RESUMEN
Liquid-liquid phase separation (LLPS) facilitates the formation of membraneless organelles within cells, with implications in various biological processes and disease states. AT-rich interactive domain-containing protein 1A (ARID1A) is a chromatin remodeling factor frequently associated with cancer mutations, yet its functional mechanism remains largely unknown. Here, we find that ARID1A harbors a prion-like domain (PrLD), which facilitates the formation of liquid condensates through PrLD-mediated LLPS. The nuclear condensates formed by ARID1A LLPS are significantly elevated in Ewing's sarcoma patient specimen. Disruption of ARID1A LLPS results in diminished proliferative and invasive abilities in Ewing's sarcoma cells. Through genome-wide chromatin structure and transcription profiling, we identify that the ARID1A condensate localizes to EWS/FLI1 target enhancers and induces long-range chromatin architectural changes by forming functional chromatin remodeling hubs at oncogenic target genes. Collectively, our findings demonstrate that ARID1A promotes oncogenic potential through PrLD-mediated LLPS, offering a potential therapeutic approach for treating Ewing's sarcoma.
Asunto(s)
Ensamble y Desensamble de Cromatina , Proteínas de Unión al ADN , Proteína EWS de Unión a ARN , Sarcoma de Ewing , Factores de Transcripción , Humanos , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patología , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Línea Celular Tumoral , Proteína EWS de Unión a ARN/metabolismo , Proteína EWS de Unión a ARN/genética , Regulación Neoplásica de la Expresión Génica , Proliferación Celular , Proteínas de Fusión Oncogénica/metabolismo , Proteínas de Fusión Oncogénica/genética , Proteína Proto-Oncogénica c-fli-1/metabolismo , Proteína Proto-Oncogénica c-fli-1/genética , Cromatina/metabolismo , Carcinogénesis/genética , Animales , Ratones , Dominios Proteicos , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Separación de FasesRESUMEN
Ewing's sarcoma (ES) represents a rare yet exceedingly aggressive neoplasm that poses a significant health risk to the pediatric and adolescent population. The clinical outcomes for individuals with relapsed or refractory ES are notably adverse, primarily attributed to the constrained therapeutic alternatives available. Despite significant advancements in the field, molecular pathology-driven therapeutic strategies have yet to achieve a definitive reduction in the mortality rates associated with ES. Consequently, there exists an imperative need to discover innovative therapeutic targets to effectively combat ES. To reveal the mechanism of the SETD8 (also known as lysine methyltransferase 5A) inhibitor UNC0379, cell death manners were analyzed with different inhibitors. The contributions of SETD8 to the processes of apoptosis and ferroptosis in ES cells were evaluated employing the histone methyltransferase inhibitor UNC0379 in conjunction with RNA interference techniques. The molecular regulatory mechanisms of SETD8 in ES were examined through the application of RNA sequencing (RNA-seq) and mass spectrometry-based proteomic analysis. Moreover, nude mouse xenograft models were established to explore the role of SETD8 in ES in vivo. SETD8, a sole nucleosome-specific methyltransferase that catalyzes mono-methylation of histone H4 at lysine 20 (H4K20me1), was found to be upregulated in ES, and its overexpression was associated with dismal outcomes of patients. SETD8 knockdown dramatically induced the apoptosis and ferroptosis of ES cells in vitro and suppressed tumorigenesis in vivo. Mechanistic investigations revealed that SETD8 facilitated the nuclear translocation of YBX1 through post-transcriptional regulatory mechanisms, which subsequently culminated in the transcriptional upregulation of RAC3. In summary, SETD8 inhibits the apoptosis and ferroptosis of ES cells through the YBX1/RAC3 axis, which provides new insights into the mechanism of tumorigenesis of ES. SETD8 may be a potential target for clinical intervention in ES patients.
Asunto(s)
Apoptosis , Ferroptosis , N-Metiltransferasa de Histona-Lisina , Ratones Desnudos , Sarcoma de Ewing , Humanos , Ferroptosis/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , Animales , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patología , Sarcoma de Ewing/genética , Ratones , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína 1 de Unión a la Caja YRESUMEN
Ewing sarcoma (EwS) is a cancer that arises in the bones and soft tissues, typically driven by the Ewing's sarcoma breakpoint region 1-Friend leukemia virus integration 1 (EWS-FLI) oncogene. Implementation of genetically modified animal models of EwS has proved difficult largely owing to EWS-FLI's high toxicity. The EWS-FLI1FS frameshift variant that circumvents toxicity but is still able to perform key oncogenic functions provided the first study model in Drosophila. However, the quest for Drosophila lines expressing full-length, unmodified EWS-FLI remained open. Here, we show that EWS-FLI1FS's lower toxicity is owed to reduced protein levels caused by its frameshifted C-terminal peptide, and report new strategies through which we have generated Drosophila lines that express full-length, unmodified EWS-FLI. Using these lines, we have found that the upregulation of transcription from GGAA-microsatellites (GGAAµSats) presents a positive linear correlation within a wide range of EWS-FLI protein concentrations. In contrast, rather counterintuitively, GGAAµSats-independent transcriptomic dysregulation presents relatively minor differences across the same range, suggesting that GGAAµSat-dependent and -independent transcriptional upregulation present different kinetics of response with regards to changing EWS-FLI protein concentration. Our results underpin the functional relevance of varying EWS-FLI expression levels and provide experimental tools to investigate, in Drosophila, the effect of the EWS-FLI 'high' and 'low' states that have been reported and are suspected to be important for EwS in humans.
