Ewing Sarcoma Progression Research Articles

Unlocking epigenetics for precision treatment of Ewing's sarcoma.

Ewing's sarcoma (EWS) is a highly aggressive malignant bone tumor primarily affecting adolescents and young adults. Despite the efficacy of chemoradiotherapy in some cases, the cure rate for patients with metastatic and recurrent disease remains low. Therefore, there is an urgent need for innovative therapeutic approaches to address the challenges associated with EWS treatment. Epigenetic regulation, a crucial factor in physiological processes, plays a significant role in controlling cell proliferation, maintaining gene integrity, and regulating transcription. Recent studies highlight the importance of abnormal epigenetic regulation in the initiation and progression of EWS. A comprehensive understanding of the intricate interactions between EWS and aberrant epigenetic regulation is essential for advancing clinical drug development. This review aims to provide a comprehensive overview of both epigenetic targets implicated in EWS, integrating various therapeutic modalities to offer innovative perspectives for the clinical diagnosis and treatment of EWS.

Trk Inhibition Reduces Tumorsphere Formation and Changes Expression of Stemness Markers in SK-ES-1 Ewing Sarcoma Cells

Introduction: Ewing sarcoma (ES) is a highly aggressive type of childhood cancer characterized by a chromosomal translocation resulting in fusions between the gene encoding EWS RNA Binding Protein 1 (EWSR1) and one gene of the ETS family, most frequently FLI-1, resulting in the EWS-FLI1 aberrant transcription factor. ES tumors can contain a subpopulation of cells showing cancer stem cell (CSC) features, which express stemness markers including CD133, OCT4 (Octamer-binding transcription factor 4), and NANOG, and display capacity to form tumorspheres likely enriched in CSCs. Neurotrophin (NT) receptors of the tropomyosin receptor kinase (Trk) family (TrkA, TrkB, and TrkC) may play a role in stimulating ES progression, but their possible role in CSCs remains unknown. Objective: To verify the effect of Trks inhibition on the formation of tumorspheres as well as the gene expression of stem markers. Method: The cells were dissociated and the formation of spheres was induced with supplemented culture medium and the K252a treatment was performed. After RNA extraction, mRNA expression levels of target genes Prom1 (CD133), OCT4 (POU5F1), SOX2, and Musashi-1 (MSI1) were analyzed by qPCR. Results: The pan-Trk inhibitor K252a (100 or 500 mM) hindered tumorsphere formation in human SK-ES-1 ES cell cultures. K252a also reduced mRNA expression of Prom1 (CD133-coding gene) while enhancing expression of OCT4. No changes in mRNA levels of SOX2 or Musashi-1 were observed. Conclusion: These findings provide the first evidence suggesting that Trk activity can influence stemness in ES cells.

Abstract 3560: Understanding alternative splicing in Ewing sarcoma progression and metastasis to inform novel therapy development

Abstract Introduction: Ewing sarcoma (ES) is the second most common pediatric bone cancer, and patients with metastases have five-year survival rates of <30%. Both local and metastatic ES tumors are characterized by the EWS-FLI1 chromosomal translocation, resulting in an aberrant transcription factor that increases ES cell proliferation and metastasis. EWS-FLI1 activity and ES transformation are reliant upon increased IGF-1 signaling, so initial therapies have centered on IGF-1R inhibition. However, compensatory upregulation of insulin receptor A (IR-A), an oncogenic spliced isoform of IR, provides a viable resistance mechanism to support ES growth and progression. IR has two spliced isoforms formed by the inclusion or exclusion of exon 11 (IR-B and IR-A respectively), and IR-A is differentially expressed in cancers due to its high affinity binding to IGF-II. We hypothesize that the pathogenesis and metastatic potential of ES is driven in part by alternative splicing of IR and other key genes. Furthermore, modulation of this splicing via antisense oligonucleotides (ASOs) aided by adeno-associated viral (AAV) delivery may be a novel therapeutic avenue for ES. Results: We performed long-read mRNA sequencing to identify differentially spliced isoforms between the matched ES cell lines CHLA-9 and CHLA-10 (derived from a localized and metastatic ES tumor respectively). Our analysis revealed global differences in alternative splicing between CHLA-9 and CHLA-10 and identified gene isoforms in the insulin receptor/IGF signaling pathway (IGFBP3) that are unique to CHLA-10. Additionally, we detected both previously reported and novel isoforms for CD99, RHOA, RACK1, splicing factors (PTBP1), and eukaryotic initiation factors, among other genes. To further understand IR alternative splicing, our labs have developed an IR ASO targeting the CUG-BP1 regulatory site upstream of exon 11, and we have previously validated its efficacy in HeLa cells transfected with an IR minigene and in pediatric sarcoma cell lines. In ES cell lines, we observed significant IR-A upregulation in a panel of ES cell lines and patient-derived xenografts derived from local and metastatic tumors, highlighting the opportunity for intervention with our ASO. To facilitate delivery, we have explored the use of AAV vectors, a clinically approved option for gene therapy, and shown successful transduction of AAV-8 over a time-course in ES cells. Conclusions: Our results indicate that alternative splicing is differential between local and metastatic ES and produces isoforms unique to the metastatic setting. Additionally, the oncogenic IR-A isoform and other spliced isoforms are viable targets for ASO therapy aided by AAV delivery. By better understanding splicing in ES, we can identify targetable interactions at the transcriptional level and develop innovative therapeutics that can improve the clinical outcomes of ES patients. Citation Format: Akila S. Venkataramany, Safiya Khurshid, Anthony R. Miller, Saranga Wijeratne, Pin-Yi Wang, Timothy P. Cripe, Dawn S. Chandler. Understanding alternative splicing in Ewing sarcoma progression and metastasis to inform novel therapy development. [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 3560.

