Therapeutic Target In Ewing Sarcoma Research Articles

Chimeric protein EWS::FLI1 drives cell proliferation in Ewing Sarcoma via aberrant expression of KCNN1/SK1 and dysregulation of calcium signaling.

Ewing sarcoma (ES) is characterized by EWS::FLI1 or EWS::ERG fusion proteins. Knowing that ion channels are involved in tumorigenesis, this work aimed to study the involvement of the KCNN1 gene, which encodes the SK1 potassium channel, in ES development. Bioinformatics analyses from databases were used to study KCNN1 expression in patients and cell lines. Molecular approaches and in vitro assays were used to study the transcriptional regulation of KCNN1 and its involvement in the regulation of ES cell proliferation. KCNN1 is overexpressed in ES patient biopsies, and its expression is inversely correlated with patient survival. EWS::FLI1, like EWS::ERG, promotes KCNN1 and SK1 expression, binding to GGAA microsatellites near the promoter of KCNN1 isoforms. KCNN1 is involved in the regulation of ES cell proliferation, with its silencing being associated with a slowing of the cell cycle, and its expression modulates membrane potential and therefore calcium flux. These results highlight that KCNN1 is a direct target of EWS::FLI1 and EWS::ERG and demonstrate that KCNN1 is involved in the regulation of intracellular calcium activity and ES cell proliferation, making it a promising therapeutic target in ES.

423 (PB411): Characterization and evaluation of the protein kinase BUB1B as therapeutic target in ewing sarcoma

423 (PB411): Characterization and evaluation of the protein kinase BUB1B as therapeutic target in ewing sarcoma

MERTK Is a Potential Therapeutic Target in Ewing Sarcoma.

Outcomes are poor in patients with advanced or relapsed Ewing sarcoma (EWS) and current treatments have significant short- and long-term side effects. New, less toxic and more effective treatments are urgently needed. MER proto-oncogene tyrosine kinase (MERTK) promotes tumor cell survival, metastasis, and resistance to cytotoxic and targeted therapies in a variety of cancers. MERTK was ubiquitously expressed in five EWS cell lines and five patient samples. Moreover, data from CRISPR-based library screens indicated that EWS cell lines are particularly dependent on MERTK. Treatment with MRX-2843, a first-in-class, MERTK-selective tyrosine kinase inhibitor currently in clinical trials, decreased the phosphorylation of MERTK and downstream signaling in a dose-dependent manner in A673 and TC106 cells and provided potent anti-tumor activity against all five EWS cell lines, with IC50 values ranging from 178 to 297 nM. Inhibition of MERTK correlated with anti-tumor activity, suggesting MERTK inhibition as a therapeutic mechanism of MRX-2843. Combined treatment with MRX-2843 and BCL-2 inhibitors venetoclax or navitoclax provided enhanced therapeutic activity compared to single agents. These data highlight MERTK as a promising therapeutic target in EWS and provide rationale for the development of MRX-2843 for the treatment of EWS, especially in combination with BCL-2 inhibitors.

Cell cycle arrest and apoptosis are early events in radiosensitization of EWS::FLI1+ Ewing sarcoma cells by Mithramycin A

