Ewing Sarcoma Cells Research Articles

Abstract 6252: HDAC inhibition increases Ewing sarcoma sensitivity to chemotherapy

Abstract Purpose: The survival rate of those with Ewing sarcoma (ES) has seen very little improvement over the past several decades and remains dismal for those with recurrent or metastatic disease. Current treatment for Ewing sarcoma includes a combination of cytotoxic chemotherapeutic agents and local control with radiation and/or surgery. The purpose of this study is to identify currently available targeted agents which may be combined with chemotherapy for use in Ewing sarcoma to improve patient outcomes. Methods: Novel targeted approaches for the treatment of ES were identified using RNA sequencing performed on ES patient tumors with bioinformatic analysis of gene expression. HDAC inhibitors were tested on patient cell lines at escalating doses and cell viability was assessed with CellTiter-Glo 2.0 and analyzed with GraphPad Prism to determine the IC50. Cells were treated with Panobinostat for 12 and 24 hours and RNA sequencing was performed. Cell cycle analysis was performed using Incucyte® Cell Cycle Lentivirus Reagent and analyzed using an Incucyte® S3. At 24- and 48-hours western blot analysis was performed to evaluate the effect on cell cycle and apoptosis. Combination studies with standard chemotherapy were performed and viability was assessed with CellTiter-Glo 2.0. At 24 hours of treatment with a combination of doxorubicin and HDAC inhibition western blot analysis was performed to evaluate changes in DNA damage and repair. Results: RNA analysis identified HDAC2 as a potential therapeutic target. The pan-HDAC inhibitors, Panobinostat and Belinostat, and the HDAC1/2 inhibitor, Romidepsin, were cytotoxic to all Ewing sarcoma cell lines tested with clinically relevant IC50s. Mechanistically, pathways involved in cell cycle progression and DNA repair were repressed by Panobinostat as shown by RNA sequencing and western blotting (Cyclin D1, pRb, CHK1). Additionally, cell cycle analysis has shown a G1 arrest in response to HDAC inhibition. HDAC inhibitors significantly decrease cell viability when combined with standard chemotherapy (upfront and relapse therapy) compared to that of chemotherapy alone. When combined with doxorubicin, CHK1 and CHK2 were significantly decreased and pH2AX was significantly increased indicating an accumulation of DNA damage. Conclusion: This work highlights that combining HDAC inhibitors with standard of care chemotherapy may be an effective treatment and improve outcomes for Ewing sarcoma patients. Future studies of HDAC inhibitors combined with chemotherapy drugs will aim to determine efficacy in xenograft models. Citation Format: Kaitlyn H. Smith, Chloe Sholler, Divya Gandra, Kimberly McKinney, Karl Dykema, Abhinav Nagulapally, Erin Trovillion. HDAC inhibition increases Ewing sarcoma sensitivity to chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6252.

Abstract 1466: Heterogeneity of the EWS-FLI1 transcriptome drives differences in the cellular phenotype of Ewing sarcoma cell lines

Abstract Background: Ewing sarcoma (ES) is dependent on the oncogenic transcription factor EWS-FLI1 for oncogenesis and progression. EWS-FLI1 binds to chromatin to both induce and repress gene expression. These gene expression changes drive proliferation, disrupt the cell cycle, and block cellular differentiation. One approach to target EWS-FLI1 is to reverse the gene signature. However, we have observed an underappreciated but striking heterogeneity of the EWS-FLI1 transcriptome among cell lines. While many common downstream targets exist, at least an equal number of cell-line specific large magnitude gene expression changes are also present. In this study, we evaluate the phenotypic importance of these cell-line specific changes and evaluate potential mechanisms that EWS-FLI1 may use to drive divergent transcription. Methods: The EWS-FLI1 transcriptome was characterized by RNA sequencing, following siRNA silencing of EWS-FLI1 at two time points in 6 cell lines. Effects on specific genes was confirmed by qPCR and western blot analysis. Cellular migration was evaluated using scratch assays on the Incucyte Zoom. EWS-FLI1binding was evaluated using Cleavage Under Targets and Tagmentation assay (CUT&Tag) and the transcriptome of Ewing subpopulations was determined by single cell RNA sequencing. Results: Next generation RNA sequencing of highly optimized siRNA knockdowns of EWS-FLI1 in 6 ES cell lines showed striking differences in gene expression. Conditions were optimized to capture the same magnitude change in EWS-FLI1 expression at the shortest time point ever reported. Fewer common targets were identified across all cell lines than previously reported, perhaps due to the short time frame and focus on direct targets. Independently generated well-established lists of targets were enriched in the data. However, there were 184 genes that changed by a Log2FC &gt 3 (P &lt 0.0001) in only one cell line including important genes like WNT7B and CTLA4. Marked heterogeneity was observed in a previously described migratory transcriptional signature including the key effectors Zyxin and Integrin Alpha-5; an observation confirmed by unbiased pathway analysis and cellular migration assays. Importantly, increased cellular migration was only seen in one cell line with siRNA silencing and not with any EWS-FLI1 targeted small molecules. To characterize the source of heterogeneity, we evaluated EWS-FLI1 binding at Zyxin and Alpha-5 integrin and single cell RNA sequencing of the migratory sub-signature. CUT&Tag demonstrates that EWS-FLI1 is found bound at these genes even in non-migratory cell lines. Conclusion: Careful consideration of Ewing sarcoma model-specific gene expression changes is important to future therapeutic approaches as these unique changes influence cellular phenotype. Citation Format: Seneca S. Kinn-Gurzo, Susan M. Kitchen-Goosen, Maggie Chasse, Marie Adams, Rebecca Kaufman, Patrick J. Grohar. Heterogeneity of the EWS-FLI1 transcriptome drives differences in the cellular phenotype of Ewing sarcoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1466.

