Ewing sarcoma (EwS) is a rare and highly malignant bone tumor occurring mainly in childhood and adolescence. Physiologically, the bone is a central hub for Ca2+ homeostasis, which is severely disturbed by osteolytic processes in EwS. Therefore, we aimed to investigate how ion transport proteins involved in Ca2+ homeostasis affect EwS pathophysiology. We characterized the expression of 22 candidate genes of Ca2+-permeable or Ca2+-regulated ion channels in three EwS cell lines and found the Ca2+-activated K+ channel KCa2.1 (KCNN1) to be exceptionally highly expressed. We revealed that KCNN1 expression is directly regulated by the disease-driving oncoprotein EWSR1-FL1. Due to its consistent overexpression in EwS, KCNN1 mRNA could be a prognostic marker in EwS. In a large cohort of EwS patients, however, KCNN1 mRNA quantity does not correlate with clinical parameters. Several functional studies including patch clamp electrophysiology revealed no evidence for KCa2.1 function in EwS cells. Thus, elevated KCNN1 expression is not translated to KCa2.1 channel activity in EwS cells. However, we found that the low K+ conductance of EwS cells renders them susceptible to hypoosmotic solutions. The absence of a relevant K+ conductance in EwS thereby provides an opportunity for hypoosmotic therapy that can be exploited during tumor surgery.

Ewing sarcoma is an aggressive bone cancer affecting children and young adults. The main molecular hallmark of Ewing sarcoma are chromosomal translocations that produce chimeric oncogenic transcription factors, the most frequent of which is the aberrant transcription factor EWSR1-FLI1. Because this is the principal oncogenic driver of Ewing sarcoma, its inactivation should be the best therapeutic strategy to block tumor growth. In this study, we genetically inactivated EWSR1-FLI1 using CRISPR-Cas9 technology in order to cause permanent gene inactivation. We found that gene editing at the exon 9 of FLI1 was able to block cell proliferation drastically and induce senescence massively in the well-studied Ewing sarcoma cell line A673. In comparison with an extensively used cellular model of EWSR1-FLI1 knockdown (A673/TR/shEF), genetic inactivation was more effective, particularly in its capability to block cell proliferation. In summary, genetic inactivation of EWSR1-FLI1 in A673 Ewing sarcoma cells blocks cell proliferation and induces a senescence phenotype that could be exploited therapeutically. Although efficient and specific in vivo CRISPR-Cas9 editing still presents many challenges today, our data suggest that complete inactivation of EWSR1-FLI1 at the cell level should be considered a therapeutic approach to develop in the future.

RNA-binding proteins (RBPs) are important transcriptomic regulators and may be important in tumorigenesis. Here, we sought to investigate the clinical impact of RBPs for patients with Ewing sarcoma (ES). ES transcriptome signatures were characterized from four previously published cohorts and grouped into new training and validation cohorts. A total of three distinct subtypes were identified and compared for differences in patient prognosis and RBP signatures. Next, univariate Cox and Lasso regression models were used to identify hub prognosis-related RBPs and construct a prognostic risk model, and prediction capacity was assessed through time-dependent receiver operating characteristics (ROCs), Kaplan-Meier curves, and nomograms. Across the three RBP subtypes, 29 significant prognostic-associated RBP genes were identified, of which 10 were used to build and validate an RBP-associated prognostic risk model (RPRM) that had a stable predictive value and could be considered valuable for clinical risk-stratification of ES. A comparison with immunohistochemistry validation showed a significant association between overall survival and NSUN7 immunoreactivity, which was an independent favorable prognostic marker. The association of RBP signatures with ES clinical prognosis provides a strong rationale for further investigation into RBPs molecular mechanisms.

