EWSR1-ATF1 dependent 3D connectivity regulates oncogenic and differentiation programs in Clear Cell Sarcoma

Möller, Emely; Praz, Viviane; Sanalkumar, Rajendran; Dong, Rui; Cauderay, Alexandra; Xing, Yu-Hang; Lee, Lukuo; Fusco, Carlo; Broye, Liliane C.; Cironi, Luisa; Iyer, Sowmya; Rengarajan, Shruthi; Awad, Mary E.; Naigles, Beverly; Letovanec, Igor; Ormas, Nicola; Finzi, Giovanna; Rosa, Stefano La; Sessa, Fausto; Chebib, Ivan; Nielsen, G. Petur; Digklia, Antonia; Spentzos, Dimitrios; Coté, Gregory M.; Choy, Edwin; Aryee, Martin J.; Stamenkovic, Ivan; Boulay, Gaylor; Rivera, Miguel N.; Riggi, Nicolò

doi:10.1038/s41467-022-29910-4

Cited by 26 publications

(33 citation statements)

References 67 publications

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“…In recent years, it has become clear that most individual gene fusions are nonspecific, being identified in neoplasms with widely different histopathologic and clinical characteristics 22. The phenotypic and biological features of tumors driven by gene fusions are likely determined by multiple factors, such as the cell of origin, the tumor microenvironment, and the presence of concurrent genomic and epigenomic alterations 23,24. This is apparently the case for neoplasms driven by EWSR1::ATF1 , a fusion that has been identified in multiple tumor types, including clear cell sarcoma of soft tissue, malignant gastrointestinal neuroectodermal tumor (also known as clear cell sarcoma-like tumor of the gastrointestinal tract), clear cell carcinoma of salivary gland, hyalinizing clear cell carcinoma of the thymus and lung, clear cell odontogenic carcinoma, myoepithelial tumors, intracranial myxoid mesenchymal tumor, primary pulmonary myxoid sarcoma, and subsets of mesothelioma, angiosarcoma, and angiomatoid fibrous histiocytoma 25–41.…”

Section: Discussionmentioning

confidence: 99%

“…In animal models, EWSR1::ATF1 is, in and of itself, sufficient to promote oncogenesis 24. The resulting fusion protein predictably consists of an N-terminal portion that contains the transactivation domain of EWSR1 and a C-terminal portion that contains the DNA-binding domain of ATF1 23,49. The latter is a conserved leucine zipper (shared by other frequent fusion partners of EWSR1 such as CREB1 and CREM ) that binds to sequences known as cAMP response element motifs 23.…”

Section: Discussionmentioning

confidence: 99%

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Inflammatory and Nested Testicular Sex Cord Tumor

Acosta

Bridge

Cin

et al. 2023

American Journal of Surgical Pathology

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A subset of malignant testicular sex cord tumors (TSCTs), heretofore interpreted as Sertoli cell tumors, not otherwise specified, exhibits distinctive morphologic features that partially overlap with those of seminoma. In this study, we evaluated the clinicopathologic and molecular characteristics of 13 such tumors. The patients were 20 to 73 years old (median, 36 y), and all with available data presented with testicular masses (median size, 3 cm), with 2 having synchronous retroperitoneal metastases. All 11 patients with available follow-up developed metastases to retroperitoneal lymph nodes, nonretroperitoneal lymph nodes, bone, contralateral testis, and/or lung. Microscopically, the tumors showed solid nests and sheets of epithelioid cells with granular, eosinophilic to clear/vacuolated cytoplasm, admixed in most (12/13) cases with variable proportions of lymphocytes, plasma cells, eosinophils, and neutrophils. Additional features included intracytoplasmic hyaline inclusions and a prominent collagenous, sometimes hyalinized stroma. Mitotic activity was relatively low (median, 1 mitosis/ 10 HPF), but tumor necrosis was frequent (11/13). Local invasion of adjacent structures and lymphovascular invasion were noted in some tumors (4/9 cases with available data for each feature). All were α-inhibin-positive and lacked nuclear reactivity for β-catenin. In addition, all tested cases were positive for epithelial membrane antigen (9/9) and steroidogenic factor-1 (8/8), and 8/10 expressed CD30. Two "index" cases were initially analyzed using a DNA sequencing panel, which identified EWSR1::ATF1 fusions in both. Subsequently, EWSR1::ATF1 fusions were demonstrated in 8 of the remaining 11 cases using fluorescence in situ hybridization or DNA sequencing. One of the 3 cases that were negative for EWSR1::ATF1 harbored ATF1 amplification. This study, therefore, shows that a group of malignant TSCTs resembling seminoma is characterized by α-inhibin and steroidogenic factor-1 positivity, no expression of nuclear β-catenin, frequent CD30 positivity and recurrent EWSR1::ATF1 fusions. We have descriptively termed these neoplasms "inflammatory and nested TSCT." Importantly, inflammatory and nested TSCTs show significant differences in morphology, immunoprofile, molecular biology, and, likely, clinical behavior from Sertoli cell tumors, not otherwise specified and should be classified separately.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Inflammatory and Nested Testicular Sex Cord Tumor