Downregulated annexin A1 expression correlates with poor prognosis, metastasis, and immunosuppressive microenvironment in Ewing's sarcoma.

Ewing's sarcoma (ES) is a common bone malignancy in children and adolescents that severely affects the prognosis of patients. The aim of this study was to identify novel biomarkers and potential therapeutic targets for ES. Highly prognosis-related hub genes were identified by independent prognostic analysis in the GSE17679 dataset. We then performed survival analysis, Cox regression analysis and clinical correlation analysis on the key gene and validated them with the GSE63157, GSE45544 and GSE73166 datasets. Differentially expressed genes (DEGs) were screened based on the high and low expression of key gene, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, Gene Set Enrichment Analysis (GSEA), and Gene Set Variation Analysis (GSVA) were performed to explore the underlying mechanisms of ES, and significant module genes were established based on protein-protein interaction (PPI) networks. Furthermore, the correlations between module genes and the immune microenvironment were analyzed and the correlations between the key gene and immune infiltration levels in sarcoma were investigated using TIMER and TISIDB. Finally, the expression levels of these key genes in ES cell lines (RD-ES and A673 cells) were further validated by real-time quantitative PCR (RT-qPCR). CCK-8 and EdU assays were performed to assess the effect of ANXA1 knockdown on RD-ES cell proliferation. ANXA1 was identified as a key gene for ES prognosis. The overall survival (OS) time of patients with low ANXA1 expression was shorter, and the expression level of ANXA1 in the metastatic group was significantly lower than that in the primary group (P<0.01). Additionally, the abundance of 12 immune cells in the ANXA1 low-expression group was significantly lower than that in the high-expression group (all P<0.05), which may be related to the inhibition of the immune microenvironment. A PPI network was constructed based on 96 DEGs to further identify the five ANXA1-related module genes (COL1A2, MMP9, VIM, S100A11 and S100A4). The expression levels of ANXA1, COL1A2, MMP9, VIM, S100A11 and S100A4 were significantly different between ES cell lines and mesenchymal stem cells after validation in two ES cell lines (all P<0.01). Among these genes, ANXA1, COL1A2, MMP9, VIM and S100A4 were significantly associated with the prognosis of ES patients (all P<0.05). Importantly, ANXA1 knockdown significantly promoted the proliferation of RD-ES cells, which may explain the susceptibility to ES metastasis in the ANXA1 low-expression group. ANXA1 may serve as an independent prognostic biomarker for ES patients and is associated with metastasis and the immunosuppressive microenvironment in ES, which needs to be validated in further studies.

Subtype-selective induction of apoptosis in translocation-related sarcoma cells induced by PUMA and BIM upon treatment with pan-PI3K inhibitors

Translocation-related sarcomas (TRSs) harbor an oncogenic fusion gene generated by chromosome translocation and account for approximately one-third of all sarcomas; however, effective targeted therapies have yet to be established. We previously reported that a pan-phosphatidylinositol 3-kinase (PI3K) inhibitor, ZSTK474, was effective for the treatment of sarcomas in a phase I clinical trial. We also demonstrated the efficacy of ZSTK474 in a preclinical model, particularly in cell lines from synovial sarcoma (SS), Ewing’s sarcoma (ES) and alveolar rhabdomyosarcoma (ARMS), all of which harbor chromosomal translocations. ZSTK474 selectively induced apoptosis in all these sarcoma cell lines, although the precise mechanism underlying the induction of apoptosis remained unclear. In the present study, we aimed to determine the antitumor effect of PI3K inhibitors, particularly with regards to the induction of apoptosis, against various TRS subtypes using cell lines and patient-derived cells (PDCs). All of the cell lines derived from SS (six), ES (two) and ARMS (one) underwent apoptosis accompanied by the cleavage of poly-(ADP-ribose) polymerase (PARP) and the loss of mitochondrial membrane potential. We also observed apoptotic progression in PDCs from SS, ES and clear cell sarcoma (CCS). Transcriptional analyses revealed that PI3K inhibitors triggered the induction of PUMA and BIM and the knockdown of these genes by RNA interference efficiently suppressed apoptosis, suggesting their functional involvement in the progression of apoptosis. In contrast, TRS-derived cell lines/PDCs from alveolar soft part sarcoma (ASPS), CIC-DUX4 sarcoma and dermatofibrosarcoma protuberans failed to undergo apoptosis nor induce PUMA and BIM expression, as well as cell lines derived from non-TRSs and carcinomas. Thus, we conclude that PI3K inhibitors induce apoptosis in selective TRSs such as ES and SS via the induction of PUMA and BIM and the subsequent loss of mitochondrial membrane potential. This represents proof of concept for PI3K-targeted therapy, particularly such TRS patients.