Purpose The oncogenic fusion protein EWS::FLI1 is an attractive therapeutic target in Ewing sarcoma (ES). Mithramycin A (MithA) is a potent and specific inhibitor of EWS::FLI1 that can selectively radiosensitize ES cells through transcriptional inhibition of DNA double-strand break (DSB) repair. Here, we evaluate temporal changes in cell cycle progression and apoptosis in ES cells treated with MithA and/or ionizing radiation (RTx), testing the hypothesis that combining MithA with ionizing radiation would synergistically impair cell cycle progression and enhance apoptotic elimination to a greater extent than either agent alone. Materials and Methods Four EWS::FLI1 + ES cell lines TC-71, RD-ES, SK-ES-1, and A673, and one EWS::ERG cell line (CHLA-25) were exposed to 10nM MithA or vehicle and followed 24 h later by exposure to 2 Gy x-radiation or sham irradiation. Reactive oxygen species (ROS) activity was evaluated by cytometric assay, and assay of antioxidant gene expression by RT-qPCR. Cell cycle changes were evaluated by flow cytometry of nuclei stained with propidium iodide. Apoptosis was assessed by cytometric assessment of Caspase-3/7 activity and by immunoblotting of PARP-1 cleavage. Radiosensitization was evaluated by clonogenic survival assay. Proliferation (EdU) and apoptosis (TUNEL) were evaluated in SK-ES-1 xenograft tumors following pretreatment with 1 mg/kg MithA, followed 24 h later by a single 4 Gy fraction of x-radiation. Results MithA-treated cells showed reduced levels of ROS, and were associated with increased expression of antioxidant genes SOD1, SOD2, and CAT. It nonetheless induced persistent G0/G1 arrest and a progressive increase of the sub-G1 fraction, suggesting apoptotic degeneration. In vitro assays of Caspase-3/7 activity and immunoblotting of Caspase-3/7 dependent cleavage of PARP-1 indicated that apoptosis began as early as 24 h after MithA exposure, reducing clonogenic survival. Tumors from xenograft mice treated with either radiation alone, or in combination with MithA showed a significant reduction of tumor cell proliferation, while apoptosis was significantly increased in the group receiving the combination of MithA and RTx. Conclusions Taken together, our data show that the anti-proliferative and cytotoxic effects of MithA are the prominent components of radiosensitization of EWS::FLI1+ ES, rather than the result of acutely enhanced ROS levels.

Exploiting the Stemness and Chemoresistance Transcriptome of Ewing Sarcoma to Identify Candidate Therapeutic Targets and Drug-Repurposing Candidates.

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.

Abstract B019: BRCA1:NRF2 pathway as a new therapeutic target in ewing sarcoma

Abstract Ewing sarcoma (EwS) constitutes a highly aggressive pediatric tumor driven by the oncogenic fusion protein EWSR1-FLI1. A critical clinical concern is EwS metastatic spread, and identification of therapeutic targets have been limited by a general lack of mechanistic understanding. However, we do know that EWSR1-FLI1 expression in EwS displays cell-to-cell heterogeneity, and low EWSR1-FLI1 expression level been proposed to increase metastatic phenotypes. This fits with an inherent program in the cells of origin where without the interference of EWSR1-FLI1 expression, these cells would have differentiated on to a more mesodermal/mesenchymal like cells that includes a migratory program. Therefore, identify this inherent program and prevent its activation may provide the key to block EwS metastatic progression. Recently our group showed that EWSR1-FLI1 traps BRCA1 with active RNAPII, resulting in an overactivation of the RNAPII transcription complex. This prevents BRCA1 to relocate to damage, providing a mechanistic basis for the sensitivity to PARP1 inhibition. Importantly, only a small amount of BRCA1 protein is required for its DNA repair function, and PARP1 inhibition only works in the context of BRCA1 absence. With only small amounts of BRCA1 protein needed to circumvent PARP1 inhibitor efficacy it should not be surprising that PARP1 inhibitors failed in EwS clinical trials. Apart from its role in DNA repair, BRCA1 also supports NRF2 transcriptional activity. Analyzing RNAseq data from different EwS cell lines, we found that, due to BRCA1 unavailability, NRF2 pathway is downregulated in EwS. NRF2 role in response to oxidative stress has been extensively studied, and involves glutathione (GSH) recycling at the expense of intracellular glutamine/glutamate. Analyzing an immunohistochemical assay of a tissue microarray, we found that EwS tumors accumulate high level of glutamine, confirming that NRF2 pathway is partially muted in EwS. GSH is a ubiquitous molecule that fulfills xenobiotic detoxification processes, and is also key to prevent ferroptosis, an iron dependent lipid peroxide induced form of cell death. Combining the clinically approved PARP1 inhibitor Olaparib, and the ferroptosis inducer Sulfasalazine, we found a synergistic effect impairing clonogenic capacity, cell growth and survival. Unfortunately given the limited bioavailability and rapid Sulfasalazine systemic clearance, we were only able to stabilize EwS PDX-tumor growth only for the first weeks. Finally, we found that either depletion of EWSR1-FLI1 or KEAP1 (resulting in high levels of NRF2) increases EwS cell motility and that this occurs in an NRF2 dependent manner. Based on our findings we propose a model where fluctuations in EWSR1-FLI1 expression levels impacts BRCA1 dependent NRF2 activity, impacting the balance of stemness on one hand and migration on the other. Our model suggests that targeting NRF2 dependent functions may provide dual benefit first as a novel therapeutic target when EWSR1-FLI1 expression is high while impairing metastatic spread when EWSR1-FLI1 is low. Citation Format: Nicklas Bassani. BRCA1:NRF2 pathway as a new therapeutic target in ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr B019.

