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Many cancers are defined by gene fusions that frequently encode oncogenic transcription factors (TFs), such as EWSR1::FLI1 in Ewing sarcoma (EwS). Here, we report that independently to its canonical roles in transcription, EWSR1::FLI1 also functions as an mRNA decay factor, reshaping mRNA stability in EwS. This function participates in EWSR1::FLI1 tumorigenicity and involves interactions of EWSR1::FLI1 with the CCR4-NOT deadenylation complex via its EWSR1-derived low-complexity domain and with the RNA-binding protein HuR/ELAVL1 via its FLI1-derived region. Strikingly, we find that EWSR1::FLI1-mediated mRNA decay antagonizes the normal mRNA protective function of HuR and renders EwS cells highly sensitive to HuR inhibition. Our findings uncover a post-transcriptional function of EWSR1::FLI1 and suggest that targeting mRNA stability mechanisms may offer therapeutic opportunities for EwS. The EWSR1::FLI1 fusion protein is the oncogenic driver of Ewing sarcoma (EwS). Here, the authors find that EWSR1::FLI1 plays a non-canonical role in mRNA decay via interactions with the CCR4-NOT deadenylation complex and the RNA-binding protein HuR. This role uncovers a new therapeutic vulnerability of EwS to HuR inhibition.

The EWS–FLI1 fusion protein, expressed in Ewing sarcoma, increases global transcription, causes accumulation of R loops and replication stress, and impairs BRCA1-mediated repair. Ewing sarcoma's sensitivity to DNA damage Ewing sarcoma, a paediatric cancer, is characterized by the presence of cells that contain an oncogene that is a fusion between the EWSR1 gene and another gene. Ewing sarcoma cells are highly sensitive to genotoxins—substances that cause DNA damage. Alexander Bishop and colleagues show that these cells have increased replication stress and elevated levels of the three-stranded nucleic acid structures known as R-loops. The cells are also deficient in their ability to repair damaged DNA, which is likely to be due to the interaction between the BRCA1 protein and the transcriptional machinery. In normal cells, these effects are counteracted by wild-type EWSR1, which suppresses R-loop formation and facilitates repair after DNA damage. Ewing sarcoma is an aggressive paediatric cancer of the bone and soft tissue. It results from a chromosomal translocation, predominantly t(11;22)(q24:q12), that fuses the N-terminal transactivation domain of the constitutively expressed EWSR1 protein with the C-terminal DNA binding domain of the rarely expressed FLI1 protein 1 . Ewing sarcoma is highly sensitive to genotoxic agents such as etoposide, but the underlying molecular basis of this sensitivity is unclear. Here we show that Ewing sarcoma cells display alterations in regulation of damage-induced transcription, accumulation of R-loops and increased replication stress. In addition, homologous recombination is impaired in Ewing sarcoma owing to an enriched interaction between BRCA1 and the elongating transcription machinery. Finally, we uncover a role for EWSR1 in the transcriptional response to damage, suppressing R-loops and promoting homologous recombination. Our findings improve the current understanding of EWSR1 function, elucidate the mechanistic basis of the sensitivity of Ewing sarcoma to chemotherapy (including PARP1 inhibitors) and highlight a class of BRCA-deficient-like tumours.

While sarcomas account for approximately 1% of malignant tumors of adults, they are particularly more common in children and adolescents affected by cancer. In contrast to malignancies that occur in later stages of life, childhood tumors, including sarcoma, are characterized by a striking paucity of somatic mutations. However, entity-defining fusion oncogenes acting as the main oncogenic driver mutations are frequently found in pediatric bone and soft-tissue sarcomas such as Ewing sarcoma (EWSR1-FLI1), alveolar rhabdomyosarcoma (PAX3/7-FOXO1), and synovial sarcoma (SS18-SSX1/2/4). Since strong oncogene-dependency has been demonstrated in these entities, direct pharmacological targeting of these fusion oncogenes has been excessively attempted, thus far, with limited success. Despite apparent challenges, our increasing understanding of the neomorphic features of these fusion oncogenes in conjunction with rapid technological advances will likely enable the development of new strategies to therapeutically exploit these neomorphic features and to ultimately turn the “undruggable” into first-line target structures. In this review, we provide a broad overview of the current literature on targeting neomorphic features of fusion oncogenes found in Ewing sarcoma, alveolar rhabdomyosarcoma, and synovial sarcoma, and give a perspective for future developments.