MS0621, a novel small-molecule modulator of Ewing sarcoma chromatin accessibility, interacts with an RNA-associated macromolecular complex and influences RNA splicing.

Ewing sarcoma is a cancer of children and young adults characterized by the critical translocation-associated fusion oncoprotein EWSR1::FLI1. EWSR1::FLI1 targets characteristic genetic loci where it mediates aberrant chromatin and the establishment of de novo enhancers. Ewing sarcoma thus provides a model to interrogate mechanisms underlying chromatin dysregulation in tumorigenesis. Previously, we developed a high-throughput chromatin-based screening platform based on the de novo enhancers and demonstrated its utility in identifying small molecules capable of altering chromatin accessibility. Here, we report the identification of MS0621, a molecule with previously uncharacterized mechanism of action, as a small molecule modulator of chromatin state at sites of aberrant chromatin accessibility at EWSR1::FLI1-bound loci. MS0621 suppresses cellular proliferation of Ewing sarcoma cell lines by cell cycle arrest. Proteomic studies demonstrate that MS0621 associates with EWSR1::FLI1, RNA binding and splicing proteins, as well as chromatin regulatory proteins. Surprisingly, interactions with chromatin and many RNA-binding proteins, including EWSR1::FLI1 and its known interactors, were RNA-independent. Our findings suggest that MS0621 affects EWSR1::FLI1-mediated chromatin activity by interacting with and altering the activity of RNA splicing machinery and chromatin modulating factors. Genetic modulation of these proteins similarly inhibits proliferation and alters chromatin in Ewing sarcoma cells. The use of an oncogene-associated chromatin signature as a target allows for a direct approach to screen for unrecognized modulators of epigenetic machinery and provides a framework for using chromatin-based assays for future therapeutic discovery efforts.

Redundant Signaling as the Predominant Mechanism for Resistance to Antibodies Targeting the Type-I Insulin-Like Growth Factor Receptor in Cells Derived from Childhood Sarcoma.

Antibodies targeting insulin-like growth factor 1 receptor (IGF-1R) induce objective responses in only 5% to 15% of children with sarcoma. Understanding the mechanisms of resistance may identify combination therapies that optimize efficacy of IGF-1R-targeted antibodies. Sensitivity to the IGF-1R-targeting antibody TZ-1 was determined in rhabdomyosarcoma and Ewing sarcoma cell lines. Acquired resistance to TZ-1 was developed and characterized in sensitive Rh41 cells. The BRD4 inhibitor, JQ1, was evaluated as an agent to prevent acquired TZ-1 resistance in Rh41 cells. The phosphorylation status of receptor tyrosine kinases (RTK) was assessed. Sensitivity to TZ-1 in vivo was determined in Rh41 parental and TZ-1-resistant xenografts. Of 20 sarcoma cell lines, only Rh41 was sensitive to TZ-1. Cells intrinsically resistant to TZ-1 expressed multiple (>10) activated RTKs or a relatively less complex set of activated RTKs (∼5). TZ-1 decreased the phosphorylation of IGF-1R but had little effect on other phosphorylated RTKs in all resistant lines. TZ-1 rapidly induced activation of RTKs in Rh41 that was partially abrogated by knockdown of SOX18 and JQ1. Rh41/TZ-1 cells selected for acquired resistance to TZ-1 constitutively expressed multiple activated RTKs. TZ-1 treatment caused complete regressions in Rh41 xenografts and was significantly less effective against the Rh41/TZ-1 xenograft. Intrinsic resistance is a consequence of redundant signaling in pediatric sarcoma cell lines. Acquired resistance in Rh41 cells is associated with rapid induction of multiple RTKs, indicating a dynamic response to IGF-1R blockade and rapid development of resistance. The TZ-1 antibody had greater antitumor activity against Rh41 xenografts compared with other IGF-1R-targeted antibodies tested against this model.

