if pharmacologic inhibition of the enzyme responsible for nonhomologous end joining, the DNA-PK holoenzyme, alters the response of EWS cells to genotoxic standard of care chemotherapy. We used analyses of cell viability and proliferation to investigate the effects of clinical DNA-PK inhibitors (DNA-PKi) in combination with six therapeutic or experimental agents for EWS. We performed calculations of synergy using the Loewe additivity model. Immunoblotting evaluated treatment effects on DNA-PK, DNA damage, and apoptosis. Flow cytometric analyses evaluated effects on cell cycle and fate. We used orthotopic xenograft models to interrogate tolerability, drug mechanism, and efficacy in vivo. DNA-PKi demonstrated on-target activity, reducing phosphorylated DNA-PK levels in EWS cells. DNA-PKi sensitized EWS cell lines to agents that function as topoisomerase 2 (TOP2) poisons and enhanced the DNA damage induced by TOP2 poisons. Nanomolar concentrations of single-agent TOP2 poisons induced G2M arrest and little apoptotic response while adding DNA-PKi-mediated apoptosis. In vivo, the combination of AZD7648 and etoposide had limited tolerability but resulted in enhanced DNA damage, apoptosis, and EWS tumor shrinkage. The combination of DNA-PKi with standard of care TOP2 poisons in EWS models is synergistic, enhances DNA damage and cell death, and may form the basis of a promising future therapeutic strategy for EWS.

Ewing sarcoma (EWS) is an aggressive bone and soft tissue tumor that affects children and young adults. Ewing sarcomas harbor few mutations beyond the chromosomal translocation that initiates disease, and the mechanistic basis for the metastasis of these tumors remains poorly understood. The epigenome of EWS cells reflects the regulatory state of genes associated with the DNA binding activity of the fusion oncoproteins EWSR1::FLI1 or EWSR1::ERG. In this study, we examined the EWSR1::FLI1/ERG’s repression of transcription factor genes, particularly those encoding transcriptional regulators of cell differentiation. To examine EWSR1::FLI1/ERG’s regulation of gene expression, we assayed DNA binding using ChIP-seq or CUT&RUN, RNA using RNA-sequencing (RNA-seq) or qRT-PCR, and protein using immunoblotting or immunofluorescence. We depleted the expression of EWSR1::FLI1/ERG proteins using RNAi and overexpressed ETS1 using a full-length cDNA or CRISPR activation.RNA-seq analysis of control and EWSR1::FLI1-silenced TC-32, TC-71, and A673 EWS cells identified 67 genes encoding proteins with DNA binding activity that exhibited significant increases in expression following depletion of EWSR1::FLI1. Comparison of the expression of these 67 transcription factor genes in EWS cell lines (n=41) and tumors (n=79) showed 37 expressed at a wider range in tumors relative to cell lines (p <0.01). This more variable expression in EWS tumors suggests that one or more of these transcription factors could exert a phenotypic effect. Focusing on one of the EWSR1::FLI1-repressed target genes, ETS1, we detected EWSR1::FLI1 binding and an H3K27me3 repressive mark at this locus. Depletion of EWSR1::FLI1 results in ETS1’s binding of promoter regions. Analysis of EWS cells in which we profiled ETS1 binding and assessed the effects of ectopically expressed ETS1 defined 265 genes as positively regulated by ETS1 of which 103 also exhibited an increase in expression following depletion of EWSR1::FLI1. Of these 103 genes, we focused on ETS1’s regulation of TNS3, as assessment of multiple EWS tumor expression profiles, showed positive correlation of ETS1 and TNS3 expression. TNS3 encodes TENSIN3, a focal adhesion protein that regulates cytoskeletal reorganization and contributes to cell migration by connecting the cytoplasmic tail of integrins to the actin cytoskeleton. EWS cell lines (SK-N-MC (EWSR1::FLI1) and ES-5838 (EWSR1::ERG), in which we activated ETS1 expression (CRISPRa) exhibited a migratory phenotype and increased TNS3 expression. Critically, the activated ETS1 EWS cell lines show TNS3 accumulation at leading cell edges, with F-actin cytoskeletal reorganization, a phenotype associated with cell migration. Our study demonstrates that EWS cells expressing ETS1 exhibit a more migratory phenotype, which, has the potential to promote the dissemination of cells and, thus, metastasis. Citation Format: Vernon Justice Ebegboni, Tamara L. Jones, Tayvia Brownmiller, Erica Pehrsson, Patrick Zhao, Soumya Sundara Rajan, Natasha J. Caplen. ETS1, a target gene of the EWSR1::FLI1 fusion oncoprotein, regulates the expression of the focal adhesion protein TENSIN3 [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 2855.

