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Gene expression signature baesd screening identifies ribonucleotide reductase as a candidate therapeutic target in Ewing sarcoma

ABSTRACT: There is a critical need in cancer therapeutics to identify targeted therapies that will improve outcomes and decrease toxicities compared to conventional, cytotoxic chemotherapy. Ewing sarcoma is a highly aggressive bone and soft tissue cancer that is caused by the EWS-FLI1 fusion protein. Although EWS-FLI1 is specific for cancer cells, and required for tumorigenesis, directly targeting this transcription factor has proven challenging. Consequently, targeting unique dependencies or key downstream mediators of EWS-FLI1 represent important alternative strategies. We used gene expression data derived from a genetically defined model of Ewing sarcoma to interrogate the Connectivity Map and identify a class of drugs, iron chelators, that downregulate a significant number of EWS-FLI1 target genes. We then identified ribonucleotide reductase M2 (RRM2), the iron-dependent subunit of ribonucleotide reductase (RNR), as one mediator of iron chelator toxicity in Ewing sarcoma cells. Inhibition of RNR in Ewing sarcoma cells led to apoptosis and cell death in vitro and attenuated tumor growth in vivo in a xenograft model. Additionally, we discovered that the sensitivity of Ewing sarcoma cells to inhibition or suppression of RNR is mediated, in part, by high levels of SLFN11, a protein that sensitizes cells to DNA damage. This work demonstrates a unique dependency of Ewing sarcoma cells on RNR and supports further exploration of clinically used inhibitors of RNR as a therapeutic approach in treating this cancer.

ORGANISM(S): Homo sapiens

PROVIDER: GSE79641 | GEO | 2016/12/01

SECONDARY ACCESSION(S): PRJNA316585

REPOSITORIES: GEO

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Reciprocal regulation of extracellular matrix proteins by EWS-FLI1 and AP-1 in Ewing sarcoma cells

Project description:Ribonucleotide reductase (RNR) is the rate limiting enzyme in the synthesis of deoxyribonucleotides and the target of multiple chemotherapy drugs, including gemcitabine and hydroxyurea. In previous work, we identified that inhibition of RNR in Ewing sarcoma tumors upregulates the expression levels of multiple members of the activator protein-1 (AP-1) transcription factor family, including c-Jun and c-Fos, and downregulates the expression of c-Myc. However, the broader functions and downstream targets of AP-1, which are highly context- and cell-dependent, are unknown in Ewing sarcoma tumors. Consequently, in this work, we used genetically defined models, transcriptome profiling, and gene set enrichment analysis to identify that AP-1 and EWS-FLI1, the driver oncogene in most Ewing sarcoma tumors, reciprocally regulate the expression of multiple extracellular matrix proteins, including fibronectins, integrins, and collagens. AP-1 expression in Ewing sarcoma cells also drives, coincident with these perturbations in gene and protein expression, changes in cell morphology and phenotype. Furthermore, we also identified that the EWS-FLI1 oncoprotein dysregulates the expression of multiple AP-1 proteins, aligning with previous reports demonstrating genetic and physical interactions between EWS-FLI1 and AP-1. Overall, these results provide novel insight into the distinct, EWS-FLI1-dependent features of Ewing sarcoma tumors and identify a novel, reciprocal regulation of extracellular matrix components by EWS-FLI1 and AP-1.

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USP1 expression driven by EWS-FLI1 transcription factor stabilizes Survivin and promotes Ewing sarcoma cell survival

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.

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Neo-peptides identification in Ewing sarcoma cell lines using proteomics approach

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2022-05-12 | PXD027309 | Pride

Genome-wide chromatin analysis of Ewing sarcoma (ChIP-seq)

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

Expression data from the inducible expression of EWS-FLI1 (EF) in embryoid bodies

Project description:Oncogenic transformation in Ewing sarcoma tumors is driven by the fusion oncogene EWS-FLI1. The inducible expression of EWS-FLI1 (EF) in embryoid bodies, or collections of differentiating stem cells, generates cells with properties of Ewing sarcoma tumors, including characteristics of transformation. These cell lines exhibit anchorage-independent growth, a lack of contact inhibition and a strong Ewing sarcoma gene expression signature. These cells also demonstrate a requirement for the persistent expression of EWS-FLI1 for cell survival and growth. We used microarrays to detail the effects of doxycycline-inducible expression of EWS-FLI1 on the gene expression signature of cells derived from differentiating stem cells, or embryoid bodies. Triplicate biological replicates were collected and analyzed.