Recent advances on anti-angiogenic multi-receptor tyrosine kinase inhibitors in osteosarcoma and Ewing sarcoma.

Osteosarcoma (OS) and Ewing sarcoma (ES) are the two most common types of primary bone cancer that predominantly affect the young. Despite aggressive multimodal treatment, survival has not improved significantly over the past four decades. Clinical efficacy has historically been observed for some mono-Receptor Tyrosine Kinase (RTK) inhibitors, albeit in small subsets of OS and ES patients. Clinical efficacy in larger groups of OS or ES patients was reported recently with several newer generation multi-RTK inhibitors. All these inhibitors combine a strong anti-angiogenic (VEGFRs) component with simultaneous inhibition of other key RTKs implicated in OS and ES progression (PDGFR, FGFR, KIT and/or MET). However, despite interesting clinical data, none of these agents have obtained a registration for these indications and are thus difficult to implement in routine OS and ES patient care. It is at present also unclear which of these drugs, with largely overlapping molecular inhibition profiles, would work best for which patient or subtype, and treatment resistance almost uniformly occurs. Here, we provide a critical assessment and systemic comparison on the clinical outcomes to the six most tested drugs in this field in OS and ES to date, including pazopanib, sorafenib, regorafenib, anlotinib, lenvatinib and cabozantinib. We pay special attention to clinical response evaluations in bone sarcomas and provide drug comparisons, including drug-related toxicity, to put these drugs into context for OS and ES patients, and describe how future trials utilizing anti-angiogenic multi-RTK targeted drugs could be designed to ultimately improve response rates and decrease toxicity.

Therapeutic targeting the oncogenic driver EWSR1::FLI1 in Ewing sarcoma through inhibition of the FACT complex.

EWS/ETS fusion transcription factors, most commonly EWSR1::FLI1, drives initiation and progression of Ewing sarcoma (EwS). Even though direct targeting EWSR1::FLI1 is a formidable challenge, epigenetic/transcriptional modulators have been proved to be promising therapeutic targets for indirectly disrupting its expression and/or function. Here, we identified structure-specific recognition protein 1 (SSRP1), a subunit of the Facilitates Chromatin Transcription (FACT) complex, to be an essential tumor-dependent gene directly induced by EWSR1::FLI1 in EwS. The FACT-targeted drug CBL0137 exhibits potent therapeutic efficacy against multiple EwS preclinical models both in vitro and in vivo. Mechanistically, SSRP1 and EWSR1::FLI1 form oncogenic positive feedback loop via mutual transcriptional regulation and activation, and cooperatively promote cell cycle/DNA replication process and IGF1R-PI3K-AKT-mTOR pathway to drive EwS oncogenesis. The FACT inhibitor drug CBL0137 effectively targets the EWSR1::FLI1-FACT circuit, resulting in transcriptional disruption of EWSR1::FLI1, SSRP1 and their downstream effector oncogenic signatures. Our study illustrates a crucial role of the FACT complex in facilitating the expression and function of EWSR1::FLI1 and demonstrates FACT inhibition as a novel and effective epigenetic/transcriptional-targeted therapeutic strategy against EwS, providing preclinical support for adding EwS to CBL0137's future clinical trials.

