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Ewing sarcoma (ES) is an uncommon mesenchymal neoplasm that typically develops as a bone mass, although up to 30% arise in extraskeletal sites. ES of the gastrointestinal (GI) and hepatobiliary tract is rare and may be misdiagnosed as other, more common neoplasms that occur in these sites. However, the correct classification of extraskeletal ES is important for timely clinical management and prognostication. We reviewed our experience of ES in the GI and hepatobiliary tract in order to further highlight the clinicopathologic features of these neoplasms and document the potential for misdiagnosis in this setting. The archives and consultation files of 6 academic institutions were retrospectively queried for cases of ES occurring in the GI and hepatobiliary tract. The histologic slides and ancillary studies were reviewed and clinical data were retrieved for each case through the electronic medical records, when available. Twenty-three patients with ES in the GI and/or hepatobiliary tract were identified from 2000 to 2022. Of these, 11 were women and 12 were men with a median age of 38 years (range, 2 to 64). Tumor locations included the pancreas (n=5), liver (n=2), stomach (n=3), colorectum (n=3), and small intestine (n=5), as well as tumors involving multiple organs, pelvis and retroperitoneum (n=5). Tumor size varied between 2 cm and 18 cm. Twenty were primary and 3 were metastases. Of the 23 cases, only 17% were initially diagnosed as ES. The most common misdiagnoses involved various forms of neuroendocrine neoplasia due to expression of synaptophysin and other neuroendocrine markers (22%). A wide variety of diagnoses including GI stromal tumor was considered due to aberrant CD117 expression (4%). The diagnosis of ES was ultimately confirmed by detection of the EWSR1 rearrangement in 22 cases. The remaining case was diagnosed using traditional immunohistochemistry. Follow-up information was available in 20 cases, with follow-up time varying between 2 and 256 months. Six patients with follow-up died of disease between 6 and 60 months following initial presentation. Our data indicate ES in the GI and hepatobiliary tract is commonly misdiagnosed leading to a delay in therapy. In light of the attendant therapeutic and prognostic implications, ES should be considered in the differential diagnosis of any GI or hepatobiliary tumor with epithelioid and/or small round cell morphology.

EWS fusion oncoproteins underlie several human malignancies including Desmoplastic Small Round Cell Tumor (DSRCT), an aggressive cancer driven by EWS-WT1 fusion proteins. Here we combine chromatin occupancy and 3D profiles to identify EWS-WT1-dependent gene regulation networks and target genes. We show that EWS-WT1 is a powerful chromatin activator controlling an oncogenic gene expression program that characterizes primary tumors. Similar to wild type WT1, EWS-WT1 has two isoforms that differ in their DNA binding domain and we find that they have distinct DNA binding profiles and are both required to generate viable tumors that resemble primary DSRCT. Finally, we identify candidate EWS-WT1 target genes with potential therapeutic implications, including CCND1, whose inhibition by the clinically-approved drug Palbociclib leads to marked tumor burden decrease in DSRCT PDXs in vivo. Taken together, our studies identify gene regulation programs and therapeutic vulnerabilities in DSRCT and provide a mechanistic understanding of the complex oncogenic activity of EWS-WT1.

Aberrant expression of the E26 transformation-specific (ETS) transcription factors characterizes numerous human malignancies. Many of these proteins, including EWS:FLI1 and EWS:ERG fusions in Ewing sarcoma (EwS) and TMPRSS2:ERG in prostate cancer (PCa), drive oncogenic programs via binding to GGAA repeats. We report here that both EWS:FLI1 and ERG bind and transcriptionally activate GGAA-rich pericentromeric heterochromatin. The respective pathogen-like HSAT2 and HSAT3 RNAs, together with LINE, SINE, ERV, and other repeat transcripts, are expressed in EwS and PCa tumors, secreted in extracellular vesicles (EVs), and are highly elevated in plasma of patients with EwS with metastatic disease. High human satellite 2 and 3 (HSAT2,3) levels in EWS:FLI1- or ERG-expressing cells and tumors were associated with induction of G2/M checkpoint, mitotic spindle, and DNA damage programs. These programs were also activated in EwS EV-treated fibroblasts, coincident with accumulation of HSAT2,3 RNAs, proinflammatory responses, mitotic defects, and senescence. Mechanistically, HSAT2,3-enriched cancer EVs induced cGAS-TBK1 innate immune signaling and formation of cytosolic granules positive for double-strand RNAs, RNA-DNA, and cGAS. Hence, aberrantly expressed ETS proteins derepress pericentromeric heterochromatin, yielding pathogenic RNAs that transmit genotoxic stress and inflammation to local and distant sites. Monitoring HSAT2,3 plasma levels and preventing their dissemination may thus improve therapeutic strategies and blood-based diagnostics.

