Homo sapiens
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ABSTRACT:
PROVIDER: PRJNA294622 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:The pathognomonic EWS/ETS fusion transcription factors drive Ewing sarcoma (EWS) by orchestrating an oncogenic transcription program. Therapeutic targeting of EWS/ETS has not been successful; therefore identifying mediators of the EWS/ETS function could offer new therapeutic targets. Here we describe the dependency of chromatin reader BET bromodomain proteins in EWS/ETS driven transcription and investigate the potential of BET inhibitors in treating this lethal cancer. Similar to EWS/ETS fusions, knockdown of BET proteins BRD2/3/4 severely impaired the oncogenic phenotype of EWS cells. Notably, EWS/FLI1 and EWS/ERG was found to be in a transcriptional complex consisting of BRD4. RNA-Seq analysis upon BRD4 knockdown or its pharmacologic inhibition by the BET inhibitor JQ1 revealed an attenuated EWS/ETS transcriptional signature. In contrast to other reports, JQ1 reduced proliferation, and induced apoptosis through MYC-independent mechanism without affecting EWS/ETS protein levels, which was further confirmed by depleting BET proteins using PROTAC-BET degrader (BETd). Interestingly, polycomb repressive complex 2 (PRC2) associated factor PHF19 was downregulated by JQ1/BETd or BRD4 knockdown in multiple EWS cells. ChIP-seq analysis revealed occupancy of EWS/FLI1 at a distal regulatory element of PHF19 and its subsequent knockdown resulted in downregulation of PHF19 expression. Furthermore, deletion of PHF19 by CRISPR-Cas9 system lead to a decreased tumorigenic phenotype and increased sensitivity to JQ1. Importantly, PHF19 expression was associated with worse prognosis of Ewing sarcoma patients. In vivo, JQ1 demonstrated anti-tumor efficacy in multiple mouse xenograft models of EWS. Together, these results indicate that EWS/ETS require BET epigenetic reader proteins for its transcriptional program including PHF19 expression, which can be mitigated by BET inhibitors. Moreover, this study provides a clear rationale for the clinical utility of BET inhibitors in treating Ewing sarcoma.
2018-06-19 | GSE113604 | GEO
Project description:Ewing sarcomas (ES) are highly malignant, osteolytic bone or soft tissue tumors, which are characterized by early metastasis into lung and bone. Genetically, ES are defined by balanced chromosomal EWS/ETS translocations, which give rise to chimeric proteins (EWS-ETS) that generate an oncogenic transcriptional program associated with altered epigenetic marks throughout the genome. By use of an inhibitor (JQ1) blocking BET bromodomain binding proteins (BRDs) we strikingly observed a strong down-regulation of the predominant EWS-ETS protein EWS/FLI1 in a dose dependent manner. Microarray analysis further revealed JQ1 treatment to block a typical ES associated expression program. The effect on this expression program could be mimicked by RNA interference with BRD3 or BRD4 expression, indicating that the EWS/FLI1 mediated expression profile is at least in part mediated via such epigenetic readers. Consequently, contact dependent and independent proliferation of different ES lines was strongly inhibited. Mechanistically, treatment of ES resulted in a partial arrest of the cell cycle as well as induction of apoptosis. Tumor development was suppressed dose dependently in a xeno-transplant model in immune deficient mice, overall indicating that ES may be susceptible to treatment with epigenetic inhibitors blocking BET bromodomain activity and the associated pathognomonic EWS-ETS transcriptional program in ES. Ewing sarcoma cell lines A673 and TC-71 were treated for 48 hours with 2 microM JQ1 or DMSO control.
2015-12-08 | E-GEOD-72673 | biostudies-arrayexpress
Project description:BET bromodomain dependency in EWS/ETS driven Ewing Sarcoma
| PRJNA453371 | ENA
Project description:Background: Previously, we used inhibitors blocking BET bromodomain binding proteins (BRDs) in Ewing sarcoma (EwS) and observed that long term treatment resulted in the development of resistance. Here, we analyze the possible interaction of BRD4 with cyclin-dependent kinase (CDK) 9. Methods: Co-immunoprecipitation experiments (CoIP) to characterize BRD4 interaction and functional consequences of inhibiting transcriptional elongation were assessed using drugs targeting of BRD4 or CDK9, either alone or in combination. Results: CoIP revealed an interaction of BRD4 with EWS-FLI1 and CDK9 in EwS. Treatment of EwS cells with CDKI-73, a specific CDK9 inhibitor (CDK9i), induced a rapid downregulation of EWS-FLI1 expression and block of contact-dependent growth. CDKI-73 induced apoptosis in EwS, as depicted by cleavage of Caspase 7 (CASP7), PARP and increased CASP3 activity, similar to JQ1. Microarray analysis following CDKI-73 treatment uncovered a transcriptional program that was only partially comparable to BRD inhibition. Strikingly, combined treatment of EwS with BRD- and CDK9-inhibitors re-sensitized cells, and was overall more effective than individual drugs not only in vitro but also in a preclinical mouse model in vivo. Conclusion: Treatment with BRD inhibitors in combination with CDK9i offers a new treatment option that significantly blocks the pathognomonic EWS-ETS transcriptional program and malignant phenotype of EwS.
