In-depth proteome analysis of brain tissue from Ewsr1 gene knock-out mice
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive Plus
ORGANISM(S): Mus Musculus (mouse)
TISSUE(S): Brain, Neuron
DISEASE(S): Disease Free
SUBMITTER: 
Dohyun Han
LAB HEAD: Dohyun Han
PROVIDER: PXD026994 | Pride | 2023-01-16
REPOSITORIES: Pride
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Project description:Cell lines have been essential for major discoveries in cancer including Ewing sarcoma (EwS). EwS is a highly aggressive pediatric bone or soft-tissue cancer characterized by oncogenic EWSR1-ETS fusion transcription factors converting polymorphic GGAA-microsatellites (mSats) into neo-enhancers. However, further detailed mechanistic evaluation of gene regulation in EwS have been hindered by the limited number of well-characterized cell line models. Here, we present the Ewing Sarcoma Cell Line Atlas (ESCLA) comprising 18 EwS cell lines with inducible EWSR1-ETS knockdown that were profiled by whole-genome-sequencing, DNA methylation arrays, gene expression and splicing arrays, mass spectrometry, and ChIP-seq for EWSR1-ETS and histone marks. Systematic analysis of these multi-dimensional data illuminated hundreds of new potential EWSR1-ETS target genes, the nature of EWSR1-ETS-preferred GGAA-mSats, and potential indirect modes of EWSR1-ETS-mediated gene regulation. Moreover, we identified putative co-regulatory transcription factors and heterogeneously regulated EWSR1-ETS target genes that may have implications for the clinical heterogeneity of EwS. Collectively, our freely available ESCLA constitutes an extremely rich resource for EwS research and highlights the power of leveraging multidimensional and comprehensive datasets to unravel principles of heterogeneous gene regulation by dominant fusion oncogenes.
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Project description:Cell lines have been essential for major discoveries in cancer including Ewing sarcoma (EwS). EwS is a highly aggressive pediatric bone or soft-tissue cancer characterized by oncogenic EWSR1-ETS fusion transcription factors converting polymorphic GGAA-microsatellites (mSats) into neo-enhancers. However, further detailed mechanistic evaluation of gene regulation in EwS have been hindered by the limited number of well-characterized cell line models. Here, we present the Ewing Sarcoma Cell Line Atlas (ESCLA) comprising 18 EwS cell lines with inducible EWSR1-ETS knockdown that were profiled by whole-genome-sequencing, DNA methylation arrays, gene expression and splicing arrays, mass spectrometry, and ChIP-seq for EWSR1-ETS and histone marks. Systematic analysis of these multi-dimensional data illuminated hundreds of new potential EWSR1-ETS target genes, the nature of EWSR1-ETS-preferred GGAA-mSats, and potential indirect modes of EWSR1-ETS-mediated gene regulation. Moreover, we identified putative co-regulatory transcription factors and heterogeneously regulated EWSR1-ETS target genes that may have implications for the clinical heterogeneity of EwS. Collectively, our freely available ESCLA constitutes an extremely rich resource for EwS research and highlights the power of leveraging multidimensional and comprehensive datasets to unravel principles of heterogeneous gene regulation by dominant fusion oncogenes.
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Project description:Cell lines have been essential for major discoveries in cancer including Ewing sarcoma (EwS). EwS is a highly aggressive pediatric bone or soft-tissue cancer characterized by oncogenic EWSR1-ETS fusion transcription factors converting polymorphic GGAA-microsatellites (mSats) into neo-enhancers. However, further detailed mechanistic evaluation of gene regulation in EwS have been hindered by the limited number of well-characterized cell line models. Here, we present the Ewing Sarcoma Cell Line Atlas (ESCLA) comprising 18 EwS cell lines with inducible EWSR1-ETS knockdown that were profiled by whole-genome-sequencing, DNA methylation arrays, gene expression and splicing arrays, mass spectrometry, and ChIP-seq for EWSR1-ETS and histone marks. Systematic analysis of these multi-dimensional data illuminated hundreds of new potential EWSR1-ETS target genes, the nature of EWSR1-ETS-preferred GGAA-mSats, and potential indirect modes of EWSR1-ETS-mediated gene regulation. Moreover, we identified putative co-regulatory transcription factors and heterogeneously regulated EWSR1-ETS target genes that may have implications for the clinical heterogeneity of EwS. Collectively, our freely available ESCLA constitutes an extremely rich resource for EwS research and highlights the power of leveraging multidimensional and comprehensive datasets to unravel principles of heterogeneous gene regulation by dominant fusion oncogenes.