Acosta

Bridge

Cin

et al. 2023

American Journal of Surgical Pathology

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“…Interestingly, although MS1360 pulled down both the EWSR1::ATF1 fusion protein and the parental ATF1 transcription factor in SU-CCS-1 cells, ATF1, which was present in the other cell lines, was not pulled down. The inclusion of ATF1 in the MS1360-interacting complex in SU-CCS-1 cells may reflect the contribution of the EWSR1 domain to the fusion ( 20 ). These data demonstrate that MS0621 interacts with a complex enriched with proteins involved in transcription and co-transcriptional processes.…”

Section: Resultsmentioning

confidence: 99%

MS0621, a novel small-molecule modulator of Ewing sarcoma chromatin accessibility, interacts with an RNA-associated macromolecular complex and influences RNA splicing

et al. 2023

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Ewing sarcoma is a cancer of children and young adults characterized by the critical translocation-associated fusion oncoprotein EWSR1::FLI1. EWSR1::FLI1 targets characteristic genetic loci where it mediates aberrant chromatin and the establishment of de novo enhancers. Ewing sarcoma thus provides a model to interrogate mechanisms underlying chromatin dysregulation in tumorigenesis. Previously, we developed a high-throughput chromatin-based screening platform based on the de novo enhancers and demonstrated its utility in identifying small molecules capable of altering chromatin accessibility. Here, we report the identification of MS0621, a molecule with previously uncharacterized mechanism of action, as a small molecule modulator of chromatin state at sites of aberrant chromatin accessibility at EWSR1::FLI1-bound loci. MS0621 suppresses cellular proliferation of Ewing sarcoma cell lines by cell cycle arrest. Proteomic studies demonstrate that MS0621 associates with EWSR1::FLI1, RNA binding and splicing proteins, as well as chromatin regulatory proteins. Surprisingly, interactions with chromatin and many RNA-binding proteins, including EWSR1::FLI1 and its known interactors, were RNA-independent. Our findings suggest that MS0621 affects EWSR1::FLI1-mediated chromatin activity by interacting with and altering the activity of RNA splicing machinery and chromatin modulating factors. Genetic modulation of these proteins similarly inhibits proliferation and alters chromatin in Ewing sarcoma cells. The use of an oncogene-associated chromatin signature as a target allows for a direct approach to screen for unrecognized modulators of epigenetic machinery and provides a framework for using chromatin-based assays for future therapeutic discovery efforts.

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“…However, case 4BC-146 revealed the presence of rare fusions of EWSR1::ATF1 in NSCLC patient ( Figure 4C ). In this case, the outcome from the NGS is more toward understanding the molecular drivers associated with disease onset and its phenotype, since EWSR1 fusions do not have targeted therapy drugs approved ( 47 ). These data elucidate the importance of RNA exome sequencing to identify rare/novel driver mutations associated with the disease ( 59 ).…”

Section: Discussionmentioning

confidence: 99%

An absolute approach to using whole exome DNA and RNA workflow for cancer biomarker testing

Malhotra¹,

Javle²,

Tanwar³

et al. 2023

Front. Oncol.

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IntroductionThe concept of personalized medicine in cancer has emerged rapidly with the advancement of genome sequencing and the identification of clinically relevant variants that contribute to disease prognosis and facilitates targeted therapy options. In this study, we propose to validate a whole exome-based tumor molecular profiling for DNA and RNA from formalin-fixed paraffin-embedded (FFPE) tumor tissue.MethodsThe study included 166 patients across 17 different cancer types. The scope of this study includes the identification of single-nucleotide variants (SNVs), insertions/deletions (INDELS), copy number alterations (CNAs), gene fusions, tumor mutational burden (TMB), and microsatellite instability (MSI). The assay yielded a mean read depth of 200×, with >80% of on-target reads and a mean uniformity of >90%. Clinical maturation of whole exome sequencing (WES) (DNA and RNA)- based assay was achieved by analytical and clinical validations for all the types of genomic alterations in multiple cancers. We here demonstrate a limit of detection (LOD) of 5% for SNVs and 10% for INDELS with 97.5% specificity, 100% sensitivity, and 100% reproducibility.ResultsThe results were >98% concordant with other orthogonal techniques and appeared to be more robust and comprehensive in detecting all the clinically relevant alterations. Our study demonstrates the clinical utility of the exome-based approach of comprehensive genomic profiling (CGP) for cancer patients at diagnosis and disease progression.DiscussionThe assay provides a consolidated picture of tumor heterogeneity and prognostic and predictive biomarkers, thus helping in precision oncology practice. The primary intended use of WES (DNA+RNA) assay would be for patients with rare cancers as well as for patients with unknown primary tumors, and this category constitutes nearly 20–30% of all cancers. The WES approach may also help us understand the clonal evolution during disease progression to precisely plan the treatment in advanced stage disease.

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EWSR1-ATF1 dependent 3D connectivity regulates oncogenic and differentiation programs in Clear Cell Sarcoma

Cited by 26 publications

References 67 publications

Inflammatory and Nested Testicular Sex Cord Tumor

Inflammatory and Nested Testicular Sex Cord Tumor

MS0621, a novel small-molecule modulator of Ewing sarcoma chromatin accessibility, interacts with an RNA-associated macromolecular complex and influences RNA splicing

An absolute approach to using whole exome DNA and RNA workflow for cancer biomarker testing

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