Abstract PR018: Neuropeptide Y as a metastatic factor

Abstract Although neuropeptide Y (NPY) is known mainly as a sympathetic neurotransmitter, growing evidence indicates its role in tumor biology. Previous studies from our laboratory and other groups suggested associations of high expression of NPY and its Y5 receptor (Y5R) with invasive and metastatic phenotypes of various tumor types; yet the direct evidence for the metastatic activity was lacking. Our recent work began to fill this gap. Using Ewing sarcoma (ES) as a model of NPY-rich tumor, we identified the NPY/Y5R axis as a crucial pathway responsible for hypoxia-induced ES bone metastasis. This osseous dissemination was driven by hypoxia-dependent over-activation of the NPY/Y5R/RhoA pathway, which led to cytokinesis defects and formation of the aneuploid ES cell population with high propensity to bone metastasis. Here, we sought to determine the impact of the Y5R signaling on overall, hypoxia-independent metastatic capabilities of ES cells. To this end, a doxycycline-inducible CRIPSR/Cas9 system was used to knockout Y5R in ES orthotopic xenografts and test its impact on metastasis and cell migration assessed by a transwell assay. The frequency of the NPY5R gene modifications was determined by sequencing and confirmed by immunohistochemistry. FISH was used to identify NPY5R gene gains in ES xenografts. RhoA activity was measured by immunocytochemistry and pull-down assay. As expected, ES/Y5R-sgRNA primary tumors in doxycycline-treated mice consisted of a heterogeneous ES cell population with variable Y5R levels. In contrast, metastases developing from these xenografts were primarily initiated by ES clones with an intact NPY5R gene. The frequency of NPY5R gene modifications in metastases from the doxycycline-treated group was markedly reduced, as compared to the primary tumors. No metastases arising from ES clones lacking intact NPY5R gene were detected. Similarly, metastasis from wild type ES xenografts was associated with a selection of clones with the NPY5R gene gain that was present in a small percent of the parental cells. Subsequent in vitro assays implicated Y5R-dependent ES cell motility driven by the activation of a key cytoskeleton regulator, RhoA, as the mechanism underlying the metastatic effects of NPY. The above results, along with our previously published data, provide direct evidence for the crucial role of the NPY/Y5R axis in metastasis. We have shown that under basal conditions the NPY/Y5R/RhoA axis promotes tumor cell motility and overall cancer cell dissemination, while over-activation of this pathway under hypoxic conditions leads to genomic instability and bone metastasis. While our study focused on ES due to its high endogenous expression of NPY and its receptors, our findings may be relevant to other malignancies rich in NPY and Y5R, such as neuroblastoma, breast and prostate cancer. Given availability of the FDA approved Y5R antagonist, NPY/Y5R pathway may be a promising target for therapies preventing cancer progression. Yet, further research is required to establish effectiveness of such pharmacological interventions. Citation Format: Mina Adnani, Sung-Hyeok Hong, Susana Galli, Akanksha Mahajan, Nouran Abualsaud, Lindsay Caprio, Jason U. Tilan, Olga Rodriguez, Hingkun Wang, Christopher Albanese, Luciane R. Cavalli, Joanna Kitlinska. Neuropeptide Y as a metastatic factor [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr PR018.

Abstract B041: Bone microenvironment cues shift Ewing sarcoma cells towards pro-metastatic, ECM-secreting cell states

Abstract Solid tumors are complex ecosystems comprised of subpopulations of heterogeneous cancer cells that are altered by interactions with diverse cellular and acellular factors present in tumor microenvironments (TMEs). Accruing evidence suggests that heterogeneity of tumor cells and the TME, and bidirectional crosstalk between these spatially and temporally dynamic components, contributes to the generation of metastatic and therapy-resistant cell subpopulations. Ewing sarcomas are highly metastatic bone and soft tissue tumors driven by pathognomonic FET:ETS transcription factor fusions, predominantly EWS::FLI1. Despite sharing this single oncogenic driver, we and others have shown that Ewing sarcoma tumor cells are highly plastic cells that exist in transcriptionally and phenotypically distinct states. Notably, the extent of EWS::FLI1 transcriptional activity, ranging from low to high, has been shown to in part define the Ewing sarcoma cell state. Cells with high levels of fusion protein activity are generally proliferative and non-motile, while cells with lower levels of EWS::FLI1 expression or activity display enhanced metastatic potential. In this study we have leveraged single cell mRNA-seq and CITE-seq of Ewing sarcoma cell lines, gene expression and immunohistochemical profiling of primary tumor samples, and 3D culture invasion assays to define the complex Ewing sarcoma ecosystem and to investigate the role of tumor cell subpopulations and tumor: TME cross talk on tumor biology. Our results show cells with low levels of EWS::FLI1 activity activate an epithelial-mesenchymal transition (EMT)-like shift in gene expression. This activation of a partial EMT program is notable for an increase in expression and secretion of extracellular matrix (ECM) proteins by subpopulations of tumor cells and by increased matrix invasion. These ECM-related genes serve as transcriptional markers of the EMT-like cell state in both Ewing sarcoma models and in primary patient tumors. Moreover, the local TME surrounding these subpopulations is rich in tumor cell-derived ECM proteins including Tenascin-C, biglycan, and SPARC, proteins implicated in promotion of metastasis. We propose that subpopulations of ECM-secreting Ewing sarcoma tumor cells remodel the TME in a manner reminiscent of cancer associated fibroblasts (CAFs) in carcinomas. Further, we propose that this CAF-like matrix deposition contributes to local tumor progression and metastatic dissemination. Significantly, this ECM-secreting tumor cell state can be induced by canonical Wnt and TGF-beta ligands, suggesting that factors present in the bone TME can serve as inducers of a metastatic cell state. Together these findings demonstrate that Ewing sarcoma metastasis may be promoted by specific cell subpopulations which respond to and remodel the bone tumor microenvironment. Citation Format: Emma Wrenn, April Apfelbaum, Trisha Lipson, Erin Rudzinski, Xuemei Deng, Katherine Braun, Nicolas Garcia, Shruti Bhise, Olivia Waltner, Scott Furlan, Beth Lawlor. Bone microenvironment cues shift Ewing sarcoma cells towards pro-metastatic, ECM-secreting cell states [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B041.