Asunto(s)
Proteínas de Fusión Oncogénica , Proteína Proto-Oncogénica c-fli-1 , Proteína EWS de Unión a ARN , Animales , Proteína EWS de Unión a ARN/metabolismo , Proteína EWS de Unión a ARN/genética , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Proteína Proto-Oncogénica c-fli-1/metabolismo , Proteína Proto-Oncogénica c-fli-1/genética , Humanos , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patología , Animales Modificados Genéticamente , Drosophila/genética , Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismoRESUMEN
BACKGROUND: While soft tissue sarcomas affect younger patients, few studies have assessed the distribution of underlying pathogenic germline variants. PATIENTS AND METHODS: We retrospectively identified all pediatric and young adult patients (0-22 years) at Haukeland University Hospital, Norway (1981-2019), through clinical and pathological records. We identified n = 46 eligible patients. From these 46 patients, adequate material representing normal tissue was available for n = 41 cases (n = 24 diagnosed with rhabdomyosarcoma, 9 with synovial sarcomas, 2 with Ewing sarcomas, and 6 without further classification), with matching tumor tissue for n = 40. Normal tissue samples were analyzed for germline pathogenic variants (PVs) by targeted sequencing of 360 cancer genes. RESULTS: Out of the 41 analyzed cases, we found PVs or likely PVs in 7 (17%). These variants were found in TP53, MUTYH, FANCC, DICER1, FANCA, MYO3A, and MYO5B. Supporting the causality of these PVs, four cases revealed loss of heterozygosity (LOH) of the wild-type allele in the tumor tissue, one patient with a PV in DICER1 had a second somatic variant in DICER1, and a patient with a PV in TP53 had the altered allele amplified in the tumor. For three out of five with available family history, a history of other cancers in relatives was recorded. Among genes with variants of uncertain significance, CHD1L was of particular interest, revealing a stop-gain and a missense variant. INTERPRETATION: A high fraction of young patients with soft tissue sarcoma harbor PVs. Among the genes affected, we substantiate a potential role of MYO5B and propose a potential role for MYO3A.
Asunto(s)
Mutación de Línea Germinal , Humanos , Masculino , Niño , Adolescente , Femenino , Adulto Joven , Estudios Retrospectivos , Preescolar , Lactante , Sarcoma/genética , Sarcoma/patología , Recién Nacido , Adulto , Noruega , Predisposición Genética a la Enfermedad , Sarcoma Sinovial/genética , Sarcoma Sinovial/patología , Rabdomiosarcoma/genética , Rabdomiosarcoma/patología , Pérdida de Heterocigocidad , Neoplasias de los Tejidos Blandos/genética , Neoplasias de los Tejidos Blandos/patología , Sarcoma de Ewing/genética , Sarcoma de Ewing/patologíaRESUMEN
EWSR1::FLI1-mediated dysregulation of cellular machinery opens up potential new avenues for Ewing sarcoma treatment. A recent study demonstrates that pharmacologic ATR kinase inhibition dramatically synergizes with low-dose cisplatin through EWSR1::FLI1-dependent rewiring of transcription, DNA repair, and translation machinery, which could maximize the therapeutic window of the combinatory therapy. See related article by Jess et al., p. 3533.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada , Cisplatino , Sinergismo Farmacológico , Sarcoma de Ewing , Sarcoma de Ewing/tratamiento farmacológico , Sarcoma de Ewing/patología , Sarcoma de Ewing/genética , Humanos , Cisplatino/farmacología , Cisplatino/uso terapéutico , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteína EWS de Unión a ARN/genética , Proteína EWS de Unión a ARN/antagonistas & inhibidores , Proteína Proto-Oncogénica c-fli-1/genética , Proteína Proto-Oncogénica c-fli-1/metabolismo , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/antagonistas & inhibidores , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/patología , Neoplasias Óseas/genética , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéuticoRESUMEN
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.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular , Janus Quinasa 1 , Proteínas Tirosina Quinasas Receptoras , Sarcoma de Ewing , Transducción de Señal , Sarcoma de Ewing/tratamiento farmacológico , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patología , Sarcoma de Ewing/genética , Humanos , Janus Quinasa 1/metabolismo , Janus Quinasa 1/antagonistas & inhibidores , Janus Quinasa 1/genética , Línea Celular Tumoral , Animales , Transducción de Señal/efectos de los fármacos , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Ratones , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Tirosina Quinasa del Receptor Axl , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Neoplasias Óseas/genética , Ensayos Antitumor por Modelo de Xenoinjerto , Tirosina Quinasa c-Mer/metabolismo , Tirosina Quinasa c-Mer/genética , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Fosforilación/efectos de los fármacos , Femenino , Factor de Transcripción STAT6RESUMEN
Ewing sarcoma is the second most common bone cancer in children, and while patients who present with metastatic disease at the time of diagnosis have a dismal prognosis. Ewing sarcoma tumors are driven by the fusion gene EWS/Fli1, and while these tumors are genetically homogenous, the transcriptional heterogeneity can lead to a variety of cellular processes including metastasis. In this study, we demonstrate that in Ewing sarcoma cells, the canonical Wnt/ß-Catenin signaling pathway is heterogeneously activated in vitro and in vivo, correlating with hypoxia and EWS/Fli1 activity. Ewing sarcoma cells predominantly express ß-Catenin on the cell membrane bound to CDH11, which can respond to exogenous Wnt ligands leading to the immediate activation of Wnt/ß-Catenin signaling within a tumor. Knockdown of CDH11 leads to delayed and decreased response to exogenous Wnt ligand stimulation, and ultimately decreased metastatic propensity. Our findings strongly indicate that CDH11 is a key component of regulating Wnt//ß-Catenin signaling heterogeneity within Ewing sarcoma tumors, and is a promising molecular target to alter Wnt//ß-Catenin signaling in Ewing sarcoma patients.
