Dissection of EWS-FLI1 oncogenic mechanisms and small molecule targeting

剖析 EWS-FLI1 致癌机制和小分子靶向

• 批准号: 10418748
• 负责人: JEFFREY A TORETSKY
• 金额: $ 36.39万
• 依托单位: GEORGETOWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10418748
• 关键词: ARID1A gene; Alternative Splicing; Apoptosis; Binding; Bioinformatics; Biophysics; CRISPR screen; Cancer Patient; Carcinoma; Cell Line; Cells; Cessation of life; Chimeric Proteins; Chromosomal translocation; Clinical Trials; Collection; Complex; Data; Development; Dissection; Dysmyelopoietic Syndromes; EWS-FLI1 fusion protein; Enhancers; Ewings sarcoma; Exhibits; Exons; Generations; Genes; Genetic; Genetic Transcription; Global Change; Guide RNA; Heterogeneous-Nuclear Ribonucleoprotein K; Human; Investigation; Knowledge; Lead; Length; Malignant Neoplasms; Mesenchymal Stem Cells; Messenger RNA; Modeling; Mutation; Names; Normal tissue morphology; Oncogenic; Outcome; Pathogenesis; Pathway interactions; Patients; Pattern; Pediatric Neoplasm; Phase I Clinical Trials; Process; Protein Isoforms; Proteins; RNA; RNA Binding; RNA Helicase; RNA Interference; RNA Sequences; RNA Splicing; RNA helicase A; RNA-Binding Proteins; Recurrence; Regulation; Reporting; Role; Series; Site; Spliceosomes; Stratification; Technology; Testing; Time; Transcriptional Regulation; Untranslated RNA; Variant; analog; anti-cancer; base; improved; inhibitor; innovation; leukemia; multidisciplinary; mutant; new therapeutic target; novel; overexpression; personalized medicine; protein protein interaction; sarcoma; senescence; side effect; single molecule; single molecule real time sequencing; small molecule; therapeutic target; tumor; tumorigenesis

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.

剪接体是蛋白质和非编码RNA亚基的集合，与结合，裂解和 法长RNA。替代剪接可以通过新颖的表达来有助于癌症的发展 蛋白质同工型。调节剪接的致癌驱动基因由突变，过表达和 白血病，癌和肉瘤中的染色体易位。 EWS-FLI1就是这样的致癌 融合蛋白源自ewing肉瘤中肿瘤特异性染色体易位（ES）。以前的 调查已经描述了EWS-FLI1转录的调节，并与转录有关 它的致癌潜力，但是一些不结合DNA的突变体仍然具有致癌活性。这暗示着 EWS-FLI1在转录调控之外具有致癌能力。我们发现EWS-FLI1 与剪接蛋白相互作用，并显着改变ES中的同工型景观。其他人已经表现出来 某些剪接因子对于EWS-FLI1肿瘤发生至关重要。但是，剪接对ES的贡献 EWS-FLI1相互作用蛋白在剪接体中的肿瘤生成以及EWS-FLI1相互作用的作用仍然未知。 EWS-FLI1通常被称为“不可能”的目标。制定治疗的替代策略 靶向EWS-FLI1，我们确定了直接与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，名为YK-4-279，它阻止了EWS- FLI1与关键蛋白质伴侣结合。用YK-4-279处理的ES细胞显示出改变的剪接模式 模拟EWS-FLI1损失。现在，在ES患者中，正在进行I期临床试验中YK-4-279，TK216的类似物。我们 因此，假设调节少数关键基因的RNA剪接是一个速率 限制EWS-FLI1的致癌机制，除了其典型作用作为转录 监管机构。我们将该提案重点放在三个目标上。 （1）我们将确定EWS-FLI1的相对效应 通过表征关键域和残基的转录和剪接的突变体。然后我们会 确定这些突变体对肿瘤发生的影响。 （2）我们将定义EWS-FLI1之间的相互作用 和差剪接所需的剪接因子。 （3）我们将研究EWS-FLI1诱导的剪接如何 靶基因的同工型切换有助于造成肿瘤。我们证明了EWS-FLI1诱导 人间充质干细胞中许多靶基因的差异剪接（HMSC）；现在我们会的 识别诱导差异剪接的EWS-FLI1中的关键域和残基。我们的方法也将 回答EWS-FLI1是否创建了从ES中独特发现的从头剪接变体，还是EWS- FLI1是通路的一部分，该途径导致剪接体具有类似于发生的剪接活动的新型剪接活动 在骨髓发育综合综合征中。在特定肿瘤中改变的剪接驱动因素的详细知识将 增强我们对肿瘤发生的理解，导致个性化医学的分层标记，并 告知新的反癌目标的方法。