Identification of Distinct and Common Subpopulations of Myxoid Liposarcoma and Ewing Sarcoma Cells Using Self-Organizing Maps

Myxoid liposarcoma and Ewing sarcoma are the two most common tumor types that are characterized by the FET (FUS, EWSR1 and TAF15) fusion oncogenes. These FET fusion oncogenes are considered to have the same pathological mechanism. However, the cellular similarities between cells from the different tumor entities remain unknown. Here, we profiled individual myxoid liposarcoma and Ewing sarcoma cells to determine common gene expression signatures. Five cell lines were analyzed, targeting 76 different genes. We employed unsupervised clustering, focusing on self-organizing maps, to identify biologically relevant subpopulations of tumor cells. In addition, we outlined the basic concepts of self-organizing maps. Principal component analysis and a t-distributed stochastic neighbor embedding plot showed gradual differences among all cells. However, we identified five distinct and robust subpopulations using self-organizing maps. Most cells were similar to other cells within the same tumor entity, but four out of five groups contained both myxoid liposarcoma and Ewing sarcoma cells. The major difference between the groups was the overall transcriptional activity, which could be linked to cell cycle regulation. We conclude that self-organizing maps are useful tools to define biologically relevant subpopulations and that myxoid liposarcoma and Ewing sarcoma exhibit cells with similar gene expression signatures.

Inhibition of the PI3K/AKT/mTOR signaling promotes an M1 macrophage switch by repressing the ATF3-CXCL8 axis in Ewing sarcoma

Ewing sarcomas are aggressive pediatric tumors of bone and soft tissues driven by in frame chromosomal translocations that yield fusion proteins guiding the oncogenic program. Promising alternative strategies to ameliorate current treatments involve inhibition of the PI3K/AKT/mTOR pathway. In this study, we identified the activating transcription factor 3 (ATF3) as an important mediator of the PI3K/AKT/mTOR pathway in Ewing sarcoma cells. ATF3 exerted its pro-tumoral activity through modulation of several chemokine-encoding genes, including CXCL8. The product of CXCL8, IL-8, acts as a pro-inflammatory chemokine critical for cancer progression and metastasis. We found that ATF3/IL-8 axis impacts macrophages populating the surrounding tumor microenvironment by promoting the M2 phenotype.Our study reveals valuable information on the PI3K/AKT/mTOR derived chemokine signaling in Ewing sarcoma cells: by promoting ATF3 and CXCL8 downregulation, inhibition of the PI3K/AKT/mTOR signaling promotes a proinflammatory response leading to upregulation of the protective anti-tumoral M1 macrophages.

Slit2 signaling stimulates Ewing sarcoma growth.

Ewing sarcoma is a cancer of bone and soft tissue in children driven by EWS::ETS fusion, most commonly EWS::FLI1. Because current cytotoxic chemotherapies are not improving the survival of those with metastatic or recurrent Ewing sarcoma cases, there is a need for novel and more effective targeted therapies. While EWS::FLI1 is the major driver of Ewing sarcoma, EWS::FLI1 has been difficult to target. A promising alternative approach is to identify and target the molecular vulnerabilities created by EWS::FLI1. Here we report that EWS::FLI1 induces the expression of Slit2, the ligand of Roundabout (Robo) receptors implicated in axon guidance and multiple other developmental processes. EWS::FLI1 binds to the Slit2 gene promoter and stimulates the expression of Slit2. Slit2 inactivates cdc42 and stabilizes the BAF chromatin remodeling complexes, enhancing EWS::FLI1 transcriptional output. Silencing of Slit2 strongly inhibited anchorage-dependent and anchorage-independent growth of Ewing sarcoma cells. Silencing of Slit2 receptors, Robo1 and Robo2, inhibited Ewing sarcoma growth as well. These results uncover a new role for Slit2 signaling in stimulating Ewing sarcoma growth and suggest that this pathway can be targeted therapeutically.

TIPE1 inhibits the growth of Ewing's sarcoma cells by suppressing Wnt/β-catenin signaling.

