Dissection of EWS-FLI1 oncogenic mechanisms and small molecule targeting

剪接体是蛋白质和非编码RNA亚基的集合，相互作用以结合、切割和 连接核糖核酸。选择性剪接可以通过表达新的基因来促进癌症的发展 蛋白质异构体。调节剪接的致癌驱动基因来自突变、过度表达和 白血病、癌症和肉瘤中的染色体易位。EWS-FLI1就是这样一种致癌基因 尤文肉瘤(ES)中肿瘤特异性染色体易位的融合蛋白。上一首 研究已经描述了EWS-FLI1对转录的调控，并将转录与 它的致癌潜力，但一些不结合DNA的突变体仍然具有致癌活性。这表明 EWS-FLI1具有转录调控外的致癌能力。我们发现EWS-FLI1 与剪接体蛋白相互作用，显著改变ES中的异构体景观。其他人已经展示了 某些剪接因子在EWS-FLI1的致癌过程中起关键作用。然而，剪接对ES的贡献 肿瘤的发生以及EWS-FLI1相互作用蛋白在剪接体中的作用尚不清楚。 EWS-FLI1通常被称为“不能下药”的靶标。开发替代治疗策略 以EWS-FLI1为靶点，我们发现了直接与EWS-FLI1结合并抑制其相互作用的化合物 与特定的合作伙伴。2009年，我们报道了一种这样的化合物，命名为YK-4-279，它可以阻断EWS- FLI1结合到一个关键的蛋白质伙伴。经YK-4-279处理的ES细胞表现出不同的剪接模式 模拟EWS-FLI1损失。YK-4-279的类似物TK216目前正处于ES患者的I期临床试验中。我们 因此，假设对少数关键基因的RNA剪接的调节是一个速率- EWS-FLI1的致癌限制机制及其转录的规范作用 调整器。我们将这一提议集中在三个目标上。(1)我们将确定EWS-FLI1的相对影响 突变体通过表征关键结构域和残基对转录和剪接的影响。到时候我们会的 确定这些突变体对肿瘤发生的影响。(2)我们将定义EWS-FLI1之间的交互 以及差异剪接所需的剪接因子。(3)我们将研究EWS-FLI1是如何诱导剪接的 靶基因的异构体转换有助于肿瘤的发生。我们证明了EWS-FLI1诱导 人间充质干细胞(HMSC)中多个靶基因的差异剪接；现在我们将 确定EWS-FLI1中诱导差异剪接的关键结构域和残基。我们的方法也将 回答EWS-FLI1是否创建了在ES中唯一发现的从头开始的剪接变体，或者EWS-FLI1是否 FLI1是通往剪接体的途径的一部分，该剪接体具有与发生的剪接类似的新的剪接活性 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 加强我们对肿瘤发生的理解，导致个性化医学的分层标志，以及 为新的抗癌目标提供信息。

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.