Roles of USP1 in Ewing sarcoma.

Rearrangements of the EWS gene with ETS transcription factor genes as a result of chromosomal translocation and high expression levels of CD99MIC2 characterize the Ewing family of tumors (EFT). This group of rather undifferentiated neoplasms affects bone and soft tissue in children and young adults mostly between 5 and 30 years of age (median, 15 years). This study reports a case of a CD99MIC2 positive small round cell tumor in the breast of a 60-year-old woman in whom a t(11;22)(q24;q12) chromosomal aberration was identified by cytogenetic analysis. Reverse transcriptase (RT)-polymerase chain reaction (PCR) followed by sequence analysis revealed expression of a chimera transcript in which EWS exon 10 was fused to FLI1 exon 6. Previously, this gene fusion has been reported to occur in approximately 3% of EFT. The specific gene rearrangement of EWS intron 10 was confirmed on Southern blot of genomic DNA. This study further contributes to the growing list of unusual neoplasms in adults that carry genotypic and phenotypic traits of the EFT.

Ewing sarcoma is described classically as a small, round cell tumor of bone and soft tissue in children and young adults. Ewing sarcoma most often is characterized by a fusion of the Ewing sarcoma breakpoint region 1 (EWSR1) and the Friend leukemia virus integration 1 (FLI1) genes, forming an EWSR1-FLI1 fusion transcript. We report an exceptional case of primary subcutaneous Ewing sarcoma in a 16-year-old female composed entirely of spindle cells with focal fascicular growth and exhibiting strong, diffuse immunohistochemical reactivity for S100, unlike classic Ewing sarcoma. However, reverse transcription-polymerase chain reaction (RT-PCR) analysis confirmed the presence of a rare variant of the EWSR1-FLI1 fusion transcript, featuring fusion of EWSR1 exon 10 to FLI1 exon 6. To our knowledge, the combined histologic, molecular, and clinical features have not been reported previously in Ewing sarcoma, and raise a broad differential diagnosis emphasizing the importance of molecular techniques in the diagnosis of this tumor.

Background: Ewing's sarcoma tumor is an aggressive malignancy of bone and soft tissue in children and young adults. Despite advances in modern therapy, metastasis occurs and results in high mortality. Intracellular molecules Yap, Akt, mTOR, and Erk are signaling pathway members that regulate the proliferation of tumor cells.Objective and Methods: We studied the immunohistochemical expression of these proteins in 36 tumor samples from adult and pediatric patients with Ewing's sarcoma tumors. Patients' age, sex, tumor site, tumor size, clinical stage and survival (overall and disease-free survival) were collected. Tissue microarrays slides were stained with antibodies against Yap, Akt, mTOR, and Erk proteins.Results: Tumors exhibited variable expression of Yap, Akt, mTOR, and Erk (from negative, low to high), with high levels of expression present in 31%, 53%, 77% and 0% respectively. Immunohistochemical expression of Akt was associated with worse overall and disease-free survival (p<0.05). The other biomarkers did not demonstrate any difference in survival between low versus high expression.Conclusion: Although Yap, Akt, mTOR, and Erk protein are all expressed in Ewing's sarcoma by immunohistochemistry, only Akt expression is associated with worse prognosis. Larger studies are needed to verify these results and plan targeted therapy, particularly against Akt.

Ewing's sarcoma (ES) is one of the most malignant tumors of bone and soft tissue in children and young adults. ES belongs to a group of small round cell tumors (SRCTs) that also includes neuroblastoma, rhabdomyosarcoma, and malignant lymphoma. However, ES exhibits several specific chimeric genes (EWS-FLI1, EWS-ERG, EWS-ETV1, EWS-E1AF, and EWS-FEV) caused by chromosomal translocations that are not shared by other SRCTs. These chimeric genes regulate the expression of various other genes; that is, they activate inhibitors of DNA binding 2 (Id2) gene expression or they suppress transforming growth factor beta II (TbetaRII) receptor gene expression. The regulation of these chimeric genes may affect critical cell signal transductions, such as signals involved in cell cycle and apoptosis in ES tumor cells. Using an antisense oligodeoxynucleotide against a sequence containing the ATG initiation codon of the EWS-FLI1 chimeric gene that specifically reacts with the EWS-FLI1 and EWS-ERG chimeric genes, we were able to regulate the cell cycle through the down-regulation of Id2. Here, we report that treatment with an antisense oligodeoxynucleotide against this chimeric gene was very useful for inducing the regression of ES tumor growth; thus, this chimeric gene may be an important target for the treatment of ES patients.