Ewing sarcoma (EWS) is an aggressive pediatric malignancy of the bone and soft tissues in need of novel therapeutic options. To identify potential therapeutic targets, we focused on essential biological pathways that are upregulated by EWS-FLI1, the primary oncogenic driver of EWS, including mitotic proteins such as Aurora kinase A (AURKA) and kinesin family member 15 (KIF15) and its binding partner, targeting protein for Xklp2 (TPX2). KIF15/TPX2 cooperates with KIF11, a key mitotic kinesin essential for mitotic spindle orientation. Given the lack of clinical-grade KIF15/TPX2 inhibitors, we chose to target KIF11 (using SB-743921) in combination with AURKA (using VIC-1911) given that phosphorylation of KIF15S1169 by Aurora A is required for its targeting to the spindle. In vitro, the drug combination demonstrated strong synergy (Bliss score ≥ 10) at nanomolar doses. Colony formation assay revealed significant reduction in plating efficiency (1-3%) and increased percentage accumulation of cells in the G2/M phase with the combination treatment (45-52%) upon cell cycle analysis, indicating mitotic arrest. In vivo studies in EWS xenograft mouse models showed significant tumor reduction and overall effectiveness: drug combination vs. vehicle control (p ≤ 0.01), SB-743921 (p ≤ 0.01) and VIC-1911 (p ≤ 0.05). Kaplan-Meier curves demonstrated superior overall survival with the combination compared to vehicle or monotherapy arms (p ≤ 0.0001).

Intracranial Ewing sarcoma (EwS) is rare and publications on primary or metastatic intracranial EwS are minimal. The aim of this study was to describe incidence, clinical behavior, treatment, and factors associated with outcome in patients with primary intracranial EwS or patients with a primary extracranial EwS and cerebral metastases at diagnosis. We reviewed all patients with primary or with metastatic intracranial EwS at diagnosis registered in the International Clinical Trial Euro-E.W.I.N.G.99 (EE99). In total, 17 of 1435 patients (1.2%) presented with primary intracranial EwS; 3 of them had metastatic disease. Four patients (0.3%) with primary extracranial EwS presented with intracranial metastatic lesions. The 3-year event-free survival (EFS) was 64% and overall survival (OS) was 70% in patients with a primary intracranial EwS. Local control in patients with primary intracranial EwS consisted of surgery (6%), radiotherapy (RT) (18%), or both modalities (76%). Univariate analysis showed that patients < 15 years of age had significantly better outcome (EFS: 72%; OS: 76%) compared to those aged above 15 years (EFS: 13%; OS: 25%). In conclusion, primary intracranial EwS and extracranial EwS with cerebral metastases at diagnosis is rare, yet survival is comparable with local and metastatic EwS elsewhere in the body. Age and stage of disease are important prognostic factors. Besides chemotherapeutic treatment, local control with surgical resection combined with RT is recommended whenever feasible.

The receptor tyrosine kinase (RTK) RON is linked to an aggressive metastatic phenotype of carcinomas. While gaining interest as a therapeutic target, RON remains unstudied in sarcomas. In Ewing sarcoma, we identified RON among RTKs conferring resistance to insulin-like growth factor-1 receptor (IGF1R) targeting. Therefore, we explored RON in pediatric sarcoma cell lines and an embryonic Tg(kdrl:mCherry) zebrafish model, using an shRNA-based approach. To examine RON-IGF1R crosstalk, we employed the clinical-grade monoclonal antibody IMC-RON8, alone and together with the IGF1R-antibody IMC-A12. RON silencing demonstrated functions in vitro and in vivo, particularly within micrometastatic cellular capacities. Signaling studies revealed a unidirectional IGF1-mediated cross-activation of RON. Yet, IMC-A12 failed to sensitize cells to IMC-RON8, suggesting additional mechanisms of RON activation. Here, RT-PCR revealed that childhood sarcomas express short-form RON, an isoform resistant to antibody-mediated targeting. Interestingly, in contrast to carcinomas, treatment with DNA methyltransferase inhibitor did not diminish but increased short-form RON expression. Thus, this first report supports a role for RON in the metastatic progression of Ewing sarcoma. While principal molecular functions appear transferrable between carcinomas, Ewing sarcoma and possibly more common sarcoma subtypes, RON highlights that specific regulations of cellular networks and isoforms require better understanding to successfully transfer targeting strategies.