Asunto(s)
Cadherinas , Sarcoma de Ewing , Vía de Señalización Wnt , beta Catenina , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patología , Sarcoma de Ewing/genética , Humanos , Cadherinas/metabolismo , Cadherinas/genética , Línea Celular Tumoral , beta Catenina/metabolismo , Animales , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Neoplasias Óseas/genética , Ratones , Proteínas de Fusión Oncogénica/metabolismo , Proteínas de Fusión Oncogénica/genética , Proteína Proto-Oncogénica c-fli-1/metabolismo , Proteína Proto-Oncogénica c-fli-1/genética , Proteína EWS de Unión a ARN/metabolismo , Proteína EWS de Unión a ARN/genéticaRESUMEN
Ewing sarcoma is an uncommon neoplasm considered in the differential diagnosis of tumors with "small round cell" morphology, but its occurrence in the gynecologic tract has only been sporadically documented. Herein, we describe the largest cohort of Ewing sarcoma localized to the female genital tract to date, and emphasize their clinicopathologic resemblance to more common gynecologic neoplasms. Ewing sarcoma (n=21) was retrospectively identified from 5 institutions. The average patient age was 35 (range 6-61) years. Tumor sites included uterus (n=8), cervix (n=4), vulva (n=5), vagina (n=1), broad ligament (n=1), inguinal area (n=1), and pelvis (n=1). Nine of 18 cases in which slides were available for review demonstrated only classic round cell morphology, with the remainder showing a variable combination and prominence of variant ovoid/spindle or epithelioid appearance. Tumors showed diffuse membranous reactivity for CD99 (20/20) and were positive for NKX2.2 (8/8, diffuse) and cyclin D1 (7/7, of which 3/7 were patchy/multifocal and 4/7 were diffuse). They were negative for ER (0/6) and CD10 (0/6). Three cases were initially diagnosed as endometrial stromal sarcomas. EWSR1 rearrangement was confirmed in 20/21 by fluorescence in situ hybridization (n=15) and/or sequencing (n=8). Of the eight tumors that underwent sequencing, 6 harbored FLI1 , 1 ERG, and 1 FEV as the fusion partner. Of 11 patients with available follow-up, 5 died of disease, 1 developed lung metastases and 5 are alive with no evidence of disease. Ewing sarcoma of the gynecologic tract is a rare, aggressive entity that shares some morphologic and immunohistochemical features with other more common gynecologic neoplasms. In addition to the typical round cell appearance, variant spindled/ovoid to epithelioid morphology may also be observed and should prompt consideration of this entity with appropriate immunohistochemical and/or molecular studies.
