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High-throughput small molecule screen identifies inhibitors of aberrant chromatin accessibility

ABSTRACT: Mutations in chromatin-modifying proteins and transcription factors are commonly associated with a wide variety of cancers. Through gain- or loss-of-function, these mutations may result in characteristic alterations of accessible chromatin, indicative of shifts in the landscape of regulatory elements genome-wide. The identification of compounds that reverse a specific chromatin signature could lead to chemical probes or potential therapies. To explore whether chromatin accessibility could serve as a platform for small molecule screening, we adapted formaldehyde-assisted isolation of regulatory elements (FAIRE), a chemical method to enrich for nucleosome-depleted genomic regions, as a high-throughput, automated assay. After demonstrating the validity and robustness of this approach, we applied this method to screen an epigenetically targeted small molecule library by evaluating regions of aberrant nucleosome depletion mediated by EWSR1-FLI1, the chimeric transcription factor critical for the bone and soft tissue tumor Ewing sarcoma. As a class, histone deacetylase inhibitors were greatly overrepresented among active compounds. These compounds resulted in diminished accessibility at targeted sites by disrupting transcription of EWSR1-FLI1. Capitalizing on precise differences in chromatin accessibility for drug discovery efforts offers significant advantages because it does not depend on the a priori selection of a single molecular target and may detect novel biologically relevant pathways.

ORGANISM(S): Homo sapiens

PROVIDER: GSE61735 | GEO | 2015/09/24

SECONDARY ACCESSION(S): PRJNA261972

REPOSITORIES: GEO

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High-throughput small molecule screen identifies inhibitors of aberrant chromatin accessibility

Project description:Mutations in chromatin modifying proteins are associated with a variety of cancers. These mutations may result in characteristic patterns of accessible chromatin indicative of altered nucleosome positioning and depletion at regulatory elements. We describe the adaptation of Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE), a chemical method to enrich for nucleosome-depleted genomic regions, as a high-throughput, automated assay. We applied this method in a screen of epigenetically targeted small molecules by evaluating regions of aberrant nucleosome depletion mediated by EWS-FLI1, the chimeric transcription factor critical for the bone and soft tissue tumor Ewing Sarcoma. We identified a novel compound, UNC0621, which exhibited a dose-dependent reduction in chromatin accessibility, decreased Ewing Sarcoma cell viability, and inhibited anchorage-independent growth. We demonstrate that chromatin accessibility can be exploited for drug discovery efforts offering the advantage that it does not depend on the a priori selection of a single molecular target. Identified compounds can then be used for biological target discovery and potential therapeutic validation. HT-FAIRE was performed on Human Umbilical Vein Endothelial Cells (HUVEC) in duplicate. No reference/control experiment is necessary.

2015-09-24 | E-GEOD-61735 | biostudies-arrayexpress

Single cell RNA-Seq of A673/TR/shEF cell line - time course

Project description:We report the application of single-molecule-based sequencing technology for high-throughput profiling of genes regulated under EWSR1-FLI1 expression in A673/TR/shEF. EWSR1-FLI1 specific small hairpin RNA was induced in A673/TR/shEF cells by adding DOX at 1 ug/mL. After 7 days, DOX was removed to allow silencing of the shRNA and induction of EWSR1-FLI1. Cells were harvested at seven different times points: 0 day (d7), 2 days (d9), 3 days (d10), 4 days (d11), 7 days (d14), 10 days (d17) and 15 days (d22) after DOX removal.

2020-02-14 | GSE130019 | GEO

RING1B recruits EWSR1-FLI1 and cooperates in the remodelling of chromatin necessary for Ewing sarcoma tumorigenesis [microarray]

Project description:EWSR1-FLI1 genome reprogramming through remodeling of enhancers is determinant for Ewing sarcoma (ES) tumorigenesis. We describe a non-canonical function of RING1B, a PRC1 subunit highly expressed in primary ES tumors, co-localizing genome wide with EWSR1-FLI1 in active enhancers. While retaining its repressive canonical activity, we find RING1B as necessary for the expression of key EWSR1-FLI1 activated targets like NKX2-2, SOX2 or IGF1 where it mainly exerts a role in promoting oncogene recruitment to enhancers. Knockdown of RING1B is sufficient to impair growth of tumor xenografts and expression of EWSR1-FLI1 induced neomorphic enhancers in vivo. Restoration of RING1B ubiquitin ligase activity by the AURKB inhibitor AZD1152 decreases expression of RING1B/EWSR1-FLI1 common targets. Overall, our findings demonstrate RING1B as a critical modulator of EWSR1-FLI1 induced chromatin remodeling.