BackgroundTyrosine kinase inhibitors (TKIs) have gained much attention in recent years as targeted agents for the treatment of a wide range of human cancers. We have investigated the effect of the TKIs gefitinib and vandetanib on tumor cell lines derived from Ewing sarcoma, a highly malignant tumor affecting bone and soft tissue in children and young adults. Gefitinib is an inhibitor of epidermal growth factor receptor tyrosine kinase activity (EGFR) and vandetanib selectively targets vascular endothelial growth factor receptor-2 (VEGFR-2) with additional activity against VEGFR-3, EGFR and RET kinase receptors.ResultsTwo Ewing sarcoma cell lines investigated showed high levels of nuclear EGFR expression as well as moderate expression in plasma membrane and cytoplasm. When treated with concentrations of 5 μM and more of either gefitinib or vandetanib, we observed a significant decrease in cell proliferation. However, there were no detectable changes in p44/42 MAPK and Akt-1 phosphorylation, or in the expression of cyclin D1 or c-Myc following gefitinib or vandetanib treatment.ConclusionWe conclude that Ewing sarcoma tumor cell proliferation is not highly sensitive to inhibition of EGFR signaling alone or the simultaneous inhibition of VEGFR receptors, EGFR and RET kinase. Decreased tumor cell proliferation could be achieved with gefitinib and vandetanib, but only at higher doses where non-specific effects of the compounds may be overriding. As Ewing tumor cells do not seem to depend on EGFR and VEGFR pathways for survival, other key factors in the cellular signaling of Ewing sarcoma should be targeted in order to obtain a potent therapeutic response.

BackgroundEwing sarcoma is a malignant tumor arising in bone and soft tissue in children and young adults. Intracranial Ewing’s Sarcoma may be found as a result of metastasis from extracranial sites. Primary Ewing’s Sarcoma affecting the central nervous system (CNS) is rare, which is commonly intraparenchymal located either supratentorial or in the spinal cord. An extra-axial /Extradural presentation of ES is extremely rare.Case presentation20-year-old female with intracranial Ewing’s sarcoma (ES) mimicking Right middle 1/3rd Parasagittal meningioma came with headache, Hemiparesis and urinary incontinence. The patient underwent craniotomy and Gross total resection of tumor. The postoperative imaging showed complete excision and underwent chemo-radiotherapy. This case highlights the diagnostic challenge that extra-axial/Dural Ewing’s sarcoma can pose for neurosurgeons as early diagnosis can prevent distant metastases.ConclusionPrimary intracranial ES is a CNS primitive neuroectodermal tumor (PNET). Primary ES tumors can mimic intracranial meningiomas. Neurological symptoms are caused by large tumors with intracranial extension. Wide local excision is the treatment of choice. Chemotherapy has been shown to improve long-term survival rates and lower recurrence rates. Radiotherapy can be given as adjunct to improve survival rates.

Ewing sarcoma belongs to the family of pediatric tumors which arise most commonly in bone. The majority of Ewing sarcoma is characterized by a balanced translocation between chromosomes 11 and 22 which encodes for the uniquely expressed fusion protein EWS/FLI1. Tumor cells are crucially dependent on expression of the fusion protein. Protein degradation is an important and highly regulated process in all cells and novel insights are beginning to be applied for cancer therapy. We aim to investigate the mechanism of turnover with the goal to diminish EWS/FLI1 protein and thereby identify novel targets for Ewing sarcoma treatment. Here, we show first that EWS/FLI1 protein stabilizes upon proteasome inhibition. Co-immunoprecipitation of EWS/FLI1 with ubiquitin shows a characteristic ubiquitination smear indicating that EWS/FLI1 is ubiquitinated and a substrate of the ubiquitin-proteasome system. We characterized the turnover itself as rather slow. Interestingly, the half-life time of the fusion protein is increased by four-fold compared to the full length protein FLI1. This increased stability possibly contributes to the oncogenic and resistant features of the fusion protein. To identify a deubiquitinating enzyme capable of regulating EWS/FLI1 turnover, we selected 20 deubiquitinating enzymes based on their expression in Ewing cell lines and tumors in-silico. Then, we established a screening approach directly measuring EWS/FLI1 protein levels as a read-out by an ELISA-like assay, allowing us to monitor the amount of 3x flag-tagged EWS/FLI1 protein. After transient knocked down of the 20 candidates, the screening revealed ubiquitin specific protease 19 (USP19) as a promising candidate. Depletion of USP19 via siRNA or shRNA was capable to diminish endogenous EWS/FLI1 protein level which led to modulation of its target gene levels. Vice versa, USP19 overexpression stabilized EWS/FLI1 protein level by twofold. Most importantly, stable knockdown of USP19 in Ewing cell lines led to a 40% reduction of cell growth and proliferation. Currently, we further characterize the influence of USP19 on EWS/FLI1 turnover and Ewing cell viability in vivo to identify novel possibilities for therapeutic interventions. Citation Format: Maria E. Gierisch, Laura A. Lopez-Garcia, Franziska Pfistner, Felix K. Niggli, Beat W. Schaefer. Identification of deubiquitinating enzyme USP19 as a regulator of EWS/FLI1 protein turnover in Ewing sarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4966. doi:10.1158/1538-7445.AM2015-4966