Asunto(s)
Biomarcadores de Tumor , Neoplasias de los Genitales Femeninos , Proteína EWS de Unión a ARN , Sarcoma de Ewing , Humanos , Femenino , Sarcoma de Ewing/genética , Sarcoma de Ewing/patología , Sarcoma de Ewing/diagnóstico , Sarcoma de Ewing/química , Neoplasias de los Genitales Femeninos/patología , Neoplasias de los Genitales Femeninos/genética , Neoplasias de los Genitales Femeninos/diagnóstico , Adulto , Diagnóstico Diferencial , Adolescente , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/análisis , Adulto Joven , Persona de Mediana Edad , Niño , Estudios Retrospectivos , Proteína EWS de Unión a ARN/genética , Inmunohistoquímica , Hibridación Fluorescente in Situ , Proteína Homeobox Nkx-2.2 , Factores de Transcripción/genética , Proteínas de Homeodominio/genética , Valor Predictivo de las Pruebas , Reordenamiento Génico , Antígeno 12E7/metabolismo , Células Epitelioides/patología , Células Epitelioides/química , Proteínas NuclearesRESUMEN
CIC-rearranged sarcoma (CRS) is a group of high-grade undifferentiated small round cell sarcomas examined as a separate entity in the current WHO classification; since it shows more aggressive clinical behavior and distinct morphological and molecular features compared to Ewing sarcoma (ES). As CCNE1 expression is associated with tumor growth in CIC::DUX4 sarcomas, we aimed to demonstrate the value of cyclin E1 expression in CRS. Cyclin E1 immunohistochemistry and break-apart FISH for EWSR1 and CIC gene rearrangements were performed on 3-mm tissue microarrays composed of 40 small round cell tumors. Five cases were classified as CRS, whereas 22 were ES and 13 were unclassified (EWSR1-/CIC-). Among all three diagnostic groups, we found cyclin E1 expression level to be higher in CRS (80 %) and unclassified groups (61.5 %) compared to ES (4.5 %, p < 0.001). In addition, high cyclin E1 expression levels were associated with higher mean age at diagnosis, presence of atypical histology and myxoid stroma, low CD99 expression, and presence of metastasis at diagnosis. The sensitivity and specificity of high cyclin E1 expression in detecting non-ES cases were 95.5 % and 66.7 %, respectively. However, the correlation between cyclin E1 expression level and survival was not statistically significant. This is the first study that shows cyclin E1 immunohistochemical expression in EWSR1-negative undifferentiated small cell sarcomas, particularly CRS.
Asunto(s)
Biomarcadores de Tumor , Ciclina E , Reordenamiento Génico , Proteínas Oncogénicas , Proteínas Represoras , Humanos , Masculino , Proteínas Oncogénicas/metabolismo , Proteínas Oncogénicas/genética , Femenino , Adulto , Ciclina E/metabolismo , Ciclina E/genética , Persona de Mediana Edad , Adolescente , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/genética , Adulto Joven , Niño , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Inmunohistoquímica/métodos , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patología , Sarcoma de Ewing/genética , Sarcoma/patología , Sarcoma/metabolismo , Sarcoma/genética , Sarcoma/diagnóstico , Hibridación Fluorescente in Situ/métodos , Anciano , Preescolar , Proteína EWS de Unión a ARN/genética , Proteína EWS de Unión a ARN/metabolismo , Sarcoma de Células Pequeñas/metabolismo , Sarcoma de Células Pequeñas/genética , Sarcoma de Células Pequeñas/patología , Sarcoma de Células Pequeñas/diagnósticoRESUMEN
PURPOSE OF REVIEW: There is an unmet need to improve outcomes for patients for Ewing sarcoma, a rare, aggressive sarcoma with a peak incidence in adolescents and young adults (AYA). Current therapy at diagnosis involves multiagent chemotherapy and local therapy, but despite intensification of treatment, those with metastases at diagnosis and recurrent disease have poor outcomes. RECENT FINDINGS: Improved understanding of Ewing sarcoma biology has identified novel targets with promising activity in Ewing sarcoma patients, including tyrosine kinase inhibitors that are now undergoing evaluation as combination and maintenance therapy. Other emerging therapies include those that target the EWSR1::FLI1 fusion oncoprotein, and act on DNA damage, cell cycle and apoptotic pathways. Immunotherapeutic approaches, particularly CAR-T-cell therapy directed at GD2, also hold promise. Recent collaborative clinical trials that have defined an international standard of care for patients with newly diagnosed Ewing sarcoma and novel platform studies with adaptive designs offer unique opportunities to investigate these therapies inclusive of all ages. SUMMARY: Close international collaboration between clinicians and biologists will allow us to prioritize promising emerging therapies and develop biomarkers to facilitate their incorporation into standard of care and more rapidly translate into benefit for Ewing sarcoma patients.
Asunto(s)
Neoplasias Óseas , Sarcoma de Ewing , Humanos , Sarcoma de Ewing/terapia , Sarcoma de Ewing/genética , Neoplasias Óseas/terapia , Neoplasias Óseas/tratamiento farmacológico , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/antagonistas & inhibidores , Terapia Molecular Dirigida , Inmunoterapia AdoptivaRESUMEN
Retroperitoneal Ewing sarcomas (RES) are very rare and mostly described in case reports. The purpose of this study was to retrospectively analyze the clinicopathology, molecular characteristics, biological behavior, and therapeutic information of 13 cases of primary RES with immunohistochemical staining, fluorescence in situ hybridization, RT-PCR and NGS sequencing detection techniques. The thirteen patients included eight males and five females with a mean age of 34 years. Morphologically, the tumors were comprised of small round or epithelial-like cells with vacuolated cytoplasm (6/13,46 %) arranged in diffuse, nested (8/13,62 %) and perivascular (7/13,54 %) patterns. Unusual morphologic patterns, such as meningioma-like swirling structures and sieve-like structures were relatively novel findings. Immunohistochemical studies showed CD99 (12/13; 92 %), CD56 (11/13; 85 %), NKX2.2 (9/13; 69 %), PAX7 (10/11;91 %) and CD117(6/9;67 %) to be positive.12 cases (92 %) demonstrated EWSR1 rearrangement and 3 cases displayed EWSR1::FLI1 fusion by FISH. ERCC4 splice-site variant, a novel pathogenic variant, was discovered for the first time via RNA sequencing. With a median follow-up duration of 14 months (6 to 79 months), 8/13 (62 %) patients died, while 5/13(38 %) survived. Three cases recurred, and five patients developed metastasis to the liver (2 cases), lung (2 cases) and bone (1 case). RES is an aggressive, high-grade tumor, prone to multiple recurrences and metastases, with distinctive morphologic, immunohistochemical, and molecular genetic features. ERCC4 splicing mutation, which is a novel pathogenic variant discovered for the first time, with possible significance for understanding the disease, as well as the development of targeted drugs.