2019-03-31 | GSE119546 | GEO
Project description:Ewing sarcomas (ES) are highly malignant, osteolytic bone or soft tissue tumors, which are characterized by early metastasis into lung and bone. Genetically, ES are defined by balanced chromosomal EWS/ETS translocations, which give rise to chimeric proteins (EWS-ETS) that generate an oncogenic transcriptional program associated with altered epigenetic marks throughout the genome. By use of an inhibitor (JQ1) blocking BET bromodomain binding proteins (BRDs) we strikingly observed a strong down-regulation of the predominant EWS-ETS protein EWS/FLI1 in a dose dependent manner. Microarray analysis further revealed JQ1 treatment to block a typical ES associated expression program. The effect on this expression program could be mimicked by RNA interference with BRD3 or BRD4 expression, indicating that the EWS/FLI1 mediated expression profile is at least in part mediated via such epigenetic readers. Consequently, contact dependent and independent proliferation of different ES lines was strongly inhibited. Mechanistically, treatment of ES resulted in a partial arrest of the cell cycle as well as induction of apoptosis. Tumor development was suppressed dose dependently in a xeno-transplant model in immune deficient mice, overall indicating that ES may be susceptible to treatment with epigenetic inhibitors blocking BET bromodomain activity and the associated pathognomonic EWS-ETS transcriptional program in ES.
2015-12-08 | GSE72673 | GEO
Project description:Ewing sarcoma (EwS) is characterized by EWSR1-ETS fusion transcription factors converting polymorphic GGAA microsatellites (mSats) into potent neo-enhancers. Although the paucity of additional mutations makes EwS a genuine model to study principles of cooperation between dominant fusion oncogenes and neo-enhancers, this is impeded by the limited number of well-characterized models. Here we present the Ewing Sarcoma Cell Line Atlas (ESCLA), comprising whole-genome, DNA methylation, transcriptome, proteome, and chromatin immunoprecipitation sequencing (ChIP-seq) data of 18 cell lines with inducible EWSR1-ETS knockdown. The ESCLA shows hundreds of EWSR1-ETS-targets, the nature of EWSR1-ETS-preferred GGAA mSats, and putative indirect modes of EWSR1-ETS-mediated gene regulation, converging in the duality of a specific but plastic EwS signature. We identify heterogeneously regulated EWSR1-ETS-targets as potential prognostic EwS biomarkers. Our freely available ESCLA (http://r2platform.com/escla/) is a rich resource for EwS research and highlights the power of comprehensive datasets to unravel principles of heterogeneous gene regulation by chimeric transcription factors.
2024-06-18 | PXD052856 | Pride
Project description:Ewing sarcomas are characterized by the presence of EWS/ETS fusion genes in the absence of other recurrent genetic alterations and mechanisms of tumor heterogeneity that contribute to disease progression remain unclear. Mutations in the Wnt/beta-catenin pathway are rare in Ewing sarcoma but the Wnt pathway modulator LGR5 is often highly expressed, suggesting a potential role for the axis in tumor pathogenesis. We evaluated beta-catenin and LGR5 expression in Ewing sarcoma cell lines and tumors and noted marked intra- and inter-tumor heterogeneity. Tumors with evidence of active Wnt/beta-catenin signaling were associated with increased incidence of tumor relapse and worse overall survival. Paradoxically, RNA sequencing revealed a marked antagonism of EWS/ETS transcriptional activity in Wnt/beta-catenin activated tumor cells. Consistent with this, Wnt/beta-catenin activated cells displayed a phenotype that was reminiscent of Ewing sarcoma cells with partial EWS/ETS loss of function. Specifically, activation of Wnt/beta-catenin induced alterations to the actin cytoskeleton, acquisition of a migratory phenotype and up regulation of EWS/ETS-repressed genes. Notably, activation of Wnt/beta-catenin signaling led to marked induction of tenascin C (TNC), an established promoter of cancer metastasis, and an EWS/ETS-repressed target gene. Loss of TNC function in Ewing sarcoma cells profoundly inhibited their migratory and metastatic potential. Our studies reveal that heterogeneous activation of Wnt/beta-catenin signaling in subpopulations of tumor cells contributes to phenotypic heterogeneity and disease progression in Ewing sarcoma. Significantly, this is mediated, at least in part, by inhibition of EWS/ETS fusion protein function that results in de-repression of metastasis-associated gene programs.