Ewing sarcoma is characterized by the expression of the chimeric EWSR1-FLI1 transcription factor. Proteomic analyses indicate that the decrease of EWSR1-FLI1 expression leads to major changes in effectors of the dynamics of the actin cytoskeleton and the adhesion processes with a shift from cell-to-cell to cell-matrix adhesion. These changes are associated with a dramatic increase of in vivo cell migration and invasion potential. Importantly, EWSR1-FLI1 expression, evaluated by single-cell RT-ddPCR/immunofluorescence analyses, and activity, assessed by expression of EWSR1-FLI1 downstream targets, are heterogeneous in cell lines and in tumours and can fluctuate along time in a fully reversible process between EWSR1-FLI1 high states, characterized by highly active cell proliferation, and EWSR1-FLI1 low states where cells have a strong propensity to migrate, invade and metastasize. This new model of phenotypic plasticity proposes that the dynamic fluctuation of the expression level of a dominant oncogene is an intrinsic characteristic of its oncogenic potential.

The EWS-FLI1 fusion oncoprotein deregulates transcription to initiate the paediatric cancer Ewing sarcoma. Here we used a domain-focused CRISPR screen to implicate the transcriptional repressor ETV6 as a unique dependency in this tumour. Using biochemical assays and epigenomics, we show that ETV6 competes with EWS-FLI1 for binding to select DNA elements enriched for short GGAA repeat sequences. Upon inactivating ETV6, EWS-FLI1 overtakes and hyper-activates these cis -elements to promote mesenchymal differentiation, with SOX11 being a key downstream target. We show that squelching of ETV6 with a dominant-interfering peptide phenocopies these effects and suppresses Ewing sarcoma growth in vivo. These findings reveal targeting of ETV6 as a strategy for neutralizing the EWS-FLI1 oncoprotein by reprogramming of genomic occupancy. Independent but complementary studies from Vakoc and Stegmaier identify and characterize a role for ETV6 in counteracting the transcriptional activity of EWS-FLI1 during Ewing sarcoma development, which may be targeted for therapeutic benefits.

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.

A distinct subset of round cell sarcomas harbors capicua transcriptional repressor (CIC) rearrangement. Diagnosing these sarcomas can be difficult owing to their resemblance to Ewing sarcoma and other 'small round blue cell tumors'; molecular techniques are generally required. Recent gene expression studies of CIC-rearranged sarcomas identified the upregulation of ETV4. We assessed the sensitivity and specificity of ETV4 and WT1 immunohistochemistry for CIC-rearranged sarcoma. We evaluated whole-tissue sections from 40 CIC-rearranged sarcomas, 40 Ewing sarcomas, 4 BCOR-CCNB3 sarcomas, 6 unclassified round cell sarcomas, and 150 histologic mimics. Moderate-to-strong nuclear immunoreactivity for ETV4 in at least 50% of cells was observed in 36 (90%) CIC-rearranged sarcomas and 10 (5%) other tumors, including 5 unclassified round cell sarcomas, 2 Wilms tumors, and 1 each desmoplastic small round cell tumor, melanoma, and small cell carcinoma. Thirty-eight (95%) CIC-rearranged sarcomas showed nuclear staining for WT1, and 34 (85%) were positive for both ETV4 and WT1. Of 182 other tumors evaluated, 34 (19%) showed nuclear WT1 positivity, including all Wilms tumors and desmoplastic small round cell tumors, 5 unclassified round cell sarcomas, and a subset of lymphoblastic lymphomas, rhabdomyosarcomas, mesenchymal chondrosarcomas, carcinomas, and melanomas. In summary, diffuse moderate-to-strong ETV4 expression is present in most CIC-rearranged sarcomas and unclassified round cell sarcomas. More limited expression is seen in small subsets of various other round cell neoplasms. Nuclear WT1 expression is also present in most CIC-rearranged sarcomas and unclassified round cell sarcomas, along with Wilms tumors and desmoplastic small round cell tumors, and subsets of various histologic mimics. The sensitivity and specificity of diffuse ETV4 expression for CIC-rearranged sarcomas are 90% and 95%, respectively, whereas the sensitivity and specificity of WT1 are 95% and 81%, respectively. Diffuse ETV4 along with at least focal WT1 expression is helpful to distinguish CIC-rearranged sarcoma from Ewing sarcoma and other histologic mimics.