'Intracranial mesenchymal tumor, FET-CREB fusion-positive' occurs primarily in children and young adults and has previously been termed intracranial angiomatoid fibrous histiocytoma (AFH) or intracranial myxoid mesenchymal tumor (IMMT). Here we performed genome-wide DNA methylation array profiling of 20 primary intracranial mesenchymal tumors with FET-CREB fusion to further study their ontology. These tumors resolved into two distinct epigenetic subgroups that were both divergent from all other analyzed intracranial neoplasms and soft tissue sarcomas, including meningioma, clear cell sarcoma of soft tissue (CCS), and AFH of extracranial soft tissue. The first subgroup (Group A, 16 tumors) clustered nearest to but independent of solitary fibrous tumor and AFH of extracranial soft tissue, whereas the second epigenetic subgroup (Group B, 4 tumors) clustered nearest to but independent of CCS and also lacked expression of melanocytic markers (HMB45, Melan A, or MITF) characteristic of CCS. Group A tumors most often occurred in adolescence or early adulthood, arose throughout the neuroaxis, and contained mostly EWSR1-ATF1 and EWSR1-CREB1 fusions. Group B tumors arose most often in early childhood, were located along the cerebral convexities or spinal cord, and demonstrated an enrichment for tumors with CREM as the fusion partner (either EWSR1-CREM or FUS-CREM). Group A tumors more often demonstrated stellate/spindle cell morphology and hemangioma-like vasculature, whereas Group B tumors more often demonstrated round cell or epithelioid/rhabdoid morphology without hemangioma-like vasculature, although robust comparison of these clinical and histologic features requires future study. Patients with Group B tumors had inferior progression-free survival relative to Group A tumors (median 4.5 vs. 49 months, p = 0.001). Together, these findings confirm that intracranial AFH-like neoplasms and IMMT represent histologic variants of a single tumor type ('intracranial mesenchymal tumor, FET-CREB fusion-positive') that is distinct from meningioma and extracranial sarcomas. Additionally, epigenomic evaluation may provide important prognostic subtyping for this unique tumor entity.

Well-preserved molecular cargo in circulating extracellular vesicles (EVs) offers an ideal material for detecting oncogenic gene alterations in cancer patients, providing a noninvasive diagnostic solution for detection of disease status and monitoring treatment response. Therefore, technologies that conveniently isolate EVs with sufficient efficiency are desperately needed. Here, a lipid labeling and click chemistry-based EV capture platform ("Click Beads"), which is ideal for EV message ribonucleic acid (mRNA) assays due to its efficient, convenient, and rapid purification of EVs, enabling downstream molecular quantification using reverse transcription digital polymerase chain reaction (RT-dPCR) is described and demonstrated. Ewing sarcoma protein (EWS) gene rearrangements and kirsten rat sarcoma viral oncogene homolog (KRAS) gene mutation status are detected and quantified using EVs isolated by Click Beads and matched with those identified in biopsy specimens from Ewing sarcoma or pancreatic cancer patients. Moreover, the quantification of gene alterations can be used for monitoring treatment responses and disease progression.