Identification of Factors Driving Doxorubicin-Resistant Ewing Tumor Cells to Survival.

Ewing sarcoma (ES) cells exhibit extreme plasticity that contributes to the cell's survival and recurrence. Although multiple studies reveal various signaling pathways mediated by the EWSR1/FLI1 fusion, the specific transcriptional control of tumor cell resistance to doxorubicin is unknown. Understanding the molecular hubs that contribute to this behavior provides a new perspective on valuable therapeutic options against tumor cells. Single-cell RNA sequencing and LC-MS/MS-based quantitative proteomics were used. A goal of this study was to identify protein hubs that would help elucidate tumor resistance which prompted ES to relapse or metastasize. Several differentially expressed genes and proteins, including adhesion, cytoskeletal, and signaling molecules, were observed between embryonic fibroblasts and control and doxorubicin-treated tumor cell lines. While several cancer-associated genes/proteins exhibited similar expression across fibroblasts and non-treated cells, upregulation of some proteins belonging to metabolic, stress response, and growth pathway activation was uniquely observed in doxorubicin-treated sarcoma cells, respectively. The novel information on differentially expressed genes/proteins provides insights into the biology of ES cells, which could help elucidate mechanisms of their recurrence. Collectively, our results identify a novel role of cellular proteins in contributing to tumor cell resistance and escape from doxorubicin therapy and contributing to ES progression.

Polymorphic variants of IGF2BP3 and SENCR have an impact on predisposition and/or progression of Ewing sarcoma.

Ewing sarcoma (EWS), the second most common malignant bone tumor in children and adolescents, occurs abruptly without clear evidence of tumor history or progression. Previous association studies have identified some inherited variants associated with the risk of developing EWS but a common picture of the germline susceptibility to this tumor remains largely unclear. Here, we examine the association between thirty single nucleotide polymorphisms (SNPs) of the IGF2BP3, a gene that codes for an oncofetal RNA-binding protein demonstrated to be important for EWS patient's risk stratification, and five SNPs of SENCR, a long non-coding RNA shown to regulate IGF2BP3. An association between polymorphisms and EWS susceptibility was observed for three IGF2BP3 SNPs - rs112316332, rs13242065, rs12700421 - and for four SENCR SNPs - rs10893909, rs11221437, rs12420823, rs4526784 -. In addition, IGF2BP3 rs34033684 and SENCR rs10893909 variants increased the risk for female respect to male subgroup when carried together, while IGF2BP3 rs13242065 or rs76983703 variants reduced the probability of a disease later onset (> 14 years). Moreover, the absence of IGF2BP3 rs10488282 variant and the presence of rs199653 or rs35875486 variant were significantly associated with a worse survival in EWS patients with localized disease at diagnosis. Overall, our data provide the first evidence linking genetic variants of IGF2BP3 and its modulator SENCR to the risk of EWS development and to disease progression, thus supporting the concept that heritable factors can influence susceptibility to EWS and may help to predict patient prognosis.

Selected Articles from This Issue

Highlights| October 14 2022 Selected Articles from This Issue Author & Article Information Online ISSN: 1557-3265 Print ISSN: 1078-0432 ©2022 American Association for Cancer Research2022American Association for Cancer Research Clin Cancer Res (2022) 28 (20): 4355. https://doi.org/10.1158/1078-0432.CCR-28-20-HI Related Content A commentary has been published: CircIPO7 Promotes Nasopharyngeal Carcinoma Metastasis and Cisplatin Chemoresistance by Facilitating YBX1 Nuclear Localization A commentary has been published: EWS::FLI1 and HOXD13 Control Tumor Cell Plasticity in Ewing Sarcoma A commentary has been published: Combined TRIP13 and Aurora Kinase Inhibition Induces Apoptosis in Human Papillomavirus–Driven Cancers View more A commentary has been published: Inhibition of LSD1 with Bomedemstat Sensitizes Small Cell Lung Cancer to Immune Checkpoint Blockade and T-Cell Killing View less Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Tools Icon Tools Get Permissions Cite Icon Cite Search Site Article Versions Icon Versions Version of Record October 14 2022 Citation Selected Articles from This Issue. Clin Cancer Res 15 October 2022; 28 (20): 4355. https://doi.org/10.1158/1078-0432.CCR-28-20-HI Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest Search Advanced Search Metastatic progression of Ewing sarcoma requires that activity of its oncogenic driver, EWS::FLI1, be moderated. Apfelbaum and colleagues have identified HOXD13 as a tumor-specific regulator of EWS::FLI1 activity. Transcriptomic and epigenomic studies showed that EWS::FLI1 directly activates HOXD13 and that antagonism between these transcription factors determines mesenchymal cell state. In particular, cells with high HOXD13 activity selectively activated mesenchymal programs that are repressed by EWS::FLI1. These studies demonstrate the importance of cell context-dependent transcription factors as moderators of fusion gene activity and phenotypic cell state. Future studies are indicated to determine if these transcriptionally distinct cell states define metastasis-initiating cells. Human papillomavirus (HPV) causes >5% of cancers worldwide leading to significant morbidity and mortality. To address an unmet need for therapies that uniquely target HPV-driven cancers, Ghosh and colleagues tested the efficacy of 864 drugs and identified Aurora kinase inhibitors as effective in HPV-positive cancers. HPV-positive cells, which have low... You do not currently have access to this content.