Membrane-type 1 matrix metalloproteinase as predictor of survival and candidate therapeutic target in Ewing sarcoma.

Ewing sarcoma (ES) is the second most common primary bone malignancy, with an urgent need for new treatments. ES is associated with high rates of progression and relapse, driven by drug-resistant cells capable of migration, self-renewal and single-cell tumorigenesis, termed cancer stem-like cells (CSCs). Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a membrane-bound proteolytic enzyme, which, via direct and indirect mechanisms, digests four of the main types of collagen. This can be hijacked in malignancy for invasion and metastasis, with high expression predicting decreased survival in multiple cancers. In this study, we have examined the hypothesis that MT1-MMP is expressed by ES cells and explored the relationship between expression and outcomes. MT1-MMP expression in ES established cell lines, primary patient-derived cultures and daughter ES-CSCs was characterised by RNA sequencing, Western blotting, immunocytochemistry and flow cytometry. Immunohistochemistry was used to detect MT1-MMP in tumour biopsies, and the relationship between expression, event-free and overall survival examined. MT1-MMP was detected at both RNA and protein levels in five of six established cell lines, all primary cultures (n = 25) and all daughter ES-CSCs (n = 7). Immunohistochemistry of treatment-naïve biopsy tissue demonstrated that high MT1-MMP expression predicted decreased event-free and overall survival (p = .017 and .036, respectively; n = 47); this was not significant in multivariate analysis. MT1-MMP is expressed by ES cells, including ES-CSCs, making it a candidate therapeutic target. The level of MT1-MMP expression at diagnosis may be considered as a prognostic biomarker if validated by retrospective analysis of a larger cohort of clinical trial samples.

Abstract B010: The NuRD subunit CHD4 is essential for ewing sarcoma cell survival as it regulates global chromatin architecture