The small arginine-rich protein protamine condenses complete genomic DNA into the sperm head. Here, we applied its high RNA binding capacity for spontaneous electrostatic assembly of therapeutic nanoparticles decorated with tumour-cell-specific antibodies for efficiently targeting siRNA. Fluorescence microscopy and DLS measurements of these nanocarriers revealed the formation of a vesicular architecture that requires presence of antibody-protamine, defined excess of free SMCC-protamine, and anionic siRNA to form. Only these complex nanoparticles were efficient in the treatment of non-small-cell lung cancer (NSCLC) xenograft models, when the oncogene KRAS was targeted via EGFR-mediated delivery. To show general applicability, we used the modular platform for IGF1R-positive Ewing sarcomas. Anti-IGR1R-antibodies were integrated into an antibody-protamine nanoparticle with an siRNA specifically against the oncogenic translocation product EWS/FLI1. Using these nanoparticles, EWS/FLI1 knockdown blocked in vitro and in vivo growth of Ewing sarcoma cells. We conclude that these antibody-protamine-siRNA nanocarriers provide a novel platform technology to specifically target different cell types and yet undruggable targets in cancer therapy by RNAi.

BackgroundDesmoplastic small round cell tumor (DSRCT) is a rare, highly aggressive, translocation-associated soft-tissue sarcoma that primarily affects children, adolescents, and young adults, with a striking male predominance. It is characterized by t(11;22) generating a novel EWSR1-WT1 fusion gene. Secondary genomic alterations are rarely described.MethodsTumor tissue from 83 DSRCT patients was assayed by hybrid-capture based comprehensive genomic profiling, FoundationOne® Heme next generation sequencing analysis of 406 genes and RNA sequencing of 265 genes. Tumor mutation burden was calculated from a minimum of 1.4 Mb sequenced DNA. Microsatellite instability status was determined by a novel algorithm analyzing 114 specific loci.ResultsComprehensive genomic profiling identified several genomically-defined DSRCT subgroups. Recurrent genomic alterations were most frequently detected in FGFR4, ARID1A, TP53, MSH3, and MLL3 genes. With the exception of FGFR4, where the genomic alterations predicted activation, most of the alterations in the remaining genes predicted gene inactivation. No DSRCT were TMB or MSI high.ConclusionsIn summary, recurrent secondary somatic alterations in FGFR4, ARID1A, TP53, MSH3, and MLL3 were detected in 82% of DSRCT, which is significantly greater than previously reported. These alterations may have both prognostic and therapeutic implications.

As the second most common malignant bone tumor in children and adolescents, Ewing sarcoma is initiated and exacerbated by a chimeric oncoprotein, most commonly, EWS-FLI1. In this study, we apply epigenomic analysis to characterize the transcription dysregulation in this cancer, focusing on the investigation of super-enhancer and its associated transcriptional regulatory mechanisms. We demonstrate that super-enhancer-associated transcripts are significantly enriched in EWS-FLI1 target genes, contribute to the aberrant transcriptional network of the disease, and mediate the exceptional sensitivity of Ewing sarcoma to transcriptional inhibition. Through integrative analysis, we identify MEIS1 as a super-enhancer-driven oncogene, which co-operates with EWS-FLI1 in transcriptional regulation, and plays a key pro-survival role in Ewing sarcoma. Moreover, APCDD1, another super-enhancer-associated gene, acting as a downstream target of both MEIS1 and EWS-FLI1, is also characterized as a novel tumor-promoting factor in this malignancy. These data delineate super-enhancer-mediated transcriptional deregulation in Ewing sarcoma, and uncover numerous candidate oncogenes which can be exploited for further understanding of the molecular pathogenesis for this disease.