The centromeric histone H3 variant CENP-A is overexpressed in many cancers. The mislocalization of CENP-A to noncentromeric regions contributes to chromosomal instability (CIN), a hallmark of cancer. However, pathways that promote or prevent CENP-A mislocalization remain poorly defined. Here, we performed a genome-wide RNAi screen for regulators of CENP-A localization which identified DNAJC9, a J-domain protein implicated in histone H3-H4 protein folding, as a factor restricting CENP-A mislocalization. Cells lacking DNAJC9 exhibit mislocalization of CENP-A throughout the genome, and CIN phenotypes. Global interactome analysis showed that DNAJC9 depletion promotes the interaction of CENP-A with the DNA-replication-associated histone chaperone MCM2. CENP-A mislocalization upon DNAJC9 depletion was dependent on MCM2, defining MCM2 as a driver of CENP-A deposition at ectopic sites when H3-H4 supply chains are disrupted. Cells depleted for histone H3.3, also exhibit CENP-A mislocalization. In summary, we have defined novel factors that prevent mislocalization of CENP-A, and demonstrated that the integrity of H3-H4 supply chains regulated by histone chaperones such as DNAJC9 restrict CENP-A mislocalization and CIN.

Abstract The mechanistic basis for the metastasis of Ewing sarcomas remains poorly understood, as these tumors harbor few mutations beyond the chromosomal translocation that initiates the disease. Instead, the epigenome of Ewing sarcoma cells reflects the regulatory state of genes associated with the DNA-binding activity of the fusion oncoproteins EWSR1::FLI1 or EWSR1::ERG. In this study, we examined the EWSR1::FLI1/ERG's repression of transcription factor genes, concentrating on those that exhibit a broader range of expression in tumors than in Ewing sarcoma cell lines. Focusing on one of these target genes, ETS1, we detected EWSR1::FLI1 binding and an H3K27me3-repressive mark at this locus. Depletion of EWSR1::FLI1 results in ETS1’s binding of promoter regions, substantially altering the transcriptome of Ewing sarcoma cells, including the upregulation of the gene encoding TENSIN3 (TNS3), a focal adhesion protein. Ewing sarcoma cell lines expressing ETS1 (CRISPRa) exhibited increased TNS3 expression and enhanced movement compared with control cells. Visualization of control Ewing sarcoma cells showed a distributed vinculin signal and a network-like organization of F-actin; in contrast, ETS1-activated Ewing sarcoma cells showed an accumulation of vinculin and F-actin toward the plasma membrane. Interestingly, the phenotype of ETS1-activated Ewing sarcoma cell lines depleted of TNS3 resembled the phenotype of the control cells. Critically, these findings have clinical relevance as TNS3 expression in Ewing sarcoma tumors positively correlates with that of ETS1. Implications: ETS1’s transcriptional regulation of the gene encoding the focal adhesion protein TENSIN3 in Ewing sarcoma cells promotes cell movement, a critical step in the evolution of metastasis.

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.

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.

EWSR1 is a member of the FET family of nucleic acid binding proteins that includes FUS and TAF15. Here, we report the systematic analysis of endogenous EWSR1's cellular organization in human cells. We demonstrate that EWSR1, which contains low complexity and nucleic acid binding domains, is present in cells in faster and slower-recovering fractions, indicative of a protein undergoing both rapid exchange and longer-term interactions. The employment of complementary high-resolution imaging approaches shows EWSR1 exists in two visual modalities, a distributed state which is present throughout the nucleoplasm, and a concentrated state consistent with the formation of foci. Both EWSR1 visual modalities localize with nascent RNA. EWSR1 foci concentrate in regions of euchromatin, adjacent to protein markers of transcriptional activation, and significantly colocalize with phosphorylated RNA polymerase II. Our results contribute to bridging the gap between our understanding of the biophysical and biochemical properties of FET proteins, including EWSR1, their functions as transcriptional regulators, and the participation of these proteins in tumorigenesis and neurodegenerative disease.

<p>Supplementary Figure S1 PDF file - 1274K, Schematic presentation of the workflow of experiments and analytical procedures</p>

Supplementary Figure S3 PDF file - 2243K, RNAi analysis and measurement of cell viability of amplified and overexpressed chromosome 13 candidate genes