2020-10-27 | GSE133425 | GEO

RING1B recruits EWSR1-FLI1 and cooperates in the remodelling of chromatin necessary for Ewing sarcoma tumorigenesis [ChIP-Seq]

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Therapeutic Targeting Ewing Sarcoma through Inhibition of the FACT Complex (ChIP-Seq)

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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Therapeutic Targeting Ewing Sarcoma through Inhibition of the FACT Complex (RNA-Seq II)

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Therapeutic Targeting Ewing Sarcoma through Inhibition of the FACT Complex (RNA-Seq I)

2022-12-31 | GSE195803 | GEO

Gene expression profile upon EWSR1::FLI1 knockdown in Ewing sarcoma cells A673 in vitro

Project description:Ewing sarcoma, a rare and aggressive pediatric cancer, is characterized by chromosomal translocations that give rise to chimeric transcription factors. The most frequent of these chromosomal translocations is the t(11;22) that produces the fusion of the EWSR1 and FLI1 genes to generate the chimeric transcription factor EWSR1::FLI1. EWSR1::FLI1 is the main oncogenic event in Ewing's sarcoma. Recently, it has been proposed that EWSR1::FLI1 levels may fluctuate in Ewing sarcoma cells, giving rise to two cell populations: cells expressing low levels of EWSR1::FLI1 are characterized by a more migratory and invasive phenotype, while cells expressing high levels of EWSR1::FLI1 are more proliferative. The identification and functional characterization of EWSR1::FLI1 gene targets is therefore relevant to understanding the pathobiology of Ewing sarcoma, which in turn could contribute to the identification of new therapeutic targets. Using this approach, we have observed that CD44, a transmembrane protein involved in cell adhesion and migration and associated with metastasis in various cancer types, is overexpressed in the EWSR1::FLI1-low phenotype. Our results suggest that CD44 may play a role in regulating cell migration in Ewing sarcoma cells and thus contribute to the spread of tumor cells.

2023-07-25 | GSE232739 | GEO

Knowk-down of EWSR1-FLI1 in Ewing sarcoma cell lines (RNA-seq)

Project description:EWSR1-FLI1 is a chimeric transcription factor resulting from the pathognomonic translocation present in Ewing sarcoma cells. Here, we silenced EWSR1-FLI1 in different Ewing sarcoma cell lines. RNA from SKNMC, TC71 and MHH-ES1 cells was extracted 96h post transfection (siCT or siEWSR1-FLI1) or prior doxycycline (day 0) and 7 days after inducing silencing of EWSR1-FLI1 with doxycycline in ASP14 cells. RNA-seq was performed for all conditions.

2022-05-09 | GSE164372 | GEO

Cancer-specific retargeting of BAF complexes by a prion-like domain [RNA-Seq]

Project description:Alterations in the function of transcriptional regulators can orchestrate oncogenic programs that are critical for the transformation and survival of cancer cells. Here we show that the BAF chromatin remodeling complex, which is mutated in over 20% of human tumors, interacts with EWSR1, a member of the FET family of proteins containing prion-like domains that are frequent partners in oncogenic fusions with transcription factors. In Ewing sarcoma, characterized by the EWS-FLI1 fusion, we find that the BAF complex is recruited by EWS-FLI1 to tumor specific enhancers at GGAA microsatellite repeats and contributes to the activation of target genes. This process depends on tyrosine residues that are necessary for the aggregation properties of the EWSR1 prion-like domain and is a neomorphic feature of EWS-FLI1 compared to the wild type ETS transcription factor FLI1. Furthermore, fusion of short fragments of the EWSR1 prion-like domain to FLI1 is sufficient to recapitulate EWS-FLI1-mediated gene expression. Our studies demonstrate that the aggregation properties of prion-like domains can retarget chromatin regulatory complexes to establish and maintain oncogenic gene expression and proliferation.

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