We present the case of a 13-year-old boy with a very unusual periventricular atypical central neurocytoma with unique molecular features treated with subtotal surgical resection and photon intensity-modulated radiotherapy. Histological features were most consistent with atypical central neurocytoma. However, next-generation sequencing analysis revealed a novel EWSR1-ATF1 gene fusion (EWSR1-ATF1) as well as a MUTYH mutation. The EWSR1-ATF1 raised the possibility of Ewing sarcoma or angiomatoid fibrous histiocytoma, however, FLI-1 immunohistochemistry was negative. MUTYH mutations have been reported in diffuse midline paediatric glioma. The role of EWSR1-ATF1 and MUTYH mutations in central nervous system tumours is not well established. We present the first case of EWSR1-ATF1 and MUTYH mutation in a rare paediatric atypical central neurocytoma. Further studies are indicated to elucidate the consequences of these gene alterations in the context of paediatric central nervous system tumours as well as to investigate the potential role for targeted therapies.

The Ewing Sarcoma protein (EWS) is a multifaceted RNA binding protein (RBP) with established roles in transcription, pre-mRNA processing and DNA damage response. By generating high quality EWS-RNA interactome, we uncovered its specific and prevalent interaction with a large subset of primary microRNAs (pri-miRNAs) in mammalian cells. Knockdown of EWS reduced, whereas overexpression enhanced, the expression of its target miRNAs. Biochemical analysis revealed that multiple elements in target pri-miRNAs, including the sequences flanking the stem-loop region, contributed to high affinity EWS binding and sequence swap experiments between target and non-target demonstrated that the flanking sequences provided the specificity for enhanced pri-miRNA processing by the Microprocessor Drosha/DGCR8. Interestingly, while repressing Drosha expression, as reported earlier, we found that EWS was able to enhance the recruitment of Drosha to chromatin. Together, these findings suggest that EWS may positively and negatively regulate miRNA biogenesis via distinct mechanisms, thus providing a new foundation to understand the function of EWS in development and disease.

Alterations in transcriptional regulators can orchestrate oncogenic gene expression programs in cancer. Here, we show that the BRG1/BRM-associated factor (BAF) chromatin remodeling complex, which is mutated in over 20% of human tumors, interacts with EWSR1, a member of a family of proteins with prion-like domains (PrLD) that are frequent partners in oncogenic fusions with transcription factors. In Ewing sarcoma, we find that the BAF complex is recruited by the EWS-FLI1 fusion protein to tumor-specific enhancers and contributes to target gene activation. This process is a neomorphic property of EWS-FLI1 compared to wild-type FLI1 and depends on tyrosine residues that are necessary for phase transitions of the EWSR1 prion-like domain. Furthermore, fusion of short fragments of EWSR1 to FLI1 is sufficient to recapitulate BAF complex retargeting and EWS-FLI1 activities. Our studies thus demonstrate that the physical properties of prion-like domains can retarget critical chromatin regulatory complexes to establish and maintain oncogenic gene expression programs.

Ewing sarcoma (ES) involves a tumor-specific chromosomal translocation that produces the EWS-FLI1 protein, which is required for the growth of ES cells both in vitro and in vivo. However, an EWS-FLI1-driven transgenic mouse model is not currently available. Here, we present data from six independent laboratories seeking an alternative approach to express EWS-FLI1 in different murine tissues. We used the Runx2, Col1a2.3, Col1a3.6, Prx1, CAG, Nse, NEFL, Dermo1, P0, Sox9 and Osterix promoters to target EWS-FLI1 or Cre expression. Additional approaches included the induction of an endogenous chromosomal translocation, in utero knock-in, and the injection of Cre-expressing adenovirus to induce EWS-FLI1 expression locally in multiple lineages. Most models resulted in embryonic lethality or developmental defects. EWS-FLI1-induced apoptosis, promoter leakiness, the lack of potential cofactors, and the difficulty of expressing EWS-FLI1 in specific sites were considered the primary reasons for the failed attempts to create a transgenic mouse model of ES.