Abstract Ewing sarcoma tumorigenesis is tightly linked to epigenetic deregulation. Indeed, the aberrant fusion transcription factor and tumor driver EWS-FLI1 produces extensive rewiring of the cancer cell epigenome. At enhancers containing canonical ETS motifs, the fusion protein represses gene expression but, when binding to GGAA repeats, EWS-FLI1 induces transcription by recruiting activating epigenetic regulators such as the acetyltransferase p300 and the BAF chromatin remodeling complex. The nucleosome remodeling and deacetylase (NuRD) complex is an ATP-dependent multi-subunit complex that modulates chromatin architecture and regulates DNA damage repair, genome stability and gene expression. In Ewing sarcoma, NuRD subunits, such as LSD1, have been suggested to interfere with EWS-FLI1 activity and prevent tumor progression. Hence, we here aimed to comprehensively study the role of NuRD in Ewing sarcoma pathogenesis. First, we performed a NuRD-centered, negative selection, CRISPR/Cas9 screen and identified the chromatin remodeling helicase CHD4 as essential for Ewing sarcoma tumor cell survival. Validation assays using two doxycycline-inducible shRNAs targeting CHD4 demonstrated that silencing of this remodeler in fact drastically reduces tumor cell proliferation and completely prevents colony formation. This cell proliferation impairment was caused by an induction of cell death by apoptosis and not by cell cycle arrest. Surprisingly, this cell death phenotype was not linked to impaired fusion protein activity. CHD4 and NuRD, despite primarily locating to enhancers and super-enhancers in Ewing sarcoma cells, did not preferentially localize to GGAA repeats, unlike EWS-FLI1. Moreover, RNA sequencing assays performed upon CHD4 silencing showed that this ATPase does not regulate the EWS-FLI1 signature. As CHD4 is a chromatin remodeler able to move nucleosomes along the DNA, we performed ATAC sequencing experiments to investigate changes in chromatin status that could explain the dependency of Ewing sarcoma cells on CHD4 for survival. We observed that CHD4 depletion from Ewing sarcoma cells causes a drastic and global chromatin relaxation which renders tumor cells prone to DNA damage and increasingly sensitive to DNA damaging agents. Interestingly, augmented sensitivity to DNA damage was not observed by silencing CHD4 in non-tumorigenic human fibroblasts. Finally, CHD4 depletion also reduced Ewing sarcoma tumor growth in vivo and prolonged mice survival. In conclusion, we demonstrate for the first time that CHD4 is crucial for Ewing sarcoma cell survival and highlight this helicase as a promising therapeutic target for Ewing sarcoma with potential for combination therapy with drugs inducing DNA damage. Importantly, this work has initiated ongoing efforts to develop first-in-class small molecules specifically targeting CHD4 which will be crucial for the future validation of this ATPase as a viable target with clinical application. Citation Format: Joana Graca Marques, Blaz Pavlovic, Quy Ai Ngo, Gloria Pedot, Michaela Roemmele, Larissa Volken, Marco Wachtel, Beat W. Schäfer. The NuRD subunit CHD4 is essential for ewing sarcoma cell survival as it regulates global chromatin architecture [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr B010.

Abstract A012: Targeting the dysregulation of transcription and splicing in Ewing sarcoma

Abstract The goals of this study are to investigate how EWS-FLI1 disrupts regulation of transcription and splicing in Ewing sarcoma, and to evaluate this dysregulation as a potential therapeutic target. Ewing Sarcoma is an aggressive pediatric bone and soft tissue cancer driven primarily by the EWS-FLI1 fusion oncogene. EWS-FLI1 acts as an oncogenic transcription factor and also interferes with normal regulation of transcription and transcription-associated RNA processing. Chemotherapy has substantially improved overall survival, but the prognosis is still very poor for those with metastatic or recurrent disease, and toxicity is a major concern in pediatric patients. Consequently, effective, less toxic, targeted treatment strategies are much needed. We performed a genome-wide RNAi screen in the Ewing sarcoma cell line TC32 and found that one of the top pathways required for cell survival was RNA processing/splicing. Previous studies have reported increased amounts of alternative splicing events in Ewing sarcoma. RNA processing, including splicing, is functionally coupled with transcription. We recently reported that Ewing sarcoma cells have high levels of phosphorylated RNA polymerase II and increased transcription activity, accumulate R-loops, and fail to regulate transcription appropriately in response to DNA damage. We proposed that EWS-FLI1 driven hyper-activation of transcription causes a targetable dependence on RNA splicing in Ewing sarcoma. Ewing sarcoma cells showed increased sensitivity to depletion of key splicing factors compared to mesenchymal stem cell controls; this was partially rescued by depleting EWS-FLI1 or by overexpressing RNASEH1, which degrades R-loops. Ewing sarcoma cells were extremely sensitive to splicing inhibition, which showed synergy with multiple chemotherapeutic agents. Splicing inhibition in Ewing sarcoma cells caused cell cycle arrest, induced apoptosis, and altered global gene expression, alternative splicing, and R-loop profiles as seen by RNA-seq and DRIP-seq. This work provides novel insight into transcription regulation and its dysregulation by EWS-FLI1. In addition, our results point to RNA splicing as a potential new therapeutic target in Ewing sarcoma, which has the potential to benefit all Ewing sarcoma patients, especially those with chemo-resistant disease. Citation Format: Liesl A. Lawrence, Henry Miller, Alexander Bishop. Targeting the dysregulation of transcription and splicing in Ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A012.