Introduction: Ewing sarcoma (ES) is an aggressive primitive cancer of the bone and soft tissue in adolescents and young adults, typified by chromosomal translocations resulting in fusions of the EWS gene and an ETS family gene, most commonly FLI1. The resulting EWS-FLI1 fusion protein is the oncogenic driver in this cancer, but its expression alone does not induce malignant transformation in human mesenchymal stem cells (hMSCs), the putative cell of origin. Instead, EWS-FLI1 induces apoptosis in most recipient cell line and animal models. The lncRNA HOTAIR is upregulated in multiple types of cancer and promote tumorigenesis. However, its expression and function have not been studied in Ewing sarcoma. Methods: We used the R2 (http://r2.amc.nl) and Oncogenomics (https://pob.abcc.ncifcrf.gov/cgi-bin/JK) platforms to examine the expression of HOTAIR in Ewing sarcomas as compared to other tumors and normal tissues, as assessed in high-throughput datasets. We directly measured HOTAIR expression by RT-qPCR on total RNA from ES cell lines (n=13), primary marrow-derived hMSCs (n=2), and primary ES tumors (n=22). We repressed HOTAIR expression in cell lines by shRNA and antisense locked nucleic acid GAPmers (Exiqon), and we overexpressed HOTAIR by plasmid transfection into hMSCs. We then evaluated the phenotype of HOTAIR in these models. We performed RNA immunoprecipitation (RIP) on parental cell line extracts to identify interacting proteins with HOTAIR. We transfected mutant forms of HOTAIR into hMSCs and evaluated the effect of loss of interaction with partner proteins on the HOTAIR phenotype. We co-transfected EWS-FLI1 and HOTAIR into hMSCs to evaluate viability and phenotype. Results: HOTAIR was found to be most highly expressed in 3 independent datasets of primary Ewing sarcomas on the R2 platform as compared to all other primary tumors. In one dataset, HOTAIR was overexpressed in Ewing sarcomas (n=68) as compared to normal tissues (n=18, p&amp;lt;0.0001). In the Oncogenomics platform, HOTAIR was overexpressed in all ES cell lines (n=50) and primary tumors (n=72). By RT-qPCR, HOTAIR was overexpressed in 11/13 ES cell lines and 17/22 primary tumors as compared to primary hMSCs. We reduced HOTAIR expression in three ES cell lines by shRNA. We saw no effects on proliferation or chemosensitivity. However, cells with reduced HOTAIR expression had a significant reduction in anchorage-independent tumorsphere formation in soft agar, as compared to nonsilencing control cells (p≤0.01 across all three cell lines). Overexpression of HOTAIR in hMSCs this allowed anchorage independent colony formation in these cells, a phenotype not observed in the parental or vector-control cells. Treatment of the ES cell lines or HOTAIR-overexpressing hMSCs with HOTAIR-specific antisense GAPmers abrogated tumorsphere formation. We confirmed by RIP that, in ES cell lines, HOTAIR interacts with the histone-modifying proteins EZH2 at its 5' domain and LSD1 at its 3' domain, and deletion of each domain disrupts the interaction with each protein specifically. Transfection of hMSCs with HOTAIR mutants lacking either the 5' or 3' domains also failed to allow anchorage-independent tumorsphere formation. Finally, co-transfection of full-length HOTAIR with EWS-FLI1 into hMSCs allowed cell viability and tumorsphere formation, whereas expression of EWS-FLI1 alone or with mutant HOTAIR did not. Conclusions: The lncRNA HOTAIR is significantly overexpressed in Ewing sarcoma and promotes malignant transformation and tumorigenesis through mechanisms dependent on interaction with the histone-modifying proteins EZH2 and LSD1. HOTAIR may promote survival in EWS-FLI1 mediated transformation, and it may be a potential therapeutic target given its high expression in tumors and low expression in most normal tissues. Citation Format: Hasan Siddiqui, Julia Selich-Taylor, Joshua Felgenhauer, Satoru Otsuru, Edwin Horwitz, Nilay Shah. The lncRNA HOTAIR is overexpressed in Ewing sarcoma and promotes malignant transformation through interactions with histone-modifying complexes. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A48.