Ewing sarcoma (EwS) is an aggressive cancer displaying an undifferentiated small-round-cell histomorphology that can be easily confused with a broad spectrum of differential diagnoses. Using comparative transcriptomics and immunohistochemistry (IHC), we previously identified BCL11B and GLG1 as potential specific auxiliary IHC markers for EWSR1-FLI1-positive EwS. Herein, we aimed at validating the specificity of both markers in a far larger and independent cohort of EwS (including EWSR1-ERG-positive cases) and differential diagnoses. Furthermore, we evaluated their intra-tumoral expression heterogeneity. Thus, we stained tissue microarrays from 133 molecularly confirmed EwS cases and 320 samples from morphological mimics, as well as a series of patient-derived xenograft (PDX) models for BCL11B, GLG1, and CD99, and systematically assessed the immunoreactivity and optimal cut-offs for each marker. These analyses demonstrated that high BCL11B and/or GLG1 immunoreactivity in CD99-positive cases had a specificity of 97.5% and an accuracy of 87.4% for diagnosing EwS solely by IHC, and that the markers were expressed by EWSR1-ERG-positive EwS. Only little intra-tumoral heterogeneity in immunoreactivity was observed for differential diagnoses. These results indicate that BCL11B and GLG1 may help as specific auxiliary IHC markers in diagnosing EwS in conjunction with CD99, especially if confirmatory molecular diagnostics are not available.

Ewing sarcoma is an aggressive pediatric cancer of enigmatic cellular origins typically resulting from a single translocation event t (11; 22) (q24; q12). The resulting fusion gene, EWSR1-FLI1, is toxic or unstable in most primary tissues. Consequently, attempts to model Ewing sarcomagenesis have proven unsuccessful thus far, highlighting the need to identify the cellular features which permit stable EWSR1-FLI1 expression. By re-analyzing publicly available RNA-Sequencing data with manifold learning techniques, we uncovered a group of Ewing-like tissues belonging to a developmental trajectory between pluripotent, neuroectodermal, and mesodermal cell states. Furthermore, we demonstrated that EWSR1-FLI1 expression levels control the activation of these developmental trajectories within Ewing sarcoma cells. Subsequent analysis and experimental validation demonstrated that the capability to resolve R-loops and mitigate replication stress are probable prerequisites for stable EWSR1-FLI1 expression in primary tissues. Taken together, our results demonstrate how EWSR1-FLI1 hijacks developmental gene programs and advances our understanding of Ewing sarcomagenesis.

Ewing sarcoma (ES) is one of the most frequent types of malignant tumors among children. The active metabolic state of ES cells presents a new potential target for therapeutic interventions. As a primary regulator of cellular homeostasis, carbonic anhydrases (CAs; EC 4.2.1.1) have emerged as promising molecular targets for the development of anticancer drugs. Within the present study, we tested the commercial drug acetazolamide and our previously discovered inhibitors to target the CAII isoform, which was overexpressed and positively correlated with ES patient relapse. We employed molecular biology tests to identify effective inhibitors of CAII that can induce ferroptosis by downregulating FTH1 expression in ES cells. In vitro, we have also demonstrated their ability to reduce cell proliferation, decrease invasion, and induce apoptosis- or autophagy-related cell death. Using Western blotting, we confirmed the induction of cathepsin B in cells treated with CA inhibitors. It was found that the suppression of cathepsin B expression during the treatment reduces the anticancer efficacy of selected CAII inhibitors. These experiments highlighted profound antitumor activity of CAII inhibitors attributive to their remarkable ability to trigger ferroptosis in Ewing sarcoma cells without causing substantial host damage. The obtained results suggest that cytosolic CAII may be a prospective target for ES treatment, and CAII inhibitors can be considered as potential single-agent or combination antitumor agents to be used in the treatment of ES.

Ewing sarcoma is an aggressive and the second most common bone tumor in adolescent and young adult patients. The 5-year survival rate is 60-70% for localized disease but 30% for patients with metastases. Here, we aimed to identify a therapeutic target for Ewing sarcoma and evaluate antibody-based therapeutic agents using in vitro and in vivo models. We identified G protein-coupled receptor 64 (GPR64) as a therapeutic target for Ewing sarcoma via next-generation RNA-sequencing. GPR64v205 mRNA was expressed in HTB166, A673, MG63, 143B, HS-Sy II, and HT1080 cell lines as well as in Ewing sarcoma, undifferentiated pleomorphic sarcoma, leiomyosarcoma, dedifferentiated liposarcoma, and synovial sarcoma tissues. GPR64 expression was observed in 62.5% of sarcoma cases and was overexpressed in 33.9% cases. GPR64-specific monoclonal antibodies were tested as near-infrared probes for in vivo imaging using subcutaneous tumor mouse xenografts. Fluorescence intensity was stronger for the AF700-labeled anti-GPR64 antibody than that for the AF700-labeled isotype control antibody. GPR64 was detected in engrafted tumors of A673, 143B, HT1080, and the epididymis but not in other resected tissues. The anti-GPR64 antibody showed excellent binding to GPR64-positive tumors but not to healthy tissues. This antibody has potential for drug delivery in the antibody-based treatment of sarcomas.