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.

Abstract 2015: The Aurora kinases are a potential therapeutic target in Ewing sarcoma

Abstract The poor survival rates and limited treatment options for patients with relapsed/metastatic Ewing sarcoma (ES) highlights the need for more personalized targeted therapeutic approaches. In this project, we have investigated the prognostic potential of Aurora kinase expression in ES patients and evaluated the activity of Aurora kinase inhibitors in ES cell lines and patient derived cultures. The prognostic value of Aurora kinase A (AURKA), Aurora kinase B (AURKB) and Aurora kinase C (AURKC) was determined through interrogation of the online GSE17618 dataset using Cox models and Kaplan Meier plots. RNA seq data was obtained from the CCRG dataset for expression analysis (Roundhill et al., 2021, Cell Oncol, 44(5), 1065-85). Viable cell number was determined using the trypan blue exclusion assay on the Vi-CELL XR. Cells were treated with increasing concentrations of inhibitor (Table 1) or DMSO vehicle control for 48h. Protein was extracted after 48h for western blotting. In ES patients, high RNA expression of AURKA and AURKB is associated with a 5- and 3-fold increased risk of death respectively (n=42). Risk of an event was increased 3- or 2-fold with high expression of AURKA or AURKB respectively (n=42). High AURKC expression was not prognostic. Expression of AURKA and AURKB was confirmed at the RNA level in ES cell lines and patient derived cultures, and at the protein level in cell lines. Aurora kinase inhibitors (Table 1) decreased viable cell number of ES cell lines and patient derived cultures. Western blotting confirmed a decrease in AURKA and MYC-C expression with increasing concentration of inhibitor. AMG900 was the most potent inhibitor in TC32 cells (Table 1) and decreased viable cell number of patient derived cultures (n=5, p<0.01). High expression of AURKA or AURKB mRNA predicts poor outcome for ES patients. Inhibitors of these kinases reduce viable cell number of both ES cell lines and patient derived cultures. We are currently investigating the mechanism of action. Table 1. Summary of the effect of Aurora kinase inhibitors in TC32 ES cells. Aurora Kinase Inhibitor Concentration Range Used (nM) Target Kinases(IC50, nM) EC50 TC32 Cells (nM) (n=3) P value (EC50 AMG900 vs EC50 another inhibitor) References AMG900 1.25-50 AurA (5), AurB (4), AurC (1) + 10 kinases 1.57 N/A Payton et al., 2010, Cancer Res, 70(23), 9846-54 MLN8237 5-300 AurA (1.2), AurB (396.5) + 22 kinases 29.48 0.0468 Manfredi et al., 2011, Clin Cancer Res, 17(24), 7614-24 CCT137690 5-300 AurA (15), AurB (25), AurC (19) + 3 kinases 97.56 0.0004 Bavetsias et al., 2010, J Med Chem, 53(14), 5213-28 LY3295668 31.25-1000 AurA (0.8) AurB (1038)AurC (98) + 2 kinases Not able to calculate N/A Gong et al., 2019, Cancer Discov, 9(2), 248-263 EC50 values were calculated by nonlinear regression (GraphPad PRISM). Statistical differences between EC50 of AMG900 and the EC50 of other inhibitors were evaluated using two-way ANOVA and Tukey’s multiple comparisons test. IC50 - half maximal inhibitory concentration, EC50 - half maximal effective concentration Citation Format: Molly McNae, Elizabeth A. Roundhill, Susan A. Burchill, Richard W. Bayliss. The Aurora kinases are a potential therapeutic target in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2015.

