Small‐molecule screen identifies modulators of EWS/FLI1 target gene expression and cell survival in Ewing's sarcoma

Boro, Aleksandar; Prêtre, Kathya; Rechfeld, Florian; Thalhammer, Verena; Oesch, Susanne; Wachtel, Marco; Schäfer, Beat W.; Niggli, Felix

doi:10.1002/ijc.27472

Cited by 66 publications

(57 citation statements)

References 45 publications

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“…In contrast to previous efforts to inhibit EWSR1-FLI1 activity that have capitalized on individual target gene expression or physical interactions (31)(32)(33)(34)(35)(36), the application of HT-FAIRE offered a strategy to identify therapeutics based on variation in chromatin accessibility, a universal genomic feature determined by the combined effects of transcriptional regulators and chromatin regulatory proteins (37). Indeed, FAIRE has been shown to detect specific chromatin changes resulting from treatment with anthracyclines that directly interact with DNA (38,39), thereby demonstrating the utility of this assay to explore the function of small molecule compounds.…”

Section: Discussionmentioning

confidence: 99%

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

Pattenden

Simon

Wali

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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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 nucleosomedepleted 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.chromatin | Ewing sarcoma | high throughput screening | FAIRE | histone deacetylase inhibitor A growing range of human cancers have been associated with mutations in genes encoding proteins that regulate chromatin, the assembly of proteins and DNA that control DNAtemplated processes, including transcription and replication (1, 2). Small molecule drugs and chemical probes offer an approach to explore the biological consequences of these mutations and are emerging as a therapeutic strategy to target disease pathways. Drugs targeting histone deacetylase (HDAC) enzymes, the bromodomain reader BRD4, and DNA methylation have already received regulatory approval or have entered clinical testing, and chemical probes have been developed against a broad range of chromatin regulators, such as the methyltransferases (3) DOT1L (4), EZH2 (5-8), and G9a (9, 10), and the reader proteins L3MBTL3 (11) and BRD4 (12-14). However, transcription factors that lack enzymatic activity or binding pockets with targetable molecular features have been considered "undruggable," and a reductionist approach based on identification of their molecular targets has largely failed.The majority of Ewing sarcomas, highly malignant pediatric bone and soft tissue tumors, harbor a chromosomal translocation that joins the amino-terminal domain of EWSR1 with the DNA binding domain of the ETS transcription factor family member FLI1 to generate the chimeric transcription factor EWSR1-FLI1 (15). Translocations with other ETS genes are detected in most of the remaining tumors, y...

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Section: Discussionmentioning

confidence: 99%

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

Pattenden

Simon

Wali

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Boro et al (31) demonstrated that EWS/FLI1 can bind to the promoter of PHLDA1, in vivo and in vitro, leading to its repression. In MEF cells derived from conditional EWS/FLI1 knockin embryos, EWS/FLI1 expression resulted in apoptosis and EWS/FLI1 was able to directly bind the caspase 3 promoter in CHP100 cells with a concomitant increase in endogenous caspase 3 expression (32).…”

Section: Phlda1 Expression and Cell Deathmentioning

confidence: 99%

Pleckstrin homology-like domain, family A, member 1 (PHLDA1) and cancer

Nagai

2016

Biomedical Reports

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Abstract. Pleckstrin homology-like domain, family A, member 1 (PHLDA1) encodes a member of an evolutionarily conserved pleckstrin homology-related domain protein family. It was first identified as a potential transcription factor required for Fas expression and activation-induced apoptosis in mouse T cell hybridomas. The exact molecular and biological functions of PHLDA1 remain to be elucidated. However, its expression is induced by a variety of external stimuli and there is evidence that it may function as a transcriptional activator that acts as a mediator of apoptosis, proliferation, differentiation and cell migration dependent on the cellular type and context. Recently, PHLDA1 has received attention due to its association with cancer. In the present review, the current knowledge of PHLDA1 protein structure, expression regulation and function is summarized. In addition, the current data in the literature is reviewed with regards to the role of PHLDA1 in cancer pathogenesis.

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“…Initial small-molecule screens identified compounds that inhibited EWS/FLI1 modulation of gene expression, including cytarabine (8), mithramycin (9), and midostaurin (10). Other screens have been used to find molecules that bind to EWS/FLI1 or disrupt its ability to bind DNA.…”

Section: Introductionmentioning

confidence: 99%

Phosphoproteomic Profiling Reveals IL6-Mediated Paracrine Signaling within the Ewing Sarcoma Family of Tumors

Anderson

Titz

Akiyama

et al. 2014

Molecular Cancer Research

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Members of the Ewing sarcoma family of tumors (ESFT) contain tumor-associated translocations that give rise to oncogenic transcription factors, most commonly EWS/FLI1. EWS/FLI1 plays a dominant role in tumor progression by modulating the expression of hundreds of target genes. Here, the impact of EWS/FLI1 inhibition, by RNAi-mediated knockdown, on cellular signaling was investigated using mass spectrometry-based phosphoproteomics to quantify global changes in phosphorylation. This unbiased approach identified hundreds of unique phosphopeptides enriched in processes such as regulation of cell cycle and cytoskeleton organization. In particular, phosphotyrosine profiling revealed a large upregulation of STAT3 phosphorylation upon EWS/FLI1 knockdown. However, single-cell analysis demonstrated that this was not a cell-autonomous effect of EWS/FLI1 deficiency, but rather a signaling effect occurring in cells in which knockdown does not occur. Conditioned media from knockdown cells were sufficient to induce STAT3 phosphorylation in control cells, verifying the presence of a soluble factor that can activate STAT3. Cytokine analysis and ligand/receptor inhibition experiments determined that this activation occurred, in part, through an IL6-dependent mechanism. Taken together, the data support a model in which EWS/FLI1 deficiency results in the secretion of soluble factors, such as IL6, which activate STAT signaling in bystander cells that maintain EWS/FLI1 expression. Furthermore, these soluble factors were shown to protect against apoptosis.

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Small‐molecule screen identifies modulators of EWS/FLI1 target gene expression and cell survival in Ewing's sarcoma

Cited by 66 publications

References 45 publications

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

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

Pleckstrin homology-like domain, family A, member 1 (PHLDA1) and cancer

Phosphoproteomic Profiling Reveals IL6-Mediated Paracrine Signaling within the Ewing Sarcoma Family of Tumors

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