Outcomes for most patients with Ewing sarcoma (ES) have remained unchanged for the last 30 years, emphasising the need for more effective and tolerable treatments. We have hypothesised that using small-molecule inhibitors to kill the self-renewing chemotherapy-resistant cells (Ewing sarcoma cancer stem-like cells; ES-CSCs) responsible for progression and relapse could improve outcomes and minimise treatment-induced morbidities. For the first time, we demonstrate that ABCG1, a potential oncogene in some cancers, is highly expressed in ES-CSCs independently of CD133. Using functional models, transcriptomics and a bespoke in silico drug-repurposing pipeline, we have prioritised a group of tractable small-molecule inhibitors for further preclinical studies. Consistent with the cellular origin of ES, 21 candidate molecular targets of pluripotency, stemness and chemoresistance were identified. Small-molecule inhibitors to 13 of the 21 molecular targets (62%) were identified. POU5F1/OCT4 was the most promising new therapeutic target in Ewing sarcoma, interacting with 10 of the 21 prioritised molecular targets and meriting further study. The majority of small-molecule inhibitors (72%) target one of two drug efflux proteins, p-glycoprotein (n = 168) or MRP1 (n = 13). In summary, we have identified a novel cell surface marker of ES-CSCs and cancer/non-cancer drugs to targets expressed by these cells that are worthy of further preclinical evaluation. If effective in preclinical models, these drugs and drug combinations might be repurposed for clinical evaluation in patients with ES.

Previously, we used inhibitors blocking BET bromodomain binding proteins (BRDs) in Ewing sarcoma (EwS) and observed that long term treatment resulted in the development of resistance. Here, we analyze the possible interaction of BRD4 with cyclin-dependent kinase (CDK) 9.

Survival rates for Ewing sarcoma (ES) patients with metastatic disease have not improved in over 20 years. Tumor growth and metastasis are dependent on tumor vasculature expansion; therefore, identifying the regulators that control this process in ES may provide new therapeutic opportunities. ES expresses high levels of repressor element 1 silencing transcription factor (REST), which is regulated by the EWS-FLI-1 fusion gene. However, the role of REST in ES growth and the regulation of the tumor vasculature have not been elucidated. To study this role, we established REST-knockout human TC71 ES cell lines through CRISPR/Cas9 recombination. While knockout of REST did not alter tumor cell proliferation in vitro, REST knockout reduced tumor growth and metastasis to the lung in vivo and altered tumor vascular morphology and function. Tumor vessels in the REST-knockout tumors had a punctate appearance with significantly decreased tumor vascular pericytes, decreased perfusion, and increased permeability. REST-knockout tumors also showed increased apoptosis and hypoxia. These results indicate that REST plays a critical role in ES vascular function, which in turn impacts the ability of ES tumors to grow and metastasize. These findings therefore provide a basis for the targeting of REST as a novel therapeutic approach in ES.

Ewing Sarcoma (ES) is a rare malignant tumor occurring most frequently in adolescents and young adults. The ES hallmark is a chromosomal translocation between the chromosomes 11 and 22 that results in an aberrant transcription factor (TF) through the fusion of genes from the FET and ETS families, commonly EWSR1 and FLI1. The regulatory mechanisms behind the ES transcriptional alterations remain poorly understood. Here, we reconstruct the ES regulatory network using public available transcriptional data. Seven TFs were identified as potential MRs and clustered into two groups: one composed by PAX7 and RUNX3, and another composed by ARNT2, CREB3L1, GLI3, MEF2C, and PBX3. The MRs within each cluster act as reciprocal agonists regarding the regulation of shared genes, regulon activity, and implications in clinical outcome, while the clusters counteract each other. The regulons of all the seven MRs were differentially methylated. PAX7 and RUNX3 regulon activity were associated with good prognosis while ARNT2, CREB3L1, GLI3, and PBX3 were associated with bad prognosis. PAX7 and RUNX3 appear as highly expressed in ES biopsies and ES cell lines. This work contributes to the understanding of the ES regulome, identifying candidate MRs, analyzing their methilome and pointing to potential prognostic factors.