Abstract 3990: MERTK is a potential therapeutic target in Ewing sarcoma

Abstract Ewing sarcoma (EWS) is the second most common pediatric bone tumor, and outcomes remain poor in patients with advanced or relapsed disease. In addition, current treatments rely on multi-modal therapy that has significant short- and long-term side effects. New, less toxic and more effective treatments are urgently needed. One potential therapeutic target is the receptor tyrosine kinase MERTK. MERTK is overexpressed in numerous cancers where it promotes tumor cell survival, metastasis, and resistance to cytotoxic and targeted therapies. We demonstrated expression and phosphorylation of MERTK protein in 5 of 5 EWS family cell lines tested. Stimulation with the MERTK ligand GAS6 resulted in activation of downstream oncogenic signaling pathways, including JAK/STAT and MAPK/ERK. Moreover, publicly available data from CRISPR-based library screens suggest that EWS cell lines are particularly dependent on MERTK. Thus, therapeutic strategies targeting MERTK may be particularly effective for treatment of EWS. To explore the therapeutic impact of MERTK inhibition in various cancers, we developed MRX-2843, a first-in-class MERTK-selective tyrosine kinase inhibitor that is currently in clinical trials. Treatment with MRX-2843 decreased phosphorylation of MERTK in a dose dependent manner, with an IC50 of 123 nM (95% CI; 53-180 nM) in A673 cells, and reduced downstream STAT6 signaling. In addition, MRX-2843 had potent anti-tumor activity against all 5 EWS cell lines, leading to reduced expansion and decreased cell density in culture with IC50 values ranging from 178 - 297 nM. Of note, inhibition of MERTK phosphorylation correlated with anti-tumor activity in both the A673 and TC106 cell lines, implicating MERTK inhibition as a mechanism of MRX-2843 anti-tumor activity. Together these data validate MERTK as a promising therapeutic target in EWS and support development of MRX-2843 for treatment of EWS, with potential to directly inform and enable a clinical trial in pediatric patients and, ultimately, to improve both outcomes and quality of life for patients with this disease. Citation Format: Sherri K. Smart, Tsz Y. Yeung, Xiaodong Wang, Stephen V. Frye, H. Shelton Earp, Douglas K. Graham, Deborah DeRyckere. MERTK is a potential therapeutic target in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3990.

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.

KDM5B promotes tumorigenesis of Ewing sarcoma via FBXW7/CCNE1 axis

Ewing sarcoma (EwS) is an aggressive tumor that affects children and young adults. Patients with relapsed/refractory diseases have limited treatment options. Targeting the driver fusion oncoproteins of EwS remains a technical problem. Epigenetic mechanisms have been pointed out as key players and alternative therapeutic targets in EwS. Here, we reported that lysine demethylase 5B (KDM5B), a histone demethylase that specifically demethylates tri- and di-methylated H3 Lys-4 (H3K4), was upregulated in EwS and overexpressed KDM5B was correlated with poor outcomes of patients. KDM5B knockdown and KDM5B inhibitor AS-8351 suppressed EwS cell proliferation and induced cell cycle arrest. Bioinformatics analysis revealed that KDM5B mainly influenced the cell cycle pathways in EwS. In mechanistic studies, we found that overexpression of KDM5B resulted in increased CCNE1 protein level, but did not affect the mRNA level of CCNE1. KDM5B upregulation blocked the degradation pathway of CCNE1 by reducing the expression of FBXW7. KDM5B downregulated FBXW7 gene by demethylation of H3K4me3 at promoter region. Moreover, AS-8351 could inhibit tumor growth in nude mice models, indicating the antitumor effect of targeting KDM5B in EwS. Our study uncovered that KDM5B in EwS attenuated FBXW7 transcription and accumulated CCNE1 protein, leading to malignant proliferation of EwS. Epigenetic drug targeting KDM5B could be a potential treatment for EwS.

GPR64, Screened from Ewing Sarcoma Cells, Is a Potential Target for Antibody-Based Therapy for Various Sarcomas.