2015-12-31 | E-GEOD-64686 | biostudies-arrayexpress

EWS-FLI1-induced osteosarcoma model unveiled a crucial role of impaired osteogenic differentiation on osteosarcoma development [ChIP-seq]

Project description:EWS-FLI1, a multi-functional fusion oncogene, is exclusively detectable in Ewing sarcomas. However, previous studies reported that a subset of osteosarcomas also harbor EWS-ETS family fusion, suggesting that the fusion gene may be involved in the development of a particular type of osteosarcomas. Here using the doxycycline inducible EWS-FLI1 system, we established an EWS-FLI1-dependent osteosarcoma model from murine bone marrow stromal cells. We revealed that the withdrawal of EWS-FLI1 expression enhances the osteogenic differentiation of sarcoma cells, leading to mature bone formation. Taking advantage of induced pluripotent stem cell (iPSC) technology, we also showed that the sarcoma-derived iPSCs with cancer-related genetic abnormalities exhibited the impaired differentiation program of osteogenic lineage irrespective of the EWS-FLI1 expression. Finally, we demonstrated that EWS-FLI1 contributed to in vitro sarcoma development from the sarcoma-iPSCs after osteogenic differentiation. These findings demonstrated that modulating cellular differentiation is fundamental principle of the EWS-FLI1-induced osteosarcoma development. Furthermore, the in vitro cancer model using sarcoma-iPSCs should provide a novel platform for dissecting relationship between cancer genome and cellular differentiation. Chip-seq in mouse EWS-FLI1-induced osteosarcoma cell lines (SCOS#2 )

2016-03-01 | E-GEOD-72897 | biostudies-arrayexpress

EWS-FLI1-induced osteosarcoma model unveiled a crucial role of impaired osteogenic differentiation on osteosarcoma development [gene expression]

Project description:EWS-FLI1, a multi-functional fusion oncogene, is exclusively detectable in Ewing sarcomas. However, previous studies reported that a subset of osteosarcomas also harbor EWS-ETS family fusion, suggesting that the fusion gene may be involved in the development of a particular type of osteosarcomas. Here using the doxycycline inducible EWS-FLI1 system, we established an EWS-FLI1-dependent osteosarcoma model from murine bone marrow stromal cells. We revealed that the withdrawal of EWS-FLI1 expression enhances the osteogenic differentiation of sarcoma cells, leading to mature bone formation. Taking advantage of induced pluripotent stem cell (iPSC) technology, we also showed that the sarcoma-derived iPSCs with cancer-related genetic abnormalities exhibited the impaired differentiation program of osteogenic lineage irrespective of the EWS-FLI1 expression. Finally, we demonstrated that EWS-FLI1 contributed to in vitro sarcoma development from the sarcoma-iPSCs after osteogenic differentiation. These findings demonstrated that modulating cellular differentiation is fundamental principle of the EWS-FLI1-induced osteosarcoma development. Furthermore, the in vitro cancer model using sarcoma-iPSCs should provide a novel platform for dissecting relationship between cancer genome and cellular differentiation. Microarray in mouse EWS-FLI1-induced osteosarcoma cell lines(SCOS#2 and SCOS#12) and sarcoma(SCOS#2)-derived iPSCs. Total 6 samples were analyzed. We can induce EWS-FLI1 expression by Doxycycline-inducible expression system in SCOS#2 and #12. We investigaed EWS-FLI1 activated genes (Dox ON-High) and EWS-FLI1 repressed genes (Dox OFF-High) in SCOS#2 and #12 sarcoma cell lines. Also, we investigated global gene expression pattern of sarcoma-derived iPSCs (iPSC#2-A1 and #2-B5). A link to this sample file can be found below.

2016-03-01 | E-GEOD-72704 | biostudies-arrayexpress

Next-generation RNA-sequencing data of models of Ewing sarcoma with modulated expression of the lncRNA HOTAIR

Project description:Purpose: Our experimental results demonstrate an essential role for the lncRNA HOTAIR in Ewing sarcoma. We have repressed HOTAIR expression in three Ewing sarcoma cell lines and overexpressed HOTAIR, alone and with EWS-FLI1, in htert-immortalized human mesenchymal stem cells to evaluate its effects on gene expression in this cancer. Methods: RNA-Seq of Ewing sarcoma cell lines with HOTAIR represssed by GapmeR or treated with nonsilencing control, and hTERT-immortalized hMSCs with expression of control GFP, HOTAIR, or HOTAIR and EWS-FLI1, was used for gene expression analysis. Results: Defined gene expression signatures were defined as driven by HOTAIR in each cell line model, with a consensus set of gene identified in the Ewing sarcoma cell lines. Additionally, a set of genes regulated by HOTAIR independently of EWS-FLI1 was identified in the hTERT-hMSC models. Conclusions: HOTAIR expression regulates a set of genes critical to viability, cell adhesion, cell motility, and other markers of EMT and metastasis in Ewing sarcoma, independently of EWS-FLI1.

2018-01-24 | GSE109483 | GEO

High-throughput RNAi cell viability screen to identify selective targets for EWS-FLI1 positive Ewing sarcoma

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2018-09-16 | GSE73092 | GEO

Expression data from the inducible expression of EWS-FLI1 (EF) in embryoid bodies

2015-12-31 | GSE64686 | GEO