Single Enantiomer of YK-4-279 Demonstrates Specificity in Targeting the Oncogene EWS-FLI1

Barber‐Rotenberg, Julie S.; Selvanathan, Saravana P.; Kong, Yali; Erkizan, Hayriye V.; Snyder, Tara M.; Hong, Sung-Hyeok; Kobs, Christina L.; South, Natalie; Summer, Steven; Monroe, Philip J.; Chruszcz, M.; Dobrev, Veselin; Tosso, Perrer N.; Scher, Lauren; Minor, Władek; Brown, Milton L.; Metallo, Steven J.; Üren, Aykut; Toretsky, Jeffrey A.

doi:10.18632/oncotarget.454

Cited by 83 publications

(82 citation statements)

References 36 publications

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“…Transcription factors were once considered undruggable, but our approach complements other experimental demonstrations for EWS:FLI1 in pediatric Ewing's sarcoma (Barber-Rotenberg et al 2012) and MLL-associated pediatric leukemias (Chen et al 2013). While additional preclinical studies remain to be done, it is exciting to consider that entinostat might reduce PAX3:FOXO1 levels sufficiently to convert fusion-positive RMS with a poor prognosis to a biological state akin to fusion-negative RMS, which portends a significantly better prognosis even when metastatic (Sorensen et al 2002;Missiaglia et al 2012).…”

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confidence: 99%

Lineage of origin in rhabdomyosarcoma informs pharmacological response

et al. 2014

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Lineage or cell of origin of cancers is often unknown and thus is not a consideration in therapeutic approaches. Alveolar rhabdomyosarcoma (aRMS) is an aggressive childhood cancer for which the cell of origin remains debated. We used conditional genetic mouse models of aRMS to activate the pathognomonic Pax3:Foxo1 fusion oncogene and inactivate p53 in several stages of prenatal and postnatal muscle development. We reveal that lineage of origin significantly influences tumor histomorphology and sensitivity to targeted therapeutics. Furthermore, we uncovered differential transcriptional regulation of the Pax3:Foxo1 locus by tumor lineage of origin, which led us to identify the histone deacetylase inhibitor entinostat as a pharmacological agent for the potential conversion of Pax3:Foxo1-positive aRMS to a state akin to fusion-negative RMS through direct transcriptional suppression of Pax3:Foxo1.

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confidence: 99%

Lineage of origin in rhabdomyosarcoma informs pharmacological response

et al. 2014

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“…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.…”

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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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“…Although the S-enantiomer has been previously demonstrated to be considerably more potent than the R-enantiomer or the racemic mixture (34,35,47), a recent report by Hong and colleagues (34,36) demonstrates little difference in antineoplastic and anti-helicase activities between the S-enantiomer and racemic mixture. In fact, only racemic YK-4-279 was capable of improving survival in their Ewing sarcoma xenograft model (34), an unexpected finding the authors ascribed to altered absorption or enhanced elimination of the S-enantiomer in the presence of the R-enantiomer.…”

Section: Discussionmentioning

confidence: 97%

“…Previous reports indicate that YK-4-279 is both potent (half-maximal IC 50 between 0.5 and 2 mmol/L) and specific for Ewing sarcoma, having 10-fold greater activity in a panel of EWS-FLI1-positive Ewing sarcoma cell lines compared with other malignant cell lines devoid of the fusion protein (32). Both the racemic and more potent S-enantiomer (S-YK) demonstrate antineoplastic activity in murine xenograft models; yet, their short half-life and limited solubility have precluded rapid adaption to earlyphase human clinical trials (34,35). In addition, Erkizan and colleagues (36) demonstrated recently that EWS-FLI1 inhibited helicase activity of RHA and this inhibition was reversed by S-YK, suggesting a novel role for EWS-FLI1 in RNA processing.…”

Section: Introductionmentioning

confidence: 99%

An Oral Formulation of YK-4-279: Preclinical Efficacy and Acquired Resistance Patterns in Ewing Sarcoma

Lamhamedi-Cherradi

Menegaz

Ramamoorthy

et al. 2015

Molecular Cancer Therapeutics

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Ewing sarcoma is a transcription factor-mediated pediatric bone tumor caused by a chromosomal translocation of the EWSR1 gene and one of several genes in the ETS family of transcription factors, typically FLI1 or ERG. Full activity of the resulting oncogenic fusion protein occurs only after binding RNA helicase A (RHA), and novel biologically targeted small molecules designed to interfere with that interaction have shown early promise in the preclinical setting. Herein, we demonstrate marked preclinical antineoplastic activity of an orally bioavailable formulation of YK-4-279 and identify mechanisms of acquired chemotherapy resistance that may be exploited to induce collateral sensitivity. Daily enteral administration of YK-4-279 led to significant delay in Ewing sarcoma tumor growth within a murine model. In advance of anticipated early-phase human clinical trials, we investigated both de novo and acquired mechanism(s) by which Ewing sarcoma cells evade YK-4-279-mediated cell death. Drug-resistant clones, formed by chronic in vitro exposure to steadily increased levels of YK-4-279, overexpressed c-Kit, cyclin D1, pStat3(Y705), and PKC isoforms. Interestingly, cross-resistance to imatinib and enzastaurin (selective inhibitors of c-Kit and PKC-b, respectively), was observed and the use of YK-4-279 with enzastaurin in vitro led to marked drug synergy, suggesting a potential role for combination therapies in the future. By advancing an oral formulation of YK-4-279 and identifying prominent mechanisms of resistance, this preclinical research takes us one step closer to a shared goal of curing adolescents and young adults afflicted by Ewing sarcoma.

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Single Enantiomer of YK-4-279 Demonstrates Specificity in Targeting the Oncogene EWS-FLI1

Cited by 83 publications

References 36 publications

Lineage of origin in rhabdomyosarcoma informs pharmacological response

Lineage of origin in rhabdomyosarcoma informs pharmacological response

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

An Oral Formulation of YK-4-279: Preclinical Efficacy and Acquired Resistance Patterns in Ewing Sarcoma

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