Repression of enhancer RNA PHLDA1 promotes tumorigenesis and progression of Ewing sarcoma via decreasing infiltrating T-lymphocytes: A bioinformatic analysis.

Background: The molecular mechanisms of EWS-FLI-mediating target genes and downstream pathways may provide a new way in the targeted therapy of Ewing sarcoma. Meanwhile, enhancers transcript non-coding RNAs, known as enhancer RNAs (eRNAs), which may serve as potential diagnosis markers and therapeutic targets in Ewing sarcoma. Materials and methods: Differentially expressed genes (DEGs) were identified between 85 Ewing sarcoma samples downloaded from the Treehouse database and 3 normal bone samples downloaded from the Sequence Read Archive database. Included in DEGs, differentially expressed eRNAs (DEeRNAs) and target genes corresponding to DEeRNAs (DETGs), as well as the differentially expressed TFs, were annotated. Then, cell type identification by estimating relative subsets of known RNA transcripts (CIBERSORT) was used to infer portions of infiltrating immune cells in Ewing sarcoma and normal bone samples. To evaluate the prognostic value of DEeRNAs and immune function, cross validation, independent prognosis analysis, and Kaplan–Meier survival analysis were implemented using sarcoma samples from the Cancer Genome Atlas database. Next, hallmarks of cancer by gene set variation analysis (GSVA) and immune gene sets by single-sample gene set enrichment analysis (ssGSEA) were identified to be significantly associated with Ewing sarcoma. After screening by co-expression analysis, most significant DEeRNAs, DETGs and DETFs, immune cells, immune gene sets, and hallmarks of cancer were merged to construct a co-expression regulatory network to eventually identify the key DEeRNAs in tumorigenesis of Ewing sarcoma. Moreover, Connectivity Map Analysis was utilized to identify small molecules targeting Ewing sarcoma. External validation based on multidimensional online databases and scRNA-seq analysis were used to verify our key findings. Results: A six-different-dimension regulatory network was constructed based on 17 DEeRNAs, 29 DETFs, 9 DETGs, 5 immune cells, 24 immune gene sets, and 8 hallmarks of cancer. Four key DEeRNAs (CCR1, CD3D, PHLDA1, and RASD1) showed significant co-expression relationships in the network. Connectivity Map Analysis screened two candidate compounds, MS-275 and pyrvinium, that might target Ewing sarcoma. PHLDA1 (key DEeRNA) was extensively expressed in cancer stem cells of Ewing sarcoma, which might play a critical role in the tumorigenesis of Ewing sarcoma. Conclusion: PHLDA1 is a key regulator in the tumorigenesis and progression of Ewing sarcoma. PHLDA1 is directly repressed by EWS/FLI1 protein and low expression of FOSL2, resulting in the deregulation of FOX proteins and CC chemokine receptors. The decrease of infiltrating T‐lymphocytes and TNFA signaling may promote tumorigenesis and progression of Ewing sarcoma.

E2F1 promotes Warburg effect and cancer progression via upregulating ENO2 expression in Ewing sarcoma.

Altered glucose metabolism is an important characteristic of cancer cells, which is referred to as Warburg effect or aerobic glycolysis. Ewing sarcoma (EWS) is a highly malignant tumor that occurs in children and adolescents. However, the functions of aerobic glycolysis in EWS remain to be elucidated. The present study identified a transcription factor, E2F transcription factor 1 (E2F1), as a new regulator of cancer the aerobic glycolysis and progression in EWS. The present study showed that E2F1 modulated aerobic glycolysis in EWS cells by effecting glucose uptake, lactate production and ATP generation. Altered E2F1 expression increased or decreased cell viability and invasion in EWS. Mechanistically, the results demonstrated that E2F1 may promote the Warburg effect and cancer progression in EWS via upregulating enolase 2 expression. Generally, these findings indicated that E2F1 involvement in the progression of EWS and could serve as a clinical therapeutic target in EWS.