Ewing sarcoma is a rare pediatric tumor characterized by chromosomal translocations that give rise to aberrant chimeric transcription factors (e.g., EWSR1-FLI1). EWSR1-FLI1 promotes a specific cellular transcriptional program. Therefore, the study of EWSR1-FLI1 target genes is important to identify critical pathways involved in Ewing sarcoma tumorigenesis. In this work, we focused on the transcription factors regulated by EWSR1-FLI1 in Ewing sarcoma. Transcriptomic analysis of the Ewing sarcoma cell line A673 indicated that one of the genes more strongly upregulated by EWSR1-FLI1 was FEZF1 (FEZ family zinc finger protein 1), a transcriptional repressor involved in neural cell identity. The functional characterization of FEZF1 was performed in three Ewing sarcoma cell lines (A673, SK-N-MC, SK-ES-1) through an shRNA-directed silencing approach. FEZF1 knockdown inhibited clonogenicity and cell proliferation. Finally, the analysis of the FEZF1-dependent expression profile in A673 cells showed several neural genes regulated by FEZF1 and concomitantly regulated by EWSR1-FLI1. In summary, FEZF1 is transcriptionally regulated by EWSR1-FLI1 in Ewing sarcoma cells and is involved in the regulation of neural-specific genes, which could explain the neural-like phenotype observed in several Ewing sarcoma tumors and cell lines.

Ewing's sarcoma (EWS), an aggressive pediatric bone and soft-tissue sarcoma, has a very stable genome with very few genetic alterations. Unlike in most cancers, the progression of EWS appears to depend on epigenetic alterations. EWS-FLI1 and CD99, the two hallmarks of EWS, are reported to severely impact the malignancy of EWS cells, at least partly by regulating the expression of several types of non-coding RNAs. Here, we identify miR-214-3p as a common mediator of either EWS-FLI1 or CD99 by in silico analysis. MiR-214-3p expression was lower in EWS cells and in clinical samples than in bone marrow mesenchymal stem cells, and this miRNA was barely expressed in metastatic lesions. Silencing of EWS-FLI1 or CD99 restored the expression of miR-214-3p, leading to a reduced cell growth and migration. Mechanistically, miR-214-3p restoration inhibits the expression of the high-mobility group AT-hook 1 (HMGA1) protein, a validated target of miR-214-3p and a major regulator of the transcriptional machinery. The decrease in HMGA1 expression reduced the growth and the migration of EWS cells. Taken together, our results support that the miR-214-3p is constitutively repressed by both EWS-FLI1 and CD99 because it acts as an oncosuppressor limiting the dissemination of EWS cells.

Ewing sarcoma (EWS) is a common pediatric solid tumor with high metastatic potential. Due to toxic effects of treatments on reproductive functions, the cryopreservation of ovarian tissue (OT) or testicular tissue (TT) is recommended to preserve fertility. However, the risk of reintroducing residual metastatic tumor cells should be evaluated before fertility restoration. Our goal was to validate a sensitive and specific approach for EWS minimal residual disease (MRD) detection in frozen germinal tissues. Thawed OT (n = 12) and TT (n = 14) were contaminated with tumor RD-ES cells (10, 100, and 1000 cells) and EWS-FLI1 tumor-specific transcript was quantified with RT-qPCR. All contaminated samples were found to be positive, with a strong correlation between RD-ES cell numbers and EWS-FLI1 levels in OT (r = 0.93) and TT (r = 0.96) (p < 0.001). No transcript was detected in uncontaminated control samples. The invasive potential of Ewing cells was evaluated using co-culture techniques. After co-culturing, tumor cells were detected in OT/TT with histology, FISH, and RT-qPCR. In addition, four OT and four TT samples from children with metastatic EWS were tested, and no MRD was found using RT-qPCR and histology. We demonstrated the high sensitivity and specificity of RT-qPCR to detect EWS MRD in OT/TT samples. Clinical trial: NCT02400970.