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Project description:Ewing sarcoma is driven by fusion proteins containing a low complexity (LC) domain that is intrinsically disordered and a powerful transcriptional regulator. The most common fusion protein found in Ewing sarcoma, EWS-FLI1, takes its LC domain from the RNA-binding protein EWSR1 (Ewing Sarcoma RNA-binding protein 1) and a DNA-binding domain from the transcription factor FLI1 (Friend Leukemia Virus Integration 1). The LC domain in EWS-FLI1 can bind RNA polymerase II (RNA Pol II) and can self-assemble through a process known as phase separation. The ability of EWSR1 and related RNA-binding proteins to assemble into ribonucleoprotein granules in cells has been intensely studied but the role of phase separation in EWS-FLI1 activity is less understood. We investigated the overlapping functions of EWSR1 and EWS-FLI1 in controlling gene expression and tumorigenic cell growth in Ewing sarcoma, and our results suggested that these proteins function closely together. We then studied the nature of interactions among EWS-FLI1, EWSR1, and RNA Pol II. We observed EWSR1 and RNA Pol II to be present in protein granules in cells. We then identified protein granules in cells associated with the fusion protein, EWS-FLI1. The tyrosine residues in the LC domain are required for the abilities of EWS-FLI1 to bind its partners, EWSR1 and RNA Pol II, and to incorporate into protein granules. These data suggest that interactions among EWS-FLI1, RNA Pol II, and EWSR1 in Ewing sarcoma can occur in the context of a molecular scaffold found within protein granules in the cell.
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Project description:EWS/ETS fusion transcription factors, most commonly EWSR1-FLI1, drive initiation and progression of Ewing sarcoma (EwS), a highly aggressive childhood cancer of bone and soft tissues. Even though direct targeting EWSR1-FLI1 is a formidable challenge, epigenetic or transcriptional modulators have been recently proved to be promising therapeutic targets for indirectly disrupting its expression and/or function. Here, we performed transcriptome and functional genomics dataset analyses, and combined with small molecule screening of EwS lines to identify novel epigenetic/transcriptional-targeted therapeutic strategies. SSRP1 and SUPT16H, two subunits of the Facilitates Chromatin Transcription (FACT) complex, are both found to be EWSR1-FLI1-induced essential oncogenes in EwS. The FACT-targeted drug CBL0137 exhibits potent therapeutic efficacy against multiple EwS preclinical models in vitro and in vivo. Mechanistically, the FACT complex and EWS-FLI1 form oncogenic positive feedback loop via mutual transcriptional regulation and activation, and cooperatively promote cell cycle/DNA replication process and IGF1R-PI3K-AKT-mTOR pathway to drive EwS oncogenesis. The FACT inhibitor drug CBL0137 effectively targets the EWSR1-FLI1-FACT circuit, resulting in transcriptional disruption of EWS-FLI1, SSRP1 and their downstream effector oncogenic signatures. Our study illustrates a crucial role of the FACT complex in facilitating the expression and function of EWSR1-FLI1 and demonstrates FACT inhibition as a novel therapeutic strategy against EwS via indirect targeting the oncogenic fusion TF, providing preclinical support for adding EwS to CBL0137’s future clinical trials.
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Project description:EWS/ETS fusion transcription factors, most commonly EWSR1-FLI1, drive initiation and progression of Ewing sarcoma (EwS), a highly aggressive childhood cancer of bone and soft tissues. Even though direct targeting EWSR1-FLI1 is a formidable challenge, epigenetic or transcriptional modulators have been recently proved to be promising therapeutic targets for indirectly disrupting its expression and/or function. Here, we performed transcriptome and functional genomics dataset analyses, and combined with small molecule screening of EwS lines to identify novel epigenetic/transcriptional-targeted therapeutic strategies. SSRP1 and SUPT16H, two subunits of the Facilitates Chromatin Transcription (FACT) complex, are both found to be EWSR1-FLI1-induced essential oncogenes in EwS. The FACT-targeted drug CBL0137 exhibits potent therapeutic efficacy against multiple EwS preclinical models in vitro and in vivo. Mechanistically, the FACT complex and EWS-FLI1 form oncogenic positive feedback loop via mutual transcriptional regulation and activation, and cooperatively promote cell cycle/DNA replication process and IGF1R-PI3K-AKT-mTOR pathway to drive EwS oncogenesis. The FACT inhibitor drug CBL0137 effectively targets the EWSR1-FLI1-FACT circuit, resulting in transcriptional disruption of EWS-FLI1, SSRP1 and their downstream effector oncogenic signatures. Our study illustrates a crucial role of the FACT complex in facilitating the expression and function of EWSR1-FLI1 and demonstrates FACT inhibition as a novel therapeutic strategy against EwS via indirect targeting the oncogenic fusion TF, providing preclinical support for adding EwS to CBL0137’s future clinical trials.