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

Ewing sarcoma (ES) tumors arise in bone or soft tissue of children, adolescents, and young adults. The fusion protein and transcription factor, EWS-FLI1 is over-expressed in >85% of ES tumors. High throughput screening with approximately 50,000 compounds identified Mithramycin (MIT) as an inhibitor of EWS-FLI1. A previous clinical trial to test the efficacy of MIT was not successful, presumably due to toxicity from the high dose required to inhibit EWS-FLI1. We hypothesize that the efficacy and safety of treatment can be enhanced if MIT is used along with standard chemotherapeutic agents. Accordingly, preclinical experiments were conducted to test combination treatment with chemotherapeutic agents used for the treatment (standard care) of ES. After screening several chemotherapeutic agents used in the standard treatment of ES, Etoposide (VP-16) was tested for further experiments. ES cells, CHLA10 and TC205 were treated in the presence of vehicle (Dimethyl sulfoxide), or optimized doses of MIT, VP-16 or MIT+VP-16. The effect of these treatments on cell growth inhibition was studied at 48-hour post-treatment using a luminescent (CellTiter-Glo®) cell viability assay kit. The effect of these treatments on programmed cell death was measured by evaluating the apoptotic cell population using Flow cytometry with Annexin V Apoptosis Detection Kit and the expression of a protein marker, Cleaved-Poly (ADP-ribose) polymerase (c-PARP), by Western blot analysis. Nude mice were injected subcutaneously with TC205 cells, treated with MIT, ETO or MIT+VP-16 and the tumor growth inhibition was assessed. As expected, all treatments showed a dose and time-dependent effect on ES cell growth inhibition; however, the combination treatment of MIT+VP-16 was significantly (p<0.01) more effective than individual agents. The cell growth inhibition in combination treatment was accompanied by an increase in apoptotic cells and c-PARP expression in both ES cell lines. The combination treatment significantly (p<0.01) decreased the tumor weight and volume when compared to control or the groups treated by either MIT or VP-16. The combination of MIT and VP-16 is effective when compared to individual treatments in the TC205 and CHLA10 cell lines and inhibited tumor growth in a mouse model. Among the two cell lines tested, CHLA10 cell line expresses EWS-FLI1 while mTC205 cell line does not express EWS-FLI1. These results demonstrate that the combination of MIT and VP-16 is inhibiting both cell lines, suggesting a novel mechanism of targeting the ES cells even in the absence of EWS-FLI1 expression. These results demonstrate that MIT in combination with the chemotherapeutic agent VP-16 increases therapeutic efficacy in a preclinical model for ES and demonstrates strong potential for translational application.Citation Format: Anish Ray, Umesh T. Sankpal, Lina Albeer, Abigail Hunter, Holly Lout, Kathryn Dunlap, W. Paul Bowman, Don Eslin, Riyaz M. Basha. Mithramycin enhances the efficacy of etoposide in Ewing sarcoma cells and a mouse xenograft tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2605.