Asunto(s)
Proteína Homeobox Nkx-2.2 , Proteína EWS de Unión a ARN , Neoplasias Retroperitoneales , Sarcoma de Ewing , Humanos , Sarcoma de Ewing/genética , Sarcoma de Ewing/patología , Sarcoma de Ewing/diagnóstico , Sarcoma de Ewing/metabolismo , Masculino , Femenino , Adulto , Neoplasias Retroperitoneales/genética , Neoplasias Retroperitoneales/patología , Neoplasias Retroperitoneales/diagnóstico , Estudios Retrospectivos , Persona de Mediana Edad , Adulto Joven , Adolescente , Proteína EWS de Unión a ARN/genética , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Hibridación Fluorescente in Situ/métodos , Reordenamiento Génico , Inmunohistoquímica/métodos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas de Fusión Oncogénica/genética , Niño , Proteínas Nucleares , Proteínas de Homeodominio , Proteínas de Pez CebraRESUMEN
Carbohydrate markers of immature cells during prenatal human development can be aberrantly expressed in cancers and deserve evaluation as immune targets. A candidate target in Ewing sarcoma is the globo-series ganglioside stage-specific embryonic antigen-4 (SSEA-4). We detected SSEA-4 expression on the cell surface of all of 14 EwS cell lines and in 21 of 31 (68%) primary EwS tumor biopsies. Among paired subpopulations of tumor cells with low versus high SSEA-4 expression, SSEA-4high expression was significantly and consistently associated with functional characteristics of tumor aggressiveness, including higher cell proliferation, colony formation, chemoresistance and propensity to migrate. SSEA-4low versus SSEA-4high expression was not related to expression levels of the EWSR1-FLI1 fusion transcript or markers of epithelial/mesenchymal plasticity. SSEA-4low cells selected from bulk populations regained higher SSEA-4 expression in vitro and during in vivo tumor growth in a murine xenograft model. T cells engineered to express SSEA-4-specific chimeric antigen receptors (CARs) specifically interacted with SSEA-4 positive EwS cells and exerted effective antigen-specific tumor cell lysis in vitro. In conclusion, with its stable expression and functional significance in EwS, SSEA-4 is an attractive therapeutic immune target in this cancer that deserves further evaluation for clinical translation.
Asunto(s)
Sarcoma de Ewing , Antígenos Embrionarios Específico de Estadio , Animales , Femenino , Humanos , Ratones , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/genética , Neoplasias Óseas/inmunología , Neoplasias Óseas/patología , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Línea Celular Tumoral , Proliferación Celular , Gangliósidos , Glicoesfingolípidos , Sarcoma de Ewing/patología , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/genética , Antígenos Embrionarios Específico de Estadio/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Ewing's sarcoma (ES) is an aggressive cancer of bone and soft tissue, most of which tend to occur in the bone. Extraosseous Ewing's sarcoma (EES) of the cervix is extremely rare. CASE PRESENTATION: In the present work, we reported a 39-year-old cervical EES patient with a 2.5*2.1*1.8 cm tumor mass. According to previous literatures, our case is the smallest tumor found in primary cervical ES ever. The patient initially came to our hospital due to vaginal bleeding, and then the gynecological examination found a neoplasm between the cervical canal and partially in the external cervical orifice. The diagnosis of EES was confirmed below: Hematoxylin & Eosin staining (H&E) revealed small round blue malignant cells in biopsy specimens. Immunohistochemistry (IHC) showed the positive staining for CD99, NKX2.2, and FLI1. Disruption of EWSR1 gene was found by fluorescence in situ hybridization (FISH), and the EWSR1-FLI1 gene fusion was determined by next-generation sequencing (NGS). The patient received laparoscopic wide hysterectomy, bilateral adnexectomy, pelvic lymphadenectomy, and postoperative adjuvant chemotherapy and remained disease free with regular follow-up for 1 year. CONCLUSIONS: Through a systematic review of previously reported cervical ES and this case, we highlighted the importance of FISH and NGS for the accuracy of ESS diagnosis, which could assist on the optimal treatment strategy. However, due to the rarity of the disease, there is no standard treatment schemes. Investigation on molecular pathological diagnosis and standardization of treatment regimens for cervical ES are critical to patients' prognosis.