CEBPB copy number gain in Ewing sarcoma was previously shown to be associated with worse clinical outcome compared to tumors with normal CEBPB copy number, although the mechanism was not characterized. We employed gene knockdown and rescue assays to explore the consequences of altered CEBPB gene expression in Ewing sarcoma cell lines. Knockdown of EWS-FLI1 expression led to a decrease in expression of all three C/EBPβ isoforms while re-expression of EWS-FLI1 rescued C/EBPβ expression. Overexpression of C/EBPβ-1, the largest of the three C/EBPβ isoforms, led to a significant increase in colony formation when cells were grown in soft agar compared to empty vector transduced cells. In addition, depletion of C/EBPβ decreased colony formation, and re-expression of either C/EBPβ-1 or C/EBPβ-2 rescued the phenotype. We identified the cancer stem cell marker ALDH1A1 as a target of C/EBPβ in Ewing sarcoma. Furthermore, increased expression of C/EBPβ led to resistance to chemotherapeutic agents. In summary, we have identified CEBPB as an oncogene in Ewing sarcoma. Overexpression of C/EBPβ-1 increases transformation, upregulates expression of the cancer stem cell marker ALDH1A1, and leads to chemoresistance.

The long-term overall survival of Ewing sarcoma (EWS) patients remains poor; less than 30% of patients with metastatic or recurrent disease survive despite aggressive combinations of chemotherapy, radiation and surgery. To identify new therapeutic options, we employed a multi-pronged approach using in silico predictions of drug activity via an integrated bioinformatics approach in parallel with an in vitro screen of FDA-approved drugs. Twenty-seven drugs and forty-six drugs were identified, respectively, to have anti-proliferative effects for EWS, including several classes of drugs in both screening approaches. Among these drugs, 30 were extensively validated as mono-therapeutic agents and 9 in 14 various combinations in vitro. Two drugs, auranofin, a thioredoxin reductase inhibitor, and ganetespib, an HSP90 inhibitor, were predicted to have anti-cancer activities in silico and were confirmed active across a panel of genetically diverse EWS cells. When given in combination, the survival rate in vivo was superior compared to auranofin or ganetespib alone. Importantly, extensive formulations, dose tolerance, and pharmacokinetics studies demonstrated that auranofin requires alternative delivery routes to achieve therapeutically effective levels of the gold compound. These combined screening approaches provide a rapid means to identify new treatment options for patients with a rare and often-fatal disease.

Ewing sarcoma (EWS) is an aggressive bone malignancy that mainly affects children and young adults. The mechanisms by which EWS (EWSR1) fusion genes drive the disease are not fully understood. CRM1 (XPO1) traffics proteins from the nucleus, including tumor suppressors and growth factors, and is overexpressed in many cancers. A small-molecule inhibitor of CRM1, KPT-330, has shown therapeutic promise, but has yet to be investigated in the context of EWS. In this study, we demonstrate that CRM1 is also highly expressed in EWS. shRNA-mediated or pharmacologic inhibition of CRM1 in EWS cells dramatically decreased cell growth while inducing apoptosis, cell-cycle arrest, and protein expression alterations to several cancer-related factors. Interestingly, silencing of CRM1 markedly reduced EWS-FLI1 fusion protein expression at the posttranscriptional level and upregulated the expression of the well-established EWS-FLI1 target gene, insulin-like growth factor binding protein 3 (IGFBP3), which inhibits IGF-1. Accordingly, KPT-330 treatment attenuated IGF-1-induced activation of the IGF-1R/AKT pathway. Furthermore, knockdown of IGFBP3 increased cell growth and rescued the inhibitory effects on IGF-1 signaling triggered by CRM1 inhibition. Finally, treatment of EWS cells with a combination of KPT-330 and the IGF-1R inhibitor, linsitinib, synergistically decreased cell proliferation both in vitro and in vivo Taken together, these findings provide a strong rationale for investigating the efficacy of combinatorial inhibition of CRM1 and IGF-1R for the treatment of EWS. Cancer Res; 76(9); 2687-97. ©2016 AACR.