Ewing's sarcoma is the second most common bone and soft tissue malignancy in children and adolescents. Tumor necrosis factor-α-induced protein 8-like 1 (TIPE1) functions as a tumor suppressor in several cancers. Activation of Wnt/β-catenin signaling in subpopulations of tumor cells contributes to phenotypic heterogeneity and disease progression in Ewing's sarcoma. The exact role of TIPE1 in Ewing's sarcoma remains to be elucidated. This study aimed to assess the expression and function of TIPE1 in Ewing's sarcoma. TIPE1 expression in Ewing's sarcoma cells was determined by qPCR and western blotting. Furthermore, the Ewing's sarcoma cell line RD-ES was transfected with a lentivirus-based TIPE1 expression system to upregulate the expression of TIPE1. The Cell Counting Kit 8 was used to assess the effect of TIPE1 on cell proliferation. The effects of TIPE1 on cell migration and invasion was detected by Transwell assay. Flow cytometry was performed to detect apoptosis. Our results suggested lower TIPE1 expression in Ewing's sarcoma cell lines compared with normal osseous cells. TIPE1 remarkably inhibited the growth and proliferation of Ewing's sarcoma cell; TIPE1 also induced apoptosis and inhibited invasion in vitro. TIPE1 inhibited Ewing's sarcoma growth, motility, and survival through regulation of Wnt/β-catenin signaling. Our results demonstrated the anti-tumor function of TIPE1 in Ewing's sarcoma and reveal a novel therapeutic target.

Optimisation of pyrazolo[1,5-a]pyrimidin-7(4H)-one derivatives as novel Hsp90 C-terminal domain inhibitors against Ewing sarcoma

Ewing sarcoma is the second most prevalent paediatric malignant bone tumour. In most cases, it is driven by the fusion oncoprotein EWS::FLI1, which acts as an aberrant transcription factor and dysregulates gene expression. EWS::FLI1 and a large number of downstream dysregulated proteins are Hsp90 client proteins, making Hsp90 an attractive target for the treatment of Ewing sarcoma. In this article, we report a new structural class of allosteric Hsp90 C-terminal domain (CTD) inhibitors based on the virtual screening hit TVS24, which showed antiproliferative activity in the SK-N-MC Ewing sarcoma cell line with an IC50 value of 15.9 ± 0.7 µM. The optimised compounds showed enhanced anticancer activity in the SK-N-MC cell line. Exposure of Ewing sarcoma cells to the most potent analogue 11c resulted in depletion of critical Hsp90 client proteins involved in cancer pathways such as EWS::FLI1, CDK4, RAF-1 and IGF1R, without inducing a heat shock response. The results of this study highlight Hsp90 CTD inhibitors as promising new agents for the treatment of Ewing sarcoma.

Abstract IA019: Ectopic levels of low-complexity domain interactions repress endogenous oncogenic transcription

Abstract Mammalian transcription factors (TFs) ubiquitously contain intrinsically disordered low-complexity domains (LCDs) that perform transactivation functions. Recent studies have shown that dynamic, multivalent, and selective interactions between TF LCDs play an essential role in transactivation, but how such interactions mediate gene transcription remains unclear. It has been proposed that liquid-liquid phase separation (LLPS) of TFs driven by extensive LCD-LCD interactions is the mechanism underlying transactivation. Here, we investigate how tuning the level and localization of LCD-LCD interactions in vivo affects transcription of endogenous human genes. By quantitative single-cell imaging, we reveal that efficient gene activation by the oncogenic TF EWS/FLI1 requires narrow optimum of LCD-LCD interactions at its target genes associated with GGAA microsatellites. Increasing LCD-LCD interactions toward putative LLPS represses transcription of these genes in Ewing sarcoma cells. Likewise, ectopically creating LCD-LCD interactions to sequester EWS/FLI1 into a well-documented LLPS compartment, the nucleolus, represses EWS/FLI1 -driven transcription and inhibits malignant transformation of Ewing sarcoma. In addition, using live-cell single-molecule imaging, we show that a TF diffuses significantly more slowly in the nucleolus than in the nucleoplasm, demonstrating an important impact of LLPS on TF dynamic behaviors and single-molecule diffusion measurement as an effective approach to diagnose LLPS in vivo. Our findings show how altering the balance of LCD-LCD interactions can influence transcriptional regulation and suggest a potential therapeutic strategy for targeting disease-causing TFs. Citation Format: Shasha Chong. Ectopic levels of low-complexity domain interactions repress endogenous oncogenic transcription. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr IA019.