Asunto(s)
Sarcoma de Ewing , Neoplasias del Cuello Uterino , Humanos , Femenino , Sarcoma de Ewing/patología , Sarcoma de Ewing/genética , Sarcoma de Ewing/diagnóstico , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/diagnóstico , Neoplasias del Cuello Uterino/cirugía , Adulto , Proteínas de Fusión Oncogénica/genética , Proteína Homeobox Nkx-2.2 , Proteína EWS de Unión a ARN/genética , Hibridación Fluorescente in Situ , Factores de Transcripción/genética , Proteína Proto-Oncogénica c-fli-1/genética , Proteínas Nucleares , Proteínas de HomeodominioRESUMEN
The mechanistic basis for the metastasis of Ewing sarcomas remains poorly understood, as these tumors harbor few mutations beyond the chromosomal translocation that initiates the disease. Instead, the epigenome of Ewing sarcoma cells reflects the regulatory state of genes associated with the DNA-binding activity of the fusion oncoproteins EWSR1::FLI1 or EWSR1::ERG. In this study, we examined the EWSR1::FLI1/ERG's repression of transcription factor genes, concentrating on those that exhibit a broader range of expression in tumors than in Ewing sarcoma cell lines. Focusing on one of these target genes, ETS1, we detected EWSR1::FLI1 binding and an H3K27me3-repressive mark at this locus. Depletion of EWSR1::FLI1 results in ETS1's binding of promoter regions, substantially altering the transcriptome of Ewing sarcoma cells, including the upregulation of the gene encoding TENSIN3 (TNS3), a focal adhesion protein. Ewing sarcoma cell lines expressing ETS1 (CRISPRa) exhibited increased TNS3 expression and enhanced movement compared with control cells. Visualization of control Ewing sarcoma cells showed a distributed vinculin signal and a network-like organization of F-actin; in contrast, ETS1-activated Ewing sarcoma cells showed an accumulation of vinculin and F-actin toward the plasma membrane. Interestingly, the phenotype of ETS1-activated Ewing sarcoma cell lines depleted of TNS3 resembled the phenotype of the control cells. Critically, these findings have clinical relevance as TNS3 expression in Ewing sarcoma tumors positively correlates with that of ETS1. Implications: ETS1's transcriptional regulation of the gene encoding the focal adhesion protein TENSIN3 in Ewing sarcoma cells promotes cell movement, a critical step in the evolution of metastasis.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Proteínas de Fusión Oncogénica , Proteína Proto-Oncogénica c-ets-1 , Proteína Proto-Oncogénica c-fli-1 , Proteína EWS de Unión a ARN , Sarcoma de Ewing , Tensinas , Humanos , Proteína Proto-Oncogénica c-ets-1/genética , Proteína Proto-Oncogénica c-ets-1/metabolismo , Tensinas/metabolismo , Tensinas/genética , Sarcoma de Ewing/genética , Sarcoma de Ewing/patología , Sarcoma de Ewing/metabolismo , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Línea Celular Tumoral , Proteína Proto-Oncogénica c-fli-1/genética , Proteína Proto-Oncogénica c-fli-1/metabolismo , Proteína EWS de Unión a ARN/genética , Proteína EWS de Unión a ARN/metabolismo , Adhesiones Focales/genética , Adhesiones Focales/metabolismoRESUMEN
Objective: To investigate the clinical application of EWSR1 gene rearrangement by fluorescence in situ hybridization (FISH) in bone and soft tissue tumors and to analyze the cases with atypical signal pattern. Methods: The cases detected for EWSR1 gene rearrangement by FISH in Beijing Jishuitan Hospital, Capital Medical University from 2014 to 2021 were collected, and the value of detecting EWSR1 gene rearrangement for diagnosing bone and soft tissue tumors was analyzed. The cases with atypical positive signals were further analyzed by next generation sequencing (NGS). Results: FISH using EWSR1 break-apart probe kit was successfully performed in 97% (205/211) of cases, 6 cases failed. Four of the 6 failures were due to improper decalcification, 1 case due to signal overlap caused by thick slices, and 1 case due to signal amplification and disorder. EWSR1 gene rearrangements were positive in 122 cases (122/205, 59%), atypical positive signal in 8 cases (8/205, 4%), and negative in 75 cases (75/205, 37%). In cases testing positive, the percentage of positive cells ranged from 34% to 98%, with 120 cases (120/122, 98%) showing a positive cell percentage greater than 50%. Among the 205 successfully tested cases, 156 cases were histologically diagnosed as Ewing's sarcoma, of which 110 were positive (110/156, 71%), 7 were atypical positive (7/156, 4%), and 39 were negative (39/156, 25%). Nine cases were histologically diagnosed as clear cell sarcoma of soft tissue, of which 6 were positive (6/9), 1 was atypical positive (1/9), and 2 were negative (2/9). Five cases were histologically diagnosed as extraskeletal myxoid chondrosarcoma, of which 2 were positive (2/5) and 3 were negative (3/5). Three cases were histologically diagnosed as angiomatoid fibrous histiocytoma, of which 2 were positive (2/3) and 1 was negative (1/3). Two cases were histologically diagnosed as myoepithelioma of soft tissue, of which 1 was positive (1/2) and 1 was negative (1/2). One case was histologically diagnosed as olfactory neuroblastoma with a positive result. The 29 other tumor cases including osteosarcoma, synovial sarcoma, and malignant melanoma and others were all negative. Basing on histology as the standard for diagnosis and considering atypical positive cases as negative, comparing with the 29 cases of other tumors as control group, the sensitivity for diagnosing Ewing's sarcoma through the detection of EWSR1 gene rearrangement was 71%, and the specificity was 100%; the sensitivity for diagnosing clear cell sarcoma of soft tissue was 67%, and the specificity was 100%; the sensitivity for diagnosing extraskeletal myxoid chondrosarcoma was 40%, and the specificity was 100%; the sensitivity for diagnosing angiomatoid fibrous histiocytoma was 67%, and the specificity was 100%; the sensitivity for diagnosing myoepithelioma of soft tissue was 50%, and the specificity was 100%; the sensitivity for diagnosing olfactory neuroblastoma was 100%, and the specificity was 100%. Four of 8 cases with atypical positive signals analyzed by NGS showed EWSR1 rearrangement, including EWSR1::FLI1 in one case of Ewing sarcoma, EWSR1::NFATC2 in one case of EWSR1::NFATC2-rearranged sarcoma, EWSR1::ATF1 in one case of clear cell sarcoma of soft tissue and EWSR1::NR4A3 in one case of extraskeletal myxoid chondrosarcoma. Conclusions: Detection of EWSR1 rearrangement by FISH is of utmost significance in the diagnosis of bone and soft tissue tumors. Cases with atypical positive signals should be further scrutinized, correlating with their histomorphology and verifying by NGS if necessary.
Asunto(s)
Neoplasias Óseas , Reordenamiento Génico , Hibridación Fluorescente in Situ , Proteína EWS de Unión a ARN , Neoplasias de los Tejidos Blandos , Humanos , Proteína EWS de Unión a ARN/genética , Neoplasias de los Tejidos Blandos/genética , Neoplasias de los Tejidos Blandos/patología , Neoplasias de los Tejidos Blandos/diagnóstico , Hibridación Fluorescente in Situ/métodos , Neoplasias Óseas/genética , Neoplasias Óseas/diagnóstico , Neoplasias Óseas/patología , Histiocitoma Fibroso Maligno/genética , Histiocitoma Fibroso Maligno/diagnóstico , Histiocitoma Fibroso Maligno/patología , Sarcoma de Ewing/genética , Sarcoma de Ewing/diagnósticoRESUMEN
The FET protein family, comprising FUS, EWS, and TAF15, plays crucial roles in mRNA maturation, transcriptional regulation, and DNA damage response. Clinically, they are linked to Ewing family tumors and neurodegenerative diseases such as amyotrophic lateral sclerosis. The fusion protein EWS::FLI1, the causative mutation of Ewing sarcoma, arises from a genomic translocation that fuses a portion of the low-complexity domain (LCD) of EWS (EWSLCD) with the DNA binding domain of the ETS transcription factor FLI1. This fusion protein modifies transcriptional programs and disrupts native EWS functions, such as splicing. The exact role of the intrinsically disordered EWSLCD remains a topic of active investigation, but its ability to phase separate and form biomolecular condensates is believed to be central to EWS::FLI1's oncogenic properties. Here, we used paramagnetic relaxation enhancement NMR, microscopy, and all-atom molecular dynamics (MD) simulations to better understand the self-association and phase separation tendencies of the EWSLCD. Our NMR data and mutational analysis suggest that a higher density and proximity of tyrosine residues amplify the likelihood of condensate formation. MD simulations revealed that the tyrosine-rich termini exhibit compact conformations with unique contact networks and provided critical input on the relationship between contacts formed within a single molecule (intramolecular) and inside the condensed phase (intermolecular). These findings enhance our understanding of FET proteins' condensate-forming capabilities and underline differences between EWS, FUS, and TAF15.