Simple SummaryNew strategies for immunotherapy have led to an increased interest in tumor-specific antigens on the cell surface in the field of oncology. Identifying markers in sarcomas is difficult because their tumor mutation burden is less than that of carcinomas. We assumed that a target protein may be acceptable as a therapeutic target, even if it is only expressed in the epididymis along with the tumor, because the epididymis has special barriers, known as the blood–epididymis barrier (BEB). We identified GPR64 as a therapeutic target for Ewing sarcoma via next-generation RNA-sequencing. GPR64 is located on the apical membranes of efferent ductules and separated from antibodies by the BEB. This study revealed, for the first time, that anti-GPR64 antibodies accumulate in various sarcomas and avoid targeting GPR64 in the epididymis in vivo. Furthermore, GPR64 is widely expressed in various sarcomas and is, therefore, a potential antibody-based therapeutic target for sarcomas.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.

RNA sequencing and functional studies of patient-derived cells reveal that neurexin-1 and regulators of this pathway are associated with poor outcomes in Ewing sarcoma

PurposeThe development of biomarkers and molecularly targeted therapies for patients with Ewing sarcoma (ES) in order to minimise morbidity and improve outcome is urgently needed. Here, we set out to isolate and characterise patient-derived ES primary cell cultures and daughter cancer stem-like cells (CSCs) to identify biomarkers of high-risk disease and candidate therapeutic targets.MethodsThirty-two patient-derived primary cultures were established from treatment-naïve tumours and primary ES-CSCs isolated from these cultures using functional methods. By RNA-sequencing we analysed the transcriptome of ES patient-derived cells (n = 24) and ES-CSCs (n = 11) to identify the most abundant and differentially expressed genes (DEGs). Expression of the top DEG(s) in ES-CSCs compared to ES cells was validated at both RNA and protein levels. The functional and prognostic potential of the most significant gene (neurexin-1) was investigated using knock-down studies and immunohistochemistry of two independent tumour cohorts.ResultsES-CSCs were isolated from all primary cell cultures, consistent with the premise that ES is a CSC driven cancer. Transcriptional profiling confirmed that these cells were of mesenchymal origin, revealed novel cell surface targets for therapy that regulate cell-extracellular matrix interactions and identified candidate drivers of progression and relapse. High expression of neurexin-1 and low levels of regulators of its activity, APBA1 and NLGN4X, were associated with poor event-free and overall survival rates. Knock-down of neurexin-1 decreased viable cell numbers and spheroid formation.ConclusionsGenes that regulate extracellular interactions, including neurexin-1, are candidate therapeutic targets in ES. High levels of neurexin-1 at diagnosis are associated with poor outcome and identify patients with localised disease that will relapse. These patients could benefit from more intensive or novel treatment modalities. The prognostic significance of neurexin-1 should be validated independently.

Exploiting Replication Stress as a Novel Therapeutic Intervention.

Ewing sarcoma is an aggressive pediatric tumor of the bone and soft tissue. The current standard of care is radiation and chemotherapy, and patients generally lack targeted therapies. One of the defining molecular features of this tumor type is the presence of significantly elevated levels of replication stress as compared with both normal cells and many other types of cancers, but the source of this stress is poorly understood. Tumors that harbor elevated levels of replication stress rely on the replication stress and DNA damage response pathways to retain viability. Understanding the source of the replication stress in Ewing sarcoma may reveal novel therapeutic targets. Ewing sarcomagenesis is complex, and in this review, we discuss the current state of our knowledge regarding elevated replication stress and the DNA damage response in Ewing sarcoma, one contributor to the disease process. We will also describe how these pathways are being successfully targeted therapeutically in other tumor types, and discuss possible novel, evidence-based therapeutic interventions in Ewing sarcoma. We hope that this consolidation will spark investigations that uncover new therapeutic targets and lead to the development of better treatment options for patients with Ewing sarcoma. IMPLICATIONS: This review uncovers new therapeutic targets in Ewing sarcoma and highlights replication stress as an exploitable vulnerability across multiple cancers.

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.

Focal adhesion kinase confers pro-migratory and antiapoptotic properties and is a potential therapeutic target in Ewing sarcoma.

