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Dissection of EWS-FLI1 oncogenic mechanisms and small molecule targeting

剖析 EWS-FLI1 致癌机制和小分子靶向

基本信息

批准号：
10058057
负责人：
JEFFREY A TORETSKY
金额：
$ 41.61万
依托单位：
GEORGETOWN UNIVERSITY
依托单位国家：
美国
项目类别：
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/10058057
关键词：
Alternative Splicing Apoptosis Binding Bioinformatics Biophysics CRISPR screen Cancer Patient Carcinoma Cell Line Cells Cessation of life Chimeric Proteins Chromosomal translocation Cleaved cell 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/antagonist 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

项目摘要

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，它们相互作用以结合、切割和整合RNA。连接RNA。选择性剪接可以通过表达新的蛋白质异构体。调节剪接的致癌驱动基因由突变、过表达和突变引起。白血病、癌和肉瘤中的染色体易位。EWS-FLI 1是一种这样的致癌基因来源于尤文肉瘤（ES）中肿瘤特异性染色体易位的融合蛋白。先前研究已经描述了EWS-FLI 1对转录的调节，并将转录与EWS-FLI 1的转录联系起来。它的致癌潜力，但一些突变体，不结合DNA仍然有致癌活性。这表明 EWS-FLI 1具有转录调控之外的致癌能力。我们发现EWS-FLI 1 与剪接体蛋白相互作用并显著改变ES中的同种型景观。其他人已经表明一些剪接因子对于EWS-FLI 1肿瘤发生是关键的。然而，剪接对ES的贡献肿瘤发生以及剪接体中EWS-FLI 1相互作用蛋白的作用仍然未知。 EWS-FLI 1常常被称为“不可治疗”的目标。开发替代治疗策略针对EWS-FLI 1，我们鉴定了直接结合EWS-FLI 1并抑制其相互作用的化合物具体的合作伙伴。2009年，我们报道了一种名为YK-4-279的化合物，它可以阻断EWS， FLI 1与关键蛋白质伴侣结合。用YK-4-279处理的ES细胞显示改变的剪接模式，模拟EWS-FLI 1缺失。YK-4-279的类似物TK 216目前正在ES患者中进行I期临床试验。我们因此，假设少数关键基因的RNA剪接调节是一个速率- EWS-FLI 1除了作为转录因子的典型作用外，调节器我们把这项建议的重点放在三个目标上。(1)我们将确定EWS-FLI 1的相对影响突变体的转录和剪接通过表征关键结构域和残基。然后我们将确定这些突变体对肿瘤发生的影响。(2)我们将定义EWS-FLI 1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-FLI 1诱导的剪接靶基因的同种型转换有助于肿瘤发生。我们证明了EWS-FLI 1诱导在人类间充质干细胞（hMSC）中的一些靶基因的差异剪接;现在我们将鉴定EWS-FLI 1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-FLI 1是否产生ES中唯一发现的从头剪接变体，或者EWS-FLI 1是否产生ES中唯一发现的从头剪接变体。 FLI 1是导致剪接体的途径的一部分，剪接体具有与发生的剪接体类似的新剪接活性。骨髓增生异常综合征详细了解在特定肿瘤中发生改变的剪接驱动程序，增强我们对肿瘤发生的理解，为个性化医疗提供分层标志物，为新的抗癌靶点提供信息。