Chromosomal translocation that results in fusion of the genes encoding RNA-binding protein EWS and transcription factor FLI1 (EWS-FLI1) is pathognomonic for Ewing sarcoma. EWS-FLI1 alters gene expression through mechanisms that are not completely understood. We performed RNA sequencing (RNAseq) analysis on primary pediatric human mesenchymal progenitor cells (pMPCs) expressing EWS-FLI1 in order to identify gene targets of this oncoprotein. We determined that long noncoding RNA-277 (Ewing sarcoma-associated transcript 1 [EWSAT1]) is upregulated by EWS-FLI1 in pMPCs. Inhibition of EWSAT1 expression diminished the ability of Ewing sarcoma cell lines to proliferate and form colonies in soft agar, whereas EWSAT1 inhibition had no effect on other cell types tested. Expression of EWS-FLI1 and EWSAT1 repressed gene expression, and a substantial fraction of targets that were repressed by EWS-FLI1 were also repressed by EWSAT1. Analysis of RNAseq data from primary human Ewing sarcoma further supported a role for EWSAT1 in mediating gene repression. We identified heterogeneous nuclear ribonucleoprotein (HNRNPK) as an RNA-binding protein that interacts with EWSAT1 and found a marked overlap in HNRNPK-repressed genes and those repressed by EWS-FLI1 and EWSAT1, suggesting that HNRNPK participates in EWSAT1-mediated gene repression. Together, our data reveal that EWSAT1 is a downstream target of EWS-FLI1 that facilitates the development of Ewing sarcoma via the repression of target genes.

Ewing sarcoma is the second-most-common bone cancer in children. Driven by an oncogenic chromosomal translocation that results in the expression of an aberrant transcription factor, EWS/FLI, the disease is typically aggressive and micrometastatic upon presentation. Silencing of EWS/FLI in patient-derived tumor cells results in the altered expression of hundreds to thousands of genes and is accompanied by dramatic morphological changes in cytoarchitecture and adhesion. Genes encoding focal adhesion, extracellular matrix, and actin regulatory proteins are dominant targets of EWS/FLI-mediated transcriptional repression. Reexpression of genes encoding just two of these proteins, zyxin and α5 integrin, is sufficient to restore cell adhesion and actin cytoskeletal integrity comparable to what is observed when the EWS/FLI oncogene expression is compromised. Using an orthotopic xenograft model, we show that EWS/FLI-induced repression of α5 integrin and zyxin expression promotes tumor progression by supporting anchorage-independent cell growth. This selective advantage is paired with a tradeoff in which metastatic lung colonization is compromised.

BackgroundThe genetics involved in Ewing sarcoma susceptibility and prognosis are poorly understood. EWS/FLI and related EWS/ETS chimeras upregulate numerous gene targets via promoter-based GGAA-microsatellite response elements. These microsatellites are highly polymorphic in humans, and preliminary evidence suggests EWS/FLI-mediated gene expression is highly dependent on the number of GGAA motifs within the microsatellite.ObjectivesHere we sought to examine the polymorphic spectrum of a GGAA-microsatellite within the NR0B1 promoter (a critical EWS/FLI target) in primary Ewing sarcoma tumors, and characterize how this polymorphism influences gene expression and clinical outcomes.ResultsA complex, bimodal pattern of EWS/FLI-mediated gene expression was observed across a wide range of GGAA motifs, with maximal expression observed in constructs containing 20-26 GGAA motifs. Relative to white European and African controls, the NR0B1 GGAA-microsatellite in tumor cells demonstrated a strong bias for haplotypes containing 21-25 GGAA motifs suggesting a relationship between microsatellite function and disease susceptibility. This selection bias was not a product of microsatellite instability in tumor samples, nor was there a correlation between NR0B1 GGAA-microsatellite polymorphisms and survival outcomes.ConclusionsThese data suggest that GGAA-microsatellite polymorphisms observed in human populations modulate EWS/FLI-mediated gene expression and may influence disease susceptibility in Ewing sarcoma.