Oncogenesis of Ewing sarcoma (EwS), the second most common malignant bone tumor of childhood and adolescence, is dependent on the expression of chimeric EWSR1‐ETS fusion oncogenes, most often EWSR1‐FLI1 (E/F). E/F expression leads to dysregulation of focal adhesions (FAs) enhancing the migratory capacity of EwS cells. Here, we show that, in EwS cell lines and tissue samples, focal adhesion kinase (FAK) is expressed and phosphorylated at Y397 in an E/F‐dependent way involving Ezrin. Employing different EwS cell lines as in vitro models, we found that key malignant properties of E/F are mediated via substrate‐independent autophosphorylation of FAK on Y397. This phosphorylation results in enhanced FA formation, Rho‐dependent cell migration, and impaired caspase‐3‐mediated apoptosis in vitro. Conversely, treatment with the FAK inhibitor 15 (1,2,4,5‐benzenetetraamine tetrahydrochloride (Y15) enhanced caspase‐mediated apoptosis and EwS cell migration, independent from the respective EWSR1‐ETS fusion type, mimicking an anoikis‐like phenotype and paralleling the effects of FAK siRNA knockdown. Our findings were confirmed in vivo using an avian chorioallantoic membrane model and provide a first rationale for the therapeutic use of FAK inhibitors to impair metastatic dissemination of EwS.

Abstract 4459: EWS-FLI1 orchestrates beta-catenin regulation by a mechanism involving post-translational KDM1A stabilization

Abstract Beta-catenin (CTNNB) fulfills different functions depending on its subcellular localization. At the membrane it is involved in homotypic cell adhesion. In the nucleus it acts as a transcriptional co-activator, while cytoplasmic CTNNB sequesters and stabilizes the transcriptional co-activator TAZ and prohibits its nuclear translocation. The most extensively characterized CTNNB function is that of a nuclear effector of canonical Wnt signaling, binding and activating TCF/LEF family transcription factors and leading to upregulation of master regulators of epithelial-to-mesenchymal transition. In Ewing sarcoma (EwS), the presence of the driver fusion oncogene EWS-FLI1 renders canonical Wnt signaling unresponsive to Wnts. The exact mechanism of EWS-FLI1 mediated suppression of this signaling pathway is still elusive, but may involve downregulation of the beta-catenin partner transcription factor TCF7L2. In the absence of Wnt/CTNNB/TCF signaling we still find nuclear localization of CTNNB in EwS cells, and demonstrate sensitivity of the tumor cells to the CTNNB targeting cell-permeable oxazole compound iCRT3, suggesting an essential TCF independent role of beta-catenin for EwS survival. The regulation of CTNNB stability is complex and involves phosphorylation and the activity of a multi-protein cytoplasmic destruction complex containing Axin, APC , GSK3B and other proteins. Here, we identify involvement of lysine specific demethylase KDM1A (LSD1) in EWS-FLI1 dependent nuclear CTNNB stabilization. We describe a mechanism, by which EWS-FLI1 directly activates expression of the ubiquitin specific protease USP1 leading to KDM1A post-translational stabilization and ultimately CTNNB nuclear accumulation. Consequently, knockdown of EWS-FLI1 resulted in modulation of USP1, depletion of KDM1A protein, activation of the LSD1 target and negative Wnt signaling regulator DKK1, and cytoplasmic sequestration of CTNNB. Strikingly, in presence of EWS-FLI1, genetic or pharmacologic inhibition of either USP1 or LSD1, or forced DKK1 expression, led to CTNNB degradation. As we find upregulation of TAZ upon EWS-FLI1 knockdown and loss of CTNNB upon modulation of TAZ by siRNA, we speculate that TAZ may be involved in CTNNB cytoplasmic stabilization in absence of EWS-FLI1, while LSD1 is required for nuclear stabilization in presence of the fusion oncogene. These findings indicate complex CTNNB regulation in EwS, orchestrated by EWS-FLI1, and suggest USP1, KDM1A and CTNNB as potential therapeutic targets in EwS. Citation Format: Jozef Ban, Dave N. Aryee, Anna M. Katschnig, Raphaela Schwentner, Maximilian O. Kauer, Heinrich Kovar. EWS-FLI1 orchestrates beta-catenin regulation by a mechanism involving post-translational KDM1A stabilization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4459.