Ewing sarcoma family tumors (ESFT) are aggressive bone and soft tissue tumors that express EWS-ETS fusion genes as driver mutations. Although the histogenesis of ESFT is controversial, mesenchymal (MSC) and/or neural crest (NCSC) stem cells have been implicated as cells of origin. For the current study we evaluated the consequences of EWS-FLI1 expression in human embryonic stem cell-derived NCSC (hNCSC). Ectopic expression of EWS-FLI1 in undifferentiated hNCSC and their neuro-mesenchymal stem cell (hNC-MSC) progeny was readily tolerated and led to altered expression of both well established as well as novel EWS-FLI1 target genes. Importantly, whole genome expression profiling studies revealed that the molecular signature of established ESFT is more similar to hNCSC than any other normal tissue, including MSC, indicating that maintenance or reactivation of the NCSC program is a feature of ESFT pathogenesis. Consistent with this hypothesis, EWS-FLI1 induced hNCSC genes as well as the polycomb proteins BMI-1 and EZH2 in hNC-MSC. In addition, up-regulation of BMI-1 was associated with avoidance of cellular senescence and reversible silencing of p16. Together these studies confirm that, unlike terminally differentiated cells but consistent with bone marrow-derived MSC, NCSC tolerate expression of EWS-FLI1 and ectopic expression of the oncogene initiates transition to an ESFT-like state. In addition, to our knowledge this is the first demonstration that EWS-FLI1-mediated induction of BMI-1 and epigenetic silencing of p16 might be critical early initiating events in ESFT tumorigenesis.

Ewing sarcomas are characterized by the presence of EWS/ETS fusion genes in the absence of other recurrent genetic alterations and mechanisms of tumor heterogeneity that contribute to disease progression remain unclear. Mutations in the Wnt/β-catenin pathway are rare in Ewing sarcoma but the Wnt pathway modulator LGR5 is often highly expressed, suggesting a potential role for the axis in tumor pathogenesis. We evaluated β-catenin and LGR5 expression in Ewing sarcoma cell lines and tumors and noted marked intra- and inter-tumor heterogeneity. Tumors with evidence of active Wnt/β-catenin signaling were associated with increased incidence of tumor relapse and worse overall survival. Paradoxically, RNA sequencing revealed a marked antagonism of EWS/ETS transcriptional activity in Wnt/β-catenin–activated tumor cells. Consistent with this, Wnt/β-catenin–activated cells displayed a phenotype that was reminiscent of Ewing sarcoma cells with partial EWS/ETS loss of function. Specifically, activation of Wnt/β-catenin induced alterations to the actin cytoskeleton, acquisition of a migratory phenotype, and upregulation of EWS/ETS–repressed genes. Notably, activation of Wnt/β-catenin signaling led to marked induction of tenascin C (TNC), an established promoter of cancer metastasis, and an EWS/ETS–repressed target gene. Loss of TNC function in Ewing sarcoma cells profoundly inhibited their migratory and metastatic potential. Our studies reveal that heterogeneous activation of Wnt/β-catenin signaling in subpopulations of tumor cells contributes to phenotypic heterogeneity and disease progression in Ewing sarcoma. Significantly, this is mediated, at least in part, by inhibition of EWS/ETS fusion protein function that results in derepression of metastasis-associated gene programs. Cancer Res; 76(17); 5040–53. ©2016 AACR.

Ewing sarcoma (ES) is a highly aggressive bone and soft-tissue tumor with peak incidence among adolescents and young adults. Despite advances in local control and systemic chemotherapy, metastatic relapse after an initial clinical remission remains a significant clinical problem. In addition, metastasis at the time of presentation or at relapse continues to be the leading cause of death for patients diagnosed with ES. Since the discovery of the pathognomonic EWS−FLI1 fusion gene more than 20 years ago, much about the molecular and cellular biology of ES pathogenesis has been learned. In addition, more recent exploitation of advances in stem cell and developmental biology has provided key insights into the cellular origins of ES and the role of epigenetic deregulation in tumor initiation and maintenance. Nevertheless, the mechanisms that drive tumor relapse and metastasis remain largely unknown. These gaps in our knowledge continue to hamper the development of novel therapeutic strategies that may improve outcomes for patients with relapsed and metastatic disease. In this article we review the current status of ES biology research, highlighting areas of investigation that we consider to have the greatest potential to yield findings that will translate into clinically significant advances.