Oncogenic transformation in Ewing sarcoma is caused by EWS/FLI, an aberrant transcription factor fusion oncogene. Glioma-associated oncogene homolog 1 (GLI1) is a critical target gene activated by EWS/FLI, but the mechanism by which GLI1 contributes to the transformed phenotype of Ewing sarcoma was unknown. In this work, we identify keratin 17 (KRT17) as a direct downstream target gene upregulated by GLI1. We demonstrate that KRT17 regulates cellular adhesion by activating AKT/PKB (protein kinase B) signaling. In addition, KRT17 is necessary for oncogenic transformation in Ewing sarcoma and accounts for much of the GLI1-mediated transformation function but via a mechanism independent of AKT signaling. Taken together, our data reveal previously unknown molecular functions for a cytoplasmic intermediate filament protein, KRT17, in coordinating EWS/FLI- and GLI1-mediated oncogenic transformation and cellular adhesion in Ewing sarcoma.

BackgroundDesmoplastic small round cell tumor (DSRCT) is a rare sarcoma in adolescents and young adults. The hallmark of this disease is a EWS-WT1 translocation resulting from apposition of the Ewing's sarcoma (EWS) gene with the Wilms' tumor (WT1) gene. We performed morphoproteomic profiling of DSRCT (EWS-WT1), Ewing's sarcoma (EWS-FLI1) and Wilms' tumor (WT1) to better understand the signaling pathways for selecting future targeted therapies.MethodologyThis pilot study assessed patients with DSRCT, Wilms' tumor and Ewing's sarcoma. Morphoproteomics and immunohistochemical probes were applied to detect: p-mTOR (Ser2448); p-Akt (Ser473); p-ERK1/2 (Thr202/Tyr204); p-STAT3 (Tyr 705); and cell cycle-related analytes along with their negative controls.Principal findingsIn DSRCT the PI3K/Akt/mTOR pathway is constitutively activated by p-Akt (Ser 473) expression in the nuclear compartment of the tumor cells and p-mTOR phosphorylated on Ser 2448, suggesting mTORC2 (rictor+mTOR) as the dominant form. Ewing's sarcoma had upregulated p-Akt and p-mTOR, predominantly mTORC2. In Wilm's tumor, the mTOR pathway is also activated with most tumor cells moderately expressing p-mTOR (Ser 2448) in plasmalemmal and cytoplasmic compartments. This coincides with the constitutive activation of one of the downstream effectors of the mTORC1 signaling pathway, namely p-p70S6K (Thr 389). There was constitutive activation of the Ras/Raf/ERK pathway p-ERK 1/2 (Thr202/Tyr204) expression in the Wilms tumor and metastatic Ewing's sarcoma, but not in the DSRCT.ConclusionMORPHOPROTEOMIC TUMOR ANALYSES REVEALED CONSTITUTIVE ACTIVATION OF THE MTOR PATHWAY AS EVIDENCED BY: (a) expression of phosphorylated (p)-mTOR, p-p70S6K; (b) mTORC 2 in EWS and DSRCT; (c) ERK signaling was seen in the advanced setting indicating these as resistance pathways to IGF1R related therapies. This is the first morphoproteomic study of such pathways in these rare malignancies and may have potential therapeutic implications. Further study using morphoproteomic assessments of these tumors are warranted.

A subset of patients with Ewing's sarcoma responds to anti-insulin-like growth factor-1 receptor (IGF-1R) antibodies. Mechanisms of sensitivity and resistance are unknown. We investigated whether an anti-IGF-1R antibody acts via a pathway that could also be suppressed by small interfering (si) RNA against the EWS/FLI-1 fusion protein, the hallmark of Ewing's sarcoma. The growth of two Ewing's sarcoma cell lines (TC-32 and TC-71) was inhibited by the fully human anti-IGF-1R antibody, R1507 (clonogenic and MTT assays). TC-32 and TC-71 cells express high levels of IGF-2, while RD-ES and A4573 Ewing's cell lines, which were less responsive to R1507 in our assays, express low or undetectable IGF-2, respectively. TC-71 cells also expressed high levels of IGF-1R, and R1507 decreased steady-state levels of this receptor by internalization/degradation, an effect which was associated with a decrease in p-IGF-1R, p-IRS-1, and p-Akt. EWS/FLI-1 siRNA also decreased p-Akt, due to its ability to increase IGF-BP3 levels and subsequently decrease IGF-1 and IGF-2 levels, thus inhibiting signaling through p-IGF-1R. This inhibition correlated with growth suppression and apoptosis. The attenuation of Akt activation was confirmed in TC-71 and HEK-293 (human embryonic kidney) cells by transfecting them with IGF-1R siRNA. We conclude that antibodies and siRNA to IGF-1R, as well as siRNA to EWS/FLI-1, act via intersecting IGF/IGF-1R signals that suppress a common point in this pathway, namely the phosphorylation of Akt.