Abstract B018: Studying the role of FLI portion of EWS FLI in transcription regulation via modulation of chromatin 3D landscape in Ewing sarcoma

Abstract Ewing sarcoma is an aggressive bone-associated tumor currently treated with dose-intense chemotherapy, radiation, and surgery. Ewing sarcoma affects adolescents and young adults with incidence rate of 3 per million. The hallmark of Ewing sarcoma is a translocated fusion transcription factor named EWS FLI that drives the oncogenic process. The lack of efficient and targeted treatment in Ewing sarcoma directly arises from the poor understanding of how EWS/FLI regulates expression of thousands of genes as well as the current lack of effective targeting of transcription factors. We hypothesize that FLI portion of EWS::FLI containing a crucial alpha-helix plays a novel role in transcription regulation of thousands of genes by modulating chromatin looping. We postulated this hypothesis based on recent evidence from our lab that established an alpha-helix immediately downstream of DNA binding domain as an important player in regulating transcriptional activity in A673 cell line. Structure-function mapping revealed that ETS domain alone was insufficient for full transcriptional regulation. The purpose of this study is to elucidate the mechanism underlying transcriptional regulation by FLI and it is likely to guide future studies on novel therapeutics for Ewing sarcoma patients as transcriptional factors remain elusive targets of therapy. We utilized knockdown/rescue experiments in which EWS FLI was depleted with shRNA and replaced with constructs containing the ETS DNA Binding Domain (DBD) alone or DBD+ (ETS DNA Binding Domain and 4th alpha-helix). The knockdown/rescue experiments were confirmed at mRNA level using qPCR, and at protein level using immunoblots. The binding pattern and transcriptional regulation of DBD and DBD+ were assessed with RNA-Seq respectively. The extent of roles each of these construct play in organization, structure, and function of chromatin are being assessed using Micro-C technique, a variation of Hi-C with improved resolution, higher signal-to-noise ratio and more information on chromatin domain boundaries and chromatin looping. The DNA binding and genomic localization of EWS FLI was unaltered by the deletion surrounding the DNA binding domain (which contains a 4th alpha-helix) in A673 cells. Despite this similarity in genomic localization and binding, the transcriptional output driven by EWS FLI was significantly diminished by the deletion. In addition, we observed this differential transcriptional output by the EWS FLI DBD and DBD+ in another Ewing sarcoma cell line called TTC466. Current work in our lab shows that EWS FLI has a substantial role in shaping the chromatin landscape of Ewing sarcoma cells. We observed this result with Hi-C technique upon the depleting EWS FLI from A673 cells. With this current study, we hope to understand if the flanking region neighboring the DNA binding domain contributes to the chromatin architecture remodeling function of EWS FLI and whether this function could be attributed to the transcriptional output differences in the DBD and DBD+ conditions. Citation Format: Ariunaa Bayanjargal. Studying the role of FLI portion of EWS FLI in transcription regulation via modulation of chromatin 3D landscape in Ewing sarcoma. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B018.

High-content drug screening in zebrafish xenografts reveals high efficacy of dual MCL-1/BCL-XL inhibition against Ewing sarcoma

Ewing sarcoma is a pediatric bone and soft tissue cancer with an urgent need for new therapies to improve disease outcome. To identify effective drugs, phenotypic drug screening has proven to be a powerful method, but achievable throughput in mouse xenografts, the preclinical Ewing sarcoma standard model, is limited.Here, we explored the use of xenografts in zebrafish for high-throughput drug screening to discover new combination therapies for Ewing sarcoma. We subjected xenografts in zebrafish larvae to high-content imaging and subsequent automated tumor size analysis to screen single agents and compound combinations. We identified three drug combinations effective against Ewing sarcoma cells: Irinotecan combined with either an MCL-1 or an BCL-XL inhibitor and in particular dual inhibition of the anti-apoptotic proteins MCL-1 and BCL-XL, which efficiently eradicated tumor cells in zebrafish xenografts. We confirmed enhanced efficacy of dual MCL-1/BCL-XL inhibition compared to single agents in a mouse PDX model.In conclusion, high-content screening of small compounds on Ewing sarcoma zebrafish xenografts identified dual MCL-1/BCL-XL targeting as a specific vulnerability and promising therapeutic strategy for Ewing sarcoma, which warrants further investigation towards clinical application.

Systematic multi-omics cell line profiling uncovers principles of Ewing sarcoma fusion oncogene-mediated gene regulation.

Ewing sarcoma (EwS) is characterized by EWSR1-ETS fusion transcription factors converting polymorphic GGAA microsatellites (mSats) into potent neo-enhancers. Although the paucity of additional mutations makes EwS a genuine model to study principles of cooperation between dominant fusion oncogenes and neo-enhancers, this is impeded by the limited number of well-characterized models. Here we present the Ewing Sarcoma Cell Line Atlas (ESCLA), comprising whole-genome, DNA methylation, transcriptome, proteome, and chromatin immunoprecipitation sequencing (ChIP-seq) data of 18 cell lines with inducible EWSR1-ETS knockdown. The ESCLA shows hundreds of EWSR1-ETS-targets, the nature of EWSR1-ETS-preferred GGAA mSats, and putative indirect modes of EWSR1-ETS-mediated gene regulation, converging in the duality of a specific but plastic EwS signature. We identify heterogeneously regulated EWSR1-ETS-targets as potential prognostic EwS biomarkers. Our freely available ESCLA (http://r2platform.com/escla/) is a rich resource for EwS research and highlights the power of comprehensive datasets to unravel principles of heterogeneous gene regulation by chimeric transcription factors.