2016-11-30 | GSE75859 | GEO
Project description:Ewing sarcoma (EWS) is a malignant pediatric bone cancer. Most Ewing sarcomas are driven by EWS-FLI1 oncogenic transcription factor that plays roles in transcriptional regulation, DNA damage response, cell cycle checkpoint control, and alternative splicing. USP1, a deubiquitylase which regulates DNA damage and replication stress responses, is overexpressed at both the mRNA and protein levels in EWS cell lines compared to human mesenchymal stem cells, the EWS cell of origin. The functional significance of high USP1 expression in Ewing sarcoma is not known. Here, we identify USP1 as a transcriptional target of EWS-FLI1 and a key regulator of EWS cell survival. We show that EWS-FLI1 knockdown decreases USP1 mRNA and protein levels. ChIP and ChIP-seq analyses show EWS-FLI1 occupancy on the USP1 promoter. Importantly, USP1 knockdown or inhibition arrests EWS cell growth and induces cell death by apoptosis. We observe destabilization of Survivin (also known as BIRC5 or IAP4) and activation of caspases-3 and -7 following USP1 knockdown or inhibition in the absence of external DNA damage stimuli. Notably, EWS cells display hypersensitivity to combinatorial treatment of doxorubicin or etoposide, EWS standard of care drugs, and USP1 inhibitor compared to single agents alone. Together, our study demonstrates that USP1 is regulated by EWS-FLI1, the USP1-Survivin axis promotes EWS cell survival, and USP1 inhibition sensitizes EWS cells to standard of care chemotherapy.
2024-01-26 | PXD041033 | Pride
Project description:We show that EWS-FLI1, an aberrant transcription factor responsible for the pathogenesis of Ewing sarcoma, reprograms gene regulatory circuits by directly inducing or directly repressing enhancers. At GGAA repeats, which lack regulatory potential in other cell types and are not evolutionarily conserved, EWS- FLI1 multimers potently induce chromatin opening, recruit p300 and WDR5, and create de novo enhancers. GGAA repeat enhancers can loop to physically interact with target promoters, as demonstrated by chromosome conformation capture assays. Conversely, EWS-FLI1 inactivates conserved enhancers containing canonical ETS motifs by displacing wild-type ETS transcription factors and abrogating p300 recruitment. ChIP-seq for of 4 histone modifications (H3K27ac, H3K4me1, H3K4me3 and H3K27me3), FLI1, p300, WDR5, ELF1 and GABPA in primary Ewing sarcomas, Ewing sarcoma cell lines (A673 and SKMNC cells), and mesenchymal stem cells (MSC). EWS-FLI1 was knocked down in Ewing sarcoma cell lines with lentiviral shRNAs (shFLI1 and shGFP control). EWS-FLI1 was expressed in MSCs with lentiviral expression vectors (pLIV EWSFLI1 or pLIV empty vector control). * Raw data not provided for the MSC and Primary Ewing sarcoma samples. *
2014-10-30 | E-GEOD-61944 | biostudies-arrayexpress
Project description:Ewing sarcoma is a highly aggressive tumor characterized by a translocation between members of the FET family of RNA binding proteins and one of several ETS transcription factors, with the most common translocation being EWS-FLI1. EWS-FLI1 leads to changes in gene expression through mechanisms that are not completely understood. We performed RNA sequencing analysis on primary pediatric human mesenchymal progenitor cells (pMPCs) expressing EWS-FLI1 in order to identify novel target genes. This analysis identified lnc277 as a previously uncharacterized long non-coding RNA upregulated by EWS-FLI1 in pMPCs. Inhibiting the expression of lnc277 diminished the ability of Ewing sarcoma cell lines to proliferate and form colonies in soft agar whereas inhibiting lnc277 had no effect on other cell types tested. By analyzing gene expression after shRNA knockdown, we found that both EWS-FLI1 and lnc277 repressed many more genes that they induced and that a significant fraction of EWS-FLI1 repressed targets were also repressed by lnc277. Analysis of primary human Ewing sarcoma RNA sequencing data further supports a role for lnc277 in mediating gene repression. We identified hnRNPK as an RNA binding protein that interacts directly with lnc277. We found a significant overlap in the genes repressed by hnRNPK and those repressed by both EWS-FLI1 and lnc277, suggesting that hnRNPK participates in lnc277 mediated gene repression. Thus, lnc277 is a previously uncharacterized long non-coding RNA downstream of EWS-FLI1 that facilitates the development of Ewing sarcoma via the repression of target genes. Our studies identify a novel mechanism of oncogenesis downstream of a chromosomal translocation and underscore the importance of lncRNA-mediated gene repression as a mechanism of EWS-FLI1 transcriptional regulation. A673 Ewing cells expressing an shRNA targeting hnRNPK or control were subjected to paired end RNA sequencing and compared to shGFP control.
2014-11-01 | E-GEOD-60890 | biostudies-arrayexpress