LncRNA FOXP4-AS1 Promotes Progression of Ewing Sarcoma and Is Associated With Immune Infiltrates.

Ewing sarcoma (ES) is a highly malignant primary bone tumor with poor prognosis. Studies have shown that abnormal expression of lncRNA influences the prognosis of tumor patients. Herein, we established that FOXP4-AS1 was up-regulated in ES and this correlated with poor prognosis. Further analysis illustrated that FOXP4-AS1 down-regulation repression growth, migration, along with invasion of ES. On the contrary, up-regulation of FOXP4-AS1 promoted the growth, migration, as well as invasion of ES. To explore the mechanism of FOXP4-AS1, Spearman correlation analysis was carried out to determine genes that were remarkably linked to FOXP4-AS1 expression. The potential functions and pathways involving FOXP4-AS1 were identified by GO analysis, Hallmark gene set enrichment analysis, GSEA, and GSVA. The subcellular fractionation results illustrated that FOXP4-AS1 was primarily located in the cytoplasm of ES cells. Then a ceRNA network of FOXP4-AS1 was constructed. Analysis of the ceRNA network and GSEA yielded two candidate mRNAs for FOXP4-AS1. Results of the combined survival analysis led us to speculate that FOXP4-AS1 may affect the expression of TMPO by sponging miR-298, thereby regulating the malignant phenotype of ES. Finally, we found that FOXP4-AS1 may modulates the tumor immune microenvironment in an extracellular vesicle-mediated manner. In summary, FOXP4-AS1 correlates with poor prognosis of ES. It promotes the growth, migration, as well as invasion of ES cells and may modulate the tumor immune microenvironment.

The miR-146b-5p promotes Ewing's sarcoma cells progression via suppressing the expression of BTG2.

Ewing sarcoma (ES) is a malignant tumor that occurs mostly in children. However, the underlying mechanisms of ES are still unknown. Analyzing the results of two previous miRNA array reports, we found that miR-146b-5p might be an onco-miRNA in ES progression. To test this hypothesis, we detected the expression levels of miR-146b-5p by real-time PCR and observed the effects of miR-146b-5p on the progression of ES cells by CCK8 and transwell assays. Bioinformatics and luciferase assays were used to identify the target genes of miR-146b-5p. It showed that the expression levels of miR-146b-5p were upregulated in ES cell lines compared with human mesenchymal stem cells (MSCs). Up- or downregulation of miR-146b-5p in ES cell lines could effectively promote or block the proliferation, migration, and invasion of ES cells, respectively. Furthermore, we demonstrated that BTG2 was one of the target genes and mediated the effects of miR-146b-5p in ES cells. Interestingly, we also found that miR-146b-5p was partly involved in the anticancer effects of pemetrexed in ES cells. Our study revealed that miR-146b-5p affected the progression of ES by suppressing BTG2, which might shed light on anticancer drug development and ES treatment in the future.

P21-activated kinases as viable therapeutic targets for the treatment of high-risk Ewing sarcoma.

Ewing sarcoma (ES) is the second most common bone tumor in children and young adults. Unfortunately, there have been minimal recent advancements in improving patient outcomes, especially in metastatic and recurrent diseases. In this study, we investigated the biological role of p21-activated kinases (PAKs) in ES, and the ability to therapeutically target them in high-risk disease. Via informatics analysis, we established the inverse association of PAK1 and PAK4 expression with clinical stage and outcome in ES patients. Through expression knockdown and small-molecule inhibition of PAKs, utilizing FRAX-597, KPT-9274, and PF-3758309 in multiple ES cell lines and patient-derived xenograft models, we further explored the role of PAKs in ES tumor growth and metastatic capabilities. In vitro studies in several ES cell lines indicated that diminishing PAK1 and PAK4 expression reduces tumor cell viability, migratory, and invasive properties. In vivo studies using PAK4 inhibitors, KPT-9274 and PF-3758309 demonstrated significant inhibition of primary and metastatic tumor formation, while transcriptomic analysis of PAK4-inhibitor-treated tumors identified concomitant suppression of Notch, β-catenin, and hypoxia-mediated signatures. In addition, the analysis showed enrichment of anti-tumor immune regulatory mechanisms, including interferon (IFN)-ɣ and IFN-α responses. Altogether, our molecular and pre-clinical studies are the first to establish a critical role for PAKs in ES development and progression, and consequently as viable therapeutic targets for the treatment of high-risk ES in the near future.