Ewing sarcoma is a pediatric bone and soft tissue tumor treated with chemotherapy, radiation, and surgery. Despite intensive multimodality therapy, ~50% patients eventually relapse and die of the disease due to chemoresistance. Here, using phospho-profiling, we find Ewing sarcoma cells treated with chemotherapeutic agents activate TAM (TYRO3, AXL, MERTK) kinases to augment Akt and ERK signaling facilitating chemoresistance. Mechanistically, chemotherapy-induced JAK1-SQ phosphorylation releases JAK1 pseudokinase domain inhibition allowing for JAK1 activation. This alternative JAK1 activation mechanism leads to STAT6 nuclear translocation triggering transcription and secretion of the TAM kinase ligand GAS6 with autocrine/paracrine consequences. Importantly, pharmacological inhibition of either JAK1 by filgotinib or TAM kinases by UNC2025 sensitizes Ewing sarcoma to chemotherapy in vitro and in vivo. Excitingly, the TAM kinase inhibitor MRX-2843 currently in human clinical trials to treat AML and advanced solid tumors, enhances chemotherapy efficacy to further suppress Ewing sarcoma tumor growth in vivo. Our findings reveal an Ewing sarcoma chemoresistance mechanism with an immediate translational value. Due to limited therapeutic options, patients with Ewing sarcoma typically receive intensive chemotherapy which limits quality of life and often resistance develops. Here, the authors identify a chemotherapy-induced JAK-STAT-GAS6-TAM kinase signaling cascade in Ewing sarcoma and therapeutically target this axis with TAM kinase inhibition.

Standard therapy of osteosarcoma (OS) and Ewing sarcoma (EW) rests on cytotoxic regimes, which are largely unsuccessful in advanced patients. Preclinical models are needed to break this impasse. A panel of patient-derived xenografts (PDX) was established by implantation of fresh, surgically resected osteosarcoma (OS) and Ewing sarcoma (EW) in NSG mice. Engraftment was obtained in 22 of 61 OS (36%) and 7 of 29 EW (24%). The success rate in establishing primary cell cultures from OS was lower than the percentage of PDX engraftment in mice, whereas the reverse was observed for EW; the implementation of both in vivo and in vitro seeding increased the proportion of patients yielding at least one workable model. The establishment of in vitro cultures from PDX was highly efficient in both tumor types, reaching 100% for EW. Morphological and immunohistochemical (SATB2, P-glycoprotein 1, CD99, caveolin 1) studies and gene expression profiling showed a remarkable similarity between patient’s tumor and PDX, which was maintained over several passages in mice, whereas cell cultures displayed a lower correlation with human samples. Genes differentially expressed between OS original tumor and PDX mostly belonged to leuykocyte-specific pathways, as human infiltrate is gradually replaced by murine leukocytes during growth in mice. In EW, which contained scant infiltrates, no gene was differentially expressed between the original tumor and the PDX. A novel therapeutic combination of anti-CD99 diabody C7 and irinotecan was tested against two EW PDX; both drugs inhibited PDX growth, the addition of anti-CD99 was beneficial when chemotherapy alone was less effective. The panel of OS and EW PDX faithfully mirrored morphologic and genetic features of bone sarcomas, representing reliable models to test therapeutic approaches.

The lack of circulating biomarkers for tumor monitoring is a major problem in Ewing sarcoma management. The development of methods for accurate tumor monitoring is required, considering the high recurrence rate of drug‐resistant Ewing sarcoma. Here, we describe a sensitive analytical technique for tumor monitoring of Ewing sarcoma by detecting circulating extracellular vesicles secreted from Ewing sarcoma cells. Proteomic analysis of Ewing sarcoma cell‐derived extracellular vesicles identified 564 proteins prominently observed in extracellular vesicles from three Ewing sarcoma cell lines. Among these, CD99, SLC1A5, and ENO‐1 were identified on extracellular vesicles purified from sera of patients with Ewing sarcoma before treatment but not on extracellular vesicles from those after treatment and healthy individuals. Notably, not only Ewing sarcoma‐derived extracellular vesicles but also Ewing sarcoma cells demonstrated proteomic expression of CD99 and ENO‐1 on their surface membranes. ENO‐1+CD63+ extracellular vesicle detection was reduced after tumor resection while both CD99+CD63+ and ENO‐1+CD63+ extracellular vesicles were detected in serum from Ewing sarcoma‐bearing mice. Finally, the accuracy of liquid biopsy targeting these candidates was assessed using extracellular vesicles from the sera of patients with Ewing sarcoma. Elevated ENO‐1+CD81+ extracellular vesicles in the serum of patients before treatments distinguished patients with Ewing sarcoma from healthy individuals with an area under the curve value of 0.92 (P < 0.001) and reflected the tumor burden in patients with Ewing sarcoma during multidisciplinary treatments. Collectively, circulating ENO‐1+CD81+ extracellular vesicle detection could represent a novel tool for tumor monitoring of Ewing sarcoma. Proteomic analysis of Ewing sarcoma cell‐derived extracellular vesicles identified 564 proteins prominently observed in extracellular vesicles from three Ewing sarcoma cell lines. Among these, elevated ENO‐1+ CD81+ extracellular vesicles in the serum of patients before treatments distinguished patients with Ewing sarcoma from healthy individuals and reflected the tumor burden in patients with Ewing sarcoma during multidisciplinary treatments. Circulating ENO‐1+ CD81+ extracellular vesicle detection could represent a new tool for tumor monitoring of Ewing sarcoma.