Ewing sarcoma is an aggressive cancer with a defective response to DNA damage leading to an enhanced sensitivity to genotoxic agents. Mechanistically, Ewing sarcoma is driven by the fusion transcription factor EWS-FLI1, which reprograms the tumor cell epigenome. The nucleosome remodeling and deacetylase (NuRD) complex is an important regulator of chromatin function, controlling both gene expression and DNA damage repair, and has been associated with EWS-FLI1 activity. Here, a NuRD-focused CRISPR/Cas9 inactivation screen identified the helicase CHD4 as essential for Ewing sarcoma cell proliferation. CHD4 silencing induced tumor cell death by apoptosis and abolished colony formation. Although CHD4 and NuRD colocalized with EWS-FLI1 at enhancers and super-enhancers, CHD4 promoted Ewing sarcoma cell survival not by modulating EWS-FLI1 activity and its oncogenic gene expression program but by regulating chromatin structure. CHD4 depletion led to a global increase in DNA accessibility and induction of spontaneous DNA damage, resulting in an increased susceptibility to DNA-damaging agents. CHD4 loss delayed tumor growth in vivo, increased overall survival, and combination with PARP inhibition by olaparib treatment further suppressed tumor growth. Collectively, these findings highlight the NuRD subunit CHD4 as a therapeutic target in Ewing sarcoma that can potentiate the antitumor activity of genotoxic agents., Significance: CRISPR/Cas9 screening in Ewing sarcoma identifies a dependency on CHD4, which is crucial for the maintenance of chromatin architecture to suppress DNA damage and a promising therapeutic target for DNA damage repair-deficient malignancies., (©2023 American Association for Cancer Research.)

Objective: To report a rare gastroblastoma; discuss its clinical features, histopathological morphology, diagnosis, differential diagnosis, treatment, and prognosis; and so as to improve the understanding on this disease and provide reference for its diagnosis, treatment, and prognosis., Methods: The diagnosis and treatment, imaging examination, pathological, and genetic data of a 19-year-old young female patient with gastroblastoma were analyzed retrospectively, and the relevant literature was reviewed and summarized., Results: The patient was found to have a "gastrointestinal stromal tumor" for 3 days by physical examination in another hospital. Abdominal CT and MRI considered "solid pseudopapilloma of pancreas" and clinically planned to perform "radical pancreatoduodenectomy." During the operation, the tumor was observed to bulge from the posterior wall of the gastric antrum, and the root was located in the gastric antrum, so it was changed to "partial gastrectomy + Ronx-y gastrojejunal anastomosis." The postoperative pathology showed that the tumor was bi-differentiated between gastric epithelium and mesenchymal. Combined with the results of IHC and the opinions of several consultation units, the diagnosis of gastric blastoma (low-grade malignancy) was supported. However, the fracture rearrangement of GLI1 and EWSR1 genes was not detected by FISH. After 19 months of follow-up, no signs of tumor recurrence and metastasis were found., Conclusion: Combined with existing literature reports, gastroblastoma occurs in young people, equally in men and women, and tends to occur in the gastric antrum. The biological behavior of the tumor tends to be inert, and the prognosis of most cases is good. Postoperative pathology and IHC are reliable methods for the diagnosis of gastric blastoma, and surgical resection of the lesion is the preferred treatment., (© 2023. BioMed Central Ltd., part of Springer Nature.)