Abstract 6158: The relevance of endoglin and MMP14 to the metastatic potential of Ewing sarcoma cells

Abstract Background: Ewing sarcoma (ES) is a developmental bone/soft tissue neoplasia, for which mesenchymal stem cell (MSC) is the putative cell of origin. Although the outcome of ES patients bearing primary tumors has improved significantly, patients with metastatic ES still have a poor prognosis. The MSC marker endoglin/CD105 (ENG) and the ENG-shedding protease MMP14 have been, respectively, described as cell migration and invasion regulators. In our attempt to better understand the mechanism of ES progression, we are focusing on the role of ENG and MMP14 in ES cells. Methods: In vitro ENG-knockdown (ENG-KD; RM82 and SKNMC cell lines) and ENG-overexpressing (pENG; TC71 cell lines) models were generated by shRNA technology and pDisplay vectors, respectively, and confirmed by qRT-PCR, western blotting and flow cytometry. In vitro MMP14-knockout (MMP14-KO; RM82 and SKNMC) models were generated by CRISPR-Cas9 and confirmed by western blotting and Sanger sequencing. Transwell migration/invasion, adhesion, clonogenicity and proliferation assays were performed in vitro. Actin filament organization was evaluated by immunofluorescence. Extracellular vesicles (EVs) were isolated by ultracentrifugation and density gradient separation. Transcriptomic profiling was performed by Clarion S Human arrays followed by Gene Set Enrichment Analysis. HiPathia tool was used to recode the transcriptomic data into measurements of cell activity by applying mechanistic models. Reverse Phase Protein Array (RPPA) analysis was evaluated on the ENG-KD RM82 model. ENG-targeting immunoprecipitations (IP) from wild type RM82 protein extracts were analyzed by mass spectrometry (MS). Results: The impaired clonogenic potential of ENG-KD RM82 and SKNMC models suggests a role of ENG on the stemness capacity of ES, without affecting the proliferation rate. The reduced migratory and adhesive phenotype of the ENG-KD RM82 model and the transcriptomic profiling of both ENG-KD RM82 and SKNMC cells, suggest a role of ENG in ES cell migration and adhesion. The altered organization of actin filaments in ENG-KD and pENG models could support a role of ENG in actin filament remodeling. The transcriptomic profiling of ENG-KD models and the RPPA results underscore the regulation of FAK signaling by ENG. The IP-MS analysis of ENG partners identifies Integrin-Linked Kinase-Associated serine/threonine Phosphatase (ILKAP) as one of the main interactors of ENG in RM82 cells, suggesting its possible association with the focal adhesion member ILK. ENG is enriched in EVs from different ES cell lines. The transcriptomic profiling of MMP14-KO SKNMC cells highlights the involvement of MMP14 activity in the extracellular matrix organization of ES cells. Conclusion: ENG favors a pro-migratory phenotype in ES cells. These findings help to better understand the molecular landscape of tumoral cells from ES patients, and might have potential clinical implications. Citation Format: Pilar Puerto-Camacho, Ana Teresa Amaral, Juan Diaz-Martin, Alfonso Bolado-Carrancio, Carmen Jordan-Perez, Carmen Salguero-Aranda, Marina Esteban-Medina, Inmaculada Alamo-Alvarez, Joaquin Dopazo, Carmelo Bernabeu, Adam Byron, Valerie G. Brunton, Enrique de Álava. The relevance of endoglin and MMP14 to the metastatic potential of Ewing sarcoma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6158.