Evaluation of geranylgeranyl diphosphate synthase inhibition as a novel strategy for the treatment of osteosarcoma and Ewing sarcoma.

Rab GTPases are critical regulators of protein trafficking in the cell. To ensure proper cellular localization and function, Rab proteins must undergo a posttranslational modification, termed geranylgeranylation. In the isoprenoid biosynthesis pathway, the enzyme geranylgeranyl diphosphate synthase (GGDPS) generates the 20-carbon isoprenoid donor (geranylgeranyl pyrophosphate [GGPP]), which is utilized in the prenylation of Rab proteins. We have pursued the development of GGDPS inhibitors (GGSI) as a novel means to target Rab activity in cancer cells. Osteosarcoma (OS) and Ewing sarcoma (ES) are aggressive childhood bone cancers with stagnant survival statistics and limited treatment options. Here we show that GGSI treatment induces markers of the unfolded protein response (UPR) and triggers apoptotic cell death in a variety of OS and ES cell lines. Confirmation that these effects were secondary to cellular depletion of GGPP and disruption of Rab geranylgeranylation was confirmed via experiments using exogenous GGPP or specific geranylgeranyl transferase inhibitors. Furthermore, GGSI treatment disrupts cellular migration and invasion in vitro. Metabolomic profiles of OS and ES cell lines identify distinct changes in purine metabolism in GGSI-treated cells. Lastly, we demonstrate that GGSI treatment slows tumor growth in a mouse model of ES. Collectively, these studies support further development of GGSIs as a novel treatment for OS and ES.

The importance of fusion protein activity in Ewing sarcoma and the cell intrinsic and extrinsic factors that regulate it: A review.

Accumulating evidence shows that despite clonal origins tumors eventually become complex communities comprised of phenotypically distinct cell subpopulations. This heterogeneity arises from both tumor cell intrinsic programs and signals from spatially and temporally dynamic microenvironments. While pediatric cancers usually lack the mutational burden of adult cancers, they still exhibit high levels of cellular heterogeneity that are largely mediated by epigenetic mechanisms. Ewing sarcomas are aggressive bone and soft tissue malignancies with peak incidence in adolescence and the prognosis for patients with relapsed and metastatic disease is dismal. Ewing sarcomas are driven by a single pathognomonic fusion between a FET protein and an ETS family transcription factor, the most common of which is EWS::FLI1. Despite sharing a single driver mutation, Ewing sarcoma cells demonstrate a high degree of transcriptional heterogeneity both between and within tumors. Recent studies have identified differential fusion protein activity as a key source of this heterogeneity which leads to profoundly different cellular phenotypes. Paradoxically, increased invasive and metastatic potential is associated with lower EWS::FLI1 activity. Here, we review what is currently understood about EWS::FLI1 activity, the cell autonomous and tumor microenvironmental factors that regulate it, and the downstream consequences of these activity states on tumor progression. We specifically highlight how transcription factor regulation, signaling pathway modulation, and the extracellular matrix intersect to create a complex network of tumor cell phenotypes. We propose that elucidation of the mechanisms by which these essential elements interact will enable the development of novel therapeutic approaches that are designed to target this complexity and ultimately improve patient outcomes.

Glutamine-dependent effects of nitric oxide on cancer cells subjected to hypoxia-reoxygenation

Limited O2 availability can decrease essential processes in energy metabolism. However, cancers have developed distinct metabolic adaptations to these conditions. For example, glutaminolysis can maintain energy metabolism and hypoxia signaling. Additionally, it has been observed that nitric oxide (NO) possesses concentration-dependent, biphasic effects in cancer. NO has potent anti-tumor effects through modulating events such as angiogenesis and metastasis at low physiological concentrations and inducing cell death at higher concentrations. In this study, Ewing Sarcoma cells (A-673), MIA PaCa, and SKBR3 cells were treated with DetaNONOate (DetaNO) in a model of hypoxia (1% O2) and reoxygenation (21% O2). All 3 cell types showed NO-dependent inhibition of cellular O2 consumption which was enhanced as O2-tension decreased. L-Gln depletion suppressed the mitochondrial response to decreasing O2 tension in all 3 cell types and resulted in inhibition of Complex I activity. In A-673 cells the O2 tension dependent change in mitochondrial O2 consumption and increase in glycolysis was dependent on the presence of L-Gln. The response to hypoxia and Complex I activity were restored by α-ketoglutarate. NO exposure resulted in the A-673 cells showing greater sensitivity to decreasing O2 tension. Under conditions of L-Gln depletion, NO restored HIF-1α levels and the mitochondrial response to O2 tension possibly through the increase of 2-hydroxyglutarate. NO also resulted in suppression of cellular bioenergetics and further inhibition of Complex I which was not rescued by α-ketoglutarate. Taken together these data suggest that NO modulates the mitochondrial response to O2 differentially in the absence and presence of L-Gln. These data suggest a combination of metabolic strategies targeting glutaminolysis and Complex I in cancer cells.