A small molecule blocking oncogenic protein EWS-FLI1 interaction with RNA helicase A inhibits growth of Ewing's sarcoma

Erkizan, Hayriye V.; Kong, Yali; Merchant, Melinda S.; Schlottmann, Silke; Barber‐Rotenberg, Julie S.; Yuan, Linshan; Abaan, Ogan D.; Chou, T.-F.; Dakshanamurthy, Sivanesan; Brown, Milton L.; Üren, Aykut; Toretsky, Jeffrey A.

doi:10.1038/nm.1983

Cited by 384 publications

(379 citation statements)

References 41 publications

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“…The possibility remains that the N-terminal dsRBD(s) interact with distal RHA domains; future experiments to test this model are warranted. The results will contribute to understanding the essential role of RHA in normal cells and the apparent contribution of RHA dysfunction to neoplastic growth (35)(36)(37). The outcomes will fuel fundamental understanding of RNA helicases across cell biology and at the virushost interface.…”

Section: Discussionmentioning

confidence: 84%

Features of Double-stranded RNA-binding Domains of RNA Helicase A Are Necessary for Selective Recognition and Translation of Complex mRNAs

Ranji

Shkriabai²,

Kvaratskhelia³

et al. 2011

Journal of Biological Chemistry

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Section: Discussionmentioning

confidence: 84%

Features of Double-stranded RNA-binding Domains of RNA Helicase A Are Necessary for Selective Recognition and Translation of Complex mRNAs

Ranji

Shkriabai²,

Kvaratskhelia³

et al. 2011

Journal of Biological Chemistry

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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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“…The inclusion of CASP3 exon 2 with EWS-FLI1 reduction places an internal ribosome entry site into the 5′-UTR of the mRNA (72). This 5′-UTR may enhance the translation of caspase-3, thus increasing its protein levels and in part explaining the increased apoptosis seen with YK-4-279 treatment (21). CASP3 exon 2 has also been recognized as containing a polymorphism that increases the likelihood of head and neck cancer (61).…”

Section: Yk-4-279 Alters Splicing Rather Than Direct Transcriptional mentioning

confidence: 99%

“…We have validated a small molecule probe, YK-4-279, an enantio-specific inhibitor that both disrupts RHA interaction from EWS-FLI1 and restores RHA helicase activity (13,21,22). Reagents that specifically disrupt spliceosomal protein interactions are useful for the characterization of spliceosomal networks as well as understanding oncogenic aspects of posttranscriptional modifications.…”

mentioning

confidence: 99%

Oncogenic fusion protein EWS-FLI1 is a network hub that regulates alternative splicing

Selvanathan

Graham

Erkizan

et al. 2015

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

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The synthesis and processing of mRNA, from transcription to translation initiation, often requires splicing of intragenic material. The final mRNA composition varies based on proteins that modulate splice site selection. EWS-FLI1 is an Ewing sarcoma (ES) oncoprotein with an interactome that we demonstrate to have multiple partners in spliceosomal complexes. We evaluate the effect of EWS-FLI1 on posttranscriptional gene regulation using both exon array and RNAseq. Genes that potentially regulate oncogenesis, including CLK1, CASP3, PPFIBP1, and TERT, validate as alternatively spliced by EWS-FLI1. In a CLIP-seq experiment, we find that EWS-FLI1 RNA-binding motifs most frequently occur adjacent to intron-exon boundaries. EWS-FLI1 also alters splicing by directly binding to known splicing factors including DDX5, hnRNP K, and PRPF6. Reduction of EWS-FLI1 produces an isoform of γ-TERT that has increased telomerase activity compared with wild-type (WT) TERT. The small molecule YK-4-279 is an inhibitor of EWS-FLI1 oncogenic function that disrupts specific protein interactions, including helicases DDX5 and RNA helicase A (RHA) that alters RNA-splicing ratios. As such, YK-4-279 validates the splicing mechanism of EWS-FLI1, showing alternatively spliced gene patterns that significantly overlap with EWS-FLI1 reduction and WT human mesenchymal stem cells (hMSC). Exon array analysis of 75 ES patient samples shows similar isoform expression patterns to cell line models expressing EWS-FLI1, supporting the clinical relevance of our findings. These experiments establish systemic alternative splicing as an oncogenic process modulated by EWS-FLI1. EWS-FLI1 modulation of mRNA splicing may provide insight into the contribution of splicing toward oncogenesis, and, reciprocally, EWS-FLI1 interactions with splicing proteins may inform the splicing code.T he alternative splicing of mRNA expands the diversity of the human proteome through evolution and ontogeny (1, 2). Spliceosomal network interactions, including proteins that recognize splice enhancer and silencer regions, are critical for the regulation of alternative splicing leading to protein isoforms with disparate functionality (3). Alternative splicing provides both a method by which to categorize subsets of cancers and an avenue for more effective targeted treatments (4). However, the spliceosomal protein interaction networks that are specific to cancer have not been systematically defined; alternative splicing can also change protein-protein interactions within networks (5). A systems biology approach can be used to study the relationship between splicing and oncogenesis by using tumor models with chromosomal translocations whose expressed fusion proteins have putative roles in splicing (6, 7).Fusion proteins produced by chromosomal translocations in sarcomas often contain the amino-terminal portion of EWS and are classified as transcription factors due to the presence of a canonical carboxyl-terminal DNA-binding domain (6). EWS-FLI1 is a well-established ES oncoprotein and is re...

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A small molecule blocking oncogenic protein EWS-FLI1 interaction with RNA helicase A inhibits growth of Ewing's sarcoma

Cited by 384 publications

References 41 publications

Features of Double-stranded RNA-binding Domains of RNA Helicase A Are Necessary for Selective Recognition and Translation of Complex mRNAs

Features of Double-stranded RNA-binding Domains of RNA Helicase A Are Necessary for Selective Recognition and Translation of Complex mRNAs

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

Oncogenic fusion protein EWS-FLI1 is a network hub that regulates alternative splicing

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