Abstract B74: Investigating the role of tumor:bone microenvironment crosstalk in Ewing sarcoma progression

Abstract Background: Ewing sarcoma is the second most common pediatric bone cancer. Metastatic disease is almost always fatal, and there is currently no method to predict which patients are at risk for metastasis. Better therapies are needed to prevent and treat metastatic disease, which means the mechanisms that drive Ewing sarcoma metastasis must be better elucidated. It is becoming increasingly clear that interactions between tumor cells and the tumor microenvironment (TME) play an essential role in metastasis, but the specific mechanisms through which the TME contributes to Ewing sarcoma progression remain largely unknown. Our previous work has demonstrated that activation of canonical Wnt and TGF-β pathways in a subset of Ewing sarcoma cells induces changes in gene expression and protein secretion that are associated with enhanced metastatic engraftment, changes in the extracellular matrix (ECM), and increased angiogenesis. As bone, the primary site for Ewing sarcoma, is an excellent source of ligands for both pathways, we hypothesize that crosstalk between Wnt/TGF-β-activated tumor cells and the local bone microenvironment contributes to osteolysis and metastatic progression. Methods: Ewing sarcoma cells were treated with control or Wnt3a media +/- recombinant TGF-β1, and then the mRNA levels of target genes were measured by Q-RT-PCR. ELISA and Luminex assays were also performed to determine the levels of secreted proteins. Conditioned media collected from stimulated cells were used to treat osteoblast precursor cells, which were then assayed for effects on differentiation and function. Subcutaneous, femur, and vossicle transplant xenograft models were established in mice and are being validated. Results: We have demonstrated that in response to Wnt3a and TGF-β1, Ewing sarcoma cells upregulate expression of canonical Wnt targets (e.g., LEF1), ECM-associated genes (e.g., TNC, COL1A1, and MMP2), and the pro-osteolytic factor PTHrP. We have also shown that osteoblast precursor cells can be induced to differentiate in the presence of conditioned media from Ewing sarcoma cell lines. Immunohistochemical staining is being used to evaluate Wnt and TGF-β pathway activation, angiogenesis, osteolysis, and the ECM in our bone tumor xenograft models. Conclusions: Wnt3a/TGF-β1-stimulated Ewing sarcoma cells upregulate expression of genes associated with the ECM and osteolysis. We are in the process of demonstrating the functional consequences of those changes using in vitro conditioned media experiments and in vivo transplant models. Understanding the mechanisms by which these pathways contribute to Ewing sarcoma phenotypes is important for the development of new therapeutics for patients with metastatic disease. Citation Format: Kelsey Temprine, Sydney Treichel, Allegra Hawkins, Tahra Suhan, Wei Jiang, Parker Acevedo, Amy Koh, Kurt Hankenson, Laurie K. McCauley, Elizabeth R. Lawlor. Investigating the role of tumor:bone microenvironment crosstalk in Ewing sarcoma progression [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B74.

The Role of Tyrosine Kinases as a Critical Prognostic Parameter and Its Targeted Therapies in Ewing Sarcoma.

Ewing sarcoma (ES) is a rare, highly aggressive, bone, or soft tissue-associated tumor. Although this sarcoma often responds well to initial chemotherapy, 40% of the patients develop a lethal recurrence of the disease, with death recorded in 75–80% of patients with metastatic ES within 5 years, despite receiving high-dose chemotherapy. ES is genetically well-characterized, as indicated by the EWS-FLI1 fusion protein encoded as a result of chromosomal translocation in 80–90% of patients with ES, as well as in ES-related cancer cell lines. Recently, tyrosine kinases have been identified in the pathogenesis of ES. These tyrosine kinases, acting as oncoproteins, are associated with the clinical pathogenesis, metastasis, acquisition of self-renewal traits, and chemoresistance of ES, through the activation of various intracellular signaling pathways. This review describes the recent progress related to cellular and molecular functional roles of tyrosine kinases in the progression of ES.

Wnt/β-catenin-activated Ewing sarcoma cells promote the angiogenic switch.

Wnt/β-catenin signaling is active in small subpopulations of Ewing sarcoma cells, and these cells display a more metastatic phenotype, in part due to antagonism of EWS-FLI1-dependent transcriptional activity. Importantly, these β-catenin-activated Ewing sarcoma cells also alter secretion of extracellular matrix (ECM) proteins. We thus hypothesized that, in addition to cell-autonomous mechanisms, Wnt/β-catenin-active tumor cells might contribute to disease progression by altering the tumor microenvironment (TME). Analysis of transcriptomic data from primary patient biopsies and from β-catenin-active versus -nonactive tumor cells identified angiogenic switch genes as being highly and reproducibly upregulated in the context of β-catenin activation. In addition, in silico and in vitro analyses, along with chorioallantoic membrane assays, demonstrated that β-catenin-activated Ewing cells secreted factors that promote angiogenesis. In particular, activation of canonical Wnt signaling leads Ewing sarcoma cells to upregulate expression and secretion of proangiogenic ECM proteins, collectively termed the angiomatrix. Significantly, our data show that induction of the angiomatrix by Wnt-responsive tumor cells is indirect and is mediated by TGF-β. Mechanistically, Wnt/β-catenin signaling antagonizes EWS-FLI1-dependent repression of TGF-β receptor type 2, thereby sensitizing tumor cells to TGF-β ligands. Together, these findings suggest that Wnt/β-catenin-active tumor cells can contribute to Ewing sarcoma progression by promoting angiogenesis in the local TME.