Ewing sarcoma, the second most common cancer of bone and soft tissue in adolescents and young adults, is an aggressive malignancy with poor long-term outcome. Ewing sarcoma is driven primarily by the EWS/Fli-1 fusion oncoprotein, which arises from the recurrent t(11,22) translocation. Our laboratory has been interested in identifying microRNAs (miRs) with growth-modulating properties in Ewing sarcoma, manipulation of which could potentially be used for novel therapies, as shown in preclinical studies for other cancers. We have previously shown that replacement of select miRs, normally repressed by EWS/Fli1, can inhibit the growth of Ewing sarcoma cells. To identify additional, and more potent, miRs with growth-suppressive properties in EWS, we have performed a phenotype-based, secondary miniscreen in Ewing sarcoma cell lines. This screen, using transfection of miR mimics into the EWS cell lines A673 and SK-ES-1, followed by growth assays as read-out, identified miR-193b as a strong candidate growth suppressive miR in Ewing sarcoma. Subsequent cloning and stable overexpression of endogenous miR-193b verified the growth suppressive phenotype. Examination of candidate targets further identified ErbB4 as potently repressed upon miR-193b overexpression. ErbB4 has recently been shown to play a key role in Ewing sarcoma, specifically as a driver of metastasis and as a mediator of resistance to cytotoxic chemotherapy, key biological parameters that define high-risk disease. With the need for therapy expansion in pediatric EWS, miR-193b replacement, through ErbB4 inhibition, may be a way to help control high-risk disease. Citation Format: Colin Moore, Paul Jedlicka. MicroRNA-193b is growth-inhibitory in Ewing sarcoma, and represses ErbB4. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4176. doi:10.1158/1538-7445.AM2013-4176

Sarcomas constitute a large group of rare, heterogeneous cancers of mesenchymal lineage arising in the bone and soft tissues of children and young adults. Each sarcoma subtype is characterized by distinct clinical, histological, and genetic characteristics. The heterogeneity of these sarcoma subtypes is a contributing factor to poor survival outcomes observed in patients. Despite their diversity, many subtypes harbor a balanced chromosomal translocation fusing the Ewing Sarcoma RNA binding protein 1 (EWSR1) gene with a gene encoding a transcription factor (TF). Balanced translocations generate two distinct fusions: [1] a canonical fusion consisting of the N-terminal transactivation domain of EWSR1 and the C-terminal DNA-binding domain of a TF and [2] a reciprocal fusion harboring the N-terminus of the TF and C-terminal RNA-binding domain of EWSR1. Ewing sarcoma is one sarcoma subtype that harbors canonical EWSR1-FLI1 and reciprocal FLI1-EWSR1 fusions. The oncogenic roles of EWSR1-FLI1 are well-established in that the fusion protein promotes tumorigenesis in precursor cells through the rewiring of transcriptional programs and modulation of the epigenetic landscape. The role of FLI1-EWSR1 in Ewing sarcoma tumorigenesis, however, is unclear. Here, we begin to characterize the functions of FLI1-EWSR1. We developed lentiviral vectors carrying HA-tagged canonical and FLAG-tagged reciprocal fusions. To date, we have introduced these vectors into the fusion-negative fibrosarcoma cell line HT-1080 and Ewing sarcoma cell lines A673 and TC-32. RNA-seq analyses show that FLI1-EWSR1 expression in A673 leads to significant changes in Notch, Wnt, and Hedgehog signaling pathways, as well as in epithelial-mesenchymal transition and RNA-related processes. These findings suggest roles for FLI1-EWSR1 in extracellular matrix remodeling and potential increases in metastatic potential of cells expressing the reciprocal fusion. Ongoing studies aim to evaluate the effects of the reciprocal FLI1-EWSR1 on Ewing sarcoma tumorigenesis and the epigenome, with hopes of uncovering potentially targetable vulnerabilities, paving the road for the development of novel treatments in a patient population in desperate need of effective therapies. Citation Format: Sarah Gawlak, Emily Isenhart, Joyce Ohm. Establishing roles for the FLI1-EWSR1 reciprocal fusion protein in Ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Fusion-Positive Cancer: From Discovery to Therapy; 2026 Jan 13-15; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(1_Suppl):Abstract nr B020.