Multi-agent chemotherapeutic regimes remain the cornerstone treatment for Ewing sarcoma, the second most common bone malignancy diagnosed in pediatric and young adolescent populations. We have reached a therapeutic ceiling with conventional cytotoxic agents, highlighting the need to adopt novel approaches that specifically target the drivers of Ewing sarcoma oncogenesis. As KDM1A/lysine-specific demethylase 1 (LSD1) is highly expressed in Ewing sarcoma cell lines and tumors, with elevated expression levels associated with worse overall survival (P = 0.033), this study has examined biomarkers of sensitivity and mechanisms of cytotoxicity to targeted KDM1A inhibition using SP-2509 (reversible KDM1A inhibitor). We report, that innate resistance to SP-2509 was not observed in our Ewing sarcoma cell line cohort (n = 17; IC50 range, 81 –1,593 nmol/L), in contrast resistance to the next-generation KDM1A irreversible inhibitor GSK-LSD1 was observed across multiple cell lines (IC50 > 300 μmol/L). Although TP53/STAG2/CDKN2A status and basal KDM1A mRNA and protein levels did not correlate with SP-2509 response, induction of KDM1B following SP-2509 treatment was strongly associated with SP-2509 hypersensitivity. We show that the transcriptional profile driven by SP-2509 strongly mirrors KDM1A genetic depletion. Mechanistically, RNA-seq analysis revealed that SP-2509 imparts robust apoptosis through engagement of the endoplasmic reticulum stress pathway. In addition, ETS1/HIST1H2BM were specifically induced/repressed, respectively following SP-2509 treatment only in our hypersensitive cell lines. Together, our findings provide key insights into the mechanisms of SP-2509 cytotoxicity as well as biomarkers that can be used to predict KDM1A inhibitor sensitivity in Ewing sarcoma. Mol Cancer Ther; 17(9); 1902–16. ©2018 AACR.

Ewing sarcoma is the second most common bone cancer in pediatric patients. Although the primary cause of death in Ewing sarcoma is metastasis, the mechanism underlying tumor spread needs to be elucidated. To this end, the role of the CXCR4/SDF-1a chemokine axis as a mediator of Ewing sarcoma metastasis was investigated. CXCR4 expression status was measured in primary tumor specimens by immunohistochemical staining and in multiple cell lines by quantitative reverse transcriptase PCR and flow cytometry. Migration and invasion of CXCR4-positive Ewing sarcoma cells toward CXCL12/SDF-1a were also determined. Interestingly, while CXCR4 status was disparate among Ewing sarcoma cells, ranging from absent to high-level expression, its expression was found to be highly dynamic and responsive to changes in the microenvironment. In particular, upregulation of CXCR4 occurred in cells that were subjected to growth factor deprivation, hypoxia, and space constraints. This upregulation of CXCR4 was rapidly reversed upon removal of the offending cellular stress conditions. Functionally, CXCR4-positive cells migrated and invaded toward an SDF-1a gradient and these aggressive properties were impeded by both the CXCR4 small-molecule inhibitor AMD3100, and by knockdown of CXCR4. In addition, CXCR4-dependent migration and invasion were inhibited by small-molecule inhibitors of Cdc42 and Rac1, mechanistically implicating these Rho-GTPases as downstream mediators of the CXCR4-dependent phenotype. Implications: This study reveals the highly plastic and dynamic nature of CXCR4 expression in Ewing sarcoma and supports a model in which stress-induced upregulation of CXCR4 contributes to tumor metastasis to lung and bone marrow, which express high levels of SDF-1a. Mol Cancer Res; 12(6); 953–64. ©2014 AACR.

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.