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Project description:EWS/ETS fusion transcription factors, most commonly EWSR1-FLI1, drive initiation and progression of Ewing sarcoma (EwS), a highly aggressive childhood cancer of bone and soft tissues. Even though direct targeting EWSR1-FLI1 is a formidable challenge, epigenetic or transcriptional modulators have been recently proved to be promising therapeutic targets for indirectly disrupting its expression and/or function. Here, we performed transcriptome and functional genomics dataset analyses, and combined with small molecule screening of EwS lines to identify novel epigenetic/transcriptional-targeted therapeutic strategies. SSRP1 and SUPT16H, two subunits of the Facilitates Chromatin Transcription (FACT) complex, are both found to be EWSR1-FLI1-induced essential oncogenes in EwS. The FACT-targeted drug CBL0137 exhibits potent therapeutic efficacy against multiple EwS preclinical models in vitro and in vivo. Mechanistically, the FACT complex and EWS-FLI1 form oncogenic positive feedback loop via mutual transcriptional regulation and activation, and cooperatively promote cell cycle/DNA replication process and IGF1R-PI3K-AKT-mTOR pathway to drive EwS oncogenesis. The FACT inhibitor drug CBL0137 effectively targets the EWSR1-FLI1-FACT circuit, resulting in transcriptional disruption of EWS-FLI1, SSRP1 and their downstream effector oncogenic signatures. Our study illustrates a crucial role of the FACT complex in facilitating the expression and function of EWSR1-FLI1 and demonstrates FACT inhibition as a novel therapeutic strategy against EwS via indirect targeting the oncogenic fusion TF, providing preclinical support for adding EwS to CBL0137’s future clinical trials.
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Project description:Cell lines have been essential for major discoveries in cancer including Ewing sarcoma (EwS). EwS is a highly aggressive pediatric bone or soft-tissue cancer characterized by oncogenic EWSR1-ETS fusion transcription factors converting polymorphic GGAA-microsatellites (mSats) into neo-enhancers. However, further detailed mechanistic evaluation of gene regulation in EwS have been hindered by the limited number of well-characterized cell line models. Here, we present the Ewing Sarcoma Cell Line Atlas (ESCLA) comprising 18 EwS cell lines with inducible EWSR1-ETS knockdown that were profiled by whole-genome-sequencing, DNA methylation arrays, gene expression and splicing arrays, mass spectrometry, and ChIP-seq for EWSR1-ETS and histone marks. Systematic analysis of these multi-dimensional data identified GATA2 and E2F2 as EWSR1-ETS-driven putative co-regulatory transcription factors. To evaluate the relevance of these transcrition factors, RNA interference in a Ewing sarcoma cell line was employed with subsequent Affymetrix Exon array profiling. The expression data were analysed in synopsis with the effects of EWSR1-FLI1 in the same cell line.
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Project description:Ewing sarcoma (EwS) is an aggressive pediatric bone and soft tissue cancer. It is driven by a fusion oncogene involving the EWSR1 gene, predominantly EWS-FLI1. EwS is exquisitely sensitive to genotoxic agents, but the molecular basis for this sensitivity is relatively underexplored. We have discovered that EwS displays alterations in damage-induced transcription regulation and an accumulation of R-loops. Consequently, we observe an increase in replication stress but impaired homologous recombination. We demonstrate an enriched interaction between BRCA1 and the elongating transcription machinery and suggest that this interaction may be the underlying cause for impaired recombination. Investigation into the unique transcription profile of EwS also revealed upregulated mechanisms (including RNASEH2, FEN1 and Fanconi Anemia pathway) to compensate for the increased EWS-FLI1 induced toxicity. Finally, we have uncovered a novel role for EWSR1 transcriptional response to damage, suppressing R-loops and promoting homologous recombination. Taken together, our findings improve the current understanding of wild-type EWSR1 function, elucidate the mechanistic basis for EwS chemosensitivity (including PARP1 inhibitors) and suggest potential targets for novel second-line therapy.
2018-03-09 | GSE87324 | GEO
Project description:Ewing sarcomas harbor few mutations beyond the chromosomal translocation that initiates disease and the mechanistic basis for the metastasis of these tumors remains poorly understood. The epigenome of Ewing sarcoma (EWS) 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 repressive activities of the EWSR1::FLI1/ERG fusion oncoproteins. Focusing on one of the repressed EWSR1::FLI1/ERG target genes, ETS1, we detected EWSR1::FLI1 binding and a H3K27me3 repressive mark at this locus. Depletion of EWSR1::FLI1 results in ETS1’s binding of promoter regions, and we show ETS1 regulates the expression of multiple proteins that function in extracellular matrix organization including TENSIN3 (TSN3). Interestingly, TSN3 expression in EWS tumors significantly correlates with that of ETS1 (0.85, FDR <0.01). TNS3 is a focal adhesion protein that contributes to tumor cell migration by connecting the cytoplasmic tail of integrins to the actin cytoskeleton. EWS cell lines, in which we activated ETS1 expression (CRISPRa) exhibited increased TNS3 expression and a migratory phenotype. Critically, the activated ETS1 EWS cell lines show TNS3 accumulation at leading cell edges, with F-actin cytoskeletal reorganization, a phenotype associated with cell migration.
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