Asunto(s)
Sarcoma de Ewing , Factores Asociados con la Proteína de Unión a TATA , Humanos , Proteína EWS de Unión a ARN/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Separación de Fases , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patología , Proteínas/metabolismo , Tirosina , Factores Asociados con la Proteína de Unión a TATA/genética , Factores Asociados con la Proteína de Unión a TATA/metabolismoRESUMEN
PURPOSE: The importance of cellular context to the synergy of DNA damage response (DDR)-targeted agents is important for tumors with mutations in DDR pathways, but less well-established for tumors driven by oncogenic transcription factors. In this study, we exploit the widespread transcriptional dysregulation of the EWS-FLI1 transcription factor to identify an effective DDR-targeted combination therapy for Ewing sarcoma. EXPERIMENTAL DESIGN: We used matrix drug screening to evaluate synergy between a DNA-PK inhibitor (M9831) or an ATR inhibitor (berzosertib) and chemotherapy. The combination of berzosertib and cisplatin was selected for broad synergy, mechanistically evaluated for Ewing sarcoma selectivity, and optimized for in vivo schedule. RESULTS: Berzosertib combined with cisplatin demonstrates profound synergy in multiple Ewing sarcoma cell lines at clinically achievable concentrations. The synergy is due to loss of expression of the ATR downstream target CHEK1, loss of cell-cycle check-points, and mitotic catastrophe. Consistent with the goals of the project, EWS-FLI1 drives the expression of CHEK1 and five other ATR pathway members. The loss of CHEK1 expression is not due to transcriptional repression and instead caused by degradation coupled with suppression of protein translation. The profound synergy is realized in vivo with a novel optimized schedule of this combination in subsets of Ewing sarcoma models, leading to durable complete responses in 50% of animals bearing two different Ewing sarcoma xenografts. CONCLUSIONS: These data exploit EWS-FLI1 driven alterations in cell context to broaden the therapeutic window of berzosertib and cisplatin to establish a promising combination therapy and a novel in vivo schedule. See related commentary by Ohmura and Grünewald, p. 3358.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada , Cisplatino , Sinergismo Farmacológico , Proteína Proto-Oncogénica c-fli-1 , Proteína EWS de Unión a ARN , Sarcoma de Ewing , Ensayos Antitumor por Modelo de Xenoinjerto , Sarcoma de Ewing/tratamiento farmacológico , Sarcoma de Ewing/patología , Sarcoma de Ewing/genética , Humanos , Cisplatino/farmacología , Animales , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Ratones , Proteína EWS de Unión a ARN/genética , Proteína EWS de Unión a ARN/antagonistas & inhibidores , Línea Celular Tumoral , Proteína Proto-Oncogénica c-fli-1/genética , Proteína Proto-Oncogénica c-fli-1/antagonistas & inhibidores , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/antagonistas & inhibidores , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Daño del ADN/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Isoxazoles , PirazinasRESUMEN
Ewing's sarcoma (ES) is the second most common bone and soft tissue malignancy in children and adolescents with a poor prognosis. The identification of genes with prognostic value may contribute to the prediction and treatment of this disease. The GSE17679, GSE68776, GSE63155, and GSE63156 datasets were downloaded from the Gene Expression Omnibus database and qualified. Prognostic value of differentially expressed genes (DEGs) between the normal and tumor groups and immune cell infiltration were explored by several algorithms. A prognostic model was established and validated. Finally, functional analyses of the DEGs were performed. Proline rich 11 (PRR11) and mast cell infiltration were noted as the key indicators for the prognosis of ES. Kaplan-Meier and scatter plots for the training and two validation sets showed that patients in the low-PRR11 expression group were associated with better outcomes than those in the high-PRR11 expression group. The concordance indices and calibration analyses of the prognostic model indicated good predictive accuracy in the training and validation sets. The area under the curve values obtained through the receiver operating characteristic analysis for 1-, 3-, 5-year prediction were ≥ 0.75 in the three cohorts, suggesting satisfactory sensitivity and specificity of the model. Decision curve analyses suggested that patients could benefit more from the model than the other strategies. Functional analyses suggested that DEGs were mainly clustered in the cell cycle pathway. PRR11 and mast cell infiltration are potential prognostic indicators in ES. PRR11 possibly affects the prognosis of patients with ES through the cell cycle pathway.
Asunto(s)
Sarcoma de Ewing , Sarcoma , Adolescente , Niño , Humanos , Algoritmos , Calibración , Pronóstico , Sarcoma de Ewing/genéticaRESUMEN
PURPOSE: CIC-rearranged sarcomas represent a type of undifferentiated small round cell sarcoma (USRCS) characterized by poor survival, rapid development of chemotherapy resistance, and high rates of metastasis. We aim to contribute to the growing body of knowledge regarding diagnosis, treatment, clinical course, and outcomes for these patients. METHODS: This case series investigates the clinical courses of ten patients with CIC-rearranged sarcoma treated at the Johns Hopkins Hospital from July 2014 through January 2024. Clinical data were retrospectively extracted from electronic medical records. RESULTS: Patients ranged from 10 to 67 years of age at diagnosis, with seven patients presenting with localized disease and three with metastatic disease. Tumors originated from soft tissues of various anatomic locations. Mean overall survival (OS) was 22.1 months (10.6-52.2), and mean progression-free survival (PFS) was 16.7 months (5.3-52.2). Seven patients received intensive systemic therapy with an Ewing sarcoma-directed regimen or a soft tissue sarcoma-directed regimen. Three patients experienced prolonged disease-free survival without systemic treatment. CONCLUSION: Most patients in this case series demonstrated aggressive clinical courses consistent with those previously described in the literature, although we note a spectrum of clinical outcomes not previously reported. The diversity of clinical courses underscores the need for an improved understanding of individual tumor biology to enhance clinical decision-making and patient prognosis. Despite its limitations, this article broadens the spectrum of reported clinical outcomes, providing a valuable addition to the published literature on this rare cancer.
Asunto(s)
Sarcoma de Ewing , Sarcoma , Humanos , Toma de Decisiones Clínicas , Hospitales , Estudios Retrospectivos , Sarcoma/genética , Sarcoma/terapia , Sarcoma de Ewing/genética , Sarcoma de Ewing/terapiaRESUMEN
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.