A phase I/II clinical trial of the reversible LSD1 inhibitor, seclidemstat, in patients with relapsed/refractory Ewing sarcoma.

TPS11567 Background: Ewing sarcoma (ES) is a rare, aggressive bone and soft tissue cancer that predominantly afflicts adolescents and young adults. Novel therapeutic agents are needed as there are no approved targeted treatments for this disease. ES is characterized by a chromosomal translocation resulting in an EWS/ETS fusion oncoprotein, a transcription factor that results in aberrant gene expression leading to ES progression. Lysine specific demethylase 1 (LSD1) associates with EWS/ETS oncoproteins to alter gene expression and contribute to disease progression. Directly inhibiting EWS/ETS is challenging and little progress has been made, though targeting LSD1 presents a viable therapeutic strategy for ES. Seclidemstat (SP-2577, Salarius Pharmaceuticals) is a first-in-class, orally bioavailable, small molecule with reversible and noncompetitive selective inhibition of LSD1 at low nanomolar concentrations (IC50: 25-50 nM). Seclidemstat inhibits LSD1’s scaffolding functions and enzymatic activity to help reverse aberrant gene expression. In vitro data show that treatment with seclidemstat, or seclidemstat analog, modulates EWS/ETS transcriptional activity, down-regulating oncogene expression and up-regulating tumor-suppressor gene expression. In in vivo xenograft studies (e.g., SK-N-MC, A673), mice treated with seclidemstat show significant tumor growth inhibition/regression vs the control vehicle group. Methods: This phase 1/2 clinical study of seclidemstat is being conducted in relapsed or refractory ES (NCT03600649). The trial is an open-label, non-randomized dose-escalation/dose-expansion study designed to determine the maximum tolerated dose through single-patient dose escalation followed by traditional 3+3 design. The primary objective is to assess seclidemstat’s safety and tolerability while secondary objectives include pharmacokinetics, efficacy and exploratory pharmacodynamic markers. Patients must be ≥12 years old, have received at least 1 prior line of therapy including a prior camptothecin-based regimen, with a life expectancy > 4 months. All patients receive seclidemstat twice-daily (BID) as oral tablets until unacceptable toxicity or disease progression. Patients are followed for survival until the end of study. The trial is currently recruiting across 8 locations in the United States. Upon identification of the recommended phase 2 dose, that cohort will be expanded to enroll a total of 20 patients. Clinical trial information: NCT03600649 .

The Receptor Tyrosine Kinase RON and Its Isoforms as Therapeutic Targets in Ewing Sarcoma

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.

A New Classifier Based on Laboratory Indicators for Early Diagnosis and Prognosis Prediction of Ewing's Sarcoma.

Ewing's sarcoma (ES) is a prevalent bone malignancy. It is critical to explore new diagnostic and prognostic indicators because of the rapid progression of ES and the low survival rate of metastatic ES patients. However, few parameters of clinical significance have been found. The aim of this study was to establish a new classifier with clinical laboratory data to help ES detection and prognosis prediction. A total of 135 ES patients, 150 healthy individuals, and 228 patients with primary benign bone lesions were included. Logistic regression on clinical laboratory indicators was conducted to establish the classifier, and then the classifier was assessed by drawing the receiver operating characteristic (ROC) curves. Patient survival was evaluated using the Kaplan-Meier method. We established the diagnostic classifier, called Ces, with clinical laboratory indicators to distinguish ES from healthy individuals. Ces showed great diagnostic performance in the test cohort (area under the receiver operating characteristic curve (AUC) 0.95) and could identify early-stage (AUC 0.93) and small-size (AUC 0.95) ES effectively. In addition, the classifier had good ability to differentiate ES from primary benign bone lesions (AUC 0.77 for Ces, AUC 0.83 for Ces + age). Furthermore, Ces was associated with tumor metastasis and event-free survival (EFS) of ES patients and showed better performance than lactate dehydrogenase (LDH) in prognosis prediction. Our study indicates that Ces has the potential to be a non-invasive biomarker for ES diagnosis and prognosis.