Our goal is to identify novel targeted therapies for Ewing sarcoma (ES). ES is a devastating malignancy that predominantly affects children and young adults. It is the second most common cancer of the bone, yet in contrast to many other malignancies, survival rates have not improved for decades. For patients with localized disease, no biomarkers exist to predict recurrence or response to chemotherapy. For those with metastatic disease, chemotherapy is largely ineffective, and 5-year survival rates have stagnated at approximately 20%. Thus, there is an urgent need to identify biomarkers that can predict recurrence and response to therapy, as well as identify novel targeted therapies to combat this lethal disease. USP6 is the key etiologic agent in several benign bone and soft tissue tumors (BSTTs), where its chromosomal translocation results in overexpression. USP6 encodes a deubiquitylating enzyme, and our prior studies identified the tyrosine kinase Jak1 as an essential substrate of USP6 during BSTT development. Jak1 levels are dramatically elevated in USP6-overexpressing cells, leading to phosphorylation and activation of STAT transcription factors. We recently discovered that USP6 is also highly expressed in multiple sarcomas, including ES. Since Jak1-STATs play a central in mediating response to interferon (IFN), we hypothesized that USP6 might cause dysregulated IFN signaling in ES. Microarray and RNA-sequencing analysis revealed that USP6 by itself induced an IFN-response gene signature, both in immortalized, patient-derived ES cells and in primary human ES tumors. USP6+ ES cells were found to be exquisitely sensitive to exogenous IFN compared to USP6- ES cells, with both prolonged and heightened STAT1 and STAT3 activation observed. Furthermore, IFN selectively induced apoptosis of USP6+ but not USP6- ES cells. Gene expression analysis confirmed that in USP6+ ES cells, IFN synergistically induced expression of numerous IFN-stimulated genes (ISGs), including the pro-apoptotic ligand TRAIL. CRISPR-mediated depletion of TRAIL completely abrogated IFN-induced death of USP6+ ES cells. In sum, these results identify USP6 as a potential novel biomarker that may predict sensitivity of ES to targeted IFN therapy. This abstract is also being presented as Poster B11. Citation Format: Ian Henrich, Rob Young, Laura Quick, Xiaoke Wang, Andre Oliveira, Margaret Chou. Ubiquitin-specific protease 6 (USP6) oncogene confers sensitivity of Ewing sarcoma to interferon cytotoxicity [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr PR08.

Ewing's sarcoma / peripheral primitive neuroectodermal tumors occur most often in bone and soft tissues of children and young adults. The intracranial manifestation of the disease is rare, and when present, this is often misdiagnosed with other varieties of primary brain tumors. We report such a case of extraosseous Ewing's sarcoma, which was initially suspected to be a case of meningioma in an 11-year-old girl.

Ewing sarcoma (EwS) is a rare and aggressive disease affecting the bone and soft tissue of children and young adults. A FET::ETS fusion protein, typically EWSR1-FLI1 is pathognomonic for this disease. EwS is genomically quiet with widespread dysregulation driven by aberrant fusion activity leading to elevated endogenous levels of replication stress and epigenetic drift. While five-year survival rates are moderate for localized primary disease (∼70%), metastasized, relapsed, and refractory disease have much lower rates of survival (15-35%) and are associated with increased epigenetic heterogeneity. Treatment options are currently limited to systemic therapies which have not seen major advancement since the development of interval compression of chemotherapy and often lead to secondary complications for these patients. Our lab has identified a novel combination of replication stress response inhibitors (RSRi), DDK (DDKi) and WEE1 (WEE1i), which sensitize EwS cells to their own elevated levels of replication stress by removing repair strategies and forcing mitotic catastrophe and apoptosis. In one in vivo study, cell line derived xenografts in NSG mice were treated with DDKi (simurosertib) and WEE1i (adavosertib) in the following design: 1) vehicle control (n=5), 2) Irinotecan + Temozolomide (n=9), 3) Irinotecan (full dose) + DDKi + WEE1i (n=7), 4) Irinotecan (half dose) + DDKi + WEE1i (n=9), for a total of 30 samples. We have shown that this RSRi combination is able to effectively limit tumor growth over time even in the context of decreased chemotherapeutic dose. As increased replication stress can cause epigenetic drift, changes in methylation associated with treatment condition were investigated by reduced representation bisulfite sequencing. Differential methylation analysis was performed on individual CpG sites, CpG structures (islands, shores) and transcriptional elements (promoters, enhancers, transcription start sites). In this context, key pathways were found to be differentially methylated between treatment conditions, including those involved in cell signaling, motility, metabolism, and neuronal processes. Overall, this targeted therapy presents a promising therapeutic option for this pediatric patient population. Citation Format: Emily Isenhart, Ajay Gupta, Joyce Ohm. The effect of a novel therapy on DNA methylation in Ewing Sarcoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Methylation, Clonal Hematopoiesis, and Cancer; 2025 Feb 1-4; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2025;85(3 Suppl):Abstract nr B012.