Dissection of EWS-FLI1 oncogenic mechanisms and small molecule targeting

The spliceosome is a collection of protein and non-coding RNA subunits, interacting to bind, cleave, and 连接RNA。 Alternative splicing can contribute to cancer development through the expression of novel 蛋白质亚型。 Oncogenic driver genes that modulate splicing arise from mutations, over-expression, and chromosomal translocations in leukemias, carcinomas, and sarcomas. EWS-FLI1就是这样一种致癌物质 fusion protein derived from a tumor-specific chromosomal translocation in Ewing sarcoma (ES).上一页 investigations have described the modulation of transcription by EWS-FLI1 and connected transcription with its oncogenic potential, yet some mutants that do not bind DNA still have oncogenic activity.这表明 EWS-FLI1 has oncogenic capacity outside of transcriptional regulation.我们发现EWS-FLI1 interacts with spliceosomal proteins and significantly alters the isoform landscape in ES.其他人已经表明 some splicing factors are critical for EWS-FLI1 oncogenesis.然而，剪接对 ES 的贡献 oncogenesis as well as the role of EWS-FLI1-interacting proteins in the spliceosome, remain unknown. EWS-FLI1 通常被称为“不可成药”的靶标。 To develop alternative strategies for therapeutic targeting of EWS-FLI1, we identified compounds that directly bind to EWS-FLI1 and inhibit its interaction 与特定的合作伙伴。 In 2009 we reported one such compound, named YK-4-279, that blocks the EWS- FLI1 与关键蛋白质伙伴结合。 ES cells treated with YK-4-279 show altered splicing patterns that 模拟 EWS-FLI1 损失。 An analog of YK-4-279, TK216, is now in phase I clinical trials in ES patients.我们 因此假设少数关键基因的 RNA 剪接调节是一个速率- 除了作为转录的典型作用之外，EWS-FLI1 的限制致癌机制 调节器。我们将此提案集中于三个目标。 (1) We will determine the relative effects of EWS-FLI1 通过表征关键结构域和残基来研究突变体的转录和剪接。我们随后将 determine the effects of these mutants on oncogenesis. (2) 我们将定义EWS-FLI1之间的交互 and splicing factors required for differential splicing. (3) We will investigate how EWS-FLI1-induced splice 靶基因的亚型转换有助于肿瘤发生。我们证明 EWS-FLI1 诱导 differential splicing of a number of target genes in human mesenchymal stem cells (hMSC);现在我们将 识别 EWS-FLI1 中诱导差异剪接的关键域和残基。我们的方法也将 回答 EWS-FLI1 是否创建 ES 中唯一发现的从头剪接变体，或者 EWS- FLI1 是导致剪接体的途径的一部分，该剪接体具有与发生的剪接活性相似的新颖剪接活性 在骨髓增生异常综合征中。对特定肿瘤中改变的剪接驱动程序的详细了解将 增强我们对肿瘤发生的理解，为个性化医疗提供分层标记，以及 为新的抗癌目标的方法提供信息。

The spliceosome is a collection of protein and non-coding RNA subunits, interacting to bind, cleave, and ligate RNA. Alternative splicing can contribute to cancer development through the expression of novel protein isoforms. Oncogenic driver genes that modulate splicing arise from mutations, over-expression, and chromosomal translocations in leukemias, carcinomas, and sarcomas. EWS-FLI1 is one such oncogenic fusion protein derived from a tumor-specific chromosomal translocation in Ewing sarcoma (ES). Previous investigations have described the modulation of transcription by EWS-FLI1 and connected transcription with its oncogenic potential, yet some mutants that do not bind DNA still have oncogenic activity. This suggests EWS-FLI1 has oncogenic capacity outside of transcriptional regulation. We have found that EWS-FLI1 interacts with spliceosomal proteins and significantly alters the isoform landscape in ES. Others have shown some splicing factors are critical for EWS-FLI1 oncogenesis. Yet, the contribution of splicing to ES oncogenesis as well as the role of EWS-FLI1-interacting proteins in the spliceosome, remain unknown. EWS-FLI1 was often termed an `undruggable' target. To develop alternative strategies for therapeutic targeting of EWS-FLI1, we identified compounds that directly bind to EWS-FLI1 and inhibit its interaction with specific partners. In 2009 we reported one such compound, named YK-4-279, that blocks the EWS- FLI1 binding to a key protein partner. ES cells treated with YK-4-279 show altered splicing patterns that mimic EWS-FLI1 loss. An analog of YK-4-279, TK216, is now in phase I clinical trials in ES patients. We therefore hypothesize that regulation of RNA splicing of a small number of critical genes is a rate- limiting oncogenic mechanism of EWS-FLI1 in addition to its canonical role as a transcription regulator. We focus this proposal on three aims. (1) We will determine the relative effects of EWS-FLI1 mutants on transcription and splicing through characterizing key domains and residues. We will then determine the effects of these mutants on oncogenesis. (2) We will define interactions between EWS-FLI1 and splicing factors required for differential splicing. (3) We will investigate how EWS-FLI1-induced splice isoform switching of target genes contributes to oncogenesis. We demonstrated that EWS-FLI1 induces differential splicing of a number of target genes in human mesenchymal stem cells (hMSC); now we will identify key domains and residues in EWS-FLI1 that induce differential splicing. Our approach will also answer whether EWS-FLI1 creates de novo splice variants that are uniquely found in ES or whether EWS- FLI1 is part of a pathway that leads to a spliceosome with novel splicing activities similar to those occurring in myelodysplastic syndromes. Detailed knowledge of splicing drivers that are altered in specific tumors will enhance our understanding of oncogenesis, lead to stratification markers for personalized medicine, and inform approaches to new anti-cancer targets.