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，相互作用以结合、切割和剪接连接RNA。选择性剪接可以通过新的表达促进癌症的发展蛋白质亚型。调节剪接的致癌驱动基因由突变、过度表达和白血病、癌和肉瘤中的染色体易位。 EWS-FLI1就是这样一种致癌物质源自尤文肉瘤 (ES) 肿瘤特异性染色体易位的融合蛋白。以前的研究描述了 EWS-FLI1 对转录的调节，并将转录与其致癌潜力，但一些不结合 DNA 的突变体仍然具有致癌活性。这表明 EWS-FLI1 具有转录调控之外的致癌能力。我们发现EWS-FLI1 与剪接体蛋白相互作用并显着改变 ES 中的亚型景观。其他人已经表明一些剪接因子对于 EWS-FLI1 肿瘤发生至关重要。然而，剪接对 ES 的贡献肿瘤发生以及 EWS-FLI1 相互作用蛋白在剪接体中的作用仍然未知。 EWS-FLI1 通常被称为“不可成药”的靶标。制定替代治疗策略针对 EWS-FLI1，我们鉴定了直接与 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，它可以阻断 EWS- FLI1 与关键蛋白质伙伴结合。用 YK-4-279 处理的 ES 细胞显示出剪接模式的改变模拟 EWS-FLI1 损失。 YK-4-279 的类似物 TK216 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- 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) 我们将确定 EWS-FLI1 的相对影响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之间的交互以及差分拼接所需的拼接因子。 (3)我们将研究EWS-FLI1如何诱导剪接靶基因的亚型转换有助于肿瘤发生。我们证明 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。