Background : Ten to fourteen percent of Ewing sarcoma (ES) study participants treated nationwide with IGF-1 receptor (IGF-1R)-targeted antibodies achieved tumor regression. Despite this success, low response rates and short response durations (approximately 7-weeks) have slowed the development of this therapy. Methods: We performed a meta-analysis of five phase-1b/2 ES-oriented trials that evaluated the anticancer activity of IGF-1R antibodies +/- mTOR inhibitors (mTORi). Our meta-analysis provided a head-to-head comparison of the clinical benefits of IGF-1R antibodies vs. the IGF-1R/mTOR-targeted combination. Available pretreatment clinical samples were semi-quantitatively scored using immunohistochemistry to detect proteins in the IGF-1R/PI3K/AKT/mTOR pathway linked to clinical response. Early PET/CT imaging, obtained within the first 2 weeks (median 10 days), were examined to determine if reduced FDG avidity was predictive of progression-free survival (PFS). Results: Among 56 ES patients treated at MD Anderson Cancer Center (MDACC) with IGF-1R antibodies, our analysis revealed a significant ~two-fold improvement in PFS that favored a combination of IGF-1R/mTORi therapy (1.6 vs. 3.3-months, p = 0.042). Low pIGF-1R in the pretreatment specimens was associated with treatment response. Reduced total-lesion glycolysis more accurately predicted the IGF-1R response than other previously reported radiological biomarkers. Conclusion: Synergistic drug combinations, and newly identified proteomic or radiological biomarkers of IGF-1R response, may be incorporated into future IGF-1R-related trials to improve the response rate in ES patients.

Ewing sarcoma (EwS) is a highly malignant sarcoma of bone and soft tissue with early metastatic spread and an age peak in early puberty. The prognosis in advanced stages is still dismal, and the long-term effects of established therapies are severe. Efficacious targeted therapies are urgently needed. Our previous work has provided preliminary safety and efficacy data utilizing T cell receptor (TCR) transgenic T cells, generated by retroviral gene transfer, targeting HLA-restricted peptides on the tumor cell derived from metastatic drivers. Here, we compared T cells engineered with either CRISPR/Cas9 or retroviral gene transfer. Firstly, we confirmed the feasibility of the orthotopic replacement of the endogenous TCR by CRISPR/Cas9 with a TCR targeting our canonical metastatic driver chondromodulin-1 (CHM1). CRISPR/Cas9-engineered T cell products specifically recognized and killed HLA-A*02:01+ EwS cell lines. The efficiency of retroviral transduction was higher compared to CRISPR/Cas9 gene editing. Both engineered T cell products specifically recognized tumor cells and elicited cytotoxicity, with CRISPR/Cas9 engineered T cells providing prolonged cytotoxic activity. In conclusion, T cells engineered with CRISPR/Cas9 could be feasible for immunotherapy of EwS and may have the advantage of more prolonged cytotoxic activity, as compared to T cells engineered with retroviral gene transfer.

Immune-inhibitory barriers in the tumor microenvironment of solid cancers counteract effective T cell therapies. Based on our finding that Ewing sarcomas (EwS) respond to chimeric antigen receptor (CAR) gene-modified effector cells through upregulation of human leukocyte antigen G (HLA-G), we hypothesized that nonclassical HLA molecules, HLA-G and HLA-E, contribute to immune escape of EwS. Here, we demonstrate that HLA-G isotype G1 expression on EwS cells does not directly impair cytolysis by GD2-specific CAR T cells (CART), whereas HLA-G1 on myeloid bystander cells reduces CART degranulation responses against EwS cells. HLA-E was induced in EwS cells by IFN-γ stimulation in vitro and by GD2-specific CART treatment in vivo and was detected on tumor cells or infiltrating myeloid cells in a majority of human EwS biopsies. Interaction of HLA-E-positive EwS cells with GD2-specific CART induced upregulation of HLA-E receptor NKG2A. However, HLA-E expressed by EwS tumor cells or by myeloid bystander cells both failed to reduce antitumor effector functions of CART. We conclude that non-classical HLA molecules are expressed in EwS under inflammatory conditions, but have limited functional impact on antigen-specific T cells, arguing against a relevant therapeutic benefit from combining CART therapy with HLA-G or HLA